Physico-chemical characterization of growth hormone from water buffaloes (Bubalus bubalis)

A purified preparation of growth hormone from pituitaries of water buffaloes (Bubalus bubalis) has been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00+/-8.44Da and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by electrophoresis and molecular sieve chromatography using 125I-labelled buffalo GH. Similar size heterogeneity was found in standard preparations of ovine and bovine growth hormones. Isoelectric focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge isoforms having pI of 7.2, 7.7 and minor forms in the pI range of 5.7 to 7.0 were found. Lectin chromatography on Concanavalin A matrix showed that less than 1% of buffalo GH was glycosylated. Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine as the NH2-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan residue indicated that a single tryptophan residue was present. Ellman's method showed presence of two disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited lambda emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of buGH was constituted by alpha-helicity. The T(M) of buGH as determined by differential scanning calorimetric (DSC) studies was found to be 63 degrees C.     

Induction of parturition in water buffaloes (Bubalus bubalis )

Parturition was induced in ten buffaloes by combining treatments of dexamethasone and vetoestrol, so that they calved about one month before the expected term. Either dexamethasone alone (Group B) or dexamethasone and vetoestrol (Group C) was used. Another five animals served as controls (Group A). Calving was induced in four animals in group B and three animals in group C after two injections of the compounds four days apart. The average time from first injection to calving for these animals was 117.22 hr and 117.66 hr for groups B and C respectively. Induced calves weighed less at birth (P < 0.05) averaging 24.4 and 26.2 kg for groups B and C respectively, than controls (Group A; 30.20 kg). The body weight gain/week among calves was not significantly different (P > 0.05). The service period, number of services/conception and the milk yield of the buffaloes induced to calve was not significantly different (P > 0.05) from their previous records.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Bio-histopathological studies of endometrium in repeat breeding buffaloes (Bubalus bubalis)

A total of 24 uterine biopsies were successfully obtained from 40 repeat-breeding buffaloes. The endometrium of 15 buffaloes showed marked neutrophilic infiltration in the epithelium, stroma and uterine glands. In some cases, lymphocyte and plasma cell infiltration, cystic dilatation of the glands and periglandular fibrosis indicated chronicity of the endometritis. Most of the animals (85.0%) had uterine cultures positive for Streptococci, Staphylococci, Gram positive variable cocci, Gram negative bacilli, Anthracoids, and Gram positive bacilli with a predominance of Corynebacteria. With the treatment based on in vitro drug sensitivity tests, 57.1% repeat-breeding buffaloes conceived.     

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).     

Serum prolactin levels of non-cycling murrah buffaloes (Bubalus bubalis )

Prolactin levels were quantified by double antibody radio-immunoassay in the blood sampled daily for 24 days, from 9 non-cyclic Murrah buffaloes during hot months (ambient temp.41 to 43 degrees C). All buffaloes were kept in open loose housing. Three buffaloes were sprinkled with water for half an hour twice daily between 1200 and 1430 h to overcome partly the effect of heat during the period of investigation. Mean prolactin levels of 6 unsprinkled buffaloes ranged on different days from 249 to 739 ng/ml serum. The range of averages in case of 3 sprinkled animals was from 152 to 342 ng/ml serum. Buffaloes subjected to sprinkling had significantly (P / 0.01) lower prolactin levels than control buffalo.     

Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis)

Altogether 218 frozen semen AI doses, prepared between 1980 and 1989 and also between 2003 and 2005 from 18 AI Thai swamp buffalo sires, were examined to determine whether seasonality affects post-thaw viability, as plasma membrane integrity (PMI, using SYBR-14/PI), plasma membrane stability (PMS, using Annexin-V/PI), or motility (Mot, using CASA). A thermoresistance test (38 degrees C for 60 min) was used to further analyze sperm survivability in vitro. All variables were compared over 3 seasons of the year (rainy: July-October; winter: November-February; and summer: March-June), with distinct ambient temperature and humidity. PMI (% of alive spermatozoa) was higher in winter (54.6%, P<0.001) than in the rainy (43.5%) or summer (46.7%) seasons. Outcomes of PMS (Annexin-V/PI assay) confirmed those of PMI, the highest PMS in spermatozoa processed in winter (55.7%, P<0.001). Spermatozoa depicting linear Mot post-thaw ranged from 48.2% to 48.8% across seasons (ns), proportions that decreased during incubation (33.5-37.9%), albeit without seasonal differences. The mean percentages of straight linear velocity (VSL), average path velocity (VAP), or curvilinear velocity (VCL) were higher (P<0.05-0.001) in the rainy season than in winter or summer, while average lateral head displacement (ALH) was higher (P<0.05) in summer, differences maintained after incubation. In conclusion, post-thaw PMS and PMI, assessed by flow cytometry, were significantly better in sperm samples processed during winter than in samples processed during the other seasons of the year, a seasonal difference not picked up by CASA, probably due to the larger number of spermatozoa assessed.     

