ABSTRACT
The aim of the present study was to investigate the effects of four equilibration times [2, 4, 8 and 16 hours] and two extenders [tris or Bioxcell] on cryopreservation of buffalo semen. In this experimental study, split pooled ejaculates [n=4], possessing more than 70% visual sperm motility were divided in two aliquots and diluted in Bioxcell and tris-citric egg yolk [TCE] extenders. Semen was cooled to 4°C within 2 hours, equilibrated at 4°C for 2, 4, 8 and 16 hours, then transferred into 0.5 ml French straws, and frozen in a programmable cell freezer before being plunged into liquid nitrogen. Postthaw motility characteristics, plasma membrane integrity, acrosome morphology and DNA integrity of the buffalo sperm were studied after thawing. There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. Post thaw sperm motility [PMOT], progressive motile spermatozoa [PROG], plasma membrane integrity [PMI] and normal apical ridge [NAR] measures were lower for sperm equilibrated for 2 hours in both TCE and Bioxcell extender compared to others equilibration times. PMOT, PMI and NAR for sperm equilibrated for 4, 8 and 16 hours showed no significant differences in either extender, although PROG measures were superior in Bioxcell compared to TCE at all equilibration times [p<0.05]. Kinematic parameters such as average path velocity, curvilinear velocity and linearity in the Bioxcell extender were superior to those in the TCE extender studied. In contrast to motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected by different equilibration times. Equilibration time is necessary for preservation of the motility and integrity of buffalo sperm membranes. Equilibration times of over than 2 hours resulted in the greatest preservation of total semen parameters during cryopreservation. There were no significant interactions between equilibration times over 4 hours and type of extender which lead to greater post thaw sperm survival
Subject(s)
Animals , Buffaloes , Chromatin , CryopreservationABSTRACT
The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws. In this experimental study semen was collected with artificial vagina [42[degree sign]C] from four buffalo bulls. Split pooled ejaculates [n=4] were extended at 37[degree sign]C with a Bioxcell[registered sign] extender. Semen was cooled to 4[degree sign]C within 2 hours, equilibrated at 4[degree sign]C for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen. Straws were thawed at water bath temperatures of 37, 50 or 70[degree sign]C for 30, 15 and 6 seconds, respectively. Semen was incubated at 37[degree sign]C for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa. Analysis of variance [ANOVA] was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan's multiple range tests. The initial postthaw motility [0 hour] averaged 62.7 +/- 7.2%, 73.1 +/- 9.77%, and 74.9 +/- 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70?C for 6 seconds were superior to other rates studied [p<0.05]. After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups [p>0.05]. A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures [p>0.05]. The percentage of spermatozoa with chromatin dispersion for the thaw rate of 70[degree sign]C for 6 seconds was significantly higher than for the to other rates studied [p< 0.05]. In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation. The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37[degree sign]C in 30 seconds to70[degree sign]C in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37[degree sign]C for two hours. A thaw rate of 70[degree sign]C for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls