In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

在本研究中，对 Barbari 的低温获能相关变化、凋亡样变化、脱蛋白、总抗氧化能力 (TAC) 和体外精子功能属性进行了评估冻融后的钱。建立了脱蛋白与精子功能特征之间的相关性。使用免疫印迹程序，检测到对应于鱼精蛋白-1 的单个 28-kDa 蛋白带的存在。通过免疫荧光测试进一步验证了精子头部区域的定位。获能 (B-) 和顶体反应 (AR-) 模式精子，具有磷脂酰丝氨酸外化和相对较小线粒体跨膜电位的精子，以及脱蛋白和 DNA 片段化较大（P < 0.05) 冻融后，分别表明存在冷冻获能和细胞凋亡样变化。此外，使用免疫印迹和免疫荧光程序检测含酪氨酸蛋白的磷酸化证实了在冻融后雄鹿精子中存在类似冷冻能力的变化。总抗氧化能力（TAC）、体外热阻反应、Vanguard 距离、孕激素敏感性和体外获能反应较少（P < 0.05) 冻融后的精子与初始稀释和平衡后的精子相比。脱蛋白（色霉素 A3 阳性细胞，CMA3+）和 DNA 片段化（TUNEL+ve）与 B 型和 AR 型精子呈正相关，而其他变量的其他值则呈负相关。总之，这项研究的结果表明巴克精子中存在鱼精蛋白 1，冻融后鱼精蛋白 1 丢失，同时巴克精子中存在与低温获能相关的变化和凋亡样变化。精子去鱼精可能是由于 DNA 断裂增加，导致降压精子的受精能力受损。