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Effects of chronic hypoxia and protein malnutrition on growth in the developing chick
Effects of chronic hypoxia and protein malnutrition on growth in the developing chick Suzanne L. Miller, PhD,a Lucy R. Green, PhD,b Donald M. Peebles, PhD,a Mark A. Hanson, PhD,b and Carlos E. Blanco, MDc London and Southampton, United Kingdom, and Maastricht, The Netherlands OBJECTIVE: The purpose of this study was to determine how chronic hypoxia and/or protein malnutrition in ovo affect growth in developing chicks. STUDY DESIGN: Chicken eggs were incubated under normoxic (21% oxygen; n = 30 eggs) or hypoxic (14% oxygen; n = 80 eggs) conditions. Hypoxia was imposed from day 0 (n = 38 eggs), day 10 (n = 22 eggs), or from day 0 to 10 (n = 20 eggs). Protein malnutrition alone (n = 20 eggs) or in combination with hypoxia (n = 24 eggs) was induced by removal of 10% of the estimated total albumin content of the egg. Embryos/chicks were killed and weighed at day 10, 15, or immediately after hatch; organs were removed and weighed. RESULTS: Embryos to which hypoxia was imposed from day 0 weighed less than control embryos at day 10, which stayed the same until hatch (64.67% ± 3.56% egg mass vs 69.36% ± 3.90% [mean ± SD]; P < .05). Malnourished chicks at day 15 and at hatch (63.42% ± 4.28%; P < .05) weighed less than control chicks, as did malnourished plus hypoxia chicks (59.74% ± 3.41%; P < .001). Malnourished plus hypoxia chicks weighed less than malnourished chicks alone (P < .05). Embryos that were hypoxic from day 0 to 10 weighed less than control embryos at day 15 (P < .05), but not at hatch. At hatch, neither hypoxia nor malnutrition decreased crown-rump length. Brain and heart weights were increased in both malnourished groups, but not chicks that were hypoxic from day 0. CONCLUSION: Chick embryos exposed to malnutrition show asymmetric growth restriction with relative “sparing” of the brain and heart. Early growth restriction that was induced by hypoxia from the beginning of incubation is reversed by the restoration of normoxia at mid incubation. (Am J Obstet Gynecol 2002;186:261-7.)
Key words: Chronic hypoxia, protein malnutrition, chick embryo, growth
Optimal prenatal growth is dependent on the adequate supply and balance of substrates and, to a lesser extent, the fetal genotype. Clinically, deficiencies in the supply of nutrients and oxygen may retard the normal pattern of growth, thus resulting in intrauterine growth retardation. Epidemiologic evidence also suggests that low birth weight babies have an increased risk of subsequent neurologic and cardiovascular impairment.1 Investigations in animal models have confirmed that chronic periods of oxygen deprivation or malnutrition restrict fetal growth. Sheep and rat experimental models have frequently been used for these studies; however, the interventions used in these animals remain extrinsic to
From the Departments of Obstetrics and Gynaecology, University College Londona; the Centre for Fetal Origins of Adult Disease, University of Southamptonb; and the Department of Pediatrics, Institute GROW, Maastricht University.c Supported by the Wellcome Trust. Received for publication December 19, 2000; revised May 17, 2001; accepted August 20,2001. Reprint requests: Carlos E. Blanco, MD, Department of Pediatrics, University of Maastricht, P Debyelaan 25, 6202 AZ Maastricht, The Netherlands; e-mail: [email protected]. Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/119629 doi:10.1067/mob.2002.119629
the fetus and commonly include an alteration in maternal substrate availability, a disruption to uteroplacental vascular function, or an interference with placental growth.2 Maternal undernutrition has been studied extensively with respect to fetal growth; in rats and sheep, maternal undernutrition throughout pregnancy reduces fetal body weight and length measurements.3,4 A decrease in fetal oxygenation produces a similar effect on fetal growth.5-7 Chronic malnutrition also results in decreased placental weight,4 and chronic hypoxia reduces the average size of placentomes.6 Moreover, disruption of maternal nutrient or oxygen supply stimulates an upregulation of both maternal3,6,8 and placental9 cardiovascular and endocrine function that may, in turn, influence the fetal response. Therefore, mammalian models that have been investigated to date cannot be used to characterize the intrinsic fetal effects of interventions in a whole animal preparation, because the fetus remains dependent on the maternoplacental unit. The aim of the present study was to investigate the separate and combined effects of reduced oxygenation and nutrient supply on developing chick embryo growth. It has been demonstrated in the chicken embryo that the distribution of cardiac output10 and cardiovascular responses, including heart rate variability and chorioallan261
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toic blood flow,11 is consistent with the well-characterized responses seen in late gestation fetal sheep under normoxic conditions and during acute hypoxia.12 The particular advantage of chick embryos is that their use eliminates the confounding influence of maternal contribution and allows the study of the intrinsic effects of reduced oxygen supply and/or malnutrition on fetal growth. Moreover, the supply of both oxygen and protein to the embryo can be accurately and independently manipulated. The avian egg comprises 2 basic components, the yolk and the albumin. The yolk contains a high proportion of fats and, during embryonic development, is the primary source of energy, whereas the albumin contains amino acids and water, which are necessary for protein synthesis.13 Known amounts of albumin can be extracted from the egg to produce protein malnutrition. During chick incubation, the diffusion of gases must occur through the pores of the eggshell, and hypoxic conditions can be imposed by a reduction of the number of available pores or by a decrease of the ambient oxygen concentration. In the current study, we have used the chicken embryo to determine (1) the effects of decreasing ambient oxygen alone or in combination with protein malnutrition on normal growth, (2) the stage at which such perturbations can produce their effects, and (3) whether early changes in growth can be reversed. Methods Fertilized white leghorn chicken eggs were acquired from a commercial hatchery (Poyndon Farm, Waltham Cross, Herts, UK) and placed in a refrigerator at 12°C until required (≤1 week). On the day of use, eggs were numbered and weighed then placed in an incubator (Brinsea, Sandford, Somerset, UK) that was maintained at 38.5°C and 50% to 60% humidity. This was termed day 0; incubation length for the chick is 21 days. Two incubators were used simultaneously: 1 incubator contained normal room air, and the other incubator provided ambient hypoxic conditions. The use of these animals and the procedures performed were in accordance with the Animals (Scientific Procedures) Act 1986, United Kingdom. Experimental protocols Control. Eggs were placed into the room air incubator, and embryos were killed by immediate decapitation on day 10 (n = 10 embryos), day 15 (n = 10 embryos), or within 12 hours after hatching, when hatching occurred on day 21 (n = 10 embryos). Hypoxia. Ambient oxygen concentration within an incubator was regulated to 14%, with a gas mixture of air and nitrogen, with a flow rate of approximately 5 L/min. The percent environmental oxygen was monitored daily with the use of an oxygen analyzer (570A; Servomex, Sussex, UK). Three protocols for hypoxia were used: hypoxia
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from day 0 (H0), hypoxia from day 0 to 10 (H0-10), and hypoxia from day 10 (H10). H0 chicks were killed on day 10 (n = 10 chicks), on day 15 (n = 19 chicks), or at hatch (n = 9 chicks); H0-10 chicks were killed on day 15 (n = 9 chicks) or at hatch (n = 11 chicks); and H10 chicks were killed on day 15 (n = 11 chicks), or at hatch (n = 11 chicks). Protein malnutrition. On day 0, eggs were weighed, and 10% of the total albumin content was removed (calculated with the equation derived by Finkler et al14). This was achieved under sterile conditions with the use of an electric hand drill to make a small hole in the pointed end of the eggshell. An 18-gauge 25-mm needle was used to withdraw the appropriate amount of albumin into a 5-mL syringe. The hole was sealed with sterile cyanoacrylate (RS Components, Corby, Northants, UK), and the egg was reweighed. Two groups of proteinmalnourished embryos were studied: after albumin removal alone (malnourished) or in combination with hypoxia (malnourished hypoxic). All albumin-reduced eggs were placed into the normal room air incubator from day 0 (n = 44 eggs); 24 of the eggs were then transferred to the 14% oxygen chamber from day 10. Malnourished chicks were killed on day 15 (n = 10 chicks) or at hatch (n = 10 chicks), and malnourished hypoxic chicks were also killed on day 15 (n = 12 chicks) or at hatch (n = 12 chicks). Table I gives a comparison of numbers of embryos/chicks used in each group. Postmortem procedures. On days 10 and 15, the embryos were removed from the shell, the chorioallantoic vessels were severed, and the embryo was immediately weighed and killed by decapitation (Compact Balance EK-200; 200 g 0.01 g; A&D Instruments, Oxford, UK). Hatched chicks were weighed and killed. Morphometric measurements were performed with electronic digital calipers with an accuracy of 0.1 mm (Zennex, The Netherlands). The measurements obtained were crown-rump length, biparietal diameter, beak length, tibia length, tarsal length, and third toe length. The brain, heart, liver, adrenals, lungs, and kidneys were then removed with the aid of a dissecting microscope and weighed. The remaining body, which included the carcass, gut, and yolk sac, was also weighed. Statistical analyses. Embryo and chick weights are expressed as absolute weight and as a percentage of the mean initial egg mass; organ weights are expressed as a percentage of individual embryo weight. Results are presented as mean ± SD. Statistical analyses were performed with SPSS software (SPSS Inc, Chicago, Ill). Control versus H0 at day 10 was analyzed with a t-test for unpaired data. Day 15 and hatch data were analyzed by 1-way analysis of variance, and least significant difference was used post hoc where significant differences were observed. Nonparametric comparisons were analyzed with the Kruskal-Wallis Test. Fisher’s Exact test was used to exam-
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Table I. Number of embryos/chicks used in each group
Day 10 (n) Day 15 (n) At hatch (n) Hatch success* (%)
Control
H0
H10
H0-10
Protein malnutrition
Protein malnutrition + hypoxia from day 10
10 10 10 56
10 19 9 34
NA 11 11 69
NA 9 11 58
NA 10 10 45
NA 12 12 48
NA, Numbers not applicable, because the embryos were not killed at day 10. *Represents the percentage of successfully hatched chicks on day 21, at which time the treatment groups are not significantly different from the control group.
