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An in vitro assay was developed to study protease activity during the maturation of beef postmortem. Myofibrils were purified from the semitendinosus and used as a sentinel for assessing the activity of endogenous proteases in longissimus thoracis et lumborum (LTL) and the extensor carpi radialis (ER) over time postmortem in beef carcasses. Samples were collected from each muscle at 0, 1, 2, 7, and 14 d of aging and snap frozen. Samples were powdered and added to an in vitro proteolysis assay containing buffer and purified myofibrils. Aliquots were collected at 0, 2, 120, 480, and 1440 min of incubation, and intact desmin and troponin-T were quantified. Digestions at 0 and 1 d using either muscle had little desmin degradation during the entire digestion period. In contrast, LTL muscle collected at 2, 7, and 14 d had the greatest proteolytic capacity as indicated by disappearance of intact desmin by 480 and 1440 min incubation. Though degradation ensued using powdered ER muscle, disappearance of intact proteins was limited. Degradation in vitro paralleled that observed in intact muscle. Addition of ethylene glycol tetra-acetic acid (EGTA), a cysteine protease inhibitor, and calpastatin inhibited proteolysis and suggest proteolytic activity observed in muscles and detected in our proteolysis assays is due to an active calpain protease. Collectively, our data show an active protease is minimal in bovine muscle until 48 h postmortem in the LTL muscle and suggest an in vitro assay containing purified myofibrils is a potential tool for studying temporal changes in proteolysis during the maturation and tenderization of beef across muscles., Competing Interests: Declaration of competing interest This work is supported in part by A1364 Novel Foods and Innovative Manufacturing Technology [grant no. 2020-67017-31269] from the USDA National Institute of Food and Agriculture., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background : Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods : Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results : Bovine oxidative muscle mitochondria contain greater mtDNA ( p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption ( p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA ( p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity ( p < 0.05) than glycolytic muscle. Conclusions : These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle.

Grass-fed beef is becoming increasingly popular and is expected to be a $14 billion industry by 2024. Even so, grass-fed beef is typically darker in appearance than that of conventional grain-fed beef. Aging has been shown to improve lean color ( L *, a *) of dark-cutting beef however little work has focused on aging as it relates to improving the lean color of grass-fed beef. Therefore, the aim of this study was to evaluate the effect of dry aging on grass-fed beef compared to varying lengths of grain-feeding. Thirty commercial Angus crossbred steers were randomly assigned to either pasture finishing (CON), short (SF), or long fed (LF) programs. The SF and LF treatments ranged from 90 to 114 d (average: 98 d) and 118 to 134 d (average: 125 d) on ad libitum high concentrate feeding, respectively. Cattle were randomly identified from each treatment group and harvested over a consecutive 3-wk span. Carcass evaluation and longissimus lumborum samples were collected 24 h postmortem. Carcasses were aged for 21 d, and steaks were collected on 1-, 3-, 7-, 10-, 14-, and 21-d postharvest, and objective color was evaluated following 1 h bloom. Our data show color ( L *, a *, b *) was improved with aging regardless of treatment. However, grass-fed (CON) showed the greatest improvement in both lightness ( L *) and redness ( a *) ultimately making grass-fed comparable to that of grain-fed beef by day 21. These data argue that dry-aging grass-fed beef improves color development similar to that of grain-fed beef., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

