A ractopamina (Rac) tem sido utilizada durante a gestação de fêmeas suínas gestantes, com objetivo de melhorar o aporte nutricional ao feto, sendo considerada como repartidor de nutrientes, promovendo a lipólise e a retenção de nitrogênio, e a síntese proteica. O aumento no fluxo de nutrientes pode apresentar impactos positivos na condição corporal da fêmea gestante e o desenvolvimento do feto. O presente trabalho avaliou os efeitos da inclusão de 10 e 20 mg/kg de ractopamina na ração de fêmeas suínas gestantes, entre os dias 25 a 50 da gestação, sobre o desempenho produtivo, reprodutivo, parâmetros fisiológicos, hemograma e bioquímica sanguínea das fêmeas e sobre a vitalidade, hemograma, bioquímica sanguínea, número de fibras musculares no músculo semitendinosus e estado metabólico dos leitões. Foram utilizadas 41 fêmeas de linhagem comercial híbrida, DB 90 (DB-Genética Suína, Patos de Minas, MG), divididas de acordo com a ordem de parto em 3 tratamentos: Controle: sem ractopamina; Rac10: 10 mg/kg de ractopamina; Rac20: 20 mg/kg de ractopamina. O tratamento foi administrado diariamente via top dressed durante os 25 a 50 dias de gestação. As avaliações e as coletas de amostras foram realizadas nos 25, 37, 50, 107 dias de gestação e nos 1, 7, 14 e 21 dias da lactação. Para a avaliação da vitalidade foi utilizado o teste de pontuação Apgar modificado por Motta-Rojas (2005). A suplementação de ractopamina durante a gestação não influenciou o desempenho reprodutivo das fêmeas, dos leitões até o desmame e no número de fibras musculares no músculo semitendinosus ao nascimento. Houve um aumento na frequência cardíaca e de leitões com vitalidade baixa nas fêmeas suplementadas com Rac. Houve alterações no hemograma, bioquímica sanguínea das fêmeas, mas não nos leitões. Os resultados obtidos a partir das análises do hemograma e bioquímica sanguínea indicam ação da ractopamina sobre o metabolismo lipídico e proteico das fêmeas sem reflexos importantes nos valores observados nos leitões. O presente trabalho concluiu que a suplementação de Rac durante os 25 a 50 dias da gestação não influencia negativamente no metabolismo das fêmeas e sua progênie até o desmame. Por outro lado, a suplementação de Rac aumenta a frequência cardíaca sem aumento de frequência respiratória e temperatura retal das fêmeas, no entanto, pode ser considerado ainda dentro dos parâmetros fisiológicos normais durante a gestação. O aumento no número de leitões de vitalidade baixa 10 pode estar relacionado a um aumento numérico do peso ao nascimento dos leitões. Contudo, a suplementação de Rac durante os 25 50 dias da gestação não influencia negativamente no desempenho produtivo das fêmeas e sua progênie até o desmame. / The ractopamine (Rac) has been used in pregnant sows, aiming to improve the nutritional contribution to the fetus, being considered as nutrient delivery, promoting lipolysis and retention of nitrogen and protein synthesis. Increased nutrient flow may have positive impacts on the body condition of sows and fetal development. The present study evaluated the effects of the inclusion of 10 and 20 mg / kg of ractopamine in the diet sows from 25 to 50 days of pregnancy. Assessing possible effects on productive, reproductive performance, physiological parameters, complete blood count and blood serum biochemistry of females and on vitality, complete blood count, blood serum biochemistry, number of muscle fibers in the semitendinosus muscle and metabolic status of their piglets. Forty one sows of hybrid commercial line, DB 90 (DB-Genetics Swine, Patos de Minas, Minas Gerais),were used and divided according to the order of parity in 3 treatments: Control: No ractopamine; Rac10: 10 mg / kg ractopamine; Rac20: 20 mg / kg ractopamine. The treatment was administered daily via top dressed during the 25 to 50 days of gestation. The evaluations and sample collections were performed at 25, 37, 50, 107 days of gestation and at 1, 7, 14 and 21 days of lactation. Assessment of vitality was used the apgar score test modified by Motta-Rojas (2005). The supplementation of ractopamine during gestation did not influence the female reproductive performance, piglets performance until weaning and muscle fibers in the semitendinosus muscle at birth. There was an increase in heart rate and low vitality in females supplemented with Rac. There were alterations in the complete blood count, blood serum biochemistry of the females, but not in the piglets. The results obtained from the hemogram and blood biochemistry analysis indicate the action of ractopamine on the lipid and protein metabolism of the females without significant reflexes in the piglets values. The present study concluded that the supplementation of Rac during the 25 to 50 days of gestation does not negatively influence the metabolism of the females and their progeny until weaning. On the other hand, supplementation of Rac increases the heart rate without increasing respiratory rate and rectal temperature of the females, however, this can still be considered within the normal physiological parameters during gestation. The increase in the numbers of low-vitality piglets may be related to a numerical increase in the birth weight of the piglets. However, Rac 12 supplementation during the 25-50 days of gestation does not negatively influence the productive performance of the females and their progeny until weaning.

