柴田 昌宏

Taste is crucial to meat quality, and free Glu is an important taste-active component in meat. Our recent study showed that the short-term feeding of a low-Lys diet increases the concentration of free Glu and other free amino acids in chicken muscle and improves its taste. Here, we investigated the mechanisms by which the feeding of a low-Lys diet increases free Glu in chicken muscle. Two groups (n = 10 per group) of 28-day-old female Ross strain broiler chickens were fed diets with a graded Lys content of 90% or 100% of the recommended Lys requirement (according to National Research Council [1994] guidelines) for 10 D. Free amino acid concentrations and the mRNA abundance of protein metabolism-related genes were measured in breast muscle, and breast muscle metabolome analysis was conducted. Free Glu in muscle was increased by 51.8% in the Lys 90% group compared with the Lys 100% group (P < 0.01). Free threonine, glutamine, glycine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, and 3-methyl-histidine concentrations in breast muscle were also increased in the Lys 90% group (P < 0.05). Metabolome analysis also showed that free amino acids were increased in the Lys 90% group. The mRNA abundance of μ-calpain, caspase-3, and 20S proteasome C2 subunit were increased in the Lys 90% group (P < 0.05). Moreover, the free Glu concentration in muscle was correlated with mRNA abundance of μ-calpain (r = 0.74, P < 0.01), caspase 3 (r = 0.69, P < 0.01), 20S proteasome C2 subunit (r = 0.65, P < 0.01), and cathepsin B (r = 0.52, P < 0.05). Our study suggests that the feeding of a low-Lys diet to chickens increased the free Glu content of breast muscle by promoting protein degradation.

Taste is a crucial factor of meat quality, and amino acids are important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free glutamate (Glu), were investigated. Twenty-eight-day-old broilers (Gallus gallus) were fed diets with graded Lys content of 90% or 100% of the recommended Lys requirement, (according to the National Research Council,) for 10 days. Free amino acid content in meat and sensory scores of meat soup were estimated. Free Glu content, the main taste-active component of meat, was significantly increased by a reduction of dietary Lys. Compared with the Lys 100% group (control), free Glu concentrations of meat were increased by 35.7% in the Lys 90% group (P &lt 0.05). In addition, free glycine, valine, isoleucine, leucine, histidine and threonine concentrations of meat were significantly increased in the Lys 90% group (P &lt 0.05). Sensory evaluation of meat soup made from the Lys 100% and 90% groups indicated different meat tastes. Sensory scores of taste intensity, umami and kokumi tastes were significantly higher in the Lys 90% group. These results suggest that a reduction of dietary lysine increased free glutamate content in meat and improved its taste.

Taste is a crucial factor of meat quality, and amino acids are important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free glutamate (Glu), were investigated. Twenty-eight-day-old broilers (Gallus gallus) were fed diets with graded Lys content of 90% or 100% of the recommended Lys requirement, (according to the National Research Council, ) for 10 days. Free amino acid content in meat and sensory scores of meat soup were estimated. Free Glu content, the main taste-active component of meat, was significantly increased by a reduction of dietary Lys. Compared with the Lys 100% group (control), free Glu concentrations of meat were increased by 35.7% in the Lys 90% group (P &lt; 0.05). In addition, free glycine, valine, isoleucine, leucine, histidine and threonine concentrations of meat were significantly increased in the Lys 90% group (P &lt; 0.05). Sensory evaluation of meat soup made from the Lys 100% and 90% groups indicated different meat tastes. Sensory scores of taste intensity, umami and kokumi tastes were significantly higher in the Lys 90% group. These results suggest that a reduction of dietary lysine increased free glutamate content in meat and improved its taste.

