Proceedings of The 7th Wildlife Assisted Reproductive Technology (ART) Workshop “Reproductive Biotechnology: from Basic to Applications”
March 7-11, 2016
the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016
Proceedings of The 7th Wildlife Assisted Reproductive Technology (ART) Workshop March 7-11, 2016
the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016 Welcome Message
By Associate Professor Dr. Chuchat Kamollerd
Dean of Faculty of Veterinary Medicine,
Khon Kaen University
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Distinguished participants
Ladies and Gentlemen.
On behalf of the Faculty of Veterinary Medicine, Khon Kaen University, it is my great pleasure to you all a very warm welcome to the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016, which is the joint conference of the Zoological Park Organization and Khon Kaen University. The conference is arranged during 7th-11st March 2016, the scientific session held at the Faculty of Veterinary Medicine, Khon Kaen University and the workshop held at Khon Kaen Zoo, Khon Kaen, Thailand.
It is a very great opportunity to have the collaboration with the Zoological Park Organization and Khon Kaen Zoo. The conference will dedicate to provide scientific forum focus on wildlife reproduction and ART for scientists, veterinarians, graduate and students to learn and exchange more knowledge. I hope that we all will enjoy the conference and best wish for the successful workshop.
I wish that you will have a pleasant time in Khon Kaen. I hope that the joint symposium of Zoological Park Organization and Khon Kaen University will continue for the future and have a strengthen relationship in animal reproductive science and technologies.
Thank you very much.
the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016 Opening Remarks
By Mr.Benjapon Nakprasert
Director of Zoological Park Organization under the Royal Patronage of H.M. the King
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Distinguished participants
Ladies and Gentlemen
On behalf of the organizing committee of the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016, I am very pleased to welcome colleagues, friends, veterinarians, scientists, students and exhibitors who participating in the joint conference of Khon Kaen University and Zoological Park Organization. This conference is a great occasion for researchers and veterinarians whose works focus for animal reproductive biology to share knowledge and experiences, which in turn will be a great contribution to the wildlife conservation.
The conference theme “Reproductive Biotechnology: from Basic to Applications” will offer relevant delegates to have opportunities to review fundamental reproductive science, access novel research finding and technologies for wildlife. The conference features special lectures given by experts in the field and sessions for free communication. The conference also provides a hand on workshop for researchers in the field to practice and share the experience for building up network in future.
I wish you have productive scientific forum, successful workshop and enjoy the time during the conference.
the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016 Organizing committee
1. Benjapon Nakprasert Advisor 2. Assoc Prof.Dr. Chuchart Kamollerd Advisor 3. Sumate Kamolnorranath Advisor 4. Tanachon Kensingh Advisor 5. Dr. Ampika Thongphakdee Chairperson 6. Asst Prof. Sarawut Sringam Vice Chairperson 7. Asst Prof. Dr. Vibuntita Chankitisakul Chairperson of Scientific Committee 8. Chavin Chaisongkarm Venue Committee 9. Nudthakamol Kajornklin Secretary 10. Saifon Yapila Assistant secretary 11. Supalak Kaitsomboon Registration Committee 12. Sunthita Karoon Treasurer 13. Dr.Saksiri Sirisathien Moderator 14. Asst Prof.Dr.Wuttigrai Boonkum Moderator
the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016 “The 7th Wildlife Assisted Reproductive Technology (ART) Workshop”
This workshop is aimed to establish one of the stages where reproductive scientist, veterinarian and relevance persons can join together sharing, talking, and giving their experiences about reproductive science and technology to each others.
The dates of the workshop are March 6-11, 2016. It will be held at Khon Kaen University (KKU) and Khon Kaen Zoo (KKZ), Khon Kaen, Thailand.
Program of the 7th ART Workshop 2016
6-Mar Participants arrive Khon Kaen
Scientific forum at Faculty of Veterinary Medicine, 7-Mar Khon Kaen University 9.00-16.30
Workshop at Khon Kaen Zoo Welcome address: Tanachon Kensingh (Director of KKZ) 8-Mar Overview of ART Workshop: Dr. Ampika Thongphakdee Semen collection and cryopreservation 8.00-16.30 2 Thamin Eld's deer and 2 Hog Deer
Embryo collection and cryopreservation 9-Mar 4 Rusa deer 8.00-16.30
Embryo collection and cryopreservation 10-Mar 4 Rusa deer 8.00-16.30
11-Mar Conclusion 8.00-12.00
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Agenda of the 7th Wildlife ART Workshop: March 7, 2016 at Faculty of Veterinary Medicine, Khon Kaen University
Time Topic Speaker 8.00-9.00 Registration 9.00-9.15 Welcome address Assoc. Prof. Dr. Chuchart Kamollerd (Dean of Khon Kaen University, KKU) Opening remarks Benjapon Nakprasert (Director of Zoological Park Organization, ZPO) Special lecture: 9.15-10.00 Prof.Dr.Mongkol Techakumphu (Vice President of Chulalongkorn University, CU) Reproductive biotechnology: basic to application 10.00-10.15 Group photo & coffee break 10.15-10.45 Why conservation and Wildlife ART in Thailand Sumate Kamolnorranart (DVM) (ZPO) 10.45-11.10 Health practice for captive breeding management Visit Arsaitumkul (DVM) (ZPO) 11.10-11.30 Best practice for wildlife anesthesia and reproductive case Chavin Chaisongkram (DVM) (KKZ, ZPO) 11.30-12.00 The role of oxidative stress in reproduction Dr.Yoswaris Semaming (DVM) (Udon Thani Rajabhat University) 12.00-13.00 Lunch 13.00-13.30 Cryopreservation of gametes and embryos: fundamental aspects Asst Prof. Dr. Theerawat Tharasanit (DVM) (CU) 13.30-14.00 Cryopreservation of sambar deer semen Assoc Prof. Dr. Thevin Vongpralub (KKU) 14.00-14.30 Semen evaluation and improvement of semen quality Asst Prof. Sarawut Sringam (DVM) (KKU) 14.30-14.50 Coffee break
