Abstract：To establish cryopreservation method of sperm for Taiwan loach Paramisgurnus dabryanus ssp. taiwan, effects of four extenders (HBSS, D-15, D-17, and Ringer), five cryoprotectants [methanol (MeOH), ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF)] at four volume concentrations (2.5%, 5.0%, 7.5%, and 10.0%), different equilibrium time (0, 10, 20, 30, 40 and 50 min), fumigation height and time (5, 7, 9, 11 cm and 5, 10, 15 min), and thawing temperature (17, 27 and 37 ℃) and time (5, 10 and 15 s) on the sperm viability of Taiwan loach was observed by microscopy and the plasma membrane integrity was measured by flow cytometry (FCM). The results revealed that the sperm with HBSS had the maximal motility (45.28±5.75)%, and the sperm with 5% methanol had the maximal motility of (81.70±2.35)% and the plasma membrane integrity of (51.25±5.03)%, with the best equilibrium time of 30 min, and the best fumigation height of 9 cm for 10 min. The better thawing effect was observed by thawing in 37 ℃ water bath for 10 s, with the fertilization rate of (68.94±6.22)% at artificial insemination with thawed Taiwan loach sperm. In summary, the fresh sperm of Taiwan loach was diluted with HBSS and mixed with MeOH to a final concentration of 5% at 4 ℃ for 30 min, then put it into straws, fumigated at 9 cm above the liquid nitrogen surface for 10 min, and then was stored in liquid nitrogen. When thawing, good results were observed when the fresh sperm of Taiwan loach was thawed in a 37 ℃ water bath for 10 s. The findings provided data foundation with artificial breeding and germplasm resource protection of Taiwan loach.
[1] 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. [2] 周洲,李世凯,赵飞,等.不同抗冻剂对西伯利亚鲟精子冷冻保存的影响[J].西南农业学报,2021,34(6):1347-1350. [3] 刘浩域,郭建华,罗兰,等.虾青素和平衡时间对猪精子冷冻保存效果的影响[J].中国畜牧杂志,2022,58(8):243-247. [4] LIU M J, YUAN S, ZHAO Z M, et al. Non-programmable cryopreservation of sperm from industrial-farmed Murray cod (Maccullochellapeelii)[J]. Aquaculture,2021,541:736811. [5] ANJOS C, SANTOS A L, DUARTE D, et al. Effect of trehalose and sucrose in post-thaw quality of Crassostrea angulata sperm[J]. Frontiers in Physiology,2021,12:749735. [6] 李景春,柯文杰,杨虹,等.超低温冷冻技术应用于鱼精子保存的研究进展[J].江苏农业科学,2019,47(18):66-69. [7] 周鑫,夏欣一,曹奇,等.SYBR-14/PI双染法流式细胞术检测精子质膜完整性的研究[J].中华男科学杂志,2010,16(7):589-593. [8] 华咏,钱宪明,陈炳官,等.流式细胞术在精子功能检测中的应用[J].中华男科学杂志,2006,12(7):608-611. [9] 胡润环,张圣朝,靳木子,等.使用荧光染色和流式细胞术检测山羊精子[J].畜牧与兽医,2012,44(8):4-7. [10] 任康,李俊杰,王亮,等.荧光探针JC-1与PI联合染色法在猪精子线粒体膜电位检测中的应用[J].中国畜牧杂志,2010,46(9):18-22. [11] WATTS J. Measurement of membrane integrity in canine spermatozoa using a fluorescent computer-assisted spermatozoal quantification method after SYBR-14/PI staining compared with manual counting after CFDA/PI staining[J]. Reproduction in Domestic Animals,2021,56(6):915-927. [12] 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):69-73. [13] 杜俊鹏,王昭萍,崔玉婷,等.PI/Rh123双染和流式细胞术作用下不同盐度对葡萄牙牡蛎精子质量及受精的影响[J].