Dog sperm cryopreservation using cryovials and different dilution steps.

IF 1 4区生物学 Q3 BIOLOGY

Cryo letters Pub Date : 2024-01-01

S Ibrahim, N A Hamad Talha, J Cho, Y Jeon, I J Yu

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引用次数: 0

Abstract

Background: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time.

Objective: To develop a more efficient freezing method using cryovials.

Materials and methods: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 10⁸ 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 LN₂ 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_2/ 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 LN₂ after freezing and their functional performance and gene expression determined.

Results: 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.

Conclusion: 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.

本刊更多论文

使用低温瓶和不同稀释步骤进行狗精子冷冻保存。

背景：狗精子的传统冷冻方法是使用吸管，包括两步稀释和较长的平衡时间：材料与方法：使用低温瓶（0.5 mL）的三种冷冻方案：第 1 组精子在冷冻瓶和扩展剂 1（E1）及扩展剂 2（E1 + 1 M 甘油）中于 4 摄氏度下冷却 50 分钟，然后在 LN2 上冷冻 20 分钟；第 2 组精子在冷冻瓶和 E1 及 E2 混合液（1：1）在深冷冻箱（-80 摄氏度）中冷冻 30 分钟；第 3 组精子在冷冻瓶中与 E1 一起在 4 摄氏度下冷却 20 分钟，加入 E2（E1:E2，1:1）后再冷却 20 分钟，然后用 LN2/ 蒸汽冷冻 20 分钟。对照组（第 4 组）是用两步稀释法冷冻吸管中的精子。冷冻后，将所有组的精子放入 LN2 中保存，并测定其功能表现和基因表达：第 2 组、第 3 组和第 4 组（仅顶体完整性）的进行性运动和顶体完整性最高（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。第 2 组精子线粒体相关富半胱氨酸蛋白（SMCP）和抗凋亡 B 细胞淋巴瘤 2（BCL2）基因的表达量最高（P < 0.05），第 4 组促凋亡 Bcl2 相关 X 蛋白（BAX）的表达量最低（P < 0.05）：结论：使用低温瓶、一步稀释法和深冷箱冷冻精子（第 2 组）被证明是一种简单且适合狗精子的冷冻保存方法。https://doi.org/10.54680/fr24110110312。

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来源期刊

Cryo letters 生物-生理学

CiteScore

1.80

自引率

10.00%

发文量

审稿时长

1 months

期刊介绍： A bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.