Surender Lal Goswami

Polymorphism of BMPR1B, BMP15 and GDF9 fecundity genes in prolific Garole sheep

Mutation studies in different prolific sheep breeds have shown that the transforming growth factor beta super family ligands viz. the growth differentiation factor 9 (GDF9/FecG), bone morphogenetic protein 15 (BMP15/FecX) and associated type I receptors, bone morphogenetic protein receptor (BMPR1B/FecB), are major determinant of ovulation rate and consequent increase in litter size. The Garole sheep is a highly prolific sheep breed of India. Characterization of fecundity genes in these animals could substantially improvise the breeding programme in these animals as well as other sheep breeds of the region. The present study was therefore designed with the objective of polymorphism study of fecundity genes in these prolific microsheep. A total of 11 point mutations were detected by polymerase chain reaction (PCR)-based method. A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type a total of ten points of two ovine fecundity genes (GDF9 and BMP15). The FecB locus of the BMPR1B gene and G1 locus of GDF9 gene were found to be polymorphic. In FecB locus, two genotypes, wild type (FecB+) and mutant (FecBB), were detected with allele frequencies of 0.39 and 0.61, respectively. At G1 locus, two genotypes, mutant (A) and wild types (G) were detected with allele frequencies of 0.18 and 0.82, respectively. This study reports Garole sheep as the fourth sheep breed after Belclare/Cambridge, Lacaune and Small-tailed Han sheep, where coexisting polymorphism has been found in two different fecundity genes (BMPRIB and GDF9 genes).

POLYMORPHISM OF FECUNDITY GENES (FECB, FECX, AND FECG) IN THE INDIAN BONPALA SHEEP

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.

Kinetics of GDF9 expression in buffalo oocytes during in vitro maturation and their associated development ability

The capacity of fully grown oocytes to regulate their own microenvironment by secreted paracrine factors contribute to their developmental competence. In spite of growing evidence about the vital role of Growth Differentiation Factor 9 (GDF9) in determination of oocyte developmental competence, there is insufficient information about time dependent behavior of its expression during in vitro maturation (IVM) to have definite understanding about at what time point during IVM it plays most crucial role. The study reports the kinetics of GDF9 expression under four different IVM supplement conditions in buffalo oocytes and their concomitant development rate up to blastocyst. Oocytes matured under an ideal media condition with all supplements and those cultured with only FSH resulted in significantly higher cumulus expansion, nuclear maturation, cleavage and blastocyst rates. GDF9 expression at both mRNA and protein levels at different time points of IVM revealed that magnitude of mRNA abundance at 8h of IVM was most important towards imparting development competence to buffalo oocytes. Appearance of GDF9 protein in maturing oocytes was found asynchronous with mRNA appearance in the time course of IVM suggesting possible posttranscriptional regulation of this gene under dynamic oocyte cumulus cell communication process. Abundance of mature GDF9 protein at 16 h was most consistently related with all oocyte development parameters.

Heterologous Expression and Characterization of Indian Sahiwal Cattle (Bos indicus) Alpha Inhibin

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T5 promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25 µg mL−1) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.

Sperm chromosome analysis of Karan Swiss and Karan Fries crossbred bulls

Abstract Zona-free hamster eggs were penetrated by Karan Swiss and Karan Fries crossbred bull spermatozoa capacitated using calcium ionophore A23187. Zona-free hamster egg penetration percentage varied between 59 and 83 among 11 crossbred bulls. Penetrated eggs were cultured in TC 199 medium with 10% fetal calf serum. Podophyllotoxin and vinblastine were used to interrupt karyogamy and tubulin polymerization, respectively. Gradual fixation air drying method was modified for the preparation of slides. Hamster cattle metaphase yield was 28 ± 5.2%. The analysis of 105 crossbred cattle sperm metaphases indicated that 51 contained a Y-chromosome and 54 had an X-chromosome. The incidence of aneuploidy was 14.28%.