Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Chemical and Life Science Engineering. Bibliography: leaves 145-164. Also available online via the Internet.

1) The carpopodite extensor muscle of Carcinus maenas and Homarus americanus demonstrated regional localization of at least three types of fibre: fast phasic, slow tonic and intermediate. A specific area of the extensor, area 1, was identifiable in extensors from different animals and comprised fast phasic fibres in Carcinus and intermediate fibres in Homarus. When the M-C joint of the leg was held at a 90° angle, the sarcomeres in area 1 fibres were at rest length. Measurements of sarcomere length taken in the middle peripheral region were indicative of sarcomere lengths in other regions of the intermoult fibre. 2) The mechanism of growth of the fibres was determined in intermoult animals of increasing size. Fibres increased in length by the addition of sarcomeres and increased in width by an increase in the number of myofybrils. Myofibrils increased in size by the addition of thick and thin filaments in the characteristic lattice arrangement. The number of myofibrils may increase by myofibrillar splitting as has been previously described for vertebrate muscle. Fibre number remained constant with growth of the animal. There was a decrease in mitochondrial density with increase in fibre area. 3) Over ecdysis, fibres lengthened by addition of short sarcomeres to the exoskeletal region of the fibre. During the 3-4 days postecdysial expansion of the exoskeleton, these short sarcomeres increased in length. hypothesis that these short sarcomeres are precursor sarcomeres being added during ecdysis was investigated. When intermoult muscle was held in a stretched position for 2 weeks, there was a regional increase in the length of the muscle similar to the increases found over ecdysis. In contrast, fibre area did not increase over ecdysis. Instead, fibre area increased during the late postmoult and intermoult. The frequency distribution of myofibril size altered over ecdsysis; large myofibrils were more commonly present during premoult than during the immediate postmoult. Lateral stretch on the muscle during ecdysial expansion of the exoskeleton may be the stimulus for myofibrillar splitting. Possible satellite cells were identified in the skeletal muscle fibres-. vitro and jLn vivo radiolabelling techniques were adapted for the carpopodite extensor muscle of Carcinus maenas. The levels of free phenylalanine, total bound protein and phenylalanine bound in the muscle protein increased with size of the muscle. The mean in vitro rate of synthesis or FSR in the intermoult animal was 0.3% per day and the in vivo rate of synthesis at 1.24% per day. The FSR remained constant with size of the extensor muscle in intermoult animals. Fixatives may have an effect on the specific activity in the bound fraction of the muscle which is attributable to binding artifacts of the free amino acids. 5) The levels of phenylalanine in the free pool fluctuated during the moult cycle. The protein/wet weight ratio of the muscle also varied with the moult cycle. Increases in protein during the early postmoult supported other evidence for growth of the muscle over ecdysis. Synthesis rates were raised during the premoult and immediate postmoult period and remained slightly elevated during the late postmoult. These increases in rate corresponded to periods of muscle growth. 6) Autoradiographic techniques combined with measurements of bound specific activity revealed an increase in activity in the exoskeletal or cuticular region of the fibre corresponding to the region of new muscle synthesis over ecdysis. No differences in grain density were found between fast phasic and slow tonic fibres or between the central and peripheral region of the myofibril in intermoult muscle. Regional localization of grains was found at the light microscope level in the Z, I and B bands but no further evidence of this differentiatial labelling was found at the electron microscope level.

Orientador: Maeli Dal Pai Silva / Coorientador: Maria Célia Portella / Banca: Fernanda Antunes Alves Costa / Banca: Leonardo Susumu Takahashi / Resumo: O crescimento muscular em peixes é controlado por vias moleculares que podem ser afetadas pela restrição alimentar e realimentação. Neste estudo, foi analisado se curtos períodos de jejum seguidos de realimentação podem influenciar o desempenho, a morfologia e a expressão de genes relacionados com o crescimento muscular, em juvenis de tilápia do Nilo (Oreochromis niloticus), linhagem chitralada. Juvenis com peso médio de 0,6 ± 0,19 g foram estocados em caixas de polietileno com sistema de fluxo contínuo de água e aeração constante. Os peixes foram divididos aleatoriamente em cinco grupos experimentais com três repetições: (AL), controle com alimentação contínua do início ao final do experimento; (J5), jejum por cinco dias e realimentação por 37 dias; (J10), jejum por dez dias e realimentação por 32 dias e (J20), jejum por 20 dias e realimentação por 22 dias; (JJ), jejum durante todo o período experimental. As análises foram realizadas no início do experimento (dia 0), aos 5, 10, 20, e 42 