Gale M. Strasburg

And the constantly changing conception of muscle which has resulted, there has not been … any striking application of the principles of modern biochemistry to the technology of handling of meat animals and meat." Marsh goes on to observe: "… one's attention is drawn … to [Bate-Smith's] thoughts on the bewildering growth of fundamental knowledge, on 'the constantly changing conception of muscle,' and on the absence of 'any striking application of these principles' to meat technology. These views are even more relevant today than they were in 1948; for although our knowledge of meat and its qualities has grown enormously in the intervening years, knowledge of muscle and its behavior has increased even more. The gap between discovery and application is still widening. The output of new information, the development of new concepts, the postulation of new theories ---these are accelerating at such a rate that we may liken muscle biology to an expanding universe and ourselves to the particles within it, fast receding from each other and suffering the inevitable consequence of greater and greater isolation. Compounding the problem, new branches of science splinter from the old, and studies which a few years ago were clearly together within one particular discipline are now so altered that they are scarcely recognizable as relatives." Since those words were published over five decades ago, we have witnessed an exponential growth of these "new branches of science" and a shift in our approach to doing science. In the following sections, we contrast the historical approach to addressing scientific questions with that which is increasingly dominant in today's culture. We then illustrate with examples the importance of using hypothesis-generating (data-driven) science to generate hypotheses that address mechanistic questions. Finally, we propose a framework for integrating the two approaches and application of knowledge.Historically, science made incremental advances by asking a question based on observations, formulating a hypothesis, explaining the observations, and testing that hypothesis with an appropriate experimental design. The results of the experiment(s) would be analyzed and interpreted, and the conclusions drawn would lead to a new hypothesis. Contrast this hypothesis-based approach with that used today by many muscle biologists (including ourselves) as we routinely employ techniques from these "new branches of science" such as nucleic acid sequencing, transcriptomics, proteomics, lipidomics, metabolomics to address broad-based biological questions. (Figure 1). For example, how do the muscles of the meat animal respond to heat or cold stress, and what are the implications for meat quality? Applying transcriptome analysis, for example, may provide information on genes that are upor down-regulated in response to a biological perturbation or stress. These genes may be organized into clusters that are associated with specific biological pathways and functions, and in turn, may suggest a hypothesis to be tested that would not only define a mechanism by which muscle responds to the stress, but also, in so doing, may suggest a dietary or other management intervention that may result in reduced incidence of myopathy due to a stress, and improvement in meat quality. We contend that collectively, we all too frequently conduct and publish our studies that conclude with pious words such as "these results provide new information that may be useful for breeders and producers for improvement of avian health and meat quality." If that is our endpoint, we have missed the opportunity to generate hypotheses that could lead to discovery of mechanisms that indeed lead to strategies that can be applied at the level of breeder and producer. This shift in research paradigms introduces both opportunities and challenges. On one hand, hypothesis-generating research allows the discovery of new mechanisms that may otherwise remain uncovered. On the other hand, it can lead to data overload, making it difficult to pinpoint the most significant findings. To fully utilize the power of -omics research, scientists must integrate these exploratory methods with traditional hypothesis-driven research. Although exploratory data alone may provide new insights, it is critical that researchers develop testable hypotheses from these findings, ensuring that molecular discoveries lead to practical, mechanistic insights. By integrating these approaches, we can narrow the gap between data collection and actionable applications. In the following section, we offer examples of work that illustrate this approach Examples of -Omics Research as Hypothesis-generating Research Example 1: Sporer et al. (3) investigated temporal changes in breast muscle gene expression in two lines of turkeys at three stages of muscle development: 18d embryo (when the breast muscle is undergoing hyperplasia), 1-day post-hatch (when muscle is undergoing hypertrophy), and 16 weeks (market age of the turkey). Of the >3000 differentially expressed genes (FDR < 0.0001), three were selected for further investigation for their role in muscle growth and development: versican (VCAN), matrix Gla protein (MGP), and death-associated protein (DAP). These selections were based on the magnitude of expression changes with developmental stage coupled with lack of information on their roles in myogenesis (4). Velleman et al. then used small interfering RNA (siRNA) in cultured satellite cells to characterize the roles of these genes in proliferation and differentiation (4). Subsequent studies further documented the critical role of DAP in satellite cell proliferation and differentiation (5,6). Although the significance of these genes is now evident, the next steps should extend this work to define the specific functions of these gene products.Example 2: Lake et al. (7) used genome-wide association and transcriptome studies to identify genes associated...

