l‐Cysteine improves antioxidant enzyme activity,post‐thaw quality and fertility of Nili‐Ravi buffalo (Bubalus bubalis) bull spermatozoa

The effects of l ‐cysteine in extender on antioxidant enzymes profile during cryopreservation, post‐thaw quality parameters and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris–citric acid‐based extender having different concentrations of l ‐cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm ) and frozen in 0.5‐ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre‐freezing and post‐thawing in extender containing 2.0 mm l ‐cysteine as compared to other groups. Post‐thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curvilinear velocity (μm s^?1), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l ‐cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l ‐cysteine than in the control. In conclusion, the addition of 2.0 mm l ‐cysteine in extender improved the antioxidant enzymes profile, post‐thaw quality and in vivo fertility of Nili‐Ravi buffalo bull spermatozoa.     

Cryoprotectant effect of trehalose in extender on post‐thaw quality and in vivo fertility of water buffalo (Bubalus bubalis) bull spermatozoa

This study was designed to ascertain the cryoprotectant effects of different concentrations of trehalose [0 (T0), 25 (T25), 35 (T35), 45 (T45) mm ], egg yolk [20% (E20), 15% (E15) v/v] and glycerol [7% (G7), 5% (G5) v/v] in Tris‐citric acid‐based extender on post‐thaw quality and in vivo fertility of buffalo bull spermatozoa. Twenty‐five ejaculates were collected from five bulls and split into four parts. After that, the split ejaculates from each of the bull were diluted either in T0E20G7 (control) or T25E20G5 or T35E15G5 or T45E15G5 extender. Finally, the sperm suspension was frozen in 0.54‐ml French straws. Post‐thaw sperm total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm/s), straightline velocity (μm/s), curvilinear velocity (μm/s), linearity (%), plasma membrane and acrosome integrities (%) were higher (p < .05) in T45E15G5 extender as compared to other treatment groups and control. The fertility rate (56.8% versus 41.3%) was higher (p < .05) in buffaloes inseminated with semen doses cryopreserved in extender containing T45E15G5 combination of cryoprotectants than the control. In conclusion, addition of 45 mm trehalose along with 15% egg yolk and 5% glycerol in extender improves the post‐thaw quality and in vivo fertility of buffalo bull spermatozoa.     

Isolation of bacteria in semen and evaluation of antibiotics in extender for cryopreservation of buffalo (Bubalus bubalis) bull spermatozoa

This study was designed to investigate the occurrence of bacterial species in water buffalo semen at the time of collection/processing and to assess the efficacy of some selected antibiotics (GTLS; gentamycin, tylosin and linco‐spectin or SP; streptomycin and penicillin) in cryodiluent on bacterial control and quality including in vivo fertility of buffalo spermatozoa. For this purpose, four experiments were conducted. In experiment 1, a total of 11 bacterial species were isolated from buffalo ejaculates. In experiment 2, total aerobic bacterial counts at post dilution and thawing were lower (P < 0.05) in GTLS than in SP or control. The majority of the bacterial isolates from ejaculates were more susceptible to GTLS than SP. In experiment 3, sperm acrosome integrity was higher (P < 0.05) in GTLS and SP compared to control. In experiment 4, the in vivo fertility results for GTLS were higher (P < 0.05) than that for SP. In conclusion, a number of bacterial species were isolated from the bubaline semen, which requires an efficient control before its use in artificial insemination program. The GTLS combination of antibiotics may be incorporated into a freezing extender/protocol without compromising the post‐thaw quality and in vivo fertility of buffalo bull spermatozoa.     

Programmable fast‐freezing method improves the post‐thaw motion dynamics,integrities of plasmalemma,mitochondrial transmembrane,DNA and,acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN₂] for 10 min, plunging in LN₂; FR2, programmable medium, +4°C to ?15°C at 3°C min^?1, from ?15 to ?80°C at 10°C min^?1 and final holding for 1 min at ?80°C, plunging in LN₂; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to ?20°C at 10°C min^?1, from ?20°C to ?100°C at 30°C min^?1, final holding for 1 min at ?100°C and plunging in LN₂) were assessed on post‐thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s^?1), straight line velocity (μm s^?1), curved line velocity (μm s^?1), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast‐freezing method (FR3) improves the post‐thaw in vitro quality and in vivo fertility of water buffalo spermatozoa.     

Use of post‐thaw semen quality parameters to predict fertility of water buffalo (Bubalus bubalis) bull during peak breeding season

This study was designed to predict the fertility of water buffalo bull using post‐thaw semen quality parameters during peak breeding season. Thirty ejaculates were collected from five bulls with artificial vagina and cryopreserved. At post‐thaw, semen was analysed for motility parameters, velocity distribution, kinematics, DNA integrity/fragmentation, viability, mitochondrial transmembrane potential, morphology, plasma membrane and acrosome integrity. Data of 514 inseminations were collected for estimation of in vivo fertility. Pearson's correlation coefficients showed that progressive motility (PM), rapid velocity, average path velocity, straight line velocity, straightness, supravital plasma membrane integrity, viable spermatozoon with intact acrosome or with high mitochondrial activity were correlated with in vivo fertility (r = .81, p < .01; r = .85, p < .01; r = .64, p < .05; r = .73, p < .05; r = .57, p < .05; r = .88, p < .01; r = .84, p < .01 and r = .81, p < .01 respectively). Step forward multiple regression analysis showed that the best single predictor of fertility was PM. However, combinations of semen quality parameters to predict fertility were better as compared to single parameter. In conclusion, fertility of buffalo bull can be predicted through some of the post‐thaw in vitro semen quality tests during peak breeding season.     

Comparison of Tris egg yolk‐based,Triladyl® and Optixell® extender on post‐thaw quality,Kinematics and in vivo fertility of Nili Ravi Buffalo (Bubalus bubalis) bull spermatozoa

Our objectives were to ascertain the comparison of Tris egg yolk‐based, Triladyl ^® and Optixell ^® extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk‐based, Triladyl ^® , Optixell ^® extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk‐based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross‐frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell ^® extender as compared to Tris egg yolk‐based and Triladyl ^® extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk‐based and Triladyl ^® extender respectively. It is concluded that Optixell ^® extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.     

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender

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.