Dark-cutting beef mitochondrial proteomic signatures reveal increased biogenesis proteins and bioenergetics capabilities.

Mitochondria remain active in postmortem muscles and can influence meat color via oxygen consumption. Previous studies have shown that dark-cutting compared with normal-pH beef has greater mitochondrial protein and DNA content per gram of muscle tissue. However, the mechanism regulating mitochondrial content in dark-cutting vs. normal-pH beef is still unknown. Therefore, the objective was to compare mitochondrial proteomes of dark-cutting vs. normal-pH beef using LC-MS/MS-based proteomics and mitochondrial respiratory capacity using a Clark oxygen electrode. Dark-cutting compared with normal-pH beef has up-regulation of proteins involved in mitochondrial biogenesis, oxidative phosphorylation, intracellular protein transport, and cellular calcium ion homeostasis. Mitochondria isolated from dark-cutting phenotypes showed greater mitochondrial complex II respiration and uncoupled oxidative phosphorylation. However, mitochondrial membrane integrity and respiration at complexes I and IV were not different between normal-pH and dark-cutting beef. These results indicate that dark-cutting beef has greater mitochondrial biogenesis proteins than normal-pH beef, increasing mitochondrial content and contributing to dark-cutting beef. SIGNIFICANCE: Defective glycogen metabolism resulting from chronic stress before slaughter coupled with the greater mitochondrial protein and DNA content per gram of muscle tissue promotes muscle darkening in dark-cutting phenotypes in beef. However, the mechanistic basis for this occurrence in dark-cutting phenotypes is still unknown. In this work, we show that dark-cutting beef phenotype is caused, in part, as a consequence of over-proliferation of mitochondria. This is supported by the up-regulation of proteins involved in mitochondrial biogenesis, mitochondrial electron transport, calcium homeostasis, and fatty acid metabolism. Hence, the study of mitochondrial proteome changes provides a set of mitochondrial biogenesis proteins that could be used as potential candidate markers for detecting changes in pre-slaughter developmental events contributing to dark-cutting phenotypes in beef.

Changes in glycolytic and mitochondrial protein profiles regulates postmortem muscle acidification and oxygen consumption in dark-cutting beef.

Dark-cutting beef is a condition in which beef fails to have a characteristic bright-red color when the cut surface is exposed to oxygen. However, the mechanistic basis for this occurrence is not clear. Protein expression profiles were compared between dark-cutting and normal-pH beef using LC-MS/MS-based proteomics. Mass spectrometry analysis identified 1162 proteins in the proteomes of dark-cutting and normal-pH beef. Of these, 92 proteins had significant changes in protein abundance between dark-cutting versus normal-pH beef. In dark-cutting beef, 25 proteins were down-regulated, including enzymes related to glycogen metabolism, glucose homeostasis, denovo synthesis of adenosine monophosphate (AMP), and glycogen phosphorylase activity. In comparison, 27 proteins were up-regulated in dark-cutting beef related to oxidation-reduction processes, muscle contraction, and oxidative phosphorylation. Down-regulation of glycogenolytic proteins suggests decreased glycogen mobilization and utilization, while the up-regulation of mitochondrial transport chain proteins indicates a greater capacity to support mitochondrial respiration in dark-cutting beef. These results showed that changes in proteins involved in glycogenolysis and mitochondrial electron transport would promote the development of high-pH and greater oxygen consumption, respectively; thus limiting myoglobin oxygenation in dark-cutting beef. SIGNIFICANCE: The current understanding indicates that defective glycolysis causes less carbon flow, leading to less postmortem lactic acid formation and elevated muscle pH in dark-cutting beef. However, to the best of our knowledge, limited research has evaluated how changes in glycolytic and mitochondrial protein abundance regulate postmortem muscle acidification and oxygen consumption in dark-cutting beef. We utilized a shotgun proteomics approach to elucidate potential differences in protein profiles between dark-cutting versus normal-pH beef that may influence differences in postmortem metabolism and muscle surface color characteristics. Our study shows that down-regulation of glycolgenolytic and IMP/AMP biosynthetic proteins results in elevated postmortem muscle pH in dark-cutting beef. In addition, the up-regulation of mitochondrial protein content coupled with the higher muscle pH are conducive factors for enhanced oxygen consumption and less myoglobin oxygenation, contributing to a dark meat color typically associated with dark-cutting beef.

