Assessment of calpain and caspase systems activities during ageing of two bovine muscles by degradation patterns of αII spectrin and PARP‐1

The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate‐ribose) polymerase‐1 (PARP‐1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P < 0.01), while SBDP120 and PARP‐1 degradation products were similar between muscles. At T1, no difference was found in the level of SBDP145 between muscles, while SBDP120 and PARP‐1 cleavage products were not detected. At T2 neither αII spectrin nor PARP‐1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period.     

Differences in calpain system,desmin degradation and water holding capacity between commercial Meishan and Duroc × Landrace × Yorkshire crossbred pork

The objective of this study was to examine the differences in calpain system, desmin degradation, pH values and water holding capacity (WHC) between muscles of commercial Meishan and Duroc × Landrace × Yorkshire crossbred pigs. Meishan pork presented better WHC evidenced by lower purge loss at days 1 and 3 and less centrifugation loss at day 1 post mortem (P < 0.05). pH values at 45 min post mortem in Meishan pork were significantly higher compared to crossbred pork (P < 0.05). Calpain‐1 messenger RNA (mRNA) expression was lower in Meishan pork compared to that from crossbred pork (P < 0.05). Additionally, calpain‐1 activity, the ratio of calpain‐1 to calpastatin activity and desmin degradation were lower in Meishan pork compared to those from crossbred pork samples (P < 0.05). The results indicate that the calpain system including mRNA expression and activity were different between commercial Meishan and crossbred pork resulting in difference in the degree of desmin degradation during post mortem aging. pH values at 45 min and 24 h post mortem rather than calpain activity and desmin degradation could explain the higher water holding capacity in commercial Meishan pork.     

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.     

Calpain activation and proteolysis in postmortem goose muscles

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0–5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10–15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain‐1 and ‐11 activities, and contents of the calpain‐1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.     

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.     

Intermuscular variation in tenderness: association with the ubiquitous and muscle-specific calpains

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.     

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.     

Tenderization potential of Hanwoo beef muscles from carcasses with differed genders and loin intramuscular fat content levels during post mortem ageing

Carcasses from Hanwoo steers (n = 15) and cows (n = 15) were classified into three groups: group 1 (G1), the carcasses had 10% to < 11.5% intramuscular fat (IMF) in loin muscles; group 2 (G2), the carcasses had 13% to < 14.5% IMF in loin muscles; and group 3(G3), the carcasses had 17% to < 18.5% IMF in loin muscles. These were used to evaluate the effects of gender and carcass group on quality traits and Warner–Bratzler shear force (WBSF) of Psoas major (PM), Longissimus thoracis (LT), Longissimus lumborum (LL), Longus colli (LC), Supraspinatus (SS), Latissimus dorsi (LAD), Semimembranosus (SM), Quadriceps femoris (QF), Biceps femoris (BF) and Semitendinosus (ST) muscles. Our results showed that pH values of LT, LL, LC, BF and QF muscles were lower in steers than in cows (P < 0.05). Water holding capacity (WHC) was found higher in LC, SS, LAD and QF muscles of steers (P < 0.05). At day 2 of ageing, gender affected the WBSF values of only PM, LD and QF muscles in G1, and QF muscle in G3; however, with additional ageing, the gender effect was observed for most of the muscles. Most muscles showed ageing responses; however, the rates of ageing response significantly varied depending on gender and carcass groups. The muscles of G1 and G2 had generally higher tenderization potentials than those of G3. Furthermore, most muscles in G3 had generally lower WBSF values than in G1 and G2. These results clearly indicate that ageing has a significant effect on quality and WBSF of beef muscles, and the classification by loin IMF level may be useful for prediction of the tenderness of other muscles.     

Satellite cells isolated from skeletal muscle will proliferate faster in WENS yellow feather chicks

This study was conducted to evaluate the differential proliferation ability of satellite cells (SCs) derived from pectoral muscles (PM) with different fiber characteristics and further to explore the underlying molecular mechanism. WENS Yellow Feather Chicks (WYFC) were chosen as the animal model, with White Plymouth Rock Chicks (WPRC) as a comparison. The results showed that WPRC had higher body and pectoral muscle weight than WYFC at 4 days old (P < 0.05). However, WYFC showed greater fiber numbers/mm² but smaller fiber cross‐sectional area compared with WPRC in PM (P < 0.05). SCs derived from PM of WYFC had a faster proliferation rate but smaller cell size compared with that from PM of WPRC (P < 0.05). The percentage of cell population in G2/M phase and the messenger RNA abundance of TSC1 (P = 0.08), Rheb (P = 0.07) and target of rapamycin (TOR, P = 0.06) in WYFC were higher than that in WPRC. In conclusion, our results demonstrated that SCs isolated from PM of WYFC had faster proliferation rate but smaller cell size than that in WPRC. The higher SC proliferation in WYFC may be due to higher gene expression of TOR signaling pathway than in WPRC, and the larger cell size of WPRC may be due to higher insulin‐like growth factor‐1 expression than in WYFC.     