dias. Nestes períodos, foram realizadas biometrias, para a avaliação do desempenho e coleta das amostras musculares para a análise morfológica. A análise morfométrica (cálculo do diâmetro das fibras) e a expressão do mRNA da MyoD, miogenina e miostatina (por RT-qPCR) foi realizada nos grupos AL, J5 e J10, nos períodos 0, 5, 10, 20 e 42 dias. Os juvenis de tilápia submetidos a cinco, dez e 20 dias de jejum mostraram um ganho de peso compensatório, entretanto somente o grupo J5 apresentou crescimento compensatório total. Os grupos AL, J5 e J10 apresentaram maior comprimento dorso lateral do que nos grupos J20 e JJ, indicando um espessamento da massa muscular dorsal. A taxa de crescimento específico foi maior após 10 dias de realimentação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Muscle growth mechanisms are controlled by molecular pathways which can be affected by food restriction and refeeding. In this study, we analyze if short periods of starvation and refeeding can influence the expression of muscle growth-related genes and muscle growth characteristics, in juvenile Nile tilapia (Oreochromis niloticus), strain chitralada. We used juvenile fish with average body weight of 0.6 ± 0.19 g randomly divided into three groups with three replicates: (AL) feeding continuously from beginning to end of the experiment; (J5) five days fasting and refeeding for 37 days and (J10) ten days fasting and refeeding for 32 days. The experiment lasted 42 days. After 5 and 10 days of food restriction, fish from J5 and J10 were fed to apparent satiation with a commercial diet. At the beginning of the experiment (day 0) and at 5, 10, 20 and 42 days, fish from all treatment (n= 14) were anesthetized and sacrificed. Muscle samples (n= 7 at each group) were collected, fixed in Karnovsky solution and transverse sections were stained by hematoxylin-eosin to evaluate muscle morphology and muscle fiber hypertrophy and hyperplasia. The muscle fibers were grouped into classes, 20 μm, >20 - 30 μm, >30 - 40 μm, >40 - 50 μm and >50 μm. MyoD, myogenin and myostatin mRNA expression was performed by RT-qPCR. At the end of the experiment, fasting for five and ten days resulted in small reduction on body weight compared with AL group. Following refeeding, fasted fish gained weight continuously; at 42 day, J5 showed an increase in body weight, which was similar to that observed in AL group. In J10, body weight increased after refeeding, but it was lower than that observed in AL group. HE stain showed white skeletal muscle making up most of the muscle mass in all groups studied; morphological characteristic were... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T20:29:31Z : No. of bitstreams: 1 nebo_c_me_botib.pdf: 12693861 bytes, checksum: 9b8f76c434e71d8e5d62937d1ee48886 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O crescimento muscular em peixes é controlado por vias moleculares que podem ser afetadas pela restrição alimentar e realimentação. Neste estudo, foi analisado se curtos períodos de jejum seguidos de realimentação podem influenciar o desempenho, a morfologia e a expressão de genes relacionados com o crescimento muscular, em juvenis de tilápia do Nilo (Oreochromis niloticus), linhagem chitralada. Juvenis com peso médio de 0,6 ± 0,19 g foram estocados em caixas de polietileno com sistema de fluxo contínuo de água e aeração constante. Os peixes foram divididos aleatoriamente em cinco grupos experimentais com três repetições: (AL), controle com alimentação contínua do início ao final do experimento; (J5), jejum por cinco dias e realimentação por 37 dias; (J10), jejum por dez dias e realimentação por 32 dias e (J20), jejum por 20 dias e realimentação por 22 dias; (JJ), jejum durante todo o período experimental. As análises foram realizadas no início do experimento (dia 0), aos 5, 10, 20, e 42 dias. Nestes períodos, foram realizadas biometrias, para a avaliação do desempenho e coleta das amostras musculares para a análise morfológica. A análise morfométrica (cálculo do diâmetro das fibras) e a expressão do mRNA da MyoD, miogenina e miostatina (por RT-qPCR) foi realizada nos grupos AL, J5 e J10, nos períodos 0, 5, 10, 20 e 42 dias. Os juvenis de tilápia submetidos a cinco, dez e 20 dias de jejum mostraram um ganho de peso compensatório, entretanto somente o grupo J5 apresentou crescimento compensatório total. Os grupos AL, J5 e J10 apresentaram maior comprimento dorso lateral do que nos grupos J20 e JJ, indicando um espessamento da massa muscular dorsal. A taxa de crescimento específico foi maior após 10 dias de realimentação... / Muscle growth mechanisms are controlled by molecular pathways which can be affected by food restriction and refeeding. In this study, we analyze if short periods of starvation and refeeding can influence the expression of muscle growth-related genes and muscle growth characteristics, in juvenile Nile tilapia (Oreochromis niloticus), strain chitralada. We used juvenile fish with average body weight of 0.6 ± 0.19 g randomly divided into three groups with three replicates: (AL) feeding continuously from beginning to end of the experiment; (J5) five days fasting and refeeding for 37 days and (J10) ten days fasting and