This study was designed to determine the effect of different osmotic pressures on spermatozoa characteristics of cryopreserved buffalo bull semen using tris egg yolk extender (TEYE). Semen of Nili-Ravi buffalo bulls (n = 4) housed at a semen production unit was collected at weekly intervals for 10 weeks. Three solutions of tris-citric acid-fructose with osmotic pressures of 255, 275 and 295 mOsm/kg were used in extender preparation. Semen straws containing 20 × 10 6 spermatozoa were processed and stored at −196°C in liquid nitrogen. Post-thaw analyses of spermatozoa included motility, viability, acrosomal integrity, plasma membrane integrity, DNA integrity and lipid per-oxidation. Significantly higher (P < .05) sperm motility, acrosomal and DNA integrity were recorded at osmotic pressures of 275 and 295 mOsm/kg compared to 255 mOsm/kg. However, differences in spermatozoa viability, plasma membrane integrity and lipid per-oxidation were non-significant among three osmotic pressures. It is concluded that osmotic pressure of the solution used in extender preparation plays an important role in post-thaw quality of cryopreserved buffalo bull semen.

In this study, efforts were made to investigate fresh semen parameters and to select a suitable extender for buffalo semen cryopreservation. Experiment I, fresh undiluted seminal parameters were determined while Experiment II, efficacy comparison of coconut water extender (CWE) with tris citric acid extender (TCAE) and skimmed milk extender (SME) was made. Four bulls single ejaculate was collected weekly for 5 and 10 weeks for Experiment I and II, respectively however osmotic pressure replicates were 16 and 6 for semen and seminal plasma, respectively. In Experiment I, each bull spermatozoa concentration, motility (%), semen volume and pooled semen percentage NAR, PMI, viability and MTT (3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction rate was checked. In Experiment II, pooled semen added to extenders and then equilibrated (4oC) and filled to obtain 20×106 spermatozoa/0.5 ml straws before plunging in liquid nitrogen. Percentage thawed spermatozoa viability, normal acrosomal ridge (NAR), motility, DNA damage, plasma membrane integrity (PMI) and lipid peroxidation (nM) recorded. In Experiment I, seminal parameters as spermatozoa concentration (1226.43±71.48 million/mL), semen volume (2.84±0.14 mL), viability (90.05±0.71%), motility (77.13±0.71%), PMI (86.23±0.34%), NAR (94.67±0.30%) and MTT reduction rate (0.290±0.06) while osmotic pressure of seminal plasma (294.83±3.87 mOsm/kg) and semen (290.87±2.58 mOsm/kg) was recorded. In Experiment II, TCAE higher (P<0.05) sperm motility noted compared to CWE and SME whereas percentage viability, NAR, PMI, DNA damage was non-significant. Lipid peroxidation compared to SME and TCAE was higher (P<0.05) in CWE. In conclusion, based on sperm motility and lipid per oxidation, TCAE was more efficient for cryopreservation of buffalo semen.