Available online at www.sciencedirect.com Respiratory Physiology & Neurobiology 159 (2007) 155–162 Organ growth in chicken embryos during hypoxia: Implications on organ “sparing” and “catch-up growth” Milène A. Azzam, Jacopo P. Mortola ∗ Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada Accepted 11 June 2007 Abstract The primary aim of this study was to establish whether or not embryonic hypoxia selectively affects the growth of specific organs. Chicken embryos were incubated either in normoxia (Nx) or in hypoxia (15% O2 from embryonic day E5, Hx). The length of the beak and third toe (as indexes of skeletal growth) and the weights of internal organs (eyes, brain, heart, lungs, liver, kidneys, stomach, and intestines) were collected at E14, E17, E19, and E20. Hypoxia reduced embryonic body weight (BW). At any given age, the specific weight (organ weight/BW) of some organs in Hx was higher, and that of others was lower, than in Nx. However, almost all differences disappeared when organ weights were compared as function of BW, rather than at fixed chronological ages. The important exception was the chorioallantoic membrane (CAM), the mass of which in Hx developed out of proportion. In a third group of embryos, hypoxic until E14 and normoxic thereafter, there was no post-hypoxic catch-up growth, differently from what known to occur postnatally. A possible interpretation is that catch-up growth does not depend on the age of the embryo but on its BW. In conclusion, at least in the chicken embryo and for the level of hypoxia tested, hypoxia has no selective effects on the growth of specific organs, except for the CAM. Qualitative differences in the weight response to hypoxia among organs observed at any given age can be explained largely by the effects of the blunted growth on the growth trajectory of the individual organs. © 2007 Elsevier B.V. All rights reserved. Keywords: Embryonic development; Heart; Hypoxia; Lungs; Chorioallantoic membrane; Normalisation 1. Introduction In mammals, hypoxia during gestation usually results in small neonates, although the maternal response to hypoxia can limit the blunting of fetal growth (Moore and Price, 1948; Metcalfe et al., 1962; Smith et al., 1969; Robinson et al., 1983; Chang et al., 1984; Faridy et al., 1988; Moore et al., 1982; Monge and León-Velarde, 1991; Moore, 2003). In the avian embryo, hypoxia during incubation consistently decreases body growth (e.g., Tazawa et al., 1971; Wangensteen et al., 1974; Metcalfe et al., 1981; McCutcheon et al., 1982; Xu and Mortola, 1989; Azzam et al., 2007). As one may expect, small embryos also have small internal organs. However, upon normalisation by body weight (BW), some organs have larger specific weights (organ weight/BW) than normoxic controls, meaning that during hypoxia the drop in their weight was not as marked as that of the whole body, at the expense of other organs where the ∗ Corresponding author. Tel.: +1 514 398 4335; fax: +1 514 398 7452. E-mail address: jacopo.mortola@mcgill.ca (J.P. Mortola). 1569-9048/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2007.06.003 opposite had occurred. Non-homogeneous effects of hypoxia among internal organs have been observed also in mammals during development (Mortola, 2001, for review). These inter-organ differences may reflect differences in metabolic sensitivity to hypoxia and the redistribution of O2 delivery, which appears to favour key vital organs, at the expense of organs less crucial for the immediate survival (Kuwahira et al., 1993; Côté and Porras, 1998). The impression that, during hypoxia, the growth of some organs may increase at the expense of others is based on the finding that, at any given developmental age, their specific weights are higher than in normoxic controls. However, BWnormalisation when comparing same-age animals with different growth rates (as it happens in hypoxia) can lead to misleading conclusions whenever the growth rate of various organs differ, which is typically the case during embryonic development. In fact, for an organ normally growing at a rate lower than the whole body (and therefore dropping its specific weight with age), in the case of stunted body growth, at any given age its specific weight must exceed the control value. The opposite has to occur for any organ normally growing at a rate higher than that of 156 M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 the whole embryo. Therefore, whenever comparisons are made at fixed chronological ages, one cannot sort out to what extent differences in organ specific weights result from the effects of hypoxia on that organ or from the generalised blunting in body growth. In this study, organ weights of hypoxic and normoxic