Effects of nutritional level of concentrate‐based diets on meat quality and expression levels of genes related to meat quality in Hainan black goats

The present study investigated the effects of the nutritional levels of diets on meat quality and related gene expression in Hainan black goat. Twenty‐four goats were divided into six dietary treatments and were fed a concentrate‐based diet with two levels of crude protein (CP) (15% or 17%) and three levels of digestive energy (DE) (11.72, 12.55 or 13.39 MJ/kg DM) for 90 days. Goats fed the concentrate‐based diet with 17% CP had significantly (P < 0.05) higher average daily gains (ADG) and better feed conversion rates (FCR). The pH _24h value tended to decrease (P < 0.05) with increasing DE levels. The tenderness of Longissimus dorsi muscle (LD) and Semimembranosus muscle (SM) reduced with increasing CP levels (P < 0.05). With increasing DE levels, tenderness was increased (P < 0.05). The heart fatty acid‐binding protein (H‐FABP) mRNA expression levels in LD and SM increased with increasing DE levels (P < 0.05), but decreased with increasing CP levels (P < 0.05). The calpastatin (CAST) and μ‐calpain mRNA expressions levels in LD and SM were affected significantly (P < 0.05) by CP and DE levels in the diet. Therefore, the nutritional levels of diets affect meat quality and expression levels of genes associated with meat quality in Hainan black goats.     

Effect of negative dietary cation‐anion differences on carcass characteristics and beef tenderness of Japanese Black steers

Lowering dietary cation‐anion differences (DCAD) can enhance responsiveness to Ca‐homeostatic hormones and increase Ca availability, which might have potential to activate a Ca‐dependent protease, calpain, and to enhance postmortem myofibrillar proteolysis. In this study, we investigated the effects of DCAD manipulation on calpain activity and beef tenderness in Japanese Black cattle which are characterized by their high marbling. Thirty‐six Japanese Black steers were allotted to one of two treatments: (i) control (CON; DCAD +6.09 mEq/100 g of dry matter (DM)) or (ii) negative DCAD (NEGD; DCAD ?8.27 mEq/100 g DM) for 70 days before slaughter. Lowering DCAD decreased DM and energy intake (P < 0.01) even though it did not negatively affect the growth performance or carcass characteristics. In NEGD, urine pH was decreased by acidification caused by the negative DCAD (P < 0.01). Calpain activities tended to be improved in NEGD (P = 0.09), but Warner‐Bratzler shear force values were not affected by treatment. Although calpain activities tended to improve, lowering DCAD to ?8.27 for 70 days before slaughter was insufficient to enhance beef tenderness in Japanese Black steers.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC-1 α) expression and the formation of slow-twitch muscle fibers in porcine and bovine skeletal muscles

The peroxisome proliferator‐activated receptor‐γ coactivator‐1 α (PGC‐1 α) induces mitochondria biogenesis in skeletal muscles. To determine the relationships between PGC‐1 α and the muscle fiber types, the expression levels of PGC‐1 α were analyzed in porcine and bovine skeletal muscles. As a first step, the nucleotide sequences of the porcine and bovine PGC‐1 α were determined. The porcine and bovine PGC‐1 α cDNA encoded 796 amino acid sequences and showed 95.1% identity between the two species. The expression levels of the PGC‐1 α mRNA were analyzed in the same 10 skeletal muscles from four pigs and three cattle. The contents of porcine and bovine PGC‐1 α were higher in the tongue, masseter and diaphragm, and lower in the Biceps femoris, semimembranosus, Longissimus thoracis and semitendinosus muscles. The contents of myosin heavy chain slow‐type protein (MyHC‐slow) were also determined in the same muscles by ELISA. The analysis of MyHC‐slow showed results similar to those for the PGC‐1 α contents in all of the muscles except for the tongue. The content of MyHC‐slow in the tongue was the lowest among the porcine muscles, and moderate among the bovine muscles. The results suggest that PGC‐1 α relates to the development of oxidative muscle fibers, but is not the principal factor in determining type I fiber content.     

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.     

