Source: AUBURN UNIVERSITY submitted to NRP
GENE EXPRESSION AND EPIGENETICS EFFECTS FROM ADMINISTRATION OF RUMEN PROTECTED METHIONINE ON RUMEN EPITHELIUM AND SKELETAL MUSCLE IN BEEF CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1012875
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jun 1, 2017
Project End Date
May 31, 2022
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Animal Sciences
Non Technical Summary
Under a constantly growing global population, beef producers are in the need to improve their animals performance with the aim to satisfy beef consumers demands. One of the ways to improve animal performance is through nutritional management. The proposed studies presented here are design to discover biomarkers present in the genome of the animals that will allow to give tools to beef producers for selection of high performance animals in terms of nutrients absorption, muscle growth and fat deposition.These biomarkers will be studied at an DNA level. There is a new research area that is focusing in changes in the way the DNA chain is folded inside the nucleous of the cells. There are molecules that tend to get attached of the DNA chain and produce changes in the way the DNA is folded. This changes will make our DNA more expose to mofidications in the expression of genes. As a result, some genes can be turn off or turned on, depending on the attachment location of these molecules in the DNA chain.In recent studies using dairy cattle, it was proved that the administration of an amino acid called Methionine in the diet could produce changes in the way the DNA chain is folded without changing the sequence of our genome, hence, our genetic code. This process is called "epigenetics". Furthermore, these changes in the genome were observed in lactating dairy cows by increments in milk yield, immune response and animal performance. The hypothesis of this project is that the administration of methionine in the diet of beef cattle will have similar improvements in animal performance by improvements in nutrient absorption at the level of the stomach (rumen), muscle growth and fat deposition.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043310104050%
3053399102025%
3053320101025%
Knowledge Area
305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3320 - Meat, beef cattle; 3310 - Beef cattle, live animal; 3399 - Beef cattle, general/other;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism; 1020 - Physiology;
Goals / Objectives
These projects aims are:Detect genes and epigenetic regulatory outcomes during the transition from two different diets in rumen epithelium due to administration of rumen-protected methionine (RPM).Detect if administration of RPM ameliorates the effects of stress due to transportation on the occurrence of muscle fatigue in beef cattle.Detect changes in gene expression and epigenetic regulatory outcomes due to administration of RPM on genotyped Angus cattle during growing and finishing phases.Detect positive effects of administration of RPM on carcass and meat quality of Angus cattle.
Project Methods
Rumen epithelium biopsies will be performed at the individual stalls where the animals are located prior to feeding. Prior to biopsy, the rumen will be manually partially emptied. Rumen contents will be returned to the rumen immediately after the biopsy is finalized. Once partially emptied, the ventral sac area of the rumen will be retracted to the cannula where papillae will be excised using surgical scissors. Approximately 0.5 to 1 gram total of papillae from each steer at the specified times will be biopsied (IACUC#2016-2993).Skeletal muscle biopsy will start with a stab incision through the skin and underlying fascia is made with a sterile scalpel. A biopsy core of muscle (~300 mg) will be removed using a biopsy needle. Up to 12 puncture biopsies will be obtained during one surgery. Tissue adhesive will be used to close the skin wound, and a topical antibiotic spray will be applied. A new biopsy site will be identified alongside the back on the left side of the spine, approximately 5 cm apart from the previous biopsy. Repeated biopsy allows the effects of nutritional or management treatments over time to be determined. This surgery does not involve penetration of the body cavity and as such is minimally invasive.Blood (20 to 30 mL) will be sampled by puncture of a tail vein during ruminal epithelium biopsies for quantification of metabolic indicators including non-esterified fatty acids, β-hydroxybutyric acid, glucose, and insulin. Serum will be obtained by centrifugation at 2,000 × g for 15 min at 25 °C.Rumen papillae and muscle tissue samples will be weighted (∼0.4 g) and immediately homogenized with Trizol reagent and linear acrylamide as coprecipitant to proceed with RNA extraction. Genomic DNA will be removed from RNA with DNase using RNeasy Mini Kit columns. The RNA concentration will be measured using a NanoDrop OneC spectrophotometer. Moreover, RNA integrity number will be measured using the Bioanalyzer platform. The final data will be normalized using the geometric mean of UXT, MTG1 and RPS15A, which were validated as suitable internal control genes in bovine skeletal muscle samples. For rumen papillae, internal control genes will be ACTB, UXT and RPS9.Primers will be designed using Primer Express 3.0 with minimum amplicon size of 100 bp. Primers will be aligned against publicly available databases using BLASTN at NCBI and UCSC's Bos taurus Genome Browser Gateway. Prior to qPCR, primers will be tested in a 20 µL PCR reaction following manufacturers recommendation. For primer testing a universal reference cDNA (RNA mixture from 5 different bovine tissues) will be used to ensure identification of desired genes. Five µL of the PCR product will be run in a 2% agarose gel stained with DNA Sybr Safe (2 µL). The remaining 15 µL will be cleaned using QIAquick® PCR Purification Kit and sequenced at the Core DNA Sequencing Facility in CASIC building at Auburn University. Only those primers that did not present primer-dimer, had a