Abstract

Avian eggs that are incubated naturally or artificially in captivity are often from valuable individuals or species that may be compromised by parental neglect or incorrect incubation parameters. These eggs may need to be salvaged through assisted hatching techniques. Additionally, eggs that fail to hatch should be opened for examination and embryos staged according to standard references to determine causes of mortality and potential future corrective actions.

Normal Hatching Process

To make sound decisions whether and when to provide hatching assistance, it is important to have a thorough understanding of the avian hatching process. At about 85% of the incubation period, the embryo has reached its maximum size and consumed any remaining fluids within the shell. Transillumination (“candling”) reveals a sudden, apparent increase in air cell size with a newly irregular margin. Air cell volume is unchanged actually, but the inner and outer shell membranes have begun to separate around the air cell and the inner membrane, previously stretched tautly, is now draped loosely over the embryo. The embryo positions itself for hatching at this time. Although the head is initially between the legs, it moves along the right side of the body to position under the right wing with the egg tooth in position to pierce the air cell. The shadow of the beak tip pushing under the air cell may be seen on candling. With its spine aligned parallel to the long axis of the egg, the embryo’s dorsal side corresponds roughly with the highest edge of the air cell and the ventral side with the lowest point. The chorioallantoic membrane (CAM) can no longer fully meet the respiratory needs of the embryo so slight hypoxia and hypercapnia result.^4,6 This metabolic state initiates a cascade of events that causes the hatching muscle (complexus muscle) along the dorsal neck to engorge with lymph and contract, causing the egg tooth to pierce the inner shell membrane. With this “internal pip,” pulmonary respiration is initiated and the embryo’s gas exchange improves so pipping contractions subside, and the embryo rests. At entrance to the air cell, the embryo also gains additional space to move within the shell, allowing it to use its legs as well as its neck for leverage during the hatching process. These body movements are essential in helping retract the remaining yolk sac into the body cavity before hatching is completed.

At this point, the embryo may be vocal and responsive to external stimuli, such as tapping on the shell and vocalizing by caretakers, by mimicking parental interactions. Respiration may be monitored by candling and should be even and rhythmic, in contrast to the irregular, jerky pipping contractions. Internal pip can be confirmed by hearing respirations through a stethoscope or by listening to the egg against one’s clean ear. However, the embryo soon exhausts the limited oxygen supply within the air cell and pipping contractions resume, causing the egg tooth to pierce the shell (“external pip”). Once the outer shell membrane and shell are pierced, the embryo can breathe outside air and it will rest for longer periods. Exposure to air and friction between the body and shell causes blood to recede from the CAM. Gradually, this membrane closes its vasculature, initially around the pip site and lastly around the umbilical seal. The confinement of the embryo within the egg prevents it from fully inflating the lungs and air sacs, so gas exchange again becomes inadequate and pipping contractions resume. The embryo first “breaks up” the pip site (often enlarging the opening), and after a brief rest, the embryo begins a counterclockwise rotation, breaking a new section of shell with each incremental shift of position. Rotation may be as little as halfway around or more than a full circumference before the embryo pushes off the shell cap and rests again. Residual umbilical vessels connecting the umbilicus to the CAM dry with full exposure to air. It is preferred to allow the embryo to separate itself completely from the shell after resting as premature removal of the shell at chick emergence can tear the vessels and create a more accessible route for umbilical infection.

Assisted Hatching Techniques

By monitoring the hatching process in many healthy, self-hatching eggs, veterinarians and technicians will develop the ability to recognize when and to what extent intervention may be needed. Much emphasis is often placed on the time of the pip-to-hatch interval as a sole gauge in determining when to intervene. Although this is an important measure, it is somewhat arbitrary. For example, artificially incubated California condor eggs have an average pip-to-hatch interval of 72 hours but have successfully self-hatched in as little as 45 hours or as much as 96 hours after external pip. Lack of expected progress is usually the first indication that an embryo may require assistance. If the embryo is lethargic and unresponsive on multiple checks, shows labored respiration, or becomes frantically hyperactive, more immediate intervention will be needed to provide air to the embryo. If, after air cell draw down, the embryo’s beak is not seen pushing under the air cell, it may be weak or malpositioned.³ Another indication of possible malposition is an air cell that appears parallel to the short axis of the egg, rather than angled towards the sharp end of the egg. However, not all malpositioned embryos have abnormal air cells. In either case, radiographs would be indicated to determine the embryo’s position within the egg. A length of surgical wire is used as a marker along the presumed ventral midline by lightly taping it to the shell at the blunt end where the air cell is located; this line should be traced on the shell surface in pencil, parallel to the long axis of the egg. Four views are imaged, starting with ventral-to-dorsal and rotating the egg 45° counterclockwise each time. These views will most clearly show the positioning of various structures.

