Evaluation of the immune responses in buffaloes vaccinated with a live-attenuated lumpy skin disease vaccine (Lumpi-ProVacInd).

Since 2019, Lumpy skin disease (LSD) has suddenly spread in many Asian countries, including India. LSD primarily occurs in cattle. However, recent LSD outbreaks in India have also revealed significant morbidity and production losses in buffaloes. This has raised concerns about the role of buffaloes in the epidemiology and transmission of LSD and necessitates the inclusion of buffaloes in the mass vaccination program for the prevention and control of the disease in the country. However, there is no significant data on the immune response in buffaloes following vaccination with the LSD vaccine. In this study, we evaluated antibody- and cell-mediated immune responses following vaccination with a newly developed live-attenuated LSD vaccine (Lumpi-ProVacInd). The detectable amount of anti-LSDV antibodies was observed at 1-2 months following vaccination, with a peak antibody titer at 3 months. Upon stimulation of the peripheral blood mononuclear cells (PBMCs) with the UV-inactivated LSDV antigen, there was a significant increase in CD8 + T cell counts in vaccinated animals as compared to the unvaccinated animals. Besides, vaccinated animals also showed a significant increase in IFN-γ levels upon antigenic stimulation of their PBMCs with LSDV antigen. In conclusion, the buffaloes also mount a potent antibody- and cell-mediated immune response following vaccination with Lumpi-ProVacInd.

The Transcript Levels and the Serum Profile of Biomarkers Associated with Clinical Endometritis Susceptibility in Buffalo Cows.

Determining the gene expression and serum profile of the indicators linked to clinical endometritis susceptibility in Egyptian buffalo cows was the aim of this investigation. The buffalo cows that were enrolled were divided into two groups: forty infected buffalo cows with clinical endometritis and forty seemingly healthy buffalo cows that served as the control group. For the purposes of gene expression and biochemical analysis, ten milliliters of blood was obtained via jugular venipuncture from each buffalo cow. TLR4, IL-8, IL-17, NFKB, SLCA11A1, NCF4, Keap1, HMOX1, OXSR1, ST1P1, and SERP1 were manifestly expressed at much higher levels in the buffaloes with endometritis. On the other hand, the genes that encode SOD, CAT, NDUFS6, Nrf2, and PRDX2 were down-regulated. There was a significant (p < 0.05) elevation of the serum levels of non-esterified fatty acids (NEFAs), beta hydroxy butyric acid (BHBA), triglycerides (TGs), globulin, creatinine, and cortisol, along with a reduction in the serum levels of glucose, cholesterol, total protein albumin, urea, estrogen (E2), progesterone (P4), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroxine (T4), prostaglandin F2 α (PGF2α), calcium, iron, and selenium, in the endometritis group in comparison with the control. However, no significant change was observed in the values of phosphorus, magnesium, copper, or zinc in either group. Within the selective breeding of naturally resistant animals, the variation in the genes under study and the changes in the serum profiles of the indicators under investigation may serve as a reference guide for reducing endometritis in Egyptian buffalo cows.

Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.

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.

Insights into mycobacteriome composition in Mycobacterium bovis-infected African buffalo (Syncerus caffer) tissue samples.

Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis, and M. komaniense. The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.

Cardiac biomarkers as tools in the prediction and diagnosis of traumatic pericarditis and traumatic reticuloperitonitis in cattle and buffaloes.

