Iranian Journal of Aquatic Animal Health 5(1) 1-16 2019 Effects of diets containing dry extracts of Achillea millefolium, Mentha piperita and Echinacea purpurea on growth, hematological and immunological indices in juvenile common carp (Cyprinus carpio) S Alinezhad* Institute of Agricultural Education and Extension, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran Received: March 2019 Accepted: April 2019 Abstract In this study, the effects of three herbal dry Mean corpuscular volume (MCV) and mean extracts (Achillea millefolium, Mentha corpuscular hemoglobin (MCH) in all groups piperita and Echinacea purpurea) were except 0.1% M. piperita group and 0.5% E. investigated on growth, hematological and purpurea were increased compare with control immunological indices in juvenile common group (P≤0.05). MCHC in 0.5% E. purpurea carp (Cyprinus carpio). 400 juvenile fish with and 0.1 and 1% M. piperita groups showed the initial weight of 14.30 ± 0.77g were studied in highest values. Levels of 0.5% M. piperita and 10 treatment groups (9 treatment groups & a 1% E. purpurea and A. millefolium make control) with four replicates for 60 days. Three significantly increases in total leukocytes and levels (0.1, 0.5 and 1%) of dry extracts of each neutrophils (P≤0.05). Significantly increases of herb were prepared according to standard lymphocytes and decrease of monocytes were method and added to the commercial common observed in levels of 0.5% E. purpurea and 1% carp feed. At the end of period twelve fish level of all herbs groups (P≤0.05). Increased collected out of each group and the parameters levels of immunoglobulin compared to control were measured. In order to the results, weight were significant only in 1% level of all herbs gain, specific growth rate (SGR) and (P≤0.05). Complement C3 was also increased complement C4 were not affected by dietary 1% of A. millefolium and M. piperita groups treatments (P>0.05). Red blood cell (RBC) compared to the control (P≤0.05). All levels of counts in 0.5 and 1%-diet groups as well as A. millefolium and M. piperita and 1% E. Hemoglobin in three levels of all herbs was purpurea groups caused a significant increase increased (P≤0.05). Hematocrit in 0.5%, 1%- in lysozyme concentration compare with the diet M. piperita and E. purpurea groups was control (P≤0.05). The results indicated all three shown significant increases (P≤0.05). herb extracts in diet can improve immune responses and hematological parameters in *Correspondence S Alinezhad, Institute of common carp. Comparing these extracts, the M. Agricultural Education and Extension, Agricultural piperita extract with a lower concentration is more efficient. Research Education and Extension Organization (AREEO), Tehran, Iran (e-mail: Keywords: Medicinal Herb, Immunology, [email protected]). Hematology, Growth indices, Common carp 1 S Alinezhad, Comparison of herbal dry extracts on juvenile common carp Introduction The immunostimulants include a group of carp species feed were reported. Mixture of biological and synthetic compounds that Ocium bacilicum, Mentha piperita, juglans enhance the innate immunity in animals and regia and Cinnamomum zeylanicum extracts protect them from pathogens (Bairwa, Jakhar, (Hajibeglou & Sudagar 2010), Mixture of Satyanarayana & Reddy 2012). Regarding the Astragalus membranaceus, Polygonum mechanism of action on immune system, these multiflorum, Isatis tinctoria and Glycyrrhiza stimulants have more effective response in glabra extracts (Yuan, Chen, Sun, Wu, Gong & lower vertebrates rather than higher vertebrates. Han 2007), Zataria multifloria (Soltani, On the other hand, the tendency to Sheikhzadeh, Ebrahimzadeh-Mousavi & eliminate antibiotics in aquaculture due to high Zargar 2010), Olea eurcpaea (Karimi pashaki, costs, drug resistance, environmental and Ghasemi, Zorrieh Zahra, Shrif Rohani & administrative problems has led to more Hosseini 2018) had positive effects on growth, attention to safe stimulants as an alternative hematological and immunological indices in method (Harikrishnan, Kim, Kim, common carp. Balasundaram & Heo 2011). The use of herbal Achillea millefolium, Mentha piperita and immune-stimulant compounds has multiple Echinacea purpurea are perennial plants that advantages, including the lesser side effects of have antioxidant, anti-bacterial, anti- organisms and environment, lack of drug inflammatory and immunostimulant activities resistance, low price, sustained and (Hajibeglou & Sudagar 2010, Sharif Rohani, affordability and attracted a lot of attention Pourgholam & Haghighi 2016, Aly & globally, especially in developed countries Mohamed 2010, Candan, Unlu, Tepe, Daferera, (Vaseeharan & Thaya 2014). Common carp Polissiou, Sökmen & Akpulat 2003). (Cyprinus carpio) is a bony fish and one of the In this research, in order to the importance most popular fish in many countries. This fish of this medicinal herbs in immunity and fish is widely cultured in Europe and Asia and also production, the effects of different Iran (Toral-Granda, Lovatelli & Vasconcellos concentrations in