Esteso Milagros C, Fernández-Santos María R, Soler Ana J, Garde José J
Dpto. Ciencia y Tecnologia Agroforestal. Campus Universitario, 02071, Albacete, Spain.
Cryo Letters. 2003 Jul-Aug;24(4):261-8.
The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or - 1.8 vs. 70.6 + or - 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.
本研究的目的是评估解冻程序对马鹿精子在冷冻和解冻后形态学上不同亚群中分布的影响。为此，使用两种不同的解冻方案（I = 37摄氏度20秒与II = 70摄氏度5秒）对附睾精子进行解冻。从10只伊比利亚雄鹿采集的精子在室温下用含20%卵黄的Triladyl培养基稀释，并在氮蒸气中冷冻。通过显微镜评估精子活力来判断标准的精子冷冻保存能力。解冻程序对精子活力百分比有影响（P = 0.05），使用方案I时总体恢复率最佳（76.8±1.8 vs. 70.6±1.8）。此外，通过精子分类分析仪（SCA）对每个样本至少200个精子头部的形态学尺寸进行分析，并记录平均测量值。当使用方案II解冻精子时，鹿精子头部明显（P = 0.01）小于使用程序I时（面积 = 30.02平方微米 vs. 30.32平方微米；宽度 = 4.47微米 vs. 4.51微米；长度 = 8.05微米 vs. 8.11微米），但并非所有雄鹿都是如此。所有精子头部测量值都存入统计数据库并进行多变量聚类分析。通过方差分析比较两种不同解冻程序主要聚类所有参数的平均测量值。解冻方案I的精子头部尺寸主要聚类中长度、宽度、面积、周长、形状因子和宽度/长度的平均值与方案II的显著不同（P = 0.001）。此外，在所有雄鹿的整个形态学参数中都发现了差异（P = 0.001），使用方案II时精子头部总体尺寸最小。此外，快速解冻方案导致异质性急剧丧失。最后，我们的结果表明，异质性丧失越大，精子冷冻损伤程度越高。
