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Semiautonomous Development of the Extraembryonic Membranes in the Chicken Embryo

N. Everaert^*,1, P. M. Coucke, F. Bamelis, B. Kemps^*,, B. De Ketelaere, V. Bruggeman^*, J. De Baerdemaeker and E. Decuypere^*

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

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

	ABSTRACT

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Based on an old paradigm that the extra-embryonic membranes develop semiautonomously from the embryo, it can also be postulated that subembryonic fluid (SEF) will be formed semiautonomously against embryonic growth, because the formation of SEF is mediated by the yolk sac membrane. In this study, we interfered in the development of SEF or the embryo. The acoustic resonance technique (which measures the resonant frequency of an excited egg) was used as a nondestructive tool to monitor the development of SEF. In the first experiment, in which the embryo was killed chemically with NaN₃, it was proven that the formation of SEF continued, even when the embryo was killed after the initiation of the growth of the yolk sac membrane. In the second experiment, in which the development of SEF was inhibited chemically with amiloride, it was shown that the embryo developed further, although SEF formation was inhibited. In the last experiment, it was shown that the age of the flock affected the development of the embryo and the sudden decrease of the resonant frequency in a different way. However, some presetting conditions, such as storage, may affect both in a similar way. Our results further strengthen the idea that the formation of SEF develops semiautonomously against embryonic development by using the nondestructive acoustic resonance technique as an indirect method to monitor yolk sac membrane formation.

Key Words: subembryonic fluid • acoustic resonance technique • chicken embryo • extraembryonic membrane

	INTRODUCTION

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In higher vertebrates, including birds, the development of the embryo occurs with the mediation of temporary appendages that persist until the embryo can start its independent existence. These appendages are the extra-embryonic membranes. An old paradigm states that although the extraembryonic membranes are living structures, derived from the blastoderm and continuous with the tissues from the embryonic body, these membranes are semiautonomous in their development (Romanoff, 1960). This was proven by Grodzinski (1934), who killed embryos from the first to fifth day of incubation and found that both the area vitellina and area vasculosa survived and continued to grow after the embryo was killed. The area vitellina is a peripheral, nonvascular region, and the area vasculosa is a medial vascularized region. Together they form the yolk sac membrane. Hence, Grodzinski (1934) proved the paradigm that this extraembryonic membrane has a semiautonomous development against the growth of the embryo.

In the line of this paradigm, it can also be postulated that subembryonic fluid (SEF) will be formed semiautonomously against embryonic growth, because the formation of SEF is mediated by the yolk sac membrane. The formation of SEF involves the active transport of Na ions by the yolk sac membrane across the vitelline membrane from the albumen into the yolk, below the embryo. The created electroosmotic gradient draws chloride ions and water from the albumen across the membrane to a new compartment under the developing embryo to form the SEF (Deeming, 2002). This compartment can be considered a temporary store that holds water and electrolytes and from which the water can be easily "channeled" through the blood vessels to other compartments. At the same time, the stored fluid disperses the condensed yolk nutrients and increases their availability to the yolk sac membrane (Schlesinger, 1958). The formation starts after the second incubation day and reaches its maximum (of 14 mL) on the seventh day in chicken eggs. Thereafter, the volume reduces, and by the 14th or 15th incubation day, the compartment has disappeared (Ar, 1991). The nondestructive acoustic resonance technique (ART) measures the resonant frequency (RF) of an egg (Coucke et al., 1997). Around the 100th h of incubation, the RF of an incubated fertile egg suddenly decreases due to the formation of SEF (Bamelis et al., 2002).

In the present research, we will show that the development of the yolk sac membrane is semiautonomous against the development of the embryo, and this was monitored indirectly by following the formation of SEF. The ART was used as a nondestructive tool to monitor the development of SEF. The biological effect of flock age on the embryonic growth and on the formation of SEF will be investigated as well.

	MATERIALS AND METHODS

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Incubation Conditions
Eggs were vertically incubated in a Pas Reform force-drought incubator (Pas Reform Hatchery Technologies, Zeddam, The Netherlands) at 37.8°C in 55% RH. The eggs were turned every hour at an angle of 90°.

