Browsing by Subject "Satellite cells"
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Item Effect of rate of gain during the stocker period on beef cattle skeletal muscle development, satellite cell activity, and marbling development(2012-05) Vaughn, Mathew; Starkey, Jessica D.; Johnson, Bradley J.; Ballou, Michael A.It is estimated that 76% of the yearling beef cattle calf crop enters a stocker management system. Stocker systems are used to prepare cattle for finishing by adapting them to drinking out of fountain waters and eating out of a bunk. Stocker programs allow producers to utilize homegrown feedstuffs, take advantage of grazing opportunities, delay finishing, target a specific market, and promote skeletal growth in small-framed cattle. Research has shown that rate of gain during the stocker period affects tissue growth and feedlot performance. Cattle that are fed at lower rates of gain during the stocker period experience a period of compensatory gain upon entering the finishing phase. Cattle that experience lower rates of gain during the stocker period require more days on feed to reach the same carcass composition as cattle that are fed diets to achieve high rates of gain during the stocker period. To determine the effects of stocker programs on skeletal muscle development, satellite cell activity and marbling development, fall-weaned angus steers (n = 72; 259 ± 28 kg) from the Oklahoma State University cow herd were randomly assigned to 1 of 4 stocker systems. Stocker systems included: 1) grazing dormant native range (NR) supplemented with cottonseed (1.0 kg∙steer-1∙d-1) followed by season-long grazing on summer pasture (CON); 2) grazing dormant NR supplemented with corn based supplement (1% of BW) followed by short season grazing on summer pasture (CORN); 3) grazing winter wheat pasture at high stocking density (3.0 steers/ha) to produce a moderate ADG (LGWP); and 4) grazing winter wheat pasture at low stocking density (1.0 steers/ha) to produce high ADG (HGWP). Steers continued grazing until the average BW of the treatment group reached a common BW of approximately 375 kg, at which time steers were transitioned into a common finishing diet and fed to a common backfat thickness of at least 1.18 cm. At the end of the stocker and finishing phases, four steers per treatment were harvested and longissimus muscle (LM) was collected for analysis. LM fiber cross-sectional area, fiber type, Pax7+ cell density, Myf-5+ cell density, Pax7+/Myf-5+ cell density, capillary density, and nuclear density were determined by immunoflourescence on cryosections. Bovine satellite cells (BSC) from the LM of each steer were cultured, satellite cell activity, fusion capacity and gene expression were analyzed. 3T3-L1 preadipocytes were grown to confluence and subsequently exposed to conditioned media from mitotically active and differentiated BSC, to detect the effects of BSC produced factors on preadipocyte differentiation. At the conclusion of the stocker phase, LGWP and HGWP steers had greater (P < 0.01) muscle fiber cross sectional area when compared to CON and CORN steers. CON steers tended (P = 0.12) to have more Type 1 muscle fibers compared to all other treatments. CORN steers had a lower (P = 0.05) percentage of cells within the LM co-expressing Pax7 and Myf-5 when compared to all other treatments. At the conclusion of the finishing phase, relative gene expression of satellite cells after 14 d in culture were similar among treatments, except HGWP steers had greater (P = 0.03) relative abundance of Pax7 than all other treatments. LGWP steers tended (P = 0.06) have greater Pax7 expression in culture and numerically lower myonuclei when compared to all other treatments. 3T3-L1 preadipocytes exposed to conditioned media from HGWP BSC at 96 h in culture had greater (P = 0.05) PPARγ and lower (P = 0.04) FABP4 mRNA abundance when compared to preadipocytes treated with media from CON and CORN BSC, with preadipocytes treated with media from LGWP BSC being intermediate. Overall, these data suggest that rate of gain and stocker management strategy during the stocker period can impact skeletal muscle characteristics, satellite cell activity, and marbling development. Further research is required to elucidate the mechanisms behind these effects, but factors from BSC do seem to affect adipocyte differentiation.Item Growth