Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan 54596, Republic of Korea. Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology, Khartoum 11111, Sudan.
College of Veterinary Medicine, Chungnam National University, 34134 Daejeon, Republic of Korea.
Cryo Letters. 2024 Jan-Feb;45(1):16-27.
The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time.
To develop a more efficient freezing method using cryovials.
Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 10 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN after freezing and their functional performance and gene expression determined.
Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4.
The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.
犬精子的传统冻存方法是使用细管，包括两步稀释和较长的平衡时间。
开发一种使用 cryovials 的更有效的冻存方法。
使用 cryovials（0.5 mL）进行了三种冻存方案，精子浓度为 1 x 10 个/mL：第 1 组精子在 cryovials 和 extender 1（E1）和 extender 2（E1 +1 M 甘油）中于 4 度冷却 50 分钟，然后在 LN 中冷冻 20 分钟；第 2 组精子在 cryovials 中冷却并与 E1 和 E2（1:1）的混合物在深冻机（-80 度 C）中冷冻 30 分钟；第 3 组精子在 cryovials 和 E1 中于 4 度冷却 20 分钟，加入 E2（E1:E2，1:1）后再冷却 20 分钟，然后使用 LN 蒸汽冷冻 20 分钟。对照组（第 4 组）由使用两步稀释的传统冻存方法的 straws 中的精子组成。所有组在冻存后都进行浸泡和储存在 LN 中，并测定其功能表现和基因表达。
第 2、3 和 4 组（仅顶体完整性）的精子运动能力和顶体完整性最高（P < 0.05）。第 3 组的活力明显优于其他组，第 2 组的活性氧（ROS）水平和磷脂酰丝氨酸（PS）转位指数明显低于其他组。精子线粒体相关含半胱氨酸丰富蛋白（SMCP）和抗凋亡 B 细胞淋巴瘤 2（BCL2）基因的表达在第 2 组中最高（P < 0.05），而促凋亡 Bcl2 相关 X 蛋白（BAX）的表达在第 4 组中最低（P < 0.05）。
使用 cryovials、一步稀释和深冻机（第 2 组）冷冻的精子被证明是一种简单且适合犬精子的冷冻保存方法。https://doi.org/10.54680/fr24110110312。