single band at the expected size in the gel, and had the right amplification product (verified by sequencing) will be used for qPCR. The accuracy of a primer pair also will be evaluated by the presence of a unique peak during the dissociation step at the end of qPCR.The cDNA will be synthesized using 100 ng RNA, 1 µg dT18, 1 µL 10 mmol/L dNTP mix, 1 µL random primers, and 10 µL DNase/RNase free water. The mixture will be incubated at 65°C for 5 min and kept on ice for 3 min. A total of 6 µL of master mix composed of 4.5 µL 5X First-Strand Buffer, 1 µL 0.1 M DTT, 0.25 µL (50 U) of SuperScriptTM III RT, and 0.25 µL of RNase Inhibitor will be added. The reaction will be performed in an Eppendorf Mastercycler® nexus using the following temperature program: 25 °C for 5 min, 50 °C for 60 min and 70 °C for 15 min. The cDNA product will be then diluted 1:4 (v:v) with DNase/RNase free water.Quantitative PCR will be performed using 4 µL diluted cDNA combined with 6 µL of a mixture composed of 5 µL 1 × SYBR Green master mix, 0.4 µL each of 10 µM forward and reverse primers, and 0.2 µL DNase/RNase free water in a MicroAmp™ Optical 384-Well Reaction Plate. Each sample will be run in triplicate and a 6 to 7 points relative standard curve plus the non-template control will be used. The reactions will be performed in an ABI Prism 7500 HT SDS instrument using the following conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C (denaturation) and 1 min at 60 °C (annealing + extension). The presence of a single PCR product will be verified by the dissociation protocol using incremental temperatures to 95 °C for 15 s plus 65 °C for 15 s. Data will be calculated with the 7500 HT Sequence Detection Systems Software. The final data will be normalized using the geometric mean of the three most stable genes among the ones tested as internal controls.Epigenetic analysis will be based on global methylation of the whole genome and methylation of a specific DNA region for the genes of interest. For global methylation, 5-mC DNA ELISA Kit will be used. This kit works with a unique anti-5-Methylcytosine (5-mC) monoclonal antibody that is sensitive and specific for 5-mC across a wide range of starting DNA amount. For methylation of specific DNA regions, OneStep qMethyl kit will be used, which is a real-time procedure for bisulfite-free determination of DNA methylation of specific sequences that contain CpG islands within a gene of interest.Quantitative PCR data will be analyzed using the MIXED procedure of SAS. Before statistical analysis, normalized qPCR data (using the geometric mean of the internal control genes) will be transformed to fold-change relative to day 0 (i.e., day on treatment). To estimate standard errors at day 0 and prevent biases in statistical analysis, normalized qPCR data will be transformed to obtain a perfect mean of 1.0 at day 0, leaving the proportional difference between the biological replicate. The same proportional change will be calculated at all other time points to obtain a fold-change relative to day 0. Fixed effects in the statistical model for each variable analyzed included treatment, time on experiment, treatment × time on experiment interactions when appropriate. Gene expression data analysis will include a repeated-measures statement with an autoregressive covariate structure. Animal performance and blood metabolites will be also analyzed using the MIXED procedure of SAS, and treatment will be the fixed effect in the statistical model. The random effect in all models was steer within treatment.The statistical model will be: Yij = µ + Ci + Tj + Sl + (C × T)ij + εij; where, Yij is the background-adjusted normalized fold change or animal performance or blood data value; µ is the overall mean; Ci is the fixed effect of time; Tj is the fixed effect of treatment; Sl is the random effect of steer nested within treatment; C × T, is the interactions of time by treatment and εij is the random error (0, σe2) associated with Yij. A likelihood ratio test will be used in order to examine if the main effects were non-significant, and if they could have an impact on the logarithm of convergence of the original model. Statistical differences for animal performance data will be declared at P < 0.10 due to the scarce degrees of freedom that came out from the subset of steers from which skeletal muscle was used for gene expression analysis. For gene expression, epigenetics and blood metabolites analysis, statistical significant differences will be declared at P < 0.05. Lastly, partial Pearson correlation analysis among genes and between genes and DNA methylation, adjusted for the fixed effects, will be conducted using the PROC CORR procedure of SAS.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Scientific community is the main target audience during the reporting period for this project. Furthermore, beef producers who are intending to acquire new nutritional products that might help to improve animal performance from a reproductive and growth standpoint are also contemplated as target audience. Efforts are focused on research, extension and outreach of new advanced beef nutriepigenomics technology expected to be considered in the future for animal selection and beef cattle performance improvement. Changes/Problems:This research project was scheduled to be concluded on 05/31/2022 but the principal investigator is not continuing her work at Auburn University. Therefore, this project is going to be finalized in 05/15/2021. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results of the calving season and transportation study where presented in a poster at the 2019 ASAS-CSAS Annual Meeting and Trade Show at the Austin Convention Center Austin, TX in July 8-11, 2019 and in Auburn University Graduate students symposium 2019. What do you plan to do during the next reporting period to accomplish the goals?Lab work related to this study is going to be concluded by March 2021 and manuscripts will be submitted for publication in PlosOne or a journal with similar impact factor during 2021.

