Effect of dexamethasone on the expression of atrogin‐1/MAFbx in chick skeletal muscle

Expression of atrogin‐1/MAFbx, a muscle‐specific E3 ubiquitin ligase, is high under catabolic conditions, that result in muscle atrophy. Messenger RNA (mRNA) expression of atrogin‐1/MAFbx is increased by the glucocorticoid dexamethasone in mammalian skeletal muscle. This study investigated the effects of dexamethasone on expression of atrogin‐1/MAFbx in skeletal muscle of neonatal chicks and in chick myotubes. Chicks were given a single intraperitoneal injection of dexamethasone at a concentration of 10 mg/kg body weight. Twenty‐four hours after dexamethasone administration, the Pectoralis muscle weight of chicks was decreased. mRNA expression of atrogin‐1/MAFbx in skeletal muscle of chicks was significantly increased by dexamethasone administration. Expression of other proteolytic‐related genes (20S proteasome C2 subunit, m‐calpain large subunit, and cathepsin B) in skeletal muscle of chicks was not increased by dexamethasone administration. Chick myotubes were incubated with dexamethasone (1, 10 or 100 µmol/L) for 6 h. Expression of atrogin‐1/MAFbx mRNA in chick myotubes was increased in the presence of all concentrations of dexamethasone. However, expression of other proteolytic‐related genes (20S proteasome C2 subunit, m‐calpain large subunit and cathepsin B) in chick myotubes was not affected by dexamethasone treatment. These results indicate that dexamethasone enhances atrogin‐1/MAFbx expression in chick skeletal muscle, resulting in increased muscle atrophy.     

Cold exposure increases slow‐type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats

The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism‐related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold‐exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow‐type MyHC1 content in the soleus muscle (a typical slow‐type fiber), while the intermediate‐type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast‐type fiber) and gastrocnemius (a mix of slow‐type and fast‐type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism‐related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus.     

Possible roles of myostatin and PGC-1α in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: Expression of myostatin and PGC-1α in chicks exposed to cold

This study examined the hypothesis that myostatin and PGC-1α are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24 h) or long-term (8 d) cold at 4 °C or kept warm at 30 °C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1α mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24 h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1α mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1α expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.     

In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding β‐galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.     

In ovo leptin administration accelerates post‐hatch muscle growth and changes myofibre characteristics,gene expression and enzymes activity in broiler chickens

To evaluate the effect of maternal leptin on muscle growth, we injected 0 μg (control, CON), 0.5 μg (low leptin dose, LL) or 5.0 μg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 μl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross‐sectional area (CSA) of myofibres. Real‐time PCR was performed to quantify leptin receptor (LEPR), insulin‐like growth factor 1 (IGF‐1), IGF‐1 receptor (IGF‐1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF‐1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down‐regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose‐dependent and sex‐specific manner. The altered expression of IGF‐1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects.     

Leucine regulates slow‐twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow‐twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow‐twitch muscle fibers and mitochondrial function‐related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho‐AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow‐twitch muscle fibers and mitochondrial function‐related gene expression. Finally, we showed that Sirt1 was required for leucine‐induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow‐twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.     

Effects of Dietary Supplementation of β‐hydroxy‐β‐methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets

The effects of dietary β‐hydroxy‐β‐methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β‐hydroxy‐β‐methylbutyrate calcium (HMB‐Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor‐4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin‐like growth factor‐1 (IGF‐1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.     

