Malama E, Zeron Y, Janett F, Siuda M, Roth Z, Bollwein H
Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
"Sion" Artificial Insemination Center, Hafetz-Haim, Israel.
Theriogenology. 2017 Jan 1;87:79-90. doi: 10.1016/j.theriogenology.2016.08.002. Epub 2016 Aug 25.
Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at -196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca levels (LOW-Ca, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca content, but not on the DNA integrity of sperm after freezing-thawing.
气候的季节性波动被认为是影响牛精子发生和精液质量的主要因素。我们的研究旨在使用计算机辅助精子分析（CASA）和流式细胞术研究季节对冻融牛精液功能参数的影响。为此，在夏季（8月至9月；n = 43）和冬季（12月至1月；n = 43）的月份里，从饲养在亚热带条件下的五头成熟荷斯坦 - 弗里生公牛采集了86份射精样本。精液用基于Tris - 蛋黄的稀释液稀释，并在 - 196°C下冷冻。在解冻后（0小时）和孵育3小时后（3小时）立即进行计算机辅助精子分析，以评估总活动精子、渐进性活动精子和快速活动精子的百分比（%）。此外，还测定了精子的平均路径速度、曲线速度和直线速度以及头部横向位移幅度。通过流式细胞术在0小时和3小时测定具有完整质膜和顶体的精子百分比（PMAI，%）、具有高线粒体膜电位的精子百分比（HMMP，%）、具有低细胞内钙水平的精子百分比（LOW - Ca，%）以及具有高DNA碎片化指数的精子百分比（DFI%，%）。分别在0小时和3小时使用流式细胞术评估精子在低渗条件下的存活率（HYPO - SURV，%）和具有诱导顶体反应的精子百分比（IAR，%）。通过应用线性混合效应模型探讨季节（冬季与夏季）对精子质量参数的固定效应。结果表明，与夏季相比，冬季未孵育和孵育后的精子所有CASA参数（直线速度除外，P > 0.05）均有所改善（所有情况下P < 0.01）。夏季采集的射精样本在0小时和3小时时IAR值较低（P < 0.001），PMAI、HMMP和LOW - Ca值也较低（所有情况下P < 0.01）。相反，HYPO - SURV和DFI%（在0小时和3小时）不受季节影响（所有情况下P > 0.05）。总之，使用CASA和流式细胞术揭示了冷冻保存的牛精子功能状态与季节相关的变化，这表明夏季热应激对精子活力、质膜和顶体完整性、顶体反应诱导性、线粒体功能和细胞内钙含量有不利影响，但对冻融后精子的DNA完整性没有影响。
