海湾扇贝精液超低温冷冻保存技术的初步研究
|
摘要
分别采用自然排放和解剖的方式获取海湾扇贝(Argopecten irradians irradians)精液,采用传统的分步降温法,从抗冻保护剂、精液稀释比例、降温程序、解冻温度和精子质量评价等方面探究海湾扇贝精液超低温冷冻保存技术。研究发现,海湾扇贝精子超低温冷冻保存效果最好的方案为:以解剖的方式获取海湾扇贝精液,用浓度20%的二甲基亚砜作为抗冻保护剂,以体积比2∶1与精液混合,控制总体积0.5~1 mL;体系经4℃平衡5 min后,于液氮面以上10cm稳定5min,投入液氮保存;解冻时,体系于30℃水浴中摇动融化。此方案下,精子解冻后活力最高,晃动和游动精子比例达到86.0%。将解冻后的海湾扇贝精子与虾夷扇贝(Patinopecten yessoensis)卵子人工授精,发现虾夷扇贝卵子可以受精并孵化,受精率为21.7%,这证实了此冻存方案的可行性。本研究完善了扇贝精液超低温保存的方法和流程,对于海湾扇贝种质资源保护、跨季杂交育种等研究有参考价值。
Sperm of Argopecten irradians irradians were collected by natural spawning and stripping,respectively,to develop a suitable method for the sperm cryopreservation of this species.Cryoprotectant,sperm dilution ratio,freezing steps,thawing temperature and quality assessment were explored with the traditional method of freezing by steps.Sperm were better when collected by stripping.The best cryoprotectant was DMSO at a concentration of 20%and in two volumes of sperm.To guarantee the freezing and thawing speed,the whole system should be kept between 0.5 mL and 1.0 mL.After being stabilized at 4℃for 5 min,the system was maintained at 10 cm above liquid nitrogen surface for 5 min,and then immersed in liquid nitrogen.This scheme was the best for the sperm cryopreservation of A.i.irradians and sperm motility reached 86.0% with being thawed at 30℃.Fresh eggs of Patinopecten yessoensis were fertilized by cryopreserved sperm of A.i.irradians to assess the freezing effect.The fertilization rate turned out to be21.7%,showing the feasibility of implementation.This study developed an efficient method of sperm cryopreservation of A.i.irradians,and could be used for germplasm preservation and trans-seasonal cross-breeding.
Sperm of Argopecten irradians irradians were collected by natural spawning and stripping,respectively,to develop a suitable method for the sperm cryopreservation of this species.Cryoprotectant,sperm dilution ratio,freezing steps,thawing temperature and quality assessment were explored with the traditional method of freezing by steps.Sperm were better when collected by stripping.The best cryoprotectant was DMSO at a concentration of 20%and in two volumes of sperm.To guarantee the freezing and thawing speed,the whole system should be kept between 0.5 mL and 1.0 mL.After being stabilized at 4℃for 5 min,the system was maintained at 10 cm above liquid nitrogen surface for 5 min,and then immersed in liquid nitrogen.This scheme was the best for the sperm cryopreservation of A.i.irradians and sperm motility reached 86.0% with being thawed at 30℃.Fresh eggs of Patinopecten yessoensis were fertilized by cryopreserved sperm of A.i.irradians to assess the freezing effect.The fertilization rate turned out to be21.7%,showing the feasibility of implementation.This study developed an efficient method of sperm cryopreservation of A.i.irradians,and could be used for germplasm preservation and trans-seasonal cross-breeding.
引文
[1]于千钧.中国海湾扇贝产业结构、特征和绩效研究[D].青岛:中国海洋大学,2014.Yu Q J.A Study on Structure,Characteristics and Performances of Bay Scallop Industry in China[D].Qingdao:Ocean University of China,2014.
[2]李成林,宋爱环,胡炜,等.山东省扇贝养殖产业现状分析与发展对策[J].海洋科学,2011,35(3):92-98.Li C L,Song A H,Hu W,et al.Status analyzing and developing counter-measure of cultured scallop industry in Shandong province[J].Marine Sciences,2011,35(3):92-98.
[3]马英杰.海湾扇贝种质退化及其对策探讨[J].海洋科学,1992(6):64-65.Ma Y J.Degeneration of genetic characterization of Argopecten irradians and solutions[J].Marine Sciences,1992(6):64-65.