We aimed to understand the roles of miRNAs in the muscle tissue maturation and those of circulating microRNAs (c-miRNAs) in beef production of Japanese Black (JB) cattle (Wagyu), a breed with genetically background of superior intermuscular fat depot, by comparing different feeding conditions (indoor grain-feeding vs. grazing on pasture). The cattle at 18 months old were assigned to pasture feeding or conventional indoor grain feeding conditions for 5 months. Microarray analysis of c-miRNAs from the plasma extracellular vesicles led to the detection of a total of 202 bovine miRNAs in the plasma, including 15 miRNAs that differed between the feeding conditions. Validation of the microarray results by qPCR showed that the circulating miR-10b level in the grazing cattle was upregulated compared to that of the grain-fed cattle. In contrast, the levels of miR-17-5p, miR-19b, miR-29b, miR-30b-5p, miR-98, miR-142-5p, miR-301a, miR-374b, miR-425-5p, and miR-652 were lower in the grazing cattle than in the grain-fed cattle. Bioinformatic analysis indicated that the predicted target genes of those c-miRNAs were enriched in gene ontology terms associated with blood vessel morphogenesis, plasma membrane, focal adhesion, endocytosis, collagen, ECM-receptor interaction, and phosphorylation. In the grazing cattle, the elevation of miR-10b expression in the plasma was coincident with its elevation in the longissimus lumborum (LL) muscle. Expression of bovine-specific miR-2478, the most plasma-enriched miRNA, tended to be also upregulated in the muscle but not in the plasma. Furthermore, grazing caused the downregulated mRNA expression of predicted miR-10b and/or miR-2478 target genes, such as DNAJB2, PTEN, and SCD1. Thus, the feeding system used for JB cattle affected the c-miRNAs that could be indicators of grain feeding. Among these, miR-10b expression was especially associated with feeding-induced changes and with the expression of the potential target genes responsible for glucose homeo-stasis and intramuscular fat depot in the LL muscle of JB cattle.

To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. (C) 2016 Elsevier Ltd. All rights reserved.

The present study investigated the influence of a diet largely comprising rice whole-crop silage (rWCS) on growth performance, carcass and meat characteristics, and expression of genes involved in muscle growth of Japanese Black steers. Steers were randomly separated into rWCS-fed (rWCS ad libitum and restricted feeding of concentrate) and concentrate-fed groups. Total digestible nutrient intake and daily gain (DG) decreased in rWCS-fed steers in comparison with concentrate-fed steers, whereas dressed carcass weight and final body weight did not significantly differ between the groups. Decreases in drip loss in the muscle of rWCS-fed steers may be caused by -tocopherol and -carotene in muscle. Feeding large amounts of rWCS to steers may maintain quantitative productivity of beef steers equally to a concentrate-based diet, and improve the qualitative productivity. Results of gene expression suggest that activation of skeletal muscle growth in rWCS-fed steers may occur at the late fattening period owing to a decrease in myostatin and increase in myosin heavy chain gene expression. Preadipocyte factor-1 and myostatin genes may be strongly involved in the control of lipid accumulation. This rearing system would allow beef production to switch to rWCS-based diets from concentrate-based diets.

Regulation of taste is important for improving meat quality and glutamate (Glu) is one of the important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free Glu, were investigated. Fourteen-day-old broiler chicks (Gallus gallus) were fed on diets containing 100% or 150% of the recommended Lys content for 10 days. Concentrations of free amino acids in plasma, muscle and liver were measured. The levels of messenger RNAs (mRNAs) for enzymes related to Glu metabolism were determined in muscle and liver. The concentration of muscle metabolites was also determined. The free Glu content in muscle of chicks fed the Lys150% diet was increased by 44.0% compared with that in chicks fed the Lys100% diet (P&lt 0.01). The mRNA level of lysine α-ketoglutarate reductase, which is involved in Lys degradation and Glu production, was significantly increased (P&lt 0.05) in the Lys150% group. Metabolome analysis showed that the Lys degradation products, muscular saccharopine, pipecolic acid and α-aminoadipic acid, were increased in the Lys150% group. Our results suggest that free Glu content in muscle is regulated by Lys degradation. These results suggest that a short-term feeding of high-Lys diet could improve the taste of meat.

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2)+light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.

