Background: Fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa through artificial insemination (AI) is reported to be affected by seasonality. The quality of frozen–thawed semen is one of the most influential factors affecting the probability of pregnancy. This study was designed to investigate various semen quality parameters to predict the in vivo fertility of buffalo bull during low and peak breeding seasons. In this study, we have also investigated the effect of seasons on structural-functional parameters and in vivo fertility of buffalo bull spermatozoa. In the last study, we have investigated the effect of hydrogen peroxide (H 2 O 2 ) on semen quality parameters having the capability of prediction of fertility with ultimate aim to validate them for buffalo bull spermatozoa. Materials and Methods: Semen was collected from five mature water buffalo bulls with artificial vagina maintained at 42 °C during low and peak breeding seasons. After collection, semen samples were transferred to the laboratory immediately for initial evaluation. Sperm progressive motility was assessed using phase contrast microscope (x 400) connected with closed circuit monitor and sperm concentration was measured by using the specific spectrophotometer at a wavelength of 546 nm. Qualifying ejaculates having > 1 mL volume, > 60 % sperm progressive motility and > 0.5 x 10 9 spermatozoa/mL concentration from each bull were diluted in Tris–citric acid egg yolk glycerol extender (TCA). For cryopreservation, semen of each bull was extended in TCA extender at 37 °C, cooled to 4 °C in 2 hr, and equilibrated for 4 hr in cold cabinet (4 °C). Extended semen was then packed in polyvinyl French straws (0.54 mL) and frozen in a programmable cell freezer. Finally, semen straws were plunged into liquid nitrogen (–196 °C) for storage until analyses. For in vivo fertility, data of at least 100 inseminations per bull were collected under controlled field conditions per season. In order to study the effect of H 2 O 2 on semen quality parameters like computer-aided sperm motion analysis (CASA) aspects, subjective motility (SM, %), supra–vital plasma membrane integrity (SV–PMI, %), viability/ mitochondrial transmembrane potential and viability/ acrosome integrity of buffalo bull spermatozoa, qualifying ejaculates from five buffalo bull were diluted at the rate of 25 x 10 6 /mL) in PBS–0.1% BSA extender containing 10 μM H 2 O 2 , equilibrated for 5 min followed by measurement of semen quality parameters at different time intervals (0 min, 30 min, 60 min, 90 min and 150 min). Furthermore, for studying the effects of H 2 O 2 on sperm DNA fragmentation indices, semen samples were diluted in 1PBS–0.1% BSA extender containing 0.0 mM, 25 mM, 50 mM, 75 mM and 100 mM H 2 O 2 respectively, equilibrated for 1 hr to induce damage and then followed by neutral comet assay protocol. Results: In experiment 1, we have investigated various semen quality parameters to predict the in vivo fertility of buffalo bull during low breeding season. Pearson’s correlation coefficients showed that sperm progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, μm/s), straight line velocity (VSL, μm/s) and rapid subpopulation 1 (%) of buffalo bull were significantly correlated with in vivo fertility during low breeding season (r = 0.64, P < 0.01; r = 0.57, P < 0.01; r = 0.52, P < 0.01; r = 0.56, P < 0.01 and r = 0.73, P < 0.001). Moreover, sperm SV–PMI and viable spermatozoa with intact acrosome (V/IACR, %) were significantly correlated with in vivo fertility (r = 0.74; P < 0.001 and r = 0.88; P < 0.001), whereas nonviable spermatozoa with damaged acrosome or low mitochondrial transmembrane potential (NV/DACR or NV/LP, %) were negatively associated with in vivo fertility during low–breeding season (r = 0.79; P < 0.001 and r = 0.75; P < 0.001). Comet length (CL, μm) parameter of neutral comet assay was negatively associated with in vivo fertility during low-breeding season (r = –0.60, P < 0.05). Step forward regression analyses showed that the prognostic value to predict the in vivo fertility by the equation of VAP, VSL, curvilinear velocity, rapid subpopulation 1, SV–PMI, V/IACR, and viable with high mitochondrial transmembrane potential (V/HP, %) accounted for 81.30 % (P < 0.001) during low breeding season. In experiment 2, we investigated various semen quality parameters to predict the in vivo fertility of buffalo bull during peak breeding season. Pearson’s correlation coefficients showed that sperm PM, RV, VAP, VSL, straightness (STR, %) and rapid subpopulation 1 (%) of buffalo bull were significantly correlated with in vivo fertility during peak breeding season (r = 0.81, P < 0.01; r = 0.85, P < 0.01; r = 0.64, P < 0.05; r = 0.73, P < 0.05; r = 0.57, P < 0.05 and r= 0.65, P < 0.05). Furthermore, sperm SM, SV–PMI, V/IACR and V/HP were positively correlated with in vivo fertility during peak breeding season (r = 0.79; P < 0.01, r = 0.88, P < 0.01, r = 0.84, P < 0.01 and r = 0.81, P < 0.01), whereas NV/DACR or NV/LP were negatively correlated with in vivo fertility during peak breeding season (r = –0.81, P < 0.01 or r = –0.81, P < 0.01). Tail length (TL, μm) were negatively correlated with in vivo fertility during peak breeding season (r = –0.70, P < 0.05). Moreover, the best predictive equation (R 2 adjusted=83.50 %, P < 0.000) of fertility 2for frozen–thawed buffalo semen included PM, RV, VAP, VSL and SV–PMI during peak breeding season. In experiment 3, we investigated the effect of season on structural-functional parameters and in vivo fertility of buffalo bull spermatozoa during peak and low breeding seasons. Analysis of variance showed that ejaculate volume (mL), concentration of spermatozoa (mL), total motility (TM, %), PM, VAP, VSL, STR, linearity (LIN, %) rapid subpopulation 1, SV–PMI, V/IACR, V/HP and DNA integrity in fresh semen were significantly higher (P < 0.05) during peak than low breeding season. At post–thawing, sperm TM, rapid subpopulation 1, SV–PMI, V/IACR, V/HP and DNA integrity were significantly higher (P < 0.05) during peak than low breeding season. In vivo fertility of frozen–thawed buffalo spermatozoa processed during peak breeding season was significantly higher (P < 0.05) than semen samples cryopreserved during low breeding season in a fertility trial carried out during low breeding season (58.98 % vs. 52.49 %). In experiment 4, we have studied the effect of H 2 O 2 on semen quality parameters having the capability of prediction of fertility with ultimate aim to validate them for buffalo bull spermatozoa. It was found that H 2 O 2 at a dose of 10 μM diminished PM, RV, motion kinematics (VAP, VSL and curvilinear velocity), SV–PMI, V/HP and V/IACR of buffalo bull in a time dependent manner as compared to control. Moreover, exposure of H 2 O 2 significantly increases (P < 0.05) comet length, tail length, tail DNA, tail moment and olive moment in a dose dependent manner as compared to control. Similarly, exposure of H 2 O 2 significantly increases (P < 0.05) DNA fragmentation in a dose dependent manner than control. Conclusion: It is concluded that assessment of CASA parameters and sperm structural and functional parameters viz. SV-PMI, viability/ acrosome integrity and viability/ mitochondrial transmembrane potential were able to predict the in vivo fertility of water buffalo bull during low and peak breeding seasons. Moreover, ejaculates collected/ processed during peak breeding season demonstrated better semen quality and in vivo fertility in a fertility trial conducted during low breeding season and are thus more suitable for AI than ejaculates collected during low breeding season. Finally, H 2 O 2 negatively modulated semen quality parameters and may be used for monitoring the effectiveness of buffalo bull semen quality and must be included in optimization procedures. It is expected that application of these findings will improve the outcome of AI in water buffalo.