Satellite cells (SCs) comprise a heterogeneous population of muscle stem cells. Thermal stress during the first week after hatch alters proliferation, myogenesis, and adipogenesis of SCs of turkey pectoralis major (p. major) muscle via mechanistic target of rapamycin (mTOR) and wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) pathways. Pivotal genes in mTOR and Wnt/PCP pathways are mTOR and frizzled-7 (Fzd7), respectively. The objective of this study was to determine the differential effects of thermal stress on SDC4 and CD44 expression in turkey p. major muscle SCs and how the expression of SDC4 and CD44 is modulated by the mTOR and Wnt/PCP pathways. Satellite cells were isolated from the p. major muscle of 1-week-old faster-growing modern-commercial (NC) turkeys and slower-growing historic Randombred Control Line 2 (RBC2) turkeys, and were challenged with hot (43°C) and cold (33°C) thermal stress for 72 h of proliferation followed by 48 h of differentiation. The NC line SCs were found to contain a lower proportion of SDC4 positive and CD44 negative (SDC4⁺CD44⁻) cells and a greater proportion of SDC4 negative and CD44 positive (SDC4⁻CD44⁺) cells compared to the RBC2 line at the control temperature (38°C) at both 72 h of proliferation and 48 h of differentiation. In general, at 72 h of proliferation, the proportion of SDC4⁺CD44⁻ cells decreased with heat stress (43°C) and increased with cold stress (33°C) relative to the control temperature (38°C) in both lines, whereas the proportion of SDC4⁻CD44⁺ cells increased with heat stress and decreased with cold stress. In general, the expression of SDC4 and CD44 in the NC SCs showed greater response to both hot and cold thermal stress compared to the RBC2 cells. Knockdown of mTOR or Fzd7 expression increased the proportion of SDC4⁺CD44⁻ cells while the proportion of SDC4⁻CD44⁺ cells decreased during differentiation with line differences being specific to treatment temperatures. Thus, differential composition of p. major muscle SCs in growth-selected commercial turkey may be resulted, in part, from the alteration in SDC4 and CD44 expression. Results indicate differential temperature sensitivity and mTOR and Wnt/PCP pathway responses of growth-selected SC populations and this may have long-lasting effect on muscle development and growth.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

Satellite cells (SCs) are stem cells responsible for post-hatch muscle growth through hypertrophy and in birds are sensitive to thermal stress during the first week after hatch. The mechanistic target of rapamycin (mTOR) signaling pathway, which is highly responsive to thermal stress in differentiating turkey pectoralis major (p. major) muscle SCs, regulates protein synthesis and the activities of SCs through a downstream effector, S6 kinase (S6K). The objectives of this study were: 1) to determine the effect of heat (43°C) and cold (33°C) stress on activity of the mTOR/S6K pathway in SCs isolated from the p. major muscle of one-week-old faster-growing modern commercial (NC) turkeys compared to those from slower-growing Randombred Control Line 2 (RBC2) turkeys, and 2) to assess the effect of mTOR knockdown on the proliferation, differentiation, and expression of myogenic regulatory factors of the SCs. Heat stress increased phosphorylation of both mTOR and S6K in both turkey lines, with greater increases observed in the RBC2 line. With cold stress, greater reductions in mTOR and S6K phosphorylation were observed in the NC line. Early knockdown of mTOR decreased proliferation, differentiation, and expression of myoblast determination protein 1 and myogenin in both lines independent of temperature, with the RBC2 line showing greater reductions in proliferation and differentiation than the NC line at 38° and 43°C. Proliferating SCs are more dependent on mTOR/S6K-mediated regulation than differentiating SCs. Thus, thermal stress can affect breast muscle hypertrophic potential by changing satellite cell proliferation and differentiation, in part, through the mTOR/S6K pathway in a growth-dependent manner. These changes may result in irreversible effects on the development and growth of the turkey p. major muscle.

