Ontogeny of heart rate responses to exogenous melatonin in Muscovy duck and chicken embryos

One of the major physiological effects of melatonin is coupling the internal clock with different organ functions. Despite the long list of functional responses to melatonin discovered in the past, it has been unclear when responsiveness to melatonin develops during ontogeny. The aim of the present study was therefore to investigate in Muscovy duck and chicken embryos, when they start to exhibit heart rate (HR) changes to exogenous melatonin. HR was recorded continuously in Muscovy duck embryos from day 24 of incubation (D24) and in chicken embryos from D17 until hatching. Every day four doses of 10 microg melatonin were injected into each egg at 30 min intervals. In Muscovy duck embryos HR responses to melatonin were first observed on D25; from D27 all embryos responded. In all cases HR decreased immediately after the injection. HR deviation from baseline values and duration of decreased HR period increased during the experimental period. Chicken embryos showed similar responses as Muscovy ducks from D17 onwards; from D18 the response rate was 100%. On D19 dose-response data revealed a partial responsiveness to exogenous melatonin at doses of 0.1 to 1 ng and full responsiveness from 10 ng to 10 microg. The time of the first occurrence of HR responses to melatonin coincides with published results on the start of periodic pineal melatonin secretion. These data suggest that the output signal of the avian internal clock, periodic plasma melatonin fluctuations, could result in periodic cardiac function already prenatally in these two avian species.     

Placing growth factor-coated beads on early stage chicken embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon. Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.     

Contractile and relaxing reactivity in carotid and femoral arteries of chicken embryos

In the embryo, hypoxemia causes redistribution of cardiac output from the periphery toward the heart and the brain. In view of this, we investigated developmental changes in the contractile and relaxing properties of the peripheral femoral artery (Fem) and the more central carotid artery (Car) at 0.7, 0.8, and 0.9 of the chicken embryo incubation time. Isolated arteries were studied in myographs and were exposed to norepinephrine or phenylephrine. High K(+) (125 mM) and electrical field stimulation (0.25-16 Hz) were used to induce receptor-independent and neurogenic contractions. Relaxing responses to ACh were evaluated in the absence and presence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and before and after endothelium removal. alpha(1)-Adrenergic contractile responses increased in a time-dependent manner and were significantly larger in Fem than in Car. Neurogenic contractions and adrenergic nerves could only be demonstrated in Fem at 0.9 incubation. ACh caused relaxation in both Fem and Car at 0.7, 0.8, and 0.9 incubation. The NO-independent part of the relaxation was more pronounced in Car than in Fem at all developmental stages. We conclude that the chicken embryo is a useful model to investigate the development of vasomotor control and vascular heterogeneity. The observed regional vascular differences may contribute to cardiac output redistribution during hypoxia in the embryo and might result from endothelial and neurogenic influences on vascular smooth muscle differentiation.     

In ovo electroporations of HH stage 10 chicken embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology. With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function. By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function. Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements. Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive. This video demonstrates first how to make a window in the eggshell to manipulate the embryo. Next, the embryo is visualized with a dilute solution of India ink injected below the embryo. A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. Finally, the egg is sealed with tape and placed back into an incubator for further development. Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.     

Thermal acclimation of heart rates in reptilian embryos

In many reptiles, the thermal regimes experienced by eggs in natural nests vary as a function of ambient weather and location, and this variation has important impacts on patterns of embryonic development. Recent advances in non-invasive measurement of embryonic heart rates allow us to answer a long-standing puzzle in reptilian developmental biology: Do the metabolic and developmental rates of embryos acclimate to local incubation regimes, as occurs for metabolic acclimation by post-hatching reptiles? Based on a strong correlation between embryonic heart rate and oxygen consumption, we used heart rates as a measure of metabolic rate. We demonstrate acclimation of heart rates relative to temperature in embryos of one turtle, one snake and one lizard species that oviposit in relatively deep nests, but found no acclimation in another lizard species that uses shallow (and hence, highly thermally variable) nests. Embryonic thermal acclimation thus is widespread, but not ubiquitous, within reptiles.     

Long-term measurement of heart rate in chicken eggs

Taking advantage of acoustocardiogram (ACG), we measured the heart rate (HR) of chick embryos continuously from day 12 until hatching and then investigated the development of HR irregularities (HRI), HR variability (HRV), and the existence of a circadian rhythm in mean HR (MHR). HRI comprised transient bradycardia and tachycardia, which first developed on day 14 and 16 in most embryos, respectively. Transient bradycardia increased in frequency and magnitude with embryonic development and occurred over periods of up to 30 min in some embryos. MHR was maximal on around days 14-15 and thereafter decreased to about 250-260 bpm on days 16-18. Baseline HRV, which is an oscillation of the MHR baseline, occurred as HR decreased from days 15-16 and became predominant on days 17-18. The magnitude of the baseline oscillations reached up to 50 bpm in some embryos and the period ranged between about 40-90 min (ultradian rhythm). A circadian rhythm of MHR was not found in late chick embryos. On days 18-19, embryonic activities were augmented and then breathing movements began to occur, disturbing ACG signals and thus making it difficult to measure the HR. Instead, the development of breathing activities was recorded. Breathing frequency was irregular at first and then increased to a maximum of about 1.5 Hz prior to hatching.     