Characterization of water intake and water efficiency in beef cattle1,2.

In the future, water may not be as readily available due to increases in competition from a growing human population, wildlife, and other agricultural sectors, making selection for water efficiency of beef cattle increasingly important. Substantial selection emphasis has recently been placed on feed efficiency in an effort to reduce production costs, but no emphasis has been placed on making cattle more water efficient due to lack of data. Thus, the objective of this study was to calculate water efficiency metrics for cattle and evaluate their relationship to growth, feed intake (FI), and feed efficiency. Individual daily FI and water intake (WI) records were collected on 578 crossbred steers over a 70-d test period. Animals with low water intake ate less feed, had lower gains, and were more water efficient (as defined by water to gain ratio, W/G, and residual water intake, RWI). However, the amount of water consumed by animals had minimal phenotypic relationship with feed efficiency (residual feed intake [RFI], R2 = 0.1050 and feed to gain ratio (F/G) ratio R2 = 0.0726). Cattle that had low DMI consumed less water, had lower gains, had lower RFI, and had higher F/G. The level of feed consumed had minimal relationship with water efficiency. WI, W/G, RWI, and ADG had moderate heritability estimates of 0.39, 0.39, 0.37, and 0.37, respectively. High heritability estimates were observed for DMI and RFI (0.67 and 0.65, respectively). Feed to gain had a low heritability estimate of 0.16. WI had a strong positive genetic correlation with W/G (0.99) and RWI (0.88), thus selecting for decreased WI should also make cattle more water efficient. The genetic correlation between WI and ADG was 0.05; thus, selecting for low WI cattle should have little effect on growth. There is a low to moderate genetic correlation between WI and DMI (0.34). RWI has a positive genetic correlation with W/G ratio (0.89) and F/G ratio (0.42) and is negatively genetically correlated with RFI (-0.57). Water to gain and F/G had a strong positive genetic correlation (0.68). RFI has a positive genetic correlation with W/G ratio (0.37) and F/G (0.88). Minimal antagonisms seem to be present between WI and ADG, although it should be noted that standard errors were large and often not significantly different from zero due to the small sample size. However, care should be taken to ensure that unintended changes do not occur in DMI or other production traits and incorporation of WI into a selection index would likely prove to be the most effective method for selection.

Frequencies and severity of injection-site lesions in muscles from rounds of cow carcasses.

The frequency and severity of injection-site lesions in the outside round muscles of both beef and dairy cattle were evaluated through a series of audits. Audits were conducted in 2017 on 1,300 rounds from dairy and beef cows from seven locations throughout the United States. Outside round muscles were butterfly cut into 1.25-cm slices and, if present, lesions were counted, measured, and categorized. Rounds from beef (7%) and dairy cattle (15%) had at least one injection-site lesion present. The most common location of injection-site lesions was quadrant 2 and 3, which contained both the biceps femoris and semitendinosus muscles. Injection-site lesions were more frequent (P < 0.05) in the biceps femoris for both beef and dairy rounds. Clear lesions accounted for 57% of injection-sites in both beef and dairy rounds, whereas metallic lesions made up 23% of the total in beef and 25% in dairy. Overall, there was a dramatic decline in the frequency (P < 0.05) of injection-site lesions since the 1998 (24 and 45 percentage units greater in beef and dairy rounds, respectively) and 2000 audits (13 and 20 percentage units greater in beef and dairy rounds, respectively). Educational programs, such as Beef Quality Assurance (BQA) and requirements for BQA training, have resulted in substantial improvements in beef management practices for both the beef and dairy industries.

Mitochondrial Degeneration, Depletion of NADH, and Oxidative Stress Decrease Color Stability of Wet-Aged Beef Longissimus Steaks.

