J. M. Reddish

Proteomic analysis of proteins associated with body mass and length in yellow perch, Perca flavescens.

The goal of commercial yellow perch aquaculture is to increase muscle mass which leads to increased profitability. The accumulation and degradation of muscle‐specific gene products underlies the variability in body mass (BM) and length observed in pond‐cultured yellow perch. Our objective was to apply a combination of statistical and proteomic technologies to identify intact and/or proteolytic fragments of muscle specific gene products involved in muscle growth in yellow perch. Seventy yellow perch randomly selected at 10, 12, 16, 20, and 26 wk of age were euthanized; BM and length were measured and a muscle sample taken. Muscle proteins were resolved using 5–20% gradient SDS‐PAGE, stained with SYPRO® Ruby and analyzed using TotalLab™ software. Data were analyzed using stepwise multiple regression with the dependent variables, BM and length and proportional OD of each band in a sample as a potential regressor. Eight bands associated with BM (R2 = 0.84) and nine bands with length (R2 = 0.85) were detected. Protein sequencing by nano‐LC/MS/MS identified 20 proteins/peptides associated with BM and length. These results contribute the identification of gene products and/or proteolytic fragments associated with muscle growth in yellow perch.

Comparative proteomic characterization of the sarcoplasmic proteins in the pectoralis major and supracoracoideus breast muscles in 2 chicken genotypes

The selection processes that have resulted in broiler (meat) and leghorn (eggs) chickens have had very different effects on the pectoralis major and supracoracoideus muscles. The objective of this study, therefore, was to analyze the one-dimensional proteomic profiles of sarcoplasmic protein fractions isolated from the p. major and supracoracoideus muscles collected from 10 chicks from each genotype to compare developmental differences. The sarcoplasmic protein fraction was analyzed by SDS-PAGE. The mean band percentages were analyzed using a mixed model, with strain and muscle type as main effects. Six bands were found to be significantly different across the 2 strains. Strain differences in glycogen phosphorylase, enolase, elongation factor 1, creatine kinase, fructose-bisphosphate aldolase, and glyceraldehyde 3-phosphate-dehydrogenase suggest a genotype-specific shift in energy metabolism during breast muscle growth and development.

Myosin heavy chain isoform expression is not altered in the pectoralis major muscle in selenium-deficient chickens recovering from exudative diathetic myopathy

The recovery of broiler chickens experiencing skeletal muscle myopathy caused by a selenium deficiency was compared with control broiler chickens in an age matched study by ultrastructural analysis of the pectoralis major (PM) muscle and examination of the temporal expression of the developmental fast skeletal myosin heavy chain (MyHC) isoforms. Selenium-deficient chicks showing signs of exudative diathesis (ED) were injected subcutaneously with sodium selenite in water and allowed to recover. At 0, 2, 5, 10, 20, and 30 d after selenium injection, a sample of the PM muscle was removed from selenium-deficient and control chicks for analysis. Ultrastructural analysis revealed vacuolization in the PM of selenium-deficient chicks with little or no visible damage to the sarcomere. Relative amounts of chicken ventricular, embryonic, neonatal, and adult fast skeletal MyHC isoforms were determined using chicken fast skeletal MyHC isoform specific monoclonal antibodies. The temporal expression of the developmental MyHC isoforms was similar in all chickens (P > 0.05). There was no expression of chicken ventricular MyHC observed in the PM of either group. These results indicate that chicken fast muscle recovering from exudative diathetic myopathy does not use the same pathways as chicken skeletal fast muscle regenerating from physical or toxic injury in which temporal expression of the MyHC isoforms is initially predominantly ventricular, then predominantly embryonic, neonatal, and finally predominantly adult developmental MyHC isoform.

