Synergistic protective effects of 3,4-dihydroxyphenylglycol and hydroxytyrosol in male rats against induced heat stress-induced reproduction damage.

Testicular heat stress disrupts spermiogenesis and damages testicular tissue. The study aims to assess 3,4-dihydroxyphenylglycol (DHPG) and hydroxytyrosol (HT) from olive oil as antioxidants to reduce heat-induced testicular damage. Seven groups of 35 male rats were used. Group I got normal saline. Group 2 had HS (43 °C for 20 min/day) and normal saline for 60 days. Groups 3-7 had HS and DHPG/HT doses (0.5 mg/kg DHPG, 1 mg/kg DHPG, 5 mg/kg HT, 0.5 mg/kg DHPG + 5 mg/kg HT, and 1 mg/kg DHPG + 5 mg/kg HT). The evaluation included tests on testicular tissue, sperm quality, oxidative status, gene activity, and fertility after 60 days. After DHPG and HT treatment, sperm motility, viability, and plasma membrane functionality, as well as levels of total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), and Bcl-2 gene expression, and in vivo fertility indexes increased. Meanwhile, abnormal morphology and DNA damage decreased, along with levels of glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA), and Bax, caspase-3, and caspase-9 gene expression, compared to the HS group. The study found that DHPG and HT have a more substantial synergistic effect when used together, improving reproductive health.

Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 µM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 µM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 µM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Effect of C-phycocyanin purified from Spirulina platensis on cooled ram semen quality and in vivo fertility.

This research sought to purify C-phycocyanin (C-PC) from Spirulina platensis and investigate its potential in enhancing the quality parameters and in vivo fertility of ram semen subjected to cooled storage at 5 °C, when using a skim milk (SM) based semen extender. The purification process of C-PC involved cold maceration, pre-purification using chitosan and activated charcoal, followed by purification through aqueous two-phase extraction (ATPE) and ion-exchange chromatography. Afterward, fifty ejaculates were collected from 4 fertile Boujaâd rams and extended using the SM extender at 37 °C, enriched with 0 µg/mL (control), 1.2 µg/mL, 2.4 µg/mL, 3.6 µg/mL, or 4.8 µg/mL of C-PC. The diluted semen was subsequently cooled to 5 °C using a controlled cooling process, with a gradual cooling rate of approximately 0.5 °C per minute, and its quality parameters were evaluated after 0, 4, 8, and 24 h of cooling storage. Then, its fertilization ability after 4 h of cooling storage was evaluated using artificial insemination. The adopted purification process yielded a grade analytical purity of 4.06. Additionally, semen extended in SM with a 2.4 µg/mL C-PC supplement displayed significant (P < 0.0001) enhancement in total motility, progressive motility, curvilinear velocity, straight-line velocity, average path velocity, viability and lipid peroxidation of ram semen at 0, 4, 8, and 24 h of cooling storage. These improvements were observed in direct comparison to both the control group and the other C-PC concentrations. Regarding fertility rates, semen extended in SM with a 2.4 µg/mL C-PC recorded a 76 % rate, a notable increment from the 63 % observed in ewes inseminated by semen extended in SM alone, although the difference was not statistically significant (p > 0.05). These findings underscore the promising potential of C-PC as a natural supplement for enhancing semen quality, warranting further investigations.

Effect of platelet-rich plasma (PRP) on post-thaw quality, kinematics and in vivo fertility of fertile and subfertile buffalo (Bubalus bubalis) spermatozoa.

