This study investigated the effect of season, age and period on semen traits in buffalo bulls. Data collected during the year 2010-2021 for seven semen traits viz. ejaculate volume (VOL), sperm concentration (SPC), mass activity (MA), initial motility (BFM), post-thaw motility (PTM), number of sperms per ejaculate (NSP) and number of motile sperm (NMSP) from Murrah buffalo bulls (n = 45; aged: 24-75 months). A general mixed model was applied to obtain the R2 value which was the proportion of variation in semen variable. With study period, the highest SPC was obtained during 2012; with the season, the highest VOL (ml) in hot humid and the highest SPC during the hot-dry season and with age, higher VOL, SPC, NSP and NMSP was obtained in older bulls. This study showed that factors i.e. period, season and age had a significant effect on semen traits viz. VOL, MA, SPC, BFM, PTM, NSP and NMSP in buffalo bulls.

The correlation between peripheral and seminal hormones and biochemical parameters with sperm kinematics across seasons in buffalo bulls were studied. Semen from buffalo bulls (n = 13) during different seasons viz. summer, comfort (spring and autumn) and winter was collected. Sperm kinematics of ejaculates (n = 39) (three ejaculates per bull) were carried out during each season and simultaneously blood (10 mL) was collected and serum was separated for hormones (leptin, testosterone and kisspeptin) and biochemical [heat shock protein 70 (HSP70), ascorbic acid and total antioxidant capacity] estimation. Total motility (TM, %), progressive motility (PM, %) and rapid motility (RM, %) showed significant differences across seasons with the highest TM, PM and RM during winter. HSP70 was higher in seminal plasma levels during summer and TAC was higher in serum than seminal plasma during all seasons. In conclusion, seminal leptin and ascorbic acid showed an association with sperm kinematics across seasons in buffalo bulls.

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

This study was aimed to decipher the inter-relationship peripheral hormones [Insulin-like growth hormone (IGF-1), testosterone and growth hormone] with body weight, body condition score and scrotal circumfermnce across age-groups in male buffalo. Male buffalo (n=20) of different age groups viz. Group 1 (0 to 8 months), 2 (9 to 16 months), 3 (17 to 24 months) and 4 (25 to 32 months) were selected and Blood was collected along with body weight, body condition score and scrotal circumference. Significant difference (P<0.05) in the body weight, body condition core and scrotal circumference was observed between the groups. Peripheral IGF-1 level increased with age, highest in Group 4 (202.4±9.36 ng/ml). Similarly, testosterone was different between Group 1, 2 and 4, highest in Group 4 (1.73±0.02 ng/ml). Growth hormone, differed (P<0.05) between Group 1 (3.65±0.50 ng/ml), Group 3 (3.65±0.50 ng/ml) and Group 4 (8.56±1.96 ng/ml). Postive correlation (P<0.05) between various parameters (body weight, body condition score and scrotal circumference, testosterone and growth hormone) was observed. In conclusion, this study reports the age-related variations and inter-relationships of peripheral hormones with body weight, body condition score and scrotal circumference in male buffalo.

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.

This study was attempted to estimate the genetic parameters of semen quality traits in buffalo bulls. The study data consisted of 10975 ejaculates from 45 Murrah buffalo bulls (aged 24–72 months) used for breeding program during year 2010 to 2020. Semen quality traits (ejaculate volume, concentration of sperm, mass activity, initial and post-thaw motility, number of sperms per ejaculate, motile sperm number and discard rates) were studied. It was observed that average ejaculate volume was 2.82 ± 1.45 mL with mean concentration of 1040.12 ± 523.26 million/mL. Higher heritability was observed for number of sperms per ejaculate, number of motile sperm and sperm concentration. Significant phenotypic correlation was obtained between volume and number of sperms per ejaculate as well as volume and number of motile sperms. Likewise, significant phenotypic correlation was evident between sperm concentration with sperm number per ejaculate. Highest phenotypic correlation was obtained between sperm count per ejaculate and motile sperm count. Estimated genetic trends showed significant change in volume and motile sperm number. In conclusion, this study ascertains that genetic parameters of semen traits can be considered during the selection of buffalo bulls in breeding program.