Interrelationship between mitochondria and myoglobin function influence beef color. NADH level in postmortem muscle is an important determinant of mitochondrial activity and metmyoglobin reduction. Increased aging time promotes discoloration of steaks; however, the mechanism of this effect is not clear. The objective was to characterize the role of wet-aging in beef longissimus lumborum muscle mitochondrial function and to characterize the global metabolome to determine the mechanism of that can regenerate NADH. Beef longissimus lumborum muscles were randomly assigned to 3, 7, 14, 21, and 28 days aging periods. Surface color, biochemical, mitochondrial, and metabolite profiles were determined at each aging period and at the end of 6-day display. During 6-day display, sections aged for 28 days had 30.4% decrease in redness than sections aged for 3 days. Aging time decreased (P <0.05) muscle oxygen consumption, mitochondrial protein content, and antioxidant capacity. Metabolites such as fumaric acid, creatinine, and fructose, that can take part in glycolytic/TCA cycle and regenerate NADH decreased (P <0.05) with aging and display time. In support, NADH levels also decreased (P <0.05) with aging time, but aging time had no effect (P = 0.44) on NADH-dependent reductase activity. The results suggest that decreased color stability in aged beef can be attributed to increased mitochondrial damage, depletion of metabolites that can regenerate NADH, and increased oxidative stress. PRACTICAL APPLICATION: Beef aging time results in increased discoloration of steaks under retail display. The current research determines the fundamental basis of lower color stability in aged beef. The results indicate that mitochondrial degeneration, depletion of metabolites that produce NADH, and increased oxidative stress can limit shelf-life of aged steaks. Hence, application of post-harvest strategies to minimize mitochondrial damage and oxidative changes may have the potential to increase shelf-life of aged beef.

Environmental effects on water intake and water intake prediction in growing beef cattle.

Water is an essential nutrient, but there are few recent studies that evaluate how much water individual beef cattle consume and how environmental factors affect an individual's water intake (WI). Most studies have focused on WI of whole pens rather than WI of individual animals. Thus, the objective of this study was to evaluate the impact of environmental parameters on individual-animal WI across different seasons and develop prediction equations to estimate WI, including within different environments and management protocols. Individual daily feed intake and WI records were collected on 579 crossbred steers for a 70-d period following a 21-d acclimation period for feed and water bunk training. Steers were fed in 5 separate groups over a 3-yr period from May 2014 to March 2017. Individual weights were collected every 14 d and weather data were retrieved from the Oklahoma Mesonet's Stillwater station. Differences in WI as a percent of body weight (WI%) were analyzed accounting for average temperature (TAVG), relative humidity (HAVG), solar radiation (SRAD), and wind speed (WSPD). Seasonal (summer vs. winter) and management differences (ad libitum vs. slick bunk) were examined. Regression analysis was utilized to generate 5 WI prediction equations (overall, summer, winter, slick, and ad libitum). There were significant (P < 0.05) differences in WI between all groups when no environmental parameters were included in the model. Although performance was more similar after accounting for all differences in weather variables, significant (P < 0.05) seasonal and feed management differences were still observed for WI%, but were less than 0.75% of steer body weight. The best linear predictors of daily WI (DWI) were dry mater intake (DMI), metabolic body weights (MWTS), TAVG, SRAD, HAVG, and WSPD. Slight differences in the coefficient of determinations for the various models were observed for the summer (0.34), winter (0.39), ad libitum (0.385), slick bunk (0.41), and overall models (0.40). Based on the moderate R2 values for the WI prediction equations, individual DWI can be predicted with reasonable accuracy based on the environmental conditions that are present, MWTS, and DMI consumed, but substantial variation exists in individual animal WI that is not accounted for by these models.

Test duration for water intake, ADG, and DMI in beef cattle.