A. millefolium, M. piperita 2008, Iran Fisheries Organization 2017). and E. purpurea extracts in diet were studied on In several studies, the positive effects of the growth, hematological and immunological medicinal plants were studied on common carp indices in common carp juveniles. growth and immunity improvements. In such Materials and methods researches, weight gain amelioration, FCR decrease, specific growth rate (SGR), Extraction method leukocytosis, lysozyme activity enhancement, The studied plants were got from the Iranian increase of phagocytic activity and plasma Institute of Medicinal Plants. After drying the globulin value after usage of herbs extracts in plants in shade and mild air, the samples were 2 Iranian Journal of Aquatic Animal Health powdered. In each extraction time, 100 g of General Culture Conditions powder was poured in a laboratory balloon The number of 400 juvenile common carp with containing the ratio of 1:1 distilled water and a weight of 14.30 ± 0.77 g were separated from 99.6% ethanol (300 ml of each substance) and the original stock and kept in ten groups in forty was gently mixed in a shaker for 48 hours. The 200-litered aquaria. mixture was then filtered with a Whatman 42 The daily water change rate was 10% for each filter paper and a Buchner funnel. After finishing aquarium. The water temperature was 26-28°C, extraction, the extracts obtained by rotary device dissolved oxygen was 5.08-7.70 ppm, pH was were concentrated at 40-50°C. The concentrated 7.9 ± 0.3 and ammonia was less than 0.2 ppm. extract was dried in oven at 40°C for two days. After a week of adaptation with commercial Then, they were kept in autoclaved glass common carp diet (without additives), the containers at 3ºC until they were mixed with the treatment diets were given to fish for 60 days. commercial diet (Sivam 2001). Fish were fed satiated by experimental diet. Feeding times were eight times per day in the Preparation of treatments first three weeks and 4 times per day from Three levels (0.1, 0.5 and 1%) of dry extracts of fourth week until the end of the period. At the each medicinal herb were added to the end of period, three fish were taken from each commercial common carp feed (Faradaneh, replicate for the tests (e.g. 12 fish from each Aquatic Animal Feed Producer) (table 1). In treatment). order to add dried extract to the diet, the total feed intake at each group was estimated, and Sample collection and analysis then the dry extract was added to the diet with Feeding was stopped one day before blood a specified volume of distilled water (40 ml) to sampling. At the end of period, three fish of each obtain a paste product. It was finally dried at replicate were randomly caught to measure the room temperature at 30°C for 48h. All diet blood parameters. They were anesthetized by groups were stored at -20°C until used (Akbary, 100 ppm clove extract (Javaheri, Nekoubin & Kakoolaki, Salehi, Zorriehzahra, Sepahdari, Haji Moradlu 2012). Blood sampling was done Mehrabi 2016). Totally ten groups with four from caudal vein that were stored in EDTA- replicates were prepared (one commercial diet containing tubes at 4°C until testing (Blaxhall & group without additives as control group). Daisley 1973). Measured growth indices Table1. Approximate analysis (%) of juvenile included initial, final weights, weight gain and common carp feed specific growth rate (SGR), which is calculated Chemical analysi juvenile feed based on the following formula (Watanabe, crude protein 35-38 fat 4-8 Ernest & Chassar 1993). Fiber 3-6 L W -L W Ash 7-11 SGR%= ( n 1 n 0/Experimental period (Day))*100 Moisture 5-11 Phosphorus 1-1.5 LnW1: Natural log of final weights, LnW0: Size of floating pellet (mm) 2 Natural log of final weights 3 S Alinezhad, Comparison of herbal dry extracts on juvenile common carp Hematological parameters including Statistical Analysis hematocrit (Rehulka 2000), red blood cell The design was carried out in ten treatments counts (RBC), hemoglobin value (Blaxhall and four replicates as a completely and Daisley 1973), mean corpuscular volume randomized design with three observations per (MCV), mean corpuscular hemoglobin (MCH) replication. and mean corpuscular hemoglobin Finally, the design was analyzed with SPSS concentration (MCHC) (Klinger, Blazer & (version 21, Chicago, USA) software by One- Echevarria 1996) were determined. Way ANOVA method to evaluate the effects The measured immune parameters consist of treatments at 0.05. The Kolmogorov- of white blood cell count (WBC) by Neubauer Smirnov (K-S) test for the normality of data chamber method (Thrall, Weiser, Allison & distribution and the Duncan's multiple range Campbell 2012), separation detection of a test comparison of the means were used at variety of white cells by the extension method 0.05. with Wright-Giemsa stain (Sharif-Rohani et al. 2016), measuring immunoglobulin and C3 Results and C4 enzymes complement system by Growth indices in juvenile common carp commercial kits (Authorized by: Eurolyser, In order to the juvenile common carp biometry Belgium) Pars Azmoon Co.