Previous studies showed that monochromatic green light stimuli during embryogenesis accelerated posthatch body weight (BW) and pectoral muscle growth of broilers. In this experiment, we further investigated the morphological and molecular basis of this phenomenon. Fertile broiler eggs (Arbor Acres, n=880) were pre-weighed and randomly assigned to 1 of the 2 incubation treatment groups: (1) dark condition (control group), and (2) monochromatic green light group (560 nm). The monochromatic lighting systems sourced from light-emitting diode lamps and were equalized at the intensity of 15 lx at eggshell level. The dark condition was set as a commercial control from day 1 until hatching. After hatch, 120 male 1-day-old chicks from each group were housed under incandescent white light with an intensity of 30 lx at bird-head level. No effects of light stimuli during embryogenesis on hatching time, hatchability, hatching weight and bird mortality during the feeding trial period were observed in the present study. Compared with the dark condition, the BW, pectoral muscle weight and myofiber cross-sectional areas were significantly greater on 7-day-old chicks incubated under green light. Green light also increased the satellite cell mitotic activity of pectoral muscle on 1- and 3-day-old birds. In addition, green light upregulated MyoD, myogenin and myostatin mRNA expression in late embryos and/ or newly hatched chicks. These data suggest that stimulation with monochromatic green light during incubation promote muscle growth by enhancing proliferation and differentiation of satellite cells in late embryonic and newly hatched stages. Higher expression of myostatin may ultimately help prevent excessive proliferation and differentiation of satellite cells in birds incubated under green light.

The partial contributions of reductions in fetal nutrition and oxygenation to slow fetal growth and a developmental origin of cardiovascular disease remain unclear. By combining high altitude with the chick embryo model, we have previously isolated the direct effects of high-altitude hypoxia on growth. This study isolated the direct effects of high-altitude hypoxia on cardiovascular development. Fertilized eggs from sea-level or high-altitude hens were incubated at sea level or high altitude. Fertilized eggs from sea-level hens were also incubated at high altitude with oxygen supplementation. High altitude promoted embryonic growth restriction, cardiomegaly and aortic wall thickening, effects which could be prevented by incubating eggs from high-altitude hens at sea level or by incubating eggs from sea-level hens at high altitude with oxygen supplementation. Embryos from high-altitude hens showed reduced effects of altitude incubation on growth restriction but not on cardiovascular remodeling. The data show that: (1) high-altitude hypoxia promotes embryonic cardiac and vascular disease already evident prior to hatching and that this is associated with growth restriction; (2) the effects can be prevented by increased oxygenation; and (3) the effects are different in embryos from sea-level or high-altitude hens.

The developing heart increases its mass predominantly by increasing the number of contained cells through proliferation. We hypothesized that addition of fibroblast growth factor-2, a factor previously shown to stimulate division of the embryonic myocytes, to the left ventricular myocardium in an experimental model of left heart hypoplasia created in the chicken would attenuate phenotypic severity by increasing cellular proliferation. We have established an effective mode of delivery of fibroblast growth factor-2 to the chick embryonic left ventricular myocardium by using adenovirus vectors, which was more efficient and better tolerated than direct injection of recombinant fibroblast growth factor-2 protein. Injection of control adenovirus expressing green fluorescent protein did not result in significant alterations in myocytic proliferation or cell death compared with intact, uninjected, controls. Co-injection of adenoviruses expressing green fluorescent protein and fibroblast growth factor-2 was used for verification of positive injection, and induction of proliferation, respectively. Treatment of both normal and hypoplastic left ventricles with fibroblast growth factor-2 expressing adenovirus resulted in to 2 to 3-fold overexpression of fibroblast growth factor-2, as verified by immunostaining. An increase by 45% in myocytic proliferation was observed following injection of normal hearts, and an increase of 39% was observed in hypoplastic hearts. There was a significant increase in anti-myosin immunostaining in the hypoplastic, but not the normal hearts. We have shown, therefore, that expression of exogenous fibroblast growth factor-2 in the late embryonic heart can exert direct effects on cardiac myocytes, inducing both their proliferation and differentiation. These data suggest potential for a novel therapeutic option in selected cases of congenital cardiac disease, such as hypoplastic left heart syndrome.

The heart rate (HR) of chick embryos before hatching is a relevant physiological parameter for the evaluation of regulation mechanisms of the cardiovascular system under different environmental conditions, e.g. oxygen supply. Movements of the embryo can be interpreted as a behavioural response to such changes. Using the PowerLab hardware and Chart software (AD Instruments), the ECG of a 14 day old chick embryo was recorded almost non-invasively by three electrodes penetrating the otherwise intact egg shell. The Chart program can be used to calculate the HR and its variability from the precisely detectable R-waves of the ECG. Artefacts in the ECG trace are derived from movements of the embryo. Under hypoxic conditions, the program can show that HR and HRV increased, whereas the embryo movements decreased. Using this technology, it can be determined that the embryo compensates for lower oxygen supply with faster blood circulation and saves energy by reducing unnecessary movements.

