Söderquist L, Madrid-Bury N, Rodriguez-Martinez H
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine Swedish University of Agricultural Sciences, Uppsala, Sweden.
Theriogenology. 1997 Nov;48(7):1115-25. doi: 10.1016/s0093-691x(97)00344-0.
Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.
从用于人工授精计划的5只2.5岁公羊采集精液，连续3天使用人工阴道采集。精液用含7%甘油（体积/体积）的脱脂乳稀释剂稀释，装入法国小型细管（约100微升/细管），并在程序控制冷冻机中冷冻。每只公羊进行三次冷冻操作。每次冷冻操作取三根细管进行以下解冻程序：1）70℃，5秒；2）50℃，9秒；3）35℃，12秒。解冻后精子活力用相差显微镜主观评估；同时使用羧基荧光素二乙酸酯和碘化丙啶（CFDA/PI）组合荧光染料、低渗肿胀试验（HOS）以及正常顶嵴（NAR）的存在情况来确定精子膜完整性程度。解冻处理之间在解冻后活力（P < 0.05）和膜完整性（P < 0.01）方面存在显著差异，公羊之间的差异具有统计学意义。在70℃解冻的细管，解冻后精子活力以及显示完整膜的精子百分比显著高于（P < 0.01）在35℃解冻的细管（分别为67.0±1.1%和63.0±1.1%，以及50.5±1.5%和41.7±1.5%）。然而，比较70℃和50℃解冻时，这些参数没有相应的统计学显著差异。得出的结论是，精子可以在50℃解冻9秒而不是在70℃解冻5秒，而不会进一步降低精子活力或膜完整性。这种较低的解冻温度将有助于在瑞典农场条件下更广泛地使用冷冻/解冻公羊精液。
