Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

Disentangling river and swamp buffalo genetic diversity: initial insights from the 1000 Buffalo Genomes Project.

More people in the world depend on water buffalo for their livelihoods than on any other domesticated animals, but its genetics is still not extensively explored. The 1000 Buffalo Genomes Project (1000BGP) provides genetic resources for global buffalo population study and tools to breed more sustainable and productive buffaloes. Here we report the most contiguous swamp buffalo genome assembly (PCC_UOA_SB_1v2) with substantial resolution of telomeric and centromeric repeats, ∼4-fold more contiguous than the existing reference river buffalo assembly and exceeding a recently published male swamp buffalo genome. This assembly was used along with the current reference to align 140 water buffalo short-read sequences and produce a public genetic resource with an average of ∼41 million single nucleotide polymorphisms per swamp and river buffalo genome. Comparison of the swamp and river buffalo sequences showed ∼1.5% genetic differences, and estimated divergence time occurred 3.1 million years ago (95% CI, 2.6-4.9). The open science model employed in the 1000BGP provides a key genomic resource and tools for a species with global economic relevance.

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples.

Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January-June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures.

Identification and molecular characterization of a novel Babesia orientalis rhoptry neck protein 4 (BoRON4).

Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109-452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.

Incubating frozen-thawed buffalo sperm with olive fruit extracts counteracts thawing-induced oxidative stress and improves semen quality.

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 106/mL in IVF medium with 0, 72, 143, and 214 µL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) µM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity.

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.

Sero epidemiological study on bovine babesiosis in cattle and buffaloes in Sharkia Governorate, Egypt.

Background: Bovine babesiosis represents a serious challenge for animal health, herd production, and profitability. Understanding the epidemiology and risk factors associated with babesiosis is critical to reduce their negative impacts. Aim: Investigation of the seroprevalence and risk factors associated with Babesia bigemina (B. bigemina) and Babesia bovis (B. bovis) in five districts in Sharkia governorate using ELISA. Methods: Across-sectional research was conducted to determine the seropositivity of babesiosis by collecting a total of 352 blood samples from 250 cattle and 102 buffaloes. A multivariate logistic regression model was implemented to evaluate the strength of the risk factors associated with both Babesia species infection. Results: The seroprevalence of B. bigemina and B. bovis was 42.6% and 17.0 %, respectively. The prevalence of babesiosis in cattle was found to be 48.8% for B. bigemina and 16.8% for B. bovis. Inclusive, in buffaloes, the prevalence was 27.5% for B. bigemina and 17.6% for B. bovis. Adult animals were more vulnerable to infection with babesia than young animals by 3-5 times, respectively. Males were more susceptible to B. bigemina and B. bovis than females by 3.7 and 3.5 times. Similarly, the odds of infection in infested animals with ticks were 2-4 times higher than in animals without ticks. Conclusion: The obtained results revealed that age, sex of the animal, and tick infestation were major risk factors for the seropositivity of both Babesia species. Inclusive, there was no evidence to support the premise that seroprevalence of babesiosis is correlated with the season and species.

Estimation of the threshold for heat stress and genetic features for milk yield in Mehsana buffaloes in India.

Heat stress is one of the primary environmental factors that harm both the productivity and health of buffaloes. The current study was conducted to estimate the threshold of temperature humidity index (THI)1 and genetic features for milk yield of first-lactation Mehsana buffaloes using an univariate repeatability test-day model. The data included 130,475 first lactation test-day milk yield (FLTDMY) records of 13,887 Mehsana buffaloes and the daily temperature and humidity. The statistical model included herd test day as fixed effects, days-in-milk (DIM) classes, age of the animal, as well as random factors such as the additive genetic effect (AGE) of animal in general conditions (intercept), AGE of the buffaloes subjected to heat stress (slope), permanent environmental effect of animal in general conditions (intercept), permanent environmental effect of animal under heat stress conditions (slope) and random residual effect. It was expected that the general effects and the heat-tolerance effects would be correlated, represented by the present investigation's repeatability models. The variance components of FLTDMY in the present study were computed using the REML method. The threshold for THI was 78. At the THI below the threshold, the heritability estimated for the FLTDMY trait was 0.29, and the additive genetic variance (AGV) for heat stress conditions was 0. At THI of 83, AGV for heat stress conditions was highest for FLTDMY. The genetic correlation of general AGE to heat-tolerant AGE was -0.40. The results indicated that a consistent selection for milk production, avoiding the thermal tolerance, may diminish the thermal tolerance capacity of Mehsana buffaloes.

