Current status of the river buffalo (Bubalus bubalis L.) gene map.

Ninety-nine loci have been assigned to river buffalo chromosomes, 67 of which are coding genes and 32 of which are anonymous DNA segments (microsatellites). Sixty-seven assignments were based on cosegregation of cellular markers in somatic cell hybrids (synteny), whereas 39 were based on in situ hybridization of fixed metaphase chromosomes with labeled DNA probes. Seven loci were assigned by both methods. Of the 67 assignments in somatic cell hybrids, 38 were based on polymerase chain reaction (PCR), 11 on isozyme electrophoresis, 10 on restriction endonuclease digestion of DNA, 4 on immunofluorescence, and 4 on chromosomal identification. A genetic marker or syntenic group has been assigned to each arm of the five submetacentric buffalo chromosomes as well as to the 19 acrocentric autosomes, and the X and Y chromosomes. These same markers map to the 29 cattle autosomes and the X and Y chromosomes, and without exception, cattle markers map to the buffalo chromosome or chromosomal region predicted from chromosome banding similarity.     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.     

Chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis)

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.     

Monosomy X and gonadal dysgenesis in a buffalo heifer (Bubalus bubalis)

Investigations on a 3-yr-old river buffalo heifer presenting anestrus revealed a chromosome make-up of 2n=49 in the lymphocyte cultures, compared with the 2n=50 characteristic of riverine buffalo. The missing chromosome was identified as one of the Xs by karyotypic analysis, and monosomy X was confirmed by C and G-banding techniques. Both ovaries of the heifer were underdeveloped, although the other components of the internal genitalia were normal. The phenotypic and karyotypic features confirmed this to be a case of ovarian dysgenesis with 49,XO karyotype similar to that of the Turner's syndrome in man and other mammals.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

Effect of vitamin E on lipid peroxidation in buffalo Bubalus bubalis L

Vitamin E significantly (P less than 0.01), inhibited lipid peroxidation as indicated by malonaldehyde (MDA) production and improved significantly (P less than 0.01) motility and percent live spermatazoa of B. bubalis semen. Bulls with higher MDA formation had lower sperm motility and percent live count. Variance due to bulls for all the three parameters were significant (P less than 0.05).     

Evidence for the presence of two different beta-globin chains in the hemoglobin of the river buffalo (Bubalus bubalis L.).

1. Hemoglobin (Hb) of the river buffalo (Bubalus bubalis L.) was studied by employing isoelectric focusing (IEF) in the 6.7-7.7 pH range and by IEF in ultra-narrow immobilized pH gradient (IPG) 7.1-7.5. 2. Three Hb BB phenotypes were identified which were characterized by sets of two or four Hbs with different isoelectric points. 3. These type were called BB, BsBs and BBs, the Bs phenotype showing Hbs with slightly slower mobility. 4. Analysis of constituent globin chains by acid urea polyacrylamide gel electrophoresis in the presence of Triton X-100, provided clear evidence of a novel polymorphism at the beta-globin level. 5. Titration curves of beta-globins from heterozygous Hb BBs indicated a single curve thus suggesting the absence of a net charge in all the pH field. 6. A neutral-to-neutral amino acid replacement probably differentiates the two beta-globins.     

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.     

A set of polymorphic DNA microsatellites useful in swamp and river buffalo (Bubalus bubalis)

DNA microsatellites have found widespread application in gene mapping, pedigree determination and population genetics. In closely related species such as bovids, heterologous polymerase chain reaction (PCR) primers may in some cases be used, bypassing the need to isolate and characterize microsatellite-containing sequences and design PCR primers. We report on the ability of a set of eighty bovine derived DNA microsatellite primers to amplify sequences in the two types (swamp and river) of water buffalo ( Bubalus bubalis ). Number of alleles and per cent heterozygosities in a large number of animals were determined on a subset of microsatellite loci selected on the robustness of the primers. These loci will form the basis of a set of polymorphic DNA markers for use in water buffalo.     

