5Th International Symposium on Sturgeon

1 International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir

Organized by: Iranian Fisheries Science Research Institute International Sturgeon Research Institute Guilan University World Sturgeon Conservation Society

Sponsored by: Agricultural Research Education and Extension organization (AREO) Iranian Fisheries Organization Governor of Guilan Province Management and Planning Guilan Province Jihad- e – Nasr Institute Fisheries Department of Guilan Province Guilan Agricultural Jihad Organization Islamic Revolution Mostazafan Anzali Free Zone Organization Agricultural Services Specialized holding Company

Conference Director Prof. Mohammad Pourkazemi

Conference Secretory Dr. Mohammadali Yazdani Sadati

Conference Secretariat Coordinator Dr. Rezvanollah Kazemi

Steering Committee Prof. Mohammad Pourkazemi Iranian Fisheries Science Research Institute, Tehran, Iran Prof. Ahmad Razi Guilan University Dr. Mohammadali Yazdani International Sturgeon Research Institute, Rasht, Iran Prof. Harald Rosenthal World Sturgeon Conservation Society, Neu Wulmstorf, Germany Dr. Paolo Bronzi World Sturgeon Conservation Society, Italy

Scientific programmed Committee Prof. Mohammad Pourkazemi (Conference chair) Dr. Mohammadali Yazdani Sadati (Conference secretary) Prof. Harald Rosenthal, FRG (Conference Co-chair WSCS President) Dr. Paolo Bronzi, Italy (Vice President WSCS) Prof. Michaeil Chebanov (Russia) Dr. Jorn Gessner (Germany) Dr. Leonardo Conjiu (Italy) Dr. Mirmasoud Sajjadi (Iran) Dr. Homayoun Hosseinzadeh sahafi (Iran) Prof. Sohrab Rezvani Gilkolaei (Iran) Prof. Wei Qwei (China) Dr. Hassan Salehi (Iran) Prof. Gobad Azari Takami (Iran) Dr. Rezvanollah Kazemi (Iran) Dr. Ayoub Yousefi Jourdehi (Iran) Dr. Zohreh Ramezanpour (Iran) Dr. Hossein Abdolhay (Iran) Prof. Mehdi Soltani (Iran) Prof. Bagher Mojazi Amiri (Iran) Prof. Mahmoud Bahmani (Iran) Dr. Ali Asghar Khanipour (Iran) Dr. Issa Sharifpour (Iran) Prof. Tooraj Valinasab (Iran) Dr. Mostafa Sharifrouhani (Iran) Prof. Bahram Falahatkar (Iran) Dr. Ali Bani (Iran) Dr. Shahram Abdolmalaki (Iran) Dr. Mahtab Yarmohammadi (Iran) Dr. Mahmoud Mohseni (Iran) Dr. Seyed Jalil Zorriehzahra (Iran) Dr. Abbas Matinfar (Iran) Dr. Yazdan Moradi (Iran) Dr. Nima Pourang (Iran) Dr. Alireza Shenavar Masoleh (Iran) Mr. Mahmoud Shakourian (Iran) Mr. Zabihollah Pajand (Iran) Dr. Ali Hosseinpour Zelati (Iran) Mr. Mahmoud Tavakoli (Iran)

Local Organizing Committee Prof. Mohammad Pourkazemi Prof. Eskandar Zand Dr. Hassan Salehi Dr. Mohammd Ali Yazdani Sadati Prof. Ahmad Razi Mr. Mohammad Hossein Asgharian Dr. Seyyed Ziaoddin Mirhosseini Dr. Keyvan Mohammadi Dr. Issa Sharifpour Dr. Rezvanollah Kazemi Mr. Eshagh Shabani Mr. Alireza Shaban Nezhad Mr. Naser Karami Rad Mr. Ahmad Ghanaatparast Mr. Samad Darvishi Mr. Iraj Effatpanah Komaei Mr. Mahmoud Shakourian Mr. Mohammadreza Behrouz Khoshghalb Mr. Seyyed Sadroddin Hozni Mr. Jalil Jalilpour Mr. Mahmoud Fallah Shojaei 5-6, September 2016 Rasht: Guilan University Secretariat: International Sturgeon Research Institute, P.O.Box 41635-3464, Rasht-Iran TelFax: ++98-13-34506142 www.ICSA2016.ir IV

International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir

Conference Secretary Committee Dr. Rezvanollah Kazemi Dr. Ayoub Yousefi Jourdehi Dr. Zohreh Ramezanpour Mr. Zabihollah Pajand Mr. Koroush Haddadi Moghadam Mr. Seyyed Ali Mosavi Miss. Bahareh Younes Haghighi Miss. Bemani Sasani Mrs. Bahareh Deldar Conference Website Committee Mr. Mahmoud Fallah Shojaei Mr. Jalil Jalilpour Mr. Mohammadreza Behrouz Khoshghalb

Conference Audiovisual and Publication Committee Mr. Hamed Yousefpour Mr. Behrouz Fadaei Mr. Mohammadreza Behrouz Khoshghalb Dr. Zohreh Ramezanpour Mrs. Fruzan Chubian Mr. Nemat Peykaran Mana Mr. Mohammadreza Safari Mr. Mohebali Pourgholam Mr. Hamidreza Pourali Fashtomi Mr. Abrahim Dehghan Mr. Amin Farahbod Roudbaraki Mrs. Bahareh Deldar Dr. Frouzan Bagherzaheh

Conference Security Committee Mr. Seyyed Sadroddin Hozni Mr. Sohrab Alimohammadi Mr. Esmaeil Farzaneh Mr. Mohammad Nazari Dr. Ali Hosseinipur ZeLti Mr. Hooshang Yeganeh Mr. Majid Poursafar Mis. Bahareh Younes Haghighi Mrs. Fatemeh Rezakhah

Coference Workshop Committee Dr. Mahtab Yarmohammadi Mr. Ali Hallajian Mr. Shahrooz Baradaran Noveiri Mr. Mohammad Hasanzadeh Saber Mr. Amin Farahbod Roudbaraki Mr. Soheil Bazarimoghaddam Mr. Javad Sayyadfar Mr. Mirhamed Seyyed Hasani

5-6, September 2016 Rasht: Guilan University Secretariat: International Sturgeon Research Institute, P.O.Box 41635-3464, Rasht-Iran TelFax: ++98-13-34506142 www.ICSA2016.ir V

International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir

Conference Posters Committee Mr. Mahmoud Tavakoli Dr. Mehdi Masoumzadeh Mr. Mehdi Alizadeh Roudposhti Dr. Mohammad Reza Nowruzfashkhami Conference Finance Committee Mr. Touraj Raoofi Mr. Seyyed Hossein Hosseini Aghoozboneh Miss. Fahimeh Azimi Conference Support Committee Mr. Alireza Alipour Joursheri Mr. Ghorban Kamranjoo Mr. Azizollah Hosseinpour Mr. Alireza Ashoori Mr. Esmaeil Hooseinnia Mr. Mehdi Izadi Mr. Hashem Joushideh Mr. Ahmad Nezami Bloochi Mr. Bahman Shafiei Conference exhibition, Marketing and booth Committee Dr. Alireza ShenavarMasouleh Dr. Tooraj Sorabi Langroudi Mr. Mohammad Pourdehghani Mr. Mahmoud Shakourian Mr. Shahram Mohammadi

Preface Considering to the importance of economic and ecological development of sturgeon aquaculture programs in the world and the need for research achievements in order to increase production efficiency and optimization methods for breeding sturgeon, the International Conference on the Future of Sturgeon Aquaculture (ICSA2016) was held on the below purposes and axes: 1- Farming management (Pond, Cage, Pen, RAS, ...) 2- Nutrition and Feeding 3- Induced Reproduction 4- Pathology and Diseases 5- Applied Breeding and Genetics 6- Ecology And Environmental Interaction 7- Processing, Economic and Marketing International conference on the future of sturgeon aquaculture was held on 16-15 September 2016 in collaboration with the International Sturgeon Research Institute, the Iranian Fisheries Research Institute, University of Guilan and the World Association of Sturgeon Protection (WSCS) with presence of more than 350 people. The national and provincial fishery managers, executives Guilan province and Iranian fisheries scientists, foreign guests and students in 9 specialized panels (nutrition, reproduction, physiology, processing, ecology and pollutants, marketing, health and diseases). The newest achievements of the sturgeon aquaculture science were presented by researchers in oral and poster papers during the two days. Simultaneously during the conference organizing three workshops such as Cage Culture, Applied Genetics, Current and Future Caviar Markets were held and more than 140 people attended on the workshops and also an exhibition was held in cooperation with 9 companies and firms. After creating dedicated web conference call in late April 2016 a total of 183 articles reached to the conference secretariat. After the referee of papers by 55 researchers, professors and cultural centers; 38 oral and 116 posters papers were accepted. Based on reviewer comments, 29 articles were rejected because of the lack of scientific quality. At the end of the conference three oral and poster papers were awarded prizes.

Contents Page Key Lectures

Sturgeon aquaculture development and the Brand of Iranian caviar in International Market 2 Mohammad Pourkazemi

Farming of sturgeon in Iran, challenge and programme 4 Hossein Ali Abdolhay, Naser Karami Rad

Sturgeon aquaculture and its achievements in Iran 6 Mohamad Ali Yazdani Sadati Aquaculture

Feasibility study to develop sturgeon culture in Chalous Shahid Kashiri catch station Kourosh Haddadi Moghaddam, Ayoub Yousefi Jourdehi, Zabihola Pajand, Fruzan Chubian, Esmail 9 Farzaneh, Esmail Hossein Nia

Rotifer and its role in feeding in Persian sturgeon (Acipenser persicus) larvae 11 Kourosh HaddadiMoghaddam, Zabihola Pajand, Fruzan Chubian, Bahare Younes Haghighi

A trigger to high survivorship of the Persian sturgeon (Acipenser persicus) larvae released into the Caspian Sea waters 13 Mahmoud Hafezieh

The impact of Tocopherol (E) and Riboflavin (B2) Vitamins on growth and nutritional Indices of Sturgeon Fingerlings (Acipenser nudiventris) Ali Reza Ashouri, Hossein Khara, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Ismael 14 Hossein Nia, Zabihollah Pajand, Nemat Peikaran Mana

Effect of ozone application to improve closed system performance for sturgeon culture 16 Azam Mohammad Salehi, Ebrahim Rajabzadeh Ghatramie

Comparison of F1 and F2 bester, big bester (back-cross of bester with beluga) and beluga growth during one year rearing period Shahrouz Baradaran Noveiri, Mohammad Pourkazemi, Mohammad Hassanzadeh Saber, 18 Mohammadali Yazdani, Mohammad Pourdehghani, Rezvanollah Kazemi, Hossein Mohammadi Parashkuh, Mohammadreza Nowrouzfashkhami

Effects of Biomin as Nutritional Supplement on the Growth and Immune Responses of Acipenser ruthenus 20 Mohammad Ali Bisheban, Abbas Ali Zamini , Mehrdad Nasri Tajan

Rearing and growth of white worm (Enchytraeus albidus) with Azolla in Comparson to Other Diets Esmaeil Hossein Nia, Ghobad Azari Takami, Koresh Hadadi Moghadam 21

Check temperature in the growth of white worms (Enchytraeus albidus) 23 Esmaeil Hossein Nia, Mohsen Poor Asadi, Ali Reza Ashoori, Nemat Peikaran Mana

The Importance of moisture on the growth of white worm (Enchytraeus albidus) 25 Esmaeil Hossein Nia, Ghobad Azari Takami, Ayoub Yousefi, Rezvanollah Kazemi Proper soil pH for the growth of white worm (Enchytraeus albidus) as live feed Esmael Hossein Nia ,Ghobad Azari Takami, Ali Reza Ashoori, Nemat Peikaran Mana 27

Effects of micro/ nano-encapsulation of Lactobasillos bulgaricus with alginat/ chitosan on growth performance and Feed efficiency of great sturgeon juveniles Huso huso. Seyed Samad Hosseini, Mojtaba Alishahi, Koroush Amini, Mohammadreza Abbaspour, Masoud 29 Ghorbanpour, Takavar mohammadian

The effect of ascorbic acid and vitamin E (tocopherol) supplementation in broodstock feed on reproduction of Persian sturgeon (Acipenser persicus, Borodin, 1897) Mahmoud Mohseni, Mohammad Pourkazeni , Mahmoud Bahmani, Hamid Reza Pourali, Ali Hallajian, Rezvanollah Kazemi , Mir Hamed Seyed Hassani , Mohammad Pourdaghan, Ayoub 31 Yousefi Jourdehi

Dietary vitamin E requirement of juvenile beluga, Huso huso, at two lipid levels, and their effects on immune responses 32 Mahmoud Mohseni, Mir Hamed Seyed Hassani, Mohammad Ali Yazdani, Hamid Reza Pourali, Rezvanollah Kazemi, Ali Hallajian, Sungchul, C. Bai

The effect of replacing fish meal with poultry by-product meal on growth rate and biochemical and immunological indices in fingerling Beluga (Huso huso Linnaeus, 1758) 35 MirHamed Sayed Hassani, Mohamad Ali Yazdani, Hamid Rez Pourali, Ayoub Yousefi Jourdehi, Mahmoud Shakourian, Hoshang Yeganeh, Javad Sayadfar

Reproductive performance in Sterlet sturgeon (Acipenser ruthenus) injected with thiamine and the resulting larval quality 37 Bahram Falahatkar, Sareh Ghiasi, Murat Arsalan, Konrad Dabrowski

Feasibility study to develop sturgeon culture at the Larim Shahid Gholami catch station Ali Hallajian, Mohammd Pourkazemi, Rezvanollah Kazemi, Ayoub Yousefi Jourdehi, Mahmoud 39 Mohseni, Mahtab Yarmohammadi.

Raising surgery operated sturgeon brood stocks using pen culture in the Gorgan Bay (Caspian Sea) Nemat Peykaran Mana, Hamid Reza Pourali Fashtomi, Masoud Seidgar. Mohammad Pourkazemi, 41 Mahmoud Shakorian, Mohammad Ali Yazdani Sadati, Esmaeil Hosseinnia, Alireza Ashoori.

Artemia cyst decapsulation and its necessity in sturgeon larvae feeding Nemat Peykaran Mana, Masoud Seidgar, Latif Esmaeili Dahesht, Hadi Asadi, Esmaeil Hosseinnia, 43 Alireza Ashoori.

Dietary administration of Lactobacillus plantarum enhances both, growth performance and innate immune response of Siberian sturgeon, Acipenser baerii Moheb Ali Pourgholam, Hossein Khara, Reza Safari, Mohammad Ali Yazdani Sadati, Mohammad 45 Sadegh Aramli, Mir Hamed Sayed Hassani

The effect of Allium sativum on the growth performance of Juvenile Siberian sturgeon (Acipenser baerii) 47 Hooshang Yegane Rastekenari, Habibvahabzade Roudsari, Mohamad Ali Yazdani Sadati

The effect of L – Carnitin on growth rate, body composition and hematological tT Indices of Juvenile Acipenser nudiventris 49 Mohamad Ali Yazdani Sadati., Mir Hamed Sayed Hassani, Mina Habibzade

Effect of fish oil and vegetable oil on growth performance fo sterlet (Acipenser ruthenus) broodstock 53 Sara Pourhosein Sarameh, Bahram Falahatkar, Amir Bahri, Mahtab Yarmohamadi, Ali Reza Salarzadeh

The feasibility of creating a database of live sturgeon brood stocks (500 breeders) in Pen culture conditions in the Gorgan Gulf 56 Nemat Peykaran Mana, Mohmmad Pourkazemi, Ayoub Yousefi Jourdehi, Seyyed Hamed Hassani, Hadi Asadi, Seyed Mustafa Aghilinejhad, Shahram Mohammadi Gholinejhad

Effect of diet composition on growth performance and hepatic metabolism in Siberian sturgeon, Acipenser baerii submitted to starvation and refeeding 59 Sedigheh Babaei, Abdolmohamad Abedian Kenari, Mehdi Hedayati, Mohamad Ali Yazdani Sadati Effects of stable barium isotope on growth and barium concentration in the muscle of Persian sturgeon (Acipenser persicus) 62 Hadis Mirali, Ali Bani, Javad Fasihi

A study on effects of light period and feeding regime on juvenile Huso huso growth performances 64 Mohsen Pourasadi, Bahram Falahatkar, Mehran Avakh Keysami, Mohammad Rahanande, Iraj efat penah Komaee Ahmad Razavi sayad, Bahman Meknatkhah

Study on the effects of Aloe vera extract on growth parameters and bacterial flora of intestine in Siberian sturgeon, Acipenser baerii Soheil Bazari Moghaddam, Masoud Haghighi, Mostafa Sharif Rohani, Mohammad Ali Yazdani, 66 Alireza Shenavar Masouleh, Jalil Jalilpour, Mehdi Masoumzadeh, Mehdi Alizadeh

Effects of organic culture on growth in juvenile Siberian sturgeon, Acipenser baerii 67 Rahin Alsadat Hasehemi, Mahmoud Bahmani, Ayoub Yousefi Jourdehi

Benefits of extruded feeds in aquaculture 69 Nigel Linley

Comparison of growth and microstructure pattern in the pectoral fin spines of reared juvenile Persian sturgeon, Acipenser persicus in the south Caspian Sea Shima Bakhshalizadeh, Ali Bani, Shahram Abdolmalaki 73

The possibility of farming fleshy Beluga Huso huso in earthen ponds in West Azerbaijan province 75 Yousef Ali Asadpour, Alireza Azarvandi, Ehsan Soleimannejad bari, Masoud Seidgar

Study on fatty acid profile in Persian sturgeon larvae (Acipenser persicus) using algae Scenedesmus dimorphus 76 Fruzan Chubian, Zohreh Ramezanpour, Mahmoud Hafeziyeh, Koroush Haddadi Moghadam, Zabihollah Pajad, Bahareh Younes Haghighi, Hamidreza Pourali, Rudabeh Rufchaie

Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Acipenser baerii juveniles. Mir Hamed Sayed Hassani, Ali Hossien pour Zelti, Alireza Shenavar, Forouzan Bagerzadeh Lakani, 78 Mohamad Ali yazdani Sadati, Mahmoud Shakourian, Mahmoud Mohseni, Hooshang Yegane, Mohebali Pourgolam, Alireza Alipour, Hamid Reza pouali, Javad Syad far,Ghorban Kamranjo

Effects of different copper nanoparticle levels on bioaccumulation in organs of Siberian sturgeon (Acipenser baerii) juvenile: Comparison of dietary and waterborne exposure 80 Forouzan Bagherzadeh Lakani, Saeid Meshkini, Mohammad Ali Yazdani Sadati, Bahram Falahatkar

Growth and survival of Persian Sturgeon, Acipenser persicus larvae in Relation to the Consumption of Different Composition of Diet 82 Zabih Ollah Pajand, Korosh Haddadi Moghadam, Fruzan Choubian, Esmaeil Farzaneh, Esmaeil Hosseinnia, Alireza Ashori

The comparison of different pond type in sturgeon aquaculture in Iran Mohammad Pourdehghani, Mohammad Ghorbani, Forouzan Bagherzadeh Lakani, Yashar 85 kohalvandi, Farnoush Alizadeh, Ismaeil Mirabdollahi, Ali Hamidoghli

Nemat Paykaran Mana*, Hamid Reza Pour Ali Fashtami, Mir Hamed Sayad Hassani , Mohamad 89 Pour Kazemi, Mohamad Ali Yazdani, Sayed Mostafa Aghili Nejad.

The use of Inland waters of Iran for sturgeon culture 91 Mahmoud Ramin, Mastooreh Doustdar, Nader Nakhaei.

Natural polymers: new candidates for antibiotics in sturgeon culture 93 Ebrahim Hossein Najdegrami , Peter Bossier

Review of aquaponics system 96 Seyedeh Yalda Bani Esmaeili

The role of phospholipids in improving growth and survival of sturgeon and teleost fish 98 Naser Agh, Fatemeh jafari, Esmaeil Paghe, Farzaneh Noori

The effect of dietary glutamine supplementation on growth performance in Acipenser baerii (Brandt, 1869) 101 Saman Darvishi, Hossein Khara, Mohaddeseh Ahmadnezhad

The effect of density on mean individual weight of Beluga (Huso huso) juveniles reared in fiberglass tanks (Voshmgir Dam Recruiting Centre) 104 Abasali Aghaei Moghadam, Kamran Aghili, Sara Haghparast..

Comparision quality of chironomid larvae on different feeding substrates to feed farmed fish fingerlings Ahmad Nezami Baluchi, Hamid Reza Pourali Fashtomi, Mohammad Ali Yazdani Sadati, Nemat 107 Peykaran Mana, MirHamed Seyed Hasani.

Mass production of Daphnia spp. using sewage outlet of fish rearing tanks Javad Syad far, Mohamad Ali Yazdani Sadati, Alireza Alipour, Mir Hamed Sayed Hassani, Hamid 110 reza Pourali, Ali Hossien pour Zelti, Ghorban Kamranjo

Comparison between the effect of different levels of dietary probiotic (Bactocel), on growth and nutrition performance and survival rate in sturgeon (Huso huso) 114 Ali Akbar jafari, Hosean mollaaghaee, Behnam zargari, Khosro Shirghazi, Pedram seraji, Behzad eslami

Mass production of Daphnia sp using and replace of (mixture of yeast and molasses and rice bream) instead of organic manure. 116 Javad Syad far, Mohamad Ali Yazdani Sadati, Mir Hamed Sayed Hassani, Ali Hossien pour Zelti

Effects of starvation and re-feeding on histological changes of intestine in Persian sturgeon Acipenser persicus 117 Mahtab Yarmohammadi, Mohammad Pourkazemi, Rezvanollah Kazemi, Ali Hallajian , Ayoub Yousefi Jourdehi, Amin Farahbod Roudbaraki ,Mamoud Shakourian

Designing of cages for culture of aquatic animals Hossein Askarinejhad, Hojjat Ahmadi, Ayoub Yousefi Jourdehi 118

Ecology:

Changes in sedimentation, sediment characteristics, and benthic macro faunal assemblages in Huso huso and Rainbow trout (Oncorhynchus mykiss) reared in sea cage in the 120 areas of Guilan and Mazandaran Provinces (Caspian Sea) Kourosh HaddadiMoghaddam, Mehdi soltani, Abolgasem kamali, Hossein Ali Abdolhay

Bioaccumulation of Toxic metal cadmium in muscle and liver of cultured Huso huso 123 Mehrnoush Norouzi, Abuzar Galianmoghadam

The bioremediation of wastewater using Nereis diversicolor in an integrated culture system with beluga, Huso huso (Linnaeus, 1758) 125 Zabih Ollah Pajand, Mehdi Soltani, Abolghasem Kamali, Mahmoud Bahmani

Semi mass culture of freshwater rotifers for Acipenser persicus larvae 128 Rudabeh Rufchaie, Reza Armodli, Sahebali Ghorbani

Geochemical Partitioning of heavy metals in surface sediment of south Caspian Sea and Anthropogenic evaluation 133

Tooraj Sohrabi, Hossein Saberi, Adel Hosseinjani, Gholamreza Mehdizadeh

Assessment of heavy metal enrichment and contamination factors in surface sediment of south coastal zone of Caspian Sea 135 Tooraj Sohrabi, Hossein Saberi, Mohammad Salvatian

Ecosystem service modeling of marine aquaculture in Mazandaran province 136 Elham Haghshenas, Mehdi Gholamalifard, NematMahmoudi

Enrichment of Daphnia spp. by algae Chlorella vulgaris in order to improve profile of polyunsaturated fatty acids (PUFA) in larvae of Persian sturgeon (Acipenser persicus) 140 Fruzan Chubian, Zohreh Ramezanpour, Mahmoud Hafeziyeh, Koroush Haddadi Moghadam, Zabihollah Pajad, Bahareh Younes Haghighi, Hamidreza Pourali, Rudabeh Rufchaie

Population dynamic of Acipenser persicus (Borodin, 1897) in the southern Caspian Sea (Guilan province) using Monte Carlo and Bootstrap methods Amirali Moradinasab, Moslem Daliri, Hadi Raeisi, Sarah Haghparast, Ayoub Yousefi Jourdehi, 142 Mahmoud Bahmani, Amin Farahbod Roudbaraki, Arezoo Vahabnejhad

The toxicity effect of Fe3O4, SiO2 and SnO2 nanoparticles on Daphnia magna in larviculture of sturgeon 145 Fatemeh Shariati, Zohreh Ramezanpour, Tahereh Pourdeljoo

The effect of Beluga (Huso huso) cages on water abiotic factors in the southern part of Caspian Sea (Jafroud region) 148 Zabih Ollah Pajand, Korosh Haddadi Moghadam, Zohreh Ramzanpor, Fruzan Choubian1, Esmaeil Farzaneh

The enrichment of Nereis diversicolor using of Bacillus and Lactobacillus probiotic in Acipenser baeri 151 Esmaiel Farzaneh, Hossein Khara, Zabihollah Pajand

An investigation on macrobenthic communities in Chabuksar Shore of the Caspian Sea and their importance in the nutrition of sturgeons 153 Shahpor Gholami , Maryam Shapoori, Zabih hollah Pajand

Qualitative and Quantitative survey of released sturgeon fingerlings in the Guilan province in 2015 158 Sayed Ali Mosavi, Shahram Abdolmalaki, Mahmoud Tavakoli, Mohammad Reza Behrouz khoshghalb and Hashem Joshideh

Conservation of Caspian south basin rivers for sustainable fish exploitation 161 Mahmoud Ramin, Mastooreh Doustdar.

The first report of Acipensiridae larvae in natural habitat of Coastal southeastern waters of Caspian Sea 162 Zohreh Mazaheri Kohanestani, Rasoul Ghorbani, Mahnaz Rabbaniha, Kourosh Amini

Biological control of algal bloom in sturgeon pond using rice straw powder 165 Zohreh Ramezanpour, Zahra Ebadi, Fruzan Chubian, Rodabe Rufchaeii, Alireza Shenavar, Kurosh Hadadi Moghadam

The role of the release on the sturgeon population in southeastern coast of the Caspian Sea (the Golestan province) 166 Gholamali Bandani, Mahmoud Tavakoli, Amin Mirhashami, Mohammad Larijani Genetics:

The influence of gamma and UV irradiation on the motility of ship sturgeon Acipenser nudiventris sperm 170 Mohammd Hassanzadeh Saber, Mohammad Pourkazemi, Sharouz Baradaran Noveiri, Gholam Reza Shahhosseini

Induction of triploidy in the hybrids obtained from ♀ ship sturgeon and ♂ Siberian sturgeon: Possibility of tetraploidy and its verification using microsatellite marker 173 Mohammad Hassanzadeh Saber, Mohammad Pourkazemi, Mahtab Yarmohammadi

Cross-species amplification of Acipenseridae microsatellite DNA markers in stellate sturgeon, Acipenser stellatus 174 Mehrnoush Norouzi

Population identification of Acipenser persicus in two main rivers of the South Caspian Sea, using microsatellite markers 178 Fereidon Chakmehdouz Ghasemi, Mohammad Pourkazemi, Mahtab Yarmohammadi

Transcription survey of P450 in response to Endosulfan in the gill of Persian Sturgeon (Acipenser persicus) Roghieh Safari, Abasali Hajibeigloo 180

Possibility of hybridization in Stellate sturgeon (Acipenser stellatus ♀) × Siberian sturgeon (Acipenser baeri ♂) using cryopreserved sperm Bahram Falahatkar, Samaneh Poursaeid, Bahman Meknatkhah, Iraj Efatpanah, Mehdi Rahmati 183

Genetic Diversity Evaluation of Great Sturgeon (Huso huso) reared in the Shahid Dr. Beheshti Restoration Center

Mahtab Yarmohammadi, Samad Darvishi, Rezvanollah Kazemi, Mohammad Ali Yazdani Sadati, Hosein Mohammadi Parashkooh, Yalda Baniesmaili 185

Molecular Identification and expression profile of the sex-candidate genes during gonad developmental stages in great sturgeon Huso huso 187 Mahtab Yarmohammadi, Mohammad Pourkazemi, Rezvanollah Kazemi, Mohammad Ali Yazdani

Sadati , Mohammad Hassanzadeh Saber, Ali Hallajian, Ayoub Yousefi Jourdehi

A Review of Isolation and molecular identification of lactic acid bacteria in sturgeon Seyedeh Yalda Bani Esmaeili, Fatemeh Alaei, Niloofar Eradatmand 189

Physiology and Biochemistry

The Impact of Tocopherol (E) and Riboflavin (B2) Vitamins on Blood Factors and Immunity of Sturgeon Fingerlings (Acipenser Nudiventris) 191 Ali Reza Ashouri, Hossein Khara, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Ismael Hossein Nia, Zabihollah Pajand, Nemat Peikaran Mana

Effects of dietary phospholipids on alkaline phosphatase activity on stelate (Acipenser stellatus) 195 Fatemeh jafari, Naser Agh, Farzaneh Noori

The effect of niacin (vitamin B3) on growth, body composition, hematology and immune parameters of Sterlet (Acipenser ruthenus) 197 Rana Haghparast, Hossein Khara, Masod Farokhroz

Effects of dietary Saccharomyces cerevisiae on growth performance and immune system of Acipenser baerii juvenile. MirHamed Sayed Hassani, Mohamad Ali Yazdani, Hoshang Yeganeh, Mahmoud Shakourian, 199 Mahmoud Mohseni, Mohebali Pourgholam, Ali Hossien pour Zelti , Hamid Rez Pourali, Ayoub Yousefi Jourdehi, Amin Farahbod Roudbaraki, Alireza alipour

Determining of some liver enzymes levels in juvenile Persian sturgeon (Acipenser persicus) captured in the southern coasts of the Caspian Sea 201 Ali Hallajian, Rezvanollah Kazemi, Ayoub Yousefi Jourdehi, Sohrab Dejandian, Mahtab Yarmohammadi, Mahmoud Mohseni.

Sturgeon Induced Reproduction Instructions 204 Liyuan Qiu

Changes in cortisol, estradiol-17β and thyroid hormones during the embryonic development of the Persian sturgeon, Acipenser persicus 206 Bahram Falahatkar, Parisa Haghparast

Caudal fin primary cell lines of Persian sturgeon Acipenser persicus Mohammad Reza Nowruzfashkhami*, Mohammad Pourkazemi, Mahmoud Bahmani, Morteza Daliri, 208 Mohammad Hassanzadeh Saber, Shahrouz Baradran Noveiri, Ahmad Ghoroghi

Primary culture of ovarian follicular cells of Sterlet Acipenser ruthenus Mohammad Reza Nowruzfashkhami, Mohammad Sudagar, Mahmoud Bahmani, Negin Salamat, 210 Mohammad Mazandrani, Mohammad Ali Yazdani Sadati

Different methods for sexing in sturgeon Rezvanollah Kazemi, Ali Hallajian, Ayoub Yousefi Jourdehi, Mahmoud Mohseni, Mahtab 214 Yarmohammadi, Mahmoud Shakourian

Effects of photoperiod and light intensity on amylase activity during early larval development of Persian sturgeon Acipenser persicus Farzaneh Noori, Elahe Niazi, Rezvanollah Kazemi, Rasul Ghorbani 216

The influence of stocking density on blood indices of ship sturgeon (Acipenser nudiventris) juveniles in Khuzestan province 218 Mohammad Yooneszadeh Feshalami, Seyed Abdolsaheb Mortazavizadeh, Farokh Amiri, Mansour Nikpey

Changes in sex steroid hormones of Siberian sturgeon Acipenser baerii during vitellogenesis in captivity Yalda Hooshyar, Ali Bani, MohammadAli Yazdani Sadati, Rezvanollah Kazemi 220

Assessing how juvenile beluga sturgeon responds to salinity exposure in terms of growth and osmoregulation Seyed Mostafa Aghilinejhad, Ali Jalali 223

Differential count of White blood cells in lethal concentration (lc 1h) of Zataria multiflora and Allium sativum in Persian sturgeon, Acipenser persicus, fingerlings 226 Soheil Bazari Moghaddam, Mostafa Sharif Rohani, Masoud Haghighi, Alireza shenavar Masouleh, Jalil Jalilpour , Mehdi Masoumzadeh, , Mehdi Alizadeh and Bahareh Younes Haghighi

Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Bester juvenile Ali Hossien pour Zelti, Mir Hamed Sayed Hassani, Forouzan Bagerzadeh Lakani, Alireza Shenavar, Mohamad Ali yazdani Sadati, Hooshang Yegane, Mohebali Pourgolam, Alireza Alipour, Javad Syad 227 far, Ghorban Kamranjo

Purification and Characterization of Lysozyme from Persian Sturgeon, Acipenser persicus 229 Roxana Badan-Ara, Mahmoud Reza Aghamaali, Mohammad Reza Nowruzfashkhami Study on applying counting number of sperm, measuring spermatocrite percent and spectrophotometry in determining sperm concentration in Huso huso of Caspian Sea 230 Alireza Alipour, Bahareh Younes Haghighi, Ali Hosseinpour, Ghorban Kamranju

Cholesterol and triglyceride level changes in farmed Acipenser nudiventris fingerlings exposed to acute toxicity of atrazine Marzieh Naji, Ayoub Yousefi Jourdehi, Sourena Abdali, Mohammad Ali, Yazdani Sadati 235

Study on the effects of Lactic acid bacteria Enterococcus faecalis on blood biochemical and serum factors in Persian sturgeon fingerlings

Mahdi Alizadeh Roud poshti, Alireza Shenavar Masouleh, Jalil Jalilpoor, Soheil Bazari Moghaddam, 238 Mehdi Masoumzadeh, Mahmoud Fallah Shojaei, Hoshang Yeganeh, Bahareh Younes Haghighi

Anatomic and morphometric study of the gastrointestinal tract in Beluga, Huso huso 240 Ala Enayati, Omid Zehtabvar, Ali Taheri Mirghaed, Hassan Morovati

Anatomical Evaluation of Cartilaginous-Skeletal Structures of Food Prehension In The Bester (Sterlet × Beluga) 242 Omid Zehtabvar, Ala Enayati, Ali Taheri Mirghaed, Khanamooei, M.

Anatomic and morphometric study of liver, spleen and pancreas in Beluga, Huso huso 243 Omid Zehtabvar, Ala Enayati, Seyed Fakhredin Borgheie, Ali Taheri Mirghaed, Hassan Morovati

Sex determination in Sturgeon 245 Abasali Hajibeglou and Roghaieh Safari

Histological evaluation of liver and some blood serum factors of farmed Beluga (Huso huso) Suspected fatty liver in sea cage Jalil Jalilpour, Ayoub Yousefi Jourdehi, Ali Halajian, Bahareh Younes Haghighi, Mehdi 247 Masoumzadeh, Soheil Bazari Moghaddam, Mehdi Alizadeh, Mohammad pourdehghani, Alireza Shenavar, Mahmoud Falah shojaee, Majid Poursafar.

Reducing the age of sex maturity in sturgeon

Ayoub Yousefi Jourdehi, Mahmoud Bahmani, Mohammad Sudagar, Mohammad Ali Yazdani Sadati, 249 Rezvanollah Kazemi, Homayoon Hosseinzadeh Sahafi, Ali Hallajian, Mohammad Pourdehghani, Mahtab Yarmohammadi

Histopathological effect of Ammonia on gill and kidney tissue in Acipenser ruthenus 254 Ayoub Yousefi Jourdehi, Sorena Abdali, Brat Ghrmanpour, Ali Hallajian

Ultrasonographic appearance of the gonads in Siberian sturgeon (Acipenser baerii) 255 Alireza Vajhi, Majid Masoudifard, Mehdi Moghim, Omid Zehtabvar

Induction of spawning in Iranian sturgeon (Acipenser persicus Borodin, 1897), by LHRH-a acute release implantation and its effects on reproductive indices. 256 Seyed Amin Mirhashemi Rostami,Kourosh Amini, Abbas Ali Aghaeemoghadam, Seyed Morteza Hosseni, Mohammad Mazandarani, M. Determination of some serum biochemical markers in sub-adult Huso huso 257 Mohammad Mazandarani, Seyyed Morteza Hoseini, Kourosh Amini, Ali Taheri Mirghaed

Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Acipenser baerii Mahmoud Shakorian, Alireza Alipour, Mohamad Ali yazdani, Mir hamed Sayed Hassani, Ali 260 Hossien pour Zelti, Alireza Shenavar, Forouzan Bagerzadeh Lakani, Hooshang Yegane,Mohebali Pourgolam, Hamid Reza pouali, Javad Syad far,Ghorban Kamranjo

Localization of mitochondria rich cells in the gill of Russian sturgeon Acipenser gueldenstaedtii by Immunohistochemistry technique 262 Salman Malakpour Kolbadinezhad, Jonathan Mark Wilson Fish Diseases and Health

A case study of skeletal deformity of Acipenser baerii in a recirculation farm in Khouzestan province 265 Mohammad Mehdi Haghparast, Mojtaba Alishahi, Reza Ghanei, Zahra tulabi

Prevention and the decontamination parasites in marine and fresh water fish at the time of harvesting, processing and post-processing 269

Mina Ahmadi, Ali Asghar Khanipour, Fatemeh Noghani, Soghra Kamali

Histopathological effects of trichodiniasis in farmed sturgeon Acipenser baerii 271 Zahra tulaby Dezfuly, AnnahitaRezaie, MojtabaAlishahi, Mohammad Mehdi Haghparastradmard

Isolation and identification of sturgeon probiotics in Beluga sturgeon (Huso huso) and Siberian sturgeon (Acipenser baerii)

Alireza Shenavar Masouleh, Mohammad Pourkazemi, Mohammadali Yazdani, Parisa Esmaeili, 273 Yalda Baniesmaeili, Mahtab Yarmohammadi, Mehdi Alizadeh, Jalil Jalilpoor, Mehdi Masoumzadeh, Soheil Bazarimoghddam, Mehdi Soltani

Monitoring of bacteria flora in intestine and water of Beluga, Huso huso, juveniles in earthen ponds in West Azerbaijan province 275 Yousef Ali Asadpour, Ehsan Soleimannejadbari, Abbas Zomorrodi

Histopathological effects of endosulfan on the gill of Persian sturgeon (Acipenser persicus Borodin, 1987) 276 Roghieh Safari, Abasali Hajibeglou

Sodium chloride as an agent of control of Saprolegniasis in Persian Sturgeon eggs 278 Abbas Ali Aghaei Moghaddam Kamran Aghili , Sara Haghparast

Histopathological effects of waterborne copper nanoparticle on gills of Siberian sturgeon (Acipenser baerii) juveniles 280 Forouzan Bagherzadeh Lakani, Saeid Meshkini, Mohammad Ali Yazdani Sadati, Bahram Falahatkar, Ali Asghar Tehrani

Effects of physicochemical parameters of water on frequency of Saprolegniceae from Persian sturgeon (Acipenser persicus) eggs 282 Milad Adel, Mohammad Jalil Zorriehzahra, Maryam Dadar

Skeletal deformities in Sturgeon fish 285 Fatemeh Hassantabar, Mohammad Jalil Zorriehzahra, Milad Adel and Maryam Dadar

Survey of intestinal parasites in sturgeon breeders caught in spring migration from Sangar dam 286 Alireza Babaalian Amiri, Milad Adel , Mohammad Jalil Zorriehzahra

Investigation on susceptibility of Sturgeon fish to Viral Nervous Necrosis (VNN) Mohammad Jalil Zorriehzahra, Alireza Shenavar Masouleh, Milad Adel, Maryam Dadar , Fatemeh 287 Hassantabar

Investigation on probable reasons for eye disaster in fingerlings of beluga (Huso huso) produced using artificial propagation 289 Amin Farahbod Roudbaraki, Ayoub Yousefi Jourdehi, Mahmoud Bahmani, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Ali Hallajian, Mohammad Pourdehghani

In vitro and In vivo Biocontrol of Saprolegniasis on Persian Sturgeon (Acipenser persicus, Pisces:Acipenseriformes) eggs by Pseudomonas aeroginosa (PTCC: 1430) 291 Abbas Ali Aghaei Moghaddam, Kamran Aghili, Maryam Ghiasi, Rasoul Ghorbani, Roghayeh Safari.

Comparison of parasites in Beluga sturgeon, Huso huso, and ship sturgeon, Acipenser nudiventris, in southern part of the Caspian Sea 293 Mohammad Mazandarani, Hamid Reza Naderi Farsani, Melika Ghelichpour, Kourosh Amini

Review of parasite in Persian sturgeon (Acipenser persicus) from Iranian coasts of the Caspian Sea 297 Kourosh Amini, Melika Ghelichpour, Amin Hashemi Rostami

Parasites of Russian sturgeon, Acipenser gueldenstaedtii, and starry sturgeon, Acipenser stellatus, in the south Caspian Sea 300 Mohammad Mazandarani, Hamid Reza Naderi Farsani, Melika Ghelichpour, Kourosh Amini

Behavioral and histopathological changes in liver tissues of Acipenser stellatus under toxicity of aflatoxin B1 303 Jalil Jalilpour, Habib Vhabzadeh, Abolfazl Sepahdari, Zabihola Pajand, Ali Halajian, Soheil Bazari Moghaddam, Mehdi Alizadeh, Mehdi Masoumzadeh.

Biochemical characterization of type I collagens from the skin of Persian sturgeon, Acipenser persicus 305 Ehsan Ahmadi Khanehsaz, Katayoon Karimzadeh, Ali Mostafaie

Changes of Microbial spoilage indices Beluga caviar processed (Huso huso) during storage at - o 2 C 307 Afshin Fahim. Ali Asghar khanipour., Ghorban Zare Gashti , Ferydon Rafiepour, Amiri.a

Marketing and Prepare feeds and food in sturgeon 308 Uranus Ghiassi

Alika Jafari kaliji, Ali Jafarpour, Reza Safari Influence of chitosan nanocomposites and rosemary Rosmarinus officinalis L. extract coating on microbial load of inoculated fillet of Huso huso with Listeria monocytogenes during refrigerated storage 313 Alika Jafarikaliji, Ali Jafarpour, Reza Safari

Advantage of the production and marketing of farmed sturgeon in Iran Mahmoud Asadollahi, Yousef Joursara 316

Key lectures

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Sturgeon aquaculture development and the Brand of Iranian caviar in International Market Mohammad Pourkazemi* *Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization (Areeo), Tehran-Iran

The Caviar production in the Caspian Sea declined from 3000 tons in 1985 to less than 40 tonnes in 2015 which all current catch supposed to be allocated for rehabilitation, restocking and research. Although a regional agreement took place for the conservation and management of unique sturgeon species by the Caspian range states, however, no improvement of wild stocks of sturgeon recorded since 2010. Considering to the historical data, Iranian caviar was the best quality and most expensive in the International market. The question is that if the current situation of the Caspian Sea continued, Can Sturgeon Aquaculture Development be considered as a solution to save Iranian Caviar Brand alive in the world Caviar market? There are two possible ways to answer this question 1) Sustainable management of Sturgeon Fisheries in the Caspian Sea through management of shared stocks, joint agreed action plan by 5 Caspian littoral states, combating illegal catch and overfishing, social and economic improvement of local communities, rehabilitation, restocking, access to spawning ground, Action against pollution, habitat degradation, etc… that seems will not happen in near future. 2) Development of a sustainable sturgeon aquaculture in Iran with several advantage such as: availability of the native and endemic species, long experience and tradition (world well known market of Iranian caviar), trade name of the Caspian Sea (cage culture, coastline land based sturgeon aquaculture) high local demand (if we suppose only 1% of 80 million population of IRAN consume100 gr. / year, it means we need 80 tons of caviar for domestic demand). In this paper, two development strategies is recommended in order to maintain Iranian Caviar alive in competition with new aquaculture origin caviar: a) Short term: Although Iran produced more than 1500 tons of sturgeon meat and 2.2 tons of caviar in 2015, however it should be consider a quick way to supply caviar into the international market. Appropriate policy to select the relevant sturgeon species (Beluga/ Persian Sturgeon, various hybrids, Sterlet…), using local adopted rearing techniques (pond, circular concrete ponds, RAS, Cage,) and most important considering the public awareness via advertising as well as development an Eco-Tourists plan. At present more than 80 private farms are involved for sturgeon aquaculture, the largest farm 2000 tons of meat and 30 tons of Caviar being established in Mazandaran province, about ten small farms producing less than 100 tons of meat and 2 tons of caviar, existing several registered caviar processing plan, traditional Caviar are harvested (no ova or unfertilized eggs removal from the live fish via micro- Cysarian technique for caviar processing). b) Medium and long term: Implication and sturgeon recovery and management plan, agreement on applied action plan (NOT on the paper), rehabilitation and restocking, create live gene bank using IUCN Red list Criteria for Critical Endangered Species. Currently huge investment on sturgeon cage culture in the Caspian Sea are operational and more the 5 companies are investing for Cage culture which Beluga is the main species for the Caspian Sea with the capacity of 25-50 tons fish per cage, fully government support with reduction of Bank interest rate. Compare to international price for sturgeon meat and caviar there is a good market internally where in 2013 the sturgeon whole fish price was quite high (15-20

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USD/Kg.) but in 2015 the price declined to 10-12 USD/Kg. For whole sale of farmed Beluga Caviar is 1000-1500 USD/Kg which 90-95% exported. In Conclusion: Considering that in near future there is no chance to harvest wild caviar from the Caspian Sea, and currently more than 340 tons of farmed caviar has already being provided in the world caviar market, Iran should take an quick steps for its caviar production through aquaculture development, since Iran produced less than 1% of world caviar in 2015 and has unique opportunity in the Caspian Sea through cage culture, high efficiency techniques, native sturgeon species, long-term experience availability, the caviar brand as well as high population number, domestic market, good quality caviar from the Caspian Sea origin. above mentioned

Keywords: Iranian Caviar, Caspian Sea, International Market, Aquaculture criteria, CAN Survive the Brand of Iranian Caviar

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Farming of sturgeon in Iran, challenge and programme

Hossein Ali Abdolhay*, Naser Karami Rad

*Iranian Fisheries Science Research Institute, Agriculture Research, Education and Extension Organization (Areeo), P.O.B 14965-149, Tehran- Iran,

In last two decade huge decline of sturgeon happened in the Caspian Sea, in which total sturgeon meat production (28500 tonnes) and Caviar (3000 tonnes) in 1985 reduced to less than 200 tonnes of meat and 10 tonnes of caviar in 2015. Since 2010 -2011 all Caspian littoral states voluntary self-moratoriumthe wild sturgeon commercial catch and caviar export and use all broodstocks for restocking and research purposes. Such decline was the main reason for Caspian Sea countries to start sturgeon aquaculture in captive condition. In Iran, Sturgeon farming started in 1988-1989 at experimental level but increased gradually and nowadays are very high interest due to lack of supply from wild origin. At present, sturgeon farming practices in more than 20 provinces and all in private farms (table 1). The larges farm started from 2015 in Mazandaran province and it has capacity of 2000 meat and 30 tonnes of caviar. Nasr company plan to establish farm for sturgeon culture with capacity of 1000 tonnes meat and 30 tonnes of caviar. Bonyad Mostaztefan plan to establish a very big farm in Guilan province with capacity of 20 tonnes of caviar and more than 1000 meat. Government provide appropriate financial support for private investors and especially in cage culture. Table1: Sturgeon Farm in different province

province Number of farm Guilan 24 Mazandaran 24 Qazvin 4 Fars 3 Khuzestan 3 Kermanshah 3 Kerman 3 Markazi 3 Khorasane Razavi 3 Yazd 2 Qom 2 Lorestan 2 Esfahan 2 Golestan 2 Others 3 Total 82

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Beluga, Persian sturgeon, Ship sturgeon, Siberian and Sterlet sturgeon are the main species in aquaculture but very rear occasion the stellate sturgeon being farmed. However, Beluga is the main species in all farms and comprise of more than 85% of production. From type of sturgeon farming point of view, normally the earthen ponds, circular concrete ponds, fibreglass tanks and recently floating cages in the Caspian Sea, inland lakes and reservoirs are the most common methods for sturgeon farming in Iran. Total sturgeon meat and Caviar production in Iran was 1000 and 2.2 tonnes in 2015 respectively. The future plan to increase to 100 tonnes of caviar and 10000 tonnes of meat. Two years ago the sturgeon meat price was quite high (15-20 USD/Kg) as whole fish but it reduced to 10-12 USD/Kg. in 2016. It seems it will face further reduction when new sturgeon products arrive to the market. The farmed caviar in very small quantities (5-10%) being used inside the country but more than 90-95% exported, while its price range 1000-1500 USD/Kg. mostly for Beluga species. Iran had historical trade of caviar in Europe, after declined of wild sturgeon it is difficult to back to market. Producer should to encourage to domestic market both in meat and caviar. According to national short time plans, the sturgeon meat and caviar production will have increased and expected to produce 10000 tonnes of meat and 100 tonnes of caviar in next 10 years (table 2). Iran population is 80 million now and there is very big market for sturgeon meat and caviar. Foreign investors and caviar traders are involved in sturgeon aquaculture and seems very high grows of sturgeon meat and caviar production occurs in Iran up to 2025. It is necessary to planning for market development not only for domestic but also for foreign market. Table 2: Production plan of meat and caviar in different years

Years 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025

Meat production (tonnes) 3363 3290 3600 3300 5000 6000 7000 8000 9000 10000

Caviar 3 3 4 10 30 45 60 75 90 100

Keywords: Sturgeon, Aquaculture, Farming, Caviar, Meat

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Sturgeon aquaculture and its achievements in Iran

Mohamad Ali Yazdani Sadati*

* International Sturgeon Research Institute (AREEO), Rasht, Iran, P.O.Box: 41635 - 3464. [email protected] Caspian Sea is the largest of brackish water sea in the world. There are 6 sturgeon species in Caspian Sea and Volga River that in the past years produced a total of 90 percent of world wild caviar. Unfortunately, Changing in rivers due to construction of hydrolic dams, suspension or stopping direction, Migration of breeders to upstream river, Reduction in spawning areas (Veshchev, 1991a,b) Inefficient fishery management (Veshchev, 2000), increase in illegal catch (Raspopov, 1992) and Water pollution (Khodrevskaya et al., 1997; 2001; 202; 2009) lead to dramatically decrease of sturgeon population. Statistics of caviar removal in Caspian Sea indicated that 261 tons- in 1992, but decreased to 2.1 ton in 2013 (FAO, 2014). Decreasing of sturgeon stock and global demand for sturgeon meat and caviar had a stimulating effect on commercial production of sturgeon in pent system such as pond, tanks and race ways (Chebanov, 2001). Nowadays, number of sturgeon farms in the world has been increased rapidly. 22 species of economical sturgeons (12 species original and 10 species hybrid) have been reared in more than 38 countries (FAO, 2015).Estimates of the total sturgeon aquaculture production indicated 17938 ton in 2005 while reached to 100000 ton in 2013, respectively. Nonetheless, before 2009, there is no commercial culture of sturgeon in Iran. In this year only 20 ton meat produced. But sturgeon culture had a rapid development in Iran. At present there are 45 active farms in Iran that able to produce 3400 ton meat and more than 16 ton caviar. International Sturgeon Research Institute was established in 1995 to aim of study about physiology, propagation, rearing and nutrition of sturgeon fish. Some of the most research obtaining this center is: - Obtaining Caviar from Siberian Sturgeon (Acipenser baerii): - Intensive rearing of Huso huso in concert Pools - Removal of eggs without killing of breeders - Technique of rearing sturgeons in cage in the Caspian Sea - Sex determination in farmed sturgeon through biopsy - Sex determination of sturgeons by laparoscopy - Reducing time of sexual maturation and caviar production in Huso huso - Mass production of live food (Chironomidae and Nereis) - Production of economical food for farmed Huso huso - Production of specific probiotic for sturgeon References Chebanov, M., Rosenthal, H., Gessner, J., Van Anrooy, R., Doukakis, Ph., Pourkazemi, M. and Williot, P. 2011. Sturgeon hatchery practice and management for release. Guideline, FAO Fisheries and Aquaculture Technical Paper 57, 110pp. (ISBN 978-92-5-107044-4). Khodorevskaya, R.P., Ruban, G.I. and Pavlov, D.S. 2009. Behavior, migrations, distribution and stocks of Sturgeons in the Volga-Caspian Basin. WSCS Spec. Publ. 3, 233pp. (ISBN 987-3-8391- 5449-6).

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Veshchev, P.V. 1991A. Efficiency of natural reproduction in the stellate sturgeon, Acipenser stellatus Pallas, under conditions of flow regulation I the Volga River (Effektinost yestesvennnogo razmnozheniya sevryugi Acipenser stellatus Pallas v usloviakh zaregulirovannogo stoka Volgi). Voprosy ikhtiology 31(2), 222-227. [In Russia]. Veshchev, P.V. 1991b. Qualitative composition of spawners and spawning in the stellate sturgeon, Acipenser stellatus, under new ecological conditions (Kachestvennyi sostav proizoditele I razmnozheniye volzhskoi sevryugi Acipensr stellatus v Novykh ecologicheskikh usloviyakh). Voprosy Ikhtiology. 31(3), 442-450. [In Russia]. Khodorevskaya, R.P., Ruban, G.I. and Pavlov, D.S. 2009. Behavior, migrations, distribution and stocks of Sturgeons in the Volga-Caspian Basin. WSCS Spec. Publ. 3, 233pp. (ISBN 987-3-8391- 5449-6).

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Aquaculture

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Feasibility study to develop sturgeon culture in Chalous Shahid Kashiri catch station Kourosh Haddadi Moghaddam*, Ayoub Yousefi Jourdehi, Zabihola Pajand, Fruzan Chubian, Esmail Farzaneh, Esmail Hossein Nia *International Sturgeon Research Institute (AREEO) - Marine ecology Department, Rasht, Iran [email protected] Introduction The Chalous Shahid Kasiri catch station in Mazandaran Province which monitors the biotic and abiotic factors for sturgeon rearing (water chemical parameters, hydrobiology, fauna and flora) and also includes the biotechnique of sturgeon rearing condition in its assessment (density, different rearing systems, rearing facilities, food, formulated diets and supply of sturgeon fingerlings). Sampling was carried out in 4 times monthly. This study was carried out in 2009 – 2010. Results Based on the results obtained the highest abundance of phytoplankton species at the sampling site was reported for Nitzschia sp. and Exuviealla sp., (table 1). Table 1.The results of the distribution of phytoplankton in project area Phytoplankton At a distance of 50 At a distance of 100 Opposite the mouth of species meters from the meters from the the Chalus river beach beach Skeletonema sp. * * - Thalassionema sp. - * - Prorocentrum sp. - * - Nitzschia sp. * * - Coscinodiscus sp. - * - Synedra sp. * - -

The most important zooplankton species were Calanoidea, Protozoa and Rotifera. Sturgeon is able to tolerate a wide range of temperatures and feed between 10° to 28 ºC. Hence, the temperature range (10 to 29ºC) recorded in this region is within the allowable range for sturgeon rearing. The dissolved oxygen concentration recorded was 7.8 mg/L while the optimum range for dissolved oxygen for Huso huso rearing under culture conditions in Iran is reported to be between 4 to 6 mg per liter. On the basis of research carried out in this region, the dissolved oxygen concentrations were 4.1-7.35 and 4.8-7.9 mg/L at 2 and 5 m depths, respectively thus indicating suitable conditions for sturgeon culture in this region. The optimum pH concentration for sturgeon larvae and fingerling stages is reported to be between 6.5 and 7 and between 6.5 and 8 for the grow-out stage. The results of studies showed that pH concentration in this area is around 8 at 2 and 5 m depths and it is suitable for Huso huso rearing. Highest salinity recorded at 2 and 5 m depths was 12.62 ppt in the spring season (table 2).

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Table2. The results of physicochemical factors determined at the Chalous Shahid Kasiri catch station (during 2009 - 2010).

P- N- N- N- Index Temperature BOD5 EC Salinity Total Total pH PO43- NH4+ NO3 NO2 (mg/l) µs/cm (ppt) hardness iron (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Well 18 2.1 584 7.5 *ND ND .015 1.985 .003 420 .087 River 15.3 2.04 1728 8 ND ND .009 .883 .065 500 ND Sea 16 3.3 7960 8.36 6 ND .017 .529 .02 2400 ND )ND = not detect) *

Discussion The results of geological studies on tectonic plates in this region indicated that there are faults which can produce earthquakes at a level up to 7 magnitudes on the Richter but records show that earthquakes in this region are negligible. It will be possible that these faults have no risks for construction of sturgeon rearing facilities. Although soil erosion occurs when the water level of the Caspian Sea rises, construction of a rearing center with the suitable area of 0.909 hectares is possible by keeping in mind the height code (– 24.7 m) and privacy code (60 m) from the coast. Huso huso fingerlings with the rearing density 1 to 2 kg/m2 were fed commercial food (Biomar and Isfahan Company) made up of 53-55 % protein, 15-19% lipid and 6-8 % carbohydrate for larval stage and 35-45% protein, 7-9% lipid and 20-25% carbohydrate for other stages. Food ingredients consist of fish meal, soybean meal, wheat meal, corn meal, yeast, blood meal, meat meal, lecithin, fish oil and other food additives for fattening stage of sturgeon which are feed 5 to 5.5 % of body weight daily. Granulated food is given to Huso huso fingerlings on the basis of mouth size. Water exchange for sturgeon rearing tanks must be without extensive turbulence and it water exchange must be carried out gradually, replacing the tank volume three times a day. Sturgeon rearing can be conducted with the decrease of water volume to 2 m3 in concrete tanks. Owing to the fluctuations in the Caspian Sea level and the fact that the water level is at its highest at present, water from the sea will have to be pumped through pipelines installed at the bottom of the sea. In our correspondence with the local water and waste water Company of Chalous city, the extraction of ground water for aquaculture purposes is not possible. Waste water treatment facilities must be designed and constructed to cut down pollution during emergencies such as unsuitable climate, electricity failure and equipment breakdowns. The results showed that On the whole Shahid Kasiri catch station is suitable for sturgeon rearing and owning to the restriction of land in this area, the maximum recommended capacity for production of meat is 85 tons annually.

References Ryder, J.A. 2013. The sturgeon and sturgeon industries of the eastern coast of the United States, with an account of experiments bearing upon sturgeon culture. U.S. Fish Commission Bull. 8: 231-328. Hung, S.S.O. 2012. Nutrition and feeding of hatchery – produced juvenile white sturgeon (A. transmontanus): an overview. In: P. Williot (Editor), Proceedings of the first Inter. Symp. Stur. Cemagref, Bordeau, France, pp. 65 – 77.

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Rotifer and its role in feeding in Persian sturgeon (Acipenser persicus) larvae

Kourosh Haddadi Moghaddam*, Zabihola Pajand, Fruzan Chubian, Bahareh Younes Haghighi *International Sturgeon Research Institute (AREEO) - marine ecology Department, Rasht, Iran [email protected] Introduction This study was conducted to apply nauplii of Artemia and rotifer cultures to achieve maximum growth and survival in A.persicus larvae.

Materials and Methods Early rearing of rotifers Brachionus plicatilis small type was carried out in test tubes using florescent lamps (2000 lux), mild aeration, 25- 30 oC, pH of 7.5- 8.3 and salinity of 25 ‰. Rotifers were fed marine algae Chlorella sacchrophilia at a rate of 6 x 106 cell ml-1. Stocking density was examined every day. Semi-intensive and intensive culture was carried out in 15 and 500 L tanks filled with water holding green algae blooms. About 500 A. persicus larvae with a mean weight of 36.9 mg and mean length of 1.9 cm were stocked in 60 L tanks. Larvae were measured on the 3th, 7th as well as 11th day of rearing. In this Experiment, 4 treatments were carried out (each treatment with 3 replicates). The treatments were as follows: Diet1: Artemia nauplius parthenogenetica 100% (control treatment), Diet2: Artemia nauplius 75%, Brachionus plicatilis 25%. Diet3: Artemia nauplius 50%, Brachionus plicatilis 50%., Diet4: Artemia nauplius 25%, Brachionus plicatilis 75%. Statgrap and Excel software were used to employ ANOVA and Multi factor Duncan test to analyze differences of various length and weight parameters obtained in the different tests.

Results and Discusiion Analyses of condition factor of larvae showed that the maximum mean condition factor was achieved on day 7 in-group 4 with 0.6 ± 0.12 and the minimum in-group 1 with 0.5 ± 0.11. ANOVA Analysis of variance revealed significant statistical differences between groups (F= 21.22, sig. level = 0.00). Monitoring of percentage of mortality of A. persicus larvae showed the maximum mortality up to the 7th day in larvae of group 4 while from the day 7 onward groups 1 experienced minimum mortality. Studies conducted on Rockfish larvae showed during the early stages of rearing that this species consumes more rotifer as compared to Artemia nauplii. Similarly up to day 7 larvae fed Diet 1 had a higher condition factor compared to larvae in other groups. This can be attributed to the density of rotifers in their diet. Subsequently, with an increase in body size and mouth size and in turn higher energy needs of larvae, rotifers should be substituted by other live food organisms. Mortality rates at the end of day 7 were also lowest (15 %) in the experimental group fed Diet 1. This clearly indicates the effects of rotifers in increasing growth and decreasing mortality during early rearing. We may thus conclude that mixed diets (75 % rotifers + 25 % Artemia nauplii) positively affect growth and survival in fish larvae up to 7 days.

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Table 1: The results of growth of Persian sturgeon larvae in the total of breeding period Treatment SGR (%/day) )2( WG (g))1( CF)3(

Witness 5.15±.64 76.61±4.08 .55±.14 2 5.07±.08 75.16±15.32 0.61±.08 3 4.06±.54 67.93±10.05 .62±.02 4 5.02±.21 73.84±4.08 .64±.13 1) Body weight index 2)Daily growth index )3)Condition factor index ) ) *The mean (± SD) is a significant sign of lack of letters in each row (p≥0.05)

References Morten, V., Torodd, Y. and Gunvor, O. 2013. Automatic measurement of rotifer Brachionus plicatilis densities in first feeding tanks. Aquacultural Engineering, 36: 115–121.

Morizane, T. 1983. A review of automation and mechanization used in the production of rotifer in Japan. Aquaculture Nansei Japan, 2(24): 39-45.

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A trigger to high survivorship of the Persian sturgeon (Acipenser persicus) larvae released into the Caspian Sea waters

Mahmoud Hafezieh*

* Iranian Fisheries Research Organization, P.O. Box: 46414-356 Tehran, Iran. Email: [email protected] Introduction

High mortality frequently occurs in larval mass production of Persian sturgeon, Acipenser persicus which are intended for release in rehabilitation programme. Nutritional deficiencies in live food, Artemia urmiana nauplii, fed to larvae could be one of the possible reasons.

Materials and Methods A series of experiments was carried out to evaluate the effects of nutritional enrichment of Artemia by oils in different concentrations with or without different levels of vitamin C during two enrichment periods (12 and 24h). Parameters investigated included survival, growth rate and salinity tolerance in larvae reared in tanks. Four types of oils (ICES30/4, sturgeon ovary, cod liver, and linseed oils) were evaluated.

Results and Discussion Artemia nauplii eicosapentaenoic (EPA), docosahexaenoic acids (DHA) contents and DHA/EPA and ω3/ω6 ratios significantly increased (P<0.05) as highly unsaturated fatty acid (HUFA) concentration and enrichment period increased. Artemia arachidonic acid (ARA) content, however, increased significantly (P<0.05) as HUFA concentration decreased and enrichment period increased. 300 ppm ICES30/4 plus 20% vitamin C with 24h enrichment period resulted in significantly higher (P<0.05) vitamin C content (1063.8± 48.00 µg/g DW ) in Artemia nauplii than other combinations. HUFA oil source with or without vitamin C did not affect the growth rate of Persian sturgeon larvae. Nevertheless, survival rates and chemical compositions were significantly improved (P<0.05) only in fish larvae fed Artemia enriched HUFA oil supplemented with vitamin C. ICES30/4 provided the best oil source for improving the chemical composition including fatty acids in Persian sturgeon larvae only when it was supplemented with vitamin C. Lower levels of vitamin C (less than 30%) and longer enrichment period (24h) were more effective (P<0.05) in improving the chemical composition of sturgeon larvae. When exposed to salinity test, those larvae fed with HUFA enriched Artemia showed a tolerance level only up to 6 ppt while those fed HUFA+vitamin C enriched Artemia demonstrated a higher tolerance (≥ 90% survival) up to 12 ppt for 120h. Although the enrichment of Artemia urmiana with 300 ppm ICES30/4 and 30% vitamin C gave the best salinity tolerance at 12ppt (100% survival), the enrichment with 300 ppm sturgeon ovary oil and 10% vitamin C was economically affective in increasing the salinity tolerance of Persian sturgeon larvae (98 % survival).

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The Impact of Tocopherol (E) and Riboflavin (B2) Vitamins on Growth and Nutritional Indices of Sturgeon Fingerlings (Acipenser nudiventris) Ali Reza Ashouri,*Hossein Khara, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Ismae Hossein Nia, Zabihollah Pajand, Nemat Peikaran Mana * International Sturgeon Research Institute (AREEO), proliferation and breeding sector, Rasht, Iran, P.O.Box: 3464-416352.2. [email protected] Introduction Farming sturgeon in Iran is developing rapidly and major farmed species in these production centers are Huso huso, Acipenser nudiventris and Acipenser ruthenus. One of the goals of producers is Economic savings, reducing mortality and losses of fry and juveniles, accelerating the growth and achieves higher production, in level unit. In a study that examined the impact of riboflavin in the diet, antioxidant defense in grouper fish with concentrations 0.9, 1.6, 4.4, 6.7, 12.9, 19.4 mg/kg, it was concluded that antioxidant activity in concentration of 1.6 and 0.9 mg/kg is poor (Huang et al., 2010). Since the ship sturgeon is one of the major species in breeding sturgeon in Iran, and should be farmed in high densities, identifying food requirements, and investigating the impact of adding water and fat soluble vitamins on the growth trend was undertaken. The results will have positive effect on increasing the production efficiency,; therefore, this research examined the impact of different levels of vitamin E and riboflavin, separately and in mixture on growth trend of ship Sturgeon fingerlings in the growth phases. Materials and Methods The steps of performing this research were conducted from May 21, 2012 to June 23, 2012, in sturgeon research station, Guilan- Chaboksar, and by using Caspian Sea brackish water. At the beginning of the experiment fish were adapted to the new breeding conditions. 150 ship sturgeons, transferred from Dr. Dadman International Sturgeon Research Institute of Caspian Sea in Rasht to Chaboksar site, were maintained in fiberglass tubs. In order to adapt to the new conditions they were fed with BioMar France feed, over two weeks. Diet1: lack of vitamins E and B2 Diet2: 300 mg per kilogram vitamins E Diet3: 20 mg per kg riboflavin Diet4: 300 + 15 mg vitamins E and riboflavin Diet5: 300 + 20 mg vitamins E and riboflavin Diet6: adding 300 + 25 mg vitamins E and riboflavin to per kilogram Diet Primary data stored in Excel as database, and after recording were analyzed by one-way ANOVA and Duncan test separator and by using SPSS at confidence interval of 95 percent. Kolmogorov-Smirnov Test was used to evaluate normal distribution of distributed fish in tubs. Results By increasing the levels of vitamins E and B2 in treatments, the final body mass indices were assessed. The percentage of increasing body mass and specific growth rate were rated in comparison to the control treatment, and the data showed significant increases. Fish fed by diet containing 300 mg vitamin E and 20 mg vitamin B2, had the highest final mass, the percentage of increasing body mass and specific growth rate, respectively were (27.5 ± 0.1 g), (135.00 ± 0.0 %) and (1.52 ± 0.004% per day) (P <0.05). However, a declining trend of the above mentioned indices was observed in fish fed Diet 6, a diet containing of 300 mg vitamin

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E and 20 mg vitamin B2; however, compare to the control diet, it was in higher levels and exhibited statistically significant differences in results (P <0.05). The lowest feed conversion efficiency was recorded to fish of Diet 5 (300 mg vitamin E and 20 mg vitamin B2). Feed conversion efficiency, compared to control diet, decreased significantly by adding vitamin supplements (P <0.05). Discussion By increasing the levels of vitamin E and B2 in diet, final body mass indices, percentage of increasing body mass and specific growth rate significantly increased compared to the control diet. Fish fed the diets containing 300 mg of vitamin E and 20 mg vitamin B2 had the highest final body mass, percentage of increasing body mass and specific growth rate (P <0.05). Similar results have been obtained by Sharif Zadeh (2011) on the impact of a mixture of vitamin E and riboflavin on the growth, survival, and blood factors of Cyprinus carpio, using 80 mg vitamin E, and 7 mg of vitamin B2 in the diet, representing levels recommended for this species. Sau et al. (2004) have studied the needs of Labeo rohita fry for diets containing vitamin E, in relation to growth trend and carcass composition. Five levels were studied: 0, 50, 100, 150 and 200 mg per kg. The authors concluded that the diet that contains less than 100 mg vitamin E supplement, caused drastic weight losses. In that experiment, the need of Labeo rohita for vitamin E, due to optimal growth and other functions of diet were considered to be 131.9 mg/kg. Huang, (2010) indicated that vitamin E and B2 affect the growth of fish. Koven et al. (1993), as well as Rainuzzo et al. 1997 demonstrated that high levels of omega-3 polyunsaturated fatty acids increases the growth rate of many marine fish larvae, such as Common bream. Kyeong et al., in 2004 have investigated long-term effects of vitamin E and C diets and their interactions on growth and reproductive performance in Perca flavescens. The results of this study suggest that suitable concentration of vitamin E in the diet of ship fry play a significant role in growth and increase of biological indices, such as mass and total length, so that, a mixture 300 mg of vitamin E and 20 mg riboflavin in the diet, increase growth, and it is concluded that adding vitamin E and riboflavin separately, in quantities of 300 and 20 mg per kg of diet, improve growth in the ship fry. Finally, adding these amounts in commercial sturgeon diet is recommended. References Sharif Zadeh, M. 2011. The effect of vitamin E and riboflavin on growth, survival, and blood factors of Cyprinus carpio. Sau, S., Paul, B., Mohanta, K. and Mohanty, S. 2004. Dietary vitamin E requirement, fish performance and carcass composition of rohu (Labeo rohita) fry. Aquaculture. Vol. 240. pp. 359-368. Liu, Y., Wang, W., Wang, A., Wang, J. and Sun, R. 2007. Effects of dietary vitamin E supplementation on antioxidant enzyme activities in Litopenaeus vannamei. Kyeong, J. M., Byun, Y. I., Sung, E. C. & Chun, M. S. 2004. Ubevijhk Photometric Study of the open cluster NGC 2849. The Astronomical Journal, 128:2331-2338. Koven, W.M., and Ler, A., Sklan, D. and Andkissil, G.W. 1993. The association of cicosapentae-noic and docos ahexaenole acids in the main phospholipids of different-age Sparus aurata larvae with growth Aduacalture, 116:71-82. Huang, J. Tian, L. Wu, X. Yang, H. Liu, Y. 2010. Effects of dietary riboflavin levels on antioxidant defense of the juvenile grouper Epinephelus coioides. Fish Physiol Biochem. Rainuzzo, J.R., Reitan, K.I., Olsen, Y. 1997. The significance of lipids at early stages of marine fish: a review. Aquaculture. 155: 103 – 115.

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Effect of Ozone application to improve closed system performance for sturgeon culture Azam Mohammad Salehi*, Ebrahim Rajabzadeh Ghatramie * Ph.D student fisheries, Aquacultre, Khorramshahr Marine Science and Technology University Corresponding author: [email protected] Introduction The use of ozone in the aquaculture industry to improve the level of annual production and increase the production per unit area can be effective and positive results in trials conducted it the past have shown reasonable results. Increased ammonia concentration in culture units is one of the major problems in aquaculture. The increase proliferation, especially in intensive culture systems and ultra-dense fish and shrimp are in use, require intensive water quality control. Finding Ways to eliminate this problem, requires continuous measure to control also the gas problem is the need for devices to make measurements exactly the same cannot be invoked aquaculture is an important practical approach (Khazraienia et al., 1379). Proteins are digested by fish resulting in the exception of ammonia. Ammonia is highly toxic to fish. If a high level of ammonia is in the water, the fish will be poisoned Wickins and Lee, 2002.The aims of this study the effects of ozone on improving surveillance systems sturgeon and ozone oxide ultrafine particles, and the ammonia in the system to prevent and increase production efficiency was also improved.

Materials and Methods In this study two pools with an area of 1200 square meters with quite the same volume of 2400 cubic meters of water similar to the water Farm wells used in a pool two oxygen generators that supplied oxygen to the ozone generator. Use as oxygen to produce ozone for treatments and subsequent pool control systems were considered. In both the pool circulation system with 25 liters per second of fresh water. The water from the pool passes the drum filters and biofilter with 400 liters per second water flow. Ozone volume was 320 g per hour by 7 devices and device maker with a rate of 5 liters per minute of oxygen in the pool tests. In both pools the 20 tons of sturgeon with 120 kg of food at the same time feeding per day for 30 days was carried out in three.

Results The maximum amount of oxygen dissolved in ammonia gas as well as at least two pools were significantly different from each other (p≤0.05), while the amount of nitrite and pH was no significant difference in two treatments (p≤0.05). The ammonia concentration before and after feeding treatments were significantly different from each other (p≤0. 05) and the amount of this concentration was significantly reduced in treatments with treatments of control.

Discussion The use of new technologies such as the use of automatic feeding, automatic control system for water physico-chemical factors, the use of intensive farming systems and high-intensity, sorting, mechanization and modern methods of food production, and other new methods can be used in the development of aquaculture for Sturgeons. These fish can tolerate a wide range of physical properties and chemical water drawn and are highly resistant to disease (Sturgeon aquaculture development roadmap country, 1390). At pH less than 5.7, most ammonia appears in ionized ammonia form (NH4) and relatively high levels are tolerable to fish. However, at higher pH, ionized ammonia equilibrium is shifted to un-ionized levels of

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir ammonia which will be problematic because of its toxicity at low levels. Hence, the oxidation of ammonia to nitrate is vital to the success of fish culture. Controlled oxidation of the specific nitrogenous waste (un-ionized ammonia) in treatment units focus on the design and operation of circulation systems with excellent biofilter performance. Among the available technologies, mainly used biological treatment (Crab et al., 2007). Ozone O3 is an oxygen atom form three most powerful oxidizers and disinfectants are available commercially after its effectiveness has not left any remainder of diatomic oxygen O2 and quickly converted. Ozone is produced on-site is the raw material of air or oxygen. Ozone instead of all the chemical solutions such as chlorine, hydrogen peroxide, Percidine, Halamide and will be used in addition to the elimination of all microorganisms saves time and energy generated and the mechanism of action of ozone on the walls of bacteria, speed is about 3000 times faster than chlorine in water, respectively.

References Crab, R., Avnimelech, Y., Defoird, T., Bossier, P. and Verstraete, W. 2007. Nitrogen removal techniques in aquaculture for sustainable production. Aquaculture, Vol. 270, No. 1- 4, pp. 1-14, ISSN 0044-8486 Khazraei neia, C., Peyghan, R. and Azari Takami, B.C. 1379. Study of some serum enzymes, urea blood cholesterol Common carp fish in acute experimental poisoning Mvnyak. Mjlh Tehran Faculty of Veterinary Medicine, Volume 55, Issue 3, 29-3. Robert, M., Crosby, D.M. and Brunson, M.W. 1997. Ammonia in Fish Ponds. Southern Regional Aquaculture Center Publication, No. 463. Wickins, J.F. and Lee, D.O.C. 2002. Crustacean Farming, Ranching and Culture.2th edition. Blackwell Science, 464 p.

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Comparison of F1 and F2 bester, big bester (back-cross of bester with beluga) and beluga growth during one year rearing period Shahrouz Baradaran Noveiri*, Mohammad Pourkazemi, Mohammad Hassanzadeh Saber, Mohammadali Yazdani, Mohammad Pourdehghani, Rezvanollah Kazemi, Hossein Mohammadi Parashkuh and Mohammadreza Nowrouzfashkhami *Agricultural Research, Education and Extension Organization (AREEO), International Sturgeon Research Institute, Genetics Department, P.O.Box: 41635-3464, Rasht, Iran

[email protected]

Introduction Most sturgeon species are close to extinction due to population overexploitation for meat and caviar. Bester is one of the promising hybrids in Acipenseridae to be produced for its meat and caviar which is able to tolerate freshwater, grows faster than beluga and get matured at shorter period of time. The F1 generation of this hybrid is fertile and could be used for further hybridization. In this study we produced F2 generation of bester from its first generation (F1), as well as a back cross with beluga (big bester) and make a comparison among produced fish with beluga as control by 12 months of rearing in Iran.

Materials and Methods Spermatozoa (2.47±1.2 × 109/ml) and eggs (85 numbers per g) were obtained from matured F1 bester population. The F2 generation of bester was fed by Artemia and Daphnia afterward and by reaching to about 1 g of weight gradually adapted to concentrated diet.

Results and Discussion Data showed that at 3 months of rearing, total length (17.3±2.2 cm) and weight (21.9±7.1 g) of F2 bester were higher than the same data obtained of other groups. Meanwhile the weight of big bester after 6 months rearing (182.79±69.9 g) was significantly higher than that of the other groups. The mean weight of big bester and F1 bester generation were the same but show differences with F2 bester and beluga after 9 and 12 months (p<0.05) (Fig.1). Total length of F1 bester and big bester after 6, 9 and 12 months of rearing, did not show significant differences with each other but were higher than the other groups (F2, beluga) (Fig.2).

Fig 2: Mean total length (cm) comparison of Fig 1: Mean weight (g) comparison of F1 and F2 bester, big bester and beluga during 12 F1 and F2 bester, big bester and beluga during months of rearing 12 months of rearing 5-6, September 2016 Rasht: Guilan University Secretariat: International Sturgeon Research Institute, P.O.Box 41635-3464, Rasht-Iran TelFax: ++98-13-34506142 www.ICSA2016.ir 19

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References Bronzi, P. and Rosenthal, H. 2014. Present and future sturgeon and caviar production and marketing: A global market overview. J. Appl. Ichthyology., 30: 1536–1546. Williot, P., Brun, R., Rouault, T., Pelard, M., Mercier, D. & Ludwig, A. 2005. Artificial spawning in cultured starlet sturgeon, Acipenser ruthenus L., with special emphasis on hermaphrodites. Aquaculture, 246: 263-273.

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Effects of Biomin as Nutritional Supplement on the Growth and Immune Responses of Acipenser ruthenus

Mohammad Ali Bisheban,* Abbas Ali Zamini, Mehrdad Nasri Tajan * Department of Aquaculture Rasht Branch, Islamic Azad University, Rasht, Iran. [email protected]

Introduction This important species produces caviar every year at the lowest cost and in the shortest time. The objective of this research was to assess the effect of Biomin, as a nutritional supplement, on the growth indices and safety factors of sterlets (Acipenser ruthenus).

Materials and Methods The research plan was designed randomly with three treatment groups and one control group. The supplement under study was added to the basic diet of the samples in the following three dosages: 1, 1.5, and 2 g/kg. A total of 72 sterlets with an average weight of 88.60  1.35 were divided into 6 test groups and were placed in twelve 100-liter tanks. The groups were fed for 60 days manually based on 3% of their body weight per day.

Results and Discussion At the end of the culture period, the study of some of the growth parameters of the samples revealed that the 1.5 g dosing of Biomin (as a nutritional supplement) had a considerable effect and the results obtained were significantly different compared to the results of the control group. However, in the examination of health safety factors it was found that the total immunoglobulin level, lysozyme (2 and 1.5 g), and IgM only increased considerably with 2-g dosing of the supplement as compared to the control group samples. Therefore, research results indicated that the growth and safety parameters were satisfactory with the 1.5 and 2 g/kg dosages of the aforementioned nutritional supplement.

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Rearing and Growth of White Worm (Enchytraeus albidus) with Azolla in Comparson to Other Diets Esmaeil Hossein Nia,* Ghobad Azari Takami, Koresh Hadadi Moghadam * International Sturgeon Research Institute (AREEO) [email protected]

Introduction To feed Sturgeon juveniles requires producing and culturing natural food. Digestion and absorption of natural food is supposed to be easy, and supplies essential body components; moreover, natural food may help to built resistance against pathogens (Kohne Shahri and Azari Takami, 1974).The most commonly used natural food, that generally are cultured to artificial breeding of sturgeon, and are available to fry include Daphnia, Artemia, Mosquito Larvae, Gammarus and White Worm (Barnes, R.D. 1963). Experts of Soviet Union were the first ones who have used white worm in the diet of sturgeon (Kohne Shahri and Azari Takami, 1974).In the diet of hybrids fry of Starry sturgeon and ship sturgeon, white worm has been employed up to 95 percent (Keivan, 1994). White worm is considered a very good natural food that is cultured to feed fry (Pennak, 1989). Because production costs are high, this study to breed white worms has used foods that considerably reduce production costs.

Materials and Methods Wheat flour, rice bran, potatoes, carrots and Azolla were used in this study to feed worms. Initially each of these ingredients were put into boiling water and cooked. The cooking time of potatoes is approximately 50-60 minutes; carrots and Azolla respectively were cooked for 40 and 25 -30 minutes. There after the fully cooked ingredients were grinded in meat grinders to produce dough. After putting them in the mixer, some water was added to completely mixed together and becomes semi-liquid. Usually every kg needs adding 700 to 800 cc water. Prepared food was poured in large basins, and transferred to the greenhouse. The soil of boxes (culture medium) was stirred by hand to distribute worms evenly and reach so that moisture and air can reach the lower parts of the boxes. Then, three transversal grooves were provided in the box and the food was poured into it and covered by soil. To provide sufficient moisture, water was evenly sprayed on the surface of the boxes. Food was added once a week (Kohne Shahri and Azari Takami, 1974). Four different diets were used: diet a (wheat flour, rice bran and carrots), diet b (wheat flour, rice bran and potatoes), diet c (wheat flour, rice bran, potatoes and carrots) and diet d (wheat flour, rice bran and Azolla).

Results The results showed that different combinations of food ingredients have resulted into different biomass gains of worms. The worms that have used mixture of wheat flour mixture, rice bran, potatoes and carrots, have had the highest growth (P <0.05) (Table 1).

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Table 1: The growth of worm biomass when different foods during three months of culture (g / m2). Biomass was determined in 30 days interval Increase of biomass 30 days (g / m2) 60 days (g / m2) 90 days (g / m2)

Type of food Wheat flour (25%), rice bran (15%), 234 363 534 carrots (60%) (control) Wheat flour (25%), rice bran (15%), 243 384 546 potatoes (60%) Wheat flour (25%), rice bran (15%), 246 417 584 potatoes (30%), carrots (30%) wheat flour (25%), rice bran (15%), 240 369 533 Azolla (60%

Discussion and Conclusion Nutritionally, the highest biomass gain that has been achieved when culturing worms in different combinations of the four ingredients was by a mixture of wheat flour, rice bran, carrots and potatoes. However, as the table shows, weight gains through different diets are very close to each other, but still statistically significant. Therefore, due to the high costs of culturing white worm, by foods that already are used, as well as, by wheat flour, rice bran, and Azolla, would be more economical. If the cost of wheat flour and rice bran, which are used in all foods, be ignored, the cost of producing white worm by Azolla will reduce the costs substantially, because Azolla can be easily gathered from the ponds and wetlands at low labor costs. According to the results based on the present nutritional and breeding conditions in this study, the combination tested can be used as a new model to breed white worms and improve its production.

References Kohne Shahri, M. and Azari Takami, Gh. 1974. Reproduction and breeding sturgeon. Tehran University Press; pp. 236-218 Barnes, R.D. 1963. Invertebrate zoology. University of Michigan, Saunders pub, P: 632. Keivan, A. 1994. Applied Technical Report of second International Symposium on sturgeon in Moscow; p. 92.

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Check temperature in the growth of white worms (Enchytraeus albidus)

Esmaeil Hossein Nia*, Mohsen Poor Asadi, Ali Reza Ashoori, Nemat Peikaran Mana

*International Sturgeon Research Institute of Caspian Sea [email protected] Introduction Sturgeons are one of the highly valuable fish species, and the Caspian Sea is the main habitat for several species. Therefore, to preserve generation of these sturgeons, controlled breeding and release seems to be necessary to maintain these populations. To feed Sturgeons fry, it is very important to produce natural (live) food through controlled culture. Digestion of natural food has been easy, and it is believed that natural food will also assist to build resistance against pathogens (Kohne Shahri and Azari Takami, 1974). Natural food generally cultured in controlled breeding of sturgeon includes Daphnia, Artemia, Mosquito Larvae, Gammarus and White Worm (Barnes, 1963). In the diet of hybrids fry of Starry sturgeon and ship sturgeon, white worm represent up to 95 percent of the diet (Keivan, 1994). White worm is one of the valuable and well accepted natural food that is cultured to feed fry (Pennak, 1989). The present study considers the control of temperature as an important environmental variable that needs to be regulated during the warm season. Materials and Methods To carry out this study, at first breeding wooden boxes were prepared (length 70 cm, width 50 cm, height15 cm). The surface of each box was 0.35 m2. Since adequate temperature control was impossible when culturing worms outdoors, the test has been performed at greenhouse temperature. Due to high temperatures during the warm season, cooling was necessary and was supplied by two coolers. Temperature of white worm breeding hall was measured daily.

Results The results indicated that white worms grow optimally at 22 °C. Due to increase of temperature, the environment for their growth will be sub-optimal and reduces feeding. White worm can also tolerate temperature up to 28 °C; but, when soil temperature exceeded 28 °C, worms try to get out from boxes (culture medium), and at a temperature of approximately 30 °C, most of the worms will leave the boxes and scatter around the floor to reach the cooler places.

Figure 1: diagram showing the average temperature changes during the breeding period

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Discussion Different sources identify the suitable temperature to breed white worm to be between 16 and 18 °C, (Kohan Shahri and Azari Takami, 1974); the results showed that white worms are able to grow optimally at higher temperature. However increasing temperature to more than 25 °C, the environment will be inappropriate for optimum growth of worms and their feeding intensity will be reduced. White worm can also tolerate temperature up to 28°C. If soil temperature exceeded from 28°C, they try to get out from culture medium, and go to cooler places. Therefore, maintaining temperature of greenhouse at an optimal level will positively affect the growth of worms; in addition feeding the worms by warm food has an impact on increasing the soil temperature (Kotpal, 1995 and 1996). References Kohan Shahri, M. and Azari Takami, Gh. 1974. Reproduction and breeding sturgeon; Tehran University Press; pp. 236-218. Barnes, R.D. 1963. Invertebrate zoology; University of Michigan, Saunders pub, P: 632. Keivan, A. 1994. Applied Technical Report of second International Symposium on sturgeon in Moscow; p. 92 Pennak, R.W. 1989. Freshwater invertebrates of the United States; Science Pub, P: 628. Brink Hurst, R.O. 1971. A guide for the identification of British aquatic oligochaeta; scientific publication, No.22, University of Toronto, P: 544. Kotpal, R.L. 1995. Annelida – A text book for college & university students, Rastogi Pub, edition 5, P: 263. Kotpal, R.L. 1996. Modern text book of zoology invertebrates for B Sc; and B Sc (Hons) classes; Rastogi Pub, edition 6, P: 648.

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The importance of moisture on the growth of white worm (Enchytraeus albidus)

Esmaeil Hossein Nia*, Ghobad Azari Takami, Ayoub Yousefi, Rezvanollah Kazemi *International Sturgeon Research Institute (AREEO) [email protected]

Introduction .To feed Sturgeons fry, producing and culturing natural food is very important. Most natural foods in breeding of sturgeon include Daphnia, Artemia, Mosquito Larvae and Gammarus and White Worm (Barnes, 1963).Experts of Soviet Union have used white worm in the diet of sturgeon since the mid 19-hundreds (Kohne Shahri and Azari Takami, 1974). Culturing hybrid fry of Starry and ship sturgeon, up to 95% white worm have been used (Keivan, 1994). White worm is a valuable and good natural food that is cultured to feed fry (Pennak, R.W. 1989). This study tried to investigate the effects of different moisture levels on growth of white worm.