Preliminary study on superovulation in rusa deer using a split- 14.50-15.20 single intramuscular administration of follicle-stimulating Asst. Prof. Dr. Vibuntita Chankitisakul (DVM) (KKU) hormone
Prof. Dr. Yan-Der Hsuuw (DVM) (National Pingtung University of Science and 15.20-15.50 Embryo development and quality assessment Technology) 15.50-16.20 Embryo cryopreservation: slow freezing aspect Dr. Saksiri Sirisathien (DVM) (KKU) 16.30-16.40 Closing scientific session 16.40-17.10 Introduction for workshop (only participants for workshop) 18.00-20.00 Welcome dinner and transport to Khon Kaen Zoo
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Special Lecture:
Reproductive Biotechnology: Basic to Application
Mongkol Techakumphu
Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10300
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Why Conservation and Wildlife ART in Thailand
Sumate Kamolnorranart
Bureau of Conservation and Research, the Zoological Park Organization, Thailand
Increasingly altered and fragmented habitats directly affected natural wildlife populations. More species are actively managed for their long-term survival. Conservation breeding of endangered species is one of the crucial missions at the ZPO. Today, total of 532 species (10,280 individuals); 3,971 individuals of mammal, 4,961 individuals of birds, 1,329 individuals of reptiles and 19 individuals amphibians are cared in ZPO zoos. Seven member zoos under the Zoological Park Organization (ZPO) are implementing ‘UN Decade on Biodiversity’ campaign by contributing to specific strategies including; 1) maintaining genetic diversity of captive wildlife population of global importance; 2) strengthen health management, research programs on reproductive science and establish genome bank; and 3) reintroduction programs for the ‘extinct-in-the-wild’ species. Prioritized species selected for IMP program are highlighted on Thai native endangered species such as Eld’s deer (Rucervus eldii), Eastern Sarus crane (Grus antigone sharpii), Malayan tapir (Tapirus indicus), goral (Naemorhedus griseus) and wild cats. The presentation will give the information “why conservation and biodiversity are important?” and “update assisted reproductive technology in wildlife in Thailand”.
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Health Practice for Captive Breeding Management
Visit Arsaithamkul Bureau of Conservation and Research, the Zoological Park Organization, Thailand
Wildlife captive breeding is a part of wildlife conservation, wildlife health is crucial for wildlife conservation. The vital role of play in conservation initiatives have to consider management ex situ wild animal relate to in situ wild animal. Zoo veterinarians and zoo animal managing staffs have been challenged to apply management, welfare and animal health not only for individuals but have more in preventive and therapeutic medicine for populations. Working group on each specific species have to design as Scientific Advisory Groups (SAGs) which comprised of zoo professionals and outside experts who research specific topics ex. behavior and husbandry, reintroduction, veterinary science, contraception, nutrition, small population management and genome resource banking, etc. and establishing Species Survival Plan (SSP) programmes which is a co-operative population management and conservation programme to facilitate the maintenance of a genetically- viable and demographically-stable population of a specific species in captivity and educate the public and foster basic veterinary, nutrition and reproductive research.
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Best Practice for Wildlife Anesthesia and Reproductive Case
Chavin Chaisongkram
Department of Research, Conservation and Animal Health, Khon Kaen Zoo, Khon Kaen,
Thailand, 40002
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The Role of Oxidative Stress in Reproduction
Yoswaris Semaming
Veterinary Technology Program, Faculty of Technology, Udon Thani Rajabhat University,
Udon Thani, Thailand 41000
Free radical has been shown to play an important role in pathophysiology of various degenerative diseases. Reactive oxygen species (ROS) are unstable and highly reactive. They acquire electron from cells to become stable which resulting in cellular damage. Under normal condition, scavenging molecules are known as antioxidants convert ROS to H2O to prevent over production of ROS. There are two systems which consist of enzyme and non-enzymatic antioxidants. Oxidative stress (OS) is the result of imbalance between ROS and antioxidants which can lead to oxidative damage. In reproduction, oxidative stress has been considered a major contributory factor to the infertility. Various factors including, disease, pollutants, chemicals, radiation, pathogens, metabolic disorder and physical disturbance promote excess free radical production. There are oxidative stress biomarkers which could be detected in certain sample type both in vitro and in vivo (blood, cell, urine, tissue and culture media) such as malondialdehyde (MDA), protein carbonyl content (PCC), 8-hydroxydeoxyguanosine (8-OHdG), nitric oxide, total antioxidant capacity, catalase, glutathione and superoxide dismutase. Recently, oxidative stress has been identified as important in assisted reproductive techniques (ART) including, intrauterine insemination, in vitro fertilization, intracytoplasmic sperm injection. The impact of oxidative stress on ART resulted in poor quality of oocyte and sperm and impaired embryonic development. In order to promote assisted reproductive techniques success and improve reproductive performance, the antioxidant supplementation should be considered.