characterization of callipyge cells(Texas Tech University, 2004-08) Fleming, Jolena NCallipyge is a mutation that causes post-natal muscle hypertrophy in the loin and hindquarters of domestic sheep. Although much research has been conducted in the past 20 years to explain this mechanism, no clear answer has been reached. This thesis contains three separate studies to try to explain the mutated phenotype of large muscle fibers and leaner carcasses. Study I consists of two sets of data on the proliferation rates of satellite cells from the muscles of normal and mutated sheep. Satellite cells are mononucleated cells located beneath the basement membrane of skeletal muscles and incorporate themselves into existing muscle fibers for muscle growth or repair. Satellite cells were isolated from the semimembranosus muscles of callipyge CLPG/+ (n = 13), maternal non-expressers +/CLPG (n = 6), homozygous CLPG/CLPG (n = 6), and normal +/-t- (n = 13) lambs ranging from 2 to 8 wks of age. Primary isolated satellite cell growth rates were taken on days five, six, and seven of culture, and calculated as a percent gain from the day five counts to standardize. A genotype effect was seen in primary counts (P = 0.0005), with the non-expressers having the highest growth rates at 231.74 + 111.44%, followed by callipyge 175.57 + 48.72%, normal 137.81 + 42.27%, and homozygous 99.81 + 22.89% gain over the day five counts. Secondary cultured satellite cell plates were made by adding lO^ cells to plates and counting 24, 48, and 72 h after plating. Genotype was also significant in the secondary counts (P = 0.0358). Proliferation rates were non-expressers 404.39 ± 150.40%, homozygous 309.34 + 88.23%, callipyge 236.41 + 49.25%, and normal 193.85+ 51.11%. Study 2 involved two experiments evaluating the plasma leptin levels of normal, callipyge. and homozygous sheep at differing physiological states. Leptin is a peptide hormone produced in white adipose tissue of mammals. This hormone is involved in a feedback loop to help maintain body composition homeostasis. Large amounts of adipose tissue produce high levels of leptin, which signal the hypothalamus to trigger a decrease in appetite and an increase in physical activity to help reduce the level of fat in the body. Experiment I quantified the plasma leptin levels in callipyge (n = 10) and normal ewes (n = 10) during mid-gestation, as well as 60 d old callipyge (n = 10) and normal (n = 10) lambs. No genotypic effect was observed in either the ewes (P = 0.78) or the lambs (P = 0.95). Experiment 2 involved quantifying the plasma leptin levels of callipyge (n = 6), homozygous (n = 8), and normal (n = 8) ewes through late gestation, lambing, and early lactation. A significant genotype effect was observed in Experiment 2 (P = 0.0005), with the normal ewes having the highest mean leptin levels at 9.13 +.0.93 ng/ml, foUowed by homozygous ewes at 8.11 + 0.70 ng/ml. The callipyge ewes showed the lowest mean leptin levels at 5.41 +_0.40 ng/ml. Study 3 consisted of quantifying the relative expression levels of five muscle regulatory factors in cultured myoblast and tissue samples from callipyge (n = 3), nonexpresser (n = 3), homozygous (n = 3), and normal (n = 3) lambs at ages 2, 4, and 8 wks. We evaluated the expression level of four positive muscle growth regulators including MyoDl, myf-5, myogenin, and MRF4. One negative regulator, myostatin, was also evaluated. Total RNA was isolated from each sample. Total cDNAs were made via reverse transcription, then specific factors were quantified using Real-Time PCR. Ribosomal RNA levels were used to standardize the amount of total RNA per sample. A significant age x genotype interaction was observed in all factors tested. In the cultured cells, the expression levels of MyoDI (p = 0.0002), myogenin (p < 0.0001), MRF4 (p < O.OOOI), and myostatin (p = 0.0125) were all highest at 4 wks of age. Myf-5 expression was lowest in the 4 wk cells (p < 0.0001). Only a few of the most prominent comparisons are reported between genotypes here. The 4 wk non-expressers showed the highest overall expression levels of MyoDl (p < 0.0001 versus all others), myogenin (p < 0.0001 versus all others), MRF4 (p < 0.0001 versus all others), and myostatin (p < 0.001 versus all others) and showed the overall lowest expression level of myf-5 (p < 0.0001). The callipyge cells showed the overall lowest