Regulation of taste is important for improving meat quality and glutamate (Glu) is one of the important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free Glu, were investigated. Fourteen-day-old broiler chicks (Gallus gallus) were fed on diets containing 100% or 150% of the recommended Lys content for 10 days. Concentrations of free amino acids in plasma, muscle and liver were measured. The levels of messenger RNAs (mRNAs) for enzymes related to Glu metabolism were determined in muscle and liver. The concentration of muscle metabolites was also determined. The free Glu content in muscle of chicks fed the Lys150% diet was increased by 44.0% compared with that in chicks fed the Lys100% diet (P&lt;0.01). The mRNA level of lysine -ketoglutarate reductase, which is involved in Lys degradation and Glu production, was significantly increased (P&lt;0.05) in the Lys150% group. Metabolome analysis showed that the Lys degradation products, muscular saccharopine, pipecolic acid and -aminoadipic acid, were increased in the Lys150% group. Our results suggest that free Glu content in muscle is regulated by Lys degradation. These results suggest that a short-term feeding of high-Lys diet could improve the taste of meat.

Myokines are muscle-secreted factors to regulate cellular functions. However, it remains elusive what type of myokine is released during muscle differentiation. Here we evaluated the dynamics of myokines. More than 400 proteins were detected in conditioned medium and approximately 8% of them were categorized as myokines. The levels of myokines which promote myotube formation, vascularization or neurogenesis peaked during early differentiation, whereas myokines contributing to repellent activity against nerve cells or suppression of adipogenesis decreased after differentiation. Our findings suggest that muscle cells secrete different types of myokines at different developmental stages to communicate with various types of cells.

MicroRNA (miRNA) are highly conserved, noncoding small RNA involved in post-transcriptional gene regulation in a variety of biological processes. To elucidate roles of miRNA in bovine muscle type specification and maintenance, we sought to determine differentially expressed miRNA between semitendinosus (STD) and masseter (MS) muscles from 3 Japanese black cattle by massively parallel sequencing. Differential gene expression of myosin heavy chain (MyHC) isoforms confirmed that STD and MS were MyHC-2x-and MyHC-1-abundant muscles, respectively. In total, 192 known miRNA and 20 potential new bovine miRNA were obtained from the sequencing. The differentially expressed miRNA with more than 2-fold difference in each muscle were identified. In particular, miR-196a and miR-885 were exclusively expressed in STD muscle, which was validated by quantitative reverse transcription-PCR (P = 0.045 and P &lt 0.001, respectively), whereas a slow type-directing miR-208b was highly expressed in MS compared with STD (false discovery rate &lt 0.05). In addition, 16 potential novel miRNA were mapped and confirmed for their precursor structures by computational analyses. The results of functional annotation combined with in silico target analysis showed that the predicted target genes of miR-196a/b and miR-885 enriched gene ontology (GO) terms related to skeletal system development and regulation of transcription, respectively. Moreover, GO terms enriched from predicted targets miRNA suggested that STD-abundant and MS-abundant-miRNA were associated with embryonic body planning and organ/ tissue pattern formation, respectively. The present results revealed that the differentially expressed miRNA between the STD and MS muscles may play key roles to determine muscle type-specific tissue formation and maintenance in cattle thorough attenuating putative target genes involved in different developmental events. © 2013 American Society of Animal Science. All rights reserved.

