肌醇类SIB对云南半细毛羊精液冷冻保护效果影响的研究

摘要

In order to decrease the mechanical injuries caused by ice formation during freeze on ram spermatozoa of Yunnan semi-fine wool sheep,a synthetic ice blocker derived from cyclohexanhexol (1,4-CHD) was firstly used in cryopreservation of ram spermatozoa in this study.Briefly,following collection by electric stimulation,equilibration at 4℃ following dilution with the freezing extender,and pre-freezing in liquid nitrogen vapor,the spermatozoa were preserved in liquid nitrogen.The results indicated that 1,4-CHD could not increase the motility of frozen spermatozoa.With the elevation of the concentration of 1,4-CHD,the motility and moving velocity of frozen spermatozoa decreased steadily.Additionally,1,4-CHD could not increase the intact acrosome and membrane percentage of frozen spermatozoa.When the isotonic binding buffer was used to dilute the thawed spermatozoa,the percentage of cells labeled with propidium iodide (PI) after cryopreservation in the presence of 1,4-CHD was significantly higher than that of spermatozoa frozen in the absence of 1,4-CHD (P＜0.05).However,the percentage of frozen-thawed spermatozoa with exposed PS in the presence of 1,4-CHD was significantly less than that of spermatozoa frozen in the absence of 1,4-CHD (P＜0.01).When the basic extenders with an osmolarity of 404 mOsm,528 mOsm,648 mOsm,or 853 mOsm were used to dilute the frozen-thawed spermatozoa respectively,there were no significant differences among the four groups with respect to the moving velocity and membrane integrity (P＞0.05).In conclusion,1,4-CHD can't improve the motility,moving velocity,acrosome staus and membrane integrity of frozen spermatozoa.However,1,4-CHD may inhibit early apoptosis caused by freezing and thawing.%为了进一步降低冷冻过程中冰晶形成对云南半细毛羊精子的机械损伤,实验首次检验了肌醇类冰晶抑制剂(SIB)对绵羊精子的冷冻保护效果.采用的SIB是1,4-环己二醇(1,4-CHD).电刺激采集精液后,经4℃平衡、液氮气相预冻后直接投入液氮保存.结果表明:1,4-CHD不能提高云南半细毛羊冷冻精子的活力,而且随着1,4-CHD浓度的增加,精子活力呈逐步下降趋势.此外,1,4-CHD也不能改善解冻后云南半细毛羊精子的顶体状态和质膜完整性.解冻后,当用等渗缓冲液稀释精子时,1,4-CHD冷冻组精子的质膜破损率明显高于不合1,4-CHD的对照组(P＜0.05).然而,SIB的存在可能有利于冷冻精子磷脂酰丝氨酸(PS)正常分布的维持.解冻后,1,4-CHD冷冻组精子的PS标记率明显低于对照组(P＜0.01).当分别采用渗透压为404 mOsm,528 mOsm,648 mOsm或853 mOsm的缓冲液去稀释解冻后的精子时,就移动速度和质膜完整性指标而言,4个处理组之间差异不显著(P＞0.05).总之,肌醇类SIB并不能改善云南半细毛羊冷冻精子的活力、移动速度、顶体以及质膜完整性等指标.然而1,4-CHD可能抑制冷冻解冻过程引起的细胞早期凋亡.