M.R. Ansari

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Effects of hydrogen peroxide (H2O2) on fresh and cryopreserved buffalo sperm functions during incubation at 37°C in vitro.

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H2O2 was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris-egg yolk-citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5 degrees C) and cryopreserved in 0.5-ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen-thawed semen was separated by centrifugation (1500 g; 15 min) and were washed with sperm TALP. The sperm cells were re-suspended in incubation TALP at the rate of 10(8) sperm cells per millilitre and incubated with 0, 10, 25, and 50 microm H2O2 per ml at 37 degrees C. Sperm motility, viability and intact acrosome percentages were assessed at 15-min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H2O2. Among the different levels of H2O2, the 50-microm H2O2-incorporated group had significantly (p<0.05) higher malonaldehyde (MDA) level than the other groups. In the 50-microm H2O2-incorporated group, the MDA levels in fresh, equilibrated and frozen-thawed semen after incubation for 60 min were 961.6+/-12.7, 991.8+/-10.3 and 1234.9+/-9.6 nm per 10(9) spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H2O2 and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H2O2 was significantly (p<0.05) higher in frozen-thawed than fresh and equilibrated spermatozoa.

Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.

Survival of goat embryos frozen and thawed rapidly

Abstract Eighty goat embryos at the 16-cell stage were frozen to −196°C following a two-step method of rapid cooling. Dimethyl-sulphoxide (DMSO) or glycerol at a concentration of 1.5 M was used as cryoprotectant. After storage for 1 month at −196°C, the embryos were rapidly thawed at ∼500°C min −1 , employing a sucrose dilution procedure for the removal of cryoprotectants. On transplantation of 16 embryos frozen in glycerol and 39 embryos frozen in DMSO into foster mothers, six and 16 kids were born, respectively. Both the cryoprotectants afforded a similar cryoprotection.

Effect of oviductal proteins on structural and functional characteristics of cryopreserved sperm in Murrah buffaloes.

The present study was undertaken to elucidate the effect of non-luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non-luteal based on ovarian morphology. Non-luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10,000 g at 4 degrees C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at -20 degrees C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris-citrate-egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non-luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5 degrees C, filled in 0.5 ml French straws, exposed to LN(2) vapour, plunged into LN(2) and then stored at -196 degrees C. The equilibrated and frozen-thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo-osmotic sperm swelling test (HOST). In frozen-thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non-luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze-thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen-thawed semen. It was inferred that incorporation of non-luteal whole oviductal fluid proteins improved the sperm quality in frozen-thawed semen in Murrah buffaloes.