Manipulating plasma thyroid hormone levels at hatching alters development of endothermy and ventilation in Pekin duck (Anas platyrhynchos domestica).

At hatching in precocial birds, there are rapid physiological and metabolic phenotypic changes associated with attaining endothermy. During the transition to ex ovo life, thyroid hormone levels naturally increase, peaking at hatching, and then decline. To better understand the role of the natural increase in thyroid hormone at hatching in regulating the developmental trajectory of the Pekin duck's endothermic phenotype, we examined development of O2 consumption (V̇O2 ) and ventilation (frequency, tidal volume and minute ventilation) while inhibiting the developmental increase in thyroid hormones that occurs at hatching via administration of the thyroid-peroxidase inhibitor methimazole (MMI) or accelerating the developmental increase via triiodothyronine (T3) supplementation. Animals were dosed only on day 24 of a 28-day incubation period and studied on incubation day 25, during external pipping (EP) and 1 day post-hatching (dph). On day 25, there was an increase in V̇O2  in the hyperthyroid treatment compared with the other two treatments. During the EP stage, there was a significant effect of thyroid status on V̇O2 , with hyperthyroid V̇O2  being highest and hypothyroid V̇O2  the lowest. By 1 dph, the supplemented T3 and control animals had similar V̇O2  responses to cooling with comparable thermal neutral zones followed by increased V̇O2 Hypothyroid 1 dph hatchlings had a lower resting V̇O2  that did not increase to the same extent as the supplemented T3 and control animals during cooling. During EP, inhibiting the rise in T3 resulted in embryos with lower ventilation frequency and tidal volume than control and supplemented T3 embryos. At 1 dph, ventilation frequency of all animals increased during cooling, but tidal volume only increased in supplemented T3 and control hatchlings. Our data support the role of the late incubation increase in T3 in regulating the systemic development of endothermic metabolic capacity and associated control of ventilation occurring at hatching of the Pekin duck.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity during the transition to endothermy in an altricial bird.

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump critical to muscle calcium cycling during contraction, and SERCA has also been proposed as the basis for a non-shivering thermogenesis mechanism in birds. Despite its potential importance to both shivering and non-shivering thermogenesis, the activity of this transporter has rarely been studied in altricial birds, and never during the developmental transition from ectothermy to endothermy. Here, we describe SERCA activity in the pectoralis muscle and heart ventricle of red-winged blackbird (Agelaius phoeniceus) nestlings, fledglings and adults. Additionally, using a diet manipulation, we tested the hypothesis that muscle SERCA activity is affected by dietary fatty acid composition, as has been shown in some previous studies. In blackbird hearts, SERCA activity increased throughout development and into adulthood, conspicuously jumping higher just prior to fledging. In pectoralis muscle, SERCA activity increased throughout the nestling period, but then declined after fledging, an effect we attribute to remodeling of the muscle from a primarily heat-generating organ to a primarily force-generating organ. SERCA activity of the pectoralis muscle was correlated with the proportion of linoleic acid in muscle phospholipids when including all ages in the control group. However, in diet-manipulated birds, there was no consistent relationship between SERCA activity and muscle membrane fatty acid composition at any tested age (5-9 days old). It is unclear whether SERCA might be affected by developmental changes in fatty acid composition at younger ages.

The membrane pacemaker hypothesis: novel tests during the ontogeny of endothermy.

The 'membrane pacemaker' hypothesis proposes a biochemical explanation for among-species variation in resting metabolism, based on the positive correlation between membrane docosahexaenoic acid (DHA) and metabolic rate. We tested this hypothesis using a novel model, altricial red-winged blackbird nestlings, predicting that the proportion of DHA in muscle and liver membranes should increase with the increasing metabolic rate of the nestling as it develops endothermy. We also used a dietary manipulation, supplementing the natural diet with fish oil (high DHA) or sunflower oil (high linoleic acid) to alter membrane composition and then assessed metabolic rate. In support of the membrane pacemaker hypothesis, DHA proportions increased in membranes from pectoralis muscle, muscle mitochondria and liver during post-hatch development. By contrast, elevated dietary DHA had no effect on resting metabolic rate, despite causing significant changes to membrane lipid composition. During cold challenges, higher metabolic rates were achieved by birds that had lower DHA and higher linoleic acid in membrane phospholipids. Given the mixed support for this hypothesis, we conclude that correlations between membrane DHA and metabolic rate are likely spurious, and should be attributed to a still-unidentified confounding variable.

