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Toxic tall fescue contains ergot alkaloids, which induce vasoconstriction in livestock, leading to fescue toxicosis. This condition can impact muscle development due to altered blood flow. Supplementation with 5-HTP may counteract the vasoconstrictive effects of fescue alkaloids. We aimed to investigate the influence of 5-HTP supplementation on satellite cells, myoblast, and preadipocyte numbers in maternal and fetal muscle during late gestation in Suffolk ewes. We hypothesized that 5-HTP supplementation would mitigate the effects of fescue toxicosis on muscle cell populations, potentially promoting muscle development and regeneration in maternal and fetal tissues. Suffolk ewes (n = 75), naïve to toxic tall fescue, were blocked by body condition score and body weight into 10 groups for synchronization. Ewes were confirmed pregnant using transabdominal ultrasound, and lamb number was estimated at gestational day (gd) 60. Pregnant ewes carrying twins (n = 30) were randomly assigned to one of three treatments: 1) negative control (CON, n = 10), 2) toxic tall fescue seed containing 1.77 mg ewe-1ּ d-1 ergovaline/ergovalinine plus 5-HTP (2.5 mg/kg BW) supplement (5HTP, n = 10), or 3) positive control, fed toxic tall fescue seed containing 1.77 mg ewe-1ּ d-1 ergovaline/ergovalinine (E+, n = 10). Ewes were individually fed a total mixed ration from gd 78 to gd 85 of pregnancy and then TMR plus treatment (CON, E+ or 5HTP) from gd 86 to d 111. On gd 112, terminal necropsies were performed, and semimembranosus muscle (SM) from the ewe and fetus was removed. Weights were obtained, and then samples were digested using pronase (1.5mg/mL) to isolate preadipocyte (PP1), myoblasts (PP2) and satellite cell (PP3) fractions using a pre-plating technique. Cells were quantified using a Neubauer-hemocytometer and trypan blue to also assess cell viability and changes in cell number. Fetal SM had a greater (P < 0.05) number of pre-adipocytes, myoblasts, and satellite cells per gram of tissue digested in comparison with the ewes. Pre-adipocyte and myoblast number per gram of tissue digested did not differ (P > 0.05) by treatment. The 5HTP group had the greater (P < 0.05) number of satellite cells per gram of tissue digested in comparison with the CON group. Satellite cell numbers tended (P < 0.10) to be greater in the 5HTP group in comparison to the E+ group. Satellite cell numbers from CON and E+ treatment groups did not differ (P > 0.05). In conclusion, 5-HTP supplementation during late gestation in Suffolk ewes exposed to toxic tall fescue significantly increased satellite cell numbers in maternal and fetal muscle tissues. This suggests a potential mitigation of the impact of fescue toxicosis on muscle cell populations. [ABSTRACT FROM AUTHOR]

Development and maintenance of healthy muscle fibers rely on the myogenic potential of satellite cells (SC), muscle stem cells that proliferate and differentiate to form myotubes. Satellite cells are indispensable for post-hatch muscle growth as well as muscle repair and regeneration when myofibers are damaged. Pectoralis major of young broiler chicks (5-d olds) is a readily available source of SC, which can be used in vitro to elucidate cellular and molecular mechanisms responsible for muscle growth and regeneration in broilers. Here, we optimized a method for efficient isolation, purification, and differentiation of SC, from young broiler chicks. This procedure includes a simple method that allows SC to be purified from other muscle cell types that can impede the fidelity of follow-on experiments, particularly highly sensitive measures such as RNAseq. The methods for culturing and differentiating SC into multinucleated myotubes were also optimized by testing serum types, concentrations, and the effects of chicken embryo extract. Using the isolation procedure, a highly pure SC population (94.6 ± 2.11% Pax7+) with high viability and yield was obtained, and their capacity to differentiate into myotubes was confirmed. Enrichment for SC and myogenic capacity were maintained through multiple passages and after cryopreservation. Analysis of gene expression over the first 48 h of differentiation confirmed that SC exhibited the expected molecular signature of myogenesis. Taken together, this method simplifies the ability to isolate and maintain a relatively pure population of SC with strong myogenic potential from young broiler chicks, and should support downstream applications for assessing the impact of nutrients, metabolites, and other physiological cues on muscle growth and development in broilers. [ABSTRACT FROM AUTHOR]

Beta-adrenergic agonists (β-AA s) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which β-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCR s) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no β-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide- p -trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by β-AA treatment in ovine or porcine cells. These findings indicate that β-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to β-AA incubation also demonstrates differing physiological responses to β-AA across species, which helps to explain the variation in its effectiveness as a growth supplement. [ABSTRACT FROM AUTHOR]

Skeletal muscle growth is largely dependent on the proliferation and differentiation of muscle-specific stem cells known as satellite cells (SC). Previous work has shown that dietary inclusion of the vitamin D3 metabolite, 25-hydroxycholecalciferol (25OHD3), also called calcidiol, can promote skeletal muscle growth in post-hatch broiler chickens. Improving vitamin D status of broiler breeder hens by feeding 25OHD3 in addition to vitamin D3 has also been shown to positively impact progeny. Yet, whether combined pre- and post-hatch supplementation with 25OHD3 produces an additive or synergistic SC-mediated, skeletal muscle growth response remains unanswered. To evaluate the effect of combined maternal and post-hatch dietary 25OHD3 supplementation on the growth and SC mitotic activity of the Pectoralis major (PM) muscles in broiler chickens, a randomized complete block design experiment with the main effects of maternal diet (MDIET) and post-hatch diet (PDIET) arranged in a 2 × 2 factorial treatment structure was conducted. From 25 to 36 wk of age, broiler breeder hens were fed 1 of 2 MDIET formulated to provide 5,000 IU D3 (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3). Their male broiler chick offspring (n = 400) hatched from eggs collected from 35 to 36 wk of age were reared in raised floor pens. Broilers were fed 1 of 2 PDIET formulated to provide 5,000 IU of D3 per kg of feed (PCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (P25OHD3). Muscle was collected at days 4, 8, 15, 22, and 29 and stored until immunofluorescence analysis. Data were analyzed as a 2-way ANOVA with SAS GLIMMIX. Dietary 25OHD3 was effectively transferred from hen plasma to egg yolks (P = 0.002) and to broiler progeny plasma (days 4 to 22; P ≤ 0.044). Including 25OHD3 in either MDIET or PDIET altered PM hypertrophic growth prior to day 29 (P ≥ 0.001) and tended to reduce Wooden Breast severity (P ≤ 0.089). Mitotic SC populations were increased in PM of MCTL:P25OHD3 and M25OHD:PCTL-fed broilers at d 4 (P = 0.037). At d 8, the PM mitotic SC populations were increased 33% by P25OHD3 (P = 0.054). The results of this study reveal that combined maternal and post-hatch 25OHD3 supplementation does not produce additive or synergistic effects on SC-mediated broiler muscle growth. However, vitamin D status improvement through dietary 25OHD3 inclusion in either the maternal or post-hatch diet stimulated broiler breast muscle growth by increasing proliferating SC populations. [ABSTRACT FROM AUTHOR]

Satellite cell (SC) activation is defined as the time frame during which the stem cell becomes poised to reenter G1 of the cell cycle. The growth factors and events leading to full mitotic activation in equine SCs remain largely unknown. Insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), and fibroblast growth factor 2 (FGF2) are sequentially transcribed during the muscle repair and recovery period following strenuous exercise in adult horses. Expression of IGF-I occurs within 24 h of the postexercise recovery period suggesting it may affect early SC actions. As a first step, gluteus medius muscle cryosections from adult horses (n = 9) were examined for the presence of central nuclei (CN), a marker of SC addition to the fiber. Results demonstrate few CN fibers prior to exercise with a 3-fold increase (P = 0.05) 24 h postexercise. Cultures of SC (n = 4 isolates) were treated with 100 ng/mL IGF-I for varying times prior to measurement of myogenic events. Results demonstrate that IGF-I does not affect the initial lag period, proliferation, or subsequent differentiation of equine SC in vitro (P > 0.05). However, media containing a combination of IGF-I and 10 ng/mL FGF2 and 25 ng/mL HGF hastens (P < 0.05) the time to S-phase entry in fresh isolates of SCs. Media supplementation with optimal concentrations of FGF2, HGF, or a combination of HGF and FGF2 suppresses (P < 0.05) the percentage of myogenin immunopositive SCs to levels below that found in control- or IGF-I-treated SCs. These results provide new insight into the combinatorial roles growth factors play during equine SC myogenesis. [ABSTRACT FROM AUTHOR]

Satellite cells (SC s) are multipotential stem cells having the plasticity to convert to an adipogenic lineage in response to thermal stress during the period of peak mitotic activity (the first week after hatch in poultry). The mechanistic target of rapamycin (mTOR) pathway, which regulates cellular function and fate of SCs, is greatly altered by thermal stress in turkey pectoralis major muscle SCs. The objective of the present study was to determine the effects of thermal stress, selection for growth, and the role of the mTOR pathway on SC intracellular lipid accumulation and expression of adipogenic regulatory genes. These effects were analyzed using SCs isolated from the pectoralis major muscle of 1-wk-old modern faster-growing commercial turkey line (NC) selected for increased growth and breast muscle yield as compared with SCs of a historic slower-growing Randombred Control Line 2 (RBC2) turkey. Heat stress (43 °C) of SCs during proliferation increased intracellular lipid accumulation (P < 0.001), whereas cold stress (33 °C) showed an inhibitory effect (P < 0.001) in both lines. Knockdown of mTOR reduced the intracellular lipid accumulation (P < 0.001) and suppressed the expression of several adipogenic regulatory genes: peroxisome proliferator-activated receptor-γ (PPARγ ; P < 0.001), CCAAT/enhancer-binding protein-β (C/EBPβ ; P < 0.001), and neuropeptide-Y (NPY ; P < 0.001) during both proliferation and differentiation. The NC line SCs showed fewer reductions in lipid accumulation compared with the RBC2 line independent of temperature. Both intracellular lipid accumulation (P < 0.001) and PPARγ expression (P < 0.001) were greater at 72 h of proliferation than at 48 h of differentiation in both the RBC2 and NC lines independent of temperature. Thus, hot and cold thermal stress affected intracellular lipid accumulation in the pectoralis major muscle SCs, in part, through the mTOR pathway in wea growth-dependent manner. Altered intracellular lipid accumulation could eventually affect intramuscular fat deposition, resulting in a long-lasting effect on the structure and protein to fat ratio of the poultry pectoralis major muscle. [ABSTRACT FROM AUTHOR]

