Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia.

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

Effect of increased embryonic temperature during developmental windows on survival, morphology and oxygen consumption of rainbow trout (Oncorhynchus mykiss).

Temperature is a crucial environmental factor that influences physiological functions in fishes, and increased temperature during development can shape an organism's phenotype. An active line of inquiry in comparative developmental physiology is whether short-term exposure to thermal changes have lasting phenotypic effects. This is the first study to apply a developmental 3-dimensional critical window experimental design for a vertebrate, using time, temperature, and phenotypic response (i.e., variables measured). Rainbow trout (Oncorhynchus mykiss) are an anadromous species for which resident populations occupy freshwater environments that are likely impacted by variable and rising temperatures, particularly during embryonic development. To assess thermal effects on fish development, we examined trout hatchling phenotypes following rearing in constant temperatures (5, 10, 15 and 17.5 °C) and following exposure to increased temperature above 5 °C during specific developmental windows. Time to 50% hatch, hatchling mass and body length showed general trends of decreasing with increasing constant temperature, and survival was highest in constant 10 °C incubation. Thermally shifting embryos into 17.5 °C during gastrulation and organogenesis reduced survival at hatch compared to 10 °C, and exposure to 15 and 17.5 °C only late in development produced lighter and shorter hatchlings. Oxygen consumption rate (V̇o2) at organogenesis differed between embryos incubated constantly in increased temperature or exposed only during organogenesis, but generally we found limited temperature effects on V̇o2 that may be due to high data variability. Collectively, these results suggest that survival of rainbow trout hatchlings is most sensitive to 17.5 °C exposure during gastrulation and organogenesis, while warm water exposure later in development has greater impacts on morphology. Thus, trait-specific critical windows of thermal sensitivity exist for rainbow trout embryos that alter the hatching phenotype.

Temperature tolerance and oxygen consumption of two South American tetras, Paracheirodon innessi and Hyphessobrycon herbertaxelrodi.

Temperature is a primary factor affecting species' ability to thrive in a particular ecological niche, but thermal conditions have changed dramatically in recent decades. Fishes shift their thermal tolerance range with a maximum and minimum temperature correlated to their recent thermal acclimation history, and species can show a reduced temperature quotient (Q10) following chronic thermal acclimation. Neon tetra (Paracheirodon innesi) and Black Neon tetra (Hyphessobrycon herbertaxelrodi) are popular hobbyist aquarium fishes, and both species are examples of freshwater teleosts native to South American river systems that are potentially affected by changing thermal conditions. We acclimated these species to three different constant temperatures (26 °C, 29 °C, and 31 °C) for 15.4 ±â€¯2.1 days, then measured acute critical thermal maxima (CTMax) and acute oxygen consumption rate (Mo2) at each acclimation temperature. We also estimated chronic lethal thermal maximum (CLT) for both species following a 2-week acclimation to 30.4 °C. Mean CTMax of both species were found to increase with acclimation temperature from 38.5 to 39.6 °C for Neon tetra and from 39.5 to 41.0 °C for Black Neon tetra, gaining 0.24 (Neon tetra) or 0.29 °C (Black Neon tetra) of tolerance per 1 °C of acclimation. However, Black Neon tetra demonstrated consistently higher CTMax (1.0-1.4 °C). CLT was lower for Neon tetra (33.5 °C), compared to Black Neon tetra (35.9 °C). Mean Mo2 were statistically similar across acclimation temperatures within species; Q10 between 26-31 °C were 1.92 and 1.22 for Neon and Black Neon tetra, respectively. Neon and Black Neon tetras physiologically acclimated to changing thermal demands, and although they demonstrate robust CTMax responses, CLT responses indicated both species are unable to survive temperatures 4-5 °C above current average natural values. The demonstrated metabolic plasticity and CTMax values provide a moderate cushion for both species to combat changing temperatures due to climate change, but CLT values suggest vulnerability to projected climate trends.

Immediate and Persistent Effects of Temperature on Oxygen Consumption and Thermal Tolerance in Embryos and Larvae of the Baja California Chorus Frog, Pseudacris hypochondriaca.

