Sarah J. Andrewartha

Embryonic control of heart rate: Examining developmental patterns and temperature and oxygenation influences using embryonic avian models

Long-term measurements (days and weeks) of heart rate (HR) have elucidated infradian rhythms in chicken embryos and circadian rhythms in chicken hatchlings. However, such rhythms are lacking in emu embryos and only rarely observed in emu hatchlings. Parasympathetic control of HR (instantaneous heart rate (IHR) decelerations) occurs at ∼60% of incubation in both precocial and altricial avian embryos, with sympathetic control (IHR accelerations) becoming more prevalent close to hatching. A large increase in avian embryonic HR occurs during hatching (presumably an energetically expensive process, i.e. increased oxygen consumption M(O) ₂), beginning during pipping when a physical barrier to O(2) conductance is removed. Alterations in ambient O(2) have little effect on early embryonic HR, likely due to the low rate of M(O)₂ of early embryos and the fact that adequate O(2) delivery can occur via diffusion. As M(O)₂ increases in advanced embryos and circulatory convection becomes important for O(2) delivery, alterations in ambient O(2) have more profound effects on embryonic HR. Early embryos demonstrate a wide ambient temperature (T(a)) tolerance range compared with older embryos. In response to a rapid decrease in T(a), embryonic HR decreases (stroke volume and blood flow are preserved) in an exponential fashion to a steady state (from which it can potentially recover if re-warmed). A more severe decrease in T(a) results in complete cessation of HR; however, depending on developmental age, embryos are able to survive severe cold exposure and cessation of HR for up to 24h in some instances. The development of endothermy can be tracked by measuring baseline HR during T(a) changes. HR patterns change from thermo-conformity to thermoregulation (reverse to T(a) changes). Further, IHR low frequency oscillations mediated by the autonomic nervous system are augmented at low T(a)s in hatchlings. Transitions of baseline HR during endothermic development are unique to individual avian species (e.g. chickens, ducks and emu), reflecting differences in life history.

Development of hematological respiratory variables in late chicken embryos: The relative importance of incubation time and embryo mass

Oxygen demand increases during embryonic development, requiring an increase in red blood cells (RBCs) containing hemoglobin (Hb) to transport O(2) between the respiratory organ and systemic tissues. A thorough ontogenetic understanding of the onset and maturation of the complex regulatory processes for RBC concentration ([RBC]), Hb concentration ([Hb]), hematocrit (Hct), mean corpuscular indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration ([MCHb])) is currently lacking. We hypothesize that during the last half of incubation when the respiratory organ (the chorioallantoic membrane) envelops most of the egg contents, mean corpuscular indices will stabilize. Accordingly, Hct, [RBC] and [Hb] must also all change proportionally across development. Further, we hypothesize that the hematological respiratory variables develop and mature as a function of incubation duration, independently of embryonic growth. As predicted, a similar increase in Hct (from 18.7±0.6% on day 10 (d10) to 34.1±0.5% on d19 of incubation), [RBC] (1.13±0.03×10(6)/μL to 2.50±0.03×10(6)/μL) and [Hb] (6.1±0.2 g% to 11.2±0.1 g%) occurred during d10-19. Both [RBC] and [Hb] demonstrated high linear correlation with Hct, resulting in constant [MCHb] (~33 g% from d10 to d19). The decrease in MCV (from ~165 μ(3) on d10 to ~140 μ(3) on d13) and MCH (~55 pg to ~45 pg) during d10-13, may be attributed to a changeover from larger primary to smaller secondary and adult-type erythrocytes with MCV and MCH remaining constant (~140 μ(3) and ~45 pg respectively) for the rest of the incubation period (d13-19). Hematological respiratory values on a given incubation day were identical between embryos of different masses using either natural mass variation or experimental growth acceleration, indicating that the hematological variables develop as a function of incubation time, irrespective of embryo growth.

