Maternal genetic diversity and phylogenetic analysis of Indian riverine and swamp buffaloes: insights from complete mitochondrial genomes.

This study explored the genetic diversity and evolutionary history of riverine and swamp buffaloes in India, utilizing complete mitochondrial genome sequences. Through comprehensive sampling across varied agro-climatic zones, including 91 riverine buffaloes from 12 breeds and 6 non-descript populations, along with 16 swamp buffaloes of the Luit breed, this study employed next-generation sequencing techniques to map the mitogenomic landscape of these subspecies. Sequence alignments were performed with the buffalo mitochondrial reference genome to identify mitochondrial DNA (mtDNA) variations and distinct maternal haplogroups among Indian buffaloes. The results uncovered the existence of 212 variable sites in riverine buffaloes, yielding 67 haplotypes with high haplotype diversity (0.991), and in swamp buffaloes, 194 variable sites resulting in 12 haplotypes, displaying haplotype diversity of 0.950. Phylogenetic analyses elucidated the genetic relationships between Indian buffaloes and the recognized global haplogroups, categorizing Indian swamp buffaloes predominantly into the SA haplogroup. Intriguingly, the haplogroup SB2b was observed for the first time in swamp buffaloes. Conversely, riverine buffaloes conformed to established sub-haplogroups RB1, RB2, and RB3, underscoring the notion of Northwestern India as a pivotal domestication site for riverine buffaloes. The study supports the hypothesis of independent domestication events for riverine and swamp buffaloes, highlighting the critical role of genetic analysis in unraveling the complex evolutionary pathways of domestic animals. This investigation contributes to the global understanding of buffalo mitogenome diversity, offering insights into this important livestock species' domestication and dispersal patterns.

Skeletal muscle transcriptomics of sheep acclimated to cold desert and tropical regions identifies genes and pathways accentuating their diversity.

The current study attempts to investigate the differences in gene expression in longissimus thoracis muscles between sheep breeds acclimated to diverse environments. Changthangi sheep inhabits the cold arid plateau of Ladakh, at an altitude above 3000 m with prevalence of rarefied atmosphere. Muzzafarnagri sheep, on the other hand is found in the sub-tropical hot and humid plains at an altitude of about 250 m. Comparative transcriptomics was used to provide a molecular perspective of the differential adaptation of the two breeds. RNA sequencing data was generated from four biological replicates of the longissimus thoracis muscles from both breeds. The common genes expressed in both breeds were involved in muscle contraction and muscle fibre organization. The most significant pathways enriched in Changthangi muscles were glycogen metabolism, reduction of cytosolic Ca++ levels and NFE2L2 regulating anti-oxidant, while those in Muzzafarnagri were extracellular matrix organization and collagen formation. The hub genes identified in Changthangi were involved in hematopoiesis and HIF signaling pathway, suggesting the molecular acclimatization of Changthangi to the high altitude cold desert of Ladakh. The nodal genes discovered in Muzzafarnagri sheep were associated with the extracellular matrix which accentuates its significance in the development, growth and repair of muscles. The observed transcriptomic differences underscore the morphological and adaptive disparity between the two breeds. The candidate genes and pathways identified in this study will form the basis for future research on adaptation to high altitude and body size in small ruminants.

Molecular insights into Pashmina fiber production: comparative skin transcriptomic analysis of Changthangi goats and sheep.

Ladakh, one of the highest inhabited regions globally, hosts the unique Changthangi goat, renowned for producing Pashmina, the world's most luxurious natural fiber. In comparison, the fiber derived from Changthangi sheep is considered next only to Pashmina. This research endeavors to compare the skin transcriptome profiles of Changthangi goats and Changthangi sheep, aiming to discern the molecular determinants behind the recognition of Changthangi goats as the source of Pashmina. Drawing upon previously conducted studies, a collective of 225 genes correlated with fiber characteristics were extracted from the differentially expressed genes noticed between the two species (p-value of ≤ 0.05 and a log2 fold change of ≥ 1.5). These genes were analyzed using DAVID software to understand their biological functions and to identify enriched KEGG and Reactome pathways. The protein-protein interaction networks were constructed using Cytoscape, cytoHubba, and STRING to focus on key genes and infer their biological significance. Comparative transcriptome analysis revealed significantly higher expression of genes involved in signaling pathways like Wnt, MAPK, PI3K-Akt, Hedgehog, associated with fiber development and quality in Changthangi goats. These pathways play crucial roles in hair follicle (HF) formation, maintenance of epidermal stem cells, and fiber characteristics. Findings also highlight the enrichment of cell adhesion molecules and ECM-receptor interaction, emphasizing their roles in HF structure, growth, and signaling. This investigation offers an in-depth understanding of the molecular intricacies governing Pashmina production in Changthangi goats, providing valuable insights into their unique genetic makeup and underlying mechanisms influencing the exceptional quality of Pashmina fibers.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues.