Materials and Methods To carry out this study, at first breeding wooden boxes were prepared (length 70 cm, width 50 cm, height 15 cm). The surface of each box was 0.35 m2. Culture medium for white worm was a mixture of sawdust and peat of equal proportions. The goal of the study was testing and evaluating higher and lower moisture values used in breeding centers, as well as available resources to obtain optimal level of moisture under Iran climatic conditions. Therefore, three levels were tested: (a) moisture less than 35%, (b) between 35-45%, and (c) more than 50 percent. During experiment, moisture content of boxes was measured once a week, and moisture loss due to evaporation was compensated by adding water.

Results The results indicated that when the moisture is less than 30 percent, no worm can survive. In examining the moisture between 35-45 percent, it became clear that white worm can grow in moisture above 40%, so that, in the first month, that moisture content of culture medium was controlled between 40 and 45 percent, and the worms gained weight. But to evaluate the least moisture content that worms can tolerate, values were kept between 35 to 40 percent in the second month. It was observed that the growth of worms stopped in this moisture level and with continued exposure, Mortality will begin. Measuring the biomass of worms, at the end of the second month revealed that worms had losses, and their biomass was reduced. In the third month, the biomass of worms, in a culture medium with moisture between 35 - 40 percent was less than 5 grams. Inmoisture levels of more than 50%, it became clear that the worms grow rapidly and are more active. In moisture content between 60-65%, the number of worms can be seen about 2 to 3 cm of the soil surface, and even sometimes appear on the soil surface. In the moisture content between 65 to 70 percent, a greater number of worms were appeared on the soil surface, and medium, in the moisture content of more than 70% is unsuitable to worms, and worms bring themselves on the soil surface. Therefore, proper humidity to growth of white worm is between 50 to 60 percent, and worms have their maximum growth in this moisture content. Discussion

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The results of measuring biomass of worms revealed that humidity is very effective in growth of these worms. According to observations worms can tolerate starvation for a while, without any losses, but, at the same time, reducing the moisture will result in their death. If the moisture content in the culture medium of white worm is too high, their living conditions will also be inappropriate, and they consume less food, because high humidity results in a decrease of the soil oxygen content, and the worms come to the soil surface to breathe (LAL, 1996). This situation can be more observed in moisture contents higher than 65%. Therefore, it can be said that through increasing humidity (required amount) producing white worms can be increased.

References Barnes, R.D. 1963. Invertebrate zoology; University of Michigan, Saunders pub, P: 632. Kohne Shahri, M. and Azari Takami, Gh. 1974. Reproduction and breeding sturgeon; Tehran University Press; pp. 236-218 Keivan, A. 1994; Applied Technical Report of second International Symposium on sturgeon in Moscow; p. 92 Pennak, R.W. 1989. Freshwater invertebrates of the United States; Science Pub, P: 628. LAL, S.S. 1996. A text book of practical zoology invertebrates for all Indian universities; Rastogi Publications, P: 512.

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Proper soil pH for the growth of white worm (Enchytraeus albidus) as live feed

Esmael Hossein Nia* ,Ghobad Azari Takami, Ali Reza Ashoori, Nemat Peikaran Mana *International Sturgeon Research Institute of Caspian Sea (AREEO) [email protected]

Introduction To feed Sturgeons fry, producing and culturing natural food is very important. Digestion and absorption of natural food has been easy, and its consumption will supply body's essential materials; moreover, natural food will improve the resistance of fish against pathogens (Kohne Shahri and Azari Takami, 1974). The best natural food, that generally are cultured under controlled breeding of sturgeons, are available to fry include Daphnia, Artemia, Mosquito Larvae and Gammarus and White Worm (Barnes, 1963). In the diet of hybrids fry of Starry sturgeon and ship sturgeon, white worm has been used up to 95 percent (Keivan, 1994). White worm is highly valuable and among the best natural foods that are cultured to feed fish fry (Pennak, R.W. 1989).

Materials and Methods Proper and applied culture medium to breed and cultivate white worm was a mixture of sawdust and peat. To evaluate the growth of white worm in acidic and basic medium, these two culture media were prepared. Therefore, the ratio of peat and sawdust was chosen in a way that results in desired culture medium. To acidic and basic pH conditions were prepared, respectively just peat and mixture of peat was selected; so that, the ratio of sawdust was more than peat. PH of acidic and basic medium respectively was between 6-7, and 7-8. During the experimental period, pH in the culture boxes was measured once a week. Results According to the obtained results it can be said that the proper pH to growth of white worm is between 6.4 and 7.6. White worm can tolerate pH higher and lower than these two measures, and grow in both media; so that measured pH in this project has been variable between 6.2 and 8.1. On the basis of results, there is no significant difference in the growth of worms in acidic and basic media (P>0.05).

Table 3 Comparison of worms' growth in acidic and basic media, over the first, second and third months

Increase of growth 30 days (g / m2) 60 days (g / m2) 90 days (g / m2) Type of medium

Acidic(6.2-7) 228 393 568 Basic (7-8) 243 384 576

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Discussion In the case of pH it can be said that with respect to this factor that white worm naturally lives in acidic media (Delinad, 1992). Therefore, it was expected that the growth of this worm being higher in acidic medium; but considering the table related to pH, it can be seen that white worm is able to grow in basic medium too, and its growth is almost the same as in the acidic medium. Some sources have mentioned proper pH to growth of white worm, between 6.5-7 and maximum pH, between 6-7.5 (Far Pour, 1973), but this experiment revealed that, white worm can have an appropriate growth in pH ranges between 6.4-7.6. In addition, during the experimental period, pH of culture medium reached to 8, but white worm has grown without losses. The least measured PH was 6.2 that did not cause a problem to worms.

References Kohne Shahri, M. and Azari Takami, Gh. 1974. Reproduction and breeding sturgeon; Tehran University Press; pp. 236-218 Barnes, R.D. 1963. Invertebrate zoology; University of Michigan, Saunders pub, P: 632. Keivan, A. 1994. Applied Technical Report of second International Symposium on sturgeon in Moscow; p. 92 Pennak, R.W. 1989. Freshwater invertebrates of the United States; Science Pub, P: 628. Delinad, L. 1992. Annelids of Caspian Sea; translation; Fisheries Research Center of Guilan Province (Bandar-e Anzali), p.19 Farpour, H. 1973. Live animals; the first volume, published by the Ministry of Science and Higher Education, pp.: 440- 437, 461- 459 and 469- 468.

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Effects of micro/ nano-encapsulation of Lactobasillos bulgaricus with alginat/ chitosan on growth performance and feed efficiency of great sturgeon juveniles Huso huso.

Seyed Samad Hosseini*, Mojtaba Alishahi, Koroush Amini, Mohammadreza Abbaspour, Masoud Ghorbanpour and Takavar mohammadian. *Ph.D Student Aquatic health, Department of Clinical Sciences, Faculty of Veterinary Medicine Shahid Chamran University of Ahvaz, Ahvaz, Iran. [email protected]

Introduction The administration of probiotic bacteria as nutraceuticals is an area that has rapidly expanded in recent years. The oral administration of most bacteria results in a large loss of viability associated with the passage through the stomach, which is attributed to the high acid and bile salt concentrations present. This loss of viability effectively lowers the efﬁcacy of the administered probiotics. Nano-encapsulation is a proper way for protecting the bacteria from early gastrointestinal degradation. In this study the effect of nano-encapsulation of lactobasillus bulgaricus by alginate/chitosan on the growth rate and feeding efficiency of great sturgeon (Huso huso) juveniles were investigated.

Materials and Methods 350 fish with the average weight of (27.28±2.86 g, Means ± SD), were randomly divided in to five equal groups in triplicates as follows: T1: the fish were fed with alginate/chitosan free bacteria, T2: the fish were fed with encapsulated bacteria using the emulsification method, T3: the fish were fed with encapsulated bacteria with Ionic gelation method, T4: the fish were fed with only bacteria and T5: control group fed with free bacteria and alginate/chitosan diet. The fish were fed at a level of 3% of body weight per day in all trials.

Results The results showed that a significant change occurred in some evaluated indices (P<0.05) over the experimental period of 60 days. The highest feed conversion ratio (FCR) was measured in T3 (1.64±0.11) which was significantly higher than T4 (1.14±0.06). The highest values of SGR, PER, FER were recorded in T4 among the groups in both sampling periods. Although oral administration of L.bulgaricus stimulate growth indices of H.huso (T4) compare to control group (T5), nanoencapsulation of bacteria with alginate/chitosan showed adverse effects on growth indices of fish (T3).

Table 1: Growth performance of great sturgeon juveniles after 8 weeks feeding on experimental diets (mean ± SD) Index Group Day30 Day60 T1 ab,A 24.05±323.28 ab,B 8.80±94.52 T2 b,A 24.64َ ±318.83 b,B 4.38±78.96 Weight Growth Percent T3 ab,A 27.56±369.52 a,B 15.69±99.98 T4 a,A 32.64±378.53 a,B 9.55±101.07 T5 ab,A 32.27±350.13 a,B 9.39±102.37 T1 b,A 0.009±0.343 ab,A 0.005±0.347 T2 b,A 0.001±0.338 b,B 0.008±0.315 CF T3 b,A 0.010±0.334 b,A 0.008±0.325 T4 b,A 0.004±0.335 ab,A 0.043±0.354

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T5 a,A 0.003±0.368 a,A 0.008±0.367 b,B ab,A T1 0.067±1.27 1.43±0.07 b,B a,A T2 0.038±1.27 1.53±0.11 T3 b,B 0.119±1.64 1.36±0.14 ab,A FCR T4 1.14±0.065 b,B 1.32±0.09 b,A T5 1.26±0.111 b,B 1.40±0.06 ab,A T1 2.08±0.08 ab,A 0.96±0.06 ab,B T2 2.07±0.08 b,A 0.84±0.03 b,B SGR T3 2.23±0.08 ab,A 1.00±0.11 a,B T4 2.26±0.09 a,A 1.01±0.06 a,B T5 2.17±0.10 ab,A 1.01±0.06 a,B T1 1.74±0.09 b,A 0.58±0.03 ab,B T2 1.74±0.05 b,A 0.54±0.03 a,B PER T3 1.35±0.09 a,A 0.61±0.06 ab,B T4 1.94±0.10 b,A 0.63±0.04 b,B T5 1.76±0.14 b,A 0.59±0.02 ab,B T1 78.65±4.14 a,A 69.99±3.85 ab,B T2 78.33±2.31 a,A 65.46±4.71 b,B FER T3 61.03±4.34 b,A 74.01±7.64 ab,B T4 87.39±4.88 a,A 75.98±5.73 a,B T5 79.36±6.71 a,A 71.19±3.21 ab,B The superscript small letters (a, b) in the same row mean the significant difference at Pb<0.05 within the same trial. The superscript capital letters (A, B) in the same row mean the significant difference at Pb<0.05 of the same dietary treatment between trial 1 and trial 2.

Discussion According to the result, nano-encapsulation of probiotic, L.bulgaricus, with chitosan/alginate decrease its probiotic effects particularly in growth promotion of H. huso. This decrease in growth indices can be probably referred to the anti-nutritional effects of alginate /chitosan in H. huso. It can be suggested that other degradable biopolymers use as alternative to alginate/chitosan.

References Huiyi, S., Weiting, Y., Meng, G., Xiudong, L. and Xiaojun, M. 2013. Microencapsulated probiotics using emulsification technique coupled with internal or external gelation process. Carbohudrate polymers, 96:181-189. Maria, A.A., Ana, I.B., Antonio, A.V. and Miguel, A.C. 2014. Alginat/chitosan nanopartices for encapsulation and controlled release of vitamin B. International Journal of Biological Macromolecules, 77.pp: 141-146. Melinda, M. 2012. Dietary Supplements for Improving Body Composition and Reducing Body Weight. International Journal of Sport Nutrition and Exercise Metabolism; 22.pp:139- 154. Shiau, S.Y. and Yu, Y.P. 1999. Dietary supplementation of chitin and chitosan depresses growth in Tilapia, Oreochromis niloticus _ O. auratus. Aquaculture; 179: 439-446. Tafi, E. and Meshkini, S. 2015. The effect of different levels of Chitosan on the growth parameters of Rainbow trout (Onchorhynchus mykiss). Journal of Animal biology: 4 (1): 35- 44.

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The effect of ascorbic acid and vitamin E (tocopherol) supplementation in broodstock feed on reproduction of Persian sturgeon (Acipenser persicus, Borodin, 1897) Mahmoud Mohseni *, Pourkazeni Mohammad, Bahmani Mahmoud, Pourali Hamidreza, Hallajian Ali, Kazemi Rezvan, Hassani Hamed, Pourdaghan Mohammad, Yousefi Jourdehi Ayoub *International Sturgeon Research Institute (AREEO), Rasht. P. O. Box: 41635 – 3464. [email protected] Introduction During the past three decades, considerable attention has been paid to the effects of vitamins C (ascorbic acid, AA) and E (tocopherol) in broodstock performance, egg quality, and fry viability of several fish species (Watanabe and Takashima, 1977; Soliman et al., 1986; Dabrowski and Blom, 1994). Observed effects of a deficiency of vitamin E include delay in ovarian development (Watanabe and Takashima, 1977), and decreased egg hatchability and fry survival (Takeuchi et al., 1981). The lack of dietary AA also resulted in lower egg hatching rates (Sandnes et al., 1994), egg strength (Mangor-Jensen et al., 1994), and poor fry survival (Soliman et al., 1986).

Materials and Methods A 728-day feeding trial was conducted to observe the influence of varying levels of dietary Soybean meal; Vitamin C and E were evaluated on growth and maturation of male and female Persian sturgeon (Acipenser persicus, Borodin, 1897). Sixteen premature Persian sturgeons (8 males in stage III and 8 females in stage II-III of sexual maturity) were stocked in fibers glass tanks in duplicate. They were fed to apparent satiation with diets containing [Diet 1 for Female, Soybean extract+250 mg vitamin C+150 mg vitamin E per kilogram feed; Diet 2 for female, Soybean extract without vitamin C and E); Diet 3 for male, 250 mg vitamin C+150 mg vitamin E per kilogram feed, without soybean extract; Diet 4 for male, Without soybean extract and without vitamin C and E)]., Fish were fed four times daily, at 08:00, 14:00, 20:00 and 02:00 h. Highest gonad somatic index in males was recorded when fed diet 3. Histological examinations carried out on these fishes at the end of the experimental period showed that 75% of them were in stage IV of sexual maturity. 450 cc of spermatozoa was extracted from these males and cryopreserved. Results and Discussion Highest somatic index in females was recorded in fish fed Diet 1 and 100% of the fish in this group had attained stage III of sexual maturity. Linear correlation was obtained between soybean extract as well as ratios of vitamin C & E used in the diets and gonadogenesis. It may be concluded that vitamin C (ascorbic acid), vitamin E (α-tocopherol) and phytoestrogen found in soybean extract in the diet are very effective in stimulating gametogenesis, fertility, fertilization, quality and viability of sperms as well as in the production of healthy larvae in farmed A. persicus. This research showed that the effect of food compounds (Soya and Vitamin E, C) on the sexual system of male and female fishes is important and positive.

References Soliman A.K., Jauncey, K. and Roberts, R.J. 1986. The effect of dietary ascorbic acid supplementation on hatchability, survival rate and fry performance in Oreochromis mossambicus (Peters). Aquaculture, 59: 197– 208. Packer, J.E., Slater, T.F. Willson, R.L. 1979. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature, 278: 737– 738. Takeuchi, M., Ishii, S. Ogino, T. 1981. Effect of dietary vitamin E on growth, vitamin E distribution and mortalities of the fertilized eggs and fry in ayu Plecoglossus altivelis. Bulletin of Tokai Regional Fisheries Research Laboratory, 104: 111 – 122.

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Dietary vitamin E requirement of juvenile beluga, Huso huso, at two lipid levels, and their effects on immune responses

Mahmoud Mohseni*, Mir Hamed Seyed Hassani, Mohammad Ali Yazdani, Hamid Reza Pourali, Rezvanollah Kazemi, Ali Hallajian, Sungchul, C. Bai

*Caspian Sea International Sturgeon Research Institute, Rasht. P. O. Box: 41635 – 3464.

[email protected]

Introduction Vitamin E functions as a lipid-soluble antioxidant, protecting biological membranes, lipoproteins and lipid stores against oxidation (Shiau and Hsu, 2002).

Materials and Methods A growth trial was conducted to investigate the vitamin E (dl-a-tocopheryl acetate) requirements of juvenile beluga, Huso huso, at two dietary lipid levels, and their effects on immune responses. Semi-purified diets containing five levels of dl-a-tocopheryl acetate (0, 50, 100, 200, 400 and 800 mg/kg diet) at either 12% or 15% lipid were each fed to triplicate groups of beluga (mean weight: 4.6 ± 0.61 g) for 12 weeks.

Results and discussion In the 12% lipid group, fish fed diets with ≥ 200 mg E/kg had significantly highest weight gain, followed by fish fed diets with 50 mg E/kg, and lowest in fish fed the vitamin E-free control diet; in the 15% lipid group, fish fed diets with ≥200 mg E/kg had significantly highest weight gain, followed by fish fed diets with 50 and 100 mg E/kg, and lowest in fish fed the control diet. Muscle a-tocopherol concentrations were increased as the dietary vitamin E supplementation level increased regardless of the dietary lipid level. In both 12% and 15% lipid groups, fish fed diets supplemented with vitamin E generally had lower hepatic and muscle thiobarbituric acid- reactive substance (TBARS) values and higher white blood cell count, plasma lysozyme and liver enzymes (Alanine aminotransferase and Aspartate aminotransferase) than in fish fed the un- supplemented control diet. Weight gain and hepatic TBARS values analyzed by broken-line regression indicated that the optimum dietary vitamin E requirements of juvenile beluga were 175 and 189 mg/kg diet in 12% and 15% lipid diets, respectively.

References Mohseni, M., Pourali, H.R., Kazemi, R. and Bai Sunhchal, C. 2014. Evaluation of the optimum dietary protein level for the maximum growth of juvenile beluga (Huso huso L. 1758). Aquaculture Research, 45: 1832–1841. Shiau, S.Y. and Hsu C.Y. 2002. Vitamin E sparing effect by dietary vitamin C in juvenile hybrid tilapia, Oreochromis niloticus- O. aureus. Aquaculture, 210: 335– 342.

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Oral resized Acipenser persicus larvae in the early days feeding

Rudabeh Rufchaie*, Armodli Reza., Daryoush parvaneh and Mohammad Salavatian *Inland Water Aquaculture Institute, Specialized Research Station of Aquatics Nutrition and live food, Bandare Anzali, Iran [email protected] Introduction Acipenser persicus showsing high mortality during larval culture. A. persicus larvae culture needs to improve survival rates and enhance resistance in these larvae at the onset of exogenous feeding. Since microscopic examination of the digestive system of Acipenser persicus fish larvae show that the full development of the digestive system takes about fifteen to twenty days, this study measured the size of the mouth (important to identify size of prey that can be ingested) to show its development in relation to the body size during the twenty first feeding days.

Materials and Methods 30 Larvae at start feeding from 20 days of heaven sinks workshop was fixed in 20% formalin. Measurements of the mouth size were assisted by using Biocom software and a Nikon camera (E- 600) attached to the loop and the physical size of the oral Sturgeon Research Institute reviewed.

Results The study measured the size of the mouth in relation to the body length during the twenty first feeding days. The performance was evaluated by f hiowrsc rr a h iw grires em 0.1 ± 0.01 mm.

Fig.1. Acipenser persicus larval: identifying the mouth size from day 1 to day 20 of start feeding. Fig.2. Acipenser persicus larvae: Measuring the increase Data points show mean values and bars represent in the body length during 20 days counted from start standard deviation feeding (day 1). Data points represent mean values and bars indicate the standard deviation

Discussion The study by Aguadoa in 2007 on the relationship between size of fish larvae and live prey size showed that with the increasing success of hunting prey fish size is smaller than the larger prey. According to the result (Fig.1 & 2) the low growth progress in the first four days, can be attributed to the ability of the larvae to feed on zooplankton such as abundant rotifers in the early days of its feeding.

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References Aguadoa, P., Nandini, S. and Sarma, S. 2006. Differences in population growth of rotifers and cladocerans raised on algal diets supplemented with yeast Limmologicca-Ecology and management of Inland waters Vol 35. Pp. 298-303. Shaibani, M. 1996. Microscopic structure of the spleen and lymph tissues along the gastrointestinal tract in Acipenser persicus, 60(1), 45.

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The effect of replacing fish meal with poultry by-product meal on growth rate and biochemical and immunological indices in fingerling Beluga (Huso huso Linnaeus, 1758)

MirHamed Sayed Hassani*, Mohamad Ali Yazdani, Hamid Rez Pourali ,Ayoub Yousefi Jourdehi, Mahmoud Shakourian, Hoshang Yeganeh and Javad Sayadfar

*International Sturgeon Research Institute (AREEO), Iran, Rasht; P.O.Box: 41635 - 3464 Introduction Sturgeon aquaculture is a relatively new industry and growing rapidly in Iran. Currently, Feed cost is the major expense in sturgeon culture (50-60%) (Mohseni et al., 2005). It is predicted that fish meal shortages will increase because of limited availability and increased competitive uses (Jackson, 2007). However, Production of meat by sturgeon aquaculture is takes a long time (2 to 4 years) that will also increase production costs compared to fast growing species, therefore Iranian farmers will have to face the challenge against expensive fish meal and foreign cheap caviar and meat and they will have to find ways to cope with future competition. Replacement by alternative proteins, including plant and animal protein, has been studied by many fish nutritionists and the feed industry (Tacon & Jackson 1985). Poultry by-products have been suggested for reasons of high protein content (55 to 68%) (NRC, 1993), relative similar amino acid profile comparable to fish meal and without anti nutritional factors present in plant proteins (Yu et al., 2004), optimum coefficient digestibility in aquatic animal (Liu et al., 2008), widespread industry processing and having cheap price (Janmohamadi et al., 2009) is one of the potential alternative protein source of diet in sturgeon compared to other animal and plant protein sources in Iran.

Materials and Methods In this study, the effect of replacement of fish meal by Poultry by-products meal on growth indices, biochemical and immune parameters of Huso huso were studied. Average initial weight was 108.83±5. 9g (means ± SD). Fish were reared in fiberglass tanks (2000L) for 18 weeks. Six test diets (containing 42% protein and 18.5 Mj kg-1 energy) were prepared. Fish meal substituted by poultry by product was at 20% (PBM20), 40% (PBM40), 60% (PBM60), 80% (PBM80) and 100% (PBM100), respectively. In the end of the test period no significant difference were detected between final weight (FW), weight gain (WG) and Specific Growth Rate (SGR) of fish fed (PBM0), (PBM20), (PBM40), (PBM60) and (PBM80), respectively (P>0.05). Catabolic enzymes activiy (Aspartat amino transferase and Alanin Amino Transferase) were not affect by inclusion of poultry by product but plasma lysozyme was significantly higher than in PBM20 compared to PBM 80 and PBM 100(P<0.05).

Results and Discussion Poultry by-products contain a high level of protein and (depending on processing method) having a proper amino acid profile compared to fish meal that has been introduced as a good protein source for fish nutrition (Yigit et al., 2006). Poultry by-product meal has been widely studied as an alternative protein source for FM in fish diets and seemed to be a good source for replacement (Yigit et al., 2006). Many researchers tested high quality PBM many species and found that some species can tolerate up to 100% replacement (Steffens, 1994; Nengas et al., 1999; Webster et al., 2000). In our study, final weight, weight gain and specific growth rate were not significantly different among fish fed control, PBM20, PBM40 and PBM60 and PBM80, respectively. We did not observe any significant difference in concentration of ALT and AST in fish fed PBM compared to control diet that it might cause be short period feeding trail. Ast and ALT are released into blood during organ damage of hepatocyte (Racicot et al., 1975). ALT also called serum glutamic pyruvate transaminase is an enzyme present in hepatocytes. When liver cell is damaged, it leaks into the blood, basically ALT rises dramatically in acute liver damage (Racicot et al., 1975). Lysozyme and complement activity indicate general health and humoral immune responses in fish (Tort et al., 1996) and both dietary protein and lipid sources

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir influence humoral immune responses in fish. Subhadra et al. (2006 a,b), for example, found complement activity and lysozyme activity also declined in carnivorous fish, largemouth bass Micropterus salmoides when FM was replaced with PBM. Therefore, we concluded PBM Incorporated in diet and hadn't any negative effect on ALT, AST and Lysozyme in feeding period. The result showed that replacing of fish meal by a high level of poultry by-product were not negative in terms of effects on growth indices, biochemical and immune parameters of Huso huso during the grow-out phase .

References Nengas, L., Alexis, M.& Davies, S.J. 1999. High inclusion levels of poultry meals and related by-product in diets for gilthead sea bream (Sparus aurata L.). Aquaculture, 179, 13 –23. Subhadra, B., Lochmann, R., Rawles, S. & Chen, R. 2006a. Effect of dietary lipid source on the growth, tissue composition andhematological parameters of largemouth bass (Micropterus salmoides).Aquaculture, 255, 210–222. Subhadra, B., Lochmann, R., Rawles, S. & Chen, R. 2006b. Effect of fish-meal replacement with poultry by-product meal on the growth, tissue composition and hematological parameters of largemouth bass (Micropterus salmoides) fed diets containing different lipids. Aquaculture, 260, 221–231. Tort, L., Balasch, S. & MacKenzie, S. 2003. Fish immune system. A crossroads between innate and adaptive responses. Imnninoiogia- .08. 9. Available online at:[http://revista .inmunologia .org/ Upload /Articles/602.pdf] (Accessed: 11.09.08). Yigit, M., Erdem, M., Koshio, S., Ergun, S., Turker, A. & Karaali, B. 2006. Substituting ﬁsh meal with poultry by-product meal in diets for Black Sea turbot Psetta maeotica. Aquac. Nutr., 12, 340–347. Webster, C.D., Thompson, K.R., Morgan, A .M., Grisby, E.J. & Gannam, A .L. 2000. Use of hempseed meal, poultry by-product meal, and canola meal in practical diets without ﬁsh meal for sunshine bass (Morone chrysops × M. saxatilis). Aquaculture, 188, 299 – 309.

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Reproductive performance in Sterlet sturgeon (Acipenser ruthenus) injected with thiamine and the resulting larval quality

Bahram Falahatkar*, Sareh Ghiasi, Murat Arsalan, Konrad Dabrowski *Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, 1144, Guilan, Iran [email protected] Introduction Micronutrients such as thiamine (vitamin B1) are important in broodstock performance and also play a role in early life stages of fish. The present study aimed at investigating the effects of thiamine injection on reproductive performance and larval quality in females Sterlet sturgeon (Acipenser ruthenus).

Materials and Methods A total of 45 fish (698.6 ± 8.9 g) were distributed in 9 fiberglass tanks with three treatments (three replicates each). Experimental fish were fed practical diets supplemented with 1 g/kg amprolium hydrochloride (as an anti-thiamine) in the diet for 5-month before spawning. Thiamine hydrochloride was injected in experimental fish with three doses at 0 (T0), 5 (T5) and 50 (T50) mg/kg body weight at three different stages of maturing specimens (days 30, 90 and 150). At the end of the experiments fish were injected with 2.5 µg/kg LHRH-A2 and the eggs were obtained by cesarean method to determine the fecundity and number of eggs per gram. After fertilization, 5 g of eggs per fish were transferred into a small container (12 × 10 × 10 cm) and placed in Youshchenko incubators. Factors determined were hatching rates, larval weight, larval length, deformity and mortality. For statistical analysis, One-way analysis of variance was applied, and differences between treatments’ means were determined by Tukeys post-hoc test.

Results The result indicated that latency time and fecundity were not significantly different among treatments, but the number of eggs increased in T0. The lowest hatching rate was observed in T5 group. Larval weight and length at 1 dph (day post hatch) and larval weight at 6 dph showed significant increase in T50. Daily larval monitoring showed disorder symptoms such as lack of yolk sac absorption, deformation, erratic pattern of swimming and loss of equilibrium at day 4 in T0 and T5. There was no significant difference in mortality rate at 6 dph among treatments.

Table1.Reproductive performance larval quality (mean ±SE) in Sterlet (Acipenser ruthenus) broodstocks after feeding with amprolium hydrochloride and periodic injection of thiamine for 5 months (n=15). T0 T5 T50 Latency time (h) 22.1 ± 2.6 27.9 ± 2.2 28.5 ± 3.0 Working fecundity (egg number per fish) 3564.6 ± 637.6 3489.4 ± 1116.9 4998.2 ± 417.3 Number of eggs/g 141.2 ± 2.2a 127.0 ± 6.4ab 113.4 ± 4.9b Hatching rate (%) 71.2 ± 12.1a 45.1 ± 15.2b 66.5 ± 17.1a Larval weight at 1 dph (mg) 10.7 ± 0.0c 11.4 ± 0.0b 11.6 ± 0.0a Larval length at 1 dph (mm) 9.7 ± 0.1c 10.4 ± 0.1b 10.8 ± 0.1a Larval weight at 6 dph (mg) 21.6 ± 0.0b 21.7 ± 0.0b 23.1 ± 0.0a Larval length at 6 dph (mm) 15.3 ± 0.1 15.4 ± 0.1 15.6 ± 0.1 Mortality at 6 dph (%) 15.1 nd 3.9 T0: 0 mg thiamine; T10: 10 mg thiamine; T50: 50 mg thiamine per kg body weight.dph: day post hatch. Numbers with different superscripts indicating significant differences between treatments (P< 0.05). nd: no data available.

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The current study revealed no significant difference in latency time, but numerically the highest and the lowest time was observed in T50 and T0 treatments, respectively. It seems that simultaneous relative increases of fecundity and egg size caused larger gonads of T50 treatment in comparison to T0 treatment. Late ovulation of fish in T50 can be due to larger gonads since a longer time is required for hormonal induction affecting all parts of the gonad to lead finally to complete ovulation. The working fecundity was not affected by thiamine injection. In this study, the relative increase of fecundity in T50 treatment might be associated with the increased fish weight since the treatment had the highest weight increase in comparison to other groups. Our results indicated that the number of eggs per gram for T50 group decreased significantly compared to those of T0 treatment. Decreased number of eggs per gram denoted that the fish produced larger eggs in T50 treatment under thiamine injection.

Discussion This study showed that thiamine has positive effects on larval size at 0 dph and 6 dph. Results from similar studies suggest that thiamine deficiency in egg causes poor growth rate and weight of larvae. Our study showed that when thiamine decreases in eggs, survival rate reduced and mortality in larva increased. In the present study in fish treated with T0 andT5 on4 dph larvae, some signs of symptom were observed. The lack of such signs in fish were fed antithiamine and treated with T0 was not excepted and more studies are needed to find it. Thiamine has a coenzyme role in carbohydrate metabolism and is important in glucose synthesis, nervous and brain. The nervous disorders causes the fish have not balance and be sensitive to environmental shocks. Our results revealed that thiamine injection has a positive effect on reproduction and larval survival and injection of thiamine in the brooders can reduce the negative impacts of antithiamine in natural environment.

References Fitzsimons, J.D., Williston, B., Amcoff, P., Balk, L., Pecor, C., Ketola, G., Hinterkopf, J.P. & Honeyfield, D.C. 2005. The effect of thiamine injection on upstream migration, survival, and thiamine status of putative thiamine deficient Coho salmon. Journal of Aquatic Animal Health, 17: 48–58. Fitzsimons, J.D., Brown, S.B., Williston, B., Williston, G., Brown, L.R., Moore, K., Honeyfield, D.C. & Tillitt, D.E. 2009. Influence of thiamine deficiency on lake trout larval growth, foraging, and predator avoidance. Journal of Aquatic Animal Health, 21: 302–314. Fynn-Aikins, K., Bowser, P., Honeyfield, D., Fitzsimons, J. & Ketola, G. 1998. Effect of dietary amprolium on tissue thiamin and Cayuga syndrome in Atlantic salmon. Transactions of the American Fisheries Society, 127: 747–757 Ketola, G., Bowser, P.R., Wooster, G.A., Wedge, L.R. & Hurst, S.S. 2000. Effects of thiamine on reproduction of Atlantic salmon and a new hypothesis for their extirpation in Lake Ontario. American Fisheries Society Symposium, 129: 607-612. Lee, B., Jaroszewska, M., Dabrowski, K., Czesny, S. & Rinchard, J. 2009. Effects of vitamin B1 (Thiamine) deficiency in lake trout alevins and preventive treatments. Journal of Aquatic Animal Health, 21: 290–301. Tillitt, D.E. & Zajicek, J. 2005. Development of thiamine deficiencies and early mortality syndrome in lake trout Salvelinus namaycusch by feeding experimental and feral fish diets containing thiaminase. Journal of Aquatic Animal Health, 17: 4–12.

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Feasibility study to develop sturgeon culture at the Larim Shahid Gholami catch station

Ali Hallajian*, Mohammd Pourkazemi, Rezvanollah Kazemi, Ayoub Yousefi Jourdehi, Mahmoud Mohseni, Mahtab Yarmohammadi.

*Agricultural Research, Education and Extension Organization (AREEO), International Sturgeon Research Organization of the Caspian Sea, Rasht, Iran, P.O. Box: 41635– 3464.

[email protected]

Introduction Shahid Gholami catch station is situated 5 kilometers away from the Larim village of Mazandaran Province. On the basis of topographic maps and latitudinal and longitudinal profiles, the total area from the catch station to the coast is 16.93 hectares of which only 4.45 hectares are considered useful for the purpose, based on the height code (– 24.7 m) and privacy code (60 m). Hence a production goal of 5 tonnes of caviar and 60 tonnes of sturgeon meat seems to be a feasible target for this station. Concentrations of chemical parameters and the abundance of planktonic and benthic organisms in the Caspian Sea water around the station are in the range of the allowable values of these parameters for sturgeon rearing. In our correspondence with the National Iranian Oil Company (NIOC) all aspects of environmental pollution and oil exploration were considered and it was clearly seen that there were no legal barriers to the construction of a sturgeon rearing center at this station as the station is located outside the legal range of gas pipelines.

Results and Discussion And also based on the Ministry of Roads and Transportation Development Plan, Larim catch station is not on the route of the country's main roads. The Department of Environment of the Mazandaran Province announced that conducting any action around the station has no legal barriers and there are no sources of pollution within a radius of 500 meters from the station. The Cultural Heritage Handcrafts and Tourism Organization of Mazandaran Province have been studying the tourism in this area. According to the Water Board of this Province, the water static level of groundwater is at 2 m. The costs for transfer of electric supply from 50 to 400 kw to this station has been estimated by the Electricity Board. A total of 6979 sturgeon fingerlings (Huso huso, Acipenser persicus, Acipenser stellatus and Acipneser nudiventris) with the average weight of 20 g are needed for the first phase of rearing and the same number will be used in the following years. The project aims to produce 5 tonnes of caviar. Production of caviar will start from the seventh year of rearing so the target harvest of 5 tonnes will be reached between the seventh to the eleventh year after start-up of the units. And also the production of 60 tonnes of sturgeon meat at the end of the rearing period will result in revenue of 13981 million Rials. The total costs for this project has been estimated as 257444 million Rilas. It is not possible to separate the production costs for each species as the equipment and facilities used as well as the current costs are shared. So the total fixed investments including construction costs, equipment and machinery is calculated at 257444 million Rials of which the maximum annual funding of 12000 million Rials for fixed and facilities costs is related to the first two years of rearing. The production cost of one kilogram caviar in this project has been estimated at 190 thousands Tomans of which 7 million Rials is for Huso huso, 5 million Rials is for Acipenser

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir nudiventris and 3 million Rials for Acipenser stellatus. The revenue obtained from meat and caviar sales (from the third year to the end of 11th year) is estimated at 13981 and 73000 million Rials, respectively during an eleven-year rearing period and the total income from meat and caviar sales is 86981 million Rials at the end of 12th year of rearing.

Conclusions With regard to the revenue earned and expenditure incurred in this project, the producer can earn 16015 million Rials during the 10 year rearing period by year. Considering that the expenses involved are 9152.6 million Rials, the net income is estimated to be about 6.5 billion Rials. From the 12th year of production, with proper management a profit of 20 billion Rials is expected apart from returns of the initial investment.

References Hamvnyan Consulting Engineers. 2001. Fisheries socio-economic development of the northern (Caspian Sea). 340 Pages. Hung, S.S.O. 1989. Choline requirement of hatchery-produced juvenile white sturgeon. Aquaculture, in press. Hung, S.S.O. 1991. Nutrition and feeding of hatchery – produced juvenile white sturgeon (A. transmontanus): an overview. In: P. Williot (Editor), Proceedings of the first Inter. Symp. Stur. Cemagref, Bordeau, France, pp. 65 – 77. Pillay, T.V.R. 2004. Aquaculture and the environment. Second edition. Blackwell Publishing. 196p. University of Stirling (Institute of Aquaculture), Institute of Freshwater Ecology and Institute of Terrestrial Ecology, 1990. Fish farming and the Scottish freshwater environment, Nature Conservancy Council, Edinburg, Scotland.

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Raising surgery operated sturgeon brood stocks using pen culture in the Gorgan Bay (Caspian Sea)

Nemat Peykaran Mana*, Hamid Reza Pourali Fashtomi, Masoud Seidgar. Mohammad Pourkazemi, Mahmoud Shakorian, Mohammad Ali Yazdani Sadati, Esmaeil Hosseinnia, Alireza Ashoori.

*International Sturgeon Research Institute of Caspian Sea [email protected] Introduction The Gorgan Bay exhibits salinities ranging between 9 and 14 g/l. A depth of 1.5 to 2.4 m has been considered to be favorable to establish pen cultures. It is possible to maintain at least half of the mature and surgery operated brood stocks under these cage conditions until next sexual maturation phase in natural brackish waters of the Caspian Sea. In this study, the time schedule to caviar extraction from 180 operated brood stocks reared in pens based on percentage of the mean number of harvested mature female brood stocks were calculated for the test period 2009-2011. The estimated area for the required pens was 2300 square meters (rectangular in shape). After acclimation period, food will be given to fish at rates of 0.5-1% of biomass twice a day. Fish diets will be prepared from high quality ingredients. The diets will contain 35-45% protein and 10-15% crude lipids. In addition, non-edible fish like Kilka and food waste will be used as nutrient sources for producing the diets.

Materials and Methods The following design and operations criteria have been employed for the operational work: (1)- Water depth: 1.5- 2.5 m; (2)- Pen area: 12000 m2 (1.2 ha), (3)- Optimal water temperature range for holding: 15 - 25 °C, (4)- Maximum permissible water temperature for spawners is 27°C and decreased feeding up to 0.25 is necessary. (5)- Permanent water movement through the pens is required to allow sufficient water exchange in order to supply oxygen and remove excretory products. 6- Fish stocking density under semi- intensive method was 0.5 kg/m2 7- Annual survival percentage of spawners should be around 95%. 8- Wooden buildings onshore or on floats with an area of 50 m2 workspace 9- The use of appropriate technology for culture 10- Combination of automatic and manual methods of feeding (food tray, splashing). After checking the oocytes for stage of ovulation and preparation of females for reproduction, the fish were anesthetized and surgery operated brood stocks were carefully transferred, placed over the table and their belly was dried with a towel and cleaned in order to prevent water and mucus contact with oocytes. Then, the oocytes of ovisac were pulled out using a scalpel regarding to fish size making a 1-3 cm incision at ovisac. In this method the brood stock is returned to culture system after antibiotic injection and these brood stocks can be spawned at least 2 years later.

Results and discussion In order to reach a suitable space to maintain and culture the operated brood stocks and increase the efficiency for the operated brood stocks, the census of recent mature female brood stocks (2009-2011) were used. It was assumed that 50 % of mature brood stocks spawned by micro- surgery and sent to pen after the handling will allow complete recovery. Therefore, in 2011 the estimated number of spawners was 180 individuals. In this study, the

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir species composition of operated spawners to maintain and culture in pens has been as follows: Acipenser persicus: 60%, Huso huso: 15% and other spices including A. gueldenstaedtii, A. stellatus and A. nudiventris: 25%. 150 females and 30 males were held as operated brood stocks and were considered for maintenance and rearing. Considering the fact that there was a 10% decrease in capture from natural resources over last 5 years means of mature female brood stocks including 327 individuals, totally 180 individuals were included for micro caesarean operation. As a result, the annual biomass will be about 3 tonnes, which requires a culture area of 23000 m2 (2.3 ha) during the next 4 years culture period. This estimate comes from brood stock transfer in Gorgan bay rearing condition and from obtained indices. The cumulative number of spawners shifts over the first year to fourth, are estimated with 180, 162 145 and 130 individuals, respectively that will be at the most - exploited for caviar extraction after 3-5 years maintenance and rearing of spawners. So, brood rearing period is up to 4 years and in the fifth year of the culture system the spawners will be withdrawn from the system. It is intended to produce 200 to 350 kg of caviar per year consecutively and 1631 kg Caspian Sea caviar during a 10- year period from reared spawners.

References - Azari Takami, Gh. 2009. Acipenceridae culture and propagation. Tehran University press. 401 pp. (In Persian). - Chebanov, M. and Billard, R. 2001. The culture of sturgeons in Russia: production of juveniles for stocking and meat for human consumption. Aquat. Living Resour., 14: 375- 381. - Liao, I-C. and Lin, C.K. 2000. Cage culture in Asia. Proceedings of International Symposium on cage Aquaculture in Asia. Asian Fisheries Society and World Aquaculture Society, Manila, Philippines. - Peykaran mana, N. 2013. Four-volume final report of techno-economic and environmental feasibility of sturgeon breeding within the island Ashuradeh (Gorgan Bay) in Pen culture, the holding company of agricultural services. - Pourali, H.R. and Mohseni, M. 2007. Quantitative and qualitative study of density, nutrition and water in breeding sturgeon. Quarterly of Abzian, 5(11): 37-48. (In Persian). - Pourali Fashtomi, H.R., Yazdani, M.A., Peykaran Mana, N., Hassani, H., Mohseni, M. and Nezami, A. 2012. Biotechnical of sturgeon fish culture in freshwater and brackish water. Quarterly of Abzian, 9 (25): 21-30. (In Persian). - Pourkazemi, M. 2008. Living resources of the Caspian Sea and Environment Convention. Journal of Central Eurasian Studies. 1 (1): 1-20. (In Persian). - Shakorian, M., Pourkazemi, M. and Yazdani, M.A. 2010. Final project report of beluga breeding in cages in freshwater. 53 pp.

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Artemia cyst decapsulation and its necessity in sturgeon larvae feeding Nemat Peykaran Mana*, Masoud Seidgar, Latif Esmaeili Dahesht, Asadi, Hadi., Esmaeil Hosseinnia, Alireza Ashoori. *International Sturgeon Research Institute of Caspian Sea (AREEO)

[email protected]

Introduction The hard capsule that encloses Artemia dormant eggs can be completely removed by short term putting the cysts in hypochlorite solution (Decapsulation). Using decapsulated cysts has many advantages including: (a) cyst shells cannot be contaminate the culture tank, (b) it is possible to easily isolate Artemia nauplii from cyst shells, (c) non hatched cysts and empty shells cannot be ingested by fish larvae and thus cannot obstruct the fish intestine. Decapsulated cysts are smaller than Artemia cysts; also, the emerging nauplii are a better food source for fish early larval stages than Artemia cyst nauplii. Economically, Artemia cyst decapsulation can produce a high cyst hatching percentage and is a good solution for incubating cysts with otherwise low hatching percentages. In this study, the hard shell or chorionic layer of inactive Artemia cyst is removed which includes: cysts hydration, chorionic layer removal by sodium hypochlorite, washing cysts with abundant freshwater and sodium tiosulphate 0.1 N to inactivate the chloride. Resistant capsules of Artemia cysts are not digestible for sturgeon larvae and cyst decapsulation cause to reduce energy consumption of nauplii during emerging the cyst. Decapsulated cysts can be used to feed sturgeon larvae, marine fish, ornamental fish, cat fish, rainbow trout, crayfish and shrimp post larval stages and this is done a large commercial scale.

Methods and Materials Decapsulation of Artemia cysts were done using methodologies described by Lavens and Sorgeloos, 1996. Selective cysts were added to decapsulation solution at a density of 100g/L and suspended for 3-5 minutes with aeration. The process were evaluated under loop and whenever the most of the cysts were decapsulated and the color of cysts turned to light red, the cysts were removed from the decapsulation solution and washed with clean water to delute and remove the chloride . The nauplii from these decapsulated cysts can be used to feed surgeon early larval stages or dried, packed and kept in 0-4 °C until use.

Results and Discussion Decapsulated cysts have many advantages including: cyst shells cannot be imported to the culture tank, it is impossible to isolate Artemia nauplii from cyst shells during usual cyst hatching (Peykaran Mana, 2006), non hatched cysts and empty shells cannot be digested by fish larvae and may be obstruct the fish intestine. The nauplii from decapsulated cysts contain higher digestible energy content and individual weight (30-55%) compared to usual Instar I nauplii and decapsulation can provide disinfected cysts while destroying the bacteria and fungi attached to the shell surface. Decapsulated cysts are smaller than Artemia cysts, Also, its emerging nauplii is a better food source for fish early stage larvae feeding than Artemia cyst nauplii. By this method the cysts with low quality can be used for fish feeding. . Decapsulated cysts can be used for the first larval stage feeding of sturgeon fish and shrimps. Nowadays, decapsulation of Artemia cyst is practically done in most Iranian sturgeon hatcheries and breeding farms. By decapsulation of cysts it can be possible to use even cysts

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir with low hatching percentage or non hatched cysts in addition of high quality ones in cases of unforeseen shortages of good quality cysts (Esmaieli Dahesht, 2007). Lavens and Sorgeloos, (1996) have considered the decapsulated cysts as one of the products of Artemia. Azarvandi, 1997, mentioned the Artemia cyst decapsulation method and its role in aquatic animals feeding. Hosseini Ghatreh (1998) determined the nutritional value, protein, fat, fatty acid profile of Urmia lake Artemia and its decapsulated cysts in different growth stages of Artemia. Artemia decapsulated cysts have been used for feeding of Cyprinus carpio (Vanhaecke, 1990) and shrimp post larvae (Ribeiro, 2004). The optimum shelf life to maintain the nutritional value of Artemia decapsulated cysts is up to 3 months after production and vacuum packaging (Esmaieli Dahesht, 2007). Decapsulated cysts can be hatched and obtained instar I nauplli and can be used directly for sturgeon fish feeding or added to manual diets for aquatic animals rations (Leger, 1986; Bengtson, 1991). However, the most problems of decapsulated cysts are that they are non- movable and non- floating. Therefore, some reared aquatics such as marine fish larvae cannot benefit from their small size; high energy content and pathogen free aspects, unless these particles are floated by aeration or water exchange in the culture tank (Azari Takami, 1990). The result of this study is beneficial for producers and consumers of this valuable product.

References - Azari Takami, Gh. 1990. Artemia as a valuable food for sturgeon fish feeding. Proceedings of the National Conference of utilization of aquatic resources of the Caspian Sea. Babolsar, 509-524. (In Persian). -Azarvandi, A.R. 1997. Artemia cyst decapsulation and its role in aquatic animals feeding. D.V.M. Thesis. Urmia University. 128 pp. (In Persian). - Bengtson, D.A., Lager, P. and Sorgeloos, P. 1991. Use of Artemia as food source in aquaculture. In: Artemia Biology, R.A. Browne, P. Sorgeloos and C.M.A Trotina (Eds), 1st Edn. (CRC press, USA), pp: 256-285. - Esmaeili Dahesht, L. 2007. The evaluation of none hatched decapsulated cysts of Artmia urmiana and its qualitative changes during one year. Final project report of Iranian fisheries research organization, 53 pp. (In Persian). - Hosseini Ghatreh, H., 1998. The study of nutritional value of Artemia from Urmia Lake stressed to determination of protein, fat and fatty acid composition in different growth stages. D.V.M.Thesis. Urmia University. 125 pp. (In Persian). - Lavens, P. and Sorgeloos, P. 1996. Manual on the production and use of live food. - Leger. P., Bengtson, D. A., Simpson. K. L. and S0rgeloos. P. 1986. The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biol. Ann. Margaret Barnes, Ed. Aberdeen university press Rev. 24, pp: 521- 623. - Peykaran Mana, N., 2007. Qualitative and quantitative evaluation of Artemia cyst, decapsulated cyst and obtained nauplii in 3 geographical regions of Iran. M.Sc. Thesis., Lahijan Islamic Azad University, 144pp. (In Persian). -Ribeiro, F.A. &Tones, D.A. 2004. The potential of dried, low-hatch, decapsulated Artemia cysts for feeding prawn post-larvae. Aquaculture international, 6(6):421-440.

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Dietary administration of Lactobacillus plantarum enhances both, growth performance and innate immune response of Siberian sturgeon, Acipenser baerii Moheb Ali Pourgholam*, Hossein Khara, Reza Safari, Mohammad Ali Yazdani Sadati, ,Mohammad Sadegh Aramli, Mir Hamed Sayed Hassani

*Caspian Sea International Sturgeon Research Institute (AREEO), Rasht, Iran.

Introduction Recently, the culture of sturgeon has developed into a very successful industry owing to the high value of the eggs (caviar) and meat. Acipenser baerii is one of the frequently used species of Acipenseridae and a frequent candidate for Aquaculture in Iran the species is usually raised in, tanks, or ponds. Intensive culture exposes the fish to different sources of stress, including high stocking densities and manipulations; therefore the development of adequate methods to enable to boost the immune system of the fish and to reduce the risk of infectious diseases is vital (Nomoto, 2005). Probiotics are defined as live microbial feed supplements that affect the host animal beneficially by improving its intestinal microbial balance. There are many of the earlier studies in fish, which have dealt with growth promoting and disease protective ability of probiotics. (Fuller, 1989). This study investigated the probiotic effects of L. plantarum, a new class of candidate probiotics, on the innate immune response, growth and survival of the Siberian sturgeon.

Materials and Methods Juvenile fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations (1 × 107, 1 × 108 and 1 × 109 colony- forming units (cfu) g–1) and a control (non-supplemented basal) diet for 8 weeks. Growth performance indices were increased in fish fed the 1 × 108 cfu g–1 L. plantarum diet compared to the other groups.

Results and Discussion There was an increased innate immune response in fish fed the experimental diets. The highest levels of lysozyme activity, total immunoglobulin (IgM) and complement component 8 3 (C3) were observed in fish fed the diet containing L. plantarum at a concentration of 1 × 10 cfu g–1 , but there was no significant difference in the level of complement component 4 (C4) in fish fed the experimental diets or the control diet. In the present study, fish fed diets containing L. plantarum at doses of 1 × 107, 1 × 108 and 1 9 × 10 cfu g–1 showed increased levels of IgM and lysozyme activity, especially at the 1 × 108 cfu g–1 level. Improvement in systemic immune parameters, including alternative complement pathway, lysozyme and phagocytic activities, has been reported for other species (E. coioides) (Girri et al., 2014) (Chang et al., 2009) and Rohu, Labeo rohita (Girri et al., 2014) a member of the carp family) fed a L. plantarum-supplemented diet. The present study indicates some positive effects (growth performance and immune indices) of dietary administration of L. plantarum at a concentration of 1 × 108 cfu g–1 in the Siberian sturgeon.

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References Nomoto, K. 2005. Prevention of infections by probiotics. J Biosci Bioeng, 100: 583–592 Geraylou, Z., Souffreau, C., Rurangwa, E., De Meester, L., Courtin, C.M., Delcour, J.A, et al. 2013. Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut microbiota of juvenile Siberian sturgeon (Acipenser baerii). Fish Shellfish Immunol 35: 766 – 775. Fuller, R. 1989. Probiotics in man and animals. A review. J Appl bacteriol 66: 365–378. Balcazar, J.L., de Blas, I., Ruiz-Zazuela, I., Vandrell, D., Girones, O. and Muzquiz, J.L. 2007. Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss). FEMS Immunol Med Microbiol 51:185–93

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The effect of Allium sativum on the growth performance of Juvenile Siberian sturgeon (Acipenser baerii) Hooshang Yegane Rastekenari1, Habib Vahabzadeh Roudsari2*, Mohamad Ali Yazdani Sadati 3

1 Department of fisheries, Rasht branch, Islamic Azad University, Rasht, Iran - P.O.BOX: 41354196 2*Lahijan Azad University, Dr. Keivan Research Center of Fishery and Marine Science of Chamkhaleh - P.O.BOX: 4471155591. 3 International Sturgeon Research Institute (AREEO) - Rasht. P.O.BOX: 41635-3464

[email protected] Introduction One of the most reared species of sturgeon in Iran is Acipenser baerrii. It is cultured at high density in fiberglass and concrete tanks. As a result of environmental problems often due to inadequate culture management, the fish suffer stress and their growth rate and resistance to infectious diseases is lowered (Shalaby et al., 2006). Several plant products have been reported to promote various activities like anti - stress, growth enhancement, appetite promotion and immune stimulation in aquaculture practices (Citarasu et al., 2001, 2002; Sivaram et al., 2004). Garlic (Allium sativam) has been proven to control pathogens, particularly bacteria and fungi, thus improving the health of fish (Corzo - Martinez et al., 2007). also, Several herbs such as garlic, onion among others have been tested and evaluated for promoting growth promoting (Citarasu et al., 2002; Sivaram et al., 2004), feed conversion ratio (Shalaby et al., 2003, enhancement of protein digestibility (El – Dakar et al., 2004) in fishes and other aquatic organisms. (Austin, 2009). Therefore, the aim of this study was to examine the stimulate growth effect of garlic (Allium sativum) as a feed additive on Acipenser baerii L.

Methods For this purpose150 fingerlings of Acipenser baerii with an average weight of (30.45±0.4 g) were stocked in fifteen fiberglass tanks without significant difference and fed by five diets that were labeled as (AS0 ،AS0.5, AS1, AS1.5, AS2) (containing protein:45%, fat:16% and fiber3.5%) for 8 weeks. Growth was followed

Results and discussion Allium sativum Supplemented diets were offered and improved fish growth performance and feed conversion ration. Highest final weight, weight gain, daily growth rate, specific growth rate were obtained for fish fed AS 0.5 and AS1 that have significant differences compared to fish fed the control diet (P<0.05). The best of food conversion ratio was recorded in fish fed by AS1 diet (P<0.05). However, diets were not affecting the condition factor (P>0.05), Nevertheless, feed efficiency and Protein conversion efficiency ratio were significantly higher in fish fed with Allium sativum than in controls (P<0.05). Results of this study are comparable with the studies of Diab et al. (2002), Farahi et al. (2010), Nya and Austin (2009) and Shalaby et al. (2006), where significant growth rates were observed in treated fish over the controls. Khalil et al., (2001) reported that garlic contains allicin, which improves the performance of intestinal flora, thus improving digestion. This consequently enhances the utilization of energy, bringing about improved growth.

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It was clear from this study that the use of garlic stimulated desired food metabolism positively, led to enhanced feed intake as well as better digestibility. Therefore, SGR and FCR were enhanced which led to higher weight-gain in treated groups.

References Citarasu, T., Babu, M. M., Punitha, S. M. J., Venket Ramalingam, K. and Marian, M. P. 2001. Control of pathogenic bacteria using herbal biomedicinal products in the larvi culture system of Penaeus monodon. International Conference on Advanced Technologies in Fisheries and Marine Sciences, MS University, India. Diab, A.S., El-Nagar, G.O. and Abd-El-Hady, Y.M. 2002. Evaluation of Nigella sativa L (black seeds; Baraka), Allium sativum (garlic) and BIOGEN as feed additives on growth performance and immunostimulants of Oreochromis niloticus fingerlings. Suez Canal Veterinary Medicine Journal 745–775. Farahi, A., Kasiri, M., Sudagar, M., Iraei, M.S. and Shahkolaei, M.D. 2010. Effect of garlic (Allium sativum) on growth factors, some hematological parameters and body compositions in Rainbow trout (Oncorhynchus mykiss). AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation 3 (4), 317–323. Siva, G.P (2001). Recent advances on the nutritional benefits associated with the use of garlic as supplement. American Society of Nutrition Science; 2(1), 1106 – 1108

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The effect of L –Carnitin on growth rate, body composition and hematological tT Indices of Juvenile Acipenser nudiventris Mohamad Ali Yazdani Sadati*, Mir Hamed Sayed Hassani, Mina Habibzade

*Caspian Sea International Sturgeon Research Institute (Areeo)-Rasht, P.O.BOX:416353464

[email protected] ntdoftcudoft L-carnitine (l-h-hydroxy-g-N, N, N- trimethylaminobutyric acid) plays a significant role in fish nutrition. It is a lysine derivative, hygroscopic, water soluble organic compound (Harpaz, 2005). L-carnitine acts as a cofactor for the transport and oxidation of long-chain fatty acids by the mitochondria while facilitating the use of fatty acids for obtaining energy, and thus sparing dietary protein for anabolic processes (Bilinski and Jonas, 1970; Emaus and Bieber, 1983). The use of L-carnitine as a growth promoter in fish is controversial. Many studies revealed significant effects of dietary L-carnitine on the performance of various culture fish (Harpaz, 2005).

Materials and Methods The present study was carried out, to aim of investigating of the effects of L-carnitine supplementation on the performance and body composition of Acipenser nudiventris fingerlings fed basal diets on 8 weeks. Experimental diets were formulated to contain L-carnitine by considering of the recommended concentrations of L-carnitine in each kg of diet which were between 100 and 1000 mg (Baumgartner & Alonso, 1998). Then four treatments with 500 mg, 750 mg, and 1000mg per kg and of feed and a witness group (no L-carnitine) each with 3 replicates (10 fish in each tank) were designed and set up. For preparation of feed desired amount of L- carnitine was weighed using a digital balance and dissolved in 50cc water and mixed homogeneously with 1 kg of diet. The obtained compound was then mixed for 15 minutes using a mixer and passed through a commercial grinder and dried in a dryer. A total of 120 Acipenser nudiventris averaging 49.36±1.27g (mean ± SD) were randomly distributed into 12 fiberglass tanks of 500 L capacity.

Experimental diets were formulated and prepared to contain 40% crude protein and 20 MJ/kg gross energy. Fish were fed 3% of body weight daily at 20 ± 2◦C. During the 71 days experimental period 5 biometry were carried out in 14 days intervals using digital balance with 0.01g precision. Weight and total length were measured and some growth indices such as FCR, SGR, were determined using the following formulas. Daily feed consumption (percent of body weight) DFC = (Feed intake / (initial weight +final weight)/ number of days) × 100 Feed conversion ratio (F.C.R( = Feed intake (gr)/weight gain (g). S.G.R (%) = (Ln final weight – Ln initial weight)/T *100 CF = 100 x (weight /Fork Lenfeh ^3) % BWI = 100 x (final weight - initial weight)/ initial weight Protein efficiency ratio (PER) = Wet weight gain in (g) / Protein intake in (g) Feed Efficiency (F.E) = Wet body weight gain × dry feed intake -1

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At the end of the feeding trial, 30% of fish population from each treatment was selected and blood samples were collected. Carcass samples were transferred into laboratory in order to investigate the effects of dietary L-Carnitine on body composition.

Results and Discussion Result indicated that there were no significant differences in fish weigh at the beginning of the rearing period. Based on the results obtained from this study, there were no significant differences (P>0.05) in final weight, body weight increase (%BWI), specific growth rate (SGR) and feed conversion ratio (FCR). However, the highest body protein were observed in 500 and 1000 mg L-Carnitine but carcass protein content improved with increasing dietary L- Carnitine levels(750 and 1000 mg L-Carnitine) (P<0.05). Total protein content and LDL of fish fed 500 and 750 mg L-Carnitine were higher than those of fish fed other experimental diets. Significant increases in hemoglobin and hematocrit were recorded in fish fed 750 and 1000 mg L-Carnitine (P<0.05). Similarly, studies on other tilapias including Mozambique tilapia (Oreochromis mossambicus) (Jayaprakas et al., 1996), hybrid tilapia Oreochromis niloticus × O. aureus (Becker et al., 1999) and Nile tilapia (Abou-Seif, 2006) revealed that dietary L- carnitine supplementation resulted in improved growth rates and feed efficiency. Similar results have also been reported with several other species reared on diets supplemented with L-carnitine, including European sea bass (Santulli and ِِD Amelio, 1986), African catfish (Clarias gariepinus) (Torreele et al ., 1993; Ozorio et al., 2001b), red sea bream (Chatzifotis et al., 1995), Indian major carp rohu (Keshavanath and Renuka, 1998); hybrid striped bass (Twibell and Brown, 2000).

Conclusions The results obtained from this study indicated that the dietary L-Carnitine had positive effect on fish biochemical index and body composition and suggested a dietary 750-1000 mg/kg for juvenile Acipenser nudiventris.

References Bilinski, E. and Jonas, R.E. 1970. Effects of coenzyme A and carnitine on fatty acid oxidation by rainbow trout mitochondria. Journal of Fish Research Board Canada, 27: 857-864 Emaus, R.K. and Bieber, L.L. 1983. A biosynthetic role of carnitine in the yeast Torulopsis bovina. Journal of Biology and Chemistry. 258, 13160-13165. Santulli, A. and DAmelio, V. 1986. Effects of supplemental dietary carnitine on the growth and lipid metabolism of hatchery reared sea bass (Dicentrarchus labrax L.). Aquaculture 59: 177- 186. Twibell, R.G. and Brown, P.B. 2000. Effects of dietary carnitine on growth rates and body composition of hybrid striped bass (Morone saxatilis male × M. Chrysops female). Aquaculture, 187: 153-161.

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Replacement of processed soy bean meal (SPM) by fish meal on growth and body composition of juvenile Acipenser baerii

Mohamad Ali Yazdani Sadati*, Mir Hamed Sayed Hassani, Ehsan Rezaii.

*Caspian Sea International Sturgeon Research Institute (AREEO)-Rasht, P.O.BOX:416353464 [email protected]

Introduction Minimizing the use of fish meal in aquaculture diets in favor of more cost-effective and sustainable ingredients is essential for the continued growth of the aquaculture industry (Naylor et al., 2009). Due to their abundance, relative low cost, high protein levels, and favorable amino acid profiles, soy derived ingredients represent a good and potential alternative to fish meal, especially for omnivorous species (Gatlin et al., 2007). Early research evaluating the applicability of SBM in diets of piscivorous species, however, suggested limited inclusion due to relatively high levels of heat-stable, non nutrient components. These include indigestible carbohydrates, such as oligosaccharides and no starch polysaccharides, as well as saponins and isoflavones (Ingh et al., 1991; Baeverfjord and Krogdahl, 1996). Soy protein concentrate (SPC), although more expensive than SBM, does not contain the alcohol soluble fraction and can be included at much higher concentrations in diets for piscivorous marine species to 67-77% respectively. The purpose of the present study was to investigate the value of incorporating SPC as a fish meal replacement in Acipenser baerii diets.

Materials and Methods Therefore, a growth study was carried out In order to test the effects of soy bean concentrate (SPH) on growth index, body composition of Siberian Sturgeon (A. baerii) for 8 weeks. A total of 120 Acipenser baerii juveniles (initial body weight 186.44 ± 31.3g; mean ± SD) (4 treatments) were reared in 12 fiberglass tanks and fed to satiation with four experimental diets in which Fish meal had been replaced by SPH at 0%, 10%, 20% and 30%. These diets were coded (SPH, SPH10, SPH20 and SPH30), respectively. Biometry of fish was carried out monthly. In the end of experiment, 30% of total fish sacrificed and transported to the laboratory to determine body composition. Results indicated that there are no significant differences in final weight (FW), specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion efficiency (FCR) in fish fed SPH20 and SPH30 compared to control diet. Also, body and lipid protein content had increased with increasing of soy bean concentrate in the diets (P<0.05). The highest body protein and lipid contents were obtained in fish fed SPH20.

Discussion Soy protein concentrate can be further processed so that levels of oligosaccharides, b- conglycinin, and protease inhibitors are negligible (Adelizi et al., 1998; USSEC 2008). Soy protein concentrate has higher essential amino acid concentrations and nutrient digestibility compared with SBM (Wilson 2002), which permits inclusion levels as high as 100% and 75% in diets of rainbow trout Oncorhynchus mykiss (Mambrini et al., 1999) and Atlantic salmon (Storebakken et al., 2000a), respectively. Tibbetts et al. (2006) determined that the protein apparent digestibility coefficient for SPC in diets for juvenile Atlantic cod Gadus morhua was 98.6%.

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Based on the growth response data calculated in this study, 30% FM could be replaced by (SPH) without significantly affecting the WGR, FCR and survival rate of juvenile Siberian sturgeon, and this fish is able to effectively utilize appropriate levels of SPH as the main protein source in the diet.

References Adelizi, P. D., R. R. Rosati, K. Warner, Y. V. Wu, T. R. Muench, M. R. White, and P. B. Brown. 1998. Evaluation of fish-meal free diets for rainbow trout, Oncorhynchus mykiss. Aquaculture Nutrition 4: 255–262. Baeverfjord, G. and A. Krogdahl. 1996. Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., and distal intestine: a comparison with the intestines of fasted fish. Journal of Fish Diseases 19: 375–387. Ingh, T. S. G. A. M. v. d., A°. Krogdahl, J. J. Olli, H. G. C. J. M. Hendriks, and J. G. J. F. Koninkx. 1991. Effects of soybean-containing diets on the proximal and distal intestine in Atlantic salmon (Salmo salar): a morphological study. Aquaculture 94: 297–305. Gatlin, D. M., F. T. Barrows, P. Brown, K. Dabrowski, T. G. Gaylord, R. W. Hardy, E. Herman, G. Hu, A. Krogdahl, R. Nelson, K. Overturf, M. Rust, W. Sealy, D. Skonberg E. JE. J. Souza, D. Stone, R. Wilson, and E. Wurtele. 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research 38: 551–579. Naylor, R. L., R. W. Hardy, D. P. Bureau, A. Chiu, M. Elliott, A. P. Farrell, I. Forster, D. M. Gatlin, R. J. Goldburg, K. Hua, and P. D. Nichols. 2009. Feeding aquaculture in an era of finite resources. Proceedings of the National Academy of Sciences of the USA 106: 15103– 15110. Gatlin, D. M., F. T. Barrows, P. Brown, K. Dabrowski, T. G. Gaylord, R. W. Hardy, E. Herman, G. Hu, A. Krogdahl, R. Nelson, K. Overturf, M. Rust, W. Sealy, D. Skonberg E. JE. J. Souza, D. Stone, R. Wilson, and E. Wurtele. 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research 38: 551–579.

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Effect of fish oil and vegetable oil on growth performance fo sterlet (Acipenser ruthenus) broodstock Sara Pourhosein Sarameh*, Bahram Falahatkar, Amir Bahri, Mahtab Yarmohamadi, Ali Reza Salarzadeh *Department of Fishery, Islamic Azad University, Bandar Abbas Branch, Bandar Abbas, Iran [email protected]

Introduction Fish oil (FO) is the main lipid source in fish diets due to its high level of n-3 highly unsaturated fatty acids (HUFA). The decrease in FO production since the mid 1990s was mainly due to dwindling fisheries resources (Turchini et al., 2011) and a drastic increase in demand which outstrips supply. Vegetable oils (VO) have been proposed as sustainable alternatives to dietary FO with various studies finding no deleterious impact on the health or growth rate of farmed fish when FO was replaced with VO (Seierstad et al., 2005). The formulation of dry compound diets for Sturgeon represents a major challenge for the development of sturgeon aquaculture and restocking. Due to early sexual maturity and its economic importance, sterlet has become one commonly cultured sturgeon species in many countries (Williot et al., 2005). Sturgeons like other teleosts use lipids and carbohydrates mainly as non-protein energy sources (Brauge et al., 1994). We focused in our study on the long term (7 months) effect of feeding FO and VO-based diets on sterlet growth and spawning performance.

Materials and Methods This study was performed in the Shahid Dr. Beheshti Sturgeon Fish Propagation and Rearing Complex (Guilan, Iran). Forty five sterlet females (990.3 ± 49.3 g; mean ± SE) were tagged, and 5 fish were introduced to each tank. Females were divided into three treatments with triplicate groups and allocated to 1063 L fiberglass tanks. The flow rate regulated to 17.5 ± 0.7 l min-1. Three experimental diets were formulated with 6% lipid originating from 100% fish oil (FO), 1:1 blend of fish oil and vegetable oil (FO + VO) and 100% vegetable oil (VO). At the end 7 months of the experiment, individual weight of female was measured. One-way analysis of variance (ANOVA) followed by Tukey's test was applied to study the effect of diet on growth using SPSS 15.0 (Chicago, IL). Differences were considered statistically significant when P<0.05.

Results Growth indices of brood stocks fed different diets are shown in Table 1. Fish in FO+VO treatment showed the highest final weight and the lowest weight observed in VO treatment. Moreover, significant differences were observed among the treatments in CF, BWI, SGR and WG. There were no significant differences in FE (table 1). Table 1. Growth indices of sterlet sturgeon fed for a period of seven months with different diets containing 100% fish oil (FO), 1:1 blend of fish oil and vegetable oil (FO + VO) and 100% vegetable oil (VO).

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Treatment Final weight (g) FE (%) CF BWI (%) SGR (% day-1) WG (g) FO 1173.3 ± 58.8ab 13.41 ± 2.5 0.44 ± 0.01ab 17.4 ± 3.4ab 0.9 ± 0.01ab 170.7 ± 32.9ab VO 1079 ± 51.1b 9.72 ± 3.8 0.42 ± 0.01b 12.3 ± 4.7b 0.06 ± 0.02b 120.1 ± 48.4b FO+VO 1281.8 ± 80.8a 24.2 ± 4.2 0.48 ± 0.01a 30.0 ± 3.7a 0.14 ± 0.01a 306 ± 27.1a

Discussion Results of the present study indicate that the inclusion of VO in the diet had adversely affected the growth performance of sterlet broodstock. Replacing FO with VO in the diet lead to a significant response in growth indices of sterlet. Regarding growth performance, in our study, there were significant differences between treatments, so that, FO + VO treatment and VO treatment had the highest and lowest levels, respectively. It is well established that FO is highly valued for high proportions of n-3 LC-PUFA (Haraldsson and Hjaltason, 2001). VO contains high levels of fatty acids (n-6) PUFA and lower ratios of n-3/n-6 compared to marine oils (rich in n-3 HUFA) (Miller et al., 2007). Therefore, blended VO and FO in the diet can balance PUFA (n-6 and n-3) (Bransden et al., 2003). Studies have shown that white sturgeon (A. transmontanus) and Russian sturgeon (A. gueldenstaedtii) need to both n-3 and n-6 fatty acids (Şener et al., 2005). This suggested that the suitable dietary oil level of sterlet is a modest amount of VO and FO. Hosseini et al. (2010) showed that H. huso have the ability to de-saturate and elongate C18 PUFA to EPA and DHA. In conclusion, the results of this study suggest that growth in sterlet can be improved by feeding by blended of fish oil and vegetable oil.

References Bransden, M.P., Carter, C.G. & Nichols, P.D. 2003. Replacement of fish oil with sunflower oil in feeds for Atlantic salmon (Salmo salar): effect on growth performance, tissue fatty acid composition. Comparative Biochemistry and Physiology, 135 B: 611-625. Brauge, C., Medale, F. & Corraze, G. 1994. Effect of dietary carbohydrate levels on growth, body composition and glycemia in rainbow trout, Oncorhynchus mykiss reared in seawater. Aquaculture, 123: 109-120. Haraldsson, G.G. & Hjaltason, B. 2001. Fish oils as sources of important polyunsaturated fatty acids. In: Gunstone, F.D. (Ed.), Structured and Modified Lipids. Marcel Dekker, New York, pp, 313-350. Hosseini, S.V., Abedian-Kenari, A., Rezaei, M., Nazari, R.M., Feas, X. & Rabbani, M. 2010. Influence of the in vivo addition of alpha-tocopheryl acetate with three lipid sources on the lipid oxidation and fatty acid composition of Beluga sturgeon, Huso huso, during frozen storage. Food Chemistry, 118: 341-348. Miller, M.R., Nichols, P.D. & Carter, C.G. 2007. Replacement of dietary fish oil for Atlantic salmon parr (Salmo salar L.) with a stearidonic acid containing oil has no effect on omega- 3 long-chain polyunsaturated fatty acid concentrations. Comparative Biochemistry and Physiology, 146 B: 197-206. Peng, S., Chen, L., Qin, J.G., Hou, J.N.A.Y., Long, Z.Y.E.J. & Sun, X. 2008. Effects of replacement of dietary fish oil by soybean oil on growth performance in juvenile black seabream, Acanthopagrus schlegeli. Aquaculture, 276: 154-161. Seierstad, S.L., Poppe, T.T., Koppang, E.O., Svindland, A., Rosenlund, G., Froyland, L. & Larsen, S. 2005. Influence of dietary lipid composition on cardiac pathology in farmed Atlantic salmon, Salmo salar L., Journal of Fish Diseases, 11: 677-690.

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Şener, E., Yildiz, M. & Savaş, E. 2005. Effects of dietary lipid on growth and fatty acid composition in Russian sturgeon (Acipenser gultenstaedtii) juvenile. Turkish Journal of Animal Sciences, 29: 1101-1107. Turchini, G.M., Nichols, P.D., Barrow, C. & Sinclair, A.J. 2011. Jumping on the omega-3 bandwagon: distinguishing the role of long-chain and short-chain omega-3 fatty acids. Critical Reviews in Food Science and Nutrition, 52: 795-803. Williot, P., Brun, R., Rouault, T., Pelard, M., Mercier, D. & Ludwig, A. 2005. Artificial spawning in cultured sterlet sturgeon, Acipenser ruthenus L., with special emphasis on hermaphrodites. Aquaculture, 246: 263-273.

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The feasibility of creating a database of live sturgeon brood stocks (500 breeders) in Pen culture conditions in the Gorgan Gulf Nemat Peykaran Mana*, Mohmmad Pourkazemi, Ayoub Yousefi Jourdehi, Seyyed Hamed Hassani, Hadi Asadi, Seyed Mustafa Aghilinejhad, Shahram Mohammadi Gholinejhad

International Sturgeon Research Institute (AREEO), Rasht, Iran [email protected] Introduction Surely if the desirable size composition and age structure of sturgeon stocks is available, the genetic diversity of the species will be reasonable and considerable. However, with the dwindling natural stocks particularly dramatic reduction in matured brood sturgeon species is obvious, threatening the genetic diversity needed to establish sustainable populations for the future. Many matured sturgeon migrated to the Caspian Sea Rivers for spawning during the period 1987 to 1990, but the spawning stock decreased substantially and reached a minimum in recent years. Thus all 5 species of the Caspian Sea sturgeons are now in red list of IUCN. Ashuradeh Gulf is one of the most important habitat and ecosystem for preservation of extricated aquatic species such as sturgeons. In order to preserve the genetic integrity and genetic resources of these valuable species, 1420 numbers of farmed juveniles with mean weights 15 to 20 grams were reared in pens up to maturity and serve as a brood stock. If the program is fully implemented, it will generate a total of 500 breeders of the five species native to the Caspian Sea and the 7th to 10th year- old will be produced that can be considered as a treasury gene pool from which the natural resource can be rebuilt without loss of genetic integrity.

Materials and Methods This project requires 12700 square meters of rearing environment with a depth of 1.8 to 2.5 meters. Sturgeon juveniles weighing 15 to 20 g will be adapted to hand feeding and transfer to pen after this stage. 1420 sturgeon juveniles will be reared in a pen conditions with densities of 0.5 up to 8 kg per square meter in the productive stage. The desirable range for the water temperature will be between 15 and 25°C and species composition in the pens will be according to table 1 with a constant flow of water provided. Combination of automatic and manual methods of feeding (food tray, sprinkle) will be used. Table 1- number and composition of sturgeon juvenile species for creating a living database for sturgeon conservation culture Number of fish Breeder Breeder Species number percentage Acipenser persicus 850 300 60 Huso huso 210 75 15 Acipenser stellatus 145 50 10 Acipenser gueldenstaedtii and 210 75 15 Acipenser nudiventris Total 1415 500 100

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Results and Discussion Due to the harsh reality while experiencing habitat destruction for sturgeon spawning in the rivers of Iran, 100% sturgeon of reserves is related to artificial reproduction and release of juveniles into the sea. In the past few years, even artificial breeding of sturgeon species was zero in Acipenser gueldenstaedtii and Acipenser nudiventris and reproduction of valuable species of beluga has been facing serious constraints. Due to the illegal and immature fish catch, even if thousands of sturgeon fingerlings are released to the Caspian Sea, there is not hope to reach maturity and provide breeders in future. In other words, more than 95% of 4.3 million sturgeon fingerlings are produced in Iran focused on Persian sturgeon (Acipenser persicus) and 4 other species are reproduced and released in the lowest possible number. Based on the studies and experiences, Russia was to create a shelter in reservoirs and natural lakes and thousands of sturgeon to be released in those areas to have relatively normal living conditions and live bank account for this valuable species until in the case of destruction of natural resources sturgeon in the Caspian Sea, an acceptable number of specimens of each species is needed to maintain the genetic integrity of the species to use them after maturating for reproducing and breeding as well as release and thus maintaining the spcies identify. Without a doubt this should be done by any of the neighboring and beneficiary countries of the Caspian Sea for native sturgeon genetic resources conservation and initial programmes in other parts of the world are on the way to do the same. One of the practical and application solutions is using the natural status of Gorgan bay that are suitable for stock protection, gene bank and sturgeon live bank keeping because of habitat for native sturgeon species. Nowadays, 300 – 400 sturgeon breeders captured in Golestan and Mazandran province that if not preserved, we will not find a fish in the future. Food required will be 65 tons with food conversion rate 1.7 for Huso huso and 2 for other species. Based on experiences, fish will matured at age 7, 8 and 8 - year old in farmed Acipenser stellatus, Acipenser persicus and Huso huso, respectively. Ovulated eggs are extracted by micro incision method without killing breeders. Production program and biological requirements of sturgeon including pen, density, feed requirement and number of breeders mentioned in table 2. Table 2. Required feed for brood stocking

Years 1 2 3 4 5 6 7 8 9 10 Species Total Acipenser persicus 0.3 1.3 2 2.7 3.9 5 5.9 3.3 - - 24.4 Huso huso 0.2 0.9 1.3 1.7 2.7 3.7 4.7 5.7 6.4 3.3 30.6 Acipenser stellatus 0.1 0.3 0.6 0.5 0.8 1 0.5 - - - 3.8 Acipenser nudiventris and 0.1 - 0.3 0.5 0.7 1 1.3 1.5 0.8 - 6.2 Acipencer gueldenstaedtii 0.7 2.8 4.4 5.6 8.4 11 12 9.8 6.4 3.3 65

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References Peykaran Mana, N. 2013. Four-volume final report of techno-economic and environmental feasibility of sturgeon breeding within the island Ashuradeh (Gorgan Bay) in Pen culture, the holding company of agricultural services. Pourkazemi, M. 2000. Sustainable stock assessment and management. Department of Iranian Fisheries proliferation. 18: 17 – 30. Tavakoli, M. and Khoshghalb, M. 2000. Supervision on autumn and winter catch of sturgeon region 1 and 2 of guillan fisheries. International Sturgeon Research Institute. 29P. Tavakoli, M. and Khoshghalb, M. 2000. Evaluation of damages in illegal dams on sturgeon stocks at the southern coasts of the Caspian Sea. International Sturgeon Research Institute. 21P.

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Effect of diet composition on growth performance and hepatic metabolism in Siberian sturgeon, Acipenser baerii submitted to starvation and refeeding

Sedigheh Babaei, Abdolmohamad Abedian Kenari*, Mehdi Hedayati, Mohamad Ali Yazdani Sadati Ph.D. graduated, Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran. [email protected] Introduction Many fish species undergo natural starvation periods. Adaptation to starvation is possible through the activation of behavioral, biochemical and physiological mechanisms. Knowledge of the effect of dietary nutrients on the intermediary metabolism during starvation and refeeding periods can be useful to improve production performance in aquaculture.

Materials and Methods In order to analyze the effect of dietary nutrients on the liver metabolism of Siberian sturgeon (Acipenser baerii) submitted to starvation and refeeding, four isoenergetic diets differing in nutrient composition were designed. A group of 180 Siberian sturgeon juveniles (initial body weight 30 g) were obtained from International Sturgeon Research Institute (Gilan, Iran) and randomly supplied in 12, 500-L circular fiberglass tanks (n=15 per tank). Temperature, dissolved O2, pH-value and debit were maintained at 22 ± 4 °C, 7.1 ± 1.5 mg L-1, 7-8 and 4.5 ± 0.5 L min-1, respectively. Diets were named LP-St (low protein- high carbohydrate), HP-St (high protein- high carbohydrate), LP-L (low protein- high lipid) and HP-L (high protein- high lipid). Four groups of fish were fed manually to satiety with the corresponding experimental diet for 3 weeks, starved for 2 weeks and then refed for 5 weeks on the same diet and conditions. Three tanks were used for each condition.

Results and Discussion Starvation led to mobilization of the hepatic lipid store to a greater extent than glycogen. Starvation increased SOD activity irrespective of the diet, while low protein diets (LP-St and LP-L) resulted in high CAT activity values. The oxidative damage decreased after 5 weeks of refeeding. Refeeding the starved fish on a high protein/high carbohydrate diet (HP-St) promoted the greatest growth performance. In addition to report for the first time the effect of diet composition on growth, liver composition and antioxidant activities in Siberian sturgeon submitted to starvation and refeeding, our finding suggest that refeeding on HP-St diet stimulated the use of dietary carbohydrates and made possible a protein sparing effect in Siberian sturgeon.

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Fig 1. Hepatosomatic Index (HSI). Sampling was Fig 2. Liver composition (glycogen) of Siberian sturgeon. performed after 3 weeks of feeding (3 F), 2 weeks of Sampling was performed after 3 weeks of feeding (3 F), 2 starvation (2 S) and 5 weeks of refeeding (5 R). weeks of starvation (2 S) and 5 weeks of refeeding (5 R).

Fig 3. Liver lipid of Siberian sturgeon. Sampling was Fig 4. Liver protein of Siberian sturgeon. Sampling was performed after 3 weeks of feeding (3 F), 2 weeks of performed after 3 weeks of feeding (3 F), 2 weeks of starvation (2 S) and 5 weeks of refeeding (5 R). starvation (2 S) and 5 weeks of refeeding (5 R).

Fig 5. Specific activity of CAT in the liver of Siberian Fig 5. Specific activity of CAT in the liver of Siberian sturgeon after 3 weeks of feeding (3 F), 2 weeks of sturgeon after 3 weeks of feeding (3 F), 2 weeks of starvation (2 S) and 5 weeks of refeeding (5 R). starvation (2 S) and 5 weeks of refeeding (5 R).

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Our findings show for the first time that Siberian sturgeon juveniles experience metabolic adjustments to both starvation and refeeding, and that diet composition has a major impact on the metabolic responses to nutritional status. Growth performance and liver composition suggest that refeeding with a high protein/high carbohydrate diet stimulates the use of dietary carbohydrates; the results of the present study may be useful to improve feed management to achieve better nutrition efficiency and fish health.

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Effects of stable barium isotope on growth and barium concentration in the muscle of Persian sturgeon (Acipenser persicus)

Hadis Mirali, Ali Bani*, Javad Fasihi *Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran & Marine Science Department, Caspian Sea Basin Research Center, University of Guilan, Rasht, Iran. E-mail: [email protected]

Introduction Marking of fish is substantially applicable to fisheries management and ecology including the monitoring of fish movement, studying population estimates, supporting age determination, following fish growth and migration as well s determining survival (Munro et al., 2008). There are several advantages of using enriched isotopes for fish marking, for instance mark creation and detection are easy and the isotopic mark will remain throughout the life of fish (Woodcock et al., 2011). Therefore, there is a growing interest in the use of stable isotopes as markers for fish. In this study, the effects of marking with stable isotope of barium on growth and barium concentration in the muscle tissue was assessed in juvenile Persian sturgeon, Acipenser persicus.

Materials and Methods Juveniles were treated with water spiked with 0 (control), 30, 60 or 90 µg L-1 137Ba (Isoflex, San Francisco) for 1 and 5 days in the Shahid Beheshti Sturgeon Hatchery. The marked fish from each treatment group were retained for up to 14 days post-labeling to evaluate the effects of this technique on fish growth and health of the post-marking days. Muscle samples were freeze-dried for 32 h. Once dry, the samples were analyzed using ICP-OES. All data were analyzed using two-way ANOVA to assess the effects of treatment and time. Additionally, a one-way ANOVA was applied to calculate the effect of isotope exposure on the growth of juveniles.

Results and Discussion Isotope marks were observed in the pectoral fin spines in the first day of experiment. There was no considerable difference in the Ba ratios of muscle during marking and post-marking process for all three concentrations and times (P>0.05). In day 10, Ba concentration declined toward natural level (Fig.1).

Fig.1. Comparison of the Ba concentration in muscles of juvenile Persian sturgeon (Acipenser persicus) reared in 30, 60 and 90 µgL-1 of 137Ba during the immersion time and after immersion.

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There were no significant relationships between 138Ba/137Ba ratios in water and Barium concentration in muscle. Such relationship, however, was considerable between pectoral fin spine and water (Fig. 2).

y=0.449601x + 3.423251 y= -0.05844x +0.037707 2 2 R = 0.5721 R =0.1496 P<0.05 P=0.05

Fig. 2. Relationship between 138Ba/137Ba in water and Ba concentration in the muscle and pectoral fine spine.

The results indicate that far more Ba replaced with Ca2+ in bony structure compared to muscle tissues as reported by Williamson et al. 2009. In humans, it has been shown that c. 90% of the barium in the body is in bone tissue, while only 1–2% of the total body burden is generally found in muscle, adipose, skin and connective tissue (Williamson et al. 2009). Barium was quickly absorbed into bone tissues such as pectoral fine spines of treated fish; therefore, soft tissues such as muscles have a negligible concentration of Barium. Isotope immersion did not negatively affect early growth. There was no difference either in length or weight between marked and control fish during the experimental period (length: F=0.44, df 3, 31, P> 0.05; weight: F = 0.55, df 3, 31, P> 0.05). Minimal risk level (MRLs) of Ba consumption to humans is 0.2 mg/kg/day (ATDSR, 2013). The highest amount of Ba concentration was observed on the day 10 at spiking levels of 90 µg L-1. At the above-used concentration of barium, a 70 kg human could consume 31.1 kg of muscle tissue per day, every day. The present study shows that the levels of Ba required to mark juveniles are far too low to represent a health risk. These observations are in agreement with findings of Williamson et al. for marking marine fish larvae, with BaCl2. In conclusion, enriched 137Ba stable isotope can safely be used at low dosages to mark larvae of commercially important marine fishes, without adverse effects on the health of fish or on humans who may consume them.

References Munro, A.R., Gillanders, B.M., Elsdon, T.S., Crook, D.A. and Sanger, A.C. 2008 “Enriched stable isotope marking of juvenile golden perch (Macquaria ambigua) otoliths”; Canadian Journal of Fisheries and Aquatic Sciences, 65: 276–285. Woodcock, S.H., Gillanders, B.M., Munro, A.R., Crook, D.A. and Sanger, A.C. 2011. “Determining Mark Success of 15 Combinations of Enriched Stable Isotopes for the Batch Marking of Larval Otoliths”; North American Journal of Fisheries Management, 31: 843– 851. Williamson, D., Jones, G., Thorrold, S.R. and Frisch, A.J. 2009. “Transgenerational marking of marine fish larvae: stable‐isotope retention, physiological effects and health issues”; Journal of Fish Biology, 74: 891-905.

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A study on effects of light period and feeding regime on juvenile Huso huso growth performances

Mohsen Pourasadi*, Bahram Falahatkar, Mehran Avakh Keysami, Mohammad Rahanande, Iraj efat penah Komaee Ahmad Razavi sayad, and Bahman Meknatkhah

*Agricultural Research, Education and Extension Organization (AREEO)- Guilan, Iran [email protected]

Introduction The species Huso huso, family Acipenseridae, can be the most profitable endemic aquaculture fish in Iran (1), due to various advantages as growth performance and resistance to different environmental conditions (2). The optimum growth rate of fish is depending on different biotic and abiotic factors. Light period in aquaculture and feeding time are important factors that can lead to better fish growth performances. Knowledge of the environmental conditions and fish physiologic characteristics are very important in aquaculture (3&4). This research was conducted to study the combined effects of light period and feeding time on juvenile Huso huso growth performances.

