Progress 03/01/10 to 02/28/13
Outputs
Target Audience: The target audience for this research was the scientific community, particularly those studying non-ruminant and equine nutrition, and the feed industry. Eventually, once more research in this area has been accomplished, we will focus our efforts on increasing the knowledge of the individual horse owners regarding amino acid metabolism, but as this type of research is still in its early stages, we have focused on these two entities. In order to reach these audiences, we have submitted abstracts to two scientific meetings (2012 Joint Annual Meeting for ADSA-CSAS-AMPA-WSAS-ASAS; 2013 Experimental Biology), which had both scientists and feed industry representatives present. Additionally, I have been an invited speaker at meetings with a large number of feed industry representative present and have outline the methods used in this research and the value of the funded research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training activities: This research was a portion of the PhD thesis research for Sara Tanner. Throughout this project Sara was mentored about how to design and conduct animal research, including learning all of the sample collection techniques, how to perform laboratory and statistical analyses and how to write a scientific manuscript describing the findings from this research. Additionally, several other graduate and undergraduate students participated in this study and received some training regarding the sampling and laboratory analysis techniques. In particular, two undergraduate students, Lindsay Good (University of Kentucky) and Elizabeth DeLuca (Cazenovia College) completed internships in my laboratory and were instrumental in the animal care and sample collection portion of this study and they also participated in some of the early laboratory analyses (primarily the plasma amino acid analysis). One technician (Lyn Ennis) also received training in the sampling and analytical techniques throughout the course of this grant. Professional development: Both myself and the PhD student trained on this grant (Sara Tanner) attended numerous scientific meetings throughout the grant period for professional development purposes. Conferences attended include the 2011 Equine Science Society Annual meeting (Urschel and Tanner), 2012 Experimental Biology (Urschel), 2012 Joint Annual Meeting for ADSA-CSAS-AMPA-WSAS-ASAS (Urschel and Tanner), and 2013 Experimental Biology (Urschel and Tanner; the meeting occurred after the end of the grant period, however the abstract with data from this project was submitted prior to the end of the funded period). This collection of scientific meetings is diverse and ranges from nutrition-focused meetings (Experimental Biology) to equine-specific (Equine Science Society). How have the results been disseminated to communities of interest? We have prepared and presented abstracts (in both poster and oral presentation format) at two large national meetings, the 2012 Joint Annual Meeting for ADSA-CSAS-AMPA-WSAS-ASAS (catering to an animal agriculture research oriented crowd) and the 2013 Experimental Biology (catering to a more basic science crowd, but with specific sections for protein and amino acid research and animal nutrition research). Additionally, I was an invited speaker at the 2010 Joint Annual Meeting for ADSA-PSA-AMPA-CSAS-ASAS and at the 2011 Alltech Annual International Animal Health and Nutrition Symposium, where my talks focused on the use of my study methodologies to improve our understanding of equine amino acid requirements. The Alltech Symposium, in particular, had a diverse and international audience, including many representatives from the feed industry and some horse owners. Manuscript preparation is in progress to make the findings from this research more widely available. After additional research has been completed (on other related, funded studies), we will begin disseminating our findings to horse owners by working with the state extension program. What do you plan to do during the next reporting period to accomplish the goals? The grant period is complete and all objectives have been met. We will complete the manuscripts resulting from this research and submit to a peer-reviewed publication by the end of 2013.
Impacts
What was accomplished under these goals?
