中国明对虾精子超低温冷冻保存方法的建立及超微结构损伤研究

doi:10.16431/j.cnki.1671-7236.2025.08.023

摘要/Abstract

摘要： 【目的】本研究旨在建立中国明对虾雄虾精子超低温冷冻保存方法，同时摸清超低温冷冻对中国明对虾精子超微结构的损伤。【方法】采用曙红B染色法检测超低温冷冻保存后精子存活率，通过研究9种稀释液(灭菌天然海水、人工海水、去钙人工海水、去镁人工海水、去钾人工海水、5%氯化钠、生理盐水、D-Hanks、Hanks)、3种不同浓度渗透性冷冻保护剂(二甲基亚砜、甘油和丙三醇)、5种非渗透性抗冻保护剂(海藻糖、葡萄糖、麦芽糖、维生素C、牛血清蛋白)、4种降温程序和4种复苏温度(25、30、35和40 ℃)对中国明对虾精子超低温冷冻保存的效果筛选最适保存方法，同时，应用透视电镜观察超低温冷冻后精子的超微结构。【结果】 ①灭菌天然海水、人工海水组冻精复苏后精子存活率显著高于其余各组(P＜0.05)。②采用甘油作为渗透性保护剂的试验组冻精存活率显著优于其他试验组(P＜0.05)，其中15%甘油组冻精存活率最高。③添加海藻糖、维生素C非渗透性保护剂的试验组冻精复苏后精子存活率显著高于其余各组(P＜0.05)。④采用慢速降温的P-1与P-2程序的试验组冻精复苏后精子存活率显著高于其余各组(P＜0.05)，其中在0 ℃至-20 ℃慢速降温、-20 ℃至-80 ℃区间中速降温、-80 ℃至-180 ℃区间快速降温的P-1试验组冻精存活率最高。⑤35 ℃水浴解冻组冻精复苏后精子存活率显著高于其余各组(P＜0.05)。⑥采用最优方法制备的冻精复苏后正常精子占79%，结构异常的精子主要表现为棘突脱落、膜间腔增大、细胞膜肿胀、顶体脱落、细胞核空泡化等。【结论】以灭菌天然海水作为稀释液、15%甘油为保护液、添加0.25 mol/L海藻糖，采用程序降温仪三步法慢速降温，冻精以35 ℃水浴解冻是最优的中国明对虾超低温冷冻保存方法，可以有效降低中国明对虾精子在超低温冷冻保存中的结构损伤。

关键词: 中国明对虾; 精子; 超低温冷冻保存; 超微结构

Abstract: 【Objective】 This study aimed to establish cryopreserving method of Fenneropenaeus chinensis and investigate the damage of sperm ultrastructure after cryopreservation. 【Method】 The sperm survival rate was detected by using eosin B staining.The suitable way was selected by comparing the effect of nine diluents (sterilized natural seawater,artificial seawater,decalcified artificial seawater,magnesium removal artificial seawater,de-potassic artificial seawater,5% sodium chloride,normal saline,D-Hanks and Hank’s balanced salt solution),three permeable cryoprotectants (DMSO,glycerol and propylene glycol),five non-permeable cryoprotectants(trehalose,glucose,maltose,vitamin C and bovine serum protein),four cooling procedures and four different resuscitation temperature (25,30,35 and 40 ℃),and the sperm ultrastructure was observed by transmission electron microscopy. 【Result】 ①The sperm survival rate after thawing of the sterilized natural seawater and artificial seawater groups were significantly higher than that of the other groups (P＜0.05).②The sperm survival rate of the experimental groups using glycerol as the permeable cryoprotectant were significantly higher than that of the other experimental groups (P＜0.05),with the best concentration being 15%.③The sperm survival rate after thawing of the experimental groups with the addition of non-permeable cryoprotectants such as trehalose and vitamin C were significantly higher than that of the other groups (P＜0.05).④The sperm survival rate after thawing of the P-1 and P-2 experimental groups using slow cooling were significantly higher than that of the other groups (P＜0.05),among which the P-1 experimental group with slow cooling from 0 ℃ to -20 ℃,medium-speed cooling from -20 ℃ to -80 ℃,and rapid cooling from -80 ℃ to -180 ℃ had the highest sperm survival rate.⑤The sperm survival rate after thawing of the 35 ℃ water bath thawing group was significantly higher than that of the other groups (P＜0.05).⑥The normal rates of the cryopreservated sperms were 79%,structural damage of injured sperm was mainly manifested as spike lost,inter membrane space augmented,membrane swelled,acrosomal cap lost,vacuolization of the cell nucleus observed. 【Conclusion】 The optimal ultra-low temperature cryopreservation method for Fenneropenaeus chinensis sperm involves using sterilized natural seawater as the diluent,15% glycerol as the cryoprotectant solution,supplemented with 0.25 mol/L trehalose.By employing a programmable cooling device with a three-step slow cooling protocol and thawing the frozen sperm in a 35 ℃ water bath,this approach effectively minimizes structural damage to Fenneropenaeus chinensis sperm during ultra-low temperature cryopreservation.

Key words: Fenneropenaeus chinensis; sperm; cryopreservation; ultrastructure

中图分类号:

S961

刘莹, 于超勇, 刘朋, 高蓓, 李泽邦, 宋爱环, 邹琰. 中国明对虾精子超低温冷冻保存方法的建立及超微结构损伤研究[J]. 中国畜牧兽医, 2025, 52(8): 3744-3752.

LIU Ying, YU Chaoyong, LIU Peng, GAO Bei, LI Zebang, SONG Aihuan, ZOU Yan. Establishment of Cryopreservation Methods for Fenneropenaeus chinensis Sperm and Investigation of Ultrastructural Damage[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3744-3752.

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