Cardiac rhythms in prenatal and perinatal emu embryos

Emu eggs weigh approximately 600 g and have an incubation duration (ID) of approximately 50 days. The egg mass is approximately 10-fold heavier than the chicken egg and the ID is approximately 2.5-fold longer. Daily changes in mean heart rate (MHR) of emu embryos were previously determined, but further measurement was needed to investigate the species-specific behavior of cardiac rhythm for comparison with other species. In the present study, we continuously measured the electrocardiogram of emu embryos while maintaining adequate gas exchange through the eggshell and determined instantaneous heart rate (IHR) during the last 2-7 days of incubation until hatching or death. The MHR over 1-min intervals was calculated from IHR data in order to present continuous developmental patterns of heart rate (HR) in a single graph and 24-h recordings of HR in a single panel, showing the HR trend over a prolonged period. However, neither circadian nor ultradian rhythms of HR were shown in these figures or by power spectrum analysis. The IHR distinctively fluctuated and the fluctuations were mainly comprised of three patterns of irregular HR accelerations in embryos that hatched. Respiratory sinus arrhythmia also occurred in perinatal embryos. During the final stages of the perinatal period, short-term, repeated, large accelerations of IHR appeared, which signaled imminent hatching and has been reported for chick embryos. IHR fluctuations in embryos that failed to hatch tended to become inactive towards death.     

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.     

Periodic cooling of bird eggs reduces embryonic growth efficiency

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.     

Avian Incubation Patterns Reflect Temporal Changes in Developing Clutches

Incubation conditions for eggs influence offspring quality and reproductive success. One way in which parents regulate brooding conditions is by balancing the thermal requirements of embryos with time spent away from the nest for self-maintenance. Age related changes in embryo thermal tolerance would thus be expected to shape parental incubation behavior. We use data from unmanipulated Black-capped Chickadee (Poecile atricapillus) nests to examine the temporal dynamics of incubation, testing the prediction that increased heat flux from eggs as embryos age influences female incubation behavior and/or physiology to minimize temperature fluctuations. We found that the rate of heat loss from eggs increased with embryo age. Females responded to increased egg cooling rates by altering incubation rhythms (more frequent, shorter on- and off- bouts), but not brood patch temperature. Consequently, as embryos aged, females were able to increase mean egg temperature and decrease variation in temperature. Our findings highlight the need to view full incubation as more than a static rhythm; rather, it is a temporally dynamic and finely adjustable parental behavior. Furthermore, from a methodological perspective, intra- and inter-specific comparisons of incubation rhythms and average egg temperatures should control for the stage of incubation.     

The functions of belly-soaking in Kentish Plovers Charadrius alexandrinus

We tested whether belly-soaking (i.e. wetting of ventral plumage) in incubating Kentish Plovers Charadrius alexandrinus in a hot environment serves to cool overheated eggs, or whether belly-soaking would mainly serve for adult thermoregulation, and egg wetting would be a mere consequence of the ventral feathers of incubating adults being wet. When ambient temperature was high (> 30 °C), body temperature of incubating Kentish Plovers increased with ambient temperatures. When adults departed from nests to belly-soak, egg temperatures were not critical for embryos. However, eggs in exposed nests overheated to levels that may be lethal for embryos when adults left the nests to belly-soak, and adults also employed belly-soaking to cool down overheated eggs when they returned to their nests. Indeed, the cooling rate of eggs was much faster when they were wetted. We conclude that the primary function of belly-soaking in the Kentish Plover is to dissipate body heat in heat-stressed incubating adults, rather than to cool overheated eggs. Eggs overheated as a consequence of short-term interruptions in nest attendance by heat-stressed adults to belly-soak, and Plovers employed secondarily belly-soaking to cool down overheated eggs. These results indicate that adult thermoregulation and egg cooling are not mutually exclusive functions of belly-soaking.     

