Understanding early reproductive failure in turtles and tortoises

Turtles and tortoises (Order Testudines) are facing an extinction crisis, and ecosystems are at risk of collapsing with the loss of key roles they play. Hatching failure is a crucial barrier to population growth and persistence, but its causes are poorly understood, and it is unknown whether fertilization rates are declining as many populations become smaller and more female-biased. Here, we show that very few studies of turtle and tortoise hatching success consider fertilization rates, and those that do use unreliable methods to determine egg fertility. We also show that studies of hatching success are biased towards marine turtles, as opposed to freshwater and terrestrial species, and wild rather than captive populations. To address the lack of reliable methods for assessing fertilization rates in turtles and tortoises, a microscopy-based method (originally designed for bird eggs) for detecting perivitelline membrane (PVM) bound sperm and embryonic nuclei in the germinal disc of unhatched eggs has been developed and tested (in turtle and tortoise eggs). We demonstrate that this method provides unequivocal evidence of egg fertilization in five different turtle and tortoise species from both captive and wild populations, even after eggs have been left in wild nests for the full incubation period. This methodological approach represents a valuable tool for monitoring egg fertility and embryo survival rates in turtles and tortoises, with the potential to provide important insights into the underlying drivers of reproductive failure in threatened captive and wild populations.