Development of respiratory rhythms in perinatal chick embryos

In chick embryos, gas exchange takes place via the chorioallantoic membrane (CAM) and the lungs at approximately 1 day prior to hatching. The present study was designed to elucidate the development of respiratory rhythms in the chick embryo during the whole pipping (perinatal) period with a condenser-microphone measuring system. The microphone was hermetically attached on the eggshell over the air cell on day 18 of incubation. It first detected a cardiogenic signal (i.e. acoustocardiogram), and then beak clapping and breathing signals (acoustorespirogram, ARG). The first signals of lung ventilation appeared intermittently and irregularly approximately once per 5 s among the clapping signals after the embryo penetrated its beak into the air cell (internal pipping, IP). The respiratory rhythm then developed irregularly, with a subsequent more regular rate. The envelope pattern of breathing from the onset of IP through external pipping (EP) to hatching was constructed by a specially devised procedure, which eliminated external and internal noises. The envelope patterns indicated that the IP, EP and whole perinatal periods of 10 embryos were 14.1+/-6.4 (S.D.), 13.6+/-4.0 and 27.6+/-5.4 h, respectively. In addition, they also indicated the period of embryonic hatching activity (i.e. climax) which was 48+/-19 min. The development of respiratory rhythm was also shown by the instantaneous respiratory rate (IRR) which was designated as an inverse value of two adjacent ARG waves.     

Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos.

During the first day of hatching, the developing chicken embryo internally pips the air cell and relies on both the lungs and chorioallantoic membrane (CAM) for gas exchange. Our objective in this study was to examine respiratory and cardiovascular responses to acute changes in oxygen at the air cell or the rest of the egg during internal pipping. We measured lung (VO2(lung)) and CAM (VO2(CAM)) oxygen consumption independently before and after 60 min exposure to combinations of hypoxia, hyperoxia, and normoxia to the air cell and the remaining egg. Significant changes in VO2(total) were only observed with combined egg and air cell hypoxia (decreased VO2(total)) or egg hyperoxia and air cell hypoxia (increased VO2(total)). In response to the different O2 treatments, a change in VO2(lung) was compensated by an inverse change in VO2(CAM) of similar magnitude. To test for the underlying mechanism, we focused on ventilation and cardiovascular responses during hypoxic and hyperoxic air cell exposure. Ventilation frequency and minute ventilation (V(E)) were unaffected by changes in air cell O2, but tidal volume (V(T)) increased during hypoxia. Both V(T) and V(E) decreased significantly in response to decreased P(CO2). The right-to-left shunt of blood away from the lungs increased significantly during hypoxic air cell exposure and decreased significantly during hyperoxic exposure. These results demonstrate the internally pipped embryo's ability to control the site of gas exchange by means of altering blood flow between the lungs and CAM.     

Influence of diminished respiratory surface area on survival of sea turtle embryos

It has been suggested that fungal presence on sea turtle eggs may impede gas exchange. To investigate the influence of diminished gas exchange surface upon embryo survivorship, flatback (Natator depressus) and green (Chelonia mydas) eggs were painted with petroleum jelly. Variable proportions of the egg surface were covered, including both respiratory and nonrespiratory domains. Embryo survival varied with site inhibited, proportion of eggshell affected, and species of turtle. If fungi on the exterior of the eggshell are able to impede respiratory gas exchange, their presence on the upper hemisphere (primary gas exchange area in early incubation) will result in the highest embryo mortality. Large eggs are likely to demonstrate a higher survivorship than small eggs, due to their larger available respiratory area and/or to variation in weight or stage-specific embryonic metabolic demands. Interspecific differences in egg size may therefore be a contributory factor to observed mortality rate differences in the natural presence of fungi.     

