Comparative expression analysis of water buffalo (Bubalus bubalis) to identify genes associated with economically important traits.

The milk, meat, skins, and draft power of domestic water buffalo (Bubalus bubalis) provide substantial contributions to the global agricultural economy. The world's water buffalo population is primarily found in Asia, and the buffalo supports more people per capita than any other livestock species. For evaluating the workflow, output rate, and completeness of transcriptome assemblies within and between reference-free (RF) de novo transcriptome and reference-based (RB) datasets, abundant bioinformatics studies have been carried out to date. However, comprehensive documentation of the degree of consistency and variability of the data produced by comparing gene expression levels using these two separate techniques is lacking. In the present study, we assessed the variations in the number of differentially expressed genes (DEGs) attained with RF and RB approaches. In light of this, we conducted a study to identify, annotate, and analyze the genes associated with four economically important traits of buffalo, viz., milk volume, age at first calving, post-partum cyclicity, and feed conversion efficiency. A total of 14,201 and 279 DEGs were identified in RF and RB assemblies. Gene ontology (GO) terms associated with the identified genes were allocated to traits under study. Identified genes improve the knowledge of the underlying mechanism of trait expression in water buffalo which may support improved breeding plans for higher productivity. The empirical findings of this study using RNA-seq data-based assembly may improve the understanding of genetic diversity in relation to buffalo productivity and provide important contributions to answer biological issues regarding the transcriptome of non-model organisms.

Effect of mitochondria-targeted antioxidant on the regulation of the mitochondrial function of sperm during cryopreservation.

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.

Genetic Parameters for First Lactation and Lifetime Traits of Nili-Ravi Buffaloes.

The data on first lactation and lifetime performance records of 501 Nili-Ravi were collected for a period from 1983 to 2017 (35 years) maintained at ICAR-Central Institute for Research on Buffaloes, Sub-Campus, Nabha, Punjab. The data were analyzed to calculate heritability, genetic and phenotypic correlation for first lactation traits, viz., Age at First Calving (AFC), First Lactation Total Milk Yield (FLTMY), First Lactation Standard (305 days or less) Milk Yield (FLSMY), First Peak Milk Yield (FPY), First Lactation Length (FLL), First Dry Period (FDP), First Service Period (FSP) and First Calving Interval (FCI), Herd Life (HL), Productive Life (PL), Productive Days (PD), Unproductive Days (UD), Breeding Efficiency (BE), Total Lifetime Milk Yield (Total LTMY), Standard Lifetime Milk Yield (Standard LTMY), Milk Yield Per Day of Productive Life (MY/PL), Milk Yield Per Day of Productive Days (MY/PD), and Milk Yield Per Day of Herd Life (MY/HL). For estimation of variance component and heritability separately for each trait, the uni-trait animal model was equipped, whereas to estimate genetic and phenotypic correlations between traits, bi-trait animal models were fitted. The estimates of heritability for production and reproduction traits of Nili-Ravi were medium, i.e., 0.365 ± 0.087, 0.353 ± 0.071, 0.318 ± 0.082, 0.354 ± 0.076, and 0.362 ± 0.086 for FLSMY, FDP, FSP, FCI, and AFC, respectively. The estimates of heritability were low, i.e., 0.062 ± 0.088, 0.123 ± 0.090, 0.158 ± 0.090, 0.155 ± 0.091, and 0.129 ± 0.091 for HL, PL, PD, Total LTMY, and Standard LTMY and high, i.e., 0.669 ± 0.096 for BE. Genetic correlation for FLTMY was high with FLL (0.710 ± 0.103), and genetic correlation of FLTMY was high and positive with HL, Total LTMY, MY/PL, and MY/PD while low and positive with PL. Genetic correlation of AFC was low and negative with PL, PD, UD, BE, Total LTMY, Standard LTMY, MY/PL, and MY/PD and negative with MY/HL. Significant positive phenotypic association of FPY was seen with FLTMY, FLSMY, FLL, AFC, HL, Total LTMY, and Standard LTMY. Higher heritability of first lactation traits especially FPY suggests sufficient additive genetic variability, which can be exploited under selection and breeding policy in order to improve overall performance of Nili-Ravi buffaloes.

Is addition or removal of seminal plasma able to compensate for the dilution effect of buffalo semen?

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.

Escherichia coli membrane-derived oxygen-reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation.

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Serum kisspeptin: New possible biomarker for sexual behaviour and sperm concentration in buffalo bulls.

