Edward M. Dzialowski

Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos

Chicken embryos were chronically exposed to hypoxia (P(O(2)) approximately 110 mmHg) during development, and assessed for detrimental metabolic and morphological effects. Eggs were incubated in one of four groups: control (i.e. 151 mmHg), or treated with continuous 110 mmHg (15% O(2)) during days 1-6 (H1-6), 6-12 (H6-12), or 12-18 (H12-18) with normoxia during the remaining incubation. Metabolism (V(O(2))), body mass, hemoglobin (Hb) and hematocrit (Hct) were measured in embryos on days 12 and 18 of incubation and in day-old hatchlings. Ability to maintain V(O(2)) was acutely measured during a step-wise decrease in P(O(2)) from normoxia to hypoxia (55 mmHg). On day 12, V(O(2)) of H1-6 eggs were significantly lower than in the control and H6-12 eggs. P(crit) in H6-12 eggs was lower than in control and H1-6 eggs. Body mass of H1-6 and H6-12 embryos on day 12 was significantly lower than in control embryos, while in H6-12 embryos, Hct and Hb were higher. On day 18, H6-12 embryos had significantly lower V(O(2)) than control eggs. Body mass of H6-12 and H12-18 embryos was significantly lower than control embryos. Hct and Hb did not differ between treatments. In hatchlings, mass, Hb and Hct had returned to values statistically identical to controls. However, H6-12 embryos had significantly lower V(O(2)). Long-term hypoxia altered V(O(2)) when hypoxic incubation occurred during the middle third of incubation, but not during earlier or later incubation. Thus, chronic hypoxic exposure during critical periods in development altered the developmental physiological trajectories and modified the phenotypes of the developing embryos.

Maternal effects of egg size on emu Dromaius novaehollandiae egg composition and hatchling phenotype.

SUMMARY Parental investment in eggs and, consequently, in offspring can profoundly influence the phenotype, survival and ultimately evolutionary fitness of an organism. Avian eggs are excellent model systems to examine maternal allocation of energy translated through egg size variation. We used the natural range in emu Dromaius novaehollandiae egg size, from 400 g to> 700 g, to examine the influence of maternal investment in eggs on the morphology and physiology of hatchlings. Female emus provisioned larger eggs with a greater absolute amount of energy, nutrients and water in the yolk and albumen. Variation in maternal investment was reflected in differences in hatchling size, which increased isometrically with egg size. Egg size also influenced the physiology of developing emu embryos, such that late-term embryonic metabolic rate was positively correlated with egg size and embryos developing in larger eggs consumed more yolk during development. Large eggs produced hatchlings that were both heavier (yolk-free wet and dry mass) and structurally larger (tibiotarsus and culmen lengths) than hatchlings emerging from smaller eggs. As with many other precocial birds, larger hatchlings also contained more water, which was reflected in a greater blood volume. However, blood osmolality, hemoglobin content and hematocrit did not vary with hatchling mass. Emu maternal investment in offspring, measured by egg size and composition, is significantly correlated with the morphology and physiology of hatchlings and, in turn, may influence the success of these organisms during the first days of the juvenile stage.

Maturation of Cardiovascular Control Mechanisms in the Embryonic Emu (Dromiceius Novaehollandiae)

SUMMARY Our understanding of avian embryonic cardiovascular regulation has been based on studies in chickens. The present study was undertaken to determine if the patterns established in chickens are generally applicable to the emu, a ratite bird species. We studied cardiovascular physiology over the interval from 60% to 90% of the emus 50-day incubation period. During this period, embryonic emus exhibit a slight fall in resting heart rate (from 171 beats min-1 to 154 beats min-1) and a doubling of mean arterial pressure (from 1.2 kPa to 2.6 kPa). Exposures to 15% or 10% O2 initially decreased heart rate during the first period of emu incubation studied [60% of incubation (60%I)] but increased heart rate in the 90%I group. Arterial pressure responded to hypoxia with an initial depression (-1.6 kPa) at 60%I and 70%I but showed no response during the later periods of incubation (80%I and 90%I). In addition, tonic stimulation of both cholinergic and adrenergic (α and β) receptors was present on heart rate at 70%I, with the cholinergic and β-adrenergic tone increasing in strength by 90%I. Arterial pressure was dependent on a constant β-adrenergic and constant α-adrenergic tone from 60%I to 90%I. A comparison with embryonic white leghorn chickens over a similar window of incubation revealed that emus and white leghorn chickens both possess an adrenergic tone on heart rate and pressure but that only emus possess a cholinergic tone on heart rate. Collectively, these data indicate that the maturation of cardiovascular control systems differs between white leghorn chickens and emus, inviting investigation of additional avian species to determine other patterns.

