Department of Population Health and Reproduction, University of California, Davis, CA, USA.
Department of Population Health and Reproduction, University of California, Davis, CA, USA; Department of Morphology, Imaging, Orthopedics, Rehabilitation and Nutrition, Faculty of Veterinary Medicine, University of Ghent, 133 Salisburylaan, 9820, Merelbeke, Belgium.
Theriogenology. 2022 Oct 15;192:1-8. doi: 10.1016/j.theriogenology.2022.08.014. Epub 2022 Aug 17.
Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there is no information available about approximate charges or ZP in equine sperm. Therefore, in this study we aimed to characterize equine sperm ZP and identify its associations with known measures of sperm quality. Additionally, we aimed to complete a comprehensive comparison of conventional sperm selection techniques as compared to the novel method of microfluidic sorting. Ejaculates (n = 22) were partitioned into fresh (∼23 °C, 0 h; n = 12) and cooled (∼4 °C, 24 h; n = 10) groups, and processed by swim up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and microfluidic chip (MF) sorting. Motility, progressive motility, cell viability, normal morphology, and ZP were evaluated for both unprocessed fractions and post-selected fractions. The ZP of both fresh and cooled samples was net negative and also correlated with motility and progressive motility for both fresh and cooled samples (P < 0.05). The ZP of cooled samples was also correlated with viability (P < 0.05). Among the compared methods of sperm selection, MF was highly effective in selecting high quality sperm as determined by the measured parameters. Percent motility, progressive motility, normal morphology, and viability of MF selected sperm were of higher quality than sperm selected by SU, and of similar to DG-SU and DGC without the use of potentially harmful centrifugation steps. Correlations between ZP, motility, and viability parameters may indicate a role of external charge on the motility and survival of sperm within the female reproductive tract. In conclusion, we identified an average net negative ZP on equine sperm and correlations between ZP and other measures of sperm quality, as well as having identified MF as a novel effective method of equine sperm selection for IVP.
胞质内精子注射（ICSI）是马体外胚胎生产（IVP）的唯一方法。除了卵母细胞发育能力外，IVP 的结果还高度依赖于精子质量。因此，不仅必须采用优良的方法选择高质量的精子，而且还必须能够确定精子质量的哪些可量化特性最能表明其生育能力。在男性中，通过 ζ 电位（ZP）估计的净负表面电荷与精子质量和体外胚胎发育结果高度相关。然而，关于马精子的近似电荷或 ZP 尚没有信息。因此，在这项研究中，我们旨在描述马精子的 ZP，并确定其与已知精子质量测量值的关联。此外，我们旨在全面比较传统的精子选择技术与新颖的微流控分选方法。将精液（n=22）分为新鲜组（约 23°C，0 h；n=12）和冷却组（约 4°C，24 h；n=10），并通过游泳法（SU）、密度梯度离心法（DGC）、密度梯度-游泳法组合（DG-SU）和微流控芯片（MF）分选进行处理。对未经处理的部分和分选后的部分评估了运动性、前向运动性、细胞活力、正常形态和 ZP。新鲜和冷却样本的 ZP 均为净负，并且与新鲜和冷却样本的运动性和前向运动性相关（P<0.05）。冷却样本的 ZP 也与活力相关（P<0.05）。在所比较的精子选择方法中，MF 是一种高度有效的方法，因为其选择的精子的测量参数具有较高的质量。MF 选择的精子的百分比运动性、前向运动性、正常形态和活力均优于 SU 选择的精子，与 DG-SU 和 DGC 相似，而没有使用潜在有害的离心步骤。ZP、运动性和活力参数之间的相关性可能表明外部电荷在精子在雌性生殖道中的运动性和生存能力方面发挥作用。总之，我们确定了马精子的平均净负 ZP，以及 ZP 与精子质量其他测量值之间的相关性，并确定 MF 是 IVP 中马精子选择的一种新颖有效的方法。
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