Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
NEW INSIGHTS INTO BEEF QUALITY DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022131
Grant No.
2020-67017-31269
Cumulative Award Amt.
$469,000.00
Proposal No.
2019-06745
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2024
Grant Year
2020
Program Code
[A1364]- Novel Foods and Innovative Manufacturing Technologies
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Animal and Poultry Sciences
Non Technical Summary
Eating satisfaction and visual fresh meat cues drive consumer purchasing decisions of fresh beef. Because tenderness 'biology' has waned in recent years and dark beef is generally considered a result of adverse animal handling events, there is a critical need to expand our understanding of dark beef development and its maturation into tender meat. Our long-term goal is to understand the molecular and biochemical events involved in developing high quality meat. The overall objective of this application is to understand how antemortem muscle energy metabolism causes dark beef development and how these changes impact postmortem proteolysis. Our central hypothesis is thatmuscle changes inresponse tomanagement strategies. As a result,fresh beef quality characteristics changes, especially color and tenderness through unknown mechanisms. We will test our central hypothesis by studying atypical dark beef development, novel proteases and naturally occurring dark beef in commercial settings. This work is novel because it challenges the central dogma that dark beef results from aberration(s) in animal handling or harvesting events. Moreover, it revisits meat tenderness from a different vantage point, the mitochondria. Our rationale is if we understand fresh beef color and tenderness more thoroughly then we can develop strategies to exploit this knowledge into practices that may improve the eating satisfaction and visual characteristics of fresh beef. We are uniquely qualified to conduct the aforementioned studies because we have considerable experience, a compelling preliminary data and have recruited one of the world's foremost experts in beef quality development.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30833201010100%
Knowledge Area
308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3320 - Meat, beef cattle;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Define the biochemistry responsible for atypical "dark-cutting" beef.The working hypothesis is that cattle fed low energy diets produce muscle with more oxidative metabolism, which alters postmortem energy metabolism and subsequent color development, independent of animal stress. Determine the role of mitochondrial proteases on postmortem proteolysis and meat tenderness.The working hypothesis is that mitochondria and(or) associated structures modulate proteolysis through an as yet unknown mechanism.Define the biochemical and cellular differences among dark beef and determine the biochemistry responsible for its altered postmortem metabolism.The working hypothesis is that changes in mitochondria impact postmortem metabolism either directly through pH declines or indirectly via associated changes in muscle proteins.
Project Methods
Aim 1. Forty cross-bred castrate calves (360 kg) will be and randomly assigned to either a high or low growth rates and high or low concentrate diets in a 2 X 2 factorial design. Calves will be staggered onto diets to ensure all cattle are harvested at a relatively constant age and a final weight of 570 kg. Samples from the longissimus muscle opposite the 12-13th rib will be collected at 0, 30, 60, 120, 240, 480 min and 24 hr. Samples will be flash frozen. Additional samples will be mounted and frozen in isopentane cooled in liquid nitrogen and stored at -80 C until cryo-sectioning. Mitochondria will be isolated using the differential centrifugation method. Briefly, muscle samples will be finely minced at 1:5 (wt/vol) in ice-cold isolation buffer (100 mM sucrose, 180 mM KCl, 50 mM Tris, 5 mM MgCl2, 10 mM EDTA, 1 mM K-ATP, pH 7.4). Protease (subtilisin A) will be added to the tissue suspension at 0.4 mg/ml before homogenization with a Potter-Elvehjem type homogenizer system. The homogenate will then be centrifuged at 1,000 × g for 10 min at 4 °C. The resulting supernatant will be centrifuged again at 8,000 × g for 10 min at 4 °C. Mitochondrial pellet will be resuspended in mitochondrial suspension buffer (220 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl, and 1 mM EGTA, pH 7.4). Mitochondrial protein concentration will be determined using a bicinchoninic acidprotein assay kit.Mitochondria function - Based on protein content, mitochondria will be subjected in triplicate to the Seahorse Bioscience Flux Analyzer to measure mitochondrial oxygen consumption rate (OCR). Assessment of state 3 (active, maximum phosphorylating) respiration will be determined in the presence of pyruvate (10 mM), malate (5 mM), and ADP (5 mM). Complex II-driven respiration will be determined in the presence of succinate (20 mM) and ADP (5 mM), and rotenone (2 µM) will be used to block complex I. State 4 non-phosphorylating, maximal leak-dependent respiration will be quantified following the addition of oligomycin (2 µM). Maximum uncoupled respiration will be determined following addition FCCP (0.3 µM).Frozen muscle samples will be pulverized under liquid nitrogen and homogenized at 1:10 (wt/vol) in reaction buffer containing 40 mM glycogen, 60 mM KCl, 5 mM MgCl2, 10 mM Na2HPO4, 30 mM creatine, 25 mM carnosine, 10 mM sodium acetate, 5 mM ATP, 0.5 mM ADP, and 0.5 mM NAD+ (pH 7.4). 0.5 mg/ml intact, mechanically disrupted, or treated with 1 M PCA mitochondria will be add to the in vitro system. Mitochondrial pellets or supernatants will be also tested after centrifugation of disrupted mitochondria at 13,000 rpm for 5 min. Mitochondrial inhibitor (combined or individual) for complexes I, III, IV, and V will be incorporated whenever needed. Aliquots will be removed at 0, 30, 120, 240, and 1440 min for pH and metabolite analysis.Myoglobin, oxidative enzymes (succinate, dehydrogenase, citrate synthase, OXPHOS), glycogen and glucose metabolism enzymes (glycogen phosphorylase, glycogen synthase, hexokinase and lactate dehydrogenase), and other proteins of interest will be measured by Western blot analysis. Muscle proteins will be separated by SDS-PAGE, transferred to membranes, and incubated with appropriate primary and secondary antibodies. Blots will be visualized using standard image acquisition and analysis software.Aim 2. Mitochondria from bovine red and white muscles will be used in the in vitro system to determine ifthey modulate proteolysis. Because of its repeatability, the modified Scopes system will serve as our bioassay. First, we will test whether mitochondria need to be functioning. This will be accomplished by mechanically disrupting mitochondria. If disrupted mitochondria produce the same effect, we will then test whether the effect resides in the matrix (soluble) or is associated with the membranes. This will be accomplished by centrifuging mechanically disrupted mitochondria. We will also determine which specific component of the mitochondria is responsible. To achieve the aforementioned, 0 or 0.5 mg/ml of fresh isolated mitochondria will be incorporated into our in vitro system with or without a mitochondrial inhibitor cocktail (20 μM rotenone, 10 mM malonic acid, 10 mM potassium cyanide, and 10 μM oligomycin, to inhibit mitochondrial complexes I, III, IV, and V, respectively). Based on the collective results, we will then conduct additional experiments to identify the causative agent.We intend to use a two-pronged approach to Aim 3. First, we will source mitochondria on oxidative phosphorylation potential, red versus white. Mitochondria will be titrated into our in vitro modified Scopes (glycolysis) system. At various times, aliquots will be collected and tested for metabolites, pH, etc., which will give us an understand of how postmortem is modulated by inherently divergent mitochondria. For example, from cattle managed or fed differently. The second prong of this study will exploit the existing variation in beef color found in US processing plants (see attached letters of support). Following the lead of McKeith et al., we will source beef samples from the longissimus of carcasses varying in color and subject these samples to our Scopes system AND assess the relative proportion of each complex in the mitochondria.