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Cryopreservation of Gametes and Embryos: Fundamental Aspects
Theerawat Tharasanit
Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10300. Corresponding author Email: [email protected]
Cryopresservation of gametes and embryos is a technique of choice for bio-banking the genetic potential of desired animals. This technique allows long-term storage with logistically acceptable if the cryopreserved materials would need to be transferred. However, this technology frequently induces cryodamage that markedly affect cell viability and functions. Several procedures during cryopreservation potentially induce cellular changes that render poor cell viability and functions. Two cryopreservation techniques that have been frequently used for cryopreservation of gametes and embryos are slow freezing and vitrification. Slow cryopreservation requires an optimal freezing rate principally to balance the intra- and extra-cellular water and cryoprotectant(s). In contrast to the slow freezing technique, vitrification is a non-equilibrium cryopreservation technique that requires high concentration of cryoprotectants and extremely fast freezing rate. Among reproductive cells studied, sperm are the most successful cell types that well survive after cryopreservation and thawing. However, successful cryopreservation is still problematic in terms of sperm longevity and fertility post-thawing. It is well to note that sperm obtained from different species differ in cryosensitivity; sperm of particular species would need to be tested for cooling sensitivity prior to apply the technology to the field practice. Oocyte cryopreservation is more challenging because overall success is currently poor. The mammalian oocytes are the largest cells in the body that have low membrane permeability to cryoprotectants and contain complexity of cell structures and organelles. The dynamic changes of the oocytes contribute to high sensitivity of cryodamage. For instance, the microtubules of meiotic spindle of mature oocytes are irreversibly damaged during freezing and thawing processes. Embryos of particular species can also be cryopreserved with variable success in terms of their viability and conception rates after embryo transfer. Indeed, several factors have been demonstrated to affect cryosurvival of embryos including stage of embryo development and culture condition. Stem cells derived either from embryos or reprogramming of somatic cells have also been tested for feasibility of genetic banking as they, in principle, have unlimitedly cell division and can differentiate into specific cell types including gametes. However, the limitation of fundamental research of this technology is lacking behind other simplified reproductive technologies.
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Cryopreservation of Sambar Deer Semen
Thevin Vongpralub1* and Wittaya Chinchiyanond2 1Department of Animal Science, Faculty of Agriculture, Khon Kaen University, 40002, Khon Kaen, Thailand 2 Former Director of the Khao Kao Wildlife Breeding Research Center, Petchaboon, Wildlife Conservation Bureau, National Park Wildlife and Plant Conservation, Department Bangkok, 10900, Thailand *Corresponding author Email: [email protected]
ABSTRACT Sambar deer (Cervus unicolor) are the large deer species native to India peninsula and are found throughout Southeast Asia and Southern China. This species is listed as vulnerable. Cryopreservation of semen and artificial insemination (AI) has played a major role in propagation and conservation of genetic resources in wildlife species. For captive populations frozen semen with AI has the potential for overcoming inbreeding depression and maintaining genetic diversity. This review we describe the methods used for semen cryopreservation and artificial insemination of deer with the main focus on main land Sambar deer (Cervus unicolor equinus), with the hope of fostering future research and fruitful area of Sambar deer reproductive technology.
INTRODUCTION Sambar deer (Cervus unicolor) are the largest species of oriental deer and are found throughout South Asia, Southeast Asia and Southern China. This species has been introduced wildly outside it native range. Of the 14 subspecies described by Whitehead (1993) Cervus unicolor equinus is the most prominent in Southeast Asia whereas Cervus unicolor swinhoei is indigenous subspecies of sambar deer found in Taiwan. Wild populations are rapidly declining in many Southeast Asian protected areas due to commercial poaching (Leslie, 2011). In captivity, this cervid species has served as not only recreational but also commercial farm deer in Chinese medicine enterprises. The used of velvet antler in as an immune booster and as an antiallergy agent has increased in Taiwan since the start of deer farming in 1963 (Kuo et al.,2012). Sambar deer is the preferred deer meat in local and international markets (Steinmetze et al., 2006). This species of deer attained slaughter weight earlier than red deer suggests that Sambar deer are worthy of
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domestication (Semiadi et al., 1995). Therefore, captive Sambar deer farming has become commercially viable but good management requires a better understanding of the animal's physiology. There is a general paucity of information on the reproductive physiology of mainland Southeast Asian Sambar deer, both in the wild and in captivity. As with domestic animals, cryopreservation of semen and artificial insemination (AI) has played a major role in propagation and conservation of genetic resources in wildlife species. For captive populations frozen semen with AI has the potential for overcoming inbreeding depression and maintaining genetic diversity. A few studies to date have examined sperm freezing and AI in the deer (Morrow et al., 2009). Until recently, there has been only one study to determine the effect of different diluents on post-thaw sperm quality in Formosan Sambar deer (Cervus unicolor swinhoie) and Formosan Sika deer (Cervus nippon taiouanua). That study found that a different diluent required different protocols for semen cryopreservation (Cheng et al. 2004). Since specific protocols are required for optimal semen freezing for each breed or species. In the past, adequate knowledge was still lacking on reproduction of Sambar deer in captivity. During 2002 to 2004, we attempt to study the knowledge related to Sambar deer production and research was carried out at the Khao Kao Wildlife Breeding Research Center, Petchaboon, Thailand (16.66°N, 101.00°E, 700 m elevation). The practical protocols for semen cryopreservation and AI in mainland Sambar deer (Cervus unicolor equinus) were developed there. The intention of the following communication paper is to inform our experience of AI technology in Sambar deer.
Semen production Individual Sambar deer stags generally exhibit annual cyclicity in their antler cycles. The males of this species exhibit alteration periods of fertility and infertility related to dramatic changes in testis size and function. During antler regeneration, stag failed to show sexual interest and gave poor semen quality (Somphol, 2004). During a period of winter and early summer is a rust period, most of mature males have hardly antler and are highly aggressive. According to our experience, high quality of semen for cryopreservation mostly obtained during February-March. After a 65-75 days of growth period, velvet was harvested. Stags were individually housed in outdoor pens (9 m x 12 m) and exposed to a
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natural photoperiod and ambient temperature. Before semen collection period, mature stags were hand-reared for gentleness in preparation for electro-ejaculation procedures.
