ELUCIDATION OF THE MOLECULAR MECHANISM THROUGH WHICH ESTRADIOL AND TRENBOLONE ACETATE IMPROVE SKELETAL MUSCLE GROWTH IN BEEF CATTLE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1014963
• Grant No.: 2018-67016-27498
• Cumulative Award Amt.: $490,000.00
• Proposal No.: 2017-05892
• Project Start Date: Feb 1, 2018
• Project End Date: Jan 31, 2023
• Grant Year: 2018
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322

Performing Department
Animal Dairy & Veterinary Scie

Non Technical Summary
As the world population increases and the amount of land available for agricultural use decreases, it is necessary that more efficient production practices are used in order to conserve our available resources. Nearly all commercially produced cattle in the United States receive at least one anabolic implant during production that results in a 5-30% increase in production efficiency. Anabolic implants are steroid hormones, naturally occurring molecules that are found in trace amounts within all animal products, as well as in some plant products, such as soybeans. Although implants are routinely administered to feedlot cattle, the cellular mechanism through which they improve production efficiency is not fully understood. As such, the goal of this research is to gain a better understanding of how steroid hormones improve growth of skeletal muscle, in order to develop consumer accepted strategies that can be used to further improve production and efficiency of beef cattle production. Specifically, the research team comprised of researchers from Utah State University and Purdue University, will analyze how steroid hormones impact expression of genes and proteins using both a cell culture model as well as a live animal model. The results of this research will provide new knowledge regarding the specific cellular mechanism through which steroid hormones improve skeletal muscle growth. This knowledge will be used to develop consumer accepted strategies to improve production and efficiency of cattle production.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
308	3310	1020	100%

Knowledge Area
308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1020 - Physiology;

Keywords

beef cattle

estradiol

proteome

satellite cell

skeletal muscle

transcriptome

trenbolone acetate

Goals / Objectives
Our long term goal is to identify the molecular signaling pathways responsible for growth of skeletal muscle, in order to develop consumer accepted strategies to improve production and efficiency of cattle production. The objective of this research is to determine the molecular mechanism through which estradiol (E2) and trenbolone acetate (TBA) promote skeletal muscle growth. Our central hypothesis is that E2 and TBA each stimulate satellite cell (SC) proliferation and protein synthesis rates through interacting genomic and non-genomic mechanisms in both in vitro SC cultures and in vivo skeletal muscle. The rationale that underlies this research is that identification of the mechanism through which E2 and TBA improve skeletal muscle growth, will allow for development of tools that can be used to increase cattle productivity, which, in turn, will translate into increased economic profitability as well as increased sustainability. We will test our hypothesis and thereby, attain the objective of this research by pursuing the following specific aims:1. Use RNA sequencing to determine the genome-wide transcriptional responses in proliferating and fused SC cultures over time.2. Determine whether E2 or TBA treatment affects protein expression and/or activation over time in proliferating and fused SC cultures.3. Use RNA sequencing and 2DE proteomics to determine the genome-wide transcriptional and proteomic responses of skeletal muscle to TBA and/or E2 implantation over time.

