Feeding Aspergillus awamori reduces skeletal muscle protein breakdown and stimulates growth in broilers

This study was conducted to show that dietary supplementation of a fungus, Aspergillus awamori called Koji in Japan, reduces skeletal muscle protein breakdown and stimulates growth in broiler chickens. A total of 30 chicks at 15 days of age was divided into control and two treatment groups (10 birds per treatment). Control group was fed basal diet and treatment groups were fed the basal diets supplemented with A. awamori at levels of 0.05% and 0.2%. The birds were raised for 12 days from 15 to 27 days of age and then the effect on growth, organ weights and plasma 3‐methylhistidine concentration and digestibilities of protein and energy was evaluated. The messenger RNAs (mRNAs) of atrogin‐1, ubiquitin, proteasome, m‐calpain, µ‐calpain, β‐actin, myosin and pax‐7 in the breast muscle were also measured. Body weight gain and breast muscle weight were increased, although feed intake was decreased by the fungus and thus feed efficiency was increased. Protein and energy digestibilities were increased. Furthermore, plasma 3‐methylhistidine concentration was decreased by the fungus. The mRNAs of atrogin‐1, ubiquitin, proteasome, m‐calpain and µ‐calpain were all decreased. The mRNA of β‐actin but not myosin and pax‐7 was slightly increased by the fungus. In conclusion, feeding A. awamori improves growth performance because skeletal muscle proteolytic activity is reduced and digestibilities of energy and protein are increased.     

Proteome analysis of whole and water‐soluble proteins in masseter and semitendinosus muscles of Holstein cows

To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.     

Mediation of acetylcholine and substance P induced contractions by myosin light chain phosphorylation in feline colonic smooth muscle

OBJECTIVES: To determine the role of myosin light chain phosphorylation in feline colonic smooth muscle contraction. SAMPLE POPULATION: Colonic tissue was obtained from eight 12- to 24-month-old cats. PROCEDURE: Colonic longitudinal smooth muscle strips were attached to isometric force transducers for measurements of isometric stress. Myosin light chain phosphorylation was determined by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stress and phosphorylation were determined following stimulation with ACh or SP, in the absence or presence of a calmodulin antagonist (W-7; 0.1 to 1.0 mM), myosin light chain kinase inhibitor (ML-9; 1 to 10 microM), or extracellular calcium free solutions. RESULTS: Unstimulated longitudinal colonic smooth muscle contained low amounts (6.9+/-3.2%) of phosphorylated myosin light chain. Phosphorylation of the myosin light chains was dose and time dependent with maximal values of 58.5% at 30 seconds of stimulation with 100 microM Ach and 60.2% at 45 seconds of stimulation with 100 nM SP Active isometric stress development closely paralleled phosphorylation of the myosin light chains in ACh- or SP-stimulated muscle. W-7 and ML-9 dose dependently inhibited myosin light chain phosphorylation and isometric stress development associated with ACh or SP stimulation. Removal of extracellular calcium inhibited myosin light chain phosphorylation and isometric stress development in ACh-stimulated smooth muscle. CONCLUSIONS AND CLINICAL RELEVANCE: Feline longitudinal colonic smooth muscle contraction is calcium-, calmodulin-, and myosin light chain kinase-dependent. Myosin light chain phosphorylation is necessary for the initiation of contraction in feline longitudinal colonic smooth muscle. These findings may prove useful in determining the biochemical and molecular defects that accompany feline colonic motility disorders.     

The effect of weight loss on protein profiles of gastrocnemius muscle in rabbits: a study using 1D electrophoresis and peptide mass fingerprinting

The study of physiological changes occurring during selection contributes to an improved understanding of relationships leading to efficiencies in animal production. To investigate the effects of food restriction in gastrocnemius muscle protein expression, 20% weight reduction was induced in New Zealand White (meat producing) and wild rabbits, using one‐dimensional gel electrophoresis and peptide mass fingerprinting. Lower expression levels of myosin heavy chains were found in the Wild Rabbits Restricted Group, while myosin light chain and α‐crystallin proteins were not detected in restricted groups. Glyceraldeyde‐3‐phosphate dehydrogenase and glycogen phosphorylase expression levels were similar for all experimental groups. Phosphopyruvate hydratase β was not detected in the wild rabbit restricted diet group. Pyruvate kinase levels were 50% lower in the New Zealand Restricted group. LIM protein detection was absent in the control New Zealand group. Results also show relevance of actin in preserving muscle structure in depressed food availability, the sensitivity of both myosin light chain and α‐crystallin protein to restricted feed and the role of PK in the resistance of New Zealand rabbits to food restriction.     