Semen collection and evaluation Semen was collected between the period in which stags exhibited reach maximum breeding behavior. According to sedate procedure for deer was prohibited at wildlife breeding research center, stags were manually restrained by experienced herdsmen under intensive care observation. The electro-ejaculator was an Electrojac IV (Gemini Inc.,MN, USA) with a rectal probe 2.5 cm in diameter and 20.5 cm in length with three longitudinal electrodes. The electrical current intensity, from 0 to 12V, was divided in a 0 to 30 scale. Electrical stimuli began at a low voltage and was gradually increased until semen was collected. Stimuli and rest periods were two seconds each. During 2002-2004, more than 120 semen ejaculations were collected by this method with none of abnormal sign had been observed in the donor stags. Chang et al.(2004) collected Formosan Sambar deer semen by the similar method of animal control and reported that no injury was observed and ejaculates were obtained with high success with the current electro-ejaculation approach. Quality of raw semen was assessed by volume, concentration, forward progressive motility (FPM) and normal sperm morphology (Evans and Maxwell, 1987). Only the semen with at least 60 % FPM and 70 % normal sperm morphology was used in the experiments. The cryopreserved semen evaluation included FPM and acrosome integrity as described by Dott and Foster (1972).
Frozen semen processing Beneficial of removal of seminal plasma on post-thaw semen quality Removal of seminal plasma is recommended in goats (Ritar and Salamon, 1982), cattle (AI-Somai et al., 1994) and buffalo (Ahmad et al., 1996). Sahni and Mohan (1990) reported that toxicity of seminal plasma was higher among buffalo than cattle, which in turn depended on the amount of seminal plasma. Maxwell et al. (2007) reported that, depending on species, seminal plasma can either inhibit or stimulate sperm function and fertility. For Iberian red deer Martinez-Postor et al. (2009) stated that seminal plasma might be detrimental to cryopreserved sperm. In contrast, for Formosan Sambar deer, Cheng et al. (2004) reported no negative effects of seminal plasma on the post-thaw quality.
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Sperm freezability of the whole semen was lower than that of the seminal plasma eliminated and additional of egg yolk diluent enhanced the quality of sperm collected by electro-ejaculation. Notwithstanding, our study on liquid stored semen of Sambar deer revealed that seminal plasma decreased the quality of diluted semen. In our study, when seminal plasma was removed prior to redilution with tris-egg yolk extender and processed for cryopreservation, the post-thaw quality was superior to aliquots frozen in the present of seminal plasma (Vongpralub et al., 2015).
Semen freezing extender for Sambar deer The effectiveness of extenders has been different between cervid species (Asher et al., 2000; Chang et al, 2004; Jabbour et al., 1997). Most diluents used successfully in deer have been adapted from sheep and goats (Asher et al., 2000). Of the various diluents used for cryopreservation of deer semen, sodium citrate-egg-yolk-glyerol (Krzywinski and Jaezewky, 1978) and Tris-glucose-citrate-egg yolk-glycerol (Evans and Maxwell, 1987) have been usedc the most. Contributing to the body of knowledge, Cheng et al. [2004] compared the efficacy of five extenders in cryopreservation of semen from Formosan Sika and Sambar deer. Their results showed that Tris-Tes-egg yolk-glycerol extender for Eld’s deer and pig frozen semen afforded better protection for Formosan Sika deer semen than the other extenders. They also showed that Tris-citric acid- egg yolk-glycerol-based extender and an extender for dog frozen semen and the previous extender were similar in their ability to cryopreserve semen of Formosan Sambar deer. A comparison of four egg yolk extenders (20%) included (1) Tris-egg yolk, (2) egg yolk citrate, (3) Illinois Variable Temperature (IVT) extender (Salisbuly et al., 1978) and (4)
Beltsville F5 extender (Pursel and Johnson, 1975) was performed. For Thai Sambar deer semen, Tris-egg yolk extender resulted in the highest post-thaw sperm progressive motility rates compared to egg yolk citrate, Illinois Variable Temperature (IVT) and Beltsville F5 extenders. Also, the use of Tris-egg yolk extender resulted in the greatest acrosome integrity (Vongpralub et al., 2015).
Optimal level of egg yolk concentration of the extender Semen preservation techniques for cervine species have mostly been learned from experience with sheep and goats. For Thai Sambar deer, no work has been reported on the
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optimal level of egg-yolk extender. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The level of egg yolk in extended semen is species dependent. In bovines (Vishwanath and Shannon, 2000) and porcines (Paquignon, 1985), about 20 % egg yolk has been commonly used in frozen semen. On the other hand, concentrations of egg yolk above 2 % have proved problematic in goats (Ritar and Salamon, 1982). But for deer semen, there are no reported detrimental effects of egg yolk on semen quality when diluted with 2.5 to 20 % egg yolk (Asher et al., 2000). In their study of five different extenders Cheng et al. [2004] used 20 % egg yolk as part of the formulations for cryopreservation of Formosan Sambar deer semen. For red deer, egg yolk extender was beneficial for semen preservation (Martinez-Postor et al., 2009). For Thai Sambar deer semen, 20% (v/v) was the best concentration (P<0.05) of Tris-egg yolk extender for freezing semen (Vongpralub et al., 2015).
