IMPROVING QUALITY OF CRYOPRESERVED BUFFALO BULL SPERMATOZOA

Abstract

Cryopreservation techniques currently in use to preserve buffalo spermatozoa demand improvements in the freezing protocols in order to overcome the cryo-damage that may occur during the process of cryopreservation. Such damage may profoundly affect the in vitro quality of spermatozoa in terms of longevity, motility and in vivo fertilization potential. In this connection, the potential of a series of cryodiluents was investigated by applying these into freezing extenders to achieve the desired quality and productivity for water buffalo bull spermatozoa. Experiment 1; The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2) semen loaded French straws (0.5 ml) for 10 min, plunging into LN2; and FR2, programmable ultra-fast, holding French straws (0.5 ml) at +4°C for 2 min, from 4 to −10°C at −10°C/min, from −10 to −20°C at −15°C/min, from −20 to −120 °C at −60°C/min, holding at −120°C for 30 sec, plunging into LN2), and thawing rates (T1, 37 °C for 30 sec; T2, 50°C for 15 sec; T3, 70°C for 7 sec) were evaluated in tris−citric acid extender on the quality of buffalo bull spermatozoa. Progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, μm sec−1), straight line velocity (VSL, μm sec−1), and the mitochondrial transmembrane potential (MMP, %) were found to be greater (P < 0.05) with E2, FR2, and T3 as compared to other groups. Sperm curvilinear velocity (VCL, μm sec−1) was greater (P < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (STR, %) and linearity (LIN, %) were also greater (P < 0.05) with E2 when compared with the other groups. Supravital-plasma membrane integrity (SV-PMI, %), viability and acrosome integrity (V-IACR, %) of spermatozoa were greater (P < 0.05) with E2 and FR2 as compared to other groups. Sperm DNA integrity (DNA-I, %) was also greater (P < 0.05) with FR2 and T1 as compared to other groups. These experiments concluded that using 4 h equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in the cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa. Experiment 2; The antioxidant effect of different concentrations of curcumin at the rate of 0.5, 1.0, 1.5 and 2.0 mM was evaluated in tris−citric acid extender on xiii freezability of buffalo spermatozoa. At pre-freezing and post-thawing, total antioxidant contents (μM/L) were noticeably higher (P < 0.05) with 1.5 and 2.0 mM curcumin as compared to 0.5 and 1.0 mM curcumin and control. At pre-freezing, lipid peroxidation levels (LPO, μM/ml) were lower with 1.5 mM curcumin, while at post-thawing, the LPO levels were lower with 1.5 mM and 2.0 mM curcumin as compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, PM (%), RV (%) and kinematics (VAP, μm sec−1; VSL, μm sec−1; VCL, μm sec−1; STR, %; LIN, %), in vitro longevity (%, PM and RV) and DNA-I (%) were significantly higher (P < 0.05) with 1.5 mM curcumin compared to control. At post-thawing, SV-PMI (%) and V-IACR (%) were higher with 1.5 curcumin compared to 2.0 mM curcumin and control. These experiments demonstrated that the freezability of water buffalo spermatozoa is appreciably improved with the addition of 1.5 mM curcumin in the extender. Experiment 3; The effect of different concentrations of UV-C irradiated chicken egg yolk plasma (EYP; v/v; 10%, P1; 15%, P2; 20%, P3) or 20% (v/v) of whole chicken egg yolk (WCEY) in tris−citric acid extender was evaluated on water buffalo sperm quality during cryopreservation (post–dilution, PD; post–equilibration, PE; post–thawing, PT). The effect of best evolved concentration of EYP in extender on in vivo fertility capability of buffalo spermatozoa was also evaluated. Overall, at post-thawing, CASA PM (%), RV (%), VAP (μm sec−1) and VSL (μm sec−1), SV-PMI (%), MMP (%), V-IACR (%), and DNA-I (%) were higher in P3 treatment as compared to other treatments and WCEY. The decline percentage (%, longevity) in PM and RV was lower in P3 as compared to WCEY during 2 h incubation under in vitro conditions at PT. The in vivo fertility rate (%) was significantly higher with P3 as compared to WCEY (76.61 vs. 64.49). This part of the study concluded that WCEY (20%) can be replaced with UV-C irradiated chicken EYP (20%) in tris–citric acid extender for cryopreservation and in vivo fertility of water buffalo spermatozoa. Experiment 4; Another objective of the study was to determine a possible cryoprotection synergism between glycerol and DMSO for water buffalo spermatozoa. The dilution of semen was made with five extenders comprising 7% glycerol at 37 °C, control; 3.5% DMSO at 37°C as well as at 4°C, Group 1; 3.5% glycerol at 37°C and 3.5% DMSO at 4°C, Group 2); 3.5% DMSO at 37°C and 3.5% glycerol at 4°C, Group 3; and 1.75% glycerol and 1.75% DMSO at 37°C as well as at xiv 4°C, Group 4). Moreover, the effect of best concentrations of glycerol and DMSO in extender was assessed on in vivo fertility of buffalo spermatozoa. At post thawing (PT), sperm PM (%), RV (%), VAP (μm sec−1), VCL (μm sec−1), in vitro longevity (%), SV-PMI (%), MMP (%), V-IACR (%) and DNA-I (% except Group 2) were higher (P < 0.05) in extenders having 1.75% glycerol and 1.75% DMSO at 37 as well as at 4°C (Group 4) as compared to other treatment groups and the control. The in vivo fertility rate (%) was significantly greater with Group 4 as compared to the control (69.45 vs. 59.81). These experiments concluded that a synergism between glycerol and DMSO (Group 4) potentially improves the quality and in vivo fertility of cryopreserved water buffalo spermatozoa. On the whole, this study concludes that 4 h equilibration time, programmable ultra-fast freezing rate (+4°C for 2 min, from 4 to −10°C at −10°C/min, from −10 to −20°C at −15°C/min, from −20 to −120°C at −60°C/min, holding at −120°C for 30 sec), and rapid thawing at 70°C for 7 sec in the cryopreservation protocol improve the post-thaw quality of buffalo bull spermatozoa. The supplementation of 1.5 mM curcumin as antioxidant in the extender provided better cryoprotection for freezability of water buffalo spermatozoa. The UV-C irradiated chicken EYP (20%) can be effectively used as an alternate to WCEY (20%) in tris–citric acid extender for cryopreservation and in vivo fertility of water buffalo spermatozoa. A synergism between glycerol and DMSO (addition of 1.75% glycerol and 1.75% DMSO at 37°C as well as at 4°C, Group 4) potentially improves the quality as well as in vivo fertility of cryopreserved water buffalo spermatozoa. The findings of this study would be helpful for optimization of freezing protocols currently practiced for the cryopreservation of buffalo spermatozoa.