Progress 06/01/20 to 05/31/24

Outputs
Target Audience:Meat processors, academicians, meat specialists, food scientists, feed manufacturers, resturant owners, ranchers ann beef producers, extension specialist, extension agents, ag educators, professors, and consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One doctoral student was educated and now works at a sister land grant institution. Multiple undergraduates were trained, some of which have joined the graduate program. How have the results been disseminated to communities of interest?All reported herein have been published in refereed journal articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Based on the totality of the results, we showed that dark-cutting (DC)results from cattle with compromised levels of muscle energy stores, which become rate-limiting postmortem, and arrestpostmortem glycolysis prematurely as suggested by lowered glucose and lactate levels, a high ultimate pH and no residual glycogen compared to normal beef and in some cases atypically (AT) dark beef. We have no information on how muscle had limited carbohydrate storage but this is the quintessential characterization of dark, firm and dry (DFD) meat that is associated with the term "dark cutting" beef. We also showed thatAT dark beef and even DC beef results from differences in inherent energy metabolism, which likely existsprior to harvest.we report that muscle glycogen concentrations, as projected from glycolytic potential calculations or the sum of all glycolytic metabolites,in AT beef as defined in this study are likewise intermediate to levels found in DC and normal beef. We also showed that while muscle from grass-fed cattle differs from that of their 'fed' counterparts in color, it also differs in energy metabolism. Specifically, muscle from grass-fed cattle possesses more myoglobin, perhaps making it darker in appearance. In addition, beef from forage fed animals havegreater mitochondrial-based oxidative enzyme content, less glycolytic enzymes, and when subjected to anin vitroglycolyzing system produces less lactate. Finally, muscle O-GlcNAcylation is lower in muscle of forage fed animals, which is a nutrient sensing pathway tosense nutrient availability in the microenvironment.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Apaoblaza, A., S.K. Matarneh, E.M. England, T.L. Scheffler, S.K. Duckett H. Shi and D.E. Gerrard. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Sci. 161:107996.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Beline, M., J. Morales, D. Antonelo, J. Silva, V.L.M. Buarque, N. Consolo, P. Leme, S. Matarneh, D. Gerrard and S. Silva. 2020. Muscle fiber type, postmortem metabolism, and meat quality of Nellore cattle with different post-weaning growth potential. Livestock Sci. https://doi.org/10.1016/j.livsci.2020.104348.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Matarneh, S.K., S.L. Silva and D.E. Gerrard. 2021. New Insights in Muscle biology that alter meat quality. Annual Review of Animal Biosciences 9:1, 355-377.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, C., S.K. Matarneh, D.E. Gerrard and J. Tan. 2021. Modeling of energy metabolism and analysis of pH variations in postmortem muscle. Meat Science, 182, 108634. https://doi.org/10.1016/j.meatsci.2021.108634.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: G�mez, J.F.M., D.S. Antonelo; M. Beline, B. Pavan, D.B. Bambil, P. Fantinato-Neto, A. Saran-Netto, P.R. Leme, R.S. Goulart, D.E. Gerrard and S.L. Silva. 2022. Feeding strategies impact animal growth and beef color and tenderness. 2022. Meat Sci., 183, 108599. https://doi.org/10.1016/j.meatsci.2021.108599
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Antonelo, D.S., J.F.M. G�mez, S.L. Silva, M. Beline, X. Zhang, Y. Wang, B. Pavan, L.A. Koulicoff, A.F. Rosa, R.S. Goulart, S. Li, D.E. Gerrard, S.P. Suman, M.W. Schilling and C.C.J. Balieiro. 2022. Proteome basis of biological variations in color and tenderness of beef longissimus thoracis muscle from cattle differing in growth rate and feeding regime. Food Res Int. 153:110947. doi: 10.1016/j.foodres.2022.110947.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kirkpatrick, L.T., J.F.M. G�mez, M. Beline, J.C. Wicks, H. Shi, S.L. Silva, J.L. Aalhus, D.A. King and D.E. Gerrard. 2023. Muscle of dark beef differs metabolically. Meat Science. 2023 Dec; 206:109344. doi: 10.1016/j.meatsci.2023.109344.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Wicks, J. C., Wivell, A. L., Beline, M., Zumbaugh, M. D., Bodmer, J. S., Yen, C. N., Johnson-Schuster, C., Wilson, T. B., Greiner, S. P., Johnson, S. E., Shi, T. H., Silva, S. L., & D.E. Gerrard. 2024. Determining muscle plasticity and meat quality development of low-input extended fed market-ready steers. Translational Animal Science, 8, txae064. https://doi.org/10.1093/tas/txae064


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Ranchers, Meat processors, feedlot operators, cattle producers, meat purveyors, meat scientists Changes/Problems:Unfortunately, the pandemic still plagues various aspects of the study, mainly because all studies were discontinued for a period of time and feeding cattle takes time. Furthermore, some reagents still remain back ordered. While the latter is not a huge issue, the former has set us back some. What opportunities for training and professional development has the project provided?These activities and the data collected herein were used as part work for a doctoral thesis. Moreover, numerous undergraduates received research training as part of these studies. 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 plan to continue our work on goals 1 and 3, namelyDefine the biochemistry responsible for atypical "dark-cutting" beef. andDefine the biochemical and cellular differences among dark beef and determine the biochemistry responsible for its altered postmortem metabolism, respectively.These studies include setting up additional studies using cattle fed different diets during the finishing phases and measuring the tissues ability to modulate postmortem metabolism. Studies will be in the carcass and in vitro (in the laboratory).