Project Methods
Primary bovine satellite cell (BSC) isolation: Six yearling steers that have never been implanted with androgenic or estrogenic compounds will be obtained and put on a full feedlot ration for 30 days. After this time, animals will be euthanized by captive bolt, followed by exsanguination. Isolation of satellite cells will be completed as previously described. BSC culture: The BSC preparations isolated using the procedures described above will be plated on culture dishes precoated with reduced growth factor basement membrane Matrigel® diluted 1:50 with DMEM as previously described. Cultures will be plated at a density of approximately 0.2 g/cm2, which has been shown to yield cultures that are approximately 70% confluent after 72 hours in culture. All cultures will be plated in DMEM containing 10% FBS and incubated at 37°C, 5% CO2 in a water-saturated environment. Cultures that are treated with actinomysin D, will be treated following previously described procedures.BSC proliferation: Proliferation rate in BSC cultures will be determined using the Delfia cell proliferation assay kit (Perkin Elmer, Waltham, MA) following the manufacturer's protocol as previously described.Protein synthesis: Determination of protein synthesis rate in fused BSC cultures will be conducted as described previously. Fused cultures will be treated with various inhibitors, growth factors or hormones as described below. Protein synthesis will then be measured using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS assay following the manufacturer's protocol. Isolation of RNA and preparation for RNAseq: Total RNA will be isolated from both cell culture and skeletal muscle samples will be obtained using the Absolutely RNA Microprep kit (Agilent Technologies, Santa Clara, CA) as per the manufacturer's protocol as previously described. Total RNA will then be used in RNA-seq analyses.RNA-seq: Sequencing libraries will be constructed from total RNA using the Stranded mRNA-Seq Library Preparation Kit from KAPA Biosystems. This kit includes reagents for poly-A RNA purification and fragmentation. Sixteen libraries will be pooled for each sequencing run. Libraries will be sequenced on the Illumina NextSeq sequencer using the High Output v2 150 cycle kit). Transcriptome analysis of RNA-sequencing data: Co-PD Chris Bidwell, Purdue University, will conduct the transcriptome analysis of the RNA-seq data. We will use genome-guided transcriptome analysis methods with Tuxedo suite of programs (Tophat/Bowtie/Cufflinks/CummeRbund) for the primary analysis of the cell culture and cattle experiments. These programs are open access and provide a full range of read mapping, transcriptome quantification, statistical analysis, visualization and summarization tools.Protein isolation and preparation for 2DE proteomics: All protein and 2DE proteomic analyses will be completed following previously established protocols.2DE proteomics: 2DE protein gels will be run using previously described protocols. Gel images will be scanned immediately following the SDS-PAGE using Typhoon TRIO (GE Healthcare). The scanned images will be analyzed by Image Quant software (version 6.0, GE Healthcare), followed by in-gel analysis using DeCyder software version 6.5 (GE Healthcare). The fold change of the protein expression levels will be obtained from DeCyder analyses.Mass Spectrometry: Spots chosen for analysis will be picked using the Ettan Spot Picker (GE Healthcare). The gel spots will be washed and digested in-gel and prepared for mass spec following previously described procedures. MALDI- TOF MS and TOF/TOF tandem MS/MS will be performed using a GCT premier mass spectrometer. MALDI-TOF mass spectra will be acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra will be acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample. Both of the resulting peptide mass and the associated fragmentation spectra will be submitted to the GPS Explorer workstation equipped with MASCOT search engine to search the database of National Center for Biotechnology Information non-redundant. Candidates with either a protein score confidence interval % (C.I.) or ion C.I.% greater than 95 will be considered significant.Cattle management and sample collections: Forty steers that have never received an anabolic implant will be obtained from the Utah State University beef herd. As yearlings, all animals will receive a background ration for approximately one month. During this time, all animals will be treated equally and co-mingled. After this month, the feedlot phase of this project will begin. Animals will be randomly assigned to one of four different treatments: 1) no implant (n=10), 2) TBA only implant, (n=10), 3) E2 only implant (n=10), and 4) a combined E2/TBA implant (n=10). After backgrounding, animals will be weighed, implanted, vaccinated, wormed and placed into individual pens to obtain individual feeding information. Two days and ten days after animals have been implanted, skeletal muscle biopsies from the biceps femoris will be collected from all of the animals following previously described methods. All procedures utilizing live animals will be approved by the Utah State University Animal Care and Use Committee.Statistical analysis: Statistical analysis of all data obtained will be conducted using the PROC MIXED procedure of SAS. In vitro experiments will be statistically analyzed by including treatment as a fixed effect and the assay number and the animal the BSC were isolated from as random effects. When treatment differences are found to be significant, least squares means will be separated using Tukey-Kramer adjustments. In vivo experiments will be performed in a similar fashion, except the model will be different. In this model, treatment will be included as a fixed effect and animal will be included as a random effect. Treatment different will be separated using the same method as in the in vitro experiments.

Progress 02/01/18 to 01/30/23

Outputs
Target Audience:animal science academicians, beef producers Changes/Problems:All work was conducted as described in the proposal. The only change that was made was that the transcriptome was analyzed using a more targeted approach that looked at abundance of 94 different genes rather than RNAseq due to difficulties in methodology. What opportunities for training and professional development has the project provided?Dr. Caleb Reichhardt, was the PhD student who has completed all of the research associated with this project. He completed all of the cell culture and live animal trials and collected and analyzed all of the data associated with this project. Through this experience, he has become a very successful and productive research who has now completed his degree. Thus far he has published three manuscripts, given two oral presentations at national animal science meetings, and presented two different abstracts at national animal science meetings as well. How have the results been disseminated to communities of interest?A total of 3 peer-reviewed manuscripts have been published in peer-reviewed journals. In addition, 4 posters have been presented at national scientific meetings and two different oral presentations. Further, 5 different invited speaker seminars have been given at various universities and the results have been presented at a regional project meeting detailing the molecular mechanisms that are responsible for regulating skeletal muscle growth. Lastly, the results were presented at the Utah Cattlemen's meetings to ensure that the information that was found from this research has been shared with stakeholders. What do you plan to do during the next reporting period to accomplish the goals?This is the final report, no more work will be done. However, there are 4 more manuscripts that are in final stages of preparation that will be submitted.

