Bos frontalis (Mithun) and Bos grunniens (yak) are crucial to the culture, food security, and economy of Southeast Asia, especially in India and China, respectively. Their genetic closeness to Bos indicus (indicine cattle) and Bos taurus (taurine cattle) necessitates precise methods for meat origin authentication. This study introduces a DNA-based technique to distinguish Mithun and yak species using the alkaline lysis (AL) protocol for DNA extraction, followed by species-specific polymerase chain reaction (PCR) to amplify unique mitochondrial D-loop regions, yielding 489 bp and 422 bp amplicons, respectively. The AL-PCR method showed high specificity for both species, with no cross-amplification with other related species. The method’s effectiveness was validated across various sample preparations, including raw, cooked, autoclaved, microwaved, and fried samples. The AL-PCR assay is highly sensitive, detecting as little as 1 pg of Mithun DNA and 100 pg of yak DNA, and can identify down to 0.1% of these species in binary mixtures. This approach is rapid and cost-effective, offering significant benefits for consumer protection, promoting Mithun and yak farming, and addressing food safety and traceability issues.

The study aimed to assess the possibility of cervical mucus changes to predict ovulation time in Mithun. Experimental Mithuns (n=6) were estrus synchronized and cervical mucus was collected in different estrus phases from three consecutive estrous cycles (n=18). Cervical mucus colour, consistency, arborization pattern and cytology were evaluated. Plasma estradiol-17β (E2) and progesterone (P4) levels were determined during different phases of estrous cycles. Ovulation time and subsequent growth of the corpus luteum were determined using transrectal ultrasonography. Results indicated thick and moderate cloudy mucus with a typical arborization pattern in midestrus. Sticky caseous or white-coloured thick consistency cervical mucus was observed during metestrus in 66.66% (n=14) estrous cycles along with ovulation or ovulation occurring within the range of -3 to + 4.5 - +7.5 h after the appearance of white mucus. Cervical mucus cytology revealed percentages of polymorphonuclear neutrophils were significantly higher in metestrus. E2 concentrations reached the peak on the day of estrus and P4 concentrations were lower than 0.5 ng/mL. It was concluded that cervical mucus shows a typical arborization pattern in midestrus and the appearance of characteristic sticky caseous cervical mucus during metestrus can be used as a tool to predict ovulation time.