Materials and methods This study was designated randomized with 4 treatments, each in 3 replications, with 12 circular concrete tanks (800 l each) each containing 120 juvenile Huso huso, average weight 51.38 g +0.39, reared for 8 weeks at Yusefpour aquaculture center, Siahkal, Guilan, Iran. Treatment 1: natural light period and day feeding time N (d) Treatment 2: natural light and 24 hrs feeding time N (nd) Treatment 3: 24 hrs light and day feeding time L (d) Treatment 4: 24 hrs light and 24 hrs feeding time L (nd) Huso huso juvenile were fed by Biomar Company pelleted commercial feed containing 48% proteins and 22% fatty acid and feeding was ad libitum. The results of this study were assessed using 2 ways ANOVA and Tukey’s test (P≤0.05), SPSS 22, USA.

Results The results are showed in table 1. Table 1. Growth parameters of juvenile Huso huso during 8 weeks of culture under different treatments (mean ± SD)

Growth parameters Treatments

1 N(d) 2 N(nd) 3 L(d) 4 L(nd)

Initial weight (g) 51.37a± 0.06 51.37 a ± 0.06 51.40 a ± 0.01 51.40 a ± 0.01 Final weight gain(g) 414.7 b± 9.78 409.07 b ± 7.37 431.27 b ± 24.97 409.47 b ± 32.27 Condition factor (%) 0.46c ± 0.01 0.46 c ± 0.00 0.47 c ± 0.01 0.47 c ± 0.00 Mean Weight gain)g) 362.7 d± 9.82 358.30 d ± 7.33 379.90 d ± 24.97 358.07 d ± 32.27 Feed conversion ratio 0.75ab ± 0.02 0.78 ab ± 0.04 0.72 ab ± 0.02 0.76 ab ± 0.04 Specific growth ratio% 3.73bc± 0.04 3.71 bc ± 0.03 3.8 bc ± 0.10 3.70 bc ± 0.14 Daily growth rate) g) 6.48 cd± 0.18 6.40 cd ± 0.13 6.78 cd ± 0.45 6.40 cd ± 0.58

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The same superscript in the row shows there are no significant differences. The results show that there are not significant differences among treatments in weight gain, survival, specific growth rate, daily growth rate, feed conversion ratio (P≥0.5).

Conclusion In conclusion, according to the similar results achieved by the different experimental conditions, the daily feeding regime in natural light period may be recommended, because of reduced costs, both of electricity and manpower to achieve the best results in juvenile Huso huso aquaculture.

References 1. Falahatkar, B., Poursaeid, S., Efatpanah, I., Meknatkhah, B. & Biswas, A. 2012. Effect of photoperiod manipulation on growth performance, physiological and hematological indices in juvenile Persian sturgeon, Acipenser persicus. Journal of the World Aquaculture Society, 43(5), 679-687. 2. Bronzi, P., Rosenthal, H., Arlati, G. & Williot, P. 1999. A brief overview on the status and prospects of sturgeon farming in Western and Central Europe. Journal of Applied Ichthyology 15 (4-5): 224-227. 3. Ghomi, M. R., Nazari, R. M., Sohrabnejad, M., Ovissipour, M., Zarei, M., Mola, A. E. & Naghavi, A. 2010. Manipulation of photoperiod in growth factors of beluga sturgeon Huso huso. African Journal of Biotechnology, 9(13), 1978-1981. 4. Doroshov, S.I. 1985. Biology and culture of sturgeon, Acipenseriformes. In Recent advances in Aquaculture, vol, 2 (y. F. Muir and Roberts, R. J.), p. 251-274.

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Study on the effects of Aloe vera extract on growth parameters and bacterial flora of intestine in Siberian sturgeon, Acipenser baerii Soheil Bazari Moghaddam*, Masoud Haghighi, Mostafa Sharif Rohani, Mohammad Ali Yazdani, Alireza Shenavar Masouleh, Jalil Jalilpour, Mehdi Masoumzadeh and Mehdi Alizadeh

* Agricultural Research Education and Extension Organization (AREEO), International Sturgeon Research Institute, Rasht, Iran [email protected] Abstract Considering the beneficial effects and benefits of the herb Aloe vera and its application in various industries such as pharmaceuticals and food industries, this study wanted to investigate the effects of Aloe vera extraction on growth parameters and bacterial flora of the Siberian sturgeon (Acipenser baerii). In this study, a total of 360 Siberian sturgeons, average weight 10.95 ± 0.04 g, randomly distributed in four treatments including a control group and three experimental groups, each with three replications were used. Aloe Vera extract powder at ratio of 0.5%, 1% and 1.5% plus a control were added to the food and given for eight weeks to the fish reared in the fiberglass vans tanks. Physicochemical parameters of the water were daily registered. Growth indicators such as weight gain, condition factor, feed conversion ratio, specific growth rate, protein efficiency ratio, and hepatosomatic index and survival rate were calculated. Results showed that all growth parameters (except hepatosomatic index) of the experimental group compared to the control group showed statistically significant (P<0.05). Total count of bacteria in intestine in the treated and control groups did not show significant differences (p>0.05), but significant increase in the count of anaerobic bacteria (lactic acid bacteria) were observed compared to the control group (p<0.05). The result showed that Aloe vera extract can be effective in improving the growth performance of Siberian sturgeon.

Keywords: Aloe vera extract, Growth, Bacterial flora, Acipenser baerii

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Effects of organic culture on growth in juvenile Siberian sturgeon, Acipenser baerii

Rahin Alsadat Hasehemi *, Mahmoud Bahmani, Ayoub Yousefi Jourdehi * M.S. Graduated, Islamic Azad University, Rasht branch, Iran [email protected] Introduction Organic aquaculture includes components that ensure that aquaculture activities are compatible with the environment and the organism is in health and wellness conditions. Today organic products in the aquatic animals industry by super supplements has found a special place among consumers, and the use of chemicals, hormones, antibiotics and bloodstocks that are genetically modified, are incompatible with the principles of organic aquaculture (Otles et al., 2010).

Materials and Methods In this study 180 Siberian sturgeon (mean weight 680.89 ± 29.93 g and mean length 63.79 ± 1.18 cm) were prepared at the International Sturgeon Research Institute and after two week of adaptation, fish were randomly introduced in 15 fibreglass tanks with a volume of 2000 litres each. 12 fish were introduced to each tank. Super supplements at 2.5 %, 5 % and 10 % were added to the diet and experiments were performed in triplicate. Fish biometry was carried out in all treatments during rearing period. Samples were collected for the measurement of growth parameters during the rearing period. Evaluation of growth trend was calculated based on the available references of mathematical equations (Bekcan et al., 2006).

 Average daily growth (ADG) (g/fish/day) (%) = [wt – wi /wi×T] ×100  Percent body weight increase (PBWI) (%) = [wt – wi / wi] ×100  Specific growth rate (SGR) (day) = [LnWt - LnWi / T] ×100  Feed Conversion Ratio = Feed intake (g)/ wf – wi

Results According to table 1, the average initial weight, initial biomass, initial length, final length and FCR showed no significant difference between the control group and the other treatments (P > 0.05), but the average final weights in the treatments with 5% and 10% of super supplement were significantly higher than other treatments (P < 0.05). The mean final biomass, mean body weight increasing (BWI%), specific growth rate (SGR), daily growth rate (DGR) and condition factor (CF) in super supplement 5% and control treatment without antibiotic were significantly higher than in other treatments (P < 0.05).

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Table.1. Growth parameters of various treatments in Siberian sturgeon during experimental period.

Growth parameters Control Without Control with Super Super Super antibiotic antibiotic supplement 2.5% supplement 5 % supplement 10 % Initial weight (g) 680.89 ± 29.93a 692.97 ± 32.10a 596.17 ±19.84a 667.56 ±19.84a 668.44 ±4.17a Final weight (g) 1839.27 ±21.13a 1935.23 ±17.48ab 1983.13 ±15.96ab 2034.78 ±30.87b 2077.30 ±20.88b Initial Biomass (g) 8170.67 ±359.27 a 8315.67 ±385.22 a 7154 ±238.12 a 8010.67 ±357.07a 8021.33 ±50.03a ±724.32 ab ±1023.44 b Final Biomass (g) 16577.82 ±1853.75b 11472.07 ±85.14 a 13309.45 ±131.89 ab 13855.27 16837.45 Initial length(cm) 63.79 ±1.18 a 63.58 ±0.66 a 61.38 ±0.37 a 64.01 ±0.94 a 63.83 ±0.26 a Final length (cm) 70.93 ±0.58 a 71.20 ±0.15 a 70.67 ±0.13 a 71.47 ±0.92 a 71.14 ±0.21 a BWI (%) 99.68 ±7.14 b 66.52 ±1.25 a 60.64 ±4.27 a 113.97 ±8.05 b 65.93 ±1.86 a FCR 1.71 ±0.49 a 2.02 ±0.03 a 2.29 ±0.14 a 1.60 ±0.51 a 2.02 ±0.08 a SGR (day) 0.73 ±0.05 b 0.57 ±0.008 a 0.53 ±0.029 a 0.79 ±0.03 b 0.56 ±0.01 a GR 7.78 ±0.24 b 5.13 ±0.31 a 3.99 ±0.16 a 8.17 ±0.08 b 4.89 ±0.03 a CF 0.71 ±0.04 b 0.59 ±0.007 a 0.54 ±0.007 a 0.73 ±0.004 b 0.54 ±0.01 a

Discussion In the present study the highest effect of super supplements on average final weight, final biomass, increased body weight (BW%), specific growth rate (SGR), daily growth rate (GR) and condition factor (CF) was observed in treatment with super supplement at 5 %. This also helps to keep the food quality which is of the basic standards in organic fish production (Otles et al. 2010). Pahang et al. (2012) studied the impact of organic culture with 2.5 and 5 % super supplement on some growth parameters in common carp (Cyprinus carpio) and found that the average final weight, weight gain, condition factor, specific growth rate and average daily growth showed significant differences (P < 0.05).

References Bekcan, S., Dogakaya, L., Cakirogullari, G. C. 2006. Growth and body composition of European catfish (Silurus glanis) fed diet containing different percentages of protein. Isr. J. Aqauculture. 58, 137-142. Otles, Y., Ozden, O., Otles, S. 2010. Organic Fish Production and the Standards. Acta. Sci. Pol. Technol. Aliment. 9, 125-131. Pahang, P., Bahmani, M. and Zamini, A. 2012. The impact of organic culture with super supplements on some biochemical parameters, growth and carcass analysis of common carp (Cyprinus carpio). Masters thesis, Islamic Azad University of Rasht. 127.

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Benefits of extruded feeds in aquaculture Nigel Linley

[email protected]

Producing high quality Aqua Feeds has a cost, not producing high quality Aqua feeds has an even bigger cost both in terms of not benefiting from raw material such as high value fish meal, fish oil, risk of environmental pollution, poor FCR and human resources time and effort. It is understood that 80% of cost in Aqua Feeds is raw materials with four vital aspects of Feed Production Technology accounting for the other 20%. • Grinding systems. • Extrusion system. • Drying • Coating

Grinding systems This is the first and potentially most vital stage of production with poor grist and a wide grist spectrum optimum use of thermal and mechanical energy to gelatinize starch will be limited. Specification targets should be 90% of the grist should be below 50% of the screen diameter in simple terms, 1mm screens, and 90% below 500 micron and with a narrow range of particle size. Todays large chamber hammer mills with 1500 RPM drives, accurate feeding and good aspiration systems are achieving these specifications even with recipes containing up to 50% fish meal and intrinsic values of 10% oil. Result optimum potential to achieve maximum starch gelatinization even with formulations of 8-10% starch.

Extrusion Systems Conditioning plays a significant part in the optimum system prior to the extrusion process. Utilizing this process reduces total energy consumption provides opportunities for further liquid addition, reduces wear cost and overall production cost. With possible starch gelatinization of 40% plus this will reduce significantly the SME (Specific Mechanical Energy) levels necessary to give maximum gelatinization of the starch molecules. Dual shaft conditioners offer the benefit of extended retention time, providing opportunities to cook the starch using thermal energy with moisture levels of 22-24% which the pellet mill process is unable to handle.

Single Vs Twin .

Always a question when selecting a system. Both systems have valuable attributes in the case of twin-screw, positive control, more flexibility to handle a wider range of raw materials, self cleaning, and accuracy with small diameter products. Against, purchase and operating costs of 50-75% in comparison with single screw systems. Single Screw, in general higher capacities, lower capital and operating costs, against less flexible in handling high oil products, poor mixing ability, residue and necessary clean out programs, low range of products regards small diameter feeds.

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Drying systems As energy consumption and the cost of energy has become a prominent factor in Aqua Feed production, more and more, drying is recognized as a high level cost, hence accuracy and efficient drying operation plays a significant part in overall cost. Vertical and Horizontal options will be determined by product range , overall plant design , energy efficiency and accuracy with today targets of + - 0.5% moisture deviation , dryer design is stretched to the limit , airflow control and air saturation can be monitored section by section in the drying process together with gentle handling of a delicate and yet not fully stable product.

Coating Today trend leans towards both batch and potential vacuum coating, again product dependent, driven by oil levels required and in some cases product density. Raw material costs have pushed the process forward with three tier batch systems, high grade accuracy mixers and lower batch times.

The Benefits In the last 30-40 years extrusion has become a dominant and first choice option in the production of Aqua Feeds. As with every developing industry initial low cost production options are prominent, in the case of Aqua Feeds, pelleting became the first possible option to serve a compound feed with all components and benefit from raw material costs. The pellet mill limitations not allowing nutritional benefits to enhance lower grade raw materials or produce a diet based on FCR (Feed Conversion Rate) immediately has impact on cost. Minimum starch gelatinization levels are factors that reduce water stability, create more waste and fines leading to actual higher production costs. As an industry progresses and more accurate evaluation takes place regards production cost and end result, then new systems require evaluation, more capital outlay much more controlled results. Hence extrusion becomes the lower cost option as raw material prices sore , maximum benefit, maximum flexibility are deciding factors, not only in producing a wide range of products, handling more diverse raw materials but environmental factors become issues for sustainable and acceptable fish farming . Extrusion flexibility provides many options on adapting the process to serve many species from omnivorous to carnivorous opening up a diverse range of protein options, density control regards floating and sinking feeds. Manipulation of pellet structure creating open cell structures for absorbency of oils or liquids. For some countries ever increasing population levels conscious of food production costs and environmental impacts. Fish Farming provides a wonderful opportunity both inland and marine production possibilities with world fish production levels increasing approximately 15% a year, available technology and resources from government schemes, university faculties dedicated to aquaculture, there is no better time than the present to turn existing resources into food production centers with healthy nutritional potential to feed our populations. Not only creating protein options but developing export opportunities in both feed production and fish production.

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Study on the effect of Spirulina platensis and Lactococcus lactis on some growth performances of South Caspian stellate sturgeon Acipenser stellatus juveniles Maryam Ahmadi Fakhabi*, Mehdi Soltani, Abbas Ali Zamini, Abolghasem Kamali *Department of Fisheries, Science and Research Branch, Islamic Azad University, Tehran, Iran [email protected] Introduction The effect of alga Spirulina platensis and bacteria Lactococcus lactis on growth indices in juvenile of stellate sturgeon (Acipenser stellatus) was studied and compared together.

Materials and Methods 210 juvenile fishes with average weight of 10.75±0.07 g at the density of 10 fishes in each fiber glass tank with the approximate volume of 1000 l were kept for 60 days. The experimental plan includes 6 treatments and one control treatment done by 3 replicates. The treatments 1, 2 and 3 include 0.2, 0.3, and 0.4 g of spirulina per kilogram of diet and the treatments 4, 5 and 6 include the probiotic at three levels of 0.2, 0.3 and 0.4 g of bacteria per kilogram (1010 CFU/g- 1). Biometry of fishes was done once every 10 days. Biological measurements were analyzed for growth performance: final weight, final length, average daily growth (ADG), specific growth rate (SGR), condition factor (CF), and body weight increase (BWI( using the following specific formulae:  Average daily growth (ADG) (g/fish/day) (%) = [wt – wi /wi×T] ×100  Percent body weight increase (PBWI) (%) = [wt – wi / wi] ×100  Specific growth rate (SGR) (day) = [LnWt - LnWi / T] ×100  Feed Conversion Ratio (FCR)= Feed intake (g)/ wf – wi

Results The result showed that final average weight, daily growth and body weight increase in first treatment comparing to other treatments (P<0.05). Statistical analysis shows no significant difference in food conversion ratio among treatments (p>0.05). Condition factor showed the highest value in the third treatment than other treatments and control treatment. Table 1- Average final weight, final length, ADG, BWI, SGR, and CF in control and different treatments at the end of experimental period Experimental groups Parameter Treat 6 Treat 5 Treat 4 Treat 3 Treat 2 Treat 1 Control 1.91ab ±44.23 0.58ab ± 46.13 ab 0.66 ±45.03 0.74ab ±46.63 ab 0.70 ±41.06 b 1.1 ±49.2 a 1.82±37.23 Final weight (gr) 0.64ab ±28.25 0.26ab ±28.40 ab 0.33 ± 28.49 0.66ab ±28.23 ab 0.48 ± 27.68 b 0.63 ± 29.21 a 1.002 Final length (cm) ±26.92 0.20ab ±5.35 0.46ab ±5.27 ab 0.29 ± 5.26 0.50ab ± 5.47 ab 0.52 ± 4.81 b 0.53 ± 6.01 a 0.64 ± 4.09 ADG (gr/day) 12.45ab ± 27.55ab ±316.79 ab 17.92 ± 30.53ab ± ab 31.73 ± b 32.02 ± a 38.70 ± BWI (%day-1) 321.02 315.61 328.54 288.6 361.05 245.47 0.03ab ±0.56 0.04ab ±0.58 ab 0.033 ± 0.57 0.048ab ±0.59 ab 0.05 ±0.50 b 0.06 ±0.64 a 0.07 ± 0.44 SGR (%day-1) FCR 0.07 ± 1.22 0.02 ± 1.19 0.02 ± 1.30 0.04 ± 1.15 0.13 ± 1.32 0.04 ± 1.21 0.16 ±1.43 ab ab ab a ab ab b CF (%) 0.003 ±0.19 0.007 ±0.20 0.003 ±0.19 0.002 ±0.21 0.01 ±0.19 0.002 ±0.20 0.004 ±0.19 (Same Latin letters in row show significant statistical difference at Duncan test at the level of 5%)

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Conclusions Obtained data showed that adding spirulina algae and probiotic to the food of juvenile of sturgeon improve the growth and efficiency of food conversion. Also for Comparing algae with probiotic, spirulina algae especially at the level of 0.2 g per kilogram has shown better performance at enhancing growth parameters than probiotic (p<0.05).

References Heo, W.S., kim, Y.R., Kim, E.Y., Bai, S.C. and Kong, I.S. 2013. Effects of dietary probiotic, Lactococcus lactis subsp. Lactis 12, supplementation on the growth and immune response of olive flonder (Paralichthys olivaceus). Aquaculture 376-379 (2013) 20-24. Kim, S.S., Rahimnejad, S., Kim, K.W. and Lee, K.J. 2013. Partial Replacement of Fish Meal with Spirulina pacifica in Diets for Parrot Fish (Oplegnathus fasciatus). Turkish Journal of Fisheries and Aquatic Sciences 13: 197-204.

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Comparison of growth and microstructure pattern in the pectoral fin spines of reared juvenile Persian sturgeon, Acipenser persicus in the south Caspian Sea

Shima Bakhshalizadeh*, Ali Bani and Shahram Abdolmalaki *Department of Biology, Faculty of Science, University of Guilan, PO Box 41335-1914, Rasht, Iran Email: [email protected]

Introduction Early studies of juveniles characterized stocks on the basis of phenotypic variation in life history and life history traits. Long-term anthropogenic effects on Persian sturgeon populations have destroyed their ability in the national rearing sites, so investigation of growth trails in the artificial juvenile of Persian sturgeon could be useful for its conservation. The aim of this study was comparison of growth and microstructure pattern in the pectoral fin spines of reared juvenile Persian sturgeon, Acipenser persicus in the south Caspian Sea.

Materials and Methods A total of 20 specimens of the Persian sturgeon, Acipenser persicus, were obtained from the major artificial rearing sites in east and west of the Caspian Sea. Total weight (W) to the nearest gram and total length (TL) to the nearest centimeter was recorded for all samples and was compared between eastern and western sites by applying the ANOVA test. The right pectoral fin spine of the specimens was taken by scalper and were placed in warm water for 10 min (Debicella 2005; Bakhshalizadeh et al. 2013) to separate soft tissues, defleshed with a stiff brush and finally placed on filter papers to dry. Parameters of the length-weight relationship were achieved by fitting the power function to length and weight data: W = a TLb Where W is the total weight, a and b are regression constants and TL is the total length. The condition factor (CF) was calculated by CF=W/ TLb, and the comparison of CF between the sites was performed by applying the ANOVA test. After weighting each spine, they were embedded in Epo-fix epoxy (Electron Microscopy Sciences Inc., Hatfield, Pennsylvania), sectioned near the articulating process (Koch et al. 2008) using an ISOMET low-speed saw, polished with lapping film (30 µm and 3 µm successively, in sequence to achieve a smooth steady. The measure of ring structure were done using a video image-analysis system linked to the stereo microscope from the natal to the edge of pectoral fin spine profiles and this distance was compared between eastern and western sites by applying the ANOVA test.

Results and Discussion No differences in the total weight and total length were observed between eastern and western juvenile rearing site (F=1.33, df 1, P = 0.26; F=0.014, df 1, P = 0.91). The relationship between length and weight was best expressed using a geometric mean functional regression (Fig. 1). The condition factor (CF) was not significantly different between the sites (F=4.04, df 1, P = 0.06). The relations between body weight and spine weight were high in the both sites (Fig. 2). There is not any difference between distance from natal to the edge of sections (F=0.66, df 1, P = 0.43) belong to the both eastern and western sites.

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6 0.00000 y = 0/0019x - 0/0021 Eastern site: y = 8E-07x3/2628 Eastern site: R² = 0/7568 5 R² = 0/8664 0.00000 4 0.00000 3 0.00000 2 Western site: y = 5E-06x2/93 0.00000 y = 0/0007x + 0/0014 1 Western site: R² = 0/8854 R² = 0/7678 0 0.00000 0 2 4 6 0 20 40 60 80 100 120

Fig.1. Length-weight relationship for Fig. 2. Spine weight-body weight eastern rearing site (open circles and relationship for eastern rearing site (open dotted line) and western rearing sites circles and dotted line) and western rearing (closed circles and solid line) of sites (closed circles and solid line) of Persian sturgeon in the south of Persian sturgeon in the south of Caspian Caspian Sea, (W = total weight (g), TL Sea. = total length (cm)).

The result indicated that there are similar conditions in the western and eastern sites. Therefore, as Wootton (1999) stated, similar conditions for feeding rate, biological conditions, and fish health resulted to similar conditions in life history traits as obtained in the juvenile of eastern and western sites of the Caspian Sea.

References Bakhshalizadeh, S., Bani, A., Abdolmalaki, S. 2013. Comparative morphology of the pectoral fin spine of the Persian sturgeon Acipenser persicus, the Russian sturgeon Acipenser gueldenstaedtii, and the Starry sturgeon Acipenser stellatus in Iranian waters of the Caspian Sea. Acta Zoologica 94: 471-477. Debicella, J. 2005. Accuracy and precision of fin-ray aging for gag grouper (Mycteroperca microlepis). Master’s thesis, University of Florida, Gainesville, FL. Koch, J., Schreck, W., Quist, M. 2008. Standardised removal and sectioning locations for shovelnose sturgeon fin rays. Fisheries Management and Ecology 15: 139-145. Wootton, R., 1999: Ecology of teleost fishes, 1990. Croom Helm, London.

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The possibility of farming fleshy Beluga Huso huso in earthen ponds in West Azerbaijan province Yousef Ali Asadpour*, Alireza Azarvandi, Ehsan Soleimannejad Bari and Masoud Seidgar *Iranian Fisheries Science Research Institute (Areeo), National Artemia Research Center, Agricultural Research Education and Extension Organization (AREEO), Urmia, Iran Abstract The aim of this study was to investigate the possibility of culture of Huso huso (beluga) in earthen pools in West Azerbaijan.1000 Huso huso with average weight of 198 g were prepared from Rasht sturgeon propagation center. The fishes were divided in two ponds. The water characteristics of the ponds were. Temperature 14.33±3º, dissolved oxygen 7.05±2 g per liter and pH= 8.00±0. The sturgeons were fed with concentrate pellet at a ratio according to water temperature and biomass weight. Biometry includes weight and total length monthly. After 2 years culture, mean weight of beluga was 3470 g, minimum and maximum weights were 1950 and 5190 g respectively. At the end of culture period the results were: survival rate 96.8%, SGR 0.41, FCR 1.74, CF 0.42 and BWI% 19.93. There was no significant difference between data of two ponds. This study showed that Beluga has good compatibility with the new environment circumstances and could be cultured in West Azerbaijan. Keywords: Breeding, Beluga, Huso huso, Earthen pool

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Study on fatty acid profile in Persian sturgeon larvae (Acipenser persicus) using algae Scenedesmus dimorphus Fruzan Chubian*, Zohreh Ramezanpour, Mahmoud Hafeziyeh, Koroush Haddadi Moghadam, Zabihollah Pajad, Bahareh Younes Haghighi, Hamidreza Pourali, Rudabeh Rufchaie *Researcher of Agriculture Research, Education & Extension Organization (AREEO), International Sturgeon Research Institute, Ecology Department, Rasht, Iran [email protected]

Introduction The aim of this study is to enhance fatty acid profile in Persian sturgeon larvae (Acipenser persicus) using algae Scenedesmus dimorphus.

Materials and Methods Sampling of Scenedesmus dimorphus algae were collected by plankton net having 25µ mesh size. Z8 culture media (Miller et al, 1978) was applied to the culture. The density of Scenedesmus dimorphus algae for enrichment of Daphnia was 5x107cell/ml. Enrichment was carried out during 24 hours. The experimental feeding was carried out with 3 repetitions and one control. 30 larvae were stocked in each rearing tank and the larvae fed up to 30% of body weight four times a day. Extraction of fatty acid from Scenedesmus dimorphus algae was carried out by method of direct methyl esterification using Gas GC Chromatography unite in Urmia Lake Research Institute. Data analysis was performed by Excel and SPSS software. During the study period, water temperature fluctuated in range of 18-24˚C, dissolved oxygen 5.8-7.2 mg/l and pH was 5.6-8.2.

Results The rates of PUFA in Daphnia enriched by Scenedesmus dimorphus algae and in the control group were 19.8±1.83% and 15.1±1.56%, respectively. The rate of fatty acids in Persian sturgeon larvae in treatment and in the control group are shown in table 1. Table 1. The rate of different fatty acids in larvae of Persian sturgeon (Acipenser persicus) in the treatment group and in the control based on weight percent. Fatty acids Treatment group Control

SFA 36.3±9.96 33.2±9.12

MUFA 32.4±7.89 22.0±7.15 PUFA 13.2±1.49 17.5±1.51

Conclusions Stanley-Samuelson (1988) stated that unsaturated fatty acids PUFAs are very essential and important for vertebrates and invertebrates. According to studies, Microcystis pyrifera has very low fat, So by adding 6% of its meal to the diet of trout, the level of unsaturated fatty acids can be increased in meat of trout (Dantagnan et al., 2009), that coincides the results of recent study.

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References Dantagnan, P., Hernandez, A., Borquez, A. et al. 2009. Inclusion of Macroalgae meal (Macrocystis pyrifera) as food Ingredient for rainbow Trout (Oncorhynchus mykiss): Effect on flesh fatty acid composition. In Aquaculture Research 41/1, pp: 87-94. Miller, W.E., Greene, J.C and Shiroyama, T. 1978. The Selenastrum capricornatum Printz Algae assay bottle test. U.S.EPA Rep.600/9-78-018. Stanley-Samuelson D. W., Jurenka R. A., Cripps C., Blomquist G. J., and de Renobales M. 1988. Fatty acids in insects: Composition, metabolism and biological significance. Arch. Insect Biochem. Physiol. 9, 1-33.

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Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Acipenser baerii juveniles. MirHamed Sayed Hassani*, Ali Hossien pour Zelti, Alireza Shenavar, Forouzan Bagerzadeh Lakani, , Mohamad Ali yazdani Sadati, Mahmoud Shakourian, Mahmoud Mohseni, Hooshang Yeganeh, Mohebali Pourgolam, Alireza Alipour, Hamid Reza pouali, Javad Syad far,Ghorban Kamranjo * Caspian Sea International Sturgeon Research Institute (AREEO), Iran, Rasht

[email protected] Introduction Siberian sturgeon, Acipenser baerii is one of the most important sturgeon species that is used for caviar and meat production in sturgeon aquaculture (Bronzi et al., 2011; Wei et al., 2011). In the recent years, efforts have been made to increase the productivity per unit of Acipenser baerii in tanks, or ponds in Iran (Yazdani et al., 2009). However, high-density rearing seems to lead to poor physiological environment that adversely affects fish health and growth rate in fingerlings, juveniles and market periods (Kim et al., 2003; Al-Harbi and Uddin, 2004) and therefore leds to high degree of mortality. Probiotics are traditionally defined as “a live microbial feed supplement which beneficially affects the host animals by improving microbial balance” (Mohapathra et al., 2012; Panigrahi, 2007; Taoka et al., 2006; Salminen et al., 1999). Lazaro et al., 2104 simplified the definition of probiotics which states, “live or dead, or even a component of the bacteria that act under different modes of action in conferring beneficial effects to the host or to its environment. In recent years, research about the potential benefit of probiotic in aquaculture included improvement of water quality, enhancement of nutrient of host species through the production of supplement digestive enzymes, lower incidence of disease and general survival, improving growth and immune system were increased (Ayoola et al., 2013). But, there are few reports available on the impact of probiotics on A. baerii (Geraylou et al., 2014). This study investigated the effects of commercial aquaculture probiotic on growth performance and immune system of Acipenser baerii juveniles.

Materials and Methods Commercial aquaculture probiotic (CA probiotic, Superzist, Zistyar Varna, Iran) comprising Bacillus subtilis, Lactobacillus acidophilus, L. delbrueckii, L. rhamnosus, L. plantarum. In this study, we considered five sturgeon commercial diet(1.9 mm pellets, Biomar, France) (protein: 45%, fat: 16% and fiber3.5%) that Commercial aquaculture probiotic supplemented −1 at levels of 0, 100, 200 and 300 mg kg and named(SR0 ،SR100 ،SR200،SR300) respectively, A total of 240 Acipenser baerii fingerlings with average weight 10.5 ±0.14 g were introduced in 12 fiber glass tank containing 350-liter fresh water, that each tank contained 20 fish . Fish were fed 3% body weight three times daily. At the end of the trial (8 weeks), humoral innate immune parameters (serum Igm levels, lysozyme activity and alternative complement activity (ACH50)) and growth parameters (final weight, weight gain (WG), specific growth rate (SGR), food conversion ratio (FCR), feed efficiency ratio (FE), body weight increase (BWI) and condition factor (CF)) were assessed.

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Results Growth performance increased with the increase in dietary probiotic levels. Highest weight gain, feed efficiency ratio, Specific growth rate and lowest Feed conversion ratio belonged to fish fed (PR300) that have significant difference with fish fed control diet (without probiotic) (P<0.05). Commercial probiotic supplemented in diet(PR100), (PR200) (PR300) were not significantly effect on the lyzozyme , ACH50 and IGM activity, but amount ofhe lyzozyme, ACH50 and IGM activity fish fed (PR300)were higher than other treatment (P >0.05). Discussion The use of probiotics as growth promoters in edible fishes has been reported A probiotic Streptococcus strain was supplemented to the diet of Nile tilapia, Oreochromis niloticus, a significant increase in the content of crude protein and crude lipid was recorded, also fish weight has boosted from 0.154 g to 6.164 g in 9 weeks culture period (Lara-Flores et al., 2003). In Cyprinus carpio, the dietary supplementation of chitosan oligosaccharides and Bacillus coagulanson in diet of koi (Cyprinus carpio koi) resulted in growth improvement (Lin et al., 2012). In diets of catfish, Abdelhamid et al (2009) evaluated the dietary beneficial effects of patent local probiotic T-Protphyt 2000 (consist of 5% dried fermentation products of Aspergillus oryzae) when added to the diet at graded levels (0, 1, 2, 3 g kg_1diet) that were similar with our investigation. Recent studies have focused on the possible role of probiotics in immune system functions. Villamil et al., 2002 found that Lactococcus lactis caused the higher increases in immune functions of turbot (S. maximus). Later, Villamil et al., 2003 proved that the whole cell, fractions whole cell and the extra cellular products of LAB such as nisin act as Immunomodulator in turbot (Scophthalmus maximus). But in this study we were not observed significantly difference between immune system fed by different level of native probiotic but Possibility, inclusion of 200- 300 mg/ kg native probiotic will have positively effect on growth performance, feed utilization and Immune system of Acipenser baerii on aquafarm system in a long period culture.

References Ayoola, S.O., Ajani, E.K. and Fashae, O.F. 2013. Effect of Probiotics (Lactobacillus and Bifidobacterium) on Growth Performance and Hematological Profile of Clarias gariepinus Juveniles. World Journal of Fish and Marine Sciences 5 (1): 01-08, Lazado, C.C. and Caipang, C.M.A. 2014. Atlantic cod in the dynamic probiotics research in aquaculture. Aquaculture, p. 424-425. Mohapatra, S., Chakraborty, T., Prusty, A.K., Das, P., Pani Prasad, K. and Mohanta, K.N. 2012a. Use of different microbial probiotics in the diet of rohu, Labeo rohita fingerlings: effects on growth, nutrient digestibility and retention, digestive enzyme activities and intestinal microflora. Aquaculture nutrition, 18(1): 1-11. Geraylou, Z., Souffreau, C., Rurangwa, E., De Meester, L. and Courtin CM, Delcour JA, et al. 2013. Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut microbiota of juvenile Siberian sturgeon (Acipenser baerii). Fish Shellfish Immunol 35: 766–775.

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Effects of different copper nanoparticle levels on bioaccumulation in organs of Siberian sturgeon (Acipenser baerii) juvenile: Comparison of dietary and waterborne exposure

Forouzan Bagherzadeh Lakani*, Saeid Meshkini, Mohammad Ali Yazdani Sadati, Bahram Falahatkar *Fisheries Department, Faculty of Natural Resources, Urmia University, Urmia, Iran. * [email protected] Introduction Bioaccumulation of heavy metals can reflect the amount of toxin ingested by an organism, pattern in which the metals are distributed through different organs and its extent to which the metals remained in organs (Senthil et al., 2008). As the presence of nanoparticles (NPs) in commercial products increases, worries about poisoning potential and its risks in environment increases (Colvin, 2003). Cu and Cu oxide NPs are already widely found in multiple products, but an actual assessment of their environmental implications is lacking (Shaw and Handy, 2011). A few studies have focused on the reversibility of damages caused by Cu exposure in fish (Cerqueira et al., 2002). Siberian sturgeon is greatly invaluable for research on sturgeon physiology (Eslamloo & Falahatkar, 2014). To date, there is no report about the effects of Cu- NPs on Siberian sturgeon. In general little is known about the potential toxicity of metals such as Cu to Siberian sturgeon. Therefore, the aim of the present study was to determine the bioaccumulation of Cu-NPs in the gill, liver, intestine and muscle of Siberian sturgeon juvenile and the elimination of Cu-NPs from these tissues.

Materials and Methods 480 Siberian sturgeon (Acipenser baerii) juveniles, with initial weight 29.2 ± 3.1 g and length 21.8 ± 1.4 cm, were randomly distributed in 24 fiberglass tanks. 3 different copper nanoparticle (Cu-NPs) treatments plus control in 3 replicates were examined in two experiments. The first concerning waterborne exposure, treatments included control (no added Cu-NPs), and 50, 100, 200 μg/l Cu-NPs in a semi-static waterborne exposure regime. The experimental period lasted 28 days, 14 days exposure to Cu-NPs and 14 days as recovery time. In the second experiment concerning dietary exposure, treatments included control (no added Cu-NPs), 250, 500 and 1000 mg/kg added to the diet. The experimental period lasted 84 days, 42 days exposure to Cu-NPs and 42 days as recovery time. Fish liver, gill, intestine and muscle were sampled at days 0, 7, 14, 21 and 28 in waterborne exposure and at days 0, 21, 42, 63 and 84 in dietary exposure. Samples were weighed, oven dried (100ºC for 48 h) then digested in concentrated nitric acid in a water bath, cooled, and analyzed for Cu concentration in the tissues with graphite furnace atomic absorption spectroscope. Data were tested for treatment, time and treatment × time interaction effects by multifactor ANOVA followed by Tukey’s post-hoc test. Differences among groups were considered significant at P < 0.05.

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Results and Discussion In both experiments, most of the Cu was accumulated in the intestine and muscle had lowest level of Cu. Shaw & Handy (2011) mentioned that NPs are too large for ion transport mechanisms into tissue, so the most likely route for the uptake and accumulation of NPs in intestinal tissue is endocytosis. Gill is the main route for the entrance of Cu into the aquatic organisms due to its direct exposure to toxicant in water and acts as a reservoir of metal accumulation. Liver is an active organ in fish and high amount of Cu accumulation can possibly be attributed to the involvement of liver in detoxification and removal of toxic substances circulating in the blood stream (Asagba et al., 2008). Muscle has the lowest amount of Cu accumulation because it is not an active organ in accumulating metals (Alam et al., 2002) and usually has the lowest essential and nonessential metal concentrations (Wen et al., 2003). The pattern of Cu tissue accumulation showed some evidences of a dose-dependent increase in accumulation. A significant increase in tissue Cu concentration over the time was also observed exhibiting the effect of time on accumulation of this metal in tissues. In the recovery period, Cu level in tissues significantly decreased in both experiments, but it was still higher than the control group. The current findings indicate that preventing the entry of Cu-NPs into the aquatic environment would seem to be essential.

References Alam, M.G.M., Tanaka, A., Allinson, G., Laurenson, L.J.B. & Stagnitt, S. 2002. A comparison of trace element concentrations in cultured and wild carp (Cyprinus carpio) of Lake Kasumigaura, Japan. Ecotoxicology and Environmental Safety, 53: 348-354. Asagba, S.A., Eriyamremu, G.E. & Igberaese, M.E. 2008. Bioaccumulation of cadmium and its biochemical effect on selected tissues of the catfish (Clarias gariepinus). Fish Physiology and Biochemistry, 34: 61–69. Cerqueira, C.C.C. & Fernandes, M.N. 2002. Gill tissue recovery after copper exposure and blood parameter responses in the tropical fish Prochilodus scrofa. Ecotoxicology and Environmental Safety, 52: 83-91. Colvin, V.L. 2003.The potential environmental impact of engineered nanomaterials. Nature Biotechnology, 21: 1166-1170. Eslamloo, K. & Falahatkar, B. 2014. Variations of some physiological and immunological parameters in Siberian sturgeon (Acipenser baerii, Brandt, 1869) subjected to an acute stressor. Journal of Applied Animal Welfare Science, 17: 29-42. Senthil, M.S., Karuppasamy, R., Poongodi, K & Puvaneswari, S. 2008. Bioaccumulation pattern of zinc in freshwater fish Channa punctatus (Bloch.) after chronic exposure. Turkish Journal of Fisheries and Aquatic Sciences, 8: 55-59. Shaw, B.J. & Handy, R.D. 2011. Physiological effects of nanoparticles on fish: a comparison of nanometals versus metal ions. Environment International, 37: 1083-1097. Wen, B.H., Tzong, H.L. & Chih, Y.C. 2003. Accumulation of heavy metals in fish. Journal of National Hualien, 17: 35–44.

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Growth and survival of Persian Sturgeon, Acipenser persicus larvae in Relation to the Consumption of Different Composition of Diet

Zabih Ollah Pajand* , Korosh Haddadi Moghadam, Fruzan Chubian, Esmaeil Farzaneh, Esmaeil Hosseinnia, Alireza Ashori, * International Sturgeon Research Institute (AREEO), P.O. Box: 41635-3464, Rasht, Iran *[email protected]

Introduction In recent years, because of dramatic declines in sturgeon stocks of Caspian Sea, the studies were focused on aquaculture of sturgeons including Persian sturgeon in order to restocking and other aquaculture goals. In this regard, nutritional studied are one of the important elements for a successful aquaculture since a suitable food can produce healthy fish. At now, live foods such as Daphnia magna and Artemia were used for nutrition of sturgeon fish during larval stage. Usage of Nereis is very important for nutrition of commercial fish and shrimps. In sea ecosystems, the Polychaete worms play a key role in providing of essential fatty acids and other nutrients for sea fish fries. Nereis diversicolor is one of the important used species of Nereidae in aquaculture (Fidalgoe Costa and Oliveria, 2006). Earlier Information on nutrition and feeding is scarce for most sturgeon species, being limited and deal mainly with Atlantic sturgeon (Acipenser axyrhynchus) (Kelly and Arnold, 1999), lake sturgeon (A. fluvescense) (Dilauro et al, 1998), Adriatic sturgeon (A. naccrii) (Randall et al, 1992), and also for Persian sturgeon (Ebrahimi and Zare, 2006) most information is generated from nutrition studies conducted on white and Siberian sturgeon, but this information is still incomplete (Webster and Lim, 2002). The main goal of this study was comparison of the growth and survival of Persian sturgeon larvae after usage of three food items. Such study may help to the estimation of current food items that are used at now in sturgeon larval culture.

Materials and Methods The experiment was carried out at the Sturgeon Research Institute (SRI), Rasht, Iran. 1080 sturgeon fries (95.66±0.97 mg) were allocated for experiment in six treatments and each treatment with three replications. For this purpose, 18 circular fish tank were considered and each thank restocked by 60 fries. Tanks condition was: 50 cm in diameter and 25 cm in depth with water flow of 6 Lit/min. Also, the physiochemical parameters of water were: temperature 22.8±0.84°C, dissolved oxygen: 7.3±1.04 mg/Lit and pH: 7.3±0.32. To control and regulation of water quality during experiment, the physiochemical parameters of water were checked up two times in day. The fries were reared in these tanks for a period of 15 days.

Results and Discussion Data represent means and standard error from 3 replicates with initially 60 individuals stocked per tank. Diets were included: Diet 1: Daphnia (Daphnia magna) as a control, Diet 2: Nereis diversicolor worm; Diet 3: Mix of Daphnia (Daphnia spp.) (50%) and Nereis diversicolor (50%), Diet 4: Mix of Nereis diversicolor (50%) and concentrate food (50%) and Nereis diversicolor (50%); Diet 5: Mix of Daphnia spp. (33.33%) and Nereis diversicolor (33.33%) and concentrate food (33.33%). In this study, Live Daphnia spp. as a control diet (diet 1) and cut Nereis worm was cut Nereis diversicolor worm cultured in laboratory.

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Concentrate food was a sturgeon larval diet which has been used with success for sturgeon starter diet. During rearing period, feeding was carried out five times in day. Table 1: Effects of different diets on growth and survival of Persian sturgeon fingerlings for a period of 15 days. Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 (Control) Initial wt (mg) 95.39±4.37 a 93.78±6.84 a 92.21±4.21 a 94.37±3.97 a 95.18±5.46 a Final wt (mg) 239.75±5.86 c 278.82±3.13 d 282.21±4.16 d 223.87±3.81 b 210.83±1.56 a BWI 144.08±1.12 c 183.16±3.69 d 186.54±3.98 d 128.20±3.98 b 115.17±1.05 a PBWI 150.63±1.89 c 191.51±4.97 d 194.99±4.04 d 134.04±4.53 b 120.38±0.62 a GR 9.60±0.07 c 12.21±0.42 d 12.43±0.26 d 8.54±0.26 b 7.67±0.12 a DGI 10.63±0.08 c 12.71±0.37 d 12.88±0.19 d 9.72±0.24 b 8.94±0.04 a CF 0.43±0.01 a 0.43±0.00 a 0.47±0.00 b 0.41±0.00 a 0.43±0.00 a SGR (% day-1) 6.12±0.05 c 7.13±0.11 d 7.21±0.09 d 5.66±0.12 b 5.26±0.01 a FCR 9.53±0.45 b 5.80±0.02 a 5.94±0.24 a 9.36±0.40 b 10.15±1.06 b Survival (%) 91.11±2 ab 96.11±1.46 b 94.44±1.46 b 87.22±2 a 85.55±3.37 a

Differences between the means were compared by Duncan test at a 95% confidence interval at the end of the feeding trials using SPSS software. Significant differences in final weights of fingerlings were found amongst the experimental tanks. There was significant difference (P<0.05) in survival of Persian sturgeon fingerlings fed diets 2 and 3 and the other diets. Significant differences (P>0.05) were not found between diets 2 and 3. Also significant differences (P>0.05) were not found between diets 4 and 5 (table 1). A comparison of mean weights shows that fish fed mix of Nereis diversicolor and Daphnia spp. (diet 3) had a significantly (P<0.05) higher weight than the other diets and significant differences (P>0.05) were not found between diets 2 and 3 (table 1). In diet 3 treatments, the mean final weight of fingerlings was 282.21±4.16 mg, while in the trial fed mix of three diets (diet 5) this value was 210.83±1.56 mg (table 1). The value for BWI, PBWI, GR, DGI, SGR was also higher in fingerling fed mix of Nereis diversicolor and Daphnia spp. (diet 3) (p<0.05) and there was significant difference between all of groups. Except diet 3 there was no significant difference in the CF of fish fed the survey diets (table 1). There was significant difference (P<0.05) in FCR of Persian sturgeon fingerlings fed diets 2 and 3 and the other diets. Significant differences (P>0.05) were not found between diets 2 and 3. Mixed feeding stage, growth rate and mortality rate of larvae were increased because of cannibalism and passing of larvae from the live food to the artificial feeds. The more attention should be paid to the transition from endogenous feeding to mixed feeding, where cannibalism and difficulties in adaptation to a new diet, as well as over or less feeding will increase mortality at the juvenile or fingerling stages could be a good candidate for nourishment of Persian sturgeon fries. In conclusion, our results show that usage of mixed Nereis diversicolor and Daphnia spp. can provide a good profile of essential unsaturated fatty acids in Persian sturgeon.

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References Fidalgo e Costa, P. and Oliveria, R. 2006. Feeding Ecology of Nereis diversicolor (O.F. Müller) (Annelida, Polychaeta) on Estuarine and Lagoon Environments in the Southwest Coast of Portugal, Journal of Aquatic Sciences, 1-114-126. Kelly, J. L.; Arnold, D. E. 1999. Effects of ration and temperature on growth of age 0 Atlantic sturgeon. North American Journal of Aquaculture. 61, 51-57. Ebrahimi, E. & Zare, P. 2006. Growth and survival of Beluga (Huso huso) and Persian sturgeon (Acipenser persicus) fingerlings fed by live food and artificial diet. Journal Applied Ichthyology. 22(1), 321-324. Dinis, M. T., 1986. Quatre Soleidae de l’estuaire du Tage.Reproduction et croissance. Essai d’elevage de Solea senegalensis Kaup. These Doctorat, Université Bretagne Occidantale, Brest. 357 pp. Randall, D. J., Mckenzie, D. J., Abrami, G., Bondiolotti, G. P., Natiello, F., Bronzi, P., Bolis, L. and Agradi, E. 1992. Effects of diet on responses to hypoxia in sturgeon (Acipenser naccarui). Journal of experimental Biology, 170, 113-125. Webster, C. D. and Lim, C. E. 2002. Nutrient requirements and feeding of Finfish for aquaculture. CAB International, CABI publishing. Pp. 344-357.

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The comparison of different pond type in sturgeon aquaculture in Iran Mohammad Pourdehghani*, Mohammad Ghorbani, Forouzan Bagherzadeh Lakani, Yashar kohalvandi, Farnoush Alizadeh, Ismaeil Mirabdollahi, Ali Hamidoghli *International Sturgeon Research Institute (AREEO), Rasht, Iran [email protected] Introduction Aquaculture is one of the simplest and most economical ways to produce animal protein. In aquatics, the feed conversion to meat is more efficient than in other animals. The aquatic production is usually more than the crops produced from agriculture in the same farmed area. Iran has more than 2700 km water borders. Also, many inland water resources including rivers and wetlands can be used which a significant part of it exploited without using. Interestingly, the production of edible fish in inland waters (aquaculture), and in the Sea (mariculture) can compete with the catch in the Caspian Sea, the Oman Sea and the Persian Gulf in Iran. Sturgeons are members of Chondrosteans of which most species are worldwide endangered because of overfishing, pollution and habitat degradation (Birstein, 1993; Billard and Lecointre, 2001). Thus, there has been an increased demand for information on all aspects of sturgeon biology and physiology (Billard and Lecointre, 2001; Baker et al., 2005). Scientists are trying to find the knowledge in aquaculture or stocks rehabilitation of these species. The aim of this work is to evaluate different type of fish pond that can be used in aquaculture. Materials and methods Pond type depends on the ground’s surface, geometry, volume of available water, ground’s textures, geographical location and its slope. Concrete pond constructed from simple concrete or reinforced concrete (Fig 1-a), or the block ponds (Fig 1-b). A new type of metal prefabricated ponds were designed which is used in aquaculture in most European countries (Fig 1-c).

a b c

Figure 1- a. Octagonal reinforced concrete ponds. b. Octagonal block ponds. c. Manufactured metal ponds with geomembrane lining.

Conclusions According to studies conducted as well as results summary presented in Table 1, using prefabricated ponds may be the best option in aquaculture. In terms of the fit between life and other investigated factors (such as time and cost of annual depreciation), prefabricated ponds are a better option for fish farming and aquaculture activities. This is according to economic indicators such as inflation, total cost and preparation.

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Table 1- The economic comparison between different pond types. Pond type Reinforced concrete Block Prefabricated Total cost (Rials) 199,599,800 176,448,000 49,500,000 Cost per meter (Rials) 3,564,282 3,150,857 798,387 Preparation time (day) 25 21 2 Shelf Life (Year) 20 20 12 Annual depreciation (Rials) 9,979,990 8,822,400 5,187,500

References Birstein, V. J. 1993. Sturgeons and paddlefishes: threatened fishes in need of conservation. Conservation Biology, 7: 773–787. Billard, R. and Lecointre, G. 2001. Biology and conservation of sturgeon and paddlefish. Reviews in Fish Biology and Fisheries, 10: 355–392. Baker, D. W., Wood, A. M., Litvak, M. K. and Kiffer, J. D. 2005. Hematology of juvenile Acipenser oxyrinchus and Acipenser brevirostrum at rest and following forced activity. Journal of Fish Biology, 66: 208–221.

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Effects of supplemental artificial and natural attractants on growth performance and survival rates of Persian sturgeon (Acipenser persicus) larvae during the acclimation period Hamid Reza Pourali Foshtomi, Mohammad Ali Yazdani Sadati, Ali Hosseinpoue, Mahmoud Mohseni, Alireza Alipour, MirHamed Seyed Hassani, Zabih Ollah Pajand, Nemat Peykaran Mana. *Research instructor of Agricultural Research, Education & Extension Organisation (AREEO)-International Sturgeon Research Institute- Aquaculture Departement, Rasht, Iran, Postal Code: 41635-3464, Email: [email protected]

Introduction Nowadays with the development of private sturgeon rearing farms to produce farmed meat and caviar in Iran, it is imperative to produce sturgeon larvae and fingerlings which are acclimated to formulated diets. Reducing quality and quantity of farmed Persian sturgeon in early life cycle, weaning period is the most important problems. The aim of this study was to assess the best adaptation diet to concentrate food in the twenty-days-old Acipenser persicus larvae.

Materials and Methods To determine the best live food and to revaluate the optimal weaning period for Persian Sturgeon (Acipenser persicus) larvae, nine experimental diets were tested: four formulated diet were supplemented by different concentrations of 1% methionine, 3% lysine and 3% alanine (M1L3A3), 3% methionine, 1% lysine and 3% alanine (M3L1A3), 3% methionine, 3% lysine and 1% alanine (M3L3A1), 1 % all three aminoacids (M1L1A1), a fifth was an Artemia+formulated diet(AFD), the sixth a Gamarid+formulated diet(GFD), the seventh a Chironimids+formulated diet (CHFD) and the eighth a live food (Artemia+Daphina). A ninth without formulated diet was the control group. All were fed in triplicate groups to twenty- days-old larvae which were captured from the stock tank and randomly distributed into 27 tanks. The microdiets contained 44-45% crud protein, 17-19% crude fat, 9-10% moisture and 7-8% ash. Thousands larvae of Persian sturgeon (Acipenser persicus) were obtained from the hatchery of Shahid Behshti Sturgeon Rearing Center. The offspring of one female were used in this study. After 20 days of stocking the sturgeon larvae with the initial mean weight of 0.4 ± 0.09 g (mean ± SD) were randomly selected and distributed in twenty seven 35 l tanks provided with a water flow rate of 0.2 litres per second. Water temperature, dissolved oxygen concentration and pH were recorded at 18.5±0.4 °C, 8.1± 0.7 mg l-1 and 7.1± 0.2, respectively. The test was carried out for 30 days.

Results After the feeding trial, weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR) of fish fed M3L1A1, M3L3A1 and the CHFD were significantly higher than those of fish fed the other diets (P≤0.05). There were significant differences in survival rates among the experimental treatments. The highest survival rate observed at the M3L1A3 (73±7%), AFD (75±8 %) and continuously was obtained at the (CHFD) (71±3%) and M3L3A1 (71±5 %).

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Conclusions Our results showed that diets supplemented with 3 percents methionine and lysine increased the growth rates of Persian sturgeon larvae and the second stage the chironomids+ formulated diet affected significantly. Results of the present study were similar to those reported for Acipenser persicus larvae and fingerling (Roozbehfar et al., 2013; Jafari et al., 2012), Huso huso (Mohseni et al., 2012) and white sturgeon (Ng and Hung, 1995).

Table 1: The effects of dietary artificial and natural attractants on growth performance and survival rates of Acipenser persicus larvae

Total length SGR Treatments Weight (g) FCR BWI (%) Gr Sur (%) (cm) (% day -1)

M1L3A3 1.5±0.2 bc 6.3±0.4 c 0.4±0.0 a 5.5±0.3 bcd 275 ± 25 bc 2.3±0.2 bc 60 ± 4 bc M3L1A3 2.5±0.5 a 7.4±0.3 a 0.2±0.1 a 7.6±0.9 a 525 ± 129 a 4.4±1.1 a 73 ± 7 a M3L3A1 2.0±0.2 ab 7.2±0.2 ab 0.3±0.0 a 6.7±0.4 ab 400 ± 50 ab 3.3±0.4 ab 71 ± 5 ab M1L1A1 1.1±0.1 c 6.0 ±0.5 c 0.6±0.1 b 4.3±0.4 d 183 ± 30 c 1.5 ± 0.2 c 63 ± 6 abc M0L0A0 1.2±0.2 b 5.9±0.9 c 0.5±0.1 b 4.4±0.7 cd 192 ± 52 c 1.6 ± 0.4 c 50 ± 14 c Atremia+M0L0A0 1.8±0.5 b 6.7±0.3 abc 0.3±0.1 a 6.2±1.1 b 358 ± 123 b 3.0 ± 1.0 b 75 ± 8 a Gamaridea+M0L0A0 1.5±0.2 bc 6.4±0.4 c 0.4±0.0 a 5.6±0.4 b 283 ± 38 bc 2.4±0.3 bc 58 ± 7 c Chironomidae+M0L0A0 2.5±0.5 a 7.2±0.4 ab 0.2±0.0 a 7.6±0.8 a 533 ± 118 a 4.4 ± 1.0 a 71 ± 3 ab Artemia+Daphnia+Chironimidae 1.6±0.1 bc 6.5±0.3 bc 0.3±0.0 a 5.8±0.0 b 300 ± 56 bc 2.5±0.6 bc 50 ± 6 c Data represents mean±SD of three replicates. Values in the same column with different superscript are significantly different (P≤0.05)

Acknowledgements We are sincerely grateful to all colleagues of the International Sturgeon Research Institute who have helped us during the experimental work.

References Jafari, M., Roozbehfar, R., Hemation, R., Kiani, F. and Jamali, H. 2012. The potential of Russian sturgeon (Acipenser gueldenstaedtii) in exploitation of Artemia urmiana in comparison with Daphnia sp. and its mixture. International Journal of Biosciences, 2(12), 7- 12. Mohseni, M., Pourkazemi, M., Hassani, H., Okorie, O.E., Minm T.S. and Bai, S. C. 2012. Effects of different three live foods on growth performance and survival rates in Beluga (Huso huso) larvae. Iranian Journal of Fisheries Sciences, 11(1), 118-131. Ng, W. K. and Hung, S.S.O. 1995. Estimating the ideal dietary essential amino acid pattern for growth of white sturgeon, Acipenser transmontanus (Richardson). Aquac. Nutr. Vol. 1. pp: 85-94. Roozbehfar, R., Jamali, H., Jafari, M. and Hemation, R. 2013. A comparative study on the growth rate of Persian Sturgeon, Acipenser persicus, larvae fed with Artemia urmiana and Daphnia sp. Global Veterinaria, 10(2). 116-120.

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Plan of a production 40 metric tons of sturgeon meat via rearing in pen culture in Gorgan Bay Nemat Paykaran Mana*, Hamid Reza Pour Ali Fashtami, Mir Hamed Sayad Hassani , Mohamad Pour Kazemi, Mohamad Ali Yazdani, Sayed Mostafa Aghili Nejad.

* Caspian Sea International Sturgeon Research Institute, Iran, Rasht Email: [email protected] Introduction Sturgeon fish has decreased sharply in recent years. The Gorgan Bay with the salinity of 9-14 g/l and depth of 1.5 to 2.4 m has favorable conditions to establish pens. It is possible to maintain at least the half of the mature surgery operated brood stocks that used for propagation until next sexual maturation at natural brackish water of Caspian Sea in pen culture condition. In this study, the time schedule to caviar extraction from 180 operated brood stock reared in pen condition based on percentage of the mean number of harvested mature female brood stocks were calculated based on 2009-2011. The estimated area for pen was 2300 square meters and was rectangular in shape. After acclimation period, food will be given to fish in feeding rates of 0.5-1% of biomass twice a day. Fish diets will be prepared from high quality ingredients. The diets will be formulated to contain 35-45% protein and 10- 15% crude lipids. In addition, non-edible fish like Kilka and food waste will be used in food preparing.

Methods and Materials Requirement plan will be : 1- pen installation and building includcd : wooden building to 50 m2, Wooden stocks, lumber and net 2- constant curreny watrt in average alternation of 15-25 oC. 3- fish density were 2 kg/m2 4- use of optimu technology included of intensive culture systems, Recycle water current in critical time, Atomatoic feeder, Automatoic alarm systems base of production: A: Juvenile fish: number of 7560 juvinle sturgeon (5-15 gr) adapted to commercial pellete. B: Food: amount of food for recived to stable prodction were be 7.7, 8.27 and 68 metric ton in first , second and third years. C: Staff: expert and technician were be 8 person.

Results and discussion Number of catch and caviar harvest in Caspian sea from 28500 mt ton meat and 3000 mt caviarin1988 were declined to 500 mt ton meat and 1 ton caviar in 1992, respectively. One of the main result of decline were be illigal catch and destruction of natural habitats. It were not seem that sturgeon stocks were improved on caspian sea. therefor, many of researcher were belived that propagation and culture of Acipenseridea in indoor is only way to survial them. in recent years the United State of America, France, Italia and China wrer challenged to production of caspian sea meat and caviar by production of culture sturgeon caviar and meat. Therefore, A plan for production of 40 metric ton cultivated sturgron in pen culture

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enviroment in theSothern beach of gorgan bay was investigated.detalied of rearing of stugeon in the plan were be: in first year, after adaptaion to commercial diet juvnile Huso huso will be transmitted to pen envirement, On miidleof year after that fish reach to 400-500 gr, will be sort and transmitt to a pen to 7000 m2. Culture density after enhancement of wieght and resistence of fish to will be increase to 1-1.5 kg/m2. Feeding rate will be 3-4% kg/per day. In table of 1 and 2, 3 number of fish, culture surface, amount of food and Average Annualy profit present ": Table1: Number of fish and culture surface of for production of 40 mt Huso huso

Row years 1 2 3

1 Number of fish 7560 4536 4082

2 Initial weight ( kg) 0.1 1-1.5 3-5

3 Mortality (%) 40 10 2

4 Number of fish in end of rearing 4536 4082 4001

5 Individual weight in end of rearing ( kg) 1-1.5 3-5 8-12

6 biomass in end of rearing ( kg) 4536 16330 40008

7 Density ( kg/m2) 0.5 1 2.5

8 Space of pen for optimum culture ( m2) 9072 16330 16000

Table2: Aggregationly Number of fish, Biomass , Food and meat prodction of Huso huso

Row years 1 2 3

1 Number of fish 7560 4536 4082

2 Aggregationly Number of fish 7560 12096 16178

3 Mortality (%) 40 10 2

4 Number of fish in end of rearing 4536 4082 4001 Aggregationly Number of fish in end of 4536 8618 12169 rearing 5 Individual weight in end of rearing ( kg) 1-1.5 3-5 8-12

6 biomass in end of rearing ( kg) 4536 16330 40008

7 The amount of food ( Metric of ton) 7.7 27.8 68

8 The amount of meat production - - 40

References Azari Takami, Gh., 2005. Artificial propagation and rearing of sturgeon. Tehran university publication. 408 p. Pourali, H, M. and Mohseni, M. 2004. Quantity and quality evaluation of density, feeding, water in rearing sturgeon. Journal Scientific, research and education of Aquaculture.11:48-37.

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The use of Inland waters of Iran for sturgeon culture

Mahmoud Ramin*, Mastooreh Doustdar , Nader Nakhaei.

* Iranian Fisheries Science Research Institute, Agricultural Education and Extension Organization (AREEO), Tehran. Iran *[email protected]

Abstract This study was carried out for identification of brackish water resources in Kerman province in 2009. Water samples were taken from brackish water resources of Ravar, Shahrbabak, Sirjan, Zarand and Rafsanjan cities in Kerman province by seasonal sampling. Samples were obtained from 15 different resources. In this survey the physical and chemical characteristics as well as heavy metals copntents were studied. Salinity changes were 13-25ppt, water temperature changes were 13.5-24.5 degree centigrade and pH 6.8-8.6. The analysis of physical and chemical characteristics of brackish water resources showed that all factors in comparison to the standards were at the standard level. The results of measurements of heavy metals including iron, copper, manganese and zinc showed that all of them were less than standard levels. Due to the obtained results brakishwater resources including lakes, rivers, wells and drains were suitable for sturgeon culture, especially Beluga. Seasonal samplings were done in 3 brakishwater resources in each of the cities The measured factors were temperature, DO, pH, EC, TDS, Cl, HCO3, CO2, NO2, NO3, SO4, Ca, Mg, Na and heavy metals were Fe, Cu, Mn and Zn. Obtained data showed that the salinity of these resources were similar to the salinity of Caspian Sea and the amount of other physical and chemical factors, also heavy metals were suitable for sturgeon culture specially Beluga.

References Boyd, C.E. 1982. Water quality management for pond fish culture. Elsevier Scientific Publishing Co., New York.318 pp. Eaton, A.D., L.S. Clesceri, E.W. Rice and A.E. Greenberg. 2007. Standard methods for the examination of water and wastewater, American public Health Association, 21ST EDITION, 1179. Rauf, A., M. Javed and M. Ubaidullah. 2009. Heavy metal levels in three major carps (Catlacatla, Labeorohita and Cirrhinamrigala) from the river Ravi, Pakistan. Pakistan Vet. J., 29(1): 24-26.

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Brackish water Lake in Nough of Rafsanjan

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Natural polymers: new candidates for antibiotics in sturgeon culture

Ebrahim Hossein Najdegrami*, Peter Bossier * Department of Biology, Faculty of Science, Urmia University, Urmia, Iran [email protected]

Introduction In the past, antibiotics were commonly used to treat many bacterial diseases in fish and in farm animals. The ban on the use of antibiotics in both aquaculture and terrestrial animal farms has led researchers throughout the world to study alternative products that can be used for health management and diseases control (De Schreyver et al., 2010). Poly-β- hydroxybutyrate (PHB), an important member of the family polyhydroxyalkanoates (PHAs), is a polymer of β-hydroxybutyrate, a short-chain fatty acid that is produced by numerous bacteria as an intracellular energy and carbon storage compound (Anderson & Dawes, 1990). Positive effects of PHB on growth performance, digestive enzymes activity, gut microbial community and immune response have been reported in several studies (Najdegerami et al., 2015).

Materials and Methods Five hundred and ninety two Siberian sturgeon (Acipenser baerii) fingerlings were randomly distributed in 16 tanks (150 L) at a density of 35 fish per tank and fed different treatments: control, degrading bacteria (Acidovorax spp. G3DM-41 107 CFU/g diet and BSB421 107 CFU/g diet), 2% PHB, degrading bacteria + 2% PHB for 60 days.

Results: At the end of the experiment, the treatment PHB or bacteria resulted the highest average values for weight gain and SGR, although no significant difference was observed between treatments (P>0.05). The significant lowest value for FCR and highest value for gut weight ratio was observed in fish fed bacteria and PHB respectively which had significant difference with those fed control (P < 0.05). Table 1: The effects of experimental diets on growth performance in Siberian sturgeon fingerlings Control Bacteria 2% PHB 2% PHB+ Bacteria Fig. 1: The effects of experimental diets on liver and

Initial weight 11.1 ± 0.9 11 ± 0.8 11 ± 0.7 11.4 ± 0.7 gut weight ratio in Siberian sturgeon fingerlings Final weight 47.9 ± 3.2 50 ± 2.1 51.1 ± 3.1 49.5 ± 2.7 Weight gain 36.8 ± 2.6 39 ± 2.2 40.2 ± 3.2 38.1 ± 2.1 FCR 0.69 ± 0.04 a 0.63 ± 0.01b 0.65 ± 0.01ab 0.69 ± 0.02 a SGR 2.87 ± 0.1 2.97 ± 0.1 3 ± 0.1 2.8 ± 0.0 Condition Factor 0.36 ± 0.0 0.35 ± 0.0 0.35 ± 0.0 0.34 ± 0.0 Survival (%) 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

PHB and bacteria supplemented diets significantly decreased lipid content on fish muscle (P < 0.05) but did not affect on protein and ash content (P>0.05).

Table 2 : The effects of experimental diets on body composition in Siberian sturgeon fingerlings Control Bacteria 2% PHB 2% PHB+ Bacteria Protein (%) 65.0 ± 5.8 61.5 ± 5.8 67.4 ± 4.1 69.0 ± 3.4 Lipid (%) 28.5 ± 2.5 a 24.1 ± 1.7 b 23.2 ± 2.1b 25.1 ± 0.7 b

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Ash (%) 5.1 ± 0.5 4.1 ± 0.5 4.3 ± 0.4 5.0 ± 1.0

Metabolic activity of gut microbial community in fingerlings was measured by Oxitop® control system (Weilheim, Germany). After 5 days the results indicated that the highest BOD5 and the lowest pH were seen in fish gut microbial community fed PHB which had significant difference with those fed control (P < 0.05).

Fig. 2: The effects of experimental diets on metabolic activity of gut microbial community (Right) and bottle pH after 5 days activity (Left)

Discussion and conclusion In this study, PHB degrading bacteria was added to PHB supplemented diets and the effects of PHB and/or degrading bacteria were investigated. Based on previous studies, PHB is degraded by gut microbial community and produce β-hydroxybutyrate, a short-chain fatty acid that is energy source for animal, microbial community and epithelial cell in intestine and lower gut pH as well. The results of this experiment showed that PHB or bacteria improves growth performance and the lowest FCR was seen in bacteria treatment. These results are in agreement with previous reports who reported PHB improves growth performance and decreases gut pH in Siberian sturgeon, sea bass and rainbow trout (Liang ping et al. 2000; De Schryver et al. 2010; Najdegerami, 2012, 2015). Also, the results indicated that combination of degrading bacteria and PHB did not intensify PHB effects. The exact reason is not clear but it seems fish intestine condition and concentration of degrading bacteria affect on bacteria function. Naturally short chain fatty acids are a product of bacterial fatty acid catabolism and PHB is a fatty acid polymer that can be degraded and used by bacterial as an energy source (Azain, 2004). In this study, we used BOD method to evaluate metabolic activity of fingerlings gut microbial community. The results indicated that the fish fed PHB treatment had the highest microbial metabolic activity and the lowest pH in BOD test. Interestingly, the highest weight gain and SGR was observed in this treatment. The similar results have been reported in previous studies by Najdegerami et al. 2011 in Siberian sturgeon. The results of this experiment confirmed the positive effects of PHB in sturgeon culture but the research regarding the PHB degrading bacteria and addition on fish diets needs further studies.

References Anderson, A. and Dawes, E. 1990. Occurrence, metabolism, metabolic role and industrial use of bacterial polyhydroxy alkanoates. Microbiological Reviews, 54, 450-472. Azain, M.J. 2004. Role of fatty acids in adipocyte growth and development. Journal of Animal Science 82: 916–924 De Schryver, P., Sinha. A.K., Kunwar, P.S., Baruah, K., Boon, N., Verstraete, W., De Boeck, G. & Bossier, P. 2010. Poly β- hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax. Applied Microbiology Biotechnology 86: 1535–1541.

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Najdegerami, E., Ngoc, Tran, T., Defoirdt, T., Marzorati, M., Sorgeloos, P., Boon, N. and Bossier, P. 2011. Affects of Poly-β-hydroxybutyrate (PHB) on Siberian Sturgeon (Acipenser baerii) Fingerlings Performance and its GI tract Microbial Community. FEMS Microbiology Ecology 79: 25-33. Najdegerami, E.H. 2012. The effects of Poly-β-hydroxybutyrate on Siberian sturgeon (Acipenser baerii) larval and juveniles culture. Ph.D thesis, Ghent University, Belgium. Najdegerami, E.H., Bakhshi, F., Tokmechi, A., Shiri Harzevili, A., Sorgeloos, P. and Bossier P. 2015. Dietary effects of poly-β-hydroxybutyrate on the growth performance, digestive enzyme activity, body composition, mineral uptake and bacterial challenge of rainbow trout fry (Oncorhynchus mykiss). Aquaculture nutrition, DOI: 10.1111/anu.12386.

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Review of aquaponics system

Seyedeh Yalda Bani Esmaeili* * International Sturgeon Research Institute (Areeo), Rasht, Iran. [email protected] Introduction Aquaponics is the integration of hydroponic plant production into recirculating aquaculture systems (RAS), and has been proposed as a method to control the accumulation of waste nutrients from ﬁsh culture, in a way that consumes less water. Aquaponics system is growing currency around the world as the most sustainable production method for fruits, fish and vegetables. Commercial aquaponics uses methods and equipment from both the hydroponics and aquaculture industries. There have been few studies of commercial-scale aquaponics production. The purpose of this research was to document the production methods, crop and ﬁsh yields.

Materials and Methods Each aquaponic unit consisted of one fish holding tank, an associated biofilter and a hydroponic growth bed. Water from the hydroponic bed was returned to the fish tank, situated at the opposite end of the hydroponic bed from the water inlet. Daily ammonia and nitrite levels were taken to determine the degree of oxidation of ammonia to nitrate, evidence that the biological filter was working. This also allowed the establishment of a steady state bacterial biomass in the biofilter in order to minimize net nutrient uptake by bacteria at the beginning of the hydroponic trials.

Conclusions There is growing interest in locally produced food that is sold directly to consumers, and aquaponics is a growing form of aquaculture that easily fits into a local and regional food system model in part because it can be practiced in or near large population centers. In summary, past research has assumed better performance from hydroponic beds in aquaponic studies by using reciprocating water delivery designs. The present study, comparing reciprocating flooding/draining with a constant flood/drain water cycle, in identical test systems, indicates that constant flooding is actually a better methodology.

References A. Lennard, W. and Leonard, B. 2004. A comparison of reciprocating flow versus constant flow in an integrated, gravel bed, aquaponic test system. Kluwer Academic Publishers. Printed in the Netherlands. Aquaculture International 12: 539–553. David, C. Love., Jillian P. Fry., Ximin Li., Elizabeth, S. Hill., Laura Genello., Ken, Semmens. and Richard E. Thompson. 2015. Commercial aquaponics production and profitability: Findings from an international survey. Aquaculture 435:67-74. Lewis, W.M., Yopp, J.H., Schramm, H.L. and Brandenburg, A.M. 1978. Use of hydroponics to maintain quality of recirculated water in a fish culture system. Transactions of the American Fish Society 197: 92–99.

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McMurtry, M.R., Sanders, D.C., Cure, J.D., Hodson, R.G., Haning, B.C. and St. Amand, P.C. 1997. Efficiency of water use of an integrated fish/vegetable co-culture system. Journal of the World Aquaculture Society 28: 420–428. Nair, A., Rakocy, J.E. and Hargreaves, J.A. 1985. Water quality characteristics of a closed recirculation system for Tilapia culture and tomato hydroponics. Second International Conference on Warm Water Aquaculture – Fin Fish. Proceedings of a conference, Hawaii, USA, pp. 223–254. Rakocy, J.E. 1989. Vegetable hydroponics and fish culture: a productive interphase. World Aquaculture 20: 42–47. Rakocy, J.E. and Hargreaves, J.A. 1993. Integration of vegetable hydroponics with fish culture: a review. In: Wang, J.K. (ed.), Techniques for Modern Aquaculture, American Society of Agricultural Engineers, St. Joseph, Michigan, USA, pp. 112–136.

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The role of phospholipids in improving growth and survival of sturgeon and teleost fish

Naser Agh*, Fatemeh jafari, Esmaeil Paghe, Farzaneh Noori *Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran [email protected]

Introduction It has been known for almost 25 years now that inclusion of intact phospholipids in the diet could improve culture performance of various freshwater and marine fish species. The primary beneficial effect was improved growth in both larvae and early juveniles, but also increased survival rates and decreased incidence of malformation in larvae, and perhaps increased stress resistance. Determination of absolute dietary requirements has been hampered by the use, in different dietary trials, of a wide range of phospholipid preparations that can vary greatly both in phospholipid content and class composition. Larval studies have been compromised further by the need on many occasions to supply phospholipid through enrichment of live feeds with subsequent re-modelling of the phospholipid and fatty acid compositions. Generally, the levels of phospholipid requirement are around 2 - 4% of diet for juvenile fish and probably higher in larval fish. The effects were restricted to young fish, as a requirement for dietary phospholipids has not been established for adult fish, although this has been virtually unstudied. As the majority of studies have used crude mixed phospholipid preparations, particularly soybean lecithin, but also other plant phospholipids and egg yolk lecithin that are enriched in several phospholipids it has been difficult to elucidate which specific phospholipid classes impart beneficial effects. Based on the few studies where single pure phospholipid species have been used, the rank order for efficacy appears to be phosphatidylcholine > phosphatidylinositol > phosphatidylethanolamine > phosphatidylserine (Tocher et al., 2008). Phospholipids (PL) have been demonstrated to significantly affect survival, growth, deformities and/or resistance to stress in several fish and crustacea (Kanazawa et al., 1985; Geurden et al., 1995; 1998; Koven et al., 1998; Cahu et al., 2003; Gisbert et al., 2005). They play a major role in maintaining the structure and function of cellular membranes (Kanazawa, 1985; Tocher, 2003). They have been reported to act as emulsifiers in the gut (Koven et al., 1993) and to improve intestinal absorption of long chain fatty acids (Fontagne et al., 2000). Moreover, they stimulate lipoprotein synthesis in intestinal enterocytes (Fontagne et al., 1998) and play an important role in the transport of dietary lipids (Kanazawa, 1991). Lecithin is known to be a consistent source of highly bioavailable phospholipids to crustaceans and fish. But there are a number of other nutritional benefits in aquaculture applications. Lecithin has an interactive role in the intestinal absorption of cholesterol, which helps improve the growth and survival of aquaculture species. In crustaceans and fish, lecithin helps mobilise dietary cholesterol. Lecithin is a nutritionally superior source of choline, an essential nutrient present in the biologically available form of phosphatidylcholine (PC) (Paibulkichakul et al., 1998).

Materials and Methods We tested the effect of phospholipid supplementation to the basal feed of a sturgeon fish, Acipenser stellatus, and a marine fish, silvery-black porgy (Sparidentex hasta) juveniles. For sturgeon fish (10 grams average weight), seven experimental feed containing seven levels of soy lecithin (0, 1, 2, 4, 6, 8 and 10% of total lipids) were prepared. While for the silvery-black

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir porgy (average weight 50 g) four feed treatments containing 0, 3, 6 and 9 percent lecithin was used for a period of two months. Results According to the results provided in tables 1 and 2, supplementation of 6% soy lecithin as source of phospholipid showed significant effect on growth parameters, indicating the crucial role of phospholipids on performance of these fishes.

Table 1: Effect of lecithin supplementation of growth parameters of Acipenser stellatus

Treatme nt 1 (0% PL) 2 (1% PL) 3 (2% PL) 4 (4% PL) 5 (6% PL) 6 (8% PL) 7 (10% PL)

index

IW (g) 11.29±0.072a 11.29±0.072a 11.31±0.060a 11.31±.1a 11.25a 11.25a 11.30±.09a

FW (g) 27.46±2.61a 32.86±2.83ab 38.55±3.04b 46.80±3.78c 51.40±5.88c 47.01±4.79 c 46.42±5.22 c

BWI (g) 18.14±1.02a 21.57±2.85ab 27.23±3.09b 35.49±3.89c 40.1±5.88c 35.76±4.79c 35.11±5.1c

PBWI 143.18±22.71a 191.07±25.65ab 240.78±28.56b 313.96±37.2c 356.91±52.29c 317.90±42.66c 310.39±42.75c (%)

SGR (%) .16±0.09a .34±0.08 b .49±0.08 b .69±.09 c .79±.11 c 0.70±10 c 0.68±.1 c

Table 2. Effect of lecithin supplementation of growth parameters of silvery-black porgy (Sparidentex hasta)

Treatments Initial Wt. Final Wt. % increase in Wt. SGR (%) FCR PER 3% PL 29.85 77.16 158.54 1.26 1.51 1.29 6% PL 29.56 86.19 191.63 1.47 1.33 1.50 9% PL 29.70 82.55 177.95 1.39 1.40 1.42 Control 29.62 74.11 150.17 1.20 1.50 1.30

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Conclusion Feed containing 4-10% lecithin showed significantly higher growth compared to control and treatments containing lower than 4% lecithin in Acipenser stellatus. The highest growth indices were observed in group fed 6% lecithin. In silvery-black porgy also the feed containing 6% lecithin showed highest growth parameters. The results indicated that 6% lecithin seems to be the optimum lecithin concentration that may be supplemented in A. stellatus and silvery-black porgy feed.

References Cahu, C.L., Zambonino Infante, J.L. and Barbosa, V. 2003. Effect of dietary phospholipid level and phospholipid: neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet. Br. J. Nutr. 90, 21-28. Fontagne, S., Geurden, I., Escaffre, A.M. and Bergot, P. 1998. Histological changes induced by dietary phospholipids in intestine and liver of common carp (Cyprinus carpio L.) larvae. Aquaculture 161, 213223. Fontagne, S., Burtaire, L., Corraze, G. and Bergot, P. 2000. Effects of medium-chain triacylglycérols (tricaprylin and tricaproin) and phospholipid supply on survival, growth and lipid metabolism in common carp (Cyprinus carpio) larvae. Aquaculture 190, 289-303. Geurden, I., Radünz-Neto, J. and Bergot, P. 1995. Essentiality of dietary phospholipids for carp (Cyprinus carpio L.) larvae. Aquaculture 131, 303-314. Geurden, I., Marion, D., Charlon, N., Coutteau, P. and Bergot, P. 1998. Comparison of different soybean phospholipidic fractions as dietary supplements for common carp, Cyprinus carpio, larvae. Aquaculture 161, 225-235. Gisbert, E., Villeneuve, L., Zambonino Infante, J.L., Quazuguel, P. and Cahu, C.L. 2005. Dietary phospholipids are more efficient than neutral lipids for long chain polyunsaturated fatty acid supply in European sea bass Dicentrarchus labrax development. Lipids 40, 1-10. Kanazawa, A. 1985. Essential fatty acid and lipid requirement of fish. In: C.B. Cowey, A.M. Mackie, J.G. Bell (Eds). Nutrition and feeding in fish. Academic Press, London. Kanazawa, A.1991. Essential phospholipids of fish and crustaceans. In: INRA (Eds.), Fish nutrition in Practice, Biarritz (France) June 24-27, 1999. Paris 1993 (Les colloques N°61). Kanazawa, A., Teshima, S.I., Kanazawa, A., Teshima, S.I. and Sakamoto, M. 1985. Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae. Aquaculture 50, 39-49. Koven W.M., Kolkovski, S., Tandler, A., Kissil, G.Wm. Sklan, D. 1993. The effect of dietary lecithin and lipase, as function of age, on n-9 fatty acid incorporation in the tissue lipids of Sparus aurata larvae. Fish Physiol. Biochem. 10, 357-364. Koven, W.M., Parra, G., Kolkovski, S. and Tandler, A. 1998. The effect of dietary phosphatidylcholine and its constituent fatty acids on microdiet ingestion and fatty acid absorption rate in gilthead sea bream, Sparus auratus, and larvae. Aquacult. Nutr. 4, 39-45. Paibulkichakul C., Piyatiratitivorakul S., Kittakoop P., Viyakarn V., Fast A.W. and Menasveta P. 1998. Optimal dietary levels of lecithin and cholesterol for black tiger prawn ‘Penaeus monodon’ larvae and postlarvae. Aquaculture 167, 273-281. Tocher, D.R. 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Reviews Fish. Sc. 11, 1-78.

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The effect of dietary glutamine supplementation on growth performance in Acipenser baerii (Brandt, 1869)

Saman Darvishi, Hossein Khara*, Mohaddeseh Ahmadnezhad * Department of Fisheries, Lahijan Branch, Islamic Azad University, Lahijan, Iran, P.O. Box: 1616 [email protected] Introduction In order to protect wild populations and meet the high demand for caviar, culture of sturgeons has now become one of the developing branches in aquaculture (Balabanova et al., 2009). In this regard, Siberian sturgeon, Acipenser baerii, is among the species which have presented a good future for farming in European countries (Rad et al., 2003; Steffens et al., 1990). Although this species was imported to Iran in March 2006, information on optimum culture conditions, nutritional needs, and appropriate formulated diets for this species is limited like other species of sturgeons (Kozlov, 1993). As one of the most expensive dietary components of the diet, protein has the largest contribution to growth (Brauge et al., 1995). On the other hand, low levels of protein in the diet will leave adverse effects on appetite, food intake, growth, product quality, and ultimately economic efficiency (Lupatsch et al., 2001). Among the non-essential amino acids, glutamine plays essential roles in vital functions of fish. Glutamine is made by glutamine syntheses through the combination of glutamate and ammonia following the intake of adenosine triphosphate. Although the highest percentage of glutamine is made in muscles, it is also secreted from the brain and gills partially (Pohlenz et al., 2012). The main functions of glutamine include protein synthesis, regulation of acid-base balance in the kidney through ammonium synthesis, providing energy for cells, donation of nitrogen in the evolution, donation of carbon in the Krebs cycle, and non-toxic transfer of ammonia in the blood circulation. Glutamine is mainly consumed by intestinal cells. Since glutamine is also used by immune cells (Waddell and Fredrick, 2005), it seems necessary to study the effect of glutamine on the growth of Siberian sturgeon.

Materials and Methods The present study was to examine the effect of glutamine on growth performance of Siberia sturgeon and also to obtain optimal values under culture conditions for eight weeks .For this purpose, the number of 126 fish with an average weight of 56.7±71.45 gr in six treatments (0, 5, 10, 15, 20 and 30 gr of glutamine per kg of food) and three replications were treated. To check growth parameters fortnightly all the fish were measured. Glutamine with a purity of 100% made by Biotech Manufacturing Co., the US was used in this research and its doses were selected based on the study of Pohlenz et al. (2012). Considering the size of fish, they were fed at a rate of 3% biomass three times a day (7 A.M., 15 P.M., and 23 P.M.). Physicochemical parameters of water including dissolved oxygen and temperature were measured three times a day by an Oximeter (OXI3230B/SET) and pH was measured once a day by a pH meter (PH330i/SET). In addition, at any biometry time, tanks were completely drained and then washed and disinfected with antiseptics. Mean temperature, pH, and dissolved oxygen of water during the trial were 17.1±0.9°C, 7.5-7.8, and 7.8±0.2 mg/l, respectively. Fish were cultured under an open system and natural light regime. All statistical analyses were performed in SPSS-18 and all charts were plotted in Excel 2010. To ensure the normal distribution of data, Kolmogorov-Smirnov test was used. Because of the normal distribution of studied data, overall difference between mean values was determined

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Results The results of adding different levels of glutamine to the diet of Siberian sturgeon showed that although no significant difference was found in total length and final weight based on one- way analysis of variance, the best performance of weight gain was observed in the fifth treatment. By contrast, the lowest weight gain belonged to the fourth treatment. According to the obtained data, mean condition factor, weight gain percentage, specific growth rate, daily growth rate, and feed conversion rate (FCR) showed no significant difference between treatments. However, the best performance of conditions factor was found in the first treatment and the best performance of weight gain percentage, specific growth rate, daily growth rate, and FCR was related to the fifth treatment. Based on results, there was a difference between treatments significant in terms of growth, so maximum total (157.50 g) and total length (36.14 cm) was seen in group 5(30 grams of glutamine per kg food). Also, the lowest feed conversion ratio and highest daily growth rate in group 5 respectively. Discussion These results suggest that different levels of glutamine added to the diet of Siberian sturgeon left no significant impact on none of growth parameters. However, the results reflect the important point that the fish fed with low levels of glutamine (5, 10, and 15 grams of glutamine per kg of food) underwent a more stable growth process compared to those fed with higher levels of glutamine (20 and 30 grams of glutamine per kg of food). In addition, the highest and the lowest values of growth parameters were related to the fifth and fourth treatments, respectively. Similar effects of diets supplemented with glutamine have been reported in different species of fish. For example, improvement in weight gain, feed efficiency, intestine weight, histological structures, and digestive enzymes activity have been reported from Cyprinus carpio var. Jian (Yan and Qiu-Zhou, 2006), hybrid sturgeons (Acipenser schenckii × Huso dauricus) (Qiyou et al., 2011), and channel catfish, Ictalurus punctatus, (Pohlenz et al., 2012). Conclusion Based on the results, dose of 30 grams of glutamine per kg food (five treatments) are recommended. Although the addition of glutamine to the diet of Siberian sturgeon had no impact on growth parameters because of the high weight of fingerlings (initial weight of 50 grams), the highest final weight was observed in the fifth treatment (30 grams of glutamine per kg of food). Keywords: Siberian sturgeon, Acienser baerii, Glutamine, Growth

References 1- Balabanova L.V., Mikryakov D.V. and Mikryakov V.R. (2009). Response of common carp (Cyprinus carpio L.) leucocytes to hormone induced stress. Inland Water Biology 2(1): 86–88. 2- Brauge C, Corraze G & Me´dale F (1995) Effect of dietary levels of carbohydrate and lipid on glucose oxidation and lipogenesis from glucose in rainbow trout, Onchorhynchus mykiss, reared in freshwater or in seawater. Comp Biochem Physiol 111A, 117–124. 3- D. Waddell, K. Fredricks., 2005. Effects of glutamine supplement on the skeletal muscle contractile force of mice, Am J Undergr Res, 4 (2005), pp. 11–18 4- Kozlov V.I., 1993. Sturgeon farming. Moscow. VNIRO. 64P.