水产学报,2018,42(11):1737-1746. [14] BAI C L, WANG X H, LU G, et al. Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining[J]. Cryobiology,2013,67(2):117-123. [15] 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. [16] 郝忱.两种淡水经济鱼类精子超低温冷冻保存技术的研究[D].南京:南京农业大学,2009. [17] 陈健,杨壮志,李良玉,等.鱼类精液保存技术研究进展与前景展望[J].渔业致富指南,2018(8):22-24. [18] 程顺,迟美丽,顾志敏,等.河川沙塘鳢精子质量的影响因素及其精子超低温冷冻[J].中国畜牧杂志,2018,54(6):69-74. [19] 王鑫伟,史应学,魏平,等.乙二醇降低超低温冷冻黄姑鱼精子的DNA损伤[J].动物学杂志,2016,51(2):261-267. [20] 程顺,顾志敏,刘士力,等.翘嘴鲌精子生理特性及超低温冷冻的研究[J].江苏农业科学,2019,47(7):175-179. [21] 丁淑燕,李跃华,黄亚红,等.刀鲚精子超低温冷冻保存技术的研究[J].水产养殖,2015,36(1):32-34. [22] 张崇英,陈脊宇,周亚,等.胭脂鱼(Myxocyprinus asiaficus)精液超低温冷冻保存及酶活性测定[J].基因组学与应用生物学,2020,39(4):1556-1564. [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] 王明华,陈友明,丁淑燕,等.美洲鲥鱼精子超低温冷冻保存技术初探[J].江苏农业科学,2015,43(7):250-251. [25] 乔瑞峰,陆专灵,高爽爽,等.大鳞副泥鳅精液超低温冷冻保存方法的优化[J].广东农业科学,2020,47(7):142-147. [26] 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. [27] PAN J L, DING S Y, GE J C, et al. Development of cryopreservation for maintaining yellow catfish Pelteobagrus fulvidraco sperm[J]. Aquaculture,2008,279(1/2/3/4):173-176. [28] 夏良萍,陈梦婷,陈悦萍,等.黄颡鱼精子超低温冷冻保存技术[J].江苏农业科学,2013,41(10):196-198. [29] BOKOR Z, MÜLLER T, BERCSÉNYI M, et al. Cryopreservation of sperm of two European percid species,the pikeperch (Sander lucioperca) and the Volga pikeperch (S.volgensis)[J]. Acta Biologica Hungarica,2007,58(2):199-207. [30] DZIEWULSKA K, RZEMIENIECKI A, CZERNIAWSKI R, et al. Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets[J]. Theriogenology,2011,76(2):300-311. [31] 刘光霞,吴兴兵,何勇凤,等.圆口铜鱼精子超低温冷冻保存[J].中国水产科学,2020,27(1):44-52. [32] 贾濮元,郭华阳,朱克诚,等.黄鳍棘鲷精子冷冻保存方法探究[J].南方水产科学,2021,17(6):58-65. [33] 邢露梅,肖伟,李兰兰,等.兰州鲇精液超低温冷冻保存技术研究及细胞损伤检测[J].水生生物学报,2021,45(3):547-556. [34] 王鑫伟,史应学,竺俊全,等.光唇鱼精子的超低温冷冻保存及酶活性检测[J].农业生物技术学报,2017,25(4):639-649. [35] LANES C F C, OKAMOTO M, CAVALCANTI P V, et al. Cryopreservation of Brazilian flounder (Paralichthys orbignyanus) sperm[J]. Aquaculture,2008,275(1/2/3/4):361-365. [36] KIM S C, HOSSEN S, KHO K H. Cryopreservation of sperm from farmed Pacific abalone, Haliotis discus Hannai[J]. Cryobiology,2020,94:49-56. [37] SMITH J F, ADAMS S L, GALE S L, et al. Cryopreservation of GreenshellTM mussel (Perna canaliculus) sperm. Ⅰ.Establishment of freezing protocol[J]. Aquaculture,2012,334/335/336/337:199-204. [38] PEREIRA V A, DE ALENCAR D B, DE ARAÚJO I W F, et al. Supplementation of cryodiluent media with seaweed or Nile tilapia skin sulfated polysaccharides for freezing of Colossoma macropomum (Characiformes:Serrasalmidae) semen[J]. Aquaculture,2020,528:735553. [39] 章龙珍,闫文罡,庄平,等.褐牙鲆精子生理特性及超低温冷冻保存[J].上海海洋大学学报,2009,18(1):21-27. [40] 闫文罡,章龙珍,庄平,等.日本黄姑鱼精子生理特性及超低温冷冻保存研究[J].海洋渔业,2008,30(2):145-151. [41] 柳凌,危起伟,鲁大椿,等.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. [42] 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.
2024, Vol. 43 