Impacts
What was accomplished under these goals? Project objective #1: A paper was published in the Journal of Animal Physiology and Animal Nutrition under the title " Jersey steer ruminal papillae histology and nutrigenomics with diet changes".Briefly, the transition from a high forage to a high concentrate diet is an important milestone for beef cattle moving from a stocker system to the feedlot. However, little is known about how this transition affects the rumen epithelial gene expression. This study assessed the effects of the transition from a high forage to a high concentrate diet as well as the transition from a high concentrate to a high forage diet on a variety of genes as well as ruminal papillae morphology in rumen fistulated Jersey steers. Jersey steers (n = 5) were fed either a high forage diet (80% forage and 20% grain) and transitioned to a high concentrate diet (20% forage and 80% grain) or a high concentrate diet (40% forage and 60% grain) and transitioned to a high forage diet (100% forage). Papillae from the rumen were collected for histology and RT?qPCR analysis. Body weight had a tendency for significant difference (p = .08). Histological analysis did not show changes in papillae length or width in steers transitioning from a high forage to a high concentrate diet or vice versa (p > .05). Genes related to cell membrane structure (CLDN1, CLDN4, DSG1), fatty acid metabolism (CPT1A, ACADSB), glycolysis (PFKL), ketogenesis (HMGCL, HMGCS2, ACAT1), lactate/pyruvate (LDHA), oxidative stress (NQO1), tissue growth (AKT3, EGFR, EREG, IGFBP5, IRS1) and the urea cycle (SLC14A1) were considered in this study. Overall, genes related to fatty acid metabolism (ACADSB) and growth and development (AKT3 and IGFBP5) had a tendency for a treatment × day on trial interaction effect. These profiles may be indicators of rumen epithelial adaptations in response to changes in diet. In conclusion, these results indicate that changes in the composition of the diet can alter the expression of genes with specific functions in rumen epithelial metabolism. Project objective #2: A paper was published in PlosOne under the title "Preconditioning beef cattle for long-duration transportation stress with rumen-protected methionine supplementation: a nutrigenetics study". Briefly, beef cattle long-distance transportation from cow-calf operations to feedlots or from feedlots to abattoirs is a common situation in the beef industry. The aim of this study was to determine the effect of rumen-protected methionine (RPM) supplementation on a proposed gene network for muscle fatigue, creatine synthesis (CKM), and reactive oxygen species (ROS) metabolism after a transportation simulation in a test track. Angus × Simmental heifers (n = 18) were stratified by body weight (408 ± 64 kg; BW) and randomly assigned to dietary treatments: 1) control diet (CTRL) or 2) control diet + 8 gr/hd/day of top-dressed rumen-protected methionine (RPM). After an adaptation period to Calan gates, animals received the mentioned dietary treatment consisting of Bermuda hay ad libitum and a soy hulls and corn gluten feed based supplement. After 45 days of supplementation, animals were loaded onto a trailer and transported for 22 hours (long-term transportation). Longissimus muscle biopsies, BW and blood samples were obtained on day 0 (Baseline), 43 (Pre-transport; PRET), and 46 (Post-transport; POST). Heifers' average daily gain did not differ between baseline and PRET. Control heifer's shrink was 10% of BW while RPM heifers shrink was 8%. Serum cortisol decreased, and glucose and creatine kinase levels increased after transportation, but no differences were observed between treatments. Messenger RNA was extracted from skeletal muscle tissue and gene expression analysis was performed by RT-qPCR. Results showed that AHCY and DNMT3A (DNA methylation), SSPN (Sarcoglycan complex), and SOD2 (Oxidative Stress-ROS) were upregulated in CTRL between baseline and PRET and, decreased between pre and POST while they remained constant for RPM. Furthermore, CKM was not affected by treatments. In conclusion, RPM supplementation may affect ROS production and enhance DNA hypermethylation, after a long-term transportation. Administration of RPM was evaluated in non-genotyped beef cattle in a 2-years project. This research study is still in progress. The study is being conducted in North Auburn Research Unit (cow-calf pairs), and Sugg Laboratory pens (offspring post-weaning), both