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Changes in Acid-Base Balance and Related Physiological Responses as a Result of External Hypercapnia During the Second Half of Incubation in the Chicken Embryo

N. Everaert^*,1, L. De Smit^*, M. Debonne^*, A. Witters^*, B. Kamers, E. Decuypere^* and V. Bruggeman^*

^* Department of Biosystems, Division Livestock-Nutrition-Quality, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium; and Department of Biosystems, Division Mechatronics, Biostatistics and Sensors, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium

¹ Corresponding author: Nadia.everaert{at}biw.kuleuven.be

	ABSTRACT

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This study investigated the effect of high CO₂ (4%) from embryonic day (ED)10 until ED16 on the acid-base balance and related parameters in the chicken embryo. From ED10 to ED16, blood was taken from a vein from the chorioallantois membrane and was analyzed for pH, partial pressure of CO₂, partial pressure of O₂ (pO₂), [HCO₃^–], [K⁺], and [Ca²⁺]. Allantoic fluid was taken for measurement of pH, NH₃-N, phosphate, and calcium concentration. The right tibia was ashed, and calcium was measured with atomic absorption spectroscopy. Embryos exposed to high CO₂ showed a consistent higher blood pH than control embryos. Notwithstanding this alkalosis, bicarbonate concentration was significantly higher in the CO₂ group from ED12 until ED16. Potassium concentration in the blood was significantly higher in the CO₂ group from ED11 until ED16. The pH of the allantois was significantly higher on ED14 and ED15. Ammonia N concentration was significantly higher in the CO₂-incubated embryos on ED12 and ED13, whereas phosphate did not differ between groups. Calcium per tibia dry weight did not differ between incubation conditions. We can conclude that embryos adapt to high CO₂ during the second half of incubation by increasing blood HCO₃^–. It appears that this increase in HCO₃^–is mainly the result of the stimulated intracellular exchange of H⁺ with K⁺, although temporary reabsorption of HCO₃^–by the kidney cannot be excluded.

Key Words: high-carbon dioxide incubation • acid-base balance • potassium • calcium • allantois

	INTRODUCTION

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In a previous study, the effect of 4% CO₂ during the second half of incubation on embryonic development, the hatching process, and posthatch performance in a modern commercial broiler line was determined (Everaert et al., 2007). This tolerance to high CO₂ suggests that the chicken embryo has adaptation mechanisms to counteract the imposed hypercapnia. Earlier studies showed already that there is an important rise in bicarbonate ions (HCO₃^–) when chicken embryos are incubated under high CO₂ levels. Tazawa et al. (1971a) coated eggs at the beginning of incubation with epoxy cement, hereby creating an accumulation of CO₂ and an O₂ deficit in the egg. From incubation d 10 until incubation d 18, the increase in blood partial pressure of CO₂ (pCO₂) of the coated group resulted in an increased concentration of HCO₃^–together with a decrease in blood pH. Dawes and Simkiss (1971) exposed White Leghorn eggs to 9% CO₂ from the ninth incubation day and compared the acid-base blood parameters with normal incubated eggs. Blood pCO₂ and HCO₃^–levels increased during development and were higher in the CO₂ group compared with the controls. Blood pH remained relatively stable during normal development; exposure to high CO₂ resulted only in a small decrease (0.1 units) in blood pH. The CO₂ embryos showed less excretion of protons into the allantoic fluid than did the control group. Because there was no evidence that the high amount of bicarbonate ions was the result of some renal mechanism, it was postulated that the increased concentration of HCO₃^–would be the result of an increased resorption of eggshell minerals (CaCO₃), thereby releasing more Ca²⁺ and HCO₃^–into the blood. Measurements of calcium of the whole embryo could, however, not confirm their hypothesis (Dawes and Simkiss, 1971). Until now, this reason of rise in bicarbonate ions as a consequence of high CO₂ remains to be elucidated.

The aim of this study was therefore to investigate further the embryonic adaptations to extra hypercapnia above the normal respiratory acidosis that chicken embryos develop during ontogeny. The physiological reactions and adaptations of the chick embryo itself on external hypercapnia (4% CO₂ from the 10th until the 18th day of incubation) were studied by measuring blood parameters related to acid-base balance. Moreover, measurements of allantoic pH (H⁺), NH₃, and phosphate concentrations were included, because changes of these parameters are an indicator of renal mechanisms.

