Sachinandan De

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).

Lactic acid bacteria as a cell factory for riboflavin production

Consumers are increasingly becoming aware of their health and nutritional requirements, and in this context, vitamins produced in situ by microbes may suit their needs and expectations. B groups vitamins are essential components of cellular metabolism and among them riboflavin is one of the vital vitamins required by bacteria, plants, animals and humans. Here, we focus on the importance of microbial production of riboflavin over chemical synthesis. In addition, genetic abilities for riboflavin biosynthesis by lactic acid bacteria are discussed. Genetically modified strains by employing genetic engineering and chemical analogues have been developed to enhance riboflavin production. The present review attempts to collect the currently available information on riboflavin production by microbes in general, while placing greater emphasis on food grade lactic acid bacteria and human gut commensals. For designing riboflavin‐enriched functional foods, proper selection and exploitation of riboflavin‐producing lactic acid bacteria is essential. Moreover, eliminating the in situ vitamin fortification step will decrease the cost of food production.

Multifaceted attributes of dairy propionibacteria: a review

Dairy propionibacteria are Generally Recognized as Safe (GRAS) status microorganisms which have been traditionally used for the manufacture of Swiss type cheeses. In the last two decades various added features and functionalities have been discovered and developed from these bacteria. Propionibacteria are robust organisms with remarkable adaptability to technological and physiological stress conditions. Besides, they also display a multitude of health promoting properties like modulation of gut microbiota, improved gut physiology and immunomodulation suggesting their promising probiotic potential. Propionibacteria produce an interestingly wide range of functional biomolecules like B group vitamins, trehalose, conjugated linoleic acid, propionic acid, bacteriocins, bifidogenic factors etc. These bacteria are thus now being explored for designing novel functional foods as well as for industrial production of nutraceuticals. Growing interest in these bacteria is fueled by the first whole genome sequencing of a Propionibacterium freudenreichii strain providing a platform for better understanding of various pathways and further improvement in related process technologies.

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination-generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.

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.

Structural insights into the MDP binding and CARD–CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)—a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2–leucine rich repeat (LRR) domain activates NF‐κB signaling through a protein–protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD–CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD–CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2–LRR confer MDP recognition by hydrophobic and hydrogen bond (H‐bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2–CARDa and the electronegative surface on zRIP2–CARD reinforced mostly by H‐bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2–CARDa and Asp494 of zRIP2–CARD. Arg11 and Lys53 of zNOD2–CARDa and Ser498 and Glu508 of zRIP2–CARD are critical residues for CARD–CARD interaction and NOD2 signaling. The 2.7 Å H‐bond between Lys104 of the linker and Glu508 of zRIP2–CARD suggests a possible role of the linker for shaping CARD–CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD–CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective. Copyright

Seasonal variations in physio-biochemical profiles of Indian goats in the paradigm of hot and cold climate

To explore the heat and cold adaptation in Indian goats by the physiological, haematological, blood biochemical parameters and their seasonal variations, this study was conducted on heat- and cold-adapted Indian goats maintained in their natural habitat. Study was carried out in three different phases coincide with the three seasons (winter, spring and summer). The levels of physiological responses, that is rectal temperature, respiration rate and pulse rate, were observed to be significantly (p < 0.01) lower in heat-adapted breeds and higher in cold-adapted breeds, whereas the levels of Hb, PCV and TEC were significantly (p < 0.01) higher in cold-adapted goats. Significantly (p < 0.01) higher levels of plasma thyroid hormones (thyroxine and triiodothyronine) and plasma stress enzyme (AST and ALT) were also observed in cold-adapted goats. Significant (p < 0.01) seasonal variations in physiological responses, haematological and blood biochemical parameters in both heat- and cold-adapted breeds were reported in this study. Physiological responses, plasma enzymes and plasma cortisol levels significantly (p < 0.01) increased during summer in all the goat breeds. The levels of haematological parameters (Hb, PCV, TEC and TLC) and plasma thyroid hormones (thyroxine and triiodothyronine) decreased during summer. The changes in physiological parameters during summer due to heat stress were higher in cold-adapted goats whereas the levels of changes in these parameters during winter due to cold were higher in heat-adapted goats. High neutrophil/lymphocyte ratio during summer in cold-adapted breeds is an indicator of higher level of stress. Decrease in plasma electrolytes (Na and K) during summer also observed in cold-adapted breeds during summer. The variations in physiological, haematological, blood biochemical parameters in heat- and cold-adapted goats may be due to their adaptation to different environmental and geographical conditions essential for their survival.