from the Veterinary School at Auburn University from October 2017 with its conclusion in May 2019. Preliminary conclusions: •RPM administration might increase growth inside the womb, observed by greater BW at birth, suggesting its possible effect in late gestation. •RPM administered directly to the offspring did not have a significant effect on animal performance (i.e., BW and ADG) when compared to control calves. •RPM might increase milk yield in beef cows, similarly to what was already proven in dairy cows under RPM supplementation. •Offspring's RPM supplementation after weaning potentially stimulates an early differentiation of adipocytes in LM samples, detected by the upregulation of adipogenesis-related genes (PPARg). Current status of this research project: Additional quantitative PCR analysis will be performed in muscle samples from fall calving season at the beginning of 2021. Additional genes related to muscle growth and early adipogenesis will be analyzed. Two research papers are expected to be published as a product of this 2-year research study. The first research paper will consider results from year 1, where beef heifers were used and the second research paper will consider results from year 2, where mature cows were used to complete this study. Project objective #4: Carcass and meat quality analysis were not considered in the study presented for project aim #3.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Garcia, M., Montgomery, S., Mois�, S. J., Hanzlicek, G., Hulbert, L., Bradford, B. 2020 Effects of Milk Feeding Strategies on Short and Long-term Productivity of Holstein Heifers. Journal of Dairy Science
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Alfaro, G. F., Rodning, S. P., Novak, T. E., Mois�, S. J. 2020 Preconditioning beef cattle for long-duration transportation stress with rumen-protected methionine supplementation: a nutrigenetics study. PLoS ONE 15(7): e0235481. https://doi.org/10.1371/journal.pone.0235481
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Gard Schnuelle, J., Blythe, G., Cole, R., Alfaro, G. F., Muntifering, R., Pacheco, W., Rodning, R. P. and Mois�, S. J. 2020 Doppler ultrasonography for evaluation of uterine blood flow on pregnant, genotyped beef cows consuming endophyte-infected tall fescue seeds supplemented with rumen-protected niacin


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientific community is the main target audience during the reporting period for this project. Furthermore, beef producers who are intending to acquire new nutritional products that might help to improve animal performance from a reproductive and growth standpoint are also contemplated as target audience. Efforts are focused on research, extension and outreach of new advanced beef nutriepigenomics technology expected to be considered in the future for animal selection and beef cattle performance improvement. Changes/Problems:For the calving season study, we have several calving difficulties (i.e., stillborn, C-section) for the winter calving season group (Year 1). This reason made us to change from heifers to mature cows in the second year of the study. This reason lead us to consider to publish results from these 2 years in two different research manuscripts, in order to avoid confounding effects between calving season and animal age (heifer or mature cows). What opportunities for training and professional development has the project provided?An undergraduate student (Lynsie Taylor) agreed to work with the aim to actively participate in the calving season research project whenexecutedin Dr. Moisa's lab. Miss Taylorwas trained to perform skeletal muscle biopsies, blood sampling and processing in the lab. How have the results been disseminated to communities of interest?Results of thecalving season and transportation study where presented in a poster at the 2019 ASAS-CSAS Annual Meeting and Trade Show at theAustin Convention Center Austin, TX in July 8-11, 2019 What do you plan to do during the next reporting period to accomplish the goals?Manuscripts for thetransportation study is under preparation for submission for publication in January 2020 and muscle samples from the second year of the calving season study are going to be analyzed by RT-qPCR during January and February 2020. Results for the calving seasonstudy are expected to be concluded by February 2020 and manuscripts submitted for publication in PlosOne or a journal with similar impact factor.