Rapid embryonic accretion of docosahexaenoic acid (DHA) in the brain of an altricial bird with an aquatic-based maternal diet.

Docosahexaenoic acid (DHA) is an important and abundant fatty acid moiety in vertebrate brains. We measured brain phospholipid composition during development in red-winged blackbirds (Agelaius phoeniceus), an altricial species that breeds in aquatic habitats. We also manipulated diet by feeding nestlings fish oil or sunflower oil. Finally, we assessed selective uptake of yolk by comparing the yolk fatty acid composition of freshly laid eggs and day-old hatchlings. Relative to other altricial species, blackbirds achieved high DHA in brain phospholipids (20% of phospholipid fatty acids in day-old hatchlings). This was not a result of selective uptake from the yolk, but rather a consequence of a high proportion of DHA in the yolk (2.5% of total lipids) at laying. Our dietary study confirmed that nestling brains are sensitive to fatty acid supply. Red-winged blackbirds may be able to advance cognitive development relative to other altricial species owing to their aquatic maternal diet.

Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition.

We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group - even when controlling for body mass - when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes.

Ventilation changes associated with hatching and maturation of an endothermic phenotype in the Pekin duck, Anas platyrhynchos domestica.

Precocial birds begin embryonic life with an ectothermic metabolic phenotype and rapidly develop an endothermic phenotype after hatching. Switching to a high-energy, endothermic phenotype requires high-functioning respiratory and cardiovascular systems to deliver sufficient environmental oxygen to the tissues. We measured tidal volume (VT), breathing frequency (ƒ), minute ventilation (V̇e), and whole-animal oxygen consumption (V̇o2) in response to gradual cooling from 37.5°C (externally pipped paranates, EP) or 35°C (hatchlings) to 20°C along with response to hypercapnia during developmental transition from an ectothermic, EP paranate to endothermic hatchling. To examine potential eggshell constraints on EP ventilation, we repeated these experiments in artificially hatched early and late EP paranates. Hatchlings and artificially hatched late EP paranates were able to increase V̇o2significantly in response to cooling. EP paranates had high ƒ that decreased with cooling, coupled with an unchanging low VT and did not respond to hypercapnia. Hatchlings had significantly lower ƒ and higher VT and V̇e that increased with cooling and hypercapnia. In response to artificial hatching, all ventilation values quickly reached those of hatchlings and responded to hypercapnia. The timing of artificial hatching influenced the temperature response, with only artificially hatched late EP animals, exhibiting the hatchling ventilation response to cooling. We suggest one potential constraint on ventilatory responses of EP paranates is the rigid eggshell, limiting air sac expansion during inhalation and constraining VT Upon natural or artificial hatching, the VT limitation is removed and the animal is able to increase VT, V̇e, and thus V̇o2, and exhibit an endothermic phenotype.

Breathing while altricial: the ontogeny of ventilatory chemosensitivity in red-winged blackbird (Agelaius phoeniceus) nestlings.

Altricial bird species, like red-winged blackbirds, hatch at an immature state of functional maturity with limited aerobic capacity and no endothermic capacity. Over the next 10-12 days in the nest, red-winged blackbirds develop increased metabolic capacity before fledging. Although ontogeny of respiration has been described in precocial birds, ontogeny of ventilatory chemosensitivity is unknown in altricial species. Here we examined developmental changes in chemosensitivity of tidal volume (Vt), breathing frequency (ƒ), minute ventilation (V̇e), and whole animal oxygen consumption (V̇o2) from hatching to just before fledging in red-winged blackbirds on days 1, 3, 5, 7, and 9 posthatching (dph) in response to hypercapnia (2 and 4% CO2) and hypoxia (15 and 10% O2). Under control conditions, there was a developmental increase in V̇e with age due to increased Vt Hypercapnic and hypoxic chemosensitivities were present as early as 1 dph. In response to hypoxia, 1, 3, and 9 dph nestlings increased V̇e at 10% O2, by increasing ƒ with some change in Vt in younger animals. In contrast to early neonatal altricial mammals, the hypoxic response of nestling red-winged blackbirds was not biphasic. In response to hypercapnia, 3 dph nestlings increased V̇e by increasing both ƒ and Vt From 5 dph on, the hypercapnic increase in V̇e was accounted for by increased Vt and not ƒ. Chemosensitivity to O2 and CO2 matures early in nestling red-winged blackbirds, well before the ability to increase V̇o2 in response to cooling, and thus does not represent a limitation to the development of endothermy.