If the embryo is correctly positioned for hatching and internally pips but fails to externally pip, minimal assistance may be sufficient. An air hole of 1–2 mm may be made at the apex of the air cell using a 16–20-gauge hypodermic needle matched to the egg size, then carefully drilling through the shell; similarly, a hole could be created with a clean, small, pointed, abrasive bit and variable speed rotary tool. Fine forceps are then used to remove any shell membrane that occludes the opening. This assistance may be all that is required, but if the expected progress does not resume, further intervention is needed.

An egg that fails to internally pip should have an opening made in the shell over the air cell where the external pip was expected (“windowing”), using the technique described above. The hole is then enlarged using forceps or hemostats, using the edge of the tool to control the size and direction of each break. The inner shell membrane overlying the CAM will be opaque, obscuring any active blood vessels, and should be moistened with sterile swabs and sterile water for injections or isotonic fluids to make the vessels visible. If CAM vessels are particularly heavy or refill rapidly after pressure is applied in the area where the artificial internal pip is to be made, potential hemorrhage can be reduced by repeated gentle massage with the moistened swab as the embryo’s own movements would do. The inner shell membrane, CAM and amnion can then be opened over the embryo’s beak tip and nares using blunt dissection technique to minimize bleeding. The amnion is still intact surrounding the embryo at this stage but as it is no longer fluid-filled and is so thin and avascular, it may already be pierced or may not be noticeable. Some bleeding is expected and may stop on its own or may require additional pressure around the edges of the opening in the CAM. To prevent membranes from becoming too dry, the opening in the shell, which is usually 1–2 cm, depending on the size of the egg, must be partially covered. Tegaderm® (3M, St. Paul, MN) or transparent tape that is not too sticky can be used to cover the window leaving a hole of 1–2 mm to allow for air exchange. The egg should be positioned in the hatcher with the window uppermost to prevent residual fluids or blood from entering the mouth or nares. The egg should be checked frequently to ensure that beak and nares remain clear of membranes.

Despite high humidity in the hatcher and a covering over the window in the shell, membranes may still become dry, especially for species with a protracted hatching period. This concern is particularly important as shell membranes shrink markedly as they dry and can constrict the embryo. An over-the-counter artificial tears product, with light mineral oil as the primary ingredient, may be sparingly applied to keep membranes moist and pliable. Even embryos that start the rotation process on their own may be prevented from hatching by dry membranes alone, so only a little moistening may be required to complete the process.

Some embryos that are malpositioned are able to hatch unaided, but the majority of them require some level of assistance. For an egg that pips away from the air cell, it is important that, first, the pip has pierced both shell membranes and shell sufficiently to allow the embryo to begin breathing and, second, embryo must have sufficient space to inflate its lungs. With a normal internal pip, the embryo gains not only the air in the air cell, but roughly 15% more space. An embryo pipped away from the air cell cannot use this space until the pressure is relieved. The air cell is vented at its apex, as described above. The egg is then positioned in the hatcher on a slant with this vent at the bottom so that the weight of the embryo will gradually push the air out and allow the embryo to gain the needed room for respiration and movement that will aid in yolk sac retraction. Such embryos are often able to complete the hatching process without additional intervention.

A malpositioned embryo that fails to pip at all requires full intervention, beginning with a manual external then internal pip in a window as described above, placed directly over the beak which has been located radiographically. This opening is made in a similar manner to a window over the air cell, with the exception that no air space exists between membranes. The shell, membranes and CAM are parted in essentially a single operation. Some bleeding will occur but will be less than might be expected as the embryo will have been rubbing against that particular part of the CAM vasculature.

Whether malpositioned or not, an embryo requiring a manual external and internal pip typically lacks the strength or ability to complete the hatching process unaided. These eggs are monitored frequently during the species-typical period needed for retraction of the yolk sac into the body, closure of the umbilical seal and complete shutdown of the CAM vasculature. For eggs that are easily candled, regression of CAM vessels can be readily monitored. With eggs that have more opaque shells, it may be necessary to gradually remove pieces of shell to assess progress and determine whether the yolk sac remains exposed. An endoscope may be used to better visualize active vessels or external yolk sac. When removing larger areas of shell, it is important to keep the air cell cap intact to prevent the embryo from pushing from the shell prematurely. Embryos that have protracted hatching periods and require invasive procedures should be treated with prophylactic antibiotics. If the embryo is large enough and a suitable injection site presents, this intervention may be administered parenterally before hatching has completed. Although antibiotics have also been administered by dripping the solution directly onto the CAM, no research has been performed to indicate whether this route results in effective absorption.

