Muiño R, Rivera M M, Rigau T, Rodriguez-Gil J E, Peña A I
Department of Animal Pathology, Faculty of Veterinary Medicine of Lugo, Unit of Reproduction and Obstetrics, University of Santiago de Compostela, 27002 Lugo, Spain.
Anim Reprod Sci. 2008 Dec;109(1-4):50-64. doi: 10.1016/j.anireprosci.2007.11.028. Epub 2007 Nov 29.
The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25-ml straws: placing the straws in a water bath at 37 degrees C for 40s, at 50 degrees C for 15s or at 70 degrees C for 5s. In a first experiment, the three thawing rates were compared in relation to post-thaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5h of incubation at 37 degrees C. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2h of incubation at 37 degrees C. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post-thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the three thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patterns of movement: (1) moderately slow and progressive sperm (27%); (2) "hyperactivated-like" sperm (15.4%); (3) poorly motile non-progressive sperm (34.3%); (4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the four subpopulations, but there was a significant effect (P<0.05) of the interaction between thawing rate and incubation time. Higher proportions of spermatozoa with fast and progressive movement were observed after 2h of post-thaw incubation when the thawing was at the faster rates (35 degrees C/40s: 8.3%, 50 degrees C/15s: 18.1% and 70 degrees C/5s: 16.5%). Whether this subtle difference might affect to the in vivo fertility of the thawed bovine semen is not known.
本研究的目的是评估三种解冻速率对0.25毫升细管中冷冻的公牛精液的影响：将细管置于37℃水浴中40秒、50℃水浴中15秒或70℃水浴中5秒。在第一个实验中，比较了这三种解冻速率对解冻后精子活力（主观测定）以及精子质膜和顶体膜完整性（通过流式细胞术检测）的影响，检测在37℃孵育0小时和5小时后进行。在第二个实验中，基于解冻后精子活力（使用CASA系统测定）评估这三种解冻速率，检测在37℃孵育0小时和2小时后进行。此外，对于活动精子，使用多元聚类程序分析个体活力描述符，以检测解冻公牛精液样本中是否存在具有特定活力特征的不同精子亚群。最后，研究解冻速率是否对不同亚群内精子的相对频率分布有任何影响。在所评估的三种解冻方法之间，解冻后总体活力或精子质膜和顶体膜完整性方面没有显著差异。统计分析将所有活动精子聚类为四个具有明确运动模式的不同亚群：(1) 中等缓慢且渐进的精子 (27%)；(2) “超激活样”精子 (15.4%)；(3) 活动不良的非渐进精子 (34.3%)；(4) 快速且渐进的精子 (23.3%)。解冻速率对四个亚群内精子的频率分布没有显著影响，但解冻速率与孵育时间之间的相互作用有显著影响 (P<0.05)。解冻后孵育2小时后，当解冻速率较快时（35℃/40秒：8.3%，50℃/15秒：18.1%，70℃/5秒：16.5%），观察到具有快速且渐进运动的精子比例更高。这种细微差异是否会影响解冻后牛精液的体内受精能力尚不清楚。