Luteinizing hormone in blood plasma of post-partum buffaloes (Bubalus bubalis)

The changes in luteinizing hormone concentration were measured by heterologous double-antibody radioimmunoassay in blood plasma of 28 Murrah buffaloes. The LH concentration fluctuated between 0.22 to 0.78 ng/ml during first 21 days post-partum. The level increased significantly (P < 0.001) at estrus. The basal LH concentration of second and third week post-partum was inversely related to the first post-partum ovulation interval.     

Serum testosterone concentrations in advanced pregnancy in water buffaloes (Bubalus bubalis)

Peripheral testosterone levels were measured by radioimmunoassay of 65 serum samples taken at various intervals from 11 buffaloes in advanced pregnancy. Average testosterone levels measured at day 210 of gestation (248 ± 39 pg/ml) remained similar up to the last week of gestation. No difference in testosterone levels was recorded between buffaloes carrying male and female fetuses. Pregnant buffaloes had significantly higher testosterone levels than non-pregnant buffaloes.     

Ultrasonographic fetometry and determination of fetal sex in buffaloes (Bubalus bubalis)

The aim of the current study was to establish ultrasonic biometric threshold of different fetal parts in buffaloes and to evaluate the feasibility and accuracy of ultrasonic fetal sex determination. Serial ultrasonographic examinations were carried out on twelve pregnant buffalo-cows, during which fetal parts were measured, and fetal sex and presentation were determined. The obtained results revealed that embryo and amniotic vesicle (AV) were detected by the forth and fifth week of pregnancy, respectively. Organization was observed by the seventh week, while ossification was indicated between the eighth and 10th week. High correlations were found between different studied parameters and gestational age, where the highest correlation was found with the crown-rump length (CRL) and amniotic vesicle diameter (AVD) at the early-gestation; the biparietal diameter (BPD) at the mid-gestation; and the eyeball diameter (EBD) at the mid- and late-gestation. The results also revealed that the best window for fetal sexing was found between the 10th and 18th week of gestations, with an overall accuracy of 97.1%. The final polarity with all fetuses in anterior presentation was adopted by the 30th week. In conclusion, the overall data indicated the feasibility and value of ultrasonographic fetometry in buffaloes for evaluation of fetal development, estimation of gestational age and determination of fetal sex.     

Effects of a single nucleotide polymorphism in the leptin gene on the productive traits of dairy buffaloes (Bubalus bubalis)

The gene responsible for coding the leptin hormone has been associated with productive and reproductive traits in cattle. In dairy cattle, different polymorphisms found in the leptin gene have been associated with several traits of economic interest, such as energy balance, milk yield and composition, live weight, fertility and dry matter consumption. The aim of this study was to detect genetic variability in the leptin gene of buffaloes and to test possible associations with milk yield, fat and protein percentages, age at first calving and first calving interval. Three genotypes (AA, AG and GG) were identified by polymerase chain reaction–restriction fragment length polymorphism, which presented genotypic frequencies of 0.30, 0.54 and 0.16, respectively. The allele frequencies were 0.57 for the A allele and 0.43 for the G allele. No significant effects were found in the present study, but there is an indicative that leptin gene affects lipid metabolism.     

The behaviour and welfare of buffaloes (Bubalus bubalis) in modern dairy enterprises

This review deals with the behaviour of river buffaloes (Bubalus bubalis), in confinement and in extensive conditions, also focusing on the effects of different housing and rearing conditions on their welfare. The behavioural repertoire expressed by buffaloes in extensive and intensive conditions is similar to those displayed by other domestic ruminants. However, through natural selection, buffaloes have also acquired several morphological, physiological and behavioural (i.e. wallowing) adaptations to hot climatic conditions. Buffaloes kept in intensive conditions and having no access to pasture and water for wallowing extend their periods of idling and are less often involved in investigative activities. Confinement is also associated with a reduction of space; however, no specific studies have been carried out to determine the specific requirements of this species. Space restriction can adversely affect various aspects of buffalo welfare, such as health (increased levels of lesions and injuries), social behaviour (increased number of agonistic interactions) and heat dissipation. The buffaloes, originating from tropical areas, are well adapted to large variations in food availability and quality, and to dietetic unbalances. As to human animal relationship, it has been observed that the incidence of stepping and kicking behaviour of buffaloes in the milking parlour is positively correlated with the frequency of oxytocin injections, whereas the frequency of positive stockperson interactions with the animals such as talking quietly, petting and gentle touching are negatively correlated with the number of kicks during milking. Data from farms where both dairy cattle and buffaloes are present show that avoidance distance measured in the pen is lower in buffaloes than in cattle. This may be attributed to the fact that buffaloes are generally recognised to be curious animals. Finally, the effects of different farming practices on animal-related indicators are described. However, these measures should be integrated into a monitoring protocol, such as the Welfare Quality® scheme, to reliably assess buffalo welfare in the current intensive farming conditions.     

