Progress 05/01/17 to 10/31/21
Outputs
Target Audience:The target audients includes students, researchers, and academicians in meat science and muscle biology. Efforts mainly focus on education and training of graduate and undergraduate students; the student audience included one minority (Latina). Graduate students are receiving training as part of their degree programs. Two MS students were involved in research activities, including developing and performing assays, analyzing and interpreting data, and communicating results. They also assisted in teaching and mentoring undergraduate students. Two undergraduate students were involved in laboratory activities. One was a volunteer and conducted image analysis, and another performed assays and data analysis through the University Scholars research program for undergraduate students. Students participated in lab meetings and journal clubs as part of their educational program. Changes/Problems:The Covid epidemic caused delays and limitations for accessing equipment and facilities. This affected timely completion of testing and optimization of protocols which primarily affected objective 2. Analyses were undertaken to use plate based assay for activity of complexes at pHs representing postmortem muscle. What opportunities for training and professional development has the project provided?The project provided training for graduate and undergraduate students. The graduate students are involved in experimental design, sample collection, laboratory analysis, interpretation, and preparing theses and/or manuscripts regarding aspects of the project. The undergraduate students are learning techniques related to laboratory procedures, experiment design, and data analysis. Students gained experience presenting data in written (abstracts, manuscripts), visual (poster), and oral (presentations) form; students presented at local (Animal Sciences Symposium), regional (Southeast Electron Microscopy Society), and national conferences (Reciprocal Meat Conference). One students also received training in electron microscopy from personnel at UF's core facility. How have the results been disseminated to communities of interest?Results were disseminated as posters at the national Reciprocal Meat Conference and American Society of Animal Science in June and July 2021. Manuscripts are being prepared for submission to journals, and will be incorporated into future extension events. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Meat quality attributes, including color and tenderness, are key determinants of product value and consumer acceptability. These attributes are largely governed by (1) inherent characteristics related to muscle function and metabolism in the live animal, and (2) the conditions during the early postmortem period, roughly the first 24 hours after death; this period includes physical, biochemical, and energetic changes that ensue as living muscle is converted to meat. In living muscle, mitochondria utilize oxygen to produce energy. At harvest, the blood supply is removed, and oxygen can no longer be delivered to muscle. Accordingly, approaches to evaluate postmortem muscle have relied heavily on methodologies which ignore the contribution of mitochondria. Yet, mitochondria regulate other cellular functions, and evidence supports that mitochondria mediated events, including apoptosis and oxidative stress, are associated with meat quality. There is a gap in knowledge regarding how mitochondria function and integrity impact the cellular environment in postmortem muscle. Understanding how mitochondria participate in postmortem metabolism may help us identify cellular targets for the development of processing strategies that improve meat quality. It is particularly challenging to evaluate mitochondria since they are functionally complex and dynamic organelles that vary in content and distribution within and across muscles. Mitochondria content varies significantly across muscles within an individual, which is related to functional demands and work performed. In addition, the same muscle across animals may exhibit differences in mitochondria content based on genetics, activity, or other factors. Both aspects may contribute to variation in postmortem metabolism and meat quality. We used distinct biological types of cattle (Bos indicus, Brahman vs Bos taurus, Angus)and muscles with divergent properties (slow-twitch oxidative vs. fast glycolytic) to investigate mitochondrial function early postmortem. Objective 1. Evaluate changes in mitochondria function in oxidative and glycolytic muscles during the first 24h postmortem. In order to evaluate relationships between mitochondria function, metabolism, and tenderization in muscles with distinct fiber type compositions, the content and activation of protease systems were analyzed in longissimus and diaphragm. Content of calpain-1, calpastatin, and caspase were determined along with autolysis of calpain-1, and degradation of calpastatin, desmin, and troponin-T.Overall, the glycolytic longissimus was associated with faster calpain-1 autolysis, lower calpastatin content, and more rapid initiation of proteolysis. Our previous data indicated that integrity of mitochondria outer membrane is compromised earlier in longissims compared with diaphragm; and that mitochondrial parameters may be related to mitochondria content as well as the rate of decline of muscle temperature. Disruption of mitochondria may be important in hastening cell death mechanisms and compromising regulation of resting sarcoplasmic calcium levels. Together, this supports that fiber type impacts protease content and suggests that mitochondria content and integrity may contribute to protease activation. These results had been previously reported in an abstract, and the manuscript was published in late 2021. Objective 2. Determine mitochondria function in oxidative and glycolytic muscles under pH and oxygen conditions that simulate postmortem muscle. Activity of mitochondrial complexes was conducted at pH conditions representing those in postmortem muscle. Complex IV activity was determined in longissimus and diaphragm at pH 7.0, 6.7, 6.4, 6.1, and 5.8. The pH affected complex IV activity differently according to muscle (pH x muscle, P=0.03). The pH did not affect complex IV activity of diaphragm, whereas within the longissimus, the activity at pH 6.1 was lower than activity at pH 6.7 and 7.0. Complex IV activity was approximately 12 times higher in the diaphragm compared with the longissimus, which is partly due to the greater mitochondria content in the oxidative diaphragm. Complex IV activity of Brahman and Angus longissimus was also evaluated at the indicated pHs. The assay pH affected complex IV activity (P<0.0001), with similar activity between pH 7.0 and 6.4. Lower pH (6.1 and 5.8) resulted in reduced complex IV activity. Complex II activity in longissimus muscle was determined at pH 7.0, 6.7, 6.4, 6.1, and 5.7. The pH affected complex II activity (P<0.0001). There was a linear decrease in activity from pH 7.0 to 6.1 (R2 = 0.99), and activity at pH 6.1 was ~75% lower than at pH 7.0. Together, this suggests that complexes in