The effects of injecting the Poulvac E. coli vaccine (PECV) into either the air cell (AC) or amnion (AM) at different dosages at 18 days of incubation (DOI) on Hy-Line W-36-layer embryo and hatchling development were investigated. Serial dilutions of the PECV in diluent provided either 6.5 × 104, 6.5 × 103, 6.5 × 102, or 6.5 × 101 CFU dosages of E. coli. A diluent only injection treatment was included as a control. A total of 19 live embryonated eggs in each of 10 treatment groups were represented on each of 16 replicate levels (3040 total) in the hatcher unit. At 19 DOI, swabs of the AM indicated that the 6.5 × 101 and 6.5 × 102 CFU dosages provided a 50% level of PECV presence, whereas the 6.5 × 103 and 6.5 × 104 CFU dosages provided a 100% level of PECV presence. Conversely, only the 6.5 × 103 and 6.5 × 104 CFU dosages provided a 50% level of PECV presence in the AC. At all E. coli dosage levels, injection in the AM led to higher percentages of live or dead embryos that failed to pip (PEIS) (p = 0.001) or complete hatch (PEPE) (p ≤ 0.001) and a lower percentage of live fully hatched chicks (HI) (p ≤ 0.001), when compared to those injected in the AC. Like HI, significantly lower percentages of female hatchlings were also observed at 22 DOI for the AM compared to the AC injection, for all dosages except for the 6.5 × 102 CFU dosage. However, at all the dosages above the 6.5 × 101 CFU dosage, the AM injection resulted in a lower mean hatchling body weight (p = 0.010) at 22 DOI. In conclusion, E. coli populations were more prevalent in the AM than in the AC after the injection of the PECV in those sites. Furthermore, the injection of the PECV in the AM at all E. coli dosages generally increased late embryo mortality and decreased hatchability and hatchling body weight in comparison to an AC injection. It is concluded that the negative impact of the in ovo administration of the PECV in the AM at 18 DOI on the hatch process is dose dependent. However, effects of an increase in AC dosages and a decrease in AM dosages should be further investigated.

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