The effect of different doses of gonadorelin on ovarian follicle dynamics in river buffalo (Bubalus bubalis)

The objective of this study was to determine the response of the ovarian dominant follicle to the different doses of GnRH in river buffalo. The estrous cycle of 12 river bufflaloes was synchronized using norgestomet implant for 12 days in association with two injections of prostaglandin F2alpha analogue on Days 0 and 7 of implant insertion. On Day 6 or 7 of the ensuing cycle (Day 0 of the experiment), females received a norgestomet implant in conjunction with two prostaglandin injections on Days 0 and 1. On Day 6 of the experiment, females were randomly allocated into three groups. At this time, Group 1 and 2 females were given an i.m. injection of 50 or 100 microg Gonadorelin, respectively. Group 3 females did not receive any further treatment and were considered as control. All females were given prostaglandin on Day 12 and implants were removed on Day 13 of the experiment. The results revealed that in the control group, ovarian dominant follicle became persistent throughout the experiment; whereas, the persistent dominant follicle in all females belonging to Group 2 (100 microg GnRH) and one female in Group 1 (50 microg GnRH) ovulated within 48 h, subsequent with an emergence of a new follicular wave and an increase in plasma progesterone concentration within 72 and 96 h after GnRH injection, respectively. In conclusion, 100 microg of Gonadorelin seems to be the most effective dose to induce ovulation followed by an emergence of a new follicular wave in river buffalo.     

Postnatal study on the histochemistry of epididymis in buffalo (Bubalus bubalis).

The histochemical localisation and development of carbohydrates, acid mucopolysaccharides, lipids and desoxyribonucleic acid has been determined in the head, body and tail segments of buffalo epididymis at different stages of postnatal development from 3-76 weeks of age. The cytoplasm contains numerous Schiff-positive, diastase-resistant granules which are abundant in the apical region of the epithelial cells at 3-52 weeks. They decrease in number at 72-76 weeks and instead, a diffuse Schiff-positive material is observed in the cytoplasm of the cells, particularly in regions III-VI of the epididymis. In all the stages of development, the basement membrane and the luminal border of the epithelial cells have strong PAS-positive reaction. The intertubular connective tissue is mildly PAS-reactive, with moderate activity on the endothelial lining and smooth muscles of blood vessels. The capsule shows an intense Schiff-positive material in the fibers while the stereocilia are mildly reactive. PAS activity is less in region I and greater in regions V-VI as compared with the other regions of the epididymis. A moderate quantity of lipids is present in the epithelial cells and smooth muscles of the tubules. Sudanophilia is more pronounced in the tail region as compared with the head and body regions of the epididymis. Acid mucopolysaccharides are present, minutely, in the epithelial cell cytoplasm, with moderate activity on the stereocilia of the tubules. The Feulgen reaction is deep in region I and light to moderate in the other regions of the epididymis.     

Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation

Keeping in view the poor freezability of bubaline semen in conventionally used extenders, this study was conducted on three Murrah bulls to improve semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ml(-1)) in routinely used egg yolk tris-glycerol (EYTG) extender. Bradykinin (2.0 ng ml(-1)) had significant (P<0.05) beneficial effect on live sperm % (81.6+/-1.8) and hypo osmotic swelling (HOS) % (63.0+/-1.3) as compared to their respective control values of 73.4+/-2.1 and 56.3+/-2.0 at 0 h post freezing. The post-thaw progressive sperm motility in semen samples diluted with EYTG containing 2.0 ng ml(-1) Bradykinin (65.5+/-1.4) was also significantly (P<0.01) higher than control (60.3+/-1.9) at 0 h post freezing. Thus incorporation of 2 ng ml(-1) Bradykinin in buffalo semen diluted in EYTG extender may be useful in improving the quality of cryopreserved bubaline semen.     