embryos were compared not only at predetermined chronological ages, but also at comparable BWs, reasoning that a unique BW–organ weight relationship between normoxic and hypoxic embryos would indicate no selective effects of hypoxia on the growth of the organ. The primary aim of this study was to establish whether or not embryonic hypoxia affects the growth of specific organs, apart from its general effects on body growth. Measurements were conducted on chicken embryos, rather than on a mammalian animal model, to eliminate the possibilities of maternal and placental contributions to the fetal outcome. Second, we asked whether or not, during the post-hypoxic return to normoxia, the “catch-up” growth of some organs might prevail over that of others. 2. Methods Freshly laid chicken eggs of the Leghorn variety were purchased from a local supplier. Eggs were stored at 15 ◦ C until the start of incubation, and for no longer than 7 days. The eggs were weighed and placed in incubators (Hova-Bator, Savannah, GA) around midday (embryonic day E0). The incubators maintained a steady temperature (T) of about 38 ◦ C and 60% relative humidity, and provided a 45◦ egg rotation four times a day. For the first 4 days all eggs were incubated in normoxic conditions. Then, at embryonic day E5, they were separated into two groups. Some continued in normoxia (21% O2 , normoxic, Nx, N = 52), others were transferred into a hypoxic incubator kept at 15–16% O2 (on average, 15.5% ± 0.01, including the periods of incubator opening for egg transfer and cleaning) (hypoxic, Hx, N = 52). A third group of eggs was treated as the Hx eggs until day E14, when they were returned to normoxia, and maintained in normoxia thereafter. This group will be referred to as the “posthypoxic normoxic” group (Hx–Nx, N = 41). The desired level of hypoxia was obtained by leaking a small stream of warmed and humidified N2 into the incubator from a pressurised tank, under the control of a flowmeter. The O2 concentration within the incubator was continuously sampled by a calibrated fuel cell gas analyser (Foxbox, Sable Systems Int., Las Vegas, NV), displayed on a computer monitor, and stored for later averaging. A T-data logger and a hygrometer were inside each incubator; the former collected the T value every 10 min, while humidity was read daily. Data collection for the Nx and Hx groups was conducted on days E14, E17, E19, and E20, with 13 embryos per group Fig. 1. (Top) Schematic representation of the growth of an organ occurring at a rate slower than that of the whole body (left panel), so that the specific organ weight declines with age (middle panel). The curves represent the conditions of normoxia (continuous line) and hypoxia (dashed line), the latter with a growth rate equal to half the normoxic rate. In these conditions, at any given age the organ specific weight in hypoxia exceeds the normoxic value (middle panel). However, when represented as function of embryo’s weight, the data fall on a unique relationship (right panel). (Bottom) Body and head weights of embryos incubated in normoxia (open circles) or in hypoxia (filled triangles). At any given age, the head absolute and specific weights were greater in hypoxia than in normoxia (left and middle panels). However, when plotted against embryo’s weight, the head weights of hypoxic and normoxic embryos fell on the same relationship. Values are group means ± 1 S.E.M. *Statistically significant difference between the two groups. M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 and per age, while that for the Hx–Nx group was at days E17 (N = 13), E19 (N = 15), and E20 (N = 13). After weighing the egg, the embryo was killed by exposure to CO2 and cold. The egg was opened, the embryo was freed from the allantois and yolk, and its weight and that of the chorioallantoic membrane (CAM) were noted. At day E20, in a few embryos, after opening the thorax, blood was sampled by inserting a heparin-treated microhematocrit tube through the wall of the ventricle, and hematocrit was determined by microcentrifugation. The length of the third toe (from phalanx one to four inclusive) and that of the beak (from its tip to the beginning of the eye socket), taken as representative of skeletal growth (Lillie, 1952), were measured by use of a fine caliper. Internal organs (eyes, brain, heart, lungs, liver, kidneys, stomach, and intestines) were dissected, lightly blotted and drained of blood or any fluid contents, and weighed with a digital scale accurate to 10−4 g. The weight of the head in toto was also collected, for the purpose illustrated in Section 3.1. All organs and the CAM were placed in an oven at 70 ◦ C for 1 week; separate measurements had indicated that this period of time was appropriate for dry weight to stabilise. Although the eyes, lungs, and kidneys of right and left sides were isolated and weighed separately, for the analysis their weights were combined; the values presented, therefore, represent the sum of the right and left organs. All group data are presented as means ± 1 S.E.M. Statistical comparisons between two sets of data were done by two-tailed t-test, and those between the three sets (Nx, Hx and Hx–Nx embryos) were performed by ANOVA with post hoc Bonferroni’s limitations for the three comparisons. In all cases a difference was considered statistically significant at P < 0.05. 