Interleukin-6 requires JAK to stimulate inner cell mass expansion in bovine embryos.

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

Reduced skeletal muscle fiber size following caloric restriction is associated with calpain-mediated proteolysis and attenuation of IGF-1 signaling.

Caloric restriction decreases skeletal muscle mass in mammals, principally due to a reduction in fiber size. The effect of suboptimal nutrient intake on skeletal muscle metabolic properties in neonatal calves was examined. The longissimus muscle (LM) was collected after a control (CON) or caloric restricted (CR) diet was cosnumed for 8 wk and muscle fiber size, gene expression, and metabolic signal transduction activity were measured. Results revealed that CR animals had smaller (P < 0.05) LM fiber cross-sectional area than CON, as expected. Western blot analysis detected equivalent amounts of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) but reduced (P < 0.05) amounts of the splice-variant, PGC1α-4 in CR LM. Expression of IGF-1, a PGC1α-4 target gene, was 40% less (P < 0.05) in CR than CON. Downstream mediators of autocrine IGF-1 signaling also are attenuated in CR by comparison with CON. The amount of phosphorylated AKT1 was less (P < 0.05) in CR than CON. The ratio of p4EBP1T37/46 to total 4EBP1, a downstream mediator of AKT1, did not differ between CON and CR. By contrast, protein lysates from CR LM contained less (P < 0.05) total glycogen synthase kinase-3ß (GSK3ß) and phosphorylated GSK3ß than CON LM, suggesting blunted protein synthesis. Smaller CR LM fiber size associates with increased (P < 0.05) calpain 1 (CAPN1) activity coupled with lower (P < 0.05) expression of calpastatin, the endogenous inhibitor of CAPN1. Atrogin-1 and MuRF expression and autophagy components were unaffected by CR. Thus CR suppresses the hypertrophic PGC1α-4/IGF-1/AKT1 pathway while promoting activation of the calpain system.

Combinatorial effects of epidermal growth factor, fibroblast growth factor 2 and insulin-like growth factor 1 on trophoblast cell proliferation and embryogenesis in cattle.

Uterine secretions are crucial for conceptus development in mammals. This is especially important for species that undergo extended preimplantation development, like cattle and other ungulates. The present study examined cooperative interactions for epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) on the proliferation of the bovine trophoblast cell line CT1 and bovine embryo development. Proliferation of CT1 cells increased after supplementation of the culture medium with 10ngmL-1 EGF, 10ngmL-1 FGF2 or 50ngmL-1 IGF1, as well as with any combination of two factors. Greater increases in CT1 cell proliferation were detected when the growth medium was supplemented with all three factors. Supplementing the culture medium with individual or multiple factors during bovine embryo culture resulted in several positive outcomes, including increased blastocyst development, expansion, and hatching to varying degrees depending on the particular factor or combination of factors. Supplementation of the culture medium with all three factors increased embryonic trophoblast cell numbers on Day 8, as well as hatching rates and blastocyst diameter on Day 12 after fertilisation. Western blot analyses and the use of pharmacological inhibitors suggest that EGF and IGF1 affect CT1 proliferation by activating mitogen-activated protein kinase 3/1, whereas FGF2 activates AKT. In conclusion, the findings of the present study indicate that there are cooperative interactions among EGF, FGF2 and IGF1 that enhance trophoblast cell development during early embryogenesis.

Tissue organization alters gene expression in equine induced trophectoderm cells.

Rapid morphological and gene expression changes occur during the early formation of a mammalian blastocyst. Critical to successful retention of the blastocyst and pregnancy is a functional trophectoderm (TE) that supplies the developing embryo with paracrine factors and hormones. The contribution of TE conformational changes to gene expression was examined in equine induced trophoblast (iTr) cells. Equine iTr cells were cultured as monolayers or in suspension to form spheres. The spheres are hollow and structurally reminiscent of native equine blastocysts. Total RNA was isolated from iTr monolayers and spheres and analyzed by RNA sequencing. An average of 32.2 and 31million aligned reads were analyzed for the spheres and monolayers, respectively. Forty-four genes were unique to monolayers and 45 genes were expressed only in spheres. Conformation did not affect expression of CDX2, POU5F1, TEAD4, ETS2, ELF3, GATA2 or TFAP2A, the core gene network of native TE. Bioinformatic analysis was used to identify classes of genes differentially expressed in response to changes in tissue shape. In both iTr spheres and monolayers, the majority of the differentially expressed genes were associated with binding activity in cellular, developmental and metabolic processes. Inherent to protein:protein interactions, several receptor-ligand families were identified in iTr cells with enrichment of genes coding for PI3-kinase and MAPK signaling intermediates. Our results provide evidence for ligand initiated kinase signaling pathways that underlie early trophectoderm structural changes.

