Urinary Cell-Free miR-99a-5p as a Potential Biomarker for Estrus Detection in Buffalo

Abstract

Accurate estrus detection method is the need of the hour to improve reproductive efficiency of buffaloes in dairy industry, as the currently available estrus detection methods/tools lack high sensitivity and specificity. Recently, circulating miRNAs have been shown as non-invasive biomarkers by various studies. Hence, in order to evaluate their potential as estrus biomarkers, the objective of this study was to identify and compare the levels of 10 hormone-responsive miRNAs in the urine collected at proestrus (PE), estrus (E), and diestrus (DE) phases of buffaloes (n = 3) pertaining to a discovery sample. Among 10 urinary miRNAs, the levels of bta-mir-99a-5p (E/PE 0.5-fold, P < 0.05; DE/PE 1.9-fold), bta-miR-125b (E/PE 0.5-fold; DE/PE 0.7-fold), bta-mir-145 (E/PE 1.5-fold; DE/PE 0.7-fold), bta-mir-210 (E/PE 1.2-fold, DE/PE 0.7-fold), mir-21 (E/PE 1.5-fold, DE/PE 2-fold), and bta-mir-191 (E/PE 1.3-fold; DE/PE 0.8-fold) were found to be altered during different phases of buffalo estrous cycle. In contrast, bta-mir-126-3p, bta-let-7f, bta-mir-16b, and bta-mir-378 were undetected in buffalo urine. Furthermore, a validation study in an independent group of 25 buffalo heifers showed the increased levels of urinary bta-mir-99a-5p during the DE (3.92-fold; P < 0.0001) phase as compared to the E phase. Receiver operating characteristic curve analyses also revealed the ability of urinary miR-99a-5p in distinguishing the E from the DE phase (area under the curve of 0.6464; P < 0.08). In silico analysis further showed an enrichment of miR-99a-5p putative targets in various ovarian signaling pathways, including androgen/estrogen/progesterone biosynthesis and apoptosis signaling, implicating the role of miR-99a-5p in ovarian physiology. In conclusion, significantly lower levels of bta-mir-99a-5p at the E phase than the DE phase in buffalo urine indicate its biomarker potential, which needs to be further explored in a large cohort in the future studies.

Keywords: bta-miR-99a-5p; buffalo; cell free miRNA; estrus; qRT-PCR; urine.

Figure 1

Typical salivary crystallization of buffalo (air dried, ×200). (A) Salivary crystallization during the proestrus phase. (B) Typical fern patterns of saliva during the estrus phase. (C) Discontinuous and improper crystallization during the diestrus phase.

Figure 2

Real-time PCR analysis of miRNAs (A) miR-125b, (B) miR-145, (C) mir-21, (D) mir-210, (E) miR-191, and (F) miR-99a-5p in proestrus (PE), estrus (E), and diestrus (DE) phases. One-way ANOVA test was done followed by post-hoc Tukey test for analysis. Each bar in the figure represents the mean ± SE of three independent experiments *P < 0.05.

Figure 3

Real-time PCR analysis of mir-99a-5p in the validation population of the animals in estrus (E) and diestrus (DE). The paired t-test was used for analysis. Each bar represents the mean ± SEM of 25 independent experiments. ***P < 0.001.

Figure 4

Receiver operating characteristic (ROC) curve of miR-99a-5p as a diagnostic marker for estrus.

Figure 5

Association of bta-miR-99a-5p target genes with different pathways as shown in the pie chart by PANTHER, an online tool.

Figure 6

Association of bta-miR-99a-5p target genes with different biological processes as shown in the pie chart by PANTHER, an online tool.