Pathology of the Avian Embryo: What Veterinarians Need to Know About Pathology as a Disease Surveillance Tool for Avian Captive Propagation Programs
Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA, USA
Artificial incubation and hand-rearing of birds allows aviculturists to greatly increase the reproductive potential of breeding populations through the process of multiple-clutching. This has been successfully used to augment or re-establish wild populations of a number of endangered species, such as the peregrine falcon (Falco peregrinus), and California condor (Gymnogyps californianus). Because it is important to identify and correct any health or management problems that limit production or survival in these avicultural programs, veterinary involvement makes good financial and conservation sense. Embryo pathology is one of the most important tools available for disease diagnosis and surveillance in these situations. Carrying out embryo pathology can facilitate the optimization of incubation and hand-rearing parameters, as well as provide an important early indicator of impending disease outbreaks. When investigating health problems in an avian captive propagation program, very detailed and comprehensive background information about management and avicultural practices is required. Much of this information has unfortunately not been published in literature that is readily accessible to veterinarians. One of the purposes of this presentation is to make some of this information, as well as selected useful references,1-8 available to clinicians who might find themselves involved in avian captive propagation programs.
Diet, Management, and Health of Breeders
Nutritional problems in adults can not only affect fertility and hatchability of eggs, but diet items can be the original source of egg-borne infections (e.g., Salmonella spp., mycoplasmas, adenoviruses, etc.). Clues to nutritional problems include high levels of infertility, poor hatchability, thin eggshells, poor semen quality, egg-binding and other reproductive problems in females, poor plumage, poor bone quality in chicks, etc. Dietary supplements should also be investigated (do not assume the supplements are appropriate or properly administered). Nutritional management that includes manipulation of levels of many individual vitamins or trace minerals should be suspect. Inbreeding can be an important problem in small captive populations but be careful about drawing firm conclusions from limited data. Suboptimal incubation parameters are also an important cause of embryo deformities and failure to hatch.
Fertility and Hatchability of Eggs
Population level data on fertility and hatchability for the current and previous seasons is essential for proper interpretation of any mortality events. A useful rule of thumb for poultry (and a few other species) is roughly 80% of the total eggs laid should hatch. Of those that fail to hatch, about 10% will be infertile and 10% will be embryo mortalities. If incubation parameters have been very well worked-out for the species in question, you should be able to establish normal fertility and hatchability percentages. Significant deviations from the norm could indicate an underlying problem that needs to be identified and addressed.
Lay to set interval is the period from lay until incubation starts. In many species, incubation by the parents doesn’t begin for several days after lay. Large poultry operations often cool eggs for 1 week or more after lay in order to synchronize hatching. This is not advisable in other most of the species we work with. Another common practice in the poultry, waterfowl and game bird industry is fumigation and/or cleaning of eggs prior to setting. Although beneficial for many precocial species, we do not recommend these practices with altricial species. Cleaning of eggs can remove or damage the cuticle, which is the primary protective barrier against bacterial entry into the egg, and fumigation can be teratogenic to developing embryos for species with thin-shelled or highly porous eggs (e.g., altricial species).
The amount of parental incubation prior to pulling for artificial incubation is critically important. Eggs with less than 3–5 days of parental incubation usually have lower hatchability and chick survivability.
Dry bulb temperature is simply the temperature at which the eggs are incubated. It will vary with the species and is generally determined by the developmental rate of the species. For example, precocial bird species that lay large eggs have a longer incubation period and slower embryonic developmental rate. They require a lower incubation dry bulb temperature than small eggs with a short incubation period and a faster developmental rate. It is important to keep in mind that as little as a degree continuous error in incubation dry bulb temperature can result in a significant increase in congenital malformations and embryo mortality. Incubation dry bulb temperature that is too high is more detrimental to avian embryos than low incubation temperature, which may simply retard development.
Relative humidity is based on the incubation dry bulb and wet bulb temperature and is an overall measurement of the humidity in the incubator. Selection of wet bulb parameters for artificial incubation is based on the natural nest microclimate of the species being incubated (e.g., rainforest species vs. desert species).
Incubation humidity significantly influences egg weight loss (water loss), which should be monitored throughout incubation. Typically, eggs are weighed every day at the same time and the weight loss is calculated and plotted on a graph daily. In most species, 12–14% weight loss (set to pip) is the target. Abnormal weight loss reduces hatchability and survivability. Excessive weight loss leads to weak, dehydrated chicks, while inadequate weight loss leads to higher mortality in the pip to hatch interval or weak, edematous chicks. Aviculturists will sometimes partially coat the eggshell with nail polish to slow weight loss, or sand eggshells to increase weight loss. Glue is also used to seal small cracks in eggs, which could otherwise lead to excessive weight loss, bacterial penetration, or partial loss of egg content.
