Satellite cells (SCs) are a heterogenous stem cell population (Schultz, 1974; Kuang et al., 2007; Biressi and Rando, 2010; Smith et al., 2013; Tierney and Sacco, 2016), functioning in hypertrophic growth of post-hatch muscle (Moss and Leblond, 1971; Cardiasis and Cooper, 1975). As multipotential stem cells (Asakura et al., 2001; Shefer et al., 2004), SCs with appropriate external stimuli have the potential to spontaneously convert to other lineages such as adipocytes (Asakura et al., 2001; Csete et al., 2001; Shefer et al., 2004; Redshaw and Loughna, 2012). Environmental temperature is an external stimulus that has been shown to alter the cellular function and fate of SCs in chickens (Halevy et al., 2001; Harding et al., 2015; Piestun et al., 2017; Patael et al., 2019) and turkeys (Clark et al., 2016, 2017; Xu et al., 2021). Piestun et al. (2017) and Patael et al. (2019) showed when chickens were challenged with chronic heat stress immediately after hatch for 2 weeks, proliferation of SCs was suppressed while fat deposition was increased in the pectoralis major (p. major; breast) muscle. In contrast, continuous cold stress for the same period of time increased chicken p. major muscle satellite cell (pmSC) proliferation without affecting fat content of the breast muscle (Patael et al., 2019). In vitro studies with cultured pmSCs from both chickens (Harding et al., 2015) and turkeys (Clark et al., 2017) have demonstrated an increased amount of intracellular lipid with heat stress. The increased lipid content was hypothesized to arise from the conversion of pmSCs to an adipogenic-like lineage under the thermal stress.

Conversion of SCs to an adipogenic-like lineage is regulated by adipogenic regulatory factors like peroxisome proliferator-activated receptor-gamma (PPARγ) (Tontonoz et al., 1994; Rosen et al., 1999; Vettor et al., 2009) and some members in the CCAAT/enhancer-binding protein (C/EBP) family (Freytag et al., 1994; Wu et al., 1995; Rosen and Macdougald, 2006) as well as the kruppel like factor (KLF) family (Rosen and Macdougald, 2006; Tahmasebi et al., 2013; Wu and Wang, 2013). The protein C/EBPβ is expressed during the initiation stages of adipogenesis leading to an adipogenic differentiation pathway (Ntambi and Young-Cheul, 2000). The expression of C/EBPβ promotes the transcription of PPARγ during intermediate stages of adipogenic differentiation (Wu et al., 1995; Clarke et al., 1997; Ntambi and Young-Cheul, 2000). The PPARγ is required to stimulate adipogenesis of multiple types of cells including myoblasts (Hu et al., 1995; Holst et al., 2003) and pre-adipocytes (Chawla et al., 1994; Bastie et al., 1999). After being challenged with chronic heat stress immediately after hatch, chicks exhibit increased intramuscular fat deposition coupled with increased C/EBPβ expression in the breast muscle (Piestun et al., 2017; Patael et al., 2019). Furthermore, both heat and cold stress modulates the adipogenic characteristics of pmSCs in chickens (Harding et al., 2015) and turkeys (Clark et al., 2017) through altering the expression of C/EBPβ and PPARγ. Taken together, these findings suggest post-hatch thermal stress may affect lipid homeostasis of pmSCs, in part, by modifying the expression of adipogenic transcriptional factors. The family of KLFs are zinc-finger transcriptional factors (Kaczynski et al., 2003) consists of members involved in adipogenesis (Wu and Wang, 2013). Some members like KLF1 (Zhang et al., 2015) and KLF7 (Kawamura et al., 2006; Cho et al., 2007) are mainly expressed in pre-adipocytes, and inhibit adipogenic differentiation. Thus, KLF1 and KLF7 are regarded as pre-adipocyte markers.

