The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 106 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferred into 0.5 mL straws, equilibrated at 5 °C for 30 min, and placed in liquid nitrogen (LN2) vapour for 15 min before being plunged into LN2. After thawing, sperm samples were evaluated for motility and velocity variables, mitochondrial membrane potential, apoptosis, and plasma membrane and DNA integrities. For sperm motility variables, there were dose- and time-dependent effects, with the largest values recorded when 12.5 and 25 ng/mL relaxin were used for 0-120 min of incubation. Furthermore, at all of the concentrations at which there were evaluations, relaxin additions to semen diluent led to a marked improvement in sperm mitochondrial membrane potential and a lesser percentage of apoptotic cells compared to the control group. Plasma membranes and DNA integrities were not affected by relaxin supplementations to the diluent. In conclusion, supplementation of relaxin in extender before semen cryopreservation, especially at 12.5 and 25 ng/mL, had a positive effect on the sperm quality variables.

中文翻译：

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松弛素对冷冻保存的种马精液精液质量变量的影响。

该研究的目的是确定在预冻潜伏期中添加到增量培养基中的不同浓度的松弛素对种马冷冻解冻的精子质量变量的影响。将从三个种马中收集的精液样品过滤，用脱脂奶稀释，并以600g离心10分钟。将精子沉淀物悬浮在BotuCrio冷冻培养基中，使其终浓度为50×106精子/ mL。将稀释的精液分为5个实验组，分别补充0（对照），12.5、25、50或100 ng / mL的松弛素。将精液样品转移到0.5 mL吸管中，在5°C下平衡30分钟，并在浸入LN2中之前在液氮（LN2）蒸气中放置15分钟。解冻后，评估精子样本的运动性和速度变量，线粒体膜电位，细胞凋亡，质膜和DNA完整性。对于精子运动变量，存在剂量和时间依赖性，当在0-1.20分钟的孵育中使用12.5和25 ng / mL松弛素时记录的最大值。此外，在所有评估浓度下，与对照组相比，在精液稀释剂中添加松弛素可显着改善精子线粒体膜电位，并减少凋亡细胞百分比。补充稀释剂的松弛素不会影响质膜和DNA完整性。总之，在精液冷冻保存之前补充补充剂中的松弛素，尤其是在12.5和25 ng / mL时，对精子质量变量有积极影响。有剂量和时间依赖性，在12.5和25 ng / mL松弛素孵育0-120分钟时记录到最大值。此外，在所有评估浓度下，与对照组相比，在精液稀释剂中添加松弛素可显着改善精子线粒体膜电位，并减少凋亡细胞百分比。补充稀释剂的松弛素不会影响质膜和DNA完整性。总之，在精液冷冻保存之前补充补充剂中的松弛素，尤其是在12.5和25 ng / mL时，对精子质量变量具有积极影响。有剂量和时间依赖性，在12.5和25 ng / mL的松弛素孵育0-120分钟时记录到最大值。此外，在所有评估浓度下，与对照组相比，在精液稀释剂中添加松弛素可显着改善精子线粒体膜电位，并减少凋亡细胞百分比。补充稀释剂的松弛素不会影响质膜和DNA完整性。总之，在精液冷冻保存之前补充补充剂中的松弛素，尤其是在12.5和25 ng / mL时，对精子质量变量有积极影响。在所有评估浓度下，与对照组相比，在精液稀释剂中添加松弛素导致精子线粒体膜电位显着改善，凋亡细胞百分比降低。补充稀释剂的松弛素不会影响质膜和DNA完整性。总之，在精液冷冻保存之前补充补充剂中的松弛素，尤其是在12.5和25 ng / mL时，对精子质量变量有积极影响。在所有评估浓度下，与对照组相比，在精液稀释剂中添加松弛素导致精子线粒体膜电位显着改善，凋亡细胞百分比降低。补充稀释剂的松弛素不会影响质膜和DNA完整性。总之，精液冷冻保存前补充补充剂中的松弛素，尤其是在12.5和25 ng / mL时，对精子质量变量有积极影响。