Impacts
What was accomplished under these goals? Work conducted during this reporting cycle focused mainly on Goal 2Determine the role of mitochondrial proteases on postmortem proteolysis and meat tenderness. To address this goal, we first used purified mitochondria from beef longissimus muscle and measure the proteolytic ability in these mitochondria. In an exhausting set of studies, we were not able to detect any protease in the mitochondria or associated with the outside of the organelle. Furthermore, we subjected mitochondriia to proteomic analyses and consistent with the aforemetioned, we were unable to detect any protease in or associated with beef mitochondria. While we cannot exclude the possibility that mitchondria indirectly contribute to proteolysis postmortem, the organelle does not directly contribute to posmortem tenderization of meat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Antonelo, D.S., P.R. Dos Santos-Donado, C.R. Ferreira, L.A. Colnago, F.M.M Ocampos, G.H. Ribeiro, R.V. Ventura, D.E. Gerrard, E.F. Delgado, C.J. Contreras-Castillo and J.C.C. Balieiro. Exploratory lipidome and metabolome profiling contributes to understanding differences in high and normal ultimate pH beef. Meat Sci. 2022 Sep 14;194:108978. doi: 10.1016/j.meatsci.2022.108978. Epub ahead of print. PMID: 36116280.
  • Type: Book Chapters Status: Published Year Published: 2023 Citation: Matarneh, S.K., Scheffler, T.L. and D.E. Gerrard. Chapter 5 - The conversion of muscle to meat. Editor(s): Fidel Toldr�, In Woodhead Publishing Series in Food Science, Technology and Nutrition, Lawrie's Meat Science (Ninth Edition), Woodhead Publishing, Pages 159-194, ISBN 9780323854085, https://doi.org/10.1016/B978-0-323-85408-5.00010-8.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Cattle producers, meat processors, other acamedicians, extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student, several others were taught select procedures. How have the results been disseminated to communities of interest?Yes, several publications have been published in refereed journals. What do you plan to do during the next reporting period to accomplish the goals?Continue to work on the second objective.

Impacts
What was accomplished under these goals? Feedlot animals had larger longissimus muscle areas, greater backfat thickness, a more desirable meat color and were more tendercompared to pasture-fed cattle. Moreover, pasture- and feedlot-finished animals with similar growth rates (GR) did not differ in the chromatic attributes of non-aged meat, regardless of endpoint. Thus, GR appeared to be the main factor driving beef chromatic parameters, while finishing regime (diet type) had a major impact on achromatic attributes and tenderness of meat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: G�mez, J.F.M., D.S. Antonelo2; M. Beline, B. Pavan1, D.B. Bambil1, P. Fantinato-Neto1, A. Saran-Netto1, P.R. Leme, R.S. Goulart, D.E. Gerrard and S.L. Silva. 2021. Feeding strategies impact animal growth and beef color and tenderness. 2022. Meat Science, 183, 108599. https://doi.org/10.1016/j.meatsci.2021.108599
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Matarneh, S.K., S.L. Silva and D.E. Gerrard. 2021. New Insights in Muscle Biology that Alter Meat Quality. Annual Review of Animal Biosciences 9:1, 355-377.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, C., S.K. Matarneh, D.E. Gerrard and J. Tan. 2021. Modeling of energy metabolism and analysis of pH variations in postmortem muscle. Meat Science, 182, 108634. https://doi.org/10.1016/j.meatsci.2021.108634.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 150. Antonelo, D.S., J.F.M. G�mez, S.L. Silva, M. Beline, X. Zhang, Y. Wang, B. Pavan, L.A. Koulicoff, A.F. Rosa, R.S. Goulart, S. Li, D.E. Gerrard, S.P. Suman, M.W. Schilling and C.C.J. Balieiro. 2022. Proteome basis of biological variations in color and tenderness of beef longissimus thoracis muscle from cattle differing in growth rate and feeding regime. Food Res Int. 153:110947. doi: 10.1016/j.foodres.2022.110947.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 154. G�mez J.F.M., N.R.B. C�nsolo1, D.S. Antonelo, M. Beline, M. Gagaoua, A. Higuera-Padilla, LA. Colnago, D.E. Gerrard and S.L. Silva. Impact of cattle feeding strategy on the beef metabolome. Metabolites, 12(7), 640. https://doi.org/10.3390/metabo12070640


