Graduate Program in Veterinary Science, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand; Department of Morphophysiology and Pharmacology, College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz, Nueva Ecija, 3120, Philippines.
Graduate Program in Veterinary Science, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand.
Cryobiology. 2022 Jun;106:39-47. doi: 10.1016/j.cryobiol.2022.04.005. Epub 2022 May 1.
As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.
作为射出精液的替代物，附睾精子 (ES) 可以被回收并冷冻保存，用于人工授精和其他辅助生殖技术。然而，精子对冷冻和解冻过程的反应存在变异性，这挑战了其内在价值。因此，本研究旨在阐明沼泽水牛 ES 的可冷冻性现象，因为相关文献并不丰富。在这里，我们使用屠宰场来源的死后尾端附睾通过切片冲洗技术从沼泽水牛中分离 ES。通过慢速冷冻进行冷冻保存后，根据解冻后的总活力和渐进活力，将 ES 样本分为高（HF）和低可冻性（LF）。评估并比较了常规精子参数和感兴趣的蛋白质，如谷胱甘肽 S-转移酶 Mu 3 (GSTM3) 和 ATP 合酶β亚基 (ATP1B1)，HF 和 LF 之间存在显著差异。计算机辅助精子分析显示，除了摆动、线性和直线性外，几乎所有的运动和运动学参数在可冻性组之间都有显著差异。荧光染色后，HF 和 LF 中均存在明显的细胞内活性氧产生，后者的丙二醛水平明显高于前者。免疫组织化学标记表明，GSTM3 和 ATP1B1 蛋白均存在于 ES 和附睾管状上皮中。尽管通过 Western blot 分析，LF 中的 GSTM3 相对含量与脂质过氧化显著高于 HF，但在 ATP1B1 方面没有观察到显著差异。运动性、运动和运动学、氧化应激状态以及特定的精子蛋白的这种变化表明它们在区分沼泽水牛 ES 的可冻性表型方面具有潜在的用途。