157 the normoxic value (middle panel). When examined as function of the embryo’s BW (right panel) the normoxic and hypoxic curves overlapped, meaning that in hypoxia the organ weight was appropriate for body size. The bottom panels of the figure present the experimental values of head weights in normoxia (open symbols) and hypoxia (filled symbols). The head was chosen because it is a section of the body renowned for decreasing its specific weight during development in many species. At any given age, both head and BW in Hx were less than in Nx (left panel), and the head specific weight significantly exceeded that of Nx (middle panel). On the other hand, when the data were presented as function of embryonic BW (right panels), the curves overlapped. Hence, like in the model presented at top, in the Hx embryos the larger specific weight of the head can be attributed solely to their blunted body growth. 3.3. W-specific organ weights at fixed postnatal ages During the last week of the chicken embryonic development, most BW-specific weights either decreased (e.g., heart, eyes, and brain) or increased (third toe, stomach, and intestines) (Fig. 2). For those organs decreasing specific weights, the values in Hx tended to be similar to, or most often significantly above, those of 3. Results 3.1. General effects of hypoxia At the start of incubation the size of the eggs used for the measurements averaged 61 g ± 0.3 for the Nx embryos and 59.4 g ± 0.6 for the Hx embryos, with no significant difference between the two sets for any of the subgroups used at the various ages. At each of the four ages the Hx embryos were significantly smaller than controls, on average 87% ± 3 (P < 0.005 from Nx), 76% ± 3 (P < 0.0001), 85% ± 3 (P < 0.0005) and 75% ± 4 (P < 0.0002) at E14, E17, E19, and E20, respectively. Hematocrit, measured only in a few embryos at E20, did not differ significantly between the Hx (28.8% ± 2, N = 5) and Nx embryos (26.7% ± 1.1, N = 7). 3.2. Expected effects of stunted body growth on W-specific organ weights The top panels of Fig. 1 present the hypothetical case of an embryonic organ growing at a rate lower than that of the whole embryo, in normoxia (continuous lines) and in hypoxia (dashed lines). In this example, hypoxic growth rate was set at 50% of the normoxic rate. Because of this combination (stunted embryonic growth and specific weight decreasing with development), at any given age the organ specific weight during hypoxia exceeded Fig. 2. Specific organ length (beak and toe, cm/g1/3 ) and specific organ weight (organ weight/embryo’s weight, %) for embryos incubated in normoxia (open circles) or hypoxia (filled triangles), at embryonic days E14, E17 and E20. Length (beak and toe) was normalised to the one-third power of body weight, assuming geometric similarity. Percentages refer to the hypoxic/normoxic average of the 3 days. Values are group means ± 1 S.E.M. *Statistically significant difference between the two groups. 158 M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 Table 1 Dry–wet weight ratio (%) Age Organs Normoxia Hypoxia Eyes Brain Heart Lungs Liver Kidneys Stomach Intestines N = 13 4.7 ± 0.1 10.5 ± 0.1 12.6 ± 0.1 9.6 ± 0.3 21.8 ± 0.3 13.1 ± 0.5 13.5 ± 0.6 11.0 ± 0.4 N = 13 4.6 ± 0.04 10.6 ± 0.2 12.3 ± 0.2 8.7 ± 0.2 (a) 20.7 ± 0.6 11.8 ± 0.5 11.2 ± 0.4 (a) 10.2 ± 0.4 Eyes Brain Heart Lungs Liver Kidneys Stomach Intestines N = 13 5.6 ± 0.1 12.2 ± 0.1 12.8 ± 0.2 13.1 ± 0.2 25.2 ± 0.4 15.7 ± 0.3 14.3 ± 0.4 12.0 ± 0.3 N = 13 5.3 ± 0.1 11.7 ± 0.1 13.0 ± 0.1 13.1 ± 0.1 24.5 ± 0.4 14.8 ± 0.1 14.5 ± 0.3 12.2 ± 0.2 N = 13 5.4 ± 0.1 11.7 ± 0.1 13.1 ± 0.1 12.8 ± 0.3 24.5 ± 0.3 15.2 ± 0.2 14.5 ± 0.5 12.2 ± 0.3 Eyes Brain Heart Lungs Liver Kidneys Stomach Intestines N = 13 5.8 ± 0.1 13.3 ± 0.1 14.5 ± 0.3 13.6 ± 0.3 29.1 ± 0.4 15.5 ± 0.1 13.6 ± 0.3 10.9 ± 0.2 N = 13 5.9 ± 0.1 13.2 ± 0.1 14.8 ± 0.2 13.3 ± 0.3 29.0 ± 0.3 15.2 ± 0.2 12.5 ± 0.4 (a) 11.1 ± 0.3 N = 15 5.8 ± 0.04 13.1 ± 0.1 14.1 ± 0.3 14.2 ± 0.2 (b) 27.8 ± 0.3 (a,b) 15.3 ± 0.2 13.8 ± 0.4 (b) 11.6 ± 0.3 Eyes Brain Heart Lungs Liver Kidneys