Sheep producers are typically paid based on body weight, so understanding the mechanisms of postnatal skeletal muscle growth is vital. Satellite cells are responsible for the capacity for postnatal skeletal muscle growth. Satellite cells proliferate and then differentiate into myoblasts before fusing with existing myotubes. The retinoid X receptor (RXR) is a transcription factor that enhances myogenic differentiation 1 (MyoD) expression, which increases differentiation and fusion of myoblasts in sheep. LG 100268 is an RXR agonist, which activates the RXR. The objective of this project is to measure the effect of LG 100268 on primary ovine satellite cell (OSC) proliferation and protein synthesis rates. Primary OSC were isolated from the hind legs of 9-mo old commercial hair sheep wethers (n = 3). For proliferation, cultures were grown to approximately 70% confluency, followed by treatment with 5% fetal bovine serum (FBS) or 300 nM of LG 100268 in 5% FBS. For protein synthesis, cultures were grown to approximately 80% confluency, at which point cultures were induced to differentiate and treated 48-h later with serum free media (SFM) or 300 nM LG 100268 in SFM. Proliferation rates were measured at 24-h post-treatment, and protein synthesis rates were measured at 6-h post-treatment. Treatment with 300 nM LG 100268 increased (P = 0.004) OSC proliferation rates compared with control cultures. Preliminary protein synthesis results suggest that LG 100268 does not alter (P = 0.22) protein synthesis rates of fused OSC. However, more research is warranted to clarify the role of the RXR in sheep skeletal muscle growth. [ABSTRACT FROM AUTHOR]

Zinc (Zn) is the most utilized metal in biological processes and supports growth through critical roles in protein turnover. The Zn requirements of skeletal muscle may be augmented when growth promoting technologies, such as anabolic hormones are being utilized. Both anabolic hormones and Zn affect protein synthesis and degradation in skeletal muscle. However, the molecular mechanisms through which Zn may interact with anabolic hormones to support skeletal muscle growth remains unknown. The objective of this study was to determine whether trenbolone acetate (TBA), estradiol (E2) and Zn interact to promote differentiation of bovine satellite cell (BSC) cultures by assessing abundance of mRNA involved in growth, mineral transport, differentiation, protein turnover and oxidative stress. To do this, primary BSC cultures were isolated from three different steers and grown to 80% confluency and induced to differentiate in 3% horse serum, at which time a factorial design was employed to assess the impact of Zn (0, 10, 20 or 40 μM) as ZnCl2 and/or the impact of hormone (10 nM TBA,10 nM E2, 10 nM TBA and 10 nM E2). Zinc concentrations represent physiologically relevant plasma Zn concentrations. Total mRNA was isolated from the treated cultures at 4 h, 12 h and 24 h post-treatment. Assays utilizing cultures from each steer were run in duplicate (n = 6). The mRNA abundance of 45 genes were examined and analyzed using the PROC MIXED procedure of SAS with time as a repeated measure to evaluate the fixed effects of hormone, mineral, time, and all interactions. No effects (P ≥ 0.19) of hormone x mineral x time, hormone x time, hormone x mineral or hormone were noted. A mineral x time effect (P ≤ 0.04) was noted for the igf1r, slc39a7, mapk1, mmp2, and G6pd. A mineral effect was found for hormone receptors (ir, ar, esr1, esr2, and gper; P ≤ 0.03), mRNA involved in mineral transport (slc30a10, slc30a7, sod1, sod2, slc2a4, and slc39a8; P ≤ 0.009), and genes related to skeletal muscle differentiation (pax7, myf5, myf6, spry1, myog, mef2a, and myod; P ≤ 0.02) and protein turnover (akt1, fox01, fox04, mstn,fbox32, fox03, mtor, trim63, and CKM; P ≤ 0.03). These differences are generally driven by abundance being increased (P ≤ 0.05) in cultures treated with 40 or 20 μM Zn compared with cultures receiving 0 or 10 μM Zn. These results indicate that while Zn and hormone do not appear to interact to influence gene expression of differentiating primary BSC cultures at the time points measured, increased concentrations of Zn appear to increase mRNA abundance of hormone receptors, mineral transporters, and genes involved in skeletal muscle differentiation and protein turnover. [ABSTRACT FROM AUTHOR]

It has been well established in both in vitro and in vivo models that the hormones found in anabolic implants [estradiol (E2) and trenbolone acetate (TBA)] positively impact skeletal muscle growth. Previous research has demonstrated that treating primary bovine satellite cells (BSC) with E2 or TBA results in increases in proliferation and protein synthesis and decreases in protein degradation, leading to enhanced skeletal muscle growth. This improved skeletal muscle growth occurs through partially defined pathways, but many unknowns remain. More recently, research has emerged demonstrating that strategic supplementation of specific trace minerals (TM) may further augment the positive effects of E2 and TBA on skeletal muscle growth. There are many ways through which the complex signaling pathways induced by the hormones found in anabolic implants may interact with TM to further enhance skeletal muscle growth. Zinc (Zn) and manganese (Mn) are two of the TM that we have recently investigated. Zinc is the most utilized metal in biological processes and supports animal growth through critical roles in the functions of IGF1, GH, and DNA synthesis. Manganese is an essential TM providing catalytic activity to enzymes involved in protein glycosylation, nitrogen metabolism, and antioxidant capacity. However, additional research is needed to determine if hormones and TM interact to impact growth of skeletal muscle, specifically. As such, the goal of recent research that has been conducted by our group is to utilize an in vitro primary BSC model to determine whether hormones and TM interact to alter differentiation and protein synthesis. Preliminary research from our group assesses the impacts of varying, physiological concentrations of Zn or Mn on differentiation and protein synthesis of primary BSC cultures that have also been treated with or without the hormones found in anabolic implants. These preliminary findings demonstrate that while there seems to be no interaction between Zn and hormones in differentiating BSC, supplemental Zn on the higher end of physiologically relevant concentrations has a positive effect on signaling pathways involved in growth, protein turnover, skeletal muscle differentiation, and mineral transporters. However, it appears that Zn and Mn each interact with hormones to positively augment protein synthesis in primary BSC cultures. A deeper understanding of the relationship these TM and others may have with hormones will allow for more strategic supplementation practices that can potentially improve skeletal muscle growth. [ABSTRACT FROM AUTHOR]

Vitamin D is an essential micronutrient most well-known for regulating calcium and phosphorus homeostasis and bone mineralization. However, over the last 20 yr, the extra-skeletal, pleotropic effects of vitamin D and its metabolites have gained greater attention. For livestock species reared indoors, vitamin D supplementation is especially important and commonly achieved by dietary addition of cholecalciferol, also known as vitamin D3 (D3 ). Cholecalciferol is hydroxylated in the liver to produce the circulating form, 25-hydroxycholecalciferol (25OHD3 ), also called calcidiol. Circulating 25OHD3 is subsequently converted to 1α,25-hydroxycholecalciferol (1,25OHD3 ) which binds a nuclear receptor found in many tissues, including skeletal muscle, to elicit its effects. This presentation will focus on the recent work regarding the effects of both maternal and postnatal 25OHD3 supplementation on porcine and avian satellite cell mitotic activity and skeletal muscle growth characteristics. Feeding broilers 25OHD3 posthatch results in a satellite cell-mediated skeletal muscle hypertrophic response in the fast-twitch pectoralis major but not the slow-twitch biceps femoris. Maternal dietary supplementation of gilts and hens with 25OHD3 in place of the majority of D3 increases circulating 25OHD3 concentrations in both dams and their offspring. The improvements in vitamin D status coincide with positive effects on skeletal muscle fiber number in fetal piglets as well as satellite cell mitotic activity and hypertrophic growth of fibers in both swine and broiler chickens. Though, interestingly, combining maternal and posthatch 25OHD3 supplementation in broilers does not appear to produce additive or synergistic effects on SC-mediated muscle growth. However, increasing circulating concentrations of 25OHD3 by supplementing either the maternal or postnatal diet with 25OHD3 has positive effects on meat yield in both porcine and avian models. Overall, these data shed light on the cellular mechanisms behind the efficacy of this vitamin D supplementation strategy using commercially available 25OHD3 sources in increasing meat yield and yet, leaves enough unanswered questions remaining to warrant further exploration. [ABSTRACT FROM AUTHOR]

microRNAs (miRNA) are small non-coding RNAs that modulate muscle satellite cell proliferation. Previous research identified miR-22-3p as differentially expressed during ovine muscle development and in vitro experiments showed that antagomir-22-3p treatment enhanced satellite cell proliferation. The objective of this study was to test in vivo vascular injection of antagomir-22-3p in lateral saphenous vein on miRNA and HDAC family mRNA expression in intrauterine growth restricted lambs. Pregnant ewes (n = 18) with twins were either fed at 100% of NRC (CON) or nutrient restricted (60% NRC; NR) from gestational d 85 to parturition. On d 12 of age, NR lambs (n = 8) were randomly selected and given a systemic injection of antagomir-22-3p in the lateral saphenous vein of the right leg (MIR-NR). CON lambs (n = 8) were also randomly selected and given a sham injection of PBS in the lateral saphenous vein of the right leg (SHAM-CON). Antagomir-22-3p was reconstituted in phosphate buffered saline (PBS). Injections were given for 3 consecutive days and lambs harvested 24 d later. Blood samples were collected from each lamb weekly to monitor miR22-3p expression. Lambs were slaughtered 24 d after the first injection and muscle mass measured. Muscle samples were removed for miR-22-3p and mRNA expression of potential targets. Cell-free circulating RNA was isolated from blood samples and miR-22-3p expression examined over time. Cell-free circulating miR22-3p expression was downregulated (P < 0.05) for MIR-NR compared with SHAM-CON on d 14 after injection. On d 21 after injection, miR-22-3p expression in plasma tended (P = 0.08) to be down-regulated in MIR-NR compared with SHAM-CON. Lamb body weight and muscle weights at harvest were similar between MIR-NR and SHAM-CON treatments. In heart and semimembranosus (SM) muscles, expression of miR-22-3p was down-regulated (P < 0.05) in MIR-NR compared with SHAM-CON. In the gastrocnemius and semitendinosus muscle, miR-22-3p expression was unchanged (P > 0.05). The number of type I and IIa muscle fibers were greater (P < 0.05) for MIR-NR than SHAM-CON. Muscle fiber area of Type I, IIa or IIx fibers did not differ (P > 0.05) by treatment. The number of type IIx fibers did not differ (P > 0.05) between treatments. MIR-NR treatment appears to shift muscle fibers towards more oxidative metabolism and down-regulate HDAC-1, 3, and 11 and SIRT6 in SM. The systemic injection of antagomir-22-3P downregulated miR-22-3p expression in circulation and in muscle tissues, which altered expression of HDAC/ SIRT genes involved in muscle fiber type conversion and hypertrophy [ABSTRACT FROM AUTHOR]