日本の食料自給率は先進国の中でも最低レベルであり,その向上のためにも飼料自給率の向上が期待されている。飼料自給率向上のためには自給粗飼料の活用が有効であるが,日本の肉用牛の大部分は舎飼で輸入濃厚飼料多給による飼養形態が一般的である。こうした背景の中,舎飼で肉用牛へ牧乾草などの粗飼料を多給した時の肥育・枝肉成績および産肉性について検討された報告は少ない。本研究では,舎飼での牧乾草多給が黒毛和種去勢牛のと体成分および肉質におよぼす影響について明らかにした。試験区はと畜前1年間,大田研究拠点産の牧乾草を飽食かつ濃厚飼料(2㎏/day)を制限給与した粗飼料区ならびに肥育全期間,濃厚飼料飽食で牧乾草(1.5㎏/day)を給与した濃厚飼料区を設定し,黒毛和種去勢牛(各区6頭)を舎飼で供試した。28ヵ月齢でと畜後,枝肉成績の測定,半腱様筋および腰最長筋を採取し,栄養成分,ドリップロス,せん断力価およびクッキングロスを分析した。1. 試験終了時体重,可消化養分総量(TDN)の摂取量および枝肉重量が濃厚飼料区と比較して粗飼料区で有意に少なかった。また,ロース芯面積も濃厚飼料区に対して粗飼料区で有意に小さかった。一方で歩留基準値は試験区間で差は認められなかった。2. 蛋白質含量は濃厚飼料区に対し粗飼料区の半腱様筋で有意な増加が認められた。また,筋肉内脂肪含量は濃厚飼料区で増加傾向が認められたが,試験区間で有意差は認められなかった。筋肉内脂肪酸組成について,濃厚飼料区の腰最長筋で1価不飽和脂肪酸含量の有意な増加が認められたが,飽和脂肪酸及び多価不飽和脂肪酸含量はいずれの筋肉においても有意差は認められなかった。n-3系多価不飽和脂肪酸は濃厚飼料区と比較して粗飼料区で増加または検出された。3. 熟成後のドリップロスは濃厚飼料区に対し,粗飼料区の半腱様筋で有意な減少が認められ,さらに,熟成後のクッキングロスは粗飼料区の半腱様筋で減少傾向が認められた。せん断力価について,熟成前では粗飼料区の半腱様筋で有意に高くなったが,熟成後の両筋肉では試験区間で有意差は認められなかった。

Messenger RNA (mRNA) expression of calpain-1 (mu-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P &lt; 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)-2x-rich muscles compared to MyHC-slow-rich muscles. The CSTN-I and III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN-I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P &lt; 0.05). Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P &lt; 0.05). Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P &lt; 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.

This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBP alpha, PPAR gamma 2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBP alpha and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes, (C) 2011 Elsevier Ltd. All rights reserved.

The major taste active component, glutamate (Glu), improves the taste of meat. In this study, we investigated the effect of a short-term high-protein (HCP) diet on the intramuscular free Glu content to improve the taste of meat. Furthermore, we elucidated how the muscle free Glu content was controlled by the HCP diet. Chicks (14 days old) were fed the control diet or HCP diet for 10 days. Plasma and muscle free amino acid concentrations, and activity and messenger RNA (mRNA) expression of muscle enzymes related to Glu metabolism were determined. Muscle free Glu content was increased (P &lt; 0.01) by 51%. Activity and mRNA expression of glutaminase (GA), which is one of the major Glu-related enzymes, were significantly decreased (P &lt; 0.05) in the HCP group because of feedback inhibition. The mRNA expression of lysine alpha-ketoglutarate reductase (LKR), which is the enzyme involved in lysine (Lys) degradation and Glu production, was significantly increased (P &lt; 0.001) in the HCP group. These results suggest that short-term dietary HCP feeding is an effective treatment for improving the taste of meat. Furthermore, our results suggest that the free Glu content in muscle is regulated by GA and LKR.

Experiments were designed to compare the adipocyte cellularity of subcutaneous adipose tissue between growing Landrace (low backfat) and Meishan (high backfat) pigs at 1 week, 3 weeks, 6 weeks, 3 months and 5 months of age. As pigs aged, body weight and backfat thickness of both breeds significantly increased. When compared at equal ages, backfat thickness adjusted to equal body weight was greater for Meishan pigs. The mean diameter of fat cell size also increased with age, and by 6 weeks adipocytes from both outer and inner layers of subcutaneous adipose tissue were larger in Meishan pigs. At 5 months, approximately 80% of the adipose tissue mass in Meishan pigs was attributable to adipocytes measuring 95-165 mu m in diameter, whereas adipocytes of 75-145 mu m comprised most of the tissue mass in the Landrace. Although the contribution of smaller adipocytes (25-45 mu m) to the tissue volume was negligible, both breeds showed a biphasic diameter distribution at all ages, suggesting that adipocyte hyperplasia is still active. Our results demonstrate that cellularity differences exist between the subcutaneous adipose tissues of Landrace and Meishan pigs, and adipocyte hypertrophy is the most overwhelming contributor to the greater backfat deposition for Meishan pigs.