Quantitative study of the heart in 2 human twin embryos at 14 mm C-R (stage 18)

Two hearts of human twin embryos at 14 mm C-R (stage 18) were studied by means of light microscopy and quantitative methods. The serial sections of the embryos, 5 micron thick, were projected by using a microprojector for the measurement of the cardiac diameters and cardiac profile areas. The calculated heart volumes were 8.34 and 8.45 mm3, respectively. Cardiac volume is more accurate than linear measurement to establish quantitative development of the heart. It can also be considered an accessory index for embryonic classification.     

Development of heart rate rhythmicity in Muscovy duck embryos

The heart rate (HR) of Muscovy duck embryos (Cairina moschata f. domestica) was continuously recorded from as early as the 21st day of incubation (D21) until hatching (D34/35). The aim of the study was to investigate the influence of phonoperiods consisting of different acoustic stimuli on the course of HR and the development of HR periodicities during this period. Incubation was carried out at a constant temperature and in constant darkness. Until D25 HR was dominated by decelerative fluctuations only, indicating a main input from the parasympathetic system on the heart. Later sympathetic influences increased progressively. HR periodicity was investigated by means of chi 2-periodogram and fast Fourier transformation. Between D26 and D30 statistically significant and stable HR periodicities developed gradually. They had periods in the range from 5 to 38 h. Ultra-, circa- and infradian rhythms (< 20, 24 +/- 4 and > 28 h, respectively) occurred in parallel in some cases in the same embryo. The for the HR course important periods were dissimilar between individual embryos and had different intensities. There was no indication that acoustic stimulation (phonoperiods) had any effect on the development of HR periodicities.     

Vascular reactivity in intrapulmonary arteries of chicken embryos during transition to ex ovo life

The present study aimed to characterize pulmonary vascular reactivity in the chicken embryo from the last stage of prenatal development and throughout the perinatal period. Isolated intrapulmonary arteries from non-internally pipped embryos at 19 days of incubation and from internally and externally pipped embryos at 21 days of incubation were studied. Arterial diameter and contractile responses to KCl, endothelin-1, and U-46619 increased with incubation but were unaffected by external pipping. In contrast, the contractions induced by norepinephrine, phenylephrine, and electric field stimulation decreased with development. No developmental changes were observed in endothelium-dependent [acetylcholine (ACh) and cyclopiazonic acid] or endothelium-independent [sodium nitroprusside (SNP)] relaxation. These relaxations were abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Endothelium-dependent relaxation was unaffected by blockade of cyclooxygenase or heme oxygenase but was significantly reduced by nitric oxide (NO) synthase inhibitors. Reduction of O2 concentration from 95 to 5% produced a marked reduction in ACh and SNP-induced relaxations. Chicken embryo pulmonary arteries show a marked endothelium-dependent relaxation that is unaffected by transition to ex ovo life. Endothelium-derived NO seems to be the main mediator responsible for this relaxation.     

Translational control in sea urchin eggs and embryos: Initiation is rate limiting in blastula stage embryos

To determine whether initiation is rate-limiting in protein synthesis during the embryogenesis of sea urchins, polyribosome profiles of unfertilized eggs and cleavage, blastula and prism stage embryos were examined after incubation of the eggs and embryos in the presence and absence of low amounts of emetine, an inhibitor of polypeptide elongation. The ribosomes were radioactively labeled with [³H]uridine by injection of the adults during oogenesis so that we could monitor emetine-dependent shifts of monoribosomes to polyribosomes. Although initiation is not rate limiting in unfertilized eggs or 2- to 16-cell embryos of Strongylocentrotus purpuratus, it is rate limiting in blastula and prism embryos. We suggest that initiation becomes rate limiting to allow the selective translation of certain classes of mRNA during later development.     

The developmental stage of chicken embryos modulates the impact of in ovo olfactory stimulation on food preferences

Like mammals, bird embryos are capable of chemosensory learning, but the ontogeny of their feeding preferences has not been examined. We tested if the timing of stimulation in chicken embryos modulates the impact of in ovo olfactory stimulation on later food preferences. We exposed chicken embryos to an olfactory stimulus for a 4-day period in the middle or toward the end of the incubation period. The chicks were tested for their preference between foods with and without the olfactory stimulus in 3-min choice tests and on a 24-h time scale. Regardless of the type of food (familiar or novel) or the duration of the test, the control chicks not exposed to the olfactory stimulus consistently showed significant preferences for non-odorized foods. Chicks that were exposed in ovo to the olfactory stimulus did not show a preference for odorized or non-odorized foods. Only those chicks that were exposed to the olfactory stimulus toward the end of the incubation period differed from the controls and incorporated a higher proportion of odorized food into their diets on a 24-h time scale. This result indicates that olfactory stimulation at the end of embryonic development has a stronger impact on later feeding preferences. Our findings contribute to the growing pool of recent data appreciating the impact of olfactory signals on behavior regulation in avian species.     