Our lack of understanding regarding how heat exposure affects major tissue growth and meat attributes impedes our ability to counteract more frequent climate extremes and promote profitability in the beef cattle industry. The objective of the study was to determine pathways that heat shock proteins (HSPs) regulate in beef cattle's skeletal muscle and adipose tissue growth and metabolism. Bovine satellite cells (BSCs) were isolated from the longissimus muscle of 3-month-old Holstein bulls (n = 5). Preadipose cells were isolated from the subcutaneous fat of 18-monthold Angus steers (n = 5) and induced adipogenic differentiation. Satellite, myofibers, and adipose cells were incubated at 38°C (control; CON), 39.5°C (moderate heat stress; MHS), and 41°C (extreme heat stress; EHS) and harvested for mRNA gene expressions, protein level, proteomics, immunostaining. Heat exposure cued bovine satellite cell proliferation and may increase the self-renewing satellite cell population. After 3 h of heat exposure, the mRNA gene expression and protein levels of Pax3 and Myf5 were downregulated, while HSPs 20, 27, 70, and 90 were upregulated. Small heat shock proteins, especially HSP27, appear to be essential in regulating satellite cell activity under heat stress. When HSP27 knock-down BSCs were exposed to EHS for 3 h, Myf5 gene expressions and protein levels returned to normal. This may indicate that HSP27 directly regulates Myf5 under heat stress and temporarily activates the self-renewal mechanism instead of committing myogenic differentiation. Results from proteomics showed that pathways related to oxidative phosphorylation, NADH dehydrogenase activity, and transmembrane transport were upregulated by EHS while MHS upregulated biosynthesis of amino acids, PI3K-Akt signaling pathway. Heat exposure during myogenic differentiation of satellite cells seemingly stimulates the commitment of myogenesis. Heat treatments increased mRNA gene expressions of IGF I, MHC I, MyoD, and myogenin and protein levels. Heat exposure also improved the muscle protein synthesis pathway by regulating the phosphorylation of mTOR, Akt, and P70S6. Heat stress triggered adipogenic differentiation and lipid storage, followed by increased expression of HSP. Extreme heat stress increased adipose cell growth by upregulating C/EBPα and PPARγ, key regulators of adipogenesis. Lipogenic genes FAS and SCD were also increased, accompanied by HSPs. Adipocytes exposed to EHS also increased intracellular lipid accumulation. Proteomic analysis revealed that carbon and pyruvate metabolism pathways were upregulated by MHS, whereas glycolysis/gluconeogenesis, protein processing in the endoplasmic reticulum, RNA transport, and biosynthesis of amino acids were upregulated by EHS. Cellular mechanisms related to skeletal muscle hypertrophy and adipose tissue growth are responsive in in-vitro settings; however, the results are controversial depending on cell type and animal species. Various factors such as metabolic, hormonal, and immunologic regulation need to be considered to accurately assess the effect of HS on the postnatal growth of major tissues at the animal level. [ABSTRACT FROM AUTHOR]

A randomized complete block design experiment with 30 yearling crossbred steers (average BW = 436.3 ± 39.8 kg) fed a steam-flaked corn-based diet was used to evaluate the effects dietary vitamin A (Rovimix A 1000; DSM Nutritional Products Ltd. Sisseln, SUI) supplementation on myogenic gene expression and skeletal muscle fiber characteristics during the finishing phase. Steers were blocked by BW (n = 5 blocks; 6 steers/block), randomly assigned to pens (n = 2 steers/pen), and one of the following treatments: no added vitamin A (0 IU ; 0.0 IU/kg of dietary dry matter intake of additional vitamin A), vitamin A supplemented at the estimated requirement (2,200 IU ; 2,200 IU/kg of dietary dry matter (DM) of additional vitamin A), and vitamin A supplemented at 5× the estimated requirement (11,000 IU ; 11,000 IU/kg of dietary DM of additional vitamin A). After all treatments underwent a 91-d vitamin A depletion period, additional vitamin A was top-dressed at feeding via a ground corn carrier. Blood, longissimus muscle, and liver biopsy samples were obtained on days 0, 28, 56, 84, and 112. Biopsy samples were used for immunohistochemical and mRNA analysis. Sera and liver samples were used to monitor circulating vitamin A and true vitamin A status of the cattle. Expression for myosin heavy chain (MHC)-I diminished and rebounded (P = 0.04) over time. The intermediate fiber type, MHC-IIA, had a similar pattern of expression (P = 0.01) to that of MHC-I. On day 84, C/EBPβ expression was also the greatest (P = 0.03). The pattern of PPARγ (P < 0.01) and PPARδ (P < 0.01) expression seemed to mimic that of MHC-I expression, increasing from days 84 to 112. Distribution of MHC-IIA demonstrated a change over time (P = 0.02). Muscle fiber cross-sectional area increased by day (P < 0.01) for each MHC with the notable increase between days 0 and 56. Total nuclei density decreased (P = 0.02) over time. Cells positive for only Myf5 increased (P < 0.01) in density early in the feeding period, then declined, indicating that satellite cells were fusing into fibers. The dual-positive (PAX7+Myf5) nuclei also peaked (P < 0.01) around day 56 then declined. These data indicated that gene expression associated with oxidative proteins may be independent of vitamin A status in yearling cattle. [ABSTRACT FROM AUTHOR]

Postexercise skeletal muscle repair is dependent on the actions of satellite cells (SCs). The signal(s) responsible for activation of these normally quiescent cells in the horse remain unknown. The objective of the experiment was to determine whether submaximal exercise or tributyrin (TB) supplementation is sufficient to stimulate SC activation. Adult geldings were fed a control diet (n = 6) or a diet containing 0.45% TB (n = 6). After 30 d, the geldings performed a single bout of submaximal exercise. Middle gluteal muscle biopsies and blood were collected on days −1, 1, 3, and 5 relative to exercise. Diet had no effect on any parameter of physical performance. Total RNA isolated from the gluteal muscle of TB fed geldings contained greater (P < 0.05) amounts of myogenin mRNA than controls. Satellite cell isolates from TB supplemented horses had a greater (P = 0.02) percentage of proliferating cell nuclear antigen immunopositive (PCNA+) SC than controls after 48 h in culture. Submaximal exercise was sufficient to increase (P < 0.05) the percentage of PCNA(+) cells in all isolates obtained during recovery period. No change in the amount of gluteal muscle Pax7 mRNA, a lineage marker of SCs, occurred in response to either diet or exercise. Our results indicate that both submaximal exercise and TB prime SCs for activation and cell cycle reentry but are insufficient to cause an increase in Pax7 expression during the recovery period. [ABSTRACT FROM AUTHOR]

We hypothesized that oleic acid (OA) in the absence of a thiazolidinedione (i.e. a synthetic peroxisome proliferator-activated receptorγ [PPARγ] agonist) would increase adipogenic gene expression in bovine muscle satellite cells (BSC). The BSC were cultured in differentiation medium containing 10 µM ciglitazone (CI), 100 µM OA, or 100 µM OA plus 10 µM CI (CI-OA). Control (CON) BSC were cultured only in differentiation media (containing 2% horse serum). The presence of myogenin, desmin, and paired box 7 proteins was confirmed in the BSC by immunofluorescence staining, demonstrating that we had isolated myogenic cells. The OA BSC had lesser paired box 3 (Pax3) and myogenic differentiation 1 expression but greater Pax7 and mygogenin (MYOG) expression (P < 0.05), than the CON BSC. The CI BSC had greater Pax3 , Pax7 , and MYOG expression than CON BSC (P < 0.05), suggesting that CI would promote BSC myogenesis under pro-myogenic conditions (i.e. when cultured with horse serum). However, both the OA and CI treatments upregulated the expression of PPARγ , CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPß , sterol regulatory element-binding protein 1 , lipoprotein lipase , and glycerol-3-phosphate acyltransferase 3 gene expression, as well as media adiponectin concentration (P < 0.05). The CI, OA, and CI-OA treatments also increased triacylglycerol and lipid droplet accumulation, in spite of upregulation (relative to CON BSC) of adenosine monophosphate -activated protein kinase alpha-1 , perilipin 2 (PLIN2), and PLIN3 in BSC and downregulation of G protein-coupled protein receptor 43 , acyl-CoA synthetase long chain family member 3 , and stearoyl-CoA desaturase (P < 0.05). These results indicate that OA in the absence of a synthetic PPARγ agonist can effectively increase adipogenic gene expression in BSC. [ABSTRACT FROM AUTHOR]

Nutrition and other external factors are known to have a marked effect on growth of skeletal muscle, modulated, at least in part, through effects on satellite cells. Satellite cells and their embryonic precursors play an integral role in both prenatal and postnatal skeletal muscle growth of mammals. Changes in maternal nutrition can impact embryonic muscle progenitor cells which ultimately impacts both prenatal and postnatal skeletal muscle development. Satellite cells are important in postnatal skeletal muscle growth as they support the hypertrophy of existing myofibers. Hypertrophy of existing fibers is the only mechanism of postnatal muscle growth because muscle fiber number is fixed at birth and fiber nuclei have exited the cell cycle. Because fiber nuclei do not divide, additional nuclei required for hypertrophy must be acquired from satellite cells. To date, little research has aimed at determining whether nutrition directly impacts satellite cell populations within skeletal muscle of livestock species. However, it is well established that nutrition alters circulating concentrations of various growth factors such as insulin-like growth factor 1, epidermal growth factor, hepatocyte growth factor, and fibroblast growth factor. Each of these different growth factors impacts satellite cell proliferation and/or activation, indicating that nutrition likely plays a large role in skeletal muscle growth through impacting the satellite cell pool in both prenatal and postnatal growth. The relationship among nutrition, growth factors, and satellite cells relative to skeletal muscle growth is an important area of research that warrants further consideration. [ABSTRACT FROM AUTHOR]

Nutrition and other external factors are known to have a marked effect on growth of skeletal muscle, modulated, at least in part, through effects on satellite cells. Satellite cells and their embryonic precursors play an integral role in both prenatal and postnatal skeletal muscle growth of mammals. Changes in maternal nutrition can impact embryonic muscle progenitor cells which ultimately impacts both prenatal and postnatal skeletal muscle development. Satellite cells are important in postnatal skeletal muscle growth as they support the hypertrophy of existing myofibers. Hypertrophy of existing fibers is the only mechanism of postnatal muscle growth because muscle fiber number is fixed at birth and fiber nuclei have exited the cell cycle. Because fiber nuclei do not divide, additional nuclei required for hypertrophy must be acquired from satellite cells. To date, little research has aimed at determining whether nutrition directly impacts satellite cell populations within skeletal muscle of livestock species. However, it is well established that nutrition alters circulating concentrations of various growth factors such as insulin-like growth factor 1, epidermal growth factor, hepatocyte growth factor, and fibroblast growth factor. Each of these different growth factors impacts satellite cell proliferation and/or activation, indicating that nutrition likely plays a large role in skeletal muscle growth through impacting the satellite cell pool in both prenatal and postnatal growth. The relationship among nutrition, growth factors, and satellite cells relative to skeletal muscle growth is an important area of research that warrants further consideration. [ABSTRACT FROM AUTHOR]