Interactions of acid-base balance and hematocrit regulation during environmental respiratory gas challenges in developing chicken embryos (Gallus gallus)

How the determinants of hematocrit (Hct) - alterations in mean corpuscular volume (MCV) and/or red blood cell concentration ([RBC]) - are influenced by acid-base balance adjustments across development in the chicken embryo is poorly understood. We hypothesized, based on oxygen transport needs of the embryos, that Hct will increase during 1 day of hypercapnic hypoxia (5%CO(2), 15%O(2)) or hypoxia alone (0%CO(2), 15%O(2)), but decrease in response to hyperoxia (0%CO(2), 40%O(2)). Further, age-related differences in acid-base disturbances and Hct regulation may arise, because the O(2) transport and hematological regulatory systems are still developing in embryonic chickens. Our studies showed that during 1 day of hypoxia (with or without hypercapnia) Hct increased through both increased MCV and [RBC] in day 15 (d15) embryo, but only through increased MCV in d17 embryo and therefore enhancement of O(2) transport was age-dependent. Hypercapnia alone caused a ≈ 14% decrease in Hct through decreased [RBC] and therefore did not compensate for decreased blood oxygen affinity resulting from the Bohr shift. The 11% (d15) and 14% (d17) decrease in Hct during hyperoxia in advanced embryos was because of an 8% and 9% decrease, respectively, in [RBC], coupled with an associated 3% and 5% decrease in MCV. Younger, d13 embryos were able to metabolically compensate for respiratory acidosis induced by hypercapnic hypoxia, and so were more tolerant of disturbances in acid-base status induced via alterations in environmental respiratory gas composition than their more advanced counterparts. This counter-intuitive increased tolerance likely results from the relatively low [Formula: see text] and immature physiological functions of younger embryos.

Acute regulation of hematocrit and blood acid-base balance during severe hypoxic challenges in late chicken embryos (Gallus gallus)

Acid-base and hematocrit (Hct) responses of vertebrate embryos to severe hypoxia are as yet unknown, but may reveal the maturation process of physiological regulatory mechanisms. The present study elucidated how acute, severe hypoxia (10% O2, with and without 5% CO2) affects Hct and blood acid-base balance in late prenatal (days 11-19) chicken embryos. The time-course of the resulting Hct changes and blood acid-base disturbances was examined in detail in day 15 (d15) embryos to further understand the magnitude and time-components of these physiological changes. We hypothesized that Hct of developing embryos increases during severe hypoxia (10% O2) and hypercapnic hypoxia (5% CO2, 10% O2), due to increased mean corpuscular volume (MCV) and red blood cell concentration ([RBC]). We additionally hypothesized that 10% O2 would induce anaerobic glycolysis and the attendant increase in lactate concentration ([La-]) would create a severe metabolic acidosis. Hct increased in all embryos (d11-d19) during severe hypoxia (2h) but, with the exception of d19 embryos, the increase was due to increased MCV and was therefore unlikely related to O2 transport. The time-course of the d15 embryonic Hct response to hypoxic or hypercapnic hypoxic exposure was very rapid with MCV increasing within 30min. Severe metabolic acidosis occurred in all developing embryos (d11-d19) during 2h hypoxic exposure. Additionally, respiratory acidosis was induced in d15 embryos during hypercapnic hypoxia, with acid-base status recovering within 120 min in air. Throughout hypoxic exposure and recovery, changes in [HCO3-] were matched by those in [La-], indicating that anaerobic glycolysis is a key factor determining the metabolic alterations and overall acid-base status. Further, the blood gas and Hct values recovered in air and unchanged embryo mass suggest that the hypoxia-induced disturbances were only transient and may not affect long-term survival.

Transgenerational variation in metabolism and life-history traits induced by maternal hypoxia in Daphnia magna.

