Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis).

Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls' spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated 'endoGene' pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that GAPDH and PP1A were the most stably expressed among the gene panel, whereas ATPSF1 and ACTB were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, HSFY2 and AKAP4, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.

Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa.

BACKGROUND: Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells. METHODS: Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated. RESULTS: H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps. CONCLUSION: Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies.

Sequence characterization and comparative expression profile of buffalo WNT10B gene in adult and fetal tissues.

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member (WNT10B) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

ASIP gene polymorphism associated with black coat and skin color in Murrah buffalo.

The melanogenesis pathway regulates pigmentation through the synergic action of various genes. We are interested in analyzing the genetic variations in the ASIP which determine eumelanin production in the dermis layer. In the present study, the ASIP gene was characterized in buffalo and 268 genetically unrelated buffaloes belonging to 10 different populations were genotyped for the non-synonymous SNP (c.292C>T) identified in the exon 3 region of the gene using Tetra-ARMS-PCR. The TT genotype occurred at a higher rate in Murrah, followed by Nili Ravi, Tripura, and Paralakhemundi (42.63%, 19.30%, 3.45%, and 3.33%). These results convey the association of the black coat color of Murrah with the ASIP gene TT genotype and the lighter shades of black coat (brown and grayish-black) color phenotype in other breeds with the CC genotype.

Genetic admixture and population structure analysis of Indian water buffaloes (Bubalus bubalis) using STR markers.

BACKGROUND: India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different buffalo populations of India, using 20 highly polymorphic microsatellite markers has been generated for the genetic diversity analysis. The buffalo populations of Eastern Odisha state, were the primary focus. METHODS AND RESULTS: The minimal spanning network based on Bruvo's distance, PCA (Principal Component Analysis) based on the Fst (Fixation Index) values, and genetic admixture analysis using both the STRUCTURE and 'snapclust' were performed. The analysis could identify the Manda population as distinct from other Odisha buffalo breeds as well as adjoining Chhattisgarhi buffalo breeds. The total observed number of alleles ranged between 143 (Manda) and 301 (Paralakhemundi) with an average of 204 alleles per breed. The Sambhalpuri buffalo population also clustered into two separate subpopulations, half of the unique sub-population located geographically south-wards, displayed no admixture with any of the adjacent buffalo populations. The Manda buffalo population has shown sufficient allelic richness and heterozygosity under random mating being practiced in the field conditions. CONCLUSIONS: The study has led to the identification of the Manda as a distinct buffalo population, and the germplasm has been registered as a new Indian buffalo breed. Whereas, the Sambhalpuri population requires elaborate analysis to confirm the existence of two distinct sub-populations.

Current status and unique attributes of Indian Chilika buffalo for adaptation to brackish water ecology.

Chilika buffalo is native to the Eastern coast of India and well adapted to the largest coastal brackish water lagoon of Asia, Chilika Lake. We present here a report on the Chilika buffalo breed emphasizing the conservational urgency based on unique biochemical and molecular evidence related to liver and kidney functions while comparing it with tropically adapted other water buffalo breeds (Bubalus bubalis) of India. It is found that the Chilika buffalo breed has a better ability to withstand a long dehydration period as evident from its better glomerular filtration and higher expression of the ion transport channel. Mitochondrial D-loop sequencing results have shown these buffaloes being closer to swamp-type buffaloes of Bangladesh and northeast India and represent a unique "hybrid zone" on the eastern coast of India. Conservation of such uniquely adapted germplasm is crucial owing to the current global trend, where the introduction of exotic breeds has negatively impact "sui-generis" germplasm and they require higher managerial resource consumption for maintaining higher productivity. Further, the introduction of unconventional fisheries activities has proved detrimental to the lagoon ecosystem, potentially causing more threat to the buffalo's population.

Morphometric and microsatellite-based comparative genetic diversity analysis in Bubalus bubalis from North India.

