Source: PURDUE UNIVERSITY submitted to NRP
PRECISION FEEDING OF AMINO ACIDS BASED ON PRE-PARTUM METABOLIC STATUS OF DAIRY CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027949
Grant No.
2022-67015-36317
Cumulative Award Amt.
$300,000.00
Proposal No.
2021-07098
Multistate No.
(N/A)
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2024
Grant Year
2022
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
College of Agriculture
Non Technical Summary
In the dairy cow, the transition from gestation to lactation is marked by extreme physiological and nutritional changes that require whole body coordinated adaptation. If an animal fails to adapt to these changes, it leads to reduced performance and increased risk of disease and removal from the herd. An approach to improving production and health outcomes is to assess the individual animal's requirements and provide the nutrients that are most required for success during the transition period based on the individual cow. The overall goal of this 2-year seed project is determine the effects of supplementing branch chain amino acids (BCAA) to dairy cattle prior to calving and evaluating the effects into lactation. Specific objectives include providing BCAA to dairy cattle prior to calving and assessing milk production, body composition changes, and metabolite and hormone concentrations. Secondly, protein analysis will be conducted from muscle samples collected from dairy cattle fed with BCAA. Lastly, animals will be followed for an entire lactation to observe when body composition changes occur throughout the lactation. The proposed research activities will combine nutritional, physiologic, and omics work to better understand the use of tissue to meet nutritional requirement during the transition period of dairy cattle. The potential impact will be to provide information on precision feeding of dairy cattle based on body composition. These results may change the way that we group, manage and feed dairy cattle based on the requirements of individual dairy cattle.
Animal Health Component
67%
Research Effort Categories
Basic
33%
Applied
67%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30234101010100%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Amino acidprofiles are different between muscle and milk proteins with milk having greater concentrations of branched-chain amino acids (BCAA). Supplementing cows with BCAA during early lactation reduces incidences of hyperketonemia, and may be related to findings that the BCAA leucine serves to stimulate pancreatic function. However, supplementing cows after initiationof lactation may still result in AA gaps that occur, thus a more efficacious approach would begin supplementation with BCAA in late gestation. Our preliminary studies support that the amount of muscle and adipose tissue in late gestation dictates tissue accretion pre-partum and tissue mobilization post-partum which are important considerations for animal production and health. We hypothesize that dairy cattle at different metabolic status prior to calving, as indicated by different relative amounts of muscle and adipose tissue, will respond differently to BCAA supplementation during the transition period. Moreover, we envision insulin sensitivity will differ between cows with high and low muscle reserves in late gestation, and will be differentially affected by BCAA supplementation. The overall goal of this 2-year seed project is to test this hypothesis and use untargeted approaches to reveal potential mechanisms underlying metabolic status and use of BCAA supplements by transition period cows.To meet this goal, we will:Objective 1: Determine the effects of supplementing BCAA during the pre-partum period on muscle and adipose tissue accretion and mobilization and insulin sensitivity and milk production in the post-partum in dairy cattle with high versus low longissimus muscle depth pre-partum. Working hypothesis - supplementing BCAA to high muscle cows will decrease accretion of adipose reserves, whereas supplementation of low muscle cows will result in accretionthe groups, as indicated by response to intravenous glucose tolerance test (IVGTT). We hypothesize that supplementing BCAA will reduce insulin resistance in early lactation with potential positive effects on health outcomes.Objective 2: Analyze if BCAA supplementation alters circulating AA and proteome profiles of muscle tissue in dairy cattle with high versus low longissimus muscle depth pre-partum. Working hypothesis - BCAA will alter circulating AA and muscle proteome, and the response willvary by metabolic phenotype as defined by muscle depth at 42 d pre-partum. Using an untargeted approach to measure metabolic pathways in muscle will enable the revelation of potential mechanism underlying the interactions between metabolic status and nutrient-AA use, which will be used to develop hypotheses for testing in future projects.Objective 3: Determine the timing and extent of tissue mobilization and accretion (muscle and adipose) throughout the lactation cycle in cows with high and low muscle depth prepartum. This information has the potential to have a direct application for nutritionists and dairyproducers. Knowing the patterns of tissue mobilization and accretion across a lactation cycle is important for the development of systems of precision feeding. The sample being followed with give us insight into the variation in the population for future studies and the relationship to physiological state and milk production levels.Precision feeding of AA during the pre-partum period to supply nutrients that will best allow dairy cattle to successfully transition into lactation without oversupply of nutrients aligns with USDA-NIFA priorities to improve the quality and efficiency of milk production while also minimizing metabolic disorders and nutritional deficiencies. Ultimately our goal is to improve animal health and production by quantifying metabolic status and supplying the nutrients that will have the largest impact on transition success. Precision feeding would prevent excessive adipose tissue deposition, which is known to predispose cattle to metabolic diseases such as hyperketonemia. Whereas, increasing skeletal muscle during the late gestation period may act as an AA store to supply AA and glucose precursors in early lactation resulting in increased production and reduced negative health outcomes.