Stomach Intestines N = 13 6.1 ± 0.1 13.9 ± 0.3 15.1 ± 0.2 15.9 ± 0.5 31.6 ± 0.5 16.7 ± 0.2 13.9 ± 0.3 12.7 ± 0.8 N = 13 5.9 ± 0.2 13.6 ± 0.3 15.2 ± 0.2 15.0 ± 0.4 29.3 ± 0.8 (a) 15.9 ± 0.3 13.4 ± 0.5 11.8 ± 0.7 N = 13 6.0 ± 0.1 14.0 ± 0.3 15.7 ± 0.3 15.9 ± 0.4 31.1 ± 0.7 (b) 16.6 ± 0.3 14.8 ± 0.7 13.8 ± 0.8 (b) E14 E17 E19 E20 Hypoxia–normoxia Values are means ± 1 S.E.M. Hypoxia was for the period embryonic days E5–E20 included. Normoxia after hypoxia started on embryonic day E15. (a) Significant difference from normoxia. (b) Significant difference from hypoxia. the Nx embryos; the average percentage of the Nx value is indicated within each panel. Differently, for those organs increasing their specific weights with development, the values of Hx were similar (intestines) or significantly lower than in Nx (third toe and stomach). The same pattern emerged when the organ dry weights, rather than the wet weights, were considered because, for the most part, the dry–wet ratios were not significantly different between Nx and Hx embryos (Table 1). In the few cases of a difference, the dry–wet ratios in Hx were consistently lower than in Nx, indicating slightly larger water content in the hypoxic organs. 3.4. Organ–body weight relationships Fig. 3 presents the data of organ weight as function of embryo’s BW for all Nx (open circles) and Hx embryos (filled triangles). A visual inspection reveals a substantial overlapping between normoxia and hypoxia for all organs. For a statistical comparison of the two groups the data were binned into four ranges of embryo weights (Fig. 4). Out of a total of 40 pairs of values (10 organs, each compared over 4 weight ranges), only three pairs differed significantly between Nx and Hx, the heart at the embryonic BW of 15 g, and the lungs and brain at 25 g. 3.5. The chorioallantoic membrane (CAM) At E14 the CAM absolute and specific weights of Hx did not differ from Nx, while they were significantly higher at embryonic days E17, E19, and E20 (Fig. 5, left and middle panels). Unlike any of the embryonic organs considered (Section 3.4.), the differences in CAM weight from Nx were large and statistically significant also when the values were compared as function of embryo’s weight (Fig. 5, panels at right). M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 159 Fig. 3. Length (beak and toe, mm) and weight (g) of various organs for embryos incubated in normoxia (open circles) or hypoxia (filled triangles), represented as function of the corresponding embryo’s body weight. Data refer to incubation days E14, E17, E19, and E20. Fig. 4. Length (beak and toe, mm) and weight (g) of various organs for embryos incubated in normoxia (open circles) or hypoxia (filled triangles), binned in four groups of body weight ranges. The data refer to incubation days E14, E17, E19, and E20 and are those represented individually in Fig. 3. Values are group means ± 1 S.E.M. *Statistically significant difference between the two groups. 3.6. Post-hypoxic normoxia days E17, E19, and E20. At each age, the BW and the wet weight of most organs were below the average Nx values (100%, dashed lines in Fig. 6), and for the most part did not differ significantly from the corresponding values of the Hx embryos. The very few These embryos (Hx–Nx) were exposed to hypoxia until day E14 and to normoxia thereafter; their organs were measured at Fig. 5. Absolute (left) and specific (middle) weights of the chorioallantoic membrane (CAM) for embryos incubated in normoxia (open circles) or hypoxia (filled triangles). At right, the data are plotted against the corresponding embryo’s body weight, either individually (top right) or binned in four groups of body weights (bottom right). Values are group means ± 1 S.E.M. *Statistically significant difference between the two groups. 160 M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 Fig. 6. Average values of embryo’s body weight (at left) and weights of various organs, expressed in percent of the corresponding mean values in normoxia (horizontal dashed line), for embryos incubated in hypoxia (filled bars) and for embryos returned to normoxia after hypoxic exposure until day E14 (“posthypoxic normoxia”, grey bars). Columns indicate group means at days E17 (top), E19 (middle), and E20 (bottom), bars are 1 S.E.M. *Statistically significant difference between the two groups. exceptions were the toe and, notably, the kidneys at E17, which significantly exceeded the Hx values, and the stomach at E19, which was significantly smaller. Similar results were obtained with the organ specific dry weights. In this case, the only significant differences between Hx–Nx and Hx were the kidneys at E17 (118% ± 7 in Hx–Nx and 79% ± 6 in Hx, P < 0.001), the brain at E19 (respectively, 88% ± 2 and 96% ± 2, P < 0.05) and the stomach at E20 (respectively, 105% ± 11 and 77% ± 7, P < 0.05). 