Ribas-Maynou Jordi, Delgado-Bermúdez Ariadna, Mateo-Otero Yentel, Viñolas Estel, Hidalgo Carlos O, Ward W Steven, Yeste Marc
Biotechnology of Animal and Human Reproduction (TechnoSperm), Faculty of Sciences, Institute of Food and Agricultural Technology, University of Girona, C/ Maria Aurèlia Campany, 69, ES-17003, Girona, Spain.
Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003, Girona, Spain.
J Anim Sci Biotechnol. 2022 Sep 17;13(1):105. doi: 10.1186/s40104-022-00754-8.
The analysis of chromatin integrity has become an important determinant of sperm quality. In frozen-thawed bovine sperm, neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood. The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics, and to assess if this parameter can predict pregnancy rates in cattle.
A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods (0-2 h and 2-4 h), analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays, chromatin deprotamination and decondensation, sperm motility, viability, acrosomal status, and intracellular levels of total ROS, superoxides and calcium. Insemination of 59,605 females was conducted using sperm from the same bulls, thus obtaining the non-return to estrus rates after 90 d (NRR). Results showed an increased rate of double-stranded breaks in the first period (0-2 h: 1.29 ± 1.01%/h vs. 2-4 h: 0.13 ± 1.37%/h; P < 0.01), whereas the rate of sperm with moderate + high single-stranded breaks was higher in the second period (0-2 h: 3.52 ± 7.77 %/h vs. 2-4h: 21.06 ± 11.69 %/h; P < 0.0001). Regarding sperm physiology, viability decrease rate was different between the two periods (0-2 h: - 4.49 ± 1.79%/h vs. 2-4 h: - 2.50 ± 3.39%/h; P = 0.032), but the progressive motility decrease rate was constant throughout post-thawing incubation (0-2 h: - 4.70 ± 3.42%/h vs. 2-4 h: - 1.89 ± 2.97%/h; P > 0.05). Finally, whereas no correlations between bull fertility and any dynamic parameter were found, there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet (Rs = - 0.563, P = 0.003), between NRR and basal progressive motility (Rs = 0.511, P = 0.009), and between NRR and sperm with high ROS at 4 h post-thaw (Rs = 0.564, P = 0.003).
The statistically significant correlations found between intracellular ROS, sperm viability, sperm motility, DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress, where viability and motility would be affected first and sperm chromatin would be altered at a later stage, thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw. Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.
染色质完整性分析已成为精子质量的重要决定因素。在冷冻解冻的牛精子中，解冻后损伤事件的顺序以及不同类型精子DNA断裂的动态变化都尚未得到很好的理解。本研究的目的是描述这种精子降解的后果，重点关注DNA损伤动态，并评估该参数是否可以预测牛的妊娠率。
在解冻后的两个时间段（0 - 2小时和2 - 4小时）对来自25头荷斯坦公牛的75份冷冻精液进行了评估，通过碱性和中性彗星试验分析了整体和双链DNA损伤、染色质脱蛋白和去浓缩、精子活力、存活率、顶体状态以及总ROS、超氧化物和钙的细胞内水平。使用来自同一公牛的精子对59,605头母牛进行授精，从而获得90天后的返情率（NRR）。结果显示，第一个时间段（0 - 2小时：1.29 ± 1.01%/小时 vs. 2 - 4小时：0.13 ± 1.37%/小时；P < 0.01）双链断裂率增加，而第二个时间段中等 + 高单链断裂精子的比例更高（0 - 2小时：3.52 ± 7.77%/小时 vs. 2 - 4小时：21.06 ± 11.69%/小时；P < 0.0001）。关于精子生理学，两个时间段的存活率下降率不同（0 - 2小时： - 4.49 ± 1.79%/小时 vs. 2 - 4小时： - 2.50 ± 3.39%/小时；P = 0.032），但解冻后孵育过程中渐进性运动能力下降率是恒定的（0 - 2小时： - 4.70 ± 3.42%/小时 vs. 2 - 4小时： - 1.89 ± 2.97%/小时；P > 0.05）。最后，虽然未发现公牛生育力与任何动态参数之间存在相关性，但NRR与碱性彗星试验评估的高度损伤精子的基础百分比之间存在相关性（Rs = - 0.563，P = 0.003），NRR与基础渐进性运动能力之间存在相关性（Rs = 0.511，P = 0.009），以及NRR与解冻后4小时具有高ROS的精子之间存在相关性（Rs = 0.564，P = 0.003）。
细胞内ROS、精子活力、精子运动能力、DNA损伤和染色质脱蛋白之间存在统计学上显著的相关性，这表明一系列事件均由氧化应激驱动，其中活力和运动能力首先受到影响，精子染色质在后期会发生改变，因此表明牛精子应在解冻后2小时内用于受精。生育力相关性支持通过彗星试验评估整体DNA损伤可能有助于预测公牛的生育力。
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