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WNT signaling is important for regulation of embryonic development. The most abundant WNT gene expressed in the bovine endometrium during the preimplantation period is WNT5A. One objective was to determine whether WNT5A regulates competence of the bovine preimplantation embryo to become a blastocyst and alters the number of cells in the inner cell mass and trophectoderm. A second objective was to delineate features of the cell-signaling mechanisms involved in WNT5A actions. WNT5A caused a concentration-dependent increase in the proportion of embryos developing to the blastocyst stage and in the number of inner cell mass cells in the resultant blastocysts. A concentration of 200 ng/mL was most effective, and a higher concentration of 400 ng/mL was not stimulatory. Bovine serum albumin in culture reduced the magnitude of effects of WNT5A on development to the blastocyst stage. WNT5A affected expression of 173 genes at the morula stage; all were upregulated by WNT5A. Many of the upregulated genes were associated with cell signaling. Actions of WNT5A on development to the blastocyst stage were suppressed by a Rho-associated coiled-coil kinase (ROCK) signaling inhibitor, suggesting that WNT5A acts through Ras homology gene family member A (RhoA)/ROCK signaling. Other experiments indicated that actions of WNT5A are independent of the canonical β-catenin signaling pathway and RAC1/c-Jun N-terminal kinase (JNK) signaling. This is the first report outlining the actions of WNT5A to alter the development of the mammalian embryo. These findings provide insights into how embryokines regulate maternal–embryonic communication.

The objective of this experiment was to determine the effects of altering the dietary cation-anion difference (DCAD) fed for the last 21 or 42 d of gestation on glucose metabolism and tissue insulin responsiveness. Ninety parous Holstein cows at 232 d of gestation were assigned randomly to dietary treatments with 2 levels of DCAD (-70 or -180 mEq/kg) fed for 2 durations (short: the last 21 d of gestation; long: the last 42 d of gestation). For the short treatments, a diet with +110 mEq/kg was fed from 232 to 254 d of gestation. Intravenous glucose tolerance tests (IVGTT) were performed at either 250 or 270 d of gestation by infusing 0.25 g of dextrose/kg of body weight within 1 min. The following day, cows underwent an insulin challenge (IC) and received 0.1 IU of insulin/kg of body weight intravenously. Blood was sampled at min -15, -5, and 0 to establish a baseline and from 5 to 180 min relative to infusions; plasma concentrations of glucose, insulin, and fatty acids were determined, and the respective areas under the curves (AUC) were calculated. Liver was sampled after the IVGTT, and adipose tissue was sampled after the IVGTT and IC for quantification of mRNA expression and protein abundance. Reducing the DCAD altered acid-base balance compatible with a compensated metabolic acidosis. At 250 d, reducing the DCAD increased the AUC for glucose and reduced that of insulin following the IVGTT, whereas during the IC, clearance rate decreased and time to half-life of insulin increased with reducing DCAD, resulting in a tendency to a larger AUC for fatty acids. At 270 d, quantitative insulin sensitivity check index and the revised quantitative insulin sensitivity check index were smaller in cows fed the acidogenic diets for the last 42 d of gestation compared with the last 21 d of gestation, thereby suggesting reduced insulin sensitivity. In addition, cows fed for the long duration tended to have greater AUC for glucose but smaller AUC for insulin following an IVGTT than those fed for the short duration, thereby suggesting reduced insulin release and glucose disposal. Treatments did not affect hepatic mRNA expression of G6PC, PCK1, PCK2, and PC or adipose tissue mRNA expression of ATGL, ACC, B2AR, HSL, and PLIN1. On the other hand, for proteins, reducing the DCAD linearly reduced abundance of rabbit anti-mouse protein kinase B (AKT) and tended to reduce rabbit anti-human phosphorylated (Ser-9) glycogen synthase kinase-3 β (pGSK) and the pGSK:rabbit anti-human glycogen synthase kinase-3 β (GSK) ratio in hepatic tissue, whereas a linear increase in rabbit anti-human hormone-sensitive lipase (HSL) and rabbit anti-mouse phosphorylated (Ser-660) hormone-sensitive lipase (pHSL) in adipose tissue was observed after the IVGTT at 250 d. Moreover, reducing the DCAD resulted in a linear reduction of AKT and tended to reduce rabbit anti-human acetyl-CoA carboxylase (ACC) but increased pHSL linearly in adipose tissue after an IC at 250 d. Cows fed acidogenic diets for a short duration tended to have less pHSL in adipose tissue than those fed for a long duration after an IVGTT at 270 d. Associations were observed between blood pH and mRNA and protein abundance in hepatic and adipose tissues. Diet-induced metabolic acidosis altered insulin release and insulin signaling, resulting in a shift in adipose tissue metabolism that would favor lipolysis over lipogenesis.

Bos taurus indicus temperament is variable and affects beef tenderization. Our objective was to investigate temperament and performance of non–castrated Nellore and identify groups based on Longissimus lumborum (LL) pH decline as well as beef characteristics produced by those groups. We investigated 94 animals with a subset of carcasses (n = 24) selected based on LL pH at 24 h postmortem (pm) to represent two groups: resistant to pH decline (> 5.8 called pH–Res; n = 10) and normal (< 5.7 called pH–Nor; n = 14). Steaks were fabricated from the LL muscle and randomly assigned to aging (2, 7, 14, and 21 days). Sarcomere length, cooking loss, myofibrillar fragmentation index (MFI), and Warner–Bratzler shear force (WBSF) were determined. Data on temperament were investigated in a multivariate approach, while beef data were compared between groups using the analysis of variance. Rectal temperature at the beginning of the finishing phase and total weight gain were greater and related to animals in the pH–Res group (p < 0.01 and p = 0.03, respectively). Temperature and pH decline curves, sarcomere length, and cooking loss revealed that pH–Res produced beef with lower quality compared to the pH–Nor group. Results for MFI and WBSF did not show differences between groups within each time pm; however, overall steaks from pH–Res were tougher (p = 0.06). Incidence of LL pH between 5.8 and 5.9 at 24 h pm did not compromise the tenderization rate or extension; however, it affected the water holding capacity in this population of Nellore cattle.