the electron transport chain have differing susceptibilities to pH decline during the postmortem period. Moreover, pH induced changes in complex activity may be more disastrous in longissimus due to its lower mitochondria content. Manuscript is in preparation. Objective 3. Assess mitochondria respiration and postmortem metabolism in longissimus muscle with varying mitochondria content. Ultrastructural morphology of mitochondria in postmortem longissimus was evaluated using transmission electron microscopy. A subset of steers (n=4-5 per group) were evaluated at 1,3,6, and 24h and 14d. Parameters of intermyofibrillar mitochondria structure included area, surface area, perimeter, Feret diameter, form factor, circularity, roundness, aspect ratio, optical density parameters, and flocculent densities per mitochondrion. Flocculent densities are believed to be precipitates of denatured matrix proteins and lipids liberated from cristae and present as black, wooly spots on images from microscopy. During the postmortem period, structural parameters differed according to breed (P values are for breed x time): surface area (P<0.0001), perimeter (P=0.0005), Feret diameter (P=0.002), and optical density (P<0.0001). Surface area increased in both breeds from 1 to 24h, but Brahman exhibited greater surface area than Angus at 3h and 24h. Time postmortem influenced form factor (P=0.0001), circularity (P=0.0001), roundness (P=0.009), and aspect ratio (P=0.009). These changes primarily reflect in increase in roundness and circularity from 1h to 14d, which may be associated with swelling of mitochondria. Time affected the number of flocculent densities per intermyofibrillar mitochondrion (P=0.0023). The number of flocculent densities per mitochondrion increased by 24h postmortem, and this ratio also increased as pH declined postmortem (R2=0.65, P<0.0001). Breed did not impact intermyofibrillar mitochondrion per µm2 (P=0.23) but there were fewer mitochondria per µm2 (P=0.01) at 14d compared with 1h. Only mitochondria with a continuous outer membrane were counted; thus, the decrease in mitochondria per µm2 at 14d does not reflect a decrease in mitochondria content, but rather that few mitochondria have a defined outer membrane. Based on image analyses, mitochondria are intact 1h postmortem and fluctuate in size at later times postmortem. Since mitochondria are still intact at 1h postmortem, they may contribute to postmortem metabolism and meat quality development. Mitochondria in Brahman longissimus exhibit more dynamic changes in mitochondria morphology, which may be related to improved energy status and greater oxygen consumption and resilience of Brahman mitochondria during the early postmortem period. The different patterns of mitochondria morphology and function in breeds may be associated with variation in color and tenderness. Brahman produce less tender steaks with reduced proteolysis. Moreover, mitochondria oxygen consumption at 24h was associated with redness. These results were reported in an MS thesis and abstract (and poster). Manuscripts for mitochondria function, morphology, proteolysis, and color are in preparation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ramos, PM, M Pedrao, LC Bell, and TL Scheffler. Early postmortem metabolism and protease activation in fast glycolytic and slow oxidative bovine muscles. Meat and Muscle Biology. 5(1):44, p. 1-10.
- Type:
Other
Status:
Accepted
Year Published:
2022
Citation:
Hawryluk, B, PM Ramos, K Kelley, and TL Scheffler. Mitochondria morphology in postmortem longissimus lumborum of Angus and Brahman. Reciprocal Meat Conference (2021); Reno, NV.
- Type:
Other
Status:
Accepted
Year Published:
2022
Citation:
Chmielewski, E, B Hawryluk, CA Gingerich, MT McKinney, PM Ramos, and TL Scheffler. Surface color variation between Angus and Brahman longissimus lumborum muscle. Reciprocal Meat Conference (2021); Reno, NV.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Hawryluk, B. Mitochondria morphology in postmortem longissimus lumborum of Angus and Brahman. Thesis. Department of Animal Sciences, University of Florida.
|
Progress 05/01/20 to 04/30/21
Outputs
Target Audience:The target audiences includes students, researchers, and academicians in meat science and muscle biology. Primarily, efforts are directed to education and scientific training of scholars and students (graduate and undergraduate). For the scholar and students, the audience included minorities (1 Asian American, 1 Latina). Two graduate students (both MS) and two undergraduate students are receiving training as part of their degree programs. Graduate students conduct research activities related to experimental design, sample collection, and laboratory analysis, and help train undergraduate students. One undergraduate student was enrolled for course credit (for research/lab experience); and all conducted sample processing and assays, and performed analysis and interpretation of data. Students participated in lab meetings and journal clubs as part of their educational program. Changes/Problems:The major complication has been delays due to COVID-19. Access to critical equipment was limited from March 2020 through spring 2021 due to spacing restrictions and access (equipment is located in Shands Hospital at University of Florida). Additional analyses were undertaken to assess individual complexes using plate based assays. Assays are being conducted using pHs representing postmortem muscle and samples collected during the first 24h postmortem are also being analyzed to evaluate time related changes in function. We have also experienced limited availability/significant shipping delays for certain reagents for assays. What opportunities for training and professional development has the project provided?This project is providing professional development for graduate students and undergraduate students. The students are mentored by the PI and co-Pi and are involved with experimental design, sample collection, laboratory analysis, and interpretation. One MS thesis is in preparation, and another was completed in December 2020. The undergraduate students are learning techniques related to sample collection and processing, as well as how to perform laboratory analyses, and interpret results. All are involved in lab meetings and journal clubs. A former research scholar continues to be involved with research discussions and lab meetings. How have the results been disseminated to communities of interest?Results from the project were presented at national/international conference in August 2020. In addition, TL Scheffler presented results to an international group in Australia and New Zealand and to a seminar course at another university in the US (both presentations were virtual due to COVID-19). Manuscripts are in preparation for submission to journals. What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Perform analysis of mitochondria function at different pH and oxygen concentration. Assess complexes at different pHs and time postmortem. Objective 3. Finalize manuscript and submit for publication. Complete assessment of mitochondria morphology changes using electron microscopy images.