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo.

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm) along with a control (0 mm) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC-I) (12-72 hpi) and IVC-II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC-I and IVC-II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.

Embryonic genome activation events in buffalo (Bubalus bubalis) preimplantation embryos

Embryonic genome activation (EGA) is the first major step towards successful initiation of preimplantation development, which culminates in the formation of implantation‐competent embryos. EGA occurs at species‐specific embryonic cell stages. In the present work, EGA was identified for buffalo embryos by studying the development rate of embryos in normal as well as imposed transcription block conditions, analyzing bromo‐uridine triphosphate (BrUTP) incorporation rates as evidence of de novo transcription initiation, and studying the expression status of eukaryotic translation initiation factor 1A (eIF1A), U2 auxiliary splicing factor (U2AF), and polyadenylate polymerase (PAP) genes at different embryonic cell stages. Under normal, in vitro fertilization and culture conditions, about 26% and 17% of oocytes could reach morula and blastocyst stages, respectively, but no embryos could progress beyond 8‐cell stages in presence of α‐amanitin. Culturing embryos in the presence of BrUTP revealed a marked increase in its incorporation between 4‐ and 8‐cell stages. All genes studied displayed an abrupt increase in expression between 4‐ and 8‐cell stages; PAP expression was upregulated earlier from 2‐ to 4‐cell stages. About 65% of PAP transcripts from the 4‐cell stage and more than 70% of eIF1A, U2AF, and PAP transcripts at 8‐cell stage embryos were found to be synthesized de novo. Together, these data suggest that a minor EGA in buffalo embryos happens from 2‐ to 4‐cell stages, while the major EGA takes place from 4‐ to 8‐cell stage transition. Mol. Reprod. Dev. 79: 321–328, 2012.

Absolute copy number differences of Y chromosomal genes between crossbred (Bos taurus × Bos indicus) and Indicine bulls

BackgroundThe Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological difference of this chromosome between Bos taurus and Bos indicus make it an ideal model for studying structural variation, especially in crossbred (Bos taurus × Bos indicus) bulls. Copy Number Variation (CNV) is a type of genomic structural variation that gives information complementary to SNP data. The purpose of this study was to find out copy number differences of four Y chromosomal spermatogenesis-related candidate genes in genomic DNA of crossbred and purebred Indicine bulls.ResultFour Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Y chromosome (SRY), DEAD box polypeptide 3-Y chromosome (DDX3Y), Ubiquitin specific peptidase 9, Y-linked (USP9Y), testis-specific protein on Y chromosome (TSPY) were evaluated. Absolute copy numbers of Y chromosomal genes were determined by standard curve-based quantitative real time PCR. Copy numbers of SRY and TSPY genes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Y and USP9Y gene copy numbers between two groups.ConclusionThe present study demonstrates that the structural organization of Y chromosomes differs between crossbred and Indicine bulls which are reproductively healthy as observed from analysis of semen attributes. The absolute copy numbers of SRY and TSPY genes in unit mass of genomic DNA of crossbred bulls are significantly higher than Indicine bulls. No alteration in absolute copies of DDX3Y and USP9Y gene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Y and USP9Y are likely to be single copy genes in the genome of crossbred and Indicine bulls and variation in Y chromosome length between crossbred and Indicine bulls may be due to the copy number variation of SRY gene and TSPY array.