Setting and turning of eggs is also very important. Non-domestic eggs should be set in the incubator in the same position the eggs would be found in the natural nest (poultry have been selected over many generations for successful artificial incubation aircell up). Each egg must be rotated hourly around the long axis. Rotation must be back and forth (continual rotation in one direction winds up the chelaza until they break, allowing embryo to float freely, resulting in mortality). Inadequate or improper turning can increase the incidence of malpositions and embryo mortality. Egg shape is an important consideration because rounder eggs are more difficult to keep properly oriented and are therefore more prone to malpositions and embryo mortality.
Ventilation is important but can be more difficult to evaluate. Some incubators are still air (without a fan to circulate the air), while others are forced air. Large still air incubators are more likely to develop temperature gradients from top to bottom and require different incubation parameters (dry and wet bulb) than forced air incubators.
Interpreting Stage of Embryo Development
There are 45 stages of chick embryo development.1 With some approximation, this staging system can be applied to altricial species as well. Determining the stage of embryo development at which death occurred (and comparing to the number of days of incubation) enables evaluation of developmental progress and facilitates formation of a differential diagnosis and diagnostic plan.
Plotting mortality curves: Based on the fertility and hatchability numbers given above, about 10% of embryos are expected to die during incubation. Typically, there are two mortality peaks, one early and one at or near hatching (with a greater proportion occurring near hatching). Plotting embryo mortalities by stage of development enables one to quickly assess whether an unusual mortality event is occurring (provided that the background population-level data is available).
Some of the causes of early deaths include nutritional problems in breeders, incubation or transport and handling problems, long lay to set interval, fluctuating temperatures, power outages, lack of turning, poor incubator ventilation, diseases in breeders, in ovo infections, lethal genetic problems. Be careful about trying to distinguish infertile eggs from fertile eggs with no development, or from very early embryonic deaths.
Keeping in mind that mid-incubation mortality rates should be fairly low, some of the more common causes of mortalities in this period include egg infections, nutritional problems (such as riboflavin deficiency), and power or equipment failures.
The peak in embryo mortality in late incubation can be largely attributed to the critical changes occurring just before and during the hatching process (see hatching process below). Common problems in this period include malpositions, membrane entrapments (see below under gross lesions), egg infections, and lethal genetic problems.
Pip and Die or Weak Chicks
There are many causes of embryo deaths during pipping and just after hatching. These include temperature problems in incubation, humidity problems in hatchers, low oxygen, poor ventilation, hitting a blood vessel during pipping, improper turning in the first few weeks of incubation, nutritional problems, yolk sac infections, umbilical abscesses, and aspiration of egg content.
The Hatching Process (In the Chicken)
The chick embryo assumes proper position to pip 2–3 days before hatch (see gross lesions below). Lung fluid is resorbed approximately 1–2 days before hatch. The aircell is then pipped and breathing begins, resulting in closure of the ductus arteriosus and interatrial foramen, and retraction of the yolk sac. At the same time, chorioallantoic membrane blood vessels begin to close down, causing chorioallantoic respiration to decline as pulmonary function increases. The shell is pipped about 16 hours before hatch in the chicken. While these parameters (and the pip-to-hatch-interval) vary across species, the data from chickens is still a helpful guideline, which can then be adjusted as data becomes available for the species of interest. Because proper orientation of the embryo in the shell and smooth transition to pulmonary respiration are essential for successful hatching in all species, the hatching period is a critical time for avian embryos.
Procedures for Embryo Pathology
Embryo pathology is different in that embryos have little or no inflammatory response until very late in incubation. The spectrum of host responses is also relatively narrow (generally limited to necrosis, heterophilic inflammation, congestion, hemorrhage, and edema). Autolysis can be a serious problem, because some aviculturists prefer to leave eggs in incubators for several days after embryo death to be absolutely sure the embryo is dead. And it should be obvious by now that considerable background knowledge of aviculture and captive management is required in order to make sense of embryo pathology. This is due in large part to the fact that most of the causes of embryo mortality are related to incubation parameters and management issues. If all the necessary background information is not available, all we may be able to do is rule-out infectious disease as best we can.