The developmental environment has significant immediate effects on phenotypes, but it may also persistently or permanently shape phenotypes across life history. This study examined how developmental temperature influenced embryonic and larval phenotypes of Baja California chorus frog (Pseudacris hypochondriaca), an abundant amphibian in southern California and northern Baja California. We collected egg clutches from native ponds in northern San Diego County within 24 h of fertilization, and clutches were separated and distributed between constant temperatures of 10, 15, 20, and 25°C for incubation. Oxygen consumption rate ( V ˙ O2), developmental stage, and embryo and yolk masses were measured throughout development. Time to 50% hatch, survival at 50% hatch, and hatch duration were determined. Development rate was strongly affected by temperature, with warmer temperatures reducing time to hatch and hatch duration. Survival to hatch was high across all temperatures, >90%. Mass-specific V ˙ O2 of embryos either remained constant or increased throughout development, and by hatching energy demand was significantly increased at higher temperatures. There were limited temperature effects on growth, with embryo and yolk dry mass similar between temperatures throughout embryonic development. To examine long-term effects of embryonic temperature, we reared hatchlings from each temperature until onset of larval feeding. Once feeding, larvae were acclimated to 20 or 25°C (>2 weeks). Following acclimation to 20 or 25°C, we measured larval mass-specific V ˙ O2 and critical thermal maximum (CTMax) at a common developmental stage (Gosner stages 32-36, "hindlimb toe differentiation"). Embryonic temperature had persistent effects on larval V ˙ O2 and CTMax, with warmer temperatures generally resulting in similar or higher V ˙ O2 and CTMax. This partially supported a "warmer is better" effect of embryonic incubation temperature. These results suggest that in a thermally robust amphibian species, temperature may program the phenotype during early development to construct traits in thermal tolerance and energy use that may persist. Overall, P. hypochondriaca displays a thermally robust phenotype, and it is possible that amphibians that possess a wider range of phenotypic plasticity will be relatively more successful mitigating effects of climate change.

Rapid metabolic compensation in response to temperature change in the intertidal copepod, Tigriopus californicus.

Animals living in the intertidal zone must adapt to thermal variability, including adjustments in metabolism. We examined metabolic responses to temperature in the copepod, Tigriopus californicus, which inhabits supratidal splash pools along the Pacific coast of North America. We maintained three populations of T. californicus at 20 °C, one from southern California (San Diego, "SD") and two from Oregon (Fogarty Creek, "FCN", Boiler Bay, "BOB") and examined possible geographic patterns in metabolism. We measured oxygen consumption rate (V̇o2) at 20 °C and following 48 h (chronic) acclimation to 25, 27.5 and 30 °C. V̇o2 was temperature-independent, with temperature quotients (Q10) values ≤1 in all populations, indicative of metabolic compensation. We detected no variation in V̇o2 or survival between populations. To explore the time course of metabolic compensation, we performed an acute acclimation experiment in which V̇o2 was measured at 20 °C, following immediate exposure to 25 °C, and following 2 h, 4 h and 6 h exposure to 25 °C. This acute acclimation experiment revealed that V̇o2 increased immediately in SD and FCN, but was no longer different than 20 °C levels by 2 h and 4 h at 25 °C, respectively. BOB showed no significant change in V̇o2, which may indicate complete temperature-independent metabolism or different mechanisms of compensation between populations. This study demonstrates a time course of rapid metabolic compensation in response to temperature that occurs in a small intertidal animal, and suggests intertidal invertebrates can thermally acclimate within a few hours of a significant temperature change.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.

Chronic captopril treatment reveals the role of ANG II in cardiovascular function of embryonic American alligators (Alligator mississippiensis).