Heat regulation during exercise with controlled cooling

During heavy sustained exercise, when sweating is usually needed to dissipate the extra metabolic heat, controlled cooling caused heat loss to match total heat production with little sweating. The total heat produced and metabolic rate were varied independently by having subjects walk uphill and down. Heat loss was measured directly with a suit calorimeter; other measurements included metabolic energy from respiratory gas exchange and body temperatures. Thermoregulatory sweating was minimized by adjusting cooling in the calorimeter suit. Heat loss rose to match total heat, not metabolic rate, and there was a slow rise in rectal temperature. In the absence of major thermoregulatory response rectal temperature correlated most closely with total heat; it also correlated with the relative oxygen cost of exercise. Heat flow or heat content appeared to be the controlled variable and body temperature rise a secondary event resulting from thermal transport lag.     

Cardiac rhythms of late pre-pipped and pipped chick embryos exposed to altered oxygen environments

During the final stages of embryonic development in chickens, diffusive gas exchange through the chorioallantoic membrane (CAM) is progressively replaced by pulmonary respiration that begins with internal pipping (IP) of the CAM. Late chick embryos going through the transition from CAM respiration to pulmonary respiration were exposed to hyperoxic (100% O(2)) and hypoxic (10% O(2)/N(2)) environments for 2-h and the responses of baseline heart rate (HR), and HR fluctuation patterns were investigated. 16- and 18-day-old (referred to as 18-d) embryos and 20-d externally pipped (EP) embryos were examined as pre-pipped embryos and pipped embryos, respectively. 19-d embryos were divided into two groups: embryos that had not yet internally pipped (Pre-IP embryos) and embryos that had internally pipped (IP embryos). IP was identified by detecting the breathing signal with a condenser microphone attached hermetically on the eggshell (i.e. acoustorespirogram) on day 19 of incubation. In the hyperoxic environment, HR baseline of pre-pipped embryos remained unchanged and that of pipped embryos was depressed. In the hypoxic environment, HR baseline of 16-d pre-pipped embryos was depressed and that of pipped (IP and EP) embryos elevated. These different responses in pipped embryos might be partially attributed to increased cholinergic input from the vagus nerve in hyperoxia and increased adrenergic response in hypoxia. While hyperoxia did not induce marked modification of instantaneous heart rate (IHR) fluctuation patterns, hypoxia tended to augment transient decelerations of IHR in late pre-pipped embryos and markedly depressed HR fluctuations in pipped embryos.     

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.     

Metabolic responses to gradual cooling in chicken eggs treated with thiourea and oxygen

1. Late prenatal chicken embryos in eggs injected with saline showed a feeble homeothermic metabolic response to gradual cooling. This response was absent in thiourea-treated eggs. This suggests that the incipient homeothermic metabolic response before paranatal life may be attributed to thyroid development. 2. The compensatory metabolic response disappeared in embryos exposed to a hypoxic environment, while it was augmented in eggs in pure O2, decreasing as ambient temperature fell. 3. These results may indicate that the homeothermic metabolic response in late embryos is O2-conductance-limited and power-limited as previously suggested.     

Heart rate responses to cooling in emu hatchlings

Among fluctuations of instantaneous heart rate (IHR) in newly hatched chicks, heart rate (HR) oscillation with a mean frequency of 0.7 Hz has been designated as Type II HR variability characterized by low frequency (LF) oscillation [Comp. Biochem. Physiol. Part A 124 (1999) 461]. In response to exposure to lowered ambient temperature (Ta), chick hatchlings raised their HR baseline accompanied with the production or augmentation of Type II HR oscillation, indicating that LF oscillation is a phenomenon relating to thermoregulation [J. Therm. Biol. 26 (2001) 281]. In emu hatchlings that are precocial like chickens, Type II HR oscillation also occurred, but less frequently in comparison with chick hatchlings [Comp. Biochem. Physiol. Part A 131 (2002) 787]. This present experiment was conducted to elucidate how IHR of emu hatchlings responds to changes in Ta. Six hatchlings were measured for IHR and skin temperature (Ts) during a 3-h period when they were exposed to controlled Ta (ca. 35 degrees C), lowered Ta (ca. 15-30 degrees C) and again the controlled Ta for individual 1-h periods. In response to all the cooling and re-warming procedures, HR baseline changed depending upon the intensity of the Ta differences; i.e. large differences of Ta produced large changes in HR. HR fluctuations tended to augment during cooling with a few exceptions, but LF oscillation was not produced. Thus, LF oscillation, which was scarce even at the controlled Ta, could not be used as a thermoregulatory indicator in emus.     

