Ogata Kazuko, Takeuchi Ayaka, Ashibe Shiori, Sugane Naoko, Nagao Yoshikazu
Faculty of Agriculture, University Farm, Utsunomiya University, Tochigi, 321-4415, Japan.
Department of Animal Production Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.
Vet Res Commun. 2025 Jan 22;49(2):86. doi: 10.1007/s11259-025-10651-w.
The purpose of this study was to improve the quality of frozen-thawed canine spermatozoa through the optimization of glycerol concentration (GC) and freezing rate in the semen freezing protocol. Ejaculates from nine dogs were diluted with an extender containing 0%, 1.5%, 3%, 6%, or 9% glycerol. The suspensions were loaded into 0.25 ml straws, frozen in nitrogen vapor in a closed box, and immersed in liquid nitrogen (LN). The freezing rate was controlled by setting the distance from the LN surface to the straws as 1, 4, 7, or 10 cm. Firstly, freezing curves for each GC and freezing rate were analyzed. The analysis showed that the temperature of ice nucleation, freezing point, and immersion were changed with a certain trend depending on the GCs and freezing rates. Secondly, the sperm motility index (MI), viability and mitochondrial (MT) activity were evaluated. At 0 h after thawing, the MI was higher in the 3% and 6% GCs than the 0% GCs (P < 0.05). At 24 h, the 3% GC with 1 cm LN distance (1 cm-3%) and the 7 cm-6% showed higher viability than the other conditions (P < 0.05), and the highest MT activity was obtained in the 1 cm-3%, which was higher than the other conditions (P < 0.05). The present findings indicate that the rapid freezing rate at 1 cm (average - 31 °C/min) with 3% GC provided the optimal condition in this study; use of this condition should reduce the detrimental damage to dog spermatozoa caused by ice crystal formation during freezing.
本研究的目的是通过优化精液冷冻方案中的甘油浓度（GC）和冷冻速率，提高冻融犬精子的质量。用含有0%、1.5%、3%、6%或9%甘油的稀释液对9只犬的射精进行稀释。将悬浮液装入0.25毫升的细管中，在密闭盒中于氮蒸气中冷冻，然后浸入液氮（LN）中。通过将LN表面到细管的距离设置为1、4、7或10厘米来控制冷冻速率。首先，分析了每种GC和冷冻速率的冷冻曲线。分析表明，冰核形成温度、冰点和浸入温度随GC和冷冻速率的变化呈现一定趋势。其次，评估精子活力指数（MI）、存活率和线粒体（MT）活性。解冻后0小时，3%和6%GC组的MI高于0%GC组（P<0.05）。在24小时时，1厘米LN距离的3%GC组（1厘米-3%）和7厘米-6%组的存活率高于其他条件（P<0.05），1厘米-3%组获得最高的MT活性，高于其他条件（P<0.05）。本研究结果表明，在本研究中，3%GC、1厘米处的快速冷冻速率（平均-31°C/分钟）提供了最佳条件；使用该条件应能减少冷冻过程中冰晶形成对犬精子造成的有害损伤。
