Background Collecting and studying live sperm is central to many important fields of biology. Yet,a simple method to collect live sperm is lacking in wild seabird species. Here,we describe a non-invasive method to collect viable sperm samples based on a simple massage technique applied to male Black-legged Kittiwakes (Rissa tridactyla).

Methods We studied a colony breeding at Kongsfjorden,Svalbard and successfully obtained sperm samples from 32 males. With a subset of samples (n = 12 males),we compared the suitability of several extenders (0.9% NaCl,PBS,Earle's balance salt solution,Dulbecco's modified Eagle medium) in maintaining sperm alive long enough for analyses. With another 18 ejaculates,we conducted computer assisted sperm analyses using the CASA plugin for ImageJ. We provide details about the settings to be used for such analyses. Lastly,droplets from 20 ejaculates were smeared on glass slides and preserved with formalin to characterize sperm morphology in terms of total sperm length,sperm head length,midpiece length and flagellum length,and percentage of abnormal sperm.

Results With this method and under field conditions,we were able to obtain sufficient amounts of live sperm to assess traits related to sperm quality (e.g. sperm morphology,percentage of motile sperm,sperm velocity). We found that two extenders,Earle's balanced salt solution and Dulbecco modified Eagle's medium,yielded similarly good results. Additionally,we investigated whether specific behaviours were associated with successful sperm collection and whether sperm collection success depended on how long before laying sperm collection was attempted. Finally,we provide mean values for sperm morphology,sperm swimming ability and percentage of motile sperm,which may prove useful for future comparative analyses,and we report high levels of sperm abnormality and within-ejaculate variation in sperm morphology.

Conclusions We discuss the high percentage of abnormal sperm and high within-ejaculate variation in sperm morphology in light of sperm competition theory and conclude that these figures are likely due to relaxed post-copulatory sexual selection,kittiwakes being strictly monogamous. Finally,we suggest that this method could be applied to other seabird species sharing similar ecology.