To understand the similarities and dissimilarities of a breed structure among different buffalo breeds of North India, it is essential to capture their morphometric variation, genetic diversity, and effective population size. In the present study, diversity among three important breeds, namely, Murrah, Nili-Ravi and Gojri were studied using a parallel approach of morphometric characterization and molecular diversity. Morphology was characterized using 13 biometric traits, and molecular diversity through a panel of 22 microsatellite DNA markers recommended by FAO, Advisory Group on Animal Genetic Diversity, for diversity studies in buffaloes. Canonical discriminate analysis of biometric traits revealed different clusters suggesting distinct genetic entities among the three studied populations. Analysis of molecular variance revealed 81.8% of genetic variance was found within breeds, while 18.2% of the genetic variation was found between breeds. Effective population sizes estimated based on linkage disequilibrium were 142, 75 and 556 in Gojri, Nili-Ravi and Murrah populations, respectively, indicated the presence of sufficient genetic variation and absence of intense selection among three breeds. The Bayesian approach of STRUCTURE analysis (at K = 3) assigned all populations into three clusters with a degree of genetic admixture in the Murrah and Nili-Ravi buffalo populations. Admixture analysis reveals introgression among Murrah and Nili-Ravi breeds while identified the Gojri as unique buffalo germplasm, indicating that there might be a common origin of Murrah and Nili-Ravi buffaloes. The study provides important insights on buffalo breeds of North India that could be utilized in designing an effective breeding strategy, with an appropriate choice of breeds for upgrading local non-descript buffaloes along with conservation of unique germplasm.

Alternate PCR assays for screening of JH1 mutation associated with embryonic death in Jersey cattle.

Jersey haplotype (JH) 1, a stop-gain lethal mutation in the CWC15 gene, causes embryonic losses in Jersey cattle. Two PCR based assays using Amplification Refractory Mutation System (T-ARMS-PCR) and restriction fragment length polymorphism (PCR-RFLP) were developed for screening of the JH1 in cattle. During the screening, seven among 30 Indian Jersey bulls were identified as carriers of the mutant JH1 allele, the first time in the country. These PCR assays are economical, rapid and accurate; and can be used separately or in combination for screening and cross-validation of the JH1 carriers in Jersey cattle.

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution.

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.

Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis).

The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes.

Exploration of the binding modes of buffalo PGRP1 receptor complexed with meso-diaminopimelic acid and lysine-type peptidoglycans by molecular dynamics simulation and free energy calculation.

The peptidoglycan recognition proteins (PGRPs) are the key components of innate-immunity, and are highly specific for the recognition of bacterial peptidoglycans (PGN). Among different mammalian PGRPs, the PGRP1 binds to murein PGN of Gram-positive bacteria (lysine-type) and also have bactericidal activity towards Gram-negative bacteria (diaminopimelic acid or Dap-type). Buffaloes are the major sources of milk and meat in Asian sub-continents and are highly exposed to bacterial infections. The PGRP activates the innate-immune signaling, but their studies has been confined to limited species due to lack of structural and functional information. So, to understand the structural constituents, 3D model of buffalo PGRP1 (bfPGRP1) was constructed and conformational and dynamics properties of bfPGRP1 was studied. The bfPGRP1 model highly resembled human and camel PGRP structure, and shared a highly flexible N-terminus and centrally placed L-shaped cleft. Docking simulation of muramyl-tripeptide, tetrapeptide, pentapeptide-Dap-(MTP-Dap, MTrP-Dap and MPP-Dap) and lysine-type (MTP-Lys, MTrP-Lys and MPP-Lys) in AutoDock 4.2 and ArgusLab 4.0.1 anticipated ß1, α2, α4, ß4, and loops connecting ß1-α2, α2-ß2, ß3-ß4 and α4-α5 as the key interacting domains. The bfPGRP1-ligand complex molecular dynamics simulation followed by free binding energy (BE) computation conceded BE values of -18.30, -35.53, -41.80, -25.03, -24.62 and -22.30 kJ mol(-1) for MTP-Dap, MTrP-Dap, MPP-Dap, MTP-Lys, MTrP-Lys and MPP-Lys, respectively. The groove-surface and key binding residues involved in PGN-Dap and Lys-type interaction intended by the molecular docking, and were also accompanied by significant BE values directed their importance in pharmacogenomics, and warrants further in vivo studies for drug targeting and immune signaling pathways exploration.