Changes of microbial spoilage,lipid‐protein oxidation and physicochemical properties during post mortem refrigerated storage of goat meat

Examined was the effect of post mortem refrigerated storage on microbial spoilage, lipid‐protein oxidation and physicochemical traits of goat meat. Seven Boer bucks were slaughtered, eviscerated and aged for 24 h. The Longissimus lumborum (LL) and Semitendinosus (ST) muscles were excised and subjected to 13 days post mortem refrigerated storage. The pH, lipid and protein oxidation, tenderness, color and drip loss were determined in LL while microbiological analysis was performed on ST. Bacterial counts generally increased with increasing aging time and the limit for fresh meat was reached at day 14 post mortem. Significant differences were observed in malondialdehyde (MDA) content at day 7 of storage. The thiol concentration significantly reduced as aging time increased. The band intensities of myosin heavy chain (MHC) and troponin‐T significantly decreased as storage progressed, while actin remained relatively stable. After 14 days of aging, tenderness showed significant improvement while muscle pH and drip loss reduced with increase in storage time. Samples aged for 14 days had higher lightness (P < 0.05) and lower (P < 0.05) yellowness and redness. Post mortem refrigerated storage influenced oxidative and microbial stability and physico‐chemical properties of goat meat.     

Gill Reaction to Pollutants from the Tamiš River in Three Freshwater Fish Species,Esox lucius L. 1758, Sander lucioperca (L. 1758) and Silurus glanis L. 1758: A Comparative Study

The study evaluated the effects of waterborne pollutants from the Tami? River on gill histology and possible differences in gill reaction patterns between three freshwater fish species, pike Esox lucius L. 1758, pike‐perch Sander lucioperca (L. 1758) and wels catfish Silurus glanis L. 1758 from the Tami? River. Gills from analysed fish species showed moderate to intense histopathological alterations. The most frequent progressive alteration was hyperplasia of epithelium, whereas the most frequent regressive alteration was epithelial lifting. Circulatory disturbances were most often manifested in the form of hyperaemia. During comparative analysis, differences in gill indices, reaction and alteration indices, as well as in gill and filament prevalence between analysed species, were observed. Although all analysed fish species did show both progressive and regressive alterations, there was a significant difference in the level of expression of these reaction patterns. Gill index obtained for pike clearly stands out as the lowest. Wels catfish showed the highest progressive reaction index, significantly higher in comparison with the other two species (P < 0.05), while pike‐perch showed the highest regressive reaction index, also significantly higher in comparison with the other species (P < 0.001). These results may implicate species‐specific gill reactions and thus present a useful tool for better understanding toxic mechanisms of various pollutants.     

Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development

The important roles of myogenic regulatory factors (MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real‐time RT‐PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues (P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non‐significant differences between genders. We provide novel evidence for MRFs expression in age‐ and gender‐dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait‐associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species.     

Influences of incorporating detoxified Jatropha curcas kernel meal in common carp (Cyprinus carpio L.) diet on the expression of growth hormone‐ and insulin‐like growth factor‐1‐encoding genes

Jatropha curcas is a drought‐resistant shrub or small tree widespread all over the tropics and subtropics. The use of J. curcas (L) kernel meal in fish feed is limited owing to the presence of toxic and antinutritional constituents. In this study, it was detoxified using heat treatment and organic solvent extraction method. The detoxification process was carried out for 60 min to obtain the detoxified meal. Cyprinus carpio L. fingerlings (n = 180; avg. wt. 3.2 ± 0.07 g) were randomly distributed in five treatment groups with four replicates and fed isonitrogenous diets (crude protein 38%) for 8 weeks. The inclusion levels of the detoxified Jatropha kernel meal (DJKM) and soybean meal (SBM) were as follows: control diet was prepared with fish meal (FM) and wheat meal, without any DJKM and SBM; diets S₅₀ and J₅₀: 50% of FM protein replaced by SBM and DJKM respectively; diets S₇₅ and J₇₅: 75% of FM protein replaced by SBM and DJKM respectively. Highest body mass gain and insulin‐like growth factor‐1 (IGF‐1) gene expression in brain, liver and muscle were observed for the control group, which were statistically similar to those for J₅₀ group and significantly (p < 0.05) higher than for all other groups, whereas growth hormone gene expression in brain, liver and muscle exhibited opposite trend. Insulin‐like growth factor‐1 concentration in plasma did not differ significantly among the five groups. Conclusively, growth performance was in parallel with IGF‐1 gene expression and exhibited negative trend with GH gene expression.     

