Direct saliva transcript analysis as a novel non-invasive method for oestrus marker detection in buffaloes.

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.

Saliva ferning, an unorthodox estrus detection method in water buffaloes (Bubalus bubalis).

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.