Bovine mastitis is currently one of the most challenging and profit limiting problems in lactating dairy cows. There is a higher incidence of suffering from repeated mastitis when teat plugs take longer to form. This is because as keratin forms in the teat canal, it acts as a natural physical barrier, which is able to prevent pathogens from entering the udder. If the cow takes longer to form this plug, more bacteria are able to enter the canal. Another barrier for protection are mucins, which aid in the protection of the epithelial cells. They also assist with epithelial renewal and differentiation and can be found in areas such as the intestines, legs and teats. In cows, there are 2 mucins in the mammary gland (MUC1, MUC15), which act as a barrier to infections. The study of milk transcriptome from healthy and mastitic Holstein cows using RNA-Seq, can provide precise measurements of transcript levels and their isoforms, as well as identify functional structural variants (i.e., SNP, indels and splice variants) associated with immunity response to infection. This will aid in understanding the development of disease. Transcriptome analysis using RNA-Seq was performed in milk somatic cells (SC) from healthy (n = 4) and mastitic (n = 4) cows. Differentially expressed (DE) genes and isoforms involved in the metabolic pathways associated with mucins development and plug formation were identified. A total of 3,566 genes were DE between milk from healthy and mastitic cows (SC). Some of these genes impact the immune response of the bovine (IL18, IL10, MUC20, PCNA), while others effect plug formation (MUC1, MUC15) and epithelial renewal and differentiation (MUC4). The prevalence of some bacteria also depended on the health status of the milk. Further analysis will be performed to study the microbiome by 16S ribosomal sequencing. In conclusion, the identification of genes and biomarkers associated with mucins and teat plug formation will aid in improving the sustainability of agricultural practices, by facilitating the selection of cows with improved immune systems and resistance to infection.