The influence of postnatal nutrition on reproductive tract and endometrial gland development in dairy calves.

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression.

The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development.

Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.

Selection for divergent body size alters rates of embryonic skeletal muscle formation and muscle gene expression patterns.

The impact of divergent selection for body size on embryogenesis is poorly understood. The objective of this experiment was to document skeletal muscle development during embryogenesis in two lines of chickens that display divergent growth as adults. Results reveal that after 54 generations of opposing selection from a common founder population, the embryos from the low weight select (LWS) line develop more rapidly during early embryogenesis than those from the high weight select (HWS) line. Muscle formation during the late embryonic period is more rapid and extensive in the HWS embryo than in the LWS contemporary. Isolated muscle progenitors from embryonic day 10 HWS embryos proliferated more rapidly, forming fibers sooner with a larger size than the LWS cells. The limited myogenic capacity of the LWS progenitor cells is not attributed to altered patterns of expression of Pax7, Pax3 or the myogenic regulatory factor genes. Members of the fibroblast growth factor family are potent mitogens and inhibitors of myoblast differentiation. Transcript abundance of FGF2 and FGF4 was measured in cultures of HWS and LWS progenitors as a function of time. The pattern of expression of FGF4 was similar between HWS and LWS with a large increase between days 1 and 3 followed by a reduction at day 5 of culture. Expression of FGF2 in LWS muscle cells did not change while a significant reduction in FGF2 expression was observed by day 5 in the HWS. Our results indicate that divergent selection for postnatal growth has altered embryonic development.

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

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

Intravenous Injection of Sodium Hyaluronate Diminishes Basal Inflammatory Gene Expression in Equine Skeletal Muscle.

Following strenuous exercise, skeletal muscle experiences an acute inflammatory state that initiates the repair process. Systemic hyaluronic acid (HA) is injected to horses routinely as a joint anti-inflammatory. To gain insight into the effects of HA on skeletal muscle, adult Thoroughbred geldings (n = 6) were injected with a commercial HA product weekly for 3 weeks prior to performing a submaximal exercise test. Gluteal muscle (GM) biopsies were obtained before and 1 h after exercise for gene expression analysis and HA localization. The results from RNA sequencing demonstrate differences in gene expression between non-injected controls (CON; n = 6) and HA horses. Prior to exercise, HA horses contained fewer (p < 0.05) transcripts associated with leukocyte activity and cytokine production than CON. The performance of exercise resulted in the upregulation (p < 0.05) of several cytokine genes and their signaling intermediates, indicating that HA does not suppress the normal inflammatory response to exercise. The transcript abundance for marker genes of neutrophils (NCF2) and macrophages (CD163) was greater (p < 0.05) post-exercise and was unaffected by HA injection. The anti-inflammatory effects of HA on muscle are indirect as no differences (p > 0.05) in the relative amount of the macromolecule was observed between the CON and HA fiber extracellular matrix (ECM). However, exercise tended (p = 0.10) to cause an increase in ECM size suggestive of muscle damage and remodeling. The finding was supported by the increased (p < 0.05) expression of CTGF, TGFß1, MMP9, TIMP4 and Col4A1. Collectively, the results validate HA as an anti-inflammatory aid that does not disrupt the normal post-exercise muscle repair process.

A Carnitine-Containing Product Improves Aspects of Post-Exercise Recovery in Adult Horses.