Ontogeny of heart rate in embryonic and nestling crows (Corvus corone and Corvus macrorhynchos)

The developmental patterns of mean heart rate (MHR) and instantaneous heart rate (IHR) were investigated in embryos and chicks of altricial Corvuscorone and Corvus macrorhynchos. The MHR of embryos increased linearly with time from 250 beats · min⁻¹ at mid-incubation to 290 beats · min⁻¹ in hatchlings. MHR during the pipping period was maximal, but only marginally higher than in hatchlings. MHR was stable at about 290–300 beats · min⁻¹ during the 1st week after hatching. Spontaneous heart rate (HR) decelerations and accelerations were found in embryos and chicks, disturbing the baseline HR with increasing frequency during development. However, the IHR accelerations developed later and were less frequent than in precocial species. IHR and body temperature decreased during mild cold exposure (23–25 °C) and IHR accelerations were reduced in nestlings during the 1st week. We suggest that the development of parasympathetic control of HR in crows occurs at 60% of incubation, similar to precocial embryos, but sympathetic control may be delayed and suppressed in contrast to precocial embryos. Accepted: 3 March 1999     

Low-frequency oscillation of instantaneous heart rate in newly hatched chicks

Instantaneous heart rate (IHR) of chickens began to fluctuate on days 13-14 of incubation and heart rate (HR) fluctuations became augmented towards hatching and increased further after hatching. IHR fluctuations of newly hatched chicks have been categorized into three types: type I HR variability (HRV), which is high-frequency oscillation; type II HRV, which is low-frequency oscillation; and type III HR irregularities (HRI), which are irregular HR accelerations. The present experiment was carried out to investigate the origin of type II HR oscillations. Following previous evidence, we assumed that the low-frequency oscillation of HR in newly hatched chicks was related to thermoregulation and changed by environmental temperature. Eventually, type II HRV was produced or augmented by exposure of hatchlings to lowered ambient temperature and was abolished by exposure to elevated environmental temperature. The hatchlings that were exposed to large temperature decreases tended to increase their HR more than those exposed to small temperature decreases, and vice versa. The HR oscillation accompanied by an elevation of HR baseline in response to cooling may be a phenomenon related to thermoregulation in chick hatchlings.     

Effect of nanoparticles of silver and gold on metabolic rate and development of broiler and layer embryos

This investigation evaluated the effects of nanoparticles of silver (AgNano) and gold (AuNano) on metabolic rate (O₂ consumption, CO₂ production and heat production-HP) and the development of embryos from different breeds of broiler and layer chicken. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP was calculated for 10, 13, 16 and 19-day-old embryos. Relative chick and muscle weights were used as a measure of growth rate and development. AgNano but not AuNano increased the rates of O₂ consumption and HP of the layer embryos. The metabolic rate of broiler embryos was not affected by either of the treatments, but it was significantly higher compared to the layer embryos. Neither of the nanoparticles promoted nor depressed growth and development of the embryos, irrespective of breed. Although the metabolic rate of AgNano-injected layer embryos was significantly increased, their BW and muscle weights at hatching were similar to those of the control group, which suggests that the concentration of AgNano used was adequate for increasing the metabolic rate but not enough to affect growth and development. The results show that AgNano could be a potential metabolic modifier for layer embryos; however, the exact mechanism of action should be elucidated in future research.     