BACKGROUND: In the livestock industry, Foreign Body Syndrome is a devastating disease condition. Feeding management, lacking of food discrimination, and eating chopped food increase the risk of swallowing sharp foreign bodies in bovine species. In addition to the honeycomb cells shape of the reticulum, the contractions of the reticular wall, gravid uterine pressure, and parturition efforts, foreign bodies can penetrate the reticular wall, causing cascade of problems including traumatic reticulitis, traumatic reticuloperitonitis, and traumatic pericarditis. The present study was carried out to evaluate the diagnostic significance of cardiac troponin I rapid test cassette and other cardiac biomarkers including serum cardiac troponin I (cTn I), creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), and aspartate aminotransferase enzyme (AST), in confirmed cases of traumatic pericarditis (TP) and/or traumatic reticuleoperitonitis (TRP) in cattle and buffaloes. METHODS: A total number of 30 animals (22 cattle and 8 buffaloes) with different signs such as anorexia, jugular distension, brisket edema, and signs of pain (reluctance to move, arching back, and abduction of the forelimbs) were included in the present study. Based on case history, clinical signs, ferroscopic, pericardiocentesis, radiographic and ultrasonographic examinations, TP were confirmed in cattle (n = 10) and buffaloes (n = 8) while TRP were confirmed only in cattle (n = 12). Additionally, 20 clinically healthy animals (n = 10 cattle and 10 buffaloes) were used as a control group. Blood samples were collected for determination of blood level of Tn-I, and activity of CK-MB, LDH, and AST. RESULTS: The obtained results revealed a highly significant increase in serum cTn I in diseased cattle with TP and TRP (P = 0.00), while buffaloes with TP showed no significant changes in serum cTn I (P = 0.111). Both diseased cattle and buffaloes showed increased serum activities of CK-MB, AST, and LDH enzyme. On the other hand, cardiac troponin I rapid test cassette failed to detect cTn I in diseased animals. CONCLUSION: The study concluded that the cardiac troponin I rapid test cassette did not have a diagnostic significance and could not be used as a point-of-care under field condition for diagnosis of TP and TRP in large ruminants. However, the serum troponin I level is helpful in diagnosis of TP and TRP in cattle. Although cardiac biomarkers have some diagnostic values in TP and TRP, the traditional diagnostic methods (clinical, radiography and ultrasonography examinations) are crucial for thorough evaluation of TP/TRP cases in bovine.

Bovine Alphaherpesvirus 1, Bovine Alphaherpesvirus 5 and Bubaline Alphaherpesvirus 1 in Palatine Tonsils from Water Buffaloes in Northern Brazil and Possible Links with the Origin of Bovine Alphaherpesvirus Type 5.

Herpesviruses are significant pathogens of ruminants. In water buffaloes (Bubalus bubalis), however, herpesviruses have not been thoroughly studied. Although bubaline alphaherpesvirus 1 (BuAHV1) and bovine alphaherpesvirus 1 (BoAHV1) have already been recovered from water buffaloes, to date, no reports on the occurrence of bovine alphaherpesvirus 5 (BoAHV5) in these animals have been published. Therefore, the aim of this study was to search for BuAHV1, BoAHV1, and BoAHV5 in palatine tonsils of apparently healthy water buffaloes from the Pará state, Northern Brazil. Tissue samples of tonsils (n = 293) were screened by a nested PCR (nPCR) targeting a region of UL44 (gC coding gene), followed by sequencing, to detect and differentiate between the viral types. Viral genome segments were detected in 18 out of 293 (6.1%) of the palatine tonsil samples. Two animals carried genomes of BoAHV1 only, eleven animals carried BoAHV5 genomes only, and four animals carried BuAHV1 only. Another animal had both BoAHV1 and BoAHV5 genomes in its tonsils. No infectious virus could be recovered from any of the samples. The BuAHV1 sequences identified here were more closely related to BuAHV1 genomes identified in India. Phylogenetic analyses suggested a closer relationship between the recovered BoAHV5 and BuAHV1 genomes. Therefore, evidence is provided here to confirm that not only BoAHV1 and BuAHV1, but also BoAHV5, can infect water buffaloes. This report highlights (i) the first detection of BoAHV5 in water buffaloes and (ii) the occurrence of coinfections with BoAHV1 and BoAHV5 in that species. Such findings and the similarity of BoAHV5 to Indian herpesvirus genomes suggest that the origin of type 5 may be linked to recombinations between bovine and bubaline herpesviruses within bubalines, since the scenario for generation of recombinants in buffaloes is potentially present.

Unravelling the impact of heat stress on daughter pregnancy rate in Mehsana buffalo through innovative breeding interventions.