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cry-opreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 mu M) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1-0.5 mu M) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2-10 mu M) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function.

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon-polylysine (s-PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk-based semen extender containing either antibiotics (strepto-penicillin) or different concentrations of s-PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that s-PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of s-PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto-penicillin. The sperm motility and kinematic parameters assessed by a computer-assisted sperm analyser showed that s-PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow-cytometric evaluation of frozen-thawed sperm revealed interesting results. The extender supplemented with s-PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto-penicillin. Further, s-PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, s-PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, s-PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm.

Hair cortisol concentration (HCC) is used to assess the long-term retrospective measurement of the hypothalamic-pituitary-adrenal axis (HPA axis). HCC reflects HPA axis activity over a period of months and years depending on hair growth rate and length of hair measured. HCC is being used as a biomarker of chronic stress in order to check the well-being of domestic animals (cattle, horses, dogs, pigs), wild animals, captive animals, human trauma victims, and post-traumatic stress disorders (PTSD) as it provides an innovative approach to measure chronic HPA activity retrospectively over months without being affected by short term acute stressors and diurnal patterns. Stress plays a significant role in affecting the fertility of animals. Activation of the HPA axis further activates the hypothalamic–pituitary–gonadal axis (HPG) i.e., glucocorticoids are produced causing apoptosis of Leydig cells and decreased testosterone levels in males. In females, increased cortisol levels can suppress the tonic secretion of Leutinizing hormone (LH) which might hamper ovulation. Thus, hair cortisol analysis for assessment of the HPA-HPG axis could be a very important tool for animal welfare.

Sperm mitochondrion is one of the major susceptible organelles that get damaged during cryopreservation. The study aimed to minimize mitochondrial dysfunction and oxidative stress during sperm cryopreservation using mitochondria-specific antioxidants. For this, semen was collected from five buffalo bulls (3 ejaculates/bull). The ejaculates were diluted in an low-density lipoprotein-based extender and divided into four equal aliquots. Mitochondria-targeted antioxidant (MitoQ) was added at a final concentration of 0 (control), 0.02, 0.2 and 2 μM separately in each aliquotes and cryopreserved. The addition of MitoQ at a concentration of 0.02 μM improved post-thaw sperm motility, plasma membrane integrity and able to sustain sperm motility for a longer time. To investigate MitoQ's effects on mitochondrial function, we measured mitochondrial membrane potential (MMP) using JC-1 dye, superoxide production using Mitosox assay, and lipid peroxidation by TBARS assay. The supplementation of 0.02 μM MitoQ in the extender prevented the significant reduction of MMP and reduced superoxide production resulting in lower lipid peroxidation of sperm plasma membrane after cryopreservation. Further, we found that a higher concentration of MitoQ decreases MMP and increases mitochondrial superoxide production. In conclusion, MitoQ @ 0.02 μM can alleviate oxidative stress by regulating mitochondrial functionality in spermatozoa during cryopreservation.

Semen ejaculates were collected from Murrah buffalo (Bubalus bubalis) bulls and were diluted with low density lipoprotein (LDL) based extender into 20 million sperm/0.25 mL and 2 million sperm/0.25 mL. Dinitrophenol (DNP) was added (@ 0, 1, 10 and 50 µM) to 20 million and 2 million sperm concentration cryopreserved. After thawing, the parameters studied were plasma membrane integrity by HOST, sperm motility and kinetics by CASA, the thermal resistance of sperm by incubation test, mitochondrial superoxide status by MitoSOX through flow cytometry, mitochondrial membrane potential (MMP) evaluation by JC-1 through flow cytometry. There was no significant (P>0.05) change in plasma membrane integrity, sperm motility and kinematics; thermal resistance of sperm, mitochondrial superoxide status and hMMP in comparison to control within the 2 and 20 million sperm doses. Two million sperm doses resulted in low plasma membrane integrity, low thermal resistance of sperm, high mitochondrial superoxide status and decreased high MeMP and no significant (P>0.05) change in sperm motility and kinematics, but reduced total motility, beat cross frequency and no change in progressive motility, straight linear velocity, average path velocity, curvilinear velocity, the amplitude of lateral head displacement, straightness, linearity, and wobble in comparison to 20 million sperm doses. Supplementation of DNP (0, 1, 10 and 50 μM) in extender failed to improve semen quality in both 2 and 20 million sperm doses in buffalo.