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5- Lupatsch, I., Kissil, Gm., Sklan, D., 2001. Optimization of feeding regimes for European sea bass Dicentrarchus labrax: a factorial approach. Aquaculture 202, 289–302. 6- Pohlenz C, Buentello A, Mwangi W, Gatlin III DM. Arginine and glutamine supplementation to culture media improves the performance of various channel catfish immune cells. Fish Shellfish Immunol 2012; 32:762e8. 7- Qiyou, X., Qing, Z., Hong, X., Chang`an, W., Dajiang, S., 2011. Dietary glutamine supplementation improves growth performance and intestinal digestion/absorption ability in young hybrid sturgeon (Acipenser schrenckii ♀× Huso dauricus♂). Journal of Applied Ichthyology 27, 721–726. 8- Rad F., Koksal G. and Kindir M. (2003). Growth Performance and Feed Conversion Ratio of Siberian Sturgeon (Acipenser baerii) at Different Dally Feeding Rates. Turkish Journal of Vetinery Animal Science, 24: 1085-90. 9- Steffens W., Jannichen H. and Fredrich F. (1990). Possibilities of sturgeon culture in central Europe. Aquaculture, 89: 101-122. 10- Yan, L., Qiu-Zhou, X., 2006. Dietary glutamine supplementation improves structure and function of intestine of juvenile Jian carp (Cyprinus carpio var. Jian). Aquaculture 256, 389–394.

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The effect of density on mean individual weight of Beluga (Huso huso) juveniles reared in fiberglass tanks (Voshmgir Dam Recruiting Centre)

Aghaei Moghadam*, Kamran Aghili, Sara Haghparast Research Aquatic Centre of Gorgan Inland Waters [email protected] Introduction It has been a long time that Beluga (Huso huso) is considered as a suitable aquaculture volunteer by many fisheries research. Factors like rapid growth in freshwater (Mohseni et al., 2005), priority of growth in brackish water of the Caspian Sea (Feshtemi et al., 2004) and its adaptability to harsh environmental conditions, made this species an economically profitable farmed fish in order to produce animal protein. Beluga culture was successfully conducted for the first time in Iran in 1990 (Yousef Poor, 1994). Fish density and biomass are critically biological criteria in intensive aquaculture systems. Fish culture density, as behavior physiological demands against available space, can result in declining fish health and physiological condition, decreased FCR, growth and increased fish mortality. Totally, fish density is known as a chronic stressful factor (Rafatnezhad et al., 2007; Trenzado et al., 2006). Hence, studies of density effect on promoting fish products and increasing production efficacy can be helpful in the development of novel aquaculture industry. With the above scenario, this study aimed to investigate the effect of density on final individual weight gain of Beluga (Huso huso) juveniles at Voshmgir Dam Recruiting Center in 2009.

Materials and Methods A total number of 270 Beluga with an average weight of 94±11.6 g was randomly introduced to 1000 L fiberglass tanks at 4 density levels (1.5, 2, 2.5 and 3 kg/m2) corresponding to the number of individuals per each tank (n=15, n=20, n=25, and n=30). Each treatment was replicated three times. Fish were fed using Biomar three times a day and reared for 120 days. Measurement of mean individual weight was conducted three times during the rearing period in monthly interval. After being checked for normality and homogeneity of variance, data were subjected to the analysis of Two-Way ANOVA in order to survey the main effect of fish density and rearing period beyond their interaction effect. When the interaction effect was significant, mean comparisons were performed by using One-Way ANOVA as levels of density factor were compared in each level of time and vice versa.

Results Results of Two-Way ANOVA indicated significant main effects of the studied factors as well as their significant interaction effect. According to Table 1 and Figure 1, comparison of different time levels in each density level reveled a similar significant trend (P<0.05) while there were no significant difference among various density levels at the end of first and second rearing period (P>0.05). Significant differences were also observed at the end of third and fourth month of rearing as the lowest and the highest densities had the least value of mean individual weight and intermediate levels of density studied here showed the highest value of mean individual level (P<0.05).

Table 1. Mean comparison of different density and time effects on mean individual weight of

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reared Beluga (Huso huso) juveniles

Time (monthly) 4 1 2 3

D A B C 380.24±7.97 a 15 93.33±6.11 a 183.33±6.11 a 282.64±8.80 a Density D A B C 438.21±13.46 b 20 95.33±5.03 a 206.16±5.00 a 321.75±10.42 b D A B C 454.81±26.87 b 25 83.27±23.97 a 206.26±23.97 a 331.81±26.87 b D A B C 403.07±6.88 a 30 98.00±7.00 a 200.67±9.60 a 305.67±9.60 ab

Different superscript (A-D) showed significant difference in each raw (P<0.05). Different subscript (a-b) showed significant difference in each column (P<0.05).

Discussion In this study, density levels of Beluga juveniles detected a significant difference in mean individual weight, especially after three months of rearing. This is in accordance with the findings of previous researcher by Rafatnezhad et al., (2007) who expressed remarkable effects of different densities (1, 2, 4, 6, and 8 kg/m2) on growth parameters of Beluga juveniles (final weight, SGR, weight gain) with average weight of 93 g during 8 weeks. Another notable result of this trial was that the upper mean individual weight was obtained in intermediate levels of density (20 and 25 ind/m2). This was in contrast with the result of Jodun et al., (2002) who described the highest level of growth in the lowest density of Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) juveniles while was in agreement with findings of Poor Ali Feshtemi et al. (2011) who claimed that Beluga (3 g average weight) at density level of 1 kg/m2 reached the highest growth than other upper level (1500 g/m2) or lower level (150 g/m2) of density during 30 days of rearing.

Acknowledgement We are grateful of all staff working at Voshmgir Dam Centre for their help in providing facilities and useful advice.

References Jodun, W., Millard, M. and Mohler, J. 2002. The effect of rearing density on growth, survival, and feed conversion of juvenile Atlantic sturgeon.North American Journal of Aquaculture 64:10-15. Mohseni, M., Bahmani, M., Poorali, P., Kazemi, R., Agh, N., and Poor Kazemi, M. 2005. Formation of broodstock schools from reared broods in fish culture workrooms (first phase- Biotechnique of Beluga culture, Huso huso, in freshwater for animal protein production). Iranian Fisheries Research Institute. 135 p. Poor Ali Feshtemi, H. R., Mohseni, M., Sadeghi, M., Arshad, A. and Alizadeh, M. 2004.Culture of Beluga (Huso huso) juveniles in brackish waters of the southern Caspian Sea. Iranian Fisheries Research Institute. 100 p. Poor Ali Feshtemi, H., Bahmani, M., Sadeghi Rad, M., HosseinNia, A., Ashori, A. and YazdaniSadati, M. A. 2011. Study on culture density of Beluga during acclimation to

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concentrated food in brakish and fresh waters. Biological Sciences Journal of Lahijan Unit.5th year, NO. 2, 23-46. Rafatnezhad, S., Falahatkar, B. and Tolouei Gilani, M.H. 2007. Effects of stocking density on haematological parameters. Growth and fin erosion of great sturgeon (Huso huso) juveniles. Aquaculure Research 39:1506-1513 Trenzado, C., Morales, A. and Higuera, M. 2006. Physiological effect of crowding in rainbow trout, Oncorhynchus mykiss. Selected for low and high stress responsiveness.Aquaculture 258: 583-593. Usef Poor, H. 1994. Sturgeon fish culture in freshwater. National conference on aquaculture of aquatics, Iranian Fisheries Joint Stock Company.68-84.

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Comparision quality of chironomid larvae on different feeding substrates to feed farmed fish fingerlings

Ahmad Nezami Baluchi*, Hamid Reza Pourali Fashtomi, Mohammad Ali Yazdani Sadati, Nemat Peykaran Mana, MirHamed Seyed Hasani. * Fisheries Expert in Breeding and Cultivation, International Sturgeon Research Institute, Rasht, Iran, Postal Code: 41635-3464. Email: [email protected]

Introduction Blood worms (Chironomidae) are the most important aquatic insects which are distributed in all different water reservoirs. Chironomid is belonged to Kingdom Animalia, Phylum Arthropoda, Class Insecta, Order Diptera and Family Chironomidae. Chironomids like the other insects have four life stages including egg, larva, pupa and adult. Chironomids have different feeding habits consuming a large variety of food such as algae, scraps and associated microorganisms, macrophytes, wood practices and invertebrates (Berg, 1995). The chironomid larvae are known as blood worms due to the presence of hemoglobin in their bodies (Borror et al., 1989). Consumption of live food by fish larvae and fingerlings is dramatically effective in decreasing larval mortalities. Culture of live food like chironomid for feeding fish larvae is now developing all over the world and has become an integral part of fish rearing (Agh, 2002, Pourali et al., 2015). The addition of high energy feeds to enrich rearing substrates of larvae is effective in improving the quality of chironomid larvae (Habashy, 2005).

Materials and Methods Three replicates were used for each treatment. Nine, 50 L plastic tanks were used, and the rearing system was supplied with well water and equipped with aeration systems. Bottom substrate in the first treatment consisted of coastal sand without any food and additives. 30% of bottom substrate in the second and third treatments was enriched with fish meal, wheat meal and soybean meal. The protein and lipid contents were considered to be 35% and 12%, respectively in the second treatment and 45% and 20%, respectively in the third treatment. Data from each treatment was subjected to one-way analysis of variance (ANOVA) using SPSS (ver.17) and Excel (ver.2010) for Windows. When there were significances, the group means were further compared with Duncan's multiple range tests.

Results The mean temperature, dissolved oxygen concentration and pH were recorded at 16.4 ± 1ºC, 6.08 ± 0.9 mg/l and 7.8 ± 0.2, respectively. The water flow rate was maintained at 0.2 liter per second. The results of this study indicated that the mean biomass in the third substrate had a two-fold increase compared to the first treatment after 15 days of rearing. There were no significant differences among treatments using one-way analysis of ANOVA. Duncan's multiple range tests showed that there was a significant difference between the third and first treatments. The chironomid larvae were completely red in color and showed gentle S-shaped movements.

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Discussion and conclusion Berg (1995) reported that the color of chironomid larvae guts depends on food type. Rasmussen (1985) recorded that the growth rate of Chironumus riparius when fed on organic enrichment with microdetritus significantly enhanced its growth, for densities less than 2.8 cm2 and in contrast, the enrichment had no effect on another species of chironomid known as Glyptotendips paripes. Data obtained from this study revealed that there were significant differences in the nutrient level of chironomid carcass. In this study crude protein content of chironomid carcass (55.7±0.3 %) was higher than that (48%) reported by Azari Takami in 2010. The food value of chironomids was studied by Sugden (1973) who reported a value of 65% protein for chironomid larvae (Habashy, 2005). The protein content was estimated at 55.7% in this study. The relatively high protein content of up to 73.6% with the high digestibility was represented by De La Noue and Choubert in 1985. It was suggested by Armitage (1995) that the preference of bottom feeder organisms for chironomid larvae and pupae as food source is related to their high energy content (% mean value; moisture content 86, protein 48 to 55, lipid 14, carbohydrate 23, chitin 4, ash 9 with an utilization energy of 4.1 to 6.1 k Cal/g). Rasmussen (1985) and Pourali et al. (2015) studied the effect of density and micro-detritus enrichment on the growth of two species of chironomid larvae (Chironumus riparius and Glyptotendips paripes) in small ponds over the 25-days experimental period and found that density effect significantly on their growth both with and without organic enrichment. The negative effect of density was observed at the end of the rearing period in this study (Nezami Baluchi, 2010). Vos et al. (2000) and Pourali et al. (2015) reared larvae of midge Chironumus riparius on different food items and found that both quality and biochemical composition of food influenced growth of midge larvae and maximum length attained by larvae reared on fish food was higher than maximum length reached on food items of plant origin (algae, living leaves of aquatic plants) after one week rearing period. Further investigations on the third substrate in this study revealed that number of deformities from chironomid pupae to adult midges was estimated to be one individual per hour in 16.5 ºC. The time of chironomid larvae and pupae maturation was not simultaneous. In a biomass of 62 g in rearing tanks or 350 g/m2, 58 % of chironomid population was in the pupae stage and 25% and 16 % of them were in larval and midge stages, respectively. The results obtained from the first two stages of experiment indicated that conducting this project with all above mentioned including food items and isolation of adult midges from the population is important.

References Agh, N., 2002. Biology and ecology of Artemia in Urmia Lake. Final report of State Scientific Research Council. National research program. No. 1443. 130p. Azari Takami, Gh. 2010. Breeding and cultivation of sturgeon caviar fish. University of Tehran Press. 401p. Berg, M.B. 1995. Larval food and feeding behavior. In: P.D. Armitage; P.S. Cranston; L.C.V. Pionder (editors) the chironomidae; Biology and ecology of Non-biting mides. Chapman and hall. London. UK. PP. 423-435. Habashy, M. 2005. Culture of Chironomid larvae (Insect- Diptera-Chironomidae) under different feeding system. Egyptian Journal of Aquatic Research, 31. (2): 403-418. Nezami Baluchi, A. 2010. Culture of chironomid larvae on different feeding substrates to feed farmed fish fingerlings. Bachelor of Science Thesis in Breeding and Cultivation

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of Aquatic. Mirza Kochak Khan Technical and Higher Education Center for Fisheries Science and Technology. 47 p. Pourali, H. R., Bahmani, M., Abdolmalaki, SH. Yazdani, M. A., Mohseni, M., Shakourian, M., Hamed, H., Yeganeh, H., Padjand, Z. and Sadeghi, M. 2015. Effects of some food additives on growth indices of Acipenser persicus larvae during adaptation period. Final Report of the project. International Sturgeon Research Institute, Aquaculture Dep. 100 p.

Rasmussen, J.B. 1985. Effect of density and micro detritus enrichment on the growth of chirinimid larvae in a small pond. Canadian Journal of Fisheries and Aquatic Science.

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Mass production of Daphnia spp. using sewage outlet of fish rearing tanks

Javad Syad far*, Mohamad Ali Yazdani Sadati, Alireza Alipour, Mir Hamed Sayed Hassani, Hamid Reza Pourali, Ali Hossien pour Zelti, Ghorban Kamranjo

[email protected] *Agricultural Research,Education and Extension Organization(AREEO), International Sturgeon Research Organization of the Caspian Sea, Rasht, Iran, P.O.Box: 41635– 3464

Daphnia spp is one of the effective live foods used in many marine larvae nutrition, especially sturgeon larvae in the propagation and rearing farm. Daphnia spp main nourishment, are micro alga and Bactria alga, thus their production is more expensive than other live food in the usual method of production, that is the use of animal manure, especially cow manure, in soil and cement pool, but this manure is not acceptable especially in such places that have not enough spaces for accepting animal manure and saving civil conditions. This because of the difficulties of its proper preparation especially for its ware housing, due to its bad smell and strong pollution. Thus the mass production of Daphnia sp is complicated and it is necessary the use of other food resources, especially concentrate. For this reason the result of this nutrition was the decrease of efficiency and weak young fish production. The aim of using and introducing recycling sewage material from fish production is to have less pollution, and also production of live food. The aim of this work is the Daphnia massive production by means of sewage. Initially, four fiber glass tanks (including 3 treatments and 1 control) were filled by ground water to amount of one hundred liter. One pipe and stone air was set per tank. A tank (1000 l) was used to rear one hundred Acipenser baerii of average weight of 30 g and was used for production of sewage. Fish were fed 3 % of body weight in two feeding rate with concentrate diet. The amount of sewage introduced to Daphnia rearing tank were 10, 20 and 30 liter per day.1 liter of sewage water in furnace electric (1950C for 20 minute ) was dried for calculating the dry matter. The water of the exit siphon of the fish tank was withdrawed daily in one step. This water contained the excretory material and remaining food that was released in water and also some sediment. It was used for 4 treatments after homogenizing in a separate pan. This solution was added respectively to the first treatment 10 l, to the second 20 l and to the third 30 l daily. The volume of control group tank was 0.5 t that was prepared with 10 kg of cow manure leachate. 20 l manure leachate was used in control group tank daily. In all the tanks (control group and 4 treatments) was introduced wet weight of Daphnia sp that was gathered from a Daphnia sp production cement tank. The experiment lasted 14 days of nourishment. At the end of nourishment period, Daphnia spp production quantities were studied and compared by tanks water filtering and measuring of wet weight of Daphnia spp. The productions resulted: 1 treatment: 68 grams, 2 treatments: 73.5 gram, 3 treatments: 128 grams and control group: 131 grams. There was an observable difference between first and second treatment than third treatment and control group (P<0.05). But there wasn’t an observable difference in control group tank production where was used organic manurend 1 treatment where was used 30 l of daily sewage outlet. This can be a suitable replacement for fertilization and mass production of Daphnia spp, provided that it was used suitable quantity. This conclusion is in accordance with Dadgar and fellows (1393).

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References Kolkovski, S., Czesny, S., Yackey, C., Moreau, R. and Chila, F. 2000. The effect of vitamin C and E in (n-3) highly unsaturated fatty acids-enriched Artemia nauplii on growth, survival and stress resistance of freshwater walleye (Stizostedion vitreum) larvae. Aquaculture, 6: 199-200. Macedo, C.F. and Pinto-Coelho, R.M. 2001. Nutritional status response of Daphnia laevis and Moina micrura from a tropical reservoir to different algal diets: Scenedesmus quadricauda and Ankistrodesmus gracilis. Brazilian Journal of Biology, 61: 555-562. De Pauw, N, Laureys, P. and Morales, J. 1981. Mass cultivation of Daphnia magna Status on rice bran. Aquaculture. 25: 141-252.

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A study on growth and survival of Acipenser baerii as a non endemic sturgeon fish in Iran

Mohsen Pourasadi*, Mohamad Ali Yazdani Sadati, Mehran Avakh Keysami, Mohammad Rahanande, Mahmud Shakorian, and Mehran Gholami *Agricultural Research, Education and Extension Organization (AREEO) - Guilan, Iran

[email protected] Introduction Sturgeon fish Acipenser baerii as an exotic sturgeon fish species entered to Iran aimed to develop aquaculture in 1983. High growth ratio, fresh water culture potential, euritherm and euryhaline, adapted to cold culture water, omnivorous are the desirable characteristics that made this sturgeon fish selected to culture in the world (1, 4). Larval mortality in surgeon fish nursery fase is the worst problem in sturgeon fish culture industry (2, 3). This experiment was conducted to determine growth and survival of Acipenser baerii larvae during exogenous feeding time to fingerling size.

Materials and Methods 4000 pieces of A. baerii larvae were reared in 2 circular cement ponds (3.2 m2). Water quality parameters including temperature (Co), pH, O2 (mg/l), were assessed using portable oxygen meter, merk, Germany 2 times daily. Sturgeon fish Acipenser baerii larvae were fed preliminary using Artemia nauplius, instar 2 then after 5 days using a mixture of Artemia nauplius and Daphnia at 25-30 percent of larval biomass. After 5 weeks larvae were fed by using Biomar concentrated powder commercial feed at 10 percent of larval biomass 4-5 times daily. During feeding trial length and weight and survival, growth parameters such as WG, SGR, and FCR of larvae were measured every week according to Tacon 1995.

Results The results of this study are presented in Table 1 and figure 1:

Table 1. Mean growth parameters of Acipenser baerii larvae during 8 weeks of culture under replications (Mean±SD)

Initial weight Final weight FCR SGR (%) Survival (%) (mg) (mg) 23.7 ±2.06 2800±0.019 3.07±0.3 2.72±0.02 32.5±0.15

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3000

2500

2000

1500

1000

500

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Biometric times Fig. 1. Mean weight of Acipenser baerii archived during 8 weeks of culture period (Mean±SD)

Conclusions In conclusion although we didn’t compare results of growth performance of this species with other sturgeon species at same time and condition but because of reports of other researchers (3) in similar works the growth performances achieved in this case study were relatively acceptable but more studies are needed to compare this psecies with Iranian endemic sturgeon species before to recommend this species as an alternative for culture in Iran, respectively.

References Azari Takami, Gh. 2009. Rearing and propagation of sturgeon fish. Tehran University publication.No.3045, 401p (in Persian) Gisbert, E. and Williot, P. 1997. Larval behavior and effect of the timing of feeding on growth and survival of Siberian sturgeon (Acipenser baerii) Larvae small scale hatchery production. Aquaculture 156 , 63-76 Shakurian. M, Pourkazemi, M Yazdani Sadati, M & et al. 2011. Effect of replacing live food with formulated diets on growth and survival rates in Persian sturgeon (Acipenser persicus) Journal of Applied Ichtyology 27, 771-774 Williot, P., Sabeau, L., Gessner, J., Arlati, G., Bronzi, P. and Berni, P. 2001. Sturgeon farming in Western Europe, recent development, Aquatic Living Resources 14, 367-374.

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Comparison between the effect of different levels of dietary probiotic (Bactocel), on growth and nutrition performance and survival rate in sturgeon (Huso huso) Ali Akbar jafari*, Hosean Mollaaghaee, Behnam zargari, Khosro Shirghazi, Pedram Seraji and Behzad Eslami *Department of Fisheries province mazandaran, Fisheries management of Khorasan Razavi, Ghareh bron Company [email protected]

Introduction This study was conducted to evaluate the effect of probiotic Bactocell on growth, feed and survival of Huso huso larvae for 84 days.

Materials and Methods The test was carried out using a randomized complete block design consisting of zero control, 0.25, and 0.35 g of probiotic Bactocell per kg of food rations in the form of two treatments with three replications. 700 fish weighting 350 ± 0.1 mg were extracted from artificial breeding Gharehbron company farm and after feeding with more food choices 100 were released in each venire. Bioassays were performed every 10 days. At the end of the experiment, growth, feeding and larval survival were calculated using data from bioassays. The results obtained by SPSS software using ANOVA and Duncan were analyzed by the test separator. a b

15 c

) ) 10

BW(g 5

0 testifier Treatment 1 Bactocell Treatment 1 0/25 Bactocell 0/35 Fig.1. the mean of final weight of Huso huso

2 a

1/5 c b

1 FCR 0/5

0 Testifier Treatment 1 Treatment 1 Bactocell 0/25 Bactocell 0/35

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Fig 2. The mean of FCR of Huso huso

Treatments BW PBWI SGR K FCR PER Control 10.6 3150 4.14 1.03 1.59 1.21 Treatment 1 Bactocell 14.2 4900 4.65 1.05 0.96 1.6 0.25 Treatment 1 Bactocell 13.5 3610 4.29 0.99 1.1 1.51 0.35 Results and discussion According to the results, the highest final body weight (WT), (BW), percentage of body weight (PBWI), Specific growth rate (SGR), protein efficiency ratio (PER) and biomass were found in treatments containing 0.25 kg and Bactocell in the diet was observed. The difference was significant with other groups (p <05) The survival rate (SR) among treatments 0.25 Bactocell was found containing antibiotics and other treatments that are meaningful descendants (p <0.05).

References Ahilan., B., Santhanam. R. Shine, G. 2004. Influence of probiotics on the growth and gut microbial load of juvenile goldfish (Carassius auratus). Asian Fisheries Science,17: 271- 278. Bongers., A. and Vander Haevel. E.G.H.M. 2003. Prebiotics and the bioavailability of mineral and trace elements. Food reviews international, 19:397-422. Ai., Q., Mai., K., Tan, B., Xu., W., Duan, Q., Ma, H. and Zhang. L. 2006. Replacement of fish meal by meat and bone meal in diets for large Yellow croaker (Pseudosciaena crocea). Aquaculture 260, 255-263.

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Mass production of Daphnia sp using and replace of (mixture of yeast and molasses and rice bream) instead of organic manure.

Javad Syad far*, Mohamad Ali Yazdani Sadati, Mir Hamed Sayed Hassani, Ali Hossien pour Zelti * Caspian Sea International Sturgeon Research Institute, Iran, Rasht [email protected] Daphnia SP is one of kinds of the effective live food in many of marine larva nurturing especially sturgeon larva in the propagation and rearing workshops Complexes. The aim of using and introducing mixture is economy and also has not any of the difficulties of animal manure and will be acceptable by farmers. First of all it was prepared four fiber glass tanks each of them was 0.5 ton. One of them was control group and others were treatments. They were determined on the base of different amount of (yeast+ bran+ molasses). For access and produce Daphnia sp, the control group tank was used with cow manure leachate because of alga fertility by 2 kilos wet manure. All of tanks were filled with 0.4 m3 of well water and were arranged one tube and air stone in each of them. Each of studied tanks was added one liter of alga water including of Chlorella, Spirulina and Scenedesmus species that were provided by one source. Then they were added the mixture of rice bran and beetroot molasses and yeast that was rested for 48 hours in anaerobe and 25 centigrade degree conditions, this mixture was added to three studied treatments. First treatment: mixture included of 0.5 kg rice bran and molasses equally, second treatment: mixture included of 30 percent molasses and 70 percent rice bran, and third treatment: mixture included of 30 percent rice bran and 70 percent molasses. In all those mixture was added 0.2 g of yeast in equal quantity. All the tanks were setting under sunlight in environment and also airing condition until they became suitable alga bloom. Then all of the tanks were storage with 5 grams wet weigh of Daphnia sp that was gathered from a production cement tank. There were not added any other materials or water during the 14 days of nourishment. At the end of nourishment period, Daphnia sp production quantities were studied and compared by tank,s water filtering and measuring the wet weight of Daphnia sp quantities. The resulted productions are: 1 treatment: 152 g, 2 treatments: 69.5 g, 3 treatments: 74.9 g and control group: 150.8 g. There was an observable bigger difference between second and third treatment than first treatment and control group, but there was not any observable difference between control group tank productions where was used organic manure and 1 treatment where was used bran and molasses in equal quantities. This conclusion is in accordance with Li and his fellows studies (2015). It can be concluded that, this mixture can be a suitable replacement for usual and ancient methods.

References Kersting, K. 1978. Growth efficiency of Daphnia magna. I. The effect of food concentration. Hydrobiol. Bull., 12 (1): 3-21. Standard Methods, 1975. For the Examination of Water, Sewage and Industrial Wastes, 14th edt., Am. Publ. Health Assoc., Washington, DC, pp. 437-440. Schindler, D.A. 1968. Feeding, assimilation and respiration rates of Daphnia magna under various environmental conditions and their relation to production estimates. J. Anim. Ecol., 37: 369-385.

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Effects of starvation and re-feeding on histological changes of intestine in Persian sturgeon Acipenser persicus Mahtab Yarmohammadi *, Mohammad Pourkazemi , Rezvanollah Kazemi , Ali Hallajian , Ayoub Yousefi Jourdehi and , Farahbod Roudbaraki , Mahmoud Shakourian * International Sturgeon Research Institute, Agricultural Research Education and Extension organization (AREEO), 41635-3464, Rasht, Iran

Abstract The effect of starvation and re-feeding (Liu et al., 2011) was investigated on growth performance and histological changes of intestine in juvenile Persian sturgeon (Acipenser persicus). Three hundred and seventy-five fish (108±0.63 g) were divided into five feeding groups. The control group (C) was fed to satiation three times a day during the experiment. The four groups were starved for 1 (W1), 2 (W2), 3 (W3), and 4 (W4) weeks respectively, and then fed to satiation during a 4 week re-feeding period. The results indicated that some parameters including final weight, specific growth rate, and body weight were significantly affected by feeding regimes. While the histological structure of the digestive tracts of starved fish affected by the starvation period. The length of the intestine in starved juveniles decreased. After re-feeding the changes was recovered to the control fish (Falahatkar, 2012, Furné et al., 2012, Yarmohammadi et al., 2012). These findings showed that short term starvations had no significant negative effects on growth performance and intestine histology in Persian sturgeon could recover when re-feeding resumed.

References Liu, W. Wei, Q. Wen, H. Jiang, M. Wu, F. and Shi, Y. 2011. Compensatory growth in juvenile Chinese sturgeon (Acipenser sinensis): effects of starvation and subsequent feeding on growth and body composition', Journal of Applied Ichthyology, 27(2), 749-754. Falahatkar, B. 2012. 'The metabolic effects of feeding and fasting in beluga Huso huso', Marine environmental research. Furne, M. Morales, A. E. Trenzado, C. E. García-Gallego, M. Carmen Hidalgo, M. Domezain, A. Sanz Rus, A. 2012. 'The metabolic effects of prolonged starvation and refeeding in sturgeon and rainbow trout', Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 182(1), 63-76. Yarmohammadi, M. Shabani, A. Pourkazemi, M. Soltanloo, H. Imanpour, M. 2012. 'Effect of starvation and re‐feeding on growth performance and content of plasma lipids, glucose and insulin in cultured juvenile Persian sturgeon (Acipenser persicus Borodin, 1897)', Journal of Applied Ichthyology.

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Designing of cages for culture of aquatic animals

Hossein Askarinejhad*, Hojjat Ahmadi and Ayoub Yousefi Jourdehi *International Sturgeon Research Institute (Areeo), Rasht. Iran. P.O.Box: 41635 - 3464

Abstract Farming of aquatic animal specially culturing fish in cage is one of the most suitable methods for fish farming in the world that has many advantages and application in the world. Considering to having suitable capacity in Iran for cage culture, using of this method is necessary. Also, it is necessary to noticed to usage of initial resources and waste matter production. During the last decade it has been much improvement in aquaculture. In this study, we will introduce structure and particles of cage and how setting it considering to correct selection of citation. Fixing and preserving of cages and required instructions have been introduced. Finally, cages management from starting fish releasing, feeding and diseases control and environment quality control have been explained.

Keywords: Cage culture, Aquatic animals, Management

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Ecology and Environmental Interactions

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Changes in sedimentation, sediment characteristics, and benthic macro faunal assemblages in Huso huso and Rainbow trout (Oncorhynchus mykiss) reared in sea cage in the areas of Guilan and Mazandaran Provinces (Caspian Sea)

Kourosh HaddadiMoghaddam*, Mehdi soltani, Abolgasem kamali, Hossein Ali Abdolhay

* Department of Fisheries, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran.

*[email protected] Introduction Marine cage culture is a popular method for rearing finfish along coastlines and plays an important role in the fish farming industry. It may pose a risk of degrading the local environment by organic pollution, but local hydrographic conditions might influence the pollution at both temporal and spatial scales. In this study, the influences of marine cage culture on sedimentation, sediment physicochemical characteristics, and benthic macro faunal assemblages are investigated. The objective of the current study was to assess site specific impact on the benthic fauna within one area of intensive aquaculture of rainbow trout (Oncorhynchus mykiss) and Huso huso reared in sea cage in the areas of Guilan and Mazandaran Provinces (Caspian Sea).

Materials and Methods Physico-chemical parameters as well as benetic and planctonic populations were investigated monthly during two phases including prior to the installation of cages and after that. The investigations were carried out in four stations in cage and one station outside the cage (20 m in the opposite direction of water flow in the west), whilst monitoring sites devoid of farming activities were also investigated. The site specific impact on the benthic fauna was assessed within a 4.64 km2 area of intensive aquaculture situated on the northeasterly (NE) and southwesterly (SW) at five sites along a transect from within a cage-culture area to the Kelarabad region, Mazandaran between January 2015 until May 2016 and Jafroud region in June 2010 to December 2010. Four sampling sites were established along a transect across this shelf: CW1 (inside the cage zone and 500 m from CW2), CW2 (at the boundary of the cages; 0 m), CW3 (500 m from the edge of the cages), CW4 (1000 m). Samples were taken near the net cages installed at distances up cage and down cage, before and after net cage installation. The choice of control sampling stations was based on their distance from aquaculture farms substrate type and depth. Macrobenthic samples were collected from all sampling sites with the use of a grab sampler (Eckman–Birge) with an area of 0.034 m2 dropped from a boat. Samples were sieved through a 1 mm mesh sieve and each sample was placed in plastic vials in 10% formaldehyde with Rose Bengal added in order to color the living organisms. Samples in the laboratory were sorted by hand into major taxa (Crustacea, Nemertea, Polychaeta, Sipunculida and miscellaneous) and specimens were identified to species level whenever possible with the use of inverted microscope. The Principal Component Analysis (PCA) was used to compare environments.

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Results There were significant differences in sedimentation, including the sedimentation rate and POM (Particulate Organic Matter) flux, among the 4 sites in two seasons. Sediment physicochemical characteristics at Klarabad region, including TOM, TOC, grain size, silt/clay fraction, and moisture content, are summarized in table 1. The benthic fauna showed marked changes in species number, diversity and faunal abundance between farm and control sites. The polychaetes Nereis diversicolor, were the most dominant species at the farm sites (35% of the total abundance), whilst the most dominant species at the control sites were the, Cerastoderma lamarcki, Bivalvia and Mollusca (Abra ovata) the (23% of the total abundance). Species richness, diversity and evenness were higher at the control sites whereas numerical abundance was higher at the farm sites that it would be huge because of bivalves in terms of length and weigh. In table 2 is specified the species composition. Table 1. Range and mean (±SE, n=15) of sediment physicochemical characteristics at four sampling sites at Kelarabd region

Sediment CW1 CW2 CW3 CW4 characteristic Total organic matter (TOM; % DW) 2.49–5.81 2.56–7.69 3.85–8.48 4.53–8.84 Total organic carbon (TOC; % DW) 0.49–0.86 0.61–1.10 0.73–1.31 0.71–1.35 Total nitrogen (TN; % DW) 0.07–0.11 0.09–0.14 0.10–0.19 0.10–0.18 Grain size (mm) 0.01–0.03 0.02–0.10 0.02–0.03 0.02–0.05 Moisture content (%) 36.94–46.81 29.17–55.80 43.64–59.58 42.17–51.74

Table 2. The combination of macro species identified in the Huso huso cage culture cage

Class Family Species

Bivalvia Cardidae Stenocuma spp. Crustacea Balanidae Balanus spp. arvhavrh Cardidae Cerastoderma lamarcki Polychaeta Nereidae Nereis sacsina Oligochaeta Tubificidae Tubifex spp. Bivalvia Semelidae Abra ovata Discussion The results showed that Macro benthos density at different depths showed that the density and biomass away from the coast were reduced and species richness was highest where the less depth and a depth of 20 meters is the highest density per square meter. Also the results obtained indicate an impact of the aquaculture farms on the environment and mainly on the benthic communities showing marked seasonal variability. Temporal changes observed for most variables, including those more evidently affected by fish cultures, followed typical seasonal patterns, characterizing also the control sites.

Conclusions The conclusion is that water dynamics in the study area contributes to dissipate or disperse the inputs originated from the fish farms resulting in statistically insignificant impacts in the surrounding environment. During the past three decades have occurred changes in the fauna and flora of the ecosystem of the Caspian Sea, mostly caused by human factors that can increase or reduce some species, removal or replacement of some species, and food

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References Sanz-Lazaro, C. and Marin, A. 2008. Assessment of finfish aquaculture impact on the benthic communities in the Mediterranean Sea. In: Russo R. (ed) Aquaculture I. Dynamic biochemistry, process. Cromey, C.J., Thetmeyer, H., Lampadariou, N., Black, K.D., Kogeler, J. and Karakassis, C. 2014. Predicting the deposition and benthic impact of aquaculture in the eastern Mediterranean Sea. Aquacult Environ Interact 2: 157–176.

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Bioaccumulation of Toxic metal cadmium in muscle and liver of cultured Huso huso Mehrnoush Norouzi*, Abuzar Galianmoghadam *Department of Marine Biology and Fisheries Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran. [email protected] Introduction Heavy metal pollution in the Caspian Sea is due to the geological structure of the area or originates from pollutant sources resulting from human activities, such as municipal, industrial and agricultural wastewater discharges and from ship traffic. Furher, metals can also enter into the water resource system via organic and inorganic substances originating from the northern coasts to the southern shores of the Caspian Sea. High concentrations of heavy metals definitely are caused by high pollution of the Caspian Sea with consequences to humans via the food chain. The accumulation of cadmium in the human body can cause nervous system impairment and kidney dysfunction. The present study intended to investigate the bioaccumulation of cadmium in muscle and liver tissues of cultured Huso huso.

Materials and Methods Samples were collected from the Chalkerud sturgeon farm (Ramsar), in southeastern part of the Caspian Sea coast, during April and May 2015. The Chalkerud sturgeon farm was selected because the water is supplied by mixture of sea water and well water. Ten samples of H. huso were taken from these farms and the bioassay indexes, including the weight and total length, were measured. Then 10 g of each tissue (muscle and liver) was separated. Each tissue was placed separately in a round-bottom flask and then 50 ml distilled water H2O2 and 50 ml nitric acid were added. Samples were filtered by Whatman filter paper #1 and then deionized water was added to the filtered solution to reach the volume of 5 ml and the solutions were separately poured into digestion tubes (Lakshmanan et al., 2009). Then, flame atomic absorption spectrophotometer (AAS4 Zeiss) was used to measure heavy metal concentration (Standard methods, 2005).

Results The results of the bioassay of the sampling showed that the mean weight and total length were 9400±614.41g and 114.16±1.89 cm, respectively. The accumulation of cadmium in liver and muscle was 0.019±0.001 µg/g and 0.012±0.0147 µg/g, respectively. The uptake of cadmium was higher in liver than in muscle. This difference was statistically significant (P<0.05). The liver is considered as an environmental indicator for assessment of water pollution because of the tendency to accumulate high levels of various contaminants (Yilmaz, 2009) and muscle tissue is used for evaluating heavy metal concentration to ensure the health of fish. In general, concentrations of various metals in the tissues depend on their physiological role. Also, behavioral and dietary habits are another factor in the difference of accumulation in different organs of the body. The difference between heavy metal concentrations in different fish tissues can be attributed to variable affinity to proteins such as metallothioneins. Also the difference between ecological requirements and metabolic activities of fish can be considered as another important influencing factor. Heavy metals choose their target organ based on its metabolic activity and this explains the reason why metals accumulate in tissues such as the liver and gills compared to muscle tissue (with low lipophilic activity).

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Pearson correlation analyses did not show any significant association between weigh and total length of the body,. These findings are similar to those in previous reports (Pourang et al., 2005). In general in fish species, small or medium body size has no influence on metal deposition and accumulation level in tissues (Gaspic et al., 2002). According to the data recorded and compared to the permissible standard limit for cadmium, the concentration was lower than the permissible limit in H. huso. The amount of toxic heavy metal cadmium was markedly below the international guidelines for human consumption (table 1).

Table 1. Comparison of heavy metal concentrations (µg/g) in muscle tissue of Huso huso with other organs WHO (1996) MAFF(1995) FEPA (2003) USEPA (1987) FAO, 2011 NHMRC (Tuzen, This study 2009) 0.2 0.05 ˂1.0 0.008 0.5 0.02 0.012±0.0147

0/25 Fig. 1.The average concentration of cadmium in tissues and compared to WHO standards. 0/2 Columns are means and bars standard deviation 0/15 Cd

µg/g 0/1 WHO In general, on the basis of the 0/05 obtained results it can be concluded 0 that the levels of cadmium of cultured Muscle Liver H. huso from the Chalkerud sturgeon farm are below the limits of the international guidelines as well as in most cases lower than the global safety standard levels. Hence, at the present, it seems that consumption of the species do not present any danger for human health. References Federal Environmental Protection Agency (FEPA). 2003. Guidelines and standards for environmental pollution control in Nigeria. Nigeria. Gaspic, Z. K., Zvonaric, T., Vrgoc, N., Odzak, N. & Baric, A. 2002. Cadmium and lead in selected tissues of two commercially important fish species from the Adriatic Sea. Water Research, 36, 5023–5028. Joint Fao/Who. 2011. Food Standards Program Codex Committee on Contaminants in Foods Fifth session the hague, the Netherlands, 21 - 25 march. Lakshmanan, R., Kesavan, K., Vijayanand, P., Rajaram, V. & Rajagopal, S. 2009. Heavy Metals Accumulation in Five Commercially Important Fishes of Parangipettai, Southeast Coast of India. Advance Journal of Food Science and Technology, 1, 63-65. MAFF., 1995. Monitoring and surveillance of nonradioactive contaminants in the aquatic environment and activities regulating the disposal of wastes at sea. Aquatic Environment Monitoring Report No. 44. Directorate of Fisheries Research, Lowestoft. NHMRC, National Health and Medical Research Council. 1987. National food standard A12: Metals and contaminants in food. Canberra, Australia Australian Government Publishing Services. Pourang, N., Tanabe, S., Rezvari, S. & Dennis, H. 2005. Trace elements accumulation in edible tissues of five Sturgeon species from the Caspian Sea. Environmental Monitoring and Assessment, 100, 89-108. Standard methods for the examination of water and waste water, 2005. USA. pp. 100-200. USEPA, 1987. Quality Criteria for Water. EPA Publication 440/5-86- 001. U.S. Gov. Prin. Office, Washington D.C., USA.

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The bioremediation of wastewater using Nereis diversicolor in an integrated culture system with beluga, Huso huso (Linnaeus, 1758)

Zabih Ollah Pajand*, Mehdi Soltani, Abolghasem Kamali, Mahmoud Bahmani * International Sturgeon Research Institute, P.O. Box: 41635-3464, Rasht, Iran *[email protected]

Introduction Aquaculture of sturgeon has made favorable progress and the high growth rate of great sturgeon or beluga Huso huso, made it a suitable candidate for commercial aquaculture (Hung & Deng, 2002). The development of intensive fish culture during the last years focused on the effects of water wastes from the farms on the environment (Wu 1995; Tsutsumi and Inoue 1996; Trovar et al., 2000) and many researchers around the world are involved with developing new technologies to reduce the environmental footprint of intensive systems and minimize the potential for environmental damage from nutrients released with aquaculture discharge (Folke et al., 1998; Crab et al., 2007). The benefit of subsequent culture systems using polychaetes as a bioremediator is that organic matter and nutrients released from the fish culture system can be converted into a biomass which may be a valuable by-product and can be easily removed (Ryther et al., 1975; Enander and Hasselstrom, 1994).

Materials and Methods N. diversicolor worms were collected from the Anzali lagoon at Guilan Province, using Van Veen grabs and transported alive and reared at the International Sturgeon Research Institute located 35km away from Rasht in northern Iran. In this experiment, juvenile worms of 0.05 g average weight were used and a total biomass of 25 g was stocked into each tank (approximately 2000 worms per m-2). 100 fingerling Huso huso (mean initial weight, 27.78 g) hatched and reared from wild spawners at the Shahid Beheshti Sturgeon Hatchery Center, Rasht, Iran, were transported to the laboratory (international sturgeon research institute). They were placed into nine 80-L fiberglass tanks, (60 cm diameter; water depth 40cm; 3 tanks per treatment; 3 replicates per treatment) and were cultured for 8 weeks. For each treatment, 5 fish (1.5 kg m-2) were stocked into tanks. The treatments 1, 2 and 3 were group FNW (Fish and Nereis waste), NW (Nereis waste) and the FW (fish waste) as control group, respectively. At this treatment sand bed was used as a sandy filter in the integrated culture system of beluga. The effect of wastewater treatments on the growth performance of the fishes and worms was assessed by the SGR, WG, survival, biomass gain and FCR. Water sample were taken from inlet and outlet of beluga and worms tanks to find out of the concentration difference between sampling points. The water samples were analyzed to determinate TSS concentrations according to the standard methods for evaluation of water and wastewaters referred by Danish Standard Methods DS 224 (1975), Timmons et al. (2002) and APHA (2005). Percentage of organic matter was determined on weight loss on ignition: %OM = ((DW-AFDW)/DW)*100%. The conditions of water quality parameters (NH3, NO2-N, NO3-N, TSS and TOM) in fish and worm boxes during the experiment were shown in the Figs. 1-6.

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Results and Discussion 3- The highest removal efficiency of PO4 belonged to TFNW with 48.1%, whereas those in TNW and TFW were 16.2% and 12.6%, respectively. The removal efficiency of NH3-N concentrations were evidenced by the significant (P<0.05) differences among treatments. The highest removal efficiency of NH3-N among treatments belonged to TFNW with 35.4% reduction in compare to TFW and TNW which those were 20.2% and 10.1%, respectively.

0/8 1

) 1

- 0/6

(mg (mg l

)

4 1 0/4 - 0/5

PO Inlet water (mg l (mg

outlet water of Beluga Inlet water 3 0/2 outlet water of Nereis Fig 1 NH Fig 2 0 0 FNW NW FW FNW NW FW -1 In the outlet water from the worms, the concentration of NO2-N in TNW was 0.08 mg L which -1 -1 was higher than TFNW (0.04 mg L ) and TFW (0.07 mg L ). The highest removal efficiency of this factor among treatments was found in TFNW (91.2%) and TNW (78.3%), whereas that in TFW was -1 44.2%. In the outlet water from the worms, NO3-N in TFNW with 0.18 mg L was higher than TFW -1 -1 (0.17 mg L ) and TNW (0.17 mg L ). The additional efficiency of NO3-N unlike other parameters occurred and no significant differences were observed among treatments (P>0.05).

0/15 0/3 Inlet water Inlet water outlet water of Beluga outlet water of Beluga outlet water of Nereis

outlet water of Nereis

) 1

- 0/1 0/2

)

(mg l (mg

0/05 0/1

(mg l (mg

Fig 3 3

NO Fig 4 0 0 FNW NW FW FNW NW FW

-1 The overall mean TSS in the outlet water of worm boxes was 0.07 mg L in TFW, which was 49.1% removal efficiency and lower than outlet water from associated fish box (25.40 mg L-1) among treatments, whereas this value for TNW was 92.2%, and for TFNW was 67.6% which had the highest removal efficiency. In this group results indicated that, in worm boxes, there were no significant differences of TOM concentration among treatments. 0/5 Inlet water 5 outlet water of Beluga 0/4 4

outlet water of Nereis

)

- Fig 6 0/3 Fig 5 3

0.02 (mg l (mg

0.02

0/2 2 1.45

TOM (%) TOM TSS 0/1 1

0 FNW NW FW FNW NW FW Treatment Treatment

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References Wu, R. S. S. 1995. The Environmental Impact of Marine Fish Culture: Towards a Sustainable Future. Marine Pollution Bulletin, Vol. 31, Nos 4-12, pp. 159-166 APHA, 2005. American Public Health Association, American Water Works Association and Water. Folke, C., Kautsky, N., Berg, H., Jansson, A. and Troell, M. 1998. The ecological footprint concept for sustainable seafood production: a review. Ecological Applications, 8 (S), 63– 71.

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Semi mass culture of freshwater rotifers for Acipenser persicus larvae Rudabeh Rufchaie *, Reza Armodli and Sahebali Ghorbani *Inland Water Aquaculture Institute, Specialized Research Station Of Aquatics Nutrition and live food, Bandare Anzali, Iran [email protected]

Introduction Survival rates and improving overall performance of artificial breeding and rearing programs with sturgeons can be considered effective to achieve their sustainable use. Acipenser persicus, a native species of the Iranian Caspian Sea coast, shows high hatching rates but experience also high mortality during larval culture. Persian sturgeon stocks play a Significant role in the annual catch figures. In the present study, pure semi mass cultures of freshwater rotifers, native to Iranian waters, were obtained and enriched with ascorbic acid palmitate to improve survival and growth rates during feeding of early developmental stages of A. persicus.

Materials and Methods Rotifers under study were from the Anzali Lagoon. They were isolated and purified under a stereomicroscope and transferred to 20 ml test tubes containing EPA culture medium (96 mg CaHCO3+ 60 mg MnSO4+4 mg KCl in 1L water). Rotifers were fed algae (Chlorella vulgaris) at a rate of 1×106 cells/ml and were enriched with ascorbic acid palmitate (Manufactured by Basel, Switzerland) for 24 h at a rate of 1000 mg/g dry weight of rotifers. Mass culture of Chlorella vulgaris was carried out in plastic bags (7 L capacity) using culture medium Z-8±N] under continuous aeration, at a temperature of 25±1ºC and 3500±350 lux light. Three experimental groups were studied; Group 1 was fed a mixed diet of decapsulated Artemia cysts, Daphnia and Rotifers, Group 2 was fed with rotifers as the starter diet and Group 3 fed a starter diet of rotifers enriched with vitamin C. A control group was also studied which used the feeding protocol followed in the hatchery.

Fig.1. Semi mass culture of freshwater rotifers Fig.2. Density of freshwater rotifer

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Results In this study with 100 L tanks rotifers reached a stocking density of 640±40 ind/ml. To harvest the required weight of rotifers to feed sturgeon larvae. Highest final body weight, percentage weight gain and SGR and lowest FCR were recorded in experimental group 3, followed by experimental group 1 (P<0.05), whereas lowest values for growth parameters were recorded in experimental control group. Survival rates recorded in experimental groups 2 and 3 were significantly higher (P<0.05) than those recorded in experimental group 1 and in the control group.

Conclusion According to recent investigations, improved growth and survival rates in larvae are related to the increased free amino acid (FAA) content of rotifers after enrichment. The presence of amino acids during the transition from endogenous to exogenous feeding and also during early exogenous feeding greatly influenced survival rate of larvae. FAA also serves as a source of energy during yolk sac absorption and at the onset of exogenous feeding affecting larval growth . In these studies the freshwater rotifer was seen to greatly influence growth parameters during the first two weeks of active feeding.

References Aragao, C., Conciao, L.E.C., Dinis, M.T. and Fyhn, H.J. 2004. Amono acid pools and Artemia under different conditions: nutritional implications for fish larvae. Aquaculture.234:429-445. Miller, W.E., Greene, J. Cand Shiroyama,T.,1978. The Selenastrum capricornuutm Printz algal assay bottle test .U.S.EPA Rep.600/9-78-018.

Moghim, M. 2003. Sturgeon stock assessment and population parameters Acipenser persicus The Iranian coast of the Caspian Sea. J. Iran. Scientific. Fisheries, 11(4):97-118. Ronnestad, I., Tonheim, S.K., Rojas, C.R., Fyhn, H.J., Kamisaka, Y., Koven, W., Finn, R.N., Barr, Y. and Conceicao, L.E.C. 2003. The supply of amino acids during early feeding stages of marine fish larvae: a review of recent findings 265, 1–4:336–342. Velez, Z., Hubbard, P.C., Hardege, J.D., Barata, E.N. and Canario, A.V.M. 2007. The contribution of amino acids to the odor of a prey species in the sole Senegalese .Aquaculture.265, pp: 336-342.

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Surveying the nutrition condition of the juvenile Persian sturgeon (Acipenser persicus) in fish ponds

Shahpoor Gholami *, Zabialla Pajand , Eshagh Rasuli * Dr. Yousefpour propagation and pisciculture centre, Siyahkal, Iran Email: [email protected]

Introduction From the aspects of biodiversity and genetic reserves, Sturgeons are considered as rare aquatic living fossils. These creatures have lived for millions of years to present. Their meat and caviar is among highly valued foods and are considered as an important commodity in economy (Pourkazemi, 2006).

Materials and Methods This research was done in Dr. Yousefpour pisciculture centre. Samples were selected from 3 ponds with area of 2 hectares during March to June 2015. Sampling was done with a manual trawl. The length and width of the trawl mouth were 90 * 90 centimetres. Totally, the number of 210 Iranian juvenile sturgeons were selected as the sample and the contents of Zooplankton and Benthos in their stomachs were checked. Ivlev's index (1961) was used for surveying the relation between zooplankton available in ponds and the nutrition of the juvenile sturgeon (Equation 1). The range of the index is between -1 to 1.

E = Equation (1)

E = food selection index = the percentage of the intended food organisms in sturgeon stomach contents relative to the total eaten food organisms by the sturgeon. = the percentage of the same organisms in environment relative to the total other organisms.

Data analysis employed the Statistical Package of Social Science (SPSS) 17. Furthermore, one way Analysis of Variance (ANOVA) and Duncan test used to comparison of means.

Results Evidence shows dominant food in stomach contents were Daphnia from Cladocera order and Chironomus from Diptera order. Table (1) shows calculated Ivlev's index (E) three categories of zooplankton including Cladosera, Copepod and Ostracoda. Based on the table, the sign of E indices related to Cladosera and Ostracoda are positive. These mean the organisms are preferred food for the juvenile surgeon. On the other hand, the sign of E index for Copepoda is negative. It means the surgeon avoids eating Copepoda.

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Table (1) Ivlev index quantities for Cladosera, Copepoda and Ostracoda in ponds Percentage in Feed percentage Result Descriptions Factors ponds by the juveniles Cladocera 33.43 59.82 38.27 126.93 0.2 E >0 Copepoda 56.53 12.46 -40.1 67.03 -0.5 E <0 Ostracoda 0.81 2.8 1.7 892.98 0.44 E >0

The surveys found the biomass of Copepoda is more than Cladocera. Figure (1) shows the food preference of Cladocera and Ostracoda relative to Copepoda by the juvenile surgeon.

Figure (1) feeding the juvenile surgeon from zooplankton in the ponds

Discussion Research findings show with gradually increasing of the biomass of Chironomus in the ponds, the juvenile sturgeon tends to feed these organisms will increase and eating Daphnia will gradually decrease. Also, feeding of Cyclops is slight and it increases when the biomasses of Chironomus and Daphnia have strongly decreased. In fact, the juvenile sturgeon replaces Chironomus and Daphnia with Cyclops in such conditions. In the case of Ostracoda, it must be noted that it is random food for the juveniles. Because its abundance relative to other foods is small. Our findings are consistent with the results of Pyka and Kolman (2003). They observed Cladocera, Diptera and Heteroptera in fish digestive tracts. Also, they found Daphnia magna and Daphnia moina were the most abundant among zooplankton species eaten. Our findings confirm these results. Adamek et al. (2007) investigated the nutrition of Acipenser ruthenus in ponds. They found Daphnia was most abundant among 18 types of zooplankton. Also, they showed Chironomus was most abundant benthos. We find that Daphnia and Chironomus most abundant food organisms in the stomach contents of the juvenile surgeon. Thus, our results are compatible with theirs.

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References Adamek, Z., Prokes, M., Barus, V. Sukop, I. 2007. Diet and Growth of 1+ Siberian sturgeon, Acipenser baerii in Alternative pond culture. Turkish Journal of Fisheries and Aquatic Sciencesi. 7, 153-160. Fieszel, J., Bogacka-kapusta, E., Kapusta, A., Szymanska, U. and Marty Niak, A. 2011. Feeding ecology of Sterlet Acipenser ruthenus L.in the Hungarian section of the Danube River. Ivlev, V.S. 1961. Experimental ecology of the feeding of fishes. Yale University Press, New Haven, Conn. Pourkazemi, M. 2006. Caspian Sea sturgeon conservation and fisheries: past present and future. Journal of Applied Ichthyology. 22(1), PP: 12-16. Pyka, J. and Kolman, R. 1999. Food Selection by pond – reared Siberian sturgeon (Acipenser baerii Brandt) Fry. Archives of polish Fisheries, 7(1), 123-128.

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Geochemical Partitioning of heavy metals in surface sediment of south Caspian Sea and Anthropogenic evaluation

Tooraj Sohrabi *, Hossein Saberi, Adel Hosseinjani, Gholamreza Mehdizadeh, Shahram Abdolmaleki. *International Sturgeon Research Institute (AREO), Rasht. Iran. P.O.Box: 41635 – 3464 Email: [email protected] Introduction Trace metals introduced into the aquatic environments by various anthropogenic activities are ultimately absorbed by deposits and incorporated into sediments. The Caspian Sea, bounded by the five littoral states of Azerbaijan, Federation of Russia, Islamic Republic of Iran, Kazakhstan and Turkmenistan, is the largest inland body of water in the world (Kosarev and Yablonskaya, 1994). As a closed-environment without an outlet, various pollutants due to effluents from coastal catchment areas and leakage from off shore oil production have accumulated in the Caspian Sea (Karpinsky, 1992). The geological characteristics of the Caspian Sea are quite variable (Krilov, 1987; Kosarev and Yablonskaya,1994).

Material &Methods In this study ,the geochemical fractions employed were “easily,freely,or leacable and exchang-eable(EFLE)’,”acid-reducible”,”oxidable-organic”and resistant,according to Badri and Aston(1983).The mathematical summation of “EFLE”,”acid-reducible”,and ”oxidisable- organic”fractions constitute the nonresistant phase (Badri and Aston,1983). Since there has been currently No information available on “resistant”and “non-resistant” phases of heavy metals in surface sediments of south of the Caspian Sea .This study was carry out for identifying possible sources and locations of anthropogenic metals.

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Results and Discussion Resistant Resistant Cd (%) Non-resistant Pb (%) Non-resistant

80.00 80.00 70.00 70.00 60.00 60.00 50.00 50.00 40.00 40.00 30.00 30.00 20.00 20.00 10.00 10.00 0.00 0.00

Kia Kia Neka Neka Astara Anzali Gohar Astara Anzali Gohar Parasar Vazivar Vazivar Noshahr Babolsar Gaz Port Parasar Noshahr Babolsar Gaz Port Chaboksar Chaboksar Cahmkhaleh Khazar Abad Cahmkhaleh Khazar Abad Bandar Torkman Bandar Torkman

Resistant Resistant Ni (%) Non-resistant Cu (%) Non-resistant

100.00 100.00 90.00 90.00 80.00 80.00 70.00 70.00 60.00 60.00 50.00 50.00 40.00 40.00 30.00 30.00 20.00 20.00 10.00 10.00 0.00 0.00

All of metal concentrations in sediments for 14 sampling Stations (Pb, Ni, Cu) except Cd were dominated by the resistant fractions. The non-resistant fractions were lower than the resistant fractions for the whole duration of the study, which indicate that there were no significant anthropogenic inputs of Pb, Ni, Cu to the surface sediments of south Caspian Sea. The finding between resistant and non-resistant geochemical fraction in the surface sediment of the different sampling stations of these present study, generally showed that sources of Cd in surface sediments of south Caspian Sea were mainly contributed from anthropogenic sources. This was indicated by higher percentages shown for non-resistant Cd compared to resistant Cd. The non-resistant fractions are potentially toxic for organisms because it is easily removed and used by organisms, whilst fractions 2 and 3 can be solubilized depending upon physical and chemical parameters, such as oxygen content and bacterial activity (Yap et al., 2002b; Morillo et al., 2004; Ramirez, 2005; Yap et al., 2005).

References Yap, C.K., Ismail, A.R., TAN, S.G. (2005). analysis of heavy metal concentration data Cd Cu Pb and Zn in different geochemical fractions of the surface sediments in the straitsnof malacca by the use of correlation and multiple linear stepwise regression analysis. Malaysian Applied Biology. 34: 51-59. Yap, C.K., Ismail, A.R., Tan, S.G. (2003d). Cd and Zn concentrations in the straits of Malacca and intertidal sediments of the west coast of peninsular Malaysia. Marine Pollution Bulletin. 46: 1348-1353.

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Assessment of heavy metal enrichment and contamination factors in surface sediment of south coastal zone of Caspian Sea

Tooraj Sohrabi*, Hossein Saberi, Mohammad Salvatian

*International Sturgeon Research Institute (AREO), Rasht. Iran. P.O.Box: 41635 - 3464 Email: [email protected]

Introduction Development of industrial activities in coastal zones brought the risk of heavy metal contamination in marine environment.

Materials and methods Surface sediments along the south of Caspian Sea were collected to evaluate the contamination of heavy metals.

Results and conclusions The result ranged (μg/g, Fe% dw): Pb (13.06–33.48); Ni (18.01–69.63); Cd (0.62–1.5); Zn (30.11–87.88); Cu (5.86–26.37) and Fe(1.8–4%) respectively. Cadmium showed higher EF when compared to other sites. Geoaccumulation Index value for Cd in most stations was classified as moderately contaminated and moderately to strongly contaminated, as well as the average of Igeo of Cd (1.77 ± 0.35) suggested that surface sediments of Caspian coast were moderately polluted by this metal. The result of the Pearson correlation showed that there were significant positive associations between Ni, Cd and Zn (r = 0.44–0.76; p < 0.01). Based on the standard quality guidelines used in the present study, it can be classified as unpolluted to moderately polluted coastal environment.

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Ecosystem service modeling of marine aquaculture in Mazandaran province Elham Haghshenas, Mehdi Gholamalifard*, NematMahmoudi *Assistant Professor, Department of Environment, Tarbiat Modares University (T.M.U.), Noor, Iran. [email protected] Introduction Marine and coastal ecosystems are one of the most productive diverse and valuable ecosystems on Earth. Ecosystem service evaluation is essential. Marine aquaculture as one of the most important services which can be easily valued and leading role in people's welfare (Boyd & Banzhaf, 2007). Important reasons such environmental issues (reduced fishing pressure on fish stocks, and the opportunity to rehabilitate them), to create sustainable jobs and the creation of export, marine aquaculture can be considered as an important benefit to increase aquaculture production in the Iran. Modeling marine aquaculture is one of models used to assess and evaluate aquaculture products of farms based on growth parameters and the effect of temperature changes and rate of metabolism of fishes (Guerry et al, 2013).

Area of study Mazandaran Province Coast is considered as the study area. Figure 1 shows the location of fish farms in the area.

Fig 1. Area of study & farms

Materials and Methods The modeling has been done in three steps in each farm (1) The growth of individual fish has been modeled to harvest weight. (2) The total weight of fish produced has been calculated in each farm as the number of fish remaining at harvest, multiplied by their harvested weight and the weight of fish lost to natural mortality. (3) Lastly, all the fish in a farm are harvested at the same time, and the farm is restocked after a user-defined fallowing period. Valuation of processed harvest is an optional fourth step in the model .Table 1 expresses the equations in this model (Sharp et al., 2014; Eastman, 2015).

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Table 1.equations in this model Where (gr.day-1) and (non-dimensional) are growth parameters, , is 1 daily water temperature (C) at farm , and (C-1) represents the change in T t W = a Wb .et-1,f +W biochemical rates in fishes with an increase in water temperature. The t,y,f t-1.y.f t-1,y,f value for Atlantic salmon (0.08) indicates a doubling in growth with an 8- 9 C increase in temperature.

where ℎ,ℎ, is the weight at date of harvest ℎ, on farm from Equation (1), is the processing scalar which is the fraction of the fish in 2 -M. th- t0  the farm that remains after processing (in here is considered equal to 1) , TPWf.c =W th,h,f .nf e is the user-defined number of fish on farm , and − ·( ℎ− ) is the daily natural mortality rate experienced on the farm from the date of out planting ( 0) to date of harvest ( ℎ)

where W , is the total weight of processed fish on farm in harvest 1 3 NPV TPW P1. c  cycle , is the market price per unit weight of processed fish, is the f., c f c t 1r  fraction of that is attributable to costs, is the daily market discount rate, and is the number of days since the beginning of the model run

Sea surface temperature (SST) been downloaded from the satellite's MODIS. Based on the existing relationships between fish weight, changes temperature, rate of metabolism, growth parameters, daily mortality rate and by reducing the cost of doing (according to feasibility report prepared by Fisheries Organization), the production and the net present value (NPV) is calculated for culture of rainbow trout and great sturgeon. Out planting time considered in October month and release weight 100 gr. Geographical location and capacity of each farms is presented in Table 2 (Part 1) Table 2. (farms of Mazandaran province coastal) Part 1 Part 2 Farm City Longitude latitude production Harvested(Rainbow Harvested NPV NPV capacity (kg) sturgeon (Rainbow) (sturgeon) (kg) ($ x 1000) ($ x 1000)

1 Juybar 52.57360 36.49297 200 862780 148046 1139 666 2 Babolsar 52.35501 36.45150 400 1734899 297423 2290 1338 3 Noshahr 51.78493 36.64064 560 2434777 408551 3214 1838 4 Noshahr 51.72670 36.72670 200 866575 148026 1144 666

5 Kelarabad 51.14500 36.44300 100 434308 76377 573 344 6 Abasabad 51.07504 36.46665 200 864620 147681 1141 665 7 Valiabad 50.5725 36.5020 200 868114 148025 1146 666 8 Ramsar 50.3843 36.5835 200 863608 146486 1140 659 Total 8929681 1520615 11787 6842

Results and Discussion The results of the marine aquaculture model are shown in Fig 2. According to the results a farm (3) in Noshahr with 16 cages has most production capacity. The calculated harvest at the

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir end of 3 years is 2434 t for trout and 408 t for sturgeon. The NPV for these species in farm (3) are 3214 dollar for trout and 1838 dollar for sturgeon. Farm (5) with 4 cages has the least production capacity with a harvest at the end of 3 years of 434 t for trout and 76 t for sturgeon. NPV in this farm (5) is of 573 dollar for trout and 344 dollar for sturgeon. Comparison of production between the two species shows that Rainbow trout has faster growth, better marketability and lower cost) food and services( so has more profitability and higher NPV. Two important and influential parameters in this evaluation are temperature changes and growth parameters of fish. Trout growth parameter is 0.23 (g/day) and sturgeon is 0.015 (gr/day). There are also differences between the temperatures of the different farms.

harvested weight in each farms the 3 year period Total harvested weight farms in Mazandaran the 3 year r

NPV in each farms the 3 year period Total NPV farms in Mazandaran the 3 year period Fig 2. Comparison of NPV and final productions of each farm for the two species, Great sturgeon and Rainbow trout

Conclusion Economic evaluation was carried out between two species, sturgeon and trout. Figure 2 shows that trout farming is more economical because: 1) it could be possible to stock more biomass in the cage.2) Trout is faster-growing, 3) the marketability of this species, which is more acceptable than sturgeon. Although there is concern that trout is not native to the Caspian Sea, but adapted to culture in farm conditions very good and are very fast-growing and is widely adopted among other species. Marine aquaculture model will be practical when used in relation to other modeling ecosystem services, because between economic benefit and environmental costs of the industry balance is maintained. The output of this model can be used as input to other models of ecosystem services such as water quality and risk assessment models (Guerry et al., 2013).

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References Boyd, J. and Banzhaf, S. 2007. What are ecosystem services? The need for standardized environmental accounting units. Ecological Economics, 63(2-3), 616–626. Eastman J. R. and Jiang H. 2000. Application of fuzzy measures in multi-criteria evaluation in GIS. International Journal of Geographic Information, 14(2), 173–184. Guerry A.D., Ruckelshaus M.H., Plummer M.L. and Holland D. 2013. Modeling Marine Ecosystem Services, 5, 329–346. Guerry A. D., Ruckelshaus M. H., Arkema K. K., Bernhardt J. R., Guannel G., Kim C. K. and Spencer J., 2012. Modeling benefits from nature: using ecosystem services to inform coastal and marine spatial planning. International Journal of Biodiversity Science, Ecosystem Services & Management, 8(1-2), 107–121. Sharp R., Chaplin-Kramer R., Wood S., Guerry A., Tallis H., Ricketts T. 2014. InVEST 3.1.0 User’s Guide, http://www.naturalcapitalproject.org/download.html.

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Enrichment of Daphnia spp. by algae Chlorella vulgaris in order to improve profile of polyunsaturated fatty acids (PUFA) in larvae of Persian sturgeon (Acipenser persicus) Fruzan Chubian*, Zohreh Ramezanpour, Mahmoud Hafeziyeh, Koroush Haddadi Moghadam, Zabihollah Pajad, Bahareh Younes Haghighi, Hamidreza Pourali, Rudabeh Rufchaie *Researcher of Agriculture Research, Education & Extension Organization (AREEO), International Sturgeon Research Institute, Ecology Department, Rasht, Iran [email protected] Introduction The aim of this study is the enrichment of Daphnia spp. by fatty acid of Chlorella vulgaris algae in order to increase survival in larvae of Persian sturgeon (Acipenser persicus). Chlorella vulgaris is a green unicellular algae with spherical shape.

Materials and Methods Sampling was carried out by plankton net with 25µ mesh size in natural reservoirs, then the algae were separated and cultured in Z8 culture media at high density (Miller et al, 1978). The used density of algae to enrich Daphnia was 5x107cell/ml. Enrichment was performed for 24 hours. This experiment has 3 repetitions and one control. In this study, 30 larvae were stocked in each rearing tank. The larvae were fed up to 30% of body weight. Feeding carried out 4 times in day and night. Survival rate of the larvae was calculated by the following formula: Survival percent = (number of total stocked larvae/number of alive larvae) × 100 (Wahil et al., 2003) Extraction of fatty acid from Chlorella vulgaris algae was done by method of direct methyl esterification using Gas GC Chromatography unite in Urmia Lake Research Institute. Data analysis performed by Excel and SPSS software. During the study period, water temperature fluctuated in range of 18-24˚C, dissolved oxygen 5.8-7.2 mg/l and pH 5.6-8.2.

Results Percent of larval survival showed significant differences between the treatment (76%) and control group (68%) (p>0.05). Comparison of fatty acids in Daphnia is shown in fig. 1.

9 Daphnia 8 enriched

7 control

5 4 3 2 1 0

Fig. 1. Daphnia fatty acids in treatment and control group based on body weight percent

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Conclusions Daphnia galeata can change docosahexaenoic acid (DHA) and 18-carbon polyunsaturated fatty acids (C18-PUFAs) to eicosapentaenoic acid (EPA), (Von Elert, 2002). Navarro et al. (1999) stated that Artemia fransiskana and Brachionus plicatilis can convert DHA to EPA in starvation time. This can enhance the quality of Daphnia and finally the larvae of Persian sturgeon. Because, fatty acid composition in sturgeon body seriously depends on fatty acid composition of diet, it can lead to increase in larval survival of Persian sturgeon (Acipenser persicus).

References Miller, W.E., Greene, J.C and Shiroyama, T. 1978. The Selenastrum capricornatum Printz Algae assay bottle test. U.S.EPA Rep.600/9-78-018. Navarro, J.C., Henderson, R.J., McEvoy, L.A., Bell, M.V., Amat, F. 1999. Lipid conversions during enrichment of Artemia. Aquaculture, 174:155-166. Von Elert, E. 2002. Determination of limiting polyunsaturated fatty acids in Daphnia galeata using a new method to enrich food algae with single fatty acids. Limnol. Ocean ogr. 47:1764–1773. Wahli, T., Verlhac, V., Girling, P., Gabaudan, J. and Aebischer, C. 2003. Influence of dietary vitamin C on the wound healing process in rainbow trout (Oncorhynchus mykiss). Aquaculture. 225, 371-386.

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Population dynamic of Acipenser persicus (Borodin, 1897) in the southern Caspian Sea (Guilan province) using Monte Carlo and Bootstrap methods Amirali Moradinasab*, Moslem Daliri, Hadi Raeisi, Sarah Haghparast, Ayoub Yousefi Jourdehi, Mahmoud Bahmani, Amin Farahbod Roudbaraki and Arezoo Vahabnejhad *Ph.D. student of Fisheries Exploitation and Production, Faculty of Marine and Atmospheric sciences, Hormozgan University, Bandar Abbas, Iran. [email protected]

Introduction Fisheries management is based on preventive approaches. Using the uncertainty in stock assessment has been suggested in several studies, because people can simulate the consequence of various management strategies and their interactions. Monte Carlo models and simulations are computer-intensive methods that can be used to test specific hypotheses and to answer less specific “what if” type questions, including projections into the future (Haddon, 2011). The sturgeon population in the Caspian Sea has strongly collapsed over recent decades. For example, the recorded catch decreased from 3000 tons in 1989 to less than 17 tons in 2010. Persian sturgeon (Acipenser persicus) is the most abundant sturgeon species in the Caspian and includes 60 percent of catch composition (Iran fisheries organization, 2014). In the present research we studied population dynamic of A. persicus using Monte Carlo and Bootstrap methods and we hope that our findings could help to promote fisheries management in the region.

Materials and Methods Catch data of fixed gill-nets, beach seines and Aquatic Conservation Unit in Guilan province were used during the years 2001-2010. L∞ and K parameters were estimated based on length frequency in FiSAT by ELEFAN 1 method. Growth was evaluated according to Von Bertalanffy growth function for fish length against age (Sparre and Venema, 1992) (Eq. 1).

Eq. 1 Where t0 was computed by Pauly formula (Pauly, 1980): Eq. 2

Natural mortality (M) was also estimated Pauly empirical equation (Eq. 3): Log (M) = -0.0066-0.279log (L∞) +0.6543log (K) +0.4634log (T) Eq. 3

Total mortality (Z) and exploitation rate (E) were obtained be Eq. 4 and Eq. 5:

Eq. 4 Z = F+M , E = F/Z Eq. 5

95% Confidence interval (CI) of Z was calculated by using n-n-parametric bootstrap method (Haddon, 2011): Eq. 5

Eq. 6

Eq. 7 Eq. 8

The influence of sampling error, by Monte Carlo algorithm and 1000 replicates were added to Mortality formula:

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Eq. 9

Eq. 10

Results Data of 4376 specimens were analyzed. L∞, K and t0 parameters were calculated 203 cm, 0.08 year-1 and -0.94 respectively. Using catch curve, total mortality (Z) was computed 0.263 year- 1 (Fig. 1). Natural mortality (M) and exploitation rate (E) were also estimated 0.14 year-1 and 0.61. Exploitation rate indicates a fishing pressure on Persian sturgeon population.

Fig. 1. Catch curve of Persian sturgeon (A. persicus) in the southern Caspian Sea (Guilan province)

95% Confidence interval (CI) of total mortality (Z) was also calculated 0.195 to 0.327 year-1 (Fig. 2). By considering influence of sampling error total mortality (Z) was obtained 0.244 year-1.

Fig. 2. estimating the 95% Confidence interval (CI) of total mortality using Monte Carlo method

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Conclusions As FAO. 2002. Stated, when management decisions are to be based on quantitative estimates from fishery assessment models, it is desirable that the uncertainty be quantified, and used to calculate the probability of achieving the desired target and/or risk of incurring undesirable events. The process of communicating this risk to decision-makers is in its early developmental stages and presents substantial challenges to both fishery technicians and managers. In turn, fishery managers and participants must develop means of objectively evaluating the potential costs of undesirable events and define acceptable levels of risk and of risk and of short-term yield which can be foregone to reduce these risks.

References FAO, 2002. Reference point for fisheries management. Fisheries and Aquaculture Department, Rome, 90 p. Haddon, M. 2011. Modeling and Quantitative Methods in Fisheries. Second Edition, Taylor and Francis press, 449 p. Iran Fisheries Organization, 2014. Fisheries Yearbook. 63 p. Pauly, D. 1980. On the inter relationships between natural mortality, growth parameters and mean environmental temperature in 175 fish stocks. Journal Du Conseil International Pour L'Exploration De La Mer, 39(2): 175-192. Sparre, P. and Venema, S.C. 1992. Introduction to tropical fish stock assessment, part 1, manual, FAO Fisheries Technical Paper No. 306/1, Rev. 2, Rome, FAO, 433 p.

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The toxicity effect of Fe3O4, SiO2 and SnO2 nanoparticles on Daphnia magna in larviculture of sturgeon Fatemeh Shariati*, Zohreh Ramezanpour, Tahereh Pourdeljoo *Department of Environment, Lahijan Branch, Islamic Azad University, lahijan, Iran [email protected]

Introduction Progress in nanotechnology and comprehensive use of nanostructures in the industry has resulted in occurrence of nano-sized particles in industrial effluents. Zooplanktons as primary consumers of phytoplanktons in the food chain are severely at the risk of exposure and biological access to nanoparticles. In this study Daphnia magna was exposed to Fe3O4, SiO2 and SnO2 nanoparticles. SiO2 noparticles are used in different industries such as ceramic, glassware, cosmetic products, medicines, magnetic mixtures, heat and electric insulators and glazing and for increasing the strength and stability of industrial coverings. Fe3O4 nano particles show special and unique features compared to Fe3O4 particles in forms of ultra- magnetic, size and quantum feature. These nano particles are considered the most important elements of iron-based mixtures, soft magnetic materials, dyes, magnetic suspended materials, magnetic vibrant recording in tissues recuperation and molecules diagnosis are regularly used in pharmaceutical industries and biotechnology. Tin dioxide nanoparticles (SnO2) are used in fuel production and solar cells. Uncontrolled use of nanoparticles results in appearance of these materials in sewages where they may become toxic for living organisms in the receiving aquatic ecosystems. Daphnia magna is a small fresh crustacean with high population in freshwater basins and ponds.

Materials and Methods In the present experiment all steps and procedures were performed according to OECD (202) methods. A plastic tank was used for the cultivation of daphnia and the optimal experimental conditions e.g. pH = 7-8, temperature between 18- 24 ° C and 12 hours light and dark photoperiods, gentle aeration were maintained throughout the experimental period. According to this method, Daphnia magna neonates were tested 24h after hatching, and 10 daphnia neonates were placed in each beaker. Daphnia magna were kept in beaker during the experiment, and then the initial test (range detection) were performed to determine the original concentrations with 5 concentration treatments and three replicates at each concentration. Based on the range detection test results, logarithmic concentration of experimental nanoparticles were prepared in beakers using nanoparticles stock solutions. Ten neonates of 24-hour Daphnia magna were placed in each beaker and were exposed to various concentrations of experimental nanoparticles and by the end every 24 hours experimental period the number of mortalities was counted. Finally by considering the mortality rate compared to control and determining the Probit number from the table of Probit value, regression curve was plotted for each 24 hours and by using line equation obtained from the curve, the LC10, LC50, LC90 values were calculated for each 24 hours. The effects of zinc oxide, and titanium dioxide nanoparticles on D. magna has been tested and their toxicity been proven at different concentrations.

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Results and Discussion In the present study, the values of 96-hour LC50 for Daphnia magna exposed to SnO2, SiO2 and Fe3O4 were 2.15, 1.73 and 654. 65 mg L-1, respectively. Momeni et al. (2011) exposed Daphnia magna to zinc oxide and dye blue 29, and 24h and 48hLC50 were reported 2.6 and 1.9 mg l-1, respectively. Zhu et al in 2009 exposed Daphnia magna to zinc oxide and aluminum oxide and reported EC50 for daphnia mortality as 1.511 and 162.392 mg L-1 respectively. Table 1. Lethal concentrations (mg l-1)of experimental nanoparticles for Daphnia magna

24 Hours 48 Hours 96 Hours SnO2 SiO2 Fe3O4 SnO2 SiO2 Fe3O4 SnO2 SiO2 Fe3O4 - - LC10 2.87×10 0.89 258.7 10 0.14 103.4 10 0.07 15.38 -4 5 4×4.31 4 1×2.04

LC50 1.7 3704 4965. 4.3 48.8 977.2 2.1 1.73 654. 92 6 4 65

LC90 10458.5 122902 86099 1109.4 1628 9330. 2265.6 39.5 27039. 42 .3 5.8 39 0 58

In a study on the toxicity of titanium dioxide nanoparticle on Daphnia magna, the nanoparticle influenced the energy transfer in the food web. In another study conducted by Asghari et al, silver nanoparticle accumulation in Daphnia magna body hampered normal swimming activities of the organism. Daphnia magna, Artemia salina and Gammarids were exposed to the TiO2 nanoparticle. Nanoparticle were accumulated in colon and stomach and gradually spread to other body parts. Xiaoshan et al studied acute toxicity of aluminum oxide, titanium oxide, carbon 60 and zinc oxide on Daphnia magna and reported 48h LC50 for 0.622 mg L-1 for zinc oxide nanoparticle and 114.35 mg L-1 for aluminum oxide. LC50 levels were obtained 1.51 mg L-1 and 162.39 mg L-1, for zinc oxide and aluminum oxide, respectively. According to the conducted studies, the toxicity of different nanoparticles on Daphnia magna can be classified as: SiO2>SnO2; C60>ZnO> Fe3O4. Therefore, so far during the conducted research, titanium dioxide and tin dioxide have the lowest and carbon 60 (fullerene) has the highest toxicity rate on Daphnia magna. Daphnia magna is a live largely used as live feed for sturgeon larviculture in earthen ponds. Nanoparticles are regularly used in various industries as mentioned earlier. Most industrial plants unfortunately release their effluents into the rivers without any prior treatments to remove or dilute these particles. Some of these rivers are main water supplier for sturgeon rearing complexes (e.g. Sefid Rud River in Guilan- Iran). Increase in the use of nanoparticles results in increased levels of nanoparticles in effluents which enter the rivers serving as water supplier for sturgeon larvae and fingerling culture. Accumulation of toxic materials in Daphnia magna (main food for sturgeon fingerlings) subjected to these nanoparticles is a potential risk with harmful effects on sturgeon rehabilitation processes.

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References Asghari, S., A, Johari, and J. Hyun lee, 2012. Toxicity of Various Silver Nano Particles Compared to Silver, Ions in Daphnia magna. Journal of Nanobiotechnology. (10): 3155-10- 14 Compos, B., C. Rivetti, P., Rpzenkranz, J. Navas and C. Baratae, 2013. Effects of TiO2 on Food Depletion and Life History Responses of Daphnia magna. Journal of Aquatic Toxicology.15:130-131:174-83 Ebert, D., 2005. Ecology, Epidemiology and Evolution of Parasitism in Daphnia magna. National center for Biotechnology Information. ISBN-10: 1-932811-06-0 Fernandes, T., N. Christofi and V.Ston, 2007. The Environmental Implications of Nanomaterials. In: Monteiro-Riviere, N.A, Lang Tran, C. (eds) Nanotoxicology: characterization, dosing and health effects. Journal of Informa Healthcare, 405–418. Hyuck Bang, S., l., l. Thai-Hoang, S. Lee, P. Kim, J. Kim and Min, J. 2011. Toxicity Assessment of Titanium (IV) Oxide Nanoparticles Using Daphnia magna (water flea). Journal of Environmental Health and Toxicology, 1-5. Rosenkranaz, P. 2011. The Ecotoxicology of NanoParticles in Daphnnia magna, Edinburch Napier University Edinborgh, 18-141. Tao, X., Y. He, B. Zhang, Y. Chen and J. Ughes, 2011. Effects of Stable Aqueous Fullerene Nanocrystal (n60) on Daphnia magna. Evaluation Hop Frequency and Accumulation Different Condition, Journal Environmental Science (2): 233-329. Xiaoshan, Z., Z., Lin, Ch., Yongsheng and T. and Shengyan. 2009. Acute toxicities of six manufactured nanomaterial suspensions to Daphnia magna. Journal of nanoparticles reasearch. 67-75.

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The effect of Beluga (Huso huso) cages on water abiotic factors in the southern part of Caspian Sea (Jafroud region)

Zabih Ollah Pajand*, Korosh Haddadi Moghadam, Zohreh Ramzanpor, Fruzan Choubian, Esmaeil Farzaneh1 * International Sturgeon Research Institute (Areeo), P.O. Box: 41635-3464, Rasht, Iran *[email protected] Introduction The environmental impacts of aquaculture activities are one of the most important challenges facing developing countries. It needs a careful plan for monitoring and an environmental evaluation. Fish cage farms release large amounts of effluents including nutrients, waste food and feces and by-products such as medications and pesticides (Jahani et al., 2011). These effluents have impacted the local environment, depending on the amounts of released waste, the time-scale over which the releases take place, and the assimilation capacity and flushing ability of the recipient water body (Gowen and Bradbury, 1987; Ackefors and Enell, 1994; Wu, 1995; Axler et al., 1996; Kelly et al., 1996). According to the climatic, ecological and hydrological conditions in the Caspian Sea and according that the Caspian Sea is an enclosed inland body of water and habitat for many valuable aquatic species and endangered sturgeons, it is necessary that comprehensive studies are conducted before any development on fish culture in the sea. This project was financially supported by the Fisheries Organization of Guilan Province and was conducted in the International Sturgeon Research Institute in order to investigate abiotic factors for sturgeon cage culture in Jafroud region of Anzali Port (depth of 50 m) for one year.

Materials and Methods Physico-chemical factors were investigated monthly. The investigations were carried out in four stations in cage and one station outside the cage (20 m in the opposite direction of water flow in the west). The abiotic conditions for fish cage culture including physico-chemical factors (temperature, dissolved oxygen concentration, pH, salinity as well as nitrite, nitrate, orthophosphate, electric conductivity and total suspended solids (TSS) were measured monthly. Sediment grain size and total organic matter (TOM) were also measured using Holme and Mcintyre (1984) method.

Results Physico- chemical factors in rearing cage significantly did not affected (p<0.05) growth rate of Huso huso. Also results obtained from this study revealed that there were no significant differences between parameters inside and outside of the cage (p<0.05). However, significant effects of variations in TSS, Total Organic Matter (TOM) and grain sediments factors levels were observed inside and outside of the cage (p<0.01). The results of abiotic factors found around the stations are in the range of the allowable concentrations of theses parameters for culture. Considering the environmental findings, the results of this study provide useful information to manage well the fish culture based on the water quality and hydrological foreseen. The results from this study can enable managers to obtain necessary data to evaluate the rearing capacity of fish in marine cage to issue the permits for sturgeon culturists. The amounts of TOM showed no significant differences between studied stations, so we can`t relate this factor to the role of marine fish cages on sediment. No-significant differences were observed in physical-chemical parameter values among stations, which shows these factors didn't have any sever effect on our data, but significant

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In Cage Out Cage In Cage Out Cage 6 0/04 0/035 5 0/03 4

0/025

N (mg/l) N

- -

3 2 0/02 NO

TOM (%) TOM 0/015 2 0/01 1 0/005 0 0

Time Time In Cage Out Cage In Cage Out Cage 0/1 0/25 0/09 0/2

0/08

0/07

(mg/l) 0/15

0/06

N (mg/l) N

- -

0/05

3 3

0/04 4 0/1 NO 0/03 PO 0/02 0/05 0/01 0 0

Time Time Figure 6: Mean values of physical-chemical parameters in sampling stations in the Caspian Sea

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References Ackefors, H. and Enell, M. 1994. The release of nutrients and organic matter from aquaculture systems in Nordic countries. Journal Applied Ichthyology, 10(4), 225–241. Jahani N., Nabavi S. N. B., Dehghan Madiseh, S., Mortezaie, S. R. S. and Fazeli, N. 2011. The effect of marine fish cage culture on benthic communities using BOPA index in Ghazale Creek (Persian Gulf). Iranian Journal of Fisheries Sciences. 11(1) 78-88 Kelly, L. A., Stellwagen, J. and Bergheim, A. 1996. Waste loadings from a fresh-water Atlantic Salmon farm in Scotland. Water Reserch Bulletin, 32 (N5), 1017– 1025. Holme, N. A. and Mcintyre, A. D. 1984. Methods for study of marine benthos, second edition, Oxford Blackwell Scientific publication. 387p. Gowen, R. J. and Bradbury, N. B. 1987. The ecological impact of salmonid farming in coastal waters: a review. Oceanography Marine Biology Annual Review, 25, 563– 575. Wu, R. S. S., 1995. The environmental impact of marine fish culture: Towards a sustainable future. Marine Pollution Bulletin, 31, 159-166.

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The enrichment of Nereis diversicolor using of Bacillus and Lactobacillus probiotic in Acipenser baeri

Esmaiel Farzaneh, Hossein Khara*, Zabihollah Pajand3

*Department of Fisheries, Lahijan Branch, Islamic Azad University, Lahijan, Iran. [email protected]

Introduction Aquaculture industry despite significant growth often faces problems such as in fish and shrimp breeding disease because of reduce water quality and the creation of stress conditions. Most of these fish are farmed under the influence of bacterial infections that their immune system is compromised by conditions stressor conditions. Using of antibiotic is one of the most regular treatment methods for this. Antibiotics often used for bacterial diseases treatment. However, unsuitable usage of antibiotics leads to creating of resistant bacteria against to antibiotics in fish rearing (Nomoto, 2005). Antibiotics damaged bacterial equilibrium in gut destroyed most of useful bacteria and lead to bacteria resistance to drug and only 20 – 30 % of antibiotics digested by fish and others entered to environment. Usage of probiotics considered as an alternative. Using of probiotics is a new technology for aquaculture and environment that improved production ability and water quality and considered as a biological control (Salminen, 1999). Probiotics improve immune system (Taoka et al., 2006).This study was carried out to investigate the capability of enrichment of Nereis diversicolor with bacillus and lactobacillus probiotics to increase growth and survival rate of Acipenser baerii fingerlings.

