Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

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.

Mitochondrial Abundance and Function Differ Across Muscle Within Species.

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.

Determining muscle plasticity and meat quality development of low-input extended fed market-ready steers.

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 (pHu). 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.

Skeletal Muscle O-GlcNAc Transferase Action on Global Metabolism Is Partially Mediated Through Interleukin-15.

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.

Mitochondria influence glycolytic and tricarboxylic acid cycle metabolism under postmortem simulating conditions.

The purpose of this study was to test mitochondrial functionality under conditions simulating postmortem metabolism. Isolated mitochondria from porcine longissimus lumborum (LLM) and masseter (MM) muscles were incorporated into an in vitro model that mimics postmortem metabolism. pH and 13C-enrichment of glycolytic and tricarboxylic acid (TCA) cycle intermediates were evaluated at 0, 15, 30, 120, 240, and 1440 min. Addition of mitochondria to the in vitro model lowered its pH at 240 min compared with control. Reactions containing mitochondria had lower pyruvate and lactate [M + 2] and [M + 3] isotopomers at 240 and 1440 min than controls. Furthermore, LLM lowered the enrichment of [M + 2], [M + 3], and [M + 4]α-ketoglutarate at 1440 min compared with MM and control. Succinate [M + 2] and [M + 3] were greater in MM than the control and LLM. [M + 3]fumarate was greater in control at 240 and 1440 min than LLM and MM treatments. Our data indicated that mitochondria are capable of mobilizing pyruvate generated though glycolysis under conditions simulating muscle postmortem metabolism.

Ractopamine changes in pork quality are not mediated by changes in muscle glycogen or lactate accumulation postmortem.

Pork quality is a product of the rate and extent of muscle pH decline paced by carbohydrate metabolism postmortem. The beta-adrenergic agonist ractopamine (RAC) alters muscle metabolism but has little impact on pork quality. The objective of this study was to determine how feeding RAC alters postmortem carbohydrate metabolism in muscle. Muscle pH was higher early postmortem in pigs fed RAC for 2 wks compared to control, while other time points and temperatures were largely unaffected. Early postmortem, muscle lactate levels were reduced (P < 0.05) after feeding RAC for 1 and 2 wks. Similarly, pigs fed RAC for 4 wks had reduced (P < 0.05) glycogen levels early postmortem compared to control pigs, but unexpectedly, L* values (lightness) increased (P < 0.05) after inclusion of RAC in the diet for 4 wk. These data show RAC feeding reduces glycogen content and changes lactate accumulation postmortem, but raise questions about the role glycolytic flux has in driving pork quality development.

Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth.

Feeding ractopamine (RAC), a ß-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P < 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P < 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P < 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, ß 1-adrenergic receptor (AR), and ß 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P < 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P < 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P < 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. ß 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the ß 2-AR gene was decreased (P < 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and ß 2-AR gene expression in porcine skeletal muscles independent of growth.

Dual effects of obesity on satellite cells and muscle regeneration.

Obesity is a complex metabolic disorder that often leads to a decrease in insulin sensitivity, chronic inflammation, and overall decline in human health and well-being. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury; however, the mechanism underlying these changes has yet to be determined. To test whether there is a negative impact of obesity on satellite cell (SC) decisions and behaviors, we fed C57BL/6 mice normal chow (NC, control) or a high-fat diet (HFD) for 10 weeks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller size (p < .001) compared to those from NC mice, and this decreased proliferation was confirmed (p < .05) by BrdU incorporation. Moreover, in vitro assays showed that HFD SCs exhibited diminished (p < .001) fusion capacity compared to NC SCs. In single fiber explants, a higher ratio of SCs experienced apoptotic events (p < .001) in HFD mice compared to that of NC-fed mice. In vivo lineage tracing using H2B-GFP mice showed that SCs from HFD treatment also cycled faster (p < .001) than their NC counterparts. In spite of all these autonomous cellular effects, obesity as triggered by high-fat feeding did not significantly impair muscle regeneration in vivo, as reflected by the comparable cross-sectional area (p > .05) of the regenerating fibers in HFD and NC muscles, suggesting that other factors may mitigate the negative impact of obesity on SCs properties.

Exploring the Factors Contributing to the High Ultimate pH of Broiler Pectoralis Major Muscles Affected by Wooden Breast Condition.