Impacts
What was accomplished under these goals?
Meat quality attributes, including color and tenderness, are key determinants of product value and consumer acceptability. These attributes are largely governed by (1) inherent characteristics related to muscle function and metabolism in the live animal, and (2) the conditions during the early postmortem period, roughly the first 24 hours after death; this period includes physical, biochemical, and energetic changes that ensue as living muscle is converted to meat. In living muscle, mitochondria utilize oxygen to produce energy. At harvest, the blood supply is removed, and oxygen can no longer be delivered to muscle. Accordingly, approaches to evaluate postmortem muscle have relied heavily on methodologies which ignore the contribution of mitochondria. Yet, mitochondria regulate other cellular functions, and evidence supports that mitochondria mediated events, including apoptosis and oxidative stress, are associated with meat quality. There is a gap in knowledge regarding how mitochondria function and integrity impact the cellular environment in postmortem muscle. Understanding how mitochondria participate in postmortem metabolism may help us identify cellular targets for the development of processing strategies that improve meat quality. It is particularly challenging to evaluate mitochondria since they are functionally complex and dynamic organelles that vary in content and distribution within and across muscles. Mitochondria content varies significantly across muscles within an individual, which is related to functional demands and work performed. In addition, the same muscle across animals may exhibit differences in mitochondria content based on genetics, activity, or other factors. Both aspects may contribute to variation in postmortem metabolism and meat quality. In order to evaluate their functional properties, mitochondria are often isolated, or taken out of their native cellular environment. However, we are utilizing permeabilized muscle cells, which preserves mitochondria in their normal habitat. Using this approach, we have demonstrated that mitochondria remain functional and intact within the first hour postmortem; mitochondrial function appears to be maintained despite differences in breed/genetics or muscle function. We used distinct biological types of cattle (Bos indicus, Brahman vs Bos taurus, Angus) to produce beef with greater variation in tenderness. In an initial study, we showed that certain mitochondrial properties related to energy utilization and efficiency may differ between breeds and may be related to heat tolerance. We have conducted follow-up study evaluating mitochondria function during the first 24h postmortem in these breeds. Evidence suggests that mitochondria function during the postmortem period may be related to breed; Brahman generally show greater ability to maintain mitochondria function, which is related to improved energy status. This suggests that Brahman muscle may possess mechanisms that protect against cellular stress thus delaying loss in mitochondrial function. Understanding resilience of mitochondria and postmortem energy metabolism in Brahman may help improve meat quality without sarcrificing their adaptability to hot climates. Further, our work with muscles differing in metabolic and contractile properties (slow, oxidative vs. fast, glycolytic) indicate that greater mitochondrial content and slow fiber type are associated with an improved capacity to maintain mitochondrial function postmortem. This may also be related to faster temperature decline. In turn, this may provide insight into variation in tenderness and color development in muscles with different metabolic and contractile characteristics, as well as impact of temperature on these traits. Objective 1. Evaluate changes in mitochondria function in oxidative and glycolytic muscles during the first 24h postmortem.Manuscript was submitted, revised, and published. Objective 2. Determine mitochondria function in oxidative and glycolytic muscles under pH and oxygen tension conditions that simulate postmortem muscle. Initial tests are underway to evaluate mitochondria function, but progress has been delayed and limited to Covid-19. Access to critical equipment was limited from Mar. 2020 through spring 2021 (spacing restrictions and access - located in Shands Hospital at UF). Additional analyses have been undertaken to assess individual complexes using plate based assays. Assays are being conducted at pHs representing postmortem muscle, and samples collected during the first 24h postmortem are also being analyzed to determine time-related changes in function. Objective 3. Assess mitochondria respiration and postmortem metabolism in longissimus muscles with varying mitochondria content. Analyses for mitochondria function, glycolytic metabolites, pH, and temperature are complete. Tenderness and color of steaks were also determined. Results were presented as abstracts (posters) and manuscripts are currently being drafted. Samples for electron microscopy were sectioned; images have been collected and are currently being analyzed for changes in mitochondria morphology.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ramos, PM, LC Bell, SE Wohlgemuth, and TL Scheffler. 2021. Mitochondrial function in oxidative and glycolytic bovine skeletal muscle postmortem. Meat and Muscle Biology. 5(1): 11, 1-16. doi: 10.22175/mmb.11698.
