云纹石斑鱼精子冷冻保存

Sperm cryopreservation of kelp grouper Epinephelus moara

中文关键词: 云纹石斑鱼  精子  冷冻保存  精子稀释液

英文关键词: Epinephelus moara  sperm  cryopreservation  diluents

中文摘要:

      以云纹石斑鱼精液为实验材料，对精子稀释液、抗冻剂种类和适宜浓度、冷冻保存液进行了筛选。结果表明，利用9 g/L NaCl、10 g/L KHCO₃和10%小牛血清配制而成的稀释液EM1-2适宜于云纹石斑鱼精子冷冻保存，以2 ml冷冻管为精子容器，在60 L液氮生物保存罐中冷冻保存精子，冷冻解冻精子活力可达56.67±5.77%，要优于TS-2、ES1-3和其它EM系列稀释液冷冻保存精子活力。利用EM1-2为基础液对抗冻保护剂进行筛选，结果显示，10%-20%的DMSO和PG冷冻保存后精子活力无显著差异（P＞0.05），其中15%的DMSO和10% PG冷冻保存精子效果最优，解冻后精子活力分别可达54.52±7.81%和57.24±3.69%。利用冷冻保存1年的精液与云纹石斑鱼卵进行受精，受精率和孵化率均达到80%以上，与新鲜精子无显著性差异(P＞0.05)。本研究表明利用EM1-2配制15%的DMSO或10%的PG可用于冷冻保存云纹石斑鱼精液。在此基础上建立了精子冷冻库，保存精子130 ml，为人工繁育和杂交育种提供了丰富的精子源。

英文摘要:

      To study the cryopreservation of Epinephelus moara sperm , mature E. moara semen was used as experimental material, suitable concentration of various semen diluents, cryoprotectant and cryopreservation solution were screened. We used the 60L liquid nitrogen jar and 2ml freezing tubes to preserve sperms by “three-step cooling procedure”. The result shows that, EM1-2 semen diluent prepared with 9g/L NaCl, 10g/L KHCO3 and 10% FBS(fetal bovine serum) was better than TS-2, ES1-3 and other EM semen diluent, which preserved 56.67±5.77% sperm motility after unfrozen. Using EM1-2 as the liquid foundation to prepare cryoprotectant, no significant difference was found in sperm motility by 10%-20% DMSO and PG cryopreservation(P>0.05). The best of all is 15% DMSO and 10% PG, which could preserve 54.52±7.81% and 57.24±3.69% sperm motility after unfrozen respectively. Using 1-year cryopreserved semen to fertilize E. moara eggs, the rate of fertilization and hatchability reached 80% or above, without significant difference with fresh sperms(P>0.05). Our study showed that using EM1-2 as liquid foundation to prepare 15% DMSO or 10% PG could cryopreserve E. moara semen. We established a frozen sperm library based on this study, and it will provide a basis for the artificial breeding and cross breeding