1. Turkey production has increased dramatically as genetic selection has succeeded in increasing body weight and muscle yield to fulfil increasing consumer demand. However, producing fast-growing, heavily muscled birds is linked to increased heat stress susceptibility and can result in pale, soft, exudative (PSE) meat. Previous studies indicated that pyruvate dehydrogenase kinase 4 (PDK4) is significantly reduced in PSE samples, suggesting this as a candidate gene associated with the development of this problem. 2. The objective of this study was to determine whether pre-market thermal challenge results in PSE meat as a result of differential expression of PDK4. Two genetic lines of turkeys were used in this study; the Randombred Control Line 2 (RBC2) and a commercial line. Turkeys were exposed to a pre-market thermal challenge of 12 h at 35°C followed by 12 h at 27°C for five days. Birds were slaughtered and processed according to industry standards. Pectoralis major samples were categorised as PSE or normal based on marinade uptake and cook loss indicators. In the first experiment, the relative expression of pyruvate dehydrogenase (PDH) and the phosphorylation state of PDH in normal and PSE turkey meat were analysed by western blotting. In the second experiment, the same samples were used to measure metabolite levels at 5 min post-mortem, comparing the normal to the PSE samples. 3. The results of the first experiment showed that PSE samples had significantly lower total PDH (P=0.029) compared to normal meat. However, there was no significant difference in the degree of phosphorylation of sites 1, 2, or 3. In the second experiment, there were no significant differences in glycogen, lactate, glycolytic potential, or ATP when comparing PSE to control samples. 4. These results suggested that a reduction in PDK4 expression alone does not explain the development of PSE meat.

Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced DAP expression on global gene expression in proliferating and differentiating turkey pectoralis major muscle satellite cells. Small interfering RNA was used to knock down expression of DAP and the transcriptome was subsequently profiled using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were corroborated using quantitative real-time PCR. In proliferating cells, 458 loci, resulting in 378 uniquely annotated genes, showed differential expression (false discovery rate, FDR < 0.05). Pathway analysis highlighted altered eukaryotic translational initiation factors (eIFs) signaling, protein ubiquitination, sirtuin signaling, and mechanistic target of rapamycin (mTOR) signaling as the primary pathways affected in the knockdown proliferating cells. The findings underpinned the potential DAP involvement in cell proliferation of turkey satellite cells through the coordination between protein synthesis and cell cycle. In differentiating cells, 270 loci, accounting for 189 unique genes, showed differential expression (FDR < 0.05). Decreased expression of genes encoding various myofibrillar proteins and proteins involved in sarcoplasmic reticulum calcium flux suggests that DAP may affect regulation of calcium homeostasis and cytoskeleton signaling. This study provides the first evidence that reduced expression of DAP significantly alters the transcriptome profile of pectoralis major muscle satellite cells, thereby reducing proliferation and differentiation.

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as “non-defective” (n = 4), “medium-WS” (n = 6), “heavy-WS” (n = 7) and “heavy-WS+WB” (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm². Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated.

