Non-fluorescent detection of perivitelline membrane-bound sperm in Gavialis gangeticus eggs

The gharial ( Gavialis gangeticus ) is a critically endangered crocodilian endemic to India and Nepal. Population declines in recent decades have necessitated the establishment of captive breeding and reintroduction programs. Unfortunately, ex-situ reproduction remains low. Due to their size and behavior, hands-on efforts to determine fecundity (eg., ultrasonography, sperm collection and analysis) can be difficult to implement in gharials. Perivitelline membrane (PVM)-bound sperm has been visualized within chelonian and crocodilian species as a method for confirming copulation and fertilization attempts. Here, we opportunistically collected five non-viable eggs from a female gharial within the Fort Worth Zoo harem and assessed them for the presence of sperm within the PVM. Eggs were opened, the PVM was gently removed from around the yolk of each egg, and sections of the PVM were assessed using a phase contrast microscope. The presence of sperm in all five eggs was confirmed. Furthermore, sperm presence was visualized without the use of fluorescent staining, demonstrating that costly fluorescent microscopy methods are not necessary for assessing sperm presence in the PVM of gharial eggs. The female in this study had never produced a viable egg while at the Fort Worth Zoo, and it was unknown whether this female was infertile or lacked mate compatibility with the resident male. While the ultimate cause of lack of viability of this female’s eggs could not be determined, these results confirm successful copulation and fertilization attempts – a first step towards assessing infertility in captive gharial females.