expression of MyoDI (p < 0.0001 versus nonexpresser, p = 0.0002 versus normal, p = 0.3346 versus homozygous), myogenin (p < 0.0001 versus non-expresser, p = 0.0205 versus homozygous, p = 0.6105 versus normal), MRF4 (p < 0.0001 versus non-expresser and homozygous, p = 0.1717 versus normal) at 4 wks. At 2 wks of age, the callipyge cells showed the highest expression level of myf-5 (p < O.OOOI versus all others) and the lowest expression of myostatin (p < 0.0001 versus non-expresser and normal, p = 0.2803 versus homozygous). For most comparisons, the homozygous and normal cells were intermediate in their expression. In the 2 wk tissue samples, the non-expresser showed the highest expression of myf-5 (P < 0.0001), myogenin (P < 0.0001), MRF4 (P < 0.0001), and myostatin (P < 0.0001). No detectable levels of MyoDl were observed in the 2 wk tissue. No data were obtained from the 4 and 8 wk tissue samples due to low RNA quality. These three studies do indicate genotypic effects in the sheep possessing the callipyge mutation. In Study 1 with the cell proliferation rates, it is puzzling to see the two non-expressing carriers of the mutation have the highest growth rates. However, the callipyge cells showed their highest growth rates at the youngest age tested, so perhaps the expressers of the phenotype begin their muscle growth earlier in life and exhibit more plasticity throughout their entire development. The leptin data in Experiment 2 of Study 2 indicate that the callipyge ewes possess lower fat stores, even during gestation. This may help explain how these sheep are more efficient in production and muscle accretion, since more of their nutrients would go toward growth versus fat deposition. In Study 3, the differences seen between genotypes in the expression levels of different muscle growth factors may provide further explanation for early post-natal growth in callipyge sheep. The fact that expression levels between callipyge and non-expresser lambs at 4 wks were so different may provide clues toward understanding the phenotypic expression.Item Influence of insulin-like growth factor-I on skeletal muscle regeneration(2012-12) Hammers, David Wayne; Farrar, Roger P; Suggs, Laura J; Adamo, Martin L; Sweeney, H. Lee; Thompson, Wesley J; Ivy, John LSkeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury.Item New models of regenerating VML injured rat skeletal muscle : single muscle fiber injection and resistance exercise(2015-12) Song, Taejeong, 1978-; Farrar, Roger P.; Baker, Aaron; Castelli, Darla M; Suggs, Laura; Brothers, Robert Matt; Thompson, WesleyVolumetric muscle loss (VML) is a traumatic soft tissue injury that is common in recent battle fields and causes permanent losses of muscle mass and function in affected muscle. Since a new VML injury model has been developed in our lab which defects large mass of rat lateral gastrocnemius (LGAS), several sources of extracellular matrix (ECM) and stem cells including adipose tissue derived stem cells and mesenchymal stem cells have been utilized to enhance the regeneration of defected muscle. In the current study, regenerative effects of resistance exercise and satellite cells injection after the VML injury were tested. Satellite cells transplanted via single muscle fiber injection significantly enhanced generation of a specific force of defected muscle possibly via suppressing fibrotic tissue build-up and inducing significant hypertrophy of regenerating muscle at the sites of injury. However it did not increase muscle mass and tetanic force of the defected LGAS. Resistance exercise intervention after the injury improved all morphological and functional recoveries. Defected muscle mass and functions (tetanic and specific forces) were significantly increased in the resistance exercised group compared to the non-exercised. Cross sectional area (CSA) of mature and regenerating muscle fibers in injured muscle were also significantly increased by the resistance training. Single muscle fiber injection with resistance exercise did not further increase muscle mass or functions, but it had an impact on decreasing accumulation of the non-functional (fibrotic tissue and residual injured) area. These results indicate that resistance exercise is a great rehabilitative intervention after traumatic muscle injury to enhance muscle mass and function with or without satellite cells injection.