Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC‐1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real‐time quantitative RT‐qPCR was used to detect the mRNA expression levels of protamine‐related genes (PRM2, PRM3), sperm acrosome‐associated genes (SPACA3), oxidative stress‐related genes (ROMO1) and apoptosis‐related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.

中文翻译：

---

番红花对延边黄牛冻融精子凋亡，鱼精蛋白表达和膜脂质氧化的影响。

甘油用作牛精液渗透性冷冻保护剂，可大大提高冷冻和解冻的牛精子的质量。但是，高甘油浓度可能会对冷冻和融化的牛精子产生毒性作用。因此，该实验研究了用番红花代替冷冻保护剂溶液中的一部分甘油对冷冻和解冻的延边黄牛精子的精子凋亡，鱼精蛋白缺乏和膜脂质氧化的影响。实验包括对照组（6％甘油）和四个治疗组：I（3％甘油），II（3％甘油+0.5 mM crocin），III（3％甘油+ 1 mM crocin）和IV（3％甘油） + 2 mM番红花）。使用计算机辅助精液分析来检测精子运动，Hoechst 33,342，碘化丙锭，PRM2，PRM3），精子顶体相关基因（SPACA3），氧化应激相关基因（ROMO1）和凋亡相关基因（BCL2和BAX）。与对照组相比，用1 mM crocrocin代替部分甘油可显着改善精子运动能力，质膜完整性，膜脂质紊乱（p  <.05）和生存能力，线粒体膜电位，鱼精蛋白缺乏（p  <.01）。的表达水平PRM2，PRM3，SPACA3和BCL 2显著增加（p  <0.05），而表达水平ROMO1和BAX显着降低（p  <.05）。因此，BCL2 / BAX比率显着增加（p  <.05）。总之，在冷冻保护溶液中用crocrocin代替一部分甘油可以改善延边黄牛冷冻和解冻后的精子质量。