超低温冷冻保存中华鲟F_1精子授精方法研究
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摘要
利用超低温冷冻保存中华鲟子一代(F_1)的精液,研究4种不同激活液、2种不同的授精方式及5种不同冻精授精量对授精效果的影响。结果表明,冷冻保存365d的中华鲟F_1冻精,用养殖水激活授精效果优于4种激活液,获得(15.04±0.58)%的受精率和(14.6±0.59)%的孵化率。相同条件下,试验1、试验2和试验3的湿法受精率和孵化率分别为(18.3±1.45)%、(13.57±0.76)%、(18.65±0.42)%和(16.82±2.01)%、(11.03±0.98)%、(15.87±1.25)%,湿法授精效果均优于干法授精。用10mL中华鲟卵与不同冻精授精量进行湿法授精,精子密度3.82×109个/mL,稀释比1∶2,冻精活力(35.45±5.26)%,冻精用量0.1~0.6mL,随着冻精用量的加大,受精率和孵化率呈现上升趋势,0.6mL时,受精率和孵化率达最高,分别为(16.43±1.57)%和(14.58±1.35)%,但随着冻精用量的继续加大,其受精率和孵化率随之下降。
The viability was studied in cryopreserved sperm of the first filial generation of Chinese sturgeon(Acipenser sinensis)(F_1)and the effects of sperm activators containing various ratios of NaCl,Tris-HCl,glucose,MgCl_2,or CaCl_2,2 insemination methods(dry and wet insemination)and 5 volume ratios of sperms to fresh eggs(0.1 mL∶10 mL,0.3 mL∶10 mL,0.6 mL∶10 mL,1.5 mL∶10 mL and 3 mL∶10 mL)on fertilization and hatching rates of F_1 Chinese sturgeon.The results showed that there was better activation of the 365 day cryopreserved sperm of the F_1 Chinese sturgeon in water medium than that in the remaining 5 activation media,with fertilization of(15.04±0.58)% and hatching rate of(14.6±0.59)%in the water medium.In three insemination method trails,the fertilization and hatching rates under wetfertilization method were shown to be(18.3±1.45)% and(13.57±0.76)%in trail 1,(18.65±0.42)%and(16.82±2.01)%in trail 2,and(11.03±0.98)%and(15.87±1.25)%in trail 3,indicating that wetfertilization method was better than dry-fertilization method.Then the wet-fertilization method was performed by different volumes of addition of frozen semen with 10 mL Chinese sturgeon eggs(semen density of 3.82×109 ind/mL,dilution ratio of 1∶2)and frozen vitality of(35.45±5.26)%.Within the range of 0.1—0.6 mL frozen semen,the fertilization rate and hatching rate were shown to be an upward trend with increasing amount of the semen,the maximal fertilization rate of(16.43±1.57)% and hatching rate of(14.58±1.35)% at 0.6 mL of frozen semen.However,with the further increasing amount of frozen semen,the fertilization rate and hatching rate were decreased.
The viability was studied in cryopreserved sperm of the first filial generation of Chinese sturgeon(Acipenser sinensis)(F_1)and the effects of sperm activators containing various ratios of NaCl,Tris-HCl,glucose,MgCl_2,or CaCl_2,2 insemination methods(dry and wet insemination)and 5 volume ratios of sperms to fresh eggs(0.1 mL∶10 mL,0.3 mL∶10 mL,0.6 mL∶10 mL,1.5 mL∶10 mL and 3 mL∶10 mL)on fertilization and hatching rates of F_1 Chinese sturgeon.The results showed that there was better activation of the 365 day cryopreserved sperm of the F_1 Chinese sturgeon in water medium than that in the remaining 5 activation media,with fertilization of(15.04±0.58)% and hatching rate of(14.6±0.59)%in the water medium.In three insemination method trails,the fertilization and hatching rates under wetfertilization method were shown to be(18.3±1.45)% and(13.57±0.76)%in trail 1,(18.65±0.42)%and(16.82±2.01)%in trail 2,and(11.03±0.98)%and(15.87±1.25)%in trail 3,indicating that wetfertilization method was better than dry-fertilization method.Then the wet-fertilization method was performed by different volumes of addition of frozen semen with 10 mL Chinese sturgeon eggs(semen density of 3.82×109 ind/mL,dilution ratio of 1∶2)and frozen vitality of(35.45±5.26)%.Within the range of 0.1—0.6 mL frozen semen,the fertilization rate and hatching rate were shown to be an upward trend with increasing amount of the semen,the maximal fertilization rate of(16.43±1.57)% and hatching rate of(14.58±1.35)% at 0.6 mL of frozen semen.However,with the further increasing amount of frozen semen,the fertilization rate and hatching rate were decreased.
引文
[1]四川省长江水产资源调查组.长江鲟鱼类生物学及人工繁殖研究[M].成都:四川科学技术出版社,1988:284.