An inherited muscle hypertrophy in sheep is caused by the callipyge gene (CLPG) located on ovine chromosome 18. It has been suggested that this gene is a single autosomal dominant gene. Animals expressing the callipyge gene show increased muscling in the pelvic and torso regions of the body and, thus, have been given the phenotype- descriptive name callipyge (from Greek calli-beautiful; pyge-buttocks). In this study 21 wether lambs, the offspring of callipyge rams (genotype CLPG /clpg) and normal Rambouillet ewes (genotype clpg/clpg), were used to determine the difference s in muscle fiber type percentages, composition, and tenderness between normal and callipyge lambs . Eleven of these lambs showed muscle hypertrophy typical of the callipyge phenotype; 10 were classified as normal . Several histochemical, biochemical , and physical measures were examined in order to study changes in the physiology and biochemistry of some economically important muscles. When compared to normal lambs, the callipyge lambs have a larger (P < .05) average percentage of fast-twitch glycolytic (FG) muscle fibers and smaller average percentages of fast-twitch oxidative and glycolytic (FOG) and slow-twitch oxidative (SO) muscle fibers in both the longissimus and gluteus medius muscles. The diameter of the fast-twitch muscle fibers was larger in the callipyge group, but slow-twitch fibers were smaller than those of normal lambs. No differences were observed in the supraspinatus muscle of the normal and callipyge groups. Thus there is an indication of a differential effect of the callipyge gene among muscles of the callipyge lambs. The semitendinosis muscles of both the callipyge and normal groups were dissected from the carcasses. This muscle was larger (P < .01) and contained more protein in the callipyge lambs than in the normal lambs. However, the callipyge semitendinosis muscle did not have a significantly higher content of DNA than the normal lamb semitendinosis, suggesting that the muscle hypertrophy is not associated with an increase in muscle nuclei. The protein-to-DNA ratio was larger (P < .05) in the semitendinosis muscle of callipyge lambs than in the normal lambs. Protein-to-RNA and RNA-to-DNA ratios were similar; this suggests that the semitendinosis muscle was enlarged without increased translational or transcriptional activity. Samples from the callipyge longissimus and gluteus medius muscles had RNA, DNA, and protein ratios similar to those of the semitendinosis muscle, suggesting a similar mode of action for muscle enlargement in other muscles affected by this gene. Loin chops from the callipyge lambs had lower tenderness scores (P < .01) as measured by the Wamer-Bratzler shear force and myofibril fragmentation index (MFI). However, aging increased MFI scores and decreased shear scores (P < .0 l) of the callipyge lamb chops . The normal lamb chops also had decreased shear and increased MFI scores following the aging period. The loin chops from the callipyge lambs also tended to be less red (P < .1) than chops from normal animals as measured by Hunter 'L,' 'a,' and 'b' colorimeter scores.