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Exploring the Transcriptome Dynamics of In Vivo Theileria annulata Infection in Crossbred Cattle.

The molecular changes occurring in the host in response to in vivo Theileria annulata parasitic infection are not well understood. Therefore, the present study investigated the differential expression profiles of peripheral blood mononuclear cells (PBMCs) across Theileria annulata-infected and non-infected crossbred cows. The differential expression profiles from PBMCs of infected and non-infected crossbred cows were generated by RNA sequencing. A marked difference in the expression of genes associated with innate immunity (FTH1, ACTB, ISG15) was observed between the two groups. The over-represented pathways in Theileria annulata-infected cows were associated with the immune system and regulation of the mitotic cycle. Enriched genes and pathways in non-infected animals were associated with the maintenance of chromatin integrity and cell structure. The highly connected genes identified in this study form potential candidates for further investigation into host-parasite interactions in cattle. An improved understanding of the transcriptomic dynamics during theileriosis would lead to underpinning molecular level differences related to the health status of cattle.

Transcriptomics reveals key genes responsible for functional diversity in pectoralis major muscles of native black Kadaknath and broiler chicken.

RNA sequencing-based expression profiles from pectoralis major muscles of black meat (Kadaknath) and white meat (broiler) chicken were compared to identify differentially expressed genes. A total of 156 genes with log2 fold change ≥ ± 2.0 showed higher expression in Kadaknath and 68 genes were expressed at a lower level in comparison to broiler. Significantly enriched biological functions of up-regulated genes in Kadaknath were skeletal muscle cell differentiation, regulation of response to reactive oxygen, positive regulation of fat cell differentiation and melanosome. Significant ontology terms up-regulated in broiler included DNA replication origin binding, G-protein coupled receptor signaling pathway and chemokine activity. Highly inter-connected differentially expressed genes in Kadaknath (ATFs, C/EPDs) were observed to be important regulators of cellular adaptive functions, while in broiler, the hub genes were involved in cell cycle progression and DNA replication. The study is an attempt to get an insight into the transcript diversity of pectoralis major muscles of Kadaknath and broiler chicken. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03682-0.

Mitogenomic phylogeny reveals the predominance of the Nubian lineage of African wild ass in Indian donkeys.

To contribute to the knowledge of maternal genetic diversity in domestic donkeys, this study investigated the mitochondrial DNA variations and analyzed the genetic structure in Indian donkeys based on 31 mitogenome sequences representing four breeds/populations (Agra, Halari, Kachchhi and Spiti). A total of 27 haplotypes with a haplotype diversity value of 0.989 were evident in the donkey genetic resources of India. The genetic differentiation between the investigated populations was evaluated using population pairwise FST values, which showed maximum differentiation between Kachchhi and Halari donkeys. The Neighbor-Joining (NJ) tree based on the whole mitogenome sequence and the Median-Joining (MJ) network for partial D-loop fragment showed clear demarcation of Indian donkeys into Nubian and Somali clades, substantiating African maternal origin of Indian domestic donkeys. The topology of the MJ network excluded the Asian wild asses as the possible progenitors of Indian donkeys. Halari and Agra donkeys showed conformity exclusively to the Nubian lineage of the African wild asses. However, representation of both the Nubian and Somali lineages was observed in Kachchhi and Spiti donkeys. Comprehensive analysis carried out by retrieving D-loop sequences from different countries representing Asia, Africa, Europe and South America revealed existence of shared haplotypes across geographically isolated regions of the globe. This observation is indicative of utility of donkeys as pack animals across inter-continental trading routes during development of human civilizations. Our results represent a valuable contribution to maternal genetic diversity of Indian donkeys and provide insights into the worldwide spread of the species following initial domestication in Africa.

Revelation of genes associated with energy generating metabolic pathways in the fighter type Aseel chicken of India through skeletal muscle transcriptome sequencing.

