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.     

Effect of feeding Aspergillus awamori and canola seed on the growth performance and muscle fatty acid profile in broiler chicken

The aim of this study was to examine effects of dietary supplementation with Aspergillus awamori and feeding canola seed on the growth and fatty acid profile in broilers. Twenty‐eight chicks (15 days old) were assigned to the following groups: (1) control, fed a basal diet; (2) awamori, fed the basal diet supplemented with 0.05% A. awamori; (3) canola, fed a diet containing 5% canola seed; and (4) canola + awamori, fed the canola diet supplemented with A. awamori (seven birds/group). Body weight gain was increased by A. awamori but not influenced by canola seed. Breast muscle weight was increased in either awamori or canola groups. Although plasma triglyceride and cholesterol were decreased by feeding A. awamori or canola seed, fat content in the breast muscle were increased, accompanied by decrease in saturated fatty acids and increase in unsaturated fatty acids. Moreover, decreased thiobarbituric acid reactive substance and increased α‐tocopherol content in the breast muscle was observed in all experimental groups. In conclusion, these results suggested that feeding canola seed and A. awamori might improve growth performance, and modified muscle fatty acid profile and α‐tocopherol content, suggesting that they may improve meat quality.     

Ubiquitin gene expression and ubiquitin conjugation in chicken muscle do not reflect differences in growth rate between broiler and layer birds.

Previous work has shown that chicken strains selected for growth (broilers) degrade muscle proteins less rapidly than those selected for egg laying. They also have decreased calpain and increased calpastatin content in breast muscle. This study aimed to test the hypothesis that these differences correlate with changes in the ATP- and ubiquitin-dependent proteolytic system. Chickens of a broiler strain (Ross 1) and a layer strain (ISABrown) were reared to the age of 4 wk under identical conditions with ad libitum access to feed and water. Mean fractional growth rates were 10.4%/d for broilers and 7.4%/d for layers. Feed intake measured in the last week of the trial was slightly greater in layer birds (.11 and .12 g x g body weight(-1) x d(-1) for broilers and layers respectively; P < .006). Polyubiquitin (UbI) messenger RNA was abundant in the muscles of these well-fed birds, but it showed little difference between strains. Muscle did not significantly express the UbII polyubiquitin gene. The ATP-dependent system conjugating ubiquitin to endogenous proteins had greatest activity in the gastrocnemius muscle of broiler birds but was not significantly different between breeds. Proteins cross-reactive with antisera to recombinant human proteasome regulatory subunits MSS1 (multicopy suppressor of SUG 1; S7) and TBP1 (tat binding protein 1; S6') were present in muscle homogenates from both strains of bird. The chick equivalent of TBP1 was more abundant in breast muscle of broiler birds than in leg muscle, or in either muscle of layers. Antiserum to recombinant yeast subunit mts2 (mitosis temperature sensitive gene 2; S4) did not react with any protein of the expected size but detected a 30-kDa peptide that was not associated with the 26S proteasome; this was found only in muscle from the layer strain. Hence, during normal growth of chickens, rates of protein degradation are not controlled by the expression of ubiquitin mRNA or the conjugation of ubiquitin. However, the composition of the 26S proteasome may be a regulatory factor.     

Myofibrillar proteolysis in chick muscle cell cultures during heat stress

The effect of heat stress on protein oxidation and myofibrillar proteolysis in chick myotubes was investigated. Myotubes were incubated at 37 or 41°C for 6 and 24 h. Protein carbonyl content, as an index of protein oxidation, increased more at 41°C than at 37°C for 6 and 24 h incubations. N^τ‐methylhistidine release as an index of myofibrillar proteolysis also increased more at 41°C than at 37°C for 6 and 24 h incubations. Proteasome activity also increased more under those same conditions. Calpain and cathepsin D but not B + L activities showed a greater increase at 41°C than at 37°C for 24 but not the 6 h incubation. These results indicate that heat stress increases protein oxidation and proteasome activity, resulting in an increase in myofibrillar proteolysis for short‐term incubation and, for long‐term incubation, it increases calpain, proteasome and cathepsin D activities, finally accelerating myofibrillar proteolysis in chick myotubes.     