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 &lt; 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 &lt; 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.

To assess the role of muscle fiber type in beef taste-traits, we analyzed cooked meats from bovine masseter, diaphragm, psoas major, longissimus thoracis, and semitendinosus muscles with an electric taste sensing system (INSENT SA402B). The system is composed of five taste sensors of polymer membranes fixing different lipids. The sensors, CT0, CA0, AAE, C00 and AE1 are designed to respond to the individual tastes of salty, sour, umami, bitter and astringent, respectively. The system found significant differences in the converted outputs of CA0 (cvCA0), C00 (cvC00) and AE1 (cvAE1) among the bovine muscles. The slow-type muscles (masseter and diaphragm) showed lower cvCA0, higher cvC00, and higher cvAE1 than did the fast-type muscles (psoas major, longissimus thoracis, and semitendinosus). Lactic acid content was different among muscle types and was highly related to the cvCA0 output and pH. carbonyl compounds and free fatty acids were higher in the slow-type muscles. Free fatty acids were major components causing the difference in the C00 output among the muscle types. Iron content was also different among the muscle types and related to the cvC00 and cvAE1 outputs. These results suggested that the muscle fiber type affects the beef taste characteristics.

To simplify the monitoring of postmortem beef aging, we established a system to detect a troponin T (TnT) peptide fragment in bovine muscle drip (natural exudates) with an original monoclonal antibody. The antibody was raised against a synthetic peptide APPPPAEVPEVHEEVH corresponding to the N-terminal region of bovine fast-type TnT. In a competitive enzyme-linked immunosorbent assay (ELISA), our antibody detected the standard peptide dose-dependently. According to the monitoring examination with a competitive ELISA during 22 days postmortem, the concentration of the peptide in both the drip and trichloroacetic acid extracts from the longissimus muscle (n = 4) significantly increased in parallel, up to 10 nmol/ml and 16.4 nmol/g at day 14 postmortem, respectively. These events were accompanied by an increase in the conventional 30 kDa fragment in western blot analysis and a decrease in the Warner-Bratzler shear force value of the beef from 5.0 to 2.4 N/cm(2). The peptide detection system using drips with the antibody has advantages applicable to a non-destructive, simple, quick, and on-site monitoring method, such as immunochromatography. (C) 2009 Elsevier Ltd. All rights reserved.

The objective of this study was to investigate the differences in the muscle proteome of grass-fed and grain-fed cattle. Eight Japanese Black Cattle 10 mo of age were separated randomly into 2 groups: 1) grazing (grass-fed) and 2) concentrate (grain-fed) groups. All cattle were first housed individually in a stall barn and fed a combination of concentrate ad libitum and Italian ryegrass hay until 21 mo of age. After this control period, the 4 grass-fed cattle were placed on outdoor pasture, whereas the other 4 grain-fed cattle continued on the concentrate diet. The cattle were slaughtered at 27 mo of age, and tissues from the semitendinosus muscle were obtained for use in proteome analysis. Differential expression of muscle proteins in the 2 groups was carried out using 2-dimensional gel electrophoresis (2DE) and Western blot analyses, with subsequent mass spectrometry. Approximately 200 individual protein spots were detected and compared in each group using 2DE, of which 20 and 9 spots, respectively, showed differences in the spot intensity for the sarcoplasmic fraction and myofibrillar fraction. In the grazing group, the relative intensity of spots was significantly greater for adenylate kinase 1 and myoglobin in the sarcoplasmic fraction, and for slow-twitch myosin light chain 2 in the myofibrillar fraction (P &lt; 0.05), than the concentrate group. The relative spot intensity of several glycolytic enzymes was significantly greater in the grazing group, such as beta-enolase 3, fructose-1,6-bis-phosphate aldolase A, triosephosphate isomerase, and heat shock 27 kDa protein (P &lt; 0.05). Moreover, significantly greater slow twitch of troponin T, troponin I, and myosin heavy chain of semitendinosus muscle was detected in the grazing group than in the concentrate group using Western blot analysis (P &lt; 0.05). Several previous reports have described that the slow-twitch muscle contents affect elements of nutrition, flavor, and food texture of meat. This study revealed muscle fiber type conversion to slow-twitch tissues from fast-twitch tissues occurring with change in the energy metabolic enzyme when cattle were grazed in the latter fattening period. Although analyses of the influence on elements of nutrition, flavor, and food texture were not done for this study, these results show that slow-twitch converted muscle resulting from the grazing of cattle might modify several meat characteristics.