In this study, comparative analysis of skeletal muscle transcriptome was carried out for four biological replicates of Aseel, a fighter type breed and Punjab Brown, a meat type breed of India. The profusely expressed genes in both breeds were related to muscle contraction and motor activity. Differential expression analysis identified 961 up-regulated and 979 down-regulated genes in Aseel at a threshold of log2 fold change ≥ ±2.0 (padj<0.05). Significantly enriched KEGG pathways in Aseel included metabolic pathways and oxidative phosphorylation, with higher expression of genes associated with fatty acid beta-oxidation, formation of ATP by chemiosmotic coupling, response to oxidative stress, and muscle contraction. The highly connected hub genes identified through gene network analysis in the Aseel gamecocks were HNF4A, APOA2, APOB, APOC3, AMBP, and ACOT13, which are primarily associated with energy generating metabolic pathways. The up-regulated genes in Punjab Brown chicken were found to be related to muscle growth and differentiation. There was enrichment of pathways such as focal adhesion, insulin signaling pathway and ECM receptor interaction in these birds. The results presented in this study help to improve our understanding of the molecular mechanisms associated with fighting ability and muscle growth in Aseel and Punjab Brown chicken, respectively.

Unraveling the genetic mechanisms governing the host response to bovine anaplasmosis.

Bovine anaplasmosis caused by Anaplasma marginale is a tick-borne disease of livestock with widespread prevalence and huge economic implications. In order to get new insights into modulation of host gene expression in response to natural infections of anaplasmosis, this study is the first attempt that compared the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) of A. marginale infected and healthy crossbred cattle. Transcriptome analysis identified shared as well as unique functional pathways in the two groups. Translation and structural constituent of ribosome were the important terms for the genes abundantly expressed in the infected as well as healthy animals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes revealed that immunity and signal transduction related terms were enriched for the up-regulated genes in the infected animals. The over-represented pathways were cytokine-cytokine receptor interaction and signaling pathways involving chemokines, Interleukin 17 (IL17), Tumour Necrosis Factor (TNF), Nuclear Factor Kappa B (NFKB) etc. Interestingly, many genes previously associated with parasite-borne diseases such as amoebiasis, trypanosomiasis, toxoplasmosis, and leishmaniasis were profusely expressed in the dataset of the diseased animals. High expression was also evident for the genes for acute phase response proteins, anti-microbial peptides and many inflammatory cytokines. Role of cytokines in mediating communication between immune cells was the most conspicuous gene network identified through the Ingenuity Pathway Analysis. This study provides comprehensive information about the crosstalk of genes involved in host defense as well as parasite persistence in the host upon infection with A. marginale.

Study on the muscle transcriptome of two diverse Indian backyard poultry breeds acclimatized to different agro-ecological conditions.

OBJECTIVE: Free-range (FR) poultry production systems are associated with quality products and improved welfare. All the 19 diverse chicken breeds of India have evolved under the FR system and are adapted to different agro-climatic conditions. It is vital to explore indigenous germplasm with modern genomic tools to have insights into genomic characteristics of production traits and adaptation. METHODS: In this study, breast tissue transcriptome profiles were generated and analyzed from four biological replicates of two indigenous backyard poultry breeds of India-Ankaleshwar, a breed of the mainland, and Nicobari, a breed adapted to islands. The read quality of sequences was checked by FASTQC and processed reads were aligned to the reference genome (bGalGal1). RESULTS: More than 94% mapping to the reference genome was observed for all samples. A total of 12,790 transcripts were common across both groups, while 657 were expressed only in Ankaleshwar and 169 in Nicobari. The highest expressed genes across both groups were associated mainly with muscle structure, contraction, and energy metabolism. The highly expressed genes identified in Ankaleshwar were involved in fatty acid catabolism and oxidative stress mitigation. Functional terms, pathways, and hub genes in Nicobari participated in muscle fiber growth, adipogenesis, and fatty acid anabolism. A key hub gene (RAC1) in Nicobari is a potential candidate affecting the laying rate in chickens. The qRT-PCR results also substantiate the RNA-seq results. CONCLUSION: The findings provide a precious molecular resource to advance understanding of the genetic basis of adaptation, meat quality, and egg production in backyard chickens.

Comparative expression profiling of cytokine genes in Theileria annulata-infected and healthy cattle.