	MATERIALS AND METHODS

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Incubation and Experimental Design
Seven hundred fifty Cobb eggs were incubated under standard incubation conditions (temperature of 37.8°C, wet bulb temperature of 29°C, turning of 90°/h; incubator Pas Reform, Zeddam, the Netherlands) during the first 9 d. On embryonic day (ED)10, the experimental group (300 eggs) was put in a closed incubator, and CO₂ in the incubator was controlled (input and output) to gradually rise until 2% CO₂ on the 11th day. The CO₂ levels continued to rise to reach 4% at ED12, and this high CO₂ level was sustained until ED18. The incubation of the control eggs (300) was done in the normal ventilated incubator. The humidity in both incubators was matched based on wet bulb temperature to prevent differences in egg weight loss. Temperature, humidity, O₂, and CO₂ levels in the CO₂ incubator were continuously measured and controlled by a computer with 2 data acquisition boards (PCI-6023E, National Instruments, Zaventem, Belgium). Software was written in the real-time module of Labview 8 PDS (National Instruments, Spring 2006). Also in the control incubator, continuous measurements were recorded through this computerized system by using the same specialized sensors as in the CO₂ incubator [CO₂: GMM221, Vaisala (Bonn, Germany); RH sensor: Hygrosmart S7000.1, Gefran (Olen, Belgium); temperature: Pt-100 direct 1/3 DIN, Gefran (Olen, Belgium); O₂: only used in the CO₂ incubator, SST Sensing, MF 010-0-LC, Honeywell (Brussels, Belgium)]. Oxygen in the CO₂ incubator did not drop below 19.7%.

Sampling
From ED10 until ED16, blood was taken daily from 15 living embryos per group from the allantoic vein (O₂-rich blood) of the chorioallantois membrane. Blood sampling and blood gas analysis were done as described in Bruggeman et al. (2007). Eggs were candled to search for a distinct blood vessel of the chorioallantois membrane at a site where the air cell bounds on the rest of the egg; a small part of the eggshell (± 1 cm²) was then carefully peeled off this site. A drop of oil was spread on the membrane to visualize better the arterial blood vessel (bright red color of the blood; arterialized blood; Piiper et al., 1980). Blood was taken with a 1-mL syringe and 30-G needle; sampling took on average 2 min. Blood (on average 350 µL) was collected in lithium-heparinized 2-mL tubes and immediately transferred to lithium-heparinized capillaries (150 µL) for presentation to the blood gas analyzer (GEM 3000, Instrumentation Laboratories, Lexington, MA). Shaking of tubes was avoided to prevent mixture with air (Bruggeman et al., 2007). Lithium-heparinized blood samples were presented in capillaries to the blood gas analyzer, which measured the pH, pCO₂, partial pressure of O₂ (pO₂), potassium, and calcium concentration of the blood and calculated the concentration of [HCO₃^–] based on the pH and pCO₂.

From ED10 until ED16, two milliliters of allantoic fluid was taken before blood sampling with a syringe (23-G needle). Allantoic fluid was also analyzed by the blood gas analyzer (GEM 3000, Instrumentation Laboratories) for pH and calcium concentration measurements. The ammonium concentration (expressed as N mg/L of NH₄⁺) was measured spectrophotometrically (Skalar, Breda, the Netherlands). In the presence of nitroprusside as a catalysator, salicylate and an active chloride solution react with ammonium via a Berthelot reaction. The formed complex was measured at a wavelength of 660 nm. Concentration of inorganic phosphate was determined spectrophotometrically with COBAS INTEGRA 400 plus (Roche, Mannheim, Germany). Inorganic phosphate forms an ammonium phosphomolybdate complex with ammonium molybdate in the presence of sulfuric acid. The concentration of phosphomolybdate formed is directly proportional to the inorganic phosphate concentration and is determined by measuring the increase in absorbance at 340 nm.