This study investigated the effect of adding platelet-rich plasma (PRP) in semen extender prior cryopreservation on post-thaw quality, kinematics, and in vivo fertility of fertile and subfertile buffalo spermatozoa. Eleven buffalo bulls were classified based on their conception rate (CR) into fertile (n = 8, CR > 55%) and subfertile (n = 3, CR < 35%) groups. Ejaculates were collected with artificial vagina, pooled, and dispensed into 6 aliquots, diluted with Tris-egg yolk-glycerol extender supplemented with different proportions of PRP [0% (control), 5%, 10%, 15%, 20%, and 25%] followed by cryopreservation using standard procedures. Post-thaw sperm quality, kinematics, antioxidant activity, cryosurvival rate, and in vivo fertility were compared between fertile and subfertile groups and among proportions of PRP within each group. The results showed that 15% PRP greatly (P < 0.001) improved sperm characteristics, average path velocity, and curvilinear velocity of the subfertile group. Interestingly, 5%, 10%, and 15% PRP greatly (P < 0.001) reduced malondialdehyde content and improved enzymatic (glutathione peroxidase and superoxide dismutase) and total antioxidant capacity in fertile and subfertile groups. However, these three proportions of PRP significantly (P < 0.001) improved the cryosurvival rate of the subfertile group; only 15% PRP greatly improved CR of subfertile (60.83% vs. 34.17%) animals to be comparable with that of fertile ones treated with 5 (59.17%) and 10% (60.83%) PRP. In conclusion, adding 15% PRP to semen extender before cryopreservation is recommended to improve post-thaw quality, antioxidant activity, and in vivo fertility of buffalo semen particularly of the subfertile animals.

Validation of double freezing protocol for Beetal buck (Capra hircus) spermatozoa.

The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris-citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer-assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, µm/s), straight line (VSL, µm/s) and curved line velocities (VCL, µm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra-vital plasma membrane integrity (SV-PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V-IACR, %) and DNA integrity (DNA-I, %) had significantly greater values (p < .05) during single freeze-thawing as compared with the double freeze-thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze-thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n = 84/141 = 59.72%) and double freeze-thawing (n = 72/136 = 52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T1 = 90°C, T2 = 100°C, T3 = 110°C, T4 = 120°C, T5 = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT1 = 143.2 µM, CT2 = 147.4 µM, CT3 = 151.5 µM, CT4 = 157.2 µM, CT5 = 161.8 µM, BT1 = 168.3 µM, BT2 = 172.5 µM, BT3 = 176.7 µM, BT4 = 196.3 µM, and BT5 = 205.7 µM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT3) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT3, CT1 (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT3 as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers.

BACKGROUND: Metabolomic approaches, which include the study of low molecular weight molecules, are an emerging -omics technology useful for identification of biomarkers. In this field, nuclear magnetic resonance (NMR) spectroscopy has already been used to uncover (in) fertility biomarkers in the seminal plasma (SP) of several mammalian species. However, NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out. Thus, this study aimed to evaluate the putative relationship between SP-metabolites and in vivo fertility outcomes. To this end, 24 entire ejaculates (three ejaculates per boar) were collected from artificial insemination (AI)-boars throughout a year (one ejaculate every 4 months). Immediately after collection, ejaculates were centrifuged to obtain SP-samples, which were stored for subsequent metabolomic analysis by NMR spectroscopy. Fertility outcomes from 1525 inseminations were recorded over a year, including farrowing rate, litter size, stillbirths per litter and the duration of pregnancy. RESULTS: A total of 24 metabolites were identified and quantified in all SP-samples. Receiver operating characteristic (ROC) curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate (area under the curve [AUC] = 0.764) while carnitine (AUC = 0.847), hypotaurine (AUC = 0.819), sn-glycero-3-phosphocholine (AUC = 0.833), glutamate (AUC = 0.799) and glucose (AUC = 0.750) showed it for litter size. Similarly, citrate (AUC = 0.743), creatine (AUC = 0.812), phenylalanine (AUC = 0.750), tyrosine (AUC = 0.753) and malonate (AUC = 0.868) levels had discriminative capacity for stillbirths per litter; and malonate (AUC = 0.767) and fumarate (AUC = 0.868) levels for gestation length. CONCLUSIONS: The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs. Moreover, supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

Measurement of Oxidative Stress Index in Seminal Plasma Can Predict In Vivo Fertility of Liquid-Stored Porcine Artificial Insemination Semen Doses.

The study evaluated the relation between the oxidative stress index (OSI) in porcine seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 °C. Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of AI-boars (area under the curve: 0.815, p < 0.05).