ine differences in hatch rates between control and treatment groups. Differences were considered significant when the probability value was <.05. Results Survival. On days 10 and 15, 91 viable embryos and 63 hatched chicks were used in these experiments. The mean success of chick hatching for all groups was 49%, with the highest survival rate for H10 chicks and the lowest for H0 chicks (Table I). Body weight. At day 10, H0 embryos weighed significantly less than control embryos, expressed as absolute weight (1.81 ± 0.17 g vs 2.29 ± 0.20 g, respectively) or as a percentage of egg mass (3.02% ± 0.28% vs 3.93% ± 0.34%, respectively; Fig). On day 15, the absolute weight of H0 (9.08 ± 1.17 g), H10 (10.86 ± 1.63 g), malnourished (9.42 ± 1.74 g), and malnourished hypoxic (9.69 ± 0.80 g) embryos was significantly less than that of control embryos (12.18 ± 1.65 g). Weight as a percentage of egg mass of H0 (15.18% ± 1.96%), H10 (18.34% ± 2.76%), H0-10 (18.31% ± 2.16%), malnourished (15.42% ± 2.84%), and malnourished hypoxic embryos (16.04% ± 1.32%) was significantly less than that observed in control embryos (20.87% ± 2.82%). Immediately after hatch, malnourished hypoxic chicks weighed less than control chicks (35.74 ± 2.06 g vs 40.46 ± 2.28 g); when expressed as a percentage of egg mass, H0 (64.67% ± 3.56%), malnourished (63.42% ± 4.28%), and malnourished hypoxic chicks (59.74% ± 3.41%) weighed less than control chicks (69.36% ± 3.90%). The H0 and malnourished chicks also weighed significantly more than malnourished hypoxic chicks (Fig). Organ weights. The data for the organ weights, as a percentage of embryo/chick weight, are detailed in Table II. At day 10, the brain, liver, heart, and lung weights of control and H0 embryos were not significantly different. The remaining body weight was also unchanged between groups at day 10. At day 15, the brain weight relative to embryo weight was significantly greater in H0 and H10 embryos, compared with controls. At hatch, the malnourished and malnourished hypoxic chick brains weighed relatively more than control brains. The relative liver weight was significantly decreased in H0-10 and malnourished embryos,
Figure. Embryo and chick weights are expressed (A) in absolute terms and (B) as a percentage of the initial egg mass. Data are expressed as mean ± SD. Control (gray column with vertical stripes) versus hypoxia from day 0 (solid black column), hypoxia from day 10 (solid white column), hypoxia from day 0 to 10 (column with diagonal stripes), malnutrition (solid gray column), and malnutrition with hypoxia (column with lattice-work stripes). Asterisk, P < .05 and triple asterisk, P < .001, data compared with control. Cross, P < .05, data compared with hypoxia from day 0 and malnutrition in normoxia at hatch.
compared with controls at day 15, but not at hatch. At day 15, the relative heart weight was significantly increased in the H0 group versus control and at hatch in H10, malnourished, and malnourished hypoxic chicks. The lung weight of H0, H0-10, malnourished, and malnourished hypoxic chicks was significantly greater than that of con-
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Table II. Organ weights, expressed as a percentage of embryo/chick weight
Brain (%) Liver (%) Heart (%) Lungs (%) Kidney (%) Rest of the body (%)
Day
Control
10 15 At hatch 10 15 At hatch 10 15 At hatch 10 15 At hatch 15 At hatch 10 15 At hatch
6.41 ± 0.76 4.23 ± 0.47 2.08 ± 0.12 1.65 ± 0.31 2.64 ± 0.22 1.92 ± 0.21 1.07 ± 0.22 1.03 ± 0.38 0.55 ± 0.08 0.93 ± 0.32 0.83 ± 0.32 0.52 ± 0.15 0.58 ± 0.15 0.45 ± 0.09 89.93 ± 1.05 90.83 ± 0.73 94.54 ± 0.53
H0
H10
6.98 ± 1.30 NA 4.79 ± 0.41* 4.77 ± 0.11* 2.05 ± 0.10 2.13 ± 0.13 1.72 ± 0.33 NA 2.69 ± 0.24 2.77 ± 0.20 1.78 ± 0.15 2.09 ± 0.17 1.11 ± 0.23 NA 1.41 ± 0.389† 1.17 ± 0.14 0.56 ± 0.09 0.68 ± 0.08† 0.99 ± 0.22 NA 1.29 ± 0.22‡ 1.03 ± 0.21 0.66 ± 0.14† 0.64 ± 0.10 0.78 ± 0.13† 0.69 ± 0.16 0.50 ± 0.06 0.64 ± 0.11‡ 89.20 ± 1.38 NA 89.05 ± 0.66‡ 89.58 ± 0.36‡ 94.45 ± 0.36 93.83 ± 0.46†
H0-10
Protein malnutrition
Protein malnutrition + hypoxia from day 10
NA 4.43 ± 0.36 2.08 ± 0.20 NA 2.32 ± 0.36† 1.99 ± 0.36 NA 1.19 ± 0.1 0.64 ± 0.13 NA 1.27 ± 0.26‡ 0.68 ± 0.18† 0.80 ± 0.09† 0.58 ± 0.09† NA 90.00 ± 0.80* 94.03 ± 0.85
NA 4.56 ± 0.49 2.23 ± 0.14† NA 2.30 ± 0.14† 2.18 ± 0.32 NA 1.20 ± 0.15 0.79 ± 0.21† NA 1.43 ± 0.24‡ 0.77 ± 0.10‡ 0.75 ± 0.16† 0.65 ± 0.14‡ NA 87.31 ± 0.42‡ 91.19 ± 1.08‡
NA 4.51 ± 0.18 2.22 ± 0.17† NA 2.49 ± 0.29 2.16 ± 0.39 NA 1.10 ± 0.12 0.69 ± 0.14† NA 1.15 ± 0.19* 0.74 ± 0.15* 0.75 ± 0.11* 0.60 ± 0.12* NA 90.00 ± 0.42* 93.60 ± 0.83*
Data are expressed as mean ± SD. NA, Organ weights are not applicable because embryos were not killed at day 10. *P < .01, compared with control. †P < .05, compared with control. ‡P < .001, compared with control.