The influence of rutin and chlorogenic acid on oxidative stress and in vivo fertility: Evaluation of the quality and antioxidant status of post-thaw semen from Azari water buffalo bulls.

BACKGROUND: The vulnerability of buffalo sperm to cryoinjury necessitates the improvement of sperm cryo-resistance as a critical strategy for the widespread use of assisted reproductive technologies in buffalo. OBJECTIVES: The main aim of the present study was to evaluate the effects of different concentrations of rutin and chlorogenic acid (CGA) on buffalo semen quality, antioxidant activity and fertility during cryopreservation. METHODS: The semen was collected and pooled from the 3 buffaloes using an artificial vagina (18 ejaculations). The pooled sperm were divided into nine different groups: control (Tris-based extender); 0.4, 0.6, 0.8 and 1 mM rutin (rutin + Tris-based extender); and 50, 100, 150 and 200 µM CGG (CGA + Tris-based extender). Sperm kinematics, viability, hypo-osmotic swelling test, mitochondrial activity, antioxidant activities and malondialdehyde (MDA) concentration of frozen and thawed buffalo sperm were evaluated. In addition, 48 buffalo were finally inseminated, and pregnancy was rectally determined 1 month after insemination. RESULTS: Compared to the control group, adding R-0.4, R-0.6, CGA-100 and CGA-150 can improve total and progressive motility, motility characteristics, viability, PMF and DNA damage in buffalo sperm. In addition, the results showed that R-0.4, R-0.6, CGA-50, CGA-100 and CGA-150 increased total antioxidant capacity, catalase, glutathione peroxidase and glutathione activities and decreased MDA levels compared to the control group. Furthermore, it has been shown that adding 150 µM CGA and 0.6 mM rutin to an extender can increase in vivo fertility compared to the control group. CONCLUSIONS: In conclusion, adding rutin and CGA to the extender improves membrane stability and in vivo fertility of buffalo sperm by reducing oxidative stress.

Assessment of Brazil's vesicular syndrome surveillance system: Profile of notifications and performance of the official veterinary service.

Foot-and-mouth disease (FMD) is an ailment that causes serious damage to the productive chain, and its control through vaccination is of utmost importance for its eradication. Brazil initiated the National Foot-and-Mouth Disease Surveillance Program (PNEFA) with the aim of making the country FMD-free by 2026. As part of the program, notifications of vesicular lesions became mandatory for the Official Veterinary Service (OVS), which is responsible for verifying them. Due to its size, border areas with countries that do not have FMD-free status pose a risk to Brazil and require greater attention. This study described the profile of notifications of suspected outbreaks of vesicular syndrome in Brazil and analyzed the performance of the surveillance system. The results showed 7134 registered notifications of suspected vesicular syndrome outbreaks from 2018 to 2022, with 2022 having the highest number (n = 2343 or 32.85 %). The species that generated the most notifications were swine (90.99 %), cattle and buffaloes (7.54 %), goats and sheep (1.44 %), and others (0.03 %). The sources of notification were "Veterinary medicine professionals" (61.82 %), "Owners or employees" (13.66 %), "Third parties" (8.90 %), "OVS" (7.20 %), and "others" (2.66 %). 41.69 % of notifications originated from non-border municipalities, and 58.32 % from border areas. Only the state of Paraná account for 51.73 % of the total notifications. This state also accounted for 66.70 % of the 32.47 % of notifications with a final diagnosis of "absence of clinically compatible signs or susceptible animals", indicating a certain lack of knowledge in the area, leading to unnecessary notifications and system overload. The performance of the OVS was evaluated based on the service response time from notification registration trough Logistic and Negative binomial regressions. A total of 27.83 % of notifications did not meet the Brazilian legally specified time, and the zone related to the state of Parana needs improvements in performance. The presence and peaks of Senecavirus A cases may have influenced an increased number of swine notifications and led to a decrease in OVS response time. The results demonstrate better performance of surveillance in border areas. Given the vast territory of Brazil, it is not expected that 100 % of responses occur within the legal timeframe, however, the performance of the surveillance system proved to be adequate, with 86 % complied to the legislation. The performance indicators could be used as a monitoring tool, along with indicators to demonstrate system overload. Continued education actions are crucial for strengthening PNEFA.

