SUMMARYThe effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN 2 ) for 10 min, plunging in LN 2 ; FR2, programmable ultra-fast, holding at +4°C for 2 min, from 4 to À10°C at À10°C/min, from À10 to À20°C at À15°C/min, from À20 to À120°C at À60°C/min, holding at À120°C for 30 sec, plunging in LN 2 ), and thawing rates (T1, 37°C for 30 sec; T2, 50°C for 15 sec; T3, 70°C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, lm/s), straight line velocity (VSL, lm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, lm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70°C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa.

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (μM/L) and lipid peroxidation levels (μM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, μm/s; straight-line velocity, μm/s; curved-line velocity, μm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender.

No abstract

The objective of the study was to devise a cryoprotection synergism between glycerol and dimethyl sulfoxide (DMSO) for water buffalo spermatozoa. Additionally, the effect of best evolved concentrations of glycerol and DMSO in extender was assessed on in vivo fertility of buffalo spermatozoa. Ejaculates (n = 30) were equally distributed into five aliquots; first aliquot was diluted at 37°C in extender having 7 % glycerol (control); second aliquot was diluted at 37°C as well as at 4°C in extender having 3.5 % DMSO (Group 1); third aliquot was diluted at 37°C in extender having 3.5 % glycerol and then at 4°C in extender having 3.5 % DMSO (Group 2); fourth aliquot was diluted at 37°C in extender having 3.5 % DMSO and then at 4°C in extender having 3.5 % glycerol (Group 3); fifth aliquot was diluted in extenders having 1.75 % glycerol and 1.75 % DMSO at 37 as well as at 4°C (Group 4). At post thawing, sperm progressive motility (%), rapid velocity (%), average path velocity (lm/s), curved line velocity (lm/s), in vitro longevity (%), structural and functional integrity of plasmalemma (%), mitochondrial transmembrane potential (%) and viable sperm with intact acrosome (%) were higher (P \ 0.05) in Group 4 compared to other treatment groups and control. Regarding sperm DNA integrity (%); it was higher (P \ 0.05) in Group 4 compared to Group 1, 3 and control. The in vivo fertility (%) of buffalo spermatozoa was significantly higher with Group 4 compared to control (69.45 vs. 59.81). In conclusion, synergism exists between glycerol and DMSO (Group 4) in improving the quality and in vivo fertility of cryopreserved water buffalo spermatozoa.