CRYOPRESERVATION OF BUFFALO SEMEN

Abstract

This study was designed to establish the best cryopreservation procedures for buffalo semen. Therefore, the experiments were conducted 1) to identify the suitable buffer among tri-sodium citrate (CITRATE), Tris-citric acid (TCA), Tris-Tes (TEST) or TrisHepes (HEPEST), 2) to examine the effects of stages of cryopreservation i.e. dilution (D), cooling to 4°C (C), equilibration at 4°C for 4 h (E), and freezing and thawing (FT), 3) if fa ster freeze rates either during initial and subsequent freezing would improve the post-thaw semen quality and where the intermediate zone of damage to spermatozoa lies, and 4) whether different concentrations of glycerol and/or dimethyl sulfoxide (DMSO), either added at 37 or 4°C, would alter sperm motion characteristics, plasma membrane integrity, and acrosome morphology in buffaloes while using computer-assisted semen analyzer (CASA), hypo-osmotic swelling (I-lOS) or flow cytometry, and phase-contrast mIcroscope, as assays. In experiment 1, post-thaw visual motility (%) of spermatozoa tended (P = 0.07) to be hi gher in I-IEPEST (6l.0 ± 2.9) and lower in CITRATE (48.0 ± 2.5). However, computerassisted motility did not differ due to buffers. Post-thaw linear motility (%) of spermatozoa tended (P = 0.09) to be higher in TCA (78 .2 ± 5.5) and lower in TEST (52.0 ± 6.9), whereas circular motility (%) was lower (P < 0.05) in TCA (11.6 ± 2.8) and higher in TEST (29.8 ± 5.6). Post-thaw sperm curvilinear velocity Cum S- I) was lower (P < 0.05) in TCA (69.4 ± 2.0) than CITRATE (79.0 ± 5.8), TEST (87.2 ± 1.6) and HEPEST (8 2.6 ± 3.0). Post-thaw sperm lateral head displacement Cum) was lowest (P < 0.05) in TCA (1.7 ± 0.2) and highest in TEST (3.7 ± 0.6). Nonetheless, plasma membrane integri ty and normal acrosomes of buffalo spermatozoa did not vary due to buffering systems. In experiment 2, visual and computer-assisted motility (%) of spermatozoa did not differ due to dilution, cooling, or equilibration (77.3 ± 2.3 and 90.5 ± 1.2, respectively), whereas these were declined (P < 0.05) after freezi ng and thawing (53.0 ± 4.6 and 48.6 ± 6.5, res pectively). Linear motility (%) of spermatozoa was lower (P < 0.05) after dilution or equi libration (56.2 ± 2.4) compared to that after cooling and freezing and thawing (79.6 ± 1.4). Sperm curvilinear velocity Cum S-I) was reduced (P < 0.05) from 11 2.4 ± 5.3 at dilution to 96.0 ± 5.8 at cooling, and from 87.6 ± 4.1 at equilibration to 69.4 ± 2.0 at freezing and thawing. Sperm lateral head displacement Cum) varied (P < 0.05) at each stage, i.e. dilution, 3.9 ± 0.2; cooling, 2.3 ± 0.2; equilibration, 3.1 ± 0.3 and freezing and thawing, 1.7 ± 0.2. Plasma membrane integrity (%) of spermatozoa was 80.2 ± 3.9 at dilution. It reduced (P < 0.05) to 60.4 ± 5.6 at equilibration and then to 32.6 ± 3.8 at freezing and thawing. Normal acrosomes (%) of spermatozoa remained higher after dilution, coolin g or equilibration (73 .2 ± 2.4) while decreased (P < 0.05) after freezing and thawing (6 1.8 ± 2.4). Experiment 3 consisted of freezing rates, a) slow or moderate, and b) moderate, fast, or very fast, changed between 4 to - 15°C and - IS to - 80°C, respectively. Freezing rates either during initial and subsequent freezing did not improve the post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of spermatozoa. In the experiment of lethal intermediate zone, visual motility, computer-assisted motility and lateral head di splacement reduced (P < 0.05) at -40°C, curvilinear velocity at -50°C, whereas plasma membrane integrity and normal acrosomes were adversely affected at -30°C. In the subsequent experiment of lethal intermediate zone, plasma membrane integrity also reduced (P < 0.05) at -30°C, either assessed by HOS or flow cytometric assay. The correlation between these assays at various temperahlres was significant (I' = 0.90; P < 0.05), that suggested the precision of flow cytometry for the evaluation of spermatozoa in buffaloes. In experiment 4, post-thaw sperm motilities (visual and computer-assisted), velocities (straight- line, average path, and curvilinear) lateral head displacement, and plasma membrane integrity were higher (P < 0.05) in extenders containing 6% glycerol than in extenders wi th 3 or 0% glycerol. However, post-thaw visual motility, computer-assisted moti lity and plasma membrane integrity of spermatozoa decreased (P < 0.01) in the extenders either having 1.5 or 3% DMSO than that of 0% DMSO. The average values of these variables were maximum (P < 0.01) in extenders containing 6% glycerol and 0% DMSO than other combinations of glycerol and DMSO. Post-thaw visual motility, computer-assisted motility, straight-line velocity, and average path velocity of spermatozoa were maximum (P < 0.05) in extenders hav ing 6% glycerol being added at 37°C than the other adding temperatures of glycerol. Post-thaw computer-assisted motility of spermatozoa vvas highest (P = 0.04) in those extenders where 6% glycerol and 0% DMSO were added at 37°C than other combinations of glycerol and DMSO additions (37 or 4°C). Coll ect ively, these experiments suggest that in buffaloes, 1) post-thaw quality of semen can be improved using the Tris-citric acid buffering system, 2) considerable damage to motility apparatus, plasma membrane and acrosomal cap of spermatozoa occurs during freezing and thawing followed by equilibration processes, 3) fast freezing either during initial or subsequent freezing did not improve the post-thaw sperm viability, whereas the intermediate zone of damage to sperm motility apparatus and membrane integrity lies somewhe re between -20 to -40°C, and 4) DMSO at the levels investi gated did not improve the post-thaw quality of spermatozoa, however, glycerol in 6%, when added at 37°C, provided the maximum cryoprotection to the motility apparatus, and pl asma membrane integrity of spermatozoa.