Cardiac rhythms in chick embryos during hatching

Avian embryos develop within a hard eggshell which permits the measurement of heart rate while maintaining an adequate gas exchange through the chorioallantoic membrane. Heart rate has been determined from cardiogenic signals detected either noninvasively, semi-invasively or invasively with various transducers. Firstly, we reviewed these previously-developed methods and experimental results on heart rate fluctuations in prenatal embryos. Secondly, we presented new findings on the development of heart rate fluctuations during the last stages of incubation, with emphasis on the perinatal period, which remained to be studied. Three patterns of acceleration of the instantaneous heart rate were unique to the external pipping period: irregular intermittent large accelerations, short-term repeated large accelerations and relatively long-lasting cyclic small accelerations. Besides these acceleration patterns, respiratory arrhythmia, which comprimised oscillating patterns with a period of 1-1.5 s, appeared during the external pipping period. Furthermore, additional oscillating patterns with a period of 10-15 min were found in some externally pipped embryos.     

Uterine and eggshell structure and histochemistry in a lizard with prolonged uterine egg retention (Lacertilia,Scincidae, Saiphos)

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Gas exchange and energy metabolism of the ostrich (Struthio camelus) embryo

We measured oxygen consumption ( ) and carbon dioxide emission ( ) rates, air-cell gas partial pressures of oxygen (P_AO₂) and CO₂ (P_ACO₂), eggshell water vapour conductance and energy content of the ostrich (Struthio camelus) egg, ‘true hatchling’ and residual yolk, and calculated RQ and total oxygen consumption ( ) for ostrich eggs incubated at 36.5°C and 25% relative humidity. The pattern showed a drop of approximately 5% before internal pipping. just prior to internal pipping agrees with allometric calculations. Despite the higher incubation temperature compared to other studies, and the resultant shorter incubation duration (42 days), (91.7 l kg⁻¹) was similar to a previously reported value. RQ values during the second half of incubation (approx. 0.68) were lower than expected for lipid catabolism. Prior to internal pipping, P_AO₂ and P_ACO₂ were 98 and 48.3 torr (13.1 and 6.4 kPa), respectively. The growth pattern of the ostrich embryo is different from the typical precocial pattern, showing a time delay in the rapid growth phase. As a result, the lowered overall energy expenditure for tissue maintenance, as compared to other species, is reflected in the low yolk utilization and high residual yolk fraction of the whole hatchling dry mass. These could also result from the relatively short incubation period of the ostrich egg, thereby evading desiccation by excess water loss.     

Fine structure of the eggshell of Ommatissus binotatus Fieber (Homoptera,Auchenorrhyncha, Tropiduchidae)

The external morphology and fine structure of the eggshell of Ommatissus binotatus Fieber (Homoptera : Tropiduchidae) was investigated by light, scanning and transmission electron microscopy. The egg surface has 2 main regions: a specialized area and an unspecialized egg capsule. The specialized area is characterized by a large respiratory plate containing the operculum and a short respiratory horn. The latter consists of an external hollow tube and an internal coneshaped projection hosting a micropylar canal. The eggshell has 4 layers: the vitelline envelope, a wax layer, the chorion and an outer mucous layer. The chorion has inner, intermediate and outer parts. The functions of the different parts of the eggshell are discussed. Characters useful to define the eggs and the oviposition habit in the family Tropiduchidae were provided. The size and morphology of the egg, plate, respiratory horn and operculum are suggested as useful characters for ootaxonomic analysis.     

Embryonic development of the domestic guinea fowl (Numida meleagris)

Inside the viable range 36–39°C, the development of the domestic guinea fowl embryo was studied during artificial incubation of about 5000 eggs. Equations giving internal and external pipping and hatching time as a function of incubation temperature were developed. Whatever temperature was used, internal and external pipping occurred at 89% and 95% of hatching time, respectively. The chronological development in ovo of the guinea fowl is illustrated by growth curves. Guinea fowl grew in ovo at the same rate as the chicken embryo. The embryonic mortality is significantly affected by incubation temperature. The thermal tolerance of the embryo follows a parabolic curve: temperatures below or above 37.2°C increase the rate of mortality. During the course of the incubation, the mortality frequency was mainly distributed around two peaks (each one third of the total), which occurred during the first days of incubation, that is, within the first 12% of the incubation time, and during the last days, that is, after 85% of the incubation time. At the optimal 37.2°C temperature, these two peaks occurred on days 1–3 and 23–27, respectively.     

