Shamsi Rahele Ranjbar, Jozani Razi Jafari, Asadpour Reza, Rahbar Maryam, Taravat Morteza
Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.
Reproductive Health Research Center, Clinical Research Institute, Urmia University of Medical Science, Urmia, Iran.
Biopreserv Biobank. 2024 Dec 26. doi: 10.1089/bio.2024.0077.
Sperm cryopreservation is a useful storage technique in artificial insemination. Nanoparticles and nanovesicles such as exosomes are widely used in sperm cryopreservation procedures to alleviate cold-induced injury inflicted during sperm freezing. The objective of the present study was to examine the impact of varying concentrations of exosomes derived from seminal plasma added to a freezing extender on the quality of post-thawed bull sperm. Five Holstein bulls were chosen based on their samples having less than 30% progressive motility. After exosome extraction, semen samples from bulls ( = 5) with progressive sperm motility ≤30% were collected, diluted with different exosome concentrations (0, 25, 50, and 100 μg/mL), and aspirated into 0.5 mL straws. After the freeze-thaw process, sperm total and progressive motility, viability, morphology, plasma membrane integrity, mitochondrial activity, and apoptosis status were assessed. Furthermore, the expression levels of annexin (ANX1), dystrophy-associated Fer-1-like protein (DYSF), fibronectin 1 (FN1), and reactive oxygen species modulator 1 (ROMO1) were evaluated via real-time polymerase chain reaction (PCR). Adding different concentrations of exosomes (25, 50, and 150 μg/mL) significantly increased the progressive motility, viability, and membrane integrity of sperm compared with the control group ( < 0.05). For the apoptosis index, treatment with 100 μg/mL exosomes significantly increased the percentage of live cells ( < 0.05), while the percentage of necrotic cells decreased significantly ( < 0.05) compared with 25 μg/mL exosome. The results of quantitative PCR showed that the expression levels of ANX1 were significantly ( < 0.05) upregulated at 50 μg/mL exosome, and the expression of ROMO1, FN1, and DYSF were downregulated upon treatment with different exosome concentrations. In conclusion, supplementing the freezing diluent with exosome-derived seminal plasma could preserve the quality parameters of the post-thaw semen of the bull with low freezeability and could be used as a helpful method for reproductive programs.
精子冷冻保存是人工授精中一种有用的储存技术。纳米颗粒和纳米囊泡（如外泌体）被广泛应用于精子冷冻保存程序，以减轻精子冷冻过程中造成的冷诱导损伤。本研究的目的是检测添加到冷冻稀释液中的不同浓度精浆来源外泌体对解冻后公牛精子质量的影响。基于其样本的前向运动性低于30%，选择了5头荷斯坦公牛。外泌体提取后，收集精子前向运动性≤30%的公牛（n = 5）的精液样本，用不同浓度的外泌体（0、25、50和100μg/mL）进行稀释，并吸入0.5mL细管中。冻融过程后，评估精子的总运动性和前向运动性、活力、形态、质膜完整性、线粒体活性和凋亡状态。此外，通过实时聚合酶链反应（PCR）评估膜联蛋白（ANX1）、营养不良相关的Fer-1样蛋白（DYSF）、纤连蛋白1（FN1）和活性氧调节剂1（ROMO1）的表达水平。与对照组相比，添加不同浓度的外泌体（25、50和150μg/mL）显著提高了精子的前向运动性、活力和膜完整性（P < 0.05）。对于凋亡指数，与25μg/mL外泌体相比，用100μg/mL外泌体处理显著增加了活细胞百分比（P < 0.05），而坏死细胞百分比显著降低（P < 0.05）。定量PCR结果显示，5othμg/mL外泌体时ANX1的表达水平显著上调（P < 0.05），不同浓度外泌体处理后ROMO1、FN1和DYSF的表达下调。总之，用精浆来源的外泌体补充冷冻稀释液可以保存冷冻能力低的公牛解冻后精液的质量参数，并可作为繁殖计划的一种有用方法。
