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 fertilisation rates are declining as many populations become smaller and more female-biased. Here, we first show that fertilisation rates are considered in only a very small proportion of studies of turtle and tortoise hatching success, and those studies that do attempt to measure fertilisation rates use unreliable methods. 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. We address the lack of reliable methods for assessing fertilisation rates in turtles and tortoises by developing and testing a microscopy-based method for detecting perivitelline membrane (PVM) bound sperm and embryonic nuclei in the germinal disc of unhatched eggs. We demonstrate that this method can provide unequivocal evidence of egg fertilisation in three different turtle and tortoise species from both captive and wild populations, even after eggs have been left in the nest for the full incubation period. This approach therefore represents a valuable new 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 populations.