Morphology and evolution of the cynipoid egg (Hymenoptera)

We describe gross egg morphology and provide the first data on eggshell ultrastructure in cynipoids (Hymenoptera) based on species representing three distinctly different life histories: internal parasitoids of endopterygote larvae, gall inducers and phytophagous inquilines (guests in galls). We then use existing phylogenetic hypotheses to identify putative changes in egg structure associated with evolutionary life-history transitions. We find four major structural changes associated with the shift from parasitoids laying their eggs inside a host larva to gall inducers laying their eggs in or on plants: (1) from a narrow and gradually tapering gross form to a distinct division into a stout body and a long and thin stalk; (2) from a thin to a thick eggshell; (3) from a flexible to a rigid endochorion; and (4) from crystal bundles with shifting orientation in the exochorion to layers of parallel crystal rods. By contrast, we find no major changes in egg structure associated with the transition from gall inducers to inquilines. Comparison between pre- and post-oviposition eggs of one gall inducer and one inquiline suggests that mechanical stress during the passage through the egg canal gives rise to numerous tiny stress fractures in the boundary separating the exo- and endochorion. In one of the gall inducers, Diplolepis rosae , that end of the egg, which is inserted into the plant, has a specialized and apparently porous shell that may permit chemical exchange between the embryo and the plant. Other structures that could facilitate chemical communication with the host plant through the eggshell were, however, not observed in the eggs of gall inhabitants.  © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society , 2003, 139 , 247–260.     

Effects of posture and bronchoconstriction on low-frequency input and transfer impedances in humans.

We simultaneously evaluated the mechanical response of the total respiratory system, lung, and chest wall to changes in posture and to bronchoconstriction. We synthesized the optimal ventilation waveform (OVW) approach, which simultaneously provides ventilation and multifrequency forcing, with optoelectronic plethysmography (OEP) to measure chest wall flow globally and locally. We applied an OVW containing six frequencies from 0.156 to 4.6 Hz to the mouth of six healthy men in the seated and supine positions, before and after methacholine challenge. We measured mouth, esophageal, and transpulmonary pressures, airway flow by pneumotachometry, and total chest wall, pulmonary rib cage, and abdominal volumes by OEP. We computed total respiratory, lung, and chest wall input impedances and the total and regional transfer impedances (Ztr). These data were appropriately sensitive to changes in posture, showing added resistance in supine vs. seated position. The Ztr were also highly sensitive to lung constriction, more so than input impedance, as the former is minimally distorted by shunting of flow into alveolar gas compression and airway walls. Local impedances show that, during bronchoconstriction and at typical breathing frequencies, the contribution of the abdomen becomes amplified relative to the rib cage. A similar redistribution occurs when passing from seated to supine. These data suggest that the OEP-OVW approach for measuring Ztr could noninvasively track important lung and respiratory conditions, even in subjects who cannot cooperate. Applications might range from routine evaluation of airway hyperreactivity in asthmatic subjects to critical conditions in the supine position during mechanical ventilation.     

Comparison of eggshell porosity and estimated gas flux between the brown‐headed cowbird and two common hosts

