Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The primary audience served by the project are animal scientists specializing in dairy production animals with a focus on mammary metabolism. Changes/Problems:We encountered an experimental design problem with the Obj. 1 experiment. The central composite design package for R is flawed for designs using partial factorials resulting in aliased treatments. This problem was discovered during data analyses after completing the experiment, and thus many of the 2-way interactions we thought could be uniquely defined were not uniqe. This results in observations for some interactions that identify 1 or more pairs that have an interaction, but we are unable to determine which one of the group actually has the interaction. Thus the scope of the work was restricted to all linear and quadratic effects, some unique 2-way interactions, and a number of groups of interaction terms. We have also observed that curation of data from the mass spectrometers is requiring much more time that was budgeted and is greatly lagging analyses. Half of a technician FTE has been assigned to the task, but it will require considerable effort to remove the backlog. What opportunities for training and professional development has the project provided?The project provided additional training opportunities for 5 undergraduate students, and training and professional development opportunities for a graduate student. Undergraduate training included advanced use of mass spectrometry to analyze isotopic tracer data, and assessment of mRNA expression by quantitative PCR. Graduate training included mathematical analyses of isotopic tracer data, quantitative PCR analyses, and writing technical papers. How have the results been disseminated to communities of interest?1 manuscript describing our preliminary work has been published in a peer-reviewed journal and a 2nd describing a portion of Obj. 2 is under review. What do you plan to do during the next reporting period to accomplish the goals?Identification and rectification of outliers will be completed for Obj. 2. The modeling analyses of the isotope data will be refined using the rectified data, and the statistical analyses updated. The outstanding sample analyses in support of Obj. 3, and the mathematical and statistical evaluations of those data will be completed during the next reporting period. A review paper and 2 additional manuscripts describing our work will be submitted for publication.
Impacts
What was accomplished under these goals?
Obj. 1: The experimental work was successfully completed. The assessments of individual transporter mRNA and protein concentrations and several other genes of interest including the milk proteins has been completed. The statistical analyses of these data are complete and an oversized manuscript has been developed. The manuscript will be split into 2 or 3 parts for publication. As hypothesized, the concentrations of mRNA for most of the transporters responded linearly and some quadratically to varying media amino acid concentrations. all of the individual amino acids across the full range of transporters, but the list of those affecting expression of indivual transporters was less. Histidine, leucine, and lysine were most often associated with changes in expression, but the responses to each were not always positive. mRNA and proteins of the signaling pathways regulating mRNA translation were also impacted by some amino acids. 1 oversized manuscript describing the mRNA and protein responses has been drafted. We are working to break this into 2 or 3 manuscripts, and to more fully consider the patterns of mRNA and protein expression responses to amino acids so that commonalities can be identified. The intial analyses of the flux measurements from the experiment has been completed. However, we subsequently discovered that column drift over time resulted in some peak identification errors that were not detected by the student. We are currently working to correct those identification errors, and when complete we will need to repeat the model analyses required to calculated fluxes and rerun the stats. Obj. 2: the experimental work and analytical work have been completed. Much of the statistical analyses were completed. A manuscript describing the net flux measurements across the mammary gland was completed and is under review at the Journal of Dairy Science. We did not observe the expected negative effects of increased isoleucine on leucine and valine net flux, but there was a trend for a negative effect of valine on isoleucine and leucine net flux as we observed in the prior in vitro trial. Also we did not observe any positive effects of the 3 dispensible amino acids on isoleucine, leucine, or valine net flux. We will gain a more complete understanding of these observations when we have completed the modeling of the isotope data which yields bi-directional flux estimates. Obj. 3: Nothing to report.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Hruby Weston, A., I. Teixeira, P. S. Yoder, T. Pilonero and M. D. Hanigan. 2024. Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells. J Dairy Sci 107(4):2026-2046. https://doi.org/10.3168/jds.2023-23447
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience includes other scientists specializing in nutrient metabolism. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training opportunities for 1 graduate student and 20 undergraduate students. Training included mammalian cell culture, animal production, animal catheterization and infusions,sample collection and preparation, and data organization and entry. Additionally the graduate student gained expertise in experimental design, management of trials, mass spectrometry, kinetic modeling, and statistical analyses of experimental data. How have the results been disseminated to communities of interest?Presentations on the work were made to members of the research community at the tri-annual international meeting on Modeling Nutritient Metabolismand at the annual American Dairy Science Association meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the sampleand data analyses work for Objectives 1 and 2, and to complete a portion of the modeling work for Obj. 3. A manuscript describing the preliminary work is currently under review and should be published in the coming year. We also plan to publish a review of mammary amino acid transport, a manuscript describing the transporter mRNA and protein responses to individual amino acids under Objective 2, and a manuscript describing the transport kinetics for the same experiment.
Impacts
What was accomplished under these goals?
Nothing to report.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hruby-Weston, A., M. Morozyuk, T. Pilonero, and M. D. Hanigan. 2023. Effects of individual and additive amino acids on intracellular concentrations in bovine mammary epithelial cells. J. Dairy Sci. 106: (Suppl. 1). (Abstr.)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Hruby, A.C., I. Geisner, M. Morozyuk, and M.D. Hanigan. 2022. Assessing interactions of essential amino acids and two groups of non-essential amino acids on transporter expression in bovine mammary epithelial cells. International Symposium on Energy and Protein Metabolism and Nutrition. Animal
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