Date of Award


Document Type


Degree Name

Master of Science (MS)


Animal Science

First Advisor

Sabrina L. Greenwood


Milk is a highly nutritious natural product and research over the last 10 years has proven that these milk proteins not only provide a rich source of amino acids to the consumer but also contains many bioactive proteins and peptides known to exert biological activity benefitting human health. In this research, proteomic methods were first used to characterize the low abundance proteome within the skim milk fraction produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions. Milk samples were collected over a seven day period from six Holstein and six Jersey dairy cows. Samples were depleted of casein (CN) by acidification and ultracentrifugation followed by ProteoMiner treatment. Extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation followed by liquid chromatography mass spectrometry (LC-MS). Over 930 low abundance proteins were identified and label-free proteomic analysis allowed for semi-quantification of proteins. Gene ontology (GO) classified proteins into various cellular localization and function categories. Forty-three low abundance proteins were differentially expressed between the two dairy breeds. Some bioactive proteins with immunomodulatory activities were present at significantly different abundance between breeds such as lactotransferrin (P <0.01) and Complement C2 (P <0.01), whereas others like osteopontin (P = 0.17) and lactoperoxidase (P = 0.29) were present at similar levels. This work has identified the highest number of low abundance proteins within the whey fraction in bovine skim milk, providing a foundation for future research exploring the bovine milk proteome.

Nutrition is a significant animal factor that has potential to alter milk protein composition. Therefore in the second phase of this work, nutritional perturbances were used to alter the bovine milk proteome by feeding Holstein dairy cows different proportions of rumen degradable (RDP) and rumen undegradable protein (RUP) to alter whole-body nitrogen (N) metabolism. Six multiparous Holstein cows in mid-lactation were randomly assigned to one of two treatment groups. The experiment was conducted as a double-crossover design consisting of three 21-day periods. Within each period, treatment groups received diets with either 1) a high RDP:RUP ratio (control: 62.4:37.6 % of CP) or 2) a low RDP:RUP ratio (RUP: 51.3:48.7 % of CP). Both diets were isonitrogenous (CP = 18.5%) and isoenergetic (NEL = 0.8 Mcal lbs-1). Feeding a diet high in RUP decreased β-casein (P = 0.06), κ-casein (P =0.04) and total milk casein concentrations in milk (P <0.001). Milk urea nitrogen (MUN) and plasma urea nitrogen (PUN) were significantly higher in the RDP group (P = 0.04; P < 0.01, respectively). Over 590 low abundance proteins were identified and only three proteins were found to be differentially expressed between the two dietary groups. The high dietary crude protein (CP) inclusion may explain the lack of treatment effect since protein synthesis within the mammary gland (MG) may not be responsive to dietary changes when total CP levels is offered in excess. Additional feeding trials are needed to alter N utilization patterns within a dairy cow while maintaining isonitrogenous and isoenergetic diets and offering normal CP levels. Nutritional perturbances offer opportunities to selectively alter the bovine proteome, providing a tool to enhance the healthfulness of milk.



Number of Pages

169 p.