Materials and Methods The experiment was conducted using Bacillus and Lactobacillus probiotics prepared by ZIST VARNA Company containing 1010 bacteria CFU/g, to determine the capability of Nereis diversicolor enrichment. In this study, Bacillus and Lactobacillus probiotics that are solvent in fat were used in concentration of 105, 106,107and 0 CFU/g and one gram of probiotic solved in 10ml sunflower oil and an even solvent obtained. Samples were collected for bacterial culture and all of these stages were performed in three replicates. In the second phase of project the fingerlings with mean weight and length of 12.5±0.4g and 14.5±0.3cm were fed by enriched Nereis diversicolor to increase growth and survival rate. Daily feeding of fingerlings and Nereis diversicolor was carried out in all treatments and control group

Results The results of the first experiment showed that the total bacteria increased, the most observed in treatment three and the least observed in control group and there are significant differences among treatments and control group (Fig. 1). The results of the second experimental phase (Fig. 2) showed, according to Duncan’s test, the total bacteria in Nereis diversicolor increased in treatment groups and the least total bacteria was observed in control group which showed significant differences between treatment and control group. Based on one way variance analysis, the total bacteria in the intestine of Acipenser baerii fingerlings enriched with probiotic showed significant differences in all treatments in MRS culture medium (P<0.05). According to Duncan’s test, the total bacteria of

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Fig1:The total of Bacillus and Lactobacillus probiotic in Fig2:The enrichment of Nereis diversicolor using Acipenser baerii and N. diversicolor of Bacillus and Lactobacillus probiotic diets in SGR factor in Acipenser baerii Conclusions In recent years probiotic application in aquaculture rapidly expanding. These compounds, which is completely in contrast to antibiotics microbial food supplement that has beneficial effects on the host by improving the intestinal microflora balance. There are several ways to use probiotics in aquaculture and certainly success in use them is directly associated with the best way. It seems the farming of larvae, especially if the purpose of the use of probiotics to improve the growth and nutrition conditions, the best way for delivery of probiotic bacteria to aquatic animal larvae is through alive food. The results of this study indicated that the bacteria Bacillus and Lactobacillus tested have good ability to connect to Nereies worm. The ability of this worm is related to the cell biology of Bacillus and Lactobacillus and in part to the nature of Nereies worm as a filter feeder. It's possible, therefore, that the Nereies worm plays as a probiotic carrier and deliver them to target larvae. This condition can be very useful for farmers. The results of this study could certainly pave the way for the private sector breeding and rearing sturgeon fish.

References Nomoto, K. 2005. Prevention of infections by probiotics. Journal of Bioscience and Bioengineering 100, 583–592. Taoka, Y., Maeda, H., Jo, J.Y., Jeon, M.J., Bai, S.C., Lee, W.J., Yuge, K. and Koshio, S. 2006. Growth, Stress tolerance and non-specific immune response of Japanese flounder Paralichthys oliraceus to probiotics in a closed recirculating system. Fisheries Science. 72 (2): 310-321. Salminen, S., Ouwehand, A., Benno, Y. and Lee, Y.K. 1999. Probiotics: how should they be defined. Trends Food Science Technology. 10: 107–110.

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An investigation on macrobenthic communities in Chabuksar Shore of the Caspian Sea and their importance in the nutrition of sturgeons

Shahpor Gholami *, Maryam Shapoori, Zabih ollah Pajand

* Dr. Yousefpour Propagation and Pisciculture Center, Siyahkal, Iran * Email: [email protected]

Introduction Seabed and aquatic ecosystems are the habitation of benthic organisms. The benthic organisms’ population combination has close relation with habitat condition (Tolonen et al., 2001). This organism lives on or in the seabed (Miller, 2004). The nutrition of sturgeon in the Caspian Sea is benthos. The reserves of sturgeon in the Azov and Caspian Sea are about the 90 percent of the international reserves (Agusa et al., 2004).

Materials and Methods For this study sampling process applied via ‘Van Veen Grap’ and three iterations through three depth, 3, 6 and 10 meters in three stations during four seasons of 2011 were carried out. In the laboratory, the samples are rinsed and then were passed through a sieve 0.5mm and, were fixed with 4% Formalin (Muniz and Pires, 2000( . Afterward, benthic organisms were observed via stereomicroscope and classified using Atlas Book of invertebrates of the Caspian Sea (Birshteina et al., 1968). Furthermore, one way Analysis of Variance (ANOVA) was used for the comparison of means. For statistical analysis of data and drawing the graphs, Excel and Spss17 programs were used.

Fig1. Area of study with indication of sampling station

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Table1. Latitude and longitude Sampled Stations Area Stations Depth (m) Longitude Latitude 1 3 50˚ 34′ 405″ 36˚ 59′ 427″ Chabuksar 2 6 50˚ 34′ 621″ 36˚ 59′ 336″ 3 10 50˚ 34′ 766″ 37˚ 09′ 759″

Results The identified macrobenthos were Hediste diversicolor, Hypania sp, Tubificoides sp, Cerastoder glaucum, Hypanis sp, Theodoxus pallasi, Petrocuma sp, Balanus sp and Pontogammarus sp. The analysis showed that the densest macrobenthos’ type is Polychaeta with a density of 42% and the lowest dense macrobenthos’ types is with a density of 2% (Fig2). These results pointed these findings out that the maximum average of density for macrobenthos was 2861±381 ind/m2 in summer and its minimum was 1014±205 ind/m2 in winter (Fig3). Furthermore, the maximum and minimum of annual density for macrobenthos were recorded in depths of 10m and 3m respectively.

Fig 2. Percentage of macrobenthos’ density in Chabuksar Shore

Fig 3. Seasonal density of macrobenthos in Chabuksar Shore

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Their density in every station and every season was calculated. One-way analysis of variance demonstrated that macrobenthos’ density in different stations and seasons were significantly different (P-value<0.05). The maximum average of density for macrobenthos was in summer and its minimum in winter

Conclusions Our findings are consistent with Sharbati et al. (2004) results. In this study, the most dense macrobenthos’ type is Polychaeta and the lowest dense macrobenthos’ types is Insects. Overall 11 species of benthic organisms were detected in the nutrition of sturgeon, and Hediste diversicolor and Pseudocumidae are of great importance in the nutrition of sturgeon (Birshteina et al., 1968; Holcik G., 1989).

References Agusa, T., Kunito, T., Tanabe, S., Pourkazemi, M. and Aubrey, D. 2004. Concentrations of trace elements in muscle of sturgeons in the Caspian Sea. Mar. Pollut. Bull., 49, PP: 789-800. Birshteina, Ya. A.,Vinogradova, L. G., Kondakova, N. N., Kun, M.S., Astahoboj , T.V. and Romanovoj , N.N. 1968. Atlas of invertebrates of the Caspian Sea. Moscow, (in Russian). 413p. Holcik, G., 1989. The fresh water fishes of Europe. Alula-vering wieshaden. Vol. 1, part 2, PP 414-417. Miller, C. B. 2004. Biological Oceanography. Library of Congress cataloging in-pubication data, 402P. Muniz P. and Pires A.M.S. 2000. Polychaeta association in a subtropical environment (Sao Sebastiao Channel, Brazil): A structural analysis. Marine Ecology, 21(2):145-160. Sharbati, S. Akrami, R. Yelghi, S. Mirdar, J. Zeid, A. 2013. Identification, abundance and biomass of benthic communities in south east Shores of the Caspian Sea (Golestan Province), 1(4): 23-32 Tolonen, K.T., Hamalainen H., Holopainen I.J. and Karjalainen J. 2001. Influences of habitattype and environmental variables on littoral macro invertebrate communities in a large lake system. Arch Hydrobiol, 152: 39–67.

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Study and evaluation of indices of Iranian sturgeon (Acipenser persicus) caught in the Caspian basin Bagher Aminian*, Seifollah Mohammadi, Ghasem Karimzadeh, Mohse Pourasadi, Nima Vahdati, Mohammad Jafari *Higher Education Center of Pardis Agriculture, Rasht, Iran Introduction Persian sturgeon (Acipenser persicus), is a species of high value mainly in the southern part of the Caspian Sea, distributed in Iran waters, Turkmenistan and Azerbaijan (Smith, 1999). There are currently active fishing stations, 45 to 18 (Gilan), 20 (Mazandaran) and 5 (Golestan) (Tavakoli, 1390). In order to study and evaluate some biological and environmental parameters and the impact on fishing sturgeon Acipenser persicus in the years 1394-1393 Probablity 2015-2016 in international cleaner, a survey in the south-eastern basin of the Caspian Sea province of Mazandaran was done.

Materials and Methods Traps for catching fish gill (GILL NETS) were used , 48 horse-power motor boat, and for the evaluation of biometry board with an accuracy of 1 mm, 1.0 g precision digital scales, as described by Blister, looped binocular, glycerin, chemical, biological evaluation forms, laboratory glassware, thermometer with an accuracy of 1°C, slides and plates. In order to analyze data and charts from Excel and SPSS 20 test Kolmogorov - Smirnov, and analysis of variance (ANOVA) was used. A total of 131 samples of fish were caught and the data were examined and statistical analysis was performed using SPSS software.

Results Results showed that the mean fork length sturgeon was 48.18 ± 145 cm and weight 55.9 ± 49.23 kg. The highest average in March, 16.151 and 26.140 and 83.140 were the lowest in the months of October and February, the weight 55.9 ± 49.23 kg with maximum and minimum 83.21 kg 23.25 kg, respectively. The length-weight relationship has been obtained for W = 0.017L2.55 and R2 = 0.761. The sturgeon population was in the age range of 28-11 years and had the highest share with 35 percent of the fish 18 have been allocated. Male population aged 20-8 years and most of the fish are accounted for 15-9 years with a 72/82 percent. Female to male sex ratio was approximately 89.0. The fish in stage 4 of maturity were with a frequency of 68.73 percent respectively. Overall conclusion illegal fishing of immature fish caught by fishermen unauthorized web procedures, especially professional and destruction of natural habitats, aquatic (marine ecosystems and rivers) has caused a sharp decline in sturgeon stocks. Which is significantly smaller than 3 (3b) and growth pattern test t test confirmed by a negative growth for this species is the allometric.

Conclusions More fish in stage 4 maturity with a frequency of 68.73 per cent to 68.23 and 63.2 respectively the second and third phase frequency, while the frequency of sexual maturity between 1 and 5 females caught there was zero visibility.

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The frequency of sexual maturation stages in sturgeon due to changes in temperature, the amount of available food and nutrition, physical, chemical, environmental, climate and water, population density, genetic differences between the populations, especially the selection of pressure from fishing and sturgeon fishing and even sex ratio of the population, from one point to another and varies from time to time. Although we can conclude that unauthorized fishing by fishermen immature sturgeon fishing practices, especially in view of Vazbyn unauthorized loss of natural habitat aquatic (marine ecosystem River) cause a sharp decline in sturgeon stocks is an effortless way to reduce the catches of sturgeon per year significantly compared to the previous year, as 8/10400 kg in 1388 was 2591 kg in 1393. As a result, if the trend continues and the lack of proper planning of resources and protection of this category of fish, in the not too distant years, we will be very bad of the current situation.

References Tavakoli, M., kindhearted, M., Hadadi Moghadam, K., Pajand, K. and Hallajian. A. 1387. Species composition of sturgeon fishing in the Caspian Sea (Iran waters, Russia, Kazakhstan and Turkmenistan) was evaluated in Zkhayrtabstan 84-1383, Journal of biological Sciences Branch unit, Issue Avl.17-9 page. Tavakoli, M., Prafkndh true, F. and B-heartedness, M. 1392. Assay sturgeon stocks in the Caspian Sea waters swept area method. Journal of Fisheries, Iranian Journal of Natural Resources. 66: 271- 283. Courage, CE, Prophet and humps, D. 1389. Determine the sex ratio of catch composition sturgeon caught in fishing and the exploitation of reserves Bzyan. Danshgah Mazndran. The first National Conference of Islamic Azad Abadan. Sfhat146-150. Yaei, D. 1384. Analysis of sturgeon caviar fish fins unions on the basis of gender segregation in the East and West coasts of Golestan province. 0.8 Fisheries Directorate General of Golestan province page. Razavi, P. 1371. Mandab marine environment of the Caspian Fisheries Research Center Press Gilan. Pages (8, 9, and 16). Biswas, S. P. 1993. Manual of methods in fish biology. South Asian publisher Pvt Ltd., New Delhi International Book Co. 157 p. Gulland, J. A. 1983. Fish stock assessment. A manual of basic methods. FAO / Wiley series on food and agriculture. PP: 223. Ivanov, V.P. 1999. Biological resources of the Caspian Sea. Astrakhan. Published in asp NIRKH. 100P.

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Qualitative and Quantitative survey of released sturgeon fingerlings in the Guilan province in 2015 Sayed Ali Mosavi*, Shahram Abdolmalaki, Mahmoud Tavakoli, Mohammad Reza Behrouz khoshghalb and Hashem Joshideh

* International Sturgeon Research Institute, Agricultural Research, Education and Extension Organization (AREEO), P.O.Box: 41635-3464, Rasht, Iran

*[email protected] Introduction The sturgeons of the Caspian Sea are considered highly (Chebanov and Billard, 2001). Given that the majority of Guilan Rivers have lost their support for natural reproduction of sturgeon due to various factors, sturgeon hatcheries and releasing fingerlings play an important role in the rehabilitation of the sturgeon stocks in the Caspian Sea. According to the decisions of the High Commission's exploitation on control of quantity and quality on the proliferation and release of sturgeon fry, identify strengths and weaknesses of this programmes is felt to release fingerlings and effective steps to improve the balance between production, harvesting and responsible management of sturgeon.

Material and Methods In June and July 2015 to investigate releasing fingerlings in two hatcheries Shahid Beheshti amd Late Dr. Yosefpour Siahkal were conducted. To count the fingerlings, three modules were counted randomly during the loading operation and the total number of fingerlings was calculated by multiplying the total number of modules loaded in the average number of juveniles in modules count. To determine the average weight of sturgeon fingerlings, 100 fingerlings were selected randomly and weight was measured using a digital scale with a precision of 1.0 grams and length was measured to the nearest 1.0. Information was recorded in the special forms and Excel 2007 version. Average, minimum, maximum and standard deviation of parameters such as length, weight and condition factor of juveniles released from each pool were calculated. After counting and recording the weight and length of fingerlings and the information gained from the release of sturgeon fingerlings and stocking time and the initial weight of larvae in stocking, growth rate and survival rate of fingerlings released was calculated (Wootton, 1998; Kroupi, 1995). Formulas for calculating growth rate and condition factor are: 2(W1W 2) G = 100 n(W1W 2) Where G= Growth rate , W1 = Weight of fish in the releasing time , W2 = Weight of fish at the beginning of rearing , n = Number of culture days

W 100 C = 3 L Where C = Condition factor, W = fish weight (gr), L = fish length (cm)

Results and Discussion Results showed that totally, over loading of 63 stages, a total of 2.5477 million sturgeon fingerlings of two hatcheries centers of and the Shahid Beheshti and Late Dr. Yosefpour Siahkal have been released in Sefidroud River in 2015. The highest percentage of released fingerlings belonged to Persian sturgeon fry by 79/8% and Stellate sturgeon with 20/2 % were

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir in second place. Statistics show that the number of juveniles released in 2015 compared to 2014, increased 42 % (753,960 fingerlings) that the release of these two hatcheries of Persian sturgeon by increasing 490 890 (32%) and stellate sturgeon by increasing 263 070 fingerlings (104%) were in the first and second stages. According to the reports of released fingerlings from 1996 to 2014 (Fig. 1), the lowest level for sturgeons released was in 2010 with 1, 042, 845 fingerlings (Fadaei et al., 2010) and the highest number of released fingerlings reported in 1998 with 8456183 fingerlings (Fadaei et al., 1998) (It is noteworthy that in 2013 releasing of fingerlings was conducted without the presence of experts of the International Sturgeon Research Institute) .

10 millions

2015 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Year Fig. 1: Releasing sturgeon fingerlings from the Shahid Beheshti amd Late Dr. Yosefpour Siahkal hatcheries based on restocking programme during 1991-2015

Despite all the problems associated with broodstock fish caught from the Sefidroud river, each year a number of experts for the purpose of catching, gathering and transporting of sturgeon brood fish were settled in the Sefidroud river. Unfortunately in recent years due to the wide spread of undesirable environmental factors and the destructive actions of humans, particularly environmental pollution and poaching and lack of protection and security for the migration of sturgeon caused to diminish the statistics number of sturgeon broods captured from the Sefidroud river sharply as only one male brood sturgeon from the river was caught in the years 2013 and 2014 (Fig. 2).

200

150 100

number 50 0 2001 2002 2003 2004 2005 2006 2007year 2008 2009 2010 2011 2012 2013 2014

Fig.2 : Number of sturgeon brood caught from Sefidroud river during 2001-2014

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According to the results of the fishery return rate project, survival of juveniles released in weights of over 3 grams are more than fingerlings released in below 3 g weights. Therefore it is recommended to obtain the final results and determine the appropriate weight, the minimum weight of 3 grams being determined for all of the hatcheries for releasing sturgeon fingerlings. Simultaneous release of sturgeon fingerlings into the Sefidroud river with spawning migration of bony fishes, particularly some small cyprinid fishes and the usage of small mesh size gill nets for catching these small fishes by illegal fishermen, caused the fry are caught inadvertently and they are killed. Despite numerous reports on this phenomena, the deployment of gill nets continue as before and it seems that strong determination among troops (Fisheries Guard of protecting aquatic resources and the environmental protection unit) stationed in the river and estuary on the prevention of this problem does not exist.

References Fadaei, B., ParanAvar, H., Joshideh, H., Imanpour Namin, J. and Behrouz Khoshghalb, M.R. 1998. Report of releasing sturgeon fingerlings from hatcheries and breeding centers of Shahid Beheshti and the late Dr. Yousefpoor in 1998. International Sturgeon Research Institute, Rasht.35 P. Fadaei, B., Joshideh, H., Tavakoli, M., Behrouz Khoshghalb, M.R. and AzadBakhsh, A. 2010. Report of releasing sturgeon fingerlings from hatcheries and breeding centers of Shahid Beheshti and the late Dr. Yousef poor in 2010. International Sturgeon Research Institute, Rasht.48 P. Kroupi, 1995. Report of educational course on Hydrobiology at Shahid Beheshti sturgeon Hatchery Complex Sangar Dam, Rasht. 59 P. WoottonRobert, J. 1998. Ecology of Teleost Fishes. Kluwer Academic Publishers, Dordrecht. 386 p.

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Conservation of Caspian south basin rivers for sustainable fish exploitation

Mahmoud Ramin. * , Mastooreh Doustdar.

Iranian Fisheries Science Research Institute, Agricultural Education and Extension Organization (AREEO), Tehran. Iran *[email protected] Introduction The Caspian south basin is one of important basins in Iran and the Aras river, Sefidroud river, Tonekabon river, Haraz river, Tajan river and Gorganroud river are the most important water resources in this basin. According to the results of a research project titled “Biodiversity of inland water fish species of Iran” which was carried out from 2005 to 2010, 47 species from 15 families were identified in Caspian south basin. Comparison of obtained data during the past ten years showed that unfortunately the exotic fishes increased. Due to illegal overfishing, pollutants, destroying of breeding and nursery grounds of fishes and dams the stocks of endemic and native species decreased and biodiversity of them were endangered. The migration of sturgeons to the Basin Rivers is very limited. Acipenser gueldenstaedtii, Acipenser nudiventris, Acipenser persicus, Acipenser stellatus, Huso huso, Luciobarbus brachycephalus and Salmo trutta caspius are critically endangered. At the same time Carassius auratus, Gamuosia holbrooki, Pseudorasbora parva, Hemiculter leucisculus increase their territories and occupy the habitat of endemic and native species. Therefore it is necessary to protect the rivers of Caspian south basin.

Materials and Methods Seasonal samplings were done in the main branches and some of the lateral branches in different rivers of Caspian south basin. Electrofishing was used as the main method for sampling . Specimens were preserved in 10% of formalin and transported to the laboratory for further biological measurements. Fishes were identified on morphologic and meristic characters.

Results and Discussion As results of this study, 47 fish species belonging to 15 families were identified and were recorded in the Caspian south basin. The rivers of this basin are habitat of endemic and native fish species. Most of species were endemic and native, but unfortunately the abundance of alien fishes increased, So for keeping the ecological balance it is necessary to protect the rivers of Caspian south basin.

References Berg, L.S. 1949. Freshwater fishes of the U.S.S.R and adjacent countries. Israel program for scientific translation jeruslem.Vol:1-3 Coad, W.B. 2016.The freshwater fishes of Iran, Available on www. Brian Coad.com.- Kottelat, M. and Freyhof, J. 2007. Handbook of European freshwater fishes. Publications Kottelat, Cornol and Freyhof, Berlin. 646 P. Winfield, I.G. and Nelson, J.S. 1991. Cyprinid fishes. Systematic, biology and exploitation. First edition. Chapman and Hall. 667P. Wootton R. J. 1990. Ecology of Teleost fishes, Chapman & Hall, USA, first edition. 404 P.

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The first report of Acipensiridae larvae in natural habitat of Coastal southeastern waters of Caspian Sea

Zohreh Mazaheri Kohanestani*, Rasoul Ghorbani1, Mahnaz Rabbaniha2, Kourosh Amini3 *PhD Student of Aquatic Ecology, Department of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources. Gorgan, Iran. [email protected]

Introduction Coastal and estuarine systems are highly productive areas that serve as nursery grounds for many marine species of commercial importance, widely distributed on the continental shelf (Peterson, 2003). During the past few years the natural population of the fishes has been rapidly declining due to various man-made and natural causes. According to IUCN (2015); among the fish species around the world, 47 are endangered, 35 have been critically endangered, 1 of them is extinct in the wild, and 4 are extinct. Moreover, the fish are also under threat due to drying up of the low lying areas and indiscriminate use of chemicals and pollutants (Aral et al., 2011). Therefore the urgent need was felt to ichthyoplankton surveys and fish larvae communities. This interest has increased noticeably during the last two decades as a tool to provide insights into the ecology and dynamics of marine zooplankton (Moser and Smith, 1993). These surveys have contributed to improving our knowledge of the ecological relations among communities based on the analysis of the species composition and its time and space variability (Koochaknejad et al., 2011). The study on life cycle, spatial and temporal distributions of larval stages of commercial fishes are important in marine biology and fishery management subjects. In addition, Ichthyoplankton surveys are conducted to increase knowledge of early life history inclusive growth, behavior, food requirement and other information. It helps to explore for new resource and locating spawning concentrations of important stocks (Rabbaniha and Owfi, 2011). Several ichthyoplankton surveys have been carried out and focused on identification, spatial and temporal dstribution of different fish larvae in Persian Golf and Oman Sea (Owfi and Mohammadnejad, 2001; Vosoughi et al., 2010; Rabbaniha and Owfi, 2011; Koochaknejad et al., 2011), However, There is no information on the early development of the Southern waters of Caspian Sea fishes. So it is necessary to undertake proper study to characterize its various stages of embryonic and larval development. Therefore, the aim of this study is to report observing Acipenseridae larvae in coastal southeastern waters of the Caspian Sea- Golestan province.

Materials and Methods 8 transects, three vertical in Gomeishan Wetland (A, B, and C), three verticals in Miankaleh Island (D, E, F) and two transects in Gorgan Bay (G, H) (including 18 stations across the coastal southeastern waters of Caspian Sea and 6 stations in Gorgan Bay), were chosen based on gradian of environmental factors such as depth, sediment type, dominant wind direction, and freshwater effluents enriching coastal waters. Field study was conducted between the 1th April and the 20th of September 2015. Fish larvae were collected using a 46 cm bongo net with a mesh size of 330 µm. A flowmeter was attached to the net to estimate the volume of filtered water. Tows were oblique (45◦) from depth to the surface, stopping 5 min, at a constant vessel speed of 2 knots; these stops permitted to focus on the depth of the water column, where larvae of coastal fish species are more abundant (Sabates et al., 2007). All

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir sampling took place during day light hours, once a month. The plankton samples were immediately fixed in a solution of 5% formalin in seawater buffered with borax. Water quality was measured at the same time. Turbidity (NTU), salinity (ppm), temprature (◦C), pH and chlorophyll a (ppm) were measured by water checker u-10 and spectrophotometer respectively. Egg and larvae were identified to the lowest taxonomic level possible using different identification keys (Makeyeva et al., 2011). Body length and egg radius were measured by a digital calliper.

Results The larvae and eggs were caugth from 3 stations in Gorgan Bay and 2 stations in coastal water in April and May 2015. Data on some water quality parameters are given in Table 1. Temperature, pH, Turbidity, Salinity and Chorophyll a varied between 14.7-15.7 ◦C, 7.85- 7.99, 49.9-670 NTU, 8.75-11.2 ppm, and 0.043-0.151 ppm in April. Salinity was higher in May and increased more than 18 ppm. Table 1. Water quality parameters in different stations Chlorophyll Station Temperature Turbidity Salinity Location Month pH a code (◦C) (NTU) (ppm) (ppm) G2 Bay April 15.4 7.85 49.9 10.9 0.043 G3 Bay April 15.7 7.87 69.9 11.2 0.044 H1 Bay April 15.7 7.87 54.4 11.2 0.046 E1 Sea May 12.5 7.86 185 18.21 0.018 D1 Sea April 14.7 7.99 670 8.75 0.151 Data were presented on mean±standard deviation.

A total number of 533 eggs and 10 larvae were caugth. Data are presented in Table 2. 55.3% of eggs were found in Gorgan Bay and 44.3% in coastal waters. Maximum number of eggs was observed in station G3 and E1, while the larvae were caugth only in station G2-Gorgan Bay. Average eggs radius was 1.7±0.22 mm, ranged between 1.1-2.14 mm. Eggs radius was lower in station D1, coastal waters. Average notochord length was 4.43±1.21 mm and it varied from 2.37 mm to 5.86 mm. The larvae have the yolk sac yet (Fig 1).

Table 2. Eggs and larvae in different stations Station Egg/ Location Month Number R/NL(mm) code Larvae G2 Bay April Larvae 10 4.43±1.21 G3 Bay April Egg 285 1.763±0.21 H1 Bay April Egg 10 1.7±0.13 E1 Sea May Egg 200 1.71±0.17 D1 Sea April Egg 38 1.58±0.28 R represented egg radius and NL represented notochord length.

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Fig 1. Egg and preflexion stage of Acipensiridae larvae

References Aral, F. 2011. Erdinc Sahınoz and Zafer DoguEmbryonic and Larval Development of Freshwater Fish, Recent Advances in Fish Farms, InTech press. 250p. IUCN. 2015. IUCN Red list of Threatened Fishes. http://www.iucnredlist.org. Koochaknejad, E., Savari, A., Dehghan-Madiseh, S., Eskandari, G. & Sakhaiee, N. 2011. Fish larvae assemblage in the northwestern coast of the Persian Gulf: Khure Mussa channel. Journal of the Persian Gulf. 2(3): 25-29. Moser, H.G. & Smith, P.E. 1993. Larval fish assemblages and oceanic boundaries (Introduction to Symposium-Advances in the Early Life History of Fishes). Science Marine of Bulletin. 53: 283-289. Owfi, F. & Mohammednejad, J. 2001. Ichthyoplankton abundance and diversity in Bushehr coastal waters-Zyarat creeks. Iran Fisheries Research Organization. 95p. Peterson, M. 2003. A conceptual view of environment-habitat production linkages in tidal river estuaries. Reviews in Fisheries Science. 11: 291-313. Rabbaniha, M. & Owfi, F. 2011. Changes in fish larvae indicators in Khark and Kharko coralline islands. Journal of the Persian Gulf. 2(4): 33-40. Sabates, A., Olivar, M.P., Salat, J., Palomera, I. & Alemany, F. 2007. Physical and biological processes controlling the distribution of fish larvae in the NW Mediterranean. Progress in Oceanography. 74: 355-376. Vosoughi, G.H., Fatemi, M.R., Rabbaniha, M., Jamili, S., Gharra, K. & Noorinejad, M. 2010. Distribution pattern of coral & non coralline fish larvae in Khark & Kharko (Persian Gulf). Iranian Journal of Fisheries Sciences. 9(1): 173-184.

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Biological control of algal bloom in sturgeon pond using rice straw powder Zohreh Ramezanpour*, Zahra Ebadi, Fruzan Chubian, Rodabeh Rufchaeii, Alireza Shenavar, Kurosh Haddadi Moghadam *Agricultural Research, Education and Extension Organization, International sturgeon research Institute, P. O. BOX. 41635-3464 - Rasht –Iran *[email protected] Introduction No Pond is ever totally free of algae, but in a balance environment, primary production will be on control. Biological control is an excellent option where algae problems exist. Green water and string algae should be controlled using repeated applications of an effective water treatment, such as: add plants, Fish feeding, Using Ultraviolet(UV), For string algae using garden Hose, Hand or net, making shad, Reduce nutrient loads, adding salt, Bareley straw,Calcium bentonite clay and Beneficial Bacteria. Adding the beneficial bacteria will allow the nutrients to be assimilated before they become available for more algae thereby starving out future algal growth.

Materials and Methods Five sturgeon fish pond selected for treatments, two of them as control pond and three ponds have been treated by rice straw powder. Each Pond area was 2 Hectare. Rice straws were air dried and Powder. 250 kg rice straw powder added to each pond. The various parameters analyzed where pH, dissolved Oxygen, Turbidity, temperature, Phosphate, Nitrate by standard method. Phyto and Zshoooplankton of all ponds have been sampled and counted. Water samples of ponds before and after treatment cultivated for Bacteria. Number of remained stock released in each pond estimated at the time releasing them to river and Caspian Sea.

Results and Discussion Range of water temperature was 24.4-29.0 .Cat the sampling time. pH was 7 to 8.9 in all ponds and no significant different show between treatment and control ponds. Dissolved oxygen change 4.8-8.1 mg.L-1 in treatment ponds and 4-13 mg.L-1in control ponds, also there were significant different between all ponds. Changes of chemical composition of water due to added straw powder were Nitrite 0.007- 0.016 mg.L-1, Ammonia (NH4+) 0.27-0.41 mg.L-1, phosphate ‘0.24-3.5 mg.L-1and Nitrate 0.007 -0.1 mg.L-1 but The results no indicated significant differences in the mean values of them. Number of bacterial colony content of water before added straw powder 330-6000 colony per ml and after that were 500- 1100 colony per ml in ponds. Type of bacteria was variable between 3 to 5 but after treatment were 4 types in ponds. Consequently no significant different have seen in number of sturgeon released in river.

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The role of the release on the sturgeon population in southeastern coast of the Caspian Sea (the Golestan province)

Gholamali Bandani*, Mahmoud Tavakoli , Amin Mirhashami , Mohammad Larijani * Inland Aquatics Stocks Research Centre-Gorgan (IASRC), P.O. Box: 139 Gorgan, Iran [email protected]

Introduction Sturgeons are the most important and valuable species in the Caspian Sea and only five among 27 species identified in the world live in this sea (Ellen et al., 2005). Statistics collected from the Caspian Sea over the past two decades indicated a catastrophic decline of sturgeons stocks. Several factors can be effective; the most important ones including, overfishing, pollution, loss of natural spawning. Sturgeon fries releasing from hatcheries in Iranian part of the coastal areas has an important role in sturgeon restoration.

Materials and Methods Seasonal sampling, in the middle of each month from autumn in 2002 until summer in 2004 was conducted in each area. There were 3 stations in each area in different depths of 2, 5 and 10 meter. To collect sturgeon samples, Nylon gillnet with mesh-size of 26, 33, 40 and 60 mm and Capron gillnet with mesh-size of 100 and 150 mm were used for 24 hours respectively. The length and height of each string was 30 m and 5.3 to 4 m. Biometry: Total length (± 1 mm) from tip of head to the end of the caudal fin with biometric board and weight (±1 gram) with a digital scale were measured (Grand court, 2005). The frequency percentage of the caught fishes was calculated from the formula p= (f*100) /N, where f is the frequency of fish and P is frequency percentage and N is the number of total caught fish (Nsft, 1374). Regarding to the length frequency, the number of classes determined by using the Storages formula K=1+3/22*logN, where K is the number class and N is the number of fish. (Biswas, 1993).

Results

18000 Catch 10000000 16000 released 9000000 14000 8000000 7000000 12000 6000000 10000 5000000 8000 4000000 6000

3000000 youngfishcatched

4000 2000000 fingerlingfishreleased 2000 1000000 0 0 80 81 82 83 84 85 86 87 years

Fig. 1: The relationship between fingerling released and young fish sturgeon caught from beach seine net

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60 53.44 50 37.78 40 30 20

10 4.37 4.34 PercentAbundance 0

TAZEABAD TURKEMAN MIYANGHALE KHAJENAFAS

Fig. 2: The percentage of sturgeon species in different regions from 2002 to 2004.

The results of sturgeon collected from Gillnet showed the highest frequency trend to the lowest as in the Tazehabad and the Turkmen, the Khajenafas sturgeon ground respectively. The Miyanghale region had the second frequency (fig.2).

Fig. 3: Sturgeon catches per unit effort in different seasons from 2002 to 2004 The changes of Catch per unit effort showed the highest (CPUE) in summer and the lowest in spring; and autumn has the second rank (fig3). 100 84.24 90 80 70 60 50 40 30 20 6.15 4.66 4.24 PercentAbundance 10 0.37 0

Huso huso Aci.persicus Aci.stellatus Aci.nudiventris Aci.gueldensaedtii species

Fig.4: The percentage of sturgeon species from 2002 to 2004. Iranian sturgeon with 84.4 percent has the highest frequency among five collected species and the lowest one (0.37%) related to beluga (Fig.4).

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Discussion The data collected from gillnets and beach seine confirm each other; in both of them the highest frequency was related to A.persicus, A.stellatus, A.nudiventris, A.gueldenstaedtii and H.huso respectively. In the west coast of the Caspian Sea of Gilan province during 2002-2004 in five locations and seven seasons, a total of 164 sturgeon fish was caught and beluga species was not caught in this area and highest abundant with 82.2 percent belonged to the A.persicus. Other species such as A.stellatus, A.nudiventris with 11, 3.4 and 5.2 percent had the next rank respectively. (Khosghalb et al.1385). Investigation in 2002-2003 showed that Iranian sturgeon had the highest frequency among the five sturgeon species (Tavakoli, 1383). Based on Ghorbani studies carried out in 2000 in southeastern coast of the Caspian Sea, among 261 investigated sturgeon fish, 7.5 percent of them belonged to A.persicus and 3.48 percent belonged to A.gueldenstaedtii. The results of the present study confirm the results of numerous other reports which emphasize the stock restoration impact on the sturgeon population structure. Species structure, length ranges also age of investigated specimens indicated that the coastal population of sturgeons in Northern provinces is as a result of released fries originated from sturgeon hatcheries and stock restoration activities.

References Tavakoli, M. and Moghim, M. 2002. Research Assessment Report was traveling to Russia and the Caspian Sea sturgeon stocks in the North Caspian Sea (summer course 2002). Koshgalb, M. 2005. Population Fluctuations of sturgeon in the Caspian Sea of Gilan province Aniceto coastal waters Sturgeon doctor away. Ghorbani, R. 2000. Investigation of morphological Morphological characteristics, age, growth, food particles and parasites juveniles of Persian sturgeon Biswas, S.P. 1993. Manual of methods in fish biology & ecology. Grandcourt, T.Z.Al Abdessalaam, F. Francis, A.T.Al Shamsi. 2005. Preliminary assessment of .barred Spanish mackerel, Scomberomorus commerson ـ the biology and fishery for the narrow

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Genetics

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The influence of gamma and UV irradiation on the motility of ship sturgeon Acipenser nudiventris sperm

Mohammd Hassanzadeh Saber*, Mohammad Pourkazemi, Sharouz Baradaran Noveiri, Gholam Reza Shahhosseini Genetics and Biotechnology Department, International Sturgeon Research Institute, Agricultural Research Education and Extension Organization (AREEO), Rasht, Iran

* Corresponding author: [email protected]

Introduction Gynogenesis is a developmental process facilitating the inheritance of maternal genetic material alone. Induction of gynogens involves two steps: (i) elimination of paternal chromosomes, i.e. activation of egg with irradiated homologous sperm, or intact or irradiated heterologous sperm and (ii) restoration of diploidy by heat shock, to retain the second polar body, or suppress the first mitotic cleavage (Pandian & Koteeswaran, 1998). Irradiation of gametes either inactivates or fractionates genomic DNA; UV-irradiation inactivates genomic DNA of sperm by the formation of pyrimidine dimers (Ijiri & Egami, 1980; Valcarcel et al., 1994) but gamma radiation partially (Chourrout & Quillet, 1982) or totally (Parsons & Thorgaard, 1985) destroys the DNA of the sperm. Exposure of fish gametes to gamma or UV radiation results in complete inactivation of chromosomes.

Materials and Methods Persian sturgeon sperm DNA damage as a heterologous sperm was studied to induce gynogenesis in ship sturgeon. Persian sturgeon sperm DNA damage was affected by gamma irradiation with different doses reaching a level of 450, 600, 750, 900 and 1050 Gy. Also, DNA damage of Siberian sturgeon sperm was performed using UV irradiation. In order to determine the best duration for irradiation, the milt was diluted (1:9) with immobilizing solution. Samples of diluted milt were placed for UV irradiation (UV lamp model UVG-54, 254 nm, made by UVP America) with constant dose 473 µw/cm2 and height of 12 cm for 0, 60, 90, 120, 180, and 210 sec. The motility of irradiated sperms and controls were examined under the light microscope and the motility curve was drawn.

Results Results of the present study showed that the best exposure duration to UV irradiation for sperm of Siberian sturgeon was 60 seconds with a constant dose 473 µw/cm2. Motility percent was 58%. The highest percentage motility using gamma irradiation was reached with 15% with a dose of 450 Gy.

Discussion Ionizing irradiation, such as gamma irridation has a high penetrance, inducing chromosome fragmentation. Therefore, a larger volume of sperm can be irradiated. UV light induces thymidine dimers, which may be repaired by photoreactivation (Iriji & Egami, 1980). The low penetrance of UV light permits the treatment of diluted (4 to 100 times), smaller volumes (2.5 ml) of sperm only, standing to a maximum height of 2 mm (Pandian & Koteeswaran, 1998).

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Ionizing radiation has the disadvantage that few laboratories are allowed to have radioactive sources outside special containment areas. Also, at lower doses, there is a substantial risk that small paternal DNA fragments persist and will be expressed in gynogenetic progeny (Chourrout and Quillet, 1982; Disney et al., 1987). In contrast to gamma-irradiation, UV-irradiation is cheap and simple to be set up. In most cases, germicidal bulbs emitting UV light at 254 nm are used. DNA inactivation is optimized by varying the distance between the lamp and the sample, and thus the intensity, and by varying the duration of irradiation. UV-254 nm has very low penetration power and only small, shallow samples of sperm can be irradiated (Palti et al., 1997). Larger volumes (up to 10 ml) can be irradiated provided that the sample is stirred and refrigerated during irradiation (Komen et al., 1988). The primary effect of UV on DNA is dimerization of adjacent pyrimidines (Friedberg, 1985), causing DNA–DNA and DNA– protein cross-linking. Other changes are the formation of hydrates of cytosines, and base-pair substitutions (Cieminis et al., 1987). The ultimate result is chromosome fragmentation, similar to gamma-irradiation. Sub-optimal UV irradiation of eggs or sperm also can produce chromosome fragments which persist in gynogenetic or androgenetic progeny (Komen & Thorgaard, 2007). However, if properly done, the yields of gynogenetic fry obtained from using UV-irradiated sperm to fertilize eggs are higher than those obtained with gamma-irradiated sperm (Foisil and Chourrout, 1992). For this reason and because of the problems relating to access to ionizing radiation sources, UV has become the favored method for irradiating sperm.

References Chou rout, D. and Quillet, E. 1982. Induced gynogenesis in the rainbow trout: sex and survival of progenies and production of all triploid populations. Theoretical and Applied Genetics 63: 201–205. Cieminis, K.G.K., Ranceliene, V.M., Prijalgauskiene, A.J., Tiunaitiene, N.V., Rudzianskaite, A.M. and Jancys, Z.J. 1987. Chromosome and DNA damage and their repair in higher plants irradiated with short-wave UV light. Mutation Research 181: 9–16. Disney, J.E., Johnson, K.R. and Thorgaard, G.H. 1987. Intergeneric gene transfer of six isozyme loci in rainbow trout by sperm chromosome fragmentation and gynogenesis. Journal of Experimental Zoology 244: 151–158. Foisil, L. and Chourrout, D. 1992. Chromosome doubling by pressure treatment for tetraploidy and mitotic gynogenesis in rainbow trout Oncorhynchus mykiss: re-examination and improvements. Aquaculture and Fisheries Management 23: 567–575. Ijiri, K. and Egami, N. 1980. Hertwig effect causes by UVirradiation of sperm of Oryzias latipes (Teleost) and its photoreactivation. Mutation Research 69: 241–248. Komen, J., Duynhouwer, J., Richter, C.J.J. and Huisman, E.A. 1988. Gynogenesis in common carp Cyprinus carpio L. I: effects of genetic manipulation of sexual products and incubation conditions of eggs. Aquaculture 69: 227–240.

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Komen, H. and Thorgaard, G.H. 2007. Androgenesis, gynogenesis and the production of clones in fishes: A review. Aquaculture 269: 150–173. Palti, Y., Li, J.J. and Thorgaard, G.H. 1997. Improved efficiency of heat and pressure shocks for producing gynogenetic rainbow trout. Progressive Fish Culturist 59: 1–13. Parsons, J.E. and Thorgaard, G.H. 1985. Production of androgenetic diploid rainbow trout. Journal of Heredity 76: 177–181. Pandian, T.J. and Koteeswaran, R. 1998. Ploidy induction and sex control in fish. Hydrobiologia 384: 167–243. Valcarcel, A., Guerrero, G. and Maggese, C. 1994. Hertwig effect caused by UV irradiation of sperm of the catfish Rhamdia sapo (Pisces, Pimelodidae) and its photoreactivation. Aquaculture 128: 21–28.

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Induction of triploidy in the hybrids obtained from ♀ ship sturgeon and ♂ Siberian sturgeon: Possibility of tetraploidy and its verification using microsatellite marker

Mohammad Hassanzadeh Saber*, Mohammad Pourkazemi, Mahtab Yarmohammadi

*Genetics and Biotechnology Department, International Sturgeon Research Institute, Agricultural Research Education and Extension Organization (AREEO), Rasht, Iran

*Corresponding author: [email protected]

Introduction

In the present study, induction of triploidy was carried out with hybrids between female ship sturgeon, Acipenser nudiventris and male Siberian sturgeon, Acipenser baerii, and heritability pattern of triploid hybrids with their parents was compared using microsatellite markers.

Materials and Methods For triploidy induction, eggs of ship sturgeon and sperm of Siberian sturgeon were mixed and shock-treated. The parents were obtained from the Propagation and Cultivation site of International Sturgeon Research Institute (Rasht-Iran). GnRh was used for sexual maturation in the parents. Cold shock duration was 30 min which started 10 min after fertilization and temperature of cold shock was 2.5°C. Afug9 microsatellite marker was used for verification of triploidy in the shock treated hybrids.

Results Allelic inheritance in the hybrids using Afug 9 microsatellite locus has shown that two alleles of one locus have been inherited from paternal breeder (212 and 197 bp) and two alleles have been inherited from female breeder. Analysis of parental genome indicated that the maternal locus has two alleles of 115 and 102 bp, but the paternal loci have two alleles of 205 and 193 bp and two other alleles of 141 and 133 bp. This microsatellite marker could verify the triploid offsprings because all offsprings have inherited both, the two alleles of maternal locus as well as the two alleles of paternal locus, but two specimens have inherited the paternal locus of 141 and 133 bp, and surviving specimens have inherited other locus of paternal origin. Therefore, all progenies of triploid treatment have four alleles that two alleles belonged to mother, and two other alleles were related to the father.

Discussion Obtained progenies from hybridization can result on gynogenesis, androgenesis, triploidy, and tetraploidy (Chevassus, 1983).This study shows that induction of triploidy in sturgeon hybrids may produce tetraploid offsprings.

References

Chevassus, B. 1983. Hybridization in fishes. Aquaculture 33: 245–262.

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Cross-species amplification of Acipenseridae microsatellite DNA markers in stellate sturgeon, Acipenser stellatus Mehrnoush Norouzi* *Department of Marine Biology and Fisheries Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran. [email protected] Introduction Stellate sturgeon, Acipenser stellatus is a valuable commercial fish in Caspian Sea and may be regarded as an indicator species for all sturgeons, featuring the tendencies of sturgeon stocks change and decline (Velikova et al., 2012). Since stellate sturgeon not specific primers, the objectives of the present study were to optimize the use of Acipenseridae primers for stellate sturgeon. Materials and Methods A total of 52 specimens of adult stellate sturgeon were sampled from Anzali coasts and samples were preserved in 95% ethanol. Genomic DNA was extracted from fin tissue. Fifteen pairs of microsatellites previously developed for Lake Sturgeon (Acipenser fulvescens) and shovelnose sturgeon Scaphirhynchus were tested for cross-species amplification on the stellate sturgeon. Genomic DNA was used as a template to amplify microsatellite loci by touchdown polymerase chain reaction (PCR). Totally 15 primer pairs were designed for Acipenser and Scaphirhynchus (LS-19, 34, 39, 54, 57, 62, 68, 69, May et al., 1997 ; Spl-104, 105, 113, 163, 168, 170, 173, McQuown et al., 2000). For all primer sets, amplification was performed in a reaction volume of 20 μL containing 0.2 mM of dNTPs, 0.5-1pμ each primer, 100 ng of template DNA; 1 units of Taq DNA polymerase and 1-2 mM MgCl2. Microsatellites were amplified using an Eppendorf thermocycler (Mastercycler ep gradient, 96 plus, Eppendorf, Germany). An initial denaturing step of 10 minutes at 95°C was followed by amplification for 25-35 cycles with the following conditions: 30 seconds at 95°C, 30–60 seconds at 49-61.2°C and 45–120 seconds at 70–72°C. A final 5-minute extension at 72°C completed the protocol. PCR products were electrophoresed on 6% polyacrylamide gels (29:1 acrylamide: bis-acrylamide; 1X TBE buffer) and followed by silver-staining. Gels were run at 170 W for 2h and 30 min (BIO-RAD sequi Gen, GT, 38 ×30 cm/ Power PAC 5000, USA). Alleles were sized using BioCapt software (http://biocapt.software.informer.com/), and each gel contained an allelic ladder (50bp) to assist in consistent scoring of alleles. Data analysis via codominant data was computed in the GenAlex 6 software (Peakall and Smouse, 2006) and Arlequin 3.5 (Excoffier and Lischer, 2010).

Results Ten sets of primers were successfully amplified and showed polymorphic pattern in the 52 individuals assayed (figure 1) and one set (LS-39) showed monomorphic pattern. 4 sets have not shown any flanking sites in stellate sturgeon genome. The average number of alleles found was 13.9 (± 1.2), and The number of alleles in LS-34, 9 (AR=11); LS-19, 17 (AR=16.25); LS-54, 12 (AR=14.25); LS-68, 11 (AR=13.5); Spl-105, 14 (AR=13.25); Spl-104, 11 (AR=13.75); Spl-163, 16 (AR=17.5); Spl-170, 17 (AR=21.5); Spl-173, 14 (AR=25.75), and Spl-113, 18 (AR=19.25). The effective number of alleles (Ne) per locus ranged from 3.48 to 10.46, with an average of 8.022 (± 0.1).

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A B Fig.1. Microsatellite banding profile of A. stellatus using primer pair LS-19 (A) and Spl-105 (B). The average number of Ho and He found was 0.624 (±0.9), and 0.863 (±1.1) respectively. The Ho (He) in LS-34, 0.442 (He =0.713); LS-19, 0.865 (He =0.900); LS-54, 0.615 (He =0.881); LS-68, 0.673 (He =0.862); Spl-105, 0.904 (He =0.885); Spl-104, 0.538 (He =0.841); Spl-163, 0.769 (He =0.898); Spl-170, 0.692 (He =0.895); Spl-173, 0.500 (He =0.886), and Spl-113, 0.500 (He =0.901). The Fis and Fit via frequency ranged from -0.020 to 0.367 and from 0.011 to 0.331, respectively (table 1).

Table 1. Fis and Fit for each locus using ten sets of microsatellite primers.

LS-19 LS-68 LS-34 LS-54 Spl-104 Spl-105 Spl-113 Spl-163 Spl-170 Spl-173 Average Fis -0.020 0.211 0.100 0.245 0.367 0.040 0.378 -0.008 0.299 0.302 0.1914 Fit 0.012 0.171 0.201 0.223 0.321 0.057 0.325 0.011 0.331 0.317 0.198

Conclusions In summary, the results of the present study showed that at least ten microsatellite primers could be used to investigate population genetics of stellate sturgeon in the Caspian Sea. Because the stellate sturgeon is a shared stock between 5 Caspian countries, it is highly recommended to develop a joint research project on this species, which would cover the entire Caspian Sea.

References Excoffier, L. and Lischer, H.E.L. 2010. Arlequin suite ver. 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resource, 10, 564–567. McQuown, E., Sloor, B. L., Sheehen, R. J. and May, B. 2000. Microsatellite analysis of genetic variation in sturgeon: new primer sequences for Scaphyrhinchus and Acipenser. American Fisheries Society, 129, 1380-1388. May, B., Charles, C., Krueger, C. and Kincaid, L. 1997. Genetic variation at Microsatellite loci in sturgeon primer sequence homology in Acipenser and Scaphirhenchus. Sciences, 54, 1542-1547. Peakall, R. and Smouse, P.E. 2006. GENALEX 6: genetic analysis in excel. Population genetic software for teaching and research. Molecular Ecology Notes, 6, 288-295. Velikova, V.N., Shaudanov, A.K., Gasimov, A., Korshenko, A., Abdoli, A., Morozov, B., Katunin, D. N., Mammadov, E., Bokova, E. B., Emadi, H., Annachariyeva, J., Isbekov, K., Akhundov, M., Milchakova, N., Muradov, O., Khodorevskaya, R., Shahifar, R., Shiganova, T., Zarbaliyeva, T. S., Mammadli, T., Velikova, V., Barale, V. and Kim Y. 2012. Review of the environment and bioresources in the Caspian Sea ecosystem 2000-2010. CaspEco Report, 423P.

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Isolation and Examination of HindIII SatDNA in Russian Sturgeon Acipenser gueldenstaedtii Mohammad Reza Nowruzfashkhami*, Bahram Kazemi, Maryam. Jahani, Leila Azizzadeh Pormehr, Mohammad Pourkazemi, Mohammad Hassanzadeh Saber, Mahtab Yarmohhamadi, Frouzandeh Mahjoubi

*Search instructor of Agriculture Research, Education & Extension Organization (AREEO), International Sturgeon Research Institute, Genetic and Biotechnology Department, Rasht, Iran *[email protected]

Introduction Satellite DNA (SatDNA) sequences are noncoding, short and tandemly repeated sequences located mainly at the heterochromatin region as centromeres and telomeres of the chromosomes (Charlesworth et al., 1994).The data on satelliteDNAs in fishes show that the sequences are mainly located in the centromeric region of chromosomes (Martins, 2007). This study was done to isolate of HindIIISatDNA from Genome of Russian sturgeon, Acipenser gueldenstaedtii, and sequence it for using it as a FISH probe on karyotype of Persian sturgeon Acipenser persicus in another study. Materials and Methods Twenty Acipenser gueldenstaedtii (Fig.1) specimens (2-3 - year old) with average length 7.60 ± 9.02 cm and average weight 578.94 ± 414.46 g were randomly separated and stocked in a 500 l fiberglass tank.

Fig. 1. Acipenser gueldenstaedtii Primers for Hind IIIS at DNA family amplification were designed on the SatDNA extracted from Acipenser gueldenstaedtii (AJ286592, De La Herran et al., 2001). Their sequences were: 5`-CTTTTTCAAACTTTCGGGGC-3`(Sat1F), 5`-CTTACAGATTCGTTCCTGTC-3` (Sat1R). After developing the required primers, genomic DNA was extracted from caudal fin tissue of A. gueldenstaedtii specimens using the phenol-chloroform method (Pourkazemi, 1996) to extract HindIIISatDNA, PCR amplification was performed. Amplification products were analyzed by electrophoresis on agarose gel and the monomeric band (168bp) extracted and purified by DNA extraction kit (Qiagen, Cat. No. 28704). The purified band was reamplified and cloned with acloning kit (Fermentas, Cat. No. 1213).

Results and Discusion Extracted HindIIISatDNA family from genome of A. gueldenstaedtii in this study contained: 168 base pairs (Fig. 2) with the following sequence registered in NCBI as FJ594465: 1 ctt acc gat tcg gtc ctg tca aga aaa cac att ttt ttg atc tca gaa ctc caa att tta 61 gct tcc cct ttt ttt gaa aaa ggg ggc tgg tcg ggt cct gac aaa aaa ata ata att ttg 121 gcc aat ttt att ttt tcg gaa ctt caa tgc ccc cag ctt ttg aaa aag

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168 bp

Fig. 2. Extracted HindIII SatDNA from A. gueldenstaedtii

The HindIII SatDNA family was isolated from the Acipenser naccarii (Garrido-Ramos et al., 1997) for the first time. Later it was extracted from the genome of some sturgeons as Acipenser baeri, Acipenser ruthenus, Acipenser transmontanus, Acipenser naccarii, Acipenser fulvescens, Acipenser gueldenstaedtii, Acipenser brevirostrum and H. Huso. The sequence of the HindIIISatDNA extracted in this study showed %88 similarity with that previously isolated from the Russian sturgeon by De La Herran et al., 2001.On the other hand they were common in151 bp and the difference between them was calculated as %12 or in 15 bp.

References Charlesworth, B., Sniegowski, P. and Stephan, W. 1994. The evolutionary dynamics of repetitive DNA in eukaryotes, Nature, 371: 215-220. De La Herran, R., Fontana, F., Lanfredi, M., Congiu, L., Leis, M., Rossi, R., Ruiz Rejon, C., Ruiz Rejon, M. and Garrido-Ramos, M. A. 2001. Slow rates of evolution and sequence homogenization in an ancient satellite DNA family of sturgeons, Journal of Molecular Biology Evolution, 18: 432-436. Garrido-Ramos, M. A., Soriguer, M. C. and De la Herran, R. 1997. Morphometric and genetic analysis as proof of the existence of two sturgeon species in the Guadalquivir River, Marine Biology, 129: 33- 39. Martins, C. 2007. Chromosomes and repetitive DNAs: a contribution to the knowledge of fish genome. In: Pisano E, Ozouf-Costaz C, Foresti F, Kapoor BG (eds) Fish Cytogenetics, Science Publisher, Inc., Enfield, pp 421– 453. Pourkazemi, M., Skibinski, D. O. F and Beardmore, J. A. 2000. A Preliminary Study on Phylogenetic Relationship between Five Sturgeon Species in the Iranian Coastline of the Caspian Sea, Iranian Journal of Fisheries Sciences, 2 (1): 1-12.

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Population identification of Acipenser persicus in two main rivers of the South Caspian Sea, using microsatellite markers Fereidon Chakmehdouz Ghasemi, Mohammad Pourkazemi, Mahtab Yarmohammadi  Iranian Fisheries Science Research Institute, Inland Water Aquaculture Research Institute, Agricultural Research Education and Extension Organization (AREEO), Bandae Anzali, Iran [email protected]

Introduction Recently, sturgeon populations in the Caspian Sea are endangered due to various factors, such as over-exploitation, habitat alternation, pollutions, barriers to migration and loss of spawning habitat (Pourkazemi, 2006). The objective of this study was to analyze the population genetic structure of the Persian sturgeon (Acipenser persicus) in Sefidrud and Gorganrud rivers watershed based on the characterization of microsatellite markers.

Materials and Methods 100 samples of Persian sturgeon were collected from two regions. Four microsatellite loci (Ls68, Spl168, Spl173 and Afu68) were analyzed for the molecular characterization of this species which resulted in polymorphic patterns. A total of 109 alleles were observed of which the maximum number of alleles (17) were found at the Spl168 locus which belonged to sturgeons from Sefidrud River watershed and the minimum number of alleles (10) in Ls68 locus belonging to the sturgeons from the Gorganrud River watershed.

Results Results of microsatellite analysis revealed that the differences between samples from the two regions were not statistically significant (p>0.05), neither for the average number of alleles per locus nor for the observed heterozygosities. The calculated Fst and Rst between two regions was 0.07 and 0.17 showing that the genetic difference was significant (p< 0.01). Samples from the Sefidrood River watershed in Spl 173, Afu68 and Spl168 loci and samples of other regions in Afu68 and Spl168 loci followed the Hardy-Weinberg equation. The genetic distance was calculated as 0.4 which represents a significant genetic difference between samples of two studied areas.

Sampling regions map: Gorganrud watershed () and Sefidrud watershed (●)

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Variebility of microsatellite loci in two areas of the Persian sturgeon (A, number of alleles; 2 Ho, Observed heterozygosity; He, Expected hetrozygosity; P, P-values of X tests for Hardy- Weinberg equilibrium).

Locus Parameters Sefidrud Gorganrud watershed watershed A 14 10 Ls68 Ho 0.670 0.520 He 0.840 0.850 P 0.00 0.000 A 17 15 Spl168 Ho 1 0.950 He 0.840 0.840 P 0.095 0.363 A 16 14 Spl173 Ho 0.708 0.710 He 0.880 0.860 P 0.1 0.008 A 11 12 Afu68 Ho 0.250 0.500 He 0.780 0.640 P 0.113 0.285 Average number of alleles per locus 14.5 ± 2.64 12.75 ± 2.22 Average Ho 0.66 ± 0.31 0.67 ± 0.21 Average He 0.83 ± 0.04 0.80 ± 0.10

Conclusions The data generated in this study showed that populations of Acipenser persicus in the two studied areas of river systems of the southern part of the Caspian Sea are genetically differentiated.

References Pourkazemi, M. 2006. Caspian Sea sturgeon conservation and fisheries: Past, Present and Future. Journal of Applied Ichthyology, 22, 12-16.

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Transcription survey of P450 in response to Endosulfan in the gill of Persian Sturgeon (Acipenser persicus)

Roghieh Safari* and Abasali Hajibeigloo

*Gorgan university of Agricultural Science and Natural Resources ([email protected])

Introduction Organochlorines are considered to be the most hazardous chemical pesticides with respect to environmental pollution, since they are very persistent, non-biodegradable and capable of bio- magnification as they moved up in the food chain (Dong et al., 2013). Endosulfan (6,7,8,9,10,10-Hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepine-3- oxide), an organochlorine, is being widely used in agriculture as well as in integrated agriculture-aquaculture farming system to protect important food crops (Piazza et al., 2015). Even exposure of fish to low concentration of this pesticide may accumulate in the body that can result in several toxic effects including tissue damage, physiological, biochemical and molecular alterations, respiratory changes, and ultimately death which could be used as environmental bio indicators (Piazza et al., 2015). The intensity and duration of these responses are influenced by several factors, including the concentration of the toxicant, the duration of exposure, and the fish species (Piazza et al., 2015). Changes in molecular and biochemical level are usually the first detectable responses to environmental perturbation which can provide information on the sublethal cellular effects of stressors in a particular species of interest, and have the potential to be applied as sensitive biomarkers in field studies to monitor fish health (Kim et al., 2009). Nowadays, changes of the amount of mRNA - P4501A induced by xenobiotics could be considered as a biomarker indicating the pollution of aquatic environment (Dong et al., 2013). Persian sturgeon (Acipenser persicus) is one of the most economically important fishes in the Caspian Sea which its stock have declined dramatically mainly due to over fishing, illegal catch, pollution and deterioration of habitats and natural spawning grounds (Pourkazemi et al., 2000). To the best of our knowledge there is no information about the comparison study of P450 in Acipenser persicus in exposure to endosulfan. Thus, the present study was performed to investigate the sublethal effects of endosulfan exposure in the gill of A. persicus on expression of gene related to stress.

Materials and Methods Persian sturgeon (3-5 g) fingerlings were obtained from Shahid Marjani Breeding and Rearing Center (Golestan, Iran) and acclimated to the experimental condition for a period of 15 days, after which fish were randomly distributed into 9 tanks of 300 L at a density of 30 fish per tank and submitted to sublethal concentrations of endosulfan 35%, using a mixture of α- and β- isomers (70:30), 10 and 40 µgl-1 for 14 days, based in the LC 50 of 80 µgl-1 reported by Safari et al. (2015). Nine fish per treatment were taken at days 0, 4, 7 and 14, rapidly anesthetized with clove powder, the gills were taken from samples immediately deep-frozen in liquid nitrogen and stored at -80°C freezer until starting experiment. Total RNA isolation was done following BIOZOL Reagent protocol (Bioflux-Bioer, China). 1μg of total RNA was used to synthesize first- strand cDNA using a Fermentase cDNA Synthesis kit for RT-PCR. The qPCR were done using A. persicus P450 1A primer.

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Relative gene expression was calculated by Pfaffl formula. The ratio between the target (P4501A) and housekeeping (β-actin) genes was analyzed by the REST software. Statistics data were subjected to one way ANOVA with α=0.05.

Results and Discussion Alterations in P450 gene expression in response to environmental contaminants were observed in variety of fish species for evaluating the extent to which an organism is stressed (Miao et al., 2014). In the present study the transcriptional expression of P450 1 showed a clear time-dependent response in the gill after the fish were exposed to endosulfan (Table 1). Although the relative mRNA- P450 level revealed an increase in all the study days compared to control, higher up- regulation was observed on the first day. The highest expression on the first day may be attributed to the important role of this enzyme in metabolism of xenobiotics. It has been reported that induction of P450 1A is mediated by the Ah receptor and the toxicity of a pollutant is related to the degree of its affinity to AhR and pollutants with a high binding ability for AhR also have a high capacity to induce P450 ( Billard et al., 2002). P450 may also be one of the early genes that activate late response genes which can be considered as another reason for higher up- regulation on the first day. The observed decrease on day 7 may be attributed to the activation of antioxidant enzyme to detoxify ROS and the reduction on day 14 may be due to gene silencing arisen from necrosis and histopathological damages. In present study Ap P450 1A mRNA expression increased in higher concentration and followed a concentration-dependent manner. Studies confirmed that the moment, intensity and level of P450 expression was closely related to xenobiotic, exposure time, concentrations, species and the study tissue as well (Huang et al., 2014). In conclusion, based on overall results obtained in this study, up- and down- regulation of gene may be the first sign of stress that may subsequently be reflected in biochemical and histopathological indicators of toxicity. Table 1. Alteration in the Relative mRNA levels to β-actin activity in the gill of Persian sturgeon exposed to 10 and 40 µgl-1 of endosulfan for 14 days. Exposure day/ 1 4 7 14 Concentration -1 a ab b C 10 µgl 6.2±1 A 5±0.31 B 4.5±0.4 B 1.5±0.12 B -1 a ab b c 40 µgl 8.7±0.9 A 7.5±0.5 A 6.3±0.5 A 2.8±0.4 A

Results are expressed as means with standard deviation (n=9). Different superscript and subscript letters denote significant (P<0.05) difference in each column (A-B) and each row (a-b), respectively. References Billard, S.M., Hahn, M.E., Franks, D.G., Peterson, R.E., Boles, N.C. and Hodson, P.W. 2002. Binding of polycyclic aromatic hydrocarbons (PHAs) to teleost arylhydrocarbon receptors (AHRs). Comp Biochem Phys B.133:55-68. Dong, M., Zhu, L., Shao, B., Zhu, Sh., Wang, J., Xie, H., Wang, J. and Wang, F. 2013. The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferase in zebrafish (Danio rerio) livers. Ecotox Environ Safe. 92: 1-9. Huang, G. Y., Ying, G. G., Liang, Y. Q., Liu, S.S. and Liu, U.S. 2014. Expression patterns of metallothionein, cytochrome P4501A and vitellogenin genesin western mosquitofish (Gambusia affinis) in response to heavy metals. Ecotox Environ Safe.105: 97–102.

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Kim, J.H., Raisuddin, S., Ki, J.S., Lee, J. S. and Han, K. N. 2009. Molecular cloning and b- naphthoflavone-induced expression of a cytochrome P450 1A (CYP1A) gene from an anadromous river pufferfish (Takifugu obscures). Mar Pollut Bull. 57:433–440. Miao, J., Cai, Y. and Pan, L. and Li, Z. 2014. Molecular cloning and characterization of MXR- related glycoprotein cDNA in scallop Chlamys farreri: transcriptional response to benzo (a) pyrene, tetrabromobisphenol A and endosulfan. Ecotoxicol Environ Safe. 110:136-142. Piazza, Y., Pandolfi, M., Da cuna, R., Genovese, G. and Nostro, F. 2015. Endosulfan affects GnRH cells in sexually differentiated juveniles of the perciform Cichlasoma dimerus. Exotoxicol Environ Saf. 116: 150-159. Pourkazemi, M. and Skibinski, D. O. F. and Beardmore, J. A. 2000. A preliminary study on phylogenetic relationship between five sturgeon species in Iranian coastline of the Caspian Sea. IJFS. 2(1): 1-12.

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Possibility of hybridization in Stellate sturgeon (Acipenser stellatus ♀) × Siberian sturgeon (Acipenser baeri ♂) using cryopreserved sperm

Bahram Falahatkar *, Samaneh Poursaeid, Bahman Meknatkhah, Iraj Efatpanah, Mehdi Rahmati

*Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, 1144, Guilan, Iran *Department of Marine Sciences, the Caspian Sea Basin Research Center, University of Guilan, Rasht, Guilan, Iran [email protected]

Introduction Sturgeons are ancient fishes which are distributed exclusively in the northern hemisphere. Several serious risks such as overfishing, pouching and habitat distraction jeopardize all sturgeon species mainly in the Caspian Sea. Depletion of sturgeon stocks in the native habitats coupled with the strong demand of international markets for meat have led to the introduction of sturgeon species into aquaculture. For commercial sturgeon aquaculture, methods are needed for maturation and spawning fish held in captivity. Both male and female are usually induced to ovulate using hormonal treatments. However, both sexes exhibit various forms of reproductive dysfunction in culture. Mostly, fish fail to complete either spermiation or final oocyte maturation in the same time. Hence, hybridization is a common practice in sturgeon aquaculture (Urbanyi et al., 2004). Rearing of hybrids has also several advantages for farmers including faster growth to a more marketable size and sterility in case of some hybrids (Chebanov and Billard, 2001). The hybridization of many sturgeon species including great sturgeon (Huso huso), Siberian sturgeon (Acipenser baerii), Russian sturgeon (Acipenser gueldenstaedtii) and green sturgeon (Acipenser medirostris) has already been reported (Kolman et al., 1997; Jähnichen et al., 1999). Another way for synchronization of gamete availability of both sexes is sperm cryopreservation. Although cryopreservation methods of sperm have been developed for different sturgeon species (Glogowski et al., 2002), most methods require several solvents and developed tools so that are not practical in fish farming. Therefore, the aim of this study was to assess the fertilization of stellate sturgeon oocytes with frozen-thawed semen (using a simple way) obtained from Siberian sturgeon.

Materials and Methods Four seven-year-old Siberian sturgeon males with an average weight of 5.5 kg were single injected with carp pituitary extract (1.75 mg kg-1). Semen was collected 39h after injection. Sperm quality was evaluated in terms of motility and duration of motility. After collection, the percentage of motile sperm was more than 85%. Then, the collected sperm was stored in a small beaker without adding any extender and cryoprotectant at 0°C and used a week after freezing. After thawing at room temperature, the percentage of motile spermatozoa found was 1%. On the same day, eggs were obtained from one six-year-old female stellate sturgeon with an average weight of 5.3 kg, after stimulation with two dosages of carp pituitary extract. The fish received the hormone in a priming (25% of total volume) and resolving (75% of total volume) doses at a 12-h interval (Chebanov and Galich, 2011). Eggs were collected by microcaesarean method (Pourasadi et al., 2008) 18h after second injection and fertilized with thawed sperm of Siberian sturgeon. Eggs were fertilized with semi-dry fertilization method (Azari Takami, 2009). Adhesiveness of eggs was eliminated with 10% clay solution. Fertilized eggs were then incubated in Veis incubator at mean temperature of 19.4 ± 0.6ºC.

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Duration of incubation was 116.2 degree-day. The hatching rate was 1% and only 10% of hatched larvae reached exogenous feeding.

Conclusions Although the fertilizing capacity of frozen-thawed semen obtained from Siberian sturgeon was very low, this study showed that the sperm is able to be alive at 0°C and fertilize eggs but in very little capacity for fertilization. In case of sperm cryopreservation, many studies showed that Tris-sucrose-KCl and Tris-NaCl are useful extenders and methanol is a useful cryoprotectant for cryopreservation of sturgeon semen (Glogowski et al., 2002). The present method in our study permits to produce a few embryos and larvae when the fish farmers do not access to either specific cryoprotectant or liquid nitrogen for semen freezing in field condition.

References Azari Takami, G. 2009. Breeding and cultivation of sturgeon caviar fish. Second edition, Tehran University Press, Tehran, 401p. Chebanov, M., Billard, R. 2001. The culture of sturgeons in Russia: production of juveniles for stocking and meat consumption. Aquatic Living Resources 14, 375-381. Chebanov, M.S., Galich, E.V. 2011. Sturgeon Hatchery Manual. FAO Fisheries and Aquaculture Technical Paper 558, FAO, Ankara, Turkey. Glogowski, J., Kolman, R., Szczepkowski, M., Horvath, Á., Urbanyi, B., Sieczynski, P., Rzemieniecki, A., Domagala, J., Demianowicz, W., Kowalski, R. and Ciereszko, A. 2002. Fertilising rate of Siberian sturgeon (Acipenser baerii, Brandt) milt cryopreserved with methanol. Aquaculture 211, 367-373. Jähnichen, H., Kohlmann K., Rennert, B. 1999. Juvenile growth of Acipenser ruthenus and 4 different sturgeon hybrids. Journal of Applied Ichthyology 15, 248-249. Kolman, R., Krylova, V.D., Szczepkowska, B. and Szczepkowski, M. 1999. Meristic studies of Siberian sturgeon (Acipenser baerii Brandt) and its crosses with green sturgeon (Acipenser medirostris Ayres). Czech Journal of Animal Science 44, 97-101. Pourasadi, M., Falahatkar, B. and Azari Takami, G. 2009. Minimally invasive surgical technique for removal of ovulated eggs from Persian sturgeon, Acipenser persicus. Aquaculture International 17, 317-321. Urbany, B., Horvath, A. and Kovacs, B. 2004. Successful hybridization of Acipenser species using cryopreserved sperm. Aquaculture International 12, 47-56.

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Genetic Diversity Evaluation of Great Sturgeon (Huso huso) reared in the Shahid Dr. Beheshti Restoration Center Mahtab Yarmohammadi*, Samad Darvishi, Rezvanollah Kazemi, Mohammad Ali Yazdani Sadati, Hosein Mohammadi Parashkooh, Yalda Baniesmaili *International Sturgeon Research Institute, AREEO, Rasht, Iran Introduction Due to drastic decline in the wild sturgeon broodstocks, hatchery broodstocks have been used as a tool in fisheries management as prime sources of population replenishment, supplement existing populations, or introduce new species. An important component of hatcheries is broodstocks management and this is obligatory in order to minimize inbreeding and maintain genetic diversity. Monitoring of genetic diversity over successive generations of broodstocks can provide important information about the efficiency of hatchery mating and management practices. Great sturgeon Huso huso populations along eastern and other parts of southern Caspian Sea are genetically differentiated (Pourkazemi, 2006). But now days their population has drastically declined. Dr. Beheshti Restoration Center currently maintains a rearing broodstocks for Great sturgeon resulting from wild – caught adult sturgeon in the Caspian Sea. Offspring resulting from captive broodstock propagation would be incorporate into stocking or breeding programs. The aim of present study is evaluating the genetic diversity among reared brood stocks of great sturgeon. Information including genetic variation (heterozigosity), relatedness (proportion of shared alleles), and genetic distance were used to asses each fish.

Materials and Methods For this purpose a total of 100 fin tissue samples were taken from reared H. huso. Genomic DNA was extracted using the ammonium acetate (Pourkazemi, 2006). Isolated DNA was resuspended in 100 µl of Ultra pure water. Microsatellite analysis was performed using 10 markers described in Pourkazemi et al., 2006 and May et al., 1997. Markers were chosen based on numbers of alleles and polymorphism. Each PCR reaction consisted Each PCR reaction (final volume 25µl) was composed of 5µl of 10X reaction buffer, dNTPs 10mM, MgCl2 50mM, primer 20pmol, genomic DNA 100ng and 1.5-2 unit of Taq polymerase. The cycling conditions were: initial denaturation in 95°C for five to ten minutes, then 94°C for 30 seconds (denaturation), 60°C for 30 seconds (annealing) and 72°C for 30 seconds to 3 minutes (extension) for 35 Cycles, followed by 72°C for 3-10 minutes. PCR products then were run on 6% polyacrylamid gel and bands were scored after visualizing fragment with silver staining (Pourkazemi, 1996). Data analysis was carried out using GENEPOP (Raymond and Russet, 1995). Pair wise comparison of Fst values (difference in allele frequencies) were calculated using FSTAT. Pair-wise genetic distances between individuals were calculated PHYLIP software.

Results A total of 100 individual great sturgeons were genotyped using 10 microsatellite loci. Most loci showed a maximum of 2 alleles per subject (diploids). Up to 3 alleles loci were not included in the analyses. The reared population of H. huso had an average of 14 alleles per locus for 10 loci. The results showed all the great sturgeon individuals assessed in the present research had unique genotype. Genetic identification was prepared based on morphological characterization and genotyping data.

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References Pourkazemi, M. 1996. Molecular and biochemical genetic analysis of sturgeon stocks from the South Caspian Sea. Ph.D. Thesis. University of Wales, Swansea. 260P. Pourkazemi, M., Skibinski, D.O.F. and Beardmore, J.A. 2000. A preliminary study on phylogenetic relationship between five sturgeon species in Iranian coastline of the Caspian Sea. Iranian Journal of Fisheries Science, 2(1):1-12. Pourkazemi, M. 2006. Caspian Sea sturgeon conservation and fisheries: past present and future. Journal of Applied Ichthyology 22, 12-16. Edwards, A.W.F. and Cavalli-Sforza, L.L. 1964. Reconstruction of evolutionary trees In phenetic and phylogenetic classification. (eds. V.H. Heywood and J. McNeill) Systematic Association, London, UK. Vol. 6, pp.67-76. May, B., Krueger, C.C. and Kincaid, H.L. 1997. Genetic variation at microsatellite loci in sturgeon: Primer sequence homology in Acipenser and Scaphyrhinchus. Canadian Journal of Fisheries Aquatic Science, 54:1542-1547.

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Molecular Identification and expression profile of the sex-candidate genes during gonad developmental stages in great sturgeon Huso huso

Mahtab Yarmohammadi *, Mohammad Pourkazemi , Rezvanollah Kazemi ,Mohammad Ali Yazdani Sadati , Mohammad Hassanzadeh Saber , Ali Hallajian , Ayoub Yousefi Jourdehi

* International Sturgeon Research Institute, Agricultural Research Education and Extension organization (AREEO), 41635-3464, Rasht, Iran

Introduction Sturgeon species are well known as the source of black caviar (Pourkazemi, 2006). In recent years, in order to minimize the impact on the wild population, sturgeon aquaculture has been developed. The mechanism underlying sex determination in sturgeons is largely unknown. To date all efforts have been made to discover sex determination genes in sturgeons were unsuccessful (Wuertz et al. 200; Keyvanshokooh et al. 2007; McCormick et al., 2008; Yarmohammadi et al., 2011). In non-mammalian vertebrates there are some sex- candidate genes which are involved in ovarian and testicular development. In order to understand the mechanism underlying gonadal development in sturgeon, we investigated sex- candidate genes including sox9 and dmrt1, cyp19a1, and foxl2 in great sturgeon gonads during different stages of sexual maturity.

Materials and Methods Forty-six great sturgeon H. huso, ranging from 1-8 years of age, were obtained from International Sturgeon Research Institute, Iran during the October to December 2014. All fish were anesthetized using clove powder (300 mg l-1), and tissue samples were collected through biopsy method Maturity stages were classified into 5 stages according to the previous studies. No sequences of sex related genes relevant to H. huso gonadal development are available in public database. The resultant sequences for H. huso foxl2, cyp19a1, sox9 and dmrt1 were characterized and deposited in the GenBank database. Relative expression of H. huso sex- candidate genes were analyzed by relative qRT-PCR using CFX-96 Real-Time PCR thermocycler (Bio-Rad, USA). RPL6 was used as the housekeeping gene for data normalization due to its constant expression through different developmental stages. The 2– ∆∆Ct method for relative gene expression analysis described by Livak and Schmittgen (2001) was used to calculate the gene expression values.

Results The transcription factor foxl2 and cyp19a1a showed sexual dimorphism at different stages of gonadal development in females, while the sox9 and dmrt1 showed dimorphism expression in male sturgeons (P<0.05). This kind of sex-specific expression of sex candidate genes during gonadal development was also reported in other species of fish (Vizziano et al., 2007; Ijiri et al., 2008; Amberg et al., 2010, Raghuveer et al., 2011). To our knowledge, present study is the first study that investigated the ovarian and testicular gene expression during different maturity stages in great sturgeon. More studies are needed under more strictly controlled conditions to confirm the findings.

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References Ijiri, S., Kaneko, H., Kobayashi, T., Wang, D.S., Sakai, F., Paul-Prasanth, B., Nakamura, M. and Nagahama, Y. 2008. Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus. Biology of reproduction 78, 333-341. Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2− ΔΔCT method. methods 25, 402-408. McCormick, C., Bos, D. and DeWoody, J. 2008. Multiple molecular approaches yield no evidence for sex‐ determining genes in lake sturgeon (Acipenser fulvescens). Journal of Applied Ichthyology 24, 643-645. Vizziano, D., Randuineau, G., Baron, D., Cauty, C. and Guiguen, Y. 2007. Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss. Developmental Dynamics 236, 2198-2206. Wuertz, S., Gaillard, S., Barbisan, F., Carle, S., Congiu, L., Forlani, A., Aubert, J., Kirschbaum, F., Tosi, E. and Zane, L. 2006. Extensive screening of sturgeon genomes by random screening techniques revealed no sex-specific marker. Aquaculture 258, 685- 688. Yarmohammadi, M., Pourkazemi, M., Ghasemi, A., Hassanzadeh, M. and Chakmehdouz, F. 2012. AFLP reveals no sex-specific markers in Persian sturgeon (Acipenser persicus) or beluga sturgeon (Huso huso) from the southern Caspian Sea, Iran. Progress in Biological Sciences 1, 55-114.

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A Review of Isolation and molecular identification of lactic acid bacteria in sturgeon Seyedeh Yalda Bani Esmaeili*, Fatemeh Alaei, Niloofar Eradatmand International Sturgeon Research Institute, Rasht, Iran. [email protected]

Probiotics are commonly defined as mono- or mixed cultures of live microbes that applied to animal or human; possess a beneficial effect on the health of the host. Lactic acid bacteria are characterized as gram-positive, usually non-motile, non-sporulating bacteria that produce lactic acid as a major or sole product of their fermentative metabolism. This review emphasizes the importance of molecular information on lactic acid bacteria (LAB) reported in the sturgeon. The 16S rRNA gene sequence has been widely used as a molecular method to estimate phylogenetic relationships among bacteria. More recently its use has also been proposed for the identification of unknown bacteria. Acid nucleic hybridization techniques are important for the detection and identification of microorganisms allowing higher resolution than those based on the 16S rRNA gene sequence. The fastest molecular techniques are based on FISH and PCR techniques which are mainly used to discriminate among wine lactic acid bacteria species/strains. RAPD technique has been described as a useful technique for both identification and typing. Denaturing and temperature gradient gel electrophoresis (DGGE and TGGE) have been developed to rapidly analyze microbial communities by sequence- specific separation of PCR-amplified fragments. Restriction analysis of amplified rDNA (ARDRA) has been used to differentiate a variety of micorganisms Real-time PCR an emerging technique that allows the fast quantification of microorganisms avoiding growth step in plates. The main objective of any microbial classification system is to identify the species level, which is the basic unit of the taxonomic grouping. Lactic acid bacteria have become a major source of concern for aquaculture in recent decades.

References Bartowsky EJ, McCarthy J. and Henscheke, P.A. 2003. Differentiation of Australian wine isolates of Oenococcus oeni using random amplified polymorphic DNA (RAPD). Aus. J. Grape Wine Res. 9: 122-126. Christian, M., Pelletier, C., Boussaha, M., Douet, D.G., Lautraite, A. & Tailliez, P. 2007. Diversity of Lactic Acid Bacteria Associated with Fish and the Fish Farm Environment, Established by Amplified rRNA Gene Restriction Analysis. American Society for Microbiology. Vol. 73 no. 9 2947-2955. Cocolin, L., Manzano, M., Cantoni, C. & Comi, G. 2000. Development of a rapid method for the identification of Lactobacillus spp. isolated from fermented Italian sausages using a polymerase chain reactiontemperature gradient gel electrophoresis. Lett. Appl. Microbiol., 30:126-129. Du Plessis, E.M., Dicks, L.M.T., Pretorius, I.S., Lambrechts, M.G. & du Toit, M. 2004. Identification of lactic acid bacteria isolated from South African brandy base wines. Int. J. Food Microbiol. 91: 19-29. Ercolini, D. 2004. PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J. Microbiol. Meth. 56: 297-314. Ghanbari, M., Rezaei, M., Jami, M. & Nazari, R. M. 2009. Isolation and characterization of Lactobacillus species from intestinal contents of beluga (Huso huso) and Persian sturgeon (Acipenser persicus). Iranian Journal veterinary. Volume 10. Issue 2: 152-157.

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Physiology and Biochemistry

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The Impact of Tocopherol (E) and Riboflavin (B2) Vitamins on Blood Factors and Immunity of Sturgeon Fingerlings (Acipenser Nudiventris) Ali Reza Ashouri*, Hossein Khara, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Esmaeil Hossein Nia, Zabihollah Pajand, Nemat Peikaran Mana * International Sturgeon Research Institute of Caspian Sea (Areeo), proliferation and breeding sector, Rasht, Iran, PO Box: 3464-416352. [email protected] Introduction Currently farming sturgeon in Iran is developing rapidly, and major farmed species in these production centers are Beluga, Siberian Sturgeon and Ship species. Adequate vitamins are particularly important in intensive culture. Vitamin e is a fat soluble vitamin, and its deficiency in Atlantic salmon (Salmo Salar) result in reduction of growth, exophthalmos, adhesion of gill filaments, waste and muscles degeneration, reduction of oviposition and fertility, and reduction of immune response (Poston, et al., 1976; Lall et al., 2000). Riboflavin is a water soluble vitamin which its deficiency results in vast and extensive disorders in Salmonidae. Webster et al, in 2002, investigated riboflavin deficiency in Channel catfish. The results were as growth reduction and food efficiency, muscular dystrophy, fatty liver, and high mortality in larval period and among fry. In this research, the impact of different levels of vitamin E and riboflavin were examined separately and in mixture, and on growth rate of blood indices and immune system of ship sturgeon in the growth period. Materials and Methods The steps of performing this research were conducted in sturgeon research station, Guilan- Chaboksar, and by using Caspian Sea brackish water. 150 ship sturgeons, transferred from International Sturgeon Research Institute of Caspian Sea in Rasht to Chaboksar site, and storage in fiberglass tubs. In order to adaptation with new conditions were fed by BioMar France, over two weeks. After this period, 120 specimens, with an average weight of 0.145 ± 30.33 g were selected and were distributed randomly, without having a significant statistical difference in body mass index, in 12 two-ton fiberglass tubs. After research period (8 weeks) and 24 hours after the last biometry, 30 percent of fish population in each treatment selected randomly and by 3 cc syringe (Raida et al., 2003) blood samples prepared from their caudal peduncle and sent to the laboratory to measure blood indices (red blood cells, white blood cells, hemoglobin, hematocrit) and immune factors (Lysozyme, total immunoglobulin, IgM and complement CH (50)). It should be mentioned, in bloodletting, anesthetic, due to the possibility of impact on blood indices, were not used. The method of measuring blood factors 1. Counting red blood cells (RBC) 2. Counting white blood cells (WBC) 3. Measuring hemoglobin (Hb) 4. Measuring hematocrit (HCT) 5. Measuring igm 6. Measuring Lysozyme 7. Measuring total immunoglobulin 8. Measuring CH50

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Results The amount of red and white blood cells, hematocrit, hemoglobin and immune system activity, by addition of vitamins e and 2b, separately and mixed together, increased significantly, compared to the control treatment (P <0.05). The greatest amount of red and white blood cells and hematocrit belonged to the fish fed by diet 2, and also, the greatest amount of hemoglobin observed in treatments 3 and 5, which contain 20 mg vitamin B2; and although in diet 5 (300 + 20) mg, activity of the total immunoglobulin in fish showed a rising trend by increasing vitamin supplements, there was no statistically significant difference to control treatment (P> 0.05); however by adding vitamin E, and B2, the activity of lysosome , IgM and compliments in fish, significantly increased. The greatest amount of Lysozyme and IgM in fish fed by diet (300 mg vitamin E) and the highest activity of complement in fish fed by diets 2 and 3, recorded respectively contain of 300 mg vitamin E and 20 mg Riboflavin (P <0.05). Discussion In the present experiment, the amount of red and white blood cells, hematocrit, hemoglobin and immune system activity increased significantly in comparison to the control treatment by adding vitamins e and b2, separately and in mixture, which is in accordance with Liu et al. (2007) who had stated vitamin e is a powerful antioxidant that can increase lifetime, durability and the number of erythrocytes, and plays a key role in cellular respiration. Lysozyme and complement (CH50) are important nonspecific immune system indices in fish (Mury et al., 2003). Meanwhile, Lysozyme is one of the major compounds in the body's immune system in vertebrates and mollusks (Song et al., 2006). Nowadays it has been found that the function of non-specific immune system, against parasites, bacterial and viral infections, and in response to infection, reveal itself as increase of non-specific immune system indices in blood (Puangkaew et al., 2004). In general, the results of this study suggest that a mixture of 300 mg vitamin E and 20 mg Riboflavin in the diet increase blood indices and the immune system; moreover, adding vitamin E and riboflavin, separately, respectively, in quantities of 300 and 20 mg per kg of diet, improve blood indices and immunity in the ship fry; and finally, adding these amounts in commercial sturgeon diet is recommended.

References Puangkaew, J., Kiron, V., Somamoto, T., Okamoto, N., Satoh, S., Takeuchi, T., Watanabe, T., 2004. Nonspecific immune response of rainbow trout (Oncorhynchus mykiss Walbaum) in relation to different status of vitamin E and highly unsaturated fatty acids. Fish Shellfish Immunol. Vol.16. pp.25-39. Raida, M.K., Larsen, J, L., Nielsen, M.E., Buchmann, K., 2003. Enhanced resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), against Yersinia ruckeri challenge following oral administration of Bacillus subtilis and B. licheniformis (BioPlus2B). Journal of Fish Diseases. 26: 495. Webster, C.D and C.E.Lim., 2002. Nutriet Requirement and Feeding of Finfish for Aquaculture. CAB International, CABI publishing. pp 418. Liu, Y., Wang, W., Wang, A., Wang, J., Sun, R.2007. Effects of dietary vitamin E supplementation on antioxidant enzyme activities in Litopenaeus vannamei (Boone, 1931) exposed to acute salinity changes.Aquaculture 265.pp351-358.

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Relationship of spermatological parameters and sperm motility indices of bester (Huso huso and Acipenser ruthenus) semen Shahrouz Baradaran Noveiri*, Ahmad Noori, Mahmoud Bahmani, Mohammad Ali Yazdani Sadati and Arash Akbarzadeh *Agricultural Research, Education and Extension Organization (AREEO), International Sturgeon Research Institute, Genetics Department, P.O.Box: 41635-3464, Rasht, Iran [email protected] Introduction Among all sturgeon hybrids, bester, (Huso huso × Acipernser ruthenus), considered as one of the best sturgeon hybrids for aquaculture (Chebanov and Billard, 2001).There is not any documented study on the relationships among spermatological parameters of male bester sturgeon with sperm motility indices (motility duration and percentage). Materials and Methods Semen of six mature males were separately collected and subjected to injection of luteinizing hormone releasing hormone. Sperm concentration, spermatocrit, percentage of motile spermatozoa, motility duration, pH and osmolality of seminal plasma has been assessed by routine laboratory instructions (Baradaran Noveiri et al., 2006; Alavi et al., 2012). Results and Discussion The mean sperm concentration, spermatocrit, total duration of sperm motility and percentage of motile spermatozoa were estimated as 2.06 ± 0.86 (×109ml-1), 2.86 ± 1.18 (%), 318.6 ± 136 sec and 81.24 ± 20.9 (%) respectively. The pH value and osmolality of seminal plasma evaluated as 8.09 ± 0.34 and 127.6 ± 20.8 (mOsmolkg-1) respectively. Correlations among the spermatological parameters, sperm motility indices, pH and osmolality of the seminal plasma are shown in Table 1. The results showed new information of studied parameters in bester semen.

