Comparative Proteome Profiling of Saliva Between Estrus and Non-Estrus Stages by Employing Label-Free Quantitation (LFQ) and Tandem Mass Tag (TMT)-LC-MS/MS Analysis: An Approach for Estrus Biomarker Identification in Bubalus bubalis

Abstract

Accurate determination of estrus is essentially required for efficient reproduction management of farm animals. Buffalo is a shy breeder and does not manifest overt signs of estrus that make estrus detection difficult resulting in a poor conception rate. Therefore, identifying estrus biomarkers in easily accessible biofluid such as saliva is of utmost interest. In the current study, we generated saliva proteome profiles during proestrus (PE), estrus (E), metestrus (ME), and diestrus (DE) stages of the buffalo estrous cycle using both label-free quantitation (LFQ) and labeled (TMT) quantitation and mass spectrometry analysis. A total of 520 proteins were identified as DEPs in LFQ; among these, 59 and four proteins were upregulated (FC ≥ 1.5) and downregulated (FC ≤ 0.5) during E vs. PE, ME, and DE comparisons, respectively. Similarly, TMT-LC-MS/MS analysis identified 369 DEPs; among these, 74 and 73 proteins were upregulated and downregulated during E vs. PE, ME, and DE stages, respectively. Functional annotations of GO terms showed enrichment of glycolysis, pyruvate metabolism, endopeptidase inhibitor activity, salivary secretion, innate immune response, calcium ion binding, oocyte meiosis, and estrogen signaling. Over-expression of SERPINB1, HSPA1A, VMO1, SDF4, LCN1, OBP, and ENO3 proteins during estrus was further confirmed by Western blotting. This is the first comprehensive report on differential proteome analysis of buffalo saliva between estrus and non-estrus stages. This study generated an important panel of candidate proteins that may be considered buffalo estrus biomarkers which can be applied in the development of a diagnostic kit for estrus detection in buffalo.

Identification of postnatal development dependent genes and proteins in porcine epididymis

“…The inhibited activities of C3 and TF may have effects on immunosuppressive that protect the epididymal sperm against autoimmune responses and the establishment of immune tolerance [ 51 , 52 ]. SERPINB1 has multiple modulatory roles in regulating the immune response and cellular homeostasis and the decreased abundance of SERPINB1 may be linked to the enhanced sex steroid hormone stimulus with the advancement of development [ 53 , 54 ].…”

Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical Review

“…Studies on differential expression of mRNA transcripts [44][45][46], heat shock proteins [47][48][49][50] and other proteins [44,[51][52][53][54] in various biological fluids and/or tissues collected at different stages of estrous cycle have been reported and the use of the same as biomarkers for the identification of estrus has been opined.…”

Taurine dynamics in serum during the oestrous cycle in buffaloes

“…Therefore, specific biomarker needs to be identified for accurate detection of estrus in buffaloes. Previous studies have shown the use of salivary RNA (Joshi et al., 2023; Onteru et al., 2016), protein biomarkers (Singh et al., 2022), urinary pheromones or faecal volatile compounds as reliable indicators of estrus in buffalo (Karthikeyan et al., 2013; Muthukumar et al., 2018). Along with that, a salivary ferning pattern has also been recommended for detecting estrus even in the absence of cervicovaginal fluid (CVF) discharge in buffaloes (Ravinder et al., 2016; Surla et al., 2021).…”