Determination of the injection deposition site in chicken embryos by a novel in ovo injection system

Lozano F.1; Gonzalez C.1; Arbe M.2; Hernandez I.2; Felfoldi B.3; Medveczki A.3; Jaouen E.1; Visagie C.4; Kertesz K.3; Palya V.3; Gardin Y.1 - Ceva Animal Health – France1; Spain2; Hungary3; South Africa4


A novel in ovo vaccination method with dual pressure injection system  (EGGINJECT®) was used to evaluate under field-like conditions the in-ovo injection deposition site in the chicken embryo compartments at three different Embryo Development Levels (EDL) (17.5; 18.5 and 19 days of incubation) using three different broiler breeder hen ages (28; 45 and 56 weeks of age) for each EDL evaluated. Embryonating chicken eggs were randomly distributed among the 9 treatment groups designed in a 3 by 3 table design study. A total of 4,050 embryonating eggs were evaluated for the injection deposition site, conducted by three different teams, assigning randomly egg trays (holding 150 embryonating eggs each) to each team for each one of the three EDLs corresponding to each breeder hen age. Each treatment group (breeder age and EDL) had 450 eggs evaluated. The injection deposition site classification in this study was divided into three embryo compartments: Air chamber/Undefined, Intra-embryonic and External-embryonic deposition. The depositions observed in the air chamber/Undefined site were nil (0.4%). The intra-embryonic plus external-embryonic deposition sites at 19; 18.5 and 17.5 days of incubation were: 99.60%; 99.52% and 99.68% respectively.  A logistic model analysis for the independent variables, EDL and breeder flock age, was used for the evaluation of the embryo deposition site.


The objective of this study was to determine the in-ovo injection deposition site of an innovative in-ovo injection equipment using eggs from three different broiler breeder flock ages and at three different incubation schedules: 17.5; 18.5 and 19 days.

Materials & Methods:

In-Ovo and Hatchery Equipment: EGGINJECT® in-ovo injection equipment. Setter: Petersime B 9870; Model Pet 168-I; Hatcher: Petersime Model Pet 84-H.

Fertile Eggs: Fertile eggs for this study were supplied by Babolna-Tetra located in Hungary. All eggs were from the same standard broiler breeder type (Ross 308) and were grouped according to flock age. Three different flock ages were used: Young age flock (YF) 28 weeks of age; Medium age flock (MF) 45 weeks of age; Old flock (OF) 56 weeks of age.

Incubation time: Fertile eggs were incubated in three different time-frames: 17.5 days; 18.5 days; 19.0 days. Embryo development level (EDL) was evaluated previously to in-ovo vaccination.

Egg storage:

Eggs were stored for a maximum of 3 days before delivery to the hatchery, where they were randomly distributed by treatment group and properly identified by age and by assigned incubation time group in separated trolleys. Any egg showing any of the following conditions was removed from the tray, such as: Egg-shell malformation; abnormal egg shape; micro-cracks or any visual crack on the egg-shell; dirty egg; contaminated egg; too small/big egg in relation with the eggs in the tray.

In-Ovo Deposition Site definition:

The in-ovo deposition sites were classified into three categories: (1) Air chamber; (2) Intra-embryonic and (3) External-embryonic depositions.

In-Ovo Injection Procedure:

Incubated eggs were manually candled right before injection to remove unfertile eggs and early dead embryos. The empty spaces were backfilled with spare eggs from the same age and incubation time groups. All groups were injected by using a mixed solution preparation: Saline solution (0.9% NaCl) - 70%; Ceva Blue Dye - 30%. Injection was performed by the calibrated EGGINJECT® equipment. A quality control plate was used before injection to validate that 100% of the injectors were disposing the right dose of the injection mix (0.05ml +/- 5%).

Article table 1

 Table 1. Treatment group distribution and coding

In-Ovo Deposition Site Operational Process:

Immediately after in-ovo Injection with the Blue Dye solution, all eggs were processed in a break-out session. A randomized egg tray distribution of the break out session was conducted by three embryo examination teams, so each team had only one tray per treatment group to ensure a more objective evaluation of the in-ovo injection deposition site.


Evaluation of the Embryo Developmental Level before in-ovo injection: The EDL observed in the longest incubation period (19 days) showed more variability and lower development than the other incubation times. The EDL observed in the treatment groups with the shortest incubation time before injection (17.5 days) showed more development in the young breeder flock. The EDL in the 18.5 days incubation time were very close to the assigned incubation time.


Article figure 2

Figure 2. Average Embryo Development Level (EDL) obtained for each treatment group.  

Article figure 3

Table 2. Percentage of the injection deposition sites for the total trays examined by treatment group.                                      

The results of the injection deposition sites in percentage per tray and per treatment group are presented in Table 2. The total average of depositions in the egg Air Chamber/Undefined was minimal (0.4% total). The combined injection deposition sites Intra-embryo with External-embryonic at 19 days EDL was 99.60% total average; 99.52% average at 18.5 days EDL and 99.68% total average at 17.5 days EDL.


The injection deposition site analyzed in this study using a novel in-ovo vaccination method with dual pressure injection system (EGGINJECT®) was evaluated considering two independent variables, Embryo Development Level (EDL) 17.5; 18.5 and 19 days of incubation and broiler breeder flock age: Young (28 weeks old), Middle age (45 weeks old) and Old breeder flock (56 weeks old).  The combined intra-embryonic and external-embryonic deposition sites at 19; 18.5 and 17.5 days of incubation were: 99.60%; 99.52% and 99.68% respectively.  A logistic model analysis for the independent variables, EDL and breeder flock age, was used for the evaluation of the embryo deposition site.


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