Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa

This study was carried out to identify the suitable buffer for cryopreservation of buffalo semen. Semen was collected with artificial vagina (42°C) from four buffalo bulls. Split pooled ejaculates (n=5), possessing more than 60% visual sperm motility, were extended at 37°C either in tri-sodium citrate (CITRATE), Tris–citric acid (TCA), Tris–Tes (TEST) or Tris–Hepes (HEPEST). Semen was cooled to 4°C in 2 h, equilibrated at 4°C for 4 h, filled in 0.5 ml straws and frozen in a programmable cell freezer before plunging into liquid nitrogen. Thawing of frozen semen was performed after 24 h at 37°C for 15 s. Sperm motion characteristics, plasma membrane integrity, and acrosome morphology of each semen sample were assessed by using computer-assisted semen analyzer (CASA), hypo-osmotic swelling (HOS) assay, and phase-contrast microscope, respectively. Analysis of variance revealed that percent post-thaw visual motility tended (P=0.07) to be higher in HEPEST (61.0±2.9) and lowest in CITRATE (48.0±2.5). Computerized motility did not vary due to buffering system. Percent post-thaw linear motility tended (P=0.09) to be higher in TCA (78.2±5.5) and lower in TEST (52.0±6.9). Circular motility (%) was significantly lower (P<0.05) in TCA (11.6±2.8) and higher in TEST (29.8±5.6). Curvilinear velocity (μm s−1) was lower (P<0.05) in TCA (69.4±2.0) than in CITRATE (79.0±5.8), TEST (87.2±1.6) and HEPEST (82.6±3.0). Lateral head displacement (μm) was lowest (P<0.05) in TCA (1.7±0.2) and highest in TEST (3.7±0.6). Plasma membrane integrity and normal acrosomes of buffalo spermatozoa did not differ due to buffering system and averaged 40.0±2.7% and 61.4±4.6%, respectively. Based upon lower circular motility, curvilinear velocity, and lateral head displacement, it is concluded that post-thaw quality of buffalo semen can be improved using the Tris–TCA buffering system.