Detection of prion gene promoter and intron1 indel polymorphisms in Anatolian water buffalo (Bubalus bubalis)

Bovine spongiform encephalopathy (BSE) is a fatal disease caused by miss folded prion protein. Studies in the cattle, comparing genetic data from BSE diseased and healthy animals have shown that indel polymorphisms in the promoter and intron 1 of PRNP gene were associated with disease susceptibility. Several studies were conducted to find out allele and genotypic frequencies of indel polymorphisms in promoter and intron 1 of the cattle PRNP gene. Unlike domestic cattle and bison, no indel polymorphisms of the PRNP promoter and intron 1 were examined in any population of the water buffalo (Bubalus bubalis). Aim of this study was to analyse frequencies of allele, genotype, and haplotype of the indel polymorphisms (23 bp indel in promoter and 12 bp indel in intron 1) in prion protein coding gene (PRNP) of water buffalo. Therefore a PCR based procedure, previously used in cattle to detect indel polymorphisms of PRNP promoter and intron 1 locus, was applied to 106 Anatolian water buffalo DNAs. Our results have revealed high frequency of in variants and in23/in12 haplotype for PRNP promoter and intron 1 indel polymorphisms in water buffalo. The results of the study have demonstrated that frequencies of allele, genotype, and haplotype of the indel polymorphisms in PRNP gene of the Anatolian water buffalo are significantly different those from cattle and bison PRNP indel polymorphisms.     

Prenatal development of retina in buffalo (Bubalus bubalis)

The development of retina in Indian buffalo (Bubalus bubalis) has not been reported previously. The aim of the present study was therefore to report the major landmarks and the time course in the development of retina. Serial histological sections of Indian buffalo embryos and foetuses were used as group1 (<20.0 cm CVRL), group2 (>20.0 but <40.0 cm CVRL) and group3 (>40.0 cm CVRL). Age estimation was made on the basis of crown vertebral‐rump length (CVRL), which ranged between 36 and 286 days (1.6–94.0 cm). The retina in Indian buffalo was developed in a similar manner to that of the other mammals with the principal differences in the time of occurrence of various layers of this nervous tunic. In 36 days (1.6 cm stage), the foetal retina was composed of pigmented layer and the layer of neuroblasts. Differentiation of layers was first observed in 47 days (4.0 cm CVRL) which became prominent in 52 days (5.1 cm stage). At 120 days (20.5 cm stage), the differentiation of inner plexiform layer and inner nuclear layer was evident. At 143 days (31.0 cm) foetal age, the faint line in neuroblastic layer was the first evidence of the future outer plexiform layer. In foetuses of group III, the retina was comprised of all 10 layers (eight cell layers and two membranes) viz. pigmented epithelium, layer of rods and cones, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, layer of nerve fibres and the inner limiting membrane.     

Subcutaneous pharmacokinetics and dosage regimen of cefotaxime in buffalo calves (Bubalus bubalis)

The pharmacokinetics and dosage regimen of cefotaxime following its single subcutaneous administration (10 mg/kg) were investigated in buffalo calves. Plasma and urine samples were collected over 10 and 24 h post administration, respectively. Cefotaxime in plasma and urine was estimated by microbiological assay technique using E. coli as test organism. The pharmacokinetic profiles fitted one-compartment open model. The peak plasma levels of cefotaxime were 6.48 ± 0.52 µg/ml at 30 min and the drug was detected upto 10 h. The absorption half-life and elimination half-life were 0.173 ± 0.033 h and 1.77 ± 0.02 h, respectively. The apparent volume of distribution and total body clearance were 1.17 ± 0.10 l/kg and 0.45 ± 0.03 l/kg/h, respectively. The urinary excretion of cefotaxime in 24 h, was 5.36 ± 1.19 percent of total administrated dose. A satisfactory subcutaneous dosage regimen for cefotaxime in buffalo calves would be 13 mg/kg repeated at 12 h intervals.     

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10¹⁰ colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.     