refeeding for 32 days. The experiment lasted 42 days. After 5 and 10 days of food restriction, fish from J5 and J10 were fed to apparent satiation with a commercial diet. At the beginning of the experiment (day 0) and at 5, 10, 20 and 42 days, fish from all treatment (n= 14) were anesthetized and sacrificed. Muscle samples (n= 7 at each group) were collected, fixed in Karnovsky solution and transverse sections were stained by hematoxylin-eosin to evaluate muscle morphology and muscle fiber hypertrophy and hyperplasia. The muscle fibers were grouped into classes, 20 μm, >20 - 30 μm, >30 - 40 μm, >40 - 50 μm and >50 μm. MyoD, myogenin and myostatin mRNA expression was performed by RT-qPCR. At the end of the experiment, fasting for five and ten days resulted in small reduction on body weight compared with AL group. Following refeeding, fasted fish gained weight continuously; at 42 day, J5 showed an increase in body weight, which was similar to that observed in AL group. In J10, body weight increased after refeeding, but it was lower than that observed in AL group. HE stain showed white skeletal muscle making up most of the muscle mass in all groups studied; morphological characteristic were... (Complete abstract click electronic access below)

Increasing interest has emerged in the field of nutrition and its role in promoting skeletal muscle growth. Recently, studies using both in vitro and in vivo models have suggested that cannabidiol – a constituent of Cannabis Sativa – can increase the growth and regenerative capacity of skeletal muscle stem cells. Other isolated compounds, such as cannabinol, have demonstrated anti-inflammatory effects in vivo. Due to the potential benefits of both compounds, our primary objective was to further elucidate the effects of cannabidiol and cannabinol on murine C2C12 myoblast proliferation and differentiation. We hypothesized that supplementation of cannabidiol and cannabinol would augment gene expression of myogenin, leading to enhanced myotube formation; as well as, induce greater gene expression of Myf5 and MyoD, accompanied by increased cell proliferation. In relation to skeletal muscle growth, myostatin and follistatin can substantially impact the regulation of hypertrophy; with down-regulation of myostatin being a potent stimulus for muscle growth, and follistatin being the antagonist to myostatin, we therefore examined if cannabidiol or cannabinol influenced these two proteins, as a possible rationale for increased myogenesis. In this study, cells were treated with either: (1) cannabidiol, (2) cannabinol, (3) or vehicle control (methanol). Cells were grown for 48 hrs in their respective media, the MTT assay was used to assess proliferation. Muscle differentiation experiments required cells to grow for seven days with media supplemented with the respective compound. The media was changed every 48 hrs. The extent of muscle differentiation was assessed via immunocytochemical and qPCR analysis. In preliminary experiments, cell proliferation was influenced by the duration of which cells were exposed to the compound and concentration of the compound within the media. It was noted that changing growth media and compound every 24 hrs augmented the proliferative response compared to leaving it on for 48 hrs for both cannabidiol and cannabinol (p<0.05). Furthermore, supplementing cells with cannabidiol at a 1 or 5 uM concentration resulted in considerable cell growth compared to vehicle control (p<0.0001). Cannabinol at 5 uM showed the same effect (p<0.0001). We also quantified the mRNA expression of genes involved in the myogenic regulatory pathway in proliferating and differentiating cells. Herein we report that using a 5 uM concentration of cannabidiol or cannabinol did not increase the expression of any of these genes in proliferating or differentiating cells. These findings help further characterize the effects of cannabidiol and cannabinol on the myogenic response. / Thesis / Master of Science (MSc) / Nutrition impacts the regulation of skeletal muscle mass, with many individuals turning to supplements as a means to improve overall health. Cannabidiol – a constituent of the cannabis plant – has been used over the past several decades for its anti-inflammatory, neuroprotective, and anxiolytic properties; however, recent evidence has revealed its potential effectiveness in promoting muscle growth. If true, there is a possibility that it can be used to target the age-related loss of muscle mass, sarcopenia, or even improve athletic performance. Other derivatives, such as cannabinol, have seldom been studied but also demonstrate anti-inflammatory effects. Therefore, this thesis further elucidates the effects of cannabidiol and cannabinol on the myogenic signaling pathway. As a model, we used the murine C2C12 cell line that recapitulates the behaviour of human myoblasts. Interestingly, the data presented herein supports the notion that cannabidiol and cannabinol only promote cell growth and have no effect on myoblast maturation and myotube formation. These findings provide a better understanding of the potential for cannabidiol and cannabinol as a nutritional supplement targeting skeletal muscle.

