Blood flow distribution in embryonic common snapping turtles Chelydra serpentina (Reptilia; Chelonia) during acute hypoxia and α-adrenergic regulation.

Embryonic turtles have four distinct vascular beds that separately perfuse the developing embryo's body and the extra-embryonic yolk sac, amnion and chorioallantoic membrane (CAM). The mechanisms enabling differential regulation of blood flow through these separate beds, in order to meet the varying demands of the embryo during development, is of current interest. The present investigation followed the changes in blood flow distribution during an acute exposure to hypoxia and after α-adrenergic blockade. We monitored heart rate (fH), mean arterial pressure (Pm), and determined relative blood flow distribution (%Q̇sys) using colored microspheres. At 70% and 90% of the incubation period hypoxia elicited a bradycardia without changing Pm while %Q̇sys was altered only at 70%, increasing to the CAM and liver. Blockade of α-adrenergic responses with phentolamine did not change fH or Pm but increased %Q̇sys to the shell. These results show the capacity of embryos to redistribute cardiac output during acute hypoxia, however α-adrenergic receptors seemed to play a relatively small role in embryonic cardiovascular regulation.

Convective oxygen transport during development in embryos of the snapping turtle Chelydra serpentina.

This study investigated the maturation of convective oxygen transport in embryos of the snapping turtle (Chelydra serpentina). Measurements included: mass, oxygen consumption (V̇O2 ), heart rate, blood oxygen content and affinity and blood flow distribution at 50%, 70% and 90% of the incubation period. Body mass increased exponentially, paralleled by increased cardiac mass and metabolic rate. Heart rate was constant from 50% to 70% incubation but was significantly reduced at 90% incubation. Hematocrit and hemoglobin concentration were constant at the three points of development studied but arteriovenous difference doubled from 50% to 90% incubation. Oxygen affinity was lower for the early 50% incubation group (stage 19) compared with all other age groups. Blood flow was directed predominantly to the embryo but was highest to the chorioallantoic membrane (CAM) at 70% incubation and was directed away from the yolk as it was depleted at 90% incubation. The findings indicate that the plateau or reduction in egg V̇O2  characteristic of the late incubation period of turtle embryos may be related to an overall reduction in mass-specific V̇O2  that is correlated with decreasing relative heart mass and plateaued CAM blood flow. Importantly, if the blood properties remain unchanged prior to hatching, as they did during the incubation period studied in the current investigation, this could account for the pattern of V̇O2 previously reported for embryonic snapping turtles prior to hatching.

Commentary on: "Vascular distensibilities have minor effects on intracardiac shunt patterns in reptiles" by Filogonio et al. (2017).

The recent study by Filogonio et al. (2017) suggested that net cardiac shunt patterns in two species of reptiles (Trachemys scripta and Crotalus durissus) were not significantly influenced by the vascular distensibilities of the systemic and pulmonary vasculatures. This is in contrast to a previously published study (Hillman et al., 2014) in the toad (Rhinella marina) in which net cardiac shunts were predicted primarily by the physical properties of vascular distensibility rather than physiological control of resistance of the systemic and pulmonary vasculature. We analyze the data and conclusions reached by Filogonio et al. (2017) regarding the role of vascular distensibilities in determining net cardiac shunt patterns in reptiles in comparison with toads. In our view, the conclusions reached by Filogonio et al. (2017) are not supported by the data primarily because vascular distensibilities were not measured in the reptiles analyzed in their study.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

Dynamics of blood viscosity regulation during hypoxic challenges in the chicken embryo (Gallus gallus domesticus).

