Different incubation patterns affect selective antimicrobial properties of the egg interior: experimental evidence from eggs of precocial and altricial birds

Abstract

Avian eggs contend with omnipresent microorganisms entering the egg interior, where they affect embryo viability and hatchling phenotype. The incubation behaviour and deposition of egg white antimicrobial proteins (AMPs) vary highly across the avian altricialprecocial spectrum. Experimental evidence of how these alterations in avian reproductive strategies affect the antimicrobial properties of the precocial and altricial egg interior is lacking, however. Here, we tested the egg white antimicrobial activity in eggs of two representative model species, from each end of the avian altricialprecocial spectrum, against potentially pathogenic and beneficial probiotic microorganisms. Eggs were experimentally treated to mimic un-incubated eggs in the nest, partial incubation during the egg-laying period, the onset of full incubation and the increased deposition of two main egg white AMPs, lysozyme and ovotransferrin. We moreover assessed to what extent egg antimicrobial components, egg white pH and AMP concentrations varied as a result of different incubation patterns. Fully incubated precocial and altricial eggs decreased their antimicrobial activity against a potentially pathogenic microorganism, whereas partial incubation significantly enhanced the persistence of a beneficial probiotic microorganism in precocial eggs. These effects were most probably conditioned by temperature-dependent alterations in egg white pH and AMP concentrations. While lysozyme concentration and pH decreased in fully incubated precocial but not altricial eggs, egg white ovotransferrin increased along with the intensity of incubation in both precocial and altricial eggs. This study is the first to experimentally demonstrate that different incubation patterns may have selective antimicrobial potential mediated by species-specific effects on antimicrobial components in the egg white.

Partial incubation-induced changes in concentrations of egg white antimicrobials do not influence trans-shell infection but affect hatchling phenotype

“…In this study, we experimentally tested if partial incubation and clutch covering with feather nest lining during pre-incubation period affect concentrations of two principal egg white AMPs -lysozyme and avidin using precocial mallard eggs exposed in natural breeding habitat. Furthermore, as in our previous study 30 experimental increase of egg white AMPs concentrations enhanced in vitro antimicrobial activity of egg white against selected bacterial strains, we hypothesized that different concentrations of egg white AMPs will affect probability and intensity of bacterial trans-shell infection in vivo.…”

“…Lysozyme concentrations (mg/mL) for each egg white sample were interpolated from a calibration curve using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego California USA). Methods for measuring of lysozyme concentration is described in detail in 30 .…”

“…Eggshell pigmentation 20 , eggshell microstructure characteristics [21][22][23] , cuticle nanostructuring 24 , deposition of antimicrobial proteins into the eggshell structures [25][26][27] and nest material 8,10,28 have been found to significantly shape eggshell microbiota. Nevertheless, mechanisms reducing microbial trans-shell infection and proliferation of microbes inside the eggs are primarily linked with egg incubation 29,30 and the concentrations of abundant egg white proteins 31,32 , especially those having antimicrobial potential 30,33,34 .…”

“…While lysozyme has strong bactericidal activity against both G+ and G-bacteria 33,36 , avidin and ovotransferrin are rather bacteriostatic 33,37,38 due to their ability to reversibly bind biotin and iron thus making them unavailable for bacterial growth [39][40][41] . Howerver, despite documented antibacterial action of egg white AMPs, egg white proteomic profile may be significantly changed during incubation 42,43 leading to alterations in egg white antimicrobial potential 29,30,44,45 . For example, changes of egg white lysozyme and ovotransferrin concentrations were found to enhance proliferation of beneficial probiotic microorganism in egg white 30 .…”

“…Howerver, despite documented antibacterial action of egg white AMPs, egg white proteomic profile may be significantly changed during incubation 42,43 leading to alterations in egg white antimicrobial potential 29,30,44,45 . For example, changes of egg white lysozyme and ovotransferrin concentrations were found to enhance proliferation of beneficial probiotic microorganism in egg white 30 . Therefore, while incubation may significnatly shift eggshell microbiota 6,[45][46][47][48] , its selective antimicrobial effect inside the egg is most probably inherent in mediation of changes in chemical and proteomic profile of the egg white.…”