The SH3 and cysteine rich domain 3 (Stac3) gene is expressed specifically in skeletal muscle and essential for skeletal muscle contraction and postnatal life in mice. In this dissertation project, I conducted two studies to further understand the role of STAC3 in skeletal muscle development, growth, and contraction. In the first study, I compared the contractile responses of hindlimb muscles of Stac3 knockout and control mice to electrical stimulation, high [K+]-induced membrane depolarization, and caffeine and 4-chloro-m-cresol (4-CMC) activation of ryanodine receptor (RyR). Frequent electrostimulation-, high [K+]-, 4-CMC- and caffeine-induced maximal tensions in Stac3-deleted muscles were approximately 20%, 29%, 58% and 55% of those in control muscles, respectively. 4-CMC- and caffeine-induced increases in intracellular calcium were not different between Stac3-deleted and control myotubes. Myosin-ATPase and NADH-tetrazolium reductase staining as well as gene expression analyses revealed that Stac3-deleted hindlimb muscles contained more slow type-like fibers than control muscles. These data together confirm a role of STAC3 in EC coupling but also suggest that defective EC coupling is only partially responsible for the significantly reduced contractility in Stac3-deleted hindlimb muscles. In the second study, I determined the potential role of STAC3 in postnatal skeletal muscle growth, fiber composition, and contraction by disrupting Stac3 gene expression in postnatal mice through the Flp-FRT and tamoxifen-inducible Cre-loxP systems. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei without affecting the total myofiber number. Postnatal Stac3 deletion decreased limb muscle strength. Postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced maximal force output in limb muscles. Similarly, postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. These results demonstrate that STAC3 is important to myofiber hypertrophy, myofiber type composition, contraction, and EC coupling in postnatal skeletal muscle. / Ph. D.

The renewal rate of stromal and myofibrillar proteins defines muscle growth, and can affect the quality of meat, by affecting collagen turnover and proteolytic rate. There is a lack of information on changes in the muscle protein remodeling process in response to the recovery weight gain rate observed during \"realimentation\" after undernutrition, which may be altered in older animals. Changes in muscle tissue during the recovery period may be indicated by the differential expression profile of genes after RNA sequencing. The objectives of this study were to evaluate transcriptome changes in the muscle of Nellore cull cows subjected to: 1) recovery weight gain under grazing conditions; and 2) recovery from undernutrition at different weight gain rates. In the first experiment, the animals were divided into two groups and subjected to one of two nutritional managements under grazing conditions: maintenance (maintenance of weight and high body condition score under grazing conditions) and recovery gain (recovery from low body condition score with moderate body weight gain of 0.6 kg/day under grazing conditions). In the second experiment, the animals were divided into three groups and subjected to one of three nutritional managements under feedlot conditions: control (slaughtered at low body condition score), moderate recovery gain (MG; 0.6 kg of daily live weight gain) during the dry season, and high recovery gain (HG; 1.2 kg of daily live weight gain) during the dry season. In both experiments, samples of longissimus dorsi muscle were collected after slaughter and immediately frozen until sequencing analysis could be performed. In the first experiment, genes related to inflammatory response, such as semaphorin 4A (SEMA4A), solute carrier family 11 member 1 (SLC11A1), ficolin-2 (FCN2), and placental growth factor (PGF), were expressed at higher levels during recovery gain. In the second experiment, osteonectin (SPARC) and collagen type IV subunits 1 (COL4A1) were expressed at higher levels in both recovery gain and connective tissue remodeling. For MG, structural myofibrillar proteins such as myosin IE (MYO1E), myosin, heavy chain 11 (MYH11), myogenin (MYOG), and actinin, alpha 4 (ACTN4) were identified. In the HG treatment, the B-cell CLL/lymphoma 9 (BCL9), peroxisome proliferator-activated receptor alpha (PPARA), diacylglycerol O-Acyltransferase 2 (DGAT2), and phosphatidylinositol 4-Kinase, catalytic, and beta (PI4KB) genes indicated more deposition of adipose tissue. In summary, we observed that muscular deposition during recovery weight gain involved the regulation of expression of several genes related to the