Abstract：Abstract Acrossocheilus fasciatus is a type of stream fish having great economic value in China. Recently, resource of A. fasciatus has declined rapidly due to overfishing and habitat destruction. Sperm cryopreservation is an effective way to protect germ cells; therefore, research in germplasm resource protection and germ cell conservation are in urgent need. In this study, the sperm was collected from cultured mature male fish and was stored in 0.25 mL straws for the study of sperm cryopreservation. Different conditions were evaluated for sperm cryopreservation, including extenders (Kurokura-1, Ringer, Cortland, D-14, D-15, D-16, D-17, D-19, D-20 and D-21), dilution ratios (1∶1, 1∶2, 1∶3, 1∶4, 1∶5, 1∶7 and 1∶9), cryoprotectants (dimethyl sulfoxide (DMSO), methyl alcohol (MeOH), ethylene glycol (EG), and propylene glycol (PG)), equilibrium times (0, 10, 20, 30, 40, 50 and 60 min), cooling heights (using two-step cooling procedures) (2, 3.5, 5, 6.5 and 8 cm), and thawing temperatures (27, 32, 37, 40 and 42 ℃). In addition, we detected the quality of fresh and corresponding frozen-thawed sperm through their enzyme activities. The experimental data were expressed as X±SD, using SPSS 17.0 statistical software. The statistically significant differences in sperm activation rate and enzyme activities of each group were detected by one-way ANOVA. P＜0.05 were considered significant. The fresh sperm density of Acrossocheilus fasciatus was (9.32±2.03)×109 cells/mL. The optimum result was obtained with the cryoprotectant comprising D-15 diluted with 10% DMSO, which was mixed with sperm at a ratio of 1∶5, equilibrated for 30 min, swung at 3.5 cm above nitrogen for 5 min, and finally stored in nitrogen. After thawing in a water bath at 37 ℃, the activation rate of the frozen-thawed sperm was (35.33±2.52)%; meanwhile, the rate of fresh sperms was (87.67±3.06)%. Enzyme activities were examined using reagent kits. Compared to the fresh sperm, the activities of the total ATPase, succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in optimized frozen-thawed sperm decreased from (9.31±0.17) U/mL, (30.33±5.69) U/mL, and (7 454.84±252.42) U/L to (7.23±1.08) U/mL, (17.67±6.03) U/mL, and (2 172.48±209.62) U/L, respectively. Thus, significant differences were observed in sperm activation rates and enzyme activities between the two groups. The decrease in enzyme activities indicated that cryopreservation caused damage to the sperm. With the given protocol, we got early success in sperm cryopreservation of A. fasciatus. This study provide a technical basis for the foundation of the cryobank for A. fasciatus; however, further studies on parametric optimization are warranted to improve the quality of frozen-thawed sperms.
[1]陈松林, 刘宪亭, 鲁大椿, 等.鲢、鲤、团头鲂和草鱼精液超低温冷冻保存的研究[J].动物学报, 1992, 38(4):413-424 [2]陈松林, 田永胜, 李军, 等.鱼类精子和胚胎冷冻保存理论与技术[M].中国农业出版社, 2007, pp.113-122. [3]陈田飞, 吴大洋, 李春峰.冷冻保存对家蚕精液乳酸脱氢酶活性的影响[J].西南农业大学学报自然科学版, 2004, 26(6):764-768 [4]程顺, 闫家强, 竺俊全, 等.大黄鱼 精子冷冻前后的活力及超微结构变化[J].海洋与湖沼, 2013, 44(1):56-61 [5]邓顺美, 李叔庚, 文建国, 等.不育症精子乳酸脱氢酶同功酶活性测定及其定位研究[J].中国组织化学与细胞化学杂志, 2001, 10(1):8-13 [6]黄晓荣, 章龙珍, 庄平, 等.超低温冷冻对日本鳗鲡精子酶活性的影响海洋渔业[J].海洋渔业, 2008, 30(4):297-302 [7]黄晓荣, 章龙珍, 庄平, 等.超低温冷冻对长鳍篮子鱼精子中几种酶活性的影响[J].海洋科学, 2009, 33(7):16-22 [8]黄晓荣, 章龙珍, 庄平, 等.超低温冷冻保存对大黄鱼精子酶活性的影响[J].海洋渔业, 2012, 34(4):439-443 [9]蒋进, 李明云, 吴尔苗.光唇鱼染色体核型分析[J].淡水渔业, 2009, 39(3):77-79 [10]冀德伟, 李明云, 史雨红, 等.光唇鱼的肌肉营养组成与评价[J].营养学报, 2009, 31(3):298-303 [11]姜建湖, 张德明, 竺俊全, 等.光唇鱼 胚胎及仔、稚鱼的发育[J].海洋与湖沼, 2012, 43(2):280-287 [12]姜建湖, 戴海平, 竺俊全, 等.养殖光唇鱼 卵巢发育的组织学观察[J].海洋与湖沼, 2013, 44(2):348-354 [13]田永胜, 姜静, 马允, 等.额尔齐斯河江鳕精子冷冻保存研究[J].农业生物技术学报, 2014, 22(9):1149-1156 [14]汪亚媛, 张国松, 李丽, 等.瓦氏黄颡鱼精子的生理特性及其超低温冷冻保存的初步研究[J].海洋渔业, 2014, 36(1):29-34 [15]王晓爱, 杨君兴, 陈小勇, 等.软鳍新光唇鱼精子的超低温冷冻保存[J].动物学研究, 2012, 33(3):283-289 [16]徐滨, 江琪, 庄平, 等.超低温冷冻对俄罗斯鲟精浆和精子酶活性的影响[J].