Development of endothermy and concomitant increases in cardiac and skeletal muscle mitochondrial respiration in the precocial Pekin duck (Anas platyrhynchos domestica).

Attaining endothermic homeothermy occurs at different times post-hatching in birds and is associated with maturation of metabolic and aerobic capacity. Simultaneous measurements at the organism, organ and cellular levels during the transition to endothermy reveal means by which this change in phenotype occurs. We examined development of endothermy in precocial Pekin ducks ( ITALIC! Anas platyrhynchos domestica) by measuring whole-animal O2consumption ( ITALIC! V̇O2 ) as animals cooled from 35 to 15°C. We measured heart ventricle mass, an indicator of O2delivery capacity, and mitochondrial respiration in permeabilized skeletal and cardiac muscle to elucidate associated changes in mitochondrial capacities at the cellular level. We examined animals on day 24 of incubation through 7 days post-hatching. ITALIC! V̇O2  of embryos decreased when cooling from 35 to 15°C; ITALIC! V̇O2  of hatchlings, beginning on day 0 post-hatching, increased during cooling with a lower critical temperature of 32°C. Yolk-free body mass did not change between internal pipping and hatching, but the heart and thigh skeletal muscle grew at faster rates than the rest of the body as the animals transitioned from an externally pipped paranate to a hatchling. Large changes in oxidative phosphorylation capacity occurred during ontogeny in both thigh muscles, the primary site of shivering, and cardiac ventricles. Thus, increased metabolic capacity necessary to attain endothermy was associated with augmented metabolic capacity of the tissue and augmented increasing O2delivery capacity, both of which were attained rapidly at hatching.

Manipulating plasma thyroid hormone levels alters development of endothermy and ventilation in nestling red-winged blackbirds.

Thyroid hormones are key regulators of development and metabolism in vertebrates. During the nestling period, young of altricial species transition from an ectothermic phenotype to an endothermic phenotype. Red-winged blackbirds are an altricial species that exhibit an increase in plasma 3,3', 5-triiodo-L-thyronine (T3) levels during the first 5 days post-hatch (dph), begin to develop endothermic metabolic responses by 7 dph, and fledge within 10 days of hatching. We propose that thyroid hormones play an important role in regulating development of endothermy during the nestling period in altricial birds. To better understand the effects of thyroid hormones on endothermic metabolic development in an altricial species, we treated nestling red-winged blackbirds on 2, 3, and 5 dph with either methimazole (MMI) to induce hypothyroidism or supplemental T3 to induce hyperthyroidism. We then measured on 5, 7, and 9 dph morphology and whole animal O2 consumption ( V ˙ o 2 ) and ventilation in the thermal neutral zone and during gradual cooling. Treatment of nestlings with MMI resulted in lower plasma T3 levels on 5 dph that recovered by 7 dph, while supplementing with T3 did not affect plasma T3 levels on 5, 7 and 9 dph. Treatment with MMI resulted in smaller nestlings with smaller hearts and structural characters such as wing chord and femur length, but larger lungs and kidneys. Treatment with T3 produced smaller nestlings with smaller body masses and shorter femur and tarsus lengths. The development of V ˙ o 2 and ventilation endothermic responses to gradual cooling in MMI treated nestlings were delayed when compared with control nestlings. In 9 dph nestlings, hypothyroidism resulted in alterations in the responses of ventilation frequency and tidal volume to cooling when compared with the control nestlings. Supplemental T3 had no effect on the development of V ˙ o 2 and ventilation in the thermal neutral zone or in response to cooling. Our data suggest plasma thyroid hormone levels play an active role in the systemic development of endothermic capacity and the development of ventilatory control. In the nestling avian, multiple systems develop in concert to produce an endothermic phenotype, but reduced thyroid hormone delays maturation of endothermic capacity.

Thyroid hormone manipulation influences development of cardiovascular regulation in embryonic Pekin duck, Anas platyrhynchos domestica.