Theileriosis is one of the top ten economically important diseases in cattle in India. Cytokines are considered important mediators and regulators of the immune response to an infection. In the present study, the gene expression profiles of fourteen cytokines (IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, TGFB1, TNFA, IFNA and IFNB) were compared in Theileria annulata-infected and healthy crossbred cattle. Blood samples were obtained from the District Disease Diagnostic Laboratory, Karnal. The presence/absence of T. annulata infection in the animals was determined on the basis of blood smear examination and molecular detection through PCR using the genus-specific primers. Total RNA was extracted from peripheral blood mononuclear cells, which was further reverse transcribed to cDNA. Primer3 software was employed to design the primers for Real-Time qPCR. The results were examined using 2-∆∆Ct method with RPS15 and GAPDH as the reference genes. The expression of IL1B, IL6, IL8, IL10, IL12A, IL12B, TNFA, IFNA and IFNB was significantly higher, whereas the expression of IL2 was lower in the infected animals. The transcript levels of IL1A and TGFB1 were also higher in the diseased animals, but the results were non-significant. This study profiles the expression kinetics of various pro-inflammatory and anti-inflammatory cytokine genes in response to bovine theileriosis.

First report on delineation of differentially expressed genes and pathways in milk somatic cells of mastitic and healthy Murrah buffaloes.

Despite immense contribution of buffaloes as dairy species, limited studies have addressed the bubaline mastitis as compared to cattle. This was the first differential transcriptomic study investigating the alterations induced by clinical mastitis in buffalo milk relative to healthy controls. Comparative gene expression profiling of three biological replicates of each group identified 1014 up-regulated and 999 down-regulated genes in the diseased buffaloes (Fold change > 2, FDR < 0.05). Activation of immune and inflammatory responses were the most enriched GO terms in the mastitic animals, with higher transcript abundance of many genes coding for anti-microbial proteins such as ß-defensins, perforin, granzymes, granulysin, cathelicidins etc. Analysis of the gene regulatory interactions of the up-regulated DEGs identified many hub genes that govern the cellular and macromolecular metabolic processes (E2F4, E2F1, RBL2, FOXM1, IRF1 and MYB). This study contributes to an insightful understanding of molecular mechanisms governing immune response of buffaloes to mastitis.

Muscle transcriptome provides the first insight into the dynamics of gene expression with progression of age in sheep.

The dynamic synergy of genes and pathways in muscles in relation to age affects the muscle characteristics. Investigating the temporal changes in gene expression will help illustrate the molecular mechanisms underlying muscle development. Here we report the gene expression changes in skeletal muscles through successive age groups in Bandur, a meat type sheep of India. RNA sequencing data was generated from the longissimus thoracis muscles from four age groups, ranging from lamb to adult. Analysis of 20 highest expressed genes common across the groups revealed muscle protein, phosphorylation, acetylation, metal binding and transport as significant functions. Maximum differentiation was observed after 2.5-3 years on transition from lambs to adult. Transcriptional regulation by the TFAP2 transcription factors, IL-6 signaling and PI3K/AKT signaling pathways were enriched in younger animals. The gene-protein network demarcated key interactive genes involved in muscle development and proliferation that can be used as candidates for future research on improvement of muscle characteristics.

Transcriptomic diversity in longissimus thoracis muscles of Barbari and Changthangi goat breeds of India.

The present study is an attempt to examine the differential expression of genes in longissimus thoracis muscles between meat and wool type Indian goat breeds. Barbari goat is considered the best meat breed while Changthangi is famous for its fine fibre quality. RNA sequencing data was generated from four biological replicates of longissimus thoracis muscles of Barbari and Changthangi goats. A clear demarcation could be observed between the breeds in terms of expression of genes associated with lipid metabolism (FASN, SCD, THRSP, DGAT2 and FABP3). Most significant genes with high connectivity identified by gene co-expression network analysis were associated with triacylglycerol biosynthesis pathway in Barbari goat. Highly interactive genes identified in Changthangi goat were mainly associated with muscle fibre type. This study provides an insight into the differential expression of genes in longissimus thoracis muscles between Barbari and Changthangi goats that are adapted to and reared in different agro-climatic regions.

Differential expression of miRNAs in skeletal muscles of Indian sheep with diverse carcass and muscle traits.

