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From Egg To Chick: Development and Preservation of Embryos STAGES OF EMBRYONIC DEVELOPMENT Shortly after the ovum has been released from the hen's ovary, it is picked up by the funnel or infundibulum. Sperm from the male are found in the folds of the infundibulum. Soon after the ovum is picked up by the infundibulum, many sperm contact the germinal disc, but only one unites with the germ. Thus fertilization occurs about 24 hours before the egg is laid. Since the fertilized germinal disc, or blastoderm, spends about 24 hours in the warmth of the hen's body (about 107o F (42 C) while the egg is being completed, certain stages of embryonic development occur during that time. About three hours after fertilization the newly formed single cell divides and makes two cells. Then there are four, eight, sixteen, and more. Cell division continues until there are many cells grouped in a small, whitish spot visible on the upper surface of the egg yolk. When the egg is laid and its temperature drops below about 80o F (27 C), cell development ceases. Cooling at ordinary temperatures will not kill the embryo, and it will begin to develop again when the egg is placed in the incubator. Keeping eggs at temperatures above about 80o F (27 C) prior to incubation will cause a slow growth which leads to a weakening and eventual death of the embryo. During incubation various processes occur. They are mainly respiration, excretion, nutrition, and protection. Extraembryonic membranes are membranes outside the embryo's body which make these functions possible (Fig. 8). The extraembryonic membranes are the yolk sac, amnion, allantois, and chorion. The yolk sac is a layer of tissue growing over the surface of the yolk. Its walls are lined with a special tissue which digests and absorbs the yolk material. The amnion is a transparent sac filled with a colorless fluid. The amnion and amniotic fluid provide protection from mechanical shock and permit the developing embryo to exercise. Respiration by the embryo is made possible by the allantois. Blood vessels in the allantois bring oxygen to the embryo and take carbon dioxide away. The allantois also stores excretions, absorbs albumen used as food by the embryo, and absorbs calcium from the shell for the structural needs of the embryo. The allantois ceases to function when the chick punctures the air cell and starts to breathe on its own. A fourth membrane (not shown in Fig. 8), the chorion, surrounds both the amnion and yolk sac. Initially the chorion has no apparent function, but later the allantois fuses with it to form the chorio-allantois membrane. None of these extraembryonic membranes become a part of the chick. STUDYING EMBRYONIC DEVELOPMENT You can study the external form of chicken embryos at various stages of development. Beginning with the 3rd or 4th day of incubation you can, by carefully breaking open an egg each day, observe many events in the development of embryos. And it is easy to preserve the embryos for display or additional study. To study the early embryonic development from the beginning to the 4th day of incubation, you will need to prepare a set of whole mounts and observe them under a microscope. This is a rather exacting procedure which requires considerable time. It should not be attempted by the younger student or 4-H member.
Bone formation and growth can be studied by clearing and staining the embryo. This process requires advanced and exacting techniques, considerable time and equipment, and some financial outlay. Therefore, clearing and staining of embryos should be at tempted only by the advanced student under the supervision of an experienced person. If you plan to study the different stages of embryonic development by one or more of the above methods, be sure to incubate enough eggs for this purpose. The stages of embryonic development are listed in the table below (see also Figs. 9 and 10). You will not be able to see all of these with the naked eye. But just by breaking and observing an egg each day you can identify many of the stages.
Preserving embryos. You can either break open an egg and preserve the embryo each day, or you can set eggs on succeeding days and do them all at one time. Most students prefer to do one egg each day, because doing all stages at one time will require several hours. Break the egg gently in the region of the air cell, which is at the large end of the egg. Use blunt tweezers to peel away the upper portion of the shell and shell membranes and to separate the embryo from the remainder of the egg. Remove all extraembryonic membranes, and with small scissors sever the umbilical stalk near the body wall. Wash the embryo gently and thoroughly in running tap water to insure a nice, clean appearance. Be careful not to lose the embryo, especially those in the early stages of development. If you want to make close-up pictures or slides, place the embryo in a small petri dish or similar container. Then to preserve the embryo, place it in a 10 percent solution of formalin (1 part of 37 percent formaldehyde with 9 parts of water) in glass jars with screw caps. Small baby food jars are excellent for this purpose. Use a gallon jug or similar container to prepare the formalin - never use a metal container. Label each jar with pertinent information. This might include species, variety, age of embryo, date and similar information. Preparing whole mounts. Early embryonic development between zero and four days of incubation can be studied by preparing whole mounts of embryo and examining them under a microscope. This is difficult procedure, so you will need to arrange with an experienced person for guidance and counsel. The first step in preparing whole mounts is fixing the embryo, that is, preserving the actual form of the embryo and producing optical differences in its structure or making it possible to produce these difference in subsequent treatment. The chick embryo, albumen, and yolk must be removed moved from the shell and the embryo then separated from the albumen and yolk before the embryo can fixed. Since the early stages of embryonic development are the most difficult to work with, you should start with a 4- or 5-day-old embryo and work back to the 1-day embryo.
Advanced students who want to prepare whole mounts of embryos can find the procedures in such works as Animal Micrology, Introduction to Vertebrate Embryology, and Handbook of Microscopal Technique (see the reference list on page 16). Clearing and staining embryos. Bone formation and growth in chick embryos can be studied by clearing and staining the embryos. First the embryo must be fixed. Then it must be cleared, that is, made more I transparent. Finally it is stained with a color such as alizarin red S, so that the skeletal structure can be readily seen. As is true of preparing whole mounts, clearing and staining requires advanced techniques. So it should not be attempted except by the advanced student under the supervision of a qualified person. Procedures for clearing and staining embryos can be found in such references as Introduction to Vertebrate Embryology, Staining the Skeleton of Cleared Embryos with Alizarin Red S, Alizarin Red S and Toluidine Blue for Differentiating Adult or Embryonic Bone and Cartilage, and Bone Development in the Chick Embryo. (See the reference list.) IMPORTANT EVENTS IN EMBRYONIC DEVELOPMENT (From A. L. Romanoff - Cornell Rural School Leaflet, September 1939.)
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