ART
During the measurement, the egg was supported by 2 rubber diabolic rolls and excited at its equator by a small plastic rod with a plastic ball glued to it. The noise of the vibration was recorded by a microphone (type 130B10, The Modal Shop Inc., Cincinnati, OH) situated on the equator at an angle of 90° to the impacter. The signal was sent to a PC, filtered by a Butterworth filter, and transformed by fast Fourier transformation to obtain the RF for the first spherical mode of the vibrating egg. This measurement was repeated 4 times on the egg at 4 different equidistant locations on the equator, and the mean RF was used as the RF of the measured egg. A program written in the graphical programming language Labview 5.1 (National Instruments, Zaventem, Belgium) was used to control the measurements and to calculate the RF (Coucke et al., 1997).

Experiment 1: Stop of Embryonic Growth
One hundred fifty fertilized Cobb (Avibel N.V., Halle-Zoersel, Belgium) (flock age of 47 wk) eggs were used. The eggs were randomly divided into 3 groups of equal size (50 eggs). The eggs were treated with NaN₃ (100 µL/egg with a 5% NaN₃ solution; Sigma-Aldrich, Steinheim, Germany) to stop embryonic growth. Injections were done at 48, 72, or 96 h of incubation so that the first and second injections were done before (first and second group) and the third was done at the beginning (third group) of the observed normal decrease in RF. At each time point, 20 eggs were injected with NaN₃, 10 eggs were injected with a saline (0.9% NaCl) solution, 10 were perforated, and another 10 control eggs did not receive any treatment. The hole was made at the blunt pole of the egg, and NaN₃ was injected with a 23-gauge needle attached to a 1-cm³ syringe. After injection, the hole was covered with paraffin, and incubation was continued. Using the ART, the RF of the eggs was measured. Measurements of RF were done at 27, 47, 71, 95, 101, 109, 113, 117, 120, and 124 h of incubation. Moreover, the RF was measured before and after the treatment. Two hours after the NaN₃ injection, a sample of eggs of this group was opened to confirm embryonic death. After the last RF measurement, all eggs were opened to confirm embryonic death, infertility, or living embryo. A graph was made through time from the measurements of the RF of each egg.

Statistical Analysis.
The data were analyzed with the SAS statistical software package (SAS Version 8.2, SAS Institute Inc., Cary, NC). The data were ordered into a 3-way contingency table before analysis (i.e., they were cross-tabulated according to treatment, injection time, and whether the sudden decrease in RF [T(f)] had occurred). Separate analyses were performed using treatment or injection time as stratum. For those conditional tables, a Cochran-Mantel-Haenszel statistic was used to test for general association, whereas a Pearson ² test was used to test the hypothesis that the proportion of eggs that showed T (f) was the same for all levels of the considered factor (treatment or injection time; Agresti, 2002).

Experiment 2: Stop of Development of SEF
Two hundred forty Cobb (Avibel N.V.; flock age of 28 wk) eggs were incubated. Eggs were randomly divided in 4 experimental groups of 60 eggs. The first experimental group received amiloride (Sigma-Aldrich), a selective blocker of the Na⁺/H⁺ antiport, which plays a crucial role in SEF formation, at 60 h after the start of incubation. In this way, it was aimed to block the continuation of the formation of SEF. Amiloride was resolved in a saline solution (0.9% NaCl), and, after making a hole at the sharp pole of the egg, amiloride solution was injected into the albumen to obtain a concentration of 10^–3 M in the albumen of the egg. The hole was then sealed with paraffin. The second experimental group received a saline solution instead of the amiloride solution on h 60 of incubation. In eggs of the third experimental group, a hole was made at the sharp pole of the egg and sealed with paraffin. This group served as a control to check if a hole or an injection influences embryonic development or the formation of SEF. The fourth group did not receive any treatment.

Nondestructive Measurements.
The RF of the eggs was measured before and after the treatment at 60 h. From the 96 to 120 h of incubation, the RF was measured every hour from each egg, except for the third experimental group. No RF was measured from this group because of time limitations. Because the RF did not change after making a very precise hole in the blunt end of the egg, we assumed that the course of the RF would be comparable to the eggs without the hole. A graph was made through time, using the measurements of the RF of each egg.