Muscle protein expression of pikeperches (Stizostedion lucioperca and S. volgense)

The purpose of the present study was to investigate the muscle protein expression in two pikeperches (Stizostedion lucioperca and S. volgense) through intra‐ and intermyomeric composition of white muscles. Using denaturing 10% sodium dodecylsulfate‐polyacrylamide gel electrophoresis, muscle protein expression was studied in relation to within‐ and between‐species morphological development, sex, maturity and age of pikeperches. Myosin, actin and troponin have a distinct role in the contraction and length tension of muscle fibers of these species. No obvious intramyomeric differences were found in the myosin heavy chain of both species. Myosin light chains (15–38 kDa) have different expression in different age groups. The muscle protein of the fingerling and adult S. lucioperca had high molecular weight (50 kDa) myosin in contrast to the other Percid species. The molecular weight of actins increased comparatively in low‐age‐group fish. ATP is stored in myosin and released to cause contraction when myosin comes in contact with actin of the experimental fish. Troponin regulates increasing concentration of light‐chain myosin in mature fish. Because troponin T has been implicated in the regulation of skeletal muscle kinetics, muscle contraction kinetics was predicted in different age groups. The muscle proteins of both sexes of these species have polymorphism in various age groups but have no difference in similar aged fish. No muscle protein dimorphism was found in these Percid species. The white muscle protein composition and contractile properties affect power production during fast, unsteady movement and swimming.     

Effect of feeding rice whole crop silage on growth rate,levels of vitamin A, β‐carotene,vitamin E and IGF‐1 in plasma and skeletal muscle protein degradation in Japanese black calves

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, β‐carotene, vitamin E and IGF‐1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma β‐carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma β‐carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.     

Dietary mannanase‐hydrolyzed copra meal improves growth and increases muscle weights in growing broiler chickens

The utilization of copra meal as a feed ingredient is limited because it contains a high level of mannan. However, recent findings indicate that the effect of copra meal on growth performance in broiler chickens can be improved by the supplementation of mannanase in the diet. In the present study, we examined the effect of mannanase‐hydrolyzed copra meal (MCM) on growth performance and muscle protein metabolism in growing broiler chickens (Gallus gallus domesticus). Forty 8‐day‐old male broiler chicks were assigned to two groups (four birds in each pen, five replicates) and fed either a commercial diet (as a control diet) or a diet containing MCM at 0.2% until 22 days of age. Dietary MCM significantly increased the weights of body, breast muscle, and thighs in chickens, whereas the weights of abdominal adipose tissue and liver were not affected. Cumulative feed intake was significantly increased by MCM. Dietary MCM significantly decreased plasma 3‐methylhistidine level. The messenger RNA and protein levels of muscle protein metabolism‐related factors were not altered by MCM. These findings suggest that the growth‐promoting effect of MCM is related to the suppression of muscle proteolysis in growing broiler chickens.     

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.     

New fermentation technique to process fish waste

The traditional method of processing fish waste is costly and usually overheats the waste, making it less digestible. In the present study, a fermentation technique using Aspergillus awamori was used to dry minced sardine fish to find a new model for processing fish waste and to evaluate its digestibility. Wheat bran (700 g), minced fresh sardines (2638 g) and A. awamori spores (0.7 g) were mixed together and kept in a ventilated incubator for 5 days at 40°C. At the end of the fermentation, a sample of fishmeal was taken to measure the nutrient content, enzyme activity and digestibility of crude protein (CP) and dry matter (DM) using an in vitro method. Furthermore, the effect of enzymes produced during the fermentation of DM and CP digestibilities of corn–soybean mixture in vitro were also examined. The percentage of moisture, CP, crude fat and nitrogen‐free extract in the fermented fish waste were 11, 39, 23 and 25%, respectively. The in vitro digestibilities of DM (69%) and CP (84%) of the fishmeal were almost the same as those of the heat‐dried sardine powder. During the fermentation, glucoamylase, α‐glucosidase, α‐amylase and acidic protease were produced, which may improve the DM digestibility of the diet. In conclusion, fish waste can be dried successfully using the fermentation technique described in the present study. Furthermore, the DM digestibility of the corn–soybean meal mixture is improved by the enzymes produced during the fermentation.     

Comprehensive evaluation of growth performance and meat characteristics of a fattening system combining grazing with feeding rice whole‐crop silage in Japanese Black steers

We investigated the effect of a system for fattening steers combining grazing with feeding rice whole‐crop silage (rWCS) on growth performance, meat characteristics, and the expression of genes involved in skeletal muscle growth. Steers were randomly assigned to grazing or concentrate‐fed groups (CT). The grazing group (GZ) was fed rWCS after grazing until 16 months of age. The final body weight was the same in the two groups, but the dressed weight was lower in the GZ than in the CT. The beef color standard was higher in the GZ than in the CT. Although beef marbling did not differ between the two groups, there was less intramuscular fat and subcutaneous fat in the GZ than in the CT. The α‐tocopherol and β‐carotene contents in the muscle were higher in the GZ than in the CT. The GZ showed a lower daily gain (DG) during the grazing period, which may have resulted from decelerating skeletal muscle growth caused by the increased expression of genes encoding myostatin and atrogin‐1. However, the DG and feed efficiency of the GZ increased after grazing. The two groups exhibited a similar level of beef productivity.     

