NASEM (2021) recently made strides in characterizing effects of 5 individual EAA on milk protein production. However, there are 15 other AA that are incorporated into milk protein, and as such, these AA likely also play significant roles in driving milk protein synthesis, but lack of data prevents their incorporation into current models. A greater supply of AA to the mammary glands does not always mirror AA absorption—the process by which absorbed AA convert into milk protein is variable, and this may be linked to the way the udder regulates AA uptake to preserve intracellular balance. AA transporters housed within the cellular membranes of mammary epithelial cells (MEC), the mammary glands' constituents, are responsible for mediating this intracellular balance. Thus, the objectives of this dissertation were to investigate how AA transport is affected by various AA concentrations using both in vitro and in vivo approaches. In study 1, we evaluated effects of valine and a group of NEAA (AQG; Ala, Gln, and Gly) on exchange transport rates of AA in bovine MEC. High AQG concentrations stimulated Leu, Phe, and Val influx rate parameters, demonstrating that AQG likely increased transport activity for these substrates through exchange transporters. Additionally, high Val concentrations decreased Ile and Leu net uptakes, which occurred via efflux stimulation and transamination downregulation. In study 2, we aimed to identify the effects of 10 EAA and 2 Tyr (CDENSPY) on transport rates and transporter regulation (mRNA expression and protein abundance). Within the physiological AA concentrations used, we were able to measure differential effects of AA on each AA transporter. For example, His stimulated SLC38A2 and SLC38A2 mRNA expression at a decreasing rate; the apex for this curve was reached at a concentration very close to mean plasma concentrations in lactating dairy cows. Therefore, we determined that these transporters may be transcriptionally regulated to regulate intracellular His concentrations. Additionally, all EAA and NEAA groups were involved in significant 2-way interactions on transporter expression and activity. Furthermore, we measured transport rates and rate constants (free of mass influence) of 12 AA to determine important AA on influx, efflux, transamination, irreversible loss, and protein synthesis. We demonstrated competitive inhibition among several AA that share transport systems such as between BCAA. Furthermore, we again demonstrated that NEAA can stimulate transport activity for AA involved in exchange transport. In study 3, we investigated the effects of jugular Lys, Ile, Val, or AQG infusion on mammary AA metabolism and production in lactating dairy cows. Interestingly, Val decreased DMI and milk protein production along with net uptakes of several AA, while the remaining treatments had little metabolic effects. In study 4, we demonstrated that both high protein and starch concentrations independently stimulated milk protein production, but glucose precursor partitioning (lactate, propionate and other) was only affected by starch. In conclusion, we anticipate that nutrition models estimating milk protein production will eventually incorporate up to 20 AA and multiple 2-way interactions; additionally, extremely high concentrations of AA should be prevented to combat negative impacts caused by AA imbalances. However, much more work is required to take steps in this direction. / Doctor of Philosophy / Overfed protein can pose a significant health and environmental risk. Unabsorbed amino acids (AA), the building blocks of protein, are released by dairy cattle into the environment as various nitrogen products. One specific risk is excess runoff of nitrates from dairy farms into nearby water bodies. This contamination can result in serious water quality issues, including eutrophication, which depletes O2 levels in aquatic ecosystems with algae overgrowth, causing dead zones where aquatic life cannot survive. Furthermore, high nitrate levels in drinking water can decreased oxygen availability in humans, in which pregnant women and babies are the most at risk. Finally, the volatilization of N compounds also contributes to air pollution and the formation of greenhouse gases like nitrous oxide, a potent climate-altering compound with global warming potential. Theoretically, feeding an AA profile to precisely match dairy cow requirements would minimize these losses. However, the udder does not take up all available AA. Thus, this research aimed to better understand different AA profiles on AA transport, the route in which AA are taken up by the milk-producing cells in the cow's udder, to ultimately increase efficiency of milk production. In our first study, we demonstrated that non-essential AA (Ala, Gln, and Gly), which can be synthesized in the mammary glands, stimulated Leu, Phe, and Val transport activity within mammary epithelial cells, which could mean that supplemental non-essential AA could increase essential AA transport efficiency. Interestingly, high Val had a negative effect on net uptake (entry minus exit) of Leu and Ile. The second study sought to understand how varying concentrations, within ranges observed in the blood of lactating dairy cows, of 10 different essential AA and 2 non-essential AA groups affected AA transporter expression and activity. We observed greater protein presence and mRNA expression levels of several transporters in response to low availability of their AA transport substrates. Additionally, some AA were involved in stimulating transporter expression and activity when present at high concentrations, such as Leu. There was a plethora of 2-way interactions among AA on transporter protein quantity, mRNA expression, and activity that indicated that the relationship between certain AA will need to be incorporated into future nutrition models. In our third study, we observed that high Val supply decreased the amount that cows ate as well as their milk protein production. This demonstrated that excessive concentrations of certain AA may negatively affect cow metabolism. Lastly, we wanted to investigate the relationship between protein and glucose production in dairy cows, as energy availability is another driver of milk protein production. Our fourth study revealed that dietary protein and starch independently increased milk protein production, yet only starch affected glucose formation. Our findings urge caution against excessive AA concentrations in diets, as imbalances can have negative effects. Overall, we have demonstrated that AA transporters are differentially affected by changes in individual AA supply and various 2-way interactions. This work unveiled the almost unlimited AA interactions that must be further explored to better integrate this knowledge into practical dietary formulations for dairy cows.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/121017 |
| Date | 26 August 2024 |
| Creators | Weston, Alexis Hruby |
| Contributors | Dairy Science, Hanigan, Mark Daniel, Daniels, Kristy Marie, Molina De Almeida Teixeira, Izabelle Auxiliadora, El-Kadi, Samer Wassim |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/x-zip-compressed |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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