García-Álvarez O, Maroto-Morales A, Jiménez-Rabadán P, Ramón M, del Olmo E, Iniesta-Cuerda M, Anel-López L, Fernández-Santos M R, Garde J J, Soler A J
SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain.
Theriogenology. 2015 Oct 1;84(6):948-55. doi: 10.1016/j.theriogenology.2015.05.032. Epub 2015 Jun 6.
Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin-hypotaurine alone, in combination with progesterone (P4), 17β-estradiol (E2), or BSA, or just β-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin-hypotaurine (alone), or in combination with P4, or E2, nor β-cyclodextrin induced capacitation-related changes in frozen-thawed ram spermatozoa. Only the medium supplemented with heparin-hypotaurine-BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin-hypotaurine, P4, E2, β-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.
获能是精子获得受精能力的关键过程。这一过程是体外受精等辅助生殖技术成功应用所必需的。本研究的目的是调查使用添加单独的肝素-牛磺酸、与孕酮（P4）、17β-雌二醇（E2）或牛血清白蛋白（BSA）联合使用、或仅添加β-环糊精的合成输卵管液培养基替代发情绵羊血清（ESS）用于公羊精子获能的效果。在相应培养基中孵育15分钟（时间0）或60分钟后，通过计算机精子活力分析和流式细胞术（质膜状态和流动性）评估精子功能。将在与获能相关的精子功能参数方面产生最佳结果的处理方法用于体外受精试验。在此，单独的肝素-牛磺酸、或与P4或E2联合使用、或β-环糊精均未诱导冻融公羊精子发生与获能相关的变化。只有添加肝素-牛磺酸-BSA的培养基能够诱导与体外获能相关的、与精子活力模式和质膜流动性相符的变化，与含ESS的培养基相当。两种培养基产生的精子参数值均与测试的其他培养基不同（P<0.05）。然而，在体外受精试验后，BSA无法支持与ESS相当的卵裂率（21.80%）（52.60%；P<0.05）。我们得出结论，肝素-牛磺酸、P4、E2、β-环糊精或BSA不适用于在公羊精子获能和受精培养基中替代ESS。