Muscle properties and metabolism influence muscle to meat conversion. Fiber type profile impacts glycolytic capacity as well as protein turnover rate in vivo. Our objective was to investigate protease content and activation during the early postmortem period using muscles with known divergent metabolism. Samples from longissimus lumborum (LL) and diaphragm (Dia) were taken from predominantly Angus steer carcasses (n = 6) at 1, 3, and 24 h postmortem and frozen. Myosin heavy chain (MyHC) isoforms, ATP, glycogen, glucose, glucose-6-phosphate (G6P), and lactate concentrations were determined. Procaspase-3, calpain-1, calpastatin, desmin, and troponin-T were assessed by immunodetection. Muscles showed contrasting MyHC profiles, with LL represented primarily by IIx and IIa isoforms (∼88%) whereas Dia contained mostly (80%) type I isoform. Glycogen degradation was more pronounced in LL and coincided with more rapid accumulation of glucose and lactate (P < 0.01). Procaspase-3 content was influenced by muscle (m: P < 0.01), being greater in Dia. Fragments indicating activation of procaspase-3 postmortem were not detected. Calpain-1 autolysis and intact calpastatin (135 kDa) content were influenced by muscle and time (m × t: P < 0.01 and P < 0.01, respectively). Calpastatin fragmentation postmortem was not associated with greater procaspase-3 content. In conclusion, fast glycolytic LL displayed faster protease activation and greater proteolysis during the first 24 h postmortem.

This publication discusses certified beef programs and carcass quality specifications. Written by Tracy Scheffler, Jason Scheffler, and Chad Carr, and published by the UF/IFAS Department of Animal Sciences, October 2021.

Reductions in basal metabolism and growth rate appear to contribute to decreased heat production of Bos indicus breeds. Lower metabolic rate may be due to smaller organ size and lower metabolic activity. Liver and heart account for high percentage of metabolic activity relative to their weight, and at a cellular level, mitochondria are responsible for producing energy for cellular maintenance. Our objective was to determine liver and heart weight and evaluate expression of key mitochondrial proteins in Bos indicus (80–100% Brahman) and Bos taurus (80–100% Angus) steers. Steers (n = 14 per breed) were harvested at the university meat laboratory. After evisceration, livers and hearts were weighed, and samples were frozen in liquid nitrogen for further analyses. Western blotting was used to determine expression of proteins in mitochondrial energy production: citrate synthase, a marker of mitochondria content; succinate dehydrogenase B (SDH-B), complex II; cytochrome c oxidase subunit IV, complex IV; and ATP synthase subunit 5A (ATP5A), complex V. Data were analyzed using an unpaired t-test in SAS-JMP. Although steers were similar age at slaughter (P = 0.84), Brahman steers weighed less (P < 0.0001) and thus produced lighter carcasses (P < 0.0001) and smaller livers and hearts (P < 0.0001). On a relative basis (kg organ/kg BW), Brahman tended to possess smaller hearts (P = 0.06) and smaller livers (P = 0.03). Expression of mitochondria proteins in heart did not differ between breeds (P > 0.3). However, Brahman exhibited lower expression of SDH-B (P = 0.005) but tended to have higher expression of ATP5A (P = 0.07) in liver. Altogether, this supports that smaller organ size is a contributing factor to lower heat production in heat-tolerant Brahman steers, and shifts in mitochondria protein expression in liver may impact energy metabolism at the cellular level.

Myosin heavy chain (MyHC) isoform composition is a primary determinant of contractile speed of muscle fibers. Currently, bovine MyHC isoforms are evaluated using time-consuming histochemical analysis by immunflourescence or ATPase activity. Electrophoretic separation of MyHC isoforms is more rapid; however, a reliable procedure without use of gradients has not been validated for cattle. Therefore, our objectives were to develop and validate a procedure for separating bovine MyHC isoforms (I, IIa, and IIx) using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and compare results to immunohistochemistry (IHC) analysis. Muscle samples were collected from masseter, sternomandibularis, diaphragm, longissimus lumborum, and cutaneous trunci within 1.5 h postmortem. To determine appropriate conditions for electrophoretic separation, several parameters of gel composition were varied. Bovine MyHC isoforms were clearly separated by increasing glycerol content of polyacrylamide gels to 37%. Identity of MyHC isoforms was confirmed using western blotting. Percent MyHC composition evaluated by gel electrophoresis was consistent with IHC (P > 0.2). Thus, SDS-PAGE produces clear separation of MyHC isoforms, and is a viable alternative to IHC-based methods.

