基于甲醇的复合性玻璃化液对斑石鲷胚胎冷冻保存的影响

Impact of methanol-based composite vitrification on Oplegnathus punctatus embryo cryopreservation

  • 摘要:
    目的 探究以甲醇为基础形成的复合性玻璃化液对斑石鲷胚胎平衡处理后,不同低温冷冻保存胚胎存活发育情况。
    方法 以肌节期、尾芽期和心跳期的斑石鲷胚胎作为实验对象,并以甲醇为基础玻璃化液,分别逐步添加冷冻保护剂,配制为不同浓度MPS(MeOH+PG+20% Suc)、MDS(MeOH+DMSO+20% Suc)、MGS(MeOH+Gly+20% Suc)对胚胎进行毒性及不同低温冷冻保存实验。
    结果 尾芽期胚胎经3种冷冻保护剂平衡后,正常发育率均显著高于其它发育阶段。渗透性冷冻保护剂浓度为2、3和4 mol/L分别对尾芽期胚胎进行平衡处理,结果显示,随着处理浓度的升高,其对培养至发育后期胚胎正常发育的抑制作用不显著。低温0℃保存后培养至心跳期,经2 mol/L MPS平衡处理的胚胎正常发育率最高,为88.24%±1.98%。-20℃保存后,3 mol/L MPS处理组的胚胎正常发育率最优,为73.38%±2.85%。-40℃保存后,三种冷冻保护剂均显示为3M浓度组的胚胎存活率高于2M与4M浓度组,其中3 mol/L MPS组存活率最高(46.72%±1.89%)。-196℃处理后,MPS平衡处理的胚胎解冻后结构完整率最佳。
    结论 采用MPS复合型冷冻保护剂对斑石鲷尾芽期胚胎平衡处理后,其冷冻保存效果优于MDS与MGS。经-40℃冷冻后,3 mol/L的冷冻保护剂保存胚胎存活率均高于其他浓度组。该研究为斑石鲷胚胎冷冻保存提供了基础参考资料,为海水鱼类遗传种质资源的保护与利用提供了重要的技术方法。

     

    Abstract: Fish embryo cryopreservation is a crucial technical approach for protecting fish genetic germplasm resources. This study aimed to investigate the survival and development of Oplegnathus fasciatus embryos after equilibrium treatment with a methanol-based composite vitrification solution and subsequent cryopreservation at different low temperatures. Three methanol-based vitrification solutions-MPS (MeOH+PG+20% Suc), MDS (MeOH+DMSO+20% Suc), and MGS (MeOH+Gly+20% Suc)-were prepared at concentrations of 2, 3, and 4 mol/L. Results indicated that tail-bud stage embryos exhibited significantly higher normal development rates after equilibrium treatment compared to other developmental stages (P<0.05). Moreover, increasing the concentration of the cryoprotectants had no significant inhibitory effect on the normal development of embryos at later stages (P>0.05). Based on this, cryopreservation experiments are carried out, frozen viable embryos were obtained at 0, −20 and −40 ℃, and MPS had the best freezing effect among the three cryoprotectants. After −40 ℃ treatment, the survival rate of the 3 mol/L MPS group was the highest (46.72%±1.89%). At −196 ℃, thawed embryos maintained relatively intact structures but showed no further development during subsequent culture. Among the three cryoprotectants, embryos treated with MPS exhibited the highest structural integrity post-thawing. The cryopreservation effect of the MPS composite cryoprotectant on tail-bud stage embryos of O. fasciatus was superior to that of MDS and MGS. After cryopreservation at −40 ℃, the embryo survival rates under the three cryoprotectants at 3 mol/L concentration were higher than those at other concentrations.

     

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