Strength training is widely used to increase performance in sports with high physical demands. The use of drugs such as anabolic steroids among athletes is a wellknown phenomenon, and the effects of these drugs on physical performance documented. The studies presented in this thesis focused on the mechanisms of muscle fiber hypertrophy in the vastus lateralis and the trapezius muscles of strength trained elite athletes. The main hypothesis was that the muscle adaptations to strength training and anabolic steroids are muscle specific. Biopsies were obtained from the trapezius and the vastus lateralis from three groups of elite power lifters. Nine used drugs, ten did not and seven had previously used drugs. Six sedentary males served as controls. The biopsies were frozen and cut in serial cross sections. Histological and immunohistochemical staining techniques were used to analyze muscle fiber morphology and pathology. Fiber type distribution, fiber area, myonuclei number and distribution, satellite cell number and proportion of split fibers were counted and compared for the two muscles within and between the groups. The main findings were that: a) Muscle fiber hypertrophy by strength training is further increased by anabolic steroids. b) The number of nuclei per muscle fiber is higher in power lifters using anabolic steroids compared to non-steroids using lifters. c) Among power lifters who have withdrawn from anabolic steroid usage and training for several years, the number of myonuclei, both subsarcolemmal and internal, remains high. d) In active power lifters, anabolic steroids have no further effect on the number of satellite cells per fiber. e) Power lifters have a high proportion of split fibers. High intensity resistance training increases muscle strength and banned substances such as testosterone and anabolic steroids can enhance the training effects. The studies on muscle cell morphology presented in this thesis reveals that anabolic steroids and testosterone increases muscle fiber size and adds more nuclei to the muscle cell. Based on the morphological appearance of muscle sections from doped and nondoped power lifters, we conclude that testosterone and anabolic steroids enhances the hypertrophic effects of training without adding new features. The addition of myonuclei by training and doping appears to be longer lasting in some muscles than in others. The high proportion of split fibers in power lifter is probably due to high mechanical stress. The findings and conclusions in this thesis raise questions regarding relevant suspension times for athletes caught with banned substances in the body.

Unilateral exercise can produce certain contralateral strength effects. Deleterious events can be cross-transferred as well, as illustrated by a strict symmetry in some chronic inflammatory diseases. To date, knowledge on the effects of marked overuse of skeletal muscles is limited, and there is largely no information if unilateral overuse affects the contralateral muscles. In view of this, the present study was undertaken to test the hypothesis that unilateral muscle overuse causes alterations in tissue structure and the tachykinin system, with a focus on substance P (SP), not only in the exercised muscles, but also in the contralateral muscles. SP is a well-known neuromodulator that is known to be proinflammatory. An experimental rabbit model with unilateral muscle overuse of the soleus and gastrocnemius muscles caused by exercise via electrical muscle stimulation (E/EMS) was used. In total, 40 rabbits were randomly divided into seven groups of which two groups served as controls. The rabbits were anaesthetized and then set on a “kicking machine” to perform exercise via EMS for 2h every second day. Experimental periods for groups 1-3 were 1, 3 and 6w, respectively, whereas groups 4-6 were exercised for 1w but also subjected to injections in the peritendinous tissue with SP, NaCL, Captopril (C), an ACE inhibitor, and DL-Thiorphan (Th) which inhibits the activity of neural endopeptidase. One group was not subjected to the experiment at all. The day after the last session of E/EMS, the soleus muscle and the gastrocnemius muscle from both legs were collected for analysis. Alterations in muscle structure and the tachykinin system were analyzed with enzyme and immunohistochemical techniques, in situ hybridization and EIA methods. After 1w of E/EMS, focal areas of the exercised muscles contained a mild infiltration of inflammatory cells (myositis) and small morphological changes. After 3 and 6w of E/EMS, distinct myositis and muscle changes were bilaterally present in focal areas of both muscles. The structural changes, which mainly were observed in myositis areas, consisted of increased fiber size variability, split fibers, internal myonuclei, necrotic fibers, fibrosis, fat infiltration, and small fibers containing developmental MyHCs. Bilateral morphological changes, such as loss of axons, were also observed in nerves. In addition, expressions of tachykinin and the SP-preferred receptor, the neurokinin-1 (NK-1R), were bilaterally upregulated in nerve structures and blood vessel walls.  Infiltrating white blood cells exhibited tachykinin–like and NK-1R immunoreactivity. NK-1R immunoreactions were also found in necrotic and regenerating muscle fibers. The concentration of tachykinin (SP) was significantly increased in both soleus and gastrocnemius muscles after E/EMS. There was a significant correlation between the two sides in concentration of tachykinin and in the intensity of tachykinin-like immunoreaction in blood vessel walls. The muscle fiber size and capillary supply of fibers were bilaterally decreased after 3w of EMS. The myositis areas contained an increased number of vessels with a larger size than capillaries, while areas with increased amount of connective tissue contained a very low number of capillaries. A bilateral fiber type shift against a lower proportion of slow MyHCI fibers and higher proportion of fast MyHCII fibers was observed in both muscles. The local injections of C+Th and SP+C+Th led to marked structural changes in the muscle tissue and marked increased NK-1R and tachykinin-like immunoreactivity in the myositis areas and increased tachykinin concentration in the tissue. In conclusion, the repetitive unilateral muscle overuse caused by E/EMS led overtime to muscle injury and myositis. The affected areas contained both degenerative and regenerative alterations in the muscle tissue and nerves, and an upregulation of the tachykinin system. Most interestingly, the changes not only occurred in the exercised side, but also in the homologous contralateral muscles. The tachykinin system appears to be an important factor in the processes of crossover effects.