The objectives were to determine the optimal feeding amount of choline in a ruminally protected form to reduce the triacylglycerol (TAG) concentration in liver and to increase TAG in blood plasma of dairy cows. Pregnant, nonlactating multiparous Holstein cows (n = 77) were blocked by body condition score (3.59 ± 0.33) and assigned to treatment at 64 ± 10 d before calculated calving date. Dietary treatments were top-dressing of 0, 30, 60, 90, or 120 g/d of ruminally protected choline (RPC; Balchem Corp., New Hampton, NY) ions to supply the equivalent of 0, 6.5, 12.9, 19.4, and 25.8 g/d of choline ions. Diets were formulated to exceed nutrient requirements for maintenance and pregnancy and fed in ad libitum amounts for the first 5 d. From d 6 to 15, cows were restricted to consume approximately 31% of their net energy requirements to simulate early lactating cows in negative energy balance. Methionine intake was maintained throughout each 15-d period. Liver was biopsied at 5 and 14 d and analyzed for TAG and glycogen. Blood was sampled on d 5 and 14 and plasma analyzed for glucose, insulin, cholesterol, β-hydroxybutyrate, long-chain fatty acids, and haptoglobin. On d 14, a mixture of saturated long-chain fatty acids, ground corn, and dried molasses (50:37:13) was offered (908 g, as-is basis) 10 h after the single daily feeding. Blood samples were collected for 19 h and plasma analyzed for TAG and cholesterol to assess apparent absorption of dietary fat. Mean dry matter intake and energy balance decreased from means of 9.5 to 3.3 kg/d and from 0.6 to −9.2 Mcal of net energy for lactation/d during the ad libitum and restricted feeding periods, respectively. Plasma concentrations of the lipid-soluble choline biomolecules, namely total phosphatidylcholines, total lysophosphatidylcholines, and sphingomyelin, increased with choline supplementation. Feed restriction increased plasma concentrations of β-hydroxybutyrate and free long-chain fatty acids, whereas those of glucose, insulin, and total cholesterol decreased. During feed restriction, concentration of hepatic TAG and plasma haptoglobin decreased linearly, whereas concentration of hepatic glycogen tended to increase quadratically with increasing intake of RPC. After fat supplementation, mean plasma concentration of TAG increased by an average of 21% with intake of RPC ions, peaking at intakes of ≥6.5 g/d of RPC ion. In summary, feeding RPC ions to cows in negative energy balance had increasing lipotropic effects on the liver when consumed up to 25.8 g/d, whereas feeding only 6.5 g/d increased concentrations of hepatic glycogen and TAG in the blood.

Anaerobic glycolysis dominates energy metabolism postmortem. Even so, however, recent studies suggest mitochondria can modify postmortem energy metabolism and may contribute to pH decline, possibly affecting the transformation of muscle to meat and fresh meat quality development. Because oxygen is a necessary component of mitochondrial function, oxygenation of porcine and bovine longissimus thoracis et lumborum was determined postmortem using NIR spectroscopy. The ratio of oxy- to deoxymyoglobin decreased with time postmortem in both species. Metabolic analyses of muscle samples collected over the same timeframe also revealed fluctuations in TCA intermediates. Finally, mitochondria collected from muscle of electrically stimulated carcasses differed from those of non-stimulated muscle. Collectively, these data support the thesis that muscle mitochondria function early postmortem and may play a more active part in pH decline and possibly meat quality development.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

Acute activation of AMP-activated protein kinase (AMPK) increases monocarboxylate transporter (MCT) expression in skeletal muscle. However, the impact of chronic activation of AMPK on MCT expression in skeletal muscle is unknown. To investigate, MCT1, MCT2, and MCT4 mRNA expression and protein abundance were measured in the longissimus lumborum (glycolytic), masseter (oxidative), and heart from wild-type (control) and AMPK γ3 pigs. The AMPK γ3 gain in function mutation results in AMPK being constitutively active in glycolytic skeletal muscle and increases energy producing pathways. The MCT1 and MCT2 mRNA expression in muscle was lower ( < 0.05) from both wild-type and AMPK γ3 animals compared to other tissues. However, in both genotypes, MCT1 and MCT2 mRNA expression was greater ( < 0.05) in the masseter than the longissimus lumborum. The MCT1 protein was not detected in skeletal muscle, but MCT2 was greater ( < 0.05) in muscles with an oxidative muscle phenotype. Monocarboxylate transporter 2 was also detected in muscle mitochondria and may explain the differences between muscles. The MCT4 mRNA expression was intermediate among all tissues tested and greater ( < 0.05) in the longissimus lumborum than the masseter. Furthermore, MCT4 protein expression in the longissimus lumborum from AMPK γ3 animals was greater ( < 0.05) than in the longissimus lumborum from wild-type animals. In totality, these data indicate that chronic AMPK activation simultaneously increases MCT2 and MCT4 expression in skeletal muscle.

The objectives of this study were to estimate variance components, genetic parameters, EBV, accuracies, and rankings for nine ultrasound and carcass traits in a multibreed Angus-Brahman population using three genomic-polygenic models and one polygenic model (PM). The genomic-polygenic models used the complete GeneSeek GPF250k SNP set (GPM), top 5% SNP (GPMR1), and 5% SNP evenly spread across the genome (GPMR2). Yearling ultrasound traits were weight (UW), ribeye area (UREA), backfat (UFAT), and percent intramuscular fat (UPIMF). Carcass traits were slaughter age (SLA), hot carcass weight (HCW), ribeye area (REA), backfat thickness (FAT), and marbling score (MAR). The 9-trait GPM, GPMR1, GPMR2, and PM contained fixed contemporary group, age of calf (ultrasound traits only), sex of calf, and direct heterosis effects, and random animal and residual effects. Variance components and genetic parameters were computed using AIREMLF90. Comparable heritabilities were obtained with GPM and PM for UW (GPM: 0.54 ± 0.05; PM: 0.51 ± 0.05), UREA (GPM: 0.36 ± 0.03; PM: 0.34 ± 0.03), UFAT (GPM: 0.12 ± 0.02; PM: 0.11 ± 0.02), UMPIMF (GPM: 0.34 ± 0.03; PM: 0.30 ± 0.03), SLA (GPM: 0.59 ± 0.07, PM: 0.61 ± 0.06), HCW (GPM: 0.58 ± 0.06, PM: 0.52 ± 0.07), REA (GPM: 0.48 ± 0.04, PM: 0.45 ± 0.05), FAT (GPM: 0.41 ± 0.05, PM: 0.30 ± 0.05), and MAR (GPM: 0.56 ± 0.07, PM: 0.51 ± 0.08). Additive genetic correlations between pairs of ultrasound and carcass traits were all between −0.31 and 0.81. The highest positive additive genetic correlations were between UW and UREA, UW and HCW, UW and REA, UREA and HCW, UREA and REA, UFAT and FAT, and between HCW and REA. The largest negative additive genetic correlations were between UREA and UPIMF, UFAT and SLA, UFAT and HCW, UPIMF and REA, and between REA and MAR. High similarity existed among predicted EBV and accuracies from GPM, GPMR1, and GPMR2 as well as high-rank correlations for sires, dams, and progenies. This indicated that the two reduced genotype sets were appropriate alternatives to the complete GPF250k set for genomic-polygenic evaluation and selection in this multibreed Angus-Brahman population. High EBV variability existed among animals of all Angus and Brahman percentages and no specific breed composition was overwhelmingly better or worse for any of the nine traits. This indicated that optimization of genetic progress through selection in multibreed Angus-Brahman populations should be based solely on genetic merit regardless of breed composition.

