Y. Piestun

Pattern of Pax7 Expression During Myogenesis in the Posthatch Chicken Establishes a Model for Satellite Cell Differentiation and Renewal

The paired‐box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7‐expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity. Developmental Dynamics 231:489–502, 2004.

Thermal Manipulations During Broiler Embryogenesis: Effect on the Acquisition of Thermotolerance

Rapid growth rate has presented broiler chickens with serious difficulties when called on to thermoregulate efficiently in hot environmental conditions. Altering the incubation temperature may induce an improvement in the acquisition of thermotolerance (AT). This study aimed to elucidate the effect of thermal manipulations (TM) during the development of the thyroid and adrenal axis of broiler embryos on the potential of broilers to withstand acute thermal stress at marketing age. Cobb broiler embryos were subjected to TM at 39.5 degrees C and 65% RH from embryonic day 7 to 16 (inclusive), either continuously (24 h) or intermittently (12 h). After hatching chicks were raised under standard conditions to 35 d of age and then subjected to thermal challenge (35 degrees C for 5 h). Continuous TM caused a significant decline in hatchability, coupled with significantly lower BW and body temperature at hatching. The intermittent (12-h) chicks showed results similar to the controls but had significantly lower body temperature. Thermal challenge at marketing age demonstrated a significant improvement in AT in both the 12- and 24-h TM-treated broilers, which was characterized by a significantly lower level of stress (as evidenced by the level of plasma corticosterone) and rate of mortality. It was concluded that TM during the portion of embryogenesis when the thyroid and adrenal axis develop and mature had a long-lasting effect and improved the AT of broiler chickens. Whereas intermittent TM had no significant effect on hatchability and performance parameters, continuous TM negatively affected these parameters.

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.

Thermal manipulations of broiler embryos—The effect on thermoregulation and development during embryogenesis

This study aimed to elucidate the effects of thermal manipulations (TM) of broiler embryos, during the development of the thyroid and adrenal axis, on embryo development and metabolism. Cobb eggs were divided into 3 treatments: control, 24H-continuous TM at 39.5 degrees C and 65% RH from embryonic day 7 to 16 inclusive, and 12H-intermittent TM for 12 h/d in the same period. Only the 24H treatment negatively affected embryo growth and development, with lower relative weights of embryo, liver, and pipping muscle. During TM, eggshell temperature, heart rate, and oxygen consumption were elevated as embryos were in their ectothermic phase, but from the end of the TM until hatch, these parameters were significantly lower in both treatments than in the control. Moreover, plasma concentrations of the thyroid hormones were significantly lower in the 2 treatments during and after TM, until hatch. Plasma corticosterone concentration of the TM-treated embryos was significantly lower after the TM but significantly higher at hatch. It was concluded that TM during the development of the thyroid and adrenal axis lowered their functional set point, thus lowering metabolic rate during embryogenesis and at hatch.

Thermal manipulations during broiler incubation alter performance of broilers to 70 days of age

Recent decades have seen significant progress in the genetic selection of fast-growing broiler chickens. Whereas in many countries the average marketing age is 5 to 6 wk, the US, French, and other markets demand heavier broilers (~4 kg) that require a longer posthatching growing period. With greater age and greater BW, the ability to cope with hot weather conditions deteriorates, which can result in increased economic losses during periods of hot weather. Recent studies have demonstrated a long-lasting effect of intermittent thermal manipulation (TM) during embryogenesis, when it was applied for 12 h/d between embryonic (E) days E7 and E16, which was shown by improved thermotolerance during acute posthatching heat stress as well as improved feed conversion ratio (FCR) and breast muscle yield. The present study was designed to elucidate the effect of TM during embryogenesis on Cobb 500 broiler performance up to 70 d of age. Hatchability and male BW were not affected by TM, but TM females demonstrated a lower (P = 0.024) BW during the entire 70-d posthatching study. However, following embryonic TM, both sexes exhibited lower (P = 0.028 and P = 0.018 for males and females, respectively) feed intake and body temperature accompanied by improved FCR and greater breast muscle weight. In light of the present and previous studies, it was concluded that intermittent TM during broiler embryonic development had a long-lasting effect on energy balance that led to improved FCR and breast muscle yield.