An insult to the health status of an animal is associated with decreased skeletal muscle growth. Furthermore, an immune challenge to a gestating dam may inhibit pre- and postnatal muscle growth of the offspring. The objective of this study was to determine if the administration of low-dose lipopolysaccharide (LPS) to gestating sows would alter muscle characteristics of the longissimus muscle (LM) in weaned pigs exposed to LPS. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n = 7) at a dose of 2.5 μg/kg BW or saline (CON; n= 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each sow received a secondary LPS challenge. Following the postnatal challenge, barrows were euthanized (31 ± 1.3 d of age), and both LM were collected. The left LM was weighed, measured for length, and measured for circumference at the cranial, middle, and caudal portion of the muscle. Two samples from the right LM were preserved for histological analyses. Morphometric, fiber type count, primary myofiber clustering, and satellite cell counts were analyzed using the MIXED procedure of SAS 9.4 with a main effect of prenatal treatment and covariate of body weight. There were no differences (P > 0.248) between any morphometric measures. The average diameter for myosin heavy chain (MHC) type I and IIB/X fibers was increased (P < 0.048) 8.6 and 5.9% respectively in LPS offspring compared with CON offspring. Average cross-sectional area (CSA) was increased 10.6% (P = 0.030) in MHC IIB/X fibers and tended to be increased 14.9% (P = 0.080) in MHC 1 fibers of LPS offspring. Number of myofibers within the LM was increased 16.1% (P = 0.032) in CON compared with LPS offspring. Diameter, CSA, and total CSA of oxidative myofibers (MHC type I and IIA) clusters was increased (P < 0.001) 13.8, 27.6, and 22.3% respectively in LPS compared with CON offspring. There were no differences (P > 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Based on the timing of the maternal challenge, it can be theorized that LPS hinders hyperplasia during secondary myogenesis following an immune challenge to the dam, with a rescue response of increased hypertrophy of MHC I and IIB/X myofibers post-weaning and glycolytic myofiber profile. However, due to the reduction in total myofibers, the impact of LPS on parameters such as average daily gain, feed efficiency, and lean skeletal muscle accretion should be studied further. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of vitamin A supplementation during the early fetal to neonatal stages on growth performance, blood metabolites, and the development of muscle and pre-adipocytes in Korean native calves. Twenty female newborn calves [day 0 of age; initial BW: 31.2 kg (SD 3.07)] were stratified based on whether they received vitamin A supplementation during the early fetal to neonatal stages, and then randomly allocated to four groups using a 2 × 2 factorial arrangement. Vitamin A supplementation was conducted as follows: fetal stage (control: 7,800 IU/kg of as-fed, treatment: 22,800 IU/kg of as-fed); neonatal stage (control: 17,000 IU/day, treatment: 53,000 IU/day). Growth performance, blood variables, mRNA expression in muscle tissue, and muscle fiber diameter were evaluated during the experimental period and analyzed using the MIXED procedure with Tukey's test by SAS 9.4 software. As a result, fetal-vitamin A treatment group exhibited higher (P < 0.05) serum vitamin A concentrations and blood glucose concentrations on the day 3 of age. Similarly, vitamin A supplementation in the neonatal stage significantly increased (P < 0.05) serum vitamin A concentrations at day 30 and 60 of age, as well as albumin and glucose concentrations at day 60 of age. Moreover, both birth and weaning BW were significantly greater (P < 0.05) in calves receiving vitamin A supplementation during the fetal stage. However, feed intake and muscle fiber diameter showed no differences (P > 0.05) between the groups. In addition, supplementation of vitamin A during the fetal stage resulted in upregulation (P < 0.05) of ZNF423 mRNA expression in the longissimus dorsi muscle, whereas neonatal supplementation upregulated (P < 0.05) the mRNA expression of PAX7 and MYF5. Our findings suggested that vitamin A supplementation during the early fetal to neonatal stages can enhance the growth performance of offspring calves and potentially promote an increase in pre-adipocyte and satellite cell storage in muscle tissue. This information can be applied in integrated fetalneonatal nutrient feeding strategies in beef cattle. [ABSTRACT FROM AUTHOR]

As we look to better understand whole body growth and development, there are still many mechanisms of individual muscle growth that we have yet to comprehend. Therefore, the objective of this study was to compare proliferative capacity and satellite cell number within the trapezius (TP), longissimus (LM), and bicep femoris (BF) muscles of fetal bovine throughout mid and late gestation. We hypothesized a difference in satellite cell number across these three muscles may further explain the differential growth pattern of each of these muscles in utero. Muscle samples were harvested from bovine fetuses at a commercial abattoir and aged using crown rump length (~150-220d in utero). These samples were then dissected into single fibers and seeded in a 3D hydrogel culture with 20% FBS media. Cells were fixed using 4% paraformaldehyde and stained for DAPI, Pax-7, Myf-5 (Invitrogen, Bedford, MA) at 72, 96, and 120h. Wells were imaged and analyzed for proliferation rate using the Cytation 5 (Agilent; La Jolla, CA) Prime Gen 5 software. Data were analyzed as a two-way ANOVA with repeated measures using GraphPad Prism (Version 9.5). For Myf-5, Pax-7, and dual positive cells (P = 0.05) there was a difference across muscle type with BF having an increased number of positive cells compared with the TP muscle and LM being intermediate (Myf-5 mean = 51, 23, and 40 respectively; Pax-7 mean = 45, 30, and 20 respectively; Dual mean = 39, 20, 16 respectively). This variation was further supported by a similar divergence in proliferation rate across muscle type (P = 0.04), with BF muscle having increased proliferative capacity over TP muscle and LM muscle being intermediary. These findings suggest contrasting growth patterns across muscle types in utero that may influence postnatal growth patterns. Further research is needed to understand how the variability in satellite cell number contributes to differences in individual muscle growth. [ABSTRACT FROM AUTHOR]

Muscle satellite cells (SC) have an essential role in post-hatch skeletal muscle growth. To evaluate the effects of thermal variation (TV) during early-stage incubation (ESI) on Pectoralis major (PM) muscle SC heterogeneity of broiler chickens reared to 28 d posthatch a randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. Yield Plus × Ross 708 broiler breeder eggs (n = 2,160) ranging from 54 to 59 g were allotted equally by weight into 90-egg trays (n = 4 trays per incubator). All eggs were pre-warmed at 24 °C for 8 h and incubated at 37.5 °C from embryonic day (ED) 0 to 4. From ED 4 to 11, eggs were incubated at 1 of 3 temperatures (n = 2 incubators per TV treatment); 37.5 °C (CTL), 36.4 °C (COLD), or 38.6 °C (HOT). On ED 11, all incubators were returned to the CTL (37.5 °C) set point until ED 18. On ED 18, all eggs were transferred to baskets in hatchers set to 36.7 °C. After 514 h of incubation, all chicks were removed simultaneously, vent sexed, and weighed. Chicks were randomly allotted to floor pens (6 pens per treatment per sex) blocked by location and reared on a common diet. On d 28, birds (n = 10 per TV x sex treatment) were injected with bromodeoxyuridine (BrdU) before PM sample collection. Cryosections were immunofluorescence stained to facilitate taxonomy of SC populations expressing the myogenic regulatory factors Pax7 and MyoD as well as the proliferation marker BrdU by fluorescence microscopy. Data were analyzed as a 2-way ANOVA using SAS PROC GLIMMIX with TV treatment and broiler sex as main effects. Means were separated with the PDIFF option at P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.10. A TV × sex interaction was observed only for the MyoD+: Pax7+ SC density (P = 0.0232); however, none of the pairwise mean comparisons were significant at P < 0.05. Though CTL male and COLD female chicks had twice the density of MyoD+:Pax7+ SC as CTL females (P = 0.0535) and HOT males (P = 0.0645), respectively. There is no clear explanation for this observation. There were no interactive or main effects on density of MyoD+, Pax7+, MyoD+:BrdU+, Pax7+:BrdU+, MyoD+:Pax7+:BrdU+ SC (P ≥ 0.1258) or nuclear density (P = 0.1017) in 28-d-old broilers. These data suggest that if TV during ESI impacts SC myogenic regulatory factor heterogeneity and mitotic activity in broilers, the effects have concluded by d 28 post-hatch in commercial broilers. Further exploration of earlier time points will be necessary to determine whether the previously observed effects of TV during ESI on growth performance and meat yield are SC-mediated post-hatch. [ABSTRACT FROM AUTHOR]

Myokines are proteins produced by muscle cells that regulate muscle growth and metabolism. In livestock species, little is known about most myokines or their physiological impact. While FGF-21 has been examined in humans and model species for its roles in muscle development, regeneration, and metabolism, it is important to characterize its expression in livestock, such as beef cattle. This study assessed the quantity and temporal abundance of fibroblast growth factor 21 (FGF-21) expression, from cultured bovine satellite cells (BSC). BSC were isolated from the semimembranosus muscle of 3-month-old (n = 3) and 11-month-old (n = 3) commercially raised angus-based beef steers. BSC were cultured on Matrigel in Dulbecco's Modified Eagle Medium with 10% Fetal Bovine Serum until 70% confluency. At this time, the media was replaced with fresh media representing time zero. Cells and media were collected at times 0-, 12-, 24-, 48-, 72-, and 96-hour with zero serving as the baseline. All experiments included three biological and three technical replicates. The content of myokines including FGF-21, were analyzed in the sample's media using a commercial ELISA kit and cells were collected and stored for gene expression analysis. Statistical analysis was completed using the GLIMMIX procedure in SAS. A repeated measures analysis was used to evaluate the fixed effects of age, time, and age×time interaction. Contrasts were used to identify whether the baseline was statistically different from the other timepoints, and Tukey-Kramer adjustments were made. Significance was determined at P = 0.05. There was an interaction effect between age and time (P < 0.0001). The media from the BSC of the 11-month-old steers had greater (P < 0.0001) FGF-21 concentrations than that of the 3-month-old steers at every time point. When evaluating each time in comparison with the baseline of each age independently, there were differences between 0 and 12 hour (P < 0.0001) and 0 and 24 hour (P = 0.002) for the 3-monthold steer BSC, as well as 0 and 12 hour (P = 0.0003) for 11-month-old steer BSC. This study confirms that the myokine FGF-21 is produced by cultured BSC, and in differing quantities depending on the age of the steer at harvest. The observed differences in FGF-21 production in cultured cells harvested from 3- to 11-month-old steers may in part be responsible for some of the differences in growth patterns in muscle cells at these ages. The authors postulate that these data represent the first report of FGF-21 expression in cultured bovine satellite cells. [ABSTRACT FROM AUTHOR]