The secretory peptide, Neuropeptide-Y (NPY), functions as an extracellular signal regulating adipogenesis by interacting with NPY receptors (Yang et al., 2008; Baker et al., 2009; Shipp et al., 2016). The expression of NPY promotes lipid deposition by promoting adipogenesis (Kuo et al., 2007; Baker et al., 2009; Zhang et al., 2015; Shipp et al., 2016) and inhibiting lipolysis (Valet et al., 1990; Park et al., 2014; Zhang et al., 2014; Liu et al., 2017). At the transcriptional level, NPY was the gene in turkey pmSCs most downregulated by cold stress during differentiation (Reed et al., 2017). With heat stress, increased expression of NPY and its receptors was observed in turkey pmSCs (Clark et al., 2018). Because NPY promotes chicken adipocyte adipogenesis through regulating PPARγ and C/EBPα expression (Zhang et al., 2015), it may act as an upstream signal regulating adipogenic transcriptional factors during thermal-stress-induced adipogenic response in avian SCs.

Birds are homotherms and maintain their body temperature in a limited range (Yahav, 2000, 2015). For newly hatched poults, it is difficult to control body temperature due to an immature thermal regulatory system (Dunnington and Siegel, 1984; Modrey and Nichelmann, 1992; Shinder et al., 2007). Furthermore, SCs exhibit their peak mitotic activity (Mozdziak et al., 1994; Halevy et al., 2000) and temperature sensitivity (Halevy et al., 2001; Piestun et al., 2017; Patael et al., 2019; Halevy, 2020) during the first week after hatch. Newly hatched birds are frequently exposed to both hot and cold temperatures during handling and transportation from hatcheries to growing facilities. Heat stress during this period increases fat deposition in chicken breast muscle (Zhang et al., 2012; Patael et al., 2019; Halevy, 2020), which parallels the increased intracellular lipid content of cultured pmSCs in heat challenged chickens (Harding et al., 2015) and turkeys (Clark et al., 2017). Thus, early age thermal stress may detrimentally affect breast muscle structure and composition by altering the adipogenic properties of pmSCs.

Faster-growing chickens produce more metabolic heat than shower-growing lines because of a higher metabolic rate (Konarzewski et al., 2000). In addition, faster-growing heavy weight meat-type chickens have reduced capacity for heat dissipation due to their reduced breast muscle capillary density (Hoving-Bolink et al., 2000; Yahav, 2000; Havenstein et al., 2003; Joiner et al., 2014). Thus, when exposed to thermal stress, faster-growing poultry have reduced thermal tolerance (Berrong and Washburn, 1998; Yahav, 2000; Chiang et al., 2008), resulting in prolonged negative effects on breast muscle structure and growth (Joiner et al., 2014; Piestun et al., 2017; Patael et al., 2019). The proliferation rate and differentiation level of pmSCs in growth-selected faster-growing turkeys is more responsive to both heat and cold stress than non-selected slower-growing turkeys (Clark et al., 2016; Xu et al., 2021). In addition, proliferating pmSCs are more sensitive to both heat and cold stress than differentiating pmSCs (Xu et al., 2021).

The objective of the current study was to determine the effects of both heat and cold stress and selection for growth on intracellular lipid accumulation and adipogenic regulatory gene expression in pmSCs from 7-day-old Nicholas Commercial (NC) turkeys compared to those from Randombred Control Line 2 (RBC2) turkeys. The commercial turkeys used in this study were selected for growth characteristics including high growth rate and heavy breast muscling, whereas the RBC2 turkeys represent commercial turkeys in the mid-1960s (Nestor et al., 1969) and are slower-growing turkeys without heavy breast muscling. The temperature regimens used in the current study included all the possible combinations of heat and cold stress during pmSC proliferation and/or differentiation as reported by Xu et al. (2021). These regimens allowed a complete assessment of the effect of thermal stress on the adipogenic characteristics of the pmSCs from the two genetic lines. Data from the current study will provide insight on how thermal stress and selection for growth characteristics affects the synthesis of lipid by pmSCs during proliferation and differentiation in vitro. These findings have potential application in the development of management strategies to control lipid production by pmSCs which has been reported to affect the fiber organization and cellular properties of the breast muscle (Velleman et al., 2014; Patael et al., 2019).