Fresh hams display significant lean color variation that persists through further processing and contributes to a less desirable cured product. In an attempt to understand the underlying cause of this color disparity, we evaluated the differences in muscle characteristics and energy metabolites across semimembranosus (SM) muscles differing in color variation. The L* (lightness) and a* (redness) values were highest and lowest (P

Fresh turkey meat color is determined by many factors that include muscle fiber type composition and heme protein concentrations. These factors either are affected by or influence biochemical events occurring postmortem. Deviations in the processing environment also can result in aberrant fresh meat quality and may ultimately change the quality characteristics of further processed products. Our objective was to describe the underlying cause and significance of the two-toning color defect in fresh turkey breast. In the first experiment, pectoralis major muscles were collected, classified as single- or two-toned, and analyzed using image processing to characterize fresh turkey color. Samples from the large and small lobes of the pectoralis major muscle were collected for pH, glycolytic intermediates, protein abundance, mRNA expression, and quality characteristics. In the second experiment, time from stun to exsanguination was tested as a promoter of fresh turkey color. Results from the first experiment showed that the turkey breast possesses two distinct lobes. The large lobe had greater (P 0.05) glycolytic potential, lactate content, lactate dehydrogenase (LDH) abundance, and centrifugal drip loss, while pH, myoglobin mRNA expression, and soluble protein levels were lower (P 0.05) compared to the small lobe. Results from the second experiment showed that reducing time from stun to exsanguination enhanced (P 0.05) fresh turkey color by mitigating the differences between the two lobes. Our results also showed that birds exsanguinated first had greater (P 0.05) muscle pH values and body temperatures. These results show inherent differences in breast muscle and processing conditions interact to establish variations in fresh turkey color.

Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.

ObjectivesThe conversion of muscle to meat is largely controlled by postmortem energy metabolism and pH decline. These biochemical changes influence activity of enzymes implicated in proteolysis and meat tenderization. Therefore, our objective was to investigate pH decline, muscle energy metabolism, and protease activation in functionally distinct bovine muscles.Materials and MethodsSteers (n = 6) were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were taken at 1, 3, and 24h postmortem, immediately frozen using liquid nitrogen, and stored in ultra-freezer until analysis. Muscle pH was obtained using a pH meter at the same time points. Myosin heavy chain composition (I, IIa, and IIx) was determined using gel electrophoresis. Substrate (residual glycogen), as well as glycolytic metabolites, glucose, glucose-6-phosphate, and lactate, were quantified by enzymatic methods; muscle ATP at 1 and 3h was also determined. Western blotting was used to evaluate protease activation (calpain-1 and caspase-3). Data were analyzed using a randomized block design, with slaughter date as block. Animal within slaughter date was considered as random effect and fixed effects of muscle, time, and the interaction tested. Time was considered a repeated measure.ResultsDiaphragm contained a greater percentage of slow myosin heavy chain compared to LL (80% vs. 12%, respectively). Consistent with fiber type, LL contained greater glycogen than Dia at 1h (P < 0.05), but not at subsequent times postmortem. Overall, a greater decline in glycogen occurred in LL. Accordingly, lactate concentration increased markedly in LL postmortem and to a lesser extent in Dia (interaction effect; P < 0.01). Although muscles exhibited similar lactate content at 1h, at 24h the LL showed elevated lactate relative to Dia (88 vs. 53 µmol/g tissue, respectively). Accumulation of glucose and glucose-6-phosphate were affected by muscle (P < 0.01) and time (P < 0.01), with greater final content in LL compared to Dia. Muscles exhibited different patterns of postmortem pH decline (muscle × time, P < 0.0001). Initially, pH of LL was higher than Dia (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 LL than in Dia 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 Dia, whereas autolysis increased in LL from 3h to 24h postmortem. Caspase-3 was identified by one band (32 kDa) that represents the zymogen (procaspase-3). Procaspase-3 content is affected by muscle (P < 0.01), with Dia containing greater content than LL.ConclusionAlthough the Dia is considered a slow muscle, it exhibited a more rapid pH decline and lower ATP levels than LL early postmortem. These parameters were expected to coincide with more rapid calpain-1 autolysis in Dia, but this was not the case. Further work is necessary to understand the interaction between pH decline, muscle type, and postmortem proteolysis.

Functional properties and integrity of skeletal muscle mitochondria during the early postmortem period may influence development of meat quality traits, such as tenderness. Angus typically produce more tender beef than Brahman, a Bos taurus indicus subspecies known for heat tolerance. Thus, our objectives were to assess mitochondrial function in muscle collected early postmortem from Angus and Brahman cattle; and to evaluate the effect of normal and elevated temperature on mitochondria function. Longissimus lumborum was collected at 1h postmortem from Angus and Brahman steers, and high resolution respirometry was used to assess mitochondrial function in permeabilized muscle fibers at two temperatures (38.5 and 40.0°C). The main effects of breed, temperature, and their interaction were tested. On a tissue weight basis, parameters of respiratory function were not influenced by breed or temperature, though Brahman exhibited numerically greater values for respiration supported by complex I, complex I+II, and complex II substrates. Citrate synthase activity, a marker of mitochondria content, was affected by breed (P = 0.049). Consequently, oxygen consumption rate (OCR) data were normalized to citrate synthase activity. After normalization for mitochondrial content, the overall mitochondria OCR pattern changed, revealing differences among breeds for proton leak respiration (P = 0.045), as well as a persistent interaction effect primarily related to reduced OCR in mitochondria from Brahman at 40.0°C. In addition, the ratio of OCR for leak relative to complex I+II phosphorylation was lower in Brahman, evidencing greater coupling. However, mean coupling ratios for both Angus and Brahman support that respiration and phosphorylation were well-coupled for both breeds even at 1h postmortem. Thus, mitochondria retain functional capacity and integrity at 1h postmortem; and mitochondria properties may be related to differences in heat tolerance and meat quality development between cattle subspecies.

