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Mycoplasmas are pathogens of different avian species, and they are able to be vertically transmitted. Even detected, Mycoplasma prevalence in raptor eggs is very low. In contrast to poultry, raptor eggs submitted for investigations are usually incubated. To investigate the influence of incubation length on the recovery of mycoplasmas from eggs, infertile specific-pathogen-free chicken eggs and embryos were infected with Mycoplasma lipofaciens (strain ML64), which had previously been isolated from an egg of a northern goshawk (Accipiter gentilis), in two different dosages. The eggs were investigated up to 12 days after infection (infertile eggs) or embryonic death. Mycoplasmas were recovered over the entire period after embryonic death by isolation. It was possible to re-isolate M. lipofaciens (strain ML64) from infertile eggs infected with 10(6) colony-forming units (CFUs) up to 12 days, but only up to 7 days if infected with 10(2) CFUs, which may be closer to the situation after natural infection. This study demonstrates that incubation of infertile eggs does have an influence on the recovery rate of mycoplasmas. This influence must be considered if interpreting results of Mycoplasma investigations in eggs of nonpoultry species. Additionally, it is recommended to use dead in shell embryos rather than infertile eggs for Mycoplasma detection.