Table 1: Correlations (r) among spermatological parameters, sperm motility indices, pH and osmolality of the seminal plasma of bester semen (*: p <0.05, ** p <0.01). Concentration Spermatocrit Motility Duration 9 -1 pH (×10 ml ) (%) (%) (Sec) Spermatocrit 0.922** Motility 0.399 0.152 Duration 0.876** 0.847** 0.154

pH - 0.804** - 0.647** 0.788** - 0.544* Osmolality 0.717** 0.633** 0.056 0.887** - 0.393 (mOsmkg-1)

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References -Alavi, S.M.H., Hatef, A., Mylonas, C.C., Gela, D., Papadaki, M., Rodina, M., Podhorec, P., Kaspar, V., Psenicka, M. and Linhart, O. 2012. Sperm characteristics and androgens in (Acipenser ruthenus) after induction of spermiation by carp pituitary extract or GnRHa implants. Fish Physiol. Biochem., 38:1655–1666. -Baradaran Noveiri, S., Alipour, A. and Pourkazemi, M. 2006. Sperm morphometry, concentration and spermatocrit study in Persian sturgeon (Acipenser persicus). J. Appl. Ichthyol., 22 (1): 380-383. -Chebanov, M. and Billard, R. 2001. The culture of sturgeons in Russia: production of juveniles for stocking and meat for human consumption. Aquat. Living Resour. 14: 375- 381.

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Effects of dietary phospholipids on alkaline phosphatase activity on stelate (Acipenser stellatus)

Fatemeh jafari*, Naser Agh, Farzaneh Noori

Urmia Lake Research Institute, Urmia University, Urmia [email protected] Introduction Among the fish species living in the Caspian Sea, sturgeons are of utmost interest from an economic perspective, not only for their caviar but also for the meat. However all sturgeon species inhabit Caspian Sea are highly vulnerable endangered and the stellate (Acipenser stellatus) is one of them.Since last few decades a lot of interest has been developed for sturgeon aquaculture. Thus there has been an increased demand for information on all aspects of sturgeon biology and physiology. Among lipid components, phospholipids (PL) are important components for maintaining the structure and function of cellular membrane, emulsifying lipids in gut and improving intestinal absorption of long chain fatty acids. Furthermore, PL could exert beneficial effects by improving diet qualities and providing essential nutrients (Tocher et al., 2008). However few studies have been reported on effects of dietary phospholipids on digestive enzymes activity. In the research we have investigated the effect of dietary phospholipids on alkaline phosphatase activity in stellate.

Matrials and Methods At the start of the feeding experiment, 336 fish with average weight of 11.25 ± 0.0 5 g were stocked in 21 polyethylene tanks (90 L) supplied with freshwater at a flow rate of 1 L min−1.The experiment was conducted in 7 treatments (T1-T7) and three replicates for each treatment including control fed on basal diet (T1) and treatments 2-7 fed on basal diet supplemented with 1, 2, 4, 6, 8 and 10 percent phospholipid respectively. The ALP activity was estimated according to Bergmeyer, 1974 using 4-nitrophenyl phosphate as substrate.

Results and Discussion After 77 days of experiment, the results indicated that alkaline phosphatase (ALP) activity was significantly increased in treatments 3 to 7 in compare to control (T1). Moreover T5 and T6 exhibited significantly higher ALP activity in compare to other treatments (p< 0.05) (Fig. 1). Alkaline phosphatase (ALP) is a kind of metal enzyme which mainly exists in root and brush border of foregut epithelial cells and promotes nutrient uptake by intestinal epithelial cell. The activity of ALP was positively correlated with the absorption of lipid, glucose, calcium and inorganic phosphorus (Tengjaroenkul et al., 2000). Thus, ALP can be used to evaluate the capacity of nutrient absorption by intestine of vertebrate larvae (Segner et al., 1989).

Conclusion In this study, ALP activity in intestine brush border of increased significantly with the increase of dietary PL. With reference to the main aim of this study and the results obtained, we may conclude that supplementation of 6 to 8 % of dietary phospholipids can improve the ALP specific activity in juvenile A. stellatus.

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Fig.1. specific of alkaline phosphatase in intestine content of Acipenser stellatus fed six different phospholipids (T2-T7) and basal diet (control) at the end of the 77 days culture.means with different superscript were significantly.

References Segner, H., Oesch, R., Schmidt, H., von Poeppinghausen, K., 1989. Digestive enzymes in larval Coregonus larvaretus L. Journal of Fish Biology 35, 249–263. Tengjaroenkul, B., Smith, B.J. and Caceci, T. 2000. Distribution of intestinal enzyme activities along intestinal tract of cultured Nile tilapia, Oreochromis niloticus L Aquaculture182, 317–327. Tocher, D.R., Bendiksen, E.A., Campbell, P.J., Bell, J.G., 2008. The role of phospholipids in nutrition and metabolism of teleost fish. Aquaculture 280, 21–34. Zhao, J.Z., Ai, Q.H., Mai, K.S., Zuo, R.T., Luo, Y.W., 2013. Effects of dietary phospholipids on survival, growth, digestive enzymes and stress resistance of large yellow croaker, Larmichthys crocea larvae. Aquaculture 410–411, 122–128.

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The effect of niacin (vitamin B3) on growth, body composition, hematology and immune parameters of Sterlet (Acipenser ruthenus) Rana Haghparast, Hossein Khara*, Masod Farokhroz1 *Departmentof Aquaculture, Lahijan Branch, Islamic Azad University, Lahijan, Iran, P.O. Box:1616 [email protected] Introduction Sterlet, Acipenser ruthenus, is one of the valuable and also the smallest species of Acipenseridae. This fish is a freshwater and potamodromous species. Vitamin is one of the food items that is small in terms of quantity but an essential component of diets for aquatic organisms. B vitamin is one of the water-soluble vitamins. Niacin is one of the B vitamins which are involved in cellular respiration and releasing energy from carbohydrates, fats, and proteins. Niacin also plays a role in the synthesis of glycogen, the carbohydrate reserve matter in the liver and muscles. Niacin also plays an important role in fat metabolism in the body. This vitamin is also used for lowering the bad triglyceride and cholesterol of blood (LDL) and reduction of the rate of cardiovascular diseases. Niacin naturally exists in many foods such as vegetables, meat, poultry, fish, egg, and foods fortified with vitamin B3. Niacin or vitamin B3 is used as a useful treatment for high blood cholesterol. Recent studies show that niacin can increase the level of good cholesterol of blood (HDL) and reduce the level of triglyceride and bad cholesterol (LDL) (Poston and Wolfe, 1985). Therefore, the present research aimed to study the effect of niacin (B3 vitamin) on growth, body composition, and some blood biochemical parameters of sterlet fingerlings.

Materials and Methods In this study, 90 sterlet fingerlings, with a mean weight of 50 grams, were introduced to 18 fiberglass tanks sized 2×2×0.5 m which were filled with 1.6 m3 water. A combination of six levels of Niacin (0, 10, 20, 30, 40, and 50 mg per kilogram of dry feed) was used for preparation of 5 diets containing vitamin at determined doses and a control with three replicates apiece for two months (Halver, 1989; Ng et al., 1997; Shaik Mohamed and Ibra, 2001). At the end of the trial, subjects were bio-assessed and various growth parameters were measured based on standard methods (Ellis, 1990). For carcasses analysis and measurement of moisture, fat, protein, and ash, 100 grams of carcasses of each sample was transferred to the laboratory. Kjeldahl method (Kjeltec Auto Analyzer) and soxhlet extraction (Kofi et al., 1992) were used for measurement of protein and fat. To assess blood parameters at the end of 60 days, three blood samples were taken from each subject. Then, white and red blood cell count, measurement of blood indices, and white blood cell differential diagnosis were done (Klontz, 1994; Houston, 1990). In addition, immunoglobulin (IgM), lysozyme, total immunoglobulin concentration, total protein, glucose, triglyceride, cholesterol, bad cholesterol (LDL), and good cholesterol (HDL) were measured. For the first trial (age study) data were analyzed using one-way ANOVA and differences between mean values were considered significant by Turkey test at 0.05% probability. For second trial, data were statistically analyzed using the Student’s t-test (p<0.05). All these statistical analyses were performed using the SPSS statistical software (Version 14 for Windows XP). All data are presented as the mean and standard deviation (SD).

Results The results showed that weight gain between treatments, condition factor, growth rate and final length was not significant (P>0.05). In the MCV and MCH there were no differences

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir between groups. There was a significant difference in blood (red blood cells, white blood cells, hemoglobin, hematocrit, mean cell volume is observed statistically significant (P<0.05). Based on the analysis of carcass significant differences between treatments in terms of moisture content, fat, protein and ash is significant (P<0.05). The protein, ash was related to T3. T3 was the least amount of fat. The biochemical composition (triglycerides, LDL, HDL and blood protein) in the treatments are significant differences (P<0.05). Discussion According to the results, no significant difference was found between treatments in terms of moisture, fat, and protein levels. Studies of Morris and Davies (1995) on sea bream showed that the carcass fat content was significantly lower proportional to the total volume of carcasses. According to studies conducted by Shaik Mohamed and Ibrahim (2001) on the body of catfish and Ahmed (2011) on Rohu, protein content increases with the addition of niacin supplement. Based on the findings of a study conducted by Ahmed on L. rohita and C. mrigala, it was shown that niacin affects biochemical composition of body. According to the results of the present study, a significant difference was observed between treatments in terms of white blood cells, red blood cells, and other blood and immunological parameters. In a study carried out by Carriquiriborde et al. (2004), no significant difference was found between treatments in red blood cells count. However, Wing-Keung et al. (1996) found that niacin improves growth, liver efficiency, and hematocrit in channel catfish. It seems that lysozyme activity level in sturgeon to be less than in bony fishes. In subjects fed with niacin- fortified food, blood glucose and cholesterol decreased and blood good cholesterol and protein increased. In the study conducted by Jiang and Huang (2014) on gift tilapia, it was shown that no significant change occurs in glucose level with the increase in niacin. Researchers have found that niacin can reduce blood fat content by influencing lipid metabolism (Zak et al., 2006).

Conclusion Finally, according to the results we can say that, as predicted in fish low fat content and protein content has been added to the carcass, according to the analysis of the levels of niacin is 30 mg per kg dry food on the basis of the least the amount of fat in T3, as well as the highest levels of protein, ash and moisture were reported in T3. The biochemical composition and triglyceride lowering the effectiveness of the treatment 2 and in terms of effectiveness in reducing bad cholesterol and can be seen better in T1 said niacin an important role in reducing fat and bad cholesterol in the blood.

References Balabanova L.V., Mikryakov D.V. and Mikryakov V.R. (2009). Response of common carp (Cyprinus carpio L.) leucocytes to hormoneinduced stress. Inland Water Biology 2(1): 86– 88. Ahmed, I. 2011. Effect of dietary niacin on growth and body composition of two Indian major carps, rohu, Labeo rohita and mrigal, Cirrhinus mrigala fingerlings based on dose-response study. Aquacult. Int. 19:567-584. Andrews JW, Murai T (1978) Dietary niacin requirements for channel catfish. J Nutr 108:1508–1511. Aoe H, Masuda I, Taked T (1967) Water-soluble vitamin requirement of carp-III. Requirement for niacin. Bull J Soc Sci Fish 33:681–685.

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Effects of dietary Saccharomyces cerevisiae on growth performance and immune system of Acipenser baerii juvenile.

Mir Hamed Sayed Hassani, Mohamad Ali Yazdani, Hoshang Yeganeh, Mahmoud Shakourian, Mahmoud Mohseni, Mohebali Pourgholam, Ali Hossien pour Zelti , Hamid Rez Pourali, Ayoub Yousefi Jourdehi, Amin Farahbod Roudbaraki, Alireza alipour Caspian Sea International Sturgeon Research Institute (Areeo), Iran,Rasht Introduction Acipenser baerii is one of the the best of Acipenseridea candidate for Aquaculture in Iran that usually raised in, tanks, or ponds. In the recent years, efforts have been made to increase the productivity per unit of this species (Yazdani et al., 2009). However, high-density rearing seems to lead to poor physiological environment that adversely affect fish health and growth rate in fingerlings and Juvenile periods (Kim et al., 2003; Al-Harbi and Uddin, 2004) and therefore led to lost high degree of fingerling and juvenile fish that are base of Acipenser Aquaculture in Iran. Probiotics are live microbial feed supplement which beneficially affect the host animal by improving its intestinal balance as supplements to improve growth (Fuller, 1989; Kesarcodi-Watson et al., 2008) and in some cases as a mean of replacing antimicrobial compounds (Moriarty, 1997). On the other hand, their effectiveness to mitigate the effects of stress, resulting in a greater production (Ghazalah et al., 2010).

Materials and Methods This study was conducted to evaluate the effect of different levels of dietary supplementation of Saccharomyces cerevisiae on growth performance, feed utilization and immune system of biochemical composition of Acipenser baerii fingerlings. five diets containing (protein: 45%, fat: 16% and fiber3.5%) that Saccharomyces cerevisiae supplemented at levels of 0, 0.75, 1/5, −1 and 3 g kg were prepared and named (SR0 ،SR0.75 ،SR1.5،SR2.25 ،SR3). 300 fingerling 2/25 Acipenser baerii with average weight of (32.18±0.51 g) were fed in five replicate tanks Thrice daily to apparent satiation for 8 weeks. In the end of feeding trail, Blood samples were collected from caudal vein with heparinized syringes, stored in vials (1.5 CC) and transferred to laboratory. Samples were centrifuged at 3000 rpm for 10 min, plasma removed and stored in a refrigerator in -20 0C and transferred to laboratory.

Results Growth performance increased significantly with the increase in dietary yeast levels. Highest weight gain, feed efficiency ratio, Specific growth rate and lowest Feed conversion ratio belonged to fish fed SR1.5 that have significant difference with fish fed control diet (P<0.05) Saccharomyces cerevisiae supplemented to level of 1.5,2.25and 3 gr/kg led to significant increase of White blood cell, neutrophil, lysozyme and compliment in fish (P<0.05).

Discussion Juvenile Acipenser baerii fed the diet containing S. cerevisiae exhibited the increase in growth performance, which was attributed to significantly higher activity of alkaline phosphatase (Gawlicka et al., 2000; German et al., 2004). Lara- Flores et al. (2003; 2010) revealed that S. cerevisiae supplementation produced significantly higher weight gain and feed utilization efficiency in tilapia fed diets containing 27% or 40% crude protein compared

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir to the controled diet. Similar pattern that improved growth performance in tilapia fed S. cerevisiae diets have been reported by Marzouk et al. (2008), Osman et al. (2010) and Ozório et al. (2012).The results of Tewary and Patra ( 2011) show ed that, yeast cell wall able to enhance the innate immunity and also have a positive co-relation with growth parameters. Through the absorption of yeast wall particle, the immune function and disease resistance of the entire organism is stimulated. This result agrees with Taoka et al. (2006b) who investigated the effect of live and dead probiotic cells on the non-specific immune system of Nile tilapia. They found that probiotic treatment enhanced non-specific immune parameters such as lysozyme activity, migration of neutrophils and plasma bacteriocidal activity, resulting in improved resistance to Edwardsiella tarda infection. Bakers’ yeast is a source of nucleic acids and β-1,3-glucans which have been recognized to effectively enhance immune functions of African catfish (Yoshida et al., 1995), Atlantic salmon rainbow trout (Jørgensen et al., 1993; Siwicki et al., 1994), and shrimp Penaeus monodon (Thanardkit et al., 2002).

References The results of the present study indicated clearly that the supplementation of S. cerevisiae enhanced the growth performance and immune system of juvenile Acipenser baerii and suggested add of 1.5-2.25 gr yeast per kg−1 diet Kesarcodi-Watson, A., Kaspar, H., Josie Lategan, M., Gibson, L., 2008. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274, 1–8. Lara-Flores, M., Olvera-Novoa, M.A., Guzman-Méndez, B.E., Lopez- Madrid, W., 2003. Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus). Aquaculture 216, 193–201. Mohanty, S.N., Swain, S.K., Tripathi, S.D., 1996. Rearing of catla (Catla catla Ham.) spawn on formulated diets. J. Aquac. Trop. 11, 253–258. Kim, K.W., Wang, X.J., Choi, S.M., Park, G.J., Koo, J.W., Bai, S.C., 2003. No synergistic effects by the dietary supplementation of ascorbic acid, α-tocopheryl acetate and selenium on the growth performance and challenge test of Edwardsiella tarda in fingerling Nile tilapia, Oreochromis niloticus L. Aquacult. Res. 34, 1053–1058. Taoka, Y., Maeda, H., Jo, J.-Y., Kim, S.-M., Park, S., Yoshikawa, T., Sakata, T., 2006b. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fish. Sci. 72, 755–766. Teuber, M., 2001. Veterinary use and antibiotic resistance. Curr. Opin. Microbiol. 4, 493– 499.

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Determining of some liver enzymes levels in juvenile Persian sturgeon (Acipenser persicus) captured in the southern coasts of the Caspian Sea

Ali Hallajian*, Rezvanollah Kazemi, Ayoub Yousefi Jourdehi, Sohrab Dejandian, Mahtab Yarmohammadi, Mahmoud Mohseni. *Agricultural Research,Education and Extension Organization(AREEO), International Sturgeon Research Organization of the Caspian Sea, Rasht, Iran, P.O.Box: 41635– 3464 [email protected]

Introduction Persian sturgeon is one of the most valuable species that its stocks have been seriously threatened because of human activity and ecological conditions of the Caspian Sea. Also, evaluating of liver enzymes can give us some important and essential data of natural status in fish. Alanin amino transaminase (ALT), Glutamic oxaloacetic transe aminase (SGOT) and Aspartat amino transferas (AST) are the most important enzymes from aminotranferas and transe aminase groups that have very important roles in naturalization of toxic agents and liver metabolically activities that are as two key enzymes for determining liver activity. Therefore, the aim of present study was detecting some liver enzymes levels at the normal status in juvenile Persian sturgeon captured from the southern coasts of the Caspian Sea.

Matrials and Methods In this study 40 fingerlings Acipenser persicus captured using trawl method from 20 – 100 meters depth of the southern coasts of Caspian Sea during autumn 2009 - 2011. Fish were biometred and divided into 6 weight groups such as <100 gr, <500 gr, <1000 gr, <2000 gr, <5000 gr and <10000 gr. Blood samples collected using 2 cc syringes and serum extracted using centrifuge 3000 rpm in 10 min and stocked in -20 oC. Enzyme levels measured using Analyzer Bt – 1500 (Instruments Biotecnica, Italy) at 37 oC and wave length 340 nm using Pas azmoon kit. 100 µl of serum used for each sample.

Results Results showed min, max and mean of SGOT (AST) in weight group <100 gr was 2, 109 and 16.4 ± 5.7; in weight group <500 gr was 2, 478 and 38.9 ± 24.9; in weight group <1000 was 3, 96 and 26.4 ± 11.2; in weight group <2000 gr was 222, 4 and 56.9; in weight group <5000 gr 643, 21 and 332 ± 31; and in group <10000 was 11, 69 and 35 ± 17.5 µl, respectively (Fig. 1). Based on one – way AOVA and Touki weight group <5000 showed significant different with other groups (P  0.05). َAlso SGOT enzyme levels was more in weith group 5000 than other groups. Min, max and mean of SGPT (ALT) in group weight <100 gr was 0.7, 3 and 1.4 ± 0.1; in group weight <500 gr was 0.7, 3 and 1.3 ± 0.2; in group weight <1000 gr was 0.7, 2 and 1.2 ± 0.2; in group weight <2000 gr was 1, 3 and 1.9 ± 0.3; in group weight <5000 was 2, 5 and 3.5 ± 0.4 and in group weight <10000 gr was 1, 2 and 1.7 ± 0.3 µl (Fig. 2). Based on one – way AOVA and Touki, weight group <5000 showed significant different with the other groups (P  0.05). .Also SGPT enzyme levels was more in weight group 5000 than other groupsَ

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5 400 b b 4 350 4

300 3 250 3 a a 200 2 a

SGOT(IU/L) a a 150 2 a SGPT(IU/L) 100 a a 1 50 a a 1 0 0 100 500 1000 2000 5000 10000 100 500 1000 2000 5000 10000

Weight groups (g) Weight groups (g)

Fig. 1: concentration of the enzyme SGOT (AST) in Fig. 2: concentration of the enzyme SGPT (ALT) in blood Persian sturgeon blood Persian sturgeon Discussion Decrease in these agents indicated outbreak liver disasters. The liver is the responsibility of detoxification process and disposal of harmful agents such as mycotoxins and also protein disruptive agents of cells such as Antigen – antibody complexes. Increase of these enzymes especially in glutamic oxaloacetic Trans aminase (AST) is one of the most important factors in liver paraclinical assay indicated it's efficiency level. The level of this enzyme decrease in infections that is associated with liver damage. Increase of enzyme activity such as Aspartat transferase SGOT (AST) and Alanin amino transferas SGPT (ALT) in serum is an indicator of liver damage. Under normal circumstances, these enzymes are present in liver cells. But when the liver is damaged these enzymes entered into blood stream and increase of these enzyme levels signaling the liver damage. Studies showed some food ingredients, toxins and chemical matters on the liver enzyme secretion and caused to decrease and increase in their levels. ALT and AST are specific enzymes that not only present in blood serum but also can present in liver, heart, gill, kidney, muscles and other organs. Thus, it may provide them specific details in tissue dysfunction. Sepulveda et al., 2012 reported AST level in wild Scaphirhynchus platorynchus male and female was 714 and 487 µl respectively. AST levels in juvenile Scaphirhynchus platorynchus 3- year- old was 596 and 487, respectively. Shi et al., 2006. Reported that ALT and AST levels in Acipenser schrenckii with weight 55 gr and 6 – year old were 548.94 and 110.85 μl/l and AST and ALT levels in Chinese sturgeon (Acipenser sinensis) with 45 gr and 6 – year old were 144.93 and 37.30 μl/l. The range of AST I was reported 18 – 714 IU/l. Based on author's reports SGOT (AST) enzyme levels was 18 – 714 IU/l in juvenile farmed sturgeon. While in present study, the level of this enzyme in wild juvenile Acipenser persicus was 42 µl. The level of SGPT (ALT) in farmed sturgeon was differed between 5.4 to 110.8 µ/l. In present study the level of this enzyme was 1.45 µl in that was lower than other results. This information can be used to assess health status in farmed and wild fish.

References Knowles, S.; Hrubec, T.C.; Smith, S.A. and Bakal, R.S, 2006. Hematology and plasma chemistry reference intervals for cultured shortnose sturgeon (Acipenser brevirostrum). Veterinary Clinical Pathology 35:434– 440. Rajabipour, F.; Shhsavani, D.; Moghimi, A.; Jamili, Sh.; Mashaii, N. 2010. Comparison of serum enzyme activity in great sturgeon, Huso huso, cultred in brackish and freshwater earth ponds in Iran. Comp.Clin.Pathol. 19:301-305.

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Sepulveda, M.S.; Sutton, T.M.; Patrick, H.K. & Amberg, J.J. 2012. Blood Chemistry Values for Shovelnose and Lake Sturgeon. Journal of Aquatic Animal Health 24:135–140. Shahsavani, D., Mohri, M., Gholipour Kanani, H. 2010. Determination of normal values of some blood serum enzymes in Acipenser stellatus Pallas. Fish Physiol Biochem 36:39–43. Shi, X., Li, D., Zhuang, P., Nie, F., Long, L. 2006. Comparative blood biochemistry of Amur sturgeon (Acipenser schrenckii) and Chinese sturgeon (Acipenser sinensis). Fish physiology and Biochemistry, Vol. 32, Pp. 63-66. Thomas, L. 1998. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). In: Thomas L, editor. Clinical Laboratory Diagnostics. 1st ed. Frankfurt.TH-Books Verlagsg

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Sturgeon Induced Reproduction Instructions Liyuan *, Qiu *SHD Aquarium and Technology Co., Ltd. Member of World Sturgeon Conservation Society - Beijing China, Tel: +8613552607973 www.carppituitary.com www.fishpituitaryhormones.com

[email protected]

Because sturgeon longevity and the slow and late maturation (long inter-annual reproductive resting period), it becomes important to properly control reproduction ones the sturgeon reach maturity. Hormone induced spawning of sturgeon has been used for almost 80 years.

General recommendations on hormonal induction for stimulation in sturgeon broodstock spawning are: • Acetone-dried sturgeon pituitary (SP); • Acetone-dried common carp pituitary gland/ extract (CCP); • LHRH-A2

Pituitary gland / extract General recommendations on injection

The pituitary extract produces and stores gonadotropin hormones (GtH), which play a decisive role in ovulation and spermiation. Injected pituitary material by passes the brain -pituitary link, acting directly on the ovaries and testes, providing the surge in blood GtH levels that normally precedes spawning (Figure 2).

LHRH-A2 is a synthetic polypeptide, which stimulates maturated animal to release gonadotrophin and to induce maturation and breeding in cultured fish. It also can be used for ovary inactivation, delay of ovulation and increase of pregnancy rate or pregnancy amount.

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Injections of mammalian Luteinizing Hormone-Releasing Hormone (LHRH) have been used experimentally to mimic the fish’s GnRH. However, a comparatively large dose and frequent injections were required.

LHRH-A2 Suggested GnRHa total dosage for induction of ovulation in sturgeon females (Chebanov, Galich and Chmyr, 2004). Injections of (LHRH) stimulates the fish’s own pituitary to produce and release the GtH necessary for spawning

SHD Aquarium and Technology Co., Ltd is specialized in hormone products on breeding for fish. After decades of professional experience, SHD has been evaluated as New and Hi-Tech Enterprise for years. For more information please go to www.carppituitary.com www.fishpituitaryhormones.com

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Changes in cortisol, estradiol-17β and thyroid hormones during the embryonic development of the Persian sturgeon, Acipenser persicus Bahram Falahatkar*, Parisa Haghparast *Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, 1144, Guilan, Iran [email protected]

Introduction A key to success of fish culture is the mass production of high quality offspring. In this regard, it is important to examine the structural and functional development of the endocrine system during embryonic development of fish. The objectives of present study was to investigate changes of cortisol, estradiol-17β (E2) and thyroid hormone (triiodothyronine; T3, thyroxin; T4) levels during the different stages of embryogenesis of Persian sturgeon, in order to better understand the early egg requirements and possible synthesis or leakage of those functional hormones.

Materials and Methods A total of four Persian sturgeon female broodstocks (25 kg weight, 1.8 m total length), that were caught from fishing station of Shafaroud in Guilan were used for this experiment. To induce the sturgeon to reach final maturation, a synthetic hormone with 2 injections at a 12-h interval for female (1 and 2 µg LHRHa2/kg body weight in the first and second injection, respectively) was used. Males were received once (1 µg/ kg of body weight) coincidence with the second injection of females. After readiness of fish, eggs were collected by cutting the abdomen and sperm with plastic syringe that inserted in to the genital pore. Coelomic fluid was removed from the eggs before insemination. About 600 g eggs were collected and fertilized with sperm of 3 males, using the wet fertilization method. A 10% silt solution was used for elimination of eggs stickiness. Fertilized eggs were then transferred to the Youshchenko incubators and incubated at water temperature of 21 ± 0.5°C. Fertilization rate was calculated after closing blastopore, which was 91 ± 1.4%. The eggs were sampled at various stages of embryonic development including unfertilized (egg), after water absorption, two-cell division, late blastula, late gastrula, late neurula, eyed stage, heartbeating and hatching. For cortisol and E2 measurements, extraction of hormones was performed according to De Jesus et al. (1991). In order to measure T3 and T4 contents in the eggs, the extraction method described by Tagawa and Hirano (1987) was applied. After separating of surface layer, E2, T3 and T4 levels were measured by radioimmunoassay. Also, the cortisol content was measured by enzyme-linked immunosorbent assay. Differences among means were analyzed by one-way ANOVA and Duncan’s test.

Results and Discussion The results showed that cortisol levels had significant differences in various stages of embryonic development and the highest value was obtained in stages of unfertilized eggs (P < 0.05). E2 level was significantly decreased during embryonic development and the lowest was observed in hatching stage (P < 0.05). There were no significant differences in T3 and T4 concentrations during various stages of embryonic development (P > 0.05). These observations indicate that hormones are transferred from the maternal circulation and deposited into the eggs during ovarian development (De Jesus and Hirano, 1992; Tanaka et al., 1995; Falahatkar et al., 2014). The present study showed that E2 concentration could be reduced during the different stages of embryogenesis, it seems that decline in steroid content during embryonic development may reflect the relative demand of the embryos for hormones at various stages of embryogenesis (Feist et al., 1990; De Jesus and Hirano, 1992). The role of cortisol and thyroid hormones seem important during developmental stages, and their presence show a general feature of egg development and larval quality (De Jesus and Hirano,

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1992; Tanaka et al., 1995; Tagawa et al., 2000). Cortisol concentration decreased from two- cell division stage to late neurula, which is perhaps because of metabolization and clearance of cortisol (Feist et al., 1990; De Jesus and Hirano, 1992). Table 1. Cortisol, estradiol-17β and thyroid hormone levels in the Persian sturgeon egg during various stages of embryonic development (mean ± SE). Stage Cortisol (ng/g) Estradiol-17β (ng/g) T3 (ng/g) T4 (ng/g) Unfertilized 6.98 ± 0.21a 0.49 ± 0.01a 0.53 ± 0.01 1.77 ± 0.03 After water absorption 6.72 ± 0.14ab 0.48 ± 0.01a 0.55 ± 0.01 1.68 ± 0.08 Two-cell division 6.97 ± 0.19a 0.47 ± 0.01a 0.55 ± 0.01 1.68 ± 0.09 Late blastula 6.02 ± 0.28b 0.46 ± 0.02ab 0.53 ± 0.02 1.70 ± 0.07 Late gastrula 5.94 ± 0.10b 0.46 ± 0.01a 0.54 ± 0.01 1.63 ± 0.03 Late neurula 5.84 ± 0.45b 0.46 ± 0.01a 0.55 ± 0.01 1.77 ± 0.03 Eyed stage 6.09 ± 0.45b 0.45 ± 0.01ab 0.55 ± 0.04 1.77 ± 0.07 Heartbeating 6.08 ± 0.27b 042 ± 0.03bc 0.56 ± 0.02 1.90 ± 0.17 Hatching 6.07 ± 0.28b 0.40 ± 0.01c 0.55 ± 0.02 1.80 ± 0.06 The data in the same column with different letters are significantly different (P < 0.05). Conclusion In conclusion, cortisol, E2 and thyroid hormones were observed at unfertilized eggs and various stages of embryonic development of Persian sturgeon with some fluctuations. It seems that combined effects of these hormones are needed generally for active utilization in order to regulate growth and endocrine control during the embryonic development of this species. Future examinations should be done in order to find the mechanism of each hormone in embryonic development of sturgeon.

References De Jesus, E.G., Hirano, T. & Inui, Y. 1991. Changes in cortisol and thyroid hormone concentrations during early development and metamorphosis in Japanese Xounder, Paralichthys olivaceus. General and Comparative Endocrinology, 82: 369–376. De Jesus, E.G. & Hirano, T. 1992. Changes in whole body concentrations of cortisol, thyroid hormones, and sex steroids during early development of the Chum Salmon, Oncorhynchus keta. General and Comparative Endocrinology, 85: 55–61. Falahatkar, B., Akhavan, S.R. & Ghaedi, G.R. 2014. Egg cortisol response to stress at early stages of development in Persian sturgeon Acipencer persicus. Aquaculture International, 22: 215–223. Feist, G., Schreck, C.B., Fitzpatrick, M.S. & Redding, J.M. 1990. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. General and Comparative Endocrinology, 80: 299–313. Tagawa, M. & Hirano, T. 1987. Presence of thyroxine in eggs and changes in its content during early development of chum salmon, Oncorhynchus keta. General and Comparative Endocrinology, 68: 129–135. Tagawa, M., Suzuki, K. & Specker, J.L. 2000. Incorporation and metabolism of cortisol in oocytes of tilapia (Oreochromis mossambicus). Journal of Experimental Zoology, 287: 485–492. Tanaka, M., Tanangonan, J.B., Tagawa, M., de Jesus, E.G., Nishida, H., Isaka, M., Kimura, R. & Hirano, T. 1995. Development of the pituitary, thyroid and interrenal glands and applications of endocrinology to the improved rearing of marine fish larvae. Aquaculture, 135: 111–126.

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Caudal fin primary cell lines of Persian sturgeon Acipenser persicus

Mohammad Reza Nowruzfashkhami*, Mohammad Pourkazemi, Mahmoud Bahmani, Morteza Daliri, Mohammad Hassanzadeh Saber, Shahrouz Baradran Noveiri, Ahmad Ghoroghi *Research instructor of Agriculture Research, Education & Extension Organization (AREEO), International Sturgeon Research Institute, Genetic and biotechnology Department, Rasht, Iran [email protected] Introduction Fish cell culture has provided a powerful tool in studying different subjects. Fin tissue is preferred for cultivation, due to its natural high regenerative ability, and because it is easy to sample. Most fin explants cultures are performed with the caudal fin. In the present study, a monolayer culture from caudal fin explants of Persian sturgeon Acipenser persicus and subsequent subculture up to two passages was developed.

Materials and Methods A two - year old Persian sturgeon (48 cm in length and 729 g weight) was used in this study. After euthanasia by clove powder, a portion of the caudal fin (2 cm) was cut off, washed in PBS containing antibiotics-antimycotic and minced into about 1 mm2 fragments. Tissue explants transferred to a cell culture flask containing 4 ml of L-15 medium antibiotic- antimycotic, 1 ml FBS and it was incubated at 22°C. When a sheet of cells appeared around the explants and fin cells grew into a confluent monolayer, the cells were washed in PBS containing antibiotics-antimycotic and dissociated with Trypsin-EDTA solution. The detached cells were transferred into a new flask to which 5 ml of growth medium was added (first subculture). A similar procedure was followed for the second subculture.

Results and Discussion Most of the explants adhered on to the substrate and the percentage of attachment for caudal fin explants was about 60%. Cell outgrowth from the caudal fin explants was initiated on the second day of culture (Figure 1). The number of proliferating and migrating cells increased and a large number of cells were seen around the periphery of the explants. Two types of adherent cells observed in the culture, that were fibroblast-like (70-80%) and epithelial-like, but during subcultures, the cells predominantly were fibroblast-like.

Fig.1. Cells emerging from the caudal fin explants on the third day (×100)

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In the present study, caudal fin explants of A. persicus were plated without enzymatic digestion. Enzymatic treatment of fish fin explants before plating them is a matter of divergence among authors. Proliferation of tail fin cells in L-15 was good. This is in agreement with other published studies (Fernandez et al., 1993a). The best growth of tail fin was obtained with 20% FBS.The investigations into temperature within this project demonstrated that the best proliferation was obtained at 22 °C. The highest growth rate of fish cell lines was observed at 20-25°C (Kang et al., 2003). Our results showed that the caudal fin of A. persicus could be a convenient source for the establishment of cell lines and can be used with a high degree of success to produce cell lines. In the early stages of the primary culture in the present study, the cell population that grew out of the fin fragments exhibited both fibroblast-like and epithelial-like cells, but as the culture progressed, the epithelial-like cells declined in number and the fibroblast-like type became the most frequent in subcultures. Usually, a predomination of fibroblastic cells over epithelioid cells in cell cultures from fish has been reported (Lai et al., 2003).

References Fernandez, R.D. Yoshimizu, M. Ezura, Y. Kimura, T. 1993a. Comparative growth response of fish cell lines in different media, temperature and sodium chloride concentrations, Fish Pathology, 28: 27-34. Kang, M.S., Oh, M.J. Kim, Y.J. Kawai, K. Jung, S.J. 2003. Establishment and characterization of two new cell lines derived from founder, Paralichtys olivaceus (Temminck & Schlegel), Journal of Fish Diseases, 26: 657- 665. Lai, Y.S. John, J.A.C. Lin, CH. Guo, I.C. Chen, S.C. Fang, K. Lin, CH. Chang, C.Y. 2003. Establishment of cell lines from a tropical grouper Epinephelus awoara (Temminck and Schlegel) and their susceptibility to grouper irido and noda viruses. Journal of Fish Diseases, 26: 31-42.

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Primary culture of ovarian follicular cells of Sterlet Acipenser ruthenus

Mohammad Reza Nowruzfashkhami*, Mohammad Sudagar, Mahmoud Bahmani, Negin Salamat, Mohammad Mazandrani, Mohammad Ali Yazdani Sadati

*Research instructor of Agriculture Research, Education & Extension Organization (AREEO), International Sturgeon Research Institute, Genetic and Biotechnology Department, Rasht, Iran [email protected] Introduction Fish ovarian follicular cells play a vital role in fish reproduction as well as in the synthesis of steroid hormones, growth and final maturation of oocytes (Nagahama 1994), remained alive and continued to secrete hormones in culture medium (Stoklosowa & Epler 1985). The purpose of the present experiment was to culture the oocytes follicular cells of Sterlet sturgeon Acipenser ruthenus to develop an in vitro system for functional studies of these cells in the future. The Sterlet, belonging to the family Acipenseridae, is a native species in Volga River. It is also found in riverine regions of the Black, Azov and Balkan Seas. This species adapts easily to rearing conditions and does not require more special attention (Holcik 1989).

Materilas and Methods A sixty years old sterlet sturgeon (729 g in weight and 47.6 cm in length) was anesthetized by clove powder, after removing a part of the ovary. Ovarian follicular cells Isolated through the protocol developed by Stoklosowa and Epler (1985) with some modifications. The interstitial connective tissue of the oocytes was removed and the oocytes were washed in PBS solution containing antibiotics for three times. After trypsinization and centrifugation, supernatant was removed and the deposited follicular cells were resuspended in the medium L-15. After assessing viability of cells by Trypan blue exclusion test (Doyle & Griffins 1998), the follicular cells were cultured in medium L-15 supplemented with 20% FBS, streptomycin sulphate (Gibco), penicillin G potassium (Gibco) and Amphotericin B (Gibco) and transferred into an incubator at 22 °C. Culture medium was collected on days 3, 5 and 7, centrifuged and supernatant was stored at -20 °C for further analysis of steroid hormones produced including testosterone (T), progesterone (P4), 17α-hydroxyprogesterone (17αOHP) and estradiol-17β (E2). Steroids analyses were carried out by Enzyme-Linked Immune Sorbent Assay (ELISA). By removing the interstitial connective tissue, separated oocytes surrounded only by the follicular layers were obtained and follicular cells were observed as a thin layer when oocytes were examined under an inverted microscope.

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Results and Discussion Trypsinization resulted in detaching of follicular layers from the rest of the oocytes and formation of the cell suspension cells (Fig. 1). The viability of cells was 92%.

Fig. 1. Follicular cells of Sterlet oocytes (×120) After 48 hours, follicular cells were attached to the bottom of the culture vessel and proliferated. The cells were mostly fibroblast-like in shape. The propagation of cells increased in the following days and after one week the cultured cells had almost completely covered the bottom of the culturevessel (Fig. 2). Based on the results, the concentrations of T and E2 secreted in the culture mediumdecreased over time. Levels of P4 and 17αOHP in the culture medium increased gradually from day 2.

Fig. 7. Fibroblast-like cells after a week (×120) Cultivated follicular cells of Sterlet in L-15 medium, containing FBS and antibiotics at 22 °C, responded and adapted to the culture, attached to the bottom of the culture vessel, proliferated and secreted steroid hormones into the culture medium. These results indicate we have successfully developed an in vitro homologous system for follicular cells of oocyte in sterlet sturgeon that resembles the in vivo situation.

References Doyle, A., Griffiths, JB. 1998. Cell and tissue culture: Laboratory procedures in biotechnology, John Wiley & Sons Ltd, 352 p. Holcik, J. 1989. The freshwater fishes of Europe, Vol I/II. General Introduction to fishes Acipenseriformes, Aula-Verlag Wiesbaden pp. 342-362. Nagahama, Y. 1994. Endocrine regulation of gametogenesis in fish, International Journal of Developmental Biology, 38: 217-229. Stoklosowa., S & Epler, P. 1985. The endocrine activity of isolated follicular cells of the carp ovary in prime culture, General and Comparative Endocrinology, 58: 386-393.

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Detection of IgM level in Persian sturgeon (Acipenser persicus) in the southern costs of Caspian Sea

Ali Hallajian*, Ayoub Yousefi Jourdehi, Rezvanollah Kazemi Agricultural Research, Education and Extension Organization (AREEO), International Sturgeon Research Organization of the Caspian Sea, Rasht, Iran, P.O.Box: 41635– 3464 [email protected]

Introduction Sturgeon is one of the oldest vertebrate in the world named a live fossil and is the most important economic aquatic species in the Caspian Sea, a habitat for five species of sturgeon specially Acipenser persicus. Blood is one of the most sensitive body tissues for animal and widely used in aquatic research. IgM concentration may be a valuable indicator of the general health of fish. Blood immunoglobulin levels are different depending on body size, temperature, season and pathogenic organisms in various fish species. However, immunoglobulin evaluation of multiple interactions in terms of body size, and pathological changes in the blood plasma concentration in wild sturgeon are not available. Also, little information is available about the extent and effects of the disease in a positive IgM levels in the blood plasma of wild fish populations. Therefore, this study aimed to determine the concentration of immunoglobulin M (IgM) in Persian sturgeon was caught by trawl in the Caspian Sea natural conditions coast.

Materials and Methods In this study, 40 Persian sturgeon fingerlings were caught using trawl at depths of 20 to 100 meters from the southern shores of the Caspian Sea in autumn during 2009 to 2010. After biometry, blood samples collected from the caudal vein using syringe 2 ml. IgM concentration stocked at -20°C for storage and was measured by Immunoturbidimetric method and using a spectrophotometer (Model 2100-VIS Manufacturing Co. Unico America) at a wavelength of 340 nm with the blank (distilled water) and the Pars kit test. Data analyzed using One- Way ANOVA and Tukey's test and for the comparison of whether or not significant difference the 95% confidence level. The software SPSS (Version20) and Excel software was used to draw graphs. The fish were divided into 6 groups based on body weight such as less than 100, less than 500, less than 1000, less than 2000, less than 5000 and less than 10000 g.

Results and Discussion Minimum, maximum and mean levels of IgM in blood of Persian sturgeon weighing 100 grams were 3.7, 117 and 43.6 ± 8.5 mg/dl; in the group weighing 500 grams were 3.5, 122 and 27.6 ± 6.3 mg/dl; in weight 1000 grams were 11, 78 and 33.9 ± 6.8 mg/dl; in weight 2000 grams were 70 and 25.4 ± 8 mg/dl; in the group weighing 5000 grams were 19, 42 and 30.5 ± 9 mg/dl and in weight group10000 g were 6, 19 and 10.8 ± 4.1 mg per deciliter, respectively (Figure 1). Based on obtained results there was no significant difference among different weight group of fish (P>0.05). The min and max level of IgM was related to group weights 100 and 10000 grams, respectively.

IgM(mg/dL) 0 100 500 1000 2000 5000 10000 Weight groups (g)

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Fig 1. IgM level at different weight groups in Acipenser persicus

Immunoglobulin has important functions such as neutralization, deposition, opozination (activity of supplement factors during phagocytosis by neutrophile and macrophage) and the activation of supplement factor and IgM as a specific immune factor. IgM is a specific immune factor that play a protection roles of antibodies such as virus neutralization attaching to bacteria and killing them, complements system activity and easier to swallow pathogens. Studies on wild Acipenser persicus activity reported IgM levels 24 ± 22.01 and 31.4 ± 9.34 mg/dl in males and females breeders, respectively. But in farmed Acipenser baerii with mean weight 26 gr was 12 mg/dl. Also IgM level in farmed Huso huso with mean weight 95 and 2 gr were 10.13 mg/dl and 0.21 g/l, respectively. IgM levels in wild, farmed, farmed infected VHS and farmed with kidney disease (PKD) Onchorhynchus mykiss were 3.3 ± 2.1, 2.2 ± 0.7, 4.2 ± 1.5 and 10.9 ± 5.3 and in the aquarium environment and in farmed trout 50 grams were reported 1.5 ± 0.4 and 0.051 ± 0.008 mg/ml, respectively. IgM level reported in Coho salmon, clupeid fish, gold fish, Tinca tinca, Cyprinus carpio and Paddlefish (Polydon spathula) were 2.1, 0.13 – 5.32, 4.7, 9.1, 1.7 and 17.2 mg/ml, respectively. In general, IgM concentrations in fish were in the range of 0.7 – 17.2 mg/ml. According to research reports, IgM concentrations measured in sturgeon between 2.0 to 32 mg/dl. Present study was also consistent with studies of other experts and the results were at the upper limit of that range; so that the amount of IgM enzyme was 32.5 mg/dl in Acipenser persicus. Data on environmental conditions showed that this quantity must consider the environmental impacts of key variables that may be affect the biochemical parameters.

References Acton, R.T.; Weinheimer, P.F.; Dupree, H.K.; Russel, T.R.; Wolcott, M.; Evans, E.E.; Schrohenloher, R.E.; Bennet, J. C., 1971. Isolation and charactenzatlon of the immune rnacroglobuhn from the paddlefish, Polyodon spathula. J. biol. Chem. 246: 6760-6769 Davis, C.R.; Marty, G.D.; Adkison, M.A.; Freiberg, E.F.; Hedrick, R.P. 1999. Association of plasma IgM with body size, histopathologic changes, and plasma chemistries in adult Pacific herring Clupea pallasi. Diseases of aguatic organisms dis. aquat. Org. Vol. 38: 125-133. Eslamloo, K.; Falahatkar, B.; Yokoyama, S. 2012. Effects of dietary bovine lactoferrin on growth, physiological performance, iron metabolism and non-specific immune responses of Siberian sturgeon Acipenser baeri. Fish & Shellfish Immunology. 32: 976-985. Estevez, J.; Leiro, J.; Santamarina, M.T.; Ubeira, F.M. 1995. A sandwich immunoassay to quantify low levels of turbot (Scophthalmus maximus) immunoglobulins. Vet Immunol Immunopatho l45: 165-174. Fuda, H.; Soyano, K.; Yamazak, F.; Hara, A. 1991. Serum immunoglobulin M (IgM) during early development of masu salmon (Oncorhynchus masou). Comp Biochem Physiol A Comp Physio l99:637-643. Olesen, N. J. & Vestergard Jsrgensen, P.E. 1986. Quantification of serum immunoglobulin in rainbow trout Salmo gairdneri under various environmental conditions. DISEASES OF AQUATIC ORGANISMS. Vol. 1: 183-189. Richter. RF.; Ambrosius, H. 1979. Nti-idiotypic antibodies of IgM-type produced in carp (Cyprinus carpio L.). Eur J Limmunol.9 (7):578–580. Vilam, C.; Wetzel, M,-C.; Du Pasquier, L.; Charlemagne, L. 1984. Structural and functional analysis of spontaneous antinitrophenyl antibodies in three cyprinid fish species: carp (Cyprinus carpio), goldfish (Carassiusa auratus) and tench (Tinca tmca). Dev. Comp. Irnrnunol. 8: 611-622. Voss, E.W.; Groberg, W. J.; Fryer, J. L. 1980. Metabolism of Coho salmon Ig. Catabolic rate of Coho salmon tetrarneric Ig in serum. Molec. Immunol. V 17: 445-452.

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Different methods for sexing in sturgeon

Rezvanollah Kazemi*, Ali Hallajian, Ayoub Yousefi Jourdehi, Mahmoud Mohseni, Mahtab Yarmohammadi, Mahmoud Shakourian Agricultural Research,Education and Extension Organization (AREEO), International Sturgeon Research Organization of the Caspian Sea, Rasht, Iran, P.O.Box: 41635– 3464

Fish are the most diverse and largest group of vertebrates having amazing reproduction process for protecting their population. Sexual maturation times are various in different fish species longed from several months to years depend on genus and species. Sturgeon matured later than other osteichthys fish and sexing is impossible at lower age morphologically. Using different technical methods for sexing in sturgeon is necessary because of impossible sex determination at lower age and led to decrease of rearing fee in farms by detecting males and removing them. Therefore, using of different methods such as surgery (biopsy), laparoscopy, ultrasonograpgy and hormonal tests are necessary. Based on results of different studies on sturgeon, endoscopy methods are less invasive and less stressor in fish and are suitable for sexing fish at age upper than 2 – 3 years. But surgery based on biopsy is more curative and suitable method for sexing fish at higher sexual maturity stages. The following images show the different methods of sex determination.

Laparoscopy surgery Ultrasonography

Biochemical Fourier transform Biopsy (Blood samples) infrared

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References Chebanov, M., Galich, E.V. 2013. Sturgeon hatchery manual. FAO. Fisheries and aquaculture technical paper. 338p Falahatkar, B., Tolouei Gilani, M.H., Falahatkar, S., Abbasalizadeh, A. 2011. Laparoscopy, a minimally-invasive technique for sex identification in cultured great sturgeon Huso huso. Aquaculture. 321 :273–279. Hallajian, A., Kazemi, R., Yousefi Jourdehi, A. , Pourdeghani, M., Application of some methods for sexual maturation stages detection in sturgeon. Journal of Fisheries Vol. 8, No. 1. Hallajian, A., Kazemi, R., Mohseni, M., Bahmani, M. and Yousefi, A. 2007. Determination of sexual maturation stages in cultured Acipenser nudiventris using biopsy method. Iranian Scientific Fisheries Journal. Vol. 16, No. 3. Hallajian, A., Kazemi, R., Mohseni, M., Dejandian, S., Yousefi Jourdehi, A., Bahmani, M., Pourdehghani, M.,Yazdani, M. A., Yeganeh, H. 2011. Biopsy and histologigal study of gonads from farmed Acipenser persicus. J.Vet.Res. 66,3:229-233 Hallajian, A., Kazemi, R., Yousefi Jourdehi, A., Pourdehghani, M., Dejandian, S., Yazdani, M. 2011. Identify the gender of laparoscopic Acipenser nudiventris farmed. Th2 Second National Conference of Fisheries and Aquaculture Science Lahijan. Iran. Webb, M.A.H., Feist. G.W., Foster. E.P., Schreck, C.B., Fitzpatrick. M.S. 2002. Potential classification of sex and stage of gonadal maturity of wild white sturgeon using blood plasma indicators. Transactions of the American Fisheries Society, 131: 132–142.

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Effects of photoperiod and light intensity on amylase activity during early larval development of Persian sturgeon Acipenser persicus

Farzaneh Noori*, Elahe Niazi, Rezvanollah Kazemi, Rasul Ghorbani

* Urmia Lake Research Institute, Urmia University, Urmia-Iran [email protected]

Introduction Sturgeon larvae are not fully developed at hatch and must undergo further development and differentiation before external food can be properly ingested and digested. The ontogenetic development of digestive enzymes reflects the development of the digestive tract and digestive capability of the organism under study and therefore be used as an indicator of nutritional status at an early life stage and can provide information for determining the appropriate time for weaning in fish culture. Alpha-amylase is produced as a zymogen granule by pancreatic acinar cells, and is responsible for the hydrolysis of the a-1,4 glycoside bonds in glycogen and starch to provide energy for the individual's metabolic activity and growth.

Materials and Methods In the present study, the effect of photoperiods and light intensity on development of amylase activity at early larval stage of Persian sturgeon was investigated. The experiment was conducted in six treatments, 24L with 50, 150 and 300 Lux (T1-T3) and 12L with 50, 150 and 300 Lux light intensity (T4-T6) and two controls (positive control, 0L and 10 Lux light intensity, PC; negative control, under natural photoperiod and light intensity condition, NC) and three replicates. To assay enzyme activities, 20 larvae at 1, 4 and 9 days post hatch (dph) were randomly collected from each tank and homogenized in 1:3 (w/v) of ice-cold 50mM Tris-HCl buffer, pH 7.5, using a Polytron PT 1300 D homogenizer. The homogenate was centrifuged and the supernatant was collected according to Chong et al., 2002. Total soluble protein of supernatant was determined by the Bradford method using bovine serum albumin as the standard (Bradford 1976). The Alpha-amylase (E.C.3.2.1.1) activity was determined according to Bernfeld (1951) using starch as substrate. The α-amylase specific activity was defined by the μmol of maltose produced per min-1 mg-1 protein.

Results and Discussion The results indicated that the amylase activity was already detected at 1 dph larvae in all treatments however the values sharply increased in 9 dph larvae. The lowest amylase activity was observed in the treatment under 24 hours photoperiod and 50 lux light intensity in day 1 that had significant difference with positive and negative control. Moreover we observed that at the end of experiment the larvae treated with 12 hours photoperiod and 300 lux light intensity showed highest amylase activity in compare to other treatments although had no significant difference with positive control (Table 1).

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Table 1. Effects of photoperiod & light intensity on amylase activity in Persian sturgeon, Acipenser persicus Light Treatment Photoperiod intensity Day 1 Day 4 Day 9 (Lux) T1 24L 50 1.979±0.088h++- 2.059±0.213gh++-- 2.674±0.118c++- T2 24L 150 2.325±0.031defgh+ 2.213±0.098efgh++-- 2.570±0.159cd++-- T3 24L 300 2.430±0.208cdef 2.282±0.072defgh+ 2.61±0.267cd++- T4 12L 50 2.694±0.182c- 2.150±0.085fgh++- 3.251±0.264b T5 12L 150 2.725±0.068c- 2.275±0.191defgh++ 3.288±0.238b T6 12L 300 2.517±0.265cde 2.392±0.236cdefg++ 3.613±0.486a Positive 0L 10 2.657±0.095B 2.975±0.234B 3.504±0.317A Control (PC) Negative Natural - 2.364±0.178B 2.653±0.425B 3.429±0.56A Control (NC) photoperiod *Small similar superscript letter indicate no significant difference between treatments and big different superscript letter indicate significant difference in the same row. *Positive (+) and negative control (-) was compared with treatments in the same column.

Conclusion In conclusion, the results indicate that continues light has negative impact on amylase activity. In addition, the detection of amylase at early larval stage indicates that the Persian sturgeon larvae are able to synthesize amylase prior to commencement of the exogenous feeding and that its secretion is genetically programmed.

References Bernfeld, P. 1951. Amylases α and β. In: Colowick P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic Press, New York, pp: 149-157. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the protein-dye binding. Analytical Biochemistry 72, 248-254. Chong, A., Hashim, R., Lee, L.C. & Ali, A. 2002. Characterization of protease activity in developing discus Symphysodon aequifasciata larva. Aquaculture Research 33, 663-672.

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The influence of stocking density on blood indices of ship sturgeon (Acipenser nudiventris) juveniles in Khuzestan province

Mohammad Yooneszadeh Feshalami*, Seyed Abdolsaheb Mortazavizadeh, Farokh Amiri, Mansour Nikpey South Iran Aquaculture Research Center, Ahwaz, Iran [email protected] Introduction The Caspian Sea sturgeons including ship sturgeon (Acipenser nudiventris Lovetsky, 1828) is considered as critically endangered species in the Red List of the IUCN, mainly due to their over exploitation, poaching, hydro-geological alteration of their habitats, environmental pollution, as well as due to the destruction of their natural spawning sites (Salehi, 2011; IUCN, 2015).

Materials and Methods In addition, stocking density is one of the most important factors, which can directly affect welfare; physiological and behavioral responses of fish in intensive aquaculture systems Sturgeons used in the present study were obtained from the International Sturgeon Research Institute and then transferred to a commercial farm (Dezful, Khuzestan, Iran) where the trial was carried out. Ship sturgeon (BWi = 93.7 ± 0.9 g) juveniles were randomly distributed in eighteen concrete square ponds at 3 different stocking densities[low stocking density 1.5 kg m-2; , 3 kg m-2and 6 kg m-2. The number of fish for each stocking density for ship was 31, 62 and 125 fish per pond respectively. Each experimental condition was tested in triplicate. Feed was offered four times every day using a commercial diet for 2 months. After 30 days and at the end of experimental period (day 60), blood was collected from the caudal vein in 9 fish (n = 3 fish per replicate) with heparinized syringes in order to evaluate several hematological and plasma biochemical parameters. Data were analyzed using SPSS ver.15.0 and these effects were evaluated separately by a one way ANOVA. Results and Discussion In the current study, hematological parameters were not affected by the stocking density in A.nudiventris (P > 0.05) (Table 1). At 30 days, stocking density did not affect the plasma cortisol levels in both species (P > 0.05); however, the plasma cortisol increased in ship sturgeon juveniles at day 60 (Table 2). Plasma glucose levels were affected by neither stocking density nor sampling times in both species (P > 0.05). However, this parameter did not change among different groups in ship sturgeon juveniles (P > 0.05).

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Table 1 Hemato-immunological parameters of Asipencer nudiventris reared in different stocking densities.

Sampling time SD RBC Hct WBC Monocytes Eosinophils Neutrophils Lymphocytes (kg/m2) (×106 μl-1) (%) (×103 μl-1) (%) (%) (%) (%)

1.5 1.4 ± 0.5 24.3 ± 0.3 22.4 ± 3.0 - 6.0 ± 2.0 B12.0 ± 5.8 81.7±7.9 30 3 1.3 ± 0.3 25.3 ± 0.7 21.3 ± 3.0 - 8.1 ± 1.6 B12.7 ± 5.4 78.7 ± 7.3 6 1.4 ± 0.1 22.6 ± 0.5 21.2 ± 2.0 1.2 ± 0.2 9.2 ± 4.5 13.4 ± 7.6 76.5 ± 8.8

1.5 1.3 ± 0.3 26.9 ± 1.6 31.6 ± 6.5 - 5.0 ± 3.0 A15.1 ± 7.0 81.2 ± 6.0 60 3 1.7 ± 0.2 28.7 ± 1.0 24.2 ± 3.3 - 4.8 ± 2.5 A15.8 ± 8.0 78.0 ± 6.6 6 1.6 ± 0.2 29.1 ± 1.9 33.4 ± 7.4 - 5.8 ± 1.5 13.0 ± 1.5 71.2 ± 18.0

Table 2 Stress indices of Asipencer nudiventris reared in different stocking densities.

Species Sampling SD Cortisol (nm ml- Glucose (g dl-1) time 1) Acipenser nudiventris 1.5 6.3 ± 0.9b 70.6 ± 4.8 30 3 6.4 ± 0.8b 64.0 ± 4.5 6 6.6 ± 0.6b 67.1 ± 5.8

1.5 17.4 ± 3.3a 73.7 ± 3.2 60 3 16.6 ± 3.5a 68.6 ± 3.9 6 17.0 ± 5.5a 70.8 ± 3.2

Reference Salehi, h. 2011.Stock enhancement of sturgeon fishes in Iran. In: Recent Advances in Fish Farms. Eds: F. Aral, InTech Publications, Rijeka, Croatia. pp. 107–116.

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Changes in sex steroid hormones of Siberian sturgeon (Acipenser baerii) during vitellogenesis in captivity

Yalda Hooshyar, Ali Bani*, MohammadAli Yazdani Sadati, Rezvanollah Kazemi *Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran & Marine Science Department, Caspian Sea Basin Research Center, University of Guilan, Rasht, Iran. E-mail: [email protected]

Introduction The role of hormones in reproductive physiology of animals is clearly known (Baranikova et al., 2004). Information and knowledge about the synthesis and effects of sex steroid hormones in different seasons, mechanisms related to the growth and maturation of gonads, secondary sexual characteristics and sensory neural control of reproduction are essential components of reproductive physiology (Nagahama et al., 1993). Previous research investigations have shown changes of steroid hormones in teleost fishes but fluctuations of sex hormones in sturgeon is not completely studied mostly due to prolonged gonadal development process (Dabrowski et al., 1996). This study investigated the changes of the steroid hormones, 11- ketotestosterone (11 KT), testosterone (T), 17β-estradiol (E2) and 17α-Hydroxyprogesterone (17α-OHP) and gonadal development of Siberian sturgeon Acipenser baerii.

Materials and Methods In the beginning of experiment, sexual maturity of 16 fish including 7 females (average weight 3.49±0.21 kg and the mean total length 88.68±1.39 cm) and 9 males (average weight 3.1±0.31 kg and the mean total length 86.70±1.71 cm) were determined using biopsy and histological survey. To evaluate gonad development of fish during the experiment, laparoscopy was used in the third season of experiment (winter). Blood samples were collected from the caudal vein. The collected plasma was stored at -20°C for further analysis. Plasma steroid concentrations, including 11-KT in males, E2 in females and T and 17α-OHP in both sexes, were measured seasonally by Radioimmunoassay (RIA).

Results and Discussion Oocyte diameter in females differed significantly at different stages of sexual maturity (Fig. 1), ranging in stage II 282≥D≥144, in stage III 394≥D≥292 and stage IV-a 394<(μm).

Figure 1: Average diameter (mean ± SE) of oocyte in ovaries at different stages of sexual maturity in Siberian sturgeon. Sex steroids in male and female Siberian sturgeon showed correlation with season and gonadal development. 11-KT levels in fish were different in different seasons for males in stage II and IV (P<0.05). The highest concentration of 11-KT (8.1±0.6 ng/ml) was observed in stage IV of maturity when spermiation was occurring (Fig. 2). The level of plasma 11-KT was much higher in males than females. Plasma E2 concentrations increased gradually in

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir stage II females before reaching to its highest level (104±21.55 ng/ml) in spring. Peak of E2 (102.66±4.33 ng/ml) in stage III occurred in summer (Fig. 2). Plasma concentration of T in both sexes differed significantly throughout the seasons. Plasma concentration of T increased with gonadal development with a peak (67±2.5 ng/ml) occurring in stage IV. The level of plasma 17α-OHP in stage II of maturity had fluctuation in both sexes and varied seasonally (P<0.05).

Fig 2: Seasonal changes (mean±SE) in plasma levels 11-KT (males) and E2 (female) in Siberian sturgeon Acipenser baerii in stages II, III and IV.

The results showed that steroid hormones (11-KT, E2, T and 17α-OHP) levels in Siberian sturgeon vary in different stages (II, III and IV) of gonad maturity. Although there is seasonal changes in 11KT levels in fish (especially in stage II and III), the level of this hormone was much higher in males compared with females suggesting that this hormone can be considered for sex differentiation in Siberian sturgeon. Barannikova et al., 2004 showed that the level 11- KT increases during gonad development in males. Results indicated that E2 plays an important role in Siberian sturgeon reproductive cycle. E2 concentrations decreased as vitellogenesis completed as it has been shown by Kime, 1993. T plasma levels in Siberian sturgeon, was lower than the level reported for white sturgeon (161.7 ng/ml) (Webb et al., 2001), stellate sturgeon (166.6 ng/ml) (Semenkova et al., 2002 ) and Russian sturgeon (184.8 ng/ml) (Barannikiva et al., 2006). In conclusion, the levels of steroid hormones in Siberian sturgeon are associated with season, gonad maturity and also gender.

References Barannikova, I. A., Bayunova, L. V and Semenkova, T. B. 2004. Serum levels of testosterone, 11-ketotestosterone and oestradiol-17b in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment. Journal of Fish Biology. 64: 1330–1338. Barannikova, I. A. and Semenkova, T. B. 2006. Serum sex steroids and their specific cytosol binding in the pituitary and gonads of Russian sturgeon (Acipenser gueldenstaetdtii Brandt) during final maturation. Journal of Applied Ichthyology. 22: 331-333.

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Dabrowski, K., Ciereszko, R. E., Ciereszko, A., Toth, G. P., Christ, S. A., El-Saidy, D and Ottobre, J. S. 1996. Reproductive physiology of yellow perch (Perca flavescens): environmental and endocrinological cues. Journal of Applied Ichthyology. 12: 139–148. Kime, D. E. 1993. Classical and non-classical reproductive steroids in fish. Reviews of Fish Biology and Fisheries. 3: 160–180. Nagahama. Y., Yoshikumi, M., Sakai, N and Tanka, M. 1993. Molecular endocrinology of oocyte growth and maturation. Fish Physiology and Biochemistry. 11: 3-14. Semenkova, T., Barannikova, I., Kime, D. E., McAllister, B. G., Bayunova, L., Dyubin, V and Kolmakov, N. 2002. Sex steroid profiles in female and male stellate sturgeon (Acipenser stellatus Pallas) during final maturation induced by hormonal treatment. Journal of Applied Ichthyology. 18: 375–381.

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Assessing how juvenile beluga sturgeon responds to salinity exposure in terms of growth and osmoregulation

Seyed Mostafa Aghilinejhad* , Ali Jalali * Agriculture Services Specialized Parent Company, Sturgeon Affairs Management, Gorgan, Golestan Province, Iran; E-mail address: [email protected]

Introduction There is a need for better understanding of how sturgeons respond to environmental challenges especially when hatchery reared juveniles are released into natural waters for restocking. There has been a variety of studies assessing osmoregulatory mechanisms and salinity tolerance in sturgeon species and it has been documented that many species of sturgeons are able to adapt to different salinity ranges (Krayushkina 1998; Jarvis & Ballantyne 2003; Allen & Cech 2007; Zhao et al. 2010). However, having detailed information about the salinity effects on different age and size classes and during a various exposure times can provide us a better understanding of sturgeon responses to environmental challenges. It can also be a useful method to aid restocking actions especially when the fish are directly released into the estuary and sea water or are kept for coastal-based-rearing and aquaculture purposes. Thus, in this study we selected filed-based salinity doses of 0, 3, 6, 9 and 12 ppt representing salinity ranges that the beluga are likely to encounter in the Iranian part of the Caspian Sea. We examined the effects of these salinities on osmoregulatory characteristics and performance of juvenile beluga sturgeon, Huso huso.

Materials and Methods Five months old beluga with a mean body weight of 22.1±1.1 g (mean ± SD) were stocked in five groups (triplicate per group) and cultured at salinities of 0, 3, 6, 9 and 12 ppt for 60 days. Acclimation to salinity was done by increasing water salinity at an approximate rate of 3 ppt per day until reaching 12 ppt. At the end of the experiment, all fish were weighed and growth parameters were calculated. The blood was collected to evaluate the haematocrit (% PCV), + + 2+ 2+ ions (sodium [Na ], potassium [K ], calcium [Ca ] and magnesium [Mg ]) and glucose concentrations. Data were analyzed by one-way analysis of variance (ANOVA) with Duncan’s new multiple range tests.

Results Survival rate was relatively high in all treatments at the end of the experiment although there was a sign of adverse effects of salinity on the survival as fish at 12 ppt salinity showed lower survival. Growth performance was better in fish reared at 3 ppt, followed by 6, 0, 9 and 12 ppt + + 2+ 2+ (Table 1). Overall, an increase in plasma Na , K , Ca and Mg levels was found in association with the increase of salinity, while the freshwater control group maintained basal levels (Table 1).

Discussion Based on the results obtained it can be concluded that juvenile beluga sturgeon are capable to acclimate and survive in brackish water. However, it should be noted that initial salinity adaptation as well as fish size play key roles in survivorship and growth rates in saline environments because it has previously been indicated that small individuals have a poor salinity tolerance and larger fish show less sensitivity due to the structural and physiological

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developments during their ontogeny (Altinok et al. 1998; Cataldi et al. 1999; Allen et al. 2011). Successful osmoregulation capability may lead the selection of beluga sturgeon as a good candidate for pen and cage culture in brackish water of the Iranian area of the Caspian Sea. In addition, the ability to tolerate brackish water may aid restocking efforts by providing managers more information about the responses of a unique Caspian sturgeon, the beluga, when exposed to field-based salinity doses.

Table 1. Responses of juvenile Huso huso after a 60-day rearing period at various salinities. Means in the same row with different superscripts are significantly different (P<0.05); Data are presented as mean ± SD.

Parameters Treatments Salinity 0 Salinity 3 Salinity 6 Salinity 9 Salinity 12

Survival (%) 93.3±1.5 a 92.3±1.5 ab 92±2.0 ab 90±1.0 b 87±1.0 c Final weight (g/fish) 102.2±3.7 a 105.5±2.6 b 103.3±3.6 a 97.8±3.1 c 97.0±2.7 c Final length (cm) 31.0±1.1a 32.3±1.1b 30.9±1.7 a 28.8±1.7 c 28.5±1.4 c Specific growth rate 2.53±0.01a 2.61±0.02 b 2.57±0.01 b 2.46±0.03 c 2.45±0.01c Condition factor 0.33±0.005 a 0.30±0.01 b 0.34±0.005 a 0.40±0.01 c 0.41±0.01c Na+ (meq/l) 137.7±4.5 a 141.7±3.4 ab 145.3±4.3 b 155.9±4.05 c 156.4±4.5 c K+ (meq/l) 2.68±0.04 a 2.72±0.05 a 2.81±0.04 b 2.83±0.04 bc 2.87±0.06 c Ca2+ (meq/l) 6.15±0.09 a 6.33±0.06 b 6.51±0.07 c 6.51±0.04 c 6.53±0.07 c Mg2+ (meq/l) 0.79±0.04 a 0.83±0.07 ab 0.9±0.06 b 0.9±0.09 b 0.9±0.1 b

References Allen PJ, McEnroe M, Forostyan T, Cole S, Nicholl MM, Hodge B, Cech JJJr. 2011. Ontogeny of salinity tolerance and evidence for seawater-entry preparation in juvenile green sturgeon, Acipenser medirostris. J Comp Physiol B. 181: 1045–1062. Allen PJ, Cech JJJr. 2007. Age/size effects on juvenile green sturgeon, Acipenser medirostris, oxygen consumption, growth, and osmoregulation in saline environments. Environ Biol Fish. 79: 211-229. Altinok I, Sara MG, Frank AC. 1998. Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchus de Sotoi. Comp Biochem Physiol. 120: 609-616. Cataldi E, Barzaghi C, Di Marco P, Boglione C, Dini L, McKenzie DJ, Bronzi P, Cataudella S. 1999. Some aspects of osmotic and ionic regulation in Adriatic sturgeon Acipenser naccarii. I: ontogenesis of salinity tolerance. J Appl Ichthyol. 15: 57-60.

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Jarvis PL, Ballantyne JS. 2003. Metabolic responses to salinity acclimation in juvenile shortnose sturgeon Acipenser brevirostrum. Aquaculture. 219:891-909. Krayushkina LS. 1998. Characteristics of osmotic and ionic regulation in marine diadromous sturgeons Acipenser brevirostrum and A. oxyrhynchus (Acipenseridae). J Ichthyol. 38: 660-668. Zhao F, Zhuang P, Zhang LZ, Hou JL. 2010. Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon (Acipenser schrenckii). Chin J Oceanol Limnol. 28: 603–608.

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Differential count of White blood cells in lethal concentration (lc 1h) of Zataria multiflora and Allium sativum in Persian sturgeon, Acipenser persicus, fingerlings

Soheil Bazari Moghaddam*, Mostafa Sharif Rohani, Masoud Haghighi, Alireza shenavar Masouleh, Jalil Jalilpour , Mehdi Masoumzadeh, , Mehdi Alizadeh and Bahareh Younes Haghighi *Researcher of Agricultural Research Education and Extension Organization (AREEO), International Sturgeon Research Institute, Rasht, Iran, P.O.Box: 41635 - 3464 [email protected]

Abstract The importance of using herbal drugs for campaigning against infectious and non infectious diseases is obviously confirmed and it is necessary that these drugs be replaced with chemical drugs in aquaculture industry. This study was carried out to determine (lc 1h) for hydroalcoholic extracts of Zataria multiflora and Allium sativum in Persian sturgeon (Acipenser persicus) fingerlings for future application studies. The research was performed on Persian sturgeon fingerlings having 3.22±0.51g weight in static environment. In order to study the effects of these extracts, 270 fingerlings were selected for each extract in 8 treatments and one control group (3 replications for each treatment) and a total of 54 tanks having 30 liter volume were used. During the experiment the temperature, dissolved oxygen and pH were determined. In this study, the rate of nitrite, nitrate, ammonium, electric conductivity and water hardness were measured in different treatments. This experiment was conducted according to OECD method during 1h. In this survey according to Probit analysis, LC10, LC50 and LC90 for hydroalcoholic extract of Zataria moltiflora were, respectively 8521.18, 9933.44, 11582.43 mg/l, also LC10, LC50 and LC90 for hydroalcoholic extract of Allium sativum were 11748.97, 12624.08 and 13326 mg/l, respectively. The results showed that the mortality of sturgeon fingerlings occurred at lower concentration of hydroalcoholic extract of Zataria moltiflora compared to hydroalcoholic extract of Allium sativum. Differential counts of white blood cells indicate a statistically significant difference in the amount of lymphocytes, neutrophils, and eosinophils between treatment and control groups.

Keywords: White blood cells, Zataria multi flora, Allium sativum, Persian sturgeon, Lethal concentration

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Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Bester juvenile Ali Hossienpour Zelti*, Mir Hamed Sayed Hassani, Forouzan Bagerzadeh Lakani, Alireza Shenavar, Mohamad Ali yazdani Sadati, Hooshang Yegane, Mohebali Pourgolam, Alireza Alipour, Javad Syad far, Ghorban Kamranjo * International Sturgeon Research Institute (Areeo), Rasht, Iran. [email protected] Introduction Bester which is produced by crossing beluga (Huso huso) female and sterlet (Acipenser ruthenus) male, is suitable for aquaculture because it shows better growth rates than its males and earlier sexual maturation than its female parents (Burtsev, 1997). In intensive culture system, improving fish performance and disease resistance are major challenges facing fish culturists. Moreover, bacterial diseases are one of the limiting factors for fish intensive culture (Rahman et al., 1997, 2001; Li et al., 2006). Also, the intensive use of antibiotics to prevent and control bacterial diseases in aquaculture has led to an increase in antibiotic-resistant bacteria (Alderman and Hastings, 1998; Teuber, 2001). Therefore, several alternative strategies to the use of antimicrobials have been proposed, such as the use of probiotics as biological control agents. Probiotics, live microbes that may serve as dietary supplements to improve fish growth and immune responses, have received some attention in aquaculture (Gatesoupe, 1999; Irianto and Austin, 2002; Kesarcodi-Watson et al., 2008). Whereas, in future, beside H. huso and Acipenser baerii, bester will be one of the most rearing species in intensive culture systems in Iran. This investigation carried out to determine effects of dietary commercial aquaculture probiotic (CA probiotic) on growth performance and immune system of Bester juvenile.

Materials and Methods In the present study, 240 Bester fingerling with average weight of 10.5±0.92 g were randomly distributed into 12 fiberglass tanks. A CA probiotic (Superzist, Zistyar Varna, Iran) comprising Bacillus subtilis, Lactobacillus acidophilus, L. delbrueckii, L. rhamnosus, L. plantarum were used. Different CA Probiotic treatments with 3 replicates were examined. Treatments included control (PR0, no added CA Probiotic), 100 (PR100), 200 (PR200) and 300 (PR300) g/kg CA Probiotic. The control diet was a commercial fish food; (1.9 mm pellets, Biomar, France). Proximate composition of the diets was (% of dry diet from manufacturer’s guidelines): Crude lipid 16; Crude protein 47; fibre 2.3. Fish were fed 3% body weight three times daily. At the end of the trial (8 weeks), humoral innate immune parameters (serum Igm levels, lysozyme activity and alternative complement activity (ACH50)) and growth factors (final weight, weight gain (WG), specific growth rate (SGR), food conversion ratio (FCR), feed efficiency ratio (FE), body weight increase (BWI) and condition factor (CF)) were assessed.

Results and Discussion The predominant bacterial species that isolated from most of the fish digestive tracts have been reported to be aerobes or facultative anaerobes (Bairagi et al., 2002; Saha et al., 2006). The isolated many lactic acid bacteria are proved to function as probiotics, which are beneficial to host health, when ingested in sufficient quantities. The colonization of the gut by

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir probiotic bacteria prevents growth of harmful bacteria by competitive exclusion and by the production of organic acid and antimicrobial compounds. In the present study, there were no significant difference in growth factors between treatments (P>0.05) but growth performance increased by using 300 mg/ kg CA Probiotic compared to control. Highest WG, FE, SGR and lowest FCR belonged to fish fed PR300. There were some reports that spores of Bacillus toyoi and other Bacillus sp. increased the growth rate of yellow tail, Seriola quinquiradiata (Kozasa, 1986), turbot Scophthalmus, maximus (Gatesoupe 1991; 1994), common snook, Centropomus undecimalis (Irianto and Austin, 2002). Similarly, the commercial preparations of Streptococcus faecium and a mixture of bacteria and yeast improved the growth and FE of Cyprinus carpio (Bogut et al., 1998) and Catla catla (Mohanty et al., 1996), respectively. In the present study, there were significant difference in serum Igm levels and ACH50 (P>0.05). PR200 treatment showed the maximum values of Igm and ACH50 but no significant difference in the lysozyme activity between treatments were observed (P>0.05). The development of an immune response toward foreign antigens of microbial and viral origin provides an essentially systematic way through which potentially pathogenic organisms are recognized and controlled. The present findings indicate that inclusion of 200 mg/ kg CA probiotic positively enhanced the growth performance and immunological parameters in bester providing them a higher resistance against stress conditions and pathogens.

References Al-Harbi, A.H. and Uddin, N., 2003. Quantitative and qualitative studies on bacterial flora of hybrid tilapia (Oreochromis niloticus×O. aureus) cultured in earthen ponds in Saudi Arabia. Aquaculture Research, 34: 43- 48. Bairagi, A., Ghosh, K.S., Sen, S.K. and Ray, A.K., 2002. Enzyme producing bacterial flora isolated from fish digestive tracts. Aquaculture International, 10: 109-121. Irianto, A., Austin, B., 2002. Probiotics in Aquaculture. Journal of Feed Diseases, 25: 1-10. Saha, S.B., Bhattacharyya, C. and Choudhury, A., 1999. Preliminary observations on culture of Penaeus monodon in low saline waters. Naga, 22 (10): 30-33. Vaseeharan, B. and Ramasamy, P., 2003. Control of pathogenic Vibrio spp. By Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Applied Microbiology, 36: 83–87.

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Purification and Characterization of Lysozyme from Persian Sturgeon, Acipenser persicus

Roxana Badan-Ara*, Mahmoud Reza Aghamaali, Mohammad Reza Nowruzfashkhami *Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran [email protected] Introduction Lysozyme, also known as muramidase or N-acetylmuramideglycanhydrolase, plays a critical role in innate immune system. The enzyme acts as an antibacterial agent by hydrolyzing glycosidic bonds of bacterial cell wall. Besides, it is a powerful antimicrobial commonly used as a preservative in foods and beverages and as a microbial cell lysis reagent[1]. The aim of the present study was purification and characterization the lysozyme from Acipenser persicus.

Materials and Methods Eighteen fries of Persian sturgeon (0.6 g weight) were used in this study. The fries were homogenized in 0.1 M phosphate buffer pH 6.2 and the suspension was centrifuged at 9000rpm for 20min at 4°C. Then, the supernatant was dialyzed and subjected to S-sepharose column chromatography and partially purified enzyme was achieved. After purification, some of the biochemical properties such as temperature and pH profile were investigated. Moreover, SDS-PAGE analysis (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was used to estimate the purity and molecular mass of the enzyme[2].

Results Based on the results, SDS-PAGE analysis confirmed the purified band is about 14kDa. Furthermore, temperature and pH profiles disclosed the optimum activity is at about 50 °C and 6.5, respectively.

100 100 80 80

60 60

40 40 (%) (%) 20 20 0 0

20 25 30 35 40 45 50 55 60 2 3 4 5 6 7 8 Remaining activity activity Remaining Remaining activity activity Remaining Temperature pH

Fig 1: Temperature profile (left) and pH profile (right) of the lysozyme.

References Misook, K., Minjeong, P. and Yoonhwa, J. 2012. Purification and characterization of lysozyme from Filipino Venus, Ruditapes Philippinarum: Food science and Biotechnology, 21(5): 4163-146. Qing-Gang Xue., Kevin, L., Aswani, K. Volety, Fu-Lin E. Chu, Jerome La Peyre, F. 2004. Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica). Comparative Biochemistry and Physiology, 139: 11 –25.

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Study on applying counting number of sperm, measuring spermatocrite percent and spectrophotometry in determining sperm concentration in Huso huso of Caspian Sea

Alireza Alipour, Bahareh Younes Haghighi, Ali Hosseinpour, Ghorban Kamranju

International Sturgeon Research Institute (Areeo), Rasht, Iran. P.O.Box: 41635 - 3464

Introduction Huso huso is one of the most valuable and economic species of sturgeon in Caspian Sea. Sturgeons have been regarded as endangered species. Unfortunately, illegal catch, destroying of natural habitats and increasing of pollutions have been lead to reduction in sturgeon stocks. Obtaining enough and high quality milt in propagation time and management of the procedure can be regarded as a determinant index to success artificial propagation in hatcheries. Nowadays, some studies have been carried out on sperm motility of fishes in different centers in order to improve fertility and control of reproduction for economic aquaculture and stocks conservation and rehabilitation. Sperm of many species is immotile in testicle. Spermatozoids can be activated only after releasing in external environment. Their movement period is very short. The most important characteristic of sperm is its motility. This trait of sperm is favorite for physiological studies and can be a simple way to assess semen quality.

Materials and Methods In this study, quantitative characteristics of Huso huso sperm were determined through investigating spermatocrit and counting of sperm number by hemacitometer (Eslambolchi, 2008). This study was carried out on 5 male of Huso huso. In order to stimulate sexual maturation, GnRH was injected to male spawners as 2-3mg per kilogram of body weight (Dettlaff & Schmalhausen; 1993). After 15-25h, sperm sample were collected considering the temperature of the water. The collected sperm samples were investigated based on the following methods: - Hemacitometer lamella was used to determine spermatozoid concentration and number of sperm in a definite volume. -To study spermatocrite in each sample, special standard capillary tubes (with 1-1.2mm inner diameter) were used (Suquet et al., 1992). The obtained data analyzed by SPSS and Excel 2000 software.

Results and Discussion The mean sperm density in 5 collected samples of Huso huso was determined to be 1.08±0.389 by microscopic investigation and hemacitometer lamella. In this study, the minimum and maximum sperm concentration was 0.49 ± 109 and 1.53×109cell/ml, respectively. The mean percent of spermatocrite in five sperm samples was 5.1±2.01%. The minimum and maximum percent of spermatocrite were 3% and 8%, respectively.

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Discussion According to the study, it was determined that Huso huso sperm is immotile in plasma of semen. Only after dilution in water or salt solutions it can be activated. This situation is reported in sperm of other sturgeon species too (Linhart et al., 1995). According to the results of this study, the mean sperm concentration of Huso huso was 1.08±0.389 milliard cell per milliliter of semen. Furthermore, the minimum and maximum number of milts obtained was in a wide range. Other researchers also reported on the great differences in changes of sperm concentration. In the recent study, the mean spermatocrite percent in Huso huso was determined as 5.1±2.01%. The change range was very wide, so as the maximum and minimum spermatocrite percent were different in sperm of various sturgeon spawners that depends on different factors. In this study, statistical analysis and mathematical relations, correlation coefficient and Pearson’s correlation coefficient showed that there is significant relation between spermatocrite percent and sperm concentration (number of sperm) which showed strong correlation (r was more than 0.97). The results showed that it is better and more confident to assess Huso huso sperm based on measuring spermatocrite percent and counting number of sperms by hemacitometer lamella in order to enhance propagation of Huso huso in private and governmental hatcheries. Also, this technique can be used as an effective and applicable method for propagation in sturgeon hatcheries and farms which leads to increase in fertility and production. The results showed that there is significant correlation between spermatocrite percent and number of sperm (sperm concentration) in Huso huso. Each of these methods can be used to assess sperm concentration of this species.

References Eslambolchi, S. 1999. Assessment of sperm concentration in carp, Ctenopharyngodon idella and Acipenser stellatus by spectrophotometry. Thesis on B.sc. degree, College of fisheries and environmental sciences, Gorgan university of Agricultural Sciences and Natural Resources, 49p. Hashemi, M. 1996. Artificial insemination in cattle (reproduction Physiology and artificial insemination), Farhan Jame Publishing, second edition.

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Effect of supernurients on some stress indices in Siberian sturgeon (Acipenser baerii) Hedieh Haghighi*, Mahmoud Bamani, Abbasali Zamini , Ayoub Yousefi Jourdehi

* Guilan Sciences and Research Islamic Azad University

Introduction Nowadays, necessity of aquatic organic production is important for protection of species and availability of protein stocks required for mankind and also for food safety and biological immunity. “Organic animals” means products without types of pollutions and "stress free". Antibiotic usage has lateral disasters in aquatic animals such as bacteria resistance or transferring some part of them to consumers. Nowadays, many different antibiotics are used for prevention, treatment and also as a nutrients growth promoter. Present research was carried out with the aim of determining super effect on some stress indices in juvenile Acipenser baerii.

Materials and Methods Practical phases of this study was carried out at the international Sturgeon Research Institute and glucose and cortisol levels measured in VIRO MED laboratory in Rash during 6 months. Blood samples obtained in days 45 (stage I) and 90 (stage II) and plasma were provided. In this study 180 Siberian sturgeon (mean weight 680.89 ± 29.93 g and mean length 63.79 ± 1.18 cm) were prepared and after two week of adaptation, fish were randomly introduced to 15 fiberglass tanks with a volume of 2000 litres. The fish were fed diet of Skretting Company. Super supplements at 2.5 % (T1), 5 % (T2) and 10 % (T3) were added to the diet and experiments were performed in triplicate.

Results Glucose level showed significant difference in different treatments compared to control at stage II (P<0.05). Cortisol level increased significantly in treatments compared with control at time stage I and II (P<0.05). Based on obtained results, super nutrients adding in diet of Acipenser baerii caused a significant difference in biochemical and stress indices and improved them. Therefore it is recommended the use of super nutrients in Siberian sturgeon diet (Fig 1, 2).

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Control -Control + T 1 T 2 T 3 100

b 80 a ab b a Golucose (mg/dl) 60

مرحله 2 مرحله 1

Fig. 1. Changes in plasma glucose levels in Acipenser baerii

Control Control T1 T2 T3 - + 350 b 300 Cortisol ab ab ab (ng/mg) 250 b a ab ab 200 ab

a 150 100 90 50

0 Stage 1 Stage 2

Fig 2. Changes in plasma cortisol levels in Acipenser baerii

Discussion Different studies showed that some factors such as age, environmental status and stress and food regime can be effective on blood biochemical indices. Changes in these levels can be used for complete evaluation in fish species. Cortisol level was measured for detection of stress acute and there was significant difference in cortisol level changes in sturgeon. Cortisol is one of the most important glycocorticoid hormones and its level is increased by inducing stress and is a suitable index in fish response to stress (Fevolden et al., 2002). Results showed acute stress led to increase in serum cortisol level in Acipenser gueldenstaedtii and Acipenser stellatus (Bukovskaya et al., 1997). Glucose and cortisol levels in blood serum are the most important indices for stress detection (Barton et al., 1989). It is confirmed that cortisol had effective role in metabolism of energy, osmoregulation and response to stress. Glucose level

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References Pahang, P. 2012. Evaluation the effect of organic culture pattern using supernutrients on some biochemical and carcass analysis in common carp (Cyprinus carpio). MS.c thesis. With guidance Mahmoud Bahmani. 128 P. Naraghizadeh, N. 2013. Evaluation the effect of organic culture pattern using supernutrients on some biochemical and carcass analysis in common carp (Cyprinus carpio). MS.c thesis. With guidance Mahmoud Bahmani. Barton, B.A. and Iwama, G.K.1991.Physiological changes in fish stress in aquaculture with emphasis on the response and effect corticosteroids .Ann.Rev.of Fish Diseases.1:3-26. Wedemeyer, G.A., Barton, B.A. and Mcleay, D.J.1981.Stress and acclimation schreck C.B.and Moyle P.B. (Eds) In: Methods for Fish Biology .451-490.