trol at day 15, and the difference was maintained until hatch in these treatment groups. The kidney weight was significantly increased compared with control at day 15 in H0, H0-10, malnourished, and malnourished hypoxic group chicks; at hatch, the kidneys weighed significantly more in H10, H0-10, malnourished, and malnourished hypoxic treatment chicks. At day 15, remaining body weight, as a percentage of total embryo/chick weight, was significantly decreased in all treatment groups versus control and at hatch in the H10, malnourished, and malnourished hypoxic chicks. Morphometry. Table III shows the data for morphometric measures that were obtained at day 10, 15, and hatch for all groups. At day 10, the crown-rump length, tibia length, and tarsal length of H0 embryos were significantly less than those of control embryos. At day 15, the crownrump length of H0, H0-10, malnourished, and malnourished hypoxic embryos was significantly less than that in control embryos. At hatch, there was no difference between control and treatment groups in crown-rump length, except for H0-10 chicks, in which crown-rump length was greater than that of controls. At day 15, the malnourished and malnourished hypoxic embryos had a significantly smaller head diameter than controls; at hatch, the head diameter of H10, H0-10, and malnourished chicks was greater than that of controls. There was no difference in beak length of control versus day 15 embryos or chicks at hatch. The data for tibia, tarsal, and third toe length are also given in Table III. The 3 measures for the H0 embryos were consistently less than observed in controls at day 15, although there were no significant differences in these
groups by the time of hatch. Interestingly, in the H0-10 and malnourished groups, the tarsal and third toe length were significantly greater than those of controls. The ratios for the liver/brain and kidney/brain were calculated to determine whether insults during incubation produced preferential growth in organs. On day 15, the liver/brain ratio in H0 (0.56 ± 0.06), H0-10 (0.53 ± 0.08), malnourished (0.51 ± 0.08), and malnourished hypoxic embryos (0.55 ± 0.07) was significantly less than the ratio in control embryos (0.62 ± 0.04). At hatch, the liver/brain ratio in control chicks (0.92 ± 0.09) was not significantly different from any of the treatment groups. The kidney/brain ratio in treatment groups was not different from that observed in control embryos at day 15 (0.14 ± 0.04), although at hatch, the kidney/brain ratio in H10 (0.30 ± 0.04), H0-10 (0.28 ± 0.04), malnourished (0.29 ± 0.06), and malnourished hypoxic (0.27 ± 0.05) chicks was significantly elevated above that of controls (0.22 ± 0.05). Comment This study has used the developing chick embryo to examine how chronic periods of reduced oxygen availability and/or protein malnutrition may directly affect prenatal growth. In these investigations, the ambient oxygen concentration was decreased to 14% to induce hypoxia in the chick embryo. In preliminary studies, chick embryos did not survive prolonged exposure to 12% oxygen. When this less severe hypoxia was imposed from the beginning of incubation, restriction of normal growth was observed by incubation day 10, as shown by reduced embryo weight and body lengths, although at this stage
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Table III. Morphometric measures obtained in embryos and chicks at day 10, 15, and hatch Day Crown-rump Body length
10 15 At hatch Head diameter 15 At hatch Beak length 15 At hatch Tibia length 10 15 At hatch Tarsal length 10 15 At hatch Third toe length 15 At hatch
Control (mm)
H0 (mm)
H10 (mm)
H0-10 (mm)
39.6 ± 1.5 65.6 ± 3.4 88.9 ± 3.1 12.8 ± 1.4 15.0 ± 0.6 12.0 ± 1.1 16.8 ± 0.7 9.6 ± 0.4 21.6 ± 2.0 33.7 ± 2.1 6.9 ± 0.5 16.8 ± 1.3 25.6 ± 1.8 13.6 ± 1.3 23.6 ± 1.5
32.7 ± 2.8* 58.2 ± 3.1† 88.8 ± 4.1 12.2 ± 0.6 15.2 ± 0.2 11.6 ± 0.7 16.9 ± 1.5 7.8 ± 0.5* 19.3 ± 1.5† 32.9 ± 1.6 5.6 ± 0.6* 15.1 ± 1.1‡ 26.3 ± 1.1 12.3 ± 0.8* 24.0 ± 0.8
NA 63.5 ± 2.7 87.2 ± 2.3 12.5 ± 0.7 15.4 ± 0.5‡ 12.0 ± 1.4 16.6 ± 0.8 NA 20.7 ± 1.3 34.3 ± 1.1 NA 16.8 ± 1.1 26.5 ± 1.2 13.5 ± 1.1 22.8 ± 1.4
NA 62.2 ± 1.5‡ 91.9 ± 2.9‡ 12.2 ± 0.8 15.4 ± 0.3‡ 12.1 ± 0.8 17.1 ± 2.0 NA 21.3 ± 1.1 34.9 ± 1.0‡ NA 16.2 ± 1.0 27.9 ± 0.7† 12.8 ± 0.8 25.3 ± 0.9*
Protein malnutrition Protein malnutrition + (mm) hypoxia from day 10 (mm) NA 58.7 ± 4.1† 88.3 ± 4.8 11.6 ± 0.8* 15.4 ± 0.5‡ 11.7 ± 1.1 16.9 ± 1.0 NA 19.8 ± 2.0* 35.1 ± 1.1‡ NA 15.3 ± 1.8 27.8 ± 0.7† 13.0 ± 1.5 24.9 ± 0.9*
NA 58.6 ± 3.5† 88.4 ± 2.9 12.0 ± 0.5‡ 15.0 ± 0.4 12.2 ± 0.6 16.5 ± 0.8 NA 19.8 ± 1.0* 32.2 ± 1.26‡ NA 15.1 ± 0.9‡ 25.7 ± 0.2 12.6 ± 1.1‡ 23.0 ± 0.9
Data are expressed as mean ± SD. NA, Measures not applicable because embryos were not killed at day 10. *P < .01, compared with control. †P < .001, compared with control. ‡P < .05, compared with control.
