Time‐coordinated prevalence of extracellular HGF,FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon‐guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early‐differentiated satellite cells up‐regulate a secreted neural chemorepellent Sema3A upon in vivo muscle‐crush injury. The study also revealed that Sema3A expression is up‐regulated in primary satellite‐cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)‐β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time‐course of extracellular HGF, FGF2 and TGF‐β3 concentrations after crush‐injury of Gastrocnemius muscle in the rat lower hind‐limb, using a combination of a non‐homogenization/non‐spin extraction of extracellular wound fluids and enhanced chemiluminescence–Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2–8 days post‐crush, whereas active TGF‐β3 increases after 12 days, providing a better understanding of the time‐coordinated levels of HGF, FGF2 and TGF‐β3 that drive regulation of Sema3A expression during regenerative intramuscular moto‐neuritogenesis.     

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.     

Duration of feeding linseed diet influences peroxisome proliferator-activated receptor γ and tumor necrosis factor gene expression, and muscle mass of growing–finishing barrows

The aim of the study was to investigate the effect of duration of feeding linseed (rich in n-3 PUFA) on peroxisome proliferator-activated receptor γ (PPARγ) and tumor necrosis factor (TNF-α) gene expression, and muscle mass of growing–finishing barrows. Two isoenergetic and isonitrogenous diets were formulated, and one of which was the basal diet and another one was the linseed diet including linseed at the level of 10%. Twenty-four Landrace × Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to four treatments with six individuals per treatment. Pigs in treatment 1 (T1) fed the control diet throughout the experimental period, while pigs in T2, T3 and T4 fed the control diet except for 30, 60, and 90 d prior to slaughter when the linseed diet were fed. The experiment was conducted for 90 days. The longissimus muscle mass and each muscle mass in the hind leg were weighted. PPARγ and TNF-α mRNA expression levels in muscle, spleen and adipose tissue, and plasma concentrations of TNF-α data were measured and analyzed. The results showed that the longissimus muscle mass, quadriceps femoris muscle mass and semitendinosus muscle mass increased linearly (P < 0.01) as prolonged the time of feeding linseed diet. The expression of PPARγ in longissimus muscle and spleen increased (P < 0.01) linearly as prolonged the time of feeding linseed diet, while the expression of PPARγ in adipose tissue were not affected (P = 0.095). Duration of linseed addition linearly decreased (P < 0.01) TNF-α gene expression levels in the longissimus dorsi muscle, adipose and spleen, and serum concentration of TNF-α as well. The expression levels of PPARγ negatively correlated with the expression of TNF-α in muscle (R² = 0.70, P < 0.001) and spleen (R²R² = 0.77, P < 0.001) respectively. Likewise, PPARγ expression level in spleen (R²R² = 0.59, P < 0.01) or muscle (R²R² = 0.52, P < 0.05) negative correlated with serum TNF-α concentration, while there were significant quadratic relation between muscular PPARγ (R²R² = 0.80, P < 0.01) or muscular TNF-α (R²R² = 0.87, P < 0.01) expression and the longissimus dorsi muscle mass. These data suggested that duration of feeding linseed diet lead to a linear decrease of TNF-α gene expression, which may increase the muscle mass in growing–finishing barrows, at least in part, through a PPARγ-dependent mechanism.     

Acute stimulation of a smooth muscle constrictor by oestradiol‐17β via GPER1 in bovine oviducts