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Cholesterol and triglyceride level changes in farmed Acipenser nudiventris fingerlings exposed to acute toxicity of atrazine Marzieh Naji*, Ayoub Yousefi Jourdehi, Sourena Abdali , Mohammad Ali Yazdani Sadati

*Tehran North Islamic Azad University

Introduction Atrazine is one of the most consuming and major herbicide in the world during last 40 years. In Iran, this toxin was used as an herbicide in farms of the Golestan and the Khuzestan provinces. These provinces are known as the most important sites for cyprinid fishes rearing. This toxin is available in Iran markets with commercial name of Gesaprim and with formulation weight purity (WP) 80%. Atrazine is a triazine white color crystals synthetic herbicide with chemical formula: Chloro-4-ethylamino-6-isopropylamino-1-3-5-triazinre that is used in 80 countries recently for destroying broad leaves grass weeds in agriculture farms. Usage of herbicides showed harmful effects on fish and changed plankton food chain (Mason, 1991). Therefore, by notice to releasing millions sturgeon fingerlings into Sefidrood River for restocking, the shortage of information about atrazine effects on sturgeon in Iranian waters, the high usage of it in agriculture farms specially cotton and cereals in the north of Iran, the present study was carried out with the aim to evaluate the effects of atrazine herbicide on cholesterol and triglyceride level in Acipenser nudiventris fingerlings.

Materials and Methods A total of 135 Acipenser nudiventris with an average weight of 50 ±10 g were purchased from the International Sturgeon Research Institute and maintained for 96 hours in 13 aquariums with volume of 200 l of water for adaptation. According to the necessary classifications, the fish were divided into 5 groups including 3 replicates and placed into 15 aquariums with the capacity of 200 l of water (completely enclosed and aerated). For acute toxicity study, a total of 135 fish were exposed to 25, 37.5, 50 and 100 mg/l of atrazine for 96 hours. PROBIT analysis method was used for showing 96h-LC50 values. No feeding was done for fish during the experiment. Sampling was performed every 24 hours. At each sampling time, 3 fish were bled repeatedly. For bleeding, the caudal vein was punctured by using 2 mm heparinated syringes. Plasma was separated by utilizing a centrifuge set at 3000 rpm for 10 min. The data were analyzed as Mean ± S.E.M. at reliability 95 % and significant level of P<0.05. T-test method was used in SPSS 16 software.

Results Results showed that LC50-96h of atrazine was estimated as 32.01 mg/l. Cholesterol level showed significant decrease in all concentrations in comparison to the control in different times. Triglyceride was minimum at concentration of 37 mg/l and showed significant difference with respect to the blank and some other treatments (P<0.05) (figs. 1, 2).

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Control 25 37.5 50 100 140 b b b b 120 100 80 a a 60 a a a 40 a a a a a 20 0

Cholesterol (mg/dl) 24 48 72 96 Hours Fig. 1. Cholesterol level changes at different concentrations of atrazine in A. nudiventris Fish were survived at concentration 25 mf/L for 96 hours. None similar letters indicated significant differences. Control 25 ab37 .5 50 100 1200 b 1000 c b b b 800 600 a 400 ab b a ab a a 200 a 0 Triglyceride (mg/dl) 24 48 72 96 Hours Fig. 2. Triglyceride level changes at different concentrations of atrazine in A. nudiventris Fish were survived at concentration 25 mf/L for 96 hours. None similar letters indicated significant differences.

Discussion It is possible to detect toxic symptom of herbicides by studying cytological and serological indices in fish. In this study, effects of atrazine acute toxicity on clinical symptoms in Acipenser nudiventris fingerlings were measured during 24, 48, 72 and 96 hours. Bioaccumulation of atrazine in liver of tilapia (Tilapia sparrmanii) at concentration was 50g/g exposed to 16 mg/l after 72 hours (Du preez and van Vuren, 1992). Cholesterol is the base material for all steroid hormones. When it increases due to cortisol synthesis, then a large amount of cholesterol is needed (Kazemi et al., 2010). Therefore, the reduction in the amount of cholesterol may be related to its utilization in the production of cortisol arising from stress created by the toxin atrazine. Tri-glyceride is the storage form of fats and major resources of oils and fat which are flowing into the blood. The reduction of tri-glyceride volumes in blood plasma at high concentrations of the toxin atrazine could be due to the imbalance created by the higher concentrations of the toxin, affecting the digestive system, liver and related enzymes as well as hormonal and natural metabolic imbalance in fish studied (Rabinson, 1990). Therefore, atrazine is toxic for Acipenser nudiventris fingerlings even at low concentrations and we must desist from using high concentration of atrazine in agriculture. According to the table of the toxicity levels, atrazine is considered as ‘toxic’ for Acipenser nudiventris and we can use the changes in the desired parameters as tools in analyzing the situation in fishes.

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References Abdali, S., Yousefi Jourdehi, A., Kazemi, R. 2012. Evaluation of atrazine effects on some immunological and blood indices in fame grass carp (Ctenopharhyngoden idella). Research project final report. Tehran North Branch Islamic Azad University. 75P. Ajani, F., 2008. Hormonal and hematological responses of Clarias gariepinus (Burchell 1822) to ammonia toxicity. African Journal of Biotechnology. 7: 3466-3471. Hossein, S., El-Nasser, MA. and Ahmed, SM. 1996. Comparative studies on the effects of herbicide atrazine on freshwater fish Oreochromis niloticus and Chrysichthyes auratus at Assiut, Egypt. Bull. Environmental. Contamination. Toxicology. 57: 503-510. Luiz, C.K., Leonardo, G.B., Ezequiel, S., Ariane, G.C., Mateus, P. and Rafael, Z. 2010. Altered immunological parameters in silver catfish (Rhamdia quelen) exposed to sublethal concentration of an atrazine based on herbicide. Ecotoxicol. Environm. Saf. 72: 1-3. Ramesh, M., Srinivasan, R. and Saravanan, M. 2009. Effect of atrazine (herbicide) on blood parameters of common carp, Cyprinus carpio (Actinopterygii: Cypriniformes). African Journal of Environmental Science and Technology. 3: 453-458.

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Study on the effects of Lactic acid bacteria Enterococcus faecalis on blood biochemical and serum factors in Persian sturgeon fingerlings Mahdi Alizadeh Roud poshti*, Alireza Shenavar Masouleh, Jalil Jalilpoor, Soheil Bazari Moghaddam Mehdi Masoumzadeh, Mahmoud Fallah Shojaei, Hoshang Yeganeh, Bahareh Younes Haghighi Ana_ [email protected] *Department of Aquatic Animal Health, International Sturgeon Research Institute, P.O. Box 41635-3464, Rasht, Iran.

Introduction This project carried out with the aim of study on LAB in Persian sturgeon fingerlings intestine and isolation and identification of Enterococcus species and its usage on feeding, also its effects on growth rate, blood & immunity factors of sturgeon fingerlings in International Sturgeon Research Institute during 2010-2014.

Materials and Methods This project was carried out with the aim to study the LAB (Lactic Acid Bacilla) in Persian sturgeon fingerlings intestine and isolation and identification of Enterococcus species and its usage on feeding, also its effects on blood and immunity factors of sturgeon fingerlings in International Sturgeon Research Institute during 2010-2014. A total of 180 sturgeon fingerlings were sampled and LAB were isolated and enumerated on MRS agar. In ranged from log 2.93 to 5.61 CFU g-1 intestine with a mean of 4.38 ± 0.58 CFU g-1. 16S rRNA sequence analysis revealed that the LAB community was dominated by Lactococcus spp. with Lactococcus garvieae and Lactococcus lactis accounting for 42.55% and 36.17% of the LAB population, respectively. Pediococcus pentosaceus (14.90%), Weissella cibaria (4.25%) and Enterococcus faecalis (2.13%) were identified as minor components of the LAB community. In an in vivo experiment the effect of Enterococcus faecalis as a supplementary diet was evaluated on growth performance of Persian sturgeon for a period of 60 days.

Results and Discussion Strong growth of all LAB was seen at 20˚C and 30˚C, as well as 4%, and 8% (w/v) NaCl. Both W. cibaria and E. faecalis had resistance to oxytetracycline, erythromycin, trimethoprim sulfamethoxazol, enrofloxacin, florfenicol, and flumequine, while, P. pentosaceus was sensitive to oxytetracycline, L. lactis was sensitive to oxytetracycline, erythromycin, enrofloxacin, florfenicol. Also L. garvieae was sensitive to all of used antibiotics except erythromycin. Use of supplemented diet containing Enterococcus faecalis (JF831161) resulted in a significant improvement of lysozyme activity, hemoglobin, white and red blood cells mean corpuscular hemoglobin, neutrophils. These results showed that use of Enterococcus faecalis can act as a positive probiotic in Persian sturgeon feed via improvement of fish immunity and health.

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References Akrami. R. and Ahmadi, A. 2011. Effect of probiotic (Inolin) in diet on haematology and bio chemical parameters of blood serum in farmed Huso huso fingerlings.Veterinary Research Journal. Period, 66 – no. 2, pp.132-136. Shenavar Masouleh, A.R. 2012. Characterization of lactic acid bacteria in intestine of Persian sturgeon (Acipenser persicus) fingerlings and their efficacy on the growth performance and some immunophysiological variables. Ph.D thesis of veterinary Medicine in the field of Aquatic animals health, university of Tehran. 140 p. Vaglio, A. and Landtiscina, C. 1999. Changes in liver enzyme activity in the teleost Sparus aurata in response to cadmium. Intoxication. Ecotoxicol Environ Saf 43,111-116.

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Anatomic and morphometric study of the gastrointestinal tract in Beluga, Huso huso

Ala Enayati*, Omid Zehtabvar; Ali Taheri Mirghaed, Hassan Morovati *Department of Aquatic animal health and disease, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran [email protected] Introduction Caspian Sea beluga (Huso huso) is belonging to the family Acipenseridea, which since millions of years ago lives in the Caspian Sea and surrounding lakes. Due to high quality and expensive caviar production has a high economic value. This study was conducted to investigation the anatomy of the gastrointestinal tract in Beluga, Huso huso.

Materials and Methods For this study, 10 Beluga (4 months old) with an average length of 27±1 cm and an average weight of 80 ± 2 g were studied. 7 morphometric parameters (total length, standard length, head length, snout length to the eye, length of snout to nose holes, the distance between the two nose holes and the distance between the two eyes) were evaluated.

Results and Discussion The morphometric results of this study are shown in table 1. The anatomical study showed that esophagus has two parts. First is a section with prominent longitudinal and then there is a section with longitudinal fold. Then the proventriculus was located and its inner surface was smooth. Only at the end of proventricle, where the ventricle is connected to it, longitudinal folds were observed. Pneumatophore duct opens on the border between the esophagus and the proventricle. The inner surface of the ventricle was smooth. Pyloric caecum is located at the junction of the ventriculus sphincter to the small intestine. In the inner surface of descending and ascending part of the small intestine were observed networking mode. Then the spiral intestine was. The longitudinal folds were observed in the inner surface of rectum. Topography of different parts of the gastrointestinal tract is shown in Figure 1 and 2.

Figure1: Digestive system of the immature beluga, A- Right lateral view, B- Left lateral view: 1. Swim bladder, 2. Proventriculus, 3. Right lobe of the liver, 4. Ventriculus, 5. Ascending part of the small intestine, 6. Spiral colon, 7. Rectum, 8. Spleen, 9. Heart, 10. Pyloric caecum, 11. Descending part of the small intestine.

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Figure 2: Digestive system of the immature beluga, A- Ventral view, B- Dorsal view (Just digestive canal): 1. Ventriculus, 2. Pyloric caecum, 3. Descending part of the small intestine, 4. Right lobe of the liver, 5. Ascending part of the small intestine, 6. Spiral colon, 7. Pancreas, 8. Rectum, 9. Esophagus, 10. Proventriculus.

Biometric values of different parts of the gastrointestinal tract are shown in Table 2. Since the study of morphological and anatomical in classification of fish, understanding of biological factors, feeding and growth is important. Further anatomical and histological studies of the gastrointestinal tract in this species and in different ages are recommended.

Table 1. Morphometric parameters of beluga (cm) morphometric total standard head snout length of distance distance parameters length length length length snout to between between to the nose the two the two eye holes nose eyes holes Huso Huso 27±1 20.5±1 6.2±0.2 3±0.15 2.5±0.15 1.7±0.1 2±0.1

Table 2. Biometric of different parts of the gastrointestinal tract in Beluga (mm) Descending Ascending Section of the section of section of Spiral Esophagus Prestomach Stomach Rectum measurement small small intestine intestine intestine Length (mm) 17.77±1 27.86±2 107.9±5 44.17±3 21.87±2 48.47±3 9.39±0.5 Diameter 4.25±0.2 6.21±0.3 10.45±0.5 5.22±0.2 3.62±0.15 5.97±0.2 1.52±0.1 (middle part of the organ)

Reference Vajhi, A.R., Zehtabvar, O., Masoudifard, M., Moghim, M. and Akhtarzade, M. 2013. Digestive system anatomy of the Acipenser persicus: New features. Iranian Journal of Fisheries Sciences, 12(4), pp.939-946.

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Anatomical Evaluation of Cartilaginous-Skeletal Structures of Food Prehension In The Bester (Sterlet × Beluga)

Omid Zehtabvar, Ala Enayati, Ali Taheri Mirghaed, Khanamooei, M.* * Students Scientific Society of Anatomy, Embryology and Reproduction technology Faculty of Veterinary medicine University of Tehran. Under-graduate student, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. [email protected]

Abstract Sturgeons are among the most important fish species of Caspian Sea. One of these species is Huso huso which has a wide head and bulgy back and big thoracic fins. Bester is a hybrid of the beluga and Sterlet. These fish are considered to be part of primary skeletal fishes. They get their food from the sea floor and for this purpose they can extend their mouth out like a trunk and then take it back in again. They differ greatly anatomically comparing to other fish species especially in this area. For this study, 5 immature (2 - year old) Bester were brought from a private fish culture center. They were decapitated and the heads were put into 10% formalin solution. The specimens were then brought to the anatomy department of Tehran veterinary university. The skin was removed with extreme caution from some parts and the anatomical evaluations were done. The studies showed that both jaws formed an articulation with a structure called Sympletic. The upper jaw is formed by connection of Palatoquadrate cartilages and adjacent skin bones. The upper and lower jaws are hanging from the Hyoid arch and the thin part of the skin between the upper jaw and ventral part of Neurocranium is the only point of their connection to this arch. Both jaws articulate with the ventral border of Sympletic. By comparing the results of this study with those of other sturgeons, it can be concluded that generally the food prehension apparatus in this specie is similar to others but there are a few differences as well. Precise studies in other species of sturgeon can be useful in discovering these unique differences between species.

Keywords: Bester, Cartilaginous jaw, Food apprehension apparatus, Anatomy

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Anatomic and morphometric study of liver, spleen and pancreas in Beluga, Huso huso Omid Zehtabvar , Ala Enayati , Seyed Fakhredin Borgheie*, Ali Taheri Mirghaed , Hassan Morovati

* DVM student, Faculty of Veterinary, University of Tehran & 4. Student scientific association of anatomy, embryology and reproductive technology of veterinary, Faculty of Veterinary, University of Tehran &

*Email: [email protected] Introduction Caspian Sea beluga (Huso huso) is belonging to the family Acipenseridae, which sibce millions of years ago lives in the Caspian Sea and surrounding lakes. Due to high quality and expensive caviar production has a high economic value. This study was conducted to investigation the anatomy of Liver, spleen and pancreas in Beluga, Huso Huso.

Materials and Methods For this study, 10 Beluga (4 months old) with an average length of 27±1 cm and an average weight of 80 ±2 g were studied. 7 morphometric parameters (total length, standard length, head length, snout length to the eye, length of snout to nose holes, the distance between the two nose holes and the distance between the two eyes) were evaluated.

Results and Discusion The morphometric results of this study are shown in Table 1. The anatomical study showed the liver has two main left and right lobes and a middle lobe. The right lobe was too long and was sustained from the posterior to the beginning of the spiral intestine, between the two parts of the small intestine. Gallbladder was located between the right lobe and middle lobe of liver in the third anterior of abdominal cavity. Middle lobe was located between the proventriculus and the ventricles, under swim bladder. Middle lobe was sustained from the posterior to the junction of the proventricule and the ventricule. Spleen has two parts, anterior that was drawn and posterior. The anterior part in the left of abdominal cavity began from the posterior of pyloric caecum and be continued to curved section of the small intestine. This section of the spleen was located between the ventricule (dorsal) and first part of the small intestine (ventral). Posterior lob of spleen was located at dorsal of curved segment of the small intestine and has a vertex to the posterior side. The pancreas has two parts, anterior and posterior. The anterior part was located in medial of the right lobe of the liver. Posterior part was placed between the terminal portion of small intestine and the beginning of the spiral intestine. Pancreas and bile duct have created. One common duct, which was evacuated at the beginning of small intestine. Topography of liver, spleen and pancreas are shown in Figure 1.

Figure1: immature beluga, A- Left lateral view, B- Right lateral view: 1. Ventriculus, 2. Cranial part of the spleen, 3. Caudal part of the spleen, 4. Pancreas, 5. Pyloric caecum, 6. Descending part of the small intestine, 7. Ascending part of the small intestine, 8. Spiral colon, 9. Pancreas, 10. Swim bladder, 11. Proventriculus, 12. Right lobe of the liver.

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Morphometric of various sections of liver, spleen and pancreas are shown in Table 2. Histological studies of the liver, spleen and pancreas in this species recommended. Table 1. Morphometric parameters of beluga (cm) morphometric total standard head snout length of distance distance parameters length length length length to snout to between between the eye nose holes the two the two nose holes eyes Huso huso 27±1 20.5±1 6.2±0.2 3±0.15 2.5±0.15 1.7±0.1 2±0.1

Table 2. Morphometric of liver, spleen and pancreas in Beluga (cm) Section of the average average average average average average average measurement length of length of length of length of length of length of length of the right the left the middle the anterior the The anterior the lobe of lobe of lobe of part of posterior part of posterior of liver liver liver spleen of spleen pancreas pancreas Length (cm) 6.5±0.5 4.3±0.3 3.4±0.15 1.7±0.05 2.2±0.1 2.3±0.05 1.5±0.03

References Vajhi, A.R., Zehtabvar, O., Masoudifard, M., Moghim, M. and Akhtarzade, M. 2013. Digestive system anatomy of the Acipenser persicus: New features. Iranian Journal of Fisheries Sciences, 12(4), pp.939-946.

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Sex determination in Sturgeon

Abasali Hajibeglou , Roghaieh Safari*

*Gorgan university of Agricultural Science and Natural Resources ([email protected])

Introduction In many fish sex-determination only through special appearance in maturity period is easily possible. But in some fish such as sturgeon, it is not easily possible. In addition, in sturgeons determination of gender based on DNA and genetic-molecular methods are not possible. Thus, in addition to the methods of the sex determination based on the characteristics and appearance, more accurate method can also be used. Among these methods laparoscopy, ultrasonography methods, blood plasma analysis techniques, biopsy and catheterization can be noted. In sturgeons final maturity is required to spend four sexual stages which each stage has its own characteristics. In sturgeons the longest stage of sexual maturation is the stage 2, which in beluga, depending on the habitat, sometimes lasts 7-8 years. In sex determination of mature sturgeon according to the morphological characteristics, attention to the shape and size of the pectoral fins, anal shape, underbelly line, the shape of the snout and caudal stem are important. The purpose of this manuscript is to review the methods of sex determination of sturgeon fish.

Laparoscopic method In laparoscopy, fish are anesthetized and disinfected. A very small gape is created between the end plates of fish (abdominal area, third or fourth page from the tail bone to the side of the head). Laparoscopic instruments were then thrown into the body cavity fish and through a monitor, gender or the sexual maturation can be detected (Halajian et al., 2014).

Ultrasonographic method In ultrasonography method, special sensor device is moved tangent to the belly of the fish. As a result, gonadal image is displayed on the screen. In this way by observing and interpreting images of the sexual organs, gender determination and sexual maturation phase detection will be possible (Colombo et al., 2004; Moghimi et al., 2002).

Biopsy method In the biopsy method, first, fish are anesthetized and then a small incision in the abdominal area is created. Required studies will then done in the context of gender determination or sexual maturation stage diagnosis using gonad sampling or direct gonad observation (Halajian, 2007).

Other methods Other methods to determine the sex are blood plasma analysis methods such as blood plasma Lipo-phospho-protein analysis, analysis of vitellogenin levels and blood steroids (Hurvitz et al., 2007). Conclusion In general, the use of various methods for identifying gender farmed sturgeon can be effective. Moreover, the biopsy to identify fish less than 2.5 years and laparoscopy are

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References Colombo, R.E., Willis, P.S. and Garvey, J.E. 2004. Using of ultrasound imaging to determine sex of shovelnose sturgeon. North Am. J. Fish. Manage. 24 (1), 322-326. Halajian, A., Kazemi, R., Mohseni, M., Bahmani, M. and Yousefi, A. 2007. Sex determination and sexual maturation stages in the Acipenser nudiventris using a biopsy of the gonad, Iranian Journal of Fisheries. 16 (3), pp. 65-72. Halajian, A., Kazemi, R.A., Yousefi Jourdehi, A. and Pourdehghan, M. 2014. The use of methods of diagnosis and determine the stage of sexual maturation in Sturgeon. Journal of Fisheries, Islamic Azad University, Azadshahr Branch. 8 (1), pp. 28-19. Hurvitz, A., Jackson, K., Degani, G. and Levavi-Sivan, B. 2007. Use of endoscopy for gender and ovarian stage determinations in Russian sturgeon (Acipenser gueldenstaedtii) grown in aquaculture. Aquaculture. 270, 158-166. Moghimi, M., Vajhi, A.R., Veshkini, A. and Masoudifard, M. 2002. Determination of sex and maturity in Acipenser stellatus by using ultrasonography. J. Appl. Ichthyol. 18, 325-328.

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Histological evaluation of liver and some blood serum factors of farmed Beluga (Huso huso) suspected fatty liver in sea cage

*Jalil Jalilpour, Ayoub Yousefi Jourdehi, Ali Halajian, Bahareh Younes Haghighi, Mehdi Masoumzadeh, Soheil Bazari Moghaddam, Mehdi Alizadeh, Mohammad pourdehghani, Alireza Shenavar, Mahmoud Falah shojaee, Majid Poursafar. *Research instructor of Agricultural Research Education & Extension Organization (AREEO) - International Sturgeon Research Institute, Rasht, Iran.

[email protected] Introduction Fatty liver is one of the important diseases in famed fishes, which is observed usually without external signs, particularly in species having low motion and high rearing density in enclosed rearing condition. In these fishes, fatty liver disease can be identified only after death of fishes during autopsy and placing one section of stripped liver in a dish containing water, and then fat layer will be seen on the surface of the water. The main factor of fatty liver disease is feeding diets having saturated fatty acids, high level of carbohydrate and not enough antioxidants such as vitamin E Zaharia (2010).

Materials and Methods This study was carried out on 20 specimen of farmed beluga (Huso huso) by transferring to cage, during 7 months from June 2011 to January 2012. After transferring of farmed fish (average weight 103.40 ± 27.5 g) to sea cages, monthly random sampling was performed and blood samples were taken from 4 fishes. Blood samples were transferred into the vial containing heparin to determine blood parameters. Necropsy sample of liver tissue for preparation of histological sections was transferred to the Buin’s solution.

Results and Discusion

2 3

Fig. 1. Fatty liver of Huso huso Fig. 2. Liver of Huso huso showing fatty degeneration (1)

Bile stagnation (2) Necrosis (3) and Cell vacuolization (*) (H&E, 40X) In the study on the internal organs of dissected fishes, pale liver with inflamed edges have been observed in 40% of these farmed fishes (fig.1). Histopathological study of liver showed fatty degeneration, bile stagnation, necrosis and hepatocit cell vacuolization (fig. 2).

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Furthermore, the range of blood serum factors such as albumin, total protein, cholesterol and triglyceride showed significant variation in different sampling periods. Cholesterol and blood triglycerides fish as the main indicators of the health status of fish is. In between and within species depending on the type of diet, exercise intensity and stage of sexual development can be varying different. Also, increased fat in the diet and the size of the fish increases (Yousefi et al., 2011).

References Yousefi, M., Abtahi, B. and Abdian Kenari, A. 2012. Hematological, serum biochemical parameters, and physiological responses to acute stress of Beluga sturgeon (Huso huso, Linnaeus 1785) juveniles fed dietary nucleotide. Comparative Clinical Pathology. 21(5), 1043-1048. Zaharia. T. Dumitrescu., T., 2010. Disease detected at sturgeon reared in fresh and salt water. National institute for Marin research and development. Grigore Antipa, Constanta, Romania.

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Reducing the age of sex maturity in sturgeon

Ayoub Yousefi Jourdehi*, Mahmoud Bahmani, Mohammad Sudagar, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Homayoon Hosseinzadeh Sahafi, Ali Hallajian, Mohammad Pourdehghani, Mahtab Yarmohammadi *International Sturgeon Research Institute (Areeo), Rasht, Iran, P.O.BoX: 41635 – 3464.

[email protected]

Introduction Due to the long reproductive cycle in sturgeon especially in beluga (18-year-old in females) of the Caspian Sea in nature, many countries such as Iran like to farm sturgeon species in fresh and brackish waters. Several methods were used for reducing the age of sex maturity in sturgeon such as adding vitamins E, C and different soy bean products to fish diet (Bahmani et al., 2009). Phytoestrogens are by definition plant-derived substances that are able to activate the estrogen receptors. The estrogenic effects of phytoestrogens are well documented in fish (Pelissero et al., 2001). Phytoestrogens can bind to steroid-binding proteins (Dechaud et al., 1999) and to estrogen receptors (ER) of target cells (Casanova et al., 1999). Therefore, considering the last maturity of beluga, this study was carried out with the aim of using dietary soy phytoestrogens for accelerating oocyte development in females of beluga.

Materials and Methods Experiments were carried out on 54 female H. huso 5-year-old with average body weight 13.25 ± 0.3 kg and total length 140.3 ± 1.2 cm that had been farmed in terrestrial ponds of the Caspian Sea International Sturgeon Research Organization during a year (from summer 2011 to summer 2012). Ovary of all fish was at sage II of sexual maturation at the start of the experiment. Fish were selected by biopsy and laparoscopy methods (using a Stema Company, model M-CAM1700, 30 degree telescope with 4 mm in size and with length 17.5 cm and with the cold light source a halogen 250 W, Germany) and were stocked randomly in 18 concrete ponds (with dimensions 3 × 2.5 × 1.2 m3) equipped with aeration system and hole mixed the Sepidroad River water together. Fishes were treated with a practical beluga diet was formulated to contain at concentrations of 0, 0.2, 0.4, 0.8 and 1.6 g/kg of purified genistein and equol (Xi, and Keen - Source Biotechnology Co. Ltd). Genistein and equol were dissolved in 75 ml ethanol and added to the diet. Diets were mixed by a mixer in 2000-g portions for 7-10 min after adding 15% of water. Food pellets were prepared in diameter 12 mm with a standard meat grinder. Each treatment includes 6 fish in two replicates with 3 fish per each concrete pond. 1% of biomass and twice daily (0800 and 1600 hours), fishes received a diet. Oocyte diameter was measured using a stereomicroscope seasonally.

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Results Oocyte diameter increased significantly at all concentrations of GE and EQ except GE 1.6 g/kg than control. At the end of experiment, it increased significantly at EQ 0.4 g/kg (reached to a maximum 3.1 ± 0.2 mm) and 1.6 g/kg than others (P<0.05) (Fig. 1).

Start End

4 **C 3 **B 2 **A **A **A **A **A A A 1 * * * * * * * * *

0 Oocyte diameter, mm diameter, Oocyte concentration, g/kg

Figure. 1 Mean ± S.E.M of oocytes diameter at different concentrations of GE and EQ (n=54, 6 fish per each group); (A,B): indicate the significant comparision between treatments at the end of experiment. *: indicate the significant comparision between the start and the end of experiment.

Discussion In this research estrogenic effects of isoflavonic phytoestrogens genistein and equol have been investigated. Results showed that equol supplemented to the diets at 0.4 g/kg improved significantly ovary maturation than genistein. This result was confirmed by measuring reproductive indices changes such as histological and morphological changes in oocytes and increase in their diameter. Phytoestrogens have been reported to have many biological activities in fertilization, immunostimulation, anabolism and balancing hormones (Leusch et al., 2006). Although their structure is not related to vertebrate steroids, they are able to produce estrogen-like effects. The biological activities of phytoestrogens are usually studied on the level of estrogen receptor (ER) binding and typically induce biological responses through interaction with ER, either agonist or antagonist (Sicuro et al., 2010). Phytoestrogens effects have been investigated in fish (Ng et al., 2006). Ko et al. (1999) reported that in yellow perch, genistein induced vitellogenin synthesis by the liver and had a growth promoting effect similar to that of estradiol - 17β. Phytoestrogens are able to stimulate VTG synthesis in vivo in Siberian sturgeon (Pelissero et al., 1991) and in vitro in trout hepatocyte (Pelissero et al., 1993). Latonnelle et al. (2002) reported that the estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A < daidzein = formononetin < genistein < equol in trout and biochanin A < genistein < daidzein < formononetin < equol in Siberian sturgeon (Acipenser baerii) that was according to present study results about genistein and equol. The levels of genistein utilized in striped bass (Morone saxatilis) affect the hepatic, gonadal, or normal somatic growth (Pollack et al., 2003).

Conclusion Soy Isoflavonic phytoestrogens can exhibit estrogenic activity on reproduction and exert female ovary development at proper dosage, and improve reproductive performance of female Huso huso. Equol can be added to the diet to advance gonadal development which would be beneficial to the production of caviar by aquaculture. Equol added to the diet was observed to increase oocyte development at certain concentrations whereas the phytoestorgens had no

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significant effect on growth parameters. Therefore, the results can be used for improving farming practices of the Beluga sturgeon.

References Bahmani, M., Yousefi Jourdehi, A., Kazemi, R., Pourdehghani, M., Hallajian, A., Dejhandian, S. and Jalilpoor J 2009. Seasonal fluctuations of testosterone (T), 17α-hydroxy progesterone (17α-OHP), 17β-estradiol (E2) during sexual maturation in Acipenser stellatus. Iranian J fisheries 4:7-16. Cassidy, A., Hanley, B., Lamuela-Raventos, R.M. 2000. Isoflavones, ligans and stilbenes-origins, metabolism and potential importance to human health. J Sci Food. Agric 80:1044-1062. Latonelle, K., Lemenn, F., Kaushik, S.J,. and Plessiero, B. 2002. Effects of dietary phytoestrogens In Vivo and In Vitro in Rainbow trout and Siberian sturgeon: interests and limits of the in vitro studies of interspecies differences general comparative endocrinology 126:39-51. Ng, Y., Hanson, S., Malison, J.A., Wentworth, B. and Barry, T.P. 2006. Genistein and other isoflavones found in soybeans inhibit estrogen metabolism in Salmonid fish. Aquaculture 254:658-665. Pelissero C, Breton B, Bennetau B, Corraze G, Le Menn F, Cuisset B, Helou C, Kaushik SJ (2001) Effect of genistein enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology 121:173–187. Pollack, S.J., Mary, A.O., Sullivan, V.C. & Woods, L.C. 2003. The Effects of the Soy Isoflavone Genistein on the Reproductive Development of Striped Bass, North American Journal of Aquaculture 65:226-234. Sicuro, B., Badino. P., Dapra, F., Gai, F., Galloni, M., Odore, R., Palmegiano, G.B. and Macchi, E. 2010. Physiological effects of natural olive oil antioxidants utilization in rain bow trout (Onchorhynchus mykiss). Aquacult Int 18:415- 431.

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The comparison of different methods anesthesia in sturgeon (Acipenser nudiventris) Ayoub Yousefi Jourdehi*, Mahmoud Bahmani., Mohammad Ali Yazdani Sadati., Rezvanollah Kazemi., Ali Hallajian., Mahtab Yarmohammadi., Mahmoud Mohsen., Amirali Moradinasab., Mahmoud Tavakoli., Arezoo Vahabnejhad., Jalil Jalilpour., Mirhamed Seyed Hassani., Amin Farahbod., Sohrab Alimohammadi., Seyed Sadrodin Hozni , Esmaiel Hosseinnia * International Sturgeon Research Institute (Areeo), Rasht, Iran, P.O.Box: 41635 – 3464

Introduction Sturgeon are the most valuable and economic fish, which their population has been led to sharp decline due to overfishing, habitat destruction, environmental pollution, no conservation, and making barrier on their reproduction place (Doukakis et al., 1999). Anesthesia is a valuable tool to achieve enough sedation, reduce stress and pain during various procedures such as the manipulation of individuals, measurement, weighing, vaccination, transportation, and blood or biopsy sampling (Kiessling et al., 2009). There are several approaches in use to anesthetize fish. To select an anesthetic method for particular purpose, different properties such as effectiveness, physiological response, cost, and safety to fish, human, and environment should be considered (Pirhonen and Schreck, 2003). Therefore, this study was carried out to investigate the different anesthetic methods on induction and recovery times in ship sturgeon.

Materials and Methods Sturgeon Fish (Acipenser nudiventris) of three treatments were anesthetized under followed anesthesia treatments as 0.5 g/l of clove powder, 0.3 bar of CO2, and 130 V of electric shock. Anesthesia treatments were based on a preliminary experiment so that the exposure time for different anesthetics was 3 min. Fish were monitored every minute after transferring into 20-l observation tanks (filled with aerated fresh water) to which anesthetic had already been added. At the point when total loss of equilibrium, mussel tune, was obtained and opercular movements became irregular, at which stage IV of anesthesia (Summerfelt and Smith, 1990), time was recorded and considered as induction time of anesthesia. Then, anesthetized fish were transferred quickly to 20-l recovery tanks with pure well water. Fish were monitored to determine the time to recovery of normal equilibrium and tactile responses, stage IV of recovery outlined by Summerfelt and Smith, 1990. Once fish reached to normal equilibrium and respond to the tactile, fish were considered fully recovered. Recovered fish were returned to acclimation tanks and monitored for 24 hours.

Results and Discussion The results showed that the shortest and longest induction time was for electric shock and clove powder, respectively. By contrast, the shortest and longest recovery time took place for electric shock and clove powder, respectively. Clove oil has traditionally been used as topical anesthetic for toothaches, headaches, and joint pain and is extracted from the buds, leaves, and stems of clove trees, Eugenia aromatic which its active compound is eugenol [2-methoxy- 4-(2- propenyl) phenol; 70–90 % by weight] (Weber et al., 2009). Fish has described carbon dioxide as an anesthetic for the first time. It can be produced by bubbling CO2 gas or by the soda-acid technique (Summerfelt and Smith, 1990). CO2 is considered as a drug of Blow regulatory priority. It can be difficult to use CO2 uniformly and is usually slow-acting and lethal after repeated exposure (Marking and Meyer, 1985).

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The use of an electric approach in order to anesthetize fish has some advantages than chemical agents such as no production of contaminant made by chemical agents, short recovery time, low cost, and ready for use. Fish response to electricity is dependent on electrical field and shockduration. Other factors such as electro conductivity, temperature, size of fish, and species can be involved (Mirzargar and Sepidgar, 2005). Our results indicated that electric shock, CO2, and clove powder are all effective in anesthetizing juvenile Ship sturgeons according to Marking and Meyer (1985) who suggested that the ideal anesthetic would have an induction time of 3 min or less and a recovery time of less than 5 min. In this study, electric shock made faster induction times and longer recovery than other anesthetics evaluated. Anesthetizing fish to the desired endpoint with higher voltage of anesthesia may result in a longer recovery period. Similar results were found in previous studies in which fish were anesthetized to stage IV in 0.1– 0.2 min using lectrosedation and in 2.5 min using CO2 (Bowzer et al., 2012). Since there is variability in induction time due to size, water temperature (Trushenski et al., 2012b), mechanism of functioning, and pharmacokinetics of each anesthetic (Weber et al., 2009), it is difficult to compare induction and recovery times during the experiments.

Conclusion Based on the results, CO2 induced the most stress response compared to other anesthetics. Electric shock may be a suitable tool for quickly inducing anesthesia in A.nudiventris sturgeon.

References Bowzer, J.C., Trushenski, J.T., Gause, B.R. and Bowker, J.D. 2012. Efficacy and physiological responses of grass carp to different sedation techniques: II. Effect of pulsed DC electricity voltage and exposure time on sedation and blood chemistry. N Am J Aquac 74: 567–574. Kiessling, A., Johansson, D., Zahl, I.H., Samuelsen, O.B. 2009. Pharmacokinetics, plasma cortisol and effectiveness of benzocaine, MS-222 and isoeugenol measured in individual dorsal aorta annulated Atlantic salmon (Salmo salar) following bath administration. Aquaculture 286: 301–308. Marking, L.L. and Meyer, F.P. 1985. Are better anesthetics needed in fisheries? Fisheries 10: 2–5. Palic, D., Herolt, D.M., Andreasen, C.B., Menzel, B.W. and Roth, J.A. 2006. Anesthetic efficacy of tricainemethanesulfonate, metomidate and eugenol: effects on plasma cortisol concentration and neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820). Aquaculture 254:675–685.

Pirhonen, J. and Schreck, C.B. 2003. Effects of anesthesia with MS-222, clove oil and CO2 on feed intake and plasma cortisol in steelhead trout (Oncorhynchus mykiss). Aquaculture 220: 507–514.

Pirhonen, J. and Schreck, C.B. 2003. Effects of anesthesia with MS-222, clove oil and CO2 on feed intake and plasma cortisol in steelhead trout (Oncorhynchus mykiss). Aquaculture 220:507–514. Weber, R.A., Peleterio, J.B., Garcia Martin, L.O. and Aldegunde, M. 2009. The efficacy of 2- phenoxyethanol, metomidate, clove oil and MS-222 as anaesthetic agents in the Senegalese sole (Solea senegalensis Kaup, 1858). Aquaculture 288:147–150.

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Histopathological effect of Ammonia on gill and kidney tissue in Acipenser ruthenus

Ayoub Yousefi Jourdehi*, Sorena Abdali., Brat Ghrmanpour and Ali Hallajian, Mahmoud Shakourian *International Sturgeon research Institute (Areeo), Rasht, Iran. P.O.Box: 41635 – 3464.

Introduction Ammonia (NH3) in one of the most widely fertilizers entered to waters and appear in aquatic animals habitats. Ammonia is a by-product of fish metabolism and accumulated in water and soil. Increase of ammonia concentration in environment waters is toxic for fish and caused to damage organs and tissues of fish at long - term exposure.

Materials and Methods Present study was carried out aim to determining of acute effect different concentrations of ammonia on gill, liver and kidney organs in farmed juvenile Acipenser ruthenus. A total of 135 fish with mean weight 47 ± 2.1 gram and mean length 25 ± 1.4 cm were selected and exposed to 0, 25, 50 and 75 mg/L of NH3 in aquariums (volume 150 liters) for 96 hours. Each treatment had triplicates including 15 fish per each aquarium.

Results and Discussion Based on results, observed disasters in gill of fish exposed to different concentrations of NH3 included adhesion of gill secondary filament, clubbing of secondary filaments to, hyperplasia, cell necrosis, damage of the epithelium layer, congestion and bleeding that intensified with increase in concentration and exposure time. In kidney, some disasters such as degradation of renal tract, net impaired glomerular capsule Bowman, Bowman's space closure, congestion, bleeding, melanomacrophage and renal tract blockage were observed that intensified and increase in concentration and long – term exposure. Considering to obtained results, non- ionized ammonia was toxic for Acipenser ruthenus and must be controlled and monitored in water by proper nutrition and water management.

References Atle, F., Albert, K., Imsland, B. R., Edward, S., Sigurd, O. & Stefansson. 2009. Effects of chronic and periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus). Aquaculture, 296: 45–50. Benli, A.C.K. and Koksal, G. 2005. The acute toxicity of ammonia on tilapia (Oreochromis niloticus L.) larvae and fingerlings. Turkish Journal of Veterinary and Animal Sciences; 29:339–344. Boudreaux, J. andFerrara, A.M.. 2007. Fontenot, Acute toxicity of ammonia to spotted gar, Lepisosteus oculatus, alligator gar, Atractosteus spatula, and paddlefish, Polyodon spathula. J.W. Aqua. Soc., 38(2): 322-335. Das, P. C., Ayyappan, S., Jena, J K. and Das, B.K. 2004. Acute toxicity of ammonia and its sub-lethal effects on selected hematological and enzymatic parameters of mrigal, Cirrhinus mrigala (Hamilton). Aquaculture Research35: 134-143. Eddy, F.B. 2005. Ammonia in estuaries and effects on fish. J. Fish. Biol. 67: 1495-1513. El-Sherif, M.S. Andel-Feky, Amal, M. Effect of ammonia on Nile Tilapia (O. niloticus) Performance and some hematological and histological measures. 8th International Symposium on Tilapia in Aquaculture.

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Ultrasonographic appearance of the gonads in Siberian sturgeon (Acipenser baerii)

Alireza Vajhi*, Majid Masoudifard, Mehdi Moghim, Omid Zehtabvar *Department of Surgery and Radiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. [email protected] Introduction While wild catches have been generally declining, the Siberian sturgeon is increasingly farmed both for meat and to produce caviar. Because A. baerii completes its lifecycle in fresh water and sexually matures relatively early, it is the most common original broodstock for captive-bred specimens. Sex identification in sturgeons is difficult owing to the absence of phenotypic differences between females and males before sexual maturity, the gonad maturation cycle and the anatomical characteristics of the reproductive system. The objective of this study was to describe the ultrasonographic appearance of immature ovaries and testes in Siberian sturgeon. Materials and Methods 120 immature cultured Siberian sturgeons underwent ultrasonographic examination using a high frequency transducer. The stage II of ovary showed a tree-root like appearance while the ovaries were seen as hyperechoic irregular strands within the hypoechoic gonadal fat (figure 1). In more advanced stage II, the ovaries showed lamellar appearance. In stage III, point like hyperechoic structures was seen within the ovaries which gradually change to small round hypoechoic follicles. The testis stage II was oval to round anechoic to hypoechoic homogenous structure within the hypoechoic surrounding fat. The testicular wall was seen as a thin hyperechoic layer. In more advanced stage II and in stage III, the testis parenchymal appearance was hyperechoic compared to the surrounding gonadal fat (figure 2).

Fig 1. Sagittal sonographic scan of the ovary Fig 2. Transverse sonographic scan of the testis stage II in Siberian sturgeons. stage II in Siberian sturgeon.

For high validity sex determination of the sturgeons by sonography, knowledge on gonadal shape and echogenicity in very important. Different gonadal shape and echogenicity in any sturgeon breed and in several stages of maturity may cause incorrect sex determination of sturgeons using ultrasonography. References Moghim, M., Vajhi, AR., Veshkini, A. & Masoudifard, M. 2002. Determination of sex and maturity in Acipenser stellatus by using ultrasonography, Journal of Applied Ichthyology, 18: 325-328. Masoudifard, M., Vajhi, AR., Moghim, M., Nazari RM., Naghavi, AR. & Sohrabnejad, M. 2011. High validity sex determination of three years old cultured beluga sturgeon (Huso huso) Using ultrasonography, Journal of Applied Ichthyology, 27: 643-647.

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Induction of spawning in Iranian sturgeon (Acipenser persicus Borodin, 1897), by LHRH-a acute release implantation and its effects on reproductive indices. Seyed Amin Mirhashemi Rostami *,Kourosh Amini, Abbas Ali Aghaeemoghadam, Seyyed Morteza Hosseni, Mohammad Mazandarani.

*Inland waters aquatic stocks research center- Gorgan. *- Correspondent Author and lecturer. Introduction The sturgeon industry has a lot of problems due to high cost of artificial propagation for fingerlings production, decreasing of the natural stocks and also low quality and immature caught broodstocks.

Materials and Methods The immature breeders do not response to hormonal therapy at the sturgeon hatcheries as most having polarization index (PI) (above 10) and large numbers of breeders caught and transported to the hatcheries were unable to reproduce. This study was attempted to find the effect of LHRHa implantation on gonadal final maturation and spawning of the Persian sturgeon. Field studies were carried out at Shahid Marjani, a sturgeon hatchery, located at Gorgan city. Broodstocks were caught from the southeast region of the Caspian Sea. The selected female broodstocks (PI>10) ranging in size from 24 to 37.5 kg were implanted with LHRHa cholesterol pellets at concentrations of 0 μg/kg (control), 10 μg/kg (treatment 1), 15 μg/kg (treatment 2), 20 μg/ kg (treatment 3) in three replicates. The LHRHa cholesterol pellets were prepared. Blood was collected from the caudal vasculature before and within 24 hours after implantation. The following hormones: 17-β-Estradiol (ng/ml), Esteriol unconjugated (ng/ml), 17-α-Hydroxy Progesterone (ng/ml), 17-β-Hydroxy-4-Androsten-3-one (Testosterone) (ng/ml), 4-Pregnene-3, 20-Dione (Progesterone) (ng/ml), were measured by using radioimmunoassay (RIA). Artificial propagation and eggs incubation were carried out according to the current method in Iranian and also Russian sturgeon hatcheries.

Results and Discussion The results from this study indicated that females treated with LHRHa hormone implantation at 10, 15, 20 µg per kg body weight reached final maturation. These results were observed for all fish from treatment numbers 2 (15 μg/ kg) and 3 (20 μg/kg), however only one fish reached final maturation in treatment 1 (10 μg/ kg). LHRHa hormones showed different effects and variation on steroid hormones before and after implantation. The results also demonstrated that estrogens (E2, E1) were primarily increased then decreased where progesterone showed a rapid increased in the fish serum. The current implantation of LHRHa was able to enhance breeders with PI above 10 to final maturation with high fertilization (78.33% ±13.87, 68.33% ±4.16 in treatment 2 and 3, respectively) and hatching rates (85.3% ±9.07, 68.33% ±7.64 in treatment 2 and 3, respectively) and also best synchronization and high larval survival which under normal condition at sturgeon’s hatcheries this is not possible. Thus, the information from this study is very useful for artificial propagation of not-fully- matured females of Persian sturgeon at sturgeon hatcheries especially in Iran.

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Determination of some serum biochemical markers in sub-adult Huso huso Mohammad Mazandarani*, Seyyed Morteza Hoseini, Kourosh Amini, Ali Taheri Mirghaed

* Department of Fisheries, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran * Corresponding author: [email protected]

Introduction Sturgeons are important species in organism evolution sciences, as they have lived in the earth since about 200 million years ago (Secor et al., 2000). Also, they are important in aquaculture because of their highly-prized caviar and outstanding growth rate. Among them, Huso huso is the most important species, because this species produces the most valued caviar and has the highest growth rate among the sturgeons. H. huso is the main sturgeon cultured in Iran, which has been adapted to a variety of culture system such as earthen ponds, concrete ponds, fiberglass tanks, cages and pens and to a variety of water sources such as freshwater, brackish water, well water, river water and the Caspian Sea water. Serum biochemical markers are useful means to estimate an animal health and nutritional status. Serum variables are a useful tool of physiological disturbances in intensively farmed fishes and can provide important information for diagnosis and prognosis of diseases (Tavares-Dias and Moraes, 2007). Several authors tried to determine normal serum markers levels in a variety of species including Ictalurus punctatus (Tavares-Dias and Moraes, 2007), Thunnus thynnus (Percin and Konyalioglu, 2008), Morone chrysops×Morone saxatilis (Herubec et al., 2001), Acipenser brevirostrum (Knowles et al., 2006) and Acipenser persicus (Asadi et al., 2006a). In H. huso, some serum markers levels have been reported by Asadi et al. (2006b). However, no update on this information has been made since 10 years ago. This paper provides an update in serum albumin, glucose, cholesterol, triglyceride, creatinine, urea, and total-bilirubin and direct- bilirubin levels in H. huso. Materials and Methods A total number of 26 sub-adult H. huso (8 males and 18 females) were studied. The fish were reared in earthen ponds until they were 3 years old. They were fed with commercial pellets during rearing. The fish were caught from the earthen pond and transferred to indoor through-flow fiberglass tanks. After 3 days, the fish were anesthetized with clove oil (100 ppm). After anesthesia, blood samples were taken from each fish using non-heparinized syringe from caudal vein. After clotting at room temperature for 4 h, serum was separated by centrifugation (3000 rpm, 10 min). The sera were frozen at -20°C until analysis. Concentrations of serum albumin, glucose, cholesterol, triglyceride, creatinine and urea were determined by spectrophotometer and commercial kits (Pars Azmun Co. Tehran, Iran). Bilirubin levels in the sera were determined using spectrophotometer and commercial kit (Zist Chem, Tehran, Iran). Normality of the data was confirmed by Shapiro-Wilk test. Significant difference (P<0.05) in different serum markers between the males and females was checked by t-test. Data are presented as mean ± SD.

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Results There was no significant difference in the levels of serum markers between the males and females (Fig. 1 and 2).

1.6 60 250 90

1.4 80 50 200 1.2 70 40 60 1.0 150 50 0.8 30 40 0.6 100

20 30 Serumalbumin(g/dl)

0.4 Serumglucose (mg/dl) Serum cholesterolSerum(mg/dl) Serum trig;yceride trig;yceride Serum(mg/dl) 20 10 50 0.2 10

0.0 0 0 0 Male Female Both sexes Male Female Both sexes Male Female Both sexes Male Female Both sexes Gender Gender Gender Gender Figure 1: Serum albumin, glucose, triglyceride and cholesterol levels in H. huso. Sample size was 8 for males and 18 for females. Serum albumin levels were 1.15±0.23, 1.04±0.22 and 1.07±0.22 g/dl respectively in the males, females and pooled individuals (Fig. 1). Serum glucose levels were 39.9±8.10, 39.4±6.03 and 39.5±6.56 mg/dl respectively in the males, females and pooled individuals (Fig. 1). Serum triglyceride levels were 168±58.3, 149±55.4 and 155±55.0 mg/dl respectively in the males, females and pooled individuals (Fig. 1). Serum cholesterol levels were 60.0±11.5, 63.4±18.7 and 62.3±16.6 mg/dl respectively in the males, females and pooled individuals (Fig. 1). Serum total-bilirubin levels were 0.34±0.06, 0.39±0.05 and 0.37±0.06 mg/dl respectively in the males, females and pooled individuals (Fig. 2). Serum direct-bilirubin levels were 0.10±0.01, 0.11±0.01 and 0.11±0.01 mg/dl respectively in the males, females and pooled individuals (Fig. 2). Serum creatinine levels were 0.16±0.04, 0.16±0.03 and 0.16±0.03 mg/dl respectively in the males, females and pooled individuals (Fig. 2). Serum urea levels were 3.34±0.64, 3.29±0.61 and 3.30±0.61 mg/dl respectively in the males, females and pooled individuals (Fig. 2).

0.5 0.1 0.3 4.5 0.5 0.1 4.0 0.4 0.2 3.5 0.4 0.1 3.0 0.3 0.1 0.2 2.5 0.3 0.1 2.0 0.2 0.1 1.5

0.2 0.0 Serumurea (mg/dl) Serumcreatinine (mg/dl)

Serum total bilirubin (mg/dl) bilirubin total Serum 1.0 0.1 Serumdirect bilirubin(mg/dl) 0.1 0.0 0.1 0.5

0.0 0.0 0.0 0.0 Male Female Both sexes Male Female Both sexes Male Female Both sexes Male Female Both sexes Gender Gender Gender Gender Figure 2: Serum total-bilirubin, direct-bilirubin, creatinine and urea levels in H. huso. Sample size was 8 for males and 18 for females.

Discussion and Conclusions The present results showed no significant difference in serum markers between the males and females, which are partially in line with previous study on H. huso (Asadi et al., 2006b). Asadi et al. (2006b) found significantly higher serum albumin and glucose in the males than the females. However, they reported no significant difference in serum urea and creatinine

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir between the males and females. Similarly, Asadi et al., (2006) found no significant difference in serum urea, cholesterol and bilirubin between the genders of A. persicus. Serum glucose and creatinine levels in the present study were lower than those reported by Asadi et al. (2006b). Serum albumin levels in the females were lower than that reported by Asadi et al. (2006b); but the levels of the males were similar to that study. Serum bilirubin has not been reported before for H. huso. The present bilirubin levels in the females are similar to that reported in female A. persicus; however, the level of the males is lower than that of the male A. persicus (Asadi et al., 2006a). The present serum cholesterol levels are similar to that reported in juvenile H. huso (Falahatkar, 2012). On the other hand, the present serum triglyceride is lower than that reported in the juvenile H. huso (Falahatkar, 2012). In conclusion, the present results showed that there is no significant difference in the measured parameters between the genders. Also, there are some difference in the measured parameters value between the present study and a previous one, suggesting the needs for standardization of sampling and analysis procedures.

References Asadi, F., Halajian, A., Pourkabir, M., Asadian, P., & Jadidizadeh, F. 2006. Serum biochemical parameters of Huso huso. Comparative Clinical Pathology, 15(4), 245-248. Asadi, F., Masoudifard, M., Vajhi, A., Lee, K., Pourkabir, M., & Khazraeinia, P. 2006a. Serum biochemical parameters of Acipenser persicus. Fish Physiology and Biochemistry, 32(1), 43-47. Falahatkar, B. 2012. The metabolic effects of feeding and fasting in beluga Huso huso. Marine environmental research, 82, 69-75. Hrubec, T. C., Smith, S. A., & Robertson, J. L. 2001. Age‐Related Changes in Hematology and Plasma Chemistry Values of Hybrid Striped Bass (Morone chrysops× Morone saxatilis). Veterinary Clinical Pathology, 30(1), 8-15. Knowles, S., Hrubec, T. C., Smith, S. A., & Bakal, R. S. 2006. Hematology and plasma chemistry reference intervals for cultured shortnose sturgeon (Acipenser brevirostrum). Veterinary Clinical Pathology, 35(4), 434-440. Percin, F., & Konyalioglu, S. 2008. Serum biochemical profiles of captive and wild northern bluefin tuna (Thunnus thynnus L. 1758) in the Eastern Mediterranean. Aquaculture Research, 39(9), 945-953. Secor, D. H., Arefjev, V., Nikolaev, A., & Sharov, A. 2000. Restoration of sturgeons: lessons from the Caspian Sea sturgeon ranching programme. Fish and Fisheries, 1(3), 215-230. Tavares‐Dias, M., & Moraes, F. R. 2007. Haematological and biochemical reference intervals for farmed channel catfish. Journal of Fish Biology, 71(2), 383-388.

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Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Acipenser baerii Mahmoud Shakourian *, Alireza Alipour, Mohamad Ali yazdani, Mir hamed Sayed Hassani, Ali Hossien pour Zelti, Alireza Shenavar, Forouzan Bagerzadeh Lakani, Hooshang Yegane,Mohebali Pourgolam, Hamid Reza pouali, Javad Syad far,Ghorban Kamranjo

* Caspian Sea International Sturgeon Research Institute (Areeo), Iran, Rasht * [email protected] Introduction In the recent years, efforts have been made to increase the productivity per unit of Acipenser baerii in tanks, or ponds in Iran (Yazdani et al., 2009). However, high-density rearing seems to lead to poor physiological environment that adversely affect fish health in fingerlings, juvenile and market periods (Kim et al., 2003; Al-Harbi and Uddin, 2004) and therefore led to high degree of mortality. During the last decades, antibiotics used as traditional strategy for fish diseases management but also for the improvement of growth and efficiency of feed conversion. However, the development and spread of antimicrobial resistant pathogens were well documented (SCAN 2003; Kim et al., 2004; Cabello, 2006; Sorum 2006). Probiotic, which are micro-organisms or their products with health benefit to the host, are used in aquaculture as dietary supplements and as a means of disease control but unfortantly studies have been focused on growth promotion of fish by probiotic supplements (Marzouk et al., 2008). Theefore, this study investigated the Effects of Commercial Aquaculture Probiotic on growth performance and immune system of Acipenser baerii juvenile.

Materials and Methods The commercial aquaculture probiotic (CA probiotic,Superzist, Zistyar Varna, Iran) comprising Bacillus subtilis, Lactobacillus acidophilus, L. delbrueckii, L. rhamnosus, L. plantarum in amount of (100, 200 and 300 mg/kg) were added to diet (4mm pellets, Skriting, Italy, protien :41% , fat: 12% and Ash: 8.5%) which named to100 (PR100), 200 (PR200) and 300 (PR300) g/kg CA Probiotic. 120 Acipenser baerii with average of weight (109.25 ±3.7 g) were introduced I to 12 fiber glass tank containing 350-liter fresh water. Fish were fed 3% body weight three times daily. At the end of the trial (8 weeks), humoral innate immune parameters (serum IgM levels, lysozyme activity and alternative complement activity (ACH50) were assessed.

Results and Discussion Probiotic supplemented in diet (PR100), (PR200) (PR300) had not significant effect on the lyzozyme , ACH50 and IgM activity, but amount of The ACH50 activity fish fed (PR200 and PR300) were higher than other treatment but amount of Lysosyme in fish fed (PR100 and PR200) was better than control (P >0.05). Lysozyme is one of the most studied innate responses in fish. Lysozyme can act on the peptidoglycan layer of bacterial cell walls resulting in the lysis of the bacteria. Lysozyme has been found in mucus and ova (38), and serum lysozyme, probably coming from peritoneal macrophages and blood neutrophils, has been used as an indicator of non-specific immune response. Complement system, a component of the non-specific immune response, plays a key role in adaptive immune responses, involved in chemotaxis, opsonization, phagocytosis

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir and degradation of pathogens and has effector mechanisms like direct killing of microorganisms by lysis. In this study we didn’t observe significant difference between performances growth of fish fed by different level of native probiotic but it’s possible that an inclusion of 200 mg/kg of native probiotic will have a positive enhance of growth performance and feed utilization of Acipenser baerii on aqua farm system in a long period culture.

References Cabello FC. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbio 2006; 8:1137-1144. Ellis AE. Immunity to bacteria in fish. Fish Shellfish Immunol 1999; 9: 291–308. Kim S, Nonaka L, Suzuki S. Occurrence of tetracycline resistance genes tet (M) and tet (S) in bacteria from marine aquaculture sites. FEMS Microbiol Letters 2004; 237:147-156. Marzouk, M.S., M.M. Moustafa and N.M. Mohamed, 2008. The influence of some probiotics on the growth performance and intestinal microbial flora of Oreochromis niloticus. Proceedings of the 8th International Symposium on Tilapia in Aquaculture 2008, Cairo, Egypt, pp: 1059-1071. Tort, L., Sunyer, J. O., Gomez, E., Molinero, A.1996. Crowding stress induces changes in serum haemolytic and agglutinating activity in the gilthead sea bream Sparus aurata. Vet Immunol Immunopathol, 51: 179-188.

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Localization of mitochondria rich cells in the gill of Russian sturgeon Acipenser gueldenstaedtii by Immunohistochemistry technique

Salman Malakpour Kolbadinezhad* , Jonathan Mark Wilson

*Melecular physiology CIIMAR, ICBAS University of Porto, Portugal [email protected] Abstract The Acipenseridae are euryhaline species origined from freshwater fish that possess capability of migration either to brackish water or seawater. Bronchial ionocytes called chloride cells are rich in mitochondria and have an amplified basolateral membrane with high Na+/K+-ATP (NKA) and NKCC expression and apical cystic ﬁbrosis transmembrane receptor (CFTR) Cl- channel expression. By using Immunohistochemistry (IHC) technique colocolazitaion of NKA immunoreactivity (αR1 and/or α5 subunit) was detected in rather round ionocytes located in the interlamellar region of the filaments or at the base of the lamellae and flat above the lamellar surface as protrusions. However there was no detectable of CFTR in the gill. Subpopulation of MRCs were present expression of both NKA and NKCC1 with the exception a few ionocytes with no NKA. This study was directed at the molecular characterization of the gill ion transport mechanisms in Acipenser gueldenstaedtii, the Russian sturgeon

Fig. Double immunoflurorescence localization of NKA with either (A) (αR1 and/or α5 subunit) or (B) NKCC1 in the gill. Note, that NKA- IR rather round (arrow) cells were present heterogeneously distributed in the interlamellar region of the filaments or at the base of the lamella and flat above the lamellar surface as protrusions (curve arrow). Subpopulation of MRCs were present expression of both NKA and NKCC1 with the exception a few ionocytes with no NKA (arrowheads). No detectable CFTR expression was found in gill. Sections were counter stained with DAPI nuclear staining and overlaid with the contrast image. Scale bar 50 µm in upper panel and 10 µm in inset.

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References Krayushkina, L. S. and Semenova, O. G. 2006. Osmotic and Ion Regulation in Different Species of Acipenserids (Acipenseriformes, Acipenseridae). Jour. Ichth. 46, 1, pp. 108– 119. Krayushkina, L. S., Semenova. Vyushinab, O. G, A. V. and Gerasimova, A.A. 2015. Morphofunctional Remodelling of the Osmoregulatory System in Starred Sturgeon Acipenser stellatus (Acipenseridae) During Transition from Hyperosmotic to Hypoosmotic Regulation1. Jour. Ichth. 55. 2, pp. 259–272. Evans, D.H. and Claiborne, J.B. 2009. Osmotic and ionic regulation in fishes. In: D.H. Evans (ed.) Osmotic and Ionic Regulation: Cells and Animals, pp. 295–366. CRC Press, Boca Raton, FL.

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Fish Diseases and Health

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A case study of skeletal deformity of Acipenser baerii in a recirculation farm in Khouzestan province

Mohammad Mehdi Haghparast*, Mojtaba Alishahi, Reza Ghanei, Zahra tulabi Department of Clinical Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran [email protected] Introduction Sturgeon ﬁsh are mainly cultured for the production of caviare, as a result of the sharp decrease in production capacity of caviare from natural resources such as the Caspian Sea. In addition, they are also an important source of commercially valuable ﬁsh meat. Recently their culture in recirculation system in inland area such as Khousestan province was commercialized.

Materials and Methods In spring of 2016, a report of multiple disorders and slight mortality is referred to fish laboratory of veterinary faculty, Shahid Chamran University of Ahvaz. The case origin was a recirculation intensive Acipenser farm in area between Shoushtar and Dezful, Khouzestan province. Three Acipenser baerii, with two or three years age (1-3 kg weight) were sampled and transferred with oxygenated plastic bag to the fish laboratory in Ahvaz. In the observation of fish, severe emaciation and muscular atrophy, skeletal deformity (scoliosis and lordosis), skin and fin lesions shortage of operculums, unilateral and bilateral exophthalmia were seen. After biometric assay, wet mount samples from skin, fins and gills prepared and examined under light and phase contrast microscope. Bacterial samples were taken in sterile condition from skin and fin lesions. For evaluation of skeletal deformities, after deep anesthesia, dorsal and lateral X-ray radiography were done. After that fishes euthanized fish dissected and any internal abnormal signs were recorded and conventional internal parasitic and fungal examination were done. For evaluation of possible systemic bacterial infections bacterial samples were taken from Kidney, liver and spleen in TSA culture media.

Results Moderate infestation of protozoan parasite, Tricodina observed in skin and gills (fig.1), along with a slight infestation of fungal disease (Saprolegnia spp.) (fig.2) in all sampled fish were confirmed via microscopic assay. No parasitic infection detected in internal organs examination. Results of bacterial assay of internal organs and skin and fin lesions showed no bacterial infection except d a light presence of non- pathogenic bacteria in skin lesions. Radiographic photos showed a distinct and typical lordosis and scoliosis problem in all samples (fig.3).

Fig 1 Fig 2 Fig 3

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Conclusion According to macroscopic and microscopic examination of samples and history of recirculation farm, it is obvious that this health problem can't be an infectious disease. Mild fungal and parasitic infestation may occur as a secondary health problem. The main reason of the problem can be referred to nutritional, environmental and water quality management of the. Finally, improves in food and water quality, decrease in fish density, and were suggested to solve the problems.

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Comparative of protein pattern and immune parameters of Persian and Russian sturgeon (Acipenser persicus and Acipenser guldenstaedtii)

Hamed Azadi*, Hamed kolangi Miandareh, Abdolmajid Hajimoradloo, Mahdi Abbasian , Mohammad Akhavan Bahabadi1 *Department of Fishery, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan [email protected] Abstract Fish epidermal mucus provides the first line of defense against pathogens beside contains many kinds of biologically active molecules. The role of epidermal mucus enzymes in the innate immune system of Persian sturgeon (A.persicus) and Russian sturgeon (A.gueldenstaedtii) is not known exactly. In the present study skin mucus was analyzed to characterize potential innate immune factors such as lysozyme, alkaline phosphatase as well as protein patterns. This information could be beneficial in developing sustainable aquaculture health management strategies for these species.

Introduction Persian and Russian sturgeon is the most vulnerable sturgeon so these species are currently included in the IUCN Red List. Controlled and successful production in aquaculture industry requires basic knowledge of the immune system of cultured fishes. The innate immune system divided into the physical barriers, humeral and cellular components. since the majority of the infectious agents affects or initiates the process of infection in the mucus surfaces, fish skin mucus is the first line of defense against infections and the mucosal immune response plays a crucial role in the course of the infection (McNeilly et al. 2008). Mucus serves as a repository of numerous innate immune factors such as lysozyme, immunoglobulin, complement proteins, lecithin, C-reactive protein, proteolytic enzymes and various other antibacterial proteins and peptides (Shepherd, 1994; Cole et al., 1997). The innate immune system of Persian sturgeon (A.persicus) and Russian sturgeon (A.gueldenstaedtii) is not known. Therefore this study was designed to provide preliminary information on these innate immune components these species. The results of this study could be beneficial in developing sustainable aquaculture health management.

Materials and methods Mucus was collected as described by Ross et al. [1] with some modifications. Mucus protein content was analyzed using the Bradford Assay (Bradford, 1976). The supernatants were analyzed by SDS-PAGE 6% topped by 5% stacking gel according to Laemmli and stained with coomassie brilliant blueR250. [2]. Lysozyme activity was measured by the turbidimetric method as described by Sankaran and Gurnani (1972). Alkaline phosphatase activity was determined by the use of kit (Pars Azmoon Company, Iran) and absorption was read at 405 nm in spectrophotometer.

Results and discussion SDS-PAGE analysis. SDS-PAGE protein profiles of two species are shown in Fig. 1. Electrophoretic comparison of the mucus proteins revealed the differences in the intensity of several protein bands between both species. Column (1) and (2) were showed the A.persicus and A.guldenstaedtii protein patterns respectively. Bands that are marked in red arrows that

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir do not exist in one of them clearly and bands that are marked Green arrows indicate bands with enhanced intensity in one of them. The bands of ‘A’ and ‘C’ do not exist in Russian sturgeon. In the other side ‘D’, ‘B’ and ‘E’ bands are more intense than in Russian and Persian sturgeon and band respectively. Enzyme activity assay The alkaline phosphatase activity was confirmed in the mucus of the samples. The activity of alkaline phosphatase was highest in mucus of A.guldenstaedtii but the activity of alkaline phosphatase was not significantly different between the A.persicus and A.guldenstaedtii. the variation in enzymes activity could be attributed to several other factors such as habitat, species-specific evolutionary adaptation to different environmental conditions, the thickness of the epidermis and the number of mucus cells (Fast et al. 2002), handling stress, maturity, diet, sex, species variation, and genetic variation (Balfry and Iwama 2004).

Overall, our data showed each fish species has one or more enzymatic activities high but never all the activities are high or low at the same time. This could indicate that the immune response is always alert and Figure 1: protein pattern of A.persicus the fish resistance is not limited to only one factor. Nonetheless, and A.guldenstaedtii further studies should be performed to deepen in the knowledge of the fish mucus enzymes and their specific role in the mucosal immunity. References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2: 248-254. Balfry, S.K. and Iwama, G.K., 2004. Observations on the inherent variability of measuring lysozyme activity in coho salmon (Oncorhynchus kisutch). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 138(3), pp.207-211. Cole, A.M., Weis, P. and Diamond, G., 1997. Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. Journal of Biological Chemistry, 272(18), pp.12008-12013. Fast, M.D., Sims, D.E., Burka, J.F., Mustafa, A. and Ross, N.W. 2002. Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 132(3), pp.645-657. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, pp.680-685. McNeilly, T.N., Naylor, S.W., Mahajan, A., Mitchell, M.C., McAteer, S., Deane, D., Smith, D.G., Low, J.C., Gally, D.L. and Huntley, J.F., 2008. Escherichia coli O157: H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infection and immunity, 76(6), pp.2594-2602. Sankaran, K. and Gurnani, S., 1972. On the variation in the catalytic activity of lysozyme in fishes. Indian journal of biochemistry & biophysics, 9(2), pp.162-165. Shephard, K.L., 1994. Functions for fish mucus. Reviews in fish biology and fisheries, 4(4), pp.401-429.

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Prevention and the decontamination parasites in marine and fresh water fish at the time of harvesting, processing and post-processing Mina Ahmadi*, Ali Asghar Khanipour, Fatemeh Noghani., Soghra Kamali. *Iranian Fisheries Science Research Institute, Inland waters Aquaculture Research Center, Agricultural Research Educationand Extension Organization (AREEO), Bandar Anzali, Iran [email protected]

Abstract Marine and freshwater fish species are a potential source of zoonotic parasites. Infection of the parasite in humans, to occur the effect of physical contact or consumption of raw or Semi baked fish as a result it know entirely related to dietary habits and intake of foods that are eaten raw. Because some of these parasites are transferred to humans through the food chain is important identify them. Physically remove or inactivate parasites of fish at the time of harvesting, processing or after processing, not feeding raw Semi baked fish or unprocessed effective methods that can be used to reduce infection risks. Most important and largest problem in pollution control in farmed fish freshwater is prevention of pools pollution with human and animals fecal, that with control surface waters environment from there the fish are caught, health aquaculture system, control and eliminate the the first intermediate host of parasites (snail), intermediary hosts treated (human or animal source) is possible. Freezing most important practical means to control parasites and applied at every stage of production, will fix the need HACCP to eliminate parasites. Freezing as a method is to kill parasites dependent on time and degree cold. For parasitic worms, in the air giver freezer will be sufficient for 15 hours in -35 ° C or 7 days in – 20 ° C. Deactivates parasites depends on the time and temperature by heating, so that for normal cooking temperature in the thickest part of product should be at least 630 C for 15 seconds or more Microwave cooking requires a higher temperature is to kill all parasites because food heating is going. Unbalanced and non- uniform recommended the temperature reach77 oC for the thickest part of product and also moving the fish during baking helps to bake uniformly Until all its parts to be heat uniformly. Control of parasites in smoked products depends on the type and method smoking. Smoking hot method can effectively disable the parasites because products are baked during the smoking process. Contrary to bacteria, flattened parasitic worms do not disappear by seasoning and flavoring agents such as Liquid smoke. In cold smoking method because the fish is smoked at a low temperature, the heat is not enough is to kill parasites. In many processing are used of fish that have been frozen previously or products after Smoked be frozen for storage or transport or for conversion into thin slices is frozen after smoking. But it should be pointed that dead larvae Anisakis in fish muscle may still be consumer risk to health due to allergic reactions. Factors that could cause problems in identifying parasites include: o The thickness of the fillets o The skin on fillets o The amount of fat o Pigments o The experience of the person performing the test Candling is the process in which the fish fillets are placed on the table that light source located under table and the table is clear so that we can detect parasites.

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Parasites seen with this action can be taken with forceps or remove the infected part of the fillet. Part of the fillet usually has the most parasites, the region is the abdominal membrane that covers the offal (where the parasite can be the first to be migrated).

References Moeini, S. et al. 1390. Safety and quality control issues in aquatic product. Iranian Fisheries Research Institute. Pazooki J., Masoumian M., Jafari N. 2007. Check list of Iranian fish parasites. First published. Publisher Iran fisheries Research organization (IFRO). Tehran, Iran. (In Persian).

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Histopathological effects of trichodiniasis in farmed sturgeon Acipenser baerii Zahra tulaby Dezfuly, AnnahitaRezaie, MojtabaAlishahi, Mohammad Mehdi Haghparast *

*Department of Clinical Sciences, ShahidChamran University of Ahvaz, Ahvaz, Iran

[email protected] The majority of fish ectoparasitic protozoa are commensals but some of them may produce serious diseases and mortalityespecially in fry and fingerlings. Rapid growth and multiplication of these parasites due to direct life cycle, greatly increase their rate of infestation among fish causing mass mortality.Trichodina is common ectoparasites which in most cases is pathogenic toboth freshwater and marine fish. The infection being stimulated by the high density of fish in ponds. Under such conditions, it may be an epizootic. In cases of stress, several species of trichodina may become pathogenicinterfering with feeding and respiration of small fish. The lesions mostly induced by this parasite are hyperplasia andnecrosis of the epidermal cells. These cells are usually subjected to severe attacks by parasites. This results in cellulargrowth and excess mucus production. In spring of 2016, a report of multiple disorders such as skeletal malformation, gill necrosis, skin ulcers and slight mortality is referred to fish laboratory of veterinary faculty, Shahid Chamran University of Ahvaz. The case origin was a recirculation intensive Acipenser farm in area between Shoushtar and Dezful, Khouzestan province. Three Acipenser baerii, with two or three years age (1-3 kg weight) were sampled and transferred with oxygenated plastic bag.After taking sample from gill and skin, one of them euthanized and necropsy were carried out. Moderate infestation of protozoan parasite, Trichodina, observed in skin and gills by light microscope. Macroscopic examination of different organs was as follow: Severe emaciation and muscular atrophy Scoliosis and lordosis Skin and fin ulcers Unilateral and bilateral exophthalmia The gills of fish were ﬁxed in 10% neutral buffered formalin and sent to histopathological lab. Tissue sections samples were sectioned at 5 µm thicknesses from paraffin blocks and stained with hematoxylin-eosin (H&E). Microscopic results revealed hyperplasia and hypertrophy of secondary lamellae epithelium (Fig 1). Also accumulation of inflammatory cells was seen in the lamellae. Also there was diffuse accumulation of inflammatory cells around vessels and between hepatocytes. Also many melanomacrophagesand eosinophils were seen in this area (Fig 2).

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Fig.1. Gill. Note to increasing the thickness of apical part Fig.2. Liver. Note accumulation of inflammatory cells of secondary lamellae (arrows). With higher magnification around central vein and between hepatocytes (arrows). With (right side) accumulation of inflammatory cells (red higher magnification (right side) eosinophils (arrows) and arrows), hypertrophy of epithelium (black arrows) and melanomacrophages (asterisk) are seen between thickening of secondary lamellae (asterisk) are obvious inflammatory cells accumulated around central vein (H&E). (H&E).

Authors have been reported a complete destruction of gill epithelium. The intensification of fish culturing creates disease problems that originate from over crowdness or deteriorating water quality such as unsuitable water temperature, pH, carbon dioxide and free ammonia concentrations. For the control of Trichodiniasis, formalin isrecommended by many authors. Based on finding of this work, it can be suggested that main reason of the problem can be referred to nutritional, environmental and water quality management of the recirculating system's of farm.

References Dujin, C.V.F. 1973. Diseases of Fishes. 3rd Ed., Life books, London, pp. 486. Lom, J. 1995. Trichodinidae and other Ciliates (Phylum Ciliophora). In ‘‘Fish Diseases and Disorders,’’ Vol. I ‘‘Protozoan and Metazoan Infections’’ (P. T. K. Woo, Ed.), pp. 229– 262. CAB Int. Lom, J. 1970. Observations on trichodinid ciliates from freshwater ﬁshes. Archiv für Protistenkunde, 112, 153–177.

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Isolation and identification of sturgeon probiotics in Beluga sturgeon (Huso huso) and Siberian sturgeon (Acipenser baerii) Alireza Shenavar Masouleh*, Mohammad Pourkazemi, Mohammadali Yazdani, Parisa Esmaeili, Yalda Baniesmaeili, Mahtab Yarmohammadi, Mehdi Alizadeh, Jalil Jalilpoor, Mehdi Masoumzadeh, Soheil Bazarimoghddam, Mehdi Soltani

*Department of Aquatic Animal Health, International Sturgeon Research Institute, Agricultural Research, Education and Extension Organization (AREEO), P.O. Box 41635-3464, Rasht,Iran.

E-mail*: [email protected]

Introduction Sturgeon aquaculture is growing in the world and the infection and non-infection diseases can be predict in the intensive or semi intensive culture conditions. Today, the use of probiotics and vaccines is widely used as an alternative application and substitution of antibiotics in aquaculture.

Materials and Methods Apparently healthy Beluga sturgeon (Huso huso) weighing 89.14g and Siberian sturgeon (Acipenser baerii) weighing 31.3g were selected randomly for this study. The skin was then washed with 70% ethanol before opening the ventral surface with sterile scissors. The intestine was emptied and thoroughly rinsed three times in sterile 0.9% saline to remove non adherent bacteria before homogenation. Homogenates were diluted in sterile 0.9% saline and 0.1ml volumes of appropriate dilutions were spread on the surface of MRS plates. The plates were incubated anaerobically at 30°C for three days. Preliminary identification and grouping was based on the cell morphology and phenotypic. Initial identification of Lactic Acid Bacteria (LAB) isolates were performed by Gram staining, catalase test, CO2 production from glucose, growth at different temperature (10, 45 °C), two pH (4.4, 9.6), and 6.5% NaCl. Specific 16S-23S gene were amplified using total extracted DNA and primers and its PCR product following 1% agaros gel electrophoresis, were sequenced.

Results and Discussion The results of present study showed that LAB counts were between 2.72- 3.04 Log CFU/g in the intestines. All isolates were gram positive and catalase negative. Weissella confusa, Lactococcus lactis and Lactococcus garviae were constituted groups in the intestines of Beluga sturgeon and Siberian sturgeon as lactic acid bacteria. LAB populations in the GI tract of fish have been widely studied in a number of fish species. In fish, LAB are usually non- pathogenic bacteria that are thought to benefit the host species through competition against pathogenic bacteria and numerous LAB species have been utilized as probiotics in feeds, hence the LAB isolated in the present study should be assessed in invitro and invivo for their probiotic properties.

Keywords : Lactococcus, Acipenser, Lactic acid bacteria, Sturgeon, Probiotics References

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Balcazar, J. L., Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D. and Muzquiz, J. L., 2006: The role of probiotics in aquaculture. Vet. Microbiol. 114, 173–186. Dimitroglou, A., Merrifield, D. L., Carnevali, O., Picchietti, S., Avella, M., Daniels, C., Guroy, D. and Davies, S. J. 2011. Microbial manipulations to improve fish health and production – a Mediterranean perspective. Fish Shellfish Immunol. 30, 1–16. Gatesoupe, F. J. 2008. Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments. J. Mol. Microbiol. Biotechnol. 14, 107–114. Merrifield, D. L., Dimitroglou, A., Foey, A., Davies, S. J., Baker, R. T. M., Bgwald, J., Castex, M. and Ringo, E. 2010. The current status and future focus of probiotic and prebiotic applications for salmonids – a review. Aquaculture 302, 1–18. Ringo, E. and Gatesoupe, F. J. 1998: Lactic acid bacteria in fish: a review. Aquaculture 160, 177–203. Soltani, M., Pourkazemi, M., Ahmadi, M.R., Taherimirghead, A., Merrifield, D.L., Masouleh, A.S. 2013. Genetic diversity of lactic acid bacteria in the intestine of Persian sturgeon fingerlings. J Appl Ichthyol. 29(3):494-498.

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Monitoring of bacteria flora in intestine and water of Beluga, Huso huso, juveniles in earthen ponds in West Azerbaijan province Yousef Ali Asadpour*, Ehsan Soleimannejadbari, Abbas Zomorrodi *Iranian Fisheries Science Research Institute, National Artemia Research Center, Agricultural Research Education and Extension Organization (AREEO), Urmia, Iran Introduction Due to the significant decrease in production and sturgeon fishing on the Caspian Sea the main habitat and fishing source of Beluga Sturgeon recently, the present study was conducted to determine the feasibility of breeding sturgeon species in the earthen pools of Urmia Artemia research center, and also to investigate bacterial flora of sturgeon Huso huso juvenile gut.

Materials and Methods 1000 Beluga (Huso huso) juveniles with average weight of 198 gram were prepared from Rasht sturgeon propagation center. The fishes divided in two ponds. Physicochemical factors of the ponds water were measured: temperature 14.33±3ºC , dissolved oxygen 7.05±2 g per liter and pH= 8.00±0 . Biometry includes weight and total length monthly. After biometry, in order to culture intestinal and water contents were streaked on TSA and Mueller Hinton Agar within 24-48 h and incubated at 36◦C. The Gram staining and biochemical diagnostic tests API 20E (BioMerieux) were used to determine and identify bacterial species.

Results At the end of culture period the results were; survival rate 96.8%, SGR 0.41, FCR 1.74, CF 0.42 and BWI% 19.93. In the identification of intestinal flora of Beluga (Huso huso) juvenile the species of Enterobacteriaceae, Aeromonas hydrophila, Staphylococcus, Acinetobacter sp. and Flavobacterium were isolated. Keywords: Bacteria isolation, Intestinal flora, Huso huso, Earthen ponds

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Histopathological effects of endosulfan on the gill of Persian sturgeon (Acipenser persicus Borodin, 1987)

Roghieh Safari* , Abasali Hajibeglou

*Gorgan university of Agricultural Science and Natural Resources ([email protected]) Introduction Endosulfan, an organochlorine, is being widely used in agriculture as well as in integrated agriculture-aquaculture farming system to protect important food crops. Even exposure of fish to low concentration of this pesticide may accumulate in the body that can result in several toxic effects including tissue damage, physiological, biochemical and molecular alterations, respiratory changes, and ultimately death which could be used as environmental bio indicators (Piazza et al., 2015). Histopathological analysis can be considered as a sensitive tool to determine microscopic morphological alterations. The gills are considered to be the most important uptake site for waterborne pollutants, whereas liver and kidney are the main storage and detoxification organs in fish (Reynders et al., 2006). Acipenser persicus, inhabiting the Caspian Sea and listed as an endangered species (Pourang et al., 2005). In addition to overfishing, contamination resulting from industrial and domestic effluent and other man- made activities (Lenhardt et al., 2006) might cause depletion in its population to noneconomic levels over the past decades. The objective of this study was to determine the toxic effect of Endosulfan on histological structure of gill in the Acipenser persicus.

Materials and Methods Persian sturgeon (3-5 g) fingerlings were obtained from Shahid Marjani Breeding and Rearing Center (Golestan, Iran) and acclimated to the experimental conditions for a period of 15 days, after which fish were randomly distributed into 9 tanks of 300 l of capacity at a density of 30 fish per tank and submitted to sublethal concentrations of endosulfan 35%, using a mixture of α- and β- isomers (70:30), at concentration of 0.05, 0.1, 0.2 and 0.5 LC50 for 14 days, based on the LC 50 of 80 µgl-1 reported by Safari et al (2015). Nine fish per treatment were taken at days 0, 4, 7 and 14, rapidly anesthetized with clove powder and the gills were taken from samples immediately. Gill samples of control and exposed fish were dissected and fixed in 10% neutral-buffered formalin, and then samples were processed for routine wax histological evaluation (dehydrated and embedded in paraffin). Sections of 5µ thickness were prepared and stained with hematoxyline and eosine stains as described by Haschek et al. (2010).

Results and Conclusion In the present study, curling of secondary lamellae, lamellar fusion, aneurism, epithelial hyperplasia, epithelial lifting and epithelial necrosis were noted in gills of all the treated fish samples. The intensity of these changes increased up to day 14 of the experimental period. This might be considered as a defensive mechanism in this species to overcome the toxic effects.These clinical signs and the others like these as well as adaptive responses were pointed out in different tissues of various species exposed to several pollutants (Ashish and Banalata, 2008). The lifting of epithelial layer from gill lamellae, necrosis and degeneration of secondary lamellae, shorting of secondary lamellae, and clup-shaped lamella in the gills of Lebistes reticulatus (Poecilia reticulata) exposed to cyphenothrin were observed by Erkmen et al. (2000). De Silva and Samayawardhena (2002) founded shorter gill lamellae, fusion,

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir complete destruction of lamellae, increased vacuolization and irregular appearance in guppy (Poecilia reticulata) exposed to chlorpyrifos. Allen et al. (1993) emphasized adaptive response exhibited by As- treated fish during long term exposure involving reduced glutathion. The present results indicated that these parameters could be used as an effective index to monitor ecotoxicological changes. Table1. Effect of endosulfan on in gills of Acipenser persicus during 14 days.

Parameters Aneurism Epithelial Epithelial Lamellar Curling of necrosis and lifting fusion secondary desquamation lamellae Control - - - - - 0.05 C50 - - - - - Day0 0.1 LC50 - - - - - 0.2 LC50 - - - - - 0.5 LC50 - - - - - Control - - - - - 0.05 C50 - - - - - Day4 0.1 LC50 + + + + + 0.2 LC50 ++ + + + + 0.5 LC50 ++ ++ ++ ++ ++ Control - - - - - 0.05 C50 + + + + + Day7 0.1 LC50 ++ + + + + 0.2 LC50 ++ ++ ++ ++ + 0.5 LC50 ++ ++ ++ ++ ++ Control - - - - - 0.05 C50 + + + ++ + Day14 0.1 LC50 ++ + + ++ ++ 0.2 LC50 ++ ++ +++ +++ ++ 0.5 LC50 +++ ++ ++ +++ ++

References Allen, P. 1993. Affects of exposure to cadmium chloride and lead chloride on the hematological profile of Oreochomis aureus. Comparative Biochemistry and Physiology 105 (2), 213-217. Ashish, K. M. and Banalata, M. 2008. Acute toxicology impacts of hexavalent chromium on behavior and histopathology of gill, kidney and liver of freshwater fish, Bloch (Channa punctatus). Environmental Toxicology and Pharmacology 26,136- 141. De Silva, P. M.C.S. and Samayawardhena, L. A. 2002. Low concentrations of lorsban in water result in far reaching behavioral and histological effects in early life stages in guppy Ecotoxicology and Environmental Safety 53, 248-254. Gernhofer, M., Pawet, M., Schramm, M., Muller, E. and Triebskorn, R. 2001. Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams. Journal of Aquatic Ecosystem Stress and Recovery 8, 241-260. Haschek, W. M, Walling, M. A. and Rousseax, C. 2010. Fundamental of Toxicological Pathology. New York, NY: Academic Press. pp: 211-686. Lenhardt, M., Jaric, I., Kalauzi, A. and Cvijanovic, G. 2006. Assessment of extinction risk and reasons for decline in sturgeon. Biodiversity and Conservation 15, 1967-1976.

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Sodium chloride as an agent of control of Saprolegniasis in Persian Sturgeon eggs Abbas Ali Aghaei Moghaddam* , Kamran Aghili , Sara Haghparast

*Golestan Fisheries Research Center (GFRC), Iranian Fisheries Science Research Institute, Agricultural Research, Education Extension Organization (AREEO) *Corresponding author’s E-mail: [email protected] Introduction Persian sturgeon (Acipenser persicus) is one of the most valuable sturgeon fishes in Caspian Sea (Baradaran Noveiri et al., 2005). Artificial propagation is now the main source of replenishment of sturgeon resources.(Barannikova et al., 2005). Saprolegniasis is one of the most important factors responsible for the loss of eggs in sturgeon hatcheries. There are some chemicals antifungal agents that try to reduce saprolegniasis in hatcheries like the Hydrogen Proxide, Formaline and Malachite Green. Sodium chloride is one of the antifungal agent that have been granted low regulatory priority drug status by the United state Food and drug Administration (FDA) (Scherier et al., 1996). Sodium chloride is a good antifungal agent because is economic and safe (Khodabandeh et al., 2006). This research was carried out to evaluate the efficacy of sodium chloride for controlling of Saprolegniasis on Persian sturgeon eggs.

Materials and Methods Thirty hours eggs were confined within 15*15*10 cm in diameter Nylonic cages placed in Yushchenco Incubator trays (50 eggs per cage)(Abtahi et al 1994). Water flow was about 2.5 l/min. Three cages were used as replicates for each treatment level and tree cage as controls. Eggs were exposed in four duration times: 15, 30, 45 and 60 minutes at five concentrations: 1, 1.5, 2, 2.5, 3 percent in sodium chloride bath. Every three cages were put in tanks containing salt in one of the concentrations for one duration time daily. The percent rate of hatch and fungal infection were calculated. This research was carried out in Shahid Marjany Center, Golestan province and was done in Completely Random Design in Factorial with two factors of concentration of salt and duration. Duncan test at significance level of α=0.05 was used for comparing of means, and then the data were compared with control groups.

