In ovo vaccination

In ovo vaccination all information on the History, Vaccination Timing with Embryonic Development, Sites for Injection, Immune Response, & Benefits.


Although the in ovo vaccination has been widely used in many countries all over the world for over 20 years, many questions still surround this technology. 

To achieve the best of in ovo vaccination not only a good machine is essential. To understand the relation between chick quality and in ovo injection, it is important to realize the factors directly influencing in ovo application. Some of those factors have expressive influence in the final expected result: a good and effective vaccination. 

Chick Quality and in ovo injection are intrinsically related when we discuss many factors such as: Embryonic age and hatchability, site of injection, vaccine preparation and immune response, hatchery hygiene and sanitation.

In ovo vaccination history

The in ovo vaccination was first proved to be efficient for Marek’s vaccine by experiments performed by Sharma and Burmester in 1982. These authors demonstrated that chicks vaccinated in ovo at 18 days of embryo development against Marek’s Disease had better protection against virulent MD challenge carried out at 3 days of age as compared to those chicks vaccinated at hatch. At seven days of age, the both groups (vaccinated at hatch or in ovo) induced equivalent level of protection. 

At that time in ovo vaccination was only a new idea, but today it is being used in commercial applications worldwide in the poultry industry. The basic laboratory concept of this vaccination, initially used for vaccination against Marek’s disease, has expanded and today we can find machines capable of inject up to 60,000 eggs per hour.

Embryonic development

To know the stage of development of the embryos is essential to determine the correct time to achieve the best hatchability and to perform in ovo injection. Moreover, embryonic development is directly associated to many aspects of the incubation process. See our Embryonic Development page  for more information.

Before in ovo injection is taken into consideration, it is important to remember that many studies have shown that eggs transferred too early (around day 17 of incubation) from Incubators to Hatchers tend to have their hatchability reduced when compared to eggs transferred later (day 18 or 19), whether they are injected or not. The differences (water loss, humidity, heat and gas exchange) in the environment of these two types of equipment are the main responsible for differences in hatchability.

Normally, a positive effect on hatchability is observed if the embryo remains in the incubator for longer time, and is transferred to the hatchers later. Also, difference in embryonic development may be observed in different types of incubators due to their different water loss and temperature standards, and to determine the correct time for in ovo injection - to achieve maximum hatchability and good immune response against diseases - the physiological characteristics of the embryo are much more important to be considered than the number of hours of incubation.

The optimal timing for in ovo vaccination is at 18.5 days of embryo development. However, there is a certain range of development which might be acceptable. The earliest suitable stage, is defined when the embryo is in hatching position (head under the right wing) and the stalk of the yolk sac starts entering the abdomen; with a mature enough yolk sac, with no visible intestines in its the stalk. The latest suitable embryo development stage – 19.2 days – is defined by internal pipping and pulmonary respiration, being limited by a maximun of 2% of pipped eggs. 

Sites of injection

We can consider that there are 5 basic compartments on an egg during the final stage of incubation: Air Cell, Allantoic sac (waste), Amniotic sac, the Embryo itself and the Yolk sac.

in ovo vaccination
  • The air cell which is basically filled with gas.
  • The allantoic sac which is filled with fluid containing embryo development by-products.
  • Amniotic sac which is composed by amniotic fluid and the embryo body.
  • The embryo itself which is located inside the amniotic sac.
  • The yolk sac which is also inside the amniotic sac

Any one of those compartments can be accessed by the needle of the machine. However, in order to achieve and maximize immune response by in ovo vaccination, it is essential to assure that the correct compartment inside the egg is accessed.

Due to its fast development in this last stage, specially when we consider the in ovo injection window (as mentioned before, between 17.5 and 19.2 days of incubation), it is important to realize that these compartments can change fast as they are utilized by the embryo. It is also essential to recognize that the specific compartments are responsible for different support during the development of the embryos, and the placement of vaccines and/or other compounds into those compartments may allow or limit their absorption by the embryo.

The position of these compartments inside of the egg, and more specifically, the position of the embryo depend on its stage of development.