Hormonal dynamics and follicular turnover in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

The aim of this study was the investigation of hormonal and ovarian follicular dynamics in prepuberal buffaloes (Bubalus bubalis) bred in Italy. Eleven 5-9-month old buffalo calves ranging in weight from 122 to 270kg, maintained under controlled nutritional and environmental conditions, underwent 50 days of ultrasonographic ovarian follicular monitoring in the months of October-December. Blood sampling for E(2) and FSH determination and ultrasonographic monitoring using a 7.5MHz linear probe and an ALOKA SSD-500 monitor were performed daily. No differences in any of the parameters under study were highlighted when calves were divided into two weight categories (<200 and >200kg) and thus data were pooled. In this study, values are reported as mean+/-S.D. A range of two-six regular follicular waves was reported among calves with an average of 4+/-1.1. Overall interval (days) between wave emergence was 9.9+/-2.8 and largest diameters (mm) of dominant and first subordinate follicles were 8.4+/-1.2 and 4.8+/-0.6, respectively (P<0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting <10 days (6.1+/-1.2) were reported. They were monitored contemporaneously on the ovary contralateral (n=7) or ipsilateral (n=3) to the main follicular wave. Growth rate (mm per day) of dominant follicles (DF) was significantly faster than for corresponding subordinate follicles (SF) and follicles of SWs (1.08+/-0.2 versus 0.79+/-0.1 and 0.83+/-0.1, respectively, P<0.05). The static phase (days) lasted longer in DF compared to SF and SW (5.4+/-1.8 versus 2.4+/-1.2 and 2.6+/-1, respectively, P<0.05). The regressing phase (mm per day) was similar among DF, SF and SW (0.86+/-0.2, 0.94+/-0.2 and 0.84+/-0.1, respectively, P=0.09). Episodic spikes of E(2) and FSH were reported, corresponding to wave development throughout the course of investigation. In conclusion, the majority of buffalo calves displayed a typical pattern of regular follicular development in conjunction with a dynamic trend of ovarian and hypophyseal hormones. Some minor follicle turnover was reported with parallel main follicular waves.     

Dominance of Methanomicrobium phylotype in methanogen population present in Murrah buffaloes (Bubalus bubalis)

Aims:  To study the diversity of rumen methanogens in Murrah buffaloes ( Bubalus bubalis ) from North India by using 16S rRNA gene libraries obtained from the pooled rumen content from four animals and using suitable software analysis.
Methods and Results:  Genomic DNA was isolated and PCR was set up by using specific primers. Amplified product was cloned into a suitable vector and the positive clones were selected on the basis of blue–white screening and sequenced. The resulting nucleotide sequences were arranged in the phylogenetic tree. A total of 108 clones were examined, revealing 17 different 16S rRNA gene sequences or phylotypes. Of the 17 phylotypes, 15 (102 of 108 clones) belonged to the genus Methanomicrobium , indicating that the genus Methanomicrobium is the most dominant component of methanogen populations in Murrah buffaloes ( Bubalus bubalis ) from North India. The largest group of clones (102 clones) was more than 98% similar to Methanomicrobium mobile . BLAST analysis of the rumen contents from individual animals also revealed 17 different phylotypes with a range of 3–10 phylotypes per animal.
Conclusion:  Methanomicrobium phylotype is the most dominant phylotype of methanogens present in Murrah buffaloes ( Bubalus bubalis ).
Significance and Impact of the Study:  Effective strategies can be made to inhibit the growth of Methanomicrobium phylotype to reduce the methane emission from rumen contents and thus help in preventing global warming.     

Biometry of frozen-thawed sperm from eight breeds of Indian buffaloes (Bubalus bubalis)

Sperm morphometry, in combination with other objective traits, can be useful for developing a fertility index. The objective of the present study was to measure various biometric end points of frozen-thawed sperm from eight breeds of Indian buffaloes (Murrah, Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari, Pandharpuri and Nili-Ravi). The sperm head of Pandharpuri buffaloes had the greatest length (10.21 microm), width (6.05 microm), area (52.31 microm(2)) and perimeter (31.86 microm). The ratio of sperm width to length was also greatest (0.61) in Pandharpuri as well as in two other breeds, viz. Nili-Ravi and Jaffrabadi. Murrah had the smallest sperm head width (4.75 microm), area (41.65 microm(2)) and perimeter (29.17 microm), but its sperm tail was longest (57.02 microm), along with that of Jaffrabadi buffaloes (56.96 microm). Based on mean values of sperm tail length, mid piece length and its width the eight buffalo breeds were categorized into three, four and five groups, respectively. Multivariate analysis and clustering put six breeds (Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari and Nili-Ravi) in one cluster, whereas Murrah and Pandharpuri appeared as separate entities.