Functional properties and integrity of skeletal muscle mitochondria (mt) during the early postmortem period may influence energy metabolism and pH decline, thereby impacting meat quality development. Angus typically produce more tender beef than Brahman, a Bos indicus breed known for heat tolerance. Thus, our objectives were to compare mt respiratory function in muscle collected early postmortem (1 h) from Angus and Brahman steers (n = 26); and to evaluate the effect of normal and elevated temperature on mt function ex vivo. We measured mt oxygen consumption rate (OCR) in fresh-permeabilized muscle fibers from Longissimus lumborum (LL) at 2 temperatures (38.5 and 40.0 °C) and determined citrate synthase (CS) activity and expression of several mt proteins. The main effects of breed, temperature, and their interaction were tested for mt respiration, and breed effect was tested for CS activity and protein expression. Breed, but not temperature (P > 0.40), influenced mt OCR (per tissue weight), with Brahman exhibiting greater complex I+II-mediated oxidative phosphorylation capacity (P = 0.05). Complex I- and complex II-mediated OCR also tended to be greater in Brahman (P = 0.07 and P = 0.09, respectively). Activity of CS was higher in LL from Brahman compared to Angus (P = 0.05). Expression of specific mt proteins did not differ between breeds, except for higher expression of adenosine triphosphate (ATP) synthase subunit 5 alpha in Brahman muscle (P = 0.04). Coupling control ratio differed between breeds (P = 0.05), revealing greater coupling between oxygen consumption and phosphorylation in Brahman. Our data demonstrate that both Angus and Brahman mt retained functional capacity and integrity 1-h postmortem; greater oxidative phosphorylation capacity and coupling in Brahman mt could be related to heat tolerance and impact early postmortem metabolism.

Insufficient acidification results in dark, firm, and dry beef. While this defect is often indicative of a stress event antemortem, muscle tissue may change in response to feeding regime. Longissimus dorsi muscle samples from 10 grain-fed and 10 grass-fed market weight, angus-crossbred beef cattle were collected postmortem. Lower (P

The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth.

Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles.

The purpose of this study was to examine the role of mitochondria in postmortem calcium homeostasis and its effect on proteolysis and tenderness. We hypothesized that mitochondria buffer cytosolic calcium levels and delay the activation of calpain-1 and subsequently the development of meat tenderness. To test this hypothesis, pre-rigor bovine longissimus thoracis et lumborum muscle samples were injected with DS16570511 to inhibit mitochondrial calcium uptake. Free calcium, tenderness, texture profile analysis (TPA), calpain-1 activity, and proteolysis were evaluated over a 336 h aging period. Inhibition of mitochondrial calcium uptake increased (P

The quality grade system used in the United States to identify carcasses with superior eating satisfaction to consumers is based on the amount of intramuscular fat (marbling) within the ribeye and the maturity of the carcass. However, the most important quality attribute for consumers is tenderness. The objective of this study was to investigate the relationship between quality grade and tenderness assessed by slice shear force in strip loin steaks from Brangus cattle. Brangus steers (n = 860) were harvested when they reached approximately 1.27 cm fat over the ribeye. Hot carcass weight (HCW), dressing percentage (DP), marbling score (MARB; 100 to 199 = practically devoid, 200 to 299 = traces, 300 to 399=slight, 400 to 499=small, 500 to 599=modest, 600 to 699=moderate, 700 to 799=slightly abundant, 800 to 899=moderately abundant, and 900 to 999=abundant), ribeye area (REA), fat over the eye (FOE), and kidney pelvic and heart fat percentage (KPH) were evaluated for each animal. Quality grades and yield grades were calculated according to industry standards. A steak from the longissimus lumborum muscle was collected from each carcass, aged for 14 d and used for slice shear force testing to determine the tenderness. Data was analyzed with a general linear model with cooking loss, ranch, and feedlot as fixed effects. The quality grades ranged from high standard to average prime and the average of slice shear force values was 26.09 ± 8.76 kg. Quality grade had a significant effect on Slice Shear Force values (P = 0.0078), showing that quality grade significantly impacts tenderness. However, there was a high level of variation in Slice Shear Force values within quality grades. This indicates that carcasses with high quality grades could have meat with unacceptable tenderness, which could affect the demand for beef products.

Pigs with the Halothane (HAL) or Rendement Napole (RN) gene mutations demonstrate abnormal muscle energy metabolism patterns and produce meat with poor quality, classified as pale, soft, and exudative (PSE) meat, but it is not well understood how HAL and RN mutations regulate glucose and energy metabolism in porcine muscle. To investigate the potential signaling pathways and phosphorylation events related to these mutations, muscle samples were collected from four genotypes of pigs, wild type, RN, HAL, and RN-HAL double mutations, and subjected to quantitative proteomic and phosphoproteomic analysis using the TiO2 enrichment strategy. The study led to the identification of 932 proteins from the nonmodified peptide fractions and 1885 phosphoproteins with 9619 phosphorylation sites from the enriched fractions. Among them, 128 proteins at total protein level and 323 phosphosites from 91 phosphoproteins were significantly regulated in mutant genotypes. The quantitative analysis revealed that the RN mutation mainly affected the protein expression abundance in muscle. Specifically, high expression was observed for proteins related to mitochondrial respiratory chain and energy metabolism, thereby enhancing the muscle oxidative capacity. The high content of UDP-glucose pyrophosphorylase 2 (UGP2) in RN mutant animals may contribute to high glycogen storage. However, the HAL mutation mainly contributes to the up-regulation of phosphorylation in proteins related to calcium signaling, muscle contraction, glycogen, glucose, and energy metabolism, and cellular stress. The increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CAMK2) in HAL mutation may act as a key regulator in these processes of muscle. Our findings indicate the different regulatory mechanisms of RN and HAL mutations in relation to porcine muscle energy metabolism and meat quality.