Impacts
What was accomplished under these goals? Project objective #1: A paper was published in the Journal of Animal Physiology and Animal Nutrition under the title "Jersey steer ruminal papillae histology and nutrigenomics with diet changes". Project objective #2:Our hypothesis was that administering rumen-protected methionine (RPM) to cattle before shipping, will attenuate muscle fatigue associated with transportation stress. This hypothesis was based on the incorporation of additional methyl groups (through RPM) on the metabolic chemical reaction that leads to the synthesis of creatine in the liver. The additional methyl groups used as a precursor for the synthesis of creatine will provide extra energy that will enhance the control of the integrity of the myofibers under a period of stress translated as muscle fatigue, generated due to a long-term transportation event. For this study, heifers where receiving RPM during a preconditioning period of 45 days before a transportation period of 24 hours. Our preliminary data showed that all the expression of genes related to the integrity of the myocyte's cell membranes were not ameliorated by the administration of RPM as compared to controls (CON). In other words, they present less variation as compared to controls. This could be interpreted as a way to dampenthe effect of stresson the integrity of the myocytes membranes as compared to control animals. Although, RPM did affect the expression of genes related to DNA methylation, i.e., DNA methyltransferase 1 and 3 alpha (DNMT1 and DNMT3A, respectively). DNMT1 had a tendency for a significant treatment × time interaction (P = 0.06) and a time effect (P < 0.01). Control heifers (CON) had DNMT3A activation during the preconditioning phase followed by an inhibition during transportation. RPM heifers presented DNMT3A inhibition during the preconditioning phase with not significant change during transportation. Stress can also directly influence the transcriptional regulation at the epigenetic level. In our study, RPM produce the inhibition of AHCY during preconditioning. The AHCY gene provides instructions for producing the enzyme S-adenosylhomocysteine hydrolase, which is involved in a multistep process that breaks down methionine. This chemical reaction also plays an important role in regulating the addition of methyl groups to other compounds (i.e., methylation). However, under the stress of transportation, AHCY had activation in RPM heifers, coincidentally with DNMT1 upregulation. If methylation plays a role in controlling the physiological response to stressors, it maybe by regulating the release of glucocorticoids in response to challenges. Our results allow us to suggest that preconditioning with RPM supplementation produce an activation of the process of DNA methylation during transportation stress. The animal's benefit due to this metabolic response to long-term transportation stress still needs to be elucidated. Our study also suggest thatexcessive stress due to long-term transportation could lead to oxidative stress. Genes related to oxidative stress were affected by incorporation of RPM in the diet. As an example, expression of superoxide dismutase 2 (SOD2), which is an important gene that controls the clearance of reactive oxigen species (ROS) that cause oxidative stress decrease in CON during transportation while in RPM heifers it stayed active. These results suggest that RPM might help to alleviate symptoms of oxidative stress due to long-term transportation but did not prove to have any possitive effects on genes related to muscle fatigue in skeletal muscle of beef heifers exposed to a long-term transportation event. In conclusion, in contrast to our hypothesis, RPM might not be providing a boosts of extra energy to cope against muscle fatigue under long-term transportation. Current status of this research project: My student Gaston Alfaro presented the results for this study during his thesis defense in October 2019. A research paper related to this particular study will be submitted to PlosOne in January 2020. Project Objective # 3: Administration of RPM was evaluated in non-genotyped beef cattle in a 2-years project. This research study is currently in progress under IACUC protocol # 2017-3154. The study is being conducted in North Auburn Research Unit (cow-calf pairs), and Sugg Laboratory pens (offspring post-weaning), both from the Veterinary School at Auburn University from October 2017 with expected conclusion in May 2019. Twenty two Angus, Angus × Simmental, and Simmental sired dams were bred between mid-March and mid-May (winter calving season - WCS) and 22 fall-calving cows (FCS) were bred between December and January. Calving season was the "environmental treatment", based on the different environmental conditions for each calving season (temperature, humidity, pasture availability, etc.). Within each "environmental treatment", dams were allotted randomly obtaining the same average body weight in both seasonal groups (WCS: 997 ± 73 lbs; FCS: 978 ± 101 lbs). At the same time, each seasonal group was split in two groups: Rumen Protected Methionine (RPM; n = 11) and Control (CON; n=11). Winter calving season dams were weighted at 1/10/17 (beginning of supplementation), 3/28/18 (pelvic area measurement), and 5/3/18 (weaning day). Fall calving season dams started receiving RPM at the beginning of July 2018 and pelvic area measured and offsprings weaned (1/7/19). The "nutritional treatment" consist on the supplementation of rumen protected methionine (RPM) which commercial name is Smartamine® (Addiseo Inc, Antony, France). The amount of dietary RPM was determined by the total of 0.07% of the offered dry matter (DM), and it was supplemented as "top dressing" during the last ~ 90 days before calving and during lactation on the dams. Offspring's