- Type:
Other
Status:
Accepted
Year Published:
2020
Citation:
Bell, LC, B Hawryluk, MT McKinney, PM Ramos, and TL Scheffler. 2020. Citrate synthase activity as a marker for mitochondrial content in postmortem muscle. International Congress of Meat Science and Technology and Reciprocal Meat Conference. (abstract and poster presentation)
- Type:
Other
Status:
Accepted
Year Published:
2020
Citation:
Ramos, PM, B Hawryluk, L Bell, MT McKinney, SE Wohlgemuth, and TL Scheffler. 2020. Mitochondrial function and integrity in postmortem muscle of Brahman and Angus steers. International Congress of Meat Science and Technology and Reciprocal Meat Conference. (abstract and poster presentation)
- Type:
Other
Status:
Accepted
Year Published:
2020
Citation:
Gingerich, CA, D Jean, E Hicks, B Hawryluk, LC Bell, M McKinney, PM Ramos, and TL Scheffler. 2020. Postmortem energy metabolism in longissimus lumborum of Brahman and Angus steers. International Congress of Meat Science and Technology and Reciprocal Meat Conference. (abstract and poster presentation)
- Type:
Other
Status:
Accepted
Year Published:
2020
Citation:
McKinney, MT, B Hawryluk, CC Carr, PM Ramos, and TL Scheffler. 2020. Proteolysis of Angus and Brahman aged steaks. International Congress of Meat Science and Technology and Reciprocal Meat Conference. (abstract and poster presentation)
- Type:
Other
Status:
Submitted
Year Published:
2021
Citation:
Hawryluk, B, MT McKinney, CA Gingerich, LC Bell, PM Ramos, and TL Scheffler. 2021. Organ and cellular contributions to heat production in Brahman and Angus steers. American Society of Animal Sciences Annual Meeting. Louisville, KY.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
TL Scheffler. Mitochondrial contribution to postmortem metabolism. ICoMST/RMC International Congress of Meat Science and Technology/Reciprocal Meat Conference. August 5, 2020 (~130 attendees)
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
McKinney, MT. Meat quality development and protein degradation between Bos taurus and Bos indicus cattle. 2020. University of Florida.
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:The target audiences includesstudents, researchers, and academicians in meat science and muscle biology. Primarily, efforts are directed to education and scientific training of scholars and students (graduate and undergraduate). For the scholar and students, the audienceincluded minorities (1 African American, 2 Asian American, 2 Latina), as well as a single mother.The research scholar receivedtraining in muscle biology and physiology, and designed experiments and performedassays, data analysis, and interpretation to evaluate the role of mitochondria inpostmortem metabolism and meat tenderization. Two graduate students (both MS) and several undergraduate students are receiving training as part of their degree programs. Graduate students conduct research activities related to experimental design, sample collection,and laboratory analysis, and help train undergraduate students. One undergraduate student served as an intern during the summer semester and participated in sample collection and processing, and laboratory analysis. The remaining undergraduate students were enrolled for course credit (for research/lab experience); all conducted sample processing and assays, and performed analysis and interpretation of data. Students participated in lab meetings and journal clubs as part of their educational program. More broadly, works were also presentedduringextension programs (Beef Cattle Short Course, Brahman Field Day, and Beef 706; all held at University of Florida). The audience was primarily beef producers for these events. Changes/Problems:Major changes and issues relate to COVID-19 and equipment availability. Limited access to respirometer equipment has delayed progress on Objective 1; progress was further delayed due to COVID-19 restrictions. Similar issues exist for electron microscopy analysis. These analysesrelyon core facilities, and we have been delayed due to limited throughput in the facility (waiting list) and COVID-19 restrictions. What opportunities for training and professional development has the project provided?This project is providing professional development and training for a research scholar, graduate students, and undergraduate students. The scholar is mentored by the PI and co-PI, and is directly involved with experimental design, sample collection, laboratory analysis, and data analysis and interpretation. The research scholar also was involved in training and mentoring undergraduate and graduate students. The graduate students are involved with experimental design, sample collection, laboratory analysis, data analysis, and interpretation, and are preparing theses regarding aspects of the project. The undergraduates students are learning techniques related to sample collection and processing, as well as how to perform laboratory analyses, and interpret results. All are involved in lab meetings and journal clubs. How have the results been disseminated to communities of interest?Results from this project were presented as posters atnational conferences in June 2019 and July 2019. Four posters will be presented at an international conference in August 2020. Manuscripts are being prepared for submission to journals. Results were also presented at extension events including Florida Beef Cattle Short Course, Brahman Field Day, and Beef 706. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Manuscripts are being revised for submission to journals.Additional analysis is underway to investigate potential mechanisms contributing to changes in complex I and complex II activity. Objective 2: Studies will be conducted in Fall 2020 through Spring 2020 to investigate effects of pH and oxygen concentration on mitochondria function. These experiments have been delayed due to issues with access to equipment, and then by facility restrictions due to COVID19. These circumstances have delayed the training of the student responsible for leading these experiments. Student will resume training when restrictions are eased. Objective 3: Electron microscopy analyses will be performed using core facilities at UF. These analyses have been delayed due to waiting time for equipment accessand COVID19. Other laboratory assaysare complete, and statistical analysis is complete. Additional analysis of data will be conducted to understand relationships between mitochondria function, glycoltyic metabolites, ATP, and proteolysis during the first 24h postmortem. Manuscripts will be prepared for submission to journals.
Impacts
What was accomplished under these goals?