Once the majority of vessels have contracted and the yolk sac is retracted, it is safe to remove the embryo from the shell. In some cases, it is necessary to remove the embryo earlier if these processes are not occurring properly or if the embryo is becoming weak. In any case, this procedure is best handled sterilely to reduce infection. The embryo should be kept in the shell until the umbilical seal can be clearly seen. The shell should be removed over this area rather than sliding the embryo out, which may result in tearing of vessels. Membrane tissue and urates should gently be teased away from the umbilical area. The umbilical vessels may be ligated or cauterized if necessary, but they are usually residual so can be crushed and severed 1 cm from the seal. This site should be treated with a povidone iodine solution. If vessels are particularly moist or rough tissue surrounds the seal, 7% iodine tincture may be applied very sparingly to dry and effectively cauterize the tissue. Care must be taken to avoid the tincture entering the umbilical seal which leads to the abdominal cavity.

Chicks from eggs that have not lost sufficient weight during incubation will likely be edematous so are more likely to be malpositioned. Hatcher humidity is normally maintained at 70% relative humidity or higher in order to prevent membrane drying, but in these cases, it is contraindicated. The excess fluid must be gradually released through the lungs, so the hatcher should not exceed 50–60% RH. Edematous chicks tend to have partially open umbilical seals and these are difficult to close with sutures as the tissue is very friable. Occasionally, the yolk sac may be fully or partially unretracted. If the umbilical seal remains at least partially open, it may be possible to reduce a partially unretracted yolk sac. Sterile swabs lightly coated with oil-based ophthalmic ointment are used to stabilize the yolk sac intra-abdominally while the seal is sutured. If the seal has closed with all or part of the yolk sac unretracted, the residual usually must be amputated. It is important to note that a loop of intestine will be normally externalized before the yolk sac is withdrawn, so care must be taken not to ligate or sever this structure which will look similar to a residual blood vessel. While it is technically possible to surgically open the abdomen to insert the yolk sac, most chicks in this state are already so weakened that they are unlikely to survive the procedure. As the yolk material is the primary nutrient and water source for the newly hatched chick, if the yolk sac is removed, feeding and supplemental fluids must be given almost immediately. Chicks requiring significant hatching assistance should be given a course of prophylactic antibiotics and may require additional supportive care.

Egg Necropsy Techniques

An egg necropsy starts with a thorough review of parental history and the individual history of the egg. Eggs that fail to hatch should be opened for examination and embryos staged according to the standard reference.³ Although this work is based on the domestic chicken as a model, avian development is highly conserved and the descriptions are sufficiently detailed to make them appropriate for use with all avian species. Results may be characterized as follows:¹

Clear on Candling

I=infertile—white, centrally dense blastodisc and not donut-shaped as with fertile blastodisc
PD=positive development—white tissue partially covering yolk but no discrete embryo
FND=fertile/no development—white ring with clear center—blastodisc fails to reinitiate development after oviposition (rare)

Blood Ring on Candling

BWE=blastoderm without embryo—blood and membranes only
ED=early dead embryo (see below)

Obvious Dead Embryo

ED=early dead embryo—stage 1–19 (15% of incubation)
MD=middle dead—stage 20–39 (55% of incubation)
LD=late dead—stage 40–45 (30% of incubation)

Embryonic mortality of 7–13% is considered typical with roughly 1/3 of these in stages 1–19 and the remainder in stages 40–45, and nearly none during middle stages.⁵ For eggs that are clear on candling, it is not possible to determine fertility or infertility without opening the egg and examining the contents. Late dead embryos may be necropsied in the same manner as for a neonate, including histopathology. Only late dead embryos that are sufficiently developed to have started the hatching process may be characterized as normally positioned or in one of the seven recognized malpositions according to the standard reference.⁵ These findings may indicate problems with incubation parameters, parental nutrition, inbreeding or numerous other factors.^2,5 Results of breakout analysis, combined with parental and egg histories, may or may not provide definitive answers for a specific case but will surely contribute to analysis on a collection or population level.

Literature Cited

1.  Ernst, R.A., F.A. Bradley, U.K. Abbott, and R.M. Craig. 2004. Egg candling and breakout analysis. University of California, Department of Agricultural and Natural Resources. http://ucanr.org/freepubs/docs/8134.pdf (VIN editor: Link no longer accessible as of 1-5-21).

2.  Kuehler, C. 1983. Causes of embryonic mortality and malformations. Proc. Amer. Assoc. Zoo Vet. 157–170.

3.  Hamburger, V., and H.L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morph. 88: 49–89. https://homepage.univie.ac.at/brian.metscher (VIN editor: Original link modified as of 1-5-21).

4.  Rahn, H., A. Ar, and C.V. Paganelli. 1975. How bird eggs breathe. In: Birds. W.H. Freeman, San Francisco. Sci. Amer. 46–55.

5.  Romanoff, A. L., and A.J. Romanoff. 1972. Pathogenesis of the Avian Embryo. New York: John Wiley & Sons.

6.  Visschedijk, A.H.J. 1968. The airspace and embryonic respiration. 1. The pattern of gaseous exchange in the fertile egg during the closing stages of incubation. Brit. Poultry Sci. 9:173–184.