Project Methods
Study design non-lactating primiparous or greater cattle (n=48), 42 d prior to expected calving animals will be assigned to 1 of 2 groups based on longissimus dorsi depth (LDD): Low muscle (LM; <4.2 cm; n=24) and high muscle (HM; ≥4.2 cm; n=24). Within in each muscle group, half (n=12) will be supplemented with BCAA in an approximate 2:1:1 ratio of Leu:Iso:Val supplying 50:25:25 g of metabolizable Leu:Iso:Val (adjusted for MP requirements for nonlactating cows) meeting Met and Lys requirements but with additional BCAA. The other half will be fed a control diet with 100% of MP meeting Met and Lys requirements. These diets will be fed for the remainder of the dry period and a common diet will be fed after parturition. Samples of all diet ingredients and orts from each cow will be collected daily and composited by period (pre or post-parturition) for analysis. Cows will be individually housed in tie-stall barns for the duration of the study 42 d prior to expected calving until 30 d in lactation, and daily feed intake measured. Weekly blood samples will be taken at 0600. The sample size (n=12 cows/group) was based on a power analysis using our metabolite data (3-MH and NEFA) to detect differences during the transition period (α > 0.80).Objective 1: Determine the effects of supplementing with BCAA during the pre-partum period on muscle and adipose tissue accretion and mobilization and insulin sensitivity and milk production in the post-partum in dairy cattle with high versus low longissimus muscle depth pre-partum. Muscle accretion and mobilization will be measured using ultrasounds measurements at the following times (-35, -21, -7, 0, 7, 14, 21, 30 d relative to calving). Ultrasound images will be recorded using ImageCapture and an Aloka 500 ultrasound machine. At the time of ultrasound measured body weight and body condition score (BCS) will be measured. Moreover, NEFA will be measured in plasma from weekly blood samples (commercial kit) as a relative measure of adipose mobilization. Plasma analysis of creatinine and 3-MH will provide insight to muscle mass and muscle turnover, respectively, and will measured in Purdue University's Metabolite Profiling facility using established methods. BHBA, glucose, and insulin will also be measured to assess relative metabolic-energy status using a glucometer and commercial kit respectively. Milk yield will be measured daily to 30 d in lactation and weekly milk samples will be collected at two consecutive milkings and analyzed for milk component analysis using DHIA. To assess the effect of tissue reserves and BCAA supplementation on insulin sensitivity, we will perform an IVGTT on d -14 and 7 DIM. We will utilize similar methods to an IVGTT performed by our group.Objective 2: Analyze if BCAA supplementation alters circulating amino acids and proteome profiles of muscle tissue in dairy cattle with high versus low longissimus muscle depth pre-partum. AA levels in weekly plasma samples will be measured in Purdue University's MetaboliteProfiling facility. AA profile of blood will indicate if supplementation of BCAA alters the free plasma AA in circulation, and whether there is a carry-over effect in the post-partum. Animals will be biopsied at -21 and 21 d relative to calving. Cattle will be restrained in a chute, hair clipped, and lidocaine HCl (20 mg/mL; 5 mL per biopsy) administered locally. Biopsies will be taken between the 9th and 13th rib on the left side utilizing a Bergstrom biopsy needle, and ~1 g of tissue obtained from the longissimus dorsi muscle. Tissue samples will be placed on liquid nitrogen then stored at -80?F until processing. For proteome analysis, proteins will be extracted from biopsied tissue and analyzed using shotgun LC-MS/MS in the Proteomics Core Facility at Purdue University's Bindley Bioscience Center. Digested protein samples (5 μL volume containing 1 μg peptides) will be subjected to liquid chromatography-tandem mass spectrometry data acquisition as published. The raw LCMS/MS data will be analyzed using MaxQuant software (v. 1.5.3.28)as published against a UniProt Bos taurus databases. Functional annotation analysis of proteins will be done using NIH Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8and Ingenuity Pathways Analysis. Dr. Theresa Casey has extensive experience in biological interpretation of high throughout omics dataand will lead this aspect of the work. ANOVA analysis will be followed by Fisher's LSD post hoc test. Significant differences betweendays will be considered at P-adjusted < 0.05. Hierarchical clustering will be performed based on Ward method. We anticipate running correlation analysis between physiological variables to include NEFA, insulin and