Early posthatch thermal stress causes long-term adverse effects on pectoralis muscle development in broilers.

Broiler chicks in the immediate posthatch handling period are exposed to thermal stress, with potentially harmful consequences for muscle growth and structure (e.g., less protein and more fat deposition). We addressed the effects of broiler chicks' exposure to various ambient temperatures during the first 13 D posthatch on their performance, as well as on muscle development and structure, up to day 35. Body weight and pectoralis muscle growth were lower throughout the entire period in the high-heat-exposed chicks (39°C, Hot) and to a lesser extent in the mild-heat-exposed chicks (35°C, Hot Mild) than in the Control chicks that were raised under a commercial protocol. In the cold-exposed chicks (29oC, Cold), BW and pectoralis muscle absolute growth were similar to the Control group throughout the entire period. The lower body and muscle growth in the Hot and Hot Mild groups were reflected in a lower number of myonuclei expressing proliferating cell nuclear cell in pectoralis major muscle cross sections sampled on day 8, in the distribution of myofibers as the experiment progressed, and in mean myofiber diameter on day 35, whereas in the Cold group, these numbers exceeded that of the Control group. However, TUNEL assay revealed similar cell survival in all groups. Hematoxylin-eosin and Oil red O staining revealed the highest fat deposition in the pectoralis muscle derived from the Hot group, whereas lower fat deposition was observed in the Control Cold group. These results were corroborated by immunostaining for CCAAT/enhancer binding protein ß in the pectoralis muscle, the levels of which were significantly higher in the Hot and Hot Mild groups on day 35 than in the Control group. Similar results were observed with Sirius red staining for collagen content in the pectoralis muscle. Together, the results imply long-term effects of chronic heat stress vs. cold stress in the early posthatch period on the broiler's body and muscle growth in general and myodegeneration of the pectoralis muscle in particular.

Early posthatch thermal stress affects breast muscle development and satellite cell growth and characteristics in broilers.

Heat or cold stress, can disrupt well-being and physiological responses in birds. This study aimed to elucidate the effects of continuous heat exposure in the first 2 wk of age on muscle development in broilers, with an emphasis on the pectoralis muscle satellite cell population. Chicks were reared for 13 d under either commercial conditions or a temperature regime that was 5°C higher. Body and muscle weights, as well as absolute muscle growth were lower in heat-exposed chicks from d 6 onward. The number of satellite cells derived from the experimental chicks was higher in the heat-treated group on d 3 but lower on d 8 and 13 compared to controls. This was reflected in a lower number of myonuclei expressing proliferating nuclear cell antigen in cross sections of pectoralis major muscle sampled on d 8. However, a TUNEL assay revealed similar cell survival in both groups. Mean myofiber diameter and distribution were lower in muscle sections sampled on d 8 and 13 in heat-treated versus control group, suggesting that the lower muscle growth is due to changes in muscle hypertrophy. Oil-Red O staining showed a higher number of satellite cells with lipids in the heat-treated compared to the control group on these days. Moreover, lipid deposition was observed in pectoralis muscle cross sections derived from the heat-treated chicks on d 13, whereas the controls barely exhibited any lipid staining. The gene and protein expression levels of CCAAT/enhancer binding protein ß in pectoralis muscle from the heat-treated group were significantly higher on d 13 than in controls, while myogenin levels were similar. The results suggest high sensitivity of muscle progenitor cells in the early posthatch period at a time when they are highly active, to chronic heat exposure, leading to impaired myogenicity of the satellite cells and increased fat deposition.

Thermal manipulation of the chicken embryo triggers differential gene expression in response to a later heat challenge.

BACKGROUND: Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge. RESULTS: Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes. CONCLUSIONS: This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat.

The effect of temperature on proliferation and differentiation of chicken skeletal muscle satellite cells isolated from different muscle types.