RPM supplementation consist on 0.05% of the offered dry matter (DM) starting after early weaning (65 days of life on average) until 100 days after weaning. Current status of this reseach project: mRNA and DNA from all skeletal muscle samples from winter and fall calving season were extracted in the laboratory. Quantitative PCR analysis will be performed in muscle samples from fall calving season at the beginning of 2020. Genes related to muscle growth and early adipogenesis will be analyzed. Epigenetics analysis focusing on DNA methylation is going to be performed on genes affected by environmental and nutritional treatments applied. Two research papers are expected to be published as a product of this 2-year research study. The first research paper will consider results from year 1, where beef heifers were used and the second research paper will consider results from year 2, where mature cows were used to complete this study. Preliminary results for this study showed that: •RPM administration might increase growth inside womb, observed by greater BW at birth, suggesting its possible effect in late gestation. •RPM administered directly to the offspring did not have a significant effect on animal performance (i.e., BW and ADG) when compared to control calves. •RPM might increase milk yield in beef cows, similarly to what was already proven in dairy cows under RPM supplementation. •Offspring's RPM supplementation after weaning potentially stimulates an early differentiation of adipocytes in LM samples, detected by the upregulation of adipogenesis-related genes (PPARg). Project objective #4: Carcass and meat quality analysis were not considered in the study presented for project aim #3. Although, a grantproposal was submitted without successto the Foundation for meat and poultry research and educationunder the title"Beef quality response to mineral and vitamin supplementation in finishing diets of beef calves exposed to high ergovaline concentration" P.I.: Dr. Jason Sawyer, Co P.I.: Dr. Sonia Moisá and Dr. Donald Mulvaney; Duration: 5/2020 to 6/2021; Amount: $177,423.46

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Novak, T.E., Rodriguez-Zas, S. L., Southey, B. R., Starkey, J. D., Stockler, R. M., Alfaro, G. F. and Mois�, S. J. 2019 Jersey steer ruminal papillae histology and nutrigenomics with diet changes. J Anim Physiol Anim Nutr. 2019;103:16941707
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Panhans, M. H., Mason, K. M., Mullenix, M. K., Prevatt, C. G., Mois�, S. J. and Muntifering, R. B. 2019 Cowcalf performance, forage utilization, and economics of warm-season annual baleage in beef cattle winter feeding systems. Transl. Anim. Sci. 2019.XX:0-0, doi: 10.1093/tas/txz171
  • Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: Alfaro, Gaston "Preconditioning for long-duration transportation stress on beef cattle with rumen-protected methionine supplementation: a nutrigenetics study"
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Alfaro, Gast�n F., Rodning, Soren P., Novak, Taylor E., Mois�, Sonia J. 2019 Gene-regulation control of muscle fatigue during beef cattle transportation with rumen protected methionine supplementation. Abstract presented orally at CSAS-ASAS Conference in Austin, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Alfaro, Gast�n F., Rodning, Soren P., Walz, Paul H., Mois�, Sonia J.2019. Fetal programming effect of rumen-protected methionine on Angus-Simmental heifer offsprings performance and PPARg gene expression. Abstract presented as poster at CSAS-ASAS Conference in Austin, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Alfaro, Gast�n F.; Muntifering, Russell B.; Rodning, Soren P.; Gard, Julie; Cole, Robert; Pacheco, Wilmer; Mois�, Sonia J. 2019 Nutrigenomics approach to dampen the negative effects of endophyte-infected tall fescue on pregnant beef cows performance. Abstract presented as poster in Auburn University Graduate Student Research Poster Showcase 2019


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientific community is the main target audience during the reporting period for this project. Furthermore, beef producers who are intending to acquire new products that might help to improve animal performance from a reproductive and growth standpoint are also contemplated as target audience. Efforts are focused on research, extension and outreach of new advanced beef nutriepigenomics technology expected to be considered in the future for animal selection and beef cattle performance improvement. Changes/Problems:One of the goals of the project was to use genotyped animals. This goal was not possible to achieve because of the genetics of the animals used in the studies were not 75% or more Angus based breed for some of the animals under study. Currently, the company which offers the genotyping test has a platform for crossbred animals. Therefore, genotyped animals could be used in future studies, no matter the genetic background they present. What opportunities for training and professional development has the project provided?An undergraduate student (Elizabeth Brown) was awarded with an Auburn University Undergraduate Research Fellowship with the aim to actively participate in the calving season research project currently under execution in Dr. Moisa's lab. Miss Brown was trained to perform skeletal muscle biopsies, blood sampling and processing in the lab. How have the results been disseminated to communities of interest?Taylor Papstein-Novak's MS Thesis present results for the rumen epithelium study and related research paper is going to be submitted to PlosOne in January 2019. What do you plan to do during the next reporting period to accomplish the goals?Manuscripts for the rumen epithelium study, transportation and calving season study are under preparation for publication.