Heat stress profoundly affects the reproductive success of buffaloes, which are vital for the dairy industry due to their unique anatomical and physiological characteristics, necessitating careful evaluation under such conditions. Hence, this guided our search for quantifying heat stress' impact on Mehsana buffaloes using the best THI model and evaluating sires' performance. Fertility records (days open converted to daughter pregnancy rate) were collected in the span of over 24 years, w.e.f. 1989 to 2012. Finally, 3070 records of first lactation cows, daughters of 117 sires from DURDA, Gujarat, India, were used in the analysis. Meteorological data were retrieved from IMD, Pune, to understand the relationship between daughter pregnancy rate (DPR) and heat stress indicators. Several heat stress models were compared based on R2, adjusted R2, AIC, and BIC values, and the impact of heat stress was quantified. The year was classified into different heat stress zones, viz., Non heat stress zone (NHSZ), Heat stress zone (HSZ), and critical heat stress zone (CHSZ), drawing from the findings of DPR and THI. The THI 4th model based on dry and wet bulb temperature was identified as the best-fit model, and DPR significantly changed (P < 0.01) by 1.14% per unit change in THI value based on the month of calving. The average EBVs of the sires for DPR were found to be 20.78% (NHSZ), 38.09% (HSZ), and 39.08% (CHSZ) using BLUP-SM and 20.78% (NHSZ), 37.30% (HSZ), and 38.87% (HSZ) using BLUP-AM. Subsequently, the optimum sire for each of the zones was prioritized. It is noteworthy that bulls that performed better in NHSZ did not perform as well in HSZ and CHSZ, and vice versa. This supports the possibility of evaluating bulls independently in each heat stress zone.

Biochemical profile differences during the transition period based on different levels of non-esterified fatty acids at 7 weeks before parturition in Mediterranean Italian dairy buffaloes (Bubalus bubalis).

Metabolic adaptations to negative energy balance, as well as lipomobilization, influence inflammatory responses, immune function, and oxidative stress in animals. This study aimed to evaluate the biochemical profile of Mediterranean buffaloes with different levels of lipomobilization from the prepartum to the postpartum period. A total of 76 Mediterranean buffaloes were enrolled, and a weekly blood sample was taken from 7 weeks before to 6 weeks after calving. The concentration of non-esterified fatty acids (NEFAs) was determined in serum and was used to categorize buffaloes into three lipomobilization groups 7 weeks before calving: mild (NEFA-I; NEFA ≤ 0.29 mEq/L; n = 18), medium (NEFA-II; 0.29 < NEFA < 0.57 mEq/L; n = 20), and severe (NEFA-III; NEFA ≥ 0.57 mEq/L; n = 38). Two-way repeated measures ANOVA was used to assess changes within and between the groups and over time. Significant differences were found in the concentration levels of NEFA, ß-hydroxybutyrate, glucose, cholesterol, protein profile, oxygen radicals, antioxidants, lysozyme, complement, and minerals. These results suggest that both medium and severe lipomobilization groups are associated with metabolic alterations. In conclusion, buffaloes with higher NEFA levels (>0.29 mEq/L; NEFA-II and NEFA-III) at 7 weeks before calving should be monitored more closely to reduce the risk of metabolic diseases. Furthermore, the medium (NEFA-II) and severe (NEFA-III) lipomobilization groups could be associated with differences in the animals' ability to manage their metabolic status. Specifically, the severe mobilization group was most associated with a greater energy deficit during both the prepartum and postpartum periods without oxidative stress. On the contrary, the medium mobilization group was associated with a less severe energy deficit but was also associated with an inflammatory status and oxidative stress during the prepartum period. These distinctions highlight the need for tailored management strategies to address varying levels of metabolic stress in dairy buffaloes.

Early Detection of Sub-clinical Mastitis in Murrah Buffaloes through Udder Thermogram Analysis during the Natural Progression of Infection.

