MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

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.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

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.     

Storage of buffalo (Bubalus bubalis) semen

Characteristics of buffalo semen, diluents used for liquid storage, aspects involved in freezing and thawing of semen are reviewed, and fertility results after artificial insemination (AI) with frozen-thawed semen are given.     

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.     

Embryo transfer in water buffalo (Bubalus bubalis)

A normal, live 35-kg water buffalo bull calf was born 300 days after it was nonsurgically collected as a 7-day blastocyst from a water buffalo donor and transferred nonsurgically to an unrelated water buffalo recipient. The development of estrus synchronization, superovulation and estrus detection methods in water buffalo are described.     

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.     

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.     

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.     

Attenuation of luteolytic response following fish meal supplementation in dairy buffaloes (Bubalus bubalis)

Luteolysis of corpus luteum, due to un-inhibited PGF(2α) secretion, has been reported to be a cause of early embryonic mortality in dairy animals. The objective of this study was to determine the effects of fish meal (FM) supplementation on the uterine secretion of PGF(2α) and hence establish its supplementation as an antiluteolytic strategy in dairy buffaloes. Five cycling Murrah buffaloes were supplemented with 250g FM daily for 55 days in addition to their routine feed and seven buffaloes were kept as non-supplemented control. After 30 days of FM supplementation, the oestrus was synchronized in all the buffaloes using Ovsynch protocol. On day 15 of synchronized cycle, animals were challenged with oxytocin (OT; 100IU) intravenously and blood samples were collected at 15min interval, 1h before to 4h after OT challenge. The PGF(2α) response was measured as the venous concentration of 13,14-dihydro-15-keto PGF(2α) (PGFM). The mean hourly concentration of PGFM in FM supplemented buffaloes was lower than in the control buffaloes at all the occasions. During peak response (1h post-OT challenge), PGFM concentration was significantly lower (P<0.05) in FM supplemented buffaloes than in the control (197.4±41.7pg/ml versus 326.3±33.5pg/ml, respectively). Also the percent rise in PGFM after OT-challenge in FM supplemented buffaloes was less than the control (11.73% versus 22.47%). The dietary supplementation did not affect the size of corpus luteum (CL) and plasma progesterone concentration. Plasma glucose and total protein concentrations remained within the normal physiological limits during FM supplementation. The present study indicated that supplementing FM decreased the concentrations of PGF(2α) without alterations in the size of CL and plasma progesterone concentrations in dairy buffaloes.     

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.     

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.     

The serum lipoprotein pattern of water buffalo (Bubalus bubalis)

1. The serum lipoprotein pattern of water buffalo was studied by means of electrophoresis and the lipoproteins were isolated by ultracentrifugation on the basis of their hydrated density. 2. High density lipoproteins (HDL) showed a higher level of cholesterol than did the other lipoproteins. Moreover, the level of phospholipids was higher in HDL than in very low density lipoproteins (VLDL). 3. The buffalo B100 apoprotein was similar to that of man and rat. Three apoproteins similar to human apo E, apo AI and AII were found in buffalo HDL, buffalo VLDL contained essentially apo B protein.     

In vitro culture of buffalo (Bubalus bubalis) preantral follicles

Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin-transferrin-selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media. Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO2 incubator (38-39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 15 days. Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 microg/ml), respectively. In Experiment 1, we placed isolated preantral follicles individually or in groups of 2-4 preantral follicles in 30 or 50 microl droplets, respectively, using two culture media: washing media and washing media + ITS (1%) + FSH (0.05 IU/ml), respectively. In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: < or = 36, 37-54, 55-72, 73-90, 90-108 and > or = 109 microm. We cultured groups of 2-4 preantral follicles in washing media + ITS (1A) + FSH (0.05 IU/ml) in a CO2 incubator for 15 days. In Experiment 3, we allocated groups of 2-4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media + FSH (0.05 IU/ml), (3) washing media + ITS (17%), (4) washing media + ITS (1%) + FSH (50 IU/ml), (5) washing media + ITS (1%) + EGF (50 ng/ml), (6) washing media + ITS (1%) + FSH (0.05 IU/ml) + EGF (50 ng/ml), (7) washing media + ITS (1%) + FGF (50 ng/ml), (8) washing media + ITS (1%) + FSH (0.05 IU/ml) + FGF (50 ng/ml), (9) washing media + ITS (1%) + VIP (50 ng/ml), and (10) washing media + ITS (1%) + FSH (0.05 IU/ml) + VIP (50 ng/ml). In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly (P < 0.05) higher growth up to 40 days. In Experiment 5, we allocated groups of 2-4 preantral follicles to two treatments: (1) washing media + PMSG (50 IU/ml), and (2) washing media + ITS (1%) + PMSG (50 IU/ml) and cultured in a CO2 incubator for 15 days. The results indicated that the preantral follicles cultured in groups had a higher growth rate (P < 0.05) than those cultured as individuals. ITS, FSH, PMSG and growth factors significantly (P < 0.05) promoted the growth of the preantral follicles. Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation. The growth rate of preantral follicles was lower in buffalo than in cattle.     

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.