[4]张国范,刘述锡,刘晓,等.海湾扇贝自交家系的建立和自交效应[J].中国水产科学,2003,10(6):441-445.Zhang G F,Liu S X,Liu X,et al.Self-fertilization family establishment and its depression in bay scallop Argopecten irradians[J].Journal of Fishery Sciences of China,2003,10(6):441-445.
[5]张守都.海湾扇贝的选择和杂交育种[D].青岛:中国科学院研究生院(海洋研究所),2013.Zhang S D.Selective and Cross Breeding of Bay Scallop,Argopecten irradians irradians(Lamarck)[D].Qingdao:University of Chinese Academy of Sciences(Institute of Oceanology),2013.
[6]毛俊霞.海湾扇贝(Argopecten irradians)♀×紫扇贝(Argopecten purpuratus)♂杂种优势遗传基础的研究[D].青岛:中国海洋大学,2015.Mao J X.Study on the Genetic Basis of Heterosis for the Hybrids of Argopecten irradians♀×Argopecten purpuratus♂[D].Qingdao:Ocean University of China,2015.
[7]孟庆磊.扇贝异源多倍体与四倍体培育技术研究[D].青岛:中国海洋大学,2009.Meng Q L.Research on Allopolyploid and Tetraploid Breeding of Scallop[D].Qingdao:Ocean University of China,2009.
[8]姜静.几种海水鱼类种质超低温冷冻保存研究[D].上海:上海海洋大学,2014.Jiang J.The Cryopreservation Research of Several fish Germplasm Resources[D].Shanghai:Shanghai Ocean University,2014.
[9]Liu Y B,Li X X,Robinson N,et al.Sperm cryopreservation in marine mollusk:a review[J].Aquaculture International,2015,23(6):1505-1524.
[10]田永胜,马允,解子牛,等.哲罗鲑精子冷冻保存[J].农业生物技术学报,2016,24(1):90-97.Tian Y S,Ma Y,Xie Z N,et al.Cryopreservation of Hucho taimen spermatozoa[J].Journal of Agricultural Biotechnology,2016,24(1):90-97.
[11]Liu Y B,Li X X,Xu T,et al.Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata[J].Aquaculture,2014,434:362-366.
[12]Adams S L,Smith J F,Roberts R D,et al.Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas(Thunberg)[J].Aquaculture Research,2008,39(13):1434-1442.
[13]Adams S L,Smith J F,Roberts R D,et al.Cryopreservation of sperm of the Pacific oyster(Crassostrea gigas):Development of a practical method for commercial spat production[J].Aquaculture,2004,242(1-4):271-282.
[14]Paniagua-Chavez C G,Tiersch T R.Laboratory studies of cryopreservation of sperm and trochophore larvae of the Eastern oyster[J].Cryobiology,2001,43:211-223.
[15]杨爱国,王清印,孔杰,等.扇贝精液超低温冷冻保存技术的研究[J].海洋与湖沼,1999,30(6):624-628.Yang A G,Wang Q Y,Kong J,et al.Studies on spermatozoa cryopreservation of scallops,Patinopecten yessonesis and Chlamys farreri[J].Oceanologia et Limnologia Sinica,1999,30(6):624-628.
[16]杨培民,杨爱国,刘志鸿,等.虾夷扇贝精子的冷冻保存及其杂交试验应用研究[J].上海水产大学学报,2007,16(4):351-356.Yang P M,Yang A G,Liu Z H,et al.Crypreservation of Patinopecten yessoensis sperm and its application to hybridization[J].Journal of Shanghai Fisheries University,2007,16(4):351-356.
[17]李纯,李军,薛钦昭.栉孔扇贝精子的超低温保存研究[J].海洋水产研究,2000,21(1):57-62.Li C,Li J,Xue Q Z.Cryopreservation of the spermatozoa of Chlamys(Azumapecten)farreri[J].Marine Fisheries Research,2000,21(1):57-62.
[18]Espinoza C,Valdivia M,DupréE.Morphological alterations in cryopreserved spermatozoa of scallop Argopecten purpuratus[J].Latin American Journal of Aquatic Research,2010,38(1):121-127.
[19]许观照,王磊光,于小洁,等.三种人类精液冷冻程序的比较性研究[J].生殖医学杂志,2005,14(6):365-366.Xu G Z,Wang L G,Yu X J,et al.Comparative research on three methods on cryopreservation of human sperm[J].Journal of Reproductive Medicine,2005,14(6):365-366.