Mastitis is a global production disease that needs an intelligent solution to tackle effectively. Infrared Thermography (IRT) is a non-invasive technology that could be incorporated into routine day-to-day farm activities to monitor the health status of the animals. In this study, the udder health status was routinely monitored for 30 days among 41 Murrah buffaloes via IRT and the California Mastitis Test (CMT). Further, somatic cell count (SCC), microbial identification, and milk quality parameters were also estimated for representative samples. The thermal imaging data obtained was tabulated and back propagated from the 0th day to the -10th day and front propagated from the 0th day to +10th day for all the udder quarters. Results revealed that on the 0th day, the mean of udder skin surface temperature (USST) and teat skin surface temperature (TSST) showed a difference (p < 0.05) in the sub-clinical mastitis (SCM) and clinical mastitis (CM) affected quarters to the healthy quarters, and their degree of difference was the highest. The indication of infection was signaled during the -9th to -5th day to the 0th day in SCM and CM cases. There was a steep increment in the temperature from -2nd and -1st day to the 0th day of infection. Sometimes, some quarters show an increment in temperature due to mastitis during morning hours but recover by evening milking due to the animal's innate immune system. Thus, the initiation period in which the udder gets assaulted is crucial in the early assessment of SCM by monitoring temperature change using IRT.

Effect of prostaglandin treatment on the estrus behaviour, follicular and luteal morphometry and serum hormone profile in sub-estrus buffaloes during non-breeding season.

Sub-estrus buffaloes do not exhibit estrus signs despite being cyclic contributing to extended service periods and inter-calving intervals causing significant economic loss. The present study described the effect of synthetic prostaglandin (PGF2α) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E2) and progesterone (P4) profile in sub-estrus buffaloes during the non-breeding season. The incidence of sub-estrus was 38.4% during the non-breeding season. The sub-estrus buffaloes (n = 33) were divided into two groups, viz., Control (n = 16) and PGF2α treatment (Inj. Cloprostenol 500 µg, i.m., n = 17). Estrus induction response was significantly greater in the treatment (100 vs. 18.75%, p < .001), and a relatively greater proportion of animals conceived in the treatment group (29.41 vs. 6.25%, p = .08). The time elapsed to induction of estrus and insemination following treatment was significantly lower in the treatment group than control. A significant increment in the follicle diameter (9.72 ± 0.45 vs. 13.00 ± 0.45 mm, P < .0001) and serum estradiol (E2) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post-treatment period in the PGF2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF2α treated buffaloes than those of control. Of the responded buffaloes, only 30% showed high-intensity estrus attributed to the expulsion of cervico-vaginal mucus (CVM), uterine tonicity, micturition, and mounting response by a teaser bull. From this study, it can be concluded that the administration of PGF2α could induce estrus in the sub-estrus buffaloes during the non-breeding season. Behavioural changes, along with sonographic observation of POF, regressing CL, and serum E2 and P4 concentration would be useful to determine the right time of insemination in sub-estrus buffaloes during non-breeding season.

Evaluation of Immune Exhaustion and Co-Inhibitory Receptor Expression in Mycobacterium avium Subspecies paratuberculosis (MAP) Seropositive Diarrhoeic Bovines.

Mycobacterium avium subspecies paratuberculosis (MAP) infection leads to chronic, persistent granulomatous enteritis, causing prolonged diarrhoea and emaciation. The disease is managed using medications such as antibiotics, live vaccines, mycobacteriophage therapies and other treatments; however, a notable proportion of affected animals do not show improvement with this approach. We hypothesise that immunoinhibitory receptors TIM-3 (T cell immunoglobulin mucin protein-3) and PD-1 (Programmed death receptor 1) may be upregulated on Peripheral blood mononuclear cells (PBMCs) of MAP-seropositive bovines, potentially contributing to immune exhaustion. Samples (blood and faeces) were collected from 32 diarrhoeic bovines suspected of MAP infection; eight apparently healthy buffaloes from the dairy farm at Hisar, Haryana and from 14 cows (suffering from chronic diarrhoea, weakness and emaciation) housed in stray cattle shed. MAP infection was estimated using indigenous ELISA (i-ELISA), faecal IS900 PCR, culture and acid-fast staining. TIM-3 and PD-1 gene expression on PBMCs were determined using qRT-PCR. TIM3 expression was relatively higher (~400-fold, 330-fold, 112-fold, 65-fold and 16-fold) in 5 chronically diarrhoeic PBMCs samples (MAP-seropositive), and higher PD-1 expression (around ~7-fold, 1.75-fold, 2.5-fold, 7.6-fold) was recorded in 4 diarrhoeic MAP-seropositive animals, compared to apparently healthy and other MAP-seronegative diarrhoeic animals. High co-expression of TIM-3 and PD-1 levels was also recorded in chronically diarrhoeic, emaciated stray cattle. Understanding immune responses in field conditions might aid in the therapeutic management of paratuberculosis.