This study was designed to examine the effects of seminal insulin-like growth factor-1 (IGF-1) supplementation on structural and functional properties of buffalo sperm post cryopreservation. Semen ejaculates from buffalo bulls (n = 6) were proportioned into four aliquots and diluted with egg yolk-based extender. Prior to equilibration, IGF-1 was added to extender as four treatments: group IGF0 (no supplementation), IGF150 (150 ng/mL), IGF250 (250 ng/mL) and IGF350 (350 ng/mL). The extended semen was transferred into 0.25 mL mini-straws, equilibrated (4 °C at 4 h), and cryopreserved. Total sperm motility was greater (P < 0.05) when there was the IGF150 treatment compared with values for other groups. Furthermore, with the IGF150 treatment there was the least and greatest (P < 0.05) mitochondrial superoxide status and membrane potential, respectively. Similarly, with the IGF150 treatment there was a greater (P < 0.05) sperm membrane integrity with a lesser (P < 0.05) calcium status compared to values for the other groups. In conclusion, seminal IGF-1 supplementation affects the structural and functional properties of buffalo sperm following cryopreservation.

The present study aimed to compensate dilution effect using additional seminal plasma (SP) in conventional (80 million (M) spermatozoa/ml) dose and low spermatozoa/dose (8M spermatozoa/ml). We also attempted to confirm whether removal of SP before the extension of ejaculates affects post-thaw sperm quality of buffalo semen. For this, semen ejaculates (N = 15) were divided into four groups: control (CON), removal of SP by centrifugation (NSP), resuspension of the centrifuged semen pellet into SP (CEN) and extra supplementation of SP (ESP). All groups were diluted into two different semen doses to 20 and 2M spermatozoa/0.25 ml using tris egg yolk extender and subsequently cryopreserved. We found that neither addition nor removal of SP affected sperm motility, kinematics, longevity, mitochondrial superoxide production and high mitochondrial membrane potential (MMP). Further, the addition or removal of SP was not able to compensate dilution effect in 2M groups resulting in a significantly (p < .05) reduction in sperm motility, kinematics, sperm longevity, membrane integrity, MMP, and an increase production of mitochondrial superoxide. In conclusion, it appears that role of SP in the sperm cryopreservation process is insignificant.

The study aim was to determine how to minimize effects of buffalo semen dilution by evaluating the use of egg yolk (EY), low-density lipoprotein (LDL), and OptiXcell (OC) extenders. Ejaculates (n = 18 from six bulls) were divided into three aliquots that were diluted separately with EY, LDL, and OC extenders corresponding to 20 million (M), 12 M, and 2 M sperm/dose, respectively, and cryopreserved. There were a lesser sperm motility, plasma membrane integrity, and percentage un-capacitated sperm with the 2 M sperm/dose, however, the LDL extender was more effective than OC and EY extender for cryopreservation of buffalo sperm. Excess semen dilution resulted in sustained sperm velocities (curvilinear velocity, average path velocity, and straight linear velocity), and these were greater with use of the OC than LDL and EY extenders. There was no change in amplitude of sperm lateral head displacement (ALH) with respect to dilution, but with regard to extender effects, ALH was greater in sperm extended in LDL and OC than EY. Semen dilution to 2 M sperm/dose resulted in a greater mitochondrial superoxide production. Conception rate (CR) was unaffected with 20 and 12, however, with the 2 M sperm/dose dilutions there was a lesser CR. In conclusion, buffalo semen dilution to the extent of 12 M sperm/dose did not affect most of the seminal variables and CR. Using LDL extender at 2 M sperm/dose protected sperm from the 'semen dilution effect' to a greater extent than with use of EY and OC extenders.