DEVELOPMENT OF GOLD-NANOPARTICLE BASED COLOUR REACTION FOR DETECTION OF ESTRUS CANDIDATE PROTEINS IN SALIVA OF BUFFALOES
| dc.contributor.advisor | RUBINA K. BAITHALU | |
| dc.contributor.author | APARNA RAJ | |
| dc.date.accessioned | 2024-08-09T08:31:43Z | |
| dc.date.available | 2024-08-09T08:31:43Z | |
| dc.date.issued | 2023 | |
| dc.description.abstract | The domestic water buffalo (Bubalus bubalis) is an important triple purpose livestock species contributes immensely to the Indian dairy industry. However, one of the major constraints in the exploitation of production potential of buffalo is its inherently poor reproductive efficiency and factors which are contributing includes delayed sexual maturity, poor expression of estrus signs, seasonal expression of estrus signs leading to higher incidence of silent estrus, wrong time insemination and conception failure. Estrus detection is a major problem in buffaloes because of its shy breeding nature and poor expression of estrus signs that leads to wrong time insemination. Therefore, developing an easy and reliable test for estrus detection in buffaloes is essential. In this regard several studies carried out in our laboratory and by others to identify estrus biomarker proteins in easily accessible body fluids like saliva by employing proteomics approaches. But till date no on-spot test is available for accurate estrus identification in buffaloes. Therefore, present study was carried out to develop a gold-nanoparticle based colour test using earlier identified estrus candidate proteins in saliva of buffaloes. For this study a total of 26 estrous cycles from 8 heifers and 6 pluriparous buffaloes were included during breeding season (November-February). The standing to be mounted behaviour by teaser bull was most reliable sign of estrus and 92.3 % and 90.81 % of heifers and pluriparous buffaloes, respectively has shown this sign during estrus. Estrus stage was further confirmed by per-rectal examination, ultrasonography, cervical mucus ferning pattern and hormone estimation (progesterone and estradiol) in blood serum. Gold nanoparticles were prepared by citrate reduction method and the average size of prepared AuNPs was 21.26 nm with zeta potential of -24.3 mV as determined by DLS and characteristic plasmon resonance peak observed at 520 nm. Antibody concentration was optimized by considering different concentrations of VMO1, HSPA1A, CYP11A1 and ENO3 antibodies, respectively for making stable conjugate with AuNPs. Further, AuNP based colour assay was standardized for detection of estrus candidate peptides viz., VMO1, HSPA1A, CYP11A1 and ENO3. Dilution of saliva samples was optimized @ 1:15 dilution and BSA concentration was optimized @ 10% and 0.1% for initial and final blocking. AuNP-based colour assay was developed for detection of estrus candidate proteins viz., VMO1, HSPA1A, CYP11A1 and ENO3 in saliva of buffaloes. Sensitivity, specificity and accuracy of AuNP-based colour assay using HSPA1A antibody was found to be highest i.e., 91%, 77.2%, & 82%, respectively for differentiating estrus from other non-estrus stages. Several protocols were tried for optimization of conjugation of antibody with AuNPs using different buffer at different pH and antibody concentration for development of competitive lateral flow assay (cLFA). For finding the optimum amount of peptide at test line for the competitive interaction of AuNP-antibody conjugate and peptide, various dilutions of peptide were made. Saliva samples from proestrus, estrus, metestrus and diestrus stages of estrous cycle were taken for checking the efficiency of developed LFA strip against HSPA1A protein. The test line was observed with higher intensity during proestrus, metestrus and diestrus stages but not for estrus stage depicting higher presence of HSPA1A protein in saliva samples during estrus. Altogether this study developed a proof of concept for developing a method for accurate estrus identification in buffaloes. | |
| dc.identifier.uri | https://krishikosh.egranth.ac.in/handle/1/5810213093 | |
| dc.language.iso | English | |
| dc.pages | 53 p. | |
| dc.publisher | ICAR-NDRI, KARNAL | |
| dc.sub | Veterinary Gynaecology and Obstetrics | |
| dc.theme | MASTER OF VETERINARY SCIENCE | |
| dc.these.type | M.V.Sc. | |
| dc.title | DEVELOPMENT OF GOLD-NANOPARTICLE BASED COLOUR REACTION FOR DETECTION OF ESTRUS CANDIDATE PROTEINS IN SALIVA OF BUFFALOES | |
| dc.type | Thesis |