Prevalence and clinico-pathology of PSOROPTIC mange in buffaloes of the Amazon region.

Scabies is an important skin disease in several species of domestic and wild animals; however, few reports in Brazil have emphasized its occurrence in buffaloes. This article describes the epidemiological, clinical and pathological aspects and diagnosis of psoroptic mange in buffaloes in a property in the municipality of Castanhal, PA, Amazon region. Of the 41 buffaloes examined, 38 males and females of the Murrah, Baio, Mediterranean and Carabao breeds and their crossbreeds, aged between 2 and 20 years, had a history of pruritus. Clinical examination was performed to map the lesions, skin scrapings were collected to identify the mites, and a biopsy was performed for histopathological examination. Clinical signs, from mild to severe intensity, varied according to the system of creation and handling of the animals and were more severe in buffaloes raised in bays than those raised under a collective regime (pastures and collective troughs). The characteristic clinical signs were intense itching, extensive areas of alopecia, periocular edema, and thickening of the epidermis with exudative crusts covering the face, chamfer, neck, scapular region, back, base of the horn, thoracic and pelvic limbs and chest. The behavior of rubbing the affected regions of the body against structures (troughs, fence posts, gates) or with the horns was frequently observed and provided relief from itching. In the most severe cases, mites were also noted in the crusts, which were identified as Psoroptes natalensis. Histological skin lesions exhibited alterations consistent with immune-mediated dermatitis, which is typical of hypersensitivity to mite-derived allergens.

Responses of nutrient utilization, rumen fermentation and microorganisms to different roughage of dairy buffaloes.

Dairy buffaloes are typically fed a high-forage, low-quality diet with high fiber. These conditions result in an inherent energy and protein inefficiency. In order to make full and rational use of feed resources and improve the production level and breeding efficiency of dairy buffaloes, the effects of various roughages on nutrient digestibility, ruminal fermentation parameters, and microorganisms in dairy buffaloes were studied in this experiment. Three ternary hybrid buffaloes, with an average body weight of 365 ± 22.1 kg, were selected and fitted with permanent rumen fistulas. They were fed six different diets, each consisting of 1 kg concentrate supplement and one of six types of roughage, including alfalfa hay (A diet), oat hay (O diet), whole corn silage (W diet), king grass (K diet), sugarcane shoot silage (S diet), and rice straw hay (R diet) according to an incomplete Latin square design of 3 × 6, respectively. The pre-feeding period of each period was 12 d. From day 13 to 15 was the official experimental period. During the prefeeding period, free feed intake for each roughage was determined, and during the experiment, the roughage was fed at 90% of the voluntary feed intake. Digestion and metabolism tests were carried out using the total manure collection method to determine the feed intake and fecal output of each buffalo, and to collect feed and fecal samples for chemical analysis. On day 15, rumen fluid samples were collected two hours after morning feeding to determine rumen fermentation parameters and bacterial 16 S rRNA high-throughput sequencing was performed. The results showed that DM and OM digestibility were greatest for the W diet and lowest for the S diet. The rumen pH of the O diet was significantly greater than that of the W diet. The concentration of rumen fluid NH3-N (mg/dL) increased with increased CP content. The concentration of total volatile fatty acids (mmol/L) in the rumen decreased with increased NDF content but increased with increased NFC content. The relative abundances of Bacteroidetes, Firmicutes, and Spirochaetes were 57.03-74.84%, 14.29-21.86%, and 0.44-1.43% in the different quality roughage groups. Bacteroidetes were mainly Prevotellaceae1 and Rikenellaceae RC_gut_group with relative abundances of 30.17-45.75% and 3.23-7.82%. The relative abundance of Patescibacteria and Spirochaetes decreased with increasing roughage quality. These results provide a theoretical and practical basis for evaluating the nutritional value of dairy buffalo feed, utilizing feed resources, matching rations, feeding scientifically, and protecting animal health.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

Investigating the relationship between heat load and shade seeking behaviour in dairy buffaloes.