Multi-stage hatching egg incubators are challenging to manage but still common throughout the US broiler industry. It is difficult to satisfy all embryonic temperature requirements in systems where late-stage embryos must be kept from overheating while ensuring early-stage embryos are not at sub-optimal temperatures. Our previous work showed improvements in performance and meat yield when incubation temperature was increased to 38.6 °C from embryonic day (ED) 4 to 11 as opposed to lowering it to 36.4 °C compared with a 37.5 °C control. The objective of this work was to assess the effect of thermal variation (TV) during earlystage incubation (ESI) on muscle satellite cell (SC) heterogeneity in broiler chicks at transfer and hatch to explore the mechanism responsible for increased meat yield in our previous study. Broiler breeder eggs (n = 2,160) were incubated at 37.5 °C from embryonic day (ED) 0 to 3. On ED 4, COLD incubator set points decreased to 36.4 °C, HOT incubator set points increased to 38.6 °C, and control incubators remained 37.5 °C (n = 2 incubators per treatment). On ED 11, all incubators were set to 37.5 °C until ED 18 when eggs were transferred to hatchers. At transfer (ED 18) and hatch (ED 21), pectoralis major (PM) and biceps femoris (BF) muscle samples were collected from 6 chicks per treatment. Samples were immunofluorescence stained to facilitate taxonomy of SC populations expressing the myogenic regulatory factors and SC markers, MyoD, MRF4, and Myf5, by fluorescence microscopy. Data were analyzed as a 1-way ANOVA with the GLIMMIX procedure of SAS. Means were separated at P ≤ 0.05 with the PDIFF option. Tendencies were declared when 0.0501 ≤ P ≤ 0.10. Chicks from COLD incubators had the greatest density of MyoD+:Myf5+ SC in their BF (P = 0.0261) and tended to have a greater density of MyoD+:MRF4+ SC in their PM muscle (P = 0.0562) compared with chicks from HOT incubators. On ED 21, chicks from HOT incubators had the greatest density of MyoD+:MRF4+:Myf5+ SC in their PM muscle (P = 0.0406). Temperature gradients as small as 1.1 °C during ESI altered BF SC populations in chicks at transfer and PM SC populations at both transfer and hatch. Overall, these results indicate that TV during ESI can influence SC population kinetics in both PM and BF muscles of modern, fast-growing, high-yielding broiler chicks at ED 18 and 21. Further investigation is needed to understand the longer-term effects on SC populations and the cell signaling pathways involved over time during the post-hatch grow out period to explain the meat yield responses observed in our previous work. [ABSTRACT FROM AUTHOR]

Guanidinoacetic acid (GAA) is an amino acid-like compound that acts as a precursor to creatine (CRE) when either made in the body or digested. Creatine is directly used by skeletal muscle tissue as an energy source with the potential to act as a growthpromoting agent. The study objectives were to evaluate the effects of various CRE concentrations on bovine satellite cells for the rate of proliferation and myogenic differentiation. Satellite cells (SC) were isolated from the Longissimus muscle of 3-month-old Holstein bulls (n = 5). Cells were seeded at a density of 1 × 104 cells/mL on 96-well plates and incubated with 0 (CON), 2.5, 5, 7.5, and 10 mM of CRE. The absorbance rate was measured at 0, 24, 48, 72, and 96 h of incubation. Cells for gene expression were plated on 6-well plates at a density of 1 × 105 in 10% FBS/DMEM. Following 24 h of adhesion, SC were incubated with 5 and 7.5mM CRE for 48 h. After which, cells were harvested after 48 h for Pax3, Pax7, and Myf5 mRNA gene expression analysis. To identify the effects of CRE during myogenic differentiation, SCs were induced myogenic differentiation. Upon reaching approximately 90% confluency, the growth medium was replaced by differentiation media (2% horse serum/DMEM) containing 0 (CON), 5, and 7.5 mM CRE. A two-way ANOVA and Tukey's HSD test were conducted regarding cell proliferation. A one-way ANOVA and Tukey's HSD test were performed for gene expression. Interaction of treatment against time was noted (P < 0.01) in Con, 2.5, 5, 7.5, and 10 mM CRE. Cell proliferation rate was influenced by CRE treatment (P < 0.01). At 72 h of incubation, all CRE treatments increased cell proliferation rate compared with Con (P < 0.05). At 96 h of incubation, cells treated with 2.5, 5, and 7.5 mM of CRE increased proliferation compared with Con (P < 0.05). At 96 h, CRE treatment 2.5, 5, and 7.5 mM increased proliferation rate compared with 10mM CRE treated cells (P < 0.05). No treatment effect was found in the mRNA gene expression of Pax7, Pax3, and Myf5 (P > 0.05). Cells incubated with CRE showed no change in mRNA gene expression of IGF-1 and Myogenin (P > 0.05), and a tendency was noted for S6KB1 increase expression under treatment (P = 0.0643). S6KB1 is a protein kinase that, through mTOR pathway expression, leads to an increase in cell growth, protein synthesis, and cell proliferation. Results suggest that the treatment of BSCs with CRE impacted proliferation, CRE at 5mM vs. 7.5 mM concentrations treatment of differentiated muscle cells led to a trend in altering S6KB1 gene expression. [ABSTRACT FROM AUTHOR]

The neonatal period in pigs is distinguished by rapid growth as a function of extensive protein synthesis. Muscle satellite cells also referred to as stem cells (SC) have an essential role in postnatal hypertrophic muscle growth by increasing myofibrillar protein synthesis potential through fusion with existing myofibers to increase the DNA template available for transcription of these important muscle proteins. Thus, the impact of protein sources and differences in their amino acid availability on SC-mediated hypertrophic growth over time in neonatal pigs is of interest. To evaluate how different dietary protein sources fed to young piglets affect muscle fiber cross-sectional area (CSA), fiber density, and SC proliferative activity, a randomized complete block design experiment was conducted using piglets (n = 123) from 5 different farrowing groups. At birth, piglets were assigned to 1 of 4 treatments consisting of a control group and 3 liquid dietary treatments. The commercial milk replacer-based diets contained: whey only (MLK), MLK + spray dried plasma (SDP), and MLK + soy protein concentrate (SPC). After birth, all piglets were allowed to nurse at least 36-h on the sow to allow colostrum intake, assigned to 1 of 3 dietary treatments, transferred to Rescue Decks, and reared until tissue collection on d 6, 14, 19, and 25. The control group was left in the farrowing crate and nursed by the sow until sampling (SOW). On sampling day, pigs were injected with 5'-bromo-2'-deoxyuridine (BrdU) to label mitotically active cells. One-hour post-injection, pigs were euthanized and longissimus dorsi (LM) and biceps femoris (BF) muscle samples were collected for immunofluorescence microscopy analysis. Cell populations were classified as either mitotically inactive SC (MyoD+:BrdU-; Pax7+:BrdU-; MyoD+:Pax7+:BrdU-) or mitotically active SC (MyoD+:BrdU+; Pax7+:BrdU+; MyoD+:Pax7:BrdU+). Data were analyzed as a 2-way ANOVA using SAS V9.4 PROC GLIMMIX and means were separated using PDIFF at P = 0.05. Tendencies were declared when 0.0501 < P = 0.10. At d 14 and 19, MLK-fed piglets had the least BWG (P = 0.0810). On d 14 and 19, MLK-fed piglet LM fibers had the smallest CSA and greatest density (P = 0.0459) though they were similar in fiber density to SOW piglets on d 14. The MLK-fed piglets exhibited the least density of Pax7+:BrdU+ SC in BF (P = 0.0185) and the greatest density of MyoD+ SC in LM (P = 0.0831) on d 6 as well as the greatest density of MyoD+ SC in LM (P = 0.0551) and proliferative MyoD+:BrdU+ SC in BF (P = 0.0799) on d 19, though these differences did not persist through d 25. Overall, these data indicate that supplementation of different dietary protein sources can impact satellite cell-mediated hypertrophic muscle growth of neonatal piglets though the mechanism will require further investigation. [ABSTRACT FROM AUTHOR]

Hepatocyte growth factor (HGF) signals mediate mouse skeletal muscle stem cell, or satellite cell (SC), reentry into the cell cycle and myoblast proliferation. Because the athletic horse experiences exercise-induced muscle damage, the objective of the experiment was to determine the effect of HGF on equine SC (eqSC) bioactivity. Fresh isolates of adult eqSC were incubated with increasing concentrations of HGF and the initial time to DNA synthesis was measured. Media supplementation with HGF did not shorten (P > 0.05) the duration of G0/G1 transition suggesting the growth factor does not affect activation. Treatment with 25 ng/mL HGF increased (P < 0.05) eqSC proliferation that was coincident with phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and AKT serine/threonine kinase 1 (AKT1). Chemical inhibition of the upstream effectors of ERK1/2 or AKT1 elicited no effect (P > 0.05) on HGF-mediated 5-ethynyl- 2′-deoxyuridine (EdU) incorporation. By contrast, treatment of eqSC with 2 μm Gö6983, a pan-protein kinase C (PKC) inhibitor, blocked (P < 0.05) HGF-initiated mitotic activity. Geneexpression analysis revealed that eqSC express PKCα, PKCδ, and PKCε isoforms. Knockdown of PKCδ with a small interfering RNA (siRNA) prevented (P > 0.05) HGF-mediated EdU incorporation. The siPKCδ was specific to the kinase and did not affect (P > 0.05) expression of either PKCα or PKCε. Treatment of confluent eqSC with 25 ng/mL HGF suppressed (P < 0.05) nuclear myogenin expression during the early stages of differentiation. These results demonstrate that HGF may not affect activation but can act as a mitogen and modest suppressor of differentiation. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 mo, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, and 1,000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA, MHC IIX, insulin like growth factor-1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARᵧ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1,000 nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1,000 nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARä and PPARᵧ were increased (P < 0.05) with 1,000 nM of ATRA. Protein level of PPARä was also affected (P < 0.05) by 1,000 nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARᵧ compared to CON. All-trans retinoic acid (10 nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1,000 nM of ATRA. In muscle cells, ATRA may cause muscle fibers to transition towards the MHC isoform that prefers oxidative metabolism, as evidenced by increased expression of genes associated with the MHC I isoform. These changes in MHC isoforms appeared to be brought about by changing PPARδ gene expression and protein levels. [ABSTRACT FROM AUTHOR]

Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was to evaluate the effect of leucine on the mTOR signaling pathway in cultured equine satellite. Protein synthesis was evaluated by measuring the incorporation of [3H] Phenylalanine (3HPhe) in equine satellite cell myotube cultures treated with a leucine titration ranging from 0 to 408 μM. Our results show a 1.8-fold increase (P < 0.02) in protein synthesis at levels slightly greater than those found in the general circulation, 204 and 408 μM when compared to a no leucine control (0 μM). Puromycin incorporation, a nonradioactive surface sensing of translation (SUnSET) methodology, was also measured in cells treated with leucine (LEU; 408 μM), a no-leucine control (CON), and a puromycin-negative vehicle (PURO-). These results demonstrated a 180% increase (P = 0.0056) in puromycin incorporation in LEU compared to CON cultures. To evaluate the mTOR signaling pathway, equine satellite cell myotube cultures were treated with leucine (LEU; 408 μM) or a no-leucine control (CON) in the presence or absence of rapamycin (LR and CR, respectively), an inhibitor of mTOR. The mTOR inhibitor, rapamycin, suppressed phosphorylation of mTOR (P < 0.01) and rS6 (P < 0.01) with an increase in phosphorylation of rS6 in leucine-treated cultures observed when compared to control cultures (P < 0.05). Similarly, there was a 27% increase (P < 0.005) in the hyperphosphorylated γ-form of 4E-BP1 compared to total 4E-BP1 in LEU compared to CON cultures with leucine-induced phosphorylation of 4E-BP1 completely blocked by rapamycin with a smaller decrease observed in CR compared to CON cultures. The major finding of this study was that leucine activated the mTOR translation initiation pathway and increased transcription of global proteins in cultured equine satellite cells. Use of the cell culture system with primary equine muscle cell lines provides the opportunity to distinguish the impact of leucine on muscle and protein synthesis, independent of systemic interactions. [ABSTRACT FROM AUTHOR]

The Texel breed is a double muscled breed of sheep that is commonly used as a terminal sire in meatproduction. The increase in muscle mass is related to a single nucleotide polymorphism in the myostatin gene, a myokine that inhibits muscle growth. There is limited information on how postnatal skeletal muscle fiber hypertrophy is altered in Texel lambs. Satellite cells or muscle stem cells have a major role in postnatal hypertrophy of muscle fibers. Satellite cells are generally found in a state of quiescence and must activate, proliferate, and differentiate to increase DNA content in myofibers during periods of postnatal muscle growth. The objective of this study was to evaluate muscle fiber hypertrophy and satellite cell proliferation and differentiation in lambs of two sire breeds, Texel or Suffolk. Male lambs of each sire breed (n = 4/sire breed/time) were harvested at d 2, 14, and 203 of age. Muscle samples were collected from the longissimus and snap frozen for examination of myofiber cross-sectional area by fluorescent immunohistochemistry. Another portion of the longissimus was used to isolate satellite cells for in vitro experiments to examine population number, proliferation, and differentiation capacity. Isolated satellite cells were counted, seeded for activation, and grown until colonies reached 80% confluence. Activated cells were seeded for a proliferation assay measuring DNA content daily for 6 days. Following proliferation, cells were differentiated for 4 days, and the number of differentiated nuclei were counted with fluorescent immunohistochemistry. Sire breed did not impact (P < 0.05) the weight of the longissimus; however, muscle ribeye area tended (P < 0.10) to be increased for Texel lambs compared with Suffolk lambs. In the early postnatal period, from d 2 to 14, longissimus mass increased (P < 0.05) by 160%, and ribeye area increased by 100%. Between d 4 and 203 longissimus mass increased (P < 0.05) by 350%, while longissimus area only increased by 100%. Muscle fiber cross sectional area expanded (P < 0.05) from d 14 to 203 of age, and cross-sectional area was greater (P < 0.05) for Texel than Suffolk lambs at d 203. Satellite cell populations per gram of tissue increased (P < 0.05) between d 2 and 14 and decreased from d 14 to 203. The number of satellite cells that activated from quiescence was reduced (P < 0.05) as animal age increased but was not impacted (P > 0.05) by sire breed. Following activation, satellite cell proliferation capacity was greater (P < 0.05) for Texel compared with Suffolk satellite cells. Differentiation capacity was not altered (P > 0.05) by sire breed. Texel-sired lambs had larger longissimus cross-sectional muscle fiber area at d 203 of age than Suffolk. These changes appear to be related to greater proliferation of satellite cells, the stem cells of muscle, to enhance muscle fiber hypertrophy during postnatal growth. [ABSTRACT FROM AUTHOR]

Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 nM TBA exhibited increased (P < 0.05) protein synthesis rates and decreased (P < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 nM TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed (P < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 nM TBA each had no (P > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 nM TBA resulted in decreased (P < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 nM TBA exhibit increased (P < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures. [ABSTRACT FROM AUTHOR]

Basic principles governing skeletal muscle growth and development, from a cellular point of view, have been realized for several decades. Skeletal muscle is marked by the capacity for rapid hypertrophy and increases in protein content. Ultimately, skeletal muscle growth is controlled by 2 basic means: 1) myonuclear accumulation stemming from satellite cell (myoblast) proliferation and 2) the balance of protein synthesis and degradation. Each process underlies the rapid changes in lean tissue accretion evident during fetal and neonatal growth and is particularly sensitive to nutritional manipulation. Although multiple signals converge to alter skeletal muscle mass, postprandial changes in the anabolic hormone insulin link feed intake with enhanced rates of protein synthesis in the neonate. Indeed, a consequence of insulin-deficient states such as malnutrition is reduced myoblast activity and a net loss of body protein. A well-characterized mechanism mediating the anabolic effect of insulin involves the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling pathway. Activation of mTOR leads to translation initiation control via the phosphorylation of downstream targets. Modulation of this pathway by insulin, as well as by other hormones and nutrients, accounts for enhanced protein synthesis leading to efficient lean tissue accretion and rapid skeletal muscle gain in the growing animal. Dysfunctional insulin activity during fetal and neonatal stages likely alters growth through cellular and protein synthetic capacities. [ABSTRACT FROM AUTHOR]

The article presents several studies related to animal health. It mentions the effects of early or mid-gestation nutrient restriction on development of bovine fetal pancreatic. It states that effect of submaximal exercise on skeletal muscle satellite cells (SCs). It highlights daily supplementation of rumen protected fats (RPF) affects fatty acid profiles in beef heifers.

Myokine abundance and function have not been thoroughly evaluated in beef cattle. Many myokines have been shown to have a substantial impact on the health and well-being of mammals. While interleukin 6 (IL-6) is a known immune cytokine, it is also classified as a myokine and in mice is produced from muscle contraction post-exercise. IL-6 is linked to muscle hypertrophy through the action of increasing glucose uptake and stimulating satellite cell proliferation. The objective of this study is to characterize temporal IL-6 production by cultured primary bovine satellite cells (BSCs) in 11-month-old beef steers. IL-6 abundance was quantified in in vitro cultured undifferentiated primary BSCs isolated from 11-month-old steers. This experiment had 3 experimental replicates where cells were plated in triplicate with 10% Fetal Bovine Serum in DMEM. At 70% confluency the growth media was changed, and this was classified as time zero. The media then remained unchanged and was collected temporally at 0, 12, 24, and 48 hours. Media was assayed using Bovine IL-6 ELISA's to quantify myokine abundance. Cells were also harvested for mRNA extraction and analyses at a future time. We are in the initial stages of the analyses, though preliminary data indicate no difference in IL-6 abundance between the BSC pools isolated from different steers. Temporally, we report that at hour 0 there is undetectable IL-6 concentration, but at 12, 24, and 48 hours there is a steady concentration of IL-6 released into the growth media by the BSCs. This confirms that IL-6 is being produced by BSCs from post-weaned, beef steers. We conclude that myokine; IL-6 is involved in post-natal skeletal muscle growth of beef cattle. [ABSTRACT FROM AUTHOR]

As the population increases, it is necessary to improve the sustainability of beef production. Bos indicus (BI) cattle are better adapted to nutritional stress, consume less water, and are better able to withstand thermal stress when compared to Bos taurus (BT) cattle. Additionally, anabolic implants, which typically contain the steroid hormones trenbolone acetate (TBA) and/or estradiol (E2), are commonly administered to beef cattle in the U.S. to improve production. The objective of this study was to determine the effects of cattle breed type and steroid hormones on proliferation of primary bovine satellite cells (BSC). Primary BSC were isolated from the Semimembranosus muscle of Angus (AN; n=2; 100% BT) and Santa Gertrudis (SG; n=2; 19% BI, 81% BT) yearling backgrounded steers and cultured. After the cells reached 70% confluency, cultures were treated with 1% fetal bovine serum (control), 10 nM TBA, 10 nM E2, or 10 nM TBA and E2 and proliferation rates were compared to control cultures. No treatment x breed interaction (P=0.17) was observed. Cultures from AN steers had increased (P=0.007) proliferation rates compared to cultures from SG steers. Steroid hormones increased (P=0.08) proliferation rates, regardless of breed. However, based on this preliminary data, trends are indicating that a breed*treatment interaction is present such that BSC isolated from AN steers are more responsive to treatment than BSC isolated from SG steers. Additional research needs to be completed to determine whether this interaction is still present once more replicates are completed, whether different hormone concentrations affect BSC from cattle of different breed types, and/or the effects of hormone treatment and breed type on BSC relative to differentiation and protein synthesis rate. [ABSTRACT FROM AUTHOR]

Human and mouse studies have shown that fibro/adipogenic progenitors (FAPs) are a major source of intramuscular fat (IMF) and extracellular matrix (ECM) proteins. IMF and ECM proteins directly influence the palatability of beef, suggesting an essential role of FAPs in beef quality determination, which is still largely unexplored. We performed single-cell RNAseq (scRNAseq) using cells isolated from full blood Wagyu and Brahman cattle and Wagyu/Brahman cross cattle, which identified 21 cell clusters representing FAPs, several endothelial cell types, vascular smooths muscle cells, satellite cells, muscle fibers, and multiple immune cell types. More abundant FAPs were identified in the muscle of Brahman cattle, while a larger number of endothelial cells were identified in the muscle of Wagyu cattle. Further analysis of FAPs identified multiple FAP subpopulations with distinct gene expression profiles and anatomic locations. GSEA analysis revealed adipogenic and fibrogenic FAP subpopulations. A comparison of FAP subpopulations among different breeds showed higher complement system activity in the adipogenic FAP subpopulation of Wagyu cattle. Forced activation of the complement system in FAPs enhanced their adipogenic efficiency in vitro. In addition, cell-cell communication analysis identified active interactions between FAPs and other cell types through direct contact and secreted factors, many of which may affect FAP activities. In conclusion, our study revealed the single-cell atlas of bovine skeletal muscle and identified mechanisms regulating bovine intramuscular adipogenesis and fibrogenesis. [ABSTRACT FROM AUTHOR]