Results As indicated in Table (1), the main effect of concentration and the interaction effect of concentration by duration were statistically significant in the first stage (P<0.05). Table 1: Analyzes of Variance of affecting factors on fungal infection (%) of Acipenser persicus eggs in late March (first stage) Sig. F Mean Square df Sum of Source Square .019 *3.26 1.52 4 6.1 concentration .078. 3.24 1.51 1 1.51 duration 0.021 *3.2 1.5 4 6 con. × dur. 0.47 47 22 Error 57 44 total *.the mean difference is significant at the level of 0.05 According to Fig. 1, there were significant differences between 1% NaCl ×15 min treatment and other treatments (P<0.05), except for the 2%NaCl×30 min treatment and the control (P>0.05). In this stage, the main effects of concentration and duration as well as their interaction effects were significant (P<0.05) on larvae hatching (Table 2).

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Table 2: Analyzes of variance of affecting factors on larvae hatching (%) of Acipenser persicus eggs in late March (first stage). Sig. F Mean Square df Sum of Source Square 0 50.8 2158.5 4 8634 concentration 0 57.5 2448.5 1 2448.4 duration 0 46.4 1969.3 4 7877.4 con. × dur. 42.5 47 1996.7 Error 57 306308 total

Remarkably, there were significant (P<0.05) differences between 3%NaCl×30 min and all the treatments including control in larvae hatching of Acipenser persicus eggs. The highest rate of larvae hatching (84.2%) was observed in the control, representing no significant (P>0.05) difference with other treatment except 1%, 1.5% NaCl× 15min.

Discussion Different results of the sodium chloride efficacies on fungal infection of the fish eggs have been reported by various researchers. Schreier and Rach (1996) demonstrated that sodium chloride (30 g 1-1) for 15 min once a day had less treating effects than formalin, hydrogen proxide on fungal infection of rainbow trout (Onchorhynchus mykiss) eggs but improved larval hatching. Khodabandeh and Abtahi (2006) studied the antifungal effect of sodium chloride, formalin and iodine on fungal infection of the common carp eggs and suggested 3.5% NaCl as the safest and cheapest treating agent. In the present study, the eggs treated 3% NaCl for 30 min showed lower larval hatching rate and approximately the whole eggs became white and degenerated, demonstrating detrimental effect of NaCl at higher concentration whereas with decrease in treating duration by NaCl at this level, larvae could hatch but were damaged while slow-turning swimming. In contrast, NaCl solution at concentration of 35 g l- 1, were effective for treating fungal infected eggs of the common carp as reported by Khodabande and Abtahi (2006), implicating the lower sensitivity threshold of sturgeon eggs than carp eggs against NaCl treating solution.This might be related to the different structure of fish egg membrane, as Persian sturgeon has thin egg membrane and therefore more vulnerable to damages.

References Khodhbandeh, S.and Abtahi, B. 2006. Effects of sodium chloride, formalin and iodine on the hatching success of common carp (Cyprinus carpio) eggs. Journal of Applied Ichthyology.V22, N1, 54-56 Schreier, T.T. and Rach, J.J. 1996. Efficacy of formalin, Hydrogen Proxide and Sodium Chloride on fungal –infected rainbow trout eggs. Aquaculture .140 (4): 323-331

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Histopathological effects of waterborne copper nanoparticle on gills of Siberian sturgeon (Acipenser baerii) juveniles Forouzan Bagherzadeh Lakani*, Saeid Meshkini, Mohammad Ali Yazdani Sadati, Bahram Falahatkar, Ali Asghar Tehrani *Fisheries Department, Faculty of Natural Resources, Urmia University, Urmia, Iran. * [email protected] Introduction Nanoparticles (NPs) are set of several atoms from a specific material. Important features of NPs are small size, wider surface, and specific optical features and their particular surface coverage which increases their activity when they enter to the body and poisoning caused by such particles lasts longer (Kagan et al., 2005; Kipen & Laskin, 2005). Understanding the effects of NPs on fish is therefore an important aspect when considering the effects of NPs on the aquatic environment (Ates et al., 2013). Fish gills come into immediate contact with the environment and they are generally considered a good indicator of water quality, being a model for studies of environmental impacts (Velmurugan et al., 2007). Copper is an essential element required for all living organisms, since at least 30 enzymes are known to have content of Cu as a cofactor. Copper is also toxic in excess and liver and gills are known to be target organs for it. So, this study was aimed to investigate the effect of different levels of copper nanoparticles (Cu-NPs) on the histopathology of the gill of Siberian sturgeon (Acipenser baerii).

Materials and Methods Two hundred and forty Siberian sturgeon juveniles with initial weight of 29.2 ± 3.1 g and length of 21.8 ± 1.4 cm were randomly distributed in 12 fiberglass tanks at 4 different Cu-NPs treatments with 3 replicates. Four Cu-NPs concentrations (0 (Control), 50, 100 and 200 µg/l) and five different times (0, 7, 14, 21 and 28 days) were considered. The experimental period was of 28 days, 14 days exposure to Cu-NPs and 14 days as recovery time. For histopathological study, six fish from each group (two fish of each replicate) were sampled. Samples were dehydrated by routine methods and embedded in paraffin wax. They were sectioned by microtome and stained with hematoxylin-eosin staining method.

Results The pathological effects were observed in the gill tissue included necrosis, hyperplasia, cell proliferation leading to fusion of primary and secondary lamellae, loss of primary and secondary lamellae, thickening of the cartilage, numerous vacuole in primary and secondary lamellae, congestion and hemorrhage in the primary and secondary lamellae. Loss of primary and secondary lamellae reduced the functional surface of the organ. Also, the edema, epithelial lifting as well as lamellar fusion are defense mechanisms that reduce the bronchial superficial area in contact with the external milieu. These mechanisms also increased the diffusion barrier to the pollutant. With increasing Cu-NPs concentration and the exposure time, pathological effects increased. In the recovery time, the lesions decreased but they didn’t disappear until the end of the experiment. The current findings indicate that Cu-NPs had chronic pathological effects on the Siberian sturgeon even though the recovery period,

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International Conference on the Future of Sturgeon Aquaculture www.ICSA2016.ir and the complications of these nanoparticles are not completely resolved. So, preventing the entry of this nanomaterial into the aquatic environment would seem to be essential. References Ates, M., Demir, V., Adiguzel, R. & Arslan, Z. 2013. Bioaccumulation, subacute toxicity, and tissue distribution of engineered titanium dioxide nanoparticles in goldfish (Carassius auratus). Journal of Nanomaterials, 9: 1-6. Kagan, V.E., Bayir, H. & Shvedova, A.A. 2005. Nanomedicine and nanotoxicology: two sides of the same coin. Nanomedicine: Nanotechnology, Biology and Medicine, 1: 313- 316. Kipen, H.M. & Laskin, D.L. 2005. Smaller is not always better: nanotechnology yields nanotoxicology. American Journal of Physiology - Lung Cellular and Molecular Physiology, 289: 696-697. Velmurugan, B., Selvanayagama, M., Cengiz, E.I. & Unlu, E. 2007. Histopathology of lambdacyhalothrin on tissues (gill, kidney, liver and intestine) of Cirrhinus mrigala. Environmental Toxicology and Pharmacology, 24: 286-291.

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Effects of physicochemical parameters of water on frequency of Saprolegniceae from Persian sturgeon (Acipenser persicus) eggs Milad Adel*, Mohammad Jalil Zorriehzahra , Maryam Dadar *Department of Aquatic animal Health & Disease, Iranian Fisheries Sciences Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O. Box: 13185-116, Tehran, Iran. [email protected]

Introduction Persian sturgeon (A. persicus) is an important species that live in the southern margin of the Caspian basin. Unfortunately, nowadays fishery has seen a remarkable decline in the fishing yields of the Iranian Caspian Sea due to over-fishing, habitat deterioration and different diseases (Bani et al., 2009). The main problem in the artificial reproduction of A. persicus is the contamination of the eggs by fungi saprophytic and of particular concern is saprolegniosis, which is an infectious fungal disease that is wide spread in all stages of the life cycle of fish. Saprolegniosis infection may contribute to heavy mortality among fishes and are widespread in freshwater fish and affect wild and cultured fishes, also involving living, dead and eggs (Hussien et al., 2010). The aim of this study was to isolate and identify saprophytic fungi from affected eggs of A. persicus at hatchery and also carry out a survey on the effect and relation of physicochemical factors of water with the frequency of fungi contamination. Materials and Methods Sampling was done in the reproductive season of A. persicus between 2010-2012 (three times in each reproductive of season). The samples were inoculated in culture media (SDA, GPA and stilled water with cotton seed culture) at a temperature similar to the temperature at the hatchery (18-24°C). Physicochemical parameters of water including dissolved oxygen (DO), pH, temperature, total dissolved solid (TDS) and electrical conductivity (EC) were measured. Results The results showed that Saprolegnia sp, and Saprolegnia parasitica were the aquatic fungi isolated during this period of study. The minimum and maximum isolation of fungi saprophytic were related to the end of period sampling in 2010 (17%) and mid-season of sampling in 2012 (33%). During this study, significant differences in contamination rates to Saprolegnia sp. and S.parasitica were observed in 2012 (P<0.05). The results of this study showed that the frequency of fungi contamination can be directly related to physicochemical parameters of water and by decreasing water quality, the percent infection to fungi saprophyte was increased. Conclusions In conclusion, results of this study and previous studies indicated that S. parasitica is one of the most pathogenic fungi of sturgeon eggs in the hatchery and were the main cause of mortality in fish eggs. Given the important role of risk factors, reduce stressful situations through improved management and Environmental conditions, such as improve the water quality, reduction of organic matter, to avoid excessive density of fish and unnecessary manipulation and the use of appropriate disinfectants in hatcheries can be useful to control and reduce economic losses caused by this pathogen.

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References Bani, A., Tabarsa, M., Falahatkar, B. & Banan, A. 2009. Effects of different photoperiods on growth, stress and hematological parameters in juvenile great sturgeon Huso huso, Aquaculture Research, 40: 1899-1907. Hussien, A. M., Osman, I. E., Walid, A., Deen N. E. & Saman, S.E. 2010. A trial for I induction of Saprolegniasis in Mugil cephalus with special reference to biological control. Journal of American Science, 6: 203-207.

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Determination of lethal concentration of hydroalcoholic extract of Zataria multiflora and its effects on different tissues of Persian sturgeon

Mehdi Masoumzadeh*, Mostafa Sharif Rohani, Soheil Bazari Moghaddam, Ali Hallajian, Mehdi Alizadeh, Jalil Jalilpoor, Alireza Shenavar Masouleh.

*International Sturgeon Research Institute (Areeo), P.O. Box 41635-3464, Rasht, Iran [email protected]

Introduction Negative effects of drugs and chemical disinfectants which lead to resistance in body of farmed aquatics, make necessary their replacement with herbal therapeutics having natural resources and this is one of the most important priorities of researchers in fish medicine arena. Zataria multiflora belongs to Labiate family, is one of the most well-known herbal drugs in Iran and traditional medicine. Its antimicrobial effects were approved in different studies. Therapeutic parts of this plant include its top branches and dried leaves. Its extract is Thymol. Results of photochemistry experiments showed components such as alkaloid, saponin, flavonoid and tannin in Zataria multiflora.

Materials and Methods This study was carried out to determine the lethal concentration (LC50 96h) of hydroalcoholic extract of Zataria multiflora in Persian sturgeon (Acipenser persicus) fingerlings of 3.22±0.51g in weight. In order to study the effects of these extracts, 240 fingerlings were selected in seven treatments and one control group with three replications for each treatment. During the experiment, all water physicochemical parameters including the temperature, dissolved oxygen, pH, nitrite, nitrate, ammonium, electric conductivity and water hardness were measured. This experiment was carried out according to O.E.C.D method in 96h and 1h. In this regard, the linear regression equation was drawn using probit analysis, and then different values of lethal concentrations were estimated. In this survey, according to probit analysis, the LC50 of hydroalcoholic extract of Zataria multiflora during 96h and 1h were recorded to be 766.65 and 9933.44 mg/l, respectively. Study on the pictures taken out from sections of gill, liver and kidney of Persian sturgeon fingerlings showed that according to different doses of Zataria multiflora hydroalchoholic extracts the treatment groups showed some microscopic damages. They are hyperemia, adhesion in the gill filaments, cell necrosis, melanin pigments in gill primary filaments, cloudy swelling of hepatocytes, liver necrosis, hyperemia and increase in melanin pigments and melano macrophage centers in liver, glomerular changes such as congestion and blocked the dilation of Bowman's space, bleeding, cell necrosis, cloudy swelling of the in kidney.

Keywords: Lethal concentration, Tissue damage, Hydroalchoholic extract, Zataria multiflora, Persian sturgeon

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Skeletal deformities in Sturgeon fish Fatemeh Hassantabar1*, Mohammad Jalil Zorriehzahra2, Milad Adel2 and Maryam Dadar3

1 Sari University of Agricultural Sciences and Natural Resources, Department of fishery sciences, Sari, Iran 2Department of Aquatic animal Health & Disease, Iranian Fisheries Sciences Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O. Box: 13185-116, Tehran, Iran. 3 Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran [email protected]

Abstract Skeletal deformities should be considered as one of important disorders between reared and wild sturgeon fish. Among the various types of skeletal deformities three basic types of axial deformities, such as lordosis (V-shaped dorsal-ventral curvature), kyphosis (Λ-shaped dorsal- ventral curvature), and scoliosis (lateral curvature with rotation of the vertebrae) are the most frequently observed in cultured sturgeon fish. Skeletal deformities are caused by numerous factors including environmental factors (temperature, photoperiod, light intensity, salinity, high water current), nutritional imbalance (amino acids, vitamins, n-3 highly unsaturated fatty acid, phospholipid, phosphorus, manganese, zinc, heavy metal), genetic background and some pathogens such as Viral Nervous Necrosis. Occurrence of skeletal anomalies can result in significant, negative effects on animal welfare, quality of product and cost of production. In this speech some of important causative agents that could be responsible for skeletal deformities in sturgeon fish will be introduced. Furthermore, one obvious sturgeon affected case that was identified in Tehran countryside in 2012 will be discussed (Figure 1, 2)

Figure 1: Sturgeon with Scoliosis Figure 2. Typical dorsal-ventral curvature

References Athanassopoulou F., Billinis, C. and Prapas, T. 2004. Important disease conditions of newly cultured species in intensive freshwater farms in Greece: first incidence of nodavirus infection in Acipenser sp. Disease Aquatic Organism, 60: 247–52. Boglione, C., Gisbert. E., Gavaia, P., Witten. P. E., Moren. M., Fontagn, S. and Koumoundouros, G. 2013. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Review Aquaculture, 5: S121– S167. Cahu, C., Zambonino Infante. J. and Takeuchi, T. 2003. Nutritional components affecting skeletal development in fish larvae Aquaculture 227: 245–258. Gjerde, B., Pante, M.J.R. and Baeverfjord, G. 2005. Genetic variation for a vertebral deformity in Atlantic salmon (Salmo salar), Aquaculture, 244: 77–87.

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Survey of intestinal parasites in sturgeon breeders caught in spring migration from Sangar dam Alireza Babaalian Amiri 1*, Milad Adel 2 and Mohammad Jalil Zorriehzahra2 1Department of Aquatic animal Health & Disease, Faculty of Veterinary Medicine, Islamic Azad University, Sciences & Research Branch, Tehran, Iran. 2Department of Aquatic animal Health & Disease, Iranian Fisheries Sciences Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O. Box: 13185-116, Tehran, Iran. [email protected] Introduction The population of the Caspian Sea sturgeon is falling drastically during recent years and the sturgeon stocks are encountering a depletion, so the study of the harmful factors on these valuable fishes is necessary (Pourkazemi, 2006). Given the relatively high life time of sturgeon, incidence of parasitic infection plays an important role in weakening the immune system of fish and causes secondary diseases. In the present study, intestinal parasites of sturgeon breeder (Acipenser nudiventris, n=3; Huso huso, n=8 and Acipenser persicus n=4) caught in spring migration (2013) from Sangar dam in Gilan province were studied and using the parasitology methods and identification keys, parasitic infection was surveyed. The collected parasites were identified according to Jalali (1999). Materials and Methods The survey was conducted in the digestive tract of all H. huso surveyed and nematodes Cucullanus sphaerocephalus was found and the infection rate was 100%. However, no parasites were found in digestive tract of A. nudiventris and A. persicus breeders. In similar study, Bazari Moghaddam (2013) and Mohammadi et al. (2010) found that C. sphaerocphlaus had the highest intensity and contamination rate between intestenial parasites in sturgeon breeders, these results was similar to our results and confirmed it. High prevalence of C. sphaerocephalus may be related to the occurrence of Nereids (polychaeta), the intermediate host for this parasite (Noei, 2011).

Conclusions Based on the high prevalence of C. sphaerocephalus in H. huso breeders, it seems necessary to consider hygienic and management measures in various stages of reproduction and rearing of beluga.

References Bazari Moghaddam, S. 2013. Study on Internal helminthes parasites in Persian sturgeon (Acipenser persicus) spawners in southwest coasts of the Caspian Sea (2009-2011). Life Sciences Journal, 10: 12-16. Jalali, B., 1999. Parasites and parasitic Diseases in the freshwater fishes of Iran. Iranian Fisheries Company, Aquaculture Department, 652pp. (in Persian). Mohammadi, A., Hossenifard, S.R, & Ghazi, A. 2010. Survey parasitic infections of breeder sturgeon caught from the southwest coast of the Caspian Sea. Journal of Veterinary Research, 5: 15-19. Noei, MR. 2011. Parasitic worms of Acipenser stellatus, A. gueldenstaedtii, A. nudiventris and Huso huso (Chondrostei: Acipenseridae) from the southwest shores of the Caspian Sea. Caspian Journal of Environment Sciences, 9: 257-266. Pourkazemi, M. 2006. Caspian Sea sturgeon Conservation and Fisheries: Past present and Future. Journal of Applied Ichthyology, 22: 12-16.

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Investigation on susceptibility of Sturgeon fish to Viral Nervous Necrosis (VNN)

Mohammad Jalil Zorriehzahra *, Alireza Shenavar Masouleh, Milad Adel, Maryam Dadar , Fatemeh Hassantabar 1Department of Aquatic animal Health & Diseases, Iranian Fisheries Sciences Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O. Box: 13185-116, Tehran, Iran. [email protected]

Introduction The Caspian Sea is the biggest enclosed body of water on Earth. The number of endemic aquatic taxa, which is over 400, is very impressive. There are 115 species of fish, of which a number are anadromous and migrate from the Caspian Sea up the rivers to spawn. The best known of these are the seven species and subspecies of Sturgeon, which have been a valuable economic resource for over a century. The Caspian fishes can be divided into 4 groups according to the ecological peculiarities. A) Sea fishes such as grey mullet (Liza aurata and L.saliens) and Caspian kilka (Clupeonella delicatula caspia). B) Anadromous fishes such as Caspian salmon (Salmonidae). C) Semi-migratory fishes such as Kutum (Rutilus frisii kutum). D) River fishes such as the pile (Esox lucius) and the tench (Tinca tinca). Viral nervous necrosis (VNN) is a worldwide disease affecting more than 50 species of cultured marine fish. Also, VNN was occurred in wild golden grey mullet Lisa aurata and Liza saliens in Iranian waters of Caspian Sea in recent years. In order to isolate and confirm it as causative agents of golden grey mullet mortality in the Caspian Sea, a complementary research investigation project was designed in 2005. Many diagnostic aspects such as Virology (Cell culture and Electron Microscopy), Hemathology, Bacteriology, Histopathology, Molecular biology (Nested-RT-PCR), heavy metals measurement and serology (IFAT and IHC) were employed in mentioned multidisciplinary project. So according to our findings it could be confirmed that VNNV was the main causative agent for disease outbreak in Golden grey mullet in Southern coastline of Caspian Sea.

Materials and Methods So, a case study was carried out randomly in sturgeon fish of Caspian Sea in 2005 in order to investigate the susceptibility of sturgeon fish to Viral Nervous Necrosis (VNN). Therefore 35 samples of 73 sturgeon fish (Acipenser gueldenstaedtii) that were collected from 32 stations in Caspian Sea on Feb. 2005 were examined by RT-PCR and Nested PCR by O.I.E. Reference Laboratory in Japan and National Taiwan University. New findings of affected fish (Liza aurata) which were collected in 2004 summer monitoring as well as 35 samples of 73 sturgeon fish that were collected from 32 stations in Caspian Sea on Feb.2005, revealed 20% positive results by RT-PCR assay in sturgeon fish and persistent infection in wild golden grey mullet by OIE Reference Laboratory in Japan and Taiwan National University (Table 1, 2, 3, 4).

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Also, in next studies in mentioned research project in pathogenicity test sensitivity of Huso huso fry was confirmed as first time in Iran. In this study pathognomonic clinical signs of VNN occurred in affected fry and with some main diagnostic tests we could approve VNN in infected sturgeon fry. Regarding to variation of aquatic biodiversity in the Caspian Sea and susceptibility and sensitivity of some economic fish to VNN virus, it could be concluded that VNNV can be considered as new threat in the aquatic ecosystem of the Caspian Sea especially for sturgeon fish.

References Bovo, G. 2007. Monography of ethiopathology of Viral encephalopathy and retinopathy, Istituto Zooprofilattico), Italy, 4: 93-146. Zorriehzahra, M.J., Nakai, T., Sharifpour, I., Gomes, D.K., Chi, S.C., Soltani, M., Mohd, D., Hj, H., Sharif Rohani, M. and Saidi, A.A. 2005. Mortality wild golden mullet (Liza aurata) in Iranian waters of the Caspian Sea associated with viral nervous necrosis like agent. Iranian Journal of Fisheries Science, 45: 43-58. Zorriehzahra, M.E.J., Ghasemi, M., Ghiasi, M., Karsidani, S.H., Bovo, G., Nazari, A., Adel, M., Arizza, V.and Dhama, K. 2016. Isolation and Confirmation of Viral Nervous Necrosis (VNN) disease in Golden grey mullet (Liza aurata) and leaping mullet (Liza saliens) of the Iranian waters of Caspian Sea, Veterinary Microbiology Journal, 190, 27-37.

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Investigation on probable reasons for eye disaster in fingerlings of beluga (Huso huso) produced using artificial propagation Amin Farahbod Roudbaraki*, Ayoub Yousefi Jourdehi, Mahmoud Bahmani, Mohammad Ali Yazdani Sadati, Rezvanollah Kazemi, Ali Hallajian, Mohammad Pourdehghani & Mahtab Yarmohammadi

International Sturgeon Research Institute (AREEO), Rasht, Iran, P.O.Box: 41635 - 3464 E.mail: [email protected] Introduction Outbreak of apparent deformities and anomalies during recent years has created worry from the following causes, its impact on growth and preserve of sturgeon stocks. To date, some studies were carried out on apparent abnormalities in fish. Incardona et al. (2014) Able to evaluate the effects of crude oil on the heart and other organs of pelagic fish to realize the impact of aromatic compounds in the incidence of abnormalities. Yousefian et al. (2005) evaluated the preliminary causes of morphological abnormalities in trout fish, the severity and types of abnormalities caused by crossing between brothers - sister inbred in a research project.

Materials and Methods Outbreak of apparent deformities and anomalies during to evaluate the incidence of ocular abnormalities (one eye and without eye) and its impact on growth a total of 534 beluga (Huso huso) juveniles were studied. A number of 9 fish (3 fish without eyes, 3 fish with single eye and 3 fish with healthy eyes) to were selected and transferred to the physiology and biochemistry laboratory aquarium equipped aeration system in the International Sturgeon Research Institute of the Caspian Sea. Types of aquarium water were well water and water temperature on the first day was 23 oC. Feeding of Huso huso juveniles was manually and were determined and carried out according to their total weight and the water temperature to the amount of half a percent of body weight (6 g in 3 times daily).

Results The results showed that 456 x (85.5 percent) were healthy, 52 (7.9 percent) has one eye and 26 (8.4 percent) had no eyes. The remarkable thing was that the fish with single eye and no eye that apparently had no eyes, But by exposing the eye area it was observed that there are eye appendages under the skin.

Discussion and Suggestions 1. Incubators and reared ponds present in restocking centers or private parts of international sturgeon research institute are equipped with to appropriate systems and water filtration standard and central heating device for preventing of entering pollutants, sediments, heavy metals, prevent thermal fluctuations and prevent be deformed fish or low percentage hatching of fertilized eggs. 2. Breeders diet purchased from reputable factory and to increase the safety factor and vitamins, minerals, quality protein, fat, carbohydrates and microelements be analyzed. Also, Juveniles should be fed sufficiently adequate food during the period of sexual maturity. 3. It would be a certain genetic birth certificates and identity to mating relationship and its related problems should be avoided. 4. To prevent such problems, fish with no eye or eye complications should be removed in the early stages of the breeding cycle.

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5. Identifying and preventing of the proliferation of breeders with genetic Abnormalities.

References Chebanov, M.S. & Galich, E.V. 2011. Sturgeon hatchery manual. FAO Fisheries and Aquaculture Technical Paper. No.558. Ankara, FAO. 303 p. Chebanov, M.S., Galich, E.V. & Chmyr, Yu.N. 2004. Sturgeon breeding and rearing handbook. Moscow, “Rosinformagrotech”. 136 pp. (In Russian) Hinton, D.E., Baumann, P. E., Gardner, G.R., Hawkins, W.E., Hendricks, J.D., Murchelano, R.A. & Okihiro, M.S. 1992. Histopathological biomarkers. In: R.J. Huggett, R.A. Kimerle, P.M. Mehrle & H.L. Bergman (Editors), Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Lewis, Boca Raton, FL, pp. 155-209. Incardona, J. P. et al. 2014. Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish. www.Pans.org.1510 -1518.

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In vitro and In vivo Biocontrol of Saprolegniasis on Persian Sturgeon (Acipenser persicus, Pisces: Acipenseriformes) eggs by Pseudomonas aeroginosa (PTCC: 1430)

Abbas Ali Aghaei Moghaddam*, Kamran Aghili, Maryam Ghiasi, Rasoul Ghorbani, Roghayeh Safari.

*Golestan Fisheries Research Center (GFRC), Iranian Fisheries Science Research Institute, Agricultural Research, Education Extention Organization (AREEO) *Corresponding author’s E-mail: [email protected] Introduction

Persian sturgeon, Acipenser persicus belonging to the family Acipenseridae is distributed throughout the Caspian watershed and also is most common in the Caspian Sea (Bakhshalizadeh et al., 2011; Pourkazemi et al., 2012). Artificial propagation is now the main source of sturgeon resources (Barannikova et al., 2005).The main reducing agent of eggs in hatcheries is fungal infection (Hanjavanit et al., 2008) which has been reported from many fish species (Rasowo et al., 2007). According to Verschere et al. (2000), one of the most significant technologies that have evolved in response to disease control problems is the use of probiotics. Antifungal activity against pathogenic Oomycetes has been tested in the genera Pseudomonas fluorescens and Aeromonas media A199 (Lategan et al., 2004) as a biocontrol agent for reduction of saprolegniasis. The present study was designed to investigate potential biological agent for biocontrol of saprolegniasis in Persian sturgeon eggs (Acipenser persicus) by Pseudomonase aeroginosa (PTCC1430) in laboratory and sturgeon hatcheries.

Materials and Methods This research was done in Sahid Marjani Centre-Golestan province between 2010 and 2011. Fungal infected eggs were selected from the Center to obtain some purified fungi (Ghiasi et al., 2010). The bacteria were obtained from Laboratory of Microbiology of the Caspian Sea Ecology Research Institute. The basic media was inoculated with bacteria at a concentration of 107 cfu. ml-1. The next treating concentrations (106,105,104, and 103 cfu.ml-1) were prepared from this main bacterial solution. For Challenge trial in Vitro each bacterial concentration (1ml) was cultured in petri dishes containing (SDA) media. In order to test bacterial ability in the control of saprolegnia growth in vitro, hyphal tips in SDA petri dishes incubated in petri dishes containing bacteria while inoculation of hyphal tips in the plates without bacteria served as a control. Challenge design in vivo was carried out in hatchery department of centre. Green eggs from Persian Sturgeon (Acipenser persicus) were placed in Yushchenco Incubator trays. The thirty- hour eggs were confined within Nylonic cages (15×10 cm; 50 eggs per cage) (Abtahi et al., 1994). During incubation and treating stages, water flow was about 2.5 l/min. Three cages were used as replicates for each treatment level and three cages used for control. Fish eggs were treated by three duration times (15, 30 and 45 min), three concentration levels of bacterial bath (105, 106 and 107 cfu. ml-1) and two times (once at first incubation day and three times during the incubation period). Infected eggs were assessed, counted and picked up from cages daily. The experiment was performed in a completely random design mean comparisons were conducted by a LSD test and paired sample T-test using statistical software package of SPSS17.

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Results The in vitro results revealed that the concentration of 107cfu.ml-1 inhibited the growth of saprolegnia. Increase in the growth and diameter of colonies started in plates containing 106 cfu.ml-1 of bacterial solution and continued to that in 103 cfu.ml-1. The fungal growth increased significantly in control treatments (P<0.05). Based on invivo results, there is only a significant effect of concentration (p< 0.01) while no significant effects of duration and times were found on egg fungal infection (p> 0.05). There is a significant interaction effect by times and bacterial concentration and also a significant interaction effect by all three studied factors (p< 0.05). The best effects were belonged to 105 × 30 min ×1time and 105 ×15min ×3 times that reduce the fungal infections to 15% (as compared to the control with 44% egg fungal infection).

Discussion It was revealed that 107cfu.ml-1 and 104 cfu.ml-1 concentrations had the maximum and the minimum inhibitory effects on fungal growth rate, respectively. By increasing the concentration of bacteria, the observations showed the reduction of hyphal growth diameter. The general mechanism of biological control can be divided into direct and indirect effects of the biocontrol agent. Direct effects include competition for nutrients or space; it is a common mechanism for the control of fungi where the antagonist and the pathogen are closely related. Since they are closely related, both will compete for the same nutrient and site of infection (Verschere et al., 2000). Production of antibiotic and lytic enzymes is one of the important mechanisms to control fungal infection. A number of highly effective disease- suppressive agents are found among the fluorescent Pseudomonads, making this group of bacteria very interesting. Indirect effects include all those aspects that produce morphological and biochemical changes in hosts (Gohel et al., 2006). In conclusion, results of this investigation showed the potential of P. aeroginosa as a biological agent to control saprolegniosis. To investigate the strategy of the bacteria in order to control fungal growth, more studies are needed.

Rreferences Bakhshalizadeh, S., Bani, A., Abdolmalaki, S., Nahrevar, R. and Rastin, R. 2011. Age, growth and mortality of the Persian Sturgeon, Acipenser persicus, in the Iranian waters of the Caspian Sea. Caspian Journal of Environmental Sciences. 9(2) 159-167 Hanjavanit , G; Kitanch- aroen N and Rakmanee C (2008) .Exprimental Infection of Aqauatic Fungi on Eggs of African Catfish (Clarias gariepinus Burch ) . K KU Sci.J.(Supplement ) 36-43. Pourkazemi1, M., Khoshkholgh, M., Nazari, S., Azizzadeh Pormehr, L. 2012.Genetic relationships among collections of the Persian sturgeon, Acipenser persicus, in the south Caspian Sea detected by mitochondrial DNA–Restriction fragment length polymorphisms. Caspian Journal of Environmental Sciences., 10 (2) :215-226. Rosowo, J., Okoth, O., Elijah, N., Charles, C. 2007. Effect of formaldehyde, Sodium chloride, Potassium Permanganate and hydrogen peroxide on hatch rate of African catfish Clarias gariepinus eggs. Aquaculture, 269 : 271-277. Verschuere, L., Geert, R., Patrick, S. and Willy, V. 2000. Probiotics Bact- eria as Biological Control Agents in Aquaculture, Microbiology and Mulecular Biology Review, 164: (4):655-671.

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Comparison of parasites in Beluga sturgeon, Huso huso, and ship sturgeon, Acipenser nudiventris, in southern part of the Caspian Sea Mohammad Mazandarani1*, Hamid Reza Naderi Farsani 1, Melika Ghelichpour2, Kourosh Amini3 1: Department of Fisheries, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran 2: Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3: Inland Waters Aquatic Stocks Research Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Gorgan, Iran * Corresponding author: [email protected]

Introduction Huso huso is the most important species, because this species produces the most valued caviar and has the highest growth rate among the sturgeons. Acipenser nudiventris, another Caspian sturgeon, has nutritional value like other sturgeons. This is a rare sturgeon in the Caspian Sea. Parasites are among the important factors responsible for weight loss, disruption of reproduction or impotency, blindness, abnormal behavior, epithelial lesions, deformities of gills and other symptoms that ultimately lead to economic loss in the fish industry. Moreover external parasites may cause secondary fungal, bacterial and viral infections or act as carriers of bacteria, virus and other pathogens (Azari Takami, 1997). Materials and Methods In present study, to aim collecting the data about parasite infestations, records of sturgeons in Iranian part of the Caspian Sea were considered and reports that had been published in this field were collected. The reports that officially have been published and data in Institute and University library were collected. Four types of documents including final reports of research projects, thesis (bachelor, master and PhD thesis), articles of congress and journals in Persian and English languages have been collected.

Results 15 species of parasite infestations including: one species of protozoa (Trichodina sp.), at least four species of nematode (Anisakis schupakovi, Anisakis sp., Anisakis simplex, Eustrongylides excisus, Cucullanus sphaerocephalus), two species of cestoda (Eubotherium acipenserinum, Bothrimonus fallax), two species of acanthocephalan (Corynosoma capsicum, Corynosoma strumosum), four species of trematoda (Nitzschia sturionis, Skrjabinopsolus semiarmatus, Skrjabinopsolus skrjabini, Skrjabinopsolus acipenseris) and one species of copepoda (Pseudotracheliastes stellatus) have been reported in Huso huso from Iran (table 1).

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Fish species Parasites Infested References tissue Nitzschia sturionis Gill Hajimoradloo (1999); Mokhayer (1981); Pyighambari (1991); Tavakol et al. (2009) Skrjabinopsolus semiarmatus Intestine Ghoroghi 1996b; Ghoroghi & Pourgholam 1996; Mokhayer 1974; Mokhayer & Anvar 1973; Noei 2011; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Skrjabinopsolus skrjabini Intestine Ghoroghi & Pourgholam (1996) Skrjabinopsolus acipenseris Mokhayer 1974; Mokhayer & Anvar 1973

Eubothrium acipenserinum Intestine Ghoroghi 1996b; Ghoroghi & Pourgholam 1996;

) Mokhayer 1974; Sattari et al., 2001; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Bothrimonus fallax Intestine Ghoroghi & Pourgholam 1996; Mokhayer 1974 Anisakis simplex intestine Ghoroghi 1996b Huso hoso Anisakis schupakovi Intestine Mokhayer 1972

Anisakis sp. Intestine Sattari et al., 2002; Sattari & Mokhayer 2005a Cucullanus sphaerocephalus Intestine Ghoroghi 1996b; Ghoroghi & Pourgholam 1996; Mokhayer 1974; Mokhayer & Anvar 1973; Noei 2011; Sattari et al., 2001; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b

Beluga Sturgeon( Eustrongylides excisus (L) Intestine Noei 2011; Sattari et al., 2001; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Corynosoma caspicum Intestine Golvin & Mokhayer 1973; Mokhayer 1974; Corynosoma strumosum Intestine Ghoroghi 1996b; Ghoroghi & Pourgholam 1996; Sattari et al., 2002; Sattari & Mokhayer 2005a Pseudotracheliastes stellatus Skin Barzegar & Jalali 2009; Mokhayer 1974; Mokhayer 1985; 56 Trichodina sp. Skin Niak et al., 1970; Shenavar Masuleh et al., 2006

Diplostomum spathaceum Eye Shenavar Masuleh et al., 2006

Diclobothrium armatum Gill Hajimoradloo 1999; Pyighambari 1991

10 species of parasites including one genus of protozoa (Trichodina sp.), three species of nematode (Eustrongylides excisus, Cucullanus sphaerocephalus, Cystoopsis acipenseris), one species of cestoda (Eubotherium acipenserium), one species of acanthocephalan (Leptorhynchoides plagicephalus), three species of trematoda (Nitzschia sturionis, Skrjabinopsolus semiarmatus, Diclobothrium armatum) and one species of copepod (Pseudotracheliastes stellatus) were reported in Acipenser nudiventris from Iran (Table 2).

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Table 2: List of parasites isolated from Acipencer nudiventris from the south Caspian Sea

Fish species Parasites Infested References tissue Diclobothrium armatum Gill Hajimoradloo 1999, Pyighambari 1991; Sattari & Mokhayer 2005a; Nitzschia sturionis Gill Hajimoradloo 1999, Mokhayer 1981; Pyighambari 1991; Tavakol et al., 2009 Skrjabinopsolus semiarmatus Intestine Khara et al., 2010; Mokhayer 1974; Mokhayer & Anvar 1973; Noei 2011; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Eubothrium acipenserinum Intestine Mokhayer 1974; Sattari et al., 2001; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Cucullanus sphaerocephalus Intestine Khara et al., 2010; Mokhayer & Anvar 1973; Noei 2011; Sattari et al., 2001; Sattari et al., 2002; Sattari &

(Acipenser nudiventris) Mokhayer 2005a; Satarri & Mokhayer 2005b Eustrongylides excisus (L) Intestine Mokhayer 1974; Noei 2011; Sattari et al., 2001; Sattari et al., 2002; Sattari & Mokhayer 2005a; Satarri & Mokhayer 2005b Cystoopsis acipenseris Under skin Hajimoradloo 1999

Ship Sturgeon Ship Leptorhynchoides Intestine Khara et al., 2010; Noei 2011; Sattari et al., 2002; Sattari plagicephalus & Mokhayer 2005a; Satarri & Mokhayer 2005b Pseudotracheliastes stellatus Skin Hajimoradloo 1999, Pazooki & Masoumian 2012 Trichodina sp. Skin Niak et al., 1970; Shenavar Masuleh et al., 2006

Discussion Based on this study 14 species of parasites including 1 species of protozoa, 6 species of nematodes, 2 species of cestodes, 3 species of acanthocephale, 5 species of trematode, and one species of copepode have been reported from two species of sturgeon in southern part of the Caspian Sea. According to this comparison, the number of parasites recorded for H. huso is more than those recorded for A. nudiventris. Results show that most of the parasites were found in intestine in both species. Also, the number of nematodes was more than the other parasites. It seems that three mentioned parasite’s infections (S. semiarmatus, C. sphaerocephalus and L. plagicephalus) have had most prevalence in A. nudiventris. For rebuilding of sturgeons stocks in the Caspian Sea from 1972, Iran has started to release sturgeon fingerlings to the sea and its related rivers and totally 142 million pieces of this fish have been released in the Caspian Sea during 1994-2002 (Kalbassi, Abdollahzadeh & Salari- Joo 2013), but practically couldn’t repair mentioned decline. Although a wide range of parasites is described in sturgeons, little is known about their ecology and natural habitats, which can be related to kind of nutrition and different nutritional habits in these fish (Hajimoradloo & Ghorbani; 2002; Hajimoradloo 2003). Parasite infestation in the natural resources is common and usually it doesn’t make a serious problem but in artificial and intensive culture can affect farms and lead to high loses.

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References Barzegar1 M. & Jalali, B. 2009. Crustacean parasites of fresh and brackish (Caspian Sea) water fishes of Iran. Journal of Agricultural Science and Technology 11, 161- 171. Hajomoradloo A.M. 2002. The prevalence of Cystoopsis acipenseris in sturgeon (Chondrostei: Acipenseridae) from east-southern part of the Caspian Sea. Journal of Agricultural Sciences and Natural Resources 8(4), 181-190. (In Persian) Hajimoradloo, A.M., Abdoli, A. & Ghorbani, R. 2002. The study of juvenile Acipenser persicus and A. gueldenstaedtii diet in southeast of the Caspian Sea Journal of Agricultural Sciences and Natural Resources 9(2), 139-151. (In Persian) Hajimoradloo A.M. 2003. Influence of micro elements zinc, nickel, cobalt, manganese, iron and copper on the level of α-amylase activity in intestine mucosa, pyloric caeca and pancreas in Beluga juvenile (Huso huso). Pajouhesh and Sazandegi 57, 83-86. (In Persian) Kalbassi M.R., Abdollahzadeh E. & Salari-Joo H. 2013. A Review on Aquaculture Development in Iran. Ecopersia 1(2), 159-178 Khara H., Sattari M., Yousefi R., Ebrahim Saeedi S., Goudarzi L. & Ahmadnezhad M. 2010. Parasitic worms of some sturgeons (Acipenseridae) from the southern coast of the Caspian Sea (west Mazandran province). Journal of Biological Sciences of Lahijan 4 (4), 29-36. (In Persian) Noei M.R. 2011. Parasitic worms of Acipenser stellatus, A. gueldenstaedtii, A. nudiventris and Huso huso (Chondrostei: Acipenseridae) from the southwest shores of the Caspian Sea. Caspian Journal of Environmental Sciences 9 (2), 257-266. Pazooki, J. & Masoumian, M. 2004. Cryptobia acipenseris and Haemogregarina acipenseris infections in Acipenser guldenstaedtii and A. persicus in the Southern part of the Caspian Sea. Journal of Agricultural Sciences and Technology 6, 95-101. Pazooki, J. & Masoumian, M. 2012. Synopsis of the Parasites in Iranian Freshwater Fishes. Iranian Journal of Fisheries Sciences 11(3), 570-589. Sattari M. & Mokhayer B. 2005a. Occurrence and Intensity of some Parasitic Worms in Acipenser gueldenstaedtii, A. nudiventris and Huso huso (Chondrostei: Acipenseridae) from the Southwest of the Caspian Sea. Turkish Journal of Veterinary Animal Sciences 29, 1279-1284.

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Review of parasite in Persian sturgeon (Acipenser persicus) from Iranian coasts of the Caspian Sea Kourosh Amini*, Melika Ghelichpour, Amin Hashemi Rostami Inland Waters Aquatic Stocks Research Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Gorgan, Iran

* Corresponding author: [email protected] Introduction Persian sturgeon (Acipencer persicus) is a very important Caspian sturgeon species for aquaculture, because, it encompasses the main portion of total annual sturgeon broodstoks catch of the Iranian coasts of the Caspian Sea. An issue in artificial propagation of wild-caught broodstocks is transferring pathogens from natural habitats to farm. Parasite infestation of wild sturgeon broodstocks is common and a variety of parasites have been reported in these fish (Tavakol et al., 2009). Parasite infestation normally brings more adverse effects on fish growth and health in the farms compared to natural habitats, because fish are reared in the farm under stressful conditions such as water ammonia elevation, high or low temperature and overcrowded. The stressful conditions make the fish more susceptible to parasite infestation (Saeij et al., 2003). This paper reviewed the parasite infestations of A. persicus from Iranian coasts of the Caspian Sea. Materials and Methods In present study, to aim collecting the data about parasite infestations, records of sturgeons in Iranian part of the Caspian Sea were considered and reports that had been published in this field were collected. The reports that officially have been published and data in Institute and University library were collected. Four types of documents including final reports of research projects, thesis (bachelor, master and Ph.D thesis), articles of congress and journals in Persian and English languages have been collected. Results According to the present study 18 species of parasites, including 3 protozoa (Cryptobia asipenseris, Haemogregarina acipenseris, Trichodina sp., Trichodina reticulate ), at least 5 species of nematode (Anisakis schupakovi, Anisakis sp, Eustrongylides excisus, Ascarophis ovotrichuria, Cucullanus sphaerocephalus, Cystoopsis acipenseris), one species of cestoda (Eubotherium acipenserinum), three species of acanthocephal (Leptorhynchoides plagicephalus, Corynosoma capsicum, Corynosoma strumosum ), five species of trematoda (Diplostomum spathaceum, Nitzschia sturionis, Skrjabinopsolus semiarmatus, Amphilina foliacea, Diclobothrium armatum) and one species of copepod (Pseudotracheliastes stellatus) have been recorded in Persian sturgeon in southern part of the Caspian Sea during last 40 years (Table1).

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Table 1: List of A. persicus parasites from the south Caspian Sea Scientific name of parasite Infested tissue References Cryptobia asipenseris skin Pazooki & Masoumian 2004; Pazooki et al., 2006 Haemogregarina acipenseris Peripheral blood Pazooki & Masoumian 2004; Pazooki et al., 2006 Diclobothrium armatum gill Hajimoradloo 1999; Hajimoradloo & Ghorbani 2003 Nitzschia sturionis gill Tavakol et al., 2009 Skrjabinopsolus semiarmatus Intestine Alimohammadi et al., 2012 Eubothrium acipenserinum intestine Bazari Moghaddam 2013 Amphilina foliacea Gonads and abdominal cavity Hajimoradloo & Ghorbani 2003 Anisakis schupakovi Liver & intestine Mokhayer 1972 Anisakis sp. (L) liver Hajimoradloo & Ghorbani 2003, Sattari et al., 2000 Cucullanus sphaerocephalus intestine Alimohammadi et al., 2012 Cystoopsis acipenseris Under skin Hajimoradloo 1999; Hajomoradloo 2002 Eustrongylides excisus Intestine Ebrahimi & Malek 2006 Leptorhynchoides plagicephalus Intestine Bazari Moghaddam 2013 Corynosoma strumosum Intestine Masoumzadeh 2007 Pseudotracheliastes stellatus Skin Hajimoradloo & Ghorbani 2003 Trichodina sp. Skin Niak et al., 1970; Shenavar Masuleh et al., 2006 Trichodina reticulata Skin Bazari Moghaddam et al., 2010a Diplostomum spathaceum Eye Bazari Moghaddam 2013 Gyrodactylus sp. Skin Shenavar Masuleh et al., 2006

Discussion Based on this study 14 species parasites including 3 species of protozoa, 5 species of nematodes, 1 species of cestodes, 3 species of acanthocephale, 5 species of trematode, and one species of copepode have been reported from Persian sturgeon in southern part of the Caspian Sea. In most records three parasites including: S. semiarmatus, C. sphaerocephalus and L. plagicephalus has high prevalence and severity infestation in Persian sturgeon. In some reports, more than 50% of parasite infection prevalence was belonged to C. sphaerocephalus and S. semiarmatus in A. persicus. Nowadays rearing of sturgeons in ponds is developing, so any information about their habits is worthwhile and can be used in aquaculture. Although a wide range of parasites is described in sturgeons but little is known about their ecology and natural habitats, which can be related to kind of nutrition and different nutritional habit in these fish (Hajimoradloo & Ghorbani; 2002). Some of the parasites’ target tissues are gonads and eggs, which may deteriorate normal reproduction function. Parasite infestation causes more serious problems in the fish farm than in natural habitats.

Concolusion Accordingly, it is suggested that the wild broodstocks of A. persicus should be checked at farm arrival to find their parasites and treat them appropriately before propagation.

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References Alimohammadi S.R., Hoseinifard S.M. & Ghazi A. 2012. Survey of intensity worm parasites digestive system Acipenser persicus in coasts of south Caspian Sea. Journal of Veterinary Medicine Research 2(5), 15-19. Bazari Moghadam S., Mokhayer B., Shenavar Masouleh A.R., Jalilpour J., Masoumzadeh, M. & Alizadeh, M. 2010b. Study on parasitic infestation on Persian sturgeon (Acipenser persicus) larvae and fingerlings in the Shahid Dr. Beheshti Hatchery. Journal of Fisheries 4(1), 132-136. (in Persian) Bazari Moghaddam, S. 2013. Study on Internal helminthes parasites in Persian sturgeon (Acipenser persicus) spawners in southwest coasts of the Caspian Sea (2009-2011). Life Science Journal 10(10), 12-16.( in Persian)

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Parasites of Russian sturgeon, Acipenser gueldenstaedtii, and starry sturgeon, Acipenser stellatus, in the south Caspian Sea Mohammad Mazandarani*, Hamid Reza Naderi Farsani, Melika Ghelichpour, Kourosh Amini

1: Department of Fisheries, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

* Corresponding author: [email protected] Introduction It is important to have knowledge about their parasites. This paper summarized the studies reporting parasites of A. gueldenstaedtii and A. stellatus from the south Caspian Sea. Ecology and behavior of sturgeon species are different in comparison to other native fish species because of their anadromy and their long age period and life styles and big sizes (Bauer, Pugachev & Voronin 2002). So for the aquaculture or restocking purpose, knowledge on any aspects of life cycles and pathogens of this valuable fish can be worthwhile. The parasites are one of pathogens that can influence growth and production also they can effect on fish health in the larval to adult stages (Gibson & Sadighian 1980). First report of parasite infestation in sturgeon in southern part of Caspian Sea goes back to 1943 (Rostami, 1943). During last decade a lot of study has been done on sturgeon parasites in Iran. So in this paper it is tried to provide overview on parasites which has isolated from sturgeons in Iran. Materials and Methods In present study, to aim collecting the data about parasite infestations, records of sturgeons in Iranian part of the Caspian Sea were considered and reports that had been published in this field were collected. The reports that officially have been published and data in Institute and University library were collected. Four types of documents including final reports of research projects, thesis (bachelor, master and PhD thesis), articles of congress and journals in Persian and English languages have been collected. Results Totally 23 parasite species including 3 species of protozoa (Cryptobia asipenseris, Haemogregarina acipenseris, Trichodina sp.), at least 6 species of nematode (Anisakis schupakovi, Anisakis sp., Contracaecum squallii, Eustrongylides excisus, Cucullanus sphaerocephalus, Cystoopsis acipenseris, Cyclozon acipenserina), one species of cestoda (Eubotherium acipenserinum), three species of Acanthocephala (Leptorhynchoides plagicephalus, Corynosoma capsicum, Corynosoma strumosum), 6 trematoda (Diplostomum spathaceum, Nitzschia sturionis, Skrjabinopsolus semiarmatus, Amphilina foliacea, Diclobothrium armatum, Skrjabinopsolus acipenseris), one species of copepod (Pseudotracheliastes stellatus) and one species of Cnidaria of family Polypodiidae (Polypodium hydriforme) were isolated from Russian sturgeon in south of the Caspian Sea (Table 1).

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Table 1: List of parasites of Acipenser gueldenstaedtii from the south Caspian Sea Fish Parasites Infested tissue References species Cryptobia asipenseris Skin Pazooki & Masoumian 2004; Pazooki et al., 2006 Haemogregarina acipenseris Peripheral blood Pazooki & Masoumian 2004; Pazooki et al., 2006 Diclobothrium armatum gill Hajimoradloo 1999, Mokhayer 1981; Pyighambari 1991 Mokhayer 1981; Pyighambari 1991; Tavakol et al., 2009

Nitzschia sturionis gill

ii) Skrjabinopsolus semiarmatus Intestine Hajimoradloo & Ghorbani 2003 Skrjabinopsolus acipenseris Intestine Mokhayer 1974 Diplostomum spathaceum Eye Barzegar et al., 2008 Eubothrium acipenserinum intestine Hajimoradloo & Ghorbani 2003, Mokhayer 1974; Sattari et al., 2001 Amphilina foliacea Gonad and Hajimoradloo & Ghorbani 2003 abdominal cavity

Anisakis schupakovi Liver Mokhayer 1972; Mokhayer 1974

Anisakis sp (L). Liver Sattari & Mokhayer 2005a (Acipenser guldenstadt (Acipenser

Contracaecum squallii Intestine Mokhayer 1974; Mokhayer & Anvar 1973 Cucullanus sphaerocephalus intestine Khara et al., 2010 Cyclozon acipenserina intestine Mokhayer 1974; Mokhayer & Anvar 1973 Eustrongylides excisus Intestine Mokhayer 1974; Noei 2011 Corynosoma caspicum Intestine Golvin & Mokhayer 1973; Mokhayer 1974; Corynosoma strumosum Intestine Sattari et al., 2002

Leptorhynchoides plagicephalus Intestine Hajimoradloo & Ghorbani 2003 Russian Sturgeon Pseudotracheliastes stellatus Skin Barzegar & Jalali 2009 Cystoopsis acipenseris Under skin Hajomoradloo 2002; Hajimoradloo & Ghorbani 2003 Trichodina sp. Skin Niak et al., 1970 Polypodium hydriforme Egg & Gonad Mokhayer 1972; Pourgholam 1994

In this review study 20 parasites species including one species of protozoa (Trichodina sp), four species of nematode (Eustrongylides excisus, Cucullanus sphaerocephalus, Cystoopsis acipenseris, Anisakis schupakovi), three species of cestoda (Eubotherium acipensernium, Bothrimonus fallax, Ligula intestinalis), four species of Acanthocephala (Leptorhynchoides plagicephalus, Corynosoma capsicum, Corynosoma strumosum, Pomphorhynchus leavis), six species of Trematoda (Diplostomum spathaceum, Nitzschia sturionis, Skrjabinopsolus semiarmatus, Skrjabinopsolus skrjabini, Amphilina foliacea, Diclobothrium armatum), one species of copepod (Pseudotracheliastes stellatus) and one species of Cnidaria of family Polypodiidae (Polypodium hydriforme) has been reported from Stellate sturgeon in southern part of Caspian Sea (Table 2).

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Table 2: List of parasites of Acipenser stellatus from the south Caspian Sea Fish Parasite Infested tissue References species Diclobothrium armatum Gill Hajimoradloo 1999, Pyighambari 1991 Nitzschia sturionis Gill Pyighambari 1991; Tavakol et al., 2009 Skrjabinopsolus semiarmatus Intestine Ebrahimi & Malek 2006 Skrjabinopsolus skrjabini Intestine Mokhayer 1974; Mokhayer & Anvar 1973

Diplostomum spathaceum Eye Barzegar et al., 2008 Eubothrium acipenserninum Intestine Mokhayer 1974 Bothrimonus fallax Intestine Mokhayer 1974; Noei 2011 Amphilina foliacea Gonad & abdomenal cavity Sattari et al., 2002 Anisakis schupakovi Liver Mokhayer 1974; Mokhayer & Anvar 1973 Anisakis sp. Liver Sattari et al., 2001a Cucullanus sphaerocephalus Intestine Noei 2011; Rajabpour et al., 2002

(Acipenser stellatus) (Acipenser Corynosoma caspicum Intestine Golvin & Mokhayer 1973; Mokhayer 1974 Corynosoma strumosum Intestine Sattari et al., 2001a; Satarri & Mokhayer 2005b Leptorhynchoides plagicephalus Intestine Khara et al., 2010 Ligula intestinalis Abdominal cavity Khara et al., 2010 Pomphorhynchus leavis intestine Pazooki et al., 2006 Pseudotracheliastes stellatus Skin Barzegar & Jalali 2009

Trichodina sp. Skin Niak et al., 1970 Stellate sturgeon Eustrongylides excisus Intestine Ebrahimi & Malek 2006 Cystoopsis acipenseris Under skin Hajimoradloo 1999; Hajomoradloo 2002; Polypodium hydriforme Egg & gonad Nechat & Mokhayer 1974; Mokhayer 1972 Gyrodactylus sp. Skin Shenavar Masuleh et al., 2006 Cyclozon acipenserina intestine Mokhayer & Anvar 1973

Discussion This study showed that a wide variety of parasites are found in A. gueldenstaedtii and A. stellatus. The infested fish suffers and this affects their growth and life. Some of the parasites’ target tissues are gonads and eggs, which may deteriorate normal reproduction function. Parasite infestation causes more serious problems in the fish farm than in natural habitats. In the fish farm, fish are reared under stressful conditions such as overcrowded, hypoxia and temperature fluctuation. These stresses suppress fish resistance to infestation (Saeij et al., 2003). Accordingly, it is suggested that the wild broodstocks of A. gueldenstaedtii and A. stellatus should be checked at farm arrival to find their parasites and treat them or their eggs appropriately before propagation because these parasite can affect farmed and cultured sturgeon via the artificial spawning of broodstocks.

References Barzegar, M., Raeisi, M., Bozorgnia, A. & Jalali, B. 2008. Parasites of the eyes of fresh and brackish water fishes in Iran. Iranian Journal of Veterinary Research. 9(3), 256- 261. Barzegar, M. & Jalali, B. 2009. Crustacean parasites of fresh and brackish (Caspian Sea) water fishes of Iran. Journal of Agricultural Science and Technology 11, 161-171. Bauer, O.N., Pugachev, O.N. & Voronin, O.N. 2002. Review Study of parasites and diseases of sturgeons in Russia: a review. Journal of Applied Ichthyology 18, 420-429 Ebrahimi, M., Malek, M. 2006. Study of helminth parasites of Acipenser persicus and A. stellatus (Chondrostei: Acipenseridae) from the southern coast of Caspian Sea. Journal of Science University of Tehran 32(4), 361-368. (In Persian) Nouri, J., Karbassi, A. & Mirkia, S. 2008. Environmental management of coastal regions in the Caspian Sea. Journal of Environmental Sciences and Technology 5(1), 43-52.

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Behavioral and histopathological changes in liver tissues of Acipenser stellatus under toxicity of aflatoxin B1 Jalil Jalilpour*, Habib Vhabzadeh, Abolfazl Sepahdari, Zabihola Pajand, Ali Halajian, Soheil Bazari Moghaddam, Mehdi Alizadeh, Mehdi Masoumzadeh.

*Research instructor of Agricultural Research, Education & Extension Organization (AREEO)-International Sturgeon Research Institute, Rasht, Iran. [email protected]

Toxicity of aflatoxin B1 can cause liver damages, carcinogenesis, mutation and weak the immune system of aquatic animals. Many aquatic species have different degrees of susceptibility to AFB1 because of inter - species differences and biotransformation process. In this study, 10 Acipenser stellatus fingerlings with mean weight 7.9 ± 0.12 gr were stocked in each fiberglass tanks (with volume 50 lit of hole water and equipped with aeration system) and were fed with 1.5, 1.85, 2.3, 2.85, and 3.5 mg/kg of Aflatoxin B1 for 56 days. Fish were fed at three times between 08:00 to 16:00 hours. Results showed that some behavioral disasters included dark color, flushing, abnormal swimming patterns and anorexia. Significant histopathological changes were observed in liver tissues of Acipenser stellatus AFB1 at levels 1.5, 1.85, 2.3, 2.85, and 3.5 mg/kg. Changes included Fatty degeneration, Hemorrhage and Cell necrosis (Fig. 1). Bile stagnation, fatty degeneration, hemorrhages and cell necrosis (Fig. 2). Hyperplasia, fatty degeneration, hemorrhage, cell necrosis and atrophy (Fig. 3).

H HY A FD E N HE BS HE

N FD A N

Fig. 1. 1500 ppb AFB1 (H&E, 40X) Fig. 2. 1850- 2850 ppb AFB1 (H&E, 40X) Fig. 3. 3500 ppb AFB1 (H&E, 40X)

In present study, all reported signs of acute, sub-acute and chronic aflatoxinosis at different concentrations of AFB1 in Acipenser stellatus were compared with other species (Raghavan, 2011; Jantrarotai & Lovell, 1990; Jantrarotai et al., 1990; Chavez-Sanchez et al., 1994). This observation when Acipenser stellatus fed low levels of AFB1 contaminated diets showed early signs of, and progression towards liver cancer (hepatoma).

Keywords: Histopathological, Liver, Clinical signs, Acipenser stellatus.

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Processing and Products Diversification

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Biochemical characterization of type I collagens from the skin of Persian sturgeon, Acipenser persicus Ehsan Ahmadi Khanehsaz*, Katayoon Karimzadeh, Ali Mostafaie

1Department of Marine Biology, Lahijan Branch, Islamic Azad University, Lahijan, Iran. 2Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran [email protected] Introduction Collagen is an important structural protein that plays a key role in maintaining the extracellular matrix. Collagen can be used for variety purposes such as industrial and pharmaceutical applications. Nowadays, many scientists have consideration to extraction collagen from marine sources especial fish waste. This type of collagen is more safely than mammalian collagen sources. We aim to extract collagen type from Acipenser persicus skin and characterized some biochemical properties. Materials and Methods Approximately 100 grams Acipenser skin was collected from local market in Lahijan, Iran. After treatment by butylalcohl, the skin was suspended in acetic acids for 3 days. The solubilized fractions precipitated with sodium chloride and characterized with SDS –PAGE electrophoresis and amino acid profile. Results and Discussion

The SDS PAGE electrophoresis results showed that the presence of two types of α chain (α1 and α2) in Acipenser skin. In amino acid profile, Gly was the most dominate amino acids like other fish skin extracted collagens, and the amounts of two amino acids Met and Tyr were very low in Acipenser skin (table 1). From SDS-PAGE electrophoresis pattern and amino acid profile, the extracted fraction from Acipenser skin might be categorized as type I collagen.

Table 1. Amino acid profile of collagens from the skin of Acipenser sturgeon (residues/1000 residues).

SSC ASC

Alanine 108.14 ± 3.50 109.71 ± 1.28 Valine 13.71 ± 1.06 13.76 ± 1.12 Methionine 3.28 ± 1.06 4.31 ± 1.29 Isoleucine 11.66 ± 0.35 11.43 ± 0.02 Leucine 15.91 ± 1.51 16.28 ± 1.28 Tyrosine 2.20 ± 0.56 2.16 ± 0.71 Phenylalanine 12.46 ± 1.83 14.11 ± 2.03 Hydroxyproline 108.04 ± 2.05 106.48 ± 2.22 Histidine 12.20 ± 0.89 10.92 ± 1.32 Lysine 22.54 ± 1.46 24.40 ± 0.14 Aspartic acid 38.13 ± 1.19 35.36 ± 2.18 Arginine 51.29 ± 1.16 52.36 ± 0.57 Threonine 24.15 ± 2.16 23.51 ± 2.23 Serine 46.54 ± 0.73 48.20 ± 3.14 Glutamic acid 64.52 ± 3.14 62.08 ± 1.84 Proline 116.15 ± 3.21 114.29 ± 2.43 Glycine 313.29 ± 11.15 304.29 ± 12.17

Salt solubilized collagen (SSC) - acid solubilized collagen (ASC)

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References Senaratne, L. S., Park, P. J. & Kim, S. K. 2006. Isolation and characterisation of collagen from brown backed toadfish (Lagocephalus gloveri) skin. Bioresource Technology, 97(2), 191–197. Singh, P., Benjakul, S., Maqsood, S. & Kishimura, H. 2011. Isolation and characterisation of collagen extracted from the skin of striped catfish (Pangasianodon hypophthalmus). Food Chemistry, 124(1), 97–105. Veeruraj, A., Arumugam, M. & Balasubramanian, T. 2013. Isolation and characterisation of thermostable collagen from the marine eel-fish (Evenchelys macrura). Process Biochemistry, 48(10), 1592–1602. Wang, L., An, X., Xin, Z., Zhao, L. & Hu, Q. 2007. Isolation and characterisation of collagen from the skin of deep-sea redfish (Sebastes mentella). Journal of Food Science, 72(8), 451– 455.

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changes of Microbial spoilage indices Beluga caviar processed (Huso huso) during storage at -2oC Afshin Fahim, Ali Asghar khanipour, Ghorban Zare Gashti , Ferydon Rafiepour, Amiri.a

Iranian fisheries science research institute, Inland waters Aquaculture research center, Agricultural research education and extension organization (Areeo), Bandar Anzali, Iran.

[email protected]

Abstract Beluga caviar processed microbial spoilage index changes (Huso huso) under the temperature of -2 °C for twenty days were examined. The results showed that with increasing storage time of mesophilic bacteria, coliform and cool significantly reduced. The mean (SD) of mesophilic bacteria from 5.4±0.14 CFU/g at the time of production to 3.1±0.14 CFU/g, The microorganisms coliform from 2.1±0.14 during the production to 1.3±0.14 and the cooling microorganisms from 2.8±0.14 to 1.6±0.14 was twenty days of cold stirage. Quality rating microbial indicators are significantly during storage has been declining. The results showed that although a significant reduction indices mesophilic bacterium, coliform and cool during a period of twenty days at a temperature of -2°C, processed caviar is still away and will be accepted limit.

Keywords: Processed caviar, Beluga, Microorganisms total count, Coliform microorganisms cool

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Marketing and Prepare feeds and food in sturgeon" Uranus Ghiassi * Shahrak gharb sepehr .St. No.12 azalia co. Email: [email protected] Caviar – More Than Just Sturgeon Eggs and marketing You either love or hate the idea of eating caviar. Basically you are eating the eggs (roe) of sturgeon, a fish found in various parts of the world but most notably in the Caspian Sea near Russia. This is where you find the largest and highest quality caviar and the most expensive. You can also find Chinese and American caviar but they are smaller and don’t have the quality or flavor of caviar coming from the Caspian Sea.

3 Main Types of Caviar The three most popular types of caviar come from three different sturgeons thus creating a common grading system for buying these highly prized eggs. Each has its own unique qualities but which one you like depends on your own personal tastes.

Beluga – the most expensive of the three and also the largest. Beluga sturgeon can reach 19 feet in length and weigh more than one ton. They produce the largest eggs that are a range from gray to black in color.

Osetra – another large sturgeon reaching an average 7 feet in length and weighing in at over 500 pounds. The eggs from the Osetra sturgeon range in color from yellowish gray to dark brown with the gold Osetra, also known as “royal caviar” being the rarest. Sevruga – the smallest with the briniest flavor. Not as expensive as the other two, the roe ranges in color from medium gray to black. It is the most abundant and can weigh up to 150 pounds.

How to Buy Caviar Caviar is processed by straining and sorting the eggs by size and color. A mild salt (Malossol) is added to the eggs to prevent freezing. The eggs are then packed in 4 pound tins containers and exported.

An importer repackages the caviar into smaller tins or jars that you can purchase at high end markets. The typical size is 1 ounce containers.

IMPORTANT – Beluga caviar always comes in blue tins, Osetra in yellow tins and Sevruga caviar in red tins.

 IMPORTANT: Fresh Beluga is not currently available.  Buy Only Fresh Caviar –  Only Buy As Much As You Are Going To Serve –  Don’t Buy Caviar in a Supermarket –  Look for the term “Malossol” – it means lightly salted and is a good indication that the caviar is high-end.  Taste Before Buying –

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 Look for Discounts – The holidays is a big caviar buying time of the year and you often find caviar on sale. Just be careful you are not buying old or inferior product.  Don’t Buy Black Market Caviar –.

What’s it going to Cost? Prices will vary depending on they type you buy, where you buy it & how much you buy. Most likely you will be buying it in the popular 1 ounce jar or tin and here’s what you can expect to pay:

 Beluga Caviar – not currently available according to the US Fish and Wildlife  Osetra Caviar – Ranges from $90 to $140 per ounce  Sevruga Caviar – Ranges from $95 to $ $125 per ounce

Storing Fresh Caviar Caviar does not have a very long shelf life.

Do not freeze caviar

Conclusion In marketing of caviar: this subject appropriate is that a challenge among the Iran and other countries in the world market of the caviar.

Reference www.sterling.com

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Influence of chitosan nanocomposites and rosemary Rosmarinus officinalis L. extract coating on chemical and biophysical properties of inoculated fillet of Huso huso with Listeria monocytogenes during refrigerated storage Alika Jafarikaliji*, Ali Jafarpour, Reza Safari *MSc. Student, Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agriculture Science and Natural Resources University (SANRU), Sari, Iran [email protected] Introduction The packaging provided from biopolymer can become barriers against humidity, steam, gas, and solutes and also suitable means for adding a wide range of additives like fungal compounds, antioxidants, antimicrobial, colors, and other food ingredients (Rhim and Ng, 2007). Therefore, the main goal of this study, was to evaluate the capability of using a coating system containing chitosan in combination with rosemary extract as chitosan nanocomposites (CS/RE nanocomposites) to enhance the biophysical properties and determine the synergistic anti-listerial and anti-oxidative effects of chitosan and rosemary extract as a nanocomposites system in the Huso huso fillet challenged with Listeria monocytogenes during refrigerated storage.

Materials and Methods Huso huso was filleted and washed. The fresh fillets were coated with chitosan 1%, rosemary 0.5%, chitosan nanocomposites (chitosan 1% + rosemary 0.5%) and the control left untreated. Samples were immersed in each solution. Then, fillets were removed from the treatments solution with a strainer. Next, fillet samples were inoculated with 1×104 cfu/g of L. monocytogenes perior to pack tightly in an airproof plastic bags and kept at refrigerator temperature (4±1ºC) for 16 days. Subsequently, the behavior of L. monocytogenes, color parameters (L*, a*, b* and W), firmness, chemical parameters (TVB-N, TBA and PV) were determined on the inoculated fillet of Huso huso. Analysis of data among all treatments was done by One-way ANOVA (SPSS 17.0).

Results and Discussion Application of rosemary alone for coating of fillets reduced the lightness parameter, whereas the highest L* was belong to nanocomposite treatment (P<0.05) (Table 1). Table 1. Effect of chitosan 1%, rosemary extract 0.5% and CS/RE nanocomposites on L* value of L.monocytogenes in huso huso fillets inoculated with L.monocytogenes during storage at 4 ± 1ºC for 16 days. Storage time(day) Treatment Control Chitosan Rosemary CS/RE nanocomposites 0 51.36 ± 1.16Babc* 55.35 ± 1.34Aa 48.82 ± 1.30Cb 54.34 ± 0.32Aabc 4 55 ± 1Ba 57 ± 1ABa 49.33 ± 0.57Ca 59 ± 1.73Aa 8 53.33 ± 1.52Aab 56.66 ± 1.52Aa 46.66 ± 2.51Bab 55.33 ± 2.08Aab 12 49.14 ± 1.37Abc 49.48 ± 3.42Ab 44.77 ± 4.60Aab 49.88 ± 1.30Ac 16 48.47 ± 4.67Ac 49.33 ± 5.65Ab 42.82 ± 1.87Ab 50.75 ± 5.20Abc *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times) According to redness values (a*) (data not shown), chitosan and nanocomposites coated fillets revealed the highest redness among all treated samples on day 16th of storage at 4ºC. The yellowness (b* value) of all samples (data not shown), was reduced and reached to its lowest value on day 16 (P<0.05). Addition of rosemary for coating of Huso huso fillets caused a significant reduction in whiteness, while chitosan increased it significantly (P<0.05).

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Firmness of samples treated with nanocomposites was slightly increased during 16-day storage compared to chitosan-coated, rosemary-coated and untreated samples (Table 2). Table 2. Effect of chitosan 1%, rosemary extract 0.5% and CS/RE nanocomposites on the firmness of the inoculated fillets of huso huso with L.monocytogenes during storage at 4 ± 1ºC for 16 days. Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 230.85 ± 10.95Db* 285.75 ± 2.49Bb 251.28 ± 0.94Cb 316.18 ± 8.91Ad 4 142.78 ± 15.27Dc 457.88 ± 15.21Ca 543.42 ± 17.05Ba 581.42 ± 20.96Ac 8 370.58 ± 15.52Ba 250.41 ± 9.84Cc 201.88 ±11.75Dc 581.34 ± 26.96Ac 12 82.27 ± 13.45Cd 136.49 ± 7.14Bd 92.60 ± 5.44Cd 691.23 ± 41.87Ab 16 52.20 ± 0.85Ce 134.20 ± 6.73Bd 111.11 ± 11.87Bd 706.34 ± 30.17Aa *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times) According to Table 3, CS/RE nanocomposites showed the greatest antimicrobial activity against L. monocytogenes. Table 3. Effect of chitosan 1%, rosemary extract 0.5% and CS/RE nanocomposites on the population of L.monocytogenes in huso huso fillets during storage at 4 ± 1ºC for 16 days. Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 4.14 ± 0.06Be* 4.10 ± 0.05Be 4.28 ± 0.05Ae 4.14 ± 0.03Ba 4 5.79 ± 0.12Ad 4.56 ± 0.07Cd 4.97 ± 0.18Bd 3.44 ± 0.09Db 8 6.44 ± 0.05Ac 3.83 ± 0.04Cc 5.61 ± 0.04Bc 2.98 ± 0.05Dc 12 7.008 ± 0.15Ab 3.67 ± 0.05Cb 6.67 ± 0.13Ba 2.41 ± 0.09Dd 16 7.73 ± 0.08Aa 4.79 ± 0.08Ca 6.43 ± 0.10Bb 2.23 ± 0.10De *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times). TVB-N content (data not shown) increased with the storage time. The PV of oil extracted from H. huso fillet that is depicted in (Table 4) increased in all treatments during storage. Table 4. Effect of chitosan, rosemary extract and CNCT nanocomposites on PV in the inoculated fillet of Huso huso with L.monocytogenes Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 0.86 ± 0.005Ae* 0.87 ± 0.007Ae 0.84 ± 0.005Be 0.86 ± 0.01Ad 4 2.40 ± 0.02Ad 2.44 ± 0.07Ad 2.32 ± 0.03ABd 2.14 ± 0.21Bc 8 4.29 ± 0.08Ac 4.03 ± 0.01Bb 4.45 ± 0.01Ac 2.74 ± 0.13Cb 12 5.90 ± 0.06Aa 4.78 ± 0.01Ca 5.04 ± 0.07Ba 3.79 ± 0.03Da 16 4.61 ± 0.04Ab 3.83 ± 0.04Bc 4.55 ± 0.08Ab 2.96 ± 0.15Cb *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times)

The result indicated (Table 5) that TBA value significantly increased (P<0.05) in all treatments during 16-days of storage. Table 5. Effect of chitosan, rosemary extract and CNCT on TBA in the inoculated fillet of Huso huso with L.monocytogenes Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 0.64 ± 0.03Ae* 0.68 ± 0.04Ae 0.66 ± 0.03Ae 0.69 ± 0.004Ad 4 1.29 ± 0.07Ad 1.21 ± 0.04Ad 1.23 ± 0.02Ad 1.11 ± 0.02Bc 8 2.71 ± 0.03Ac 2.58 ± 0.04Bc 2.72 ± 0.02Ac 2.39 ± 0.03Cb 12 3.62 ± 0.07Ab 3.03 ± 0.08Bb 3.53 ± 0.09Ab 2.84 ± 0.33Ba 16 4.83 ± 0.04Aa 4.09 ± 0.24Ba 4.02 ± 0.11Ba 2.99 ± 0.08Ca

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*mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time). Small superscript letters in the same column represent significant difference at α=0.05) effect of each treatment in different times CS/RE nanocomposites coating efficiently improved texture properties of Huso huso fillets which was inoculated with L. monocytogenes during 16 days of refrigerated storage. Most importantly, addition of CS/RE nanocomposites showed the highest antilisterial effect among all trialed treatments. This coating efficiently improved most of the chemical properties of H.huso fillets which inoculated with L. monocytogenes compared to the control during 16 days of refrigerated storage.

References Rhim, J .W., & Ng, P. K. 2007. Natural biopolymer-based nanocomposite films for packaging applications. Critical Review in Food Science and Nurition, 47(4), 411-433.

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Influence of chitosan nanocomposites and rosemary Rosmarinus officinalis L. extract coating on microbial load of inoculated fillet of Huso huso with Listeria monocytogenes during refrigerated storage Alika Jafarikaliji*, Ali Jafarpour, Reza Safari * M.Sc. Student, Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agriculture Science and Natural Resources University (SANRU), Sari, Iran [email protected] Introduction Chitosan and rosemary was able to reduce bacterial growth. Antimicrobial nanocomposites and nanoscale materials have been investigated for their antimicrobial activity as growth inhibitors, killing agent (Qi et al., 2004). Therefore, the present research was done to evaluate the capability of using a coating system containing chitosan in combination with rosemary extract as chitosan nanocomposites (CS/RE nanocomposites) to inhibit microbial activity of Huso huso fillet challenged with Listeria monocytogenes during refrigerated (4 ± 1ºC) storage.

Materials and Methods Huso huso was filleted and washed. The fresh fillets were coated with chitosan 1%, rosemary 0.5%, chitosan nanocomposites (chitosan 1% + rosemary 0.5%) and the control left untreated. Samples were immersed in each solution. Then, fillets were removed from the treatments solution with a strainer. Next, fillet samples were inoculated with 1×104 cfu/g of L. monocytogenes perior to pack tightly in an airproof plastic bags and kept at refrigerator temperature (4±1ºC) for 16 days. Subsequently, total viable count (TVC), psychrotropic count (TPC), pseudomonas count and the behavior of L. monocytogenes, were determined on the inoculated fillet of Huso huso. Analysis of data among all treatments was done by One-way ANOVA (SPSS 17.0).

Results and Discussion Based on the results, the population of Listeria in control and rosemary samples increased during 16-days storage at 4±1ºC, while CS/RE nanocomposites showed the highest inhibition effect on populations of Listeria (P<0.05). The antibacterial effect of chitosan 1% was revealed from day 4 up to day 12 and afterward its efficiency was reduced as the population of Listeria was increased on day 16 (Table 1). Table1. Effect of chitosan 1%, rosemary extract 0.5% and CS/RE nanocomposites on the population of L.monocytogenes in huso huso fillets during storage at 4 ± 1ºC for 16 days. Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 4.14 ± 0.06Be* 4.10 ± 0.05Be 4.28 ± 0.05Ae 4.14 ± 0.03Ba 4 5.79 ± 0.12Ad 4.56 ± 0.07Cd 4.97 ± 0.18Bd 3.44 ± 0.09Db 8 6.44 ± 0.05Ac 3.83 ± 0.04Cc 5.61 ± 0.04Bc 2.98 ± 0.05Dc 12 7.008 ± 0.15Ab 3.67 ± 0.05Cb 6.67 ± 0.13Ba 2.41 ± 0.09Dd 16 7.73 ± 0.08Aa 4.79 ± 0.08Ca 6.43 ± 0.10Bb 2.23 ± 0.10De *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times)

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The initial TVC in Huso huso fillet was recorded as 4.81 log cfu/g in control, 4.76 log cfu/g in chitosan-coated, 4.70 log cfu/g (Table 2) in rosemary coated samples and 4.69 log cfu/g in samples treated with CS/RE nanocomposites. Table2. Effect of chitosan, rosemary extract and CS/RE nanocomposites on TVC of Huso huso fillets inoculated with L.monocytogenes Storage time(day) Treatment Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites 0 4.81 ± 0.01Ac* 4.76 ± 0.04ABc 4.70 ± 0.03Bd 4.69 ± 0.05Bd 4 6.06 ± 0.09Ab 5.69 ± 0.03Bb 5.90 ± 0.03ABc 5.55 ± 0.05Cc 8 7.11 ± 0.24Aab 5.88 ± 0.09Cb 6.33 ± 0.20Bb 5.79 ± 0.07Ca 12 7.84 ± 0.05Aa 5.96 ± 0.11Cb 7.05 ± 0.30Ba 5.71 ± 0.08Cab 16 6.83 ± 1.32Aab 6.57 ± 0.06Aa 6.86 ± 0.04Aa 5.63 ± 0.07Abc *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times) In this study, the initial TPC counts of samples were 4.53 log cfu/g in CS/RE nanocomposites, 4.52 log cfu/g in rosemary, 4.67 log cfu/g in chitosan and 4.58 log cfu/g in control (P>0.05) which increased to 8.64, 6.62, 6.89 and 5.71 log cfu/g in control, rosemary, chitosan and CS/RE nanocomposites, respectively, during 16 days of refrigerated storage (Table 3). Table3. Effect of chitosan 1%, rosemary extract 0.5% and CS/RE nanocomposites on TPC in huso huso fillets inoculated with L.monocytogenes during storage at 4 ± 1ºC for 16 days. Storage time(day) Treatment

Control Chitosan 1% Rosemary 0.5% CS/RE nanocomposites Be* Ad Be Bb 0 4.58 ± 0.04 4.67 ± 0.05 4.52 ± 0.04 4.53 ± 0.01 4 6.34 ± 0.13Ad 5.66 ± 0.13Bc 5.82 ± 0.08Bd 5.67 ± 0.04Ba 8 7.63 ± 0.11Ac 5.89 ± 0.01Cb 6.47 ± 0.10Bc 5.65 ± 0.13Da 12 7.86 ± 0.06Ab 6.53 ± 0.08Ca 7.65 ± 0.06Ba 5.59 ± 0.05Da 16 8.64 ± 0.09Aa 6.62 ± 0.10Ca 6.89 ± 0.01Bb 5.71 ± 0.08Da *mean ± standard error Capital superscript letters in the same row represent significant difference at α=0.05 (effect of treatments in each time) Small superscript letters in the same column represent significant difference at α=0.05 (effect of each treatment in different times) CS/RE nanocomposites coating efficiently improved microbial load and texture properties of Huso huso fillets which was inoculated with L. monocytogenes during 16 days of refrigerated storage. Most importantly, addition of CS/RE nanocomposites showed the highest antilisterial effect among all trialed treatments. This coating also reduced TVC, TPC and pseudomonas counts of the fillets in comparison with the control at the end of storage period. The same result was observed in Abdollahi et al. (2014) reported that the best inhibitory effect on TPC and TVC related to samples coated with nanocomposites activated with rosemary essential oil TPC in comparison with control.

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References Abdolahi, M., Rezaei, M. & Farzi, A. 2014. Influence of chitosan/clay functional bionanocomposite activated with rosemary essential oil on the shelf life of fresh silver carp. International Journal of Food Science and Technology, 49, 811-818. Qi, L., Xu, Z., Jiang, X., Hu, C. & Zou, X. 2004. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research, 339, 2693-2700.

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Advantage of the production and marketing of farmed sturgeon in Iran

Mahmoud Asadollahi and Yousef Joursara Director of sturgeon fish in the Mazandaran province- [email protected] Introduction The catches of sturgeon fishes declined from 14959 t in1991 to about 422 t in 2013. Research institutes predicted that the amount in 2021 will be zero. The reduction of sturgeon fishes in the natural environment has stimulated the growth of aquaculture of this species, with a production increased from 385 t in 1991 to 75000 t in 2013. Sturgeon aquaculture is growing in Iran in recent years. Material and Methods: This work and used the analytical methods and statistics and information taken from authentic sources as the FAO, Cites, Iran Fisheries Organization, mother company of agriculture services and expert opinions.

Discussion According to these studies, the range of protein, fat, fatty acid, amino acid, moisture and ash in breeding caviar is near to sea caviar. The global market demand for caviar and related products is on the rise. This amount is about 451 t and the supply is about 228 tones in Iran in 2014. There were 45 farms with capacity of 2500 t of meat and 32 t of caviar. In Mazandaran province in 2016 there are 10 farms with capacity of 2500 t of meat and 32 t of caviar that now product 250 t of meat and 1.5 t caviar. (1) More than 3500 hectares of the land are suitable for sturgeon aquaculture. At the end sixth development plan forecasted 5000 t of meat and 50 t of caviar. (6) Due to several advantages, Iran is one of the best countries for caviar production: -Records of Iran in trade caviar in the world -Experience and skill producing caviar in Iran -Buyers and markets are willing to buy Iranian caviar -Suitable sturgeon species with fast growth -Low cost of production input Difficulties of sturgeon breeding in Iran: -Lack of presence on the market of meat products as fillets, smoked, canned -The lack of market information Due to the rapid growth of breeding sturgeon in Iran and in the word, the production will be higher than demand in the coming years. Base on the study is inversely proportional to the price of exports, and it would be uneconomical for producers, therefore the following guidelines are offered to protect the industry. -Diversify in production and supply -Investment by government and domestic and foreign corporation -Exhibiting supply-Identify target market -Develop supply store-the production of biological products -Advertising-introduced sturgeon as ornamental fish -The creation of the union of farmers and exporters of sturgeon -Encourage domestic sturgeon farmers to produce sturgeon broodstock -Using of skin and waste products of sturgeon.

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Haghparast,S 104, 142, 278 Mehdizadeh 133 Haghshenas 136 Meknatkhah 64,183 Hajibeigloo 180 Meshkini 80,280 Hajimoradloo 267 Mirabdollahi 85 Hallajian 31, 32, 39, 117, 187, 201, 212, 214, Mirali 62 249, 252, 289, 284, 254 Mirhashemi Rostami 256 Hamidoghli 85 Moghim 255 Hashemi Rostami 256, 297 Mohammad Salehi 16 Hassantabar 285, 287 Mohammadi Gholinejhad 56 Hassanzadeh Saber 18, 170,173, 176, 187, 208 Mohammadi Parashkuh 18 Hedayati 59 Mohammadi,S 156 Hooshyar 220 Mohammadian 29 Hossein Nia 9,14,21,23,25,27,191 Mohseni 31,32,39,78,87,199,201,214 Hosseini 29 Mollaaghaee 114 Hosseinjani133 Moradinasab 12, 142, 252 Hosseinzadeh Sahafi 249 Morovati 240, 243, Hossien pour Zelti 78,110,116,199,260 Mortazavizadeh 218, Hozni 252 Mosavi 158 Jafari M 156 Mostafaie 305 Jafari,A.A 114 Naderi Farsani 293, 300 Jafari,F 98,195 Najdegrami 93 Jafarikaliji 310,313 Naji 236, 11 Jafarpour 310,313 Nakhaei 91 Jahani 176 Nasri Tajan 20 Jalali 223 Nezami Baluchi 5, 107 Jalilpour 66,226,247,252,303 Niazi 216 Joshideh 158 Nikpey 216 Kamali,A 71,120,125 Noghani 269 Kamali,S 269 Noori,A 193 Kamranjo 78,110,227,260 Noori,F 98, 195, 216 Karami Rad 4 Norouzi 123, 174 Karimzadeh G. 156 Nowrouzfashkhami 18, 10, 11, 176, 208, 210, Karimzadeh K. 305 229 Kazemi, B. 176 Paghe 98 Kazemi, R. Pajand 125, 130,148, 151,153,191,303 14,18,25,31,32,39,117,185,187,191,201,212,2 Parvaneh 33 14,216, 220,249 Peikaran Mana 14, 23, 27, 191 Khanamooei 242 Pour Asadi 23,64, 112, 156 Khanipour 269,307 Pourali 31, 32, 35, 41, 76, 87, 107, 110, 140, Khara 14,45,101,151,191,197 199 Kohalvandi 85 Pourdehghani 18, 85, 247, 249, 289 kolangi Miandareh 267 Pourdeljoo 145, Larijani 166 Pourgholam 45, 199 Linley 69 Pourhosein Sarameh 53 Mahjoubi 176 Pourkazemi 18, 39, 41, 56, 117, 170,173,176, Mahmoudi,N. 136 187, ,208 Malakpour Kolbadinezhad 262 Poursafar 247 Mark Wilson 262 Qiu 204 Masoudifard 255 Rabbaniha 162 Masoumzadeh 66,226,238,247 Raeisi 142 Mazaheri Kohanestani 162 Rafiepour 307

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Rahanande 64, 112 Soleimannejadbari 275 Rahmati 183 Soltani 71, 120, 125, 273, Rajabzadeh Ghatramie16, Sudagar 210, 249 Ramezanpour 76, 140, 145, 165 Sungchul 32 Ramin 91, 161 Taheri Mirghaed 240, 242, 243, 257 Rasuli 130 Tavakoli 252 Rezaie 271 Tehrani 280 Rezaii 51 Tulaby Dezfuly 271 Rufchaie 33, 76, 128, 140 Vahabnejhad 142, 252 Saberi 133, 135 vahabzade Roudsari 47 Safari, Reza. 309, 312 Vahdati 156 Safari, Roghayeh. 180, 245, 276, 291 Vajhi 257 Salamat 210 Yarmohammadi 39, 117, 173, 178, 185, 187, Salarzadeh 53 201, 214, 249, 252, 273, 289 Salavatian 33 Yazdani Sadati 6, 14, 41, 45,47, 49, 51, 59, 78, Sayadfar 35 80, 87, 107, 110, 112, 116, 185, 187, 191, 193, Seidgar 41, 43,75 210, 220, 227, 235, 249, 252, 280, 289 Sepahdari 303 Yeganeh 35, 78, 199, 238 Seraji 114 Yooneszadeh Feshalami 218 Seyed Hassani, 31, 32, 35, 45, 49, 51, 56, 78, Younes Haghighi 11, 76,140, 226,230, 238, 247 87, 89, 110, 116, 199, 227, 252, 260, Yousefi Jourdehi 9, 31, 35, 39, 56, 67, 117, Shahhosseini 170 118, 142, 187, 199, 201, 212, 214, 232, 235, Shakourian 35, 78, 117, 199, 214, 254, 260 247, 249, 252, 254, 289 Shapoori 153 Zamini 20, 71, 232 Shariati 145, Zare 307 Sharif Rohani 66, 226, 284 Zargari 114 Shenavar Masouleh 66,78, 165, 226, 227, 238, Zehtabvar 240, 242, 243, 255 247, 260, 273, 284, 287, Zomorrodi 275 Shirghazi 114, Zorriehzahra 282, 285, 286, 287 Sohrabi 135, 133,

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