Epigenetic characteristics of cloned and in vitro-fertilized swamp buffalo (Bubalus bubalis) embryos

Swamp buffalos are becoming endangered due to reproductive inefficiencies. This is of concern because many countries depend heavily on their products. Somatic cell nuclear transfer (SCNT) is a potential strategy for preserving endangered species. To date, SCNT in swamp buffalo has succeeded in the creation of blastocyst embryos. However, development to term of SCNT swamp buffalos is extremely limited, and only 1 live birth has been reported. An abnormal epigenetic mechanism is suspected to be the cause of developmental failure, as is also seen in other species. The DNA methylation and histone acetylation are key players in epigenetic modification and display marked variability during embryonic preimplantation development. Knowledge of epigenetic modifications will aid in solving the developmental problems of SCNT embryos and improving reproductive technology in the swamp buffalo. The objective of this study was to determine the relationship between preimplantation embryonic development and 2 epigenetic patterns, global DNA methylation and histone acetylation, in SCNT and in vitro-fertilized (IVF) swamp buffalo embryos. In addition, we examined the correlations between those 2 mechanisms in the SCNT and IVF swamp buffalo embryos throughout the developmental stages using double immunostaining and quantification of the emission intensities using confocal microscopy. We discovered an aberrant methylation pattern in early preimplantation-stage swamp buffalo SCNT embryos. In addition, greater variability in the DNA methylation levels among nuclei within SCNT embryos was discovered. Hyperacetylation was also observed in SCNT embryos compared with IVF embryos at the 4- and 8-cell stages (P < 0.05). Dynamic changes and interplay between these 2 epigenetic mechanisms could be crucial for embryonic development during the early preimplantation period. The aberrancies uncovered here may contribute to the low efficiency of SCNT.     

Normal haematological and biochemical values for the swamp buffalo (Bubalus bubalis) in Australia

SUMMARY: Blood samples were collected from 24 immature male, 55 immature female and 99 mature female water buffalo kept at an experimental farm in the Northern Territory. Haematological analysis was performed on blood collected in dipotassium — ethylene diamine tetra acetic acid while biochemical analysis was performed on serum and plasma (for glucose) samples. Haematological values of mature buffalo were similar to those recorded for swamp buffalo in Malaysia. Blood cell appearances were similar to those reported for adult Indian river buffalo though values recorded for red cell components were higher. Statistical analysis revealed no significant differences between immature male and female buffalo. Red cell components, eosinophils, total plasma and serum proteins, albumin, γ globulins, inorganic phosphate and the enzyme gamma-glutamyl transferase were significantly higher for mature female buffalo when compared to immature females. Reasons for the differences were not fully determined but the effect of age and nutritional status in combination with a variable period of domestication were considered.     

Malignant catarrhal fever in a swamp buffalo (Bubalus bubalis) calf in New Zealand

Malignant catarrhal fever was diagnosed in a newly imported swamp buffalo calf. This disease is prevalent in deer and cattle in New Zealand and causes sporadic losses in these species. The disease is recognised as a problem in buffaloes overseas and this paper records the first case in a swamp buffalo in New Zealand.     

The clinico-pathology of a malignant catarrhal fever syndrome in the Indonesian swamp buffalo (Bubalus bubalis)

Between September 1979 and May 1982, 50 of 177 swamp buffaloes became sick and died S3 to 719 days (mean 271) after being transferred to a research facility and housed in quarters adjacent to where sheep and goats were kept. The major clinical signs were anorexia, fever, conjunctivitis, scleral hyperaemia, ocular discharge, hyperaemia of skin, enlargement of lymph nodes and depression. Postmortem and histopathological lesions were observed in most of the tissues and organs of the body. A vasculitis with adventitial mononuclear cell infiltration and lymphoreticular reactions suggested a relationship to malignant catarrhal fever. The occurrence of serofibrinous epicarditis and myocarditis, which was seen in buffaloes in all cases, has not been a common finding in ruminants with malignant catarrhal fever.     

Immunoreactive prolactin, progesterone and luteinizing hormone in the seminal plasma of buffalo (Bubalus bubalis).

The presence of immunoreactive prolactin, luteinizing hormone (LH) and progesterone in buffalo seminal plasma is reported for the first time. Correlations were obtained between various semen attributes and the levels of the above immunoreactive hormones. Statistically significant (P < 0.01) negative correlations were found between the levels of immunoreactive prolactin in semen and sperm motility and viability. The coefficient of multiple linear correlation (R2) between the levels of immunoreactive progesterone, prolactin and LH in the seminal plasma and the various attributes of semen revealed that immunoreactive progesterone and prolactin showed stronger interactions than did LH. The biological significance of these immunoreactive hormones in semen is discussed.     

The effect of gonadotropin releasing hormone on superovulation in elite swamp buffalo cows (Bubalus bubalis)

The effect of gonadotropin releasing hormone (GnRH) supplement was investigated in twenty eight FSH-treated buffalo cows. Animals were assigned to three groups; Group I: GnRH was given at standing heat (n=9), Group II: GnRH was given 8-12 hr after standing heat (n=8) and Group III: Control group with FSH alone (n=11). The responses (no. of corpora lutea and no. of anovulatory follicles), the number of recovered embryos and transferable embryos among the three groups were compared following slaughter of the animals on days 6 to 7 after first mating. The results indicated that the application of GnRH in FSH-treated animals gave no advantage by increasing in the number of ovulations or recovered embryos in all the treatment groups (P>0.05): 4.33 +/- 3.35 vs 3.88 +/- 4.09 vs 4.5 +/- 2.68 for corpora lutea, and 2.33 +/- 2.24 vs 2.0 +/- 3.20 vs 1.91 +/- 2.74 for recovered embryos respectively. GnRH treatment tended to reduce the number of anovulatory follicles but the finding was not significant; 6.11 +/- 3.3 vs 7.38 +/- 4.84 vs 10.18 +/- 2.74 follilcles (P>0.05). The supplementation of GnRH at 8-12 hr after standing heat seemed to produce more transferable embryos than those of treated at standing heat or the controls 1.63 +/- 2.77 vs 1.25 +/- 1.67 vs 1.36 +/- 1.69 embryos respectively.     