Feed restriction delays developmental fast skeletal muscle myosin heavy chain isoforms in turkey poults selected for differential growth

Genetic selection has been very successful at significantly increasing BW and breast muscle proportion in commercial broiler and turkey strains. The mechanisms of breast muscle growth in poultry and the interactive effects of nutritional status and selection are not fully understood. The hypothesis underlying the current study is that feed restriction, simply as a vehicle for controlling early growth, would delay the temporal expression pattern of neonatal (nMyHC) and adult (aMyHC) fast skeletal muscle myosin heavy chain (MyHC) isoforms in the pectoralis major muscle of turkey poults. The poultry growth model used to evaluate this hypothesis consisted of a randombred control turkey line (RBC2) that represents commercial turkeys of the 1960s and a line developed from the RBC2 by selection for BW at 16 wk of age (F line). The F line has significantly heavier breast muscles than the RBC2 concomitant with increased BW, but the proportion of breast muscle relative to BW is similar. A quantitative indirect ELISA using fast skeletal MyHC isoform specific monoclonal antibodies revealed no significant line differences in the temporal expression of posthatch fast skeletal muscle MyHC in ad libitum fed poults. Feed restriction, however, altered the temporal expression patterns of nMyHC and aMyHC in both F line and RBC2 poults compared with the poults fed ad libitum.

Multivariate evaluation of 1-dimensional sarcoplasmic protein profile patterns of turkey breast muscle during early post-hatch development

Proteins are the main participants in metabolic pathways. However, the analysis of protein abundance patterns associated with those pathways is complicated by the large number of proteins involved. In this study, the objective was to present the application of principal component analysis (PCA) to permit the visualization of developmental proteomic patterns of sarcoplasmic proteins found in breast muscle. Different turkey genotypes and nutritional regimens were used to potentially increase the variability within the sarcoplasmic protein profile. Sarcoplasmic protein fractions from turkey breast muscle samples were collected at 6 ages between 7 to 24 d. Breast muscle samples were collected from 2 distinctly different turkey lines. The poults within each line were either ad libitum or restrict fed. Proteomic PCA plots showed a visual developmental pattern from 7 until 17 d. Multivariate ANOVA highlighted the effect of time point and feeding regimen among profile patterns. The use of different genotypes and feeding regimens influenced variability, which was measured by mean Euclidean distances and ellipses of the PCA plots. These treatment effects, however, did not mask the developmental patterns. After 17 d, the proteomic patterns converged, suggesting that a level of biological stability was achieved regardless of the genotype or treatment. The developmental pattern obtained by the PCA methodology can aid in the planning of more efficient experimental designs so the developmental stage of individuals can be more accurately assessed.

Abstract 3984: Effect of zeranol on beef skeletal muscle growth by differential image gel electrophoresis (DIGE)

Zeranol is a resorcylic acid lactone produced by fungi of the genus Fusarium that grows on corn, wheat, barley, oats and sorghum. As such Zeranol is a virtually unavoidable contaminant of crops used to feed animals that are consumed by humans. Zeranol has been shown to have a positive impact on muscle growth during the finishing phase of beef cattle production, was first approved and licensed for use as a growth promotant in cattle and sheep in the USA by FDA in 1969. However, the mechanism of this interaction is unknown but is likely related to changes in muscle specific proteins observable during the increased growth phase during finishing. To determine the affect of Zeranol on beef cattle muscle growth, we performed four DIGE experiments. We compared protein expression level changes between beef cattle treated with Zeranol and untreated beef cattle. On day zero, 10 cattle were implanted with 72 mg of Zeranol pellets, the other half (n = 10) were implanted with vehicle. The beef cattle were raised at the Ohio State University Department of Animal Sciences Beef Barn in accordance with industry standards for 120 days. At 60 days, half the implanted beef cattle and half of the control beef cattle were harvested and the remaining beef cattle were harvested at day 120. Muscle samples were analyzed by DIGE and bioinformatics Seven proteins associated with energy metabolism (creatine kinase, glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase I, aconitase 2, enolase I, and triosephophate isomerase), 5 proteins with regulation of muscle contraction (slow skeletal muscle troponin T, calsequestrin I, myosin light chains 2 and 3, tropomyosin 3), 3 structural proteins (alpha actin, myosin binding protein C and kelch repeat and BTB domain), a protein associated with myotube formation, dihydrolipoamide dehydrogenase, and HSP70, a protein associated with meat quality, and albumin were identified. The differential regulation of these proteins indicates that muscle growth in cattle implanted with Z is due to fast skeletal muscle. (Supported by NIH grant R01 ES 015212). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3984.