[2]危起伟,杨德国,柯福恩.长江鲟鱼类的保护对策[G]//黄真理.21世纪长江大型水利工程建设中的生态与环境保护—中国科协第十九次“青年科学家论坛”论文集,北京:中国环境科学出版社,1998:208-216.
[3]张四明,邓怀,危起伟,等.中华鲟天然群体蛋白质水平遗传多样性贫乏的初步证据[J].动物学研究,1999,20(2):93-98.
[4]刘鉴毅,危起伟,陈细华,等.葛洲坝下中华鲟繁殖生物学特性及其人工繁殖效果[J].应用生态学报,2007,18(6):1397-1402.
[5]危起伟,陈细化,杨德国,等.葛洲坝截流24年来中华鲟产卵群体结构的变化[J].中国水产科学,2005,12(4):452-457.
[6]陈松林.鱼类精子和胚胎冷冻保存理论与技术[M].北京:中国农业出版社,2007.
[7]苏天凤,艾红.鱼类精子活力及其超低温保存研究综述[J].上海水产大学学报,2004,13(4):343-347.
[8]于海涛,张秀梅,陈超.鱼类精液超低温冷冻保存的研究展望[J].海洋湖沼通报,2004(2):66-72.
[9]柳凌,危起伟,鲁大椿.中华鲟精子低温保存的相关因子[J].水产学报,1999,23(增刊):86-89.
[10]厉萍.中华鲟精子结构特征及其精液超低温冷冻保存技术研究[D].武汉:华中农业大学,2007.
[11]郭柏福,常剑波,肖慧,等.中华鲟初次全人工繁殖的特性研究[J].水生生物学报,2011,35(6):940-945.
[12]舒德斌,郭柏福.史氏鲟精子超低温冷冻保存研究[J].水产科学,2012,31(4):232-234.
[13]孙菊香,范丽君,贾林芝,等.伊红、PI染色法检测中华绒螯蟹活精子率[J].水产学报,2007,31(增刊1):69-73.
[14]柳凌,Otomar L,危起伟,等.计算机辅助对几种鲟鱼冻精激活液的比较[J].水产学报,2007,31(6):711-720.
[15]刘建康,何碧梧.中国淡水鱼类养殖学[M].3版.北京:科学出版社,1992:119-120.
[16]中国葛洲坝集团公司中华鲟研究所.中华鲟人工繁殖技术规程[M].北京:中国水利水电出版社,1998:13-18.
[17]陈松林,刘宪亭,鲁大椿,等.家鱼冷冻精液激活、授精方法的研究[J].水产学报,1992,16(4):337-345.
[18]严安生,宋贵文,闫拥军.鲤、团头鲂精子生理生态特性的研究Ⅲ.单糖和渗透压对精子活力的影响[J].淡水渔业,1995,25(2):3-6.
[19]陈松林,刘宪亭,鲁大椿,等.鲢、鲤、团头鲂和草鱼精液冷冻保存的研究[J].动物学报,1992,38(4):413-424.
[20]Dzuba B B,Kopeika F F,Cherepanov V V,et al.Sturgeon sperm quality after 6years of cryopreservation[J].J Appl Ichthyol,1999,15(4/5):312.
[21]Tsvetkova L I,Cosson J,Linhart O,et al.Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser baeri and A.ruthenus[J].J Appl Ichtyol,1996,12(2):107-112.
[22]Jhnichen H,Warnecke D,Trlsch E,et al.Motility and fertilizing capability of cryopreserved Acipenser ruthenus L.sperm[J].J Appl Ichthyol,1999,15(4/5):204-206.
[23]Kopeika E F,Williot P,Goncharov B F.Cryoconservation of Atlantic sturgeon Acipenser sturio L.,1758sperm:first results and associated problems[J].Bol Inst Esp Oceanogr,2000,16(1/4):167-173.
[24]刘鉴毅,甘芳,危起伟,等.几种不同浓度的离子及单糖对中华鲟精子活力的影响[J].水生生物学报,2007,31(6):849-854.
[25]柴毅,龚进玲,田梦,等.金属离子和葡萄糖对中华鲟子一代精子活力的影响[J].贵州农业科学,2013,41(12):134-137.
[26]郑跃平.中华鲟精子生理生态特性研究[D].武汉:华中农业大学,2007.
[27]甘芳,杜浩,危起伟,等.Na+、K+、Mg2+、Ca2+和葡萄糖溶液作为授精—激活介质对中华鲟精子受精率的影响[J].动物学杂志,2009,44(1):106-111.
[28]肖志忠,陈雄芳,丁福红,等.大黄鱼精液的高效超低温保存[J].海洋科学,2007,31(4):1-4.
[29]刘鹏,庄平,章龙珍,等.人工养殖西伯利亚鲟精子超低温冷冻保存研究[J].海洋渔业,2007,29(2):120-127.
