Purdy P H
USDA-ARS-NCGRP, National Animal Germplasm Program, 1111 South Mason Street, Fort Collins, CO 80521-4500, USA.
Anim Reprod Sci. 2006 Jun;93(1-2):114-23. doi: 10.1016/j.anireprosci.2005.07.002. Epub 2005 Aug 3.
The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.
研究了在冷冻保存前将稀释的公羊精液在5℃下保存长达48小时的效果。秋季通过电刺激法从6只公羊采集精液，春季又从6只不同的公羊采集精液。分别使用分光光度法和计算机自动精液分析仪测定精子浓度和活力。样品在23℃下用一步法Tris-蛋黄-甘油（5%，v/v）培养基稀释至400×10⁶个细胞/ml，在2小时内冷却至5℃，并在实验期间保持在5℃。冷却后0、24或48小时将等分试样装入0.5ml法式细管中，在液氮上方4.5cm处的液氮蒸汽中冷冻12 - 13分钟，然后投入液氮中保存。解冻后，秋季采集的精液在保存0、24或48小时后冷冻，其活力百分比（分别为29%、31%、36%）、渐进性活力（分别为16%、15%、17%）、质膜完整性（分别为28%、35%、29%）和活的顶体反应细胞（分别为0.4%、0.6%、0.8%；P>0.05）相似。此外，在5℃下保存0、24或48小时后，与鸡蛋卵周膜结合的精子数量，以精子数量平均值表示时（分别为155、177、106个精子）或受精精子比例表示时（分别为0.39、0.49、0.34；P>0.05），差异均不显著。同样，春季采集的公羊精液在冷却后0、24和48小时冷冻，其总活力（分别为21%、25%、20%）、渐进性活力（分别为14%、15%、11%）、质膜完整性（分别为26%、33%、31%）和活的顶体反应细胞（分别为3.7%、3.5%、3.2%；P>0.05）相似。与48小时保存时间相比，0小时保存时间与鸡蛋卵周膜结合的精子明显较少（分别为250和470个精子），尽管24小时保存时间与0或48小时保存时间无差异（281个精子；P<0.05），但分析受精精子的比例未观察到显著差异（P>0.05）。这些结果表明，公羊精子在冷冻前可在5℃下保存长达48小时，对活力、膜完整性或受精潜力（如膜结合能力所示）无有害影响。