The study was designed to decipher the inter-relationship between peripheral hormones (kisspeptin and testosterone), sexual behaviour and seminal variables of Murrah buffalo bulls (n = 134). In this study, we recorded that 13%, 37%, 40%, 6% and 4% Murrah buffalo bulls had reaction time of <30, 31-60, 61-180, 181-300 and >300 s, respectively. Further, it was observed that 4%, 85% and 10% buffalo bulls were sexually aggressive, active and dull, respectively, during semen collection. The courtship behaviour was not found to be desirable for the bulls used for the semen collection. Mean of ejaculate volume, sperm concentration and mass motility (0-5 scale) were 3.57 ml, 977.11 million/ml, 2.7, respectively. Correlation studies revealed that the reaction time was positively correlated with courtship behaviour and body weight, and negatively correlated with sexual aggressiveness and sperm concentration. Serum kisspeptin in buffalo bulls, measured for the first time, was found to 3.8 ± 0.7 ng/ml. Serum kisspeptin and testosterone level are negatively correlated to each other and kisspeptin level influenced the sexual behaviour (reaction time, sexual aggressiveness and penile erection) of study bulls. Serum kisspeptin was higher in the buffalo bulls with higher sperm concentration indicating its role in spermatogenesis. In conclusion, for the first time basic information related to sexual behaviour of Murrah buffalo bulls in large population along with its inter-relationship with peripheral hormones (kisspeptin and testosterone) has been documented.

Abatement of enteric methane production from lactating Murrah buffaloes (Bubalus bubalis) with improving production performance and immune status through dietary supplementation of composite feed additive.

Ruminant livestock production processes are the major sources of methane production in agriculture sector triggering global environmental pollution. Above 90% of world buffalo population present in Asian countries, India ranks first and contributes significantly to the environmental pollution by enteric methane emissions. In this study, we examined the effect of dietary composite feed additive supplementation on ruminal methane production, nutrient utilization, milk production and immune status of buffaloes (Bubalus bubalis). Eighteen lactating Murrah (Bubalus bubalis) buffaloes at early stage of lactation were divided into two groups of nine animals and fed a composite feed additive [consisted of (%, w/w) dried and ground leaves of Cordia dichotoma and Holoptelea integrifolia, 31.4 each; garlic oil, 0.6; sodium nitrate, 3.1; magnesium sulphate, 8.4; mustard oil, 12.6 and cottonseed oil, 12.5] which contained an ideal combinations of methane inhibitors, alternate hydrogen sinks and rumen stimulating agents to treatment (CFA) group animals along with basal feed of chaffed green sorghum (Sorghum bicolor) fodder, chaffed wheat straw and concentrate mixture for maintenance and milk production. The results showed a decrease (44.6%) in methane concentration in exhaled air of CFA group buffaloes with increase (p < 0.05) in digestibility of feed in comparison to control (CON). Total digestible nutrient (TDN) content of the ration fed to buffaloes of CFA group was significantly (p < 0.05) increased. The daily milk yield, 6% fat corrected milk (FCM) yield and immune response were also increased (p < 0.05) in CFA group. The study suggests that the supplementation of composite feed additive was effective to reduce enteric methane emissions and improvement in production performance and immune status of buffaloes.

Effect of rumen-protected nutrients on feed intake, body weights, milk yield, and composition in Murrah buffaloes during early lactation.

The effect of rumen-protected nutrients (bypass fat, BPF; bypass protein, BPP; or their combination, BPPF) was investigated in Murrah buffaloes during the early stage of lactation. Forty Murrah buffaloes (BW 531.92 ± 10.85 kg) just after parturition were randomly distributed into four groups according to parity and milk production. Buffaloes individually fed ration from day 0 to 90 postpartum according to feeding group and nutrient requirement. Control and BPF fed groups received a concentrate mixture, CM1 with 25% rumen-protected protein (using barley, wheat bran, and mustard oil cake), BPP and BPPF groups received a second concentrate mixture, CM2 with 40% rumen-protected protein (using barley, de-oiled rice bran, and cottonseed cake). Bypass fat fed groups (BPF and BPPF) additionally were supplemented with 15 g BPF (Ca salt of long-chain fatty acids) per kg milk yield in their respective concentrate mixtures. Dry matter intake, body weights, body condition score, and total milk yield were similar between groups (P > 0.05). Fat-corrected milk (FCM) production was improved (14.5%, P > 0.05) in groups fed BPP and BPPF, while significant (19.45%, P < 0.05) improvement was observed in BPF-fed group. Overall mean values of milk fat, solid not fat, protein, lactose, and total solids were found to be high (P < 0.05) in treatment groups as compared with control values. It may be concluded that supplementation with BPP or BPF either alone or in combination positively influences the quality of milk produced in Murrah buffaloes during early lactation and BPF additionally had improvement on the quantitative trait of milk as well.