Meat quality attributes, including color and tenderness, are key determinants of product value and consumer acceptability. These attributes are largely governed by (1) inherent characteristics related to muscle function and metabolism in the live animal, and (2) the conditions during the early postmortem period, roughly the first 24 hours after death; this period includes physical, biochemical, and energetic changes that ensue as living muscle is converted to meat. In living muscle, mitochondria utilize oxygen to produce energy. At harvest, the blood supply is removed, and oxygen can no longer be delivered to muscle. Accordingly, approaches to evaluate postmortem muscle have relied heavily on methodologies which ignore the contribution of mitochondria. Yet, mitochondria regulate other cellular functions, and evidence supports that mitochondria mediated events, including apoptosis and oxidative stress, are associated with meat quality. There is a gap in knowledge regarding how mitochondria function and integrity impact the cellular environment in postmortem muscle. Understanding how mitochondria participate in postmortem metabolism may help us identify cellular targets for the development of processing strategies that improve meat quality. It is particularly challenging to evaluate mitochondria since they are functionally complex and dynamic organelles that vary in content and distribution within and across muscles. Mitochondria content varies significantly across muscles within an individual, which is related to functional demands and work performed. In addition, the same muscle across animals may exhibit differences in mitochondria content based on genetics, activity, or other factors. Both aspects may contribute to variation in postmortem metabolism and meat quality. In order to evaluate their functional properties, mitochondria are often isolated, or taken out of their native cellular environment. However, we are utilizing permeabilized muscle cells, which preserves mitochondria in their normal habitat. Using this approach, we have demonstrated that mitochondria remain functional and intact within the first hour postmortem; mitochondrial function appears to be maintained despite differences in breed/genetics or muscle function. We used distinct biological types of cattle (Bos indicus, Brahman vs Bos taurus, Angus)to investigate mitochondrial function early postmortem. Brahman are well-known to possess greater heat tolerance, but tend to produce beef with greater variation in tenderness. In an initial study, we showed that certain mitochondrial properties related to energy utilization and efficiency may differ between breeds and may be related to heat tolerance. We have conducted follow-up study evaluating mitochondria function during the first 24h postmortem in these breeds. Evidence suggests that mitochondria function during the postmortem period may be related to breed; Brahman generally show greater ability to maintain mitochondria function, which is related to improved energy status. This suggests that Brahman muscle may possess mechanisms that protect against cellular stress thus delaying loss in mitochondrial function. Understanding resilience of mitochondria and postmortem energy metabolism in Brahman may help improve meat quality without sarcrificing their adapatability to hot climates. Objective 1. Evaluatechanges in mitochondria function in oxidative and glycolytic muscles during the first 24h postmortem.Manuscript is in preparation for submission to journal. Further analysis revealed that complex II activity partially compensates for reduced complex I function. Objective 2. Determine mitochondria function in oxidative and glycolytic muscles under pH and oxygen tension conditions that simulate postmortem muscle. Graduate student is receiving training in muscle sample collection and mitochondria analysis and experiments will be conducted in Fall 2020 through Spring 2021. Objective 3. Assess mitochondria respiration and postmortem metabolism in longissimus muscles with varying mitochondria content. Steers were harvested in summer 2019, and longissimus samples were collected during the first 24h postmortemfor assessment of mitochondria function, mitochondria structure and morphology, enzyme activity, protein expression, energy metabolism, and proteolysis.Temperature and pH were measured during this time period. More specifically, longissimus muscle was collected at 1, 3, 6, and 24h postmortem from cattle of different biological types (80-100% Angus or 80-100% Brahman; n = 13 per breed). For mitochondria analysis,permeabilized fibers were evaluated using a high resolution oxygraph for respiration measurements (oxygen consumption rate, OCR). Glycolytic metabolites (glycogen, glucose, glucose 6-phospate, and lactate), along with ATP and phosphocreatine, were quantified. In order to evaluate potential relationships between mitochondria function and meat quality development, Western blotting was conducted for calpain autolysis, calpastatin content, and degradation of Troponin T and desmin. Data were analyzed using the main effects of breed, time, and their interaction; time was considered a repeated measure. For mitochondria function, complex Imediated oxidative phosphorylation (OXPHOS) capacity decreased over time (P<0.001), but was not affected by breed. Curiously, complex I+II mediated OXPHOS capacity depended on both breed and time (P = 0.01). At 6h, complex I+II function was higher in Brahman. Cytochrome c response increased during the postmortem period (P<0.001), indicating that the outer membrane was becoming compromised. Citrate synthase activity, a marker of mitochondria content, was not different between breeds. As expected, glycogen decreased during the postmortem period (P<0.001), and glucose and lactate increased (P<0.001), but glycolytic metabolites were not affected by breed. Brahman exhibited higher ATP (breed: P=0.02), and ATP decreased from 1h to 6h (P<0.0001). The pH decline tended to differ between breeds (breed x time: P=0.07), with Brahman exhibiting higher pH than Angus at 6h and 9h (P=0.02). Temperature decline differed between breeds (breed x time: P=0.0008), with Brahman possessing lower longissimus temperature at 1, 3, 6, and 9h postmortem. With regard to tenderization, Brahman showing less u-calpain autolysis(P=0.004) and reduced degradation of troponin T (P=0.003) and desmin (P=0.02) at 24h postmortem. Previously, we had observed greater citrate synthase acitivity in longissimus of Brahman compared to Angus, and we had proposed that differences in mitochondria content and function in breeds impacted meat quality development. However, citrate synthase activity and protein expression results do not support breed differences in mitochondria content. However, Angus and Brahman exhibited differences in mitochondria function parameters at 1h postmortem and thereafter, indicating there are breed-specific responses to the cellular stresses (hypoxia, declining pH, etc.) that ensue postmortem. This is consistent with elevated ATP levels in Brahman muscle, suggesting improved maintenance of energy status and resilience in response to a changing cellular milieu. We expect this may be linked to enhanced thermotolerance of Brahman compared toAngus, but further work is necessary to support this. Meanwhile, improved energy status in Brahman does not appear to be related to glycolytic metabolism, but it may be associated with delayed progression of protein degradation and tenderization. Regardless of breed, mitochondria function data support that mitochondria are relatively well-coupled and retain OXPHOS capacity early postmortem. Together with results from Objective 1, these datasupport that complex I function decreases postmortem, whereas complex II activity partially compensates. Further work is required to determine the mechanisms underlying these shifts in mitochondria function.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