Effect of L‐lysine on expression of selected genes,serum concentration of amino acids,muscle growth and performance of growing pigs

Lysine (Lys) is the first limiting amino acid (AA) in most feed formulations for pigs and most abundant, along with leucine, in muscle proteins. An experiment was conducted with 17 pigs (17.7 ± 0.05 kg initial BW) to identify a role of dietary Lys in the control of protein synthesis in pigs. Fourteen pigs were randomly assigned to one of the two wheat‐based dietary treatments: Lys‐deficient, 3.0 g/kg (DEF) and Lys‐adequate, 10.8 g/kg (ADE). Samples from jejunum mucosa, liver, Longissumus and Semitendinosus muscles, and blood were collected. The other three pigs were sacrificed at the beginning of the trial to measure basal carcass composition. Weight gain, gain:feed ratio, Lys intake and loin eye area were greater in ADE than in DEF pigs (p < 0.01). Muscle‐related carcass characteristics were better, and myosin heavy chain IIb expression (MyHC IIb) in Semitendinosus was higher in ADE than in DEF pigs. Expression of AA transporters CAT‐1 was lower (p < 0.05), serum Lys was higher and serum Val was lower in pigs fed the ADE diet. The higher muscularity, MyHC IIb expression in Semitendinosus muscle and Lys serum of pigs fed the ADE diet suggest that Lys increases growth rate not only by functioning as protein construction unit but also as potential control of the protein synthesis process.     

Involvement of μ/m‐calpain in the proteolysis and meat quality changes during postmortem storage of chicken breast muscle

The objective of this study was to investigate the role of calpain isotypes, especially poultry‐specific μ/m‐calpain in the proteolysis and meat quality changes of chicken breast muscle during postmortem storage. Calpain activity was detected by casein zymography, while the degradation of titin, desmin and Troponin‐T was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and western blot. Meat quality indicators such as water holding capacity and tenderness were also studied. The correlation analysis between calpain activity, proteolysis and the changes in meat quality indicators indicated that there were strong correlations for μ‐calpain during the first 12 h of storage, while such strong correlations for μ/m‐calpain were only found in samples stored from 12 h to 7 days. Our study suggested that μ‐calpain played a major role in meat quality changes while μ/m‐calpain could also be involved but played a limited role in the proteolysis and meat quality changes during 12 h to 7 days postmortem storage of chicken breast muscle.     

Maternal l-arginine supplementation during early gestation affects foetal skeletal myogenesis in pigs

To study the impact of maternal l-arginine supplementation during early gestation on skeletal muscle formation in the offspring, gilts were fed 3 kg of a standard diet (control group, n=10) or standard diet and additionally 26 g/d l-arginine from d 14 to 28 of gestation (arginine group, n=10). The gilts were sacrificed at d 75 of gestation. From each litter the lightest, the heaviest, and one medium-weight foetus per sex were selected and three different muscle samples were collected. In the longissimus dorsi muscle of all foetuses (n=99) biochemical properties were assessed. In the same muscle of a subset of the lightest and heaviest female foetuses (n=28), mRNA expression of selected genes involved in myogenesis were measured. In all three muscles, myosin heavy chain (MyHC) expression analyses (n=99) were performed. The protein/DNA ratio tended to be lower (P=0.08) in the offspring of the arginine group. Treatment×sex interactions revealed lower creatine kinase activity (P<0.05) and protein concentration (P=0.07) in female's of the arginine group. The MYF5 mRNA expression was lower (P<0.01) in the lightest, whereas IGF2 and IGFBP5 expressions (P<0.001) were lower in the heaviest females of the arginine group. The proportion of MyHC mRNA expression revealed an increase in the embryonic isoform (P=0.05) and a decrease by tendency in the Slow/I and IIx isoforms (P<0.10) in response to arginine supplementation. The results suggest that arginine supplementation during early gestation stimulates myogenic proliferation and delays muscular differentiation at d 75 of gestation.     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

Relationships among muscle fiber type composition,fiber diameter and MRF gene expression in different skeletal muscles of naturally grazing Wuzhumuqin sheep during postnatal development

The aim of this study was to determine the relationships among muscle fiber‐type composition, fiber diameter, and myogenic regulatory factor (MRF) gene expression in different skeletal muscles during development in naturally grazing Wuzhumuqin sheep. Three major muscles (i.e. the Longissimus dorsi (LD), Biceps femoris (BF) and Triceps brachii (TB)) were obtained from 20 Wuzhumuqin sheep and 20 castrated rams at each of the following ages: 1, 3, 6, 9, 12 and 18 months. Muscle fiber‐type composition and fiber diameter were measured using histochemistry and morphological analysis, and MRF gene expression levels were determined using real‐time PCR. In the LD muscle, changes in the proportion of each of different types of fiber (I, IIA and IIB) were relatively small. In the BF muscle, a higher proportion of type I and a 6.19‐fold lower proportion of type IIA fibers were observed (P < 0.05). In addition, the compositions of type I and IIA fibers continuously changed in the TB muscle (P < 0.05). Moreover, muscle diameter gradually increased throughout development (P < 0.05). Almost no significant difference was found in MRF gene expression patterns, which appeared to be relatively stable. These results suggest that changes in fiber‐type composition and increases in fiber size may be mutually interacting processes during muscle development.