# 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

## TL;DR

This study identifies and validates potential biomarkers for negative energy balance in buffaloes during the transition period using in silico analysis and qRT-PCR.

## Contribution

The study validates novel biomarkers for negative energy balance in buffaloes using molecular techniques and in silico analysis.

## Key findings

- Two genes (acetyl serotonin o-methyl transferase like and guanylate cyclase activator 1B) were validated as putative markers for negative energy balance.
- Significant changes in BHBA, glucose, and oxidative stress markers were observed during the transition period.
- qRT-PCR results aligned with in-silico predictions, supporting the potential for early diagnosis.

## Abstract

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.

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 qRT-PCR. Concentrations of BHBA, glucose and oxidative stress markers were identified with their respective optimized protocols.

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.

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.