Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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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 Kour (Division of Veterinary Medicine, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu) ;
  • Neelesh Sharma (Division of Veterinary Medicine, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu) ;
  • Praveen Kumar Guttula (Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela) ;
  • Mukesh Kumar Gupta (Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela) ;
  • Marcos Veiga dos Santos (Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of Sao Paulo) ;
  • Goran Bacic (Clinic for Reproduction and Theriogenology, Faculty of Veterinary Medicine, University of Zagreb) ;
  • Nino Macesic (Clinic for Reproduction and Theriogenology, Faculty of Veterinary Medicine, University of Zagreb) ;
  • Anand Kumar Pathak (Division of Animal Nutrition, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu) ;
  • Young-Ok Son (Department of Animal Biotechnology, Faculty of Biotechnology, College of Applied Life Sciences and Interdisciplinary Graduate Program in Advanced Convergence Technology and Science, Jeju National University)
  • Received : 2023.08.02
  • Accepted : 2023.11.20
  • Published : 2024.03.01
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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

Acknowledgement

This work was supported by Department of Biotechnology (DBT), (Grant No. BT/PR26321/SPD/9/1307/2017) Government of India, and National Research Foundation of Korea (Grant No.: 2020R1A2C2004128), and Basic Science Research Program, NRF (Grant No.: 2019R1A6A1A10072987), Ministry of Education, Government of South Korea.

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