This study evaluated degradable nutrient supply effects on VFA concentrations, fluid flux and pool sizes, rumen epithelial metabolic and absorptive genes, and post-absorptive muscle and blood responses. Six ruminally cannulated Holstein heifers (BW=330 ± 11.3 kg) were used in a partially replicated Latin Square experiment with four treatments consisting of beet pulp or timothy hay and barley or corn grain. Periods were18 d with 3 d diet adaptation and 15 d of treatment. During each period, d 10 to 14 was used for in situ nutrient degradation assessment, d 16 to 18 was used for rumen fluid sampling, and d 18 was used for rumen papillae and skeletal muscle biopsies and blood sampling. In situ ruminal starch disappearance rate (barley 7.61 to 10.5 %/h vs corn 7.30 to 8.72%/h; P = 0.05) and extent of fiber disappearance (timothy hay 22.2 to 33.4 % DM vs beet pulp 34.4 to 38.7 % DM P=0.0007) differed significantly among diets. Acetate (P = 0.02) and isovalerate (P = 0.008) molar percentages (% mol) were increased by timothy hay, but propionate (P = 0.06) and valerate (P = 0.10) molar percentages were decreased. Corn increased propionate (P = 0.02) and valerate (P = 0.049) molar percentage, but decreased butyrate (P = 0.04) molar proportion. Fluid volume and fluid passage rate, and individual VFA pool sizes were not influenced by diet (P > 0.05). Four epithelial genes, two metabolic and two absorptive, had increased expression on timothy hay diets (P < 0.15). Blood acetate concentration was influenced by treatment (P = 0.067) but no other blood metabolites were. Skeletal muscle metabolic rate was significantly increased on corn diets (P = 0.023). The results of this study provide a whole-system snapshot of how the rumen environment changes on diets differing in nutrient degradability and how the post-absorptive system adapts in response. / Master of Science / Over the last 50 years, dairy cattle have been bred to optimize milk production to meet growing population demands for milk and dairy products. The world population continues to grow and is projected to reach 9.7 billion people by 2050. Because of this growing population, there is an overwhelming need for dairy nutritionists to optimize the conversion of human inedible fibers into human edible food. The ruminant animal accomplishes this conversion through microbial fermentation of feedstuffs into volatile fatty acids (VFA), which account for approximately 70% of total energy available for meat, milk, and fiber production. Because rumen fermentation is a complex biochemical system, it is influenced by myriad factors including the substrate provided, the pH of the environment, and the absorptive and metabolic capacity of the rumen wall, among others. Although we understand how diet influences individual aspects of rumen fermentation, few studies have concurrently evaluated how diet influences the rumen chemical environment, the epithelium, and the resulting shifts in postabsorptive metabolism. Our study sought to understand the impacts of feedstuffs with different expected ruminally available starch and fiber supplies on these aspects of ruminant physiology. Six ruminally cannulated Holstein heifers were fed four different diets which used either beet pulp (low fiber ingredient) or timothy hay (high fiber ingredient), and ground corn (low starch ingredient) or ground barley (high starch ingredient). Heifers were fed each diet for a period of 18 days. From day 10 to day 14 of the period, nutrient degradability was assessed by incubating bags of feed in the rumen and conducting feed analysis after removed from the rumen. During the last four days of each period, rumen fluid samples, blood samples, muscle biopsies, and rumen papillae biopsies were collected. Feed analysis indicated that the starch sources differed in degradation rates (i.e. the speed of degradation) and fiber sources different in extent of rumen degradation (i.e. the percentage of feed degraded). Timothy hay caused greater concentrations of Total VFA, Total branched-chain VFA, acetate isobutyrate, and isovalerate. Timothy hay caused greater molar proportions of acetate and isovalerate. Corn caused greater molar proportions of propionate and valerate when barley caused greater molar proportions of butyrate. Rumen papillae biopsies were used to evaluate gene expression. Out of 14 genes, four were impacted by diet. Two rumen transporters responsible for the absorption of VFA had greater expression when animals were fed timothy hay diets versus beet pulp diets. Two metabolic genes also had greater expression due to timothy hay. The changes of both absorptive genes and metabolic genes is likely connected to the increased presence of VFA in the rumen. Lastly, blood acetate was increased, but there was not a specific ingredient or combination that caused the change. These results provide an overall snapshot of rumen fermentation characteristics and how changes in the rumen affect other biology.