[20]于非非,余祥勇,郑娇,等.栉江珧和近江牡蛎的精子超低温冷冻保存[J].农业生物技术学报,2016,24(2):305-312.Yu F F,Yu X Y,Zheng J,et al.Cryopreservation of spermatozoa in Pen Shell(Atrina pectinata)and Suminoe Oyster(Crassostrea rivularis)[J].Journal of Agricultural Biotechnology,2016,24(2):305-312.
[21]Vitiello V,Carlino P A,Del P F,et al.Effects of cooling and freezing on the motility of Ostrea edulis(L.1758)spermatozoa after thawing[J].Cryobiology,2011,63(2):118-124.
[22]Kawamoto T,Narita T,Isowa K,et al.Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster,Pinctada fucata martensii[J].Cryobiology,2007,54(1):19-26.
[23]王梅芳,余祥勇,刘永,等.马氏珠母贝精子的超低温保存[J].水产学报,2006,30(2):170-174.Wang M F,Yu X Y,Liu Y,et al.Study on cryopreservation of spermatozoa in Pinctada martensii[J].Journal of Fisheries of China,2006,30(2):170-174.
[24]Gwo J C,Chen C W,Cheng H Y.Semen cryopreservation of small abalone(Haliotis diversicolor supertexa)[J].Theriogenology,2002,58(8):1563-1578.
[25]Dong Q,Huang C,Eudeline B,et al.Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters,Crassostrea gigas[J].Theriogenology,2006,66(2):387-403.
[26]刘晓赫.氨海水和5-羟色胺对海湾扇贝(Argopecten irradians Lamarck)解剖卵的体外促熟研究[D].青岛:中国海洋大学,2012.Liu X H.Improving Maturation of Oocyte Stripped from Scallop Argopecten irradians Lamarck by Ammonia Seawater and Serotonin Creatinine Sulfate Complex(5-HT)[D].Qingdao:Ocean U-niversity of China,2012.
[27]陈春华,沙艳伟,李萍.稀少精子冷冻研究进展[J].中华男科学杂志,2013,19(8):753-757.Chen C H,Sha Y W,Li P.Current progress in cryopreservation of small numbers of sperm[J].National Journal of Andrology,2013,19(8):753-757.
[28]吴永根,郑九嘉,胡黎川,等.精子DNA冷冻损伤易感性与精子活动力相关性[J].生殖医学杂志,2016,25(12):1095-1099.Wu Y G,Zheng J J,Hu L C,et al.The association between susceptibility of sperm DNA damage to freezing and sperm motility[J].Journal of Reproductive Medicine,2016,25(12):1095-1099.
[29]黄晓荣,张涛,冯广朋,等.超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响[J].海洋渔业,2016,38(5):487-494.Huang X R,Zhang T,Feng G P,et al.Effects of cryopreservation on acrosin activity and DNA damage of Russian sturgeon(Acipenser gueldenstaedti)semen[J].Marine Fisheries,2016,38(5):487-494.
[30]韩龙江,刘清华,许飞,等.长牡蛎精子超低温冷冻后超微结构损伤研究[J].水生生物学报,2017,41(1):220-217.Han L J,Liu Q H,Xu F,et al.Study on ultrastructure observation of fresh and cryopreservated sperm in Crassostrea gigas[J].Acta Hydrobiologica Sinica,2017,41(1):220-217.
[31]丁兆坤,朱豪磊,杨春玲,等.不同稀释液对香港牡蛎精子冷冻的保存效果[J].水生态学杂志,2013,34(3):67-74.Ding Z K,Zhu H L,Yang C L,et al.Effects of different extenders on spermatozoa cryopreservation of Crassostrea hongkongensis[J].Journal of Hydroecology,2013,34(3):67-74.
[32]陈来钊,王子臣.温度对海湾扇贝与虾夷扇贝及其杂交受精、胚胎和早期幼体发育的影响[J].大连水产学院学报,1994,9(4):1-9.Chen L Z,Wang Z C.Effects of temperature on the fertilization and embryonic development of Argopecten irradians Lamarck,Patinopecten yessoensis Jay and their hybrids[J].Journal of Dalion Fisheries College,1994,9(4):1-9.
[2]李成林,宋爱环,胡炜,等.山东省扇贝养殖产业现状分析与发展对策[J].海洋科学,2011,35(3):92-98.Li C L,Song A H,Hu W,et al.Status analyzing and developing counter-measure of cultured scallop industry in Shandong province[J].Marine Sciences,2011,35(3):92-98.