extracellular matrix (ECM), corroborating the inflammatory and -like models observed in mature animals. Moreover, in the HG group, genes related to collagen synthesis and fat deposition were also found, indicating the important contribution of connective tissue during muscle growth. These results are important for understanding tissue development as a whole, and will assist in the progress of scientific knowledge on muscle remodeling during recovery weight gain and its influence on protein structures and intracellular routes. / A taxa de renovação do estroma e proteínas miofibrilares define o crescimento muscular, e pode afetar a qualidade da carne, afetando turnover de colágeno e taxa proteolítica. Há uma falta de informação sobre as mudanças no processo de remodelação de proteína muscular em resposta à taxa de recuperação do ganho de peso, observada durante a \"realimentação\" depois de subnutrição, o que pode ser alterado em animais mais velhos. Alterações no tecido muscular durante o período de recuperação pode ser indicada pelo perfil de expressão diferencial de genes, pela técnica de sequenciamento de RNA. Os objetivos deste trabalho foram avaliar as alterações do transcriptoma na musculatura de vacas de descarte Nelore submetidas a: 1) a recuperação do peso em condições de pastejo; e 2) a recuperação da desnutrição em diferentes taxas de ganho de pesos. No primeiro experimento, os animais foram divididos em: grupo de manutenção (manutenção de peso e de alta escore corporal); Recuperação do ganho (recuperação de baixo escore corporal com o ganho de peso corporal moderado em 0,6 kg / dia sob pastejo). No segundo experimento, os animais foram divididos em três grupos em confinamento: Controle (abatidos com baixo escore de condição corporal), ganho moderado (0,6 kg de ganho de peso vivo por dia) durante a estação seca e alta recuperação de ganho (1,2 kg de ganho de peso vivo por dia) durante a estação seca. Nos dois estudos, amostras do Longissimus dorsi foram coletadas após o abate e imediatamente congeladas até à análise de sequenciamento ser realizada. No primeiro experimento, os genes encontrados no tratamento de recuperação do ganho foram relacionados com a resposta inflamatória como: 4A semaphorin (SEMA4A), solute carrier family 11 member 1 (SLC11A1), Ficolin 2 (FCN2) e placental growth factor (PGF). No segundo experimento, o osteonectina (SPARC), colágeno tipo IV subunidades 1 (COL4A1) foram alguns dos genes mais expressos para ambas as taxas de ganho de recuperação de remodelação do tecido conjuntivo. Para ganho moderado, foram identificadas proteínas miofibrilares estruturais como: Myosin IE (MYO1E), Myosin, Heavy Chain 11 (MYH11), (MYOG) and actinin, alpha 4 (ACTN4). No tratamento de alta recuperação de ganho, genes como CLL de célula B / 9 linfoma (BCL9), peroxisome proliferator-activated receptor alpha (PPARA), Diacylglycerol O-Acyltransferase 2 (DGAT2) and Phosphatidylinositol 4-Kinase, Catalytic, Beta (PI4KB) indicam maior deposição de tecido adiposo. Em resumo, observou-se que a deposição muscular durante a recuperação do ganho de peso envolve a regulação da expressão de vários genes relacionados com a matriz extracelular (ECM), corroborando com modelos de inflamação e similar a fibrose observada em animais maduros. Além disso, no grupo HG, genes relacionados com a síntese de colágeno e a deposição de gordura, também foram encontrados, indicando contribuição do tecido conjuntivo durante o crescimento muscular. Estes resultados são importantes para a compreensão do desenvolvimento do tecido como um todo, e auxiliam no progresso do conhecimento científico sobre a remodelação muscular durante a recuperação do ganho de peso e sua influência sobre estruturas de proteínas e vias intracelulares.

O agonista beta-adrenérgico ractopamina (RAC) modifica a composição da carcaça suína por aumentar a massa muscular e reduzir a deposição de gordura. O objetivo neste estudo foi avaliar os efeitos do tempo de fornecimento de RAC sobre o desempenho, concentração de ureia plasmática (CUP), características de carcaça e expressão gênica dos receptores beta- adrenérgicos (beta-AR) e das isoformas da cadeia pesada de miosina (MyHC) em suínos em terminação. Oitenta suínos, machos castrados (PV inicial = 69,42 ± 1,24 kg), foram utilizados em um experimento em blocos completos casualizados com cinco tratamentos, oito repetições por tratamento e dois animais por unidade experimental (baia). Os tratamentos consistiram de rações sem RAC (controle) ou com 10 ppm de RAC fornecidas por 7, 14, 21 ou 28 dias préabate. O PV individual e o consumo de ração por baia foram obtidos para determinar o ganho diário de peso (GDP), o consumo diário de ração (CDR) e a