Many factors influence the efficiency of muscle growth including genetics, nutrition, and environment. The neonatal period is characterized as a time of rapid growth. Growth rate is reduced during neonatal nutrient restriction possibly due to altered satellite cell activity which can permanently alter growth potential. Therefore, optimal nutrition is important for maximizing the growth potential of the animal. Heat stress leads to changes in digestion and metabolism, thus alters nutrient availability to muscle. Heat stress is a prevalent problem in the agriculture industry resulting in great economic losses due to reduced growth, fertility, and increased morbidity. The use of functional feed additives is a potential strategy to mitigate these negative effects. The objective of this dissertation was to investigate nutritional strategies to improve growth in pigs during key malleable periods. Three nutritional studies were conducted to discern the optimal inclusion levels of calcium phosphate, energy, and protein in the diet to maximize neonatal muscle growth. Adequate dietary calcium phosphate was most efficient for satellite cell function which may be mediated by micro-RNA. Differentiation promoting miR-206 and correspondingly the fusion rate was highest in adequate calcium phosphate diets. Excess protein diets enhanced body and muscle growth, while deficient protein was detrimental to growth. Dietary protein treatments altered energy metabolism genes, and genes regulating protein degradation were upregulated in deficient protein diets. Dietary energy levels did not influence body weight, however feed efficiency improved with energy balance. Excess energy diets had the lowest fusion rates and the lowest differentiation promoting miR-1 expression. These data suggest that nutrient inclusion levels are important for satellite cell function and may mediate satellite cell activity through the expression of micro-RNAs. The final study sought to discern the ability of supplementation of an artificial high-intensity sweetener and capsicum oleoresin to mitigate the negative effects of heat stress on pig performance. Heat stress leads to increased body temperature and respiration and was detrimental to metabolic flexibility. Supplementation helped improve feed efficiency and maintain metabolic flexibility. These data indicate that supplementation may be an efficient strategy to mitigate heat stress. / Doctor of Philosophy / Muscle is an important tissue to consider when optimizing growing conditions in feed animals due to its function as a consumer good. Many factors influence the efficiency of muscle growth including genetics, nutrition, and environment. Fractional growth rates are highest during the neonatal period and animals require adequate nutrients to facilitate this growth. Nutrient restriction reduces growth rate and can lead to permanent changes the animals' body size and composition later in life. Therefore, optimal nutrition is important for maximizing the growth potential of the animal. While the nutrients in feed can be controlled to improve growth, other factors are more difficult to regulate. Heat stress is a prevalent problem in the agriculture industry resulting in great economic losses due to reduced growth, fertility, and increased morbidity. The use of functional feed additives is a potential strategy to alleviate these negative effects. The objective of this dissertation was to investigate nutritional strategies to improve growth in pigs during key malleable periods. Three nutritional studies were conducted to determine the optimal inclusion levels of calcium phosphate, energy, and protein in the diet to maximize neonatal muscle growth. Satellite cells are muscle-specific stem cells that help facilitate the growth of muscle. Altering the ability of satellite cells to proliferate and fuse impairs the ability of muscle to grow and repair. Adequate dietary calcium phosphate was most efficient for satellite cell function. Excess protein diets enhanced body and muscle growth, while deficient protein was detrimental to growth. Dietary protein treatments altered energy metabolism genes, and genes regulating protein degradation were upregulated in deficient protein diets. Dietary energy levels did not influence body weight, however, feed efficiency improved with energy balance. Satellite cells from excess energy diets had the lowest fusion rates. These data suggest that nutrient inclusion levels are important for satellite cell function and growth. The final study sought to discern the ability of the supplementation of an artificial high-intensity sweetener and capsicum oleoresin to mitigate the negative effects of heat stress on pig performance. Heat stress leads to increased body temperature and respiration and was detrimental to metabolic flexibility. Supplementation helped improve feed efficiency and maintain metabolic flexibility. These data indicate that supplementation may be an efficient strategy to mitigate heat stress.

The neonatal period in mammals is characterized by high rates of growth, attributed to rapid myonuclear accretion and protein deposition in muscle. Low-birth-weight (LBWT) neonates experience restricted muscle development, which leads to impaired postnatal growth and metabolic disorders later in life. The overall hypothesis of this dissertation was that dysfunction of myogenic satellite cells and aberrant regulation of protein synthesis and degradation signaling predispose LBWT neonatal pigs to slower postnatal growth. We sought to determine the proliferation and differentiation of satellite cells (SCs) derived from skeletal muscle of LBWT neonatal pigs and to elucidate the cellular mechanisms that regulate protein synthesis and degradation in LBWT pig muscles. Newborn pigs were considered as normal-birth-weight (NBWT) or LBWT when weight at birth was within 0.5 SD and below 2 SD of litter average respectively. SCs isolated from longissimus dorsi (LD) muscle of NBWT and LBWT neonatal pigs displayed similar proliferation rates. Fusion was modestly diminished in SCs from muscle of LBWT pigs compared with their NBWT siblings, suggesting SCs were not intrinsically different between the two groups and were unlikely a major contributor to the impaired muscle growth of LBWT pigs. Plasma and muscle insulin-like growth factor (IGF)-I was diminished in LBWT compared with NBWT pigs. In addition, reduced activation of key components of IGF-I downstream signaling pathway in LBWT pigs muscle may lead to diminished translation initiation signaling and thus decreased protein synthesis in these animals. However, IGF-I receptor expression and myostatin signaling inversely correlated to LBWT, indicating they may participate in compensatory responses for the reduction in protein synthesis signaling. Expression of eukaryotic initiation factor (eIF) 4F complex subunits, eIF4E, eIF4G, and eIF4A was reduced in LBWT compared with NBWT pigs. This would suggest that diminished translation initiation signaling in skeletal muscle of LBWT pigs is the main factor that predisposes LBWT pigs to slower growth rates in the neonatal period. In contrast, changes in protein degradation signaling do not appear to affect protein turnover in LBWT neonatal pigs. / PHD

