African Swine Fever (ASF) causes severe economic losses in the global pig industry, characterized by high mortality rates and lack of an effective vaccine. Interestingly, India's indigenous Doom pig breed has displayed tolerance to ASF, remaining seropositive without detectable viremia. This study investigated the genetic basis of ASFV tolerance, focusing on the STING1 gene, a key component of the cGAS-STING antiviral signaling pathway. In-silico docking identified a potential ASFV protein binding site within exon 5 of STING1. A non-synonymous SNP (CGG/TGG; R→W) at position 148 in this region was selected for genotyping. We genotyped 119 pigs, representing tolerant (Doom), susceptible, and ASFV-infected groups, using allele-specific PCR and Sanger sequencing. Doom pigs and most other breeds, including ASFV-infected samples, displayed a conserved GG genotype. Susceptible Manipuri Black and Ghoongroo pigs, however, displayed both AA and GG genotypes, yet still succumbed to the disease. A significant downregulation (0.436-fold) of STING1 was observed in ASFV-infected spleen tissue, indicating active immune evasion by the virus. The lack of a unique allele in Doom pigs compared to susceptible breeds indicates that ASFV tolerance is unlikely to be associated with this STING1 SNP. Therefore, genome-wide studies are recommended to identify markers truly associated with ASFV tolerance in this resilient breed.

Background: Mastitis caused by Staphylococcus aureus remains a significant challenge in dairy livestock due to the emergence of multidrug-resistant strains and the limited effectiveness of conventional therapeutic approaches. Lysostaphin, a staphylolytic enzyme with high specificity against Staphylococcus species, has emerged as a promising alternative antimicrobial agent. In this study, we aimed to establish a preliminary mammalian expression strategy for production of biologically active recombinant lysostaphin in buffalo fibroblast cells. Methods: A modified lysostaphin gene, designed to eliminate predicted N-glycosylation sites, was synthesized and initially cloned into the pUC57 vector followed by subcloning into the pcDNA3.1/CT-GFP-TOPO expression vector. Recombinant constructs were confirmed by colony PCR and Sanger sequencing before transfection into buffalo fibroblast cells. Recombinant expression was qualitatively assessed using GFP fluorescence and antibacterial activity was evaluated using a qualitative plate inhibition assay against Staphylococcus aureus. Result: The lysostaphin gene (~798 bp) was successfully amplified and cloned into the mammalian expression vector. Screening and sequencing confirmed the correct insertion and orientation without mutations. Transfected buffalo fibroblast cells exhibited GFP fluorescence, indicating successful expression of the recombinant construct. Lysates from transfected cells produced visible zones of inhibition against S. aureus, indicating antibacterial activity of the expressed lysostaphin.

Information on global transcriptomic changes in the porcine ampulla after ovulation is crucial for understanding of oviductal physiology at the molecular level. The objective of the present study was to investigate the differentially expressed genes (DEGs) and signalling pathways regulating the functionality of ampulla in pigs post-ovulation. The RNA-sequencing of the post-ovulatory ampulla (POA) and early luteal ampulla (ELA) tissues was conducted using Illumina NextSeq2000. The R package NOISeq was used to obtain significantly differentially expressed genes (DEGs) with the probability of differential expression (1-FDR) value ≥ 0.95 and log2 fold change (log2FC) ≥ 1, which revealed 817 DEGs (657 up- and 160 down-regulated) in the POA vs. ELA group comparison. These DEGs were functionally annotated with various gene ontology terms like sterol biosynthetic process, growth, cell migration, and Reactome pathways like signal transduction, metabolism, and cell cycle, indicating key role of these molecular events in POA. The WNT, TNFR2 non-canonical NF-kB, and hedgehog signalling pathways along with the activation of the immune system process, were enriched in the POA vs. ELA group, which indicates their role in cell–cell interactions and cell fate determination in remodelling the oviductal microenvironment during transition from estrogen to progesterone domination. The highly connected upregulated hub genes ESR1, RAD51, YARS1, TYMS and CDK2 can be regarded as key regulatory factors in synchronizing the changes in POA at the molecular level in the oviduct. The present study revealed several DEGs, signalling pathways and novel modulatory factors associated with the ampullary physiology during early embryonic development in the POA, which may influence fertility and litter size in pigs.