conversão alimentar (CA). Amostras de sangue foram coletadas para determinação da CUP. No final do experimento, os animais (PV final = 102,46 ± 1,44 kg) foram abatidos e amostras de pelos e do músculo Longissimus dorsi coletadas. As carcaças foram avaliadas 24 horas post-mortem. As amostras de pelos foram utilizadas para detecção da mutação no gene do receptor de rianodina do tipo 1 (RYR1). A expressão gênica dos beta-AR (subtipos beta1 e beta2) e das isoformas MyHC (I, IIa, IIx/d e IIb) foi quantificada nas amostras de músculo. As análises estatísticas foram realizadas apenas com os animais homozigotos dominantes para a mutação no gene do RYR1. O aumento no período de fornecimento de RAC não afetou (P > 0,05) o PV final, o GDP e o CDR, porém resultou em melhora linear (P < 0,01) na CA. Melhoras (P < 0,05) nas médias semanais de GDP e CA foram observadas durante os primeiros 21 dias de fornecimento de RAC, no entanto, o crescimento animal declinou (P < 0,05) na 4ª semana de tratamento. A CUP apresentou efeito quadrático (P < 0,01) com o aumento na duração do fornecimento de RAC. Houve aumento linear (P <= 0,01) no peso da carcaça quente, na profundidade do músculo Longissimus dorsi, na área de olho de lombo e na relação carne:gordura com o aumento na duração do tratamento com RAC. Não foram detectados efeitos da RAC (P > 0,05) sobre a expressão gênica dos beta1-AR e das isoformas MyHC IIa e MyHC IIx/d, porém o aumento no período de fornecimento de RAC tendeu a reduzir linearmente (P = 0,08) a expressão gênica dos beta2-AR. Embora os níveis de RNAm da isoforma MyHC I tenham sido reduzidos linearmente (P < 0,01), a expressão gênica da isoforma MyHC IIb aumentou linearmente (P < 0,01) com o aumento na duração do tratamento com RAC. Portanto, as melhores respostas de desempenho e carcaça ocorreram quando a RAC foi fornecida por 21 e 28 dias, respectivamente. Além disso, o agonista alterou a expressão gênica das isoformas MyHC, e é possível que a ação da RAC esteja relacionada com a população de beta2-AR. / The beta-adrenergic agonist ractopamine (RAC) modifies the swine carcass composition by increasing muscle mass and decreasing fat deposition. The objective in this study was to evaluate the effects of RAC feeding duration on performance, plasma urea nitrogen (PUN) concentration, carcass traits, and gene expression of beta-adrenergic receptors (beta-AR) and myosin heavy chain (MyHC) isoforms in finishing pigs. Eighty barrows (initial BW = 69.42 ± 1.24 kg) were used in a randomized complete block design experiment with five treatments, eight replicates per treatment, and two animals per experimental unit (pen). The dietary treatments consisted of diets containing no RAC (control) or 10 ppm RAC fed for 7, 14, 21, or 28 days before slaughter. Individual pig BW and pen feed disappearance were obtained to determine average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (F:G). Blood samples were collected for determination of PUN concentrations. At the end of the experiment, pigs (final BW = 102.46 ± 1.44 kg) were slaughtered and hair and Longissimus dorsi muscle samples collected. The carcasses were evaluated 24 hours postmortem. Hair samples were used to detect the mutation of the ryanodine receptor type 1 (RYR1) gene. Gene expression of beta-AR (beta1- and beta2-subtypes) and MyHC isoforms (I, IIa, IIx/d, and IIb) was quantified in the muscle samples. Statistical analyses were performed using only the homozygous dominant pigs for the RYR1 gene mutation. Increasing RAC feeding period did not affect (P > 0.05) final BW, ADG, and ADFI, but resulted in a linear improvement (P < 0.01) in F:G. Average weekly improvements (P < 0.05) in ADG and F:G were observed during the first 21 days of RAC feeding, however, animal growth declined (P < 0.05) in the 4th week of treatment. The PUN concentrations showed a quadratic effect (P < 0.01) as RAC feeding duration increased. There were linear increases (P <= 0.01) in hot carcass weight, Longissimus dorsi muscle depth, loin eye area, and muscle to fat ratio as RAC treatment duration increased. No effects of RAC feeding (P > 0.05) were detected for beta1-AR and for isoforms of MyHC IIa and MyHC IIx/d gene expression, but increasing RAC feeding period tended to linearly decrease (P = 0.08) beta2-AR gene expression. Even though mRNA levels of MyHC I isoform decreased linearly (P < 0.01), gene expression of MyHC IIb isoform increased linearly (P < 0.01) as RAC treatment duration increased. Therefore, greater growth and carcass responses occurred when RAC was fed for 21 and 28 days, respectively. Furthermore, the agonist altered the MyHC gene expression and the RAC action may be related to the beta2-AR population.

