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Estrus or sexual receptivity determination is utmost important for efficient breeding programs for female buffaloes. Prominent estrus behavioral symptoms are the result of several molecular and neuroendocrine events involving the ovary and the brain. Expression of estrus behavior is poor in buffaloes during the summer season. Hence, the discovery of biomarkers specific to the estrus stage or its related ovarian events, like the presence of dominant ovarian follicle, is helpful for developing an easy estrus determination method. MicroRNA are small non-coding RNA with a potential to be biomarkers. Therefore, the present study targeted to investigate the potential of estrogen responsive miRNAs (miR-24, miR-200c, miR-16, miR-191, miR-223 and miR-203) as estrus biomarkers in buffalo saliva, a non-invasive fluid representing animals' pathophysiology. There was a significant (P < 0.05) increase in the salivary presence of the miR-16, miR-191 and miR-223 at 6th and 18th-19th days than the 0 day (estrus), 10th day and the following consecutive estrus day. These observations may indicate an association between the representative lower presence of these miRNA in saliva and the presence of dominant ovarian follicles. To test this association, pathway analysis, target gene identification, functional annotation and protein-protein interaction networks (PPI) were performed for miR-16, miR-191 and miR-223 by different bioinformatics tools. Interestingly, the top pathways (fatty acid biosynthesis and oocyte meiosis), target genes (FGF, BDNF and IGF1) and PPI hub genes (KRAS, BCL2 and IGF1) of these miRNAs were found essential for ovarian follicular dominance. In conclusion, the miR-16, miR-191 and miR-223 may not be the perfect estrus stage-specific biomarkers. However, their lower presence in saliva at estrus and 9th-10th day of estrous cycles, when the ovary usually has a dominant follicle in buffaloes, may intuitively indicate the follicular dominance. Further studies are needed to prove this association in a large population.

Estrus detection is a major problem in buffalo husbandry because of inconsistent expression of estrous signs at different seasons, and a high prevalence of the silent heat and postpartum anestrus in this species. Around 50% of the estrus events in buffaloes are currently undetected in the field conditions, resulting in a huge economic loss. Although the cervicovaginal fluid fern patterns confirm the estrus for a breeding decision, the fluid discharge is absent during the silent-heat condition. Therefore, the present study focused on the crystallization patterns of the saliva as an alternative method for estrus detection in buffaloes. Saliva is a body fluid available regularly, and its ferning ability before ovulation was established in women. In this study, eight female nonpregnant Murrah buffaloes (Bubalus bubalis) were considered during two experimental periods of 3 months each. One period was in summer with five animals, and another period was in rainy season with three animals. Estrus was determined by the estrus symptoms, ovarian ultrasonography, and salivary estradiol (E2) to progesterone (P4) ratio. A total of 450 saliva samples were collected from these animals on the daily basis. The salivary smear was prepared with 20 μL of the cell-free saliva on a clean glass slide, and its microscopic images were captured at a magnification of × 200. The images were used for fractal analysis as the salivary crystallization or fern patterns follow the fractal geometry. Saliva at estrus showed a typical symmetrical fern-like crystallization patterns with significantly (P < 0.05) lower fractal dimension values. Salivary estradiol levels and E2/P4 ratio were significantly (P < 0.05) higher at the estrus stage than those at the diestrus stage. An average period of an estrous cycle was 21.7 ± 2.7 days (n = 18 estrous cycles) in buffaloes on the basis of distinct salivary crystallization patterns. The proportion of estrus detection by the salivary fern patterns was very significantly (P < 0.01) higher (0.84) than the proportion of estrus detection (0.5) in the field conditions. Altogether, salivary fern patterns along with the current methods can help reduce estrus detection problem in buffaloes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Salivary RNA-based biomarkers are not available for any physiological condition in farm animals. Hence, an objective of this study was to perform salivary transcript analysis in buffaloes. Saliva, after removal of the cells and particulate matter, was directly used for RT-PCR without RNA isolation. Direct saliva transcript analysis (DSTA) showed a suggestively significant higher expression of the Heat shock protein 70 (HSP70) and Toll-like receptor 4 (TLR4) at oestrus than the diestrous period in buffaloes by a non-parametric Mann-Whitney U test. Therefore, DSTA without RNA isolation is an easy method to identify salivary RNA markers for oestrus detection in buffaloes.