In the present study Fourier Transform Infrared Spectroscopy was used to examine the effects of streptozotocin-induced diabetes mellitus on the structural components of slow- and fast-twitch rat skeletal muscles, at molecular level. Diabetes mellitus is a chronic disorder of carbohydrate, fat and protein metabolism, which is characterized by hyperglycemia caused by a defective or deficient insulin secretory response. The effect of diabetes is seen on a variety of tissues leading to important secondary complications such as kidney failure, liver dysfunction, cardiac disorders, etc. Skeletal muscle is one of the major tissues determining carbohydrate and lipid metabolism in the body / therefore, is one of the target tissues of diabetes. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. The FTIR spectra revealed a considerable decrease in lipid and protein content of diabetic skeletal muscles, indicating an increased lipolysis and protein breakdown or decreased protein synthesis. Moreover changes in protein structure and conformation were observed. In diabetes, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. Diabetes caused a decrease in the content of nucleic acids, especially RNA, and hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated slow-twitch muscle was more severely affected from diabetes. Thus, FTIR spectroscopy appears to be a useful method to evaluate the effect of diabetes on skeletal muscle tissues at molecular level.

The purpose of the study was to compare the physiological responses of skeletal muscle to a resistance training (RT) program using repetition maximum (RM) or relative intensity (RISR). Fifteen well-trained males underwent RT 3 d·wk−1 for 10 weeks in either an RM group (n = 8) or RISR group (n = 7). The RM group achieved a relative maximum each day, while the RISR group trained based on percentages. The RM group exercised until muscular failure on each exercise, while the RISR group did not reach muscular failure throughout the intervention. Percutaneous needle biopsies of the vastus lateralis were obtained pre-post the training intervention, along with ultrasonography measures. Dependent variables were: Fiber type-specific cross-sectional area (CSA); anatomical CSA (ACSA); muscle thickness (MT); mammalian target of rapamycin (mTOR); adenosine monophosphate protein kinase (AMPK); and myosin heavy chains (MHC) specific for type I (MHC1), type IIA (MHC2A), and type IIX (MHC2X). Mixed-design analysis of variance and effect size using Hedge’s g were used to assess within- and between-group alterations. RISR statistically increased type I CSA (p = 0.018, g = 0.56), type II CSA (p = 0.012, g = 0.81), ACSA (p = 0.002, g = 0.53), and MT (p < 0.001, g = 1.47). RISR also yielded a significant mTOR reduction (p = 0.031, g = −1.40). Conversely, RM statistically increased only MT (p = 0.003, g = 0.80). Between-group effect sizes supported RISR for type I CSA (g = 0.48), type II CSA (g = 0.50), ACSA (g = 1.03), MT (g = 0.72), MHC2X (g = 0.31), MHC2A (g = 0.87), and MHC1 (g = 0.59); with all other effects being of trivial magnitude (g < 0.20). Our results demonstrated greater adaptations in fiber size, whole-muscle size, and several key contractile proteins when using RISR compared to RM loading paradigms.

京都大学 / 0048 / 新制・論文博士 / 博士(人間・環境学) / 乙第12883号 / 論人博第40号 / 新制||人||169(附属図書館) / 26||論人博||40(吉田南総合図書館) / 31601 / (主査)教授 石原 昭彦, 教授 船橋 新太郎, 教授 林 達也, 准教授 神﨑 素樹, 准教授 久代 恵介 / 学位規則第4条第2項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

Pre-training muscle characteristics of subjects who are obese determine how well exercise training will improve their insulin responsiveness. J Strength Cond Res 31(3): 798–808, 2017—Only half of prediabetic subjects who are obese who underwent exercise training without weight loss increased their insulin responsiveness. We hypothesized that those who improved their insulin responsiveness might have pretraining characteristics favoring a positive response to exercise training. Thirty nondiabetic subjects who were obese volunteered for 8 weeks of either strength training or endurance training. During training, subjects increased their caloric intake to prevent weight loss. Insulin responsiveness by euglycemic clamps and muscle fiber composition, and expression of muscle key biochemical pathways were quantified. Positive responders initially had 52% higher intermediate muscle fibers (fiber type IIa) with 27% lower slow-twitch fibers (type I) and 23% lower expression of muscle insulin receptors. Whether after weight training or stationary bike training, positive responders' fiber type shifted away from type I and type IIa fibers to an increased proportion of type IIx fibers (fast twitch). Muscle insulin receptor expression and glucose transporter type 4 (GLUT4) expression increased in all trained subjects, but these moderate changes did not consistently translate to improvement in whole-body insulin responsiveness. Exercise training of previously sedentary subjects who are obese can result in muscle remodeling and increased expression of key elements of the insulin pathway, but in the absence of weight loss, insulin sensitivity improvement was modest and limited to about half of the participants. Our data suggest rather than responders being more fit, they may have been less fit, only catching up to the other half of subjects who are obese whose insulin responsiveness did not increase beyond their pretraining baseline.

