Suneel Kumar Onteru

Buffaloes do not exhibit overt estrus signs particularly during summer, leading to a significant economic loss to farmers. Previous studies have identified several candidate transcripts (HSP70, TIMP1, TLR4 and HSD17B1), abundant in buffalo saliva during estrus stage. However, there is no widely applicable technology for estrus detection targeting these transcripts. Therefore, the present study aimed to develop reverse transcription loop mediated isothermal amplification (RT-LAMP) assays for these candidate transcripts using buffalo saliva. Saliva samples were collected from 10 cyclic buffaloes and RT-LAMP assays were optimized for salivary RNA as well as direct saliva. Among the four candidate transcripts, HSP70 showed a statistically significant colour change (p-value = 0.0191) at the estrus stage compared to the diestrus stage. This abundance of HSP70 was also supported in large simulated population datasets (10,000 animals) generated using R. Further, the RT-LAMP assays were tested using direct saliva without RNA isolation, and the colour change in the samples during estrus suggested the feasibility of estrus identification using direct saliva, overcoming the tedious step of RNA isolation. The detection of HSP70 using either direct saliva or salivary RNA indicated its potential as a marker for estrus identification. Similarly, TLR4 appeared to be another potential biomarker for RT-LAMP reaction using direct saliva, but it needs further validation in both RNA and direct saliva samples. Overall, the proof-of-concept on RT-LAMP assays optimized for salivary transcripts in the present study would be useful for estrus identification in tropical production systems following further validation on a larger sample size.

Improving disease resistance in cattle relies on informed breeding and vaccine development, both depend on our understanding of immune mechanisms in cattle. However, transcriptomic studies of bovine immune responses often show considerable variability due to differences in tissue type, pathogen, time point, and experimental design, limiting the generalizability. Meta‐analysis integrates multiple transcriptomic studies to identify consistent gene expression patterns and enhance statistical power. We integrated bovine RNA‐seq datasets using immune‐response specific keywords, species constraints, and high‐throughput sequencing filters to prioritize biologically comparable and meta‐analysis‐ready studies. Specifically, in this study, we performed a meta‐analysis of four bovine transcriptomic datasets to identify immune‐related differentially expressed genes (DEGs) in Bos taurus . These datasets showed consistent results across analyses and represent immune responses related to mycobacterial infections ( Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis ), making them suitable for combined analysis. Our pipeline included FastQC, Trimmomatic, Bowtie2, SAMtools, FeatureCounts, DESeq2, and MetaRNASeq, identifying 28 DEGs (12 upregulated and 16 downregulated). We identified key immune‐related genes ( IL1A , RGS2 , RCAN1 , ZBP1 , TIMD4 , PPARG , TLR10 , and ACP5 ) with known regulatory roles in immunity. KEGG enrichment analysis revealed involvement in necroptosis, osteoclast differentiation, oxytocin signaling, and cGMP–PKG signaling pathways, associated with inflammatory cell death, cytokine signaling, and immune cell differentiation. Using reproducible transcriptomic signals across systematically selected bovine immune datasets rather than relying on single‐experiment analyses, we provide a robust meta‐analytic framework. This meta‐analysis enhances our understanding of conserved immune signaling mechanisms in cattle for identifying conserved immune mechanisms with broader biological and translational relevance.

Indicine cattle (Bos indicus) show notable resilience and disease resistance compared with taurine breeds, but the genomic basis of these traits remains largely unexplored. Identification of genomic elements for immunity will enable future controlled crossbreeding programs using molecular breeding methods. Therefore, we performed a genome-wide comparison among Nelore, Gir, and Hereford breeds using their whole-genome sequences, majorly focusing on immune-related structural and sequence variation. Our aims were to catalog insertions, deletions, and single nucleotide variants (SNVs) that intersect immune loci and known quantitative trait loci (QTLs), identify runs of homozygosity and selective-sweep signals, and prioritize candidate genes for follow-up functional studies. We retrieved whole-genome sequencing data for Nelore breed (n = 14) and Gir breed (n = 20) from NCBI using the SRA toolkit. Reads were checked with FastQC v0.12.1, filtered with Fastp 0.23.4 to remove low-quality bases and adaptors, and retained high-quality reads based on Q20 and Q30. The reads were mapped to the Bos taurus reference (ARS-UCD2.0) with Burrows-Wheeler Aligner - Maximal Exact Matches (BWA-MEM) v0.7.19-r1273; alignments were processed with Samtools 1.19.2 for sorting, duplicate marking, and MAPQ ≥ 20 filtering. Variants (insertions, deletions, SNVs) were called with GATK HaplotypeCaller (GATK v4.4.0.0), hard-filtered, normalized with Bcftools 1.19, and annotated with SnpEff 5.2b and SnpSift v5.2 f. Common variants were identified via in-house Python scripts; immune loci were detected from InnateDB and keyword searches; QTL overlaps were identified using Animal QTLdb; and DAVID was used for GO and KEGG enrichment (P < 0.05). ROH islands were defined in PLINK as regions shared by > 50% of individuals or samples, and selective sweeps were detected with RAiSD; genes overlapping ROH islands and RAiSD peaks were prioritized as candidate selection signatures. GATK v4.4.0.0 identified 1,884,058 indels and 13,997,533 SNVs in Nelore breed, and 1,457,337 indels and 11,627,881 SNVs in Gir breed, with Ti/Tv ratios of ~ 2.26 and ~ 2.25, respectively. Nelore breed has more number of variants than Gir. We observed frameshift insertions in TLR3 and LOC508441 (CD33) in both the breeds and frameshift deletions in JAM3 in Nelore breed and PAX5 in Gir breed. The variants were also identified in the regulatory regions of both breeds. The high-impact SNVs were in CD46 and IL26 genes in Nelore breed, and PLG gene in Gir breed. Genome-wide scans using RAiSD identified selective sweeps in 707 candidate genes in Nelore breed and 165 in Gir breed. Comparing the ROH and RAiSD results, we prioritized the genes ANKRD11, MAGI2, LOC132345096, FOXP2, TCF12, and ATP5PO in Nelore breed, and the genes MEFV and ORIF1 in Gir breed. These genes are found in QTLs linked to milk and health traits. Functional enrichment showed that the genes exhibiting all the three variants belong to immune pathways such as, NF-kappaB signaling, T-cell receptor signaling, and MAPK signaling in both breeds. These results reveal breed-specific genomic variation locating immune loci and its associated QTLs and provide a list of candidate genes and regions for experimental validation and marker development to improve disease resistance and productivity in Indicine cattle.