Strenuous exercise can cause tissue damage, leading to an extended recovery period. To counteract delayed post-exercise recovery, a commercial product containing L-carnitine (AID) was tested in adult horses performing consecutive exercise tests to exhaustion. Fit Thoroughbreds were administered an oral bolus of placebo (CON) or AID prior to performing an exercise test to exhaustion (D1). The heart rate (HR) and fetlock kinematics were captured throughout the exercise test. Blood was collected before, 10 min and 1, 4 and 6 h relative to exercise for the quantification of cytokine (IL1ß, IL8, IL10, TNFa) gene expression and lactate concentration. Horses performed a second exercise test 48 h later (D2), with all biochemical and physiological measures repeated. The results demonstrate that the horses receiving AID retained a greater (p < 0.05) amount of flexion in the front fetlock on D2 than the horses given CON. The horses presented a reduced (p < 0.05) rate of HR decline on D2 compared to that on D1. The expression of IL1ß, IL8 and IL10 increased at 1 h post-exercise on D1 and returned to baseline by 6 h; the cytokine expression pattern was not duplicated on D2. These results provide evidence of disrupted cytokine expression, HR recovery and joint mobility in response to consecutive bouts of exhaustive exercise. Importantly, AID may accelerate recovery through an undetermined mechanism.

Short Communication: Supplementation with calcium butyrate causes an increase in the percentage of oxidative fibers in equine gluteus medius muscle.

Optimal athletic performance requires meeting the energetic demands of the muscle fibers, which are a function of myosin ATPase enzymatic activity. Skeletal muscle with a predominant oxidative metabolism underlies equine athletic success. Sodium butyrate, a short-chain fatty acid, can affect muscle fiber composition in pigs. To determine if a similar scenario exists in horses, 12 adult Thoroughbred geldings (7.4 ± 0.6 yr of age; mean ± SEM) were fed 16 g of calcium butyrate (CB) or an equivalent amount of carrier (CON) daily for 30 d in a crossover design. Middle gluteal muscle biopsies were collected before and after the feeding trial for immunohistochemical determination of fiber type, and RNA and protein isolation. After 30 d, CB increased (P < 0.05) the percentage of type IIA fibers and tended (P = 0.13) to reduce the numbers of type IIX fibers in comparison to control (CON). No changes (P > 0.05) in type I, IIA, or IIX fiber size were observed in response to CB. No differences (P > 0.05) were noted in the abundance of succinate dehydrogenase (SDH) protein or activity between horses receiving CB or CON. Myogenin mRNA abundance was unaffected (P > 0.05) by 30 d of CB supplementation. The increase in type IIA fibers in the absence of altered mitochondrial SDH enzymatic activity suggests that CB affects myosin ATPase expression independent of altered metabolism.

The largest tissue in the body, skeletal muscle, is a heterogeneous mix of fibers that are categorized based on their primary source of energy production and speed of contraction. Evidence suggests that Thoroughbred horses with a greater percentage of type IIA, fast-twitch, oxidative fibers are more successful than those with fewer. Pigs fed a diet supplemented with butyrate contained a greater percentage of oxidative muscle fibers. This study examined the ability of calcium butyrate (CB), a short-chain fatty acid, to alter muscle fiber composition in horses. Adult Thoroughbred geldings were supplemented with a placebo or CB for 30 d, and gluteus medius muscle biopsies were retrieved and analyzed for fiber type, myogenin expression, and succinate dehydrogenase (SDH) activity. Results demonstrate a small increase in the percentage of type IIA fibers without a change in SDH activity, a marker of oxidative metabolism. Myogenin expression remained unaffected by CB supplementation. These efforts underscore the need for further research to validate improved exercise performance in response to CB supplementation and identify a mechanism of action for the fatty acid in the equine skeletal muscle.

The initial delay to mitotic activity in primary cultures of equine satellite cells is reduced by combinations of growth factors.

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.