Cooling rates on post-thaw semen quality Cooling bovine spermatozoa from room temperature to 5 o C is generally done over a period of 1.5 to 4 hr (Coulter and Foote, 1977; Foote and Kaproth, 2002; Rodriquez et al., 1975; Senger et al., 1976; Wall and Foote, 1999; Wiggin and Almquist, 1975ab). Rapid cooling to 5°C decreased the recovery rates of post-thaw spermatozoa (Dhami et al., 1992). Prolonging the incubation and equilibration period before freezing also decreased the survival rates (Pickett and Berndtson, 1974). To our knowledge, for Thai Sambar deer, no previous studies have investigated different cooling rates on the viability of frozen semen. There is also little information on the optimal cooling methods in other cervids. Most reported procedures satisfactorily cooled deer semen to 5 o C with equilibration periods modified from those for domestic ungulates. Soler et al. (2003) reported that diluted semen of mature red deer, Père David’s deer and fallow deer stags were refrigerated at 5 o C for 4 hr before freezing in liquid nitrogen vapour. For Formosan Sika deer and Formosan Sambar deer, diluted semen was loaded into 0.25 ml straws, slowly cooled to 5 o C in 2.5 hr and subsequently frozen in a programmable freezer (Cheng et al., 2004). To evaluate the effect of cooling rate on post-thaw survival of spermatozoa, semen cooling rate, from 35 o C to 5 o C was accomplished over 1, 2, 3 or 4 hours. Semen samples
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were cooled by dropping of ice cubes manually into ice bath. The cooling rates were approximately 0.41, 0.21, 0.14 and 0.10o C/min respectively. For Thai Sambar deer semen, 3 hours was the most beneficial cooling time (P<0.05) from 35 o C to 5 o C prior to freezing (Vongpralub et al., 2015).
Glycerol concentrations on post-thaw semen quality The discovery of glycerol as a cryoprotectant marked a quantum advance in semen cryopreservation; however, a high level of glycerol can be detrimental to spermatozoa and it is worth noting that inter-species variation in glycerol tolerance can be very marked (Holt, 2000). Loss of fertility was found when the concentration of glycerol exceeded 3% in boars (Watson, 1979) , whereas the concentration in bull frozen semen ranged between 7% and 13%, depending on the extenders used (Salisbury et al., 1978). Rodriguez et al. (1975) suggested that fast freezing resulted in a post-thaw motility equal to or greater than that of the slower methods when the spermatozoa were suspended between 5% and 9% glycerol, but the slower rates of freezing were superior when 11% glycerol was used. Matinez-Pastor et al. (2009) found that 4% glycerol gave a better result for post-thaw quality of red deer frozen semen, whereas Nally et al. (2011) reported that there was no difference when even 10% to 14% glycerol was used in Timor deer. In red deer, 4% to 6% glycerol has been used for freezing semen (Garde et al., 2006). As reported by Cheng et al. (2004), glycerol concentrations in successive extender used for semen cryopreservation of Formosan sambar deer ranged between 5-8%. In addition, Lin et al.(2014) report that extender supplemented with 6% glycerol was better for sperm viability, acrosomal integrity and motility in Formosan Sambar deer. For Thai Sambar deer semen, 3% was the best concentration (P<0.05) for freezing semen compared to 5%, 7% and 9% glycerol (v/v) (Vongpralub et al., 2015).
Freezing rates on the post-thaw semen quality No studies to date have examined the influence of different freezing rates on post- thaw viability of Sambar deer spermatozoa. In cervines, the freezing methods have been developed from ruminant species. In the freezing procedure for bull spermatozoa, semen straws are generally held in static nitrogen vapor 2 to 4 cm above the liquid nitrogen or at -100 o C to -160 o C (Landa and Almquis, 1979; Dhami et al., 1992; Johnson et al., 1995;
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Verma et al., 1994). The temperature at which the semen straw is held before immersing it into the liquid nitrogen is between -80 o C and -150 o C(Jabbour et al., 1993; Garde et al., 1998; Asher et al., 2000; Soler et al., 2003). To evaluate different freezing rates on frozen thaw semen quality, the straws were divided to three groups and maintained horizontally in the liquid nitrogen vapor at 2, 4 and 6 cm above the liquid nitrogen for 15 min before plunging into the liquid nitrogen. For Thai Sambar deer semen, 4 cm was the best height (P<0.05) to suspend semen straws in the liquid nitrogen vapor before plunging into the liquid nitrogen. Estimate of cooling rate were 12, 10, and 5o C/min for 2,4,and 6 cm heights respectively. Final temperature of cooling rate were -180o C, 150o C, and -100o C respectively (Vongpralub et al., 2015).
Thawing temperature and time on post-thaw semen quality In previous studies with cervid species, thawing temperature generally varied between 35 o C and 70 o C for between 8 and 60 sec. (Haigh et al., 1993; Monfort et al., 1993 Krogenase et al., 1994; Zomborszky et al., 1999; Hishinuma et al., 2003). Soler et al. (2003b) compared the effect of thawing at 70 o C for 5sec, 50 o C for 8 sec and 37 o C for10 sec and reported that the second and third protocols were the most beneficial for frozen semen among the different cervid species. Although spermatozoa from Sambar deer were cryopreserved and the thawing procedure was conducted at 37 o C (Cheng et al., 2004), the effect of this procedure on semen quality has not been compared until now. The freezing rates in this study can be considered rapid freezing. It has been argued that thawing should also be done rapidly in order to avoid the re-crystallisation of large intracellular ice crystals that can be harmful to the cell (Mazur, 1984). For Thai Sambar deer semen, an intermediate thawing rate (50 o C for 8 sec) was better than the slow (37 o C for 10 sec) or fast (70 o C for 5 sec) thawing rates (P<0.05) (Vongpralub et al., 2015).
Freezing equipment Diluted semen was loaded into 0.5 ml straws and held at 5 o C for few hr for equilibration and then frozen in static liquid nitrogen vapor. Semen was frozen in styrofoam boxes (25.5 x 37.5 x 29.0 cm) containing a 10 cm of liquid nitrogen. The temperature was measured by TC 200® (Dickson, Il, USA), a digital thermometer.