A velocidade de renovação das proteínas musculares define o crescimento muscular, e afetam a qualidade da carne, destacando-se a renovação de colágeno e taxa proteolítica que tem implicação no fenótipo de maciez. No entanto, existe pouca informação na literatura sobre mudanças no processo de remodelação de proteínas musculares em resposta à taxa de recuperação do ganho de peso, após um período de subnutrição em animais fisiologicamente maduros. Dessa forma, o uso de sequenciamento de RNA pode indicar mudanças no tecido muscular através de um perfil de expressão diferencial que explicam mudanças nos fenótipos associados ao crescimento muscular. Portanto, o objetivo deste estudo foi avaliar as mudanças no transcriptoma associado a fenótipos de carne de vacas adultas Nelore submetidas a diferentes taxas de recuperação de peso. Neste experimento foram utilizadas 28 vacas adultas de 5 a 16 anos, sendo que 23 animais, por apresentarem baixo escore corporal no inicio do experimento como resultado de perda de peso durante uma estação seca, foram aleatoriamente distribuídas em 3 grupos baseados em ganho diário de peso vivo: GB - Base (abatidos com baixo escore corporal, n=4); GL - Lento (0,6 kg/dia, n=9); GR - Rápido (1,2 kg/dia, n=10). Um quarto grupo (GM - Manutenção, n=5), foi formado de animais com alto escore corporal do início ao final do experimento. Os animais foram abatidos em quatro pontos definidos por dias (d) de confinamento (A1(0d) = GB; A2(51d) = GL e GR; A3(74d) = GR; A4(104d) = GL e GM), e as amostras do musculo Longissimus thoracis foram coletadas para obtenção do RNA total. Após a obtenção do mRNA e a preparação das bibliotecas de cDNA, o sequenciamento das amostras foi feito em um equipamento HiScanSQ. Vinte e quatro horas após o abate, foram determinados o pH e a cor instrumental (L*, a*, b*) medidos no músculo Longissimus thoracis. Dois bifes foram amostrados para a análise de força de cisalhamento às 24h e 21 dias. Na comparação entre fenótipos e genes, os contrastes utilizaram o grupo GB como referência. As análises estatísticas foram realizadas no programa R, usando ANOVA e teste de Tukey. Menores força de cisalhamento aos 21 dias (P<=0,05) foram encontradas para o grupo GL (A4) e GM (A4) comparadas ao GB. Valores de L*, a*, b* no músculo do GR (A3) também foram maiores em relação ao GB. Somente o valor de b* na gordura foi diferente (P<=0,05) em GR (A2 e A3) em relação ao GB. Foram identificados o total de 578 genes diferencialmente expressos (DE; FDR 5%) nos cinco contrastes avaliados. A análise funcional de genes DE entre contrastes identificou uma via KEEG para o grupo GL (\"Ribosome\") e cinco para o grupo GR (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). Para o enriquecimento GO, foram obtidas quatro categorias, sendo \"Semaphorin receptor activity\" para o grupo GL, enquanto \"Cellular response to amino acid stimulus\", \"Collagen trimer\" e \"Extracellular matrix structural constituent\" foram identificadas para GR. As vias em geral estão associadas a alterações no tecido conjuntivo do músculo. As diferenças encontradas entre fenótipos e genes indicam que as taxas de crescimento afetam de forma diferente os fenótipos associados à qualidade da carne. A realimentação em animais adultos, ao alterar o transcriptoma com possíveis impactos no tecido conjuntivo, parece mostrar um cenário de fibrose muscular no ganho rápido de peso, diferentemente do ganho lento, que mostrou ser uma alternativa para melhorar a qualidade da carne. Todavia, os impactos na estrutura muscular e proteica que possam explicar incremento em aspectos de textura da carne precisam ser determinados. / The speed of renewal of muscle proteins defines muscle growth, and affect meat quality, especially the renewal the of collagen turnover and proteolytic rate that has implication in the tenderness phenotype. There is little information in the literature about changes in the muscle protein remodeling process in response to the rate of recovery of weight gain after malnutrition in physiologically mature animals. In this way, the use of RNA sequencing may indicate changes in muscle tissue through a differential expression profile that explains changes in phenotypes associated with muscle growth. Therefore, the objective of this study was to evaluate changes in the transcriptome associated with meat phenotypes of Nellore adult cows submitted to different rates of weight recovery. In this experiment, 28 adult cows from 5 to 16 years old were used, being that twenty-three animals were distributed randomly in 3 groups based on daily gain of live weight, since they presented low body score at the beginning of the experiment as a result of weight loss during a dry season: GB - Base (slaughtered with low body score, n=4); GL - Slow (0.6kg/day, n=9); GR - High (1.2 kg/day, n=10). A fourth group (GM - Maintenance, n = 5) was formed from animals with high body score from the beginning to the end of the experiment. The animals were slaughtered at four points defined by confinement days(d) (A1(0d) = GB; A2(51d) = GL and GR; A3(74d) = GR; A4(104d) = GL and GM), and Longissimus thoracis muscle samples were collected to obtain the total RNA. After obtaining the mRNA and preparation of the libraries, the sequencing of the samples was done in a HiScanSQ equipment. Twenty-four hours after slaughter, the pH and instrumental color (L*, a*, b*) were determined in the Longissimus thoracis muscle. Two steaks were collected for shear force analysis at 24h and 21 days. In the comparison of the phenotypes and genes, contrasts were used the GB group as reference. Statistical analysis were performed in R software, using ANOVA and Tukey test. Lower shear forces at 21 days (P<=0.05) were found for the GL (A4) and GM (A4) groups compared to GB. Values of L*, a*, b* in the GR (A3) muscle were also higher in relation to GB. Only the value of b* in fat was different (P<=0.05) in GR (A2 and A3) in relation to GB. A total of 578 differentially expressed genes (DE; false discovery rate 5%) in the five contrasts evaluated. Functional analysis of DE genes between contrasts identified a KEEG pathway for the GL group (\"Ribosome\") and five for the GR group (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). For GO enrichment, four categories were obtained, being \"Semaphorin receptor activity\" for GL, while \"Cellular response to amino acid stimulus\", \"Collagen trimer\" and \"Extracellular matrix structural constituent\" were identified for GR. Pathways in general are associated with changes in connective tissue of muscle. Re-feeding in adult animals by altering the transcriptome with possible impacts on connective tissue, seems to show a scenario of muscle fibrosis in the fast gain of weight, unlike the slow gain, which proved to be an alternative to improve meat quality. However, the impacts on muscle and protein structure that may explain the increase in meat texture aspects need to be determined.

