Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm.

本研究旨在评估低浓度天然及冻干鸡蛋黄来源低密度脂蛋白（low density lipoprotein, LDL）用于犬精液冷冻保存的应用效果。本研究通过设置不同浓度梯度的硫酸铵溶液从蛋黄中提取LDL：先对蛋黄进行离心处理，随后分别采用10%、20%、40%、45%及50%浓度的硫酸铵溶液（ammonium sulphate solution, ASS）分离LDL，收集富含LDL的漂浮组分开展化学特性表征分析。检测结果显示，经50% ASS提取的LDL干物质含量最低（P<0.05），且LDL的纯化程度随硫酸铵浓度升高而显著提升。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，50% ASS溶液可从蛋黄中分离得到纯度更高的LDL组分。在精液冷冻保存实验环节，本研究以TRIS稀释液为基础体系，分别以1%、2%、3%（w/v）的天然或冻干LDL替代20%的全蛋黄（对照组）；将采集的精液以755×g离心7分钟，随后用上述稀释液进行稀释，分装至0.5mL细管中（精子浓度设置为100×10^6个/mL），并置于程序冷冻仪中完成冷冻程序。解冻后的精液（37℃水浴30秒）被用于检测精子活力、形态，并通过低渗肿胀试验与荧光染色试验（CFDA/PI）开展评估。实验结果表明，当采用低浓度添加时，经50% ASS提取的天然LDL对犬冷冻精子的保护效果与全蛋黄相当；而冻干LDL，尤其是在本次实验采用的两个较高添加浓度（2%与3%）下，无法维持LDL应有的犬精子冷冻保护效果。