Hypoxia in chicken embryos increases hematocrit (Hct), blood O2 content, and blood viscosity. The latter may limit O2 transport capacity (OTC) via increased peripheral resistance. Hct increase may result from increased nucleated red blood cell concentration ([RBC]) and mean corpuscular volume (MCV) or reduced plasma volume. We hypothesized changes in Hct, hemoglobin concentration ([Hb]), [RBC] and MCV and their effects on viscosity would reduce OTC. Five experimental treatments that increase Hct were conducted on day 15 embryos: 60min water submergence with 60min recovery in air; exposure to 15% O2 with or without 5% CO2 for 24 h with 6 h recovery; or exposure to 10% O2 with or without 5% CO2 for 120 min with 120 min recovery. Control Hct, [Hb], [RBC], MCV, and viscosity were approximately 26%, 9g%, 2.0 10(6)µL(-1), 130µm(3), and 1.6mPas, respectively. All manipulations increased Hct and blood viscosity without changing blood osmolality (276mmolkg(-1)). Increased viscosity was attributed to increased [RBC] and MCV in submerged embryos, but solely MCV in embryos experiencing 10% O2 regardless of CO2. Blood viscosity in embryos exposed to 15% O2 increased via increased MCV alone, and viscosity was constant during recovery despite increased [RBC]. Consequently, blood viscosity was governed by MCV and [RBC] during submergence, while MCV was the strongest determinant of blood viscosity in extrinsic hypoxia with or without hypercapnia. Increased Hct and blood O2 content did not compensate for the effect of increased viscosity on OTC during these challenges.

Critical Windows of Cardiovascular Susceptibility to Developmental Hypoxia in Common Snapping Turtle (Chelydra serpentina) Embryos.

Environmental conditions fluctuate dramatically in some reptilian nests. However, critical windows of environmental sensitivity for cardiovascular development have not been identified. Continuous developmental hypoxia has been shown to alter cardiovascular form and function in embryonic snapping turtles (Chelydra serpentina), and we used this species to identify critical periods during which hypoxia modifies the cardiovascular phenotype. We hypothesized that incubation in 10% O2 during specific developmental periods would have differential effects on the cardiovascular system versus overall somatic growth. Two critical windows were identified with 10% O2 from 50% to 70% of incubation, resulting in relative heart enlargement, either via preservation of or preferential growth of this tissue, while exposure to 10% O2 from 20% to 70% of incubation resulted in a reduction in arterial pressure. The deleterious or advantageous aspects of these embryonic phenotypes in posthatching snapping turtles have yet to be explored. However, identification of these critical windows has provided insight into how the developmental environment alters the phenotype of reptiles and will also be pivotal in understanding its impact on the fitness of egg-laying reptiles.

Net cardiac shunts in anuran amphibians: physiology or physics?

Amphibians have a single ventricle and common conus arteriosus that produces an equal pressure to the parallel pulmocutaneous and systemic vascular circuits. The distribution of blood flows between the pulmocutaneous (Qpul) and systemic (Qsys) circuits (net cardiac shunt) varies with a number of environmental conditions and behaviours; although autonomic regulation of pulmonary vascular resistance conductance has been emphasized, little attention has been paid to the possible contribution of the passive physical characteristics of the two circuits to pressure changes associated with variation in cardiac output. In this study, we re-analysed three recent studies that recorded net cardiac shunts in the cane toad (Rhinella marina) under a variety of conditions and treatments. In all three studies, Qpul and Qsys were linearly related to cardiac output (Qtot), but the slope was threefold higher for Qpul compared with Qsys as predicted by relative conductance increases associated with increases in pressure from perfused preparations where autonomic regulation and humoral control were eliminated. Our analysis indicates that the net cardiac shunt in the cane toad is predicted primarily by the physical, rather than physiological, characteristics of the parallel pulmonary and systemic vascular circuits.

Separating the contributions of vascular anatomy and blood viscosity to peripheral resistance and the physiological implications of interspecific resistance variation in amphibians.