Sequence and topological characterization of Toll-like receptor 8 gene of Indian riverine buffalo (Bubalus bubalis).

In this study, buffalo (Bubalus bubalis) Toll-like receptor 8 (TLR8) gene has been characterized by sequence analysis and detecting polymorphism. Complete ORF of buffalo TLR8 gene was amplified using the RNA isolated from spleen tissue, which was found to be 3,102 nucleotides long encoding a 1,033 amino acid protein. Buffalo TLR8 had 10 nucleotide changes as compared to other livestock species resulting in six unique amino acid changes, four of them lying within leucine-rich repeat (LRR) domains. As compared to cattle (Bos indicus and Bos taurus), out of fifteen cysteine residues, fourteen were conserved and Cys at position 521 was replaced by Arg. Nine of the LRR domains had no amino acid change as compared to cattle, whereas LRR-C-terminus had maximum, five amino acid changes. Sequence characterization of 12 riverine and swamp buffaloes revealed presence of four polymorphic nucleotides, two of them were non-synonymous, one synonymous and one site in 3'UTR. PCR-RFLP genotyping of non-synonymous SNP 2758A>G (ILeu920Val) in Toll-interleukin-1 receptor domain of 463 swamp and riverine buffaloes showed a higher frequency of allele A in swamp (95 %) as compared to riverine (9.84 %) buffaloes.

PCR-SSCP analysis of leptin gene and its association with milk production traits in river buffalo (Bubalus bubalis).

Leptin gene has been found to be associated with various economic traits including milk production and fat quality in dairy animals. In the present study, we investigated genetic variations in intron 1 region of leptin gene in riverine buffaloes (Bubalus bubalis) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing methods and associated them with milk traits. The study revealed three SSCP variants A, B and C among a total of 301 buffaloes from nine breeds. The frequency of variant C was found invariably high among all the breeds except in Marathwada buffalo. Variant A was found to be absent in Chilika, Nili-Ravi, Nagpuri and Pandharpuri breeds and also had the lowest frequencies in Mehsana, Jaffarabadi, Murrah and Toda breeds. Sequencing of SSCP variants revealed a total of five polymorphic sites, with three haplotypes. Statistical analysis revealed significantly high fat percentage at 150 days in SSCP variant B in Mehsana buffaloes. However, the associations of SSCP variants of leptin gene with total milk yield, 305 days milk yield and total fat yield were found to be non-significant. The present study is the first report on association analysis of leptin gene polymorphisms with milk production and milk quality traits in river buffalo.

Semi-quantitative RT-PCR analysis of fat metabolism genes in mammary tissue of lactating and non-lactating water buffalo (Bubalus bubalis).

Understanding the mechanism of milk fat synthesis and secretion is important for dairy industry, as the nature of the cream fraction influences the manufacturing properties and organoleptic qualities of milk and dairy products. So, there is a need to understand the mechanism of milk fat synthesis and to elucidate the key genes regulating milk fat synthesis by studying the expression of genes involved in milk fat synthesis. Present manuscript reports the expression of genes involved in milk fat synthesis and metabolism in buffalo mammary tissue. The expression of lipogenic genes was studied in lactating and non-lactating mammary tissue of water buffalo by semi-quantitative reverse transcription PCR expression analysis. The genes studied were acetyl-CoA carboxylase (ACACA), stearoyl-CoA desaturase (SCD), 3 hydroxybutyrate dehydrogenase (BDH), LIPIN, lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding protein (SREBF). The expression of ACACA, BDH, LIPIN, PPARG, LPL, and SREBF was higher in lactating as compared to non-lactating buffalo whereas no difference was found in the expression of SCD between both the stages.