Impacts
What was accomplished under these goals? Project objective #1: Preliminary data obtained to partially fulfilled this project aim #1, consist on the transition from a high forage diet to a high grain diet (Treatment A) and vice versa (Treatment B). RPM was not administered at this time. Rumen epithelium gene expression results for this project indicated that the majority of the genes tested responded to transitioning diets. Significant differences in gene expression occurred with time due to the variation in the proportion of grain in the diet. Treatment A steers experienced a decrease in body weight at the beginning of the study due to an excessive feed refusal. This feed intake behavior response was reflected in altered expression of genes related to rumen epithelium cell membrane integrity (DSG1 and CLDN1), fatty acids metabolism (CPT1A and ACADSB), ketogenesis (HMGCL, HMGCS2 and ACAT1) and urea cycle (SCL14A1). Furthermore, using cracked corn as opposed to whole corn-based diets also had a significant effect on the expression of genes related to fatty acid metabolism (ACADSB), ketogenesis (HMGCS2 and ACAT1) and oxidative stress (NQO1) in treatment A steers. In Treatment A steers, a slightly increase in rumen papillae length was also observed. Following studies will focus in these diferentially expressed genes looking further into a possible epigenetic regulation situation by administration of rumen-protected methionine on the steer's diet. Project objective #2: Our hypothesis was that administering rumen-protected methionine (RPM) to cattle before shipping, will attenuate muscle fatigue associated with transportation stress. This hypothesis was based on the incorporation of additional methyl groups (through RPM) on the metabolic chemical reaction that leads to the synthesis of creatine in the liver. The additional methyl groups used as a precursor for the synthesis of creatine will provide extra energy that will enhance the control of the integrity of the myofibers under a period of stress translated as muscle fatigue, generated due to a long-term transportation event. For this study, heifers where receiving RPM during a preconditioning period of 45 days before a transportation period of 24 hours. Our preliminary data showed that all the expression of genes related to the integrity of the myocyte's cell membranes were not ameliorated by the administration of RPM as compared to controls (CON). In other words, they present less variation as compared to controls. This could be interpreted as a way to dampers the effect of stress on the integrity of the myocytes membranes as compared to control animals. Although, RPM did affect the expression of genes related to DNA methylation, i.e., DNA methyltransferase 1 and 3 alpha (DNMT1 and DNMT3A, respectively). DNMT1 had a tendency for a significant treatment × time interaction (P = 0.06) and a time effect (P < 0.01). DNMT1 had activation in control (CON) heifers during the preconditioning period while RPM heifers present activation during transportation. DNMT3A had a significant treatment × time interaction (P < 0.01), a time effect (P < 0.01) and a treatment effect (P = 0.05). Control heifers (CON) had DNMT3A activation during the preconditioning phase followed by an inhibition during transportation. RPM heifers presented DNMT3A inhibition during the preconditioning phase with not significant change during transportation. Furthermore, excessive stress due to long-term transportation could also lead to oxidative stress. Genes related to oxidative stress were affected by incorporation of RPM in the diet. As an example, expression of superoxide dismutase 2 (SOD2), which is an important gene that controls the clearance of reactive oxigen species (ROS) that cause oxidative stress decrease in CON during transportation while in RPM heifers it stayed active. These results suggest that RPM might help to alleviate symptoms of oxidative stress due to long-term transportation but did not prove to have any possitive effects on genes related to muscle fatigue in skeletal muscle of beef heifers exposed to a long-term transportation event. In conclusion, in contrast to our hypothesis, RPM might not be providing a boosts of extra energy to cope against muscle fatigue under long-term transportation. Project Objective # 3: Administration of RPM was evaluated in non-genotyped beef cattle in a 2-years project. This research study is currently in progress under IACUC protocol # 2017-3154. The study is being conducted in North Auburn Research Unit (cow-calf pairs), and Sugg Laboratory pens (offspring post-weaning), both from the Veterinary School at Auburn University from October 2017 with expected conclusion in May 2019. Twenty two Angus, Angus × Simmental, and Simmental sired dams were bred between mid-March and mid-May (winter calving season - WCS) and 22 fall-calving cows (FCS) were bred between December and January. Calving season was the "environmental treatment", based on the different environmental conditions for each calving season (temperature, humidity, pasture availability, etc.). Within each "environmental treatment", dams were allotted randomly obtaining the same average body weight in both seasonal groups (WCS: 997 ± 73 lbs; FCS: 978 ± 101 lbs). At the same time, each seasonal group was split in two groups: Rumen Protected Methionine (RPM; n = 11) and Control (CON; n=11). Winter calving season dams were weighted at 1/10/17 (beginning of supplementation), 3/28/18 (pelvic area measurement), and 5/3/18 (weaning day). Fall calving season dams started receiving RPM at the beginning of July 2018 and pelvic area measured and offsprings