Postmortem lactate accumulation in skeletal muscle is linearly associated with the extent of pH decline. Yet, pigs harboring the AMPKγ3(R200Q) mutation produce meat with similar lactate levels to that of wild-type pigs but have a lower ultimate pH. We hypothesized that lower initial lactate levels and (or) lower buffering capacity in muscle of these pigs may help explain this discrepancy. Longissimus lumborum muscle samples were harvested at 0 and 1440 min postmortem from AMPKγ3(R200Q) and wild-type pigs. As expected, AMPKγ3(R200Q) muscle exhibited a lower ultimate pH but similar lactate levels to that of wild-type pigs at 1440 min postmortem. However, the total net lactate produced postmortem was greater in the AMPKγ3(R200Q) muscle due to lower initial lactate levels at 0 min postmortem. Buffering capacity measured over the pH range of 5.5-7.0 was also lower in AMPKγ3(R200Q) muscle. Greater net lactate accumulation postmortem (i.e., glycolytic flux) coupled with a lower buffering capacity explains the lower ultimate pH of meat from AMPKγ3(R200Q) pigs.

Our objective was to determine the influence of mitochondria on metabolites and pH decline using an in vitro model of postmortem muscle metabolism. Mitochondria were isolated from porcine longissimus lumborum and added (0, 0.5, or 2.0 mg) to powdered muscle in reaction media containing either a combination of inhibitors for mitochondria complexes (I, IV, and V) or diluent (without inhibitors). In the absence of inhibitors, adding mitochondria (0.5 and 2.0 mg) reduced ATP loss from 30 to 120 min, but did not alter glycogen or lactate during this time. In reactions with mitochondria, inhibitors decreased ATP levels by 30 min and increased glycogen degradation by 60 min. Regardless of mitochondria content, inhibitors enhanced lactate accumulation from 15 to 240 min, and decreased pH from 15 min to 1440 min. In the in vitro model, mitochondria influence the maintenance of ATP, and inhibition of mitochondria enzyme activity contributes to accelerated metabolism and pH decline.

The objective of this study was to determine the influence of Brahman genetics on muscle contractile and metabolic phenotype and postmortem proteolysis. Cattle used in this study represent a continuous spectrum of Angus-Brahman genetic variation. Steers were harvested and Longissimus samples were collected at 1.5h, 24h, and 14d postmortem. Proteolysis during the 14d aging period was evaluated, along with Warner-Bratzler shear force (WBSF) and trained sensory panel tenderness. Myosin heavy chain composition and enzymatic activity were used to evaluate fiber type characteristics. As Brahman influence increased, WBSF increased and sensory tenderness decreased. Calpain-1 autolysis decreased as Brahman percentage increased, and corresponded with reduced degradation of troponin-T, desmin, and titin. Increasing Brahman percentage was associated with greater citrate synthase activity and greater cross-sectional area of type IIx fibers. Brahman-influenced cattle produced tougher steaks and exhibited decreased protein degradation. Thus, Brahman genetics impacted not only the calpain-calpastatin system, but also muscle fiber size and metabolic properties.

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.

Beef from Bos taurus indicus is associated with toughness compared to Bos taurus taurus, suggesting there is antagonism between adaptability to heat and beef quality. Resistance to cellular stress in muscle may be protective postmortem, thereby delaying its conversion to meat. Therefore, our objective was to determine pH decline, calpain-1 and caspase 3 activation, and proteolysis in different biological cattle types. Angus, Brangus, and Brahman steers (n = 18) were harvested, and Longissimus lumborum were assessed postmortem for pH decline, ATP content, protease activation, and calpastatin content; and myofibrillar protein degradation was evaluated in beef aged to 14d. Brahman Longissimus lumborum exhibited resistance to pH decline, greater ATP content at 1 h, and delayed calpain-1 autolysis. Although content of caspase-3 zymogen was lower in Brahman, there was no evidence of caspase-3 mediated proteolysis. Greater resistance to energetic and pH changes early postmortem in Brahman Longissimus lumborum are associated with calpain-1 autolysis but not mitochondria mediated apoptosis.

Fresh meat quality development is influenced by pH decline that results from muscle glycolyzing energy substrates postmortem. The exact reason why glycolysis stops in the presence of residual glycogen remains unclear. We hypothesized that a critical glycolytic enzyme loses activity near the ultimate pH of meat. Porcine longissimus muscle samples were subjected to an in vitro system that mimics postmortem anaerobic metabolism at buffered pH values (7.0, 6.5, 6.0, 5.5 or 5.0). At pH7.0, 6.5, and 6.0, glycogenolysis and glycolysis proceeded normally while pH5.5 stopped lactate formation. Additional experimentation indicated that phosphofructokinase lost activity at pH5.5 while all other glycolytic enzymes remained active. A similar inactivation of phosphofructokinase was observed when using chicken and beef muscle. Elevated temperature hastened pH decline and phosphofructokinase activity loss. Thus, pH inactivates phosphofructokinase and arrests postmortem glycolysis, which may explain the similar ultimate pH across meat of different species.

Pigs with the AMP-activated protein kinase γ3 R200Q (AMPKγ3(R200Q)) mutation generate pork with low ultimate pH (pHu). We hypothesized that reducing muscle creatine (Cr) and phosphocreatine (PCr) may accelerate postmortem ATP consumption and prevent extended pH decline in AMPKγ3(R200Q) longissimus muscle. Wild type and AMPKγ3(R200Q) pigs were assigned to control diet or diet supplemented with the creatine analog β-guanidinopropionic acid (β-GPA, 1%) for 2 wk. β-GPA reduced muscle PCr (P = 0.006) and total Cr (P0.0001). In general, AMPKγ3(R200Q)+β-GPA exhibited more rapid metabolism than control, AMPKγ3(R200Q), and β-GPA treatment, evidenced by more rapid loss of ATP, more rapid increase in IMP, and decreased pH during the first 90 min postmortem. Overall, pHu was similar despite elevated glycogen (AMPKγ3(R200Q)), reduced total Cr (β-GPA) or both (AMPKγ3(R200Q)+β-GPA). Thus, reducing muscle phosphagens did not affect pHu in AMPKγ3(R200Q) muscle, but it hastened ATP depletion and pH decline.

