使用不含卵黄的聚乙烯醇添加剂中的必需和非必需氨基酸溶液对狗精子进行冷冻保存。
Cryopreservation of Dog Spermatozoa Using Essential and Non-Essential Amino Acids Solutions in An Egg Yolk-Free Polyvinyl Alcohol Extender.
机构信息
Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan, Republic of Korea. Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology, Khartoum, Sudan.
Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan, Republic of Korea.
出版信息
Cryo Letters. 2021 Jan-Feb;42(1):44-52.
BACKGROUND
Amino acids (AAs) have been indicated to have cryoprotective and antioxidative effects on sperm freezing using egg yolk (EY)-based extender. However, EY-based extender is difficult to be standardized for the effect of amino acids because the EY composition varies with the animal's diet.
OBJECTIVE
To test the effect of AAs in EY-free polyvinyl alcohol (EY-free PVA) extender and develop a chemically defined extender for dog sperm cryopreservation.
MATERIALS AND METHODS
In the first experiment (E1), dog spermatozoa (1ｘ10 sperms/mL) were frozen with EY-free PVA extender without AAs or supplemented with essential (EAAs, 50 x: 1, 2, 4 %) or non-essential amino acids (NEAAs, 100 x: 1, 2, 4 %). In the second experiment (E2), spermatozoa were frozen with EY-free PVA extender supplemented with 0, 0.5, 1 or 2 % of an EAA-NEAA mixture. Motility, viability and acrosome integrity were evaluated after thawing in E1 and E2. In the third experiment (E3), spermatozoa were frozen using an extender supplemented with 2 % EAAs, 2 % NEAAs or a 0.5 % EAA-NEAA mixture. Reactive oxygen species (ROS) and phosphatidylserine (PS) translocation were assessed. Expression of genes for motility-related sperm mitochondrial-associated cysteine-rich protein (SMCP), apoptosis-related B-cell lymphoma 2 (BCL2) and BCL2 associated X protein (BAX) was measured.
RESULTS
Addition of EAAs, NEAAs or an EAA-NEAA mixture to EY-free PVA extender significantly increased sperm motility without affecting viability. Only 1 % NEAAs significantly increased the acrosome membrane. EAA-NEAA mixture (0.5 %) significantly increased SMCP, BCL2 and BAX expression compared to the control group without significant effect on PS translocation or ROS.
CONCLUSION
EAAs and NEAAs addition in EY-free PVA extender improved sperm motility, with limited effect on acrosome integrity and gene expression of SMCP, BCL2 and BAX during dog sperm cryopreservation.
背景
已有研究表明，在基于卵黄（EY）的稀释液中添加氨基酸（AAs）可对精子冷冻起到抗冷冻损伤和抗氧化作用。然而，由于 EY 的成分随动物的饮食而变化，因此基于 EY 的稀释液很难标准化，这会影响 AAs 的效果。
目的
测试 EY 无聚乙烯醇（EY 无 PVA）稀释液中 AAs 的作用，并开发一种用于犬精子冷冻保存的化学定义的稀释液。
材料和方法
在第一个实验（E1）中，以不含 AAs 的 EY 无 PVA 稀释液或添加必需氨基酸（EAAs，50 x：1、2、4%）或非必需氨基酸（NEAAs，100 x：1、2、4%）对犬精子（1×10 个精子/mL）进行冷冻。在第二个实验（E2）中，以 EY 无 PVA 稀释液补充 0、0.5、1 或 2%的 EAA-NEAA 混合物对精子进行冷冻。在 E1 和 E2 中解冻后评估精子的活力、活力和顶体完整性。在第三个实验（E3）中，以添加 2%EAAs、2%NEAAs 或 0.5%EAA-NEAA 混合物的稀释液对精子进行冷冻。评估活性氧（ROS）和磷脂酰丝氨酸（PS）转位。测量与运动相关的精子线粒体相关含半胱氨酸丰富的蛋白（SMCP）、凋亡相关 B 细胞淋巴瘤 2（BCL2）和 BCL2 相关 X 蛋白（BAX）相关基因的表达。
结果
在 EY 无 PVA 稀释液中添加 EAAs、NEAAs 或 EAA-NEAA 混合物可显著提高精子活力，而对活力无影响。只有 1%的 NEAAs 可显著增加顶体膜的完整性。与对照组相比，EAA-NEAA 混合物（0.5%）显著增加了 SMCP、BCL2 和 BAX 的表达，而对 PS 转位或 ROS 无显著影响。
结论
在 EY 无 PVA 稀释液中添加 EAAs 和 NEAAs 可改善精子活力，在犬精子冷冻保存过程中对顶体完整性和 SMCP、BCL2 和 BAX 的基因表达影响有限。
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