To study the interaction between hemodynamics and morphology in abnormal development of the heart, it is essential to have an animal model with specific and reproducible cardiac malformations. A standardized method was developed for inducing such an anomaly in the chick embryo. For this purpose, all-trans retinoic acid, dissolved in 2% dimethylsulphoxide, was applied on the vitelline membrane of the embryo in concentrations of 1.0 μgm and 0.3 μgm. A total of 207 embryos, staged according to Hamburger and Hamilton, were treated at stages 10 (33 hours of incubation) and 15 (50–55 hours of incubation). Three control groups consisted of embryos solely treated with dimethylsulphoxide, sham operated chicks, and untreated embryos, respectively. The embryos were sacrificed on the 9th, 10th and 11th days of incubation and hearts were examined by microdissection. Additional histologic studies were undertaken in 110 cases. Embryos treated with retinoic acid at both stages of development showed a high percentage of lesions falling within the general spectrum of double outlet right ventricle. The abnormality varied from merely a rightward shift of the aorta to cases with an additional subaortic ventricular septal defect, which, in combination with the rightward positioned aorta, was classified as a double outlet right ventricle. Histologic examination showed the presence of muscular tissue between the leaflets of the aortic and mitral valves. No other types of cardiac malformations were seen. Our results, with respect to timing, resemble data from studies made of hearts after surgical ablation of neural crest at comparable stages. Both method and timing of treatment with retinoic acid are important factors for the induction of specific and reproducible cardiac anomalies. This chick model allows for further detailed study of development of double outlet right ventricle, as well as the assessment of early detectable hemodynamic changes.

The embryonic heart and vascular bed interact dynamically to support rapid growth of the embryo during cardiovascular development. Pressure-volume relations define ventricular function during alterations in loading conditions. We analyzed these relationships in the embryonic heart in order to define ventricular function and the response of the ventricle and vascular bed to acute changes in preload. We simultaneously measured ventricular pressure and recorded 60 video images per second in n≥6 stage 16, 18 and 21 white Leghorn chick embryos at baseline and during the infusion of 1–2 microliters of physiologic buffer into the venous sinus (sinus venosus). Ventricular tetany was then induced with the topical application of 2 Molar sodium chloride. Video fields were traced for ventricular pressure and epicardial cross-sectional area. Cross-sectional area was converted to volume assuming ellipsoid geometry, and cavity volume was calculated as total volume minus wall volume derived from the tetanized heart. We defined end-diastole at the onset of ventricular contraction and end-systole at maximum pressure/volume ratio. Stroke volume increased linearly with end-diastolic volume. End-diastolic pressure-volume relations were positive and linear, and end-systolic pressure-volume relations were curvilinear downward. Arterial elastance decreased with growth of the embryo and with volume infusion. Pressure-volume loop area, an index of consumption of energy, doubled between the embryonic stages. Thus, embryonic ventricular pressure-volume relations define diastolic and systolic function at rest and in response to altered preload.

Specialized conduction tissues mediate coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, after which it spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricles being activated from the apex toward the base, which is a hallmark of HPS function. The development of mature HPS function follows significant phases of cardiac morphogenesis. Initially, the cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases as it travels toward the anterior regions of the heart tube, the actual sequence of ventricular activation in the looped heart proceeds in the same direction as blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, changing to the mature apex-to-base pattern. Using an optical mapping approach, we demonstrate that the timing of this last transition shows striking dependence on hemodynamic loading of the ventricle, being accelerated by pressure overload and delayed in left ventricular hypoplasia. Comparison of chick and mammalian hearts revealed some striking similarities as well as key differences in the timing of such events during cardiac organogenesis.

Vertebrate somitogenesis has been shown to be associated with a molecular oscillator, the segmentation clock, whose periodicity matches that of the process of somitogenesis. The existence of such a clock in presomitic mesoderm (PSM) cells was originally proposed in theoretical models such as the ‘clock and wavefront’. Molecular evidence for the existence of this clock in vertebrates has been obtained on the basis of the periodic expression of several genes, most of which are related to the Notch signalling pathway. These genes are expressed in a dynamic sequence which appears as a wave sweeping caudo-rostrally along the whole PSM once during each somite formation. Notch-pathway mouse and fish mutants lose the dynamic expression of the cycling genes, indicating that Notch signalling is required for their periodic expression, or is required to coordinate the oscillations between PSM cells. Therefore Notch signalling is either part of the mechanism of the oscillator itself or acts as a cofactor required for cycling gene expression. A further potentially important role for the segmentation clock is to periodically activate Notch signalling in the rostral presomitic mesoderm, thereby generating the periodic formation of somite boundaries.

The lipids of the yolk represent the primary nutrient source for the chick embryo, providing over 90% of the energy required for development as well as supplying a range of structural components for membrane biogenesis. Since the energy needs and the requirements for particular fatty acids differ greatly among the various embryonic tissues and between successive developmental stages, the regulated distribution of lipid components from the yolk to the different body sites forms a central feature of this system. As a consequence, the various tissues of the newly hatched chick display a range of highly characteristic lipid and fatty acid compositions in accordance with their functions but which, in many cases, also reflect the intensity and complexity of the lipid transfer process in the embryo. For example, the liver of the chick contains very high levels of cholesteryl ester as a result of the mechanism of lipid transfer. Moreover, the triacylglycerol fraction of the liver is highly enriched in docosahexaenoic acid, possibly reflecting hepatic involvement in the transfer of this fatty acid to the brain. The accretion of large amounts of both docosahexaenoic and arachidonic acids in the phospholipids of the brain and retina is a key feature of chick embryo development, the importance of which is underlined by the expression of a series of mechanisms, possibly mediated by the liver and adipose tissue, designed to promote the transfer of these fatty acids to the neural tissues.

The earliest stages of development in most animals are under the control of maternally inherited information. The initiation of embryonic gene expression has been reported at the mid-blastula in amphibians and the mid-2-cell stage to the early morula in mammals. In chick embryos, embryonic gene expression was detectable at stage X (morula) and showed marked activation at stage XIII (blastula) with a gradual increase thereafter. Synthesis of rRNA and tRNA was low at stage X and was already the major class of RNA at stage XIII in chick embryos. The observed upregulation of RNA synthesis seems to coincide with a period of extensive fine structural differentiation when the first major cellular migrations start and signal the formation of the primitive streak in the chick embryo.