Kumaresan A, Ansari M R, Garg Abhishek
Division of Animal Reproduction, Indian Veterinary Research Institute, Izat Nagar, Bareilly, Uttar Pradesh.
Anim Reprod Sci. 2005 Nov;90(1-2):73-84. doi: 10.1016/j.anireprosci.2005.01.009. Epub 2005 Jun 9.
A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.
本研究旨在确定从发情周期不同阶段获得的输卵管蛋白对水牛精子特性的影响。从外观健康的水牛生殖道（发情周期的非黄体期和黄体期）采集输卵管，并分为峡部和壶腹部。每个输卵管段（非黄体期和黄体期）用PBS（pH 7.4）冲洗。将获得的冲洗液离心（3000转/分钟；30分钟），过滤（0.2微米），并在-20℃冷冻。合并的非黄体期峡部和壶腹部以及黄体期峡部和壶腹部液体中的蛋白质用硫酸铵沉淀过夜，离心（10000转/分钟；30分钟）并透析（>10 kDa）。蛋白质定量后，将含有10毫克蛋白质的样品等分试样在冻存管中冻干，并以冷冻形式保存在-20℃。通过人工阴道法从两头穆拉水牛公牛收集的六个合并的优质射精用于该研究。新鲜精液分析后，将每个合并的射精分成五部分，并用Tris-蛋黄-柠檬酸盐稀释液（20%蛋黄；7%甘油）稀释，以使最终稀释液每毫升产生约6000万个精子细胞，并在0.5毫升法式细管（3000万个精子细胞/细管）中于液氮（-196℃）中冷冻保存。冷冻前，将非黄体期峡部和壶腹部以及黄体期峡部和壶腹部蛋白质以1毫克/毫升的延长精液的比例加入。对平衡并冻融（37℃ 30秒）的精液进行活力、活率、顶体完整率、牛宫颈黏液穿透试验和低渗精子肿胀试验评估。此外，将处理组和对照组的精子在精子TALP中于37℃孵育6小时。在非黄体期和黄体期输卵管蛋白中，前者解冻后精子活力、活率和顶体完整性高于对照组（P<0.05）。在峡部蛋白和壶腹部蛋白之间，加入峡部蛋白组解冻后精子活力、活率和顶体完整性更高（P<0.05）。同样，在加入非黄体期峡部蛋白组中，记录到精子在宫颈黏液中的穿透距离更长。但是，无论发情周期阶段如何，加入峡部蛋白组的精子膜完整性更高，这表现为对低渗溶液反应时肿胀精子百分比高于加入壶腹部蛋白组和对照组（P<0.05）。同样，在孵育的任何时间，加入峡部蛋白处理组的精子活力和存活率高于壶腹部蛋白组和对照组（P<0.05）。但是，在顶体完整率方面未观察到相同趋势。据推断，冷冻前在稀释液中加入输卵管蛋白可改善解冻后精液质量。输卵管蛋白根据输卵管区域和获得蛋白时的发情周期阶段对精子功能有不同影响。