Impacts
What was accomplished under these goals? 1. We have completed all of the research we had proposed, all of the data has been analyzed, some of the data has been presented at national scientific meetings, and manuscripts detailing the findings are in final stages of preparation. 2. All of the objectives have been met 3. We have gained important insight into the mechanism through which estradiol and trenbolone acetate increase growth of skeletal muscle in beef muscle. In addition, we also have new information regarding the synergistic effects of providing a combined (estradiol and trenbolone acetate) implant to beef cattle that result in marked performance improvements beyond administration of one hormone alone. 4. The key impact of this research project has been a change in knowledge.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Reichhardt, C. C., Messersmith, E. M., Brady, T. J., Motsinger, L. A., Briggs, R. K., Bowman, B. R., Hansen, S. L., & Thornton, K. J. (2021, June). Anabolic implants varying in hormone type and concentration influence performance, feeding behavior, carcass characteristics, plasma trace mineral concentrations, and liver trace mineral concentrations of Angus sired steers. Animals, 11(7).
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Presentations Thornton, K. J. (Invited Lecture), University of Idaho Seminar Series, "Molecular pathways through which estradiol and trenbolone acetate contribute to skeletal muscle growth," University of Idaho, Moscow, Idaho. (October 15, 2021)
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Presentations Thornton-Kurth, K. J. (Invited Lecture), Utah Valley University Seminar Series, "Understanding the mechanism through which anabolic implants improve skeletal muscle growth of cattle," Utah Valley University, Virtual. (February 11, 2021)

Progress 02/01/21 to 01/31/22

Outputs
Target Audience:animal science academicians, beef producers Changes/Problems:We requested a one-year no-cost extension in this past reporting period that was approved. We had disruptions in getting supplies to finish our project because of Covid-19. Right now, it appears that we are on track to have everything completed in this reporting period. What opportunities for training and professional development has the project provided?Mr. Caleb Reichhardt, a PhD student, has continued working on this project over the past year. He has gained a significant amount of knowledge in conducting cell culture experiments, designing experiments, conducting statistical analyses and preparing manuscripts. In addition, he also gained a significant amount of experience in conducting various laboratory techniques. How have the results been disseminated to communities of interest?A manuscript was published detailing the results of the live animal trial relative to feedlot performance and carcass quality. Additionally, some of the findings of this research were presented in both an oral and poster presentation at the 2021 meeting of the American Society for Animal Science as well as at a department student research symposium in Logan, Utah. What do you plan to do during the next reporting period to accomplish the goals?We will complete objectives 1, 2 and 3 over the next reporting period. We also anticipate that several manuscripts will be submitted in the next year detailing our findings.

Impacts
What was accomplished under these goals? 1. We completed proteomic analysis on the samples from the live animal trial. In addition, we have also complete mRNA abundance data on these samples. we are currently working on a manuscript detailing this work. A manuscript has been published detailing the results of the live animal performance and carcass quality. We are still working on completing the transciptomic and proteomic analyses from the cell culture trial. Most of the transcriptomic data is collected, it just need analyzed. The proteomic data is still in progress. 2. none of the objectives have been met yet, but we have made progress on all of them. in objectives 1 and 2, we have completed the cell culture and need to analyze the transcriptomic data and complete collection of the proteomic data. In objective three we just need to analyze the proteomic data. 3. The main significant result we have is that we have gained more detail into the mechanism through which E2, TBA and E2+TBA improve growth of skeletal muscle. 4. The key findings of this reported period are changes in knowledge.

Publications

Progress 02/01/20 to 01/31/21

Outputs
Target Audience:animal science academicians, beef producers Changes/Problems:Currently, no changes are being made to the outlined project. What opportunities for training and professional development has the project provided?Mr. Caleb Reichhardt, a PhD student, has continued working on this project over the past year. He has gained a significant amount of knowledge in conducting cell culture experiments, designing experiments, conducting statistical analyses and preparing manuscripts. In addition, he also gained a significant amount of experience in conducting various laboratory techniques. How have the results been disseminated to communities of interest?Some of the findings of this research were presented in both an oral and poster presentation at the 2020 meeting of the American Society for Animal Science as well as at a department student research symposium in Logan, Utah. What do you plan to do during the next reporting period to accomplish the goals?During the next data reporting period, we will complete aims 1, 2 and 3. We also anticipate that several manuscripts detailing this work will be submitted over the next year. One is currently in final stages of preparation.