A seleção de linhagens de frangos de corte com altas taxas de crescimento e aumento de rendimento muscular vem contribuindo para o surgimento de perdas significativas na indústria avícola em decorrência de modificações anatômicas. Dentre estas alterações, destaca-se uma miopatia que se caracteriza pelo aparecimento de graus de estrias esbranquiçadas no músculo Pectoralis major, as quais seguem a direção da fibra muscular, referida como white striping. A etiologia da white striping ainda é desconhecida, mas avaliações histológicas demonstraram que esta alteração é usualmente associada à degeneração muscular e alterações miopáticas. Problemas de qualidade da carne podem ser resultantes de mudanças estruturais, morfométricas e bioquímicas do tecido muscular, com consequente alteração nas fibras musculares. Para tanto este trabalho, conduzido através de dois experimentos, teve como objetivos caracterizar histopatologicamente a miopatia white striping em diferentes graus de severidade (NORM, MOD, SEV) no músculo Pectoralis major de frangos de corte e as características morfométricas das fibras musculares, bem como avaliar a incidência desta condição em frangos de corte de linhagem Cobb abatidos aos 42 dias de idade. No primeiro experimento, a avaliação histopatológica demonstrou uma miopatia degenerativa nos graus de severidade classificados como MOD e SEV, entretanto as amostras de grau MOD apresentaram necrose ou necrose multifocal moderada, enquanto as de grau SEV apresentaram necrose difusa e acentuada, sugerindo avanço do processo degenerativo. Não foi observada proliferação de tecido conjuntivo em nenhum dos graus de white striping analisados, caracterizando este caso como de ocorrência aguda. No segundo experimento, foram verificados menor área de tecido muscular, menor diâmentro e maior densidade de fibras nos músculos Pectoralis major de frangos acometidos com white striping (MOD e SEV) quando comparado aos peitos sem esta condição (NORM). Conforme os resultados obtidos nesta pequisa, pode-se concluir que as fibras com os graus MOD e SEV de white striping demonstraram o início de um processo de regeneração muscular, de característica hiperplásica. / Genetic selection of broiler chickens for growth rate and increase of muscle yield has contributed to the appearance of significant losses in the poultry industry caused by anatomical changes. Among these alterations stands out a myophathy characterized by the occurrence of white striations on Pectoralis major muscle, seen parallel to the direction of muscle fibers, referred as white striping. The etiology of white striping is still unknown but histological changes demonstrated that this alteration is usually associated with muscle degeneration and myopatic changes. Meat quality problems may result from structural, morphological and biochemical changes of muscle tissue with consequent alterations in muscle fibers. To this study, two experiments were conducted, and aimed to histopathologically characterize the myopathy white striping at varying degrees of severity (NORM, MOD, SEV) on broilers Pectoralis major muscle and the morphometric muscle fibers characteristics as well as evaluate the incidence of this condition on Cobb 500 broiler breast fillets slaughtered at 42 days of age. The histopathological evaluation on experiment 1 showed a degenerative myopathy in both MOD and SEV degrees, however the samples classified as MOD showed microscopically necrosis or necrosis multifocal moderate, while the classified as SEV showed necrosis diffuse and strong suggesting an advancement in the degenerative process. The proliferation of connective tissue was not observed for either of the degrees of white striping investigated, characterizing this profile as an acute occurrence. In experiment 2, small area and diameter and higher densities of muscle fiber were observed in broilers Pectoralis major muscle with white striping (MOD and SEV) compared to NORM. According to the results, we can conclude that muscle fibers with white striping showed the beginning of a muscle regeneration process with hyperplasic characteristics.