Ear-tag retention and identification methods for extensively managed water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo were studied to evaluate ear-tag retention during an epidemiologic field trial. Plastic ear-tags were placed in both ears before the start of the trial, which was implemented in an extensively managed domestic water buffalo herd of approximately 1000 animals in Trinidad from 1999–2001. The presence or absence of ear-tags was recorded at the times of animal handling. The rate of ear-tag loss was modeled using a parametric survival analysis assuming an exponential rate of loss. A gamma distribution was used to estimate the amount of time that each animal would be positively identified based only on the presence or absence of one or more ear-tags. The overall median ear-tag retention was 272 days. The estimated rate of ear-tag loss was 0.0024 ear-tags lost per day. The use of ear-tags alone might not be sufficient for long-term identification of extensively managed animal populations.     

Disposition kinetics and urinary excretion of gentamicin in buffalo bulls (Bubalus bubalis)

The disposition kinetics and urinary excretion of gentamicin sulphate were studied in young buffalo bulls following a single intramuscular administration of the drug at 5 mg kg^-1 body weight. The time course of the serum gentamicin concentration was adequately described by the one-compartment open model. The values of the absorption and elimination halflives were 12.2±2.2 and 167.0±29.7 min respectively. The apparent volume of distribution was 0.29±0.01 L kg^-1. During the first 12 h, 63% of the total administered dose was excreted in urine. On the basis of the kinetic data, a satisfactory intramuscular dosage regimen for gentamicin sulphate would be at least 6 mg kg^-1 body weight repeated at 8 h intervals.     

Prevalence of antibodies against Bubaline herpesvirus (BuHV-1) among Mediterranean water buffalo (Bubalus bubalis) with implications in buffalo trade

Background: Both Bovine herpesvirus (BoHV-1) and Bubaline herpesvirus (BuHV-1) have been reported to cross the species barrier. Antibody seroconversion in glycoprotein E (gE) blocking ELISA during BuHV-1 infection has been documented. Recent diagnostic efforts have focused on the development and application of discriminatory tests to distinguish between infections with BoHV-1 and BuHV-1.

Objective: To evaluate the impact and distribution of these two infections in water buffalo farms in two regions (Piedmont (n = 3) and Campania (n = 10), Italy) where infectious bovine rhinotracheitis control programs have been implemented.

Animals and methods: Sampling was carried out on 13 buffalo farms comprising 1089 animals using specific gE-indirect ELISA's test able to discriminate among BoHV-1 and BuHV-1 infections.

Results: 59.0% of animals reacted positive to ELISA (irrespective of whether BoHV-1 or BuHV-1 antigen was used) and 86.4% of these were reactive to BuHV-1 only, whereas 11.8% showed absorbance values for both antigens and were classified as inconclusive. There was a statistically significant age-related difference in BuHV-1 infection rates but not in overall individual (47% vs. 58%) or herd prevalence (100% vs. 90%) of infection between the two regions.

Conclusion: The low percentage of sera reactive to BoHV-1 (1.8%, 12/643) indicates that BuHV-1 may be the main circulating alphaherpesvirus infection in Mediterranean water buffalo in the two study areas. Since Bubalus bubalis is included in Directive 64/432/EEC on animal health problems affecting intra-community trade in bovine animals, diagnostic testing with nonspecific ELISA for BoHV-1 infection in buffalo may yield false-positive reactions. This scenario could lead to economic losses and hamper buffalo trade and movement, particularly for reproduction purposes.     

Label‐free proteome of water buffalo (Bubalus bubalis) seminal plasma

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label‐free shotgun UDMS^E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Efficiency of uterine fluid cytology in the diagnosis of subclinical endometritis in the water buffalo (Bubalus bubalis)

This study compared endometrial cytology vis‐a‐vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)‐affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE‐affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.     

Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand

Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5–10 years of age (13.4%), 3–5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level.     