The brown‐headed cowbird Molothrus ater is a brood‐parasite that lays eggs in nests of a wide range of host species, including the closely‐related red‐winged blackbird Agelaius phoeniceus and the dickcissel Spiza americana. Although cowbird eggs have accelerated development and hatch sooner than similar‐sized host eggs, this development takes place within a thickened eggshell that could impede gas flux to the developing embryo. We tested the hypothesis that the accelerated development of the cowbird embryo relative to hosts is enabled by an increase in eggshell porosity that allows increased fluxes of respiratory gases to and from the developing embryo. We found cowbird eggshell thickness was significantly greater than the eggshells of these two common hosts. Although the number of pores per egg was similar among all three species, the total pore area per egg in cowbirds was significantly greater than that of either host, despite having a smaller eggshell surface area than the red‐winged blackbird. Cowbird egg pore area was 1.9×larger than that of the red‐winged blackbird. Cowbird eggshells had a significantly greater gas flux than those of the red‐winged blackbird and the dickcissel. When conductance was normalized to published values of egg mass, cowbird eggs had a higher mass‐specific conductance than red‐winged blackbird or dickcissel eggs. These results are consistent with the hypothesis that the rapid development of brown‐headed cowbird embryos is facilitated by increased eggshell porosity, and that changes in eggshell porosity represent an adaptation that enables cowbird eggs to hatch earlier than equivalently‐sized host eggs.     

In ovo electroporations of HH stage 10 chicken embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology. With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function. By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function. Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements. Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive. This video demonstrates first how to make a window in the eggshell to manipulate the embryo. Next, the embryo is visualized with a dilute solution of India ink injected below the embryo. A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. Finally, the egg is sealed with tape and placed back into an incubator for further development. Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.     

Development of cardiac rhythms in birds

Heart rate (HR) in avian embryos developing inside an eggshell has been measured by various means while maintaining adequate gas exchange through the eggshell. This is an important requirement in order to avoid adverse effects of impeding gas exchange on the cardiac rhythms of developing embryos. The present report is a review of our ontogenetic study on embryonic HR, which was measured with fulfillment of the above requirement and also hatchling HR measured non-invasively. Firstly, we reviewed measurements of daily changes (developmental patterns) in embryonic mean heart rate (MHR), which were determined from a short-term measurement of HR once a day, in 34 species of altricial and precocial birds. The allometric relationship between the MHR during pipping in altricial birds and their fresh egg masses was the same as that between the MHR at 80% of incubation duration and fresh egg masses in pre-cocial birds. Secondly, we presented the developmental patterns of MHR in chick embryos and hatchlings, which were determined from long-term, continuous measurement of HR before, during and after hatching. The ultradian and circadian rhythms of HR were clearly shown in embryos and hatchlings, respectively. Thirdly, we summarized instantaneous HR fluctuations: HR variability and HR irregularities, in chick embryos and hatchlings. The distinctive patterns were shown in pre-pipped and pipped embryos and newly hatched chicks, individually, which were partly related to autonomic nervous functions and physiological functions.     

Gas exchange and energy metabolism of the ostrich (Struthio camelus) embryo.

We measured oxygen consumption (V(O(2))) and carbon dioxide emission (V(CO(2))) rates, air-cell gas partial pressures of oxygen (P(A)O(2)) and CO(2) (P(A)CO(2)), eggshell water vapour conductance and energy content of the ostrich (Struthio camelus) egg, 'true hatchling' and residual yolk, and calculated RQ and total oxygen consumption (V(O(2)tot)) for ostrich eggs incubated at 36.5 degrees C and 25% relative humidity. The V(O(2)) pattern showed a drop of approximately 5% before internal pipping. V(O(2)) just prior to internal pipping agrees with allometric calculations. Despite the higher incubation temperature compared to other studies, and the resultant shorter incubation duration (42 days), V(O(2)tot) (91.7 l kg(-1)) was similar to a previously reported value. RQ values during the second half of incubation (approx. 0.68) were lower than expected for lipid catabolism. Prior to internal pipping, P(A)O(2) and P(A)CO(2) were 98 and 48.3 torr (13.1 and 6.4 kPa), respectively. The growth pattern of the ostrich embryo is different from the typical precocial pattern, showing a time delay in the rapid growth phase. As a result, the lowered overall energy expenditure for tissue maintenance, as compared to other species, is reflected in the low yolk utilization and high residual yolk fraction of the whole hatchling dry mass. These could also result from the relatively short incubation period of the ostrich egg, thereby evading desiccation by excess water loss.     

