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.

本研究旨在评估采用鸡蛋蛋黄来源的低浓度天然及冻干低密度脂蛋白（low density lipoprotein, LDL）用于犬精液冷冻保存的应用效果。实验通过设置不同硫酸铵浓度梯度从蛋黄中提取LDL：将蛋黄进行离心处理，分别使用10%、20%、40%、45%及50%浓度的硫酸铵溶液（ammonium sulphate solution, ASS）分离得到LDL，随后收集富含LDL的漂浮组分开展化学特性分析。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提升。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示，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中，以三羟甲基氨基甲烷（TRIS）稀释液为基础，设置20%蛋黄为对照组，分别采用1%、2%、3%（w/v，质量体积比）浓度的天然或冻干LDL替代20%蛋黄开展实验组处理。将精液经离心处理（755×g，离心7分钟）后，使用上述稀释液之一进行稀释，分装至0.5mL细管中（浓度为100×10^6精子/mL），随后置于程序化冷冻仪中完成冷冻。解冻后的精液（37℃水浴30秒）被用于检测精子活力、形态学特征，并开展低渗肿胀试验及落射荧光试验，采用羧荧光素二乙酸酯/碘化丙啶（CFDA/PI）进行染色。结果表明，当使用低浓度时，经50% ASS提取的天然LDL在保存犬冷冻精子方面的效果与全蛋黄相当；而冻干LDL，尤其是在所测试的两个较高浓度（2%和3%）下，无法维持LDL对犬精子的冷冻保护效果。