Exposure to Gynura japonica (Thunb.) Juel plants induces hepatoxicity in rats and Buffalo rat liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura japonica (Thunb.) Juel is often confused with the non-pyrrolizidine alkaloid-producing herbs, Tu-San-Qi (Sedum aizoon L.) and San-Qi (Panax notoginseng L.), due to similarities in name, appearance, and medicinal use. It contains pyrrolizidine alkaloids, which cause over 50% of cases of hepatic sinus obstruction syndrome. However, the mechanisms underlying G. japonica-induced hepatotoxicity remain poorly understood. AIM OF THE STUDY: In this study, we aimed to investigate the toxic effects of a G. japonica decoction on liver and Buffalo rat liver (BRL) cells and elucidate the associated mechanisms. MATERIALS AND METHODS: This study employed G. japonica decoction and examined its effects on liver function and tissue damage in Sprague-Dawley rats. Bioinformatics analysis was employed to identify gene expression and enriched pathways related to hepatotoxicity. Laser scanning confocal microscopy and flow cytometric annexin V/PI labeling assays were utilized to observe apoptosis in BRL cells induced by G. japonica. Transmission electron microscopy and JC-1 staining were used to determine the effects of G. japonica on mitochondrial ultrastructure and membrane potential in BRL cells. The bicinchoninic acid method and enzyme-linked immunosorbent assays were used to detect the expression of apoptosis-related proteins and caspase-3 activity, respectively. RESULTS: Comparisons of body weight, liver histopathology, and serum liver function-related indices in rats, t showed that exposure to G. japonica may cause liver damage. Bioinformatics analysis indicated that hepatotoxicity might be related to apoptotic signaling pathways, the positive regulation of programmed cell death, and responses to toxic substances. BRL cells exposed to the G. japonica decoction exhibited mid-to late-stage apoptosis and necrosis, along with alterations in mitochondrial morphology and membrane potential. Furthermore, expression of cytochrome C (Cyt C) and pro-apoptotic proteins was increased, anti-apoptotic proteins decreased, and caspase-3 activity elevated. CONCLUSIONS: These findings indicate that G. japonica-induced hepatotoxicity involves the activation of mitochondria-mediated apoptosis. Our research enhances the scientific and theoretical foundation for clinical therapy and improves public awareness of the potential toxicity of herbal remedies.

Meta-Genomic Analysis of Different Bacteria and Their Genomes Found in Raw Buffalo Milk Obtained in Various Farms Using Different Milking Methods.

Milking methods have significant impacts on the microbiological composition, which could affect the quality of raw buffalo milk. Hence, the current study was conducted on the impact of milking methods on microorganisms in buffalo tank raw milk from 15 farms in Guangxi, China. The farms were divided into two groups based on the milking method: mechanical milking (MM, n = 6) and hand milking (HM, n = 9). Somatic cell counts, bacterial cell counts and nutrients of the raw buffalo milk samples were analyzed. The comparison of raw buffalo milk samples was analyzed using metagenomic sequencing to detect any differences between the two groups. There was no significant difference in the basic nutritional compositions and somatic cell count of raw buffalo milk between the two milking methods. However, the HM samples had significantly higher bacterial counts and diversity compared to the MM samples. The results showed that Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Pseudomonas spp. were the major microbes present in canned raw buffalo milk. However, the differences between the two milking methods were the relative abundance of core microorganisms and their potential mastitis-causing genera, including the content of antibiotic-resistance genes and virulence genes. Our study revealed that Staphylococcus spp. and Streptococcus spp. were significantly more abundant in the MM group, while Klebsiella spp. was more abundant in the HM group. Regardless of the milking method used, Pseudomonas spp. was identified as the primary genus contributing to antibiotic resistance and virulence genes in canned raw buffalo milk. These findings affirm that there are differences in the microbial and genomic levels in canned raw milk. To prove the functional roles of the discovered genes and how these genes affect milk quality, further research and experimental validation are necessary.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 µM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 µM naringenin compared to 200 µM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 µM naringenin compared to all groups except 100 µM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 µM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 µM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 µM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 µM concentration.

Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Exploring the Nexus of Steroidal Hormone Receptor, Uterine VEGF Expression and NADPH-d Interaction in Buffalo Uterus During Oestrous Cycle With Seasonal Variation.

The reproductive efficiency in buffalo is highly influenced by seasonal variability. Angiogenesis in the reproductive cycle is important for optimal physiological functioning of uterus. Estrogen receptor-α (ERα), vascular endothelial growth factor (VEGF) and reduced nicotinamide adenine dinucleotide phosphatase diaphorase (NADPH-d) are vital indicators for the uterine angiogenic process. This study was conducted to see the effect of season on the expression of different uterine angiogenic factors. Season wise (winter and summer) and phase wise (follicular and luteal), immune staining intensity of buffalo uterus was measured by calculating the optical density value (OD) for ERα and VEGF. Percentage of immuno-positive cell count for ERα was done. Histoenzymic NADPH-d expression was analysed. Expression of all these factors increased during follicular phase of oestrous cycle in order to support the angiogenesis; however, the expression was significantly lower (p ≤ 0.05) in term of OD value as well as percentage count of immuno-positive cells during summer season indicating lower angiogenic activity that subsequently affected reproduction in buffalo.

Microbiome signatures associated with flavor development differentiate Protected Designation of origin water Buffalo Mozzarella cheese from different production areas.

Water Buffalo Mozzarella (BM) is a typical cheese from Southern Italy with unique flavor profile and texture. It is produced following a traditional back-slopping procedure and received the Protected Designation of Origin (PDO) label. To better understand the link between the production area, the microbiome composition and the flavor profile of the products, we performed a multiomic characterization of PDO BM collected from 57 different dairies located in the two main PDO production area, i.e. Caserta (n = 35) and Salerno (n = 22). Thus, we assessed the microbiome by high-throughput shotgun metagenomic sequencing and the Volatile Organic Compounds (VOCs) by gas chromatography/mass spectrometry (GC/MS). Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. delbrueckii were identified as the core microbiome present in all samples. However, the microbiome taxonomic profiles resulted in a clustering of the samples based on their geographical origin, also showing that BM from Caserta had a greater microbial diversity. Consistently, Caserta and Salerno samples also showed different VOC profiles. These results suggest that the microbiome and its specific metabolic activity are part of the terroir that shape BM specific features, linking this traditional product with the area of production, thus opening new clues for improving traceability and fraud protection of traditional products.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

Mitochondrial DNA D-loop SNPs unveil molecular signatures of milk production variation in Murrah buffalo.

BACKGROUND: The Murrah buffalo, pivotal in Asian agriculture, faces challenges in maximizing milk production despite significant breeding efforts. Recognizing its economic importance, this study investigates mtDNA D-loop variations in Murrah buffalo as potential indicators of milk production variability, addressing challenges in maximizing yield despite significant breeding efforts. METHODS AND RESULTS: Analyzing mtDNA D-loop sequences from 50 buffaloes, we categorized them into Low (Group 1), Medium (Group 2), and High ECM (Group 3) groups based on milk yields, fat and protein percentage of a 30-day period data. Somatic cell mtDNA D-loop analysis revealed distinct genetic variations, with significant differences among ECM groups. Group 2 showed higher SNP prevalence, group 3 had more insertions/deletions, and Group 1 exhibited the highest transition frequency. Notably, a consistent "C" deletion at the 714th position occurred in Groups 1 and 3, prevalent in 68% of Group 2. A G-A variation at the 93rd position was specific to the medium ECM group. Negative Tajima D values indicated unique variations in each group, with Group 1 having the highest number, and a specific SNP linked to Group 2 was identified. These SNPs in the D-loop region could impact mtDNA replication, influencing mitochondrial content among animals. Our results provide valuable insights into the role of mtDNA D-loop polymorphisms in milk production traits in Murrah buffalo. CONCLUSIONS: Our research highlights the potential for valuable markers of cellular energy efficiency in Murrah buffalo. Exploring diverse cytoplasmic backgrounds opens avenues for mtDNA-based selection strategies, enhancing milk production and optimizing genetic traits for the dairy industry.

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.