Water is an essential nutrient, but the effect it has on performance generally receives little attention. There are few systems and guidelines for collection of water intake (WI) phenotypes in beef cattle, which makes large-scale research on WI a challenge. The Beef Improvement Federation has established guidelines for feed intake (FI) and ADG tests, but no guidelines exist for WI. The goal of this study was to determine the test duration necessary for collection of accurate WI phenotypes. To facilitate this goal, individual daily WI and FI records were collected on 578 crossbred steers for a total of 70 d using an Insentec system at the Oklahoma State University Willard Sparks Beef Research Unit. Steers were fed in five groups and were individually weighed every 14 d. Within each group, steers were blocked by BW (low and high) and randomly assigned to one of four pens containing approximately 30 steers per pen. Each pen provided 103.0 m2 of shade and included an Insentec system containing six feed bunks and one water bunk. Steers were fed a constant diet across groups and DMI was calculated using the average of weekly percent DM within group. Average FI and WI for each animal were computed for increasingly large test durations (7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 d), and ADG was calculated using a regression formed from BW taken every 14 d (0, 14, 28, 42, 56, and 70 d). Intervals for all traits were computed starting from both the beginning (day 0) and the end of the testing period (day 70). Pearson and Spearman correlations were computed for phenotypes from each shortened test period and for the full 70-d test. Minimum test duration was determined when the Pearson correlations were greater than 0.95 for each trait. Our results indicated that minimum test duration for WI, DMI, and ADG were 35, 42, and 70 d, respectively. No comparable studies exist for WI; however, our results for FI and ADG are consistent with those in the literature. Although further testing in other populations of cattle and areas of the country should take place, our results suggest that WI phenotypes can be collected concurrently with DMI, without extending test duration, even if following procedures for decoupled intake and gain tests.

National Beef Quality Audit-2016: assessment of cattle hide characteristics, offal condemnations, and carcass traits to determine the quality status of the market cow and bull beef industry.

To continue the series that began in 1994, the National Beef Quality Audit (NBQA) - 2016 was conducted to quantify the quality status of the market cow and bull beef sector, as well as determine improvements made in the beef and dairy industry since 2007. The NBQA-2016 was conducted from March through December of 2016, and assessed hide-on carcasses (n = 5,278), chilled carcasses (n = 4,285), heads (n = 5,720), and offal items (n = 4,800) in 18 commercial processing facilities throughout the United States. Beef cattle were predominantly black-hided; 68.0% of beef cows and 67.2% of beef bulls possessed a black hide. Holstein was the predominant type of dairy animal observed. Just over half (56.0%) of the cattle surveyed had no mud contamination on the hide, and when mud was present, 34.1% of cattle only had small amounts. Harvest floor assessments found 44.6% of livers, 23.1% of lungs, 22.3% of hearts, 20.0% of viscera, 8.2% of heads, and 5.9% of tongues were condemned. Liver condemnations were most frequently due to abscess presence. In contrast, contamination was the primary reason for condemnation of all other offal items. Of the cow carcasses surveyed, 17.4% carried a fetus at the time of harvest. As expected, mean carcass weight and loin muscle area values observed for bulls were heavier and larger than cows. The marbling scores represented by cull animal carcasses were most frequently slight and traces amounts. Cow carcasses manifested a greater amount of marbling on average than bull carcasses. The predominant fat color score showed all carcasses surveyed had some level of yellow fat. Only 1.3% of carcasses exhibited signs of arthritic joints. Results of the NBQA-2016 indicate there are areas in which the beef and dairy industries have improved and areas that still need attention to prevent value loss in market cows and bulls.

Novel nitrite-embedded packaging improves surface redness of dark-cutting longissimus steaks.