Major activities completed: We conducted a study in yearling Thoroughbred colts (~400 d old and 360 kg at the time of study), where each colt was studied while receiving each of six different levels of lysine intake: 76, 90, 104, 118, 127 and 136 mg/kg BW/d, based on the analyzed composition of the forage and concentrate portions of the diet. Each colt received each diet for a week, in a randomly determined order and was studied at the end of this week period. Sampling included the collection of pre- and post-feeding blood samples to measure plasma amino acid and urea concentration, post-feeding gluteus medius muscle biopsy collections to measure the activation of the signaling pathways that regulate muscle protein synthesis, and the measurement of phenylalanine oxidation and whole-body protein synthesis using stable isotope infusions. The sample collection period of this study was completed in the Summer of 2011, sample analysis was completed for the remainder of 2011 and in 2012, data entry and analysis was also completed in 2012. This research is part of the PhD thesis of a graduate student and therefore the final manuscript (and thesis chapter) is under preparation with the goal of submitting the resulting manuscript by the end of 2013. Specific objectives met: The research conducted addressed each of the 4 objectives outlined in the original grant submission. We worked to validate the indicator amino acid oxidation (IAAO) method in horses, we conducted a study with the goal of determining the dietary lysine requirement of yearling horses, we compared the IAAO measurements with the data concerning the activation of muscle protein synthesis signaling pathways and we looked at the effect of lysine intake on plasma amino acid and urea concentrations. Significant results achieved, including major findings, developments, or conclusions Objective 1- To further develop the IAAO method for use in horses: We successfully adapted our IAAO isotope infusion and sampling methods for use in these yearling horses. We were able to measure detectable amounts of 13CO2 in the breath during both [13C]sodium bicarbonate and [1-13C]phenylalanine infusions and stable plateau were reached by the half-way point of both infusions. Therefore, we concluded that this method could be applied to studying amino acid metabolism in growing horses. Objective 2- To measure the lysine requirement in horses using the IAAO methodology: According to the IAAO methodology, as lysine intake increases towards requirement, there should be an increase in whole-body protein synthesis and a decrease in phenylalanine oxidation. Once the lysine requirement is met, whole-body protein synthesis is maximized and phenylalanine oxidation will be low and both of these parameters will no longer change based on lysine intake. Therefore, the lysine requirement would be defined as the break-point between these two response lines. In this study, there was no effect of lysine intake (from 76 – 136 mg/kg BW/d) on phenylalanine oxidation (range of 5.0 to 7.1 umol/kg/h, pooled SE of 1.0 umol/kg/h; P = 0.69) and using statistical analysis, a breakpoint could not be determined. Therefore, the IAAO applied in this particular study could not be used to determine a lysine requirement in yearling horses. Possible reasons for the inability to measure the lysine requirement are discussed in the ‘Key outcomes’ section and are not believed to be related to a problem with the IAAO methodology, but rather the diet formulation and the lack of knowledge of equine amino acid requirements. Objective 3- To compare the effects of lysine intake on rates of whole-body protein synthesis and the activation of muscle protein synthesis: Phenylalanine use for whole-body protein synthesis was not affected by lysine intake (40.5 – 45.7 umol/kg/h, pooled SE of 2.5 umol/kg/h; P = 0.72). The measured rates of whole-body protein synthesis are lower than what we have previously measured in weanling horses, but slightly higher than what we have previously measured in mature horses, confirming that rates of protein synthesis decrease as growth rate decreases. We also measured the activation of many of the signaling proteins in the mTOR pathway (pathway that stimulates muscle protein synthesis) in the gluteus medius biopsy samples. We were not able to measure