Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.