Hatching failure in an aerial insectivore, the tree swallow Tachycineta bicolor, is driven by embryonic mortality, not infertility

Egg development is a crucial stage in the life cycle of birds, and across wild species up to 17% of eggs fail to hatch under normal conditions. Hatching failure is due to either infertility or embryonic mortality; however, methods to distinguish between these two causes have been underused, partly due to the assumption that eggs must be refrigerated and tested relatively quickly. In 2022 and 2023, we monitored 1090 eggs laid by tree swallows Tachycineta bicolor from 191 nests at three study sites in British Columbia, Canada. Using fluorescence microscopy to identify embryonic and sperm nuclei, we conservatively estimated that 98.3–100% of eggs were fertile, and most cases of hatching failure were therefore due to embryonic mortality, not infertility. Embryonic mortality was most likely to occur during early incubation, before development is usually macroscopically visible. We also used fluorescence microscopy to examine 114 eggs laid by tree swallows kept in long-term frozen storage and found that not only could fertility be assessed, but the fertility rate of these eggs was also close to 100%. To our knowledge, this study is the first to quantify fertility using embryonic nuclei in tree swallows, and provides the basis for further investigation into factors contributing to hatching failure in this species. Our success using frozen eggs for fertility testing is especially relevant for conservation groups and captive breeding facilities that may already have unhatched eggs stored in freezers, or are limited by capacity or funding and need to place eggs in long-term storage until resources are available for testing.