The use of natural farming inputs is considered a viable alternative to synthetic and inorganic chemicals in agriculture, aligning with the principles of organic cultivation and sustainability. The present study was undertaken to evaluate the effect of natural farming inputs on the growth of French bean (Phaseolus vulgaris L.). The field experiment was conducted during 2024 at Department of Horticulture, School of Agriculture, ITM University, Gwalior. The experiment consisted of nine treatments in randomized block design with three replications. The treatments were used T1- Control, T2- RDF (60:120:50 kg ha-1), T3- Vermicompost + 70% R.D.F., T4- Beejamrit (1 litre/kg seed) + Jeevamrit (100 %) at weekly interval, T5- Beejamrit (1 litre/kg seed) + Ghanjeevamrit (100 %), T6- Beejamrit (1 litre/kg seed) + Jeevamrit (75 %) + Ghanjeevamrit (25 %), T7- Beejamrit (1 litre/kg seed) + Jeevamrit (50 %) + Ghanjeevamrit (50 %), T8- Beejamrit (1 litre/kg seed) + Jeevamrit (25 %) + Ghanjeevamrit (75 %) and T9- Beejamrit (1 litre/kg seed) + Jeevamrit (80 %)+ Ghanjeevamrit (80%). Treatment no. 7 i.e. Beejamrit (1 litre/kg seed) + Jeevamrit (50 %) + Ghanjeevamrit (50 %) showed fastest 50 % germination and 50 % flowering days. Beejamrit (1 litre/kg seed) + Jeevamrit (50 %) + Ghanjeevamrit (50 %) stands out with the highest mean among all treatments for leaf area and number of pods per plant.

Investigations on heat stress induced transcriptomic changes is critical to characterization of candidate genes for thermal adaptability in livestock. Continues spells of high ambient temperature due to climate change has amplified reproductive dysfunctions, necessitating immediate attention. The present study aimed to explore the transcriptomic signature of heat stressed granulosa cells (GCs) and signalling pathways regulating their adaptability to thermal challenge. The GCs were collected from small follicles (3-6 mm) of pig ovary. The GCs primary culture was subjected to in vitro heat stress challenge at 42 OC for 6 h. RNA sequencing was conducted for heat stress (treated) and non-heat stress (control) groups using Illumina NextSeq2000 sequencing platform. The significant DEGs were selected using NOISeq R package with cut-offs, probability value >= 0.95 and log2 fold change >= 1. Bioinformatics analysis was conducted for exploring gene ontology enrichment, functional pathways, hub genes in protein-protein interaction network and functional clusters regulating cellular homeostasis and survivability during heat stress challenge. The analysis pipeline yielded a total of 12156 protein coding transcripts, which were expressed during heat stress challenge in GCs, out of which 4904 were differentially (prob. >= 0.95) expressed; 2936 were upregulated and 1968 were downregulated. The large number of DEGs and gene ontologies in the study specifies the concerted mechanisms involving multiple signalling pathways like MAPK, HIPPO, WNT, PI3-AKT, NFKB, NOTCH and many more operating in the cell to maintain cellular homeostasis. Thermal stress induced differentially expressed hub genes HSP90, HSPA8, HSPA5, TGFB1 and PPARG are key elements in stress, regulating multiple pathways and expression of transcription factors. The TNF signalling pathway, phosphatidyl inositol signalling system and DERL3 gene network linked ubiquitin-dependent endoplasmic reticulum associated protein degradation pathway, which regulates cell viability, proliferation, apoptosis and estrogen synthesis, can be regarded as novel regulators involved in stress adaptation in pigs.