The objective of this research was to determine the effects of nitrite-embedded/FreshCase packaging on lean color of dark-cutting beef. Eight dark-cutting (pH > 6.0) and eight USDA Low Choice (normal-pH; mean pH = 5.6) beef strip loins (longissimus lumborum) were selected 3 day after harvest. Each dark-cutting loin was sliced into five 2.5-cm thick steaks and randomly assigned to 1) dark-cutting steak packaged in polyvinyl chloride film (PVC) overwrap, 2) dark-cutting steak packaged in nitrite-embedded film, 3) dark-cutting steaks dipped in 0.2% rosemary solution and packaged in nitrite-embedded film, and 4) dark-cutting steak dipped in deionized water and packaged in nitrite-embedded film. The fifth dark-cutting steak was used to determine pH and proximate composition. Normal-pH choice loins were used as a control and each loin was randomly assigned to either PVC overwrap for retail display or to determine pH and proximate composition. Packages were placed in coffin-style retail display cases under continuous fluorescent lighting for 3 days. A HunterLab MiniScan XE Plus spectrophotometer was utilized to characterize steak color every 24 h. There was a significant treatment × storage time interaction (P < 0.05) for a* values and nitric oxide myoglobin formation. On days 1, 2, and 3 of the display, nitrite-embedded treatment improved (P < 0.05) redness compared to other dark-cutting steaks in PVC. A 45% increase in redness (P < 0.05) was observed for nitrite-embedded rosemary treatment over dark-cutting steak in PVC on day 3 of display. Nitric oxide myoglobin formation on day 0 was less for all dark-cutting steaks in nitrite-embedded packaging. Metmyoglobin content was greater (P < 0.05) on day 0 for dark-cutting steaks packaged in nitrite-embedded treatments than dark-cutting steaks in PVC. However, metmyoglobin level in dark-cutting steaks packaged in nitrite-embedded treatments decreased (P < 0.05) on day 1 compared with day 0. Dark-cutting steaks packaged in PVC had greater (P < 0.05) L* values on day 0 than other dark-cutting steaks in nitrite-embedded packaging. Conversely, on days 1, 2, and 3, there were no differences (P > 0.05) in L* values between dark-cutting treatments. Dark-cutting steaks in nitrite-embedded packaging had lower total plate count (P < 0.05) than dark-cutting steak packaged in PVC. The current research indicated that nitrite-embedded packaging has the potential to improve surface color of dark-cutting beef.

Effects of Muscle-Specific Oxidative Stress on Cytochrome c Release and Oxidation-Reduction Potential Properties.

Mitochondria play a significant role in beef color. However, the role of oxidative stress in cytochrome c release and mitochondrial degradation is not clear. The objective was to determine the effects of display time on cytochrome c content and oxidation-reduction potential (ORP) of beef longissimus lumborum (LL) and psoas major (PM) muscles. PM discolored by day 3 compared with LL. On day 0, mitochondrial content and mitochondrial oxygen consumption were greater in PM than LL. However, mitochondrial content and oxygen consumption were lower (P < 0.05) in PM than LL by day 7. Conversely, cytochrome c content in sarcoplasm was greater on days 3 and 7 for PM than LL. There were no significant differences in ORP for LL during display, but ORP increased for PM on day 3 when compared with day 0. The results suggest that muscle-specific oxidative stress can affect cytochrome c release and ORP changes.

Effects of Metmyoglobin Reducing Activity and Thermal Stability of NADH-Dependent Reductase and Lactate Dehydrogenase on Premature Browning in Ground Beef.

Premature browning is a condition wherein ground beef exhibits a well-done appearance before reaching the USDA recommended internal cooked meat temperature of 71.1 °C; however, the mechanism is unclear. The objectives of this study were: (1) to determine the effects of packaging and temperature on metmyoglobin reducing activity (MRA) of cooked ground beef patties and (2) to assess the effects of temperature and pH on thermal stability of NADH-dependent reductase, lactate dehydrogenase (LDH), and oxymyoglobin (OxyMb) in-vitro. Beef patties (lean: fat = 85:15) were packaged in high-oxygen modified atmosphere (HiOX-MAP) or vacuum (VP) and cooked to either 65 or 71 °C. Internal meat color and MRA of both raw and cooked patties were determined. Purified NADH-dependent reductase and LDH were used to determine the effects of pH and temperature on enzyme activity. MRA of cooked patties was temperature and packaging dependent (P < 0.05). Vacuum packaged patties cooked to 71 °C had greater (P < 0.05) MRA than HiOX-MAP counterparts. Thermal stability of OxyMb, NADH-dependent reductase, and LDH were different and pH-dependent. LDH was able to generate NADH at 84 °C; whereas NADH-dependent reductase was least stable to heat. The results suggest that patties have MRA at cooking temperatures, which can influence cooked meat color.