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When performing ovo vaccination, the vaccine delivery at the right site is paramount to succeed because vaccines will behave differently according to which compartment inside the egg they are delivered into. Therefore, embryo development assessments are a key control factor in the vaccination efficacy monitoring. For successful field protection, target locations are intra-embryo or amniotic fluid. See more about the factors impacting the right injection site.

NATIVE_Ceva_Egg diagram

Last days of incubation

During the last days of incubation significant changes in embryo metabolism and positioning take place. Progressively, the yolk is internalised into the abdomen, the existing amniotic fluid is swallowed, and the embryo positioning changes from the head located between the legs, to the hatching position with the head under the right wing. As a consequence, the proportion of the different compartments inside the eggs varies and, depending on the in ovo injection time, the probability of delivering the vaccine into one compartment or another also changes. 

Optimum embryo development and flock homogeneity at in ovo injection time

To maximise in ovo vaccine delivery in target protective locations, the optimum injection time is 18.5-19.0 days of development (Mean >18.5), being a range between 18-19 days of development suitable for in ovo. 

At this range, intra-embryo and/or amniotic locations are maximised ensuring field protection. Embryos out of the mentioned range of optimum development, either below or above will have a higher risk of poorly located vaccination. Below range, there will be a higher risk of vaccine delivered into the allantoic fluid. Above range, there will be a higher risk of delivering vaccine into the feathers and skin. Neither location is considered as protective for the vaccination efficacy.


Producers should also note that, in ovo vaccination is conducted in a population of embryos, therefore flock embryo development homogeneity is critical as well.

Flocks with the same embryo development mean might have different distribution curves and it will influence the percentage of embryos out of the recommended embryo development range. In other words, the higher the heterogeneity of embryo development within a population, the higher the risk of delivering vaccines in an undesirable and not protective location. Therefore, embryo development homogeneity is key to ensure vaccination efficacy. 

Incubation hours and physiological development 

Many factors influence the incubation hours needed to reach the recommended physiological development at injection time. For example, 18.5 days of incubation does not necessarily result in embryos with 18.5 days of development because it depends on the type and breeder flock age, single or multi stage incubators, incubation parameters, hatching egg storage time and conditions, among others. Consequently, in ovo vaccination timing should be ruled by the physiological development of the embryos, not necessarily by the incubation hours given. 


Ensuring vaccine delivery at the right embryo site is paramount when performing in ovo vaccination and it depends on the embryo development and flock homogeneity at injection time. While the embryo development average will define in ovo vaccination timing, flock homogeneity will define the in ovo vaccination quality. This is why regular embryo age diagnosis as done by the regular hatchery visits of the C.H.I.C.K Program during in ovo vaccination is a critical control point to monitor the efficacy of the process and consequently the protection of flocks in production.