This research aimed to characterize the mitochondrial genome of the Ghoongroo (GH) pig, a notable breed in India, along with its crossbred varieties, to elucidate their matrilineal components, evolutionary history, and implications for conservation. Seven pigs (5 GH, 2 crossbred, namely Rani and Asha) were sequenced for complete mitochondrial genome, while 24 pigs (11 GH, 6 Rani, and 7 Asha) were sequenced for the complete D-loop of the mitochondrial genome. The genome size of these pigs was determined to be 16,690 bp. Analysis of the mitochondrial sequences and phylogenetics uncovered two distinct matrilineal components within the GH population, a phenomenon also observed in its crossbred counterparts, Rani and Asha. Phylogenetic analysis demonstrated a clear clustering of GH sequences into two clades, indicating the presence of two independent maternal lineages. The phylogenetic study using complete mitogenome also indicated that GH pigs were originated locally from Indian wild boar independently from Asian and European pig population. Haplotype analysis from complete D-loop sequences revealed 10 different haplotypes, with some sequences shared among GH, Rani, and Asha, while others differed due to varying matrilineal origins. The haplotype analysis using complete mitogenome sequences revealed 16 different haplotypes with some shared sequences among GH. Furthermore, examination of tRNA genes and nucleotide composition of different genes namely rRNAs, COX1, COX2, ATP6, ND4, ND5, ND6, Cytb offered insights into genetic diversity within these pigs. The findings suggest that geographical isolation and historical events likely contributed to the emergence of distinct maternal lineages within the GH breed. This study underscores the significance of mitochondrial DNA analysis in uncovering hidden genetic diversity within seemingly uniform populations. The molecular insights gained into the genetic makeup of GH pigs could aid in designing effective breeding programs for conservation efforts and highlight its significance in understanding the broader context of pig domestication in India.

Researchers have widely undertaken targeted genome editing in pigs to optimize pig productivity, disease tolerance and for biomedical research. The present study aimed to investigate research advancements, focus areas, gaps, and challenges in genome editing in pigs using bibliometric analysis. The bibliographic information of publications on genome editing in pigs from 2010 to 2023 was retrieved from the Scopus database. Bibliometric parameters, such as coauthorship, keyword co-occurrence, citation, bibliographic coupling, and cocitation, was analyzed using VOSviewer. Literature mining was conducted to evaluate the emerging areas and challenges in the development of genome-edited pigs. We found 725 documents on genome editing in pigs, 407 of which were research articles authored by 2826 researchers from 1359 research organizations across 40 countries. The two countries, China and the United States, account for more than 50% of the research publications on genome editing in pigs. Investigations on the optimization of the procedure, delivery methods, editing efficiency, and reducing off-target effects dominated the early phase of research, which has shifted to its application for generating knockout (KO) or knockin (KI) pigs in recent years. Areas such as xenotransplantation, disease resistance, higher muscling, and disease models have dominated the research horizon for genome editing in pigs. Emerging areas in gene editing include base editing, CRISPR-based screens, diagnostics, and therapeutics. However, investigations on reducing heat stress and environmental footprint through genetic alterations need more attention from scientists. Challenges such as off-target effects and regulatory, ethical and societal issues related to channelizing gene-edited pigs from lab to land and then from farm to fork continue to restrain this field.

African Swine Fever (ASF) is a highly contagious transboundary viral disease affecting domestic pigs worldwide, often resulting in nearly 100 % mortality due to the lack of effective vaccines. However, wild species such as warthogs (Phacochoerus sp.) and bush pigs (Potamochoerus sp.) do not exhibit clinical symptoms of the disease, previous studies showed that amino acid substitutions in a proto-oncogene, RelA, a subunit of NF-κB found in warthogs might be responsible for their resistance. Expanding this study over an Indian breed named Doom which was considered tolerant due to lack of much information regarding their ASFV-positive cases and identifying the genetic basis of tolerance might help in creating a tolerant breed, thereby controlling the spread of the disease. So, this study was initiated to investigate the polymorphic signature in RelA gene of Doom breed similar to warthogs. Initially molecular docking studies identified a potential interaction between the N-terminal sub-domain of Rel homology domain of porcine RELA and the African Swine Fever Viral (ASFV) protein A238L, a hypothetical viral protein of unknown function. This amino acid interaction corresponds to a nucleotide sequence of exon 10 of porcine RelA gene. Further, ARMS-PCR (Amplification Refractory Mutation System-PCR) and PCR-RFLP (Restriction Fragment Length Polymorphism) were optimized to target a single nucleotide polymorphism (SNP, CCT/GCT: Pro→Ala) identified in the exon 10 region through Ensemble genome browser. PCR results showed the presence of homozygous CC genotype, coding for proline, in all pig breeds tested, including Doom. Sanger sequencing confirmed the CC genotype across all breeds, indicating that amino acid substitutions in this RELA domain were not present in Doom and therefore may not be responsible for ASF tolerance. Since disease tolerance is a polygenic trait relying on one gene might not reveal the exact cause for their tolerance against ASFV. Future research can be targeted on other set of genes playing important role in innate immune pathways or Genome Wide Association Study can be preferred to identify and associate SNPs with Doom's tolerance against ASFV.

