Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
Theriogenology. 2013 Sep 15;80(5):443-50. doi: 10.1016/j.theriogenology.2013.05.006. Epub 2013 Jun 14.
Sperm cryopreservation is the most efficient method for storing boar sperm samples for a long time. However, one of the inconveniences of this method is the large variation between and within boars in the cryopreservation success of their sperm. The aim of the present work was thus to find reliable and useful predictive biomarkers of the good and poor capacity to withstand the freeze-thawing process in boar ejaculates. To find these biomarkers, the amount of proteins present in the total proteome in sperm cells were compared between good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) using the two-dimensional difference gel electrophoresis technique. Samples were classified as GFE and PFE using progressive motility and viability of the sperm at 30 and 240 minutes after thawing, and the proteomes from each group, before starting cryopreservation protocols, were compared. Because two proteins, acrosin binding protein (ACRBP) and triosephosphate isomerase (TPI), presented the highest significant differences between GFE and PFE groups in two-dimensional difference gel electrophoresis assessment, Western blot analyses for ACRBP and TPI were also performed for validation. ACRBP normalized content was significantly lower in PFE than in GFE (P < 0.05), whereas the TPI amounts were significantly lower in GFE (P < 0.05) than in PFE. The association of ACRBP and TPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ACRBP and TPI can be used as markers of boar sperm freezability before starting the cryopreservation procedure, thereby avoiding unnecessary costs involved in this practice.
精子冷冻保存是长期储存猪精子样本的最有效方法。然而，这种方法的一个不便之处是，不同公猪之间以及同一头公猪的精子冷冻保存效果存在很大差异。因此，本研究旨在寻找可靠且有用的预测生物标志物，以预测猪精子在冷冻-解冻过程中的良好和不良耐受能力。为了找到这些生物标志物，使用二维差异凝胶电泳技术比较了良好冷冻可育性精液（GFE）和不良冷冻可育性精液（PFE）中总蛋白质组中存在的蛋白质数量。使用解冻后 30 分钟和 240 分钟时精子的运动性和活力将样品分类为 GFE 和 PFE，然后比较每组在开始冷冻保存方案之前的蛋白质组。由于在二维差异凝胶电泳评估中，精浆蛋白酶结合蛋白（ACRBP）和磷酸丙糖异构酶（TPI）这两种蛋白质在 GFE 和 PFE 组之间表现出最高的显著差异，因此还进行了 ACRBP 和 TPI 的 Western blot 分析以进行验证。与 GFE 相比，PFE 中的 ACRBP 标准化含量显着降低（P <0.05），而 TPI 含量在 GFE 中显着降低（P <0.05）比 PFE 中的含量。使用 Pearson 线性相关性确认了 ACRBP 和 TPI 与解冻后精子活力和运动性的相关性。总之，ACRBP 和 TPI 可以在开始冷冻保存程序之前用作猪精子冷冻可育性的标志物，从而避免这种做法所涉及的不必要成本。