Anabolic implants containing estradiol (E2) and trenbolone acetate (TBA) promote skeletal muscle growth by increasing protein synthesis of primary bovine satellite cells (BSC). Recent data indicate skeletal muscle growth induced by anabolic implants may be positively augmented by trace mineral (TM) supplementation. Currently, the effects that E2, TBA or TM have on differentiation of primary BSC cultures is unknown. The objective of this study was to determine whether BSC differentiation is altered when cultures are treated with E2, TBA, zinc (Zn) or manganese (Mn). Primary BSC were grown to 80% confluency and treated (n=2) with 3% horse serum (CON), 10 nM TBA, 10 nM E2, 10 nM TBA and 10 nM E2, or 0.05, 0.10 or 0.25 µM of Mn, or 10, 20 or 40 µM of Zn. Creatine kinase activity was measured at 0, 24 and 72 h post-treatment to assess BSC differentiation. Creatine kinase is a muscle specific enzyme that is upregulated during satellite cell differentiation. A series of contrasts were constructed to compare CON and treated cultures at each time point. Creatine kinase activity increased (P< 0.05) over time indicating all cultures were differentiating. There was no difference (P >0.10) between the CON and any of the treatments at either 0 or 24 h post-differentiation. Treatment with 10 nM TBA increased (P=0.03) creatine kinase activity at 72 h compared to CON cultures. However, treatment with Zn, Mn, E2 or E2 and TBA did not have an effect (P >0.15) on BSC differentiation as measured by creatine kinase activity. This preliminary data indicates TBA might increase BSC differentiation, but further experiments are needed to validate this finding. Further, additional research analyzing the impacts of Zn and Mn in the presence of different steroid hormones is warranted to better understand the relationship that exists between steroid hormones and TM. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine how the use of specific miRNA mimics or inhibitors alters ovine satellite cell proliferation in vitro. Satellite cells were isolated from a Texel x Suffolk cross lamb at d 2 of age and cell purity was assessed with PAX7 protein staining and the population was 95% pure. miRNA candidates were selected from miRNA sequencing of the longissimus muscle at several time points from gestational d85 to market weight. Satellite cells were plated and allowed to reach 60% confluence, then treated with a miRNA mimic (miR-127 or 299a) at 50 or 100nM or a miRNA AntagomiR (miR-22-3p, 29a, 133, or 27) at 500nM using lipofectamine transfection reagent. Cells were allowed to proliferate for 4d following transfection and samples were collected daily for proliferation analysis using DNA quantification. Experiments were replicated and data analyzed using a priori contrasts with miRNA treatment in the model. DNA quantification showed that miR-127 mimic-100nM and miR-22-3p AntagomiR increased (P< 0.05) DNA content after 4d of proliferation following transfection. miR-127 mimic-100nM treatment increased (P< 0.05) miR-127 expression relative to controls at d1 and d4 post transfection. miR-22-3p AntagomiR transfection tended (P< 0.10) to reduce miR-22-3p expression on d4 post-transfection. miR-22-3p AntagomiR did not alter (P>0.05) PAX7, MYOD, or MYOG expression compared with the control. miR-127 mimic-100nM treatment did not alter (P>0.05) PAX7 expression, while it did increase (P< 0.05) MYOD expression on d4 and reduced (P< 0.05) MYOG expression compared to the control. mRNA sequencing was performed on d4 samples to determine possible miR-127 and 22-3p targets. IGF1, fibroin and other extra cellular matrix proteins were identified from miR-127 treated samples and CNDP2, involved in carnosine hydrolysis, was identified from miR-22-3p treated samples. miR-127 and 22-3p enhanced satellite cell proliferation and therefore could be used to promote muscle fiber hypertrophy. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin pathways have an important role in porcine skeletal muscle growth. Nevertheless, the mechanism for how it respond to environmental stimulations, especially nutrients, has not been wellinvestigated. In this study, piglets and skeletal muscle satellite cells (MuSCs) were used to investigate the work process of the lysine (Lys) signal transmitted to β-catenin in governing skeletal muscle growth. Briefly, the piglets and MuSCs were divided into the control group, the Lys deficiency group, and the Lys rescue group, taken together with the specific inhibitor and target gene knockdown for further study. We found that the transmembrane frizzled7 (FZD7) receptor displayed the same changes with the Wnt/β-catenin pathway and positively correlated with Lys levels in skeletal muscle and MuSCs. Meanwhile, the molecular docking analyses showed that FZD7 and Lys form connections at Gly17, Phe18, Cys19, and Asp84. In contrast, FZD7 specific inhibitor Fz7-21-TFA suppressed Lys rescued MuSC viability (P< 0.05). The distribution of Ki67 and active β-catenin in the Lys rescue group were also decreased by FZD7 inhibitor (P< 0.05). Furthermore, the decreased FZD7 level caused Lys re-activated TCF4/LEF activity and the Wnt/β-catenin pathway suppression (P< 0.05). Additionally, FZD7 knockdown restricted C2C12 viability and proliferation ability (P< 0.05). Besides, FZD7 knockdown inhibited the Wnt/β-catenin pathway and Lys deficiency caused much more serious inhibition of the Wnt/β-catenin pathway (P< 0.05), and these restrictions fail to be rescued by re-supplemented Lys or Wnt3a (P< 0.05). Moreover, we titrated Lys solution into recombinant pig FZD7 (rpFZD7) protein solution by using isothermal titration calorimetry, and the heat release during titration demonstrated there was an interaction between Lys and rpFZD7. Collectively, these results indicated that Lys is not only a molecular block for protein synthesis, but is also a ligand that binds to FZD7, directly activating the Wnt/β-catenin pathway to stimulate MuSCs to promote skeletal muscle growth. [ABSTRACT FROM AUTHOR]

Understanding the myogenic and self-renewal capability of satellite cells under heat stress (HS) is fundamental to coping with global warming in the beef cattle industry. Study objectives were to determine transcriptomic changes and proliferation capability of bovine satellite cells (BSCs) under moderate versus acute levels of HS and exposure times. Satellite cells were isolated from four three-month-old Holstein calves (semitendinosus) and cultured with 10% FBS/DMEM. Following 24h of attachment period, BSCs were incubated under the following temperature conditions 1) control (38°C; Con), 2) moderate (39.5°C; MHS) and extreme (41°C; EHS) and for various exposure times (0, 1, 3, 6, 12, 24, and 48 h). Cells were harvested and used for mRNA gene expression via RT-qPCR at each time point. Cell proliferation was measured using the ELISA kit. Data were analyzed by two-way ANOVA and Tukey HSD test. The expression of HSP70 was greatest (P < 0.05) in cells treated EHS at 1 and 3 h. Cells treated EHS overexpressed (P < 0.05) HSPB1 at 3, 6, 12, and 24 h. A similar pattern was observed (P < 0.05) for MyoD expression at 24 h after heat exposure. Contrarily, Myf5 and PAX3 were decreased (P < 0.05) by EHS compared to the Con at 3 h. Cell proliferation rate was enhanced (P < 0.01) in both MHS and EHS treated BSCs compared withthe Con at 48 h. Our results suggest that extreme heat exposure stimulates the HSPs (HSP70 and HSPB1) expression while modulating muscle regeneration by regulating myogenic regulatory factors (MyoD, Myf5) and PAX3. The transcriptional effect peaked after 3h of heat exposure, but the long-term (48h) exposure may activate the proliferation of BSCs. [ABSTRACT FROM AUTHOR]

Approximately 90% of beef cattle on feed in the U.S. receive an anabolic implant to improve production. The mechanisms implants operate through to improve growth have not been deciphered. Estradiol (E2) is one of the primary hormones found in anabolic implants. The objective of this study was to determine the effects of E2 on mRNA abundance of genes associated with skeletal muscle growth in proliferating and fused bovine satellite cells (BSC). To accomplish this, BSC were isolated from six backgrounded steers and cultured. Proliferation experiments used BSC cultures grown to 75% confluency, followed by treatment with 1% fetal bovine serum (FBS) or 10 nM E2 in 1% FBS. Protein synthesis experiments used cultures grown to 80% confluency, at which point cultures were induced to differentiate and treated 48-h later with serum free media (SFM) or10 nM E2 in SFM. Isolation of RNA occurred 30-min and 12-h post-treatment in both experiments. The mRNA abundance of 94 genes associated with skeletal muscle growth were examined. Treatment with E2 increased (P< 0.05) both proliferation and protein synthesis rates compared with controls. In proliferating BSC, 30-min post-treatment four genes had abundance altered (P< 0.10) by E2, while 12-h post treatment two genes had abundance altered (P< 0.10). Fused BSC, 30-min post-treatment, E2 altered (P< 0.10) abundance of five genes, while 12-h post treatment eight genes were altered (P< 0.10). A subset of significant genes include Zip8 abundance increasing (P=0.03) 30-min post-treatment in E2 treated proliferating BSC, arginase-2 abundance increasing (P=0.03) 30-min post-treatment in E2 treated fused cultures, Retinoid X Receptor-gamma abundance increasing (P=0.05) 12-h post-treatment in E2 treated proliferating BSC, and superoxide dismutase-2 abundance increasing (P=0.05) 12-h post-treatment in E2 treated fused BSC. These results indicate that E2 may improve proliferation and protein synthesis rates of BSC by altering mRNA abundance related to amino acid and micronutrient metabolism. [ABSTRACT FROM AUTHOR]

Creatine (CRE) is an energy-storing molecule of skeletal muscle and is reported as potential growth promotants. With Guanidinoacetic acid (GAA) being a precursor to creatine, the potential to evaluate supplemental GAA's role in the body from a skeletal muscle focus is needed within the beef industry. Study objectives were to evaluate the effects of CRE and GAA on bovine satellite cells (BSCs) proliferation and myogenic potential. BSCs were obtained from three-month-old Holstein calves (n=3), harvested, and incubated with 10% FBS/DMEM. Following 24h of cell adhesion, BSCs were incubated with treatments: 1) Control, 2) 10mM GAA, and 3) 5mM CRE. Cell proliferation rate was measured at 0, 24, 48, 72, and 96h. In order to identify the effects of CRE and GAA during myogenic differentiation, satellite cells were incubated in a differentiation medium (2% horse serum/DMEM) containing 10mM GAA and 5mM CRE. After 48h, cells were harvested and used for mRNA gene expression (Myh7, MyoG, IGF-1, and S6KB1). One-way ANOVA and Tukey's HSD test were used for gene expression. Two-way ANOVA and Tukey's HSD test were conducted for cell proliferation assay. No interaction effect was noted (P > 0.05) between the effects of treatment against time in end output of cell proliferation. Cell proliferation rate was not altered (P > 0.05), with proliferation rate time measurement having a greater result of cell proliferation (P < 0.001). The expression of IGF-1 was overexpressed (P < 0.05) in GAA treated BSC during the myogenic differentiation. CRE increased (P < 0.01) MyoG expression. GAA and CRE did not alter Myh7 orS6KB1 mRNA gene expression. Results suggest that the treatment of BSCs with creatine or GAA may improve myogenic differentiation and promote skeletal muscle growth via IGF-1, respectively. [ABSTRACT FROM AUTHOR]