weaned (1/7/19). The "nutritional treatment" consist on the supplementation of rumen protected methionine (RPM) which commercial name is Smartamine® (Addiseo Inc, Antony, France). The amount of dietary RPM was determined by the total of 0.07% of the offered dry matter (DM), and it was supplemented as "top dressing" during the last ~ 90 days before calving and during lactation on the dams. Offspring's RPM supplementation consist on 0.05% of the offered dry matter (DM) starting after early weaning (65 days of life on average) until 100 days after weaning. Body weight of the calves is schedule to be measured at birth, weaning and at the time of skeletal muscle biopsy. Fall calving season group first biopsy is scheduled for 01/07/2019. Current status of this reseach project: mRNA and DNA from all skeletal muscle samples from winter calving season were extracted in the laboratory. Quantitative PCR analysis will be performed as soon as samples from fall calving season are collected and mRNA and DNA extracted. Genes related to muscle growth and early adipogenesis will be analyzed. Epigenetics analysis focusing on DNA methylation is going to be performed on genes affected by environmental and nutritional treatments applied. Preliminary results: Animal performance data collected for the winter calving season group suggest that the effect of RPM supplementation on the offspring is more pronounced in utero, through the cow, than when administered directly to the calf's diet. This was observed by a great body weight at birth for RPM offspring. Another hypothesis based on this research outcomes is that the offspring responded more effectively to the maternal RPM supplementation during fetal stage and throughout lactation due to a probably greater milk production in RPM cows. Project objective #4: Carcass and meat quality analysis were not considered in the study presented for project aim #3. To fulfill this objective, meat quality analysis was contemplated in a submitted NIFA Grant Proposal # 2018-07053.

Publications

  • Type: Book Chapters Status: Accepted Year Published: 2019 Citation: Osorio, J. S. and Moisa, S. J. 2019 CHAPTER TITLE: Gene Regulation in Ruminants: A Nutritional Perspective; BOOK TITLE: Gene Expression and Control. Editor: IntechOpen The Shard, 32 London Bridge Street, London SE1 9SG, United Kingdom
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Moisa, S. J. 2018 Beef nutriepigenomics: how to program the genome to get more marbling. Argentinean Society of Animal Production Conference 2018, Mar del Plata, Buenos Aires, Argentina
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Papstein-Novak, T. 2017 Jersey Steer Ruminal Papillae Histology and Nutrigenomics. MS Thesis http://hdl.handle.net/10415/6310


Progress 06/01/17 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this reported period, I trained my graduate student to perform rumen epithelium biopies on the Jersey steers of her study and also laboratory techniques like extraction of RNA, primer design, bioanalyzer analysis (RNA quality) and agarose gel electrophoresis. 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?By the next reporting period, I will have outcomes to report. The final results for the rumen epithelium study and the transportation study. I am expecting to submit a paper for each study in the near future.

Impacts
What was accomplished under these goals? During the reported period between 06/01/2017 and 09/30/2017, we perfomed histological analysis of rumen papillae that belong to a preliminar study finalized on 5/20/2017, where we aim to examined the effect of transition from a high-forage diet to a high-grain diet, and vice versa, on the rumen epithelium of fistulated Jersey steers. More specifically the objective was to determine effects of changes in diet on gene expression of important receptors and transporters in the process of absorption of nutrients in the ruminal papillae. Results of histological analysis revealed greater rumen papillae length with time on animals that transitioned from a high-forage diet to a high-grain diet. Currently, rumen epithelium samples are analyzed in the laboratory in order to detect differences in gene expression of selected genes. During this reported period, I coordinated with people from the National Center of Asphalt Technology (NCAT), a brief beef cattle transportation study to analyze effects of administration of rumen-protected methionine before transportation on the occurrence of muscle fatigue due to transportation stress. This study started on 10/16/17 and currently we are extracting RNA from the muscle samples obtained by skeletal muscle biopsy at 45 days before transportation, 3 days before transportation and, right after a transportation period of 24 hours around NCAT test track. Finally, I coordinated another study with the aim to evaluate the effect of administration of rumen-protected methionine during late gestation in performance of cow with winter and fall calving seasons. The main goal of this study is to suggest a calving season to cow-calf producers based on performance outcomes after administration of rumen-protected methionine on cows in late gestation. Furthermore, we want to assess epigenetic regulation of growth of winter- and fall- born calves due to variations on DNA methylation of the grazing pasture and maternal supplementation of rumen-protected methionine and determine carryover effects of maternal administration of rumen-protected methionine on offspring performance after weaning. This ongoing study started in October 2017. Currenty, cows that belong to the winter-calving season group are calving.

Publications