In March 2020, the World Health Organization declared COVID-19 a pandemic, which ultimately led to many meat processors temporarily shutting down or reducing processing capacity. This backlog in processing capacity forced many feedlots to retain cattle for longer periods of time and assume the risk of major market fluctuations. The aim of this study was to understand how a dietary insult affects meat quality and muscle metabolism in market-ready steers (590 kg). Sixteen market-ready (590 kg) commercial Angus crossbred steers were subjected to a maintenance diet of either forage or grain for 60 d. Longissimus lumborum (LL) muscle samples were collected immediately postmortem and processed for characteristics reflecting the underlying muscle fiber type and energy state of the tissue. Despite cattle being subjected to a 60-d feeding period, there were no detectable differences ( P  > 0.05) in carcass characteristics, color of lean, or ultimate pH (pH u ). Moreover, our data show that muscle plasticity is rather resilient, as reflected by lack of significance ( P  > 0.05) in oxidative and glycolytic enzymes, myosin heavy chain isoforms (MyHC), myoglobin, and mitochondrial DNA (mtDNA) contents. These data show that market-ready steers are capable of withstanding a low-input feeding strategy up to 60 d without dramatically impacting underlying muscle characteristics and meat quality development., Competing Interests: There is no conflict of interest for all authors that could be perceived as prejudicing the impartiality of the research reported., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry beef, often referred to as 'dark-cutting'. However, the relationship between atypical dark (AT) beef, postmortem energy metabolism and underlying tissue characteristics remains somewhat unclear. Cattle harvested in the US and Canada representing normal (pH < 5.6), AT dark (pH 5.6-5.8) and dark cutting (DC; pH > 5.8) beef were analyzed for tissue characteristics related to energy metabolism. Results show AT dark beef is more oxidative but similar to normal beef in glycolytic potential and nucleotide abundance. Mitochondria DNA content (P < 0.05, Canada; P < 0.005, US) and oxidative enzymes for DC and AT dark beef were greater (P < 0.01; Canada and US) compared to normal beef. Myoglobin tracked (P < 0.01) with color classification. These findings show both DC and AT beef are inherently more oxidative and raise the possibility that more oxidative muscle may be more prone to develop dark beef., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Variations in postmortem metabolism in muscle impact pork quality development. Curiously, some genetic lines are more refractile to adverse pork quality development than others and may regulate energy metabolism differently. The aim of this study was to challenge pork carcasses from different genetic populations with electrical stimulation (ES) to determine how postmortem metabolism varies with genetic line and explore control points that reside in glycolysis in dying muscle. Three genetic populations (GP) were subjected to ES (100 V or 200 V, 13 pulses, 2 s on/2 s off) at 15- or 25-min post-exsanguination, or no stimulation (NS). Genetic population affected relative muscle relative abundance of different myosin heavy chains, glycogen, G6P, and lactate concentrations. Genetic lines responded similarly to ES, but a comparison of ES treatment groups revealed a trend for an interaction between voltage, time of ES, and time postmortem. Higher voltage accelerated pH decline at 20 min up to 60 min postmortem. Trends in color and firmness scores and L* values were consistent with pH and metabolite data. These data show that genetic populations respond differently to postmortem perturbation by altering glycolytic flux and suggest differences in postmortem glycolysis may be partially responsible for differences in meat quality between genetic populations, though not entirely.

Background: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence., Methods: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes., Results: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex., Conclusions: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells., (© 2022. The Author(s).)

Variations in color, though a quality frustration, are common across the face of fresh and processed hams. Herein, we measured objective color across the semimembranosus (SM) muscle early postmortem and at 1440 min, then compared these differences against biochemical and metabolic characteristics responsible for pork quality development. Color (L*, a*) differed (P < 0.001) by zone and time but no interaction was evident. Lactate content and pH were highly correlated (R 2  = 0.92) at 30 min, but weakened (R 2  = 0.161412) by 1440 min. Lactate anaplerosis was not responsible for this lack of relationship. Glycolytic potential also differed across zone (P < 0.001) and time (P < 0.005). Differences in myoglobin expression and abundance, as well as mitochondrial DNA were notable (P < 0.05) across zone. These data suggest inherent differences in SM muscle are key determinants of ham color variation, while postmortem metabolism may play a lesser role in driving this quality attribute., (Copyright © 2021. Published by Elsevier Ltd.)

Besides its roles in locomotion and thermogenesis, skeletal muscle plays a significant role in global glucose metabolism and insulin sensitivity through complex nutrient sensing networks. Our previous work showed that the muscle-specific ablation of O-GlcNAc transferase (OGT) led to a lean phenotype through enhanced interleukin-15 (IL-15) expression. We also showed OGT epigenetically modified and repressed the Il15 promoter. However, whether there is a causal relationship between OGT ablation-induced IL-15 secretion and the lean phenotype remains unknown. To address this question, we generated muscle specific OGT and interleukin-15 receptor alpha subunit (IL-15rα) double knockout mice (mDKO). Deletion of IL-15rα in skeletal muscle impaired IL-15 secretion. When fed with a high-fat diet, mDKO mice were no longer protected against HFD-induced obesity compared to wild-type mice. After 22 weeks of HFD feeding, mDKO mice had an intermediate body weight and glucose sensitivity compared to wild-type and OGT knockout mice. Taken together, these data suggest that OGT action is partially mediated by muscle IL-15 production and provides some clarity into how disrupting the O-GlcNAc nutrient signaling pathway leads to a lean phenotype. Further, our work suggests that interfering with the OGT-IL15 nutrient sensing axis may provide a new avenue for combating obesity and metabolic disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zumbaugh, Yen, Bodmer, Shi and Gerrard.)