4. Discussion As on many previous occasions (e.g., Smith et al., 1969; Metcalfe et al., 1981; McCutcheon et al., 1982; Adair et al., 1987; Stock and Metcalfe, 1987; Xu and Mortola, 1989; AssonBatres et al., 1989; Dzialowski et al., 2002; Azzam et al., 2007), also the current measurements indicated that hypoxia during incubation blunted embryonic growth. The effects on hematocrit were not significant. Previous data too have shown only small or no increase in hematocrit in hypoxic embryos (Jalavisto et al., 1965; Burton and Smith, 1969; Tazawa et al., 1971; Xu and Mortola, 1989; Dzialowski et al., 2002), probably because of the modest erythropoietic response and hemoconcentration at these developmental stages. Several authors had reported on the effects of hypoxia on the growth of internal organs. For example, McCutcheon et al. (1982) found that the three internal organs that they examined, brain, heart and liver, were smaller in hypoxic than in normoxic embryos; the decreases in heart and brain were less than that of the whole embryo, while the liver had the opposite response. Hence, they concluded that heart and brain were spared by hypoxia, at the expense of the liver. Similar results and conclusions were reached by other studies examining these or other organs at predetermined embryonic ages in avian (Stock and Metcalfe, 1987; Xu and Mortola, 1989; Asson-Batres et al., 1989; Dzialowski et al., 2002; Chan and Burggren, 2005) and non-avian embryonic models (Crossley and Altimiras, 2006). Also the current data, when analysed at fixed chronological ages (Fig. 2), would give the impression that some organs may be protected (e.g., brain, heart, and eyes) possibly at the expense of skeletal growth (as exemplified by the toe) or of the abdominal viscera (intestines and stomach). However, we believe that such interpretation of the data is incorrect. As mentioned in Section 1 and illustrated in Fig. 1, the approach of comparing organ-specific weights between animals at fixed chronological times can lead to misleading conclusions whenever embryos are growing at different rates and the specific weights of the organs under consideration change with development. Both situations occur in the comparison between hypoxic and normoxic embryos. When compared as function of BW, rather than age, the conclusion was reached that hypoxia had minimal selective effects on the growth of specific organs; therefore, the inter-organ differences in specific weights observed at any given age, for the most part, are attributable to the blunted growth. In young and adult mammals, the most effective responses to sustained hypoxia are those aiming to save energy and protect O2 delivery. However, many of those responses (e.g., drop in thermogenesis, peripheral vasoconstriction, increased pulmonary ventilation, cardiac output, and hematocrit) are minimally or not functional in the early embryonic phases. The autonomic control of blood flow becomes operational only in the second half of incubation (Mulder et al., 1998, 2002). Cardiac hypertrophy, which is a characteristic of sustained hypoxia after birth, is small or absent before hatching (Burton and Smith, 1969; AssonBatres et al., 1989), presumably because sustained hypoxia in the embryo causes vasodilatation and decreases systemic vascular resistance (Adair et al., 1987). Hence, the major survival strategy against hypoxia is the decrease in body growth. It is interesting to note that the slow rate of body growth not only saves energy but also, by delaying organ growth, achieves some degree of protection for key organs like the heart and the brain. In fact, the heart and the brain being among the organs that drop their specific weights during development, a slow growth implies that they will retain a greater proportion of BW for a longer period of time; the opposite occurs to organs with delayed development, like the stomach and the guts. Hence, even with limited mechanisms to redistribute blood flow, during embryonic development the inter-organ differences in time trajectories (Fig. 2) are such that solely by blunting body growth the relative proportion among organs shifts in favour of the heart and the brain. M.A. Azzam, J.P. Mortola / Respiratory Physiology & Neurobiology 159 (2007) 155–162 The CAM is an extra-embryonic organ that becomes fully formed at E12. After that age, its specific weight declines (Fig. 5, middle panel). The CAM weight and structural changes in hypoxia have been reported by several authors (Strick et al., 1991; Burton and Palmer, 1992; Wagner-Amos and Seymour, 2003; Chan