Perivitelline Membrane-bound Sperm Detection: A Tool for Chelonian Conservation and Research

Endangered species propagation is an important component of wildlife conservation. Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, verify breeding or artificial insemination, and to evaluate male reproductive status. Due to similarities between avian and chelonian egg structure and development, 191 eggs representing 17 turtle and tortoise species were used to determine PVM-bound sperm presence and the potential for use in breeding management and research. Presumed fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were acquired to evaluate the effect of incubation and storage on the ability to detect sperm. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Mitochondrial DNA was isolated, amplified, and sequenced from freshly laid Astrochelys radiate, C. sulcata, and S. pardalis eggs exhibiting PVM-bound sperm. Sperm were positively identified for up to 206 days post-oviposition following storage, cooling, and/or incubation. Microbial infection frequently hindered the ability to detect sperm. Storage at -20 degrees Celsius was found to be the best method for preserving eggs prior to analysis. PVM-bound sperm detection is a promising tool for the reproductive management of both captive and wild populations of turtles and tortoises. Sperm-derived mtDNA has applications for species identification, parentage analysis, and the study of sperm competition. This project is the first to successfully demonstrate the presence and use of PVM-bound sperm detection in chelonian eggs.