individual organ weights were not different from controls. By day 15, significant effects on organ weights were observable in that liver and carcass weights were reduced. At the time of hatch, the H0 chicks still weighed significantly less than control chicks, although body length and vital organs were not different. These observations correspond well with a human study in which chronic hypoxia induced by high altitude, without concomitant undernutrition, was associated with low birth weight.15 Similarly, in guinea pigs16 and sheep,6 maternal hypoxia for most of the pregnancy induced a decrease in fetal weight, with brain and heart weights either maintained or increased. However, the study by Gilbert et al16 also noted alterations in maternal weight and the placental-weight-tofetal-body-weight ratio, 2 effects of the treatment that may independently induce fetal weight changes. In the developing chick, respiratory gases are transferred through the chorioallantoic membrane (CAM), a role analogous to that of the placenta in mammalian gas exchange.17 It has previously been demonstrated in the chick embryo that reduced ambient oxygen concentration from incubation day 7 induces an increased capillary density of the CAM and elevated hematocrit by day 14.18 Such responses might account for the observed differences in morphometric measures and organ weights in the longterm hypoxic group of embryos, although further investigations of the structure of the CAM are required to elucidate changes in oxygen and/or protein delivery at varying times in incubation. Interestingly, embryos that had been incubated in low oxygen from day 0, but moved to normal oxygen at day 10 (H0-10), had weights and liver/brain ratios similar to control chicks at hatch, despite differences observed at day 15. This suggests that growth retardation in early in-
cubation can be reversed and that “catch up” body growth can be achieved. At hatch, embryos that were exposed to hypoxia in only the second half of incubation had similar body weights and body length measures to controls; relative brain weight was maintained, and heart weight was increased above control values, although carcass weight was significantly decreased. These data suggest that, although the insult was not severe enough to alter body weight, it may be associated with a hypoxiainduced redistribution of cardiac output. Malnutrition was induced in these experiments by the removal of 10% of the available albumin on day 0. The proportion of albumin removed was selected to represent an upper level of malnutrition. Hill19 found hatchling success to be correlated inversely with the proportion of albumin removed, with 8% albumin removal resulting in only 15.2% hatching. This survival is considerably lower than our mean hatch survival rate of 45% with 10% albumin removal. Increase in hatching yield may be due to the careful use of sterile techniques. In the present experiment at day 15, the body length of malnourished embryos was significantly decreased compared with controls, with no change in brain and heart weights. Muramatsu et al20 demonstrated that removal of 1 to 2 mL of albumin, corresponding to approximately 3% to 7% of the albumin,19 produced a decrease in protein synthesis at day 12 of incubation without an accompanying change in embryo weight. This, combined with observations from the present study, indicates that albumin deficiency begins to affect growth early in the second half of incubation, which, in the chick, corresponds to the onset of the rapid increase in embryonic growth.21 At hatch, growth restriction was observed in malnourished chicks, with a significant decrease in body weight com-
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pared with controls, although there was no difference in crown-rump body lengths. Moreover, the brain, heart, lungs, and kidneys of malnourished chicks weighed more than controls, relative to chick body weight, whereas the liver was not significantly different. The rest of the body (comprising mainly carcass, gut, and yolk sac) weighed significantly less than controls. These observations indicate a redistribution of nutrients and gases away from nonvital organs, muscle, and skin to maintain perfusion to the essential organs, although it is not yet known whether the relative increase in organ weight is due to an increase in normal tissue growth or tissue edema. Maternal dietary restriction for most of gestation results in decreased fetal weight in pregnant sheep4 and decreased fetal weight and body length in fetal rats.3 It is interesting to note that nutrient restriction in early pregnant sheep does not decrease fetal weight or body measures in late gestation, although fetal hypothalamo-pituitary-adrenal responses are reduced and cardiovascular function is altered, which suggests a “reprogramming” mediated by substrate availability.22,23 In a further group of chick embryos, protein malnutrition was combined with hypoxia from day 10. At day 15 this group of embryos weighed less than control embryos but were not different from the protein-restriction-alone group. However, at the time of hatch, malnourished hypoxic chicks weighed significantly less than both controls and the malnourished normoxic chicks. The weight of the brain, heart, lungs, and kidneys of malnourished hypoxic chicks was increased in comparison with control chicks, although the rest of the body weighed less. This pattern of differences in organ weights is the same as that seen in malnourished chicks alone. Thus, chicks that were protein malnourished with superimposed hypoxia from midway through incubation were more growth restricted than chicks subjected to malnutrition alone, although both groups showed the same degree of centralization of nutrients and oxygen to vital organs. A reduction in ambient oxygen alone, from day 10, did not affect chick weight at the time of hatch. Malnutrition plus hypoxia in the chick embryo may be likened to experiments in the fetal sheep in which placental transfer is disrupted, thereby reducing the supply of oxygen and nutrients, but where the effects on the placenta cannot be dissociated from the effects on the fetus. In such experiments, fetal growth rate and pattern are decreased, with a reduction in body weight that is greater than observed changes in body length.2 This is similar to the asymmetric developmental changes seen in malnourished chicks, with or without additional hypoxia, although the reduction in chick body weight is more pronounced when malnutrition is combined with low oxygen. During mammalian pregnancy, the fetal response to acute and chronic hypoxia and malnutrition is well characterized12,24; however, this response is probably influenced by concomitant cardiovascular and endocrine
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changes in the mother and placenta. The present study has demonstrated that the responses to long-term hypoxia or malnutrition in the developing chick are qualitatively similar, both producing an asymmetric decrease in body weight at the time of hatch without a reduction in body length. However, hypoxia for the duration of incubation did not alter vital organ weights relative to chick weight, whereas malnutrition in normoxia or hypoxia resulted in relative increases in brain, heart, lungs, and kidney weights, with an accompanying decrease in the weight of the rest of the body. This finding suggests that malnutrition stimulates compensatory circulatory centralization to favor substrate delivery and the growth of vital organs. This study is the first to examine the intrinsic developmental effects of disruption to substrate supply during the incubation period in the chick. Results have demonstrated that long-term nutrient restriction or hypoxia imposes an asymmetric limitation on chick embryo growth; however, the mechanism by which these individual insults act may not be the same, because of the differences observed in relative organ weights. This investigation has also shown that hypoxia-induced growth restriction may be reversed if normoxic conditions are restored. Future experiments in the chick embryo could provide information about the mechanisms that mediate these developmental responses to substrate limitation and determine how the fetus adapts to such stressors. We thank Dr David Walker for his help in the preparation of this article. REFERENCES
1. Barker DJP. Mothers, babies and health in later life. 2nd ed. Edinburgh: Churchill Livingstone; 1998. 2. Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: metabolic and endocrine aspects. In: Gluckman PD, Johnston BM, Nathanielsz PW, editors. Advances in fetal physiology, research in perinatal medicine. vol 8. Ithaca (NY): Perinatology Press; 1989. p. 263-86. 3. Woodall SM, Breier BH, Johnston BM, Gluckman PD. A model of intrauterine growth retardation caused by chronic maternal undernutrition in the rat: effects on the somatotrophic axis and postnatal growth. J Endocrinol 1996;150:231-42. 4. Mellor DJ. Nutritional and placental determinants of foetal growth rate in sheep and consequences for the newborn lamb. Br Vet J 1983;139:307-24. 5. De Grauw TJ, Myers R, Scott WJ. Fetal growth retardation in rats from different levels of hypoxia. Biol Neonate 1986;49:85-9. 6. Jacobs R, Robinson JS, Owens JA, Falconer J, Webster MED. The effect of prolonged hypobaric hypoxia on growth of fetal sheep. J Dev Physiol 1988;10:97-112. 7. Smolich JJ, Esler MD. Total body catecholamine kinetics before and after birth in spontaneously hypoxemic fetal lambs. Am J Physiol 1999;277:R1313-20. 8. Owens JA, Falconer J, Robinson JS. Effect of restriction of placental growth on oxygen delivery to and consumption by the pregnant uterus and fetus. J Dev Physiol 1987;9:137-50. 9. Bauer MK, Harding JE, Bassett NS, Breier BH, Oliver MH, Gallaher BH, et al. Fetal growth and placental function. Mol Cell Endocrinol 1998;140:115-20. 10. Mulder ALM, van Golde JC, Prinzen FW, Blanco CE. Cardiac output distribution in response to hypoxia in the chick embryo in the second half of the incubation time. J Physiol 1998;508:281-7.
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11. Van Golde J, Mulder T, Blanco CE. Changes in mean chorioallantoic artery blood flow and heart rate produced by hypoxia in the developing chick embryo. Pediatr Res 1997;42:293-8. 12. Jensen A, Berger R. Fetal circulatory responses to oxygen lack. J Dev Physiol 1991;16:181-207. 13. Romanoff AL, Romanoff AJ. The avian embryo. New York: Wiley; 1949. 14. Finkler MS, Van Orman JB, Sotherland PR. Experimental manipulation of egg quality in chickens: influence of albumen and yolk on the size and body composition of near-term embryos in a precocial bird. J Comp Physiol [B] 1998;168:17-24. 15. Giussani DA, Phillips PS, Anstee S, Barker DJP. Effects of altitude versus economic status on birth weight and body shape at birth. Pediatr Res 2001;49:490-4. 16. Gilbert RD, Cummings LA, Juchau MR, Longo LD. Placental diffusing capacity and fetal development in exercising or hypoxic guinea pigs. J Appl Physiol 1979;46:828-34. 17. Metcalfe J, Stock MK. Current topic: oxygen exchange in the chorioallantoic membrane, avian homologue of the mammalian placenta. Placenta 1993;14:605-13. 18. Dusseau JW, Hutchins PM. Hypoxia-induced angiogenesis in chick chorioallantoic membranes: a role for adenosine. Respir Physiol 1988;71:33-44.
