Comparing Methods to Quantify Sperm Storage in Female Blue Crab (Callinectes sapidus) Spermathecae

Potential sperm limitation has become a concern for blue crabs Callinectes sapidus in Chesapeake Bay. However, methods to quantify sperm in blue crabs are time intensive, which limits the sample size that can be obtained for sperm limitation studies. Our goal was to develop an efficient method for rapidly preparing spermathecae samples that more quickly quantifies sperm in blue crabs. Ultimately such a method will help address questions of sperm limitation in the Chesapeake Bay. In this study, we tested two methods for grinding spermathecae, and we tested whether DNA fluorometry is an accurate method for determining sperm quantity in blue crabs. We processed spermathecae using a dounce homogenizer and a Polytron, and compared sperm counts using a paired t-test. We compared fluorometry with direct counting of sperm cells under a microscope, a more time consuming yet already tested process, to determine if the sperm quantities obtained from fluorometry correspond with sperm counts. Samples processed using the dounce homogenizer had significantly higher sperm counts (p=0.007). Fluorescence was negatively related to sperm count (p=0.003), which was opposite the expected pattern. Our results indicate that the dounce homogenizer should be used to process samples and that the fluorometry methods we tested should not be used to quantify sperm in blue crabs.