The study presented in this Research Communication aimed to investigate the relationship between physiological responses, body surface temperature and shade-seeking behaviour in Nili Ravi dairy buffaloes during summer months. We enrolled 60 buffaloes, and each animal was observed for three consecutive days starting before sunrise until they moved towards the shade structures. A repeated measures ANOVA was employed to assess the changes in physiological parameters and body surface temperature between the early morning and the occurrence of shade-seeking behaviour. The average temperature humidity index and heat load index during the behavioural monitoring period (0400 to 1200 h) were 81.3 ± 6.5 and 92.9 ± 17, respectively (mean ± sd). There was no significant difference in core body temperature between sunrise and the time of shade-seeking event. However, the buffaloes had a slightly higher respiration rate at the time of shade-seeking (19.2 vs. 22.4 breaths/min). In addition, body surface temperature, measured at the flank region, shoulder, base of the ear and forehead was significantly higher at the occurrence of shade-seeking behaviour compared to the early morning. On average, the buffaloes sought shade when the surface temperature was 2°C higher than the temperature recorded before sunrise. Overall, the current findings suggest that body surface temperature, rather than core body temperature was strongly associated with shade-seeking behaviour in dairy buffaloes. These findings could be useful in developing strategies to mitigate the effects of heat stress in dairy buffalo herds and thereby improve animal welfare.

Clinico-haematobiochemical and cardiac alterations in Trypanosoma evansi infected buffaloes of Andhra Pradesh, India.

The present research aimed to document the incidence, clinical signs, haematological, and serum biochemical alterations, as well as electrocardiography and echocardiography findings in 62 buffaloes (selected from a total of 240) infected with Trypanosoma evansi. The study spanned one year, from January 2022 to December 2022. Morphological identification of Trypanosoma evansi was done by the presence of a centrally positioned nucleus with a small sub-terminal kinetoplast at the posterior position through microscopic examination of Giemsa stained peripheral blood smears. The incidence of trypanosomosis were determined to be 26% (62/240) using stained blood smear examination and 41% (98/240) through polymerase chain reaction assay. Clinical signs exhibited by buffaloes with trypanosomosis included the lack of rumination (94%; 58/62), anorexia (90%; 56/62), emaciation (87%; 54/62), loss of milk yield (84%; 52/62), ocular discharges (82%; 51/62), depressed demeanour (81%; 50/62), sunken eye balls (61%; 38/62), fever (60%; 37/62), scleral congestion (56%; 35/62) and intermittent fever (42%; 26/62). Cardiovascular clinical findings in affected buffaloes included tachycardia (44%; 27/62), cardiac arrhythmia (24%; 15/62), cardiac murmurs (19%; 12/62) and muffled heart sounds (18%; 11/62). In the present study, buffaloes with trypanosomosis exhibited significant reduction in haemoglobin (p = 0.008), packed cell volume (p = 0.004), total erythrocyte count (p = 0.003), mean corpuscular volume (p = 0.042), total leucocyte count (p = 0.048) and absolute neutrophil count (p = 0.012); a significant increase in absolute eosinophil count (p = 0.011) and absolute monocyte count (p = 0.008) compared to the apparently healthy buffaloes. Additionally significant decrease in albumin (p = 0.001), A/G ratio (p = 0.007), calcium (p = 0.008), glucose (p = 0.007), phosphorous (p = 0.048), sodium (p = 0.008), potassium (p = 0.041) and chloride (p = 0.046) were observed in buffaloes with trypanosomosis compared to healthy ones. Buffaloes with trypanosomosis also showed significant increase in globulin (p = 0.004), aspartate aminotransferase (p = 0.008), bilirubin (p = 0.034), blood urea nitrogen (p = 0.071), creatinine (p = 0.029), cholesterol (p = 0.046), lactate dehydrogenase (p = 0.009), gamma-glutamyl transferase (p = 0.004) and creatine kinase-myoglobin binding levels (p = 0.005). Electrocardiography explorations in buffaloes with trypanosomosis revealed sinus tachycardia, low voltage QRS complex, ST segment elevation, wide QRS complex, sinus arrhythmia, sinus bradycardia, wandering pace maker, first degree atrio ventricular block, biphasic T wave and tall T wave. Echocardiography examination unveiled cardiac chamber dilatation, ventricular wall thickening and indications of pericarditis/cardiac tamponade. Necropsy was carried on the dead buffaloes during the study period disclosed severely congested blood vessels on epicardial surface, endocardial haemorrhages, and presence of pericardial fluid. Histopathological examination of the heart revealed hyaline degeneration, haemorrhages in the cardiac muscles and varying degrees of degenerative changes. Additionally, the pericardium displayed increased thickness due to presence of more elastic fibres, fibroblast cells in the myocardium, discontinuity of muscle layers, vascular congestion, perivascular mono nuclear cell infiltration and augmented thickness of the endocardium with fibroblast cell proliferation. The study's conclusion highlights cardiac alterations as secondary complications in buffaloes infected with Trypanosoma evansi. Further investigations are recommended to elucidate therapeutic modifications and refine the treatment paradigm.