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19. Hill WL. Importance of prenatal nutrition to the development of a precocial chick. Dev Psychobiol 1993;26:237-49. 20. Muramatsu T, Hiramoto K, Koshi N, Okumura J, Miyoshi S, Mitsumoto T. Importance of albumen content in whole-body protein synthesis of the chicken embryo during incubation. Br Poultry Sci 1990;31:101-6. 21. Starck JM. Structural variants and invariants in avian embryonic and postnatal development. In: Starck JM, Ricklefs RE, editors. Avian growth and development. Oxford (UK): University Press; 1998. p. 59-88. 22. Hawkins P, Steyn C, McGarrigle HHG, Saito T, Ozaki T, Stratford LL, et al. Effect of maternal nutrient restriction in early gestation on development of the hypothalamic-pituitary-adrenal axis in fetal sheep at 0.8-0.9 of gestation. J Endocrinol 1999;163:553-61. 23. Hawkins P, Steyn C, Ozaki T, Saito T, Noakes DE, Hanson MA. Effect of maternal undernutrition in early gestation on ovine fetal blood pressure and cardiovascular reflexes. Am J Physiol 2000;279:R340-8. 24. Hoet JJ, Hanson MA. Intrauterine nutrition: its importance during critical periods for cardiovascular and endocrine development. J Physiol 1999;514:617-27.
Bound volumes available to subscribers Bound volumes of the American Journal of Obstetrics and Gynecology are available to subscribers (only) for the 2001 issues from the publisher, at a cost of $122.00 for domestic, $156.22 for Canada, and $146.00 for international for Vol. 184 (January-June) and Vol. 185 (July-December). Shipping charges are included. Each bound volume contains a subject and author index, and all advertising is removed. The binding is durable buckram with the Journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Mosby, Subscription Customer Service, 6277 Sea Harbor Dr, Orlando, FL 32887. Telephone (800)654-2452 or (407)345-4000. Fax (407)363-9661. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular Journal subscription.
Key words: Chronic hypoxia, protein malnutrition, chick embryo, growth
Optimal prenatal growth is dependent on the adequate supply and balance of substrates and, to a lesser extent, the fetal genotype. Clinically, deficiencies in the supply of nutrients and oxygen may retard the normal pattern of growth, thus resulting in intrauterine growth retardation. Epidemiologic evidence also suggests that low birth weight babies have an increased risk of subsequent neurologic and cardiovascular impairment.1 Investigations in animal models have confirmed that chronic periods of oxygen deprivation or malnutrition restrict fetal growth. Sheep and rat experimental models have frequently been used for these studies; however, the interventions used in these animals remain extrinsic to
From the Departments of Obstetrics and Gynaecology, University College Londona; the Centre for Fetal Origins of Adult Disease, University of Southamptonb; and the Department of Pediatrics, Institute GROW, Maastricht University.c Supported by the Wellcome Trust. Received for publication December 19, 2000; revised May 17, 2001; accepted August 20,2001. Reprint requests: Carlos E. Blanco, MD, Department of Pediatrics, University of Maastricht, P Debyelaan 25, 6202 AZ Maastricht, The Netherlands; e-mail: [email protected]. Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/119629 doi:10.1067/mob.2002.119629
the fetus and commonly include an alteration in maternal substrate availability, a disruption to uteroplacental vascular function, or an interference with placental growth.2 Maternal undernutrition has been studied extensively with respect to fetal growth; in rats and sheep, maternal undernutrition throughout pregnancy reduces fetal body weight and length measurements.3,4 A decrease in fetal oxygenation produces a similar effect on fetal growth.5-7 Chronic malnutrition also results in decreased placental weight,4 and chronic hypoxia reduces the average size of placentomes.6 Moreover, disruption of maternal nutrient or oxygen supply stimulates an upregulation of both maternal3,6,8 and placental9 cardiovascular and endocrine function that may, in turn, influence the fetal response. Therefore, mammalian models that have been investigated to date cannot be used to characterize the intrinsic fetal effects of interventions in a whole animal preparation, because the fetus remains dependent on the maternoplacental unit. The aim of the present study was to investigate the separate and combined effects of reduced oxygenation and nutrient supply on developing chick embryo growth. It has been demonstrated in the chicken embryo that the distribution of cardiac output10 and cardiovascular responses, including heart rate variability and chorioallan261
262 Miller et al
toic blood flow,11 is consistent with the well-characterized responses seen in late gestation fetal sheep under normoxic conditions and during acute hypoxia.12 The particular advantage of chick embryos is that their use eliminates the confounding influence of maternal contribution and allows the study of the intrinsic effects of reduced oxygen supply and/or malnutrition on fetal growth. Moreover, the supply of both oxygen and protein to the embryo can be accurately and independently manipulated. The avian egg comprises 2 basic components, the yolk and the albumin. The yolk contains a high proportion of fats and, during embryonic development, is the primary source of energy, whereas the albumin contains amino acids and water, which are necessary for protein synthesis.13 Known amounts of albumin can be extracted from the egg to produce protein malnutrition. During chick incubation, the diffusion of gases must occur through the pores of the eggshell, and hypoxic conditions can be imposed by a reduction of the number of available pores or by a decrease of the ambient oxygen concentration. In the current study, we have used the chicken embryo to determine (1) the effects of decreasing ambient oxygen alone or in combination with protein malnutrition on normal growth, (2) the stage at which such perturbations can produce their effects, and (3) whether early changes in growth can be reversed. Methods Fertilized white leghorn chicken eggs were acquired from a commercial hatchery (Poyndon Farm, Waltham Cross, Herts, UK) and placed in a refrigerator at 12°C until required (≤1 week). On the day of use, eggs were numbered and weighed then placed in an incubator (Brinsea, Sandford, Somerset, UK) that was maintained at 38.5°C and 50% to 60% humidity. This was termed day 0; incubation length for the chick is 21 days. Two incubators were used simultaneously: 1 incubator contained normal room air, and the other incubator provided ambient hypoxic conditions. The use of these animals and the procedures performed were in accordance with the Animals (Scientific Procedures) Act 1986, United Kingdom. Experimental protocols Control. Eggs were placed into the room air incubator, and embryos were killed by immediate decapitation on day 10 (n = 10 embryos), day 15 (n = 10 embryos), or within 12 hours after hatching, when hatching occurred on day 21 (n = 10 embryos). Hypoxia. Ambient oxygen concentration within an incubator was regulated to 14%, with a gas mixture of air and nitrogen, with a flow rate of approximately 5 L/min. The percent environmental oxygen was monitored daily with the use of an oxygen analyzer (570A; Servomex, Sussex, UK). Three protocols for hypoxia were used: hypoxia
February 2002 Am J Obstet Gynecol
from day 0 (H0), hypoxia from day 0 to 10 (H0-10), and hypoxia from day 10 (H10). H0 chicks were killed on day 10 (n = 10 chicks), on day 15 (n = 19 chicks), or at hatch (n = 9 chicks); H0-10 chicks were killed on day 15 (n = 9 chicks) or at hatch (n = 11 chicks); and H10 chicks were killed on day 15 (n = 11 chicks), or at hatch (n = 11 chicks). Protein malnutrition. On day 0, eggs were weighed, and 10% of the total albumin content was removed (calculated with the equation derived by Finkler et al14). This was achieved under sterile conditions with the use of an electric hand drill to make a small hole in the pointed end of the eggshell. An 18-gauge 25-mm needle was used to withdraw the appropriate amount of albumin into a 5-mL syringe. The hole was sealed with sterile cyanoacrylate (RS Components, Corby, Northants, UK), and the egg was reweighed. Two groups of proteinmalnourished embryos were studied: after albumin removal alone (malnourished) or in combination with hypoxia (malnourished hypoxic). All albumin-reduced eggs were placed into the normal room air incubator from day 0 (n = 44 eggs); 24 of the eggs were then transferred to the 14% oxygen chamber from day 10. Malnourished chicks were killed on day 15 (n = 10 chicks) or at hatch (n = 10 chicks), and malnourished hypoxic chicks were also killed on day 15 (n = 12 chicks) or at hatch (n = 12 chicks). Table I gives a comparison of numbers of embryos/chicks used in each group. Postmortem procedures. On days 10 and 15, the embryos were removed from the shell, the chorioallantoic vessels were severed, and the embryo was immediately weighed and killed by decapitation (Compact Balance EK-200; 200 g 0.01 g; A&D Instruments, Oxford, UK). Hatched chicks were weighed and killed. Morphometric measurements were performed with electronic digital calipers with an accuracy of 0.1 mm (Zennex, The Netherlands). The measurements obtained were crown-rump length, biparietal diameter, beak length, tibia length, tarsal length, and third toe length. The brain, heart, liver, adrenals, lungs, and kidneys were then removed with the aid of a dissecting microscope and weighed. The remaining body, which included the carcass, gut, and yolk sac, was also weighed. Statistical analyses. Embryo and chick weights are expressed as absolute weight and as a percentage of the mean initial egg mass; organ weights are expressed as a percentage of individual embryo weight. Results are presented as mean ± SD. Statistical analyses were performed with SPSS software (SPSS Inc, Chicago, Ill). Control versus H0 at day 10 was analyzed with a t-test for unpaired data. Day 15 and hatch data were analyzed by 1-way analysis of variance, and least significant difference was used post hoc where significant differences were observed. Nonparametric comparisons were analyzed with the Kruskal-Wallis Test. Fisher’s Exact test was used to exam-
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Table I. Number of embryos/chicks used in each group
Day 10 (n) Day 15 (n) At hatch (n) Hatch success* (%)
Control
H0
H10
H0-10
Protein malnutrition
Protein malnutrition + hypoxia from day 10
10 10 10 56
10 19 9 34
NA 11 11 69
NA 9 11 58
NA 10 10 45
NA 12 12 48
NA, Numbers not applicable, because the embryos were not killed at day 10. *Represents the percentage of successfully hatched chicks on day 21, at which time the treatment groups are not significantly different from the control group.