[3]马英杰.海湾扇贝种质退化及其对策探讨[J].海洋科学,1992(6):64-65.Ma Y J.Degeneration of genetic characterization of Argopecten irradians and solutions[J].Marine Sciences,1992(6):64-65.
[4]张国范,刘述锡,刘晓,等.海湾扇贝自交家系的建立和自交效应[J].中国水产科学,2003,10(6):441-445.Zhang G F,Liu S X,Liu X,et al.Self-fertilization family establishment and its depression in bay scallop Argopecten irradians[J].Journal of Fishery Sciences of China,2003,10(6):441-445.
[5]张守都.海湾扇贝的选择和杂交育种[D].青岛:中国科学院研究生院(海洋研究所),2013.Zhang S D.Selective and Cross Breeding of Bay Scallop,Argopecten irradians irradians(Lamarck)[D].Qingdao:University of Chinese Academy of Sciences(Institute of Oceanology),2013.
[6]毛俊霞.海湾扇贝(Argopecten irradians)♀×紫扇贝(Argopecten purpuratus)♂杂种优势遗传基础的研究[D].青岛:中国海洋大学,2015.Mao J X.Study on the Genetic Basis of Heterosis for the Hybrids of Argopecten irradians♀×Argopecten purpuratus♂[D].Qingdao:Ocean University of China,2015.
[7]孟庆磊.扇贝异源多倍体与四倍体培育技术研究[D].青岛:中国海洋大学,2009.Meng Q L.Research on Allopolyploid and Tetraploid Breeding of Scallop[D].Qingdao:Ocean University of China,2009.
[8]姜静.几种海水鱼类种质超低温冷冻保存研究[D].上海:上海海洋大学,2014.Jiang J.The Cryopreservation Research of Several fish Germplasm Resources[D].Shanghai:Shanghai Ocean University,2014.
[9]Liu Y B,Li X X,Robinson N,et al.Sperm cryopreservation in marine mollusk:a review[J].Aquaculture International,2015,23(6):1505-1524.
[10]田永胜,马允,解子牛,等.哲罗鲑精子冷冻保存[J].农业生物技术学报,2016,24(1):90-97.Tian Y S,Ma Y,Xie Z N,et al.Cryopreservation of Hucho taimen spermatozoa[J].Journal of Agricultural Biotechnology,2016,24(1):90-97.
[11]Liu Y B,Li X X,Xu T,et al.Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata[J].Aquaculture,2014,434:362-366.
[12]Adams S L,Smith J F,Roberts R D,et al.Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas(Thunberg)[J].Aquaculture Research,2008,39(13):1434-1442.
[13]Adams S L,Smith J F,Roberts R D,et al.Cryopreservation of sperm of the Pacific oyster(Crassostrea gigas):Development of a practical method for commercial spat production[J].Aquaculture,2004,242(1-4):271-282.
[14]Paniagua-Chavez C G,Tiersch T R.Laboratory studies of cryopreservation of sperm and trochophore larvae of the Eastern oyster[J].Cryobiology,2001,43:211-223.
[15]杨爱国,王清印,孔杰,等.扇贝精液超低温冷冻保存技术的研究[J].海洋与湖沼,1999,30(6):624-628.Yang A G,Wang Q Y,Kong J,et al.Studies on spermatozoa cryopreservation of scallops,Patinopecten yessonesis and Chlamys farreri[J].Oceanologia et Limnologia Sinica,1999,30(6):624-628.
[16]杨培民,杨爱国,刘志鸿,等.虾夷扇贝精子的冷冻保存及其杂交试验应用研究[J].上海水产大学学报,2007,16(4):351-356.Yang P M,Yang A G,Liu Z H,et al.Crypreservation of Patinopecten yessoensis sperm and its application to hybridization[J].Journal of Shanghai Fisheries University,2007,16(4):351-356.
[17]李纯,李军,薛钦昭.栉孔扇贝精子的超低温保存研究[J].海洋水产研究,2000,21(1):57-62.Li C,Li J,Xue Q Z.Cryopreservation of the spermatozoa of Chlamys(Azumapecten)farreri[J].Marine Fisheries Research,2000,21(1):57-62.
[18]Espinoza C,Valdivia M,DupréE.Morphological alterations in cryopreserved spermatozoa of scallop Argopecten purpuratus[J].Latin American Journal of Aquatic Research,2010,38(1):121-127.
