Browsing by Author "KUMAR, SUDARSHAN"
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ThesisItem Open Access ANNOTATION OF HIGHLY ABUNDANT PREGNANCY ASSOCIATED GLYCOPROTEIN ISOFORMS IN BUFFALO AND DEVELOPMENT OF PAG BASED IMMUNOLOGICAL ASSAY FOR PREGNANCY DIAGNOSIS(ICAR-NDRI, KARNAL, 2021) SRAVANI, MUDAMALA; KUMAR, SUDARSHANThe early pregnancy diagnosis is the major challenge in the livestock sector. One of the molecules that gives scope to diagnose pregnancy at early stages of gestation is PAGs (pregnancy associated glycoproteins), as they are directly secreted from conceptus to maternal circulation around 22 to 28 days after fertilization. Thus, PAGs serve as marker for pregnancy diagnosis and early embryonic mortality. So far, 12 PAG isoforms and 61 variants were found in buffaloes at mRNA level. But there is inadequate knowledge about PAG genes in buffaloes and also, we do not know the source for existence of so many PAG variants at mRNA level. Whether these variants come from single gene loci or have multiple genes or all these variants are products of splicing mechanism is not known. In order to gain insight about these conundrums, the PAG genes were annotated in buffalo by using latest genome assembly of water buffalo. During the course of following study, 39 PAG genes were identified, all of which were found to be clustered on chromosome 5. Later by using genome data viewer of NCBI, nucleotide position of known PAG isoforms and their mRNA variants were identified; where it was observed that all 61 PAG variants were falling only under 9 gene loci. Multiple sequence alignment of PAG protein sequences was done by using MUSCLE tool, in which exon skipping events were not observed. From the following study it was found that PAG variants do not have independent gene loci and occurrence of these variants is not due to splicing. So, one of the possible answers for existence of these large number of PAG variants might be; rapid karyokinesis in trophoblasts during gestation, that is not equally followed by cytokinesis, which is why we see BNCs and multinucleated cells in trophoblast layer of placenta. So, this faster DNA replication is followed by rapid transcription and translation due to which there might be improper proof- reading activity resulting in error prone transcription in trophoblast cells of placenta. In the second part of the research, an ELISA based assay using antibodies against BuPAG 2 isoform to screen pregnant and non-pregnant sera samples of buffaloes was developed. And from the results it can be inferred that BuPAG 2 can serve as pregnancy biomarker for detection of pregnancy in buffaloes significantly at day 35 post AI by ELISA.ThesisItem Open Access SCREENING OF POTENTIAL LIGANDS FOR SPERM SURFACE PROTEINS AND VALIDATION OF THEIR INTERACTION WITH SELECTED TARGET PROTEINS IN SAHIWAL BREED OF CATTLE(ICAR-NDRI, KARNAL, 2021) MATHUR, MANYA; KUMAR, SUDARSHANIndia takes pride in being the world‟s largest milk producers. The milk and milk based industries largely depend on the female cattle and heifers to meet this large and ever increasing demand for milk. This makes gender pre-selection and important aspect of animal farming and in different animal husbandry industries. The use of sexed semen has revolutionized the dairy industries. The only commercially available method for this selection is flow cytometry in which X and Y chromosomes are separated based on their DNA content. However, this approach, although successful, has its own drawbacks including reduced fertility and lower cell count in sexed semen. This raised questions regarding the efficiency of this approach as well as prompted the need for development of new methods. In India, the use of FACS for semen sorting is still under protected intellectual property rights which hinders its wider application for Indian breeds semen sorting. Over years immunological approaches have been considered to have the potential of being developed as a non-invasive but efficient method for semen sexing. One of the approach that has been highlighted these days is the biomarker based approach involving the use of differentially or uniquely expressed X and Y sperm specific surface proteins. The results from the present study suggest the possibility of the presence of certain sperm surface proteins that are uniquely expressed on either X or Y chromosome bearing sperm cells. For this different databases were screened and ultimately 3 proteins, Toll like receptor- 7 (TLR7), GABA Receptor subunit epsilon (GABAE) and Gastrin releasing peptide receptor (GRPR), were shortlisted as the potential candidate proteins that can be used as a unique sperm surface biomarker. For each protein, a separate ligand was identified, whose interaction resulted in the changes the properties of selected sperm cells which could be used for separation. These proteins were evaluated using extensive bioinformatics based screening, we were able to shortlist three sperm surface proteins that have the potential of being used as biomarkers and may have an application in sperm sex sorting. Through our work we were able to show the effect of TLR7 and R848 interaction on Y sperm enrichment in upper layers as well as X sperm enrichment in lower layers. We were also able to show the effect of interaction of receptor GABAE and its ligand clonazepam on sperm cell sample and validated the results to check for X sperm cell enrichment in upper layer and Y sperm enrichment in lower layers. We validated the effect of these ligands on sperm cell separation using Real-time PCR where we compared the sperm cell enrichment in different fractions with a control (with treatment) sample.