Detomidine-diazepam-ketamine anaesthesia in buffalo (Bubalus bubalis) calves

Eight buffalo calves (8-12 months, 70-100 kg) were randomly assigned to two groups of four animals each. Animals of group I were given detomidine (100 micrograms/kg), whereas animals of group II received a mixture of detomidine (100 micrograms/kg), diazepam (100 micrograms/kg) and ketamine (3 mg/kg) (DDK) intravenously. Various clinical parameters, such as weak time, down time, pedal and pinprick reflexes, muscle relaxation and extent of sedation, as well as heart and respiratory rates and electrocardiograms were measured before (time 0) and 15, 30, 45, 60, 75 and 90 min after treatment. In all the animals of group II (DDK), the pedal reflex was completely abolished (score: 3.00 +/- 0.00) within 5 min, the pinprick response was either very weak or it was completely abolished at this interval. Muscle relaxation and sedation were excellent within 5 min of DDK administration. The depth of sedation and analgesia was maximum from 5 to 15 min postinjection. Detomidine alone, however, failed to produce appropriate depression of the pedal and pinprick reflexes, sedation was mild and muscle relaxation was inadequate. Heart rate showed a significant (P < 0.05) decrease in group I, but the decrease was non-significant in group II. A more pronounced increase in respiratory rate was observed in group I as compared to group II. Animals of both groups recovered within 90 min without any complication. Minimal changes in the cardiovascular system in the group given the DDK combination were an advantage over the group given detomidine. The results indicated that DDK combination is safe and suitable for 15 min of anaesthesia with excellent muscle relaxation and has only limited cardiorespiratory effects in buffaloes.     

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.     

Transmission and virological studies of a malignant catarrhal fever syndrome in the Indonesian swamp buffalo (Bubalus bubalis)

A malignant catarrhal fever-like syndrome in indonesian swamp buffalo was experimentally transmitted to one of 2 Bos indicus and 3 of 3 Bos javanicus cattle by intravenous inoculation of 250 ml of citrated, whole blood from affected buffaloes. The 4 cattle developed clinical signs of disease on average 32.5 days after receiving the inoculation of blood. The 4 cattle died after a variable period of illness. None of a further 3 B. javanicus cattle inoculated intravenously with a spleen homogenate prepared from another affected buffalo developed the disease. The experimental disease was clinically and pathologically similar to the natural disease in buffaloes although differences were noted. Attempts to adapt the agent to mice, guinea pigs and rabbits failed. A cytopathic agent (Japanese encephalitis virus) was isolated from the spleen of one buffalo with clinical signs but was not considered significant. Sixty-three B. indicus, 7 B. javanicus (and 6 of their crosses), 3 B. taurus and 4 Bubalus bubalis (Murrah buffalo) were kept in the same quarters where 50 of 177 swamp buffaloes died between September 1979 and May 1982. Four of the 7 B. javanicus cattle developed the clinical signs of disease and died. All the other cattle in contact remained healthy.     

Pharmacokinetics and plasma protein binding (in vitro) of oxytetracycline in buffalo (Bubalus bubalis)

The pharmacokinetics of oxytetracycline given in a single dose (22 mg/kg) either IV or IM was studied in 4 female buffalo calves. The half-life (t1/2) after IV administration varied between 169.02 and 216.56 minutes and that after IM administration, between 630 and 990 minutes. The drug was distributed well in the body after IM administration (Vdarea 1.18 to 2.15 L/kg). The total body clearances varied between 1.02 and 1.45 and between 1.17 and 1.49 ml/kg/min after the IV and the Im dosings, respectively. It has been proposed that oxytetracycline is excreted mainly by glomerular filtration in the buffalo species, but tubular reabsorption also may have a small part. About 42% of the drug was bound to plasma proteins at concentrations of 2 to 20 micrograms of oxytetracycline/ml. The drug dosage schedules to maintain serum levels of 0.5, 1, 2, and 5 micrograms/ml also are determined.     

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.     

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.     

Neosporosis in water buffalo (Bubalus bubalis) in Southern Italy

A study was carried on 1377 water buffalo serum samples from 50 farms in southern Italy to test the presence of Neospora caninum antibodies by indirect fluorescence antibody test (IFAT). Rabbit anti-buffalo immunoglobulins conjugated to fluorescein were used in the test. Fluorescence in sera dilutions above 1:200 was considered as indicative of the presence of N. caninum antibodies. The overall prevalence of infection in the animals was 34.6%. The prevalence increased in relation to the age of subjects and most of the herds examined (82%) were found infected. In two farms abortions and neurological signs were reported. No suppurative inflammatory lesions were seen, but few protozoan-like cysts were observed on foetal tissues by histology.     

Toxocara vitulorum in the milk of buffalo (Bubalus bubalis) cows

The duration of excretion of Toxocara vitulorum larvae in the milk of buffalo cows determines the optimum time for treating calves. Studies on 10 cows showed that a few larvae occur in the colostrum of some cows before the calf has suckled, but most are present from the day after calving and for a further five days. From day 9 onwards, very few larvae were found in the milk. The total number of larvae found was comparable with the number of adult parasites collected from the calves of cows with similar histories. The larvae were 1254 +/- 60 microns long and 36 +/- 6.7 microns in diameter at the ventriculus, figures which are substantially different from some published results.