Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus–oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

低氧对水牛卵泡内膜细胞雄激素合成能力的影响

本研究采用低氧(5％O2)条件培养水牛内膜细胞,探讨低氧对水牛内膜细胞雄激素合成的影响.结果 显示,低氧能显著提高水牛内膜细胞雄激素合成相关基因(CYP11A1、CYP17A1和3β-HSD)表达和睾酮分泌水平(P＜0.05);低氧能显著增强水牛内膜细胞对促黄体素(LH)的敏感性(P＜0.05).结果 表明,低氧(5％...     

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10^-3, 10^-5, 10^-7, and 10^-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10^-5 M SNP + 1 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10^-3, 10^-5, 10^-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10^-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.     

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.     

Histology,histochemistry and ultrastructure of the nasopharyngeal tonsil of the buffalo (Bubalus bubalis)

The light microscopic appearance and ultrastructure of the nasopharyngeal tonsil (tonsilla pharyngea), collected from 12 adult buffaloes of local mixed breed, were explored for the distribution of different types of epithelia, lymphoid tissue and high endothelial venules. The tonsillar mucosa was lined by pseudostratified columnar ciliated epithelium having goblet cells. The respiratory epithelium associated with the underlying lymphoid tissue formed the lymphoepithelium. The epithelium was further modified into follicle‐associated epithelium (FAE) characterized by reduced epithelial height, presence of a few dome‐shaped cuboidal cells equivalent of the M‐cells and absence of goblet and ciliated cells. The lymphoid tissue was distributed in the form of isolated lymphoid cells, diffuse lymphoid tissue and lymphoid follicles, mainly distributed within the propria‐submucosa along with the sero‐mucous glandular tissue. The goblet cells of the respiratory epithelium and the acinar cells contained different mucopolysaccharides. Scanning electron microscopy of the surface mucosa demonstrated a dense mat of cilia, island‐like arrangement of microvillus cells, M‐cells and a few brush‐like cells. The transmission electron microscopy revealed the different cell organelles of the respiratory epithelium and the FAE. Lymphocyte migration via the high endothelial venules in the propria‐submucosa was also observed.     

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.     

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.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

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.     

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.     

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.     

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase‐3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.     

Effect of exogenous growth-hormone-releasing factor on blood metabolites and minerals in late maturing buffalo heifers (Bubalus bubalis)

Previous studies have suggested that growth-hormone-releasing factor (GRF) enhanced growth and advanced puberty onset along with hormonal changes in buffalo heifers (Bubalus bubalis). However, it is not known to what extent exogenous GRF could influence blood metabolites and minerals to bring about puberty in buffalo heifers. Therefore, we planned to investigate the effect of exogenous bovine GRF (bGRF) on blood metabolites and minerals in buffalo heifers during a 3-month pre-treatment period, 9-month treatment period and 1-month post-treatment period. Six buffalo heifers were treated intravenously with bGRF (10 mug per 100 kg body weight) at 15-day interval for 9 months. Another six buffalo heifers of weight- and age-matched received requisite amount of vehicle (0.9% NaCl solution) during the same period. Exogenous bGRF enhanced (p < 0.01) plasma non-esterified fatty acids (NEFA) concentrations in treatment group when compared with control group during the treatment and post-treatment period, while plasma alpha-amino nitrogen (AAN) concentrations showed a decreasing trend (p < 0.05) in the treatment group when compared with the control group during the treatment and post-treatment periods. The plasma inorganic phosphorus (Pi) was found to be higher (p < 0.05) in the treatment group animals in comparison with the levels recorded in the control group animals during the treatment as well as post-treatment periods. However, there was no change (p > 0.05) in plasma glucose and calcium concentrations between the two groups. Plasma NEFA was found to be positively correlated with plasma growth hormone (GH); however, it was only significant for the treatment group (r = + 0.76; p < 0.05). Plasma AAN in the treatment group exhibited negative correlation with plasma GH (r = 0.72; p < 0.05), while plasma AAN and GH were recorded to be positively correlated in the control group (r = 0.47; p < 0.05). The present findings suggest that exogenous bGRF induces GH release that increases plasma NEFA and Pi and decreases AAN concentrations, which probably help to reach a certain physiological state that initiates events necessary for bringing about puberty in buffalo heifers.     

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.