PM Ramos, CC Li, MA Elzo, SE Wohlgemuth, and TL Scheffler. Mitochondrial oxygen consumption in early postmortem permeabilized skeletal muscle fibers is influenced by cattle breed. Journal of Animal Science. 98:1-10 doi: 10.1093/jas/skaa044
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
TL Scheffler. Heat adaptability and tenderness. Florida Beef Cattle Short Course Proceedings. May 8-10, 2019. p. 32-34. (Paper and presentation)
|
Progress 05/01/18 to 04/30/19
Outputs
Target Audience:The target audience is mainly students, researchers, and academicians in meat science and muscle biology. One research scholar is receiving training in muscle biology and physiology; the research scholar is designing experiments and performing assays, data analysis and interpretation to evaluate the role of mitochondria in postmortem metabolism and meat tenderization. One graduate student (PhD) is participating in research activities related to sample collection and laboratory analysis. Undergraduate students are assisting with sample collection and laboratory assays. These students are either employed as undergraduate laboratory assistants,completing undergraduate supervised research for credit, or are volunteering their time in the lab to gain research experience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project is providing professional development and training for a research scholar, a graduate student, and an undergraduate student. The scholar is mentored by the PI and co-PI, and is directly involved with experimental design, sample collection, laboratory analysis, and data analysis and interpretation. The research scholar is also involved in helping train a graduate student and several undergraduates. The graduate student is involved with benchtop analysis. The undergraduate students are learning techniques related to sample collection and processing, as well as how to perform laboratory and data analyses, and interpret results. How have the results been disseminated to communities of interest?Results from this project were submitted as four abstracts to two professional conferences; these will be presented as posters at the conferences in summer 2019. Manuscripts are being prepared for submission to journals. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Manuscripts are in preparation. Objective 2: Studies will be initiated in Fall 2019to investigate effects ofpH and oxygen concentration on mitochondria function Objective 3: One manuscript is in preparation from study evaluatingmitochondria function in muscle from Brahman and Angus. Another study has been initiated to assesschanges in mitochondria functionduring the early postmortem period in Brahman and Angus longissimus and its potential relationships to tenderization and color. Cattle are on feed and will be harvested in Summer 2019.
Impacts
What was accomplished under these goals?
Meat quality attributes, including color and tenderness, are key determinants of product value and consumer acceptability. These attributes are largely governed by (1) inherent characteristics related to muscle function and metabolism in the live animal, and (2) the conditions during the early postmortem period, roughly the first 24 hours after death; this period includes physical, biochemical, and energetic changes that ensue as living muscle is converted to meat. In living muscle, mitochondria utilize oxygen to produce energy. At harvest, the blood supply is removed, and oxygen can no longer be delivered to muscle. Accordingly, approaches to evaluate postmortem muscle have relied heavily on methodologies which ignore the contribution of mitochondria. Yet, mitochondria regulate other cellular functions, and evidence supports that mitochondria mediated events, including apoptosis and oxidative stress, are associated with meat quality. There is a gap in knowledge regarding how mitochondria function and integrity impact the cellular environment in postmortem muscle. Understanding how mitochondria participate in postmortem metabolism may help us identify cellular targets for the development of processing strategies that improve meat quality. It is particularly challenging to evaluate mitochondria since they are functionally complex and dynamic organelles that vary in content and distribution within and across muscles. Mitochondria content varies significantly across muscles within an individual, which is related to functional demands and work performed. In addition, the same muscle across animals may exhibit differences in mitochondria content based on genetics, activity, or other factors. Both aspects may contribute to variation in postmortem metabolism and meat quality. In order to evaluate their functional properties, mitochondria are often isolated, or taken out of their native cellular environment. However, we are utilizing permeabilized muscle cells, which preserves mitochondria in their normal habitat. Using this approach, we have demonstrated that mitochondria remain functional and intact within the first hour postmortem; mitochondrial function appears to be maintained despite differences in breed/genetics or muscle function. We used distinct biological types of cattle (Bos indicus, Brahman vs Bos taurus, Angus) to investigate mitochondrial function early postmortem. Brahman are well-known to possess greater heat tolerance, but tend to produce beef with greater variation in tenderness. We showed that certain mitochondrial properties related to energy utilization and efficiency may differ between breeds and may be relatedto heat tolerance. In another experiement, we used muscles that vary in their functional properties to demonstrate declines in mitochondrial function postmortem. By 24h postmortem, the more glycolytic longissimus muscle showed compromised integrity and function, which is associated with greater activation of enzymes involved in protein degradation and tenderization. The loss in structural integrity is related to inherent muscle functional properties. Ongoing studies are aimed at pinpointing the time when mitochondria structure begins to deteriorate, and evaluate its progression in distinct muscles and genetic groups. These changes influence the cellular environment, and thus are expected to modulate the development of meat color and tenderness. Objective 1. Evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem. Longissimus (glycolytic) and diaphragm (oxidative) muscles were collected from market steers (n=6) at 1,3, and 24h postmortem. Fresh tissue was evaluated in a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. Another portion of tissue was frozen in liquid nitrogen and used for enzyme activity analysis, metabolites, and Western blotting for protein content and degradation.Citrate synthase (CS) activity was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity to account for differences in mitochondria content. Coupling efficiency of oxidative