[19]许观照,王磊光,于小洁,等.三种人类精液冷冻程序的比较性研究[J].生殖医学杂志,2005,14(6):365-366.Xu G Z,Wang L G,Yu X J,et al.Comparative research on three methods on cryopreservation of human sperm[J].Journal of Reproductive Medicine,2005,14(6):365-366.
[20]于非非,余祥勇,郑娇,等.栉江珧和近江牡蛎的精子超低温冷冻保存[J].农业生物技术学报,2016,24(2):305-312.Yu F F,Yu X Y,Zheng J,et al.Cryopreservation of spermatozoa in Pen Shell(Atrina pectinata)and Suminoe Oyster(Crassostrea rivularis)[J].Journal of Agricultural Biotechnology,2016,24(2):305-312.
[21]Vitiello V,Carlino P A,Del P F,et al.Effects of cooling and freezing on the motility of Ostrea edulis(L.1758)spermatozoa after thawing[J].Cryobiology,2011,63(2):118-124.
[22]Kawamoto T,Narita T,Isowa K,et al.Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster,Pinctada fucata martensii[J].Cryobiology,2007,54(1):19-26.
[23]王梅芳,余祥勇,刘永,等.马氏珠母贝精子的超低温保存[J].水产学报,2006,30(2):170-174.Wang M F,Yu X Y,Liu Y,et al.Study on cryopreservation of spermatozoa in Pinctada martensii[J].Journal of Fisheries of China,2006,30(2):170-174.
[24]Gwo J C,Chen C W,Cheng H Y.Semen cryopreservation of small abalone(Haliotis diversicolor supertexa)[J].Theriogenology,2002,58(8):1563-1578.
[25]Dong Q,Huang C,Eudeline B,et al.Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters,Crassostrea gigas[J].Theriogenology,2006,66(2):387-403.
[26]刘晓赫.氨海水和5-羟色胺对海湾扇贝(Argopecten irradians Lamarck)解剖卵的体外促熟研究[D].青岛:中国海洋大学,2012.Liu X H.Improving Maturation of Oocyte Stripped from Scallop Argopecten irradians Lamarck by Ammonia Seawater and Serotonin Creatinine Sulfate Complex(5-HT)[D].Qingdao:Ocean U-niversity of China,2012.
[27]陈春华,沙艳伟,李萍.稀少精子冷冻研究进展[J].中华男科学杂志,2013,19(8):753-757.Chen C H,Sha Y W,Li P.Current progress in cryopreservation of small numbers of sperm[J].National Journal of Andrology,2013,19(8):753-757.
[28]吴永根,郑九嘉,胡黎川,等.精子DNA冷冻损伤易感性与精子活动力相关性[J].生殖医学杂志,2016,25(12):1095-1099.Wu Y G,Zheng J J,Hu L C,et al.The association between susceptibility of sperm DNA damage to freezing and sperm motility[J].Journal of Reproductive Medicine,2016,25(12):1095-1099.
[29]黄晓荣,张涛,冯广朋,等.超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响[J].海洋渔业,2016,38(5):487-494.Huang X R,Zhang T,Feng G P,et al.Effects of cryopreservation on acrosin activity and DNA damage of Russian sturgeon(Acipenser gueldenstaedti)semen[J].Marine Fisheries,2016,38(5):487-494.
[30]韩龙江,刘清华,许飞,等.长牡蛎精子超低温冷冻后超微结构损伤研究[J].水生生物学报,2017,41(1):220-217.Han L J,Liu Q H,Xu F,et al.Study on ultrastructure observation of fresh and cryopreservated sperm in Crassostrea gigas[J].Acta Hydrobiologica Sinica,2017,41(1):220-217.
[31]丁兆坤,朱豪磊,杨春玲,等.不同稀释液对香港牡蛎精子冷冻的保存效果[J].水生态学杂志,2013,34(3):67-74.Ding Z K,Zhu H L,Yang C L,et al.Effects of different extenders on spermatozoa cryopreservation of Crassostrea hongkongensis[J].Journal of Hydroecology,2013,34(3):67-74.
[32]陈来钊,王子臣.温度对海湾扇贝与虾夷扇贝及其杂交受精、胚胎和早期幼体发育的影响[J].大连水产学院学报,1994,9(4):1-9.Chen L Z,Wang Z C.Effects of temperature on the fertilization and embryonic development of Argopecten irradians Lamarck,Patinopecten yessoensis Jay and their hybrids[J].Journal of Dalion Fisheries College,1994,9(4):1-9.