Angiotensin II (ANG II) is a powerful vasoconstrictor of the renin-angiotensin system (RAS) that plays an important role in cardiovascular regulation in adult and developing vertebrates. Knowledge of ANG II's contribution to developmental cardiovascular function comes from studies in fetal mammals and embryonic chickens. This is the first study to examine the role of ANG II in cardiovascular control in an embryonic reptile, the American alligator (Alligator mississippiensis). Using chronic low (~ 5-mg kg embryo-1), or high doses (~ 450-mg kg embryo-1) of captopril, an angiotensin-converting enzyme (ACE) inhibitor, we disrupted the RAS and examined the influence of ANG II in cardiovascular function at 90% of embryonic development. Compared to embryos injected with saline, mean arterial pressure (MAP) was significantly reduced by 41 and 72% under low- and high-dose captopril treatments, respectively, a greater decrease in MAP than observed in other developing vertebrates following ACE inhibition. Acute exogenous ANG II injection produced a stronger hypertensive response in low-dose captopril-treated embryos compared to saline injection embryos. However, ACE inhibition with the low dose of captopril did not change adrenergic tone, and the ANG II response did not include an α-adrenergic component. Despite decreased MAP that caused a left shifted baroreflex curve for low-dose captopril embryos, ANG II did not influence baroreflex sensitivity. This study demonstrates that ANG II contributes to cardiovascular function in a developing reptile, and that the RAS contributes to arterial blood pressure maintenance during development across multiple vertebrate groups.

Dynamics of acid-base and hematological regulation in day 15 chicken embryos (Gallus gallus domesticus) exposed to graded hypercapnia and hypoxia.

Most experiments examining acid-base regulation of chicken embryos have employed static, single time point measurements rather than dynamic, multiple time point measurements that might reveal additional components of developing acid-base regulation. Thus, we studied blood acid-base balance and hematology of day 15 chicken embryos under 24h exposure to graded hypercapnia (1%-7% CO2) accompanied by graded hypoxia (20% O2 down to 13% O2). Across all hypercapnic/hypoxic environments, respiratory acidosis occurred 2h after exposure in proportion to the magnitude of hypercapnia. An additional metabolic alkalosis occurred in ≥16% O2, and metabolic acidosis in ≤14% O2. As exposure progressed, compensatory metabolic alkalosis occurred in all groups at 6h, but partial metabolic compensation could not be preserved as hypoxia increased (≤18% O2). Across all hypercapnic/hypoxic groups, hematocrit, mean corpuscular volume and red blood cell concentration significantly increased by 24h, most likely due to hypoxia rather than hypercapnia. Overall, day 15 chicken embryos cannot maintain even partial compensation in their acid-base physiology after 24h exposure to hypercapnic/hypoxic environments of ≥5% CO2+≤15% O2.

Lipid content and fatty acid profile during lake whitefish embryonic development at different incubation temperatures.

Lipids serve as energy sources, structural components, and signaling molecules during fish embryonic development, and utilization of lipids may vary with temperature. Embryonic energy utilization under different temperatures is an important area of research in light of the changing global climate. Therefore, we examined percent lipid content and fatty acid profiles of lake whitefish (Coregonus clupeaformis) throughout embryonic development at three incubation temperatures. We sampled fertilized eggs and embryos at gastrulation, eyed and fin flutter stages following chronic incubation at temperatures of 1.8, 4.9 and 8.0°C. Hatchlings were also sampled following incubation at temperatures of 3.3, 4.9 and 8.0°C. Fertilized eggs had an initial high percentage of dry mass composed of lipid (percent lipid content; ~29%) consisting of ~20% saturated fatty acids (SFA), ~32% monounsaturated fatty acids (MUFA), ~44% polyunsaturated fatty acids (PUFA), and 4% unidentified. The most abundant fatty acids were 16:0, 16:1, 18:1(n-9c), 20:4(n-6), 20:5(n-3) and 22:6(n-3). This lipid profile matches that of other cold-water fish species. Percent lipid content increased during embryonic development, suggesting protein or other yolk components were preferentially used for energy. Total percentage of MUFA decreased during development, which indicated MUFA were the primary lipid catabolized for energy during embryonic development. Total percentage of PUFA increased during development, driven largely by an increase in 22:6(n-3). Temperature did not influence percent lipid content or percent MUFA at any development stage, and had inconsistent effects on percent SFA and percent PUFA during development. Thus, lake whitefish embryos appear to be highly adapted to low temperatures, and do not alter lipids in response to temperature within their natural incubation conditions.

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos.

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2°C, 5°C or 8°C water) and weekly, 1-h heat shocks (+3°C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148d at 2°C, 92d at 5°C, 50d at 8°C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3°C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8°C and 5°C embryos were significantly smaller and had larger yolks than 2°C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.