Somatic cell nuclear transfer (SCNT) holds promise for animal cloning but remains limited by low efficiency and phenotypic abnormalities, often attributed to incomplete nuclear reprogramming. This study presents an integrative genomic and epigenomic analysis of cloned buffaloes and their respective donors using long-read Oxford Nanopore sequencing. Our results showed a high degree of genomic similarity between clones and donors, with most variations located in non-coding regions and structural variants (SV) distributions highly correlated at the chromosomal level. Gene and protein level overlap of SV-affected loci revealed 70.9-73.3% gene-level and 69.7-72.5% protein-level similarity. Despite this genetic similarity, DNA methylation analysis identified differentially methylated regions (DMRs), particularly in intergenic and promoter regions. Clones exhibited slightly lower CpG methylation than the donors. The DMRs in donor vs. clone comparisons indicated higher hypomethylated regions than hypermethylated regions. Functional enrichment of DMR-associated genes highlighted pathways linked to mitochondrial function, oxidative phosphorylation, and reproductive processes. Although clones showed moderate genome-wide methylation correlation with donors, key differences in methylation suggest incomplete epigenetic reprogramming. Despite these epigenetic differences, all clones were phenotypically normal and healthy into adulthood. This study offers the first comprehensive SV and methylome profile of SCNT-derived buffaloes and emphasizes the role of epigenetic mechanisms in clone development and health, providing valuable insights to enhance cloning efficiency.

Mitochondrial DNA (mtDNA) serves as a valuable molecular marker for constructing matrilineal genealogies and tracing the evolutionary history of animals. This study aimed to characterize the complete mitochondrial genome of the Indian wild pig (IWB) (Sus scrofa cristatus) and identify IWB-specific DNA sequences that could be used as genomic signatures to differentiate IWB from domestic Indian pigs (IDP) in forensic cases. For the purpose, three wild IWB from a rescue centre were used for the characterization of the mitochondrial genome of the IWB. The mitochondrial genome was sequenced by the primer walking technique using 30 overlapping primers. The mitochondrial genome of the IWB was found to be 16,689 bp long containing 37 genes coding for 2 rRNAs, 22 tRNAs, 13 protein coding genes, and 1 D-loop region similar to the mitogenome of other pigs. Sequence analysis of the D-loop of IWB with other IDP indicated some signature sequence for IWB like duplication and transition event from 1090th to 1099th position, deletion of a 10 bp sequence at the 755th position, insertion of (CA) at the 137th position, and substitution of AT to GA at the 638th position. These variations specially the duplication along with transition event causes creation of unique signature sequence (-ACACAAACCT-) in the IWB that could serve as signature sequences for the IWB and be used as markers for differentiation of IWB from IDP breeds in academic as well as forensic or vetero-legal cases. Overall, a total of 36 polymorphic positions were identified in the IWB, with 29 sites being unique to the IWB only and seven being common to the Doom and HDK75 pig breeds. None of the common polymorphic sites were identified in prevailing domestic pig populations. Phylogenetic analysis of the mitochondrial genome revealed the distinct separation of the IWB from IDP. The results of genetic distance evaluation showed that the Doom pig breed was the closest to the IWB. This study provides valuable insights into the mitogenome characterisation, signature sequence and genetic distance analysis of the IWB and establishes a foundation for future studies on the conservation of this protected species.

The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein–protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Ghoongroo pig is the first recognized pig breed in India from the North Bengal and adjoining districts of Assam. This breed has been evolved through continuous breeding and selection within its native breeding tract. The present investigation aimed to evaluate the genetic performance of Ghoongroo pigs across generations, utilizing data collected over a 13-year period (2008-2021) from the Nucleus Pig Breeding Farm at the ICAR-National Research Centre on Pig in Guwahati, Assam. The analysis of productive, reproductive and carcass traits revealed continuous improvements in litter size, litter weight, weaning weight and other traits over successive generations due to selective breeding. The study highlighted the consistent genetic progress achieved through selective breeding within the Ghoongroo pig breed. The scientifically managed nucleus herd of Ghoongroo pigs at the research institute serves as a valuable repository for preserving the breed’s genetic resources and acts as a conservation unit for this unique indigenous breed.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

