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

Abstract

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

CLC Number:

S961

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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Establishment of Cryopreservation Methods for Fenneropenaeus chinensis Sperm and Investigation of Ultrastructural Damage

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