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 (n = 6) or saline (n = 6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period (P &lt; 0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle. (C) 2009 Elsevier Inc. All rights reserved.

The composition of tropomyosin (TPM) and myosin heavy chain (MyHC) isoforms was analyzed in 10 physiologically different bovine muscles (masseter, diaphragm, tongue, semispinalis, pectoralis profundus, biceps femoris, psoas major, semimembranosus, longissimus thoracis and semitendinosus) to clarify the relationships between TPM and MyHC isoforms in different muscle fiber types. The content of TPM1 and TPM3 was different in muscles according to their function in muscle contraction, although the content of TPM2 was constantly about 50% of the total TPM in all muscles. The content of TPM1 was higher in semimembranosus, longissimus thoracis and semitendinosus, while that of TPM3 was higher in masseter and diaphragm. The high positive correlation between MyHC-slow content and TPM3 content (r = 0.92) suggested a coexpression of TPM3 and MyHC-slow isoforms in a muscle fiber. MyHC-slow and TPM3 were expressed at the same level in masseter and diaphragm, whereas there was more TPM3 than MyHC-slow in tongue and semispinalis, so it appears that the excess TPM3 in tongue and semispinalis is expressed with other MyHC isoforms. MyHC-2a was the only fast type isoform expressed in tongue and semispinalis. Therefore, the excess TPM3 was composed of myofibrils with MyHC-2a. The results suggested that a fiber expressing MyHC-2a would be regulated delicately by changing the TPM isoform types.

The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non-synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non-synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher&apos;s exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.

To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis.

Myostatin is a specific negative regulator of skeletal muscle growth and is regarded as one of the most important factors for myogenesis. The aim of the current study was to analyze the developmental change in the gene expression of myostatin and an adipogenic transcription factor (peroxisome proliferator-activated receptor gamma 2) in the semitendinosus muscle of Japanese Black Cattle throughout the whole life cycle. An additional aim was to compare the temporal expression patterns of myostatin and relevant myogenic regulatory factors (MRF) mRNA during muscle regeneration after frostbite injury at 16 mo of age. The developmental pattern of myostatin gene expression exhibited 2 peaks: the greatest expression occurred in utero (P &lt; 0.05) and the second greatest occurred at 16 mo of age (P &lt; 0.05). The greatest level of peroxisome proliferator-activated receptor gamma 2 expression was observed at 16 mo of age (P &lt; 0.05), which paralleled myostatin expression. During frostbite-induced muscle regeneration, gene expression for myostatin and 4 MRF; i.e., Myf5, MyoD, myogenin and MRF4, showed contrasting responses. Myostatin mRNA dramatically declined by 68.1 and 82.6% at 3 and 5 d after injury (P &lt; 0.05), respectively, which paralleled its protein expression, and was restored at 10 d. In contrast, the expressions of all 4 MRF mRNA were low initially but increased by 5 d after injury (P &lt; 0.05) and then remained constant or decreased slightly. These results suggest that myostatin may play a role in muscle marbling in the fattening period by decreasing myogenesis and increasing adipogenesis, and that the interaction between myostatin and MRF genes may take place at an early stage of skeletal muscle regeneration.

