Abstract：
Hypothermic liquid storage at 4-5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l-cysteine (LC) and N-acetyl-l-cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen.
摘要：
在4-5°C的低温液体储存已成为一种保存公猪精液的新方法，为精液保存提供创新的可能性。然而,这种方法也带来了挑战，包括冷激和过量的活性氧（ROS）产生。因此,减少由低温引起的氧化损伤变得必不可少，同时补充适当的保护剂。在这项研究中,我们使用计算机辅助精子分析(CASA)研究了牛血清白蛋白(BSA)与聚乙烯吡咯烷酮(PVP)和脱脂奶粉(SMP)相比在维持公猪精子活力和进行性运动方面的功效.在测试的浓度中，4g/L的BSA表现出最好的保护效果。随后，我们补充了不同浓度的1-半胱氨酸（LC）和N-乙酰-1-半胱氨酸（NAC）作为添加剂，在BSA作为保护剂的存在下。我们的结果表明，与其他浓度相比，1mmol/L的LC和0.5mmol/L的NAC对精子质量具有更好的保护作用。此外，与对照组相比，1mmol/LLC和0.5mmol/LNAC组显示出显著改善的质膜完整性和顶体完整性。这些组还表现出增强的抗氧化能力，线粒体膜电位（MMP）增加，ATP生产，总超氧化物歧化酶(T-SOD)活性,总抗氧化能力(T-AOC)，谷胱甘肽(GSH),谷胱甘肽过氧化物酶（GSH-PX），和GPX-4级别。此外,它们显示出活性氧（ROS）和丙二醛（MDA）水平降低，以及降低的氧化型谷胱甘肽（GSSG）和谷胱甘肽还原酶（GR）水平。此外，LC和NAC处理增强了AMP激活的蛋白激酶(AMPK)磷酸化。然而,使用化合物C抑制AMPK并不抑制LC和NAC对低温保存的公猪精子的保护作用。这些发现表明，4g/LBSA可以作为猪精液低温液体储存的有效保护剂。此外,LC和NAC补充通过增强抗氧化能力而不是通过AMPK介导的ATP补充来减少氧化损伤。这些结果有助于推进LC和NAC在猪精液低温液体储存中的应用。