glucose area under the curves in response to IVGTT, muscle and fat depth at study enrollment, and extent of loss of tissue over study to determine if there are underlying relying relationships to be explored for future studies launched by this seed grant.Objective 3: Determine the timing and extent of tissue mobilization and accretion (muscle and adipose) throughout the lactation cycle.We will perform ultrasound scans on the same 48 cows from objective 1 every 2 weeks for the duration of their lactation and non-lactating period to assess when during the lactation cycle animals begin to accrete tissue. Ultrasound scans will be used to quantify longissimus dorsi muscle and subcutaneous backfat thickness by taking 3 images per time point per cow and averaging those images using a cutoff of CV ≤ 10% between the images. We will use factors including feed efficiency in early lactation and milk production throughout lactation, to determine if relationships exist between timing and extent of tissue mobilization and accretion with production parameters. Data and Statistical Analysis. Differences between treatments will be determined using the MIXED procedure of SAS. Post-hoc comparisons will utilize a Bonferroni or Tukey correction. Data will be expressed as a least squares means ± SE and will be determined statisticallydifferent if P ≤ 0.05. Analysis of data collected for Objective 3 will be analyzed using a generalized linear model to assess relationships between tissue mobilization and production and health parameters.

Progress 01/01/22 to 12/31/24

Outputs
Target Audience:Our target audience for this research project is dairy nutritionists and consultants, dairy producers, and animal scientists. Changes/Problems:We changed from supplementing branched-chain amino acids to branched-chain volatile fatty acids due to the availability of the product. Other than this change, there were no major changes or problemsrelated to this project. What opportunities for training and professional development has the project provided?We have trained 3 graduate students, 8 undergraduate students, and 1 post-doctoral researcher. Students have presented their findings at multi-state meetings and the American Dairy Sciece Association Annual Meeting. How have the results been disseminated to communities of interest?Results have been disseminated through presentations at dairy science meetings, journal articles, popular press articles, and conference proceedings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We successfully executed a transition cow study where we supplemented branched-chain volatile fatty acids during the prepartum period. We assessed changes in skeletal muscle reserves prepartum into early lactation and the associated lactation responses. We assessed the effects of muscle reserves on calf body weight and body composition. 2. We performed muscle biopsies on pre and post-partum dairy cows and measured muscle fiber type and size. We performed transcriptomics on muscle samples to determine which RNA transcripts are altered by muscle reserves or branched-chain volatile fatty acid supplementation. 3. We assessed the changes in skeletal muscle reserves from calving until300 days in lactation to determine the timing and extent of skeletal muscle depletion and accretion. We published 4 manuscripts with 1 more under review. We have developed methods for measuring muscle fiber type and size frombovine skeletal muscle biopsies.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Gouveia, K. M., Beckett, L. M., Casey, T. M., & Boerman, J. P. (2024). Production responses of multiparous dairy cattle with differing prepartum muscle reserves and supplementation of branched-chain volatile fatty acids. Journal of Dairy Science.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Hanno, S. L., Casey, T. M., de Oliveira, H. R., & Boerman, J. P. (2024). Assessment of skeletal muscle dynamics and milk production across a 300-day lactation in multiparous dairy cattle. Journal of Dairy Science.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Casey, T. M., Gouveia, K. M., Beckett, L. M., Markworth, J. F., & Boerman, J. P. (2024). Molecular signatures of longissimus dorsi differ between dairy cattle based on prepartum muscle reserves and branched-chain volatile fatty acid supplementation. Physiological Genomics, 56(9), 597-608.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Gouveia, K. M., Beckett, L. M., Flinders, M. N., Casey, T. M., & Boerman, J. P. (2024). Prepartum Skeletal Muscle Reserves and Branched-Chain Volatile Fatty Acid Supplementation have Minimal Effects in Response to Intravenous Glucose Tolerance Tests in Periparturient Dairy Cattle. JDS Communications.