Diospyros mafiensis F. White is a medicinal shrub or small tree (6 m tall) widely distributed in the Zanzibar-Inhambane regional mosaic and traditionally used to treat leprosy, diarrhoea, and skin fungal infections in Tanzania and Mozambique. Our objective was to determine the anti-aflatoxigenic properties of compounds fromD. mafiensis root bark against vegetative growth, sporulation and aflatoxin production byAspergillus flavus andAspergillus parasiticus. Bioassay-guided extraction, fractionation, and isolation of bioactive compounds usingA. parasiticus B62 were employed. The bioactive compounds were elucidated using¹H and¹³CNMR and LC-MS. Growth inhibition was determined by measuring the colony diameter ofA. flavus AF3357 andA. parasiticus SU-1 ATCC56775. Inhibitory effects on sporulation were estimated using a haemocytometer. Total aflatoxin was quantified by direct competitive enzyme-linked immunosorbent assay (ELISA). Bioactive compounds diosquinone (DQ) and 3-hydroxydiosquinone (3HDQ) were identified. DQ weakly inhibitedA. flavus andA. parasiticus vegetative growth (MIC₅₀ > 100 µg/ml) and 3HDQ strongly inhibitedA. flavus (MIC₅₀ = 14.9 µg/ml) andA. parasiticus (MIC₅₀ = 39.1 µg/ml). DQ strongly reduced total aflatoxin production byA. flavus from 157 to 36 ng/plate, and byA. parasiticus from 1,145 ng/plate to 45 ng/plate at 100 µg/ml. 3HDQ reduced total aflatoxin production byA. parasiticus from 1,145 to 32 ng/plate; stimulated production byA. flavus from 157 to 872 ng/plate at 12.5 µg/ml but reduced to 45 ng/plate at 100 µg/ml. In summary, DQ and 3HDQ could be used as natural antifungal compounds to prevent mould growth and aflatoxin accumulation in food and feed.

Climate change poses a multi-dimensional threat to food and agricultural systems as a result of increased risk to animal growth, development, health, and food product quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells cultured under cold or hot thermal challenge to better define molecular mechanisms by which thermal stress alters breast muscle ultrastructure. Satellite cells isolated from the pectoralis major muscle of 7-weeks-old male turkeys from two breeding lines (16 weeks body weight-selected and it’s randombred control) were proliferated in culture at 33 °C, 38 °C or 43 °C for 72 h. Total RNA was isolated and 12 libraries subjected to RNAseq analysis. Statistically significant differences in gene expression were observed among treatments and between turkey lines with a greater number of genes altered by cold treatment than by hot and fewer differences observed between lines than between temperatures. Pathway analysis found that cold treatment resulted in an overrepresentation of genes involved in cell signaling/signal transduction and cell communication/cell signaling as compared to control (38 °C). Heat-treated muscle satellite cells showed greater tendency towards expression of genes related to muscle system development and differentiation. This study demonstrates significant transcriptome effects on turkey skeletal muscle satellite cells exposed to thermal challenge. Additional effects on gene expression could be attributed to genetic selection for 16 weeks body weight (muscle mass). New targets are identified for further research on the differential control of satellite cell proliferation in poultry.

Aflatoxin, a mycotoxin found commonly in maize and peanuts worldwide, is associated with liver cancer, acute toxicosis, and growth impairment in humans and animals. In Tanzania, sunflower seeds are a source of snacks, cooking oil, and animal feed. These seeds are a potential source of aflatoxin contamination. However, reports on aflatoxin contamination in sunflower seeds and cakes are scarce. The objective of the current study was to determine total aflatoxin concentrations in sunflower seeds and cakes from small-scale oil processors across Tanzania. Samples of sunflower seeds (n = 90) and cakes (n = 92) were collected across two years, and analyzed for total aflatoxin concentrations using a direct competitive enzyme-linked immunosorbent assay (ELISA). For seed samples collected June-August 2014, the highest aflatoxin concentrations were from Dodoma (1.7–280.6 ng/g), Singida (1.4–261.8 ng/g), and Babati-Manyara (1.8–162.0 ng/g). The highest concentrations for cakes were from Mbeya (2.8–97.7 ng/g), Dodoma (1.9–88.2 ng/g), and Singida (2.0–34.3 ng/g). For seed samples collected August-October 2015, the highest concentrations were from Morogoro (2.8–662.7 ng/g), Singida (1.6–217.6 ng/g) and Mbeya (1.4–174.2 ng/g). The highest concentrations for cakes were from Morogoro (2.7–536.0 ng/g), Dodoma (1.4–598.4 ng/g) and Singida (3.2–52.8 ng/g). In summary, humans and animals are potentially at high risk of exposure to aflatoxins through sunflower seeds and cakes from micro-scale millers in Tanzania; and location influences risk.