Physiological effects and bioconcentration of triclosan on amphibian larvae.

We examined the acute effects of triclosan (TCS) exposure, a common antimicrobial found as a contaminant in the field, on survival and physiology of amphibian larvae. LC50 values were determined after 96h for North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a developmental model: Xenopus laevis. Amphibian larvae were most sensitive to TCS exposure during early development based upon 96-h LC50 values. Heart rates for X. laevis and North American larvae exposed to TCS were variable throughout development. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to TCS were significantly affected in larvae exposed to [50% LC50] and [LC50]. Tissue uptake and tissue bioconcentration factor (BCF) of TCS were investigated in X. laevis, B. woodhousii woodhousii, and R. sphenocephala. In general, a significant increase was observed as exposure concentration increased. Tissue BCF values were dependent upon stage and species. While TCS concentrations used here are higher than environmental concentrations, exposure to TCS was dependent upon species and developmental stage, with early developmental stages being most sensitive to TCS exposure.

Physiological Control of Warming and Cooling during Simulated Shuttling and Basking in Lizards

Differences in warming and cooling rates in basking lizards have long been thought to be brought about by adjustments in heart rate and blood flow. We examined the physiological control of warming and cooling in Iguana iguana, Sceloporus undulatus, and three species of Cordylus by measuring time constants, heart rate, and superficial capillary blood flow. Previously, techniques have not been available to measure time constants in shuttling animals. Using a combination of rapid measurements of temperature and blood flow and numerically intensive parameter‐fitting methods, we measured dominant and subdominant time constants in lizards subjected to periods of both simulated basking and simulated shuttling. Cutaneous blood flow and heart rate were measured using laser Doppler flowmeters. Of the three, only the larger I. iguana measurably altered rates of warming and cooling during basking. During shuttling, none of the species effectively controlled warming and cooling. During both basking and shuttling, blood flow and heart rate tended to change in predicted directions. Superficial blood flow correlated with surface temperature while heart rate correlated more closely with core temperature. Changes in superficial blood flow and heart rate varied relatively independently in I. iguana. The techniques used here provide a better understanding of the ability of these species to control thermoregulation.

Maturation of the contractile response of the Emu ductus arteriosus.

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor l-NAME. However, prostaglandin E2 (PGE2) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48–49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE2, locally produced PGE2 are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.

Morphological Changes in the Chicken Ductus Arteriosi During Closure at Hatching

The chicken embryo has two functioning ductus arteriosi (DA) during development. These blood vessels connect the pulmonary arteries to the descending aorta providing a right‐to‐left shunt of blood away from the nonrespiring lungs and to the systemic circuit and chorioallanotic membrane. The DA consists of two distinct tissue types along its length, a muscular proximal portion and an elastic distal portion. During hatching, the DA must close for proper separation of systemic and pulmonary circulation. We examined the morphological changes of the chicken DA before, during, and after hatching. Occlusion of the proximal DA began during external pipping and was complete at hatching. Anatomical remodeling began as early as external pipping with fragmentation of the internal elastic lamina and smooth muscle actin appearing in the neointimal zone. By day 2 posthatch, the proximal DA lumen was fully occluded by endothelial cells and smooth muscle actin positive cells. In contrast, the distal DA was not fully occluded by day 2 posthatch. Increases in Po2 of the blood serves as the main stimulus for closure of the mammalian DA. The responsiveness of the chicken proximal DA to oxygen increased during hatching, peaking during external pipping. This peak correlated with an increase in blood gas Po2 and the initial occlusion of the vessel. The distal portion remained unresponsive to oxygen throughout hatching. In conclusion, the chicken DA begins to close during external pipping when arterial Po2 increases and vessel tone is most sensitive to oxygen. Anat Rec, 291:1007–1015, 2008.

Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo

The avian embryo provides a novel model for studying the ductus arteriosus (DA) during the transition from in ovo to ex ovo life. Here we examined the mechanisms regulating the vasoreactivity of the two morphologically distinct portions of the chicken DA (proximal and distal) in response to O(2). Oxygen-induced contraction is redox sensitive and reversed by the reducing agent dithiothreitol and the H(2)O(2) scavenger N-mercaptopropionylglycine. As in the mammalian DA, inhibiting mitochondrion-derived reactive oxygen species production with rotenone and antimycin A relaxed the O(2)-constricted DA. The contractile response to O(2) matures during hatching and is mimicked by the K(v) channel inhibitor 4-aminopyridine (4-AP) on day 19 and externally pipped (EP) embryos. Together, O(2) and 4-AP significantly increase DA tone above that observed with either alone. The O(2)-induced contraction is mediated by influx of extracellular Ca(2+) through l-type Ca(2+) and store-operated channels. Inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores play a minor role in the O(2)-induced contraction. The O(2)-induced contraction is mediated by the Rho kinase pathway, as fasudil and Y-27632 significantly relax the O(2) contracted DA. Prostaglandins E(2), F(2alpha), and D(2) produce significant contraction of the proximal DA. The O(2)-induced relaxation of the distal portion of the DA is mediated by an endothelial-derived nitric oxide/cGMP pathway. Both 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and endothelial cell removal inhibit O(2)-induced relaxation in the distal segment. Mechanisms regulating O(2)-induced contraction in chicken proximal DA are similar to those found in mammalian DA, making the chicken a useful model for studying development of this O(2)-sensitive vessel.

Development of endothermic metabolic response in embryos and hatchlings of the emu (Dromaius novaehollandiae)

During hatching, there is a maturation of the mechanisms controlling the respiratory physiology involved in endotherm in precocial avian species. Here we examined the timing of the development of an endothermic response of oxygen uptake (MO2) to an alteration of ambient temperature (T(a)) in a model precocial species, the preterm and hatching emu (Dromaius novaehollandiae). Late stage pre-pipped and pipped embryos and hatchlings were measured for responses of MO2 and shell or skin temperature (T(s)) to altered T(a) (DeltaT(a)). MO2 remained unchanged in pre-pipped and internally pipped (IP) embryos at the end of 1.5h exposure to DeltaT(a) of +/-10 degrees C. Externally pipped (EP) embryos responded to a cooling and a warming exposure with marked increase and decrease in MO2, as hatchlings responded to DeltaT(a) with an endothermic change in MO2. The demonstration of the endothermic inverse metabolic response first appearing in EP embryos suggests that pre-EP embryos may also possess the ability to produce the endothermic inverse metabolic response, but they are restricted by the eggshell gas conductance. Late pre-pipped and IP embryos were measured again for responses of [Formula: see text] to DeltaT(a) in air and then in a 40% O(2) environment. The metabolic response of pre-pipped embryos at 90% of incubation was partially altered by switching from air to hyperoxia. IP embryos responded to DeltaT(a) in 40% O(2) with apparent inverse changes in MO2. The late stage emu embryo possesses the ability to produce an endothermic metabolic response at an earlier stage of development than in chickens, but this response is limited by the eggshell gas conductance.

Prenatal cardiovascular shunts in amniotic vertebrates

During amniotic vertebrate development, the embryo and fetus employ a number of cardiovascular shunts. These shunts provide a right-to-left shunt of blood and are essential components of embryonic life ensuring proper blood circulation to developing organs and fetal gas exchanger, as well as bypassing the pulmonary circuit and the unventilated, fluid filled lungs. In this review we examine and compare the embryonic shunts available for fetal mammals and embryonic reptiles, including lizards, crocodilians, and birds. These groups have either a single ductus arteriosus (mammals) or paired ductus arteriosi that provide a right-to-left shunt of right ventricular output away from the unventilated lungs. The mammalian foramen ovale and the avian atrial foramina function as a right-to-left shunt of blood between the atria. The presence of atrial shunts in non-avian reptiles is unknown. Mammals have a venous shunt, the ductus venosus that diverts umbilical venous return away from the liver and towards the inferior vena cava and foramen ovale. Reptiles do not have a ductus venosus during the latter two thirds of development. While the fetal shunts are well characterized in numerous mammalian species, much less is known about the developmental physiology of the reptilian embryonic shunts. In the last years, the reactivity and the process of closure of the ductus arteriosus have been characterized in the chicken and the emu. In contrast, much less is known about embryonic shunts in the non-avian reptiles. It is possible that the single ventricle found in lizards, snakes, and turtles and the origin of the left aorta in the crocodilians play a significant role in the right-to-left embryonic shunt in these species.