Effect of low‐ or high‐CP diet with cold‐pressed oil supplementation on growth,immunity and antioxidant indices of growing quail

This study was to investigate the effect of dietary protein levels and supplementation with two cold‐pressed oils on the performance, immunity and antioxidant indices of growing Japanese quails. A 3 × 4 factorial experiment, using 3 dietary crude protein (CP) levels (22%, 24% and 26%) and 4 supplemental herbal oils levels: control without additives, 250 mg marjoram (Mar), 250 mg thyme (Thy) and 125 mg Mar + 125 mg Thy per kg diet. A total of 720 unsexed, 7‐day‐old quail chicks were randomly distributed into twelve treatment groups (4 replicates per group of 15 chicks in each). The live body weight (LBW) on 42 days and body weight gain (BWG) from 7 to 42 days were significantly improved (p < 0.01 and 0.05) in chicks fed 24% or 26% CP compared with those of the 22% CP group. The groups fed diets supplemented with Mar or in combination with Thy oil exhibited significantly better LBW, BWG and feed conversion ratio (FCR) than the control at all periods except BWG from 21 to 42 days and FCR from 7 to 21 days were not significant. Feed intake was gradually decreased with increasing dietary CP level or adding herbal oils during the periods 21–42 and 7–42 days of age. Plasma triglycerides, cholesterol, total lipids and malondialdehyde were decreased by cold‐pressed oils supplementation, but the activity of reduced glutathione and superoxide dismutase was increased (p < 0.01) compared with the control. In conclusion, quails fed a diet containing 24% or 26% CP or diets supplemented with Mar or Thy oils or both exhibited improvement in the performance, lipid profile, immunity and antioxidant capacity without any detrimental impacts on the other studied parameters.     

不同时长冷暴露下C2C12细胞中O-GlcNAc和IL-6表达的变化

冷暴露下骨骼肌中O-GlcNAc（O-linked β-N-acetylglucosamine）糖基化修饰水平显著升高，作为"应激感受器"调控细胞信号转导及基因转录等；而骨骼肌作为分泌器官通过收缩产生的肌源性IL-6具有免疫作用，更能够调节骨骼肌中糖、脂代谢。本试验旨在检测不同时长冷暴露下小鼠成肌细胞系C2C12中O-GlcNAc相关蛋白和IL-6表达变化，初步探究冷暴露下O-GlcNAc与IL-6之间的潜在联系。本试验通过体外培养小鼠成肌细胞系C2C12，并随机分为不做冷暴露处理的对照组（cold 0 h组，也称为control组，37℃±0.5℃）和冷暴露（32℃±0.5℃）3、6、9、12 h处理组，采用Western blot方法检测不同时长冷暴露下O-GlcNAc糖基化水平和OGT、OGA及IL-6表达水平。结果显示，与对照组相比，冷暴露3 h，OGT表达水平显著升高（P<0.05），冷暴露6 h，OGA表达水平显著升高（P<0.05）；冷暴露3 h，O-GlcNAc糖基化修饰水平和IL-6表达水平极显著升高（P<0.01）。不同时长冷暴露下，C2C12细胞中O-GlcNAc糖基化修饰水平及IL-6表达水平显著升高，并且，不同时长冷暴露下，O-GlcNAc糖基化修饰和IL-6表达变化趋势高度相似，提示冷暴露下O-GlcNAc糖基化修饰可能参与IL-6表达的调控。     

Long non‐coding RNA analysis of muscular responses to testosterone deficiency in Huainan male pigs

Long non‐coding RNAs (lncRNAs) participated in growth and development of skeletal muscle; however, little is known about their response to testosterone deficiency in porcine skeletal muscle. We compared lean mass related carcass traits and lncRNAs expression files in Longissimus dorsi (LD) muscle between intact and castrated Huainan male pigs. The results showed that castration significantly reduced eye muscle area and lean meat percentage (P < 0.05), but increased the fat mass weight (P < 0.05). Meanwhile, 8946 lncRNAs, including 6743 intergenic lncRNAs (lincRNAs), 498 anti‐sense lncRNAs, and 1705 intronic lncRNAs, were identified in porcine LD, among which, 385 lncRNAs were considered as the differentially expressed candidates between intact groups and castrated groups (q‐value < 0.05). Functional analysis indicated that these differently expressed lncRNAs and their target genes were involved in the estrogen receptor signaling pathway and skeletal and muscular system development and function. We first detect porcine muscular lncRNA response to castration, and the results suggested that lncRNAs and their target genes participated in the regulation of testosterone deficiency‐related skeletal muscle growth.     