Improvement of cold resistance and performance of broilers by acute cold exposure during late embryogenesis

The aim of this study was to fine-tune previous acute cold exposure treatments of broiler embryos during late embryogenesis to improve lifelong cold resistance and performance. Six hundred Cobb hatching eggs were incubated under standard conditions and then exposed to 3 treatments: control; cold treatment in which embryos were exposed to 15°C for 30 min on d 18 and 19 of incubation (30 × 2); and cold treatment similar to 30 × 2 but with 60-min exposures (60 × 2). Egg shell temperature (T(egg)) and heart rate (HR) were monitored pre- and posttreatment. Upon hatching, hatchability, body weight, and body temperature were recorded. From 14 to 35 d of age, three quarters of the chickens in each treatment were raised under ascites-inducing conditions (AIC) and the remaining birds were raised under standard brooding conditions (SBC). The T(egg) and HR decreased significantly in response to increased exposure time on d 18 of incubation. On d 19 of incubation, before the second cold exposure, the 30 × 2 group showed greater T(egg) and HR than the controls, and during the second exposure they maintained these parameters better than the 60 × 2 embryos. No treatment effect on hatchability was observed. At 35 d of age ascites incidence among 30 × 2 chickens under AIC was significantly less than that among the controls (P < 0.01), and body weight of these chickens under either SBC or AIC was significantly higher than that of the controls. Under SBC relative breast muscle weight was significantly higher in 60 × 2 chickens, whereas the relative heart weight was higher in both cold-treated groups than in the controls. It can be concluded that repeated short acute cold exposures during late embryogenesis significantly reduced ascites incidence and improved growth rate under either SBC or AIC. These results may be related to a prenatal epigenetic adaptation of the thermoregulatory and cardiovascular systems to low ambient temperature.

The effect of monochromatic photostimulation on growth and development of broiler birds

The only light source for chickens in environmentally controlled houses is an artificial one. Thus, source, spectra, intensity and regimen of light supplementation became major factors in modern meat type bird management. Light spectra affect growth in meat type birds both in ovo and post hatch. Broilers photostimulated in ovo with green light gained significantly more weight than birds incubated under dark conditions. Furthermore, we defined the cellular and molecular events associated with the effect of in ovo green photostimulation on muscle growth. We found that in ovo photostimulation have a stimulatory effect on the proliferation and differentiation of satellite cells and a promoting effect on the uniformity of the muscle fibers in the early post-hatch period. How does in ovo photostimulation affect intracellular events, such as proliferation and differentiation of muscle cells, leading to post-hatch muscle growth? It is possible that the monochromatic green light penetrates the eggshell and has a direct effect on the embryos muscle. We were unable to detect any proliferative effect of monochromatic green light on cultured myoblasts derived from standard (un-illuminated) E17 embryos and 3-day-old chicks. A more likely explanation is that green light indirectly affects myoblast proliferation by activating the endocrine system; the latter receives photic cues from the retinal or extra-retinal photoreceptors. We gathered some evidence to support these findings; we have shown a higher expression of growth hormone (GH) receptor mRNA in satellite cells derived from green light illuminated chicks. In addition, plasma GH levels and IGF-I levels in muscle tissue, were higher in the green group relative to the dark one in early post-hatch. Another possible explanation for this phenomenon could be that growth factor secretion is activated in response to green light photostimulation. Both retinal and extra-retinal photoreceptors are active during embryogenesis and can be first detected at E14. Combinations of in ovo and post-hatch green light photostimulation to broilers and turkeys did not cause synergetic effect on growth. In a recent study, we found that in ovo green light photostimulation suppresses the green and red opsin receptors gene expression in the last three days before hatching, while red light enhances their expression. Furthermore, we found that the down-regulation of the green and red opsins in response to incubation under monochromatic green lighting lasted up to 9days post hatch, suggesting a possible epigenetic effect.

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.

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

&NA; 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 &bgr; 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.