Maturation of the homeothermic response of heart rate to altered ambient temperature in developing chick hatchlings (Gallus gallus domesticus)

On the basis of evidence showing that instantaneous heart rate (IHR) of chick hatchlings responds to exposure to altered ambient temperature (Ta; Tazawa H, Moriya K, Tamura A, and Akiyama R. Comp Biochem Physiol A 131A: 797-803, 2002), we elucidate here the developmental timeline for the homeothermic response of HR in newly hatched chicks (days 0-7) maintained at room temperature ( approximately 24-27 degrees C). Hatchlings were exposed to Ta of 25, 35, and 25 degrees C for 1-h periods, respectively, and IHR was measured together with skin temperature (Ts) during this warming and cooling bout. Early 0-day-old (0 day) chicks responded to warming and cooling exposures with various changes in HR baseline. In newly hatched chicks (0-7 h old), HR baseline was elevated during warming (Delta126 beats/min, n = 13) and declined during cooling (-Delta94 beats/min). With progress of development on day 0, the elevation of HR baseline during warming decreased and advanced 0-day chicks tended to decrease HR baseline during warming rather than increase HR. The more developed 1- to 7-day-old chicks exhibited the expected homeothermic decrease in HR during warming. The diurnal variations of HR responses during warming and cooling on the first day of post-egg life indicate that pronounced development of thermoregulatory competence occurs during the day of hatching (day 0). The response of IHR fluctuations to altered Ta was observed in the form of low- and high-frequency oscillations. High-frequency oscillations corresponding to respiratory sinus arrhythmia developed as the hatchlings aged. There was a significant increase in the number of chicks exhibiting both low- and high-frequency oscillations that depended on age and the development of thermoregulatory competence of hatchlings.     

The behavioural and physiological strategies of bird and reptile embryos in response to unpredictable variation in nest temperature

Temperature profoundly affects the rate and trajectory of embryonic development, and thermal extremes can be fatal. In viviparous species, maternal behaviour and physiology can buffer the embryo from thermal fluctuations; but in oviparous animals (like most reptiles and all birds), an embryo is likely to encounter unpredictable periods when incubation temperatures are unfavourable. Thus, we might expect natural selection to have favoured traits that enable embryos to maintain development despite those fluctuations. Our review of recent research identifies three main routes that embryos use in this way. Extreme temperatures (i) can be avoided (e.g. by accelerating hatching, by moving within the egg, by cooling the egg by enhanced rates of evaporation, or by hysteresis in rates of heating versus cooling); (ii) can be tolerated (e.g. by entering diapause, by producing heat‐shock proteins, or by changing oxygen use); or (iii) the embryo can adjust its physiology and/or developmental trajectory in ways that reduce the fitness penalties of unfavourable thermal conditions (e.g. by acclimating, by exploiting brief windows of favourable conditions, or by producing the hatchling phenotype best suited to those incubation conditions). Embryos are not simply passive victims of ambient conditions. Like free‐living stages of the life cycle, embryos exhibit behavioural and physiological plasticity that enables them to deal with unpredictable abiotic challenges.     

Can Reptile Embryos Influence Their Own Rates of Heating and Cooling?

Previous investigations have assumed that embryos lack the capacity of physiological thermoregulation until they are large enough for their own metabolic heat production to influence nest temperatures. Contrary to intuition, reptile embryos may be capable of physiological thermoregulation. In our experiments, egg-sized objects (dead or infertile eggs, water-filled balloons, glass jars) cooled down more rapidly than they heated up, whereas live snake eggs heated more rapidly than they cooled. In a nest with diel thermal fluctuations, that hysteresis could increase the embryo’s effective incubation temperature. The mechanisms for controlling rates of thermal exchange are unclear, but may involve facultative adjustment of blood flow. Heart rates of snake embryos were higher during cooling than during heating, the opposite pattern to that seen in adult reptiles. Our data challenge the view of reptile eggs as thermally passive, and suggest that embryos of reptile species with large eggs can influence their own rates of heating and cooling.     

Dynamical systems analysis of arterial blood pressure signals in relation to heart rate fluctuations in chick embryos

We attempted a new approach based on a modern dynamical system theory to reconstruct the arterial blood pressure signals in relation to heart rate fluctuations of developing chick embryos. The dynamical systems approach in general is to model a phenomenon that is presented by a single time series record and approximate the dynamical property (e.g. heart rate fluctuations) of a system based only on information contained in a single-variable (arterial blood pressure) of the system. The time-series data of the arterial blood pressure was reconstructed in 3-dimensional space to draw characteristic orbits. Since the reconstructed orbits of the blood pressure should retain information contained in the pressure signals, we attempted to derive instantaneous heart rate (IHR) from the reconstructed orbits. The derived IHR presenting HR fluctuations coincided well with the IHR obtained conventionally from the peak-to-peak time intervals of the maximum blood pressure. Movements of the reconstructed orbits of the arterial blood pressure in 3-dimensional space reflected HR fluctuations (i.e. transient decelerations and accelerations).     