Oviducts play roles in reproductive processes, including gametes transport, fertilization and early embryo development. Oviductal transport is controlled by various factors such as endothelins (EDNs) and nitric oxide (NO), smooth muscle contracting and relaxing factor, respectively. EDNs and NO production depend on an ovarian steroid hormone, oestradiol‐17β (E2) and E2 quickly exerts their biological functions through G protein‐coupled oestrogen receptor 1 (GPER1), which mediates rapid intracellular signalling. Because follicular fluid which contains a high concentration of E2 enters the oviduct, we hypothesized that E2 in the follicular fluid participates via GPER1 in producing EDNs and NO. To test this hypothesis, we investigated 1) the expression and localization of GPER1 in bovine oviductal tissues and 2) rapid effects of E2 via GPER1 on EDN1, EDN2 and inducible NO synthase (iNOS) expression in cultured bovine oviductal isthmic epithelial cells. GPER1 was observed in the oviductal epithelium, stroma and smooth muscle, and its expression was highest in the isthmus. Short‐term treatments (≤1 hr) of E2 increased EDN2 mRNA expression in the isthmic epithelial cells, although E2 did not affect EDN1 and iNOS mRNA expressions. Results of GPER1‐selective agonist G‐1 and GPER1‐selective antagonist G‐15 treatments revealed acute stimulation by E2, which is mediated via GPER1. The overall findings suggested that E2 in follicular fluid rapidly stimulates EDN2 expression via GPER1 in the isthmic epithelial cells. Follicular fluid may play a role in retention of the ovulated oocyte in the end of ampulla by contracting the isthmus for successful fertilization.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

Effect of dietary fish oil intake on ubiquitin ligase expression during muscle atrophy induced by sciatic nerve denervation in mice

Dietary fish oil intake improves muscle atrophy in several atrophy models however the effect on denervation‐induced muscle atrophy is not clear. Thus, the aim of this study was to investigate the effects of dietary fish oil intake on muscle atrophy and the expression of muscle atrophy markers induced by sciatic nerve denervation in mice. We performed histological and quantitative mRNA expression analysis of muscle atrophy markers in mice fed with fish oil with sciatic nerve denervation. Histological analysis indicated that dietary fish oil intake slightly prevented the decrease of muscle fiber diameter induced by denervation treatment. In addition, dietary fish oil intake suppressed the MuRF1 (tripartite motif‐containing 63) expression up‐regulated by denervation treatment, and this was due to decreased tumor necrosis factor‐alpha (TNF‐α) production in skeletal muscle. We concluded that dietary fish oil intake suppressed MuRF1 expression by decreasing TNF‐α production during muscle atrophy induced by sciatic nerve denervation in mice.     

Effects of transforming growth factor‐β1 treatment on muscle regeneration and adipogenesis in glycerol‐injured muscle

Transforming growth factor (TGF)‐β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF‐β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF‐β1 on muscle regeneration and adipogenesis in glycerol‐injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF‐β1 was either co‐injected with glycerol, as an ‘early treatment’ group, or injected at day 4 after glycerol, as a ‘late treatment’ group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol‐injured group (without TGF‐β1 treatment). Moreover, the Oil red O‐positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol‐injured group. Furthermore, TGF‐β1 treatment increased endomysial fibrosis and induced immunostaining of α‐smooth muscle actin. The greater inhibitory effects of early TGF‐β1 treatment than that of late TGF‐β1 treatment during regeneration in glycerol‐injured muscle suggest a more potent effect of TGF‐β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF‐β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies.     

Effect of dietary fish oil on fatty acid deposition and expression of cholesterol homeostasis controlling genes in the liver and plasma lipid profile: comparison of two animal models