Functional morphology of embryonic development in the Port Jackson shark Heterodontus portusjacksoni (Meyer)

The oviparous Port Jackson shark Heterodontus portusjacksoni embryo has a long incubation of 10–11 months during which it undergoes major morphological changes. Initially the egg capsule is sealed from the external environment by mucous plugs in either end of the capsule. Four months into incubation, the egg capsule opens to the surrounding sea water. Fifteen stages of development are defined for this species, the first 10 occur within the sealed capsule, the remaining five after capsule opening to hatching. The functional significance of major definitive characters such as circulation within the yolk membrane and embryo, rhythmic lateral movement of the embryo, external gill filaments, heart activity, internal yolk supplies, egg jelly and the significance of the opening of the egg capsule are described. The egg jelly in the sealed capsule functions to mechanically protect the embryo during early development, however, it eventually creates a hypoxic environment to the embryo as the available oxygen is used up. This generates several physiological challenges to the developing embryo. It is able to overcome these problems by morphological changes such as increasing the effective surface area for gaseous exchange with the development of external gill filaments, fins and extensive circulation in both the embryo and attached external yolk sac. These adaptations become limiting as the embryo grows and respiratory needs outweigh the available oxygen. At this time, the mucous plugs dissolve and the capsule becomes open to the external environment.     

Embryonic heart rate and oxygen pulse in two procellariiform seabirds,Diomedea immutabilis and Puffinus pacificus

Developmental patterns of embryonic heart rate were measured non-invasively in two procellariiform seabirds, the Laysan albatross (Diomedea immutabilis) and wedge-tailed shearwater (Puffinus pacificus), during prepipping and after pipping. The O₂ pulse, defined as the O₂ consumption per single heart beat, was calculated using the previously reported O₂ consumption for these species. The embryonic heart rate of the albatross was not changed by internal pipping (initial pipping event in this species), remained unchanged during the prolonged internal pipping period and tended to increase, although insignificantly, with the initiation of external pipping (second pipping event). Heart rate in the shearwater remained unchanged during the late prepipping stages, did not change with external pipping (initial pipping event), but increased during the prolonged internal pipping period (second pipping event) and reached a maximum on the last day of incubation. The developmental pattern of heart rate in the shearwater was very similar to that reported previously for the brown noddy, a member of the order Charadriiformes but with the same pipping sequence. Developmental patterns of embryonic O₂ pulse were also different between the two procellariiform seabirds. However, those of the shearwater and the noddy were similar. The sequence of access to atmospheric O₂ during development and hatching may be an important factor determining the developmental patterns of embryonic heart rate and O₂ pulse.Abbreviations A-VO₂ difference, arteriovenous oxygen difference - BCG ballistocardiogram of egg - CV coefficient of variation - EP external pipping - HR heart rate - IP internal pipping - mass mass of freshly laid egg - SD standard deviation     

Water Relations of Chelonian Eggs and Embryos: Is Wetter Better?

The exchange of water between a chelonian egg and its subterraneanenvironment is influenced by numerous factors, the most importantof which are (1) structure of the calcareous layer of the eggshell,(2) water potential and temperature in the nest, and (3) fractionof the eggshell that actually contacts soil in the nest cavity.Eggs with relatively porous shells tend to absorb large quantitiesof water from cool, moist environments and to lose large amountsof water to warm, dry ones. Net water-exchange by such eggsalso tends to be more favorable (= positive) when soil contactsthe entire eggshell than when a large fraction of the shellis exposed to air trapped inside the nest cavity. In contrast,eggs with relatively impermeable shells usually exchange onlysmall amounts of water with their environment, regardless ofthe physical conditions that prevail inside the nest. The patternof net water-exchange, together with size (and water content)of the freshly laid egg, determines the amount of water thatis available to sustain the embryo. An embryo having accessto a relatively large reserve of water will consume more ofits yolk and grow to larger size before hatching than will anembryo having access to a smaller reserve of water. Large, well-hydratedhatchlings may survive better than small, dehydrated animalsduring the trek overland from nest to water. If so, a cooler,wetter nest will also be a better nest.     

Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos

During the first day of hatching, the developing chicken embryo internally pips the air cell and relies on both the lungs and chorioallantoic membrane (CAM) for gas exchange. Our objective in this study was to examine respiratory and cardiovascular responses to acute changes in oxygen at the air cell or the rest of the egg during internal pipping. We measured lung (V·_O2lung) and CAM (V·_O2CAM) oxygen consumption independently before and after 60 min exposure to combinations of hypoxia, hyperoxia, and normoxia to the air cell and the remaining egg. Significant changes in V·_O2total were only observed with combined egg and air cell hypoxia (decreased V·_O2total) or egg hyperoxia and air cell hypoxia (increased V·_O2total). In response to the different O₂ treatments, a change in V·_O2lung was compensated by an inverse change in V·_O2CAM of similar magnitude. To test for the underlying mechanism, we focused on ventilation and cardiovascular responses during hypoxic and hyperoxic air cell exposure. Ventilation frequency and minute ventilation (V_E) were unaffected by changes in air cell O₂, but tidal volume (V_T) increased during hypoxia. Both V_T and V_E decreased significantly in response to decreased P_CO2. The right-to-left shunt of blood away from the lungs increased significantly during hypoxic air cell exposure and decreased significantly during hyperoxic exposure. These results demonstrate the internally pipped embryo's ability to control the site of gas exchange by means of altering blood flow between the lungs and CAM.     

Carbon dioxide-oxygen relationships in gas exchange of animals. In memory of Hermann Rahn.

In external gas exchange of vertebrates, behavior of the respiratory gases CO2 and O2 can in many cases adequately be explained by the different physico-chemical properties of the gases, including solubility, chemical combination in blood and tissue, and diffusivity. In particular, the differences in behavior between CO2 and O2 are often of particular relevance. This is demonstrated on a number of examples of gas exchange mechanisms in vertebrates, including (1) exchange ratio after changes in ventilation, (2) local variations of pulmonary ventilation/perfusion ratio, (3) absorption of gas from gas pockets, (4) water vs. air breathing, (5) multimodal breathing, (6) skin breathing, (7) gas exchange of avian eggs, (8) anomalous gas/blood CO2 equilibration, (9) blood/gas CO2 equilibration in avian lungs, (10) pulmonary diffusing capacity, (11) blood/water CO2 equilibration in fish gills, (12) deposition of gas into fish swim bladder.     

Oxygenation and establishment of thermogenesis in the avian embryo

The production of heat (or thermogenesis) and its response to cold improve very quickly around birth in both mammals and birds. The mechanisms for such rapid perinatal development are not fully understood. Previous experiments with hyperoxia suggested that, during the last phases of incubation, eggshell and membranes might pose a limit to oxygen availability. Hence, it was hypothesized that an improvement in oxygenation by opening the eggshell may contribute to the establishment of thermogenesis. Thermogenesis and its response to cold were measured by indirect calorimetry, in warm (38 degrees C) conditions and during 1-h exposure to 30 degrees C. Both improved throughout the various phases of the hatching process. During the latest incubation phases (internal pipping, IP, and star fracture of external pipping, EP), the removal of the eggshell in the region above the air cell raised metabolic rate both in warm and cold conditions (in IP) or the thermogenic response to cold (in EP). Adding hyperoxia after opening the eggshell caused no further increase in the thermogenic response. In cold-incubated embryos thermogenesis during the EP phase was much less than normal; in these embryos, increasing the oxygen availability did not improve thermogenesis. We conclude that oxygenation contributes to the maturation of the thermogenic mechanisms in the perinatal period as long as these mechanisms have initiated their normal developmental process.