Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes

Abstract

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 μm) and frozen at −20 °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 °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 LN 2 (−196 °C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1 mg/ml of extended semen. The equilibrated and frozen thawed (37 °C for 30 s) 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.