Skeletal muscle satellite cells are a muscle stem cell population that mediate posthatch muscle growth and repair. Satellite cells respond differentially to environmental stimuli based upon their fiber-type of origin. The objective of this study was to determine how temperatures below and above the in vitro control of 38°C affected the proliferation and differentiation of satellite cells isolated from the chicken anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b.femoris) muscles. The satellite cells isolated from the p. major muscle were more sensitive to both cold and hot temperatures compared to the b.femoris satellite cells during both proliferation and differentiation. The expressions of myogenic regulatory transcription factors were also different between satellite cells from different fiber types. MyoD expression, which partially regulates proliferation, was generally expressed at higher levels in p. major satellite cells compared to the b.femoris satellite cells from 33 to 43°C during proliferation and differentiation. Similarly, myogenin expression, which is required for differentiation, was also expressed at higher levels in p. major satellite cells in response to both cold and hot temperatures during proliferation and differentiation than b. femoris satellite cells. These data demonstrate that satellite cells from the anaerobic p. major muscle are more sensitive than satellite cells from the aerobic b. femoris muscle to both hot and cold thermal stress during myogenic proliferation and differentiation.

The effect of temperature on apoptosis and adipogenesis on skeletal muscle satellite cells derived from different muscle types.

Satellite cells are multipotential stem cells that mediate postnatal muscle growth and respond differently to temperature based upon aerobic versus anaerobic fiber-type origin. The objective of this study was to determine how temperatures below and above the control, 38°C, affect the fate of satellite cells isolated from the anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b. femoris). At all sampling times, p. major and b. femoris cells accumulated less lipid when incubated at low temperatures and more lipid at elevated temperatures compared to the control. Satellite cells isolated from the p. major were more sensitive to temperature as they accumulated more lipid at elevated temperatures compared to b. femoris cells. Expression of adipogenic genes, CCAAT/enhancer-binding protein ß (C/EBPß) and proliferator-activated receptor gamma (PPARγ) were different within satellite cells isolated from the p. major or b. femoris. At 72 h of proliferation, C/EBPß expression increased with increasing temperature in both cell types, while PPARγ expression decreased with increasing temperature in p. major satellite cells. At 48 h of differentiation, both C/EBPß and PPARγ expression increased in the p. major and decreased in the b. femoris, with increasing temperature. Flow cytometry measured apoptotic markers for early apoptosis (Annexin-V-PE) or late apoptosis (7-AAD), showing less than 1% of apoptotic satellite cells throughout all experimental conditions, therefore, apoptosis was considered biologically not significant. The results support that anaerobic p. major satellite cells are more predisposed to adipogenic conversion than aerobic b. femoris cells when thermally challenged.

Thermal manipulation during embryogenesis affects myoblast proliferation and skeletal muscle growth in meat-type chickens.

Thermal manipulation (TM) of 39.5°C applied during mid-embryogenesis (embryonic d 7 to 16) has been proven to promote muscle development and enhance muscle growth and meat production in meat-type chickens. This study aimed to elucidate the cellular basis for this effect. Continuous TM or intermittent TM (for 12 h/d) increased myoblast proliferation manifested by higher (25 to 48%) myoblast number in the pectoral muscles during embryonic development but also during the first week posthatch. Proliferation ability of the pectoral-muscle-derived myoblasts in vitro was significantly higher in the TM treatments until embryonic d 15 (intermittent TM) or 13 (continuous TM) compared to that of controls, suggesting increased myogenic progeny reservoir in the muscle. However, the proliferation ability of myoblasts was lower in the TM treatments vs. control during the last days of incubation. This coincided with higher levels of myogenin expression in the muscle, indicating enhanced cell differentiation in the TM muscle. A similar pattern was observed posthatch: Myoblast proliferation was significantly higher in the TM chicks relative to controls during the peak of posthatch cell proliferation until d 6, followed by lower cell number 2 wk posthatch as myoblast number sharply decreases. Higher myogenin expression was observed in the TM chicks on d 6. This resulted in increased muscle growth, manifested by significantly higher relative weight of breast muscle in the embryo and posthatch. It can be concluded that temperature elevation during mid-term embryogenesis promotes myoblast proliferation, thus increasing myogenic progeny reservoir in the muscle, resulting in enhanced muscle growth in the embryo and posthatch.

Thermal manipulation during embryogenesis has long-term effects on muscle and liver metabolism in fast-growing chickens.