Destructive Measurements.
After 132 h of incubation, 30 eggs from each group were broken, and the length of the embryos was measured to check if normal embryonic growth continued after injection. The embryo was taken out of the egg and stretched on a paper. The length of the embryo, lying on its side, was defined from head to tail and measured with a sliding caliper of 0.01-mm accuracy.

After 132 h of incubation, the remaining 30 eggs from each group were used to measure the quantity of SEF based on the method of Deeming (1989). A hole was made at the blunt pole of each egg before they were put into heating water. When the water boiled, eggs were kept in the water for 10 more minutes. After cooling down, the eggs were opened at the blunt end. Because there are fewer proteins in the SEF compartment compared with the yolk or albumen, SEF is more liquid than yolk or albumen and can be separated using a syringe and a needle and can be weighed. Infertile eggs were not used for the calculations.

Statistical Analysis.
The data were analyzed with the SAS statistical software package (SAS Version 8.2, SAS Institute Inc.). The effect of treatment on SEF, embryonic length, and T(f) were elucidated using a multivariate ANOVA. Wilks’ lambda was used to investigate overall effects of treatment on the 3 parameters given above. Positive overall treatment effects were followed by a separate 1-way ANOVA for each of the 3 parameters. Positive omnibus tests for these separate analyses were followed by post hoc tests to find differences among treatment groups. Tukey’s multiple comparison correction was applied, with an overall level of significance fixed at 5% (Neter et al., 1990).

Experiment 3: Influence of Flock Age on Embryonic Growth and SEF Formation
Four hundred Ross eggs (Avibel N.V.) were collected from the same flock at 3 ages: 31, 40, and 50 wk. The eggs of each age group were divided into 2 subgroups: 200 eggs were incubated 1 d after laying, and another 200 eggs were first stored in an acclimatized room at 16°C with 30% RH for 19 d. Unfortunately, during storage of the 31-wk-old hens, a technical problem with the acclimatization occurred, so the results from this subgroup could not be used for evaluation. So the final experiment was based on 5 different experimental groups.

Nondestructive Measurements.
The RF of the same 120 eggs per experimental group was measured every 24 h from the start of incubation. From 96 to 120 h of incubation, this measurement was done every hour. A graphical overview of the RF evolution in time (96 to 120 h) was made for each egg to record the time when the abrupt change of the RF occurred. The last hour before the RF started to decrease was defined as T(f).

Parameters.
The weight of the eggs was measured before the start of incubation, using a balance with 0.1 g of accuracy. On the fourth (96 h) and fifth (120 h) incubation days, 10 eggs per group were broken, and the length of the embryos was measured from head to tail, with a sliding caliper of 0.01 mm of accuracy.

Statistical Analysis.
The data were analyzed with the SAS statistical software package (SAS Version 8.2, SAS Institute Inc.). Differences among the experimental groups were analyzed using a 2-way ANOVA treating hen age, storage, and their interaction as possible covariates (Neter et al., 1990). Differences among means were examined with Tukey’s multiple testing procedure, fixing the significance level at 5%.

	RESULTS

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Experiment 1: Injection with NaN₃
Comparing the RF before and after the injection, only a very small decrease in RF (4 to 12 Hz) was observed after the injection. Overall, treatment and time of injection had a significant effect on the proportion of eggs that showed a sudden decrease in RF (P < 0.001). For the treatments given at 48 h of incubation (Table 1), the ² test showed a clear influence of treatment on the course of the RF (P < 0.0001). When eggs were NaN₃-injected at 48 h of incubation, the course of the RF was comparable to that of an infertile egg, showing no clear decrease in RF around 100 h of incubation. Only 2 NaN₃-injected eggs (of the 17 fertile eggs) showed the typical RF pattern for a fertile egg, with a sudden decrease of RF between 96 and 120 h of incubation. In contrast, all eggs injected with saline or perforated eggs showed a decrease of RF, which was similar to the RF drop of control eggs. When the NaN₃ injection was performed at 72 h of incubation, the RF of most eggs (12 out of 17 fertile eggs) did not decrease, whereas all eggs of the saline, perforated, and control groups showed the decrease. The statistical analysis confirmed the significant effect of treatment (P < 0.0001). When the different treatments were given at 96 h of incubation, there was no effect of treatment (P = 0.59). Even when the NaN₃ injection was given, the course of the RF showed the same pattern (14 of the 16 eggs) as for all other groups. When controlling for treatment, the statistical analysis also showed that the time of injection of NaN₃ was crucial for the course of the RF (P < 0.001); whereas injection at 48 and 72 h of incubation had a clear effect on T(f), this effect vanished when the injection had taken place at 96 h of incubation. This time effect was not encountered for all other treatment groups.

