Temperature-induced changes in egg white antimicrobial concentrations during pre-incubation do not influence bacterial trans-shell penetration but do affect hatchling phenotype in Mallards

Microbiome formation and assemblage are essential processes influencing proper embryonal and early-life development in neonates. In birds, transmission of microbes from the outer environment into the egg’s interior has been found to shape embryo viability and hatchling phenotype. However, microbial transmission may be affected by egg-white antimicrobial proteins (AMPs), whose concentration and antimicrobial action are temperature-modulated. As both partial incubation and clutch covering with nest-lining feathers during the pre-incubation period can significantly alter temperature conditions acting on eggs, we experimentally investigated the effects of these behavioural mechanisms on concentrations of both the primary and most abundant egg-white AMPs (lysozyme and avidin) using mallard (Anas platyrhychos) eggs. In addition, we assessed whether concentrations of egg-white AMPs altered the probability and intensity of bacterial trans-shell penetration, thereby affecting hatchling morphological traits in vivo. We observed higher concentrations of lysozyme in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg-white AMP concentration and we observed no association between concentration of egg-white lysozyme and avidin with either the probability or intensity of bacterial trans-shell penetration. The higher egg-white lysozyme concentration was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds can alter concentrations of particular egg-white AMPs, though with no effect on bacterial transmission into the egg in vivo. Furthermore, a higher egg white lysozyme concentration compromised hatchling body condition, suggesting a potential growth-regulating role of lysozyme during embryogenesis in precocial birds.