Relationships between muscle growth potential,intramuscular fat content and different indicators of muscle fibre types in young Charolais bulls

Genetic selection in favor of muscle growth at the expense of fat should affect characteristics of muscles, and therefore beef quality. This study was conducted with two extreme groups of six animals selected among 64 Charolais young bulls ranked according to their genetic potential for muscle growth. Muscle characteristics were assessed in Rectus abdominis (RA, slow oxidative) and Semitendinosus (ST, fast glycolytic) muscles. Intramuscular fat content and proportions of myosin heavy chains I (slow) and IIA (fast oxido‐glycolytic) and certain indicators of oxidative metabolism (activities of citrate synthase (CS), isocitrate dehydrogenase and cytochrome‐c oxidase (COX); expression of H‐fatty acid binding protein (FABP)) were higher in RA than in ST muscle. Genetic selection for muscle growth reduced intramuscular fat content and the activities of some oxidative metabolism indicators (namely CS, COX only). The positive correlation between muscle triacylglycerol content and A‐FABP messenger RNA level (a marker of adipocyte differentiation) (r = 0.53, P < 0.05) suggests that A‐FABP may be a good marker of the ability of bovines to deposit intramuscular fat. In conclusion, the metabolic muscle characteristics which respond to the selection process in favor of muscle growth clearly differ from the muscle characteristics which allow muscle types to be differentiated.     

Determination of sulfadiazine,trimethoprim, and N4‐acetyl‐sulfadiazine in fish muscle plus skin by Liquid Chromatography–Mass Spectrometry. Withdrawal‐time calculation after in‐feed administration in gilthead sea bream (Sparus aurata L.) fed two different diets

This study presents a depletion study for sulfadiazine and trimethoprim in muscle plus skin of gilthead sea bream (Sparus aurata L.). N⁴‐acetyl‐sulfadiazine, the main metabolite of sulfadiazine (SDZ), was also examined. The fish were held in seawater at a temperature of 24–26 °C. SDZ and trimethoprim (TMP) were administered orally with medicated feed for five consecutive days at daily doses of 25 mg SDZ and 5 mg TMP per kg of fish body weight per day. Two different diets, fish oil‐ and plant oil‐based diets, were investigated. Ten fish were sampled at each of the days 1, 3, 5, 6, 8, 9, 10, and 12 after the start of veterinary medicine administration. However for the calculation of the withdrawal periods, sampling day 1 was set as 24 h after the last dose of the treatment. Fish samples were analyzed for SDZ, TMP, and acetyl‐sulfadiazine (AcSDZ) residues by liquid chromatography–mass spectrometry. SDZ and TMP concentrations declined rapidly from muscle plus skin. Considering a maximum residue limit of 100 μg/kg for the total of sulfonamides and 50 μg/kg for TMP residues in fish muscle plus skin, the withdrawal periods of the premix trimethoprim‐sulfadiazine 50% were calculated as 5 and 6 days, at 24–26 °C, in fish oil (FO) and plant oil (PO) groups, respectively. The investigation of this work is important to protect consumers by controlling the undesirable residues in fish.     

Effects of dietary leucine on antioxidant activity and expression of antioxidant and mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets

The study was conducted to investigate the effects of dietary leucine on antioxidant activity and expression of antioxidant‐ and mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets. Three diets were formulated with different levels of supplemented leucine (0%, 0.25%, 0.5%). Results showed that supplementation of 0.25% leucine significantly increased antisuperoxide anion (ASA) and antihydroxyl radical (AHR) levels and activities of total superoxide dismutade (T‐SOD), glutathione peroxidase (GPx), glutathione S‐transferase (GST), and total antioxidant capacity (T‐AOC) in serum, longissimus dorsi muscle and liver of piglets as compared with the control group. The SOD2, catalase (CAT), GPx, GST, glutathione reductase (GR), and nuclear factor erythroid 2‐related factor 2 (Nrf2) mRNA levels in longissimus dorsi muscle and liver were significantly increased by 0.25% leucine supplementation. However, the malondialdehyde (MDA) content and the mRNA level of Kelch‐like ECH‐associated protein 1 (Keap1) exhibited an opposite tendency. Additionally, supplementation of 0.25% leucine significantly increased the mRNA levels of mitochondrial‐related genes in longissimus dorsi muscle and liver of piglets. Results suggested that supplementation of 0.25% leucine improved antioxidant activity and mitochondrial biogenesis and function of piglets, which was related to the increase in antioxidant enzymes activities and upregulation of expression of antioxidant‐ and mitochondrial‐related genes.     