  • Type: Other Journal Articles Status: Under Review Year Published: 2024 Citation: Beckett, L., Gast, B., Tobolski, E., Jones, L., Gouveia, K., Han-Hallett, Y., Casey, T., and Boerman, J. (2024). Dam Prepartum Skeletal Muscle Reserves and Supplementation with Branched-Chain Volatile Fatty Acids During Late Gestation Influences Calf Birth Weight and Calf Muscle Metabolic Activity. JDS Communications.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Boerman, J. P. (2024). Timing and Extent of Skeletal Muscle Accretion and Depletion. Proceedings from the Cornell Nutrition Conference, Syracuse, NY. October 22-24, 2024.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:We have reached several target audiences including dairy scientiests through presentations at the American Dairy Science Association and dairy nutritionists through presentations at the Penn State Dairy Nutrition Conference in 2023. Additionally, we have reached thus far is graduate and undergraduate researchers.They have learned new techniques around animal husbandry and animal research procedures. Changes/Problems:We decided to perform RNA sequencing rather than proteomics in order to have a more complete idea of what was happening in the muscle. We have added muscle histology analysis to the paper to compliment what was described in the original proposal. We have added calf measurements and are working to deterimine where these results will fit. What opportunities for training and professional development has the project provided?2 graduate students, 1 post-doctoral researcher, and 1 undergraduate student were able to attend and present at the American Dairy Science Association Annual Meeting in 2023 due to this project. How have the results been disseminated to communities of interest?Results have been disseminated at the American Dairy Science Association Annual Meeting in 2023. What do you plan to do during the next reporting period to accomplish the goals?We have approximately a month of additional laboratory analysis and have a minimum of five manuscripts to finish preparation for and submit. This upcoming year will be focused on finalizing manuscripts and publishing results.

Impacts
What was accomplished under these goals? Objective 1: Determine the effects of supplementing BCVFAduring the pre-partum period on muscle and adipose tissue accretion and mobilization and insulin sensitivity and milk production in the post-partum in dairy cattle with high versus low longissimus dorsi muscle depth pre-partum. We have concluded the animal portion of this project and laboratory analysis in 2023 related to this objective. We are in the manuscript preparation phase where we have the following publications in preparation: 1. Effects of branched-chain volatile fatty acids and pre-partum muscle reserves on response to intravenous glucose tolerance tests pre- and postpartum - Short communication 2. Production responses to prepartum muscle reserves and branched-chain volatile fatty acid supplementation during the prepartum period 3. Tissue mobilization is impacted by muscle researves and branched-chain volatile fatty acid supplementation during the prepartum period Objective 2: Analyze if BCVFA supplementation alters circulating AA and proteome profiles of muscle tissue in dairy cattle with high versus low longissimus dorsi muscle depth pre-partum. We have concluded the RNAseq portion and analysis portion of this objective and are finalizing details on the following publication: 1. Differential experssion of genes related to prepartum muscle reserves and branched-chain volatile fatty acid supplementation in the prepartum period Objective 3: Determine the timing and extent of tissue mobilization and accretion (muscle and adipose) throughout the lactation cycle in cows with high and low muscle depth prepartum. We have concluded following these animals for 300 days into lactation and have to complete the laboratory analysis associated with those samples. The manuscript that will be written regrading this objective is: 1. Effect of muscle reserves of tissue mobilization and accretion throughout lactation

Publications

  • Type: Other Status: Published Year Published: 2023 Citation: Gouveia, K. M., Beckett, L. M., Markworth, J. F., Casey, T. M., and Boerman, J. P. 2023. Changes in body measurements, blood glucose and ?-hydroxybutyrate concentrations, and milk yield due to prepartum muscle reserves and branched-chain volatile fatty acid supplementation of transition dairy cattle. J. Dairy Sci. Vol. 106, Suppl. 1: 175.