Hypoxic stress can alter conspecific phenotype and additionally alter phenotypes of the filial generation, for example, via maternal or epigenetic processes. Lasting effects can also be seen across development and generations even after stressors have been removed. This study utilized the model of rapidly developing, parthenogenetic Daphnia to examine the intraspecific variability of response of exposure of a parental generation to hypoxia (4 kPa) within a single clone line across development, across broods, and across generations. Body mass across development and reproductive output were monitored in the parental generation and the first three broods of the first filial generation (which were not directly exposed to hypoxia). O2 consumption across a wide Po2 range (normoxia to anoxia) was assessed to determine whether exposure of the parental generation to hypoxia conferred hypoxia tolerance on the offspring and whether this transgenerational, epigenetic phenomenon varied intraspecifically. Differences in mass occurred in both the parental generation (hypoxia-exposed smaller during brood 1 and brood 2 neonate production) and the filial generation (e.g., brood 1 and 2 neonates from hypoxic mothers were initially smaller than control neonates). However, differences in mass were not accompanied by changes in reproductive output (assessed by brood number and neonate size). At day 0, first filial generation brood 1 neonates from hypoxia-exposed mothers had a higher metabolic rate than control neonates. However, this effect, like that of body mass, dissipated with development within a brood but also with subsequent broods. An isometric scaling exponent for was repeatedly observed across a wide Po2 range (21–2 kPa) throughout neonatal development.

Hematocrit and blood osmolality in developing chicken embryos (Gallus gallus): In vivo and in vitro regulation

Hematocrit (Hct) regulation is a complex process involving potentially many factors. How such regulation develops in vertebrate embryos is still poorly understood. Thus, we investigated the role of blood pH in the regulation of Hct across developmental time in chicken embryos. We hypothesized that blood pH alterations in vitro (i.e., in a test tube) would affect Hct far more than in vivo because of in vivo compensatory regulatory processes for Hct. Large changes in Hct (through mean corpuscular volume (MCV)) and blood osmolality (Osm) occur when the blood was exposed to varying ambient temperatures (T(a)s) and P(CO2) in vitro alongside an experimentally induced blood pH change from ~7.3 to 8.2. However, homeostatic regulatory mechanisms apparently limited these alterations in vivo. Changes in blood pH in vitro were accompanied by hydration or dehydration of red blood cells depending on embryonic age, resulting in changes in Hct that also were specific to developmental stage, due likely to initial blood gas and [HCO(3)(-)](v) values. Significant linear relationships between Hct and pH (Hct/ΔpH=-21.4%/(pH unit)), Hct and [HCO(3)(-)] (ΔHct/Δ[HCO(3)(-)]=1.6%/(mEq L(-1))) and the mean buffer value (Δ[HCO(3)(-)]/ΔpH=-13.4 (mEq L(-1))/(pH unit)) demonstrate that both pH and [HCO(3)(-)] likely play a role in the regulation of Hct through MCV at least in vitro. Low T(a) (24°C) resulted in relatively large changes in pH with small changes in Hct and Osm in vitro with increased T(a) (42°C) conversely resulting in larger changes in both Hct and Osm. In vivo exposure to altered T(a) caused age-dependent changes in Hct, demonstrating a trend towards increased Hct at higher T(a). Further, exposing embryos to a gas mixture where P(CO2) = 5.1 kPa for >4 h period at T(a) of 37 or 42°C also did not elicit a change in Hct or Osm. Presumably, homeostatic mechanisms ensured that in vivo Hct was stable during a 4-6 h temperature and/or hypercapnic stress. Thus, although blood pH decreases (induced by acute T(a) increase and exposure to CO(2)) increase MCV and, consequently, Hct in vitro, homeostatic mechanisms operating in vivo are adequate to ensure that such environmental perturbations have little effect in vivo.

Acute regulation of hematocrit and acid-base balance in chicken embryos in response to severe intrinsic hypercapnic hypoxia.