Comparison of carcass and meat characteristics of Brahman grade cattle (Bos indicus) and crossbred water buffalo (Bubalus bubalis)

This study was conducted to determine and compare the live traits, carcass yield and the physical, chemical, processing and sensory properties of meat from 20 Brahman grade cattle (crossbred cattle) and crossbred water buffalo (Philippine carabao × Murrah, Bulgarian or Indian) with an average age and weight of 29 months and 434 kg, respectively. The animals were fed a similar diet for 180 days before slaughter. A survey was performed to establish consumer meat preference between the two species. Live weights before slaughter of the two species were found to be different (P < 0.05) with the crossbred water buffalo showing higher bodyweight. However, the crossbred water buffalo had a lower (P < 0.05) dressing yield based on hot carcass weight and chilled carcass weight. The lower dressing yield of crossbred water buffalo can be attributed to its higher (P < 0.05) weights of edible and non‐edible slaughter by‐products. The chilled carcass yield of the forequarters and hindquarters from crossbred cattle and crossbred water buffalo were comparable when expressed as percent of live weight and chilled carcass weight. The estimated lean yield was higher in crossbred cattle than crossbred water buffalo. Crude protein, ash, fat, cholesterol, myofibrillar, sarcoplasmic and insoluble protein contents of the beef and carabao meat were all similar. Water holding capacity, pH, muscle fiber diameter, tenderness, firmness and marbling score in carabeef were all comparable to the beef. Redness was found to be higher (P < 0.05) in carabeef than the beef as measured with a color meter. The consumer meat preference survey showed that 55.9% selected the beef while 44.1% preferred carabeef. The color and amount of fat on the outside of the beef sample were primary in buyer selection.     

Theca cell-conditioned medium enhances steroidogenesis competence of buffalo (Bubalus bubalis) granulosa cells

Theca cells (TCs) play a crucial role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to oestrogens needed for follicular growth. However, the effects of TCs in the form of conditioned medium on steroidogenesis in buffalo GCs remain unclear. In the present study, the impacts of TC-conditioned medium (TCCM) on oestrogen synthesis in buffalo GCs were examined. The results showed that TCs secreted principally testosterone, but almost no androstenedione or oestradiol into TCCM. TCs at passage 3 had a stronger secretion capacity of testosterone in TCCM. Furthermore, TCCM collected at 72 hr improved both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1, 3β-HSD and 17β-HSD) and the secretion levels of estradiol in GCs. The treatment of 72 hr in TCCM promoted both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 3β-HSD) and the secretion levels of estradiol in GCs. Besides, TCCM that was collected at 72 hr and applied to GCs for 72 hr (72 & 72 hr) improved the sensitivity of buffalo GCs to FSH. This study indicates that TCCM (72 & 72 hr) enhances the steroidogenesis competence of GCs mainly through facilitating the responsiveness of GCs to FSH in buffalo.     

Genetic analysis of river,swamp and hybrid buffaloes of north‐east India throw new light on phylogeography of water buffalo (Bubalus bubalis)

This study analysed buffaloes from north‐east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South‐East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north‐east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise F_ST revealed the clustering of all swamp‐type buffaloes of north‐east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D‐loop region of 530‐bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north‐east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise F_ST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median‐joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north‐east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north‐east India to South‐East Asia.     

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis)

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)‐affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis‐affected buffaloes. The extent of difference between normal and subclinical endometritis‐affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.     

Ultrastructure changes in buffalo (Bubalus bubalis) oocytes before and after maturation in vitro with sericin

The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis ). Oocytes were matured in vitro in tissue culture medium?199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P  < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS ). In contrast, the oocytes matured either with or without sericin showed the formation of PVS , erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P  < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin.     

Epidural anesthesia in Egyptian water buffalo (Bubalus bubalis): a comparison of lidocaine,xylazine and a combination of lidocaine and xylazine

Objective

To evaluate and compare the analgesic effects of a combination of lidocaine and xylazine to lidocaine or xylazine administered alone for epidural anesthesia in Egyptian water buffalo (Bubalus bubalis).