Satellite cells are the resident stem cells found within skeletal muscle. Following strenuous exercise, the cells become mitotically active to supply progenitors for muscle repair. The signals responsible for their exit from the dormant state are largely unknown. Hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor I (IGF-I) are located within the local environment postexercise suggesting their involvement in mitotic activation. Treatment of satellite cells in vitro with optimal concentrations of HGF, FGF2, or IGF-I did not affect transition into the cell cycle. By contrast, inclusion of all three growth factors in the media caused an increase in the numbers of activated satellite cells. The combination of factors suppressed expression of myogenin, the requisite transcriptional mediator of differentiation. Although IGF-I stimulates myogenin expression in other muscle cell types, a similar response was not observed in equine satellite cells. These results support a role for HGF, FGF2, and IGF-I during the initial postexercise repair period in horses.

Molecular and biochemical regulation of skeletal muscle metabolism.

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.

Skeletal muscle is a heterogenous population of cells that are classified into muscle types based on contractile speed and metabolism. The various types of muscle cells utilize different biochemical pathways to produce energy to support cellular functions. These complex biochemical pathways are unique in their subcellular localization, substrate source, energy production capacity, and regulatory mechanisms. The purpose of this review is to describe the major metabolic pathways in skeletal muscle and the associated regulatory mechanisms.

Protein kinase C delta mediates fibroblast growth factor-2-induced interferon-tau expression in bovine trophoblast.

Interferon-tau (IFNT) is the trophoblast-secreted factor responsible for establishing and maintaining pregnancy in ruminants. Several uterine- and embryo-derived factors, including fibroblast growth factor-2 (FGF2), regulate IFNT production. The objective of the present study was to decipher the intracellular signaling mechanisms employed by FGF2 to regulate IFNT production. In bovine trophoblast cells (CT1), mitogen-activated protein kinase kinase-dependent pathways mediated constitutive IFNT mRNA concentrations. However, FGF2-mediated increases in IFNT mRNA levels occurs independent of mitogen-activated protein kinase. Exposure to the pan-protein kinase C (PKC) inhibitor, calphostin C, did not affect basal IFNT mRNA levels but limited the ability of FGF2 to increase IFNT mRNA abundance. Also, supplementation with phorbol 12-myristate 13-acetate (PMA) stimulated IFNT mRNA levels to the same extent as with FGF2. PMA and FGF2 cosupplementation did not elicit an additive effect on IFNT mRNA abundance. Pharmacological antagonists for classic PKCs (Gö6976) or novel PKCs, including PKC delta (rottlerin), were used to identify the specific PKC isoform utilized by FGF2. Supplementation of CT1 cells with Gö6976 did not affect FGF2 or PMA activities, whereas rottlerin prevented FGF2- and PMA-dependent increases in IFNT mRNA abundance in CT1 cells. Rottlerin also prevented FGF2 from increasing IFNT mRNA levels in Vivot trophoblast cells and primary trophoblast outgrowths. Modifications in PRKCD phosphorylation status were evident following FGF2 and PMA treatment. Also, reducing PRKCD expression by RNA interference attenuated FGF2-dependent increases in IFNT mRNA abundance. In conclusion, these results provide evidence that FGF2 regulates IFNT production in bovine trophectoderm by acting through PRKCD.

Fibroblast growth factor 2 promotes primitive endoderm development in bovine blastocyst outgrowths.

Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.

ß-Hydroxy ß-methylbutyrate supplementation to adult Thoroughbred geldings increases type IIA fiber content in the gluteus medius.

Consumption of ß-hydroxy ß-methylbutyrate (HMB) alters muscle composition and metabolism leading to strength and agility improvements in human athletes. To determine if HMB affects athletic performance and muscle function in horses, Thoroughbred geldings were fed a control (CON; n = 5) or HMB (n = 6) supplement for 6 wk prior to completing a standardized exercise test (SET). Gluteus medius (GM) muscle biopsies were obtained before the SET for fiber typing. Heart rate, biceps femoris (BF) and semitendinosus (ST) surface electromyograms (EMG), and fore and hind limbs metacarpophalangeal joint angles were captured at the gallop of the SET. Results demonstrate that HMB supplementation increased (P < 0.05) the percentage of type IIA and IIA/X muscle fibers in the GM with a corresponding decrease (P < 0.05) in type IIX fibers. The percentage of type I fibers was unaffected by diet. Supplementation with HMB did not result in any measurable effects on performance or biomechanical properties by comparison to CON. Supplementation with HMB resulted in an increase (P < 0.05) in ST median frequency at speeds of 10 m/s and greater. Increasing treadmill speed resulted in an increase (P < 0.05) in stride length and the maximal proximal forelimb fetlock angle, and a decrease (P < 0.05) in stance phase time of the gait cycle. Integrated EMG (iEMG) increased (P < 0.05) with increasing treadmill speeds for both the BF and ST with the BF exhibiting greater (P < 0.05) iEMG values than the ST. In summary, HMB increased the percentage of type IIA GM fibers, which did not translate into improved performance.

