海藻糖对施氏鲟精子冷冻保存效果的影响及其冷冻损伤机理的初步探究  

Effect of trehalose on sperm cryopreservation of Acipenser schrenckii and preliminary study on the mechanism of its cryoinjury

作　　者：陈张帆[2,3] 程鹏 胡谋[4] 王洁 张婷婷 黄红涛[1] 杜合军 陈松林 CHEN Zhangfan;CHENG Peng;HU Mou;WANG Jie;ZHANG Tingting;HUANG Hongtao;DU Hejun;CHEN Songlin(Hubei Key Laboratory of Three Gorges Proyject for Comseration of Fishes,Chinese Sturgeon Research Inuitute,China Three Gurges Corporation,Yichang Hubei 443100,China;Iaborawory Jor Maine Science and Food Production Press,Laoshan Latboratory,Yllosr Sea Fisheries Research Institude,Chinese Academy of Fishery Sciences(CAFS),Qingedo Shandong 266071,China;Shandong Key Laboralory of Marine Fishenies Biotechnolgy and Genetic Breeding,Qingedoo Shandong 266071,China;Hangzhou iandaohu Xunlong Scitech Co.,Ltd.,Hangzhou 311701,China;College of Fisheries and Life Science,Shanghai Oean Unirersity,Shanghai 201306,China)

机构地区：[1]三峡工程鱼类资源保护湖北省重点实验室,中国长江三峡集团有限公司中华鲟研究所,湖北宜昌443100 [2]中国水产科学研究院黄海水产研究所,崂山实验室,海洋渔业科学与食物产物过程功能实验室,山东青岛266071 [3]山东省海洋渔业生物技术与遗传育种重点实验室,山东青岛266071 [4]杭州千岛湖鲟龙科技股份有限公司,杭州311701 [5]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋渔业》2023年第1期1-13,共13页Marine Fisheries

基　　金：三峡工程鱼类资源保护湖北省重点实验室主任基金开放课题(2020048-ZHX);山东省重点研发计划(2021LZGC028);中央级公益性科研院所基本科研业务费(No.2020TD20);泰山学者攀登计划。

摘　　要：为了解抗冻剂对施氏鲟(Acipenser schrenckii)精子冷冻保存效果的影响及其冷冻损伤机理,比较了添加不同浓度海藻糖和蔗糖作为冷冻保护剂的精子稀释液处理后施氏鲟精子冷冻复苏的活力、快速运动时间和寿命。结果表明,添加60 mmol·L^(-1)海藻糖1.0 mmol·L^(-1)氯化钾的稀释液处理后,精子冻后快速运动时间和寿命与鲜精相比无显著差异(P>0.05);且活力较其他处理组有所提高,达到(26.67±3.32)%,但仍显著低于鲜精(P<0.05)。利用透射电镜和扫描电镜对施氏鲟精子超低温冷冻保存前后的超微结构进行观察,并对其能量代谢酶和抗氧化酶活进行测定和比较,结果表明,低温冻存造成施氏鲟精子的膜系统和细胞器(主要为线粒体和轴丝)损伤;能量代谢酶[总ATP酶、肌酸激酶(CK)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)]和抗氧化酶[过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)]活性均显著下降(P<0.05),而抗氧化酶谷胱甘肽还原酶(GR)活性显著上升(P<0.05),表明冷冻精子的生理功能损伤、内环境稳定性下降,能量供给系统被破坏。未添加抗冻剂组精子的总ATP酶、CK、CAT、SOD及GR酶活性相比添加抗冻剂组有显著差异,说明含有海藻糖的抗冻剂可对冷冻精子起到一定的保护作用。研究结果可为后期进一步优化施氏鲟抗冻剂的配方提供理论基础与研究思路。Cryopreservation is of importance for the genetic improvement of resources,conservation of species diversity,and breeding of improved varieties in aquaculture industry.The techniques of sperm cryopreservation have been established and optimized in numerous kinds of sturgeons;however,the motility,fast movement,and durations of frozen-thawed semen were significantly lower than those of fresh semen.In this study,we added various concentrations of trehalose or sucrose as cryoprotectants in the extender for sperm cryopreservation of Acipenser schrenckii and evaluated the motility,fast movement,and duration of the treated groups and the control group(fresh sperm).The extender diluted with 10% MeOH was mixed with sperm at a ratio of 1∶2 for sperm cryopreservation.The diluted semen was dispensed into 2 mL cryovials,placed into cloth bags,suspended 15 cm above nitrogen vapor for 10 min,then 5 cm above nitrogen vapor for 5 min,and finally stored in liquid nitrogen.After thawing at 37℃,the motility,fast movement and durations of frozen-thawed semen was observed and recorded under the microscope,compared with fresh semen.Results showed that ST5 solution(1 mM NaCl,20 mM Tris,60 mM trehalose,pH 8.0)worked most effectively during sperm cryopreservation of A.schrenckii.The motility of frozen-thawed spermatozoa was(26.67±3.32)%,significantly lower than that of fresh spermatozoa.The fast movement of fresh spermatozoa could last(93.17±4.34)s,while frozen-thawed spermatozoa could last(75.63±6.05)s.The duration of fresh spermatozoa[(246.61±2.29)s]had no significant difference with frozen-thawed spermatozoa[(239.35±1.49)s].To explore the mechanisms of cryoinjury in A.schrenckii semen,we fixed the fresh and frozen-thawed spermatozoa with 2.5%glutaraldehyde and observed their ultrastructure by using transmission electron microscope(TEM)and scanning electron microscope(SEM).Meanwhile,enzyme activities were compared between fresh and frozen-thawed sperm.Results showed that mature spermatozoa were comprised of acrosome,head,midpie

关 键 词：施氏鲟 精子 超低温冷冻 海藻糖 冷冻损伤 

分 类 号：S961[农业科学—水产养殖]