Indicine cattle ( Bos indicus ) are known for resilience to infectious diseases and environmental stress. However, the genomic basis underlying this advantage remains poorly understood. To characterize variations in immune-related genetic elements in four indicine breeds (Kangayam, Gir, Tharparkar, and Sahiwal), a taurine breed (Holstein Friesian), and a taurine–indicine crossbreed (Karan Fries), we performed integrative whole-genome analysis. Using whole-genome data representing 108 animals, we identified structural variants, copy number variants, single-nucleotide variations, and insertions/deletions. High-impact single-nucleotide variations in the key innate immune genes, CARD9 and NLRP8 , shared across all indicine breeds, were absent in the other breeds. Genetic differentiation analysis identified several innate immune genes showing strong divergence between the indicine breeds and the taurine breed. Selective sweep detection analysis highlighted multiple breed-specific immune-related sweep regions. Functional enrichment analysis showed significant enrichment of immune pathways in indicine breeds. A comparison of the candidate genes with basal gene expression profiles of unchallenged peripheral blood mononuclear cells indicated that genomic variation influences the differential expression of several genes in indicine breeds. We synthesized the data from population genome structure analysis, nucleotide diversity, genetic differentiation, selective sweep analysis, and correlations with gene expression profiles. Indicine breeds exhibited a higher number of immune-related variants and stronger signals of selection in immune pathways. These findings provide a curated set of innate immune gene candidates in indicine breeds for future functional studies and breeding programs.

Exosome-encapsulated miRNAs have been identified as the bioactive ingredient in milk, which provides immunological and nutritional advantages. Milk is processed through various treatments to ensure safe consumption, including pasteurisation, ultraviolet (UV) treatment, and ultra-heat treatment (UHT). Trends in the consumption of such processed milk are increasing across the world. In the present study, the effect of UV radiation as well as UHT processing on exosomes and their encapsulated miRNAs was investigated. The presence and size of exosomes in the UV-treated and UHT milk were confirmed by Zetasizer analysis. Bicinchoninic acid (BCA) protein assay showed that there was no significant loss of exosomes after UV radiation treatment, whereas after UHT processing, a significant loss was observed. As analysed by qPCR, a significant loss of exosomal miRNAs was observed after UV radiation as well as UHT processing, as compared to pasteurized milk. Since milk miRNAs have been shown to possess physiological effects, such loss due to UV and UHT processing could be of concern.

Innate immunity, the primary defence mechanism, encompasses a range of protective processes like anatomical barriers, cytokine secretion, and the action of various immune cells. Cattle breeds might differ in these processes because of their genetic differences such as copy number variations (CNVs). Therefore, the present investigation employed an array comparative genomic hybridisation (aCGH) approach on breed representative pooled DNA samples to evaluate CNVs across six cattle breeds: four indigenous Indian breeds, Kangayam (KNG), Tharparkar (TP), Sahiwal (SW), Gir (GIR), one crossbred Karan Fries (KF), and one exotic breed, Holstein Friesian (HF). In aCGH, HF DNA was used as control, while test DNA was from the other breeds. Each pooled test DNA sample was a representative of 18 animals belonging to three distinct geographical locations of India. The study using Aberration Detection Method 2 (ADM-2) of Agilent Genomic Workbench revealed the highest number of duplications in KNG (1189 genes), followed by TP (534 genes), and the greatest number of deletions in SW (774 genes). Among these genes, 183 and 76 innate immune genes with hub genes TGF-β1, CD79A, and IL4 showed duplications in KNG and TP, respectively. In SW, 113 innate immune genes with hub genes PSMC5, MAPK1, and AXIN1 showed deletions. In contrast, KF and HF showed no genes with deletions and fewer duplicated innate immunity genes, reflecting either lower genetic variability in their immune gene repertoire or a potential bias due to HF DNA as a control in aCGH. Functional enrichment of innate immune genes revealed duplications in KNG enriched in interleukin-1 receptor (IL1R) activity (p = 9.9 × 10^–3) and nucleobase metabolism (p = 2.88 × 10⁻¹¹), while duplications in TP were linked to DNA-binding transcription factor activity (p = 2.34 × 10⁻¹⁴). The KEGG pathway analysis highlighted Th17 cell differentiation (p = 1.3 × 10⁻⁴) in KNG and Hippo signalling (p = 3.7 × 10^–2) in TP. Overall, these findings highlight the importance of genetic diversity in shaping innate immunity in indigenous Indian cattle breeds, favouring a balanced crossbreeding to sustain the Indian dairy sector.