Theriogenology Laboratory, Southern Regional Station of ICAR-National Dairy Research Institute, Bengaluru, India.
ICAR-National Research Centre on Equines, Hisar, Haryana.
Biopreserv Biobank. 2024 Feb;22(1):82-87. doi: 10.1089/bio.2022.0197. Epub 2023 Jul 19.
Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased ( < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased ( < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced ( < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.
尽管精液在维持精子功能和促进配子相互作用方面起着至关重要的作用，但在冷冻保存马精子之前，通常会去除精液以提高精子的冷冻存活率。本研究评估了从生产高质量精液的种马中补充异源精液是否可以改善遗传上优越但产生低质量精液的种马的重要精子功能参数。将来自低质量精液产生的种马的精子分为三份：两份分别用高质量精液产生的种马的精液补充 20%和 30%，而第三份保持对照（0%精液）并在 37°C 下孵育 30 分钟。通过流式细胞术在孵育过程中的不同时间间隔评估精子膜完整性、线粒体膜电位（MMP）、线粒体超氧化物（mtROS）生成和细胞内钙状态。观察到在 20%和 30%精液补充组中，孵育过程中死亡精子的比例增加（<0.01）。然而，在不同的时间间隔内，对照组和处理组的 MMP 没有观察到显著变化。有趣的是，发现与对照组相比，在精液补充组中，精子 mtROS 的产生在孵育过程中增加（<0.01）。在精液孵育组中，具有高细胞内钙的活精子比例减少（<0.01）。总之，异源精液的添加不能修复冷冻保存造成的损害，进一步导致精液质量恶化，如我们的研究中观察到的，通过降低活力、增加活性氧（ROS）产生来实现，这可能是由于死细胞比例较高，或者是一些（尚未确定）诱导种马精子氧化应激的因素。