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa.

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 µ M l-tryptophan, D2 = 50 µ M l-tryptophan, D3 = 75 µ M l-tryptophan, and D4 = 100 µ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.

Semen evaluation and in vivo fertility in a Northern Italian pig farm: Can advanced statistical approaches compensate for low sample size? An observational study.

The evaluation of sperm functionality and morphology allows discerning between high and low quality ejaculates, but does not give detailed predictive information regarding in vivo fertility. The current developments in statistical modeling have helped in carrying out reproductive studies, but their biggest limitation is in the size of the dataset to be used. The aim of the present observational study was to evaluate whether advanced statistical approaches, such as mixed effects regression models and bootstrap resampling, can help in assessing the predictive ability of semen parameters in terms of in vivo fertility (farrowing rate and litter size), on a small/medium farm with a limited number of animals. Data regarding 33 ejaculates, including viability, subjective motility and acrosome reaction, were collected. Two hundred and thirty-five sows were inseminated with an outcome of 167 deliveries and 1734 newborn piglets. In order to evaluate the relationships among the parameters measured and fertility, mixed effects regression statistical models were used. Once the covariates to be included in the final models were identified, non-parametric bootstrapping was used. The results showed that the farrowing rate was highly associated with the total number of spermatozoa and subjective motility, while litter size was associated with percentage of acrosome reaction. In conclusion, the proposed statistical approach seemed to be suitable for studies regarding reproduction and fertility, even for relatively small sample sizes. Nonetheless, larger data sets are still preferable and required in order to achieve higher reliability.

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.

Chicken egg yolk plasma in tris-citric acid extender improves the quality and fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa.

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (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 (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C-irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (µm/s) and straight line velocity (µm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C-irradiated chicken EYP (20%) in TCA extender for cryopreservation of water buffalo 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.

Semen quality parameters as fertility predictors of water buffalo bull spermatozoa during low-breeding season.

The present study was carried out to assess various postthaw semen quality parameters for the prediction of fertility in buffalo bull during low-breeding season. Semen (30 ejaculates) was collected from five adult buffalo bulls with artificial vagina (42 °C). Sperm motility parameters, velocity distribution, motion kinematics, and subpopulations were analyzed by computer-aided sperm motion analyzer (CASA). Moreover, sperm visual motility, supravital plasma membrane integrity, viability/acrosome integrity, viability/mitochondrial transmembrane potential, DNA fragmentation/integrity, and morphology were analyzed by phase-contrast microscope, supravital hypoosmotic swelling test, Trypan blue/Giemsa staining, propidium iodide/"5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide" (JC-1) fluorochromes, neutral comet assay/acridine orange assay and wet mount technique, respectively. Outcome of 528 inseminations was analyzed for in vivo fertility. Pearson's correlation coefficients revealed that sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), straight line velocity (µm/s), subpopulation one (most rapid, and progressive) of motile spermatozoa (%), supravital plasma membrane integrity (%), and viable spermatozoa with intact acrosome (%) were significantly correlated with in vivo fertility (r = 0.64, P < 0.01; r = 0.57, P < 0.01; r = 0.52, P < 0.01; r = 0.56, P < 0.01; r = 0.73, P < 0.001; r = 0.74, P < 0.001; r = 0.88, P < 0.001); whereas nonviable spermatozoa with damaged acrosome or low-mitochondrial transmembrane potential and comet length (µm) of neutral comet assay were negatively associated with in vivo fertility (r = -0.79, r = -0.75, P < 0.001, and r = -0.60, P < 0.05, respectively). Multiple regression analysis reported that combination of semen quality parameters as predictor of fertility were better (R(2) adjusted = 81.30%, P < 0.001) as compared with single parameter (R(2) adjusted = 50.20%, P < 0.007). It is concluded that assessment of CASA parameters and some other sperm structural and functional parameters, that is, integrity of plasma membrane and acrosome, and transmembrane potential of mitochondria were able to predict the in vivo fertility of water buffalo bull during low-breeding season.