乌克兰鳞鲤精子超低温冷冻保存方法研究
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摘要
为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种"3步冷冻法"以及3种解冻温度(20、30、40℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12℃/min,解冻温度为30℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。
The motility was determined in both fresh and post-thawed sperm preserved at 4 ℃ in three extender solutions Hank′s, Cortland, and Freezefish at 3 dilution ratios(1∶1, 1∶3, 1∶5) and 3 cryoprotectants dimethyl sulphoxide DMSO, propylene glycol, glycerin at 10% volume fraction in Ukraine scale carp(Cyprinus carpio) in order to optimize the sperm cryopreservation, and the cryopreservation protocol for Ukrainian scale carp was subsequently tested with 3 "3-steps"cooling methods and 3 thawing temperature(20, 30 and 40 ℃) by using the screening cryoprotectant and dilution ratio. The optimal post-thawed motility(>68%) was observed in the sperm preserved at sperm to Hank′s ratio=1∶3 with 10% DMSO with mean 12 ℃/min cooling rate, and 30 ℃ water bath. The findings had significant advantages in germplasm protection, and provided reference for other fish sperm cryopreservation.
The motility was determined in both fresh and post-thawed sperm preserved at 4 ℃ in three extender solutions Hank′s, Cortland, and Freezefish at 3 dilution ratios(1∶1, 1∶3, 1∶5) and 3 cryoprotectants dimethyl sulphoxide DMSO, propylene glycol, glycerin at 10% volume fraction in Ukraine scale carp(Cyprinus carpio) in order to optimize the sperm cryopreservation, and the cryopreservation protocol for Ukrainian scale carp was subsequently tested with 3 "3-steps"cooling methods and 3 thawing temperature(20, 30 and 40 ℃) by using the screening cryoprotectant and dilution ratio. The optimal post-thawed motility(>68%) was observed in the sperm preserved at sperm to Hank′s ratio=1∶3 with 10% DMSO with mean 12 ℃/min cooling rate, and 30 ℃ water bath. The findings had significant advantages in germplasm protection, and provided reference for other fish sperm cryopreservation.
引文
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[28] 刘洪军,官曙光,刘清华,等.美洲黑石斑精子超低温保存技术[J].海洋科学,2013,37(1):76-80.
[29] 杨春玲,赵永贞,陈秀荔,等.南美白对虾精子超低温冷冻保存技术研究[J].2013,44(8):1382-1389.
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[31] 程顺,闫家强,竺俊全,等.大黄鱼(Pseudosciaena crocea)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.
[32] 赵维信,姜仁良,刘修英,等.几种鲤科鱼类精子和胚胎冷冻损伤的扫描电镜研究[J].淡水渔业,1992,22(5):3-5.
[33] 金春华,闫家强,竺俊全.黄姑鱼精子的超低温冻存及细胞结构损伤的检测[J].水产学报,2011,35(6):846-853.
[2] 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.
[3] Cabrita E,Sarasquete C,Martínez-Páramo S,et al.Cryopreservation of fish sperm:applications and perspectives[J].Journal of Applied Ichthyology,2010,26(5):623-635.
[4] Liu Y,Li X,Robinson N,et al.Sperm cryopreservation in marine mollusk:a review [J].Aquaculture International,2015,23(6):1505-1524.
[5] 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.
[6] 顾凤林,吴会民,张韦.乌克兰鳞鲤早繁试验[J].天津水产,2010(3):27-28.
[7] 赵立明,东世民,朱洪燕.乌克兰鳞鲤(俄罗斯鲤)健康养殖技术[J].齐鲁渔业,2007,24(6):29-30.
[8] 赵小光,滕淑芹,朱洪利.乌克兰鳞鲤池塘健康养殖实用技术[J].当代水产,2012(2):58-60.
[9] 缴建华.乌克兰鳞鲤无公害养殖技术(一)[J].科学养鱼,2012(9):17-18.