Grazing‐induced changes in muscle microRNA‐206 and ‐208b expression in association with myogenic gene expression in cattle

To investigate the roles of microRNAs (miRNAs) in muscle type conversion, the effects of 4 months of grazing on the expression levels of miRNAs and mRNAs associated with skeletal muscle development were analyzed by quantitative RT‐PCR using the Biceps femoris muscle of Japanese Shorthorn cattle. After 4 months of grazing, the expression of muscle fiber type‐associated miR‐208b was higher in the grazed cattle than in the housed. In concordance with the pattern in miR‐208b expression, the expression of MyoD, a myogenic regulatory factor associated with the shifting of muscle property to the fast type, was lower in the grazed cattle after 4 months of grazing than in the housed cattle. In addition, the expression of MyHC‐2x (a fast type) was higher in the housed cattle than in the grazed, after 4 months of grazing. During the grazing period, miR‐206 expression decreased in the housed cattle, whereas expression in the grazed cattle did not change, but rather remained higher than that of the housed cattle even at 3 months after the grazing ended. These miRNAs including miR‐206 persisting with muscles of grazed cattle may be associated with regulation of muscle gene expression during skeletal muscle adaptation to grazing.     

Leucine‐induced promotion of post‐absorptive EAA utilization and hepatic gluconeogenesis contributes to protein synthesis in skeletal muscle of dairy calves

The high rate of protein synthesis in skeletal muscle of dairy calves can benefit their first lactation even lifetime milk yield. Since the rate of protein synthesis is relatively low in the post‐absorptive state, the aim of this research was to determine whether leucine supplementation could increase the post‐absorptive essential amino acid (EAA) utilization and protein synthesis in the skeletal muscle. Ten male neonatal dairy calves (38 ± 3 kg) were randomly assigned to either the control (CON, no leucine supplementation, n = 5) or supplementation with 1.435 g leucine/L milk (LEU, n = 5). Results showed that leucine significantly increased the length and protein concentration in longissimus dorsi (LD) muscle, whereas it decreased creatinine concentration and glutamic‐oxalacetic transaminase (GOT) activity. Compared to the control group, leucine supplementation also reduced the glutamic‐pyruvic transaminase (GPT) activity. Supplementation of leucine improved the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E‐binding protein 1 (4EBP1) and substrates ribosomal protein S6 kinase 1 (p70S6K). Supplementation of leucine resulted in increased concentrations of glucose, methionine, threonine, histidine and EAAs and decreased concentration of arginine in serum. Liver glucose concentration was higher and pyranic acid was lower in LEU compared to CON. In conclusion, leucine supplementation can promote post‐absorptive EAA utilization and hepatic gluconeogenesis, which contributes to protein synthesis in skeletal muscle of dairy calves.     

Effects of rice feeding and carnitine addition on growth performance and mRNA expression of protein metabolism‐related genes in broiler grower chicks

This study was carried out to evaluate the nutritional effects of rice feeding and carnitine addition to a diet for broiler chicks. Thirty‐six male 10‐day‐old broiler chicks were assigned to one of the following four treatment groups: corn‐based diet (corn group), rice‐based diet (rice group), and each diet with added carnitine (100 ppm). The experimental period was 2 weeks. Rice feeding resulted in significantly higher growth performance (body weight gain and feed efficiency) compared to corn feeding. Carnitine addition also resulted in higher growth performance. Breast muscle and thigh muscle weight (g) were significantly higher in broiler chicks fed rice and those fed diets with added carnitine. Liver mRNA expression of IGF‐I was significantly higher in broiler chicks fed rice compared to those fed corn. There was no significant difference in mRNA expression of muscle atrogin‐1 or liver CPT‐I between broiler chicks fed rice and those fed corn, not between broilers chicks fed diets containing carnitine or not. Overall, these results show that rice feeding and carnitine addition improve the growth performance of broiler chicks by increasing mRNA expression of liver IGF‐I. In addition, carnitine action is not affected by different cereals (corn and rice).     