Amphibian pulmonary and systemic vascular circuits are arranged in parallel, with potentially important consequences for resistance (R) to blood flow. The contribution of the parallel anatomic arrangement to total vascular R (R T), independent of blood viscosity, is unknown. We measured pulmonary (R P) and systemic (R S) vascular R with an in situ Ringer's solution perfusion technique using anesthetized anuran and urodele species to determine: (1) relative contributions of vascular anatomy and blood viscosity to R T; (2) distensibility index (%Δ flow kPa(-1)) of the pulmonary and systemic vascular circuits; and (3) interspecific correlates of variation in these parameters with red blood cell size, cardiac power output, and aerobic capacities. R P was lower than R S in anurans, while R P of the urodeles was greater than R S and significantly greater than anuran R P. Anuran R T was lowest and did not vary interspecifically, whereas urodele R T was significantly greater than anuran, and varied interspecifically. Pulmonary and systemic circuit distensibility differences may explain cardiac shunt patterns in toads with changes in cardiac output from rest to activity. When blood viscosity was taken into account, vascular resistance accounted for about 25 % of R T while blood viscosity accounted for the remaining 75 %. Owing to lower R T, terrestrial anuran species required lower cardiac power outputs when moving fluid through their vasculature compared to aquatic species. These results indicate that physical characteristics of the vasculature can account for interspecific differences in cardiovascular physiology and suggest a co-evolution of cardiac and vascular anatomy among amphibians.

Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression.

Reptile embryos tolerate large decreases in the concentration of ambient oxygen. However, we do not fully understand the mechanisms that underlie embryonic cardiovascular short- or long-term responses to hypoxia in most species. We therefore measured cardiac growth and function in snapping turtle embryos incubated under normoxic (N21; 21% O2) or chronic hypoxic conditions (H10; 10% O2). We determined heart rate (fH) and mean arterial pressure (Pm) in acute normoxic (21% O2) and acute hypoxic (10% O2) conditions, as well as embryonic responses to cholinergic, adrenergic, and ganglionic pharmacological blockade. Compared with N21 embryos, chronic H10 embryos had smaller bodies and relatively larger hearts and were hypotensive, tachycardic, and following autonomic neural blockade showed reduced intrinsic fH at 90% of incubation. Unlike other reptile embryos, cholinergic and ganglionic receptor blockade both increased fH. ß-Adrenergic receptor blockade with propranolol decreased fH, and α-adrenergic blockade with phentolamine decreased Pm. We also measured cardiac mRNA expression. Cholinergic tone was reduced in H10 embryos, but cholinergic receptor (Chrm2) mRNA levels were unchanged. However, expression of adrenergic receptor mRNA (Adrb1, Adra1a, Adra2c) and growth factor mRNA (Igf1, Igf2, Igf2r, Pdgfb) was lowered in H10 embryos. Hypoxia altered the balance between cholinergic receptors, α-adrenoreceptor and ß-adrenoreceptor function, which was reflected in altered intrinsic fH and adrenergic receptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a developing reptile embryo.

Alternative developmental pathways associated with diapause regulated by temperature and maternal influences in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus enter a state of developmental arrest termed diapause as part of their normal developmental program. Diapause can occur at two distinct developmental stages in this species, termed diapause II and III. When incubated at 25°C, most embryos enter diapause II, whereas a small percentage of 'escape' embryos develop continuously past diapause II and enter diapause III. Control of entry into diapause II can be altered by maternal influences and the incubation environment experienced by the embryos. Young females produce a higher proportion of escape embryos than do older females. In addition, increasing the incubation temperature from 25 to 30°C induces all embryos to escape from diapause. Surprisingly, escape embryos follow a different morphological and physiological developmental trajectory than do embryos that enter diapause II. Development of anterior structures is advanced compared with that of posterior structures in escape embryos when compared with embryos that will enter diapause II. The difference in timing of development for these two trajectories is consistent with changes observed between two species but is very atypical of variation observed within a species. Importantly, the two developmental pathways diverge early in development, during the segmentation period, when, according to evolutionary theory, constraint on developmental pathways should be relatively high. The possession of alternative developmental pathways in a vertebrate embryo is a novel finding, the ecological and evolutionary importance of which is still unknown, but potentially significant in terms of life-history evolution.