ABSTRACT
Dead and damaged spermatozoa cells present universally in the ejaculates of all eutherian mammals exert toxic effect on contemporary healthy cells mostly through generation of excessive free radicals. This is much more evident during extended period of processing, resulting in poor ejaculate quality. The solution lies in depletion of dead/damaged spermatozoa from the neat ejaculates itself. Thus the objective of the study was to evaluate the efficiency of the protocols such as discontinuous PercollTM density gradient centrifugation (PDGC) and glass wool filtration (GWF) for depletion of dead/damaged spermatozoa from fresh semen in buffalo. Random ejaculates (n=6) of Murrah buffalo bulls were divided into two aliquots after quality assessment: PDGC and GWF protocols (Group I and II, respectively). At the end of the purification protocol, efficiency of the protocols in depleting dead/damaged spermatozoa as reflected by certain quality parameters were evaluated. The mean efficiency (%) of purification protocols based on recovery of spermatozoa was 44.68 and 40.02% for PDGC and GWF, respectively. Moreover significantly (p<0.05) greater values for quality parameters was observed in the Group II (26.4+6.8 vs 68.8+4.4 for acrosome integrity (%); 12.68+6.6 vs 57.7+7.5 for functional plasma membrane integrity (%); 20.3+5.8 vs 80.75+6.7 for viability (%) in Group I and II, respectively). It was concluded that GWF is a better technique than PGDC to filter out dead/damaged spermatozoa from fresh semen with improvement in semen quality and can be a valuable tool in assisted reproductive technology
ABSTRACT
The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.