2.3. Spermatophore and sperm quality
The spermatophore and sperm quality of the males in the intermolt stage were measured at the beginning (day zero) and at the end (day 30) of the experiment. Both spermatophores from each male were manually extruded (Nakayama et al., 2008b), and one was randomly selected and weighed to the nearest 0.001 g. The spermatophore was then homogenized in 2 mL of calcium-free saline solution. Sperm counts were estimated by counting the cells present in the resulting sperm-saline solution using a hemacytometer under a light microscope according to the method described by Leung-Trujillo and Lawrence (1987). Abnormal cells identified by malformations of the main body or the absence of
the spike were counted, and the percentage of normal sperm was estimated. Melanization were checked by visually examining the coxae of the fifth pereiopod pair and the extruded spermatophore