添加胰岛素-转铁蛋白-亚硒酸钠对牛体外胚胎生产、低温保存及常规冷冻效果的影响

doi:10.16431/j.cnki.1671-7236.2023.10.021

摘要/Abstract

摘要： 【目的】探究添加胰岛素-转铁蛋白-亚硒酸钠(insulin-transferrin-sodium selenite,ITS)对牛体外胚胎生产、4 ℃低温保存及常规程序冷冻效果的影响,以期应用ITS提高牛体外胚胎的生产及保存效果。【方法】对屠宰场卵巢来源的牛卵母细胞进行体外受精,①研究在胚胎体外培养后期液M2中添加ITS对囊胚率的影响,试验分为M2中添加ITS组(M2+ITS)和M2中未添加ITS的对照组(M2-control);②研究在M2中添加ITS后进一步在前期培养液M1中添加ITS对早期胚胎发育的影响,试验分为M1和M2中均添加ITS组(M1+M2+ITS)和仅M2中添加ITS的对照组(M1-control);③在三气培养条件下对ITS改善胚胎发育的效果进行评估,试验分为添加ITS组(ITS)和未添加ITS的对照组(control),以上各组均统计卵裂率、7 d囊胚率及总囊胚率;④将获得的7 d牛囊胚在添加ITS的M2保存液(Hypo-ITS组)中低温保存(4 ℃)24 h后复苏培养,或者在添加ITS(Cryo-ITS组)的冷冻保存液中常规程序冷冻解冻后培养,以低温保存液中未添加ITS(Hypo-control组)和冷冻液中未添加ITS(Cryo-control组)为对照,统计胚胎的存活率和孵化率。【结果】与对照组相比,M2+ITS和M1+M2+ITS组的7 d囊胚率均显著提高(P＜0.05);三气培养条件下,ITS组7 d囊胚率显著提高(P＜0.05)。Hypo-ITS组4 ℃低温保存复苏的牛胚胎存活率与对照组差异不显著(P＞0.05),但孵化率显著高于对照组(P＜0.05);Cryo-ITS组的常规程序冷冻解冻的牛胚胎存活率及孵化率与对照组均无显著差异(P＞0.05)。【结论】ITS可作为添加剂及低温保护剂用于提高牛体外胚胎生产及低温保存效果,但在常规程序化冷冻液中添加ITS对牛体外胚胎常规程序冷冻效果的提升并不明显。

关键词: 牛体外胚胎; 胰岛素-转铁蛋白-亚硒酸钠(ITS); 低温保存; 常规程序冷冻

Abstract: 【Objective】 This study was aimed to evaluate the effect of adding insulin-transferrin-sodium selenite (ITS) to production,4 ℃ hypothermic storage and programmed freezing system of bovine in vitro embryos,so as to improve the production and preservation effect of in vitro bovine embryos via ITS.【Method】 After in vitro fertilization of bovine oocytes derived from slaughterhouse ovaries,the following experiments were carried out:① An experiment to study the effect of adding ITS to anaphase culture medium M2 on blastocyst rate,with M2+ITS and M2-control (without ITS,control) groups. ② An experiment to further investigate the effect of adding ITS to prophase culture medium M1 on early embryonic development,with M1+M2+ITS(both M1 and M2 with ITS addition) and M1-control (without ITS addition but M2,control) groups. ③ An experiment to further evaluate the effect of ITS in improving embryonic development under three gases culture conditions,with ITS (with ITS) and control (without ITS) groups.The cleavage rate,day-7 blastocyst rate,and total blastocyst rate were calculated for each of the above groups.④ The day-7 bovine embryos were stored at 4 ℃ for 24 h by M2 storage solution with ITS (Hypo-ITS group),or day-7 in vitro embryos in frozen solution with ITS were frozen by programmed procedure (Cryo-ITS group),with Hypo-control (without ITS) and Cryo-control (without ITS) group,and the survival and hatching rates of the recovered and thawed embryos were counted,respectively.【Result】 Compared with control group,the day-7 blastocyst rates of M2+ITS and M1+M2+ITS groups were significantly increased (P＜0.05);Under the three gas culture conditions,the day-7 blastocyst rate in ITS group was also significantly increased (P＜0.05).There was no significant difference of the survival rate between Hypo-ITS and Hypo-control groups (P＞0.05),but the hatching rate of Hypo-ITS group was significantly higher than that of Hypo-control group (P＜0.05).There was no significant difference of the survival and hatching rates of programmed freezing between Cryo-ITS and Cryo-control groups (P＞0.05).【Conclusion】 ITS could be used as additive and hypo-protectant to improve the effect of bovine in vitro embryo production and hypo-preservation.However,the addition of ITS in the freezing solution did not significantly improve the programmed freezing effect of bovine in vitro embryos.

Key words: bovine in vitro embryo; insulin-transferrin-sodium selenite(ITS); hypothermic storage; routine programmed freezing

中图分类号:

S814.6

张浩波, 赵善江, 王欢, 徐茜, 冯志强, 王婉洁, 巩建飞, 庞云渭, 杜卫华, 朱化彬. 添加胰岛素-转铁蛋白-亚硒酸钠对牛体外胚胎生产、低温保存及常规冷冻效果的影响[J]. 中国畜牧兽医, 2023, 50(10): 4077-4084.

ZHANG Haobo, ZHAO Shanjiang, WANG Huan, XU Xi, FENG Zhiqiang, WANG Wanjie, GONG Jianfei, PANG Yunwei, DU Weihua, ZHU Huabin. Effects of Addition of Insulin-Transferrin-Sodium Selenite on Effect of Production, Hypothermic Storage and Routine Freezing of Bovine in vitro Embryos[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4077-4084.

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