Crockett E C, Graham J K, Bruemmer J E, Squires E L
Department of Physiology, Colorado State University, Fort Collins 80523, USA.
Theriogenology. 2001 Feb 1;55(3):793-803. doi: 10.1016/s0093-691x(01)00444-7.
The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.
将冷却后的种马精液运送到专门从事精子冷冻保存的机构，这一能力将使种马能够留在原地，而其精液则在具备适当冷冻设备和专业技术的机构进行冷冻保存。为实现这一目标，必须开发出冷冻冷却后运输精液的方法。为此进行了三项实验，以确定最合适的精子稀释液、包装、离心时间、精子浓度以及采集后在冷冻保存前精子能够冷却的时间长度。在第一项实验中，将精子离心以去除精浆，然后重悬于脱脂乳稀释液、脱脂乳 - 蛋黄 - 糖稀释液或脱脂乳 - 蛋黄 - 盐稀释液中，冷却至5℃，并在冷却后2.5小时或24小时分别装入0.5毫升或2.5毫升的细管中进行冷冻。冷却后2.5小时冷冻的样本中，进行性运动（PM）精子的百分比（27%）高于冷却后24小时冷冻的样本（10%；P < 0.05）。在含有蛋黄的脱脂乳稀释液中冷却后2.5小时冷冻的样本，其PM精子的百分比（平均32%）高于仅在含有脱脂乳的稀释液中冷冻的精子（平均16%；P < 0.05）。装入0.5毫升或2.5毫升细管中冷冻的PM精子百分比相似（分别为21%和28%；P > 0.05）。在第二项实验中，精子在冷却前（25℃）或冷却后（5℃）离心以去除精浆，然后在冷却至5℃ 2、6或12小时后进行冷冻。冷却前离心的精子（30%）的PM精子百分比高于冷却后离心的精子（19%；P < 0.05）。在25℃离心然后冷却12小时至5℃的精子，其解冻后的前进运动能力（23%）高于冷却12小时后在5℃离心的精子（13%；P < 0.05）。在第三项实验中，精子离心以去除精浆，重悬于精子浓度为50、250或500×10⁶/mL，冷却至5℃ 12小时后进行冷冻。包装精子浓度为50或250×10⁶/mL的样本冷冻后PM精子的百分比（25%和23%）高于包装精子浓度为500×10⁶/mL的样本（17%；P < 0.05）。我们建议精液在25℃离心以去除精浆，重悬至250×10⁶精子/ml，并在冷冻前于5℃保存12小时。