Myogenic SCs are partially differentiated stem cells with multiple cellular fates (Asakura et al., 2001; Shefer et al., 2004) that respond to extrinsic stimuli including oxygen concentration (Csete et al., 2001; Redshaw and Loughna, 2012), nutritional status (Powell et al., 2014), and environmental temperature (Harding et al., 2015; Clark et al., 2017) resulting in changes in SC adipogenic characteristics (adipogenic gene expression or lipid synthesis). Post-hatch heat and cold stress has been shown to affect intramuscular fat content in the poultry breast muscle (Zhang et al., 2012; Piestun et al., 2017; Patael et al., 2019). The changes in intramuscular fat deposition may be, in part, attributable to the altered adipogenic potential of SCs (Smith and Johnson, 2014; Piestun et al., 2017; Patael et al., 2019).

During the immediate 7 days after hatch, SCs exhibit peak mitotic activity (Mozdziak et al., 1994; Halevy et al., 2000) and responsiveness to extrinsic temperatures (Halevy et al., 2001; Patael et al., 2019; Xu et al., 2021). Furthermore, selection for growth has increased the proliferation (Merly et al., 1998; Velleman et al., 2000) and thermal sensitivity (Clark et al., 2016; Xu et al., 2021) of pmSCs. Rossi et al. (2010) showed that faster-proliferating SCs have a greater potential to spontaneously express adipogenic characteristics than slower-proliferating populations. Thus, the present study determined the extent to which selection for higher growth rate and increased breast muscle yield at a processing age has altered the adipogenic potential of cultured 7-day-old pmSCs, as well as to identify differential adipogenic gene expression in pmSCs following a hot or cold thermal stress. A previous study by Clark et al. (2017) assessed the effect of both temperature and selection for growth on the adipogenic properties of 7-week-old pmSCs from non-commercial F-line turkeys that were selected from the RBC2 line only for increased 16-week body weight. The current study extends the previous work by comparing 7-day-old pmSCs from a contemporary commercial line with that of the RBC2 line. In addition, the effect of timing of the thermal stress during proliferation and/or differentiation on adipogenic potential of pmSCs was also examined.

Due to an asymmetric division strategy, not all daughter cells are committed to a myogenic pathway during proliferation (Kuang et al., 2007). Thus, proliferation is a critical period in which the cellular fate of SCs is determined by environmental temperature. Xu et al. (2021) showed that both hot and cold temperatures had a greater effect on myogenic differentiation if the thermal stress occurred during proliferation rather than only during differentiation. In the present study, heat-treated pmSCs from both lines had more intracellular lipid during late proliferation than late differentiation. In support of this finding, Clark et al. (2017) reported increased lipid synthesis in 7-week-old turkey pmSCs with hot temperatures during proliferation. Lipid content of both lines was increased to a higher extent during the earlier stages of differentiation (24 and 48 h) if the heat stress was applied during proliferation than only during differentiation. Heat stress-induced increases in intracellular lipid have been shown to result in a concomitant increase in intramuscular fat content and to have a prolonged effect on the structure and composition of the breast muscle (Piestun et al., 2017; Patael et al., 2019; Halevy, 2020).

Under cold temperatures, intracellular lipid decreased in both lines with the NC line showing a greater reduction than the RBC2 line during proliferation. In previous in vitro studies, a reduction in lipid content was observed in pmSCs from both chickens (Harding et al., 2015) and turkeys (Clark et al., 2017) with cold stress. This parallels the results from an in vivo study of Patael et al. (2019) that showed no significant intramuscular fat content in the p. major muscle of cold-stressed chickens. The decreased lipid content may arise from reduced pmSC proliferation (Clark et al., 2016; Harding et al., 2016; Xu et al., 2021). Furthermore, if the cold stress was applied during proliferation rather than only during differentiation, the reduction in lipid synthesis was greater in both lines. Thus, lipid synthesis in proliferating pmSCs was more sensitive to both heat and cold stress than differentiating cells. In addition, the lack of detectable expression of the pre-adipocyte markers KLF1 and KLF7 during both proliferation and differentiation suggests a lack of pre-adipocytes in the turkey p. major muscle. This is not unexpected as most fat is deposited in abdominal and subcutaneous adipose depots and not as intramuscular fat in poultry (Cahaner et al., 1986; Crespo and Esteve-Garcia, 2002; Fereidoun et al., 2007). This finding also demonstrates the isolated pmSCs used in this study do not contain pre-adipocytes, and lipid synthesis is directly from the SCs. These data are supported by Shefer et al. (2004) who showed the spontaneous conversion of SCs to an adipogenic lineage. Taken together, these results suggest thermal stress may affect the properties of SCs including the synthesis of lipids, and proliferating SCs are more sensitive to temperature stimuli than differentiating SCs. Since poultry skeletal muscle SCs have their highest mitotic activity during the immediate 7 days after hatch (Mozdziak et al., 1994; Halevy et al., 2000), these data support that extrinsic temperature needs to be maintained at an optimal level to avoid possible harmful effects on breast muscle composition.

