Kumaresan A, Ansari M R, Garg Abhishek, Kataria Meena
Division of Animal Reproduction, Indian Veterinary Research Institute, Izat Nagar, Bareilly 243122, Uttar Pradesh, India.
Anim Reprod Sci. 2006 Jul;93(3-4):246-57. doi: 10.1016/j.anireprosci.2005.06.030. Epub 2005 Sep 29.
A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen 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 split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN2 (-196 degrees C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1mg/ml of extended semen. The equilibrated and frozen thawed (37 degrees C for 30s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly (P < 0.05) higher sperm penetration distance in cervical mucus (23.00+/-1.15 mm) than the control group (15.00+/-3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly (P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly (P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.
开展了一项研究，以探究添加输卵管蛋白对水牛精子功能和脂质过氧化（LPO）水平的影响。从外观健康的水牛生殖道（发情周期的非黄体期和黄体期）收集输卵管冲洗液，离心（3000转/分钟；30分钟），过滤（0.2微米），并在-20℃下冷冻。将合并的非黄体期和黄体期输卵管液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（分子量大于10 kDa）。蛋白质定量后，将含有10毫克蛋白质的样品等分试样冻干于冻存管中，并在-20℃下冷冻保存。通过人工阴道法从两头摩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三部分，并用Tris-蛋黄-柠檬酸盐稀释液（20%蛋黄：7%甘油）进行稀释，以使最终稀释液产生约6000万个精子细胞/毫升，并在0.5毫升法式细管（每管3000万个精子细胞）中于液氮（-196℃）中冷冻保存。冷冻前，将非黄体期和黄体期输卵管蛋白（NLOP和LOP）以1毫克/毫升的稀释精液浓度加入。对平衡和冻融（37℃，30秒）后的精液进行活力、存活率和顶体完整性评估、牛宫颈黏液穿透试验和低渗精子肿胀试验。除了这些试验外，还评估了平衡和冻融精液中精子和精浆中的LPO水平。结果显示，冷冻前向精液中添加输卵管蛋白在精子活力、存活率和顶体完整性方面具有有益作用。在冻融精液中，非黄体期输卵管蛋白组的精子在宫颈黏液中的穿透距离（23.00±1.15毫米）显著高于对照组（15.00±3.46毫米）（P<0.05）。同样，NLOP处理组的肿胀精子百分比也显著高于包含LOP的组和对照组（P<0.05）。在冻融精子中，添加NLOP组的LPO水平显著低于添加LOP组和对照组（P<0.05）。据推断，冷冻前在稀释液中加入输卵管蛋白可降低水牛精子在冷冻保存期间的脂质过氧化水平，从而提高解冻后精液质量。