脂肪細胞分化制御因子PPARγ2の遺伝的変異(Ala18Val)は黒毛和種の産肉形質に影響することが示唆されていることから，但馬系黒毛和種，高知系褐毛和種，見島牛の主な種雄牛およびその産子を用いて，変異体を含む家系の調査を行った。遺伝的変異体の検出には既に報告済のミスマッチプライマーを用いるPCR-RFLP法を用いた。その結果，但馬系種雄牛において1頭のヘテロ型変異体(Ala/Val型)に由来する家系の存在が明らかとなり，家系内には52頭の種雄牛が存在し，それらは21道府県で繋養されていた。さらに，この家系に属する4頭の種雄牛がAla/Val型であることが確認され，それら4頭の種雄牛からは約13,000頭の産子が生産されていた。また，残る48頭の種雄牛については，約半数がAla/Val型であると推定された。一方，高知系褐毛和種と見島牛については，Ala/Val型の存在する可能性は低いと思われた。

Myostatin, a member of the transforming growth factor-β superfamily, is a well known negative regulator of skeletal muscle growth. In the present study, the 6660 bp nucleotide sequence of the myostatin gene in Japanese Black cattle (JBC), including the entire coding region of 1128 bp, was determined. The amino acid sequence deduced from the nucleotide sequence of JBC was well conserved with its sequence of other cattle, although it was found that an A→G transition at nucleotide position 641 results in the substitution of asparagine by serine at amino acid position 214. In order to examine the expression pattern of the myostatin gene in the skeletal muscles of JBC, its expression in three skeletal muscles, Semitendinosus (ST) muscle, Biceps femoris muscle and Longissimus lumborum muscle, of fetal and calf stages was analyzed by real time polymerase chain reaction. The highest level of the myostatin expression was observed in the fetal stage. In calf stages the highest expression was observed in ST muscle compared with the other two muscles. These results suggest that a higher expression of myostatin gene, especially in the fetal stage and in ST muscle during calf stages, is involved in the arrest in skeletal muscle growth and that its functional domains and genomic structure in JBC are well conserved with those in other mammals.

Effect of slaughter age (24, 28 and 38 months of age) on beef color stability during display of m. serratus ventralis, m. psoas major, m. semitendinosus and m. longissimus thoracis from Japanese Black steers was studied. Steak samples from muscles were over-wrapped with PVC film and displayed under fluorescent lights at 4degreesC for 12 days. Percentages of metmyoglobin of steak samples were determined at days 0, 3, 6, 9 and 12. The percentage of metmyoglobin of m. psoas major at day 3 of display in the 24 months group was lower (p&lt;0.05) than that in the 38 months group. The percentage of metmyoglobin of m. semitendinosus at day 6 of display in the 38 months group was higher (p&lt;0.05) than that in the other groups. The percentage of metmyoglobin of m. longissimus thoracis at day 3 of display in the 24 months group was lower (p&lt;0.01) than that in the other groups. The percentage of metmyoglobin of m. longissimus thoracis at day 6 (p&lt;0.01), 9 (p&lt;0.01) and 12 (p&lt;0.05) of display in the 38 months group were higher than those in the other groups. Crude fat concentration in m. longissimus thoracis increased (p&lt;0.05) after 28 months of age. alpha-Tocopherol concentration in m. serratus ventralis in the 38 months group was higher (p&lt;0.001) than that in the other groups. In m. psoas major the alpha-tocopherol concentration in the 38 months group was higher (p&lt;0.05) than that in the 24 months group. The alpha-tocopherol concentration in m. longissimus thoracis increased (p&lt;0.001) with age. These results suggested that in spite of increase in both the crude fat and the alpha-tocopherol concentrations in m. longissimus thoracis, the beef color stability during display became short with age.