Another finding in the present study is that selection for growth for increased breast muscling may have enhanced adipogenic differences between the faster-growing NC pmSCs and the slower-growing RBC2 pmSCs. At 38°C during proliferation, the NC line pmSCs had a greater linear increase in lipid content than the RBC2 line. Similar to the findings of Velleman (2014) and Clark et al. (2017), pmSCs from the growth selected F-line turkeys expressed higher levels of the adipogenic genes PPARγ and C/EBPα than the RBC2 line during proliferation. The increased adipogenic potential may be partially attributed to the greater proliferation rate of the pmSCs in faster-growing turkeys (Velleman et al., 2000; Clark et al., 2016; Xu et al., 2021). Thus, it is possible that the increased proliferation rates of the growth-selected NC line pmSCs creates a larger SC pool, having a greater potential to express adipogenic properties.

During myogenic differentiation, most SCs are committed to a myogenic pathway, as reflected in the increased myogenic differentiation and enhanced expression of myogenic genes as previously shown (Clark et al., 2016; Xu et al., 2021). This may explain the gradual decrease in lipid synthesis observed in the hot and cold treatment groups in the current study. Expression of PPARγ was significantly reduced during differentiation compared to proliferation in all the treatment groups independent of temperature. However, if the heat stress was continued throughout proliferation and differentiation (43°→43°C), expression of NPY and C/EBPβ increased in both lines compared to other treatment groups (38°→38°C, 38°→43°C, and 43°→38°C), and the NC line showed the highest increase. Taken together with the results by Xu et al. (2021), the NC line pmSCs tended to generate a larger cell pool than the RBC2 line with heat stress. This suggests that a larger pool of SCs may have a greater adipogenic potential even during myogenic differentiation. Furthermore, these results also suggest an intrinsic difference in adipogenic potential between the NC and RBC2 lines during myogenic differentiation, as the NC line pmSCs expressed more NPY and C/EBPβ than the RBC2 line in the control group.

In addition to the intrinsic difference in adipogenic potential, selection for growth may have altered the sensitivity of turkey breast muscle to temperature stimuli. Faster-growing poultry lines tend to have larger diameter myofibers from excessive hypertrophic SC-mediated muscle growth in the breast muscle than slower-growing lines (Wilson et al., 1990; Dransfield and Sosnicki, 1999; Velleman et al., 2003; Joiner et al., 2014). As an anaerobic muscle (Smith and Fletcher, 1988), chicken breast muscle has a reduced capillary supply compared to aerobic muscles, and this is exacerbated in faster-growing chickens (Hoving-Bolink et al., 2000; Yahav, 2000; Havenstein et al., 2003; Joiner et al., 2014). The circulatory supply is necessary to remove lactic acid, the byproduct of anaerobic respiration. The presence of excessively large myofibers typically found in growth-selected breast muscles further limits circulatory supply resulting in breast muscle degradation (Wilson et al., 1990; Velleman et al., 2003). Furthermore, because of a higher metabolic activity (Konarzewski et al., 2000), faster-growing chickens also produce more metabolic heat. Since birds are homeotherms and only keep body temperature within a limited interval (Yahav, 2000, 2015), heat cannot be as readily dissipated due to the reduced circulatory support in faster growing poultry. Therefore, faster-growing poultry has an impaired ability to cope with external thermal stress. In the current study, intracellular lipid synthesis in pmSCs from the faster-growing NC turkeys showed greater variations in response to the heat stress during both proliferation and differentiation than the RBC2 line. However, since the lipid content in pmSCs was higher in the NC line than the RBC2 line at 43°C, the intrinsic difference in lipid content between the two cell lines during proliferation was not affected by the heat stress. In contrast, cold stress eliminated this intrinsic difference between the two cell lines during proliferation. The 7-day-old pmSCs from the NC line tend to have increased myogenic differentiation and wider diameter myotubes than the RBC2 line with heat stress (Clark et al., 2016; Xu et al., 2021). Therefore, if faster-growing turkeys are exposed to a heat stress immediately after hatch, giant muscle fibers coupled with increased intramuscular fat content may occur in the breast muscle, resulting in prolonged negative effects on breast muscle structure and composition.

