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• A systematic assessment of 18 potential reference genes (RGs) in 10 different ovine tissues was carried out to identify internal control genes for qPCR analysis. • Four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), were employed to rank the stability of these RGs. • RefFinder was used for a comprehensive ranking, identifying ACTB, PPIB, BACH1 , and B2M as the most stable RGs. • RPS9, RPS15 , and PGK1 showed variable expression across different tissues. • Validation was performed using qPCR analysis of four target genes in the skin from two sheep breeds. 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. [ABSTRACT FROM AUTHOR]

• Global skin transcriptome profiles of Changthangi and Muzzafarnagri sheep were compared. • Changthangi sheep had 149 up-regulated and 2139 down-regulated genes (Fold change > 1.5, FDR < 0.05). • Enriched pathways in Changthangi sheep involve melanin biosynthesis, tyrosine metabolism, and developmental pigmentation. • High expression of melanogenesis genes like TYR, TYRP1, DCT, SLC45A2, PMEL, and others was noted in the Changthangi breed. • The study reveals how genetic factors affect phenotypic traits and environmental adaptability in various sheep breeds. Changthangi sheep, which inhabit the high-altitude regions of Ladakh, are known for their fine fiber production and are characterized by grey skin and either black or white coats. In contrast, Muzzafarnagri sheep from the plains of Uttar Pradesh produce coarse wool and have white skin and coats. We conducted comparative global gene expression profiling on four biological replicates of skin from each breed. Notably, our analysis identified 149 up-regulated genes and 2,139 down-regulated genes in Changthangi sheep compared to Muzzafarnagri sheep, with a p-adjusted value (padj) of ≤0.05 and a Log2 fold change of ≥1.5. Gene Ontology analysis of the up-regulated genes revealed an enrichment of terms related to melanin biosynthesis and developmental pigmentation. Additionally, enriched KEGG pathways included tyrosine metabolism and metabolic pathways. Among the melanogenesis-related genes that exhibited higher expression in Changthangi sheep were TYR, TYRP1, DCT, SLC45A2, PMEL, MLANA, and OCA2. These findings confirm melanin's role in both the animals' black coat color and UV protection at high-altitude. Furthermore, we observed more pronounced expression of genes related to fiber quality, namely KRTAP6, KRTAP7, KRTAP13, and KRTAP2, in the fine wool-producing sheep from Ladakh. The results of the RNA sequencing were validated using real-time PCR on 10 genes governing fiber quality and coat color, with ACTB and PPIB serving as reference genes. In conclusion, our comparative skin transcriptome analysis of Changthangi and Muzzafarnagri sheep sheds light on the genetic differences associated with distinct phenotypic traits and environmental adaptability, offering valuable insights into the underlying mechanisms. [ABSTRACT FROM AUTHOR]

• The transcriptional landscape of milk somatic cells of Murrah buffaloes with clinical mastitis was compared with the healthy controls. • A total of 1014 up-regulated and 999 down-regulated genes were observed in the diseased buffaloes (Fold change > 2, FDR < 0.05). • The expression of major milk protein genes (CSN2, CSN1S1, CSN1S2, PAEP and LALBA) was higher in the healthy buffaloes. • Mastitic buffaloes had higher transcript abundance of many genes coding for anti-microbial proteins. 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 β-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. [ABSTRACT FROM AUTHOR]

• This is the first study that compares gene expression profiles of cattle and buffalo milk somatic cells. • Enrichment of immune response related GO terms was observed in buffaloes. • Higher expression of genes for some host defense peptides was evident in buffaloes. • Genes for PI3K-Akt pathway showed higher expression in cattle milk. India is the world's largest milk producing country because of massive contribution made by cattle and buffaloes. In the present investigation, comprehensive comparative profiling of transcriptomic landscape of milk somatic cells of Sahiwal cattle and Murrah buffaloes was carried out. Genes with highest transcript abundance in both species were enriched for biological processes such as lactation, immune response, cellular oxidant detoxification and response to hormones. Analysis of differential expression identified 377 significantly up-regulated and 847 significantly down-regulated genes with fold change >1.5 in Murrah buffaloes as compared to Sahiwal cattle (p adj <0.05). Marked enrichment of innate and adaptive immune response related GO terms and higher expression of genes for various host defense peptides such as lysozyme, defensin β and granzymes were evident in buffaloes. Genes related to ECM-receptor interaction, complement and coagulation cascades, cytokine-cytokine receptor interaction and keratinization pathway showed more abundant expression in cattle. Network analysis of the up-regulated genes delineated highly connected genes representing immunity and haematopoietic cell lineage (CBL, CD28, CD247, PECAM1 and ITGA4). For the down-regulated dataset, genes with highest interactions were KRT18, FGFR1, GPR183, ITGB3 and DKK3. Our results lend support to more robust immune mechanisms in buffaloes, possibly explaining lower susceptibility to mammary infections as compared to cattle. [ABSTRACT FROM AUTHOR]