淡水渔业, 2013, 43(1):9-13 [17]熊承良, 黄勋彬, 沈继云, 等.弱精子症患者精子中和琥珀酸脱氢酶的含量与精子活率的关系[J].同济医科大学学报, 1999, 28(4):289-291 [18]闫秀明, 张小雪.超低温冷冻对黄鳝精子中几种酶活性的影响[J].水生生物学报, 2011, 35(5):882-886 [19]章龙珍, 江琪, 庄平, 等.超低温冷冻对俄罗斯鲟精子抗氧化酶活性的影响[J].大连水产学院学报, 2009, 24(6):504-508 [20]张玉明, 闫家强, 姜建湖.光唇鱼精子的活力观察[J].浙江海洋学院学报自然科学版, 2009, 28(4):393-397 [21]张玉明, 姜建湖.光唇鱼人工繁殖研究[J].浙江海洋学院学报自然科学版, 2010, 29(3):211-214 [22]张玉明, 程顺, 姜建湖, 等.养殖光唇鱼生长的初步研究[J].上海海洋大学学报, 2012, 21(4):542-548 [23]周磊, 罗渡, 卢薛, 等.斑鳜精液超低温冷冻保存及其效果分析[J].中国水产科学, 2014,21(2):250-259 [24]Afromeev V I, Tkachenko V N.Change in the percent of lactate dehydrogenase isoenzyme level in testes of animals exposed to superhigh frequency radiation[J].Biofizika, 2014,44(5):931-932 [25]Bilgeri Y R, Winckelmann A, Berzin M, et al.Denosine triphosphate levels in human spermatozoa[J].Archives Andrology, 1987, 18(3):183-188 [26]Babiak I, Glogowski J, Goryczko K, et al.Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout spermatozoa[J].Theriogenology, 2001, 56(1):177-192 [27]Chan S Y, Wang C.Correlation between semen adenosine triphosphate and sperm fertilizing capacity[J].Fertility Sterility, 1987, 47(4):717-719 [28]Comhaire F H, Vermeulen L, Schoonjans F.Reassessment of the accuracy of traditional sperm characteristics and adeno-sine triphosphate(ATP)in estimating the fertilizing potential of human semen in vivo[J].International Journal of An-drology, 1987,10(5):653-662 [29]Ciereszko A, Dietrich G J, Nynca J, et al.Cryopreservation of rainbow trout semen using a glucose-methanol extender[J].Aquaculture, 2014, s 420-421(3):275-281 [30]Dietrich G J,Nynca J,Dobosz S,et al.Application of glucose–methanol extender to cryopreservation of semen of sex-reversed females rainbow trout results in high post-thaw sperm motility and fertilizing ability [J].Aquaculture,2014,434:27-32 [31]Ding S Y, Ge J C, Hao C, et al.Long-term cryopreservation of sperm from Mandarin fish Siniperca chuatsi[J].Animal Reproduction Science, 2009, 113(1-4):229-235 [32]Fu S X,jiang J H,Yang W X,et al.A histological study of testis development and ultrastructural featuresof spermatogenesis in cultured Acrossocheilus fasciatus[J].Tissue and Cell,2016,48(1):49-62 [33]Irawan H, Vuthiphandchai V, Nimrat S.The effect of extenders,cryoprotectants and cryopreservation methods on common carp(Cyprinus carpio)sperm[J].Animal Reproduction Science, 2010, 122(3-4):236-243 [34]Linhart O, Rodina M, Cosson J.Cryopreservation of Sperm in Common Carp Cyprinus carpio: Sperm Motility and Hatching Success of Embryos[J].Cryobiology, 2000, 41(3):241-250. [35]Linhart O, Rodina M, Flajshans M, et al.Cryopreservation of European catfish Silurus glanis sperm:Sperm motility,viability,and hatching success of embryos[J].Cryobiology, 2005,51(3):250-261 [36]Liu G D, Sheng Z, Wang Y F, et al.Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride[J].Gene, 2016,578(2):210-218. [37]Nahiduzzamana M, Hassan M M, Roy P K, et al.Sperm cryopreservation of the Indian major carp,Labeo calbasu:Effects of cryoprotectants,cooling rates and thawing rates on egg fertilization[J].Animal Reproduction Science, 2012, 136(1-2):133-138 [38]Nynca J, Dietrich G J, Dobosz S, et al.Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen[J].Aquaculture, 2014,433:62-65. [39]Piasecka M, Wenda-Rozewicka L, Ogonski T.Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa[J].Andrologia, 2001,33(1):1-12 [40]Vuthiphandchai V, Thadsri I, Nimrat S.Chilled storage of walking catfish(Clarias macrocephalus) semen[J].Aquaculture, 2009,296(1-2):58-64 [41]Warnecke D, Pluta H J.Motility and fertilizing capacity of frozenthawed common carp (Cyprinus carpio L)sperm using dimethyl-acetamide as the main cryoprotectant[J].Aquaculture, 2003, 215(1):167-185 [42]Zhao Y Q, Liu G D, Hou C C, et al.Effect of cadmium exposure on antioxidant enzyme catalase in different tissues[J].Mol Cell Toxicol, 2016,12:255-263.
