Semen Cryobiology Laboratory, Division of Animal Physiology and Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar, via- Jaipur, Rajasthan, 304 501, India.
Semen Cryobiology Laboratory, Division of Animal Physiology and Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar, via- Jaipur, Rajasthan, 304 501, India.
Cryobiology. 2020 Oct;96:76-84. doi: 10.1016/j.cryobiol.2020.07.013. Epub 2020 Aug 7.
Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10 sperm mL) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at -25 °C min up to -125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.
冷冻精液的宫颈内人工授精失败阻碍了绵羊在田间条件下实施人工授精。在这里，研究了平衡时间和过氧化氢酶对公羊精液解冻后质量的影响。将精液混合（800×10 个精子/mL）后用 TES-Tris-果糖稀释液（6%甘油、15%卵黄）稀释，添加 0、50、100 和 200 U/mL 过氧化氢酶，并分装到 0.25 mL 细管中。在实验 1 中，细管在 5°C 的冰箱中平衡 3 小时（E3）或在冰箱中平衡 10 小时（E10）和 22 小时（E22）。在实验 2 中，所有细管都在冰箱中平衡 22 小时。细管在 -25°C 下以每分钟-125°C 的速度冷冻，然后放入液氮中。与 E3 和 E10 相比，E22 的解冻后总运动和快速运动活力更高（P<0.05）。E3 和 E22 之间的精子动力学相似，但 E10 较低。同样，顶体完整性、功能膜完整性、高胆固醇百分比（mCHO）和活线粒体膜潜能（MMP）更高（P<0.05），而活高细胞内钙和顶体反应精子则更低在 E22 与 E3 和 E10 相比。与对照组相比，过氧化氢酶处理的样本中的快速运动活力百分比、高 mCHO 和活高 MMP 显著降低（P<0.05），而膜完整性在组内相似。总之，与 3 或 10 小时相比，冷冻前平衡 22 小时可提高解冻后精子的功能，而过氧化氢酶对公羊精液的冷冻保存有负面影响。