phosphorylation was calculated as 1 - (Leak / ADP-stimulated oxidative phosphorylation capacity). Raw and normalized OCR were analyzed in a randomized block design, with slaughter date as block and fixed effects of muscle, time, and the interaction. Time was considered a repeated measure. Muscle type affected (P = 0.0002) leak OCR, with diaphragm showing a higher rate than longissimus. After ADP was added, mitochondria from diaphragm exhibited higher OCR at all times tested and at all steps, with OCR being 4 times higher after FCCP addition. Mitochondrial content, evidenced by greater (P < 0.0001) CS activity in diaphragm, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from diaphragm and longissimus were similar (P > 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P < 0.05) in diaphragmmitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR <10%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased over 50%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with diaphragm exhibiting greater efficiency. Glycolytic metabolites, pH, and ATP were also determined at the same time points to understand potential relationships with mitochondrial function.Overall, a greater decline in glycogen occurred in longissimus. Accordingly, lactate concentration increased markedly in longissimuspostmortem and to a lesser extent in diaphragm(interaction effect; P < 0.01).Muscles exhibited different patterns of postmortem pH decline (muscle × time, P < 0.0001). Initially, pH of longissimuswas higher than diaphragm(P < 0.01) and remained different at 3h (P < 0.05); but by 24h, pH values were similar. Content of ATP was influenced by muscle (P < 0.01) and time (P < 0.01). Initial ATP was greater (P < 0.01) in longissimusthan in diaphragm, and remained greater (P = 0.002) at 3h postmortem. From 1 to 24h, the pattern of calpain autolysis differed between muscles (interaction effect; P = 0.01). Calpain-1 autolysis was similar at all times in diaphragm, whereas autolysis increased in longissimusfrom 3h to 24h postmortem.While the diaphragmis considered a slow muscle, it exhibited a more rapid pH decline and lower ATP levels than longissimusearly postmortem.Despite inherent metabolic differences between the longissimusand diaphgram, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of longissimus mitochondria are reduced compared to diaphgram. Declining mitochondrial function may be associated withprotease activation. Objective2. Determine mitochondrial function in oxidative and glycolytic muscles under pH and oxygen tension conditions that simulate postmortem muscle.We will start these experiments in Fall 2019. Objective 3. Assess mitochondrial respiration and postmortem metabolism in longissimus muscles with varying mitochondrial content. Longissimus will be collected at 1, 3, 6, and 24h from cattle of different biological types (80-100% Angus or 80-100% Brahman) and permeabilized fibers will be used for mitochondria function analysis. Temperature and pH will also be evaluated along with glycolytic metabolites, ATP, and protease activation. Location and morphology of mitochondria will be evaluated via electron microscopy.
Publications
- Type:
Other
Status:
Accepted
Year Published:
2019
Citation:
Ramos, PM, LC Bell, SE Wohlgemuth, and TL Scheffler. 2019. Mitochondrial function in oxidative and glycolytic bovine skeletal muscle. Reciprocal Meat Conference. Loveland, CO. (abstract and poster)
- Type:
Other
Status:
Accepted
Year Published:
2019
Citation:
Ramos, PM, CC Li, MA Elzo, SE Wohlgemuth, and TL Scheffler. 2019. Mitochondrial function of skeletal muscle early postmortem is influenced by cattle breed and temperature. American Society of Animal Science. Austin, TX. (abstract and poster)
- Type:
Other
Status:
Accepted
Year Published:
2019
Citation:
Ramos, PM, M Pedrao, LC Bell, and TL Scheffler. 2019. Early postmortem metabolism and protease activation in bovine muscles. Reciprocal Meat Conference. Loveland, CO. (abstract and poster)
- Type:
Other
Status:
Accepted
Year Published:
2019
Citation:
B Hawryluk, PM Ramos, DD Johnson, MA Elzo, CC Carr, and TL Scheffler. 2019. Comparing heat shock proteins in Angus and Brahman cattle and their effect on tenderness. Reciprocal Meat Conference. Loveland, CO. (abstract and poster)
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Progress 05/01/17 to 04/30/18
Outputs
Target Audience:The target audience is mainly students, researchers, and academicians in meat science and muscle biology. Currently, one research scholar is receiving training in muscle biology and physiology; the research scholar isdesigning experiments and performing assays, data analysis and interpretationtoevaluate the role of mitochondria in postmortem metabolism and meat tenderization.Undergraduate students are assisting with sample collection and laboratory assays. These students are either completing undergraduate supervised research for credit, or are volunteering their time in the lab to gain research experience. Preliminary findings werediscussedat an extension event (Florida Beef Cattle Short Course, 2017). The oral presentation entitled "Exploring factors that contribute to beef tenderness" was given to ~200 participants, primarily cattle producers that are members of the Florida Cattlemen's Association. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project is providing professional development and training forseveral individuals, including aresearch scholar and undergraduate students. The scholar (Patricia Ramos)is mentored by the PI and co-PI, and is directly involved with experimental design, sample collection, laboratory analysis, and data analysis and interpretation. This project is also providing training toundergraduate students, whoare performing sample collection and benchtop analysis; this isgiving them hands-on opportunities to learn techniques and procedures, gain lab experience, and interpret data. How have the results been disseminated to communities of interest?Preliminary results were included in an oral presentation at an event mainly attended by beef cattle producers (Beef Cattle Short Course; Gainesville, FL; 2017). Factors influencing beef tenderness were discussed, including the potential role for mitochondria in influencing postmortem metabolism and development of meat quality attributes. What do you plan to do during the next reporting period to accomplish the goals?During the next period, we will continue to conduct experiments and analysispertaining to Objectives 1-3. Specific actions to address issues: Objective 1: We will collect samples from oxidative and glycolytic muscles during the first 24h postmortem. Initially, we proposed that all studies would be conducted in bovine muscle. However, in running practicetrials with porcine muscle, we have found intriguing results andplan to incorporate porcine muscle in orderto draw comparisons between species and metabolic type. Objectives 1 & 3:We have not been able to collect samples at 30 minutes, and instead are utilizing samples collected at 1h. Although we do not have a pre-harvest or 0 min sample for comparison, the samples collected at 1h do not show evidence of compromised mitochondrial integrity. Forobjective 1, weare utilizing diaphragm as an oxidative muscle (instead of sternomandibularis).The orientation of fibers in the diaphragm faciliates repeated sampling because samples can beremoved with relatively less impact to the remaining muscle.