The elevated ultimate pH (pH u ) found in wooden breast (WB) meat suggests an altered muscular energetic status in WB but also could be related to a prematurely terminated post-mortem pH decline. The aims of this study were to explore the factors contributing to the elevated pH u and establish whether the occurrence of WB defect alters muscle post-mortem carbohydrate metabolism and determine if the contractile apparatus reflects such changes. A total of 24 carcasses from Ross 308 male chickens were obtained from a commercial producer and harvested using commercial processing procedures. Carcasses were categorized into unaffected (NORM) and WB groups (n = 12 each), and samples were collected from cranial bone-in pectoralis major (PM) muscles at 15 min and 24 h post-mortem for the determination of pH, glycolytic metabolites, adenonucleotides, buffering capacity, phosphofructokinase (PFK) activity, and in vitro pH decline. Twenty-four additional deboned PM samples (12 NORM and 12 WB) were collected from the same processing plant to assess muscle histology and sarcomere length at four different locations throughout the PM muscle. Data show that the reduced glycolytic potential of WB muscles only partially explains the higher (P < 0.001) pH u of WB meat, as residual glycogen along with unaltered PFK activity suggests that neither glycogen nor a deficiency of PFK is responsible for arresting glycolysis prematurely. The dramatic reduction in ATP concentrations in the early post-mortem period suggests a defective ATP-generating pathway that might be responsible for the reduced pH decline in WB samples. Further, the addition of excess of ATPase extended post-mortem glycolysis of WB meat in an in vitro glycolytic system. WB-affected samples have longer (P < 0.001) sarcomeres compared to NORM, indicating the existence of compromised energy-generating pathways in myopathic muscles that may have had consequences on the muscle contraction and tension development, as in vivo, also during the post-mortem period. Considering the overall reduced glycolytic potential and the myodegenerative processes associated with WB condition, we speculate that the higher pH u of WB meat might be the outcome of a drastically impaired energy-generating pathway combined with a deficiency and/or a dysfunction of muscle ATPases, having consequences also on muscle fiber contraction degree.

Phosphofructokinase and mitochondria partially explain the high ultimate pH of broiler pectoralis major muscle.

During postmortem metabolism, muscle pH gradually declines to reach an ultimate pH near 5.6 across most meat species. Yet, broiler pectoralis major (P. major) muscle generates meat with high ultimate pH (pH ∼ 5.9). For better understanding of the underlying mechanism responsible for this phenomenon, we evaluated the involvement of breast muscle chilling on the extent of postmortem metabolism. Broiler breast muscles were either subjected to chilling treatment (control) or left at room temperature (RT) for 120 min. P. major muscle from the RT treatment had lower ultimate pH, greater glycogen degradation and lactate accumulation. While these findings suggest that carcass chilling can contribute to the premature termination of postmortem metabolism, chilling did not fully explain the high ultimate pH of P. major muscle. Our results also revealed that glucose-6-phosphate (G6P) was very low at 24 h, and therefore we hypothesized that G6P was limiting. To test this hypothesis, muscle samples from P. major and porcine longissimus lumborum (LL) muscle were homogenized into a reaction buffer that mimics postmortem glycolysis with or without 0.5 mg/mL isolated mitochondria. While samples containing porcine LL muscle reached the normal level of ultimate pH, P. major muscle samples reached a value similar to that observed in vivo even in the presence of excess G6P, indicating that G6P was not limiting. Mitochondria enhanced the glycolytic flux and pH decline in systems containing muscle from both species. More importantly, however, was that in vitro system containing chicken with mitochondria reached pH value similar to that of samples containing LL muscle without mitochondria. To investigate further, phosphofructokinase (PFK) activity was compared in broiler P. major and porcine LL muscle at different pH values. PFK activity was lower in P. major muscle at pH 7, 6.5, and 6.2 than LL muscle. In conclusion, carcass chilling can partially contribute to the high ultimate pH of broiler P. major muscle, while low PFK activity and mitochondria content limit the flux through glycolysis.

A mitochondrial protein increases glycolytic flux.

The purpose of this study was to determine the role of mitochondria in postmortem muscle metabolism. Isolated mitochondria were incorporated into a reaction buffer that mimics postmortem glycolysis with or without mitochondrial electron transport chain inhibitors. Addition of mitochondria lowered pH values at 240 and 1440min regardless of inhibitors. Reduction in pH was accompanied by enhanced glycogen degradation and lactate accumulation. To explore the mechanism responsible for this exaggerated metabolism, mitochondrial preparations were mechanically disrupted and centrifuged. Resulting supernatants and pellets each were added to the in vitro model. Mitochondrial supernatants produced similar effects as those including intact mitochondria. To narrow further our target of investigation, mitochondrial supernatants were deproteinized with perchloric acid. The effect of mitochondrial supernatant was lost after perchloric acid treatment. These data indicate that a mitochondrial-based protein is capable of increasing glycolytic flux in an in vitro model and may partially explain acid meat development in highly oxidative AMPKγ3R200Q mutated pigs.