Fast-growing chickens have a limited ability to tolerate high temperatures. Thermal manipulation during embryogenesis (TM) has previously been shown to lower chicken body temperature (Tb) at hatching and to improve thermotolerance until market age, possibly resulting from changes in metabolic regulation. The aim of this study was to evaluate the long-term effects of TM (12 h/d, 39.5°C, 65% RH from d 7 to 16 of embryogenesis vs. 37.8°C, 56% RH continuously) and of a subsequent heat challenge (32°C for 5 h at 34 d) on the mRNA expression of metabolic genes and cell signaling in the Pectoralis major muscle and the liver. Gene expression was analyzed by RT-qPCR in 8 chickens per treatment, characterized by low Tb in the TM groups and high Tb in the control groups. Data were analyzed using the general linear model of SAS considering TM and heat challenge within TM as main effects. TM had significant long-term effects on thyroid hormone metabolism by decreasing the muscle mRNA expression of deiodinase DIO3. Under standard rearing conditions, the expression of several genes involved in the regulation of energy metabolism, such as transcription factor PGC-1α, was affected by TM in the muscle, whereas for other genes regulating mitochondrial function and muscle growth, TM seemed to mitigate the decrease induced by the heat challenge. TM increased DIO2 mRNA expression in the liver (only at 21°C) and reduced the citrate synthase activity involved in the Krebs cycle. The phosphorylation level of p38 Mitogen-activated-protein kinase regulating the cell stress response was higher in the muscle of TM groups compared to controls. In conclusion, markers of energy utilization and growth were either changed by TM in the Pectoralis major muscle and the liver by thermal manipulation during incubation as a possible long-term adaptation limiting energy metabolism, or mitigated during heat challenge.

Thermal manipulations in late-term chick embryos have immediate and longer term effects on myoblast proliferation and skeletal muscle hypertrophy.

We investigated the cellular and molecular bases for the promotion of muscle development and growth by temperature manipulations (TMs) during late-term chick embryogenesis. We show that incubation at 39.5 degrees C (increase of 1.7 degrees C from normal conditions) from embryonic days 16 to 18 (E16 to E18) for 3 or 6 h daily increased diameter of myofibers as of day 13 of age and enhanced absolute muscle growth relative to controls, until day 35 of age. TMs had immediate (E17) and later (up to 2 wk posthatch) effects in elevating muscle cell proliferation relative to controls. This was indicated by higher DNA incorporation of thymidine and a higher number of cells expressing PCNA in intact muscle, accompanied by higher Pax7 levels, all reflecting a higher number of myogenic cells, and suggesting that the increased hypertrophy can be attributed to a higher reservoir of myogenic progeny cells produced in response to the TM. IGF-I levels were higher in the TM groups than in controls, implying a mechanism by which heat manipulations in chicks affect muscle development, with locally secreted IGF-I playing a major role. Whereas hypertrophy was similar in both TM groups, cell proliferation and Pax7 levels were more robust in the 6-h muscle, mainly posthatch, suggesting a differential effect of various TM periods on cell reservoir vs. hypertrophy and a high sensitivity of myoblasts to relatively small changes in heat duration with respect to these processes, which is manifested in the short and long term.

AlphaMUPA mice: a transgenic model for longevity induced by caloric restriction.

Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the underlying mechanisms of this phenomenon are still poorly understood. To get more insight into these mechanisms, we took advantage of the alphaMUPA transgenic mice that previously were reported to spontaneously eat less and live longer compared with their wild-type control mice. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, i.e. the urokinase-type plasminogen activator (uPA), in causing the reduced appetite. This phenotypic change could have resulted from the ectopic transgenic expression that we detected in the adult alphaMUPA brain, or alternatively, from a transgenic interference in brain development. Here, we have summarized similarities and differences so far found between alphaMUPA and calorically restricted mice. Recently, we noted several changes in the alphaMUPA liver, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, alphaMUPA mice showed a reduced level of serum IGF-1 and a reduced incidence of spontaneously occurring or carcinogen-induced tumors in several tissues. In contrast, alphaMUPA did not differ from wild type mice in the levels of low molecular weight antioxidants when compared in several tissues at a young or an old age. Overall, the alphaMUPA model suggests that fine-tuning of the threshold for apoptosis, possibly linked in part to modulation of serum IGF-1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.

Long-lived alpha MUPA transgenic mice exhibit increased mitochondrion-mediated apoptotic capacity.

Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the mechanisms underlying this phenomenon are still poorly understood. To study this issue, the transgenic model of alpha MUPA mice, which previously were reported to spontaneously eat less and live longer compared with their wild-type (WT) control mice, were used. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, that is, the urokinase-type plasminogen activator (uPA), in causing the reduced appetite. Recently, several changes in the alpha MUPA liver were noted, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, alpha MUPA mice showed a reduced level of serum IGF-1 and a reduced incidence of spontaneously occurring or carcinogen-induced tumors in several tissues. Overall, the alpha MUPA model suggests that long-lasting, moderately increased apoptotic capacity, possibly linked in part to modulation of serum IGF-1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.

Mitochondrion-mediated apoptosis is enhanced in long-lived alphaMUPA transgenic mice and calorically restricted wild-type mice.

Caloric restriction (CR) can extend the life-span of multiple species and is the only intervention known to attenuate aging in mammals. Mechanisms mediating the CR influence are as yet unclear. To get insight into these mechanisms we took advantage of alphaMUPA transgenic mice that have previously been reported to spontaneously eat less and live longer compared with their wild-type (WT) control. Here we report that mitochondria isolated from young adult alphaMUPA livers showed increased susceptibility to calcium-induced high-amplitude swelling, increased cytochrome c release and enhanced glutathione levels. Furthermore, young adult alphaMUPA mice showed significantly enhanced caspase-3 activity in liver homogenates, increased fraction of apoptotic hepatocytes, and a lower level of serum IGF-1. In addition, alphaMUPA mice showed a decreased rate of spontaneously occurring lung tumors at an old age. Short-term (8 weeks) calorically restricted WT mice also showed an increase of mitochondrial swelling and caspase-3 activity compared with ad libitum (AL) fed WT mice. These results provide the first indication that CR can enhance mitochondrion-mediated apoptotic capacity. Collectively, the results are consistent with the possibility that long lasting, moderately increased apoptotic capacity, possibly linked in part to IGF-1 and GSH modulation, could play a role in the CR-induced anti-aging influence in mice.

Hyper- or hypothyroidism: its association with the development of ascites syndrome in fast-growing chickens.

The ascites syndrome in broiler chickens is attributed to the progress in genetic selection for rapid growth, coupled with the metabolic burden imposed by exposure to a relatively low-ambient temperature (T(a)). The syndrome is mainly characterized by hematocrit elevation, decline in blood oxygen saturation, accumulation of fluid in the abdominal cavity, and finally, death. Ascitic chickens have demonstrated hypothyroidism coupled with a marked stress response (high corticosterone concentration) and reduction in the hemoglobin content. The objective of the present study was to examine the role of thyroid and corticosterone hormones in the development of the syndrome. Ascites was induced by exposure to a gradually declining T(a) and supplementation of a pellet-form diet. Exogenous thyroxin (T(4)) and propylthiouracil (PTU) (in Experiments 1 and 2, respectively) were supplemented in drinking water to induce hyper- or hypothyroidism, respectively. Ascites syndrome was developed in 21.5% and 23% of the birds exposed to ascites-induced conditions (Exps. 1 and 2, respectively). Excess T(4) (Exp. 1) significantly reduced the percentage of ascites (down to 7%), whereas PTU (Exp. 2) significantly increased the appearance of the syndrome (35%). In the T(4)-treated chickens, although the T(4) concentration reached pharmacological levels, the triiodothyronine (T(3)) concentration remained within physiological levels, whereas T(3) in the ascitic birds exhibited a reduction pattern similar to that observed in the ascitic non-supplemented ones. In the PTU-treated chickens, however, both ascitic and non-ascitic birds demonstrated significant reductions in both T(4) and T(3) concentrations. In both experiments, ascitic chickens exhibited a considerable stress response, characterized by a significant and persisted elevation in plasma corticosterone concentration, which was in accordance with a similar elevation of hematocrit, and the PTU-treated non-ascitic birds exhibited a similar stress response. At 5 weeks of age, ascitic birds and the PTU-treated non-ascitic ones exhibited significant reductions in the hemoglobin content of their red blood cells. It may be concluded that deficiency in the thyroid hormones and elevated corticosterone may play a key deleterious role in the development of the ascites syndrome.