Impacts
What was accomplished under these goals?
It is particularly challenging to evaluate mitochondria since they are functionally complex and dynamic organelles that vary in content and distribution within and across muscles. Mitochondria content varies significantly across muscles within an individual, which is related to functional demands and work performed. In addition, the same muscle across animals may exhibit differences in mitochondria content based on genetics, activity, or other factors. Both aspects may contribute to variation in postmortem metabolism and meat quality attributes. In order to evaluate their functional properties, mitochondria are often isolated, or taken out of their native cellular environment. However, we are utilizing permeabilized muscle cells, which preserves mitochondria in their normal habitat. Using this approach, we have demonstrated that mitochondria remain functional and intact within the first hour postmortem; mitochondrial function appears to be maintained despite differences in breed/genetics or muscle function. However, within the first 24 hours mitochondrial integrity is significantly disrupted. The loss in structural integrity is related to inherent muscle functional properties. Ongoing studies are aimed at pinpointing the time when mitochondria structure begins to deteriorate, and evaluate its progression in distinct muscles and genetic groups. Moreover,our results support that breedtype influences mitochondrial content of longissimus muscle; this may relate to delayed pH decline and slower progression of tenderization in Bos indicus influenced cattle.Overall,changes in mitochondrial integrity and function influence the cellular environment, and thus are expected to modulate the development of meat quality attributes, such as color and tenderness. Objective 1. Evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Muscles from functionally distinct muscles (diaphragm, oxidative; superficial pectoralis, intermediate) were collected at 1h and 24h postmortem. Muscle samples were permeabilized and oxygen consumption was determined in presence of substrates. Integrity was evaluated using response to cytochrome c, and <15% increase in oxygen consumption was used as a benchmark for intact mitochondria. Mitochondria are intact within the first hour, but surpass the 15% standard at 24h. However, the increase in oxygen consumption with cytochrome c at 24h was not as high as expected in the small set analyzed thus far. The longissimus will be incorporated into future analyses, and additional samples at more timepoints will be collected in order to delineate a time course for loss in integrity and function. Objective 2. Determine mitochondrial function in oxidative and glycolytic muscles under pH and oxygen tension conditions that simulate postmortem muscle. We have not started these experiments; however, our data collected with 24h samples from bovine muscles suggest that the disruption of mitochondrial integrity and function may be slower than expected in bovine muscles. Objective 3. Assess mitochondrial respiration and postmortem metabolism in longissimus muscles with varying mitochondrial content. Samples from longissimus muscle were collected from Angus (80-100% Angus, 0-20% Brahman; n=13) and Brahman steers (0-20% Angus, 80-100% Brahman; n=13) at 1h postmortem. Our preliminary data suggested breed affects mitochondria content, as evidenced by the mitochondria marker citrate synthase. Consistent with this, longissimus from Brahman exhibited higher citrate synthase activity (P<0.05), which was approximately 20% greater than Angus. We evaluated parameters of mitochondria function, including leak respiration, state 3 respiration (ADP-stimulated respiration), state 3 (complexes I+II), state 3 (complex II only), and electron transport system capacity (determined with an uncoupler). Cytochrome c was also used to evaluate mitochondrial integrity, with all samples considered acceptable (<15% increase in oxygen flux). Data for oxygen flux were analyzed on a tissue unit basis, as well as per mitochondrial unit (normalized to citrate synthase activity). On a tissue basis, there was a trend for breed (P=0.11) to influence state 3 respiration (complex I+II). Brahman showed numerically greater state 3 values, which may be partly attributed to differences in mitochondrial content. In agreement, the effect of breed on state 3 (complex I+II) disappeared once data were adjusted for citrate synthase activity. However, on a per mitochondria unit basis, leak respiration was lower in Brahman, suggesting that there are qualitative differences in mitochondrial components related to breed type. The pH decline of Brahman longissimus tends to be slower than Angus, and we suggest this prolonged postmortem metabolism may delay loss in mitochondrial function, and also delay tenderization in Brahman. Brahman (Bos indicus) generally exhibited reduced proteolysis, and subsequently, generate beef with higher shear force and lower sensory tenderness scores. Next, we will determine if breed impacts the time course for loss of mitochondria function in longissimus.
Publications
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