Immune response

Scientists of the University of California at Davis (Wakenell et al., 2002) conducted some trials in order to evaluate the ability to induce immune response when a MD vaccine is delivered in different compartments of the egg (Fig. 1).

Immune response new 2

The results have shown that the deposition of MD vaccine in the air cell or the allantoic sac does not induce effective immune response. Those studies reported that MD vaccine injected into the air cell resulted in no protection of the bird while injection in the allantoic sac induced only 28.3% of protection. Injection in the amniotic fluid or in the embryo body resulted in about 94% protection of the vaccinated birds.

In conclusion, in order to be able to induce effective immunity against the Marek’s Disease, from those 5 basic compartments described above, only 2 of them have the ability to promote positive immune response: the amniotic fluid and/or the embryo itself. The vaccine must be delivered deep enough inside the egg to reach the amniotic sac (amniotic fluid and embryo). 

It is expected that injections in the initial stage of the in ovo injection window (closer to 18 days of incubation), most of the eggs would be normally injected in the amniotic fluid. When the vaccine is delivered in the amniotic fluid, the substance will be swallowed by the embryo prior to hatch. On later injections (around 19 days of incubation), as the embryo is more mature, a bigger percentage of embryos would receive the injection in their body (expected to happen in the right breast). This injection in the embryo body is normal and acceptable, however excessive depth penetration can result in embryo damage.

As noted, the site of injection is a critical point to induce effective immune response and precision is needed to allow the vaccine to be delivered in the correct site inside the egg, therefore resulting in effective vaccination.


• Safe, consistent and accurate method of vaccine application
• Significant labour costs reduction vs subcutaneous or field vaccination
• No Post-vaccination reaction
• Improvement of the chick quality of your flocks
• Early access to food and water. Day-old-chicks are not stressed after hatch
• Better development of the immune system at hatch.

In ovo vaccination can be used for Marek's disease,  Gumboro disease Newcastle disease  or  Avian Influenza disease .


In ovo injection has become an important tool to administer vaccines in the hatcheries. As it was aforementioned, everything started around 25 years ago with Marek’s Disease vaccine and today there are several other products to be injected through this equipment. Products like Transmune®, Vectormune® IBD,  Vectormune® ND,  Vectormune® AIAvian Influenza are already commercially available and many others are being developed, clearly demonstrating an even brighter future for this technology.

Nevertheless, some basic pre-cautions must be taken into account in order to achieve the best results with this tool. Good sanitation of the hatchery, proper disinfection of the hatching eggs are among these special cares. Furthermore, precise maintenance of the machine is compulsory. By ensuring that these procedures are properly followed, companies can sure benefit from this interesting technology.


  • Avakian, A., P.S.Wakenell, T.Bryan, J.L.Schaeffer, C.J.Williams, and C.E.Whitfill, 2002. In ovo Administration of Marek’s Disease Vaccine: Importance of Vaccine Deposition Site in the Fertile Egg. Proceeding of the 51st Western Poultry Disease Conference, May 1-4, 2002, pp.119-121, Puerto Vallarta, Mexico.
  • Meijerhof, R., “Maximising in ovo vaccination success”
  • Phelps, P., “The in ovo Administration of Antibiotics into Broiler Eggs at Transfer”, Ph.D Dissertation, North Carolina State University, 1995.
  • Sharma, J.M and Burmester, B.R., Resistance to Marek’s Disease at hatching in chickens vaccinated as embryos with the turkey herpesvirus, Avian Diseases, v. 26, p. 134-149, 1982
  • Wakenell, P.S., T.Brian, J.Schaeffer, A.Avakian, C.Williams, and C.Whitfill, 2002. Effect of In ovo Vaccine Delivery Route on Herpes Virus of Turkey/SB-1 Efficacy and Viraemia. Avian Disease 46(2); 274-280.
  • Williams, C.J., “In ovo Vaccination and Chick Quality”, International Hatchery Practice – Volume 19 Number 2.
  • Williams, C.J., “In ovo vaccination for disease prevention”, Pfizer/Embrex Web Home Page

Find out more about Egginject in ovo vaccination system here