ine differences in hatch rates between control and treatment groups. Differences were considered significant when the probability value was <.05. Results Survival. On days 10 and 15, 91 viable embryos and 63 hatched chicks were used in these experiments. The mean success of chick hatching for all groups was 49%, with the highest survival rate for H10 chicks and the lowest for H0 chicks (Table I). Body weight. At day 10, H0 embryos weighed significantly less than control embryos, expressed as absolute weight (1.81 ± 0.17 g vs 2.29 ± 0.20 g, respectively) or as a percentage of egg mass (3.02% ± 0.28% vs 3.93% ± 0.34%, respectively; Fig). On day 15, the absolute weight of H0 (9.08 ± 1.17 g), H10 (10.86 ± 1.63 g), malnourished (9.42 ± 1.74 g), and malnourished hypoxic (9.69 ± 0.80 g) embryos was significantly less than that of control embryos (12.18 ± 1.65 g). Weight as a percentage of egg mass of H0 (15.18% ± 1.96%), H10 (18.34% ± 2.76%), H0-10 (18.31% ± 2.16%), malnourished (15.42% ± 2.84%), and malnourished hypoxic embryos (16.04% ± 1.32%) was significantly less than that observed in control embryos (20.87% ± 2.82%). Immediately after hatch, malnourished hypoxic chicks weighed less than control chicks (35.74 ± 2.06 g vs 40.46 ± 2.28 g); when expressed as a percentage of egg mass, H0 (64.67% ± 3.56%), malnourished (63.42% ± 4.28%), and malnourished hypoxic chicks (59.74% ± 3.41%) weighed less than control chicks (69.36% ± 3.90%). The H0 and malnourished chicks also weighed significantly more than malnourished hypoxic chicks (Fig). Organ weights. The data for the organ weights, as a percentage of embryo/chick weight, are detailed in Table II. At day 10, the brain, liver, heart, and lung weights of control and H0 embryos were not significantly different. The remaining body weight was also unchanged between groups at day 10. At day 15, the brain weight relative to embryo weight was significantly greater in H0 and H10 embryos, compared with controls. At hatch, the malnourished and malnourished hypoxic chick brains weighed relatively more than control brains. The relative liver weight was significantly decreased in H0-10 and malnourished embryos,
Figure. Embryo and chick weights are expressed (A) in absolute terms and (B) as a percentage of the initial egg mass. Data are expressed as mean ± SD. Control (gray column with vertical stripes) versus hypoxia from day 0 (solid black column), hypoxia from day 10 (solid white column), hypoxia from day 0 to 10 (column with diagonal stripes), malnutrition (solid gray column), and malnutrition with hypoxia (column with lattice-work stripes). Asterisk, P < .05 and triple asterisk, P < .001, data compared with control. Cross, P < .05, data compared with hypoxia from day 0 and malnutrition in normoxia at hatch.
compared with controls at day 15, but not at hatch. At day 15, the relative heart weight was significantly increased in the H0 group versus control and at hatch in H10, malnourished, and malnourished hypoxic chicks. The lung weight of H0, H0-10, malnourished, and malnourished hypoxic chicks was significantly greater than that of con-
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Table II. Organ weights, expressed as a percentage of embryo/chick weight
Brain (%) Liver (%) Heart (%) Lungs (%) Kidney (%) Rest of the body (%)
Day
Control
10 15 At hatch 10 15 At hatch 10 15 At hatch 10 15 At hatch 15 At hatch 10 15 At hatch
6.41 ± 0.76 4.23 ± 0.47 2.08 ± 0.12 1.65 ± 0.31 2.64 ± 0.22 1.92 ± 0.21 1.07 ± 0.22 1.03 ± 0.38 0.55 ± 0.08 0.93 ± 0.32 0.83 ± 0.32 0.52 ± 0.15 0.58 ± 0.15 0.45 ± 0.09 89.93 ± 1.05 90.83 ± 0.73 94.54 ± 0.53
H0
H10
6.98 ± 1.30 NA 4.79 ± 0.41* 4.77 ± 0.11* 2.05 ± 0.10 2.13 ± 0.13 1.72 ± 0.33 NA 2.69 ± 0.24 2.77 ± 0.20 1.78 ± 0.15 2.09 ± 0.17 1.11 ± 0.23 NA 1.41 ± 0.389† 1.17 ± 0.14 0.56 ± 0.09 0.68 ± 0.08† 0.99 ± 0.22 NA 1.29 ± 0.22‡ 1.03 ± 0.21 0.66 ± 0.14† 0.64 ± 0.10 0.78 ± 0.13† 0.69 ± 0.16 0.50 ± 0.06 0.64 ± 0.11‡ 89.20 ± 1.38 NA 89.05 ± 0.66‡ 89.58 ± 0.36‡ 94.45 ± 0.36 93.83 ± 0.46†
H0-10
Protein malnutrition
Protein malnutrition + hypoxia from day 10
NA 4.43 ± 0.36 2.08 ± 0.20 NA 2.32 ± 0.36† 1.99 ± 0.36 NA 1.19 ± 0.1 0.64 ± 0.13 NA 1.27 ± 0.26‡ 0.68 ± 0.18† 0.80 ± 0.09† 0.58 ± 0.09† NA 90.00 ± 0.80* 94.03 ± 0.85
NA 4.56 ± 0.49 2.23 ± 0.14† NA 2.30 ± 0.14† 2.18 ± 0.32 NA 1.20 ± 0.15 0.79 ± 0.21† NA 1.43 ± 0.24‡ 0.77 ± 0.10‡ 0.75 ± 0.16† 0.65 ± 0.14‡ NA 87.31 ± 0.42‡ 91.19 ± 1.08‡
NA 4.51 ± 0.18 2.22 ± 0.17† NA 2.49 ± 0.29 2.16 ± 0.39 NA 1.10 ± 0.12 0.69 ± 0.14† NA 1.15 ± 0.19* 0.74 ± 0.15* 0.75 ± 0.11* 0.60 ± 0.12* NA 90.00 ± 0.42* 93.60 ± 0.83*
Data are expressed as mean ± SD. NA, Organ weights are not applicable because embryos were not killed at day 10. *P < .01, compared with control. †P < .05, compared with control. ‡P < .001, compared with control.