Effects of mid-gestational l-citrulline supplementation to twin-bearing ewes on umbilical blood flow, placental development, and lamb production traits.

The objective of the study was to examine how l-citrulline supplementation to ewes during mid-gestation influences placental activity, placental blood flow, lamb body weight, and carcass characteristics. Two studies were completed. A pharmacokinetic study to compare circulating plasma amino acid concentrations after a single intravenous injection of 155 µmol/kg BW l-citrulline or after an isonitrogenous amount of l-alanine (control; 465 µmol/kg BW). Increases (P < 0.05) in circulating citrulline concentrations were detected for 8 h after l-citrulline injection versus the control. Similarly, increases (P < 0.05) in circulating arginine concentrations were detected for 24 h after l-citrulline treatment. The second study used 12 ewes with twin pregnancies. Daily intravenous injections of either l-citrulline or l-alanine were administered for 39 d from d 42-45 to 81-84 of gestation. Ewes were limit-fed at 85% daily energy requirements during the injection period. A decrease (P < 0.0001) in body weight was observed in both treatment groups during this period. No treatment differences were observed in circulating pregnancy-specific protein B concentrations or placental blood flow during the treatment and post-treatment gestational period. No treatment differences were observed in lamb survival nor in lamb birth, weaning and slaughter weights. Treatment did not influence lamb carcass composition or organ weights. However, there was a tendency (P = 0.10) for an increase in antral follicle numbers in ovaries from ewe lambs derived from ewes treated with l-citrulline. In summary, a daily l-citrulline injection increased both circulating citrulline and arginine concentrations in ewes, but daily l-citrulline injections during mid-gestation did not produce any detectable changes in placental activity and blood flow, neonatal and postnatal lamb development, and lamb carcass composition at slaughter. In conclusion, no benefits in placental function and lamb development were observed after providing l-citrulline during mid-gestation in ewes exposed to a mild energy restriction, but there was an indication that follicle numbers in ewe lambs were positively influenced by l-citrulline treatment during fetal development.

Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation.

Niche localized HGF plays an integral role in G(0) exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

Satellite cells and their regulation in livestock.

Satellite cells are the myogenic stem and progenitor population found in skeletal muscle. These cells typically reside in a quiescent state until called upon to support repair, regeneration, or muscle growth. The activities of satellite cells are orchestrated by systemic hormones, autocrine and paracrine growth factors, and the composition of the basal lamina of the muscle fiber. Several key intracellular signaling events are initiated in response to changes in the local environment causing exit from quiescence, proliferation, and differentiation. Signals emanating from Notch, wingless-type mouse mammary tumor virus integration site family members, and transforming growth factor-ß proteins mediate the reversible exit from growth 0 phase while those initiated by members of the fibroblast growth factor and insulin-like growth factor families direct proliferation and differentiation. Many of these pathways impinge upon the myogenic regulatory factors (MRF), myogenic factor 5, myogenic differentiation factor D, myogenin and MRF4, and the lineage determinate, Paired box 7, to alter transcription and subsequent satellite cell decisions. In the recent past, insight into mouse transgenic models has led to a firm understanding of regulatory events that control satellite cell metabolism and myogenesis. Many of these niche-regulated functions offer subtle differences from their counterparts in livestock pointing to the existence of species-specific controls. The purpose of this review is to examine the mechanisms that mediate large animal satellite cell activity and their relationship to those present in rodents.

Dietary tributyrin supplementation and submaximal exercise promote activation of equine satellite cells.

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.