Correlation of udder thermogram and somatic cell counts as a tool for detection of subclinical mastitis in buffaloes.

This study aimed to monitor the mammary health of 37 multiparous Murrah buffaloes through infrared thermography (IRT). Based on the California Mastitis Test (CMT) and milk somatic cell counts (SCC), buffaloes were grouped into healthy (H, n = 16), subclinical mastitis (SCM, n = 10), and clinical mastitis (CM, n = 11). Buffaloes were milked twice daily in the morning (5:00-6:00 AM) and evening (5:00-6:00 PM). Rectal temperature and respiratory rates were recorded, CMT was performed and thermal images of the mammary gland of all the buffaloes were taken before and after each milking. Milk samples were analysed after each milking for SCC, fat, Solids-Not-Fat (SNF), density, protein, lactose, salts, conductivity, and pH immediately in the laboratory from fresh milk samples. The surface temperature of the periocular region of both the eyes, muzzle, flank, and vagina were also taken. Thermal images were used to assess the surface temperature of the udder (USST), teat apex (TAT), teat barrel (TB1T), teat base (TB2T), and teat skin surface (TSST). Eye and USST showed significantly higher temperatures (p < 0.05), whereas skin surface temperatures (SST) of different body parts were non-significant in both SCM and CM animals than buffaloes in the H group. Milk SCC showed a positive correlation with conductivity (r > 0.7), salts, and pH (r < 0.6) and a negative correlation with fat, SNF, density, protein, and lactose. TAT, TB1T, TB2T, TSST, and USST were positively correlated with milk SCC. Receiver Operating Characteristic (ROC) analysis of H and SCM groups showed that USST before milking had optimum sensitivity (Se = 0.80) and specificity (Sp = 0.906) among the various skin temperatures recorded. Thermal images captured during the morning showed higher sensitivity compared to images taken in the evening. Results indicate IRT can be used to monitor the mammary health of buffaloes but using IRT in conjunction with milk SCC can help in the accurate prediction of SCM in dairy buffaloes.

Milking Temperament and Its Association with Test-Day Milk Yield in Bulgarian Murrah Buffaloes.

The goal of this research was to evaluate milking temperament and its relationship with test-day milk (TDMY0) yield in Bulgarian Murrah buffaloes. This study involved 90 buffalo cows reared under a tie-stall production system which were milked twice a day with a milking pipeline. The behavioral responses of the buffaloes were reported during preparation for milking and during actual milking. The average temperament score during preparation for milking was 1.83, and 1.93 during milking itself. The most common reaction was leg lifting (18.9%), followed by cows moving on the stall bed (10%), definite kicking (9.9%), and 13.3% managing to remove the milking cluster during milking. The frequency of buffaloes showing adverse reactions (scores 4 and 5) increased considerably during milking compared to preparation for milking. Repeated scoring of temperament during the same lactation did not show a significant difference in the frequency of temperament assessments both in preparation for milking and during milking. The minimal difference may be due to the accuracy of the assessment or a momentary change in the condition of the animals during the two scorings. Cows with the most unwanted milking behavior (scores 5 and 4) had the highest LS means for TDMY, 8.18 kg and 7.65 kg, respectively. The reasons for these buffaloes remaining until later lactations was their high milk yield and the injection of oxytocin before milking, which helps them to be fully milked.

