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Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm 2 , gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96 h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168 h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature ( p ≥ 0.1148), but culturing broiler SC at 41°C for 96 h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations ( p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C ( p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage ( p < 0.0001) and tended to result in the formation of thinner myotubes ( p = 0.061) without impacting the density of differentiated cells ( p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gregg, Hutson, Flees, Starkey and Starkey.)

Muscle satellite cells (MSCs) are myogenic stem cells that play a critical role in post-hatch skeletal muscle growth and regeneration. Activation of regeneration pathways to repair muscle fiber damage requires both the proliferation and differentiation of different MSC populations as well as the function of resident phagocytic cells such as anti-inflammatory and pro-inflammatory macrophages. The Wooden Breast (WB) phenotype in broiler chickens is characterized by myofiber degeneration and extensive fibrosis. Previous work indicates that the resident MSC populations expressing the myogenic regulatory factors, Myf-5 and Pax7 are larger and more proliferative in broilers severely affected with WB vs. unaffected broilers. To further characterize the cellular and molecular changes occurring in WB-affected muscles, samples from pectoralis major (PM) muscles with varying severity of WB (WB score 0 = normal; 1 = mildly affected; 2 = severely affected) were collected at 25 and 43 days post-hatch ( n = 8 per score per age) and processed for cryohistological and protein expression analyses. Collagen per field and densities of macrophages and MyoD+, Myf-5+, and Pax7+ MSC populations were quantified on immunofluorescence-stained cryosections. Relative collagen protein expression was quantified by fluorescent Western Blotting. In both 25 and 43-days-old broilers, the proportion of collagen per field ( P ≤ 0.021) and macrophage density ( P ≤ 0.074) were greater in PM exhibiting severe WB compared with normal. At day 43, populations of MyoD+, Myf-5+:MyoD+ MSC were larger and relative collagen protein expression was greater in WB-affected vs. unaffected broilers ( P ≤ 0.05). Pax7+ MSC relative to total cells was also increased as WB severity increased in 43-days-old broilers ( P ≤ 0.05). Densities of Myf-5+ ( P = 0.092), MyoD+ ( P = 0.030), Myf5+:MyoD+ ( P = 0.046), and Myf-5+:MyoD+:Pax7+ ( P = 0.048) MSC were greater in WB score 1 birds compared with WB score 0 and 2 birds. Overall, alterations in the resident MSC and macrophage populations and collagen protein content were observed in WB-affected muscle. Further investigation will be required to determine how these changes in cell population kinetics and local autocrine and paracrine signaling are involved in the apparent dysregulation of muscle maintenance in WB-affected broilers., (Copyright © 2020 Ferreira, Kindlein, Flees, Shortnacy, Vieira, Nascimento, Meloche and Starkey.)