Meat quality development, or the transformation of muscle to meat, involves a myriad of biochemical pathways that are largely well-studied in living muscle tissue. However, these pathways are less predictable when homeostatic ranges are violated. In addition, there is far less known about how various management or environmental stimuli impact these pathways, either by substrate load or altered cellular environment. Likewise, it is largely accepted that oxygen plays little to no role in the conversion of muscle to meat, as anaerobic metabolism predominates in the muscle tissue. Even so, the oxygen tension within the tissues does not fall precipitously at exsanguination. Therefore, transition to an anaerobic environment may impact energy metabolism postmortem. Antemortem handling, on the other hand, clearly impacts meat quality development, yet the exact mechanisms remain a mystery. In this paper, we will attempt to review those factors known to affect postmortem energy metabolism in muscle and explore those areas where additional work may be fruitful.

Rate and extent of postmortem metabolism control pork quality development. Our objective was to evaluate the role of the phosphagen system (phosphocreatine, PCr; and creatine, Cr) on metabolism and pork quality. Muscle PCr and Cr were manipulated by feeding pigs the creatine analogue, β-guanidinopropionic acid (β-GPA). In experiment 1, pigs received standard (control) diet or β-GPA supplemented (2%) diet (1 wk or 2 wk). Supplementation with β-GPA (2 wk) decreased total Cr (PCr+Cr; P=0.02) and improved pork color (decreased reflectance, P=0.003); however, β-GPA supplementation reduced growth performance (P=0.007). To separate effects of phosphagen system and growth, a second experiment was conducted with control, pair-fed, and 2 wk β-GPA (1%) supplementation; pigs were also offered a control or β-GPA supplemented flavored beverage. Neither treatment influenced pork quality. Immediately postmortem, ATP/ADP was higher in control compared to pair-fed (P0.05); subsequently, ATP/ADP was similar among all groups. Loss of the phosphagen system may lead to adaptive changes that promote conservation of cellular ATP.

Extent of postmortem pH decline influences meat quality development. To better understand physiological determination of ultimate pH (pH u ), we utilized female and castrated male pigs from a line whose selection index includes differentiated pH u . All genotypes of AMP-activated protein kinase γ3 subunit (AMPKγ3) V199I site were present. The mutant 199II genotype increased pH u , but only in castrated males. Genotype affected glycolytic potential (GP), but GP was weakly associated with pH u . A subset of animals was selected based on low (− Gly) and high (+ Gly) residual glycogen content, and compared with AMPKγ3 200Q, which is associated with low pH u . Both + Gly and 200Q muscle contained glycolytic substrate at 24 h; however, 200Q muscle generated low pH u and greater lactate compared to + Gly. Additionally,− Gly and + Gly groups exhibited similar pH u despite a large difference in GP. In conclusion, high GP does not appear to directly impact the extent of postmortem pH decline.

Postmortem changes that occur during the process of converting muscle to meat play a critical role in the development of quality traits and the overall acceptance of the fresh product. After an animal is harvested, muscle glycogen along with high-energy phosphate compounds is anaerobically mobilized in an effort to maintain adenosine triphosphate (ATP) homeostasis. As a consequence of postmortem glycolysis, lactate and H+ accumulate in the muscle and cause the drop in pH. Ultimately postmortem metabolism is arrested due to substrate depletion or the inactivation of the glycolytic enzyme phosphofructokinase-1, resulting in ATP depletion and the development of rigor mortis. The muscle subsequently undergoes proteolytic disruption of structural proteins, leading to an improvement of meat tenderness and flavor. Factors such as genetics, nutrition, environmental conditions, and pre- and postmortem handling can drastically influence the conversion of muscle to meat.

Muscle tissue experiences profound changes during its conversion to meat. Indeed, these changes are necessary and requisite for meat to assume those unique properties are keenly coveted by consumers of this highly nutritious and palatable food source. However, when this process is perturbed, the resulting quality is dramatically impacted. Therefore, understanding those reactions forming the basis of postmortem metabolism is incumbent to those interested in meat animal production. Initially, the biochemistry responsible for postmortem metabolism is simply an extension of those processes responsible for supplying energy to functioning skeletal muscle, responsible for locomotion. However, at some yet-to-be defined point, muscle tissues broach the physiological threshold where they begin to die and at this juncture, biochemical reactions dysregulate according to physiological normalcy. Even with the seemingly dearth of information available on the topic, our intent is to outline the biochemical processes responsible for postmortem metabolism and the conversion of muscle to meat as currently known.

Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPK gamma 3 subunit (AMPK gamma 3(R200Q)); this may decrease the glycogen storage capacity of AMPK gamma 3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPK gamma 3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPK gamma 3(R200Q), RyR1(R615C), and AMPK gamma 3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPK gamma 3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPK gamma 3(R200Q) relative to all other genotypes, whereas both AMPK gamma 3(R200Q) and AMPK gamma 3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPK gamma 3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPK gamma 3(R200Q) mutation than activity. NINDS NIH HHS [R01 NS056454]; NIDDK NIH HHS [R01 DK027221]

Calcium is important for muscle contraction and controls many cellular processes. Although there is evidence that calcium-mediated signals regulate AMP-activated protein kinase (AMPK) activity, the molecular mechanisms by which calcium regulates AMPK are poorly understood. To compare the function of sustained vs. intermittent calcium oscillations on AMPK activity and define specific signals in this pathway, we administered mice with aminoimidazole-carboxamide-ribonucleotide (AICAR) and caffeine with or without dantrolene. AMPK activity was increased by 10 d AICAR treatment (P < 0.01). Ten day caffeine treatment decreased AICAR-induced AMPK activity to control level. This repressed AMPK activity was blocked by dantrolene. Different calcium frequencies were simulated in C2C12 myotubes by alternating media containing caffeine and dantrolene. Intermittent calcium oscillation increased AMPK activity compared to control (P < 0.05), whereas sustained calcium oscillation decreases AICAR-induced AMPK activity to control level. This result suggests a biphasic control of AMPK activity by calcium. Knockdown of CaMKII expression by short-hairpin RNA resulted in increased AMPK phosphorylation by AICAR even in the presence of caffeine. These data show different calcium oscillations elicit distinct responses in muscle cells suggesting that the negative effects of chronic calcium treatment on AMPK activity is partly mediated through the CaMKII signals.

ObjectivesHeat shock proteins (HSP) are biomarkers of stress and perform chaperoning functions to fold, unfold, and refold proteins after heat stress. Brahman are more heat tolerant than Angus, while Angus beef has been associated with greater palatability than Brahman. The objectives were to determine if HSP content in the longissimus lumborum differs between Angus and Brahman and examine how HSP content relates to the eating quality of beef.Materials and MethodsAngus and Brahman steers (n = 12 per breed) were finished during summer in Florida and harvested at approximately 17 mo of age. Samples of longissimus lumborum were collected at 1 h after exsanguination and were immediately immersed in liquid nitrogen. Samples were then pulverized, diluted in extraction buffer, and homogenized. The protein samples were assayed to assess protein concentration and subsequently diluted to equal concentrations for loading into acrylamide gels. Proteins were separated by gel electrophoresis, and western blotting was used to evaluate content of aβ-crystallin, HSP27, HSP60, HSP70, and HSP90. Target bands were detected and quantified using LI-COR Odyssey and target signal was normalized to total protein stain. Tenderness was evaluated in 14d-aged steaks using Warner-Bratzler shear force (WBSF) and a trained sensory panel. Data were analyzed using one-way ANOVA and Pearson correlations were conducted for content of HSPs and objective and subjective tenderness.ResultsHSP27, HSP60, and HSP70 did not differ between breeds (P > 0.05); however, HSP90 and aβ-crystallin were greater (P = 0.005) in the longissimus lumborum of Angus compared to Brahman. Even though WBSF did not differ (P = 0.29) between breeds, breed affected (P < 0.0001) sensory tenderness. Content of aβ-crystallin was associated with sensory tenderness (r2 = 0.52, P = 0.0098).ConclusionLongissimus dorsi from the Angus were contained more aβ-crystallin and HSP90 than Brahman. Elevated concentrations of both aβ-crystallin and HSP90 could be breed related or may have been influenced by the season they were harvested. While WBSF was not affected by breed, panelists rated Angus steaks as more tender after aging for 14d. Content of aβ-crystallin is associated with tenderness; however further work is necessary to determine if this small HSP affects proteolysis.

ObjectivesMitochondrial function in postmortem muscle is affected by decreasing oxygenation. Functional properties relating to energy production and integrity of mitochondria may influence development of meat quality characteristics. Therefore, the objective was to evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Materials and MethodsSteers (n = 6) of primarily Angus (80 to 100%) genetics were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were collected at 1, 3, and 24h postmortem. Fresh-preserved muscle samples were permeabilized using saponin, and muscle bundles (2–4 mg) were transferred to a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. First, pyruvate and malate were added to support the TCA cycle and assess leak respiration. Then, ADP was added to support electron flow through complex I. The influence of glutamate on NADH production (complex I) was tested, followed by complex II activation by succinate. Integrity of the mitochondria outer membrane was tested with cytochrome c. Next, an uncoupler (FCCP) was added to force the electron transport system (ETS) to maximum capacity. Citrate synthase (CS) activity (nmol/min/mg tissue) was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity (pmol/sec/U CS) 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.ResultsMuscle type affected (P = 0.0002) leak OCR, with Dia showing a higher rate than LL. After ADP was added, mitochondria from Dia 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 Dia, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from Dia and LL were similar (P > 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P < 0.05) in Dia mitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR by 6.6%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased 52.4%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with Dia exhibiting greater efficiency.ConclusionDespite inherent metabolic differences between the LL and Dia, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of LL mitochondria are reduced compared to Dia. Declining mitochondrial function may be associated with calcium overload, mitochondrial fragmentation, and protease activation.

Pale, soft and exudative (PSE) pork represents considerable economic losses for the industry due to its limited functionality and undesirable appearance. During the past several decades, exhaustive research covering various aspects of the food chain has established genotyping procedures, recommended handling practices, and quality indicators in order to reduce the incidence of inferior pork quality. Despite these efforts, there is still a relatively high occurrence of PSE pork. Development of pork quality attributes is largely governed by the rate and extent of postmortem pH decline. The combination of high temperature at low pH or abnormally low ultimate pH causes denaturation of sarcoplasmic and myofibrillar proteins, resulting in paler color and reduced water holding capacity. The pH decline is closely related to muscle energy availability and demand at or around slaughter. The postmortem degradation of glycogen through glycogenolysis and glycolysis provides ATP to help meet energy demand and decreases pH by generating lactate and H+. Therefore, the flux of metabolites through glycolysis, the involvement of energy signaling pathways that modulate glycolytic activity, and the inherent metabolism of different fiber types are critical factors influencing pH decline and pork quality. Further, recent work implicates adenosine monophosphate-activated protein kinase (AMPK) as a major energy sensor for the cell, and thus may be involved in the control of postmortem metabolism. The intent of this paper is to review the biochemistry controlling postmortem energy metabolism in pig muscle and explore new information generated using genetic mutations in order to define the fundamental mechanisms controlling the transformation of muscle to meat.