Cloning and heterologous expression of the ovine (Ovis aries) P‐glycoprotein (Mdr1) in Madin–Darby canine kidney (MDCK) cells

Zahner, D., Alber, J., Petzinger, E. Cloning and heterologous expression of the ovine (Ovis aries) P‐glycoprotein (Mdr1) in Madin–Darby canine kidney (MDCK) cells. J. vet. Pharmacol. Therap. 33 , 304–311. P‐glycoprotein (P‐gp) plays a crucial role in the multidrug resistance of pathogenic helminths in sheep (Ovis aries) as well as in antiparasitic drug pharmacokinetics in the host. We cloned sheep P‐gp cDNA and expressed it stably in Madin–Darby canine kidney (MDCK) cells. The open reading frame consists of 3858 nucleotides coding for a 1285 amino acids containing protein. The sequence shows high homology to the orthologs of other mammalian species, especially cattle. Both ruminant DNA sequences show a 9 bp insertion that is lacking in all other investigated sequences. Expressed in MDCK cells, the protein displays a size of 170 kDa on Western analysis. Transfection of MDCK cells with sheep P‐gp resulted in 10‐ to 50‐fold resistance to the cytotoxic P‐gp substrates colchicin and daunorubicin, and in reduced digoxin accumulation.     

Characterization of myofibrillar adenosine triphosphatase activity and liberation of actin from myofibrils upon heating chicken breast meat

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg²⁺‐ATPase (adenosine triphosphatase) and Ca²⁺‐ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine‐5′‐monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat‐stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.     

Effects of methionine on muscle protein synthesis and degradation pathways in broilers

This study investigated the hypothesis that supplementation of methionine (Met) to broiler diets increases muscle growth due to regulation of molecular pathways related to protein synthesis and degradation depending on the Met source. Day‐old male Cobb‐500 broilers (n = 240) were phase‐fed three different wheat–soya bean meal‐based basal diets during days 1–10, 11–21 and 22–35. Basal diets (Met‐ group, Met + Cys concentration 15% below NRC recommendations) were supplemented with 0.10% or 0.40% Met either as DL‐Met (DLM) or DL‐2‐hydroxy‐4‐(methylthio) butanoic acid (DL‐HMTBA) (equimolar comparison). Breast muscle weights were lower in the Met‐ group compared to all Met‐supplemented groups and were lower in broilers supplemented with 0.10% of DL‐HMTBA compared to the other groups fed Met‐supplemented diets. However, the expression of genes or relative phosphorylation and thus activation state of proteins involved in the somatotropic axis, the mammalian target of rapamycin (mTOR) pathway of protein synthesis, the ubiquitin–proteasome pathway (UPP) and autophagy–lysosomal pathway of protein degradation, the GCN2/eIF2a pathway involved in the inhibition of protein synthesis and in the myostatin–Smad2/3 pathway involved in myogenesis were not affected by Met source. Feeding diets with suboptimum Met + Cys concentrations, however, decreased expression of GHR and IGF1 in liver and muscle and increased that of MURF1 involved in the UPP in the broiler's muscle at day 10 and 21, while that of FOXO and atrogin‐1 and FOXO phosphorylation remained unaffected. Additionally, suboptimum dietary Met concentrations increased expression of the autophagy‐related genes ATG5 and BECN1 at day 35. Met supplementation neither affected gene expression nor phosphorylation of proteins involved in the GNC2/eIF2a and mTOR pathways. These data indicate that protein synthesis was not affected on the molecular level, while protein degradation was marginally affected by dietary Met dosage.     

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.     

An Immunohistochemical Observations on the Oviduct of the Goat

The oviduct has an important role in regulating transport of gametes and fertilization. The main role in these functions has a smooth muscle cells and ciliated epithelium lining the oviduct. All functions are under the influence of hormonal and nervous system. The objective of this study was immunohistochemically to examine the following structures: lining epithelium, smooth muscle cells, elastic fibres and nerve fibres. For this purpose, the following antibodies were used: cytokeratin 18, S‐100 protein, acetylated α‐tubulin, smooth muscle actin, desmin and elastin. Ciliary and secretory cells of the lining epithelium were positive for cytokeratin 18 and S‐100 protein. Cilia and the basal body‐associated structures of ciliary cells were positive to acetylated α‐tubulin. Smooth muscle cells (SMC) in mucosa and of the muscular layer were positive for α‐smooth muscle actin (SMA) and desmin. High density of nerve fibres positively reacted to acetylated α‐tubulin and S100 protein was present in the mucosa, muscular layer and serosa. Elastic fibres positive for elastin form a dense network at the base of the mucosal folds and in the muscle layer. A dense network of these fibres is accompanying the blood vessels. It is supposed that together with smooth muscle cells they are involved in the transport of ovum and in blood flow regulation.     

Myosins 1 and 6, myosin light chain kinase,actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.