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.

Quantitative proteomics profiling of spermatozoa and seminal plasma reveals proteins associated with semen quality in Bos indicus bulls.

Cattle breeding approaches are an evolving field of research in veterinary science. Certain factors such as Ejaculate Rejection Rate (ERR) pose a limitation to such approaches. In this regard, we sought to investigate the spermatozoa and seminal plasma proteome of Hallikar bulls with low (n = 3) and high (n = 3) ERR. Through the Tandem mass spectrometry approach, we identified a total of 2409 proteins, in which 828 proteins were common in both the semen components, whereas 375 and 378 proteins were unique to spermatozoa and seminal plasma respectively. Tandem mass tags (TMT) based protein quantification resulted in 75 spermatozoal, and 42 seminal plasma proteins being differentially regulated between high and low ERR bulls. Proteins such as SPADH2, TIMP-2, and PLA2G7 which are negative regulators of motility were upregulated in the seminal plasma of high ERR bulls. Proteins such as OAZ3, GPx4, and GSTM3 whose upregulation leads to reduced motility were upregulated in the spermatozoa of high ERR bulls. Caltrin and ADM proteins that enhance sperm motility were downregulated in the seminal plasma of high ERR bulls. The regulation of ACE, a negative regulator of sperm motility was upregulated in both the spermatozoa and seminal plasma of high ERR bulls. SIGNIFICANCE: The saying "Bull is more than half of the herd" signifies the importance of bull in the genetic improvement of the herd. Traditionally used semen quality tests will provide limited information about the potential fertility of bulls. The proteomics approach is a promising omics technology to understand the factors involved in male fertility. The present study identified the spermatozoal and seminal plasma proteins that are differentially regulated between high and low ERR bulls. Sperm motility-associated proteins are differentially regulated. This study if improved further, can be used to develop markers associated with semen quality which is useful for the selection of bulls.