Yes

Yes / Objectives: To assess the test-retest variability of both diffusion parameters and fat fraction (FF) estimates in normal muscle, and to assess differences in normal values between muscles in the thigh. Methods: 29 healthy volunteers (mean age 37 years, range 20-60 years, 17/29 males) completed the study. Magnetic resonance images of the mid-thigh were acquired using a stimulated echo acquisition mode-echoplanar imaging (STEAM-EPI) imaging sequence, to assess diffusion, and 2-point Dixon imaging, to assess FF. Imaging was repeated in 19 participants after a 30 min interval in order to assess test-retest variability of the measurements. Results: Intraclass correlation coefficients (ICCs) for test-retest variability were 0.99 [95% confidence interval, (CI): 0.98, 1] for FF, 0.94 (95% CI: 0.84, 0.97) for mean diffusivity and 0.89 (95% CI: 0.74, 0.96) for fractional anisotropy (FA). FF was higher in the hamstrings than the quadriceps by a mean difference of 1.81% (95% CI:1.63, 2.00)%, p < 0.001. Mean diffusivity was significantly lower in the hamstrings than the quadriceps (0.26 (0.13, 0.39) x10-3 mm2s-1, p < 0.001) whereas fractional anisotropy was significantly higher in the hamstrings relative to the quadriceps with a mean difference of 0.063 (0.05, 0.07), p < 0.001. Conclusions: This study has shown excellent test-retest, variability in MR-based FF and diffusion measurements and demonstrated significant differences in these measures between hamstrings and quadriceps in the healthy thigh. Advances in knowledge: Test-retest variability is excellent for STEAM-EPI diffusion and 2-point Dixon-based FF measurements in the healthy muscle. Inter- and intraobserver variability were excellent for region of interest placement for STEAM-EPI diffusion and 2-point Dixon-based FF measurements in the healthy muscle. There are significant differences in FF and diffusion measurements between the hamstrings and quadriceps in the normal muscle. / ICA-CL-2016-02-017/DH_/Department of Health/United Kingdom; NIHR

<p dir="ltr">The production cycle of lactating dairy cattle consists of physiological adaptations and changes in body tissue reserves as energy and protein requirements shift and may not be able to be met by feed intake. Tissue reserves are mobilized in early lactation and accreted thereafter, which can be used as a pool of labile amino acids and energetic substrates used at the beginning of the subsequent lactation. Nutritional strategies can be employed to mitigate extensive tissue loss and gain as a management tool to enhance dairy performance. The first study's objective was to evaluate the effects of high oleic soybean oil (HOSO) supplementation on milk production, body composition, and apparent total tract digestibility variables. A cross-over design with 21-d periods was employed with thirty Holstein cows (n = 16 primiparous, n = 14 multiparous at 87 ± 26 DIM at start of trial). Treatments consisted of a control (CON) with no added soybean oil and a HOSO diet with 1.5% diet dry matter (DM) of high oleic soybean oil added. Milk production and DM were collected on the last 7 d of each period. Body weights (BW), milk, and fecal samples were collected on the last 3 d of each period. Ultrasound scans of the <i>longissimus dorsi </i>muscle and body condition scores (BCS) were collected on the last d of each period. Dry matter intake, milk production, and milk component yields were not impacted by HOSO supplementation, although milk fat concentration tended to be greater for HOSO cows. A treatment by parity interaction and treatment by parity tendency were observed for BCS and BW, respectively, with multiparous HOSO cows having increased BCS and BW compared to CON with no effect on primiparous cows. Compared with CON, HOSO increased backfat depth (BFD) by 0.44 mm as well as apparent total tract fat digestibility by 12 percentage units with no differences observed in muscle<i> </i>depth. The second study’s objective was to evaluate changes in <i>longissimus dorsi </i>depth and its impact on production parameters in cows with two muscle phenotypes. Forty multiparous dairy cows were categorized into two muscle phenotype groups of high muscle (HM; n = 18) and low muscle (LM; n = 22) as determined by <i>longissimus dorsi </i>depth immediately after parturition. Ultrasound images of the <i>longissimus dorsi </i>and body weights were collected monthly from 0 to 300 DIM. Blood samples were collected from the coccygeal vessels on d 7, 150, and 300 and analyzed for 3-methylhistidine (3-MH), creatinine, non-esterified fatty acids (NEFA), and insulin. Milk weight data was collected daily using AfiFarm and milk components were collected from monthly DHIA test day data. Neither BW nor BFD were different between HM and LM cows at any timepoint, although, a DIM effect was observed. Cows lost BW from 0 to 60 DIM and BFD from 0 to 90 DIM and began increasing thereafter until 300 DIM. A muscle group by DIM interaction was observed where HM cows had more <i>longissimus dorsi </i>depth (LDD) at 0 and 300 DIM and tended to have more at 60 DIM compared to LM cows, with no differences at any other timepoint. In both muscle groups, LDD was decreased from 0 to 60 DIM but substantial LDD accretion did not occur until 240 DIM. No differences were observed between muscle groups for blood analytes; however, significant DIM effects were observed and corresponded to the observed changes in body tissue reserves. Daily milk production was grouped by stage of lactation with DIM groups consisting of early lactation from 0 to 60 DIM, mid-lactation from 60-240 DIM, and late lactation from 240 to 300 DIM. There was a muscle group by DIM group interaction for daily milk yield as HM cows produced more milk/d in early and mid-lactation but produced less milk in late lactation compared to LM cows. The results of these studies indicate that oleic acid can be supplemented to lactating dairy cattle to increase adipose tissue reserves with no effect on muscle reserves. This could potentially be caused by the lack of muscle accretion throughout mid-lactation whereas adipose tissue is gained. These results also describe that nutrition and muscle phenotype play a role in body composition throughout lactation.</p>