Dynamic effects of acute cooling in body temperature regulation of the euthermic warm-acclimated golden hamster (Mesocricetus auratus)

During the dynamic phase of external cooling of euthermic golden hamsters in the initial period of metabolic response, peripheral body temperature is the decisive control variable determining the level of metabolic heat production. Under these conditions the rate as well as the magnitude of the peripheral body temperature change constitute the effectual input to the controller of body temperature. The apparent sensitivity with which the regulator drives the metabolic response to unit change of the peripheral temperature is in an inverse relation to the rate of peripheral temperature change. This parameter, despite its limited significance can serve as a working index characterising the thermoregulatory system in different groups of experimental animals of the same species providing that the actual conditions of the experiment are comporting.     

Response of egg temperature,heart rate and blood pressure in the chick embryo to hypothermal stress

Summary Chicken eggs incubated for 12–18 days were catheterized via the allantoic artery and temperature was monitored simultaneously using a probe positioned in the allantoic fluid adjacent to the embryo. Fluid temperature (referred to as egg temperature), arterial pressure and heart rate were measured following abrupt exposure to a lower environmental temperature (ca., 2628°C). Egg temperature and heart rate diminished exponentially: The rate of decline of egg temperature approximated Newton's law of cooling, the rate coefficient being 0.0220.025°C/min·°C throughout the incubation period from 12 to 18 days. The half time of temperature response of the egg was 2728 min. The response was much slower than that of fertile unincubated eggs (Kaplan et al. 1978), suggesting that the extraembryonic fluids act as a thermal buffer in embryonated eggs. The heart rate response in older embryos (1718 days) changed in the same manner as egg temperature, while in younger embryos (1216 days) the heart rate diminished more quickly than the change in egg temperature. During development the cardiac pacing of the embryo suggests that it becomes resistive to mild cold stress. The systolic pressure remained almost unchanged or even increased during one hour of exposure as the embryo developed, while the diastolic pressure decreased steadily after exposure irrespective of development. The 18-day-old embryos retained the systolic pressure unaltered during 3-hour exposure. In embryos 34 days prior to hatching the functional capacity of the heart apparently allows continued pumping even after prolonged exposure to low environmental temperature. Symbols and Abbreviations: See definitions at the end of the Materials and methods section     

Egg color as an adaptation for thermoregulation

ABSTRACT.   Avian embryos are incubated at temperatures only 2–6 °C below that at which hyperthermia begins to influence survival. In habitats where sunlight directly strikes the eggs, even for short periods, heat gain may be a substantial threat to survival, and reflective pigmentation may reduce the rate of heat gain. The results of previous studies suggest that light-colored eggs acquire heat slower than dark eggs, but artificial pigments were used to create differences in egg coloration. This approach is problematic because natural eggshell pigments have low absorbance in the near-infrared waveband that encompasses about half of incident solar radiation. We used naturally-pigmented eggs to measure the influence of egg coloration on heat gain. Triads ( N = 18) of eggs from Brewer's ( Euphagous cyanocephalus ), Red-winged ( Agelaius phoeniceus ), and Yellow-headed ( Xanthocephalus xanthocephalus ) blackbirds were crossed with six nests of each species and either exposed to full sunlight or placed under a diffusing umbrella. Thermisters recorded internal egg temperature every minute until an asymptotic temperature was reached. Eggs in full sunlight acquired heat more rapidly than eggs in the shaded environment, but heat gain did not vary with egg color in either environment. Eggs placed in Yellow-headed Blackbird nests took longer to reach asymptotic temperature, but there was no significant egg-by-nest interaction. Thus, it appears that differences in reflectivity of eggshell pigments in the visible range (400–700 nm) do not result in different rates of heat acquisition. The thermoregulation hypothesis was not supported.