On ED12, ED14, and ED16, the right tibia of each embryo was dissected and dried overnight at 105°C to measure dry tibia weight. Thereafter, tibias were ashed at 550°C for 5 h. Ashed tibias were dissolved in 10 mL of HCl (4N) and heated until boiling. When the ashes were dissolved in the acid, the solution was filtered with a Whatman 589/ 3 ashless filterpaper (Schleicher & Schuell, Dassel, Germany). Samples were diluted with Milli-Q water (Millipore, Milford, MA) and a solution of lanthanum nitrate to obtain a final solution of 1% lanthanum nitrate. The concentration of calcium was measured by atomic absorption spectroscopy (Solaar 969, atomic absorption spectrometer, Thermo Optek, Bornem, Belgium) within a range of 0 to 25 mg/L. The amount of calcium present in the tibia was expressed relative to dry tibia weight (mg of Ca²⁺/g of dry tibia weight).

Statistical Analysis
The data were processed using the statistical software package SAS version 8.2 (SAS Institute Inc., Cary, NC). A GLM was used to analyze the effect of embryonic age and incubation condition (normal or CO₂-incubated) on pH, pCO₂, pO₂, [HCO₃^–], [K⁺], and [Ca²⁺] of the blood and on pH, ammonium, phosphate, and calcium concentration of the allantoic fluid from ED11 to ED16. Calcium per dry tibia weight was analyzed in the same way. When the means of the GLM were statistically different, then these means were further compared between the control and the experimental group by Tukey’s test, per embryonic day. Significance was based on P < 0.05. All values are expressed as mean ± SEM.

	RESULTS

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From ED10 until ED16, pH of the blood of the allantoic vein from the chorioallantois membrane did not change significantly with embryonic age in both groups (Figure 1). The average pH was 7.69 and 7.61, respectively, for CO₂ and control embryos. The blood pH was significantly higher in the CO₂ group than in the control group from ED11 until ED16 (P < 0.0001). The concentration of bicarbonate ions increased during embryonic development and reached a plateau from ED15 onwards in the CO₂ group (38 mmol/L), whereas the bicarbonate levels of the control group further increased until ED16 (34 mmol/L; Figure 1). The HCO₃^–levels were significantly higher in the CO₂ group from ED12 until ED16 (P < 0.0001). The pCO₂ increased in both groups with embryonic age, reaching maximum levels at ED15 to ED16 (32 to 34 mmHg; Figure 2). The pCO₂ did not differ during embryonic development between the control and CO₂ group (P = 0.7664). The pO₂ decreased from 104.55 mmHg on ED10 to 64.16 to 66.07 mmHg on ED16 (Figure 2). Incubation treatment had a significant effect on the pO₂ in the blood from ED11 to ED17 (P = 0.0080); pO₂ was significantly lower only at ED13 in the CO₂ group compared with the control group. The potassium concentration increased with embryonic age until ED16 (Table 1). From ED11 until ED16, embryos of the CO₂ group had significantly higher blood concentrations of potassium.

View larger version (12K): [in this window] [in a new window]   Figure 1. The course of pH and concentration of bicarbonate ions (mmol/L) from embryonic day (ED)10 until ED16 from blood from the allantoic vein of the CO2 group and the control group. Filled symbols: CO2 group; open symbols: control group; dashed line: pH; solid line: [HCO3–]. Asterisk: means between experimental groups differ (P < 0.05) per embryonic day. All values are expressed as mean ± SEM.

View larger version (11K): [in this window] [in a new window]   Figure 2. The course of the partial pressure of CO2 (pCO2) and partial pressure of O2 (pO2; mmHg) from embryonic day (ED)10 until ED16 from blood from the allantoic vein of the CO2 group and the control group. Filled symbols: CO2 group; open symbols: control group; dashed line: pO2; solid line: pCO2. Asterisk: means between experimental groups differ (P < 0.05) per embryonic day. All values are expressed as mean ± SEM.

View larger version (12K): [in this window] [in a new window]   Figure 3. Davenport diagram of the blood from the allantoic vein of the CO2 group and the control group per embryonic day (ED). Filled symbols: CO2 group; open symbols: control group. Solid and dashed isobars of partial pressure of CO2 with indication level of partial pressure of CO2.

View larger version (10K): [in this window] [in a new window]   Figure 4. The course of pH and concentration of NH3-N (mg of N/L) from embryonic day (ED)10 until ED16 from the allantoic fluid of the CO2 group and the control group. Filled symbols: CO2 group; open symbols: control group; dashed line: pH; solid line: NH3-N; asterisk: means between experimental groups differ (P < 0.05) per embryonic day. All values are expressed as mean ± SEM.