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein- coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparinbinding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (³H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 nM TBA showed (P < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures (P < 0.05). Treatment with GDPβS, MMPI, CRM197, AG1024, AG1478, and/or AG879 all suppressed (P < 0.05) TBA-induced increases in proliferation. These data indicate that TBA likely initiates a nongenomic response involving GPCR, MMP2 and MMP9, hbEGF, EGFR, erbB2, and IGF-1R, which may play a role in TBA-mediated increases in BSC proliferation. [ABSTRACT FROM AUTHOR]

With improvements in care, the equine population is living longer, remaining active, and competing at increasingly older ages. Both advancing age and exercise result in increased concentrations of circulating and local cytokines, including IL-1β, IL-6, and tumor necrosis factor-α. Athletic endeavors in the aged horse may further increase the proinflammatory environment in muscle, decreasing the ability to react appropriately to exercise. Poor response to exercise limits the athletic ability of geriatric horses, thus reducing their useful life span and potentially increasing the risk of injury. Satellite cells are muscle stem cells that reside adjacent to muscle fibers in skeletal muscle and are at least partially responsible for maintenance of muscle mass and muscle hypertrophy. In the adult animal, these cells normally exist in a quiescent state, becoming active, proliferating, and differentiating in response to specific stimuli. Growth factors and cytokines present during hypertrophy and following exercise affect satellite cell activity. Whereas the specific effects of cytokines on equine satellite cells are not well established, cytokines can influence satellite cell and myoblast proliferation and differentiation both positively and negatively. Understanding the effects of cytokines on equine satellite cell function will provide insight into the mechanisms responsible for the poor response to exercise in the aged horse. The proinflammatory environment in aged horses may inhibit exerciseinduced satellite cell activity, thereby diminishing exercise-induced hypertrophy. As more horses are surviving and competing into their 20s, more research is required to understand the response of these animals to exercise during normal aging. [ABSTRACT FROM AUTHOR]

If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose-and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed. [ABSTRACT FROM AUTHOR]

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myo genin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells. [ABSTRACT FROM AUTHOR]

Skeletal muscle satellite cells (SC) play a critical role in the hypertrophic growth of postnatal muscle. Increases in breast meat yield have been consistently observed in broiler chickens fed 25-hydroxycholecalciferol (250HD3), but it is unclear whether this effect is mediated by SC. Thus, our objective was to determine the effect of vitamin D status improvement by replacing the majority of dietary vitamin D3 (D3) with 250HD3 on SC activity and muscle growth characteristics in the pectoralis major (PM) and the biceps femoris (BF) muscles. Day-old, male Ross 708 broiler chickens ( n = 150) were fed 1 of 2 com and soybean meal-based diets for 49 d. The control diet (CTL) contained 5,000 IU D3 per kg of diet and the experimental diet (250HD3) contained 2,240 IU D3 per kg of diet + 2,760 IU 250HD3 per kg of diet. Ten birds per treatment were harvested every 7 d. Two hours before harvest, birds were injected intraperitoneally with 5'-bromo-2'deoxyuridine (BrdU) to label mitotically active cells. Blood was collected from each bird at harvest to measure circulating concentrations of 250HD3, a marker of vitamin D status. The PM and BF muscles were weighed and processed for cryohistological determination of skeletal muscle fiber cross-sectional area, enumeration of Myf-5+ and Pax7+ SC, and mitotically active (BrdU+) SC using immunofluorescence microscopy. Circulating 250HD3 concentrations were greater in 250FlD3-fed birds on d 7, 14,21,28,35,42, and 49 when compared with CTL (P < 0.001). Growth performance and feed efficiency did not differ among dietary treatments (P > 0.10). Improved vitamin D status as a result of feeding 250HD3 increased the number of mitotically active (Pax7+;BrdU+) SC (P = 0.01) and tended to increase the density of Pax7+ SC = (P 0.07) in the PM muscles of broilers on d 21 and 35, respectively. Broiler chickens fed 250FID3 also tended to have greater Myf-5+ SC density (P=0.09) on d 14, greater total nuclear density (P = 0.05) on d 28, and a greater muscle fiber cross-sectional area (P = 0.09) on d 49 in their PM muscles compared with CTL birds. Collectively, these results suggest that improvement of vitamin D status by replacing the majority of D3 in the diet with 250HD3 can stimulate SC activity in the predominantly fast-twitch PM muscle and provide evidence toward understanding the mechanism behind previously observed increases in breast meat yield in 25QHD3-fed commercial broiler chickens. [ABSTRACT FROM AUTHOR]

Both androgenic and estrogenic steroids are widely used as growth promoters in feedlot steers because they significantly enhance feed efficiency, rate of gain, and muscle growth. However, despite their widespread use relatively little is known about the biological mechanism by which androgenic and estrogenic steroids enhance rate and efficiency of muscle growth in cattle. Treatment of feedlot steers with a combined estradiol (E2) and trenbolone acetate (TBA) implant results in an increased number of muscle satellite cells, increased expression of IGF-1 mRNA in muscle tissue, and increased levels of circulating IGF-1. Similarly, treatment of bovine satellite cell (BSC) cultures with either TBA or E2 results in increased expression of IGF-1 mRNA, increased rates of proliferation and protein synthesis, and decreased rates of protein degradation. Effects of E2 on BSC are mediated at least in part through the classical E2 receptor, estrogen receptor-ɑ (ESRİ), the IGF-1 receptor (IGFR1), and the G protein-coupled estrogen receptor-1 (GPER-1), fonnerly known as G protein-coupled receptor-30 (GPR30). The effects of TBA appear to be primarily mediated through the androgen receptor. Based on current research results, it is becoming clear that anabolic steroid-enhanced bovine muscle growth involves a complex interaction of numerous pathways and receptors. Consequently, additional in vivo and in vitro studies are necessary to understand the mechanisms involved in this complex process. The fundamental information generated by this research will help in developing future, safe, and effective strategies to increase rate and efficiency of muscle growth in beef cattle. [ABSTRACT FROM AUTHOR]

Growth hormone, also called somato-tropin (ST), is a polypeptide hormone produced by the anterior pituitary. The major functions of GH include stimulating bone and skeletal muscle growth, lipolysis, milk production, and expression of the IGF-I gene in the liver. Based on these functions, recombinant bovine ST (bST) and recombinant porcine ST (pST) have been used to improve milk production in dairy cows and lean tissue growth in pigs, respectively. However, despite these applications, the mechanisms of action of GH are not fully understood. Indeed, there has been a lot of controversy over the role of liver-derived circulating IGF-I and locally produced IGF-I in mediating the growth-stimulatory effect of GH during the last 15 yr. It is in this context that we have conducted studies to further understand how GH stimulates skeletal muscle growth in cattle. Our results do not support a role of skeletal muscle-derived IGF-I in GH-stimulated skeletal muscle growth in cattle. Our results indicate that GH stimulates skeletal muscle growth in cattle, in part, by stimulating protein synthesis in muscle through a GH receptor-mediated, IGF-I-independent mechanism. In this review, besides discussing these results, we also argue that liver-derived circulating IGF-I should be still considered as the major mechanism that mediates the growth-stimulatory effect of GH on skeletal muscle in cattle and other domestic animals. [ABSTRACT FROM AUTHOR]

Muscles in most domestic animals dif-fer in function and growth potential based largely on muscle fiber type composition. Though much is known about satellite cells (SC), information is limited regard-ing how populations of SC differ with muscle fiber type, especially in pigs. Therefore, the objective of this study was to isolate and culture SC from red (RST) and white (WST) portions of the semitendinosus muscle of neonatal and adult pigs and determine their capacity to proliferate, differentiate, and express various myosin heavy chain (MyHC) isoforms in vitro. Porcine satel-lite cells were isolated from RST and WST muscles of 6-wk-old and adult (>6-mo-old) pigs and cultured under standard conditions. Muscle from neonatal pigs yielded nearly 10 times more (P < 0.001) presumptive satellite cells as those from adult pigs, with fusion percentages close to 60% for the former. The RST yielded more (P < 0.001) SC per gram muscle compared to WST, 8.1 ± 0.2 x 104 cells versus 6.7 ±0.1 x 104 cells/gram muscle in young pigs, and 9.7 ± 0.4 x 10³ cells versus 5.5 ± 0.4 x 10³ cells/gram muscle in adult pigs, respectively. Likewise, satellite cells from RST proliferated faster (P < 0.001) than those from WST across both ages, as indicated by a shorter cell doubling time, 18.6 ± 0.8 h versus 21.3 ± 0.9 h in young pigs, and 23.2 ± 0.7 h ver-sus 26.7 ± 0.9 h in adult pigs, respectively. As a result of shorter times to confluence, satellite cells from RST also formed myotubes earlier than those SC originating from WST. Once induced, however, SC from WST differenti-ated and fused faster (P < 0.05) as evidenced by fusion percentage within the first 24 h, 41.6% versus 34.3%, respectively; but reached similar ultimate fusion per-centages similar to WST by 48 h. Over 90% of MyHC expressed in maximally fused SC cultures from both RST and WST was restricted to the embryonic isoform. Type IIX MyHC mRNA was not detected in any culture. Myotube cultures from RST expressed more (P <0.01) Type I MyHC isoform mRNA than those from WST, whereas those cultures from WST expressed more (P < 0.05) Type II (including Types IIA and IIB) MyHC tran-scripts. These data show SC cultures from porcine fast and slow muscles express MyHC profiles largely reflec-tive of their muscle of origin and suggest satellite cells are partially restricted to a particular muscle phenotype in which they are juxtapositioned. Understanding the molecular nature of these intrinsic control mechanisms may lead to improved strategies for augmenting meat animal growth or muscle regeneration. [ABSTRACT FROM AUTHOR]

Skeletal muscle hypertrophy is a culmination of catabolic and anabolic processes that are interwoven into major metabolic pathways, and as such modulation of skeletal muscle metabolism may have implications on animal growth efficiency. Muscle is composed of a heterogeneous population of muscle fibers that can be classified by metabolism (oxidative or glycolytic) and contractile speed (slow or fast). Although slow fibers (type I) rely heavily on oxidative metabolism, presumably to fuel long or continuous bouts of work, fast fibers (type IIa, IIx, and IIb) vary in their metabolic capability and can range from having a high oxidative capacity to a high glycolytic capacity. The plasticity of muscle permits continuous adaptations to changing intrinsic and extrinsic stimuli that can shift the classification of muscle fibers, which has implications on fiber size, nutrient utilization, and protein turnover rate. The purpose of this paper is to summarize the major metabolic pathways in skeletal muscle and the associated regulatory pathways., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)