Salt sensitivity of the morphometry of Artemia franciscana during development: a demonstration of 3D critical windows.

A 3D conceptual framework of 'critical windows' was used to examine whether the morphometry of Artemia franciscana is altered by salinity exposure during certain key periods of development. Artemia franciscana were hatched at 20 ppt (designated control salinity) and were then exposed to 10, 30, 40 or 50 ppt either chronically (days 1-15) or only on days 1-6, 7-9, 10-12 or 13-15. On day 15, maturity was assessed and morphometric characteristics, including mass, total body length, tail length and width, length of the third swimming appendage and eye diameter, were measured. Maturation and morphometry on day 15 were influenced by the exposure window and salinity dose. Artemia franciscana were generally larger following exposure to 10 and 40 ppt during days 1-6 and 7-9 when compared with days 10-12 and 13-15, in part due to a higher percentage of mature individuals. Exposure to different salinities on days 1-6 produced the greatest differences in morphometry, and thus this appears to be a period in development when A. franciscana is particularly sensitive to salinity. Viewing the developmental window as three-dimensional allowed more effective visualization of the complex interactions between exposure window, stressor dose and the magnitude of morphometric changes in A. franciscana.

Hypercapnic thresholds for embryonic acid-base metabolic compensation and hematological regulation during CO2 challenges in layer and broiler chicken strains.

Time specific acid-base metabolic compensation and responses of hematological respiratory variables were measured in day 15 layer (Hyline) and broiler (Cornish Rock) chicken embryos during acute hypercapnic challenges (3, 6, 10 and 20% CO2). Control acid-base status and hematology differed between two strains. Broiler embryos were relatively respiratory acidotic and had higher hematocrit (Hct) and hemoglobin concentration. The partial metabolic compensation for respiratory acidosis produced by ≤ 10% CO2 exposures occurred in proportion to CO2 concentrations in both strains, but metabolic compensation for 20% CO2 respiratory acidosis was depressed at 2, 6 and 24h, particularly in broiler embryos. Exposure to ≤ 10% CO2 induced the same hematological responses across CO2 concentrations; i.e., Hct and mean corpuscular volume (MCV) increased while RBC concentration remained unchanged. In response to 20% CO2 exposure, Hct and MCV increased dramatically in both stains. Consequently, altered acid-base and hematology responses to 20% CO2 exposure compared to ≤ 10% CO2 suggest that the hypercapnic threshold to compensation for acidosis and regulation of hematology is >10% CO2.

Developmental critical windows and sensitive periods as three-dimensional constructs in time and space.

A critical window (sensitive period) represents a period during development when an organism's phenotype is responsive to intrinsic or extrinsic (environmental) factors. Such windows represent a form of developmental phenotypic plasticity and result from the interaction between genotype and environment. Critical windows have typically been defined as comprising discrete periods in development with a distinct starting time and end time, as identified by experiments following an on and an off protocol. Yet in reality, periods of responsiveness during development are likely more ambiguous that depicted. Our goal is to extend the concept of the developmental critical window by introducing a three-dimensional construct in which time during development, dose of the stressor applied, and the resultant phenotypic modification can be utilized to more realistically define a critical window. Using the example of survival of the brine shrimp (Artemia franciscana) during exposure to different salinity levels during development, we illustrate that it is not just stressor dose or exposure time but the interaction of these two factors that results in the measured phenotypic change, which itself may vary within a critical window. We additionally discuss a systems approach to critical windows, in which the components of a developing system--whether they be molecular, physiological, or morphological--may show differing responses with respect to time and dose. Thus, the plasticity of each component may contribute to a broader overall system response.

Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis).