The objective of the present study was to compare hepatic fatty acid deposition, plasma lipid level and expression of cholesterol homeostasis controlling genes in the liver of rats (Wistar Albino; n = 32) and pigs (Large White × Landrace; n = 32) randomly assigned into two groups of 16 animals each and fed 10 weeks the diet with either 2.5% of fish oil (F; source of eicosapentaenoic and docosahexaenoic acid, EPA+DHA) or 2.5% of palm oil (P; high content of saturated fatty acids; control). F‐rats deposited in the liver three times less EPA, but 1.3 times more DHA than F‐pigs (p < 0.05). Dietary fish oil relative to palm oil increased PPARα and SREBP‐2 gene expression much strongly (p < 0.01) in the pig liver in comparison with the rat liver, but expression of Insig‐1 and Hmgcr genes in the liver of the F‐pigs relative to the expression of these genes in the liver of the P‐pigs was substantially lower (p < 0.01 and p < 0.05 respectively) as compared to rats. When plasma lipid concentration in the F‐animals was expressed as a ratio of the plasma concentration in the P‐counterparts, dietary fish oil decreased HDL cholesterol less (p < 0.01), but LDL cholesterol and triacylglycerols more (p < 0.05 and p < 0.001 respectively) in rats than in pigs: more favourable effect of fish oil on rat plasma lipids in comparison with pigs can therefore be concluded. Concentration of total cholesterol and both its fractions in the rat plasma was negatively correlated (p < 0.01) with hepatic DHA, but also with unsaturated myristic and palmitic acid respectively. It has been concluded that regarding the similarity of the plasma lipid levels to humans, porcine model can be considered superior; however, using this model, dietary fish oil at the tested amount (2.5%) was not able to improve plasma lipid markers in comparison with saturated palm oil.     

Fbxw7β, E3 ubiquitin ligase,negative regulation of primary myoblast differentiation,proliferation and migration

Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7β, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant‐negative, F‐box deleted Fbxw7β, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7β.     

Fast‐to‐slow shift of muscle fiber‐type composition by dietary apple polyphenols in rats: Impact of the low‐dose supplementation

Our previous studies demonstrated that an 8‐week intake of 5% (w/w) apple polyphenol (APP) in the diet improves muscle endurance of young‐adult rats. In order to identify a lower limit of the dietary contribution of APP to the effect, the experiments were designed for lower‐dose supplementation (8‐week feeding of 0.5% APP in AIN‐93G diet) to 12‐week‐old male Sprague‐Dawley rats. Results clearly showed that the 0.5% APP diet significantly up‐regulates slower myosin‐heavy‐chain (MyHC) isoform ratios (IIx and IIa relative to total MyHC) and myoglobin expression in lower hind‐limb muscles examined (P < 0.05). There was a trend to increased fatigue resistance detected from measurements of relative isometric plantar‐flexion force torque generated by a stimulus train delivered to the tibial nerve (F(98, 1372) = 1.246, P = 0.0574). Importantly, there was no significant difference in the animal body‐phenotypes or locomotor activity shown as total moving distance in light and dark periods. Therefore, the present study encourages the notion that even low APP‐intake may increase the proportions of fatigue‐resistant myofibers, and has promise as a strategy for modifying performance in human sports and improving function in age‐related muscle atrophy.     

Expression of receptor tyrosine kinase targets PDGFR‐β, VEGFR2 and KIT in canine transitional cell carcinoma

Transitional cell carcinoma (TCC) is the most common neoplasia of the canine urinary tract. It tends to be locally invasive and has a moderate metastatic rate. Receptor tyrosine kinases (RTKs) play an important role in promoting cell growth, differentiation and regulation of cell function. RTK inhibitor toceranib phosphate has been used anecdotally to treat TCC. The goal of this study was to evaluate archived normal urinary bladder, TCC and cystitis bladder samples for expression of toceranib phosphate targets: VEGFR2, PDGFR‐β and stem cell factor receptor (KIT). A significant number of TCC samples expressed PDGFR‐β compared with cystitis and normal bladder samples (P<.0001). While all the tumour samples stained positively for VEGFR2, there was no significant difference between tumour, cystitis and normal bladder samples in intensity scores or staining distribution. Minimal positive staining for KIT was noted in the tumour samples. Based on this proof of target study, further investigation is warranted to determine clinical response of TCC to toceranib phosphate.     

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/β‐catenin signalling pathway in the Angora rabbit

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene β‐catenin and lymphoid enhancer‐binding factor 1 (LEF1) in Wnt/β‐catenin pathway. The levels of β‐catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of β‐catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle‐regulatory genes, insulin‐like growth factor‐1 (IGF‐1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/β‐catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/β‐catenin signalling pathway that promotes the proliferation of DP cells.     