Adipogenic regulatory genes mediate the response to a thermal stress. The expression of both NPY and PPARγ expression was downregulated in the NC line during proliferation than the RBC2 line at the control temperature, and the expression of NPY and PPARγ was decreased with heat treatment during proliferation in both lines. A reduction in PPARγ and C/EBPβ expression in the heat-treated proliferating pmSCs was observed by Clark et al. (2017) as well. In chickens, a high fat diet downregulated NPY, PPARγ, and C/EBPα expression in both abdominal and subcutaneous adipose tissue (Wang et al., 2017). Furthermore, obesity resistance can be stimulated with a diet that is rich in fat and inhibits the expression of NPY and PPARγ in a rodent model (Cifani et al., 2015). In the current study, the increased intracellular lipid resulting from heat stress may act as an intracellular signal inhibiting NPY expression to suppress further lipid synthesis. As a key adipogenic transcription factor, NPY promotes lipid synthesis through the PPARγ pathway in pre-adipocytes in mice (Tang et al., 2015) and chickens (Zhang et al., 2015; Liu et al., 2017). In turkeys, PPARγ may also be a potential downstream regulator within the NPY-induced adipogenesis pathway in pmSCs, as both NPY and PPARγ showed a similar expression pattern at 72 h of proliferation in both lines. Alternatively, with cold stress, the expression of NPY, PPARγ, and C/EBPβ was decreased in the RBC2 line during proliferation. This reduction in expression may be stimulated by low intracellular lipid to prevent further loss of intracellular lipids in the RBC2 line. Furthermore, pmSCs from the NC line appear to have a reduced response to the cold-induced lipid loss than the RBC2 line, as neither NPY nor C/EBPβ showed significant change in the NC line at 33°C compared to the 38°C temperature during proliferation (PPARγ expression only showed a slight increase). Thus, when intracellular lipid was decreased with cold stress, pmSCs from the NC line may have difficulty regulating the lipid loss. This may explain why the cold-treated pmSCs from the NC line had a greater reduction in intracellular lipid than the RBC2 line during proliferation.

In summary, the current study showed that thermal stress alters the adipogenic potential of turkey pmSCs at 7-days of age. During pmSC proliferation, hot temperatures increased the intracellular lipid content in pmSCs while cold temperatures had the opposite effect. Proliferating pmSCs were more sensitive to both heat and cold stress in the determination of intracellular lipid content than differentiating pmSCs. The pmSCs from the faster-growing NC turkeys showed a higher lipid synthesis potential and their cellular fate was more sensitive to both heat and cold stress than the slower-growing RBC2 turkeys. The increased intracellular lipid accumulation of the heat-treated pmSCs may eventually promote intramuscular fat deposition in vivo, resulting in prolonged effects on turkey breast muscle structure and composition. Future studies will need to focus on defining the signal transduction pathways involved in the regulation of adipogenesis and their response to heat and cold stress.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical review and approval was not required for the animal study because the study did not use any birds. Cell lines used in the study were previously isolated. Only satellite cells lines were used.

All authors have made a substantial and intellectual contribution to the work, and approved the submitted article. SV conceived and designed the experiments with GS and KR. JX performed the experiments and statistical analysis, and wrote the manuscript. SV, GS, and KR revised the manuscript.

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.