and Burggren, 2005). Differently from all embryonic organs, the CAM weight increased not only at fixed developmental ages but also when examined as function of embryo’s BW (Fig. 5, panels at right). This indicates that hypoxia had a specific positive effect on the mass of the CAM, most likely providing an improvement of its O2 diffusion capacity, and reminiscent of the changes in placenta structure seen in mammals during hypoxic gestation (Faridy et al., 1988; Monge and León-Velarde, 1991; Zamudio, 2003). The second goal of the study was to consider the possibility that, during post-hypoxic normoxia, some organs may catchup at a faster pace than others. However the results, globally considered, indicated that neither the whole embryo nor its individual organs showed a significant post-hypoxic catch-up growth. There were very few exceptions to this pattern, and the most notable one was the increase in kidney size at E17, i.e. 3 days after termination of hypoxia. This increase, however, was only transient, and its basis is not clear; it cannot be ascribed to a post-hypoxic surge in blood flow, because the increase was approximately the same for the wet (121%) and dry kidneys (118%). Postnatally, the return to normoxia after several days in hypoxia stimulates an increased speed of growth. For example, human infants growth-retarded because of cardiogenic cyanotic right-to-left shunts recover to the normal, or quasi-normal, W percentile after surgical correction of the disease (Prader and Tanner, 1963; Gingell et al., 1981). Catch-up growth has been observed also in numerous animal experiments upon resolution of the original growth restriction, including the return to normoxia after hypoxia (Okubo and Mortola, 1988; Sant’Anna and Mortola, 2003). The current data, on the other hand, show a lack of catch-up growth in the hypoxic avian embryo returned to normoxia. The mechanisms behind the phenomenon of catchup growth are still obscure, but it is worth noting that the growth trajectory upon termination of hypoxia can be an important factor in determining the magnitude of catch-up growth. This is schematically illustrated in Fig. 7, where the growth curves are depicted as convex (A) or as concave (B) relative to the timeaxis. In these examples, the growth curves in hypoxia (dashed lines) are chosen to be half of the normoxic curves. Upon restoration of normoxia (filled triangles), if the growth rate resumed at the rate pertinent to that body W, catch-up growth will be minimal in condition A and almost complete in condition B. B is the familiar growth trajectory after birth, while A is the common growth pattern during embryonic development for many species, including the chicken embryo (e.g., Wangensteen et al., 1974; Vleck et al., 1980; Azzam et al., 2007). Hence, if the return to normoxia after a period of hypoxia consisted primarily in resuming body growth at the rate pertinent to that BW, irrespective of chronological age, then, the degree of catch-up growth could depend largely on the body growth trajectory. In this case, therefore, catch-up growth in the embryonic period is expected to be minimal, and lower than postnatally. 161 Fig. 7. Hypothetical growth curves in normoxia (continuous lines), hypoxia (dashed lines), and post-hypoxic normoxia (filled triangles). In (A), the growth curves are convex with respect to the time-axis (growth rate increasing with time); in (B), they are concave toward the time-axis (growth rate declining with time). In either case, the growth rate of the hypoxic curve is assumed to occur at half the normoxic rate. From day 14, the hypoxic growth curve resumes at the rate that the normoxic curve had at that body weight (filled triangles), mimicking the situation of the hypoxic embryos returned to normoxia. Catch-up growth (expressed as percent of normoxia, bottom panels, and filled triangles) in (A) is virtually non-existent, while in (B) is almost complete by day 24. In conclusion, we confirm previous data that, during embryonic hypoxia, at any given age, the specific weight of some organs may exceed, and that of others may be below, the normoxic values. However, at least in the chicken embryo and for the level of hypoxia tested, these differences are largely explained by the generalised blunting in body growth, and there is no indication that hypoxia has selective effects on the growth of specific organs. An important exception is the CAM, the mass of which in hypoxia develops out of proportion. Upon return to normoxia, differently from the postnatal life, catch-up growth is almost absent, presumably because embryonic growth rate does not depend on age but on the embryo’s BW. 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