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Artificial insemination Sambar deer hinds estrous cycle In Thailand, female Sambar deer (Cervus unicolor equinus) estrus is not seasonal, normally 19 to 21 days estrous cycle and a gestation of 265.4 days are found (Chinchiyanoon, personal communication (n=30)). In comparison, the estrous cycle and gestation of female Formosan Sambar deer (Cervus unicolor swinhoei) were 18.2 and 258 days, respectively (Chan et al., 2009)
Estrous synchronization To our knowledge, there is no published information on estrus synchronization and timed AI in Sambar deer. Previous studies on other cervids have resulted in variable behavioral estrus rates following various methods of estrous synchronization. Fallow deer exhibited 50% to 100% estrus rates at 48 hr following CIDR-G (with PG) removal. Behavioral estrus rates depended on the treatment duration, increasing from 50% for 8 days CIDR-G insertion to over 90% for periods of at least 14 days (Morrow et al., 1995). In sika deer, a 13 day CIDR-G treatment followed by 50 IU of eCG at time of CIDR-G removal resulted in 36.6 2.9 hr to the onset of estrus (Willard et al., 1996). In most cases of estrus synchronized farmed deer, the timing of AI is performed from 44 to 75 hr post-synchronization, depending on the species (Willard et al., 2002). In induced estrus of red deer hinds with 150 IU of eCG following CIDR removal, 96% were observed in estrus ranging from 29 to 91 hr (mean 47.5 ± 3.3 hr) (Bowers et al., 2004). In Eld’s deer (Cervus eldi thamin), after 14 days of CIDR-G insertion, behavioral estrus was detected in 12 of 20 (60%) hinds (Monfort et al., 1993). In our study (Vongpralub et al., 2015), estrus was synchronized on ten hinds by using 13 days of the controlled progesterone releasing intra-vaginal device for goats (CIDR-G) (Inter Ag, Hamilton, New Zealand) followed at the time of withdrawal by IM injection of 200 IU equine Chorionic Gonadotropin (eCG) (Folligon; Intervet International BV, Holland) and
PGF2α (375 µg cloprostenol; 1.5 ml estroplan; Parnell laboratories Pty, Ltd, Australia). Protocol for timed insemination was determined by preliminary estrus synchronization studies (n=8) in which estrus was detected by closely observing the behavior of a trained, mature stag as he interacted with hinds in 12 hr intervals. Onset of estrus was determined when the hinds allowed a 5 minute one-to-one interaction with the stag. Estrus was observed 30 hr after CIDR-G withdrawal and lasted 24 to 60 hr.
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AI with frozen semen in Sambar deer In our study, the highest quality semen from one stag was used to inseminate ten hinds. Quality was determined by semen volume, percentage of motile sperm, percentage of live sperm, percentage of normal sperm, and sperm concentration which was 1 ml, 85%, 95%, 91% and 1,400 million cells/ml, respectively. The best methods, as determined from the results of above experiments, were used to freeze semen with 20 x 106 cells/straw. Progressively motile sperm of the thawed semen was always more than 65 % motility. To achieve insemination a bovine breeding gun was inserted into a foam type catheter for pig AI (pig champ). The modified catheter was then inserted through the vagina, close to the cervix. Adjustments were then made to the tip of the catheter, and its correct position in the cervix was confirmed by rectal examination, so that the bovine gun could be inserted through the first ring of the cervix to deposit the semen. One time of AI, only one straw was used for each female. According to sedated procedure was prohibited, the hinds were also manually restrained with the soft net and under intensive care. All ten Sambar hinds (100%) exhibited estrus at 28.8 ± 11.2 hr following removal of the CIDR-G and injections with 200 IU eCG and PGF2α. The duration of estrus was 28.0 ± 6.1 hr. The hinds were manually restrained and duration of restraint including insemination for each hind was less than 10 min. Artificial insemination was performed in 2.8 ± 1.0 min. Pregnancy rates and fawns born were not different when comparing single versus double timed AI (48 hr versus 48 hr + 12 hr, respectively) following estrus synchronization (P>0.05). After 60 days, non-return to estrus was 60% for both treatments. The fawning rate for single AI was 40% versus 60% for double AI (P>0.05).
CONCLUSION Since specific protocols are required for optimal semen freezing for each breed or species. Little information has been reported regarding semen cryopreservation and AI technology in Thai Sambar deer. Our studies demonstrated for the first time that the most appropriate cryopreservation protocol for the highest percentage of viable/motile Sambar deer semen (spermatazoa). (1) Discard the seminal plasma immediately after semen collection. (2) Use Tris-egg yolk at 20% concentration with 3% glycerol for the most suitable extender. (3) Cool from room temperature to 5 o C over a period of 3 hours. (4) Place the
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semen straws in the vapor 4 cm above the liquid nitrogen, before freezing, (5) Thaw the 0.5 ml frozen semen straws at 50o C for 8 sec for optimal motility. Estrus was successfully synchronized in Thai Sambar deer using CIDR-G for 13 days followed by 200 IU eCG and PGF2α. Using the high quality frozen semen, a single timed insemination at 48 hr post-CIDR removal resulted in a accetable pregnancy rate of 60 %. The knowledge gained from this study could serve as a useful model for cryopreservation and AI in Cervus unicolor or other critically endangered deer species in captivity or under field conditions.
ACKNOWLEDGMENTS Appreciation is expressed to the National Park, Wildlife and Plant Conservation Department for financial support. We also thank the staff of the Khao Kao Wildlife Breeding Research Center for their assistance in all aspects of raising, handling behavioural observation, and artificial insemination of the deer.
