Department of Medicine and Animal Surgery, University of Murcia, Murcia, Spain.
Anim Reprod Sci. 2013 Aug;140(3-4):173-9. doi: 10.1016/j.anireprosci.2013.06.015. Epub 2013 Jul 5.
The goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.
本实验旨在评估单层离心（SLC）作为一种常规的猪精子冷冻保存选择方法的适用性和效果，使用猪特异性胶体 Androcoll-P。本研究特别关注 SLC 处理整个富含精子的精液部分的有效性，以及在冷冻前使用 SLC 选择冷冻解冻（FT）精子的受精能力。13 个富含精子的精液部分（每个公猪一个）被分成 3 份。两份 15 和 150mL 的精液分别用 15 和 150mL（v/v）的 Androcoll-P-Large 和 Androcoll-P-XL 进行 SLC 处理（500×g 离心 20min）。第三份精液作为对照不进行处理。无论使用何种 Androcoll-P，SLC 处理后，形态正常且表现出快速和渐进运动（通过 CASA 评估）的精子比例更高（P<0.01），而核 DNA 碎片化（精子染色质弥散试验）的比例更低（P<0.01）。总精子、活动精子、存活精子（用 H-42、PI 和 FITC-PNA 染色后通过流式细胞术评估）和形态正常精子的回收率在 20%至 100%之间，核 DNA 完整的精子回收率在 60%至 100%之间，无论使用何种 Androcoll-P 都是如此。此后，精液样本按照标准的 0.5mL straw 冷冻方案进行冷冻保存。解冻后，无论使用何种 Androcoll-P，SLC 处理组的精子活力（总活力和快速直线运动）、活力和核 DNA 完整性百分比均高于对照组（P<0.05）。SLC 处理还提高了 FT 精子的体外受精能力（用 300:1 的活精子：卵比例受精的 679 个体外成熟卵，共孵育 6 小时），表现为穿透卵的百分比以及穿透卵中肿胀精子头部和/或雄性原核的平均数量。然而，SLC 处理对可能的受精卵发育为囊胚的体外能力没有影响。总的来说，这些结果表明，使用 Androcoll-P 对猪精液进行 SLC 处理可以提高冷冻保存猪精子的质量和受精能力。然而，在考虑将 SLC 纳入猪精液冷冻保存方案的常规程序之前，应努力确保持续获得高回收率。