Effects of delaying post‐hatch feeding on lipid peroxidation and antioxidant enzyme mRNA expression in the pectoralis major muscle of newly hatched chicks

Excessive lipid peroxidation negatively affects the physiological response and meat quality of chickens. Delaying post‐hatch feeding was previously found to increase lipid peroxidation in the skeletal muscle of finishing broiler chickens. The aims of this study were to investigate the effects of delayed post‐hatch feeding on lipid peroxidation and the mRNA expressions of antioxidant enzymes in the pectoralis major muscle of broiler chicks during the post‐hatching period. Newly hatched chicks either had immediate free access to feed (freely‐fed chicks) or had no access to feed from 0 to 2 days old (delayed‐fed chicks), after which both groups were fed ad libitum until 4 or 13 days old. The lipid peroxidation level was higher in the delayed‐fed than freely‐fed chicks at 2, 4, and 13 days old. At 2 days old, the mRNA expressions of Cu/Zn‐SOD, Mn‐SOD, and GPX7 were lower in the delayed‐fed than freely‐fed chicks, while catalase mRNA levels did not differ. Furthermore, at 4 and 13 days old, lower mRNA expressions of Cu/Zn‐SOD and Mn‐SOD were observed in the delayed‐fed than freely‐fed chicks. These results suggest that delaying post‐hatch feeding reduces the mRNA levels of Cu/Zn‐SOD and Mn‐SOD, consequently affecting muscle lipid peroxidation in chicks during subsequent growth.     

In ovo carbohydrate supplementation modulates growth and immunity‐related genes in broiler chickens

A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune‐related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth‐related genes: chicken growth hormone (cGH), insulin‐like growth factor‐I & II (IGF‐I & II) and mucin were studied in hepatic and jejunum tissues of late‐term embryo and early post‐hatch chicks. Expression of candidate immune genes: Interleukin‐2, 6, 10 and 12 (IL‐2, IL‐6, IL‐10 and IL‐12), Tumour necrosis factor‐alpha (TNF‐α) and Interferon gamma (IFN‐γ) were studied in peripheral blood monocyte cells of in ovo‐injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con‐A). Glucose injection significantly increased the expression of IGF‐II gene during embryonic period and both cGH and IGF‐II in early post‐hatch period, while ribose‐injected chicks had higher expression of IGF‐II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose‐injected chicks during embryonic age. Glucose‐injected chicks had higher expression of IL‐6 or IL‐10, while those injected with fructose or ribose had higher expression of IL‐2, IL‐12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late‐term embryos and chicks. In ovo glucose could modulate humoral‐related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens.     

Single injection of clenbuterol into newly hatched chicks decreases abdominal fat pad weight in growing broiler chickens

The aim of the current study was to examine the effects of clenbuterol injection into newly hatched chicks on both the abdominal fat pad tissue weight and the skeletal muscle weight during subsequent growth. Twenty‐seven 1‐day‐old chicks were divided into two groups, receiving either a single intraperitoneal (i.p.) injection of clenbuterol (0.1 mg/kg body weight) or phosphate‐buffered saline (PBS). Body weight gain, feed intake and feed conversion ratio were not affected by clenbuterol injection during the 5‐week experimental period, while the abdominal fat pad tissue weight of the clenbuterol‐injected chicks was lower than that of the control chicks at 5 weeks post‐injection. Plasma non‐esterified fatty acid concentrations were significantly increased in the clenbuterol‐injected chicks, while plasma triacylglycerol concentrations did not differ. Additionally, the enzymatic activity of fatty acid synthase was lower in the liver of the clenbuterol‐injected chicks. Conversely, the skeletal muscle weights were not affected by clenbuterol injection. These results suggest that a single clenbuterol injection into 1‐day‐old chicks decreases the abdominal fat pad tissue weight, but may not affect skeletal muscle weights during growth. © 2015 Japanese Society of Animal Science     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.