In vitro culture of somite stage rat embryos: A new technique for maintaining growth and continuously monitoring heart rate

Summary Rat embryos removed during somite stages were attached to small rafts by using the technique described by D. A. T. New (J. Embryol. Exp. Morphol. 17: 513–525, 1967). The embryos were maintained in a viable growing state in circulating human serum. Twenty-four-hour cultures of day 11 rat embryos resulted in nearly normal increases in somite count, but length and protein increments were about one-half of in vivo values. After 24 hr of culture, the embryos were generally translucent and showed no apparent areas of gross or microscopic tissue necrosis. The apparatus is extremely compact and requires only about 9 ml of medium, which is sufficient to support three embryos. The small volume allows measurement of metabolites consumed or liberated with reasonable accuracy. By use of a technique of transillumination, using a laser and photomultiplying tube, a detailed and continuous record of heart rate can be made. Addition of drugs to, and sampling of, the culture medium is done easily. With this technique, a linear dependence of heart rate on temperature between 30 and 40°C has been shown. Studies of energy metabolism indicated that the earliest stage studied (about 11 somites) was characterized by a relatively high glucose utilization rate, with the major end product appearing as lactate. Further evidence that this stage was functioning anaerobically was shown by the presence of a resistance to aerobiosis. Embryos with 25 or more somites utilized less glucose, produced less lactate, and were damaged by lack of oxygen. Supported by United States Public Health Service grants HD02392, HD00180, and HD00836 and by the Graduate School Research Fund, University of Washington.     

Ventricular myosin light chain-2 gene expression in developing heart of chicken embryos

Recent gene knock-out studies in mice have suggested that ventricular myosin light chain-2 (vMLC2) has a role in the regulation of cardiogenic development and that perturbation in expression of vMLC2 is linked to the onset of dilated cardiomyopathy. In an attempt to develop an avian model for such studies, we examined the expression pattern of vMLC2 in chicken embryos at various stages and analyzed the effect of antisense oligonucleotide-mediated interference of vMLC2 function in cultures of whole embryos. Our results showed vMLC2 to be a specific marker for ventricular chamber throughout chicken embryonic development and antisense vMLC2 treatment of primitive streak stage (stage 4) embryos to produce pronounced dilation of heart tube with severe deficiency in formation of striated myofibrils. Further studies with antisense mRNA techniques of whole embryo cultures should, therefore, be useful to evaluate the role of vMLC2 and other putative regulatory factors in cardiac myofibrillogenesis.     

Intramyocardial pressure measurements in the stage 18 embryonic chick heart

Intramyocardial pressure (IMP) and ventricular pressure (VP) were measured in the trabeculating heart of the stage 18 chick embryo (3 days of incubation). Pressure was measured at several locations across the ventricle using a fluid-filled servo-null system. Maximum systolic and minimum diastolic IMP tended to be greater in the dorsal wall than in the ventral wall, but transmural distributions of peak active (maximum minus minimum) IMP were similar in both walls. Peak active IMP near midwall was similar to peak active VP, but peak active IMP in the subepicardial and subendocardial layers was four to five times larger. These results suggest that the passive stiffness of the dorsal wall is greater than that of the ventral wall and that during contraction the inner and outer layers of both walls generate more contractile force and/or become less permeable to flow than the middle part of the wall. Measured pressures likely correspond to regional variations in wall stress that may influence morphogenesis and function in the embryonic heart.     

Development of heart rate responses to acoustic stimuli in Muscovy duck embryos

Heart rate (HR) of Muscovy duck embryos (Cairina moschata f. domestica) was continuously recorded from the 21st day of incubation (E21) until hatching (E35). During that period, embryos were exposed to different acoustic stimuli (species-specific maternal and duckling calls, music, rectangular and sine waves, white noise). Sudden HR changes occurred at the onset of acoustic stimulation (on-response), as well as spontaneously. From E27 onwards, the response rate was significantly higher than the rate of spontaneous HR changes. The on-response rate increased further until E30. Most responses were elicited by maternal calls and music, but rarely by duckling calls. On-responses could be classified into: HR increase (36.4%), HR decrease (37.9%) and an increase in instantaneous HR variability (23.2%). The increase in HR variability occurred only in response to sounds, but not spontaneously. HR increases were mainly observed when the baseline HR was lower than the long-term HR trend. On-response duration was no longer than 3 min in 90% of all observations. The hourly mean HR and standard deviation did not change, even during phonoperiods composed of several sound patterns and lasting several hours. We conclude that Muscovy duck embryos are able to perceive exogenous acoustic stimuli, and that the acousto-sensory-->cardiac axis is functional from E27.