View larger version (9K): [in this window] [in a new window]   Figure 5. The course of phosphate concentration (mg/dL) from the allantoic fluid from embryonic day (ED)10 until ED16. Filled symbols: CO2 group; open symbols: control group; asterisk: means between experimental groups differ (P < 0.05) per embryonic day. All values are expressed as mean ± SEM.

View larger version (9K): [in this window] [in a new window]   Figure 6. The course of calcium concentration (mmol/L) from the blood and the allantoic fluid from embryonic day (ED)10 until ED16. Calcium per dry tibia weight (mg/g) at ED12, ED14, and ED16. Filled symbols: CO2 group; open symbols: control group. All values are expressed as mean ± SEM.

	DISCUSSION

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The age-observed patterns of the measured blood parameters are in general in accordance with literature (Dawes and Simkiss, 1969; Erasmus et al., 1970–1971; Freeman and Misson, 1970; Girard, 1971; Boutilier et al., 1977). The pCO₂ from the blood increased due to increased metabolism and the limited diffusion rate, due to eggshell resistance. The pO₂ in the blood on the other hand decreased during embryonic development. During normal development, the blood pH stayed relatively constant in the allantoic vein, in accordance with results of Dawes and Simkiss (1969). As in their study, a small drop of the blood pH can be seen from ED13 to ED14. Tazawa et al. (1971b), Freeman and Misson (1970), and Erasmus et al. (1970–1971) on the other hand found a decrease with time of blood pH during the second week of incubation. Differences in absolute values between our experiment and other studies might be due to strain or flock differences, which are known to affect embryonic growth and eggshell permeability (Tazawa et al., 1971b). The concentration of bicarbonate ions increased during development, which probably stabilized the pH, hereby buffering for the respiratory acidosis that arises during the second half of development (Erasmus et al., 1970–1971; Freeman and Misson, 1970). The maximal level of HCO₃^–concentration was reached at ED16 in the control group or possibly even later, because no blood samples were taken thereafter. The attainment of the highest levels of HCO₃^–, however, was accelerated in the CO₂ group. Girard (1971) suggested that the plateau phase reached around ED15 indicates a steady state of acid-base balance when a maximal degree of respiratory acidosis is reached. Dawes and Simkiss (1969), Erasmus et al. (1970–1971), and Freeman and Misson (1970) suggested that bicarbonate ions are provided from the shell, are conserved by the kidney when created by the activity of carbonic anhydrase, or both. The protons emerging from the interaction of CO₂ and H₂O, catalyzed by carbonic anhydrase, could then be excreted into the allantoic fluid (Boutilier et al., 1977). As a result, a decrease in the allantoic pH from 7.71 on the tenth incubation day to 6.82 and 7.13 on ED16 in the control and CO₂ group, respectively, occurred as in the study of Dawes and Simkiss (1971). The Davenport diagram showed a relative respiratory acidosis occurring during development, seen by a shift to the upper left in the control group. From ED14 to ED16, compensation by increased HCO₃^–can be seen by a shift to the upper right.

Exposure of embryos to high CO₂ did surprisingly not increase blood pCO₂ nor air cell pCO₂ (Everaert et al., 2007; experiment 2) and contradict previous findings of similar experimental design (Everaert et al., 2007; experiment 1), although an additional rise in HCO₃^–was observed. Differences in eggshell permeability between eggs of different experiments might explain the differences observed in partial pressure of gases in the air cell and blood between the experiments. Still, the bicarbonate concentration was significantly higher in the CO₂-incubated group. Three different mechanisms could explain the higher bicarbonates in the blood; renal compensations, increased eggshell resorption, and changes at the cellular level.