Resumo: O presente estudo teve como objetivo a comparação do desempenho produtivo e do crescimento muscular (morfometria das fibras musculares e expressão gênica dos fatores de regulação miogênica) de três linhagens de tilápia-do-nilo (GIFT, ―Supreme‖ e Tailandesa), alimentadas com dieta comercial contendo ou não o hormônio 17α-metiltestosterona, durante a fase inicial, quando ocorre o processo de diferenciação sexual. O trabalho foi conduzido em esquema fatorial (2 dietas X 3 linhagens), constituído por seis tratamentos com quatro repetições de cada, por 29 dias. As larvas (com quatro dias pós eclosão) foram estocadas na densidade de 4 larvas.L-1 e receberam dieta comercial farelada, em seis arraçoamentos diários. Foram realizadas cinco avaliações biométricas durante o experimento para verificação dos resultados de desempenho. Além disso, foram calculadas as taxas de sobrevivência, de masculinização e a homogeneidade dos animais no final do experimento. Para a avaliação morfométrica das fibras musculares (n = 8 indivíduos por tratamento) foram realizadas amostragens aos 4, 19 e 33 dias pós-eclosão, para confecção das lâminas de tecido muscular. Mensurou-se o diâmetro e a área de 200 fibras musculares para cada amostra, que foram distribuídas nas seguintes classes de diâmetro (D): classe 10 (D ≤10μm), classe 20 (10< D ≤20 μm), classe 30 (20< D ≤30 μm), classe 40 (30< D ≤40μm) e classe 50 (D > 40 μm). As análises de expressão gênica da MyoD e Miogenina foram realizadas por PCR em tempo real, com uma amostragem aos 4 dpe e outra aos 33 dpe. A linhagem ―Supreme‖ apresentou melhores respostas de desempenho produtivo e de sobrevivência, e menores valores do coeficiente de variação. As taxas de masculinização em todas as linhagens estiveram acima de 97%. A masculinização, por meio da adição de 17α-metiltestosterona, interferiu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to compare the performance and muscle growth (muscle fiber morphometry and gene expression of myogenic regulatory factors) from three Nile tilapia strains (GIFT, "Supreme" and Thai), fed commercial diet with or without the hormone 17α-methyltestosterone (MT) during the initial phase, when does the process of sexual differentiation. The experiment was conducted in a factorial scheme (3x2), making up six treatments with four replicates each. The larvae (4 days post-hatching, dph) were stocked at 4 larvae.L-1 and were fed a commercial diet six times a day, during 29 days. Fish growth was evaluated along the experiment, as well as the survival rates, masculinization rates and homogeneity of the animals at the end (29 days). For the morphometric analysis of the muscle fibers (n = 8 per treatment), samples were taken at 4, 19 and 33 days post-hatching (dph) and transversal sections of the epiaxial muscle were obtained. The diameter (d) and the area of 200 muscle fibers were measured in each sample, and the diameters were classified as follow: class 10 = d ≤ 10 μm, class 20 = 10 < d ≤ 20 μm, class 30 = 20 < d ≤ 30 μm, class 40 = 30 < d ≤ 40 μm, and class 50 = d > 40μm. The analysis of gene expression of MyoD and Myogenin were performed by real time PCR (sampling at 4 and 33 dph). The "Supreme" strain showed better growth performance, survival rates, and size homogeneity. All strains displayed masculinization rates above 97%. The use of MT affected the productive performance of Nile tilapia larvae, and the effects were different among the strains. The proportion of males and females of the juveniles that did not receive MT suggests a certain dependence on the strain used, requiring further studies in this direction. The ―Supreme‖ strain performed better, characterized by hyperplasia and hypertrophy of the muscle fibers... (Complete abstract click electronic access below) / Orientadora: Maria Célia Portella / Coorientador: Maeli Dal Pai-Silva / Banca: Fernanda Antunes Alves da Costa / Banca: Vander Bruno dos Santos / Mestre