Nearly (?) sterile avian egg in a passerine bird

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity and survival in vertebrates. Bird egg has been suggested to be either (1) initially sterile ( Sterile egg hypothesis ) and (2) colonized through horizontal trans-shell migration after egg laying, or (3) initially seeded with bacteria through vertical transfer from mother’ ss oviduct. Little empirical data illuminate so far the contribution of these mechanisms to gut microbiota formation in avian embryos. We evaluated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit ( Parus major ), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that avian egg is (nearly) sterile , barely distinguishable in microbial composition from negative controls. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in controls), E13 gut and female samples, which might indicate its possible vertical transfer . Unlike in poultry, we have shown in passerines that major bacterial colonisation of the gut does not occur before chick’s hatching. We stress that protocols carefully checking for environmental contamination are critically important in studies using samples with low bacterial biomass.