Arias María Elena, Andara Katherine, Briones Evelyn, Felmer Ricardo
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile; Department of Animal Production, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile.
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine Universidad de La Frontera, Temuco, Chile.
Reprod Biol. 2017 Jun;17(2):126-132. doi: 10.1016/j.repbio.2017.03.002. Epub 2017 Mar 28.
This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (ΔYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high ΔΨm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p<0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression.
本研究评估了使用密度梯度（Percoll和BoviPure）和上游法对牛精子（从三头公牛获取）进行分离对精子功能和基因表达的影响。精子评估包括使用流式细胞术检测质膜完整性（SYBR14/PI）、顶体完整性（PNA-FITC/PI）、氧化应激（ROS；CH2FDDA）、DNA片段化（TUNEL检测）和线粒体膜电位（ΔYm；TMRM）。通过计算机辅助精子分析（CASA）评估精子活力，并使用RT-qPCR分析基因表达。结果表明，与未分离的对照组（分别为70.3%和62.4%）相比，Percoll分离法获得的具有完整质膜和顶体膜的精子比例更高（分别为89.8%和87.5%），上游法（分别为74.9%和63.3%）和BoviPure法（分别为83.3%和80.4%）也是如此。Percoll法和BoviPure法之间具有高ΔΨm的精子比例没有差异（分别为84.3%和83.5%），这高于上游法和未分离的对照组（分别为72.8%和43.8%）。Percoll分离的精子中ROS水平较高，所有组之间精子DNA完整性没有差异。活力分析表明，与对照组相比，分离方法可提高（p<0.05）总活力和前向运动活力，Percoll在这方面最为有效。最后，在验证6个参考基因后，对瘦素（LEP）、芳香化酶细胞色素P450（CYP19）和鱼精蛋白I（PRM1）的基因表达分析表明，各组之间没有差异。总之，使用密度梯度分离牛精子可改善精子活力和功能参数，而不影响基因表达。