Impacts
What was accomplished under these goals? 1.The major activities that were completed during this reporting period is the completion of the animal trial. We have also begun to conduct RNA-seq analyses in both the cell culture and the live animal trial studies. Proteomic analyses will begin in the next couple months. 2.None of the specific objectives have been met yet, however progress was made on all 3 over the past year. All of the cell cultures in aims 1 and 2 have been completed, as well as the animal trial in aim 3. We are not finishing up analyses associated with each of these aims. 3.The main significant result that was achieved during this reporting period was the findings describing how different implants (E2 only, TBA only, or E2+TBA) impacts feedlot performance, feeding behavior, and carcass quality. 4.They key impacts of the findings of this reporting period are changes in knowledge.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Presentations Messersmith, E. M. (Presenter & Author), Reichhardt, C. C. (Author Only), Thornton-Kurth, K., Hansen, S. L. (Author Only), American Society of Animal Science Annual Meeting, "Hormone content of anabolic implants differentially affects plasma and liver trace mineral concentrations," American Society of Animal Science, Virtual. (July 19, 2020 - July 23, 2020)

Progress 02/01/19 to 01/31/20

Outputs
Target Audience:animal science academicians Changes/Problems:Currently, no changes are being made to the outlined project. What opportunities for training and professional development has the project provided?Mr. Caleb Reichhardt, a PhD student, has continued working on this project over the past year. He has gained a significant amount of knowledge in conducting cell culture experiments, designing experiments, conducting statistical analyses and preparing manuscripts. In addition, he also gained a significant amount of experience in conducting various laboratory techniques. How have the results been disseminated to communities of interest?Some of the findings of this research were presented at an invited seminar presentation at Iowa State University. Additionally, these findings were also presented at the annual meeting of the NC1184. Mr. Reichhardt also present some of this work in a poster presentation at the annual meeting of the American Society for Animal Science. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the data to complete aims 1 and 2 will completed. In addition, the animal trial associated with aim 3 will be completed and we will begin to analyze that data. We anticipate that at least one manuscript detailing our findings will be submitted in the next year.

Impacts
What was accomplished under these goals? 1. The major activities that were completed during this reporting period is the finding that TBA and E2 improve proliferation of bovine satellite cells through a non-genomic mechanism. In addition, all cell culture for the project has been completed and transcriptomic and proteomic analyses will begin on these samples shortly. In addition, we have organized and prepared the animal trials and they began in January 2020. 2. None of the specific objectives have been met yet, however progress was made on all 3 over the past year. All of the cell cultures in aims 1 and 2 was completed and the animal trial in aim 3 was started. 3. The main significant result that was achieved during this reporting period was the findings that TBA and E2 work to increase proliferation of bovine satellite cells through a non-genomic mechanism. 4. They key impacts of the findings of this reporting period are changes in knowledge.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Presentations Reichhardt, C. (Presenter & Author), Ahmadpour, A. (Author Only), Bidwell, C. (Author Only), Thomas, A. (Author Only), Thornton-Kurth, K. (Author Only), Annual Meeting of the American Society for Animal Science, "Determining the molecular mechanism through which estradiol and trenbolone acetate improve skeletal muscle growth in beef cattle," American Society for Animal Science, Austin, TX, US. (July 7, 2019 - July 11, 2019)
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Presentations Thornton-Kurth, K., Seminar Presentation, "Understanding the mechanism through which anabolic implants improve skeletal muscle growth of cattle.," Iowa State University, Ames, Iowa. (February 1, 2019)

Progress 02/01/18 to 01/31/19

Outputs
Target Audience:We are in the process of collecting our data so we do not have an audience to target currently. Once we have a more complete data set, we will be able to disseminate this data to appropriate audiences. Changes/Problems:There are currently no changes or problems to address What opportunities for training and professional development has the project provided?A PhD student has been recruited and trained in primary satellite cell isolation and culture. How have the results been disseminated to communities of interest?We are currently still collecting data and do not yet have a complete data set. As such, none of this data has been disseminated yet. What do you plan to do during the next reporting period to accomplish the goals?We will continue to collect the in vitro proliferation and protein synthesis data over the next year. We will also plan to begin our animal trial in December 2019

Impacts
What was accomplished under these goals? We have isolated all of the bovine satellite cells that are needed for the in vitro experiments. All of the proliferation experiments are currently underway and that dataset is almost complete. We have also just begun to collect the data relative to protein synthesis rates in the fused satellite cell cultures.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Thornton-Kurth, K., USDA-NIFA Annual PD meeting, "Elucidation of the mechanism through which estradiol and trenbolone acetate improve growth of skeletal muscle in beef cattle," USDA-NIFA, Washington, DC. (June 12, 2018 - June 13, 2018)