trol at day 15, and the difference was maintained until hatch in these treatment groups. The kidney weight was significantly increased compared with control at day 15 in H0, H0-10, malnourished, and malnourished hypoxic group chicks; at hatch, the kidneys weighed significantly more in H10, H0-10, malnourished, and malnourished hypoxic treatment chicks. At day 15, remaining body weight, as a percentage of total embryo/chick weight, was significantly decreased in all treatment groups versus control and at hatch in the H10, malnourished, and malnourished hypoxic chicks. Morphometry. Table III shows the data for morphometric measures that were obtained at day 10, 15, and hatch for all groups. At day 10, the crown-rump length, tibia length, and tarsal length of H0 embryos were significantly less than those of control embryos. At day 15, the crownrump length of H0, H0-10, malnourished, and malnourished hypoxic embryos was significantly less than that in control embryos. At hatch, there was no difference between control and treatment groups in crown-rump length, except for H0-10 chicks, in which crown-rump length was greater than that of controls. At day 15, the malnourished and malnourished hypoxic embryos had a significantly smaller head diameter than controls; at hatch, the head diameter of H10, H0-10, and malnourished chicks was greater than that of controls. There was no difference in beak length of control versus day 15 embryos or chicks at hatch. The data for tibia, tarsal, and third toe length are also given in Table III. The 3 measures for the H0 embryos were consistently less than observed in controls at day 15, although there were no significant differences in these
groups by the time of hatch. Interestingly, in the H0-10 and malnourished groups, the tarsal and third toe length were significantly greater than those of controls. The ratios for the liver/brain and kidney/brain were calculated to determine whether insults during incubation produced preferential growth in organs. On day 15, the liver/brain ratio in H0 (0.56 ± 0.06), H0-10 (0.53 ± 0.08), malnourished (0.51 ± 0.08), and malnourished hypoxic embryos (0.55 ± 0.07) was significantly less than the ratio in control embryos (0.62 ± 0.04). At hatch, the liver/brain ratio in control chicks (0.92 ± 0.09) was not significantly different from any of the treatment groups. The kidney/brain ratio in treatment groups was not different from that observed in control embryos at day 15 (0.14 ± 0.04), although at hatch, the kidney/brain ratio in H10 (0.30 ± 0.04), H0-10 (0.28 ± 0.04), malnourished (0.29 ± 0.06), and malnourished hypoxic (0.27 ± 0.05) chicks was significantly elevated above that of controls (0.22 ± 0.05). Comment This study has used the developing chick embryo to examine how chronic periods of reduced oxygen availability and/or protein malnutrition may directly affect prenatal growth. In these investigations, the ambient oxygen concentration was decreased to 14% to induce hypoxia in the chick embryo. In preliminary studies, chick embryos did not survive prolonged exposure to 12% oxygen. When this less severe hypoxia was imposed from the beginning of incubation, restriction of normal growth was observed by incubation day 10, as shown by reduced embryo weight and body lengths, although at this stage
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Table III. Morphometric measures obtained in embryos and chicks at day 10, 15, and hatch Day Crown-rump Body length
10 15 At hatch Head diameter 15 At hatch Beak length 15 At hatch Tibia length 10 15 At hatch Tarsal length 10 15 At hatch Third toe length 15 At hatch
Control (mm)
H0 (mm)
H10 (mm)
H0-10 (mm)
39.6 ± 1.5 65.6 ± 3.4 88.9 ± 3.1 12.8 ± 1.4 15.0 ± 0.6 12.0 ± 1.1 16.8 ± 0.7 9.6 ± 0.4 21.6 ± 2.0 33.7 ± 2.1 6.9 ± 0.5 16.8 ± 1.3 25.6 ± 1.8 13.6 ± 1.3 23.6 ± 1.5
32.7 ± 2.8* 58.2 ± 3.1† 88.8 ± 4.1 12.2 ± 0.6 15.2 ± 0.2 11.6 ± 0.7 16.9 ± 1.5 7.8 ± 0.5* 19.3 ± 1.5† 32.9 ± 1.6 5.6 ± 0.6* 15.1 ± 1.1‡ 26.3 ± 1.1 12.3 ± 0.8* 24.0 ± 0.8
NA 63.5 ± 2.7 87.2 ± 2.3 12.5 ± 0.7 15.4 ± 0.5‡ 12.0 ± 1.4 16.6 ± 0.8 NA 20.7 ± 1.3 34.3 ± 1.1 NA 16.8 ± 1.1 26.5 ± 1.2 13.5 ± 1.1 22.8 ± 1.4
NA 62.2 ± 1.5‡ 91.9 ± 2.9‡ 12.2 ± 0.8 15.4 ± 0.3‡ 12.1 ± 0.8 17.1 ± 2.0 NA 21.3 ± 1.1 34.9 ± 1.0‡ NA 16.2 ± 1.0 27.9 ± 0.7† 12.8 ± 0.8 25.3 ± 0.9*
Protein malnutrition Protein malnutrition + (mm) hypoxia from day 10 (mm) NA 58.7 ± 4.1† 88.3 ± 4.8 11.6 ± 0.8* 15.4 ± 0.5‡ 11.7 ± 1.1 16.9 ± 1.0 NA 19.8 ± 2.0* 35.1 ± 1.1‡ NA 15.3 ± 1.8 27.8 ± 0.7† 13.0 ± 1.5 24.9 ± 0.9*
NA 58.6 ± 3.5† 88.4 ± 2.9 12.0 ± 0.5‡ 15.0 ± 0.4 12.2 ± 0.6 16.5 ± 0.8 NA 19.8 ± 1.0* 32.2 ± 1.26‡ NA 15.1 ± 0.9‡ 25.7 ± 0.2 12.6 ± 1.1‡ 23.0 ± 0.9
Data are expressed as mean ± SD. NA, Measures not applicable because embryos were not killed at day 10. *P < .01, compared with control. †P < .001, compared with control. ‡P < .05, compared with control.
