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Identification and validation of putative biomarkers by in silico analysis, mRNA expression and oxidative stress indicators for negative energy balance in buffaloes during transition period |
Savleen Kour1 
, Neelesh Sharma1,*
, Praveen Kumar Guttula2
, Mukesh Kumar Gupta2
, Marcos Veiga dos Santos3
, Goran Bacic4
, Nino Macesic4
, Anand Kumar Pathak5
, Young-Ok Son6,*
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1Division of Veterinary Medicine, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu, R.S. Pura, Jammu, UT of J&K 181 102, India
2Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha 769 008, India
3Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga, SP 13635-900, Brazil
4Clinic for Reproduction and Theriogenology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb 100 00, Croatia
5Division of Animal Nutrition, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu, R.S. Pura, Jammu, UT of J&K 181 102, India
6Department of Animal Biotechnology, Faculty of Biotechnology, College of Applied Life Sciences and Interdisciplinary Graduate Program in Advanced Convergence Technology and Science, Jeju National University, Jeju 690756, Korea |
Correspondence:
Neelesh Sharma, Tel: +91-94192-48061, Email: drneelesh_sharma@yahoo.co.in
Young-Ok Son,Email: sounagi@jejunu.ac.kr
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Received: 2 August 2023 • Revised: 7 November 2023 • Accepted: 20 November 2023 |
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| Abstract |
Objective
Transition period is considered from 3 weeks prepartum to 3 weeks postpartum, characterized with dramatic events (endocrine, metabolic, and physiological) leading to occurrence of production diseases (negative energy balance/ketosis, milk fever etc). The objectives of our study were to analyze the periodic concentration of serum beta-hydroxy butyric acid (BHBA), glucose and oxidative markers along with identification, and validation of the putative markers of negative energy balance in buffaloes using in-silico and quantitative real time-polymerase chain reaction (qRT-PCR) assay.
Methods
Out of 20 potential markers of ketosis identified by in-silico analysis, two were selected and analyzed by qRT-PCR technique (upregulated; acetyl serotonin o-methyl transferase like and down regulated; guanylate cyclase activator 1B). Additional two sets of genes (carnitine palmotyl transferase A; upregulated and Insulin growth factor; downregulated) that have a role of hepatic fatty acid oxidation to maintain energy demands via gluconeogenesis were also validated. Extracted cDNA (complementary deoxyribonucleic acid) from the blood of the buffaloes were used for validation of selected genes via qRTPCR. Concentrations of BHBA, glucose and oxidative stress markers were identified with their respective optimized protocols.
Results
The analysis of qRT-PCR gave similar trends as shown by in-silico analysis throughout the transition period. Significant changes (p<0.05) in the levels of BHBA, glucose and oxidative stress markers throughout this period were observed. This study provides validation from in-silico and qRT-PCR assays for potential markers to be used for earliest diagnosis of negative energy balance in buffaloes.
Conclusion
Apart from conventional diagnostic methods, this study improves the understanding of putative biomarkers at the molecular level which helps to unfold their role in normal immune function, fat synthesis/metabolism and oxidative stress pathways. Therefore, provides an opportunity to discover more accurate and sensitive diagnostic aids. |
| Keywords:
Beta-hydroxy Butyric Acid (BHBA); Buffaloes; Gene Expression; In-silico; Ketosis; Oxidative Stress Markers |
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