PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION 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 ABSTRACT Based on an old paradigm that the extraembryonic 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 2006 Poultry Science 85:1626–1631
INTRODUCTION 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 extraembryonic 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. ©2006 Poultry Science Association Inc. Received January 26, 2006. Accepted May 20, 2006. 1 Corresponding author:
[email protected]
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).
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NEW PROOF OF AN OLD PARADIGM
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 Incubation Conditions Eggs were vertically incubated in a Pas Reform forcedrought 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., HalleZoersel, 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 L/ 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
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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 3way 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 χ2 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
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EVERAERT ET AL. Table 1. The number of eggs (injected with NaN3 or saline, perforated or noninjected) that showed a sudden decrease of resonant frequency (RF) from the 96 to 120 h of incubation1 Time of treatment
NaN3 group
Saline group
Perforated group
Control group
h 48 of incubation h 72 of incubation h 96 of incubation χ2
2/17 5/17 14/16