any differences in activation of any of the signaling factors studied (rpS6: P = 0.42; 4E-BP1: P = 0.21; Akt: P = 0.88), which is consistent with the lack of measured changes in whole-body protein synthesis. Objective 4- To determine the effects of increasing lysine intake on plasma amino acid and urea concentrations: As lysine intake increased, there was a linear increase in plasma lysine concentrations post-feeding (R2 = 0.65; P < 0.0001 ) and a linear decrease in plasma glycine concentrations (R2 = 0.42; P < 0.0001), reflecting the different intakes of these two amino acids (as lysine content in the diet increased, glycine content was reduced to ensure isonitrogenous diets). There was no effect of lysine intake (P = 0.70) on either pre-feeding (3.1 – 3.9 mmol/L, pooled SE = 1.2 mmol/L) or post-feeding (11.4 – 13.0 mmol/L; pooled SE = 1.2 mmol/L) plasma urea concentrations, although for all treatments, the post-feeding plasma urea concentration was significantly greater than the pre-feeding concentration (P < 0.0001). Previous studies in humans and pigs have shown a ‘break-point’ in both the plasma lysine and the plasma urea nitrogen response to increasing dietary lysine concentrations, but a breakpoint in the response of either of these variables could not be determined in the present study. The possible reasons for that are described in the following section. Key outcomes or other accomplishments realized The key outcome from this study was the realization that there is still much to learn about amino acid requirements in horses. Unfortunately, the lysine requirement could not be concretely determined in this study using the IAAO methodology or from the plasma metabolite (lysine, urea) concentrations. There are one of two plausible explanations for the lack of measureable requirement: 1) the lysine requirement was below the lowest level of lysine intake fed in this study or 2) another dietary amino acid was even more limiting in the diet and so the addition of additional lysine to the diet had no effect on protein synthesis as the rate was limited by another amino acid. The lowest level of lysine intake provided in the current study was substantially lower than the NRC estimate of the lysine requirement (113 mg/kg BW/d) and represented the lowest level of lysine intake that could be formulated, while still meeting energy and total crude protein requirements. Given that lysine is reasonably well documented as a limiting amino acid in growing horses, it does not seem likely that the 76 mg/kg BW/d provided by the lowest lysine diet was already above the lysine requirement. It seems more likely that the diet was limiting in an amino acid other than lysine. Although we ensured that our diets were formulated to meet the requirements that can be estimated using their ratio to lysine in the skeletal muscle, the estimated threonine requirement (~68 mg/kg BW/d) was only just met, and based on research in other species, the skeletal muscle profile may not be the best amino acid profile to use for estimating threonine requirements, because of the extensive gut metabolism of threonine. Threonine has also been reported as a potentially limiting amino acid in other studies in horses and we have already secured USDA NIFA AFRI funding to measure threonine requirements in both growing and mature horses to address the findings from this current study (project # 2012-67015-19448).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Tanner SL, Good LR, DeLuca EA, Coleman RJ and Urschel KL. Feeding graded amounts of lysine to yearling thoroughbred colts does not activate the mTOR signaling pathway. 2012 ADSA-CSAS-AMPA-WSAS-ASAS Joint Annual Meeting, Phoenix AZ, July 15-19, 2012. J Anim Sci 90(Suppl. 3): Abstract #364.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Tanner S, Good L, DeLuca E, Coleman R and Urschel K. Free amino acid content of equine muscle changes in response to graded lysine intake. Experimental Biology 2013, Boston MA, April 20 24, 2013. FASEB J 27: Abstract #631.18.
- Type:
Other
Status:
Accepted
Year Published:
2011
Citation:
Invited presentation: May 24, 2011: "Lessons from human nutrition: how isotope methods are helping to define protein requirements"; Alltechs 27th Annual International Animal Health and Nutrition Symposium, Lexington KY, United States.