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Semen Evaluation and Improvement of Semen Quality
Sarawut Sringam
Department of Veterinary Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Thailand 40002. Corresponding author Email: [email protected]
Electroejaculation (EE) is the only technique useful for collecting good quality of semen from wild animals, and the postmortem sperm recovery may be an alternative technique in some situation. Semen consists of spermatozoa mixed with secretions from testes, epididymides and accessory organs of the male reproductive system. The method to evaluate semen fertilizing capability is through the results obtained by in vivo fertility. However, as it is difficult to apply this method in practice, especially in wild animals. It is agreed that spermatozoon is multifunctional cell that must possess a large number of attributes that make it potentially fertile. A fertile ejaculate must meet certain semen parameter quality standards (?), such as: progressive motility, normal morphology, active energy metabolism, structural integrity and functionality of the membrane, penetration capacity and optimum transfer of genetic material. The analysis of a single sperm parameter cannot predict the outcome of a process as complex as fertilization. In order to increase the predictive power of the test, simultaneous analysis of multiple sperm attributes should be done.
Some of male animals cannot give the standard quality of semen. Semen collected by EE is often more dilute and may appear to be less motile than that collected by artificial vagina. Some different techniques are used to collect the spermatozoa before frozen or use for artificial insemination, but the choice strongly depend on the quality of the semen that is on the concentration, motility and morphology, in order to obtain the higher number of good spermatozoa. The principle techniques of sperm preparation consist of washing, migration and density gradient centrifugation techniques. In addition, the further studies about extender and various antioxidants on frozen semen procedures will increase the success in ART.
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Preliminary Study on Superovulation in Rusa Deer using a Split-single Intramuscular Administration of Follicle-stimulating Hormone
Vibuntita Chankitisakul1*, Ampika Thongphakdee2, Chavin Chaisongkram3, Mongkol Techakumphu4
1Department of Animal Science, Faculty of Agriculture, Khon kaen University, Khon Kaen, Thailand, 40002 2 Wildlife Reproductive Innovation Center, Bureau of Conservation and Research, Zoological Park Organization, 71 Rama 5, Dusit Bangkok 10300 3 Department of Research, Conservation and Animal Health, Khon Kaen Zoo, Khon Kaen, Thailand, 40002 4 Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, 10300 *Corresponding author Email: [email protected]
Cervids represent more than 200 subspecies divided amongst 17 genera, characterized by striking differences in geographical distribution, morphology and physiology. Within cervid subspecies, almost 40 are considered to be threatened with extinction by the International Union for Conservation of Nature and Natural Resources (IUCN Red List). In Thailand, Schomburgk’s Deer (Rucervus schomburgki) was extinct around 1932, with the last captive individual being killed in 1938. None of the indications that the species inhabited other countries can be shown to have any compelling basis and some are clearly in error. Thus the species is considered a single-country endemic, to Thailand, where it is certainly extinct. Besides Schomburgk’s Deer, others wildlife species such as Eld’s deer (two subspecies of Eld’s deer, thamin (Rucervus eldii thamin) and siamensis (Rucervus edlii siamensis)) have been counted as reserved species under the Thai Wildlife Preservation and Protection Act (1992) and are also classified as endangered species by IUCN Red List because there are no appropriate conservation measures. Therefore, ex situ conservation programs have been strongly proposed to prevent genetic loss and to increase the prolificacy of remaining individuals. The application of biotechnologies developed for domestic ruminants, such as superovulation and embryo transfer is a rapidly growing biotechnology, and had already been successfully.
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Superovulation for induction of multiple follicular developments in embryo transfer program normally requires administration of an exogenous gonadotropin preparation that mimics the effect of FSH. The gonadotropin must be available long enough for the follicle to grow and attain final maturation of the oocyte. The biological half-life of exogenous FSH normally has a relatively short and multiple treatments are needed to induce superstimulation. Generally FSH is injected intramuscularly twice daily over 3-5 day period. The administration of FSH therefore requires constant attention by personnel, increasing the possibilities of failures due to mishandling and errors with the administration. Especially in case of wildlife animals, capture strategies for immobilization of wildlife is a procedural stress of the operator and can cause undue stress and harmful to animals. Subsequently suppressive to the superstimulatory response could possibly occur. Thus simplified protocols for superstimulation of follicular development may be expected to reduce costs, handling and improve response in deer. The development of a single dose protocol of FSH would be a useful alternative to the traditional twice daily. A single injection of FSH was achieved by diluting the hormone with a biodegradable polymer such as polyvinylpyrrolidone (PVP) or/and a hyaluronan based slow-release formulation (SRF) that resulting in sustained, slow release of the hormone over several days. To the best of my knowledge, few studies of superovulation and embryo transfer in deer were reported, particularly only one study of superovulation with a single dose of FSH injection was found unsuccessfully in year 2012. Therefore this study was conducted in order to develop an effective superovulatory protocol for deer that minimizes animal handing. We hypothesized that the slow release FSH provides the same ovarian response in superovulation program in rusa deer which would be a new fundamental knowledge of superovulation and could improve assisted reproduction technologies for other deer species in the future.
Materials and Methods Rusa deer (n=13) were synchronized by inserting a controlled internal drug release (CIDR; Pfizer, USA) for 12 days. The date of CIDR insertion is designated Day 0 (initial day of treatment). Then they were assigned randomly into two treatments for superstimulation as follows; Treatment 1; Multiple injection treatment: Superovulation with multiple injection was conducted with a total dose of 180 mg of NIH-FSH-P1 (Folltropin-V, Bioniche,
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Belleville,ON, Canada). Intramuscular injections are given every 12 h for 8 injections, (22.5 mg at each injection), beginning on the afternoon of Day 9. The CIDRs were removed from donors on Day 12, concurrently with the 7th injection of FSH. Treatment 2; Split-single dose treatment: Superovulation with slow release FSH injection: The slow-release FSH treatment was prepared in a diluent (MAP-5 50 mg) and given split into two; 120 mg of FSH was administered intramuscular on the afternoon of Day 9 and the remaining 60 mg of FSH was administered 48 hours later. The CIDRs were removed from donors on Day 12. The number of corpus luteum were recorded and compared between two superovulation treatments by laparoscopy.