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Beef producers, beef cattle processors, meat scientists, general public, Extension Agents/Educators and small meat processors. Changes/Problems:COVID has and continues to give us challenges for harvesting animals and processing samples. We expect the situation to continue to improve. What opportunities for training and professional development has the project provided?A portion of these studies were used to educate a PhD student that has now accepted a position at a sister land grant institution. Also, several (5) undergraduate students were exposed to bench science during this project. How have the results been disseminated to communities of interest?Yes, all data to date has been published or is in the process of being assembled for publication. What do you plan to do during the next reporting period to accomplish the goals?Continuing to dissect the mechanism controlling the rate and extent of postmortem metabolism in bovine muscle.

Impacts
What was accomplished under these goals? Insufficient acidification results in dark, firm, and dry beef (DFD), however, dark beef is often thought related toa stress event antemortem. Our thesis is thatmuscle tissuechanges in response to feeding regime. Our results show thatsamples from 10 grain-fed and 10 grass-fed market weight, angus-crossbred beef cattle differ signficantly in quality characteristics. Grass-fed cattle had lower L* and a* values,higher ultimate pH compared to grain-fed cattle, yet differences in lactate, glycogen and glucose were not detected. Further, increasedultimate pH values a were noted when samples from grass-fed cattlesubjected to anin vitroglycolysis system. Muscle from grass-fed beef possessed nearly two-fold moremitochondrial based enzymes and greater muscle pigments. These data documentlean from grass-fed beef is indeed different than that from traditional feedlot fed cattle, yet not stressed antemortem.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Apaoblaza, A., S.K. Matarneh, E.M. England, T.L. Scheffler, S.K. Duckett H. Shi and D.E. Gerrard. 2020. Muscle from grass- and grain-fed cattle differs energetically. Meat Sci. 161:107996.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gagaoua, M., C. Terlouw, A.M. Mullen, D. Franco, R.D. Warner, J.M. Lorenzo, P.P. Purslow, D. Gerrard, D.L. Hopkins, D. Troy and B. Picard. 2020. Molecular signatures of beef tenderness: Underlying mechanisms based on integromics of protein biomarkers from multi-platform proteomics studies. Meat Sci. 172:108311
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Antonelo, D.S, N.R.B. C�nsolo, J.F.M. G�mez, M. Beline, R.S. Goulart, R.R.P.S. Corte, L.A. Colnago, M.W. Schilling, D.E. Gerrard and S.L. Silva. Metabolite profile and consumer sensory acceptability of meat from lean Nellore and Angus x Nellore crossbreed cattle fed soybean oil. Food Res Inter. 132:109056
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: 125) Matarneh, S.K., S.L. Silva and D.E. Gerrard. New Insights in Muscle Biology that Alter Meat Quality Annual Review of Animal Biosciences 2021 9:1, 355-377
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Beline, M., J. Morales, D. Antonelo, J. Silva, V.L.M. Buarque, N. Consolo, P. Leme, S. Matarneh, D. Gerrard and S. Silva. 2020. Muscle fiber type, postmortem metabolism, and meat quality of Nellore cattle with different post-weaning growth potential. Livestock Sci. https://doi.org/10.1016/j.livsci.2020.104348.