[10] 樊铮,土鹏,土震,等.陕西省关中沿黄海滩区乌克兰鳞鲤规模化人工繁殖的技术[J].渔业致富指南,2013(1):37-39.
[11] 舒德斌,郭柏福,万建义,等.匙吻鲟精子超低温冷冻保存实验[J].水产科学,2013,32(6):353-356.
[12] 王小刚,骆剑,尹绍武,等.点带石斑鱼精子超低温冷冻保存研究[J].海洋科学,2014,38(9):13-19.
[13] Suquet M,Dreanno C,Fauvel C,et al.Cryopreservation of sperm in marine fish [J].Aquaculture Research,2010,31(3):231-243.
[14] 史东杰,孙砚胜,孙向军,等.缺帘鱼精子超低温保存的初步研究[J].吉林农业大学学报,2009,31(4):467-471.
[15] 采克俊,曹访,叶金云.鱼类精子低温冷冻保存研究进展[J].湖州师范学院学报,2012,34(2):26-30.
[16] 廖馨,严维辉,唐建清,等.淡水鱼类精子的冷冻与保存[J].生物学通报,2006,41(8):16-17.
[17] 陈雄芳.大黄鱼精子和胚胎的超低温冷冻保存研究[D].青岛:中国海洋大学,2004.
[18] Ye T,Zhu J Q,Yang W X,et al.Sperm cryopreservation in Sparus macrocephalus and DNA damage detection with SCGE [J].Zoological Research,2009,30(2):151-157.
[19] 史应学,程顺,竺俊全,等.中国花鲈精子的超低温冷冻保存及酶活性检测[J].水生生物学报,2015,39(6):1241-1247.
[20] 魏平,竺俊全,闫家强,等.真鲷精子的超低温冻存及DNA损伤的检测[J].水生生物学报,2010,34(5):1049-1055.
[21] 汪小锋,樊延俊.鱼类精子冷冻保存的研究进展[J].海洋科学,2003,27(7):28-31.
[22] Magnotti C,Cerqueira V,Lee-Estevez M,et al.Cryopreservation and vitrification of fish semen:a review with special emphasis on marine species [J].Reviews in Aquaculture,2016,10(1):1-11.
[23] Viveiros A T,Godinho H P.Sperm quality and cryopreservation of Brazilian freshwater fish species:a review[J].Fish Physiol Biochem,2009,35(1):137-150.
[24] Huang C J,Dong Q X,Walter R B,et al.Initial studies on sperm cryopreservation of live-bearing fish,the green swordtail Xiphophorus helleri[J].Theriogenology,2004,62(1/2):179-194.
[25] 徐革锋,韩英,白淑艳,等.鲑鳟鱼类精子超低温保存研究进展[J].水产学杂志,2013,26(5):57-60,64.
[26] 郑乃中,张博.斑马鱼精子冻存与复苏实验方法与流程[J].遗传,2012,34(9):1211-1216.
[27] 李喜龙,季维智.动物种质细胞的超低温冷冻保存[J].动物学研究,2000,21(5):407-411.
[28] 刘洪军,官曙光,刘清华,等.美洲黑石斑精子超低温保存技术[J].海洋科学,2013,37(1):76-80.
[29] 杨春玲,赵永贞,陈秀荔,等.南美白对虾精子超低温冷冻保存技术研究[J].2013,44(8):1382-1389.
[30] 林丹军,尤永隆.大黄鱼精子生理特性及其冷冻保存[J].热带海洋学报,2002,21(4):69-75.
[31] 程顺,闫家强,竺俊全,等.大黄鱼(Pseudosciaena crocea)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.
[32] 赵维信,姜仁良,刘修英,等.几种鲤科鱼类精子和胚胎冷冻损伤的扫描电镜研究[J].淡水渔业,1992,22(5):3-5.
[33] 金春华,闫家强,竺俊全.黄姑鱼精子的超低温冻存及细胞结构损伤的检测[J].水产学报,2011,35(6):846-853.