中国水产科学2025，Vol.32Issue(10)：1393-1403,11.DOI:10.12264/JFSC2025-0160
基于非渗透性抗冻剂筛选的混合玻璃化液对斑石鲷胚胎冷冻保存的影响
Effects of screened non-permeating cryoprotectants-based mixed vitrification solution on cryopreservation of Oplegnathus punctatus embryos
摘要
Abstract
Oplegnathus punctatus,a highly valued marine fish species in China's aquaculture industry,faces major challenges in maintaining genetic diversity and sustainable propagation due to the lack of effective cryopreservation protocols.To address this,we developed an optimized embryo cryopreservation methodology integrating non-permeating cryoprotective agent(CPA)selection with developmental stage-specific vitrification strategies.Comparative evaluation of four non-permeating CPAs(sucrose,trehalose,glucose,and fructose;1 mol/L each)across three critical embryonic stages(somite,tail-bud,and heart-beating)identified sucrose and trehalose as superior,with sucrose demonstrating the highest biocompatibility.After 1 h exposure,sucrose-treated embryos achieved(89.20±3.10)％normal development rates,statistically equivalent to those of fresh control embryos[(87.50±2.90)％;P＞0.05].This finding is consistent with the established membrane-stabilizing properties of sucrose via hydrogen bonding and water replacement mechanisms.Based on these results,two proprietary CPA cocktails were formulated:PMG3S and PMG3T.Dose-response studies at 30％,35％,40％,45％,and 50％CPA concentrations revealed developmental stage-specific tolerance thresholds.Tail-bud stage embryos demonstrated exceptional resilience,maintaining normal development rates similar to those of controls at 35％CPA concentration(P＞0.05).This developmental window coincides with critical morphological transitions(e.g.,chorion hardening,yolk sac resorption,and myotome formation)that likely improve structural resistance to cryoinjury.Equilibration time optimization at 35％PMG3S concentration demonstrated a biphasic response:PMG3S achieved(87.07±5.37)％viability after 35 min exposure,which was statistically comparable with that of untreated controls[(93.52±3.89)％;P＞0.05].However,prolonged exposure beyond 45 min induced significant mortality(P＜0.05).This temporal sensitivity highlights the delicate balance between CPA penetration and osmotic toxicity.Cryopreservation trials at 0 and-20℃ using optimized PMG3S protocols revealed significant stage-specific survival.Tail-bud stage embryos demonstrated exceptional cryoresistance,achieving survival rates of(72.58±2.21)％(0℃)and(62.90±4.24)％(-20℃),respectively,which were significantly higher than those of somite and heart-beating stage embryos(P＜0.05).The survival rate of spotted grouper embryos at the same developmental stage decreased with decreasing temperature.Three factors contributed to embryo survival:(1)sucrose-mediated enhanced membrane stability through vitrification;(2)mixed cryoprotectants collaboratively mitigated temperature effects on embryos;(3)the specificity of developmental stages reduced intracellular water content.This study provides clear cryopreservation parameters for O.punctatus,identifying 35％PMG3S as the optimal CPA formulation for a 35-min equilibration treatment of tail-bud stage embryos.The study of cryopreservation techniques for O.punctatus embryos enhanced genetic resource preservation for embryo banks,promoted the implementation of controlled breeding programs,and minimized pathogen spread during transportation.Future optimization efforts may focus on enhanced freezing vehicles,improved cooling/heating rates,and novel cryoprotectants.This work bridges fundamental cryobiological principles with applied aquaculture needs,offering a replicable model for marine species conservation.Climate change and overfishing pose considerable risks to global fisheries;therefore,cryopreservation technologies are essential for ensuring food security and conserving aquatic biodiversity.关键词
斑石鲷/胚胎冷冻保存/非渗透性冷冻保存剂/玻璃化液Key words
Oplegnathus punctatus/embryo cryopreservation/non-permeating cryoprotectant/vitrification solution分类
农业科技引用本文复制引用
YANG Fengfan,LI Shengzhong,LI Linlin,WANG Shihao,ZHANG Chunbai,DING Tangtang,LI Zhentong,LIU Yang,WANG Linna,TIAN Yongsheng..基于非渗透性抗冻剂筛选的混合玻璃化液对斑石鲷胚胎冷冻保存的影响[J].中国水产科学,2025,32(10):1393-1403,11.基金项目
国家重点研发计划项目(2022YFD2400502,2022YFD2400103) （2022YFD2400502,2022YFD2400103）
现代农业产业技术体系(CARS-47-G31) （CARS-47-G31）
山东省重点研发计划项目(2022LZGC016) （2022LZGC016）
海南省重点研发计划项目(ZDYF2025SXLH002) （ZDYF2025SXLH002）
青岛市科技惠民示范专项(24-1-8-xdny-3-nsh) （24-1-8-xdny-3-nsh）
中央公益性科研院所基本科研业务费(20603022024013,2020TD19). （20603022024013,2020TD19）