[2]危起伟,杨德国,柯福恩.长江鲟鱼类的保护对策[G]//黄真理.21世纪长江大型水利工程建设中的生态与环境保护—中国科协第十九次“青年科学家论坛”论文集,北京:中国环境科学出版社,1998:208-216.
[3]张四明,邓怀,危起伟,等.中华鲟天然群体蛋白质水平遗传多样性贫乏的初步证据[J].动物学研究,1999,20(2):93-98.
[4]刘鉴毅,危起伟,陈细华,等.葛洲坝下中华鲟繁殖生物学特性及其人工繁殖效果[J].应用生态学报,2007,18(6):1397-1402.
[5]危起伟,陈细化,杨德国,等.葛洲坝截流24年来中华鲟产卵群体结构的变化[J].中国水产科学,2005,12(4):452-457.
[6]陈松林.鱼类精子和胚胎冷冻保存理论与技术[M].北京:中国农业出版社,2007.
[7]苏天凤,艾红.鱼类精子活力及其超低温保存研究综述[J].上海水产大学学报,2004,13(4):343-347.
[8]于海涛,张秀梅,陈超.鱼类精液超低温冷冻保存的研究展望[J].海洋湖沼通报,2004(2):66-72.
[9]柳凌,危起伟,鲁大椿.中华鲟精子低温保存的相关因子[J].水产学报,1999,23(增刊):86-89.
[10]厉萍.中华鲟精子结构特征及其精液超低温冷冻保存技术研究[D].武汉:华中农业大学,2007.
[11]郭柏福,常剑波,肖慧,等.中华鲟初次全人工繁殖的特性研究[J].水生生物学报,2011,35(6):940-945.
[12]舒德斌,郭柏福.史氏鲟精子超低温冷冻保存研究[J].水产科学,2012,31(4):232-234.
[13]孙菊香,范丽君,贾林芝,等.伊红、PI染色法检测中华绒螯蟹活精子率[J].水产学报,2007,31(增刊1):69-73.
[14]柳凌,Otomar L,危起伟,等.计算机辅助对几种鲟鱼冻精激活液的比较[J].水产学报,2007,31(6):711-720.
[15]刘建康,何碧梧.中国淡水鱼类养殖学[M].3版.北京:科学出版社,1992:119-120.
[16]中国葛洲坝集团公司中华鲟研究所.中华鲟人工繁殖技术规程[M].北京:中国水利水电出版社,1998:13-18.
[17]陈松林,刘宪亭,鲁大椿,等.家鱼冷冻精液激活、授精方法的研究[J].水产学报,1992,16(4):337-345.
[18]严安生,宋贵文,闫拥军.鲤、团头鲂精子生理生态特性的研究Ⅲ.单糖和渗透压对精子活力的影响[J].淡水渔业,1995,25(2):3-6.
[19]陈松林,刘宪亭,鲁大椿,等.鲢、鲤、团头鲂和草鱼精液冷冻保存的研究[J].动物学报,1992,38(4):413-424.
[20]Dzuba B B,Kopeika F F,Cherepanov V V,et al.Sturgeon sperm quality after 6years of cryopreservation[J].J Appl Ichthyol,1999,15(4/5):312.
[21]Tsvetkova L I,Cosson J,Linhart O,et al.Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser baeri and A.ruthenus[J].J Appl Ichtyol,1996,12(2):107-112.
[22]Jhnichen H,Warnecke D,Trlsch E,et al.Motility and fertilizing capability of cryopreserved Acipenser ruthenus L.sperm[J].J Appl Ichthyol,1999,15(4/5):204-206.
[23]Kopeika E F,Williot P,Goncharov B F.Cryoconservation of Atlantic sturgeon Acipenser sturio L.,1758sperm:first results and associated problems[J].Bol Inst Esp Oceanogr,2000,16(1/4):167-173.
[24]刘鉴毅,甘芳,危起伟,等.几种不同浓度的离子及单糖对中华鲟精子活力的影响[J].水生生物学报,2007,31(6):849-854.
[25]柴毅,龚进玲,田梦,等.金属离子和葡萄糖对中华鲟子一代精子活力的影响[J].贵州农业科学,2013,41(12):134-137.
[26]郑跃平.中华鲟精子生理生态特性研究[D].武汉:华中农业大学,2007.
[27]甘芳,杜浩,危起伟,等.Na+、K+、Mg2+、Ca2+和葡萄糖溶液作为授精—激活介质对中华鲟精子受精率的影响[J].动物学杂志,2009,44(1):106-111.
[28]肖志忠,陈雄芳,丁福红,等.大黄鱼精液的高效超低温保存[J].海洋科学,2007,31(4):1-4.
[29]刘鹏,庄平,章龙珍,等.人工养殖西伯利亚鲟精子超低温冷冻保存研究[J].海洋渔业,2007,29(2):120-127.