Zhang Weijing, Min Lingjiang, Li Yajing, Lang Yaning, Hoque S A Masudul, Adetunji Adedeji Olufemi, Zhu Zhendong
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China.
Department of Animal Breeding and Genetics, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh.
Animals (Basel). 2022 Sep 30;12(19):2626. doi: 10.3390/ani12192626.
Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.
精子冷冻保存有助于人工授精（AI）在日常畜牧业中的广泛应用。然而，由于解冻后山羊精子中具有良好生物学功能的精子数量较少，其在人工授精中的应用受到限制。因此，对其进行改进一直是许多研究的重点。本研究旨在探讨在冷冻培养基中添加脯氨酸对山羊精子的影响。用添加不同浓度脯氨酸（0、0.5、1、2和4 mM）的冷冻培养基对山羊精液进行冷冻保存。评估解冻后精子的运动模式、膜完整性、顶体完整性、脂质过氧化（LPO）水平、丙二醛（MDA）水平、总抗氧化能力（T-AOC）、脯氨酸脱氢酶（PRODH）活性、超氧化物歧化酶（SOD）活性、谷胱甘肽（GSH）水平和GSH/GSSG。同样，还检测了解冻后山羊精子中PRODH的表达和免疫荧光定位。结果发现，在冷冻培养基中添加2 mM脯氨酸可显著提高解冻后山羊精子的总活力、前进活力、直线速度（VSL）、曲线速度（VCL）、平均路径速度（VAP）、直线性（STR）、线性度（LIN）、膜完整性和顶体完整性。有趣的是，PRODH在解冻后山羊精子中表达，尤其是在顶体后和精子尾部。与对照组相比，添加2 mM脯氨酸还显著提高了解冻后精子的PRODH活性。此外，添加2 mM脯氨酸可降低解冻后山羊精子的LPO水平和MDA水平。此外，与对照组相比，2 mM脯氨酸处理组解冻后山羊精子的T-AOC、SOD活性、GSH水平和GSH/GSSG值也显著增加。在2 mM脯氨酸处理中还观察到解冻后山羊精子凋亡减少，因为添加2 mM脯氨酸可降低Caspase3和Caspase9的水平。这些观察结果表明，在冷冻培养基中添加2 mM脯氨酸可通过降低冷冻保存期间的氧化应激来提高解冻后山羊精子的质量。这些发现也为脯氨酸作为一种有效的添加剂在冷冻保存期间提高解冻后山羊精子质量的应用提供了新的见解。