Expression analyses of candidate genes related to meat quality traits in squabs from two breeds of meat‐type pigeon

In this study, meat quality traits were compared between squabs from two pigeon breeds: one Chinese indigenous breed, the Shiqi (SQ) meat‐type pigeon, and an imported breed, the white king (WK) meat‐type pigeon. Breed differences were detected in the content of intramuscular fat (IMF) in the breast muscle. SQ squabs had significantly higher IMF content than the WK birds. The shear force value (an objective measure of meat tenderness) of SQ birds was also relatively lower than that of the WK squabs. Further analysis of fatty acids profile revealed that SQ squabs exhibited significant advantage in the synthesis of polyunsaturated fatty acids, while WK squabs were significantly higher in the sum of monounsaturated fatty acids. Breast muscle in the SQ squabs was also significantly higher in the ratio of polyunsaturated fatty acids to saturated fatty acids, as well as the sum of omega 6 fatty acids. Variability of expression levels of functional genes in relation to fat accumulation and meat tenderness was analysed by qRT‐PCR. Gene expression analyses showed that the hepatic expression of LPL (lipoprotein lipase), FABP4 (fatty acid‐binding protein 4), and CAPN2 (calpain‐2) were significantly higher in the SQ squabs. In the breast muscle tissue, the FABP3 (fatty acid‐binding protein 3) and CAPN2mRNA abundance was significantly higher in SQ squabs. Our results suggested that these differentially expressed genes might be candidate genes used in the programmes of targeted selection for squabs with higher IMF content, tender meat, and more favourable fatty acids composition.     

A comparison of amino acid digestibility bioassays for broilers

1. Apparent amino acid digestibilities determined in the lower ileum of broilers given free access to a diet containing normal or heat‐damaged soya protein were similar to “true” digestibilities determined with excreta using the Sibbald rapid bioassay.

2. Higher ileal digestibilities were obtained with the Farrell rapid bioassay than with birds given free access to food. This pattern was reversed when measurements were made on excreta.

3. Maximum absorption of amino acids had occurred by the time the di‐gesta had reached the upper ileum in birds fed on normal but not heat‐damaged soyabean protein.

4. Heat damage to the soyabean protein was detected with all the bioassay procedures.

     

Early post mortem expression of genes related to tenderization in two Italian Simmental young bulls' skeletal muscles differing in contractile type

The early post mortem expression of eight genes potentially involved in meat ageing process and the tenderness of two Italian Simmental young bulls' (Bos taurus) skeletal muscles differing in their contractile type were evaluated. Samples of Longissimus lumborum (LL) and Infraspinatus (IS) muscles were collected from 17 bulls. The messenger RNA (mRNA) abundances of calpain‐1, calpain‐2, calpastatin, caspase 3, caspase 9, heat shock protein 27 (Hsp27), Hsp40 and Hsp70 were detected by quantitative PCR. The myosin heavy chain‐slow and ‐fast isoform content, the pH_48h and the lipid content of the muscles were in line with the contractile and metabolic type. In comparison with the fast LL, the slow IS showed a lower calpain‐1/calpastatin mRNA content ratio after slaughtering and a higher Warner‐Bratzler Initial Yield value after 7 days of ageing. Hsp27 and Hsp70 mRNA abundances were significantly lower in LL than IS, highlighting their potential role in the ageing process of bovine muscles.     

Comparison of calpain and calpastatin activities in skeletal muscle of broiler and layer chickens

1. The objective of this study was to estimate the difference between broiler and layer chicks in the activities of calpain and calpastatin (inhibitor of calpain) in breast muscle. Differences between broilers and layers in body weight, daily gain at 3 weeks of age and fractional growth rate (FGR) during 2 and 3 weeks of age were statistically significant (P < 0.01).

2. Calpain and calpastatin activities were measured at three weeks of age with alkali‐denatured casein as a substrate. The m‐calpain (calpain activated by millimolar calcium concentration) activities in units/g muscle and units/ mg extractable muscle protein were 0.779 and 0.353 for broilers, and 1.042 and 0.440 for layers, respectively. The calpastatin activities in units/g muscle and units/mg extractable muscle protein were 0.332 and 0.153 for broilers, and 0.262 and 0.112 for layers, respectively.