In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF. The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness. The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine. In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.

<p>The main objective of the study was to investigate skeletal muscle force production during isometric contractions, active muscle stretches and shortenings. The motivation behind this work is to improve the dominant model of muscle contraction force generation based on the theories of Hill. The effect of force modification was observed after concentric and eccentric contractions and also stretch-shortening cycles. It has been shown that this force modification is not related to lengthening/shortening velocity, and the steady-state force after non-isometric contractions can be well described by initial isometric force and mechanical work performed by and on the muscle during length variations. The time constants calculated for isometric force redevelopment appeared to be in certain relations with those for initial isometric force development, an observation which extended our basis for ongoing muscle modeling. The main method of the project consists in two extensive series of experiments on mouse skeletal muscles. Analysis of the first series of experiments, concentric contractions, with an emphasis on the force depression has been presented in Paper 1. Paper 2 is based on contractions with various stretches and shortenings as well as their combination, force modification and its predictor are the quantities of interest. The third part of the project is also based on the second series of experiments. Timing aspects of the force production were calculated there.</p>

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is a condition in which peripheral skeletal muscle undergoes myopathic changes which impair muscle function, limit physical performance, and can lead to significant disability. While the etiology of the dysfunction is unknown, this study was conducted to test the hypothesis that chronic hypoxemia leads to alterations in oxygen transport and muscle function. A primary objective was to validate elastase-induced emphysema in rats as an animal model of skeletal muscle dysfunction in COPD.Arterial blood gases were used to determine the severity of hypoxemia and sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to determine the proportions of myosin heavy chain isoforms I, IIa, IIx, and IIb. Measures of microvascular oxygenation and blood flow in the spinotrapezius muscle allowed for determination of both convective and diffusive oxygen supply to the muscle, as well as calculation of muscle oxygen consumption at rest and during electrically stimulated three-minute muscle contractions. Muscle performance measures included peak force, force-time integral, and fatigue index. Due to a presumed rat respiratory virus, which likely resulted in the control group being nearly as hypoxemic as the elastase-induced emphysema group, this study was not able to definitively test the hypothesis that chronic hypoxemia leads to both a diminished supply and demand of oxygen in skeletal muscle. Although many of the results of the present study were not statistically significant, they exhibited consistent trends over time and are likely of physiological significance. All measures of muscle performance were lower in the emphysema group. In addition, spinotrapezius muscle oxygen consumption and blood flow were lower in the emphysema group. The addition of supplemental oxygen during isolated, small-muscle mass exercise did increase the force-time integral by ~18% in both groups, suggesting that muscle work in these hypoxemic animals may be limited by oxygen supply. Thus, the data on muscle fiber type, oxygen consumption and muscle performance suggest that elastase-induced emphysema in rats leads to a similar skeletal muscle dysfunction that is observed in humans with COPD, and indicates that it is a valid animal model of skeletal muscle dysfunction in COPD.