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: The sample collection portion of this research was conducted from May - July 2011. Each of the six horses were studied on all six levels of lysine intake and there were no unexpected problems during the sample collection phase. All laboratory analyses have been completed, with the exception of the determination of the isotope enrichment of the collected blood samples. Sample and data analysis will be completed by the end of 2012, and we expect a manuscript to be prepared for submission by mid-2013. One graduate student (Sara Tanner) and two undergraduate interns (Elizabeth DeLuca and Lindsay Good) were trained in various research methods including muscle biopsy collection, jugular vein catheterization, blood and breath sample collection, stable isotope infusion procedures, HPLC analysis, spectrophotometric assays and Western immunoblot procedures. Results from this research will be presented at the American Society of Animal Science and Equine Science Society annual meetings. PARTICIPANTS: Individuals: Kristine Urschel (PI, University of Kentucky), Sara Tanner (PhD student, University of Kentucky), Ashley Wagner (PhD student, University of Kentucky), Rosalyn Ennis (Lab Technician Senior, University of Kentucky), Lindsay Good (Undergraduate Intern, University of Kentucky), Elizabeth DeLuca (Undergraduate Intern, Cazenovia College), Rose Digianantonio (Undergraduate student, University of Kentucky). Students and technicians that assisted with this project learned skills such as jugular vein catheterization, muscle biopsy collection, isotope infusion techniques, blood and breath sample collection procedures, HPLC analysis and Western blot procedures. Sara Tanner will present the results from this study at scientific meetings in 2012 and 2013. TARGET AUDIENCES: Our target audience is the scientific community and the feed industry. Dissemination to the scientific audience will be achieved through the publication of abstracts and peer-reviewed papers and through presentations at conferences. It is expected that individuals in the feed industry will receive our results through attendance at meetings related to animal nutrition (Animal Science and Equine Science meetings) and through personal communications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Until we have completed our sample analysis, it is difficult to comment on the outcomes/impacts of this study. From our plasma amino acid analysis, we can confirm that the formulated diets resulted in changes in only plasma lysine (and glycine- the amino acid that was adjusted along with lysine to keep the diets isonitrogenous) concentrations, so any changes that we observe in indicator amino acid oxidation will be attributable to differences in lysine intake. We have analyzed muscle samples for the activation of factors in the signaling pathways that regulate protein synthesis (the mTOR pathway) and did not find any effect of lysine intake on these pathways, which indicates that lysine is not an independent activator of mTOR signaling when diets are isonitrogenous. It is expected that once we have determined a lysine requirement for the yearling horses used in this research, that this information will be used for improving diet formulation in growing horses. Lysine is the first limiting amino acid in the diet of growing horses and so having a measured value (rather than an estimated value) for its requirement will be a valuable addition to the field of equine nutrition.
Publications
- Tanner SL, Good LR, DeLuca EA, Coleman RJ and Urschel KL. 2012. Feeding graded amounts of lysine to yearling thoroughbred colts does not activate the mTOR signaling pathway. ADSA-AMPA-ASAS-CSAS-WSASAS Joint Annual Meeting, abstract accepted March 12, 2012 (accepted).
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Progress 03/01/10 to 02/28/11
Outputs
OUTPUTS: Activities: The sample collection portion of this research is scheduled to begin in May 2011. At this time, the diets have been formulated to allow for a range of lysine intakes of 80 - 145 mg/kg BW/day and we have worked with horses to adapt them to isotope infusion and breath sampling procedures. Sample collection will terminate in July 2011, and samples will be analyzed from August to December 2011. At that point in time, there will more tangible outputs (results and evidence of dissemination) to report. There is one PhD student, Sara Tanner, that is working on this project and has already learned about the principles of the methodology and worked to formulate the diets. Additionally, 2 undergraduate students will also be involved in this study. There are no events, services or product outputs to report at this time. PARTICIPANTS: Principle investigator: Dr. Kristine L. Urschel (University of Kentucky) Graduate students: Sara L. Tanner (part of her PhD research; University of Kentucky), Ashley L. Wagner (PhD student assisting with research; University of Kentucky) Technicians: Rosalyn Ennis (University of Kentucky) Undergraduate students: Lindsay Good (University of Kentucky), Elizabeth DeLuca (Cazenovia College) TARGET AUDIENCES: Because this research will determine the lysine requirement of yearling horses, the results of the research efforts will be relevant to any individual responsible for feeding horses of this age. Additionally, this research will provide valuable information to those responsible for formulating diets for horses or developing concentrate rations. The validation of the indicator amino acid oxidation will be of interest to other individuals in the area of Equine Nutrition, because it will provide a research tool for measuring additional amino acid requirements in the future. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Because we are just beginning the sample collection process, there are no impacts to report at this time. We do anticipate that the validation of the indicator amino acid oxidation methodology in horses will provide us with a valuable research tool to advance our knowledge of amino acid requirements in horses. Knowledge of the lysine requirements in yearlings will improve diet formulation for this age of horses.
Publications
- No publications reported this period
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