  • Type: Other Status: Published Year Published: 2023 Citation: Gast, B. L., Beckett, L. M., Tobolski, E., Jones, L., Gouveia, K., Boerman, J. P., and Casey, T. M. 2023. Impact of feeding branched-chain volatile fatty acids during the dry period on colostrum composition and neonatal calf muscle metabolic activity. J. Dairy Sci. Vol. 106, Suppl. 1: 219.
  • Type: Other Status: Published Year Published: 2023 Citation: Gouveia, K. M., Beckett, L. M., Casey, T. M., and Boerman, J. P. 2023. Muscle reserves in the prepartum period impact the response to pre- and postpartum intravenous glucose tolerance tests. J. Dairy Sci. Vol. 106, Suppl. 1: 349.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:As we are still in the active animal feeding portion of the research trial and do not have results to share with the larger audience yet, the target audience we have reached thus far is graduate and undergraduate researchers. They have learned new techniques around animal husbandry and animal research procedures. Changes/Problems:Although funding was awarded in January 2022, I was unable to start a graduate student to work on this project until August, 2022. This has delayed the timeline but we are 60 days from being complete with the animal portion with all cows enrolled and some cows completed. We worked with a commercial company to source branched-chain volatile fatty acids (BCVFA) which can be converted to some degree to branched-chain amino acids (BCAA) in the rumen. I contacted 5 companies to see if they could provide BCAA and they all said that they were unable to at this point because of the cost, difficulty in protecting that product, and/or restrictions to assist with research projects at universities due to COVID. I made the choice to supplement BCVFA instead as they are converted to BCAA in the rumen, they may also have some beneficial effects on fiber digestibility in the dairy cow. Little work has been done with these BCVFA in non-lactating cows but we worked with a commercialcompany to discuss how to best administer and any additional analysis that may be beneficial. We have added additional analysis to the muscle biopsies to evaluate if the muscle fiber size and type changes from late gestation to early lactation. We believe this will tell a more complete story about what is happening to muscle during this transition period and will contribute to our understanding and may lead to future research projects. We have added analysis of the calves born from these cows as well to provide a more holistic view of supplementation during the late gestation period. What opportunities for training and professional development has the project provided?Both undergraduate students and graduate students were trained to perform muscle biopsies and glucose tolerance tests as well as perform daily checks on animal health and deviations from that animal's normal conditions. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will complete the animal portion of this project within the next 60 days. We will submit an abstract to the American Dairy Science Association in February 2023. We will start laboratory work on these samples in January of 2023 to be able to complete Objective 2. We will begin the routine sampling of cows throughout lactation to be able to complete Objective 3.

Impacts
What was accomplished under these goals? We have enrolled 48 non-lactating pregnant cows into a study approximately 42 days before they are expected to calve. Thus far, 10cows have completed the 72 days of the trial with 20 in the lactating phase and 18 in the non-lactating phase. Cows were measured at 42 days prior to expected calving and designated and high muscle or low muscle. They were then split into a control group or a group that was supplemented in rumen protected branch-chain volatile fatty acids that was fed during the non-lactating period. We have collected daily intake data, performed muscle biopsies on cows 21 days before and 21 days after calving. and have performed glucose tolerance tests to evaluate insulin sensitivity 14 days before and 7 days after calving. We have added additional analysis to the muscle biopsies to determine changes in muscle fiber size and type from prior to calving to post calving. We have added measurements of the calves that are born from these animals to determine if treatment or high muscle vs. low muscle impacts the calf. We are very much focusing on objective 1 at this time with the animal work scheduled to be complete in February 2023. At that point, we will complete the laboratory analysis for objective 2 and continue to measure these cows for muscle depth throughout their lactation for objective 3.

Publications