individual organ weights were not different from controls. By day 15, significant effects on organ weights were observable in that liver and carcass weights were reduced. At the time of hatch, the H0 chicks still weighed significantly less than control chicks, although body length and vital organs were not different. These observations correspond well with a human study in which chronic hypoxia induced by high altitude, without concomitant undernutrition, was associated with low birth weight.15 Similarly, in guinea pigs16 and sheep,6 maternal hypoxia for most of the pregnancy induced a decrease in fetal weight, with brain and heart weights either maintained or increased. However, the study by Gilbert et al16 also noted alterations in maternal weight and the placental-weight-tofetal-body-weight ratio, 2 effects of the treatment that may independently induce fetal weight changes. In the developing chick, respiratory gases are transferred through the chorioallantoic membrane (CAM), a role analogous to that of the placenta in mammalian gas exchange.17 It has previously been demonstrated in the chick embryo that reduced ambient oxygen concentration from incubation day 7 induces an increased capillary density of the CAM and elevated hematocrit by day 14.18 Such responses might account for the observed differences in morphometric measures and organ weights in the longterm hypoxic group of embryos, although further investigations of the structure of the CAM are required to elucidate changes in oxygen and/or protein delivery at varying times in incubation. Interestingly, embryos that had been incubated in low oxygen from day 0, but moved to normal oxygen at day 10 (H0-10), had weights and liver/brain ratios similar to control chicks at hatch, despite differences observed at day 15. This suggests that growth retardation in early in-
cubation can be reversed and that “catch up” body growth can be achieved. At hatch, embryos that were exposed to hypoxia in only the second half of incubation had similar body weights and body length measures to controls; relative brain weight was maintained, and heart weight was increased above control values, although carcass weight was significantly decreased. These data suggest that, although the insult was not severe enough to alter body weight, it may be associated with a hypoxiainduced redistribution of cardiac output. Malnutrition was induced in these experiments by the removal of 10% of the available albumin on day 0. The proportion of albumin removed was selected to represent an upper level of malnutrition. Hill19 found hatchling success to be correlated inversely with the proportion of albumin removed, with 8% albumin removal resulting in only 15.2% hatching. This survival is considerably lower than our mean hatch survival rate of 45% with 10% albumin removal. Increase in hatching yield may be due to the careful use of sterile techniques. In the present experiment at day 15, the body length of malnourished embryos was significantly decreased compared with controls, with no change in brain and heart weights. Muramatsu et al20 demonstrated that removal of 1 to 2 mL of albumin, corresponding to approximately 3% to 7% of the albumin,19 produced a decrease in protein synthesis at day 12 of incubation without an accompanying change in embryo weight. This, combined with observations from the present study, indicates that albumin deficiency begins to affect growth early in the second half of incubation, which, in the chick, corresponds to the onset of the rapid increase in embryonic growth.21 At hatch, growth restriction was observed in malnourished chicks, with a significant decrease in body weight com-
266 Miller et al
pared with controls, although there was no difference in crown-rump body lengths. Moreover, the brain, heart, lungs, and kidneys of malnourished chicks weighed more than controls, relative to chick body weight, whereas the liver was not significantly different. The rest of the body (comprising mainly carcass, gut, and yolk sac) weighed significantly less than controls. These observations indicate a redistribution of nutrients and gases away from nonvital organs, muscle, and skin to maintain perfusion to the essential organs, although it is not yet known whether the relative increase in organ weight is due to an increase in normal tissue growth or tissue edema. Maternal dietary restriction for most of gestation results in decreased fetal weight in pregnant sheep4 and decreased fetal weight and body length in fetal rats.3 It is interesting to note that nutrient restriction in early pregnant sheep does not decrease fetal weight or body measures in late gestation, although fetal hypothalamo-pituitary-adrenal responses are reduced and cardiovascular function is altered, which suggests a “reprogramming” mediated by substrate availability.22,23 In a further group of chick embryos, protein malnutrition was combined with hypoxia from day 10. At day 15 this group of embryos weighed less than control embryos but were not different from the protein-restriction-alone group. However, at the time of hatch, malnourished hypoxic chicks weighed significantly less than both controls and the malnourished normoxic chicks. The weight of the brain, heart, lungs, and kidneys of malnourished hypoxic chicks was increased in comparison with control chicks, although the rest of the body weighed less. This pattern of differences in organ weights is the same as that seen in malnourished chicks alone. Thus, chicks that were protein malnourished with superimposed hypoxia from midway through incubation were more growth restricted than chicks subjected to malnutrition alone, although both groups showed the same degree of centralization of nutrients and oxygen to vital organs. A reduction in ambient oxygen alone, from day 10, did not affect chick weight at the time of hatch. Malnutrition plus hypoxia in the chick embryo may be likened to experiments in the fetal sheep in which placental transfer is disrupted, thereby reducing the supply of oxygen and nutrients, but where the effects on the placenta cannot be dissociated from the effects on the fetus. In such experiments, fetal growth rate and pattern are decreased, with a reduction in body weight that is greater than observed changes in body length.2 This is similar to the asymmetric developmental changes seen in malnourished chicks, with or without additional hypoxia, although the reduction in chick body weight is more pronounced when malnutrition is combined with low oxygen. During mammalian pregnancy, the fetal response to acute and chronic hypoxia and malnutrition is well characterized12,24; however, this response is probably influenced by concomitant cardiovascular and endocrine
February 2002 Am J Obstet Gynecol
changes in the mother and placenta. The present study has demonstrated that the responses to long-term hypoxia or malnutrition in the developing chick are qualitatively similar, both producing an asymmetric decrease in body weight at the time of hatch without a reduction in body length. However, hypoxia for the duration of incubation did not alter vital organ weights relative to chick weight, whereas malnutrition in normoxia or hypoxia resulted in relative increases in brain, heart, lungs, and kidney weights, with an accompanying decrease in the weight of the rest of the body. This finding suggests that malnutrition stimulates compensatory circulatory centralization to favor substrate delivery and the growth of vital organs. This study is the first to examine the intrinsic developmental effects of disruption to substrate supply during the incubation period in the chick. Results have demonstrated that long-term nutrient restriction or hypoxia imposes an asymmetric limitation on chick embryo growth; however, the mechanism by which these individual insults act may not be the same, because of the differences observed in relative organ weights. This investigation has also shown that hypoxia-induced growth restriction may be reversed if normoxic conditions are restored. Future experiments in the chick embryo could provide information about the mechanisms that mediate these developmental responses to substrate limitation and determine how the fetus adapts to such stressors. We thank Dr David Walker for his help in the preparation of this article. REFERENCES
1. Barker DJP. Mothers, babies and health in later life. 2nd ed. Edinburgh: Churchill Livingstone; 1998. 2. Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: metabolic and endocrine aspects. In: Gluckman PD, Johnston BM, Nathanielsz PW, editors. Advances in fetal physiology, research in perinatal medicine. vol 8. Ithaca (NY): Perinatology Press; 1989. p. 263-86. 3. Woodall SM, Breier BH, Johnston BM, Gluckman PD. A model of intrauterine growth retardation caused by chronic maternal undernutrition in the rat: effects on the somatotrophic axis and postnatal growth. J Endocrinol 1996;150:231-42. 4. Mellor DJ. Nutritional and placental determinants of foetal growth rate in sheep and consequences for the newborn lamb. Br Vet J 1983;139:307-24. 5. De Grauw TJ, Myers R, Scott WJ. Fetal growth retardation in rats from different levels of hypoxia. Biol Neonate 1986;49:85-9. 6. Jacobs R, Robinson JS, Owens JA, Falconer J, Webster MED. The effect of prolonged hypobaric hypoxia on growth of fetal sheep. J Dev Physiol 1988;10:97-112. 7. Smolich JJ, Esler MD. Total body catecholamine kinetics before and after birth in spontaneously hypoxemic fetal lambs. Am J Physiol 1999;277:R1313-20. 8. Owens JA, Falconer J, Robinson JS. Effect of restriction of placental growth on oxygen delivery to and consumption by the pregnant uterus and fetus. J Dev Physiol 1987;9:137-50. 9. Bauer MK, Harding JE, Bassett NS, Breier BH, Oliver MH, Gallaher BH, et al. Fetal growth and placental function. Mol Cell Endocrinol 1998;140:115-20. 10. Mulder ALM, van Golde JC, Prinzen FW, Blanco CE. Cardiac output distribution in response to hypoxia in the chick embryo in the second half of the incubation time. J Physiol 1998;508:281-7.
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11. Van Golde J, Mulder T, Blanco CE. Changes in mean chorioallantoic artery blood flow and heart rate produced by hypoxia in the developing chick embryo. Pediatr Res 1997;42:293-8. 12. Jensen A, Berger R. Fetal circulatory responses to oxygen lack. J Dev Physiol 1991;16:181-207. 13. Romanoff AL, Romanoff AJ. The avian embryo. New York: Wiley; 1949. 14. Finkler MS, Van Orman JB, Sotherland PR. Experimental manipulation of egg quality in chickens: influence of albumen and yolk on the size and body composition of near-term embryos in a precocial bird. J Comp Physiol [B] 1998;168:17-24. 15. Giussani DA, Phillips PS, Anstee S, Barker DJP. Effects of altitude versus economic status on birth weight and body shape at birth. Pediatr Res 2001;49:490-4. 16. Gilbert RD, Cummings LA, Juchau MR, Longo LD. Placental diffusing capacity and fetal development in exercising or hypoxic guinea pigs. J Appl Physiol 1979;46:828-34. 17. Metcalfe J, Stock MK. Current topic: oxygen exchange in the chorioallantoic membrane, avian homologue of the mammalian placenta. Placenta 1993;14:605-13. 18. Dusseau JW, Hutchins PM. Hypoxia-induced angiogenesis in chick chorioallantoic membranes: a role for adenosine. Respir Physiol 1988;71:33-44.
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