Dietary supplementation of β‐hydroxy‐β‐methylbutyrate in animals – a review

The leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) has been studied by many researchers over the last two decades. In particular, the utility of HMB supplementation in animals has been shown in numerous studies, which have demonstrated enhanced body weight gain and carcass yield in slaughter animals; positive immunostimulatory effect; decreased mortality; attenuation of sarcopenia in elderly animals; and potential use in pathological conditions such as glucocorticoid‐induced muscle loss. The aim of this study was to summarize the body of research on HMB supplementation in animals and to examine possible mechanisms of HMB action. Furthermore, while the safety of HMB supplementation in animals is well documented, studies demonstrating efficacy are less clear. The possible reasons for differences in these findings will also be examined.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

Effect of Megasphaera elsdenii NCIMB 41125 dosing on rumen development,volatile fatty acid production and blood β‐hydroxybutyrate in neonatal dairy calves

Thirty calves were randomly assigned to two treatments and fed until weaning [42 days (d) of age]. Treatments were a control group (n = 15), which did not receive Megasphaera elsdenii (Me0) and a M. elsdenii group, which received a 50‐ml oral dose of M. elsdenii NCIMB 41125 (10⁸ CFU/ml) at day 14 day of age (Me14). Calves were given colostrum for the first 3 day followed by limited whole milk feeding. A commercial calf starter was offered ad libitum starting at day 4 until the end of the study. Fresh water was available throughout the study. Feed intake and growth were measured. Blood samples were collected via jugular venipuncture to determine β‐hydroxybutyrate (BHBA) concentrations. Fourteen male calves (seven per group) were euthanised on day 42 and digestive tracts harvested. Reticulo‐rumen weight was determined and rumen tissue samples collected from the cranial and caudal sacs of the ventral and dorsal portions of the rumen for measurements of papillae length, papillae width and rumen wall thickness. Dosing with M. elsdenii NCIMB 41125 improved starter dry matter intake (DMI), weaning body weight (BW) and tended to improve average daily gain. Calves in Me14 group had greater plasma BHBA concentration than Me0‐calves during the last 3 weeks of the trial and had at day 42 greater reticulo‐rumen weight, papillae width and papillae density compared to Me0. No differences in rumen wall thickness or papillae length were observed between the two groups. Total volatile fatty acids, acetate and propionate production did not differ between treatments, but butyrate production was greater in Me14 than Me0. Dosing M. elsdenii NCIMB 41125 showed benefit for calves with improved feed intake and rumen development suggesting increased epithelium metabolism and improved absorption of digestive end products.     

Influence of dietary fat source and supplementary α‐tocopheryl acetate on pulmonary hypertension and lipid peroxidation in broilers

An experiment was conducted to examine pulmonary hypertension and lipid peroxidation of broilers as affected by dietary fat source and α‐tocopheryl acetate. Two hundred and forty day‐old male chicks were used in a completely randomized design with five treatments consisted of four replicates and 12 chicks per replicate. Treatments included a control group that received no supplemental fat (treatment 1) or groups that received diets supplemented with beef tallow, soybean oil, a 50:50 blend of beef tallow and soybean oil, or soybean oil plus α‐tocopheryl acetate added at 220 mg/kg (treatments 2 to 5). All diets were kept isoenergetic and isonitrogenous and diet treatments 2 to 5 had 50 g/kg of fat supplement. Results showed that weight gain and feed consumption were significantly (p ≤ 0.05) increased by adding fat to the diet during the starter stage. However, birds that received fat‐supplemented diets gained less (p ≤ 0.05) during the grower period. Serum malone dialdehyde concentration and glutathione peroxidase activity were not affected by dietary treatments with the exception that inclusion of α‐tocopheryl acetate to the diet supplemented with soybean oil significantly (p < 0.05) reduced the activity of the enzyme when measured at 21 days of age. The relative weights of heart and liver and the right ventricle weight to total ventricle weight ratio were greater in broilers fed fat‐supplemented diets (p < 0.05).