Power of exclusion of 19 microsatellite markers for parentage testing in river buffalo (Bubalus bubalis)

In the present study, 19 microsatellite markers were assessed for their power of exclusion to test parentage in river buffalo. Microsatellite genotypes of 216 unrelated buffaloes belonging to five different breeds were utilized for the study. The probabilities of exclusion were calculated for three hypothetical situations viz. paternity testing (PE1), one parental genotype unavailable (PE2) and exclusion of both parents i.e. substituted offspring (PE3). The mean probability of exclusion across 19 investigated markers in buffalo was 0.578 (PE1), 0.405 (PE2) and 0.764 (PE3) respectively. The probability of exclusion for paternity (PE1) ranged between 0.297 and 0.814 across different markers. The exclusion probability for the cases one parent unavailable (PE2) and substituted offspring (PE3) varied from 0.143 to 0.688 and 0.465 to 0.946 respectively. Polymorphism information content and expected heterozygosity were found to have significantly high correlation with probability of exclusion of microsatellite markers. The cumulative PE1 of nine marker loci was estimated to be 0.9999 while in case of absence of one of the parental genotypes, a minimum of 11 markers were required to achieve a cumulative PE2 of 0.999. In conclusion, the present study proposes two multiplex sets with four and five markers respectively for routine parentage testing in buffalo and an additional set of four markers for doubtful cases of paternity.     

The postnatal histology of the epididymis in buffalo (Bubalus bubalis).

The epididymis of buffalo is differentiated into 6 regions. The head represents the first 3 regions, the body the 4th region and the tail the 5th and 6th region. At 3-30 weeks, the epithelium is simple high columnar with few basal cells in region I, simple low columnar with few basal cells in region II, pseudostratified low columnar in regions III, IV, V, and pseudostratified low columar in region VI. As age advances, the epithelium increases in height and shows a tendency toward advanced pseudostratification. The basal cells are greater in number in regions III, IV and V than in the other regions of the epididymis. The epithelial cells contain stereocilia in region I at 3 weeks and regions III, IV and V at 30 weeks, whereas they are absent in regions II and VI. The tubules in region I are the smallest in diameter while the tubules in region VI have the largest diameter.     

A study on the different types of spermatogonia in buffalo (Bubalus bubalis).

As a first step to understanding spermatogenesis in the buffalo bull the cytological details of different types of spermatogonia were determined in adult buffalo bulls. Morphological changes in the nuclear details were used as a basis for classifying the different types of spermatogonia. The type A spermatogonia had a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm and having one to two nucleoli adhering to the nuclear membrane. The type A0 spermatogonia were characterized by nuclei containing moderately stained, finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, on the other hand, have pale stained, finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resembled those of type A1, but contained coarse granular chromatin dispersed in the pale nucleoplasm. The intermediate type of spermatogonia acquired a central position of the nucleolus, but the chromatin remained coarsely granulated and non-clumped. Three classes of type B (B1-B3) spermatogonia were determined on the degree of clumping of the chromatin and the central position of the nucleolus. The type B1 cells were characterized by nuclei containing a few flakes of lightly stained chromatin and a centrally located nucleolus. The type B2 cells showed comparatively more clumping of chromatin than type B1 spermatogonia, which was dispersed at random in the pale nucleoplasm and along the nuclear envelope. The type B3 spermatogonia demonstrated chromophilic chromatin dispersed in the slightly grey nucleoplasm and adhering along the nuclear membrane. Since there seems to be a succession of events following differentiation of type A1 spermatogonia till the last type B cell differentiates into resting primary spermatocytes, may intermediate stages between the presently described classes of type A (A0-A2) and type B (B1-B3) could also be located in sections of the seminiferous tubules.     

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.     

Quantitative histomorphological studies on the spinal ganglia of the Indian buffalo (Bubalus bubalis)

Spinal ganglia of C3, C7, T5, T12, L2 and L5 spinal dorsal roots were studied. These ganglia vary in their size. Their greatest cross-sectional area was at C7 (2.12 cm2) in the cervical region and at L5 (1.39 cm2) in the lumbar region. The neurons were either spherical or oval in contour. The smallest neurons (mean size 68.50 X 55.64 micrometer) were located in the T5 ganglion, while some of the largest neurons (mean size 79.17 X 63.34 micrometer) were noticed in the C3 ganglion. As for their cross-sectional area, the ganglionic neurons at the L5 level were the largest (3,713.16 micrometer2), while the least value (2,994.60 micrometer2) was noted in T5 ganglionic cells. The amphicytes (satellite cells) were clearly defined. The neuronal cytoplasm revealed finely granular Nissl material. The neurons exhibit a pale-staining, centrally located vesicular nucleus with a prominent nucleolus. Few multipolar cells with eccentric nuclei were noted in the L5 ganglion.