The timing, success and energetics of fish embryonic development are strongly influenced by temperature. However, it is unclear if there are developmental periods, or critical windows, when oxygen use, survival and hatchling phenotypic characteristics are particularly influenced by changes in the thermal environment. Therefore, we examined the effects of constant incubation temperature and thermal shifts on survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis) embryos. We incubated whitefish embryos at control temperatures of 2, 5, or 8 °C, and shifted embryos across these three temperatures at the end of gastrulation or organogenesis. We assessed hatch timing, mass at hatch, and yolk conversion efficiency (YCE). We determined cost of development, the amount of oxygen required to build a unit of mass, for the periods from fertilization-organogenesis, organogenesis-fin flutter, fin flutter-hatch, and for total development. An increase in incubation temperature decreased time to 50 % hatch (164 days at 2 °C, 104 days at 5 °C, and 63 days at 8 °C), survival decreased from 55 % at 2 °C, to 38 % at 5 °C, and 17 % at 8 °C, and hatchling yolk-free dry mass decreased from 1.27 mg at 2 °C to 0.61 mg at 8 °C. Thermal shifts altered time to 50 % hatch and hatchling yolk-free dry mass and revealed a critical window during gastrulation in which a temperature change reduced survival. YCE decreased and cost of development increased with increased incubation temperature, but embryos that hatched at 8 °C and were incubated at colder temperatures during fertilization-organogenesis had reduced cost. The relationship between cost of development and temperature was altered during fin flutter-hatch, indicating it may be a critical window during which temperature has the greatest impact on energetic processes. The increase in cost of development with an increase in temperature has not been documented in other fishes and suggests whitefish embryos are more energy efficient at colder temperatures.

The actions of the renin-angiotensin system on cardiovascular and osmoregulatory function in embryonic chickens (Gallus gallus domesticus).

Using embryonic chickens (Gallus gallus domesticus), we examined the role of the renin-angiotensin system (RAS) in cardiovascular and osmotic homeostasis through chronic captopril, an angiotensin-converting enzyme (ACE) inhibitor. Captopril (5 mg kg⁻¹ embryo wet mass) or saline (control) was delivered via the egg air cell daily from embryonic day 5-18. Mean arterial pressure (MAP), heart rate (ƒ(H)), fluid osmolality and ion concentration, and embryonic and organ masses were measured on day 19. Exogenous angiotensin I (ANG I) injection did not change MAP or ƒ(H) in captopril-treated embryos, confirming ACE inhibition. Captopril-treated embryos were significantly hypotensive, with MAP 15% lower than controls, which we attributed to the loss of vasoconstrictive ANG II action. Exogenous ANG II induced a relatively greater hypertensive response in captopril-treated embryos compared to controls. Changes in response to ANG II following pre-treatment with phentolamine (α-adrenergic antagonist) indicated a portion of the ANG II response was due to circulating catecholamines in captopril-treated embryos. An increase in MAP and ƒ(H) in response to hexamethonium indicated vagal tone was also increased in the absence of ACE activity. Captopril-treated embryos had lower osmolality, lower Na⁺ and higher K⁺ concentration in the blood, indicating osmoregulatory changes. Larger kidney mass in captopril-treated embryos suggests disrupting the RAS may stimulate kidney growth by decreasing resistance at the efferent arteriole and increasing the fraction of cardiac output to the kidneys. This study suggests that the RAS, most likely through ANG II action, influences the development of the cardiovascular and osmoregulatory systems.

ANG II and baroreflex control of heart rate in embryonic chickens (Gallus gallus domesticus).

ANG II alters the short-term blood pressure buffering capacity of the baroreflex in many adult animals. In embryonic chickens, high plasma ANG II levels contribute to baseline mean arterial pressure (MAP, kPa) without changing heart rate (ƒH, beats/min). We hypothesized, on the basis of these features, that an ANG II-induced reduction in baroreflex sensitivity is present in embryonic chickens as in adults. We examined baroreflex function in day 19 embryonic chickens (Gallus gallus domesticus) after chronic depletion of endogenous ANG II via angiotensin-converting enzyme (ACE) inhibition with captopril (5 mg/kg) from days 5-18 of incubation. The correlation between MAP and ƒH was assessed using increasing doses of sodium nitroprusside, a vasodilator, and phenylephrine, a vasoconstrictor. We used two analytical methods to evaluate baroreflex function: a conventional "static" method, in which maximal MAP and ƒH responses were examined, and a "dynamic" method that assessed beat-to-beat changes during the response to pharmacological manipulation. Captopril-treated embryos were hypotensive by 19% with baroreflex slopes ∼40% steeper and normalized gains ∼50% higher than controls, and differences across treatments were similar using either analytical method. Furthermore, reintroduction of ANG II via infusion raised MAP back to control levels and decreased the baroreflex gain in captopril-treated embryos. Therefore, during typical chicken development, ANG II dampens the baroreflex regulatory capacity and chicken embryos can be used as a natural model of elevated ANG II for studying developmental cardiovascular function. This study is the first to demonstrate that reduction of embryonic ANG II alters normal baroreflex function.

