Dept. Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
Anim Reprod Sci. 2013 Jun;139(1-4):62-8. doi: 10.1016/j.anireprosci.2013.04.005. Epub 2013 Apr 18.
The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino-2-phenylindole (DAPI) and examining for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. In a second experiment, aliquots of frozen-thawed stallion sperm were subjected to DL-DGC selection - or not - and then further subjected to the two best capacitation treatments (0.1μM and 0.05μM ionomycin). The fertilising capacity of the semen was then determined as above. The DL-DGC/capacitated sperm samples showed the highest mean penetration rates: 24.16% following capacitation with 0.1μM ionomycin, and 12.21% following capacitation with 0.05μM ionomycin. The capacitated but non-DL-DGC-selected sperm returned significantly lower values: 6.26% and 7.02% for the same ionomycin treatments respectively. These findings suggest that combining DL-DGC selection with ionomycin capacitation improves the fertilising capacity of frozen-thawed stallion sperm.
采用体外成熟去透明带牛卵母细胞的异种检测方法，研究了双层密度梯度离心（DL-DGC）与不同获能处理相结合对冻融马精子受精能力的影响。在第一个实验中，将冷冻解冻的马精子等分试样用 5 种不同的获能处理方法处理，而不进行 DL-DGC：1.0μM、0.1μM、0.05μM 或 0.01μM 的离子霉素，或 200μg/mL 的咖啡因。然后，通过用 4,6-二脒基-2-苯基吲哚（DAPI）染色上述卵母细胞，并检查精子穿透情况、每个卵母细胞穿透的精子数量以及雄性原核形成情况，在 18 小时评估精液的受精能力。在第二个实验中，将冷冻解冻的马精子等分试样进行 DL-DGC 选择-或不选择-，然后进一步进行两种最佳的获能处理（0.1μM 和 0.05μM 离子霉素）。然后如上所述确定精液的受精能力。DL-DGC/获能的精子样品显示出最高的平均穿透率：用 0.1μM 离子霉素获能后为 24.16%，用 0.05μM 离子霉素获能后为 12.21%。而未经 DL-DGC 选择的获能精子则返回明显较低的值：对于相同的离子霉素处理，分别为 6.26%和 7.02%。这些发现表明，将 DL-DGC 选择与离子霉素获能相结合可提高冻融马精子的受精能力。
