Improving our understanding of milk protein production regulation and AA transport is important for successfully formulating diets for AA and improving N efficiency. The objectives were to study protein synthesis regulation and AA transport using in vitro and in vivo models. In the first experiment, the objective was to evaluate the ability of five distinct AA profiles and balancing Lys to Met ratio to 3:1 to stimulate protein translation. No single AA profile uniquely stimulated phosphorylation of translational machinery related proteins suggesting identification of a single optimal AA profile as unlikely. In the second experiment, an in vitro method using three different AA isotopes was developed to trace AA movement. The method assesses bi-directional transport of multiple AA simultaneously enabling evaluation of unidirectional uptake kinetics. This method was used to evaluate AA concentrations representing 16, 100, 186, and 271% of cow plasma AA concentrations. Amino acid uptake was not saturable within the in vivo range for eleven AA. Arginine, Val, and Pro exhibited saturation with the Michaelis-Menten km being 95, 49, and 65% of in vivo concentrations. Results suggest that AA transport is generally non-saturable and that high bi-directional transport exists which enables a mechanism for mitigating AA shortages. In experiment 3, the objective was to evaluate milk protein production and regulation from infusing Met, Lys, and His (MKH) or Ile and Leu (IL). The two EAA groups independently and additively increased milk protein yield. This finding contradicts the single limiting AA theory that a single nutrient will limit milk protein yield. Changes in udder AA extraction and blood flow from supplemental EAA reveal flexible delivery mechanisms. The phosphorylation state of proteins associated with the mTOR pathway was impacted by both EAA treatments. Changes in the udder proteome suggest negative feedback on mTOR pathway activation when milk protein yield was increased by the EAA groups separately but when supplemented together, negative feedback was lessened. Results indicate that multiple EAA can stimulate milk protein production, the ability of AA transport to match intracellular needs, and that the single limiting AA theory or existence of a unique optimal AA profile is likely irrelevant in dairy cows. / Doctor of Philosophy / Post absorptive metabolism of dietary protein and conversion to milk protein is not well described in dairy nutrition models resulting in poor predictions of response to changing protein supply. This partially constrains diet formulation with respect to successfully balancing diets for protein and amino acids or for improving N efficiency. The efficiency of absorbed AA into milk protein varies and udder AA uptake may contribute to this varying efficiency through transport regulation in an attempt to maintain intracellular AA homeostasis. Amino acid transport was assessed when AA supplies were varied below and above in vivo supplies. High bi-directional AA transport was saturating uptake for Arg, Pro, and Val within the normal in vivo range for lactating dairy cows. The high AA exchange suggest strong ability to manage changes in AA supply to meet needs for milk protein translation. When intracellular AA supply declines, efflux of the limiting AA out of the cell declines which results in greater uptake of the limiting AA by the cell. The theory that milk protein yield is limited by a single most limiting amino acid (e.g., the barrel and stave analogy) and that a single optimal EAA profile exists predominates in the field of animal nutrition, but implementation of this theory has not greatly improved N efficiency or been adopted widely. We observed that various AA profiles can equally stimulate milk protein translational machinery in mammary epithelial cells and that balancing Lys to Met ratio to 3 to 1 only had a minor effect. Multiple EAA can regulate milk protein production through signaling to synthesis machinery and delivery of AA to the udder. Supplementation of two iv groups of EAA, 1) methionine, lysine, and histidine and 2) isoleucine and leucine, independently and additively increased milk protein yield in dairy cows. These increases were achieved by changes in blood flow in the udder, AA uptake, and nutrient signaling related to protein translation regulation. Hence, results of this dissertation tend to not support the idea of a single limiting amino acid or a unique optimal profile of AA for milk protein production in dairy cattle.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/100897 |
| Date | 28 May 2019 |
| Creators | Yoder, Peter Samuel |
| Contributors | Dairy Science, Hanigan, Mark D., Ferreira, Gonzalo, El-Kadi, Samer Wassim, Ruiz Cortes, Zulma Tatiana, Cant, John |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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