Dynamics of metabolic compensation and hematological changes in chicken (Gallus gallus) embryos exposed to hypercapnia with varying oxygen.

In day 15 chicken embryos, we determined the time course responses of acid-base balance and hematological respiratory variables during 24h exposure to 15, 20, 40 or 90% O(2), in the presence of 5% CO(2). Hypercapnic respiratory acidosis was initially (2h) only slightly (∼20%) compensated by metabolic alkalosis in normoxic/hyperoxic embryos. After 6h, respiratory acidosis was partially (∼40-50%) compensated not only in normoxic/hyperoxic embryos, but also in hypoxic embryos. However, partial metabolic compensation in 15% O(2) could not be preserved after 24h. Preservation of metabolic compensation required oxygen concentration ([O(2)]) above 20%, but the magnitude of partial metabolic compensation was unrelated to [O(2)]. Hematocrit (Hct), together with mean corpuscular volume (MCV), markedly increased in hypercapnic hypoxia, and was maintained at 24h due to a subsequent increase in red blood cell concentration ([RBC]). In contrast, Hct, together with MCV, decreased in hypercapnic normoxia/hyperoxia accompanied by a subsequent decrease in [RBC] at 24h. Regulation of variables takes place similarly irrespective of environmental [O(2)] above 20%, matching acid-base regulation.

The trade-off between maturation and growth during accelerated development in frogs.

Developmental energetics are crucial to a species' life history and ecology but are poorly understood from a mechanistic perspective. Traditional energy and mass budgeting does not distinguish between costs of growth and maturation, making it difficult to account for accelerated development. We apply a metabolic theory that uniquely considers maturation costs (Dynamic Energy Budget theory, DEB) to interpret empirical data on the energetics of accelerated development in amphibians. We measured energy use until metamorphosis in two related frogs, Crinia georgiana and Pseudophryne bibronii. Mass and energy content of fresh ova were comparable between the species. However, development to metamorphosis was 1.7 times faster in C. georgiana while P. bibronii produced nine times the dry biomass at metamorphosis and had lower mass-specific oxygen requirements. DEB theory explained these patterns through differences in ontogenetic energy allocation to maturation. P. bibronii partitioned energy in the same (constant) way throughout development whereas C. georgiana increased the fraction of energy allocated to maturation over growth between hatching and the onset of feeding. DEB parameter estimation for additional, direct-developing taxa suggests that a change in energy allocation during development may result from a selective pressure to increase development rate, and not as a result of development mode.

Analysis of cutaneous and internal gill gas exchange morphology in early larval amphibians, Pseudophryne bibronii and Crinia georgiana.

This study uses stereological techniques to examine body, internal gill and cardiovascular morphology of two larval amphibians, Pseudophryne bibronii and Crinia georgiana, to evaluate the roles of diffusive and convective gas exchange. Gosner stage 27 specimens were prepared for light microscopy and six parallel sections of equal distance taken through the body as well as a further six through the heart and internal gills. Body, internal gill and heart volume as well as body and internal gill surface areas were determined. The harmonic mean distance across the internal gills was also measured and used to estimate oxygen diffusive conductance, DO2. The species were of similar body size and surface area, but the heart and internal gills were larger in P. bibronii, which may represent precursors for greater growth of the species beyond stage 27. The much larger surface area of the skin compared to the internal gills in both species suggests it is the main site for gas exchange, with the gills supplementing oxygen uptake. The sparse cutaneous capillary network suggests diffusion is the main oxygen transport mechanism across the skin and directly into deeper tissues. A numerical model that simplifies larval shape, and has an internal (axial vessels) and external oxygen source, confirms that diffusion is able to maintain tissue oxygen with limited convective input.