It is very helpful to have a complete history, including parental and incubation data, before starting the postmortem exam. Record egg weight, length and width (measured with precision calipers). Assess eggshell shape and quality, smoothness of the shell, degree of surface fecal contamination, and integrity of the cuticle. Then open the blunt (aircell) end of the egg with old, dull scissors. Examine the air cell membrane and blood vessels, looking for evidence of inflammation, fungal growth, or hemorrhage (from vessels lacerated during pipping). Open the aircell and obtain a bacterial culture of egg content. For late stage (near hatching) embryo deaths, carefully evaluate the rotational and postural position of the embryo as it is removed from the shell (see gross lesions below).
For the external embryo exam, determine the stage (by comparing appropriate developmental features with Hamburger and Hamilton’s staging chart),1 evaluate the degree of subcutaneous or generalized edema, and look for developmental defects. If the yolk sac is still external, open it with a sterile blade and swab the yolk for bacterial culture and cytology (be careful not to aggressively swab the internal surface of the yolk sac, as hematopoietic cells will contaminate the smears, making it difficult to accurately diagnose an inflammatory process).
For the internal embryo exam, limit yourself (in most species) primarily to looking for deformities, evaluating the quantity and nature of the upper gastrointestinal content (which consists of swallowed residual egg content), the color and degree of inflation of the lungs, and gross appearance of the kidneys and liver. Opening very small hollow organs can cause sufficient tissue damage to render histopathology useless. Obtain the yolk culture and cytology at this time if sac is internal.
Gross Lesions in Embryos
The normal hatching position in all species is head under right wing, with the body rotated so that the beak is pointed toward the lower slope of the aircell. The most common malpositions are: 1) head over right wing (not usually lethal), 2) head under left wing (causes moderate to high mortality), 3) rotation away from aircell (moderate to high mortality), 4) leg over the head (high mortality), 5) upside down (high mortality), and 6) head between the legs (high mortality). Keep in mind that positioning is generally only critical in the last 24–48 hours before hatch (i.e., malpositions are lethal because they prevent pipping and/or hatching; a malpositioned embryo that dies 4 days before hatch most likely died from something other than the malposition alone).
Fetal membrane entrapment: We sporadically see late embryo deaths due to wrapping of the yolk or allantoic sac around the head or body. We speculate that rough handling or improper turning could contribute to this. We have seen this in parent incubated eggs also, but at lower incidence.
Edema or dehydration are non-specific findings but can be an indicator of weight loss problems during incubation.
There are many potential causes of embryo deformities, but incorrect incubation temperature is an often-overlooked cause. Retarded or accelerated development can be due incorrect incubation temperature (too low or too high, respectively). Yolk sac infections are very important as a cause of embryo and chick mortality but are difficult to diagnose grossly because yolk color and consistency is highly variable. Yolk cytology is very helpful in making a rapid diagnosis.
In the absence of infection, chorioallantoic membrane necrosis (and the reaction to it) most likely represents the morphologic consequences of normal blood vessel shutdown during hatch. Extramedullary hematopoiesis (EMH) can be very widespread (including myocardium for example). Loose aggregates of mature heterophils in a tissue more often reflects regressing EMH than infection. A small amount of residual lung fluid is normal, but this must be distinguished from aspirated egg content.
Congestion, hemorrhage, and edema are relatively common non-specific lesions. An infectious process should be ruled out in these cases through cultures and ancillary diagnostics. In the absence of other evidence of infection, these changes only point to the broad categories of incubation and management parameters, genetics (e.g., inbreeding), and nutrition. Because the brain of embryos and young chicks is very soft, we recommend sectioning the entire head for histopathology. This also facilitates evaluation of the nasal cavity and sinuses for evidence of inflammation or aspiration.
Yolk sac infections are one of the most common and most important histologic findings. These infections are typically acquired in one of three ways: in the oviduct of the hen before the shell is formed, immediately after lay (air is sucked in through pores in the shell as the egg cools, which can facilitate bacterial penetration), or during the pip and hatch stages when the umbilicus may be incompletely sealed.
Proper interpretation of gross and histologic findings requires their integration with clinical and management data at the individual case level, as well as the population level. Detailed egg necropsy protocols and sample egg necropsy request forms can be obtained by e-mailing the author at email@example.com
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2. Brown, A.F.A. 1979. The Incubation Book. Spur Publications Company, Saiga Publishing Ltd, Surrey, England.
3. Freeman, B.M. and M.A. Vince. 1974 Development of the Avian Embryo: A Behavioral and Physiological Study. Chapman and Hall, London (later reprinted by John Wiley and Sons, New York).
4. Hamburger, V. and H.L. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49–91.
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