Srivastava N, Srivastava S K, Ghosh S K, Jerome A, Das G K, Mehrotra S
Animal Reproduction Division, Indian Veterinary Research Institute, Izatnagar, UP, India.
Reprod Domest Anim. 2013 Oct;48(5):724-31. doi: 10.1111/rda.12151. Epub 2013 Feb 26.
PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures.
PDC - 109是牛精浆中含量最丰富的蛋白质之一，对精子具有时间和浓度依赖性的有害作用。因此，我们推测在精子细胞冷冻保存后，从射精中隔离有害蛋白质将是有益的。为此，我们评估了通过抗PDC - 109抗体（Ab）或单独的蛋黄（EY）或EY + Ab的协同作用来隔离PDC - 109对减少公牛精子冷冻损伤的效果。通过两步色谱法纯化PDC - 109蛋白。将纯化的蛋白注射到兔子体内以产生抗体，然后使用离子交换色谱法分离这些抗体。在检查抗体交叉反应性后，对其进行定量并添加到射精中，单独添加或与Tris - 甘油（TG）稀释剂中的EY一起添加。因此，射精在含有EY + TG（I组）、Ab + TG（II组）或EY + Ab + TG（III组）的稀释剂中进行处理。评估精液质量参数（SQPs），即活力和顶体完整性（FITC - PSA）、精子的冷冻损伤（金霉素，CTC测定）以及蛋白质隔离精液的体外受精能力（透明带穿透试验）。与其他组相比，III组在冷冻保存的预冻和解冻阶段观察到解冻后SQPs以及未获能精子有显著（p < 0.05）改善，这表明蛋白质介导的冷冻损伤减少。从这项研究可以得出结论，与单独使用EY或Ab相比，EY + Ab的协同作用隔离PDC - 109可显著提高精子质量，并在超低温储存时将公牛精子的冷冻损伤降至最低。