Thyroid hormones are key regulators of avian metabolism and may play a significant role in development at hatching. To better understand the role of thyroid hormones in avian development, we examined autonomic control of heart rate and blood pressure while manipulating thyroid hormone levels in the late stage embryonic Pekin duck (Anas platyrhynchos domestica). Thyroid hormone levels were manipulated on day 24 of a 28-day incubation period with the thyroperoxidase inhibitor methimazole (MMI), triiodothyronine (T3), or saline. On day 25 of incubation, autonomic tone on cardiovascular function was studied by injections of cholinergic and adrenergic receptor antagonists. Embryos from all treatment groups expressed a cholinergic and ß-adrenergic tone on heart rate at this age. Cholinergic blockade with atropine produced a larger change in heart rate in the hyperthyroid animals compared with euthyroid animals. In response to ß-adrenergic blockade, hyperthyroid conditions produced a larger decrease in heart rate compared with euthyroid animals, with no change in mean arterial blood pressure. In response to α-adrenergic blockade, mean arterial blood pressure decreased in the euthyroid animals and more developed hyperthyroid animals. Collectively, the data indicate that elevated levels of T3 can influence maturation of cholinergic and adrenergic receptor-mediated cardiovascular regulation in developing Pekin ducks near the end of incubation.

The 12-day thermoregulatory metamorphosis of Red-winged Blackbirds (Agelaius phoeniceus).

We examined development of endothermy in altricial Red-winged Blackbirds (Agelaius phoeniceus) by measuring oxygen consumption [Formula: see text], body temperature and ventilation at ambient temperatures from 35 to 15 °C. Mitochondrial respiration of permeabilized skeletal muscle was also measured from breast (pectoralis) and thigh (femorotibialis) muscles. Animals were studied from the first day of hatching through fledging (12 days post-hatch, dph). Nestling whole-body metabolic rate began to show an endothermic response to cold temperature midway between hatching and fledging. Nestlings less than 5 dph were unable to maintain elevated [Formula: see text] and body temperature when exposed to gradually decreasing temperature, whereas 7 dph nestlings maintained [Formula: see text] until ~25 °C, after which [Formula: see text] decreased. From 10 dph to fledging, animals maintained elevated [Formula: see text] and body temperature when exposed to gradual cooling; full endothermic capacity was achieved. Ventilation followed a similar developmental trend to that of [Formula: see text], with increases in 10 dph fledglings occurring in tidal volume rather than ventilation frequency. LEAK respiration and oxidative phosphorylation (OXPHOS) through complex I of breast muscle mitochondria increased significantly after 3 dph. Expression of avUCP and PCG-1α mRNA increased significantly at 3 dph and remained elevated in both skeletal muscle types. Increased metabolic capacity at the cellular level occurred prior to that of the whole animal. This change in whole animal metabolic capacity increased steadily upon hatching as evidenced by the shift of metabolic rate from an ectothermic to endothermic phenotype and the increase of mitochondrial OXPHOS activity of the shivering muscles of this altricial avian species.

Post-hatching development of mitochondrial function, organ mass and metabolic rate in two ectotherms, the American alligator (Alligator mississippiensis) and the common snapping turtle (Chelydra serpentina).

The ontogeny of endothermy in birds is associated with disproportionate growth of thermogenic organs and increased mitochondrial oxidative capacity. However, no similar study has been made of the development of these traits in ectotherms. For comparison, we therefore investigated the metabolism, growth and muscle mitochondrial function in hatchlings of a turtle and a crocodilian, two ectotherms that never develop endothermy. Metabolic rate did not increase substantially in either species by 30 days post-hatching. Yolk-free body mass and heart mass did not change through 30 days in alligators and heart mass was a constant proportion of body mass, even after 1 year. Yolk-free body mass and liver mass grew 36% and 27%, respectively, in turtles during the first 30 days post-hatch. The mass-specific oxidative phosphorylation capacity of mitochondria, assessed using permeabilized muscle fibers, increased by a non-significant 47% in alligator thigh and a non-significant 50% in turtle thigh over 30 days, but did not increase in the heart. This developmental trajectory of mitochondrial function is slower and shallower than that previously observed in ducks, which demonstrate a 90% increase in mass-specific oxidative phosphorylation capacity in thigh muscles over just a few days, a 60% increase in mass-specific oxidative phosphorylation capacity of the heart over a few days, and disproportionate growth of the heart and other organs. Our data thus support the hypothesis that these developmental changes in ducks represent mechanistic drivers for attaining endothermy.