View larger version (21K): [in this window] [in a new window]   Figure 1. Course of the resonant frequency (RF) of control, saline-injected, and amiloride-injected eggs. The RF of control egg no. 98 decreased after 103 h; the RF of control egg no. 109 decreased after 97 h. Saline-injected egg no. 195 showed a decrease of RF after 99 h, and saline-injected egg no. 187 showed a decrease after 104 h of incubation. The 2 amiloride-injected eggs did not show a sudden decrease in RF.

	DISCUSSION

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Experiment 2
Most eggs injected with amiloride did not have a sudden decrease of RF. This is caused by the blocking effect of amiloride on the Na⁺/H⁺ antiport, and, hence, no SEF will be formed in the egg. However, 20% of the amiloride-injected eggs still had a sudden decrease of RF. The action of amiloride on embryonic and extraembryonic development probably differed among eggs due to variation in their stage of development when amiloride was injected. The 6 eggs that still had a sudden decrease of RF were probably too far developed to sense the inhibiting influence of amiloride on SEF formation. Injection slightly retarded the development of the yolk sac membrane compared with noninjected eggs and showed a later drop of RF. Because the length of the embryos of the amiloride-injected eggs did not differ from the embryo length of the saline-injected eggs, it seems that injection of amiloride did not harm subsequent embryonic development. These findings illustrate that embryonic growth can continue while the SEF formation, and, indirectly, the yolk sac development, is artificially stopped. On the other hand, it also illustrates the fact that the decrease in RF is causally related to SEF formation.

Experiment 3
Due to the long storage period, many embryos did not survive or could not develop properly to hatch, which resulted in a lower hatchability percentage in the storage groups, which is in accordance with literature. At the beginning of the embryonic growth, no difference was seen in the length of the embryos from hens of different ages. These findings are in agreement with a study of Peebles et al. (2001), who found no difference in percentage of dry embryo weight on d 6 of incubation. However, Mather and Laughlin (1979) found an increase in the developmental stage of embryos after 42 h of incubation with parental age. They defined the developmental stage by Hamburger and Hamilton staging, which is a more precise way than measuring the length or the weight of the embryos. In contrast to the fact that embryos of hens of different age are equal or further in their development, the moment when water is withdrawn from the albumen near the shell was later in eggs from older hens, as can be seen by a later occurrence of the decrease of RF with age of the hens. In contrast with the findings that the time of SEF formation is not linked with the speed of embryonic development as a function of a hen’s age, storage duration postponed both embryonic growth and the formation of SEF, compared with the results of fresh eggs. In agreement with these findings are the measurements of Mather and Laughlin (1979), in which shrinkage of the blastoderm was seen after a storage period.

The results of our study confirm the old paradigm about the semiautonomous development of the extraembryonic membranes against the avian embryo, first proven by Grodzinski (1934). In the presented research, the ART was used to follow in a noninvasive way this SEF formation. The first experiment showed that SEF formation continues even after the embryonic development was arrested. The second experiment showed that the embryo continues to develop even with inhibited SEF formation. The final experiment demonstrated that biological variability in the starting material (i.e., incubating egg) due to flock age affects embryonic development as well as the time of the decline in RF.

	ACKNOWLEDGMENTS

 
N. Everaert was supported by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (grant no. G.0286.04). F. Bamelis, B. De Ketelaere, and V. Bruggeman are all postdoctoral fellows of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. B. Kemps was supported by the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie-Vlaanderen.

Received for publication January 26, 2006. Accepted for publication May 20, 2006.

	REFERENCES

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Grodzinski, Z. 1934. Zur kenntnis der Wachtstumvorgänge der Area Vasculosa beim Hühnchen. Bull. Int. Acad. Polon. Sci. Classe Sci. Math. Nat. Ser. B II:415–427.

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