From our results, it is difficult to conclude whether renal compensations played a major role in increasing HCO₃^–. If more HCO₃^–would have been produced due to conversion by carbonic anhydrase, the concomitantly formed H⁺ are normally buffered in the allantois by NH₃ or phosphate. Allantoic pH was slightly more acid in the CO₂ group from ED11 to ED13, and in this period, NH₄⁺ concentration was also higher in the CO₂ group, suggesting that the extra created protons are buffered as NH₃ in the allantoic fluid. No changes of inorganic phosphate concentration were observed after CO₂ exposure. It is, however, not necessary that absolute concentrations of these buffers increase for neutralizing protons. Therefore, buffer capacity by measuring titratable acid should be performed in future research to draw a firm conclusion on the renal buffering in CO₂- exposed embryos. The pH increased to a higher pH from ED14 onwards compared with control embryos, possibly due to a simultaneous escape of HCO₃^–from the blood into the allantois. This was supported by a study of Carter et al. (1959), in which adult rats were chronically exposed to high CO₂ causing respiratory acidosis. Surprisingly, a more alkaline pH in the urine was observed, suggesting that a portion of the filtered bicarbonate escaped into the urine to buffer for high protons, especially when the serum bicarbonate concentration was maximal. This could also be the case in our study, because the concentration of bicarbonate ions in the blood of the CO₂ group was higher and had reached a plateau from ED15. Also, Rowlett and Simkiss (1989) suggested that the increase in HCO₃^–and base excess seen during normal development of shell-less embryos was the result of metabolic compensation acting via the embryonic kidney driven by the pCO₂ of the blood. Still, a note of caution is needed when interpreting data on allantoic fluid, because the allantois would be a difficult index of renal compensation, which is reflected in a large scatter (large SE) among individual eggs, probably due to the influx of uric acid (Dawes, 1974).

Besides the possible renal adaptations, eggshell compensations might have occurred. The reaction of more H⁺, resulting from hypercapnia, with calcium carbonate from the shell would lead to a higher release of calcium and bicarbonate ions. However, calcium concentrations in the blood and in the allantoic fluid and calcium per dry tibia weight were not different between treatments. Compensation by an increased release of calcium and bicarbonate ions from the shell is therefore not considered to occur as a result of hypercapnia. Crooks and Simkiss (1974) even suggested that high CO₂ would inhibit calcium resorption, when embryos are exposed to 9% CO₂.

A third possibility to explain the absence of protons together with higher HCO₃^–seen in the blood of the CO₂- incubated group might be due to an exchange with intra-cellular electrolytes. In general, respiratory acidosis is accompanied with normal or elevated concentrations of potassium (Emmett and Seldin, 1989), as seen in the increase in potassium concentration during embryonic development in our study. During the period in which embryos were exposed to CO₂, exchange of protons with intracellular potassium occurred, resulting in the significantly higher blood potassium concentrations in the CO₂ group, generating HCO₃^–in the extracellular fluid (Emmett and Seldin, 1989). This adaptation could contribute to the high bicarbonate concentration in the blood of CO₂ embryos. More-over, these bicarbonate ions might be excreted by the kidney into the allantoic fluid, causing an increased pH.

Besides the bicarbonate buffering system, hemoglobin acts as the most important nonbicarbonate buffer in blood. During normal development, hematocrit (and hemoglobin) increased, causing an increase in buffering capacity of the blood (Erasmus et al., 1970–1971; Tazawa and Piiper., 1984). Hassanzadeh et al. (2002) showed that CO₂ (0.4% from ED15 until ED20)-incubated embryos had higher hematocrit values at external pipping. In our CO₂ studies, however, an additional increase of hematocrit due to CO₂ incubation was not observed (N. Everaert, unpublished data). Therefore, it is unlikely that hemoglobin buffering would contribute to buffer protons originating from CO₂ hydration.

In conclusion, embryos exposed to 4% CO₂ during the second half of incubation adjusted to the induced acidosis, hereby creating a blood alkalosis, which was illustrated by higher blood pH combined with a sharp increase of HCO₃^–but without changes in blood pCO₂. The main mechanism to explain the extra amount of bicarbonate ions in the blood is most probably the stimulated intracellular exchange of H⁺ with K⁺, although temporary contributions of renal metabolism cannot be excluded.

	ACKNOWLEDGMENTS

 
N. Everaert and L. De Smit are supported by the Fund for Scientific Research Flanders (Fonds Wetenschappelijk Onderzoek–Vlaanderen, Belgium; G0286.04). V. Bruggeman is a postdoctoral fellow from the Fonds Wetenschappelijk Onderzoek—Vlaanderen, Belgium.

Received for publication August 20, 2007. Accepted for publication October 31, 2007.

	REFERENCES

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