Objective Optimizing reproductive efficiency in terms of estrus induction and production performance using simplified hormonal intervention using GnRH, gonadotropins and progesterone at farmers’ field conditions to bred animals with superior quality semen and also to reduce the maintenance cost. Method T he present study was planned for optimizing reproductive efficiency using four different easily available hormonal agents with simplified protocols at farmers’ field. A total of 126 females were used in the study. Hormonal protocols I, II, III and IV using GnRH + (PMSG+ hCG), (PMSG+ hCG), PMSG alone and prepared progesterone gel respectively were used for estrus induction in gilts and sows. Results The estrus induction/rate was (77.77%, 81.36%, 78.57% and 50% respectively. The corresponding figures for interval (hrs) of heat exhibition from hormonal administration (121.33±4.93, 121.54±3.60, 78.52±4.52 and 192±24), conception rate (%) (72.22%, 81.25%, 68.18% and 33.33%) farrowing rate (55.55%, 68.29%, 68.18% and 33.33 %), total litter size at birth (7.2±0.64, 7.90±0.47, 8.90±0.47 and 7.00±0) and hormonal cost (Rs.) per animal (570, 335, 280 and 250 Rs.) were respectively. The easier to use protocol was III followed by II, I and IV. Conclusion It was found that protocol II and III are effective and easier so can be used for optimizing reproductive efficiency in pigs at farmers’ field.

Ovarian follicular development is a critical determinant of reproductive performance in litter bearing species like pigs, wherein economic gains depend on litter size. The study aimed to gain insight into the differentially expressed genes (DEGs) and signalling pathways regulating follicular growth and maturation in Ghoongroo pigs. Transcriptome profiling of porcine small follicles (SF) and large follicles (LF) was conducted using NovaSeq600 sequencing platform and DEGs were identified using DESeq2 with threshold of Padj. < 0.05 and log2 fold change cut off 0.58 (LF vs. SF). Functional annotations and bioinformatics analysis of DEGs were performed to find out biological functions, signalling pathways and hub genes regulating follicular dynamics. Transcriptome analysis revealed 709 and 479 genes unique to SF and LF stages, respectively, and 11,993 co-expressed genes in both the groups. In total, 507 DEGs (284 upregulated and 223 downregulated) were identified, which encoded for diverse proteins including transcription factors (TFs). These DEGs were functionally linked to response to stimulus, lipid metabolic process, developmental process, extracellular matrix organisation along with the immune system process, indicating wide-ranging mechanisms associated with follicular transition. The enriched KEGG pathways in LF stage consisted of ovarian steroidogenesis, cholesterol and retinol metabolism, cell adhesion molecules, cytokine receptor interaction and immune signalling pathways, depicting intra-follicular control of varied ovarian function. The hub gene analysis revealed APOE, SCARB1, MMP9, CYP17A1, TYROBP as key regulators of follicular development. This study identified candidate genes and TFs providing steroidogenic advantage to LFs which makes them fit for selection into the ovulatory pool of follicles.

Luteal steroidogenesis is critical to implantation and pregnancy maintenance in mammals. The role of androgen receptors (AR) in the progesterone (P4) producing luteal cells of porcine corpus luteum (CL) remains unexplored. The aim of the present study was to establish AR gene knock out (KO) porcine luteal cell culture system model by CRISPR/Cas9 genome editing technology and to study the downstream effects of AR gene deficiency on steroidogenic potential and viability of luteal cells. For this purpose, genomic cleavage detection assay, microscopy, RT-qPCR, ELISA, annexin, MTT, and viability assay complemented by bioinformatics analysis were employed. There was significant downregulation (p < 0.05) in the relative mRNA expression of steroidogenic marker genes STAR, CYP11A1, HSD3B1 in AR KO luteal cells as compared to the control group, which was further validated by the significant (p < 0.05) decrease in the P4 production. Significant decrease (p < 0.05) in relative viability on third passage were also observed. The relative mRNA expression of hypoxia related gene HIF1A was significantly (p < 0.05) downregulated in AR KO luteal cells. Protein-protein interaction analysis mapped AR to signaling pathways associated with luteal cell functionality. These findings suggests that AR gene functionality is critical to luteal cell steroidogenesis in porcine.