3. Broilers with high FGR showed low m‐calpain and high calpastatin activities. In contrast, layers with low FGR showed high m‐calpain and low calpastatin activities.

4. These results suggest that m‐calpain and calpastatin activities in skeletal muscle vary between breeds which have different rates of muscle production.     

Effect of graded levels of dietary corn steep liquor on growth performance,nutrient digestibility,haematology and histopathology of broilers

This experiment was conducted to assess nutritional potential of corn steep liquor (CSL) as an alternative protein source in broiler's diet. A total of 280‐day‐old unsexed broiler chicks were randomly distributed into four experimental groups in a simple randomised experimental design (each group had seven replicates; 10 birds per each). Four iso‐nitrogenous and iso‐caloric broiler starter and finisher diets (C, LCSL, MCSL and HCSL) were formulated containing 0%, 5%, 10% and 15% CSL respectively. Results revealed that chicks fed MCSL diet had significantly (p ≤ .05) the heaviest Body weight, highest Body weight gain and the lowest feed conversion ratio followed by those fed HCSL diet. In the starter period, LCSL and MCSL diets resulted in better (p ≤ .05) dry matter (DM) (89.57%) and crude protein (67.67%) digestibilities respectively. In the finisher period, DM, crude fibre and fat digestibilities were not affected by CSL inclusion levels; while crude protein digestibility was significantly (p ≤ .05) high in the groups fed LCSL and HCSL diets. The CSL inclusion had no effect on blood haematology except serum uric acid, which tended to increase with increasing CSL levels. No microscopic changes were exhibited on liver, heart, kidneys and intestine of birds across all dietary treatments. However, crops in birds fed HCSL diet showed moderate to severe hypertrophy and hyperplasia of the mucosal lining. The study showed that CSL could be added up to 15% in broiler's diet without any adverse effect on bird's performance.     

Resveratrol beneficially affects meat quality of heat‐stressed broilers which is associated with changes in muscle antioxidant status

This study was conducted to evaluate the effect of dietary resveratrol (Res) supplementation on serum parameters, meat quality and muscle antioxidant status of broilers under heat stress (HS). A total of 270 21‐day‐old male Cobb broilers were randomly assigned to three treatment groups with six replicates of 15 birds each. The three treatment groups were as follows: the control group, in which birds were reared at 22 ± 1°C, and the HS and HS + Res (400 mg/kg) groups, in which birds were reared at 33 ± 1°C for 10 h (08.00–18.00 h) and 22 ± 1°C for the rest of the time. Compared with birds in the control group, birds in the HS group exhibited increased serum corticosterone (CORT) and triacylglycerol contents, L*, drip loss and muscle malondialdehyde content, and decreased serum glucose content, pH_24 h, muscle total antioxidant capacity (T‐AOC), catalase (CAT) and glutathione peroxidase (GSH‐PX) activities (P < 0.05). Compared with birds in the HS group, birds in HS + Res group exhibited increased serum glucose content, a*, pH_24 h, muscle T‐AOC and CAT activities, and decreased serum CORT and triacylglycerol contents, L*_24 h, drip loss and muscle malondialdehyde content (P < 0.05). In conclusion, Res beneficially protects against HS‐impaired meat quality of broilers through regulating muscle antioxidant status.     

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.     

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 electrical stimulation and pre‐rigor conditioning temperature on aging potential of hot‐boned beef M. longissimus lumborum

The objective of this study was to create various pH/temp decline rates in hot‐boned bull beef M. longissimus lumborum (LL) through a combination of electrical stimulation (ES) and pre‐rigor holding temperature. The relationship between the pre‐rigor interventions, the activities of µ‐calpain and small heat shock proteins (sHSP), and the impacts on meat product quality were determined. Paired LL loins from 13 bulls were hot‐boned within 40 min of slaughter, immediately ES and subjected to various holding temperatures (5, 15, 25, and 35°C) for 3 hr. The rate of muscle pH decline, sarcomere length, shear force, and proteolysis of muscle proteins were measured. ES‐25°C had a longer sarcomere length compared to non‐electrical stimulation samples. ES‐25°C and ES‐35°C samples had lower shear force values, higher µ‐calpain activity and higher desmin, troponin‐T, and sHSP degradation. The above findings suggest that pH/temp decline rates created in hot‐boned muscle impacted muscle protein proteolysis by increasing the activity of proteases and degradation of sHSP.