Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
Theriogenology. 2020 Jan 15;142:15-25. doi: 10.1016/j.theriogenology.2019.09.030. Epub 2019 Sep 19.
Although semen cryopreservation is an important, widely used technique for long-term sperm storage, it not only induces partially irreversible damages to sperm but might also deteriorate anatomical, biochemical, and structural organelles. These cellular and epigenetic modifications are the main reasons underlying the decline in sperm motility and fertility during the freeze-thaw process. Using the two Lake and Beltsville semen extenders, the present study aims to evaluate the epigenetic patterns (DNA methylation and histone modification), cellular parameters (e.g., membrane integrity, viability, DNA stability, mitochondria activity, and apoptosis status), and fertility potential of rooster semen collected from six mature roosters before and after cryopreservation according to a standard protocol. The results show that cryopreservation leads to significantly (P < 0.05) reduced values of the parameters examined when compared with those of fresh sperms. While the extenders used exhibit no difference with respect to DNA methylation (DNMT), the Lake extender leads to significant reductions (P < 0.05) in H3K9 acetylation (17.4 ± 1.8) and H3K4 methylation (42 ± 2.3) compared to the Beltsville (9.2 ± 1.8 and 23 ± 2.3, respectively). Compared to the Beltsville extender, the Lake one is also observed to yield a significantly (P < 0.05) superior sperm quality in terms of total motility (TM; 77.2 ± 1.6 vs. 68.3 ± 1.6), average path velocity (VAP; 71 ± 1.4 vs. 53 ± 1.4), and straight-line velocity (VSL; 52 ± 1.5 vs. 34 ± 1.5) as well as significantly (P < 0.05) higher viability (60 ± 1.69 vs. 51 ± 1.69) and membrane functionality (55 ± 3.2 vs. 46 ± 3.2). The Lake extender is also found to outperform the Beltsville one due to its significantly (P < 0.05) higher fertility rate (59.5% vs. 47.2%). The two extenders, however, exhibit no differences in DNA fragmentation, mitochondrial activity, or hatchability rate. The Beltsville extender showed to be superior to the Lake one due to its significantly greater reactive oxygen species percentage (ROS; 45.9 ±.3.2 vs. 28.5 ± 3.2) and apoptosis (29 ± 2.3 vs. 27 ± 2.3). It may be concluded that the Lake extender is capable of improving the cellular and epigenetic parameters of rooster sperms during cryopreservation due to the crucial roles it plays in the protection of sperms against cryo-damages.
虽然精液冷冻保存是一种重要的、广泛应用的长期精子储存技术，但它不仅会对精子造成部分不可逆的损伤，还可能使解剖学、生化和结构细胞器恶化。这些细胞和表观遗传修饰是冷冻-解冻过程中精子活力和生育力下降的主要原因。本研究使用两种 Lake 和 Beltsville 精液 extender，根据标准方案评估了六只成熟公鸡采集的精子在冷冻保存前后的表观遗传模式（DNA 甲基化和组蛋白修饰）、细胞参数（如膜完整性、活力、DNA 稳定性、线粒体活性和凋亡状态）和生育能力。结果表明，与新鲜精子相比，冷冻保存导致检查参数显著（P < 0.05）降低。虽然两种 extender 在 DNA 甲基化（DNMT）方面没有差异，但 Lake extender 导致 H3K9 乙酰化（17.4 ± 1.8）和 H3K4 甲基化（42 ± 2.3）显著降低（P < 0.05）与 Beltsville（分别为 9.2 ± 1.8 和 23 ± 2.3）相比。与 Beltsville extender 相比，Lake extender 在总活力（TM；77.2 ± 1.6 vs. 68.3 ± 1.6）、平均路径速度（VAP；71 ± 1.4 vs. 53 ± 1.4）和直线速度（VSL；52 ± 1.5 vs. 34 ± 1.5）方面也表现出更好的精子质量，以及显著更高的活力（60 ± 1.69 vs. 51 ± 1.69）和膜功能（55 ± 3.2 vs. 46 ± 3.2）。由于 Lake extender 的受精率（59.5% vs. 47.2%）显著更高（P < 0.05），因此它也优于 Beltsville extender。然而，两种 extender 在 DNA 碎片化、线粒体活性或孵化率方面没有差异。由于 Beltsville extender 的活性氧百分比（ROS；45.9 ±.3.2 vs. 28.5 ± 3.2）和凋亡（29 ± 2.3 vs. 27 ± 2.3）显著更高，因此它优于 Lake extender。可以得出结论，由于 Lake extender 在保护精子免受冷冻损伤方面发挥着重要作用，因此它能够在冷冻保存过程中改善公鸡精子的细胞和表观遗传参数。