The regulation of blood acid-base balance and hematology in day 15 chicken embryos in response to partial water submersion (with eggs air cell in air) and complete submersion producing severe intrinsic hypercapnic hypoxia and recovery in air was studied. The acid-base disturbance during submersion was characterized by initial rapid respiratory changes and then superseded by metabolic processes, resulting in a large progressive hysteresis. Throughout submersion and recovery, blood lactate concentration changed swiftly along with the changes in bicarbonate concentration ([HCO3(-)]), indicating that anaerobic glycolysis determined overall acid-base disturbances. Both partial and complete submersion produced large, rapid increases in hematocrit through proportional increases in mean corpuscular volume and red blood cell concentration. Death ensued once the internal pool of O2 was exhausted and/or the acid-base disturbance became too severe for survival (i.e., [HCO3(-)]a<∼10mmolL(-1)). However, embryos recovered from acid-base and hematological disturbances within 120min recovery in air after short bouts of complete (20min) or partial (60min) submersion, suggesting that shorter severe intrinsic hypercapnic hypoxia does not compromise viability of embryos.

The accessory role of the diaphragmaticus muscle in lung ventilation in the estuarine crocodile Crocodylus porosus

SUMMARY Crocodilians use a combination of three muscular mechanisms to effect lung ventilation: the intercostal muscles producing thoracic movement, the abdominal muscles producing pelvic rotation and gastralial translation, and the diaphragmaticus muscle producing visceral displacement. Earlier studies suggested that the diaphragmaticus is a primary muscle of inspiration in crocodilians, but direct measurements of the diaphragmatic contribution to lung ventilation and gas exchange have not been made to date. In this study, ventilation, metabolic rate and arterial blood gases were measured from juvenile estuarine crocodiles under three conditions: (i) while resting at 30°C and 20°C; (ii) while breathing hypercapnic gases; and (iii) during immediate recovery from treadmill exercise. The relative contribution of the diaphragmaticus was then determined by obtaining measurements before and after transection of the muscle. The diaphragmaticus was found to make only a limited contribution to lung ventilation while crocodiles were resting at 30°C and 20°C, and during increased respiratory drive induced by hypercapnic gas. However, the diaphragmaticus muscle was found to play a significant role in facilitating a higher rate of inspiratory airflow in response to exercise. Transection of the diaphragmaticus decreased the exercise-induced increase in the rate of inspiration (with no compensatory increases in the duration of inspiration), thus compromising the exercise-induced increases in tidal volume and minute ventilation. These results suggest that, in C. porosus, costal ventilation alone is able to support metabolic demands at rest, and the diaphragmaticus is largely an accessory muscle used at times of elevated metabolic demand.

An Algorithm for the Automatic Analysis of Signals From an Oyster Heart Rate Sensor

An in situ optical oyster heart rate sensor generates signals requiring frequency estimation with properties different to human ECG and speech signals. We discuss the method of signal generation and highlight a number of these signal properties. An optimal heart rate estimation approach was identified by application of a variety of frequency estimation techniques and comparing results to manually acquired values. Although a machine learning approach achieved the best performance, accurately estimating 96.8% of the heart rates correctly, a median filtered autocorrelation approach achieved 93.7% with significantly less computational requirement. A method for estimating heart rate variation is also presented.

Phenotypic differences in terrestrial frog embryos: effect of water potential and phase

SUMMARY The terrestrial embryos of many amphibians obtain water in two ways; in a liquid phase from the substrate on which eggs are deposited, and in a vapour phase from the surrounding atmosphere. We tested whether the mode of water flux (liquid or vapour) affected the morphology and metabolic traits of the terrestrial Victorian smooth froglet (Geocrinia victoriana) embryos by incubating eggs both with a liquid water source and at a range of vapour water potentials. We found that embryos incubated with a liquid water source (ψπ=0 kPa) were better hydrated than embryos incubated with a vapour water source (ψv=0 kPa), and grew to a larger size. Eggs incubated in atmospheres with lower ψv values showed significant declines in mass and in the thickness of the jelly capsule, while embryos primarily showed reductions in dry mass, total length, tail length and fin height. The most significant deviations from control (ψv=0 kPa) values were observed when the ψv of the incubation media was less than the osmotic water potential (ψπ) of the embryonic interstitial fluid (approximately –425 kPa). Despite the caveat that a ψv of 0 kPa is probably difficult to achieve under our experimental conditions, the findings indicate the importance for eggs under natural conditions of contacting liquid water in the nesting substrate to allow swelling of the capsule.