Animal Reproduction Group, Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Cordoba, Córdoba, Spain.
Anim Reprod Sci. 2013 Aug;140(3-4):232-40. doi: 10.1016/j.anireprosci.2013.06.012. Epub 2013 Jul 4.
The aim of this study was to investigate whether single-layer centrifugation (SLC) with PureSperm® 80 could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were divided into two aliquots: one of them was used as control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed SLC treated samples. A multivariate clustering procedure separated 26,051 motile spermatozoa into three subpopulations (sP): sP1 consisting of highly active but non-progressive spermatozoa (40.3%), sP2 consisting of spermatozoa with high velocity and progressive motility (30.0%), and sP3 consisting of poorly active and non-progressive spermatozoa (29.7%). SLC with PureSperm® 80 yielded sperm suspensions with improved motility, morphology, viability and acrosome integrity (P<0.001). The frozen-thawed SLC treated samples were enriched in sP2, reaching a proportion of 44.1% of the present spermatozoa. From these results, we concluded that SLC with PureSperm® 80 may be an alternative and successful method for improving the quality of frozen-thawed dog spermatozoa. Moreover, sP2 (high-speed and progressive spermatozoa) was more frequently observed after SLC. Finally, this study also demonstrated that the general motile sperm structure present in dogs remained constant despite the effect caused by either cryopreservation or separation by SLC through PureSperm® 80.
本研究旨在探讨单层离心（SLC）与 PureSperm®80 是否能从冷冻犬精液中选择具有特定运动模式的优质精子，包括那些具有特定运动模式的精子。收集 5 只犬的精液，按照标准方案进行冷冻保存。解冻后，将精液样本分为两份：一份作为对照，另一份通过 SLC 处理。使用计算机辅助精液分析评估精子活力，Diff-Quick 染色评估精子形态，碘化丙啶/异硫氰酸荧光素标记的花生凝集素/罗丹明 123 三重荧光染色评估精子活力，分别对新鲜精液、冷冻解冻对照和冷冻解冻 SLC 处理样本的精液进行评估。多元聚类程序将 26051 条活动精子分为三个亚群（sP）：sP1 由高度活跃但非渐进性精子组成（40.3%），sP2 由具有高速度和渐进性运动的精子组成（30.0%），sP3 由活动能力差且非渐进性精子组成（29.7%）。PureSperm®80 与 SLC 联合使用可提高精子活力、形态、活力和顶体完整性（P<0.001）。SLC 处理后的冷冻精液样本富含 sP2，达到存在精子的 44.1%。从这些结果可以得出结论，PureSperm®80 与 SLC 联合使用可能是提高冷冻犬精子质量的一种替代和成功的方法。此外，SLC 后更常观察到 sP2（高速和渐进性精子）。最后，本研究还表明，尽管冷冻或通过 PureSperm®80 的 SLC 分离会产生影响，但犬精子的一般运动结构仍然保持不变。