Results and Discussion The mean numbers of corpus luteum (Fig.1A) were higher in the multiple injection treatment compared with split-single injection treatment (4.25 vs. 2.5) (P<0.05). In addition, we noticed that the ovaries which unresponsive to gonadotropins had smaller size (Fig.1B) compared with the others (P<0.05). However, we could not make the summary at the moment because this study was only preliminary study. The results of hormonal profiles and more sample size are needed to conduct further. The preliminary result provides the primary information that is possible to apply the biotechnology to other endangered deer species in the future.
(A) (B)
Figure.1 the corpus luteum on ovary (A); the inactive ovary (B)
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Acknowledgements
This study was financially supported by the Thailand Research Fund and the Zoological Park Organization. We are grateful to Asst.Prof.Dr.Wuttigrai Boonkum for his assistance in statistical analysis. We would like to thanks for Dr. Yoswaris Semaming, Dr. Kanda Ponsrila and Dr. Aunchisa Phojun for their surgical assistance and anesthesia procedure during the experiments. Thank you to keepers of Khon Kaen zoo and technicians of the Wildlife Reproductive Innovation Center for their kind attempt on animal care and collaboration.
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Embryo Development and Quality Assessment
Yan-Der Hsuuw
Department of Biological Science and Technology Director, Laboratory Animal Center, Embryo development and Stem Cell Laboratory, National Pingtung University of Science and Technology, Neipu, Pingtung, 912 Taiwan
The development of embryo is critically controlled by a precise cooperation of hormones and regulators (cytokines and growth factors) present in the reproductive tract. Learn more about the biological effects of hormones or regulators on the preimplantation embryo development, implantation as well as on embryogenesis and pregnancy maintenance, could therefore provide and important trail to understand the interactions between maternal receptivity and embryo viability during pregnancy. To evaluate the viability of preimplantation embryo in vitro, we have performed several methods on embryonic cell growth, differentiation or cell death at the time or after implantation. Differential staining, is based on immunosurgery, considering the impermeability of the trophectoderm (TE) layer which protects the inner cell mass (ICM) from the exposure to the antibody and complement reaction. Two cell lineages can be distinguished following the dual fluorochromes staining, bisbenzimide (Hochest 33258) and propidium iodide (PI). Cells containing fragmented nuclei (karyorrhexix) are identified as dead cells. Outgrowth assay, the ability of blastocyst to implant and develop can be assessed in the light of an outgrowing culture model. The hatched blastocysts will attach onto the fibronectin and outgrow with a cluster of ICM over the TE outgrowth. The proliferation of outgrowths is examined by the counting the nuclei directly on the dish following the BrdU incorporation and the cell spreading technique. TUNEL assay, Terminal transferase-mediated dUTP nick end labeling is based on the binding of dUPT to each 3’-hydroxyl terminal of DNA strands. Cells containing fragmented DNA (karyolysis) is identified as apoptosis. In conclusion, a sufficient number of ICM cells are imperative in the process of implantation, and the excessive reduction in this cell lineage may impair embryo viability in spite of a normal TE population or a normal implantation rate. Monitoring the embryo quality by different approaches will be helpful to estimate the optimum system in embryo culture, and to promote a successful pregnancy following the embryo transfer.
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Improvement in cryopreservation of in vitro produced bovine embryos
Saksiri Sirisathien
Dept. of Surgery and Theriogenology, Fact. of Vet. Med., Khon-Kaen University
Cryopreservation of embryos has become an essential part of assisted reproductive technology (ART) allowing embryos to be used with its full potential. Cryopreservation of in vivo derived bovine embryos is now a routine procedure in embryo transfer program. However, in vitro produced (IVP) bovine embryos differ from the vivo derived embryos in several aspects, especially its cryotolerance. The cryopreservation of IVP bovine embryos remains to be improved even though it was established for decades. Experiments were conducted to improve slow freezing technique for IVP bovine embryos using simple empirical approach. In experiment 1, effect of different base media and types of cryoprotective agents were examined. Bovine expanded blastocysts (8 days after fertilization) were cryopreserved in the freezing media consisted of 10 % ethylene glycol (EG) alone or 5% EG + 5% dimethyl sulfoxide (DMSO) diluted in one of the three base media (TCM-199, D-PBS, or cytomix) plus 0.1 M sucrose and 10% bovine serum. The initial survival rate at 24 h post thawed of embryos cryopreserved with 5% EG + 5% DMSO in three base media combined was higher than that of embryos cryopreserved with 10% EG alone (87.1% vs. 71.4%, respectively, p< 0.05). No effect of base media on the cryosurvival of embryos was detected. In experiment 2, embryos were cryopreserved with 5% EG+ 5% DMSO in D-PBS. The temperature was cooled down to reach -30 or -40ºC before plunged into liquid nitrogen. The plunging temperature had no effect on the initial survival rate at 24 h post thawed. However, hatching rate at 72h post thawed was higher in -40ºC group compared to that of -30ºC group (70.6% vs. 39.4%, respectively). In experiment 3, embryos were cryopreserved with 0.1 M sucrose or 0.1 M trehalose or 0.1 M raffinose. Replacing sucrose with trehalose or raffinose had no effect on cryosurvaival of embryos. In conclusion, a slow freezing technique using 5% EG + 5% DMSO in D-PBS plus 0.1 M sucrose and 10% bovine serum with the plunging temperature at -40ºC was found to be an efficient procedure for cryopreservation of IVP bovine embryos.
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