Milk miRNA expression in buffaloes as a potential biomarker for mastitis.

BACKGROUND: Buffaloes have the highest potential for production due to a promising gene pool that is being enhanced and upgraded. Mastitis is a significant health impediment that greatly diminishes milk yield and quality, affecting rural farmers' livelihoods. The traditional gold standard used for diagnosing mastitis or subclinical mastitis is CMT, but it has the drawback of false positive or negative results. Subclinical mastitis, if not treated promptly, can lead to mammary tumors. To address the gap in early diagnosis of subclinical mastitis in CMT-negative milk of buffaloes, we performed a retrospective analysis and evaluated the milk miRNA expression profiles as potential biomarkers. RESULTS: Thirty buffalo milk samples based on clinical signs and CMT were divided into normal, subclinical, and clinical mastitis. SCC evaluation showed significant differences between the groups. The data analysis demonstrated that the elevation of miR-146a and miR-383 differed substantially between normal, subclinical, and clinical mastitis milk of buffaloes with 100% sensitivity and specificity. The relationship of SCC with miR-146a and miR-383 in normal/healthy and subclinical mastitis was positively correlated. CONCLUSION: The overexpression of miR-146a and miR-383 is associated with inflammation. It can be a valuable prognostic and most sensitive biomarker for early mastitis detection in buffaloes with SCC below 2 lakhs and CMT-ve, enhancing the accuracy of subclinical mastitis diagnosis.

Effectiveness of near-infrared spectroscopy to predict the chemical composition of feces and total-tract apparent nutrients digestibility estimated with undigestible neutral detergent fiber or acid-insoluble ash in lactating buffaloes' feces.

Following a comparison of nutrient total-tract digestibility estimates in lactating buffaloes using single-point undigestible NDF (uNDF) or acid-insoluble ash (AIA) as internal markers, the potential of fecal near-infrared spectroscopy (NIRS) to provide calibration equations for the assessment of the chemical composition of feces and nutrient total-tract digestibility estimated with internal markers was explored. Chemical analyses were performed on 147 fecal samples from lactating buffaloes reared on 5 farms in central Italy (Naples). Each farm fed a silage-based TMR to the buffaloes, and the TMR was sampled in the 2 d before the fecal collection. The TMR and individual fecal samples were collected and analyzed for DM, OM, ash, AIA, ether extract (EE), starch, fiber fractions (amylase-treated NDF without residual ash [aNDFom], amylase-treated NDF inclusive of residual ash [aNDF], ADF without residual ash [ADFom], ADF, hemicellulose, cellulose, ADL, uNDF), N, CP and CP bound to aNDF (NDICP) and to ADF (ADICP). The uNDF content was determined through a 240-h in vitro fermentation and employed, together with AIA as markers, to estimate the total-tract apparent digestibility and total-tract digestibility of DM, OM, ash, N, CP, EE, aNDFom, aNDF, NDIP, ADFom, and ADF, ADIN, ADL, hemicellulose, cellulose, starch, NFC, and the B3 fraction of N (NB3). No correlation was found between DM and OM digestibility estimated with AIA and uNDF as internal markers. Weak correlations were detected for all the other nutients digestibilities, and strong correlations were observed for EE, ADFom, hemicellulose, NDIN, ADIN, NB3, NFC, and starch. The sample set (n = 147) was divided in a calibration set (n = 111) and a validation set (n = 36) to "train" and "validate" the fecal NIRS curve through an external validation process. An estimation usable for preliminary or initial evaluation was obtained for N, CP, and aNDF fecal content. An excellent prediction was obtained for total tract digestibility of ADIN (R2 = 0.90) when estimated with uNDF as the internal marker. The NIRS technology was not able to accurately predict all the other traits and the estimated nutrient digestibility of lactating buffalo diets from fecal spectra.