太平洋牡蛎(Crassostrea gigas)精液的超低温保存研究

CRYOPRESERVATION FOR SPERMATOZOA OF CRASSOSTREA GIGAS

筛选了六种抗冻保护剂(GLY[甘油],DMSO[二甲基亚砜],PG[丙二醇],EG[乙二醇],DMA[二甲基乙酰胺],MeOH[甲醇])及其五种不同浓度(6%,8%,10%,12%,14%,V/V)、三种稀释液(HBSS溶液,人工海水[ASW],过滤海水[FSW])、四种稀释比例(1︰1,1︰2,1︰4,1︰6)、三种添加剂(蔗糖,葡萄糖,海藻糖)、三个分步降温程序(程序A,程序B,程序C)对太平洋牡蛎(Crassostrea gigas)精液冷冻保存的影响,成功建立了太平洋牡蛎精液超低温保存方法:以10%DMSO为抗冻保护剂,HBSS溶液为稀释液,1:4的稀释比例,添加海藻糖,采用分布降温法冷冻保存太平洋牡蛎的精液,37°C水浴解冻后精液运动率达到71.27%±3.24%,显著高于其它实验组(P<0.05),受精率和孵化率分别达到95.04%±1.99%、93.33%±1.33%,与鲜精(88.89%±15.16%,94.90%±0.95%)、程序冷冻前精液(95.96%±2.15%,92.67%±14.83%)差异不显著(P>0.05),证明该太平洋牡蛎精液超低温保存方法可以应用于生产实践和科学研究中。

We selected five cryoprotectants GLY(glycerin), DMSO(dimethyl sulfoxide), PG(propylene glycol), EG(ethylene glycol), DMA(dimethyl sulfoxide), MeOH(methanol) at five concentrations(6%, 8%, 10%, 12%, and 14%, V/V) in three types of diluents: Ca2+ free Hank's, artificial seawater(ASW), filtering seawater at four dilution ratios(1︰1, 1︰2, 1︰4, 1︰6), three additives(sucrose, glucose, and trehalose), in three step-wise cooling schemes, to find the influence on the cryopreservation of Crassostrea gigas sperm. Results show that the best cryopreservation protocol for C. gigas sperm is: 10% DMSO as antifreeze protective agent with Ca2+ free Hank's; dilution ratio at 1︰4; trehalose addition at 0.45 mol/L; and cooling scheme of cooling from 0°C to –60°C at 15°C /min, holding for 2 min, further cooling to –150°C at –20°C /min, waiting for 5 min. After 37°C water bath thawed, motility of the sperm reached 71.27%±3.24%, which is significantly higher than other combination groups(P<0.05). Fertilization rate and hatching rate after thawing were 95.04%±1.99% and 93.33%±1.99%, respectively, as better as those of fresh sperm(88.89%±15.16%) and those before cooling program(95.96%±2.15%), in no significant difference(P>0.05). Therefore, the C. gigas sperm cryopreservation protocol can be applied in the aquaculture practice.