The study presents the miRNA profiles of two Indian sheep populations with divergent carcass and muscle traits. The RNA sequencing of longissimus thoracis muscles from the two populations revealed a total of 400 known miRNAs. Myomirs or miRNAs specific to skeletal muscles identified in our data included oar-miR-1, oar-miR-133b, oar-miR-206 and oar-miR-486. Comparison of the two populations led to identification of 100 differentially expressed miRNAs (p < 0.05). A total of 45 miRNAs exhibited a log2 fold change of ≥ ( ±) 3.0. Gene Ontology analysis revealed cell proliferation, epithelial to mesenchymal transition, apoptosis, immune response and cell differentiation as the most significant functions of the differentially expressed miRNAs. The differential expression of some miRNAs was validated by qRT-PCR analysis. Enriched pathways included metabolism of proteins and lipids, PI3K-Akt, EGFR and cellular response to stress. The microRNA-gene interaction network revealed miR-21, miR-155, miR-143, miR-221 and miR-23a as the nodal miRNAs, with multiple targets. MicroRNA-21 formed the focal point of the network with 42 interactions. The hub miRNAs identified in our study form putative regulatory candidates for future research on meat quality traits in Indian sheep. Our results provide insight into the biological pathways and regulatory molecules implicated in muscling traits of sheep.

Milk somatic cell derived transcriptome analysis identifies regulatory genes and pathways during lactation in Indian Sahiwal cattle (Bos indicus).

BACKGROUND: The present study is an effort to understand the genomic drivers of lactation in Sahiwal (Bos indicus), the best milch cattle breed of the tropics. METHODS: RNA sequencing of four animals from early, mid and late lactation stages was performed using milk somatic cells as source of RNA. RESULTS: The genes encoding the milk casein and whey proteins showed highest expression in early and mid lactation, with a declining trend towards the late stage. The enhanced expression of PLIN2, FABP5 and FABP3 genes in mid lactation suggests enrichment of the PPARα pathway which is linked to fatty acid metabolism. A gradual decline in the percentage of genes involved in metabolism of proteins, mRNA and insulin synthesis from early to late lactation reflected transition from lactogenesis to involution. Major biological pathways maintained throughout lactation were adaptive immune system, FGF signaling, EGFR signaling, activated TLR4 signaling, NFkB and MAP kinases activation mediated by TLR4 signaling repertoire. Differential expression analysis revealed 547, 1010 and 1313 differentially expressed genes (p < 0.05) between early-late, early-mid and mid-late stages, respectively. The topmost regulatory genes identified by network analysis from the differentially expressed genes, were involved in Chemokine receptor, GPCR and EGFR1 pathways. CONCLUSION: The genes and pathways delineated in this study have regulatory implications in cell morphogenesis, lipid droplet formation and protein synthesis in the course of lactation. The study provides an insight into the expression profile of genes influencing milk properties and lactation in Sahiwal cattle.

Pharmacodynamic and pharmacokinetic profile of RBx 343E48F0: a novel, long acting muscarinic receptor antagonist.

RBx 343E48F0 is a novel, potent, selective and long acting muscarinic receptor antagonist with a potential for use in the treatment of Chronic Obstructive Pulmonary Disease (COPD). The aim of the present study was to describe the in vitro and in vivo profile of RBx 343E48F0 and to compare the results with the present day benchmark therapy, tiotropium. Radioligand binding and isolated tissue based functional assays were used to evaluate the affinity, potency and receptor subtype selectivity of RBx 343E48F0. Inhibition of carbachol-induced bronchoconstriction in the anaesthetized rat and acetylcholine-induced bronchoconstriction in the conscious rat were used to assess the extent and duration of the bronchospasmolytic activity of RBx 343E48F0. In vitro and in vivo pharmacokinetic studies were conducted to evaluate the pharmacokinetic and lung retention properties of the compound. In vitro radioligand binding studies using human recombinant muscarinic receptors showed that RBx 343E48F0 had a pKi of 9.6 at the M(3) receptor and a 60-fold selectivity for the M(3) receptor over the M(2) receptor. In isolated tissue bioassays, it exhibited surmountable antagonism at the guinea pig trachea with a pK(B) of 9.5. Intratracheal administration to anaesthetized rats demonstrated a dose-dependent inhibition of carbachol-induced bronchoconstriction with an ED(50) value of 110 ng/kg. RBx 343E48F0 also exhibited a fast onset of action and long duration of action of greater 24h.