Characterisation of European eel (Anguilla anguilla) spermatozoa morphometry using Trumorph tool in fixed and non-fixed samples

Currently, the techniques used to analyse fish spermatozoa head morphology are not only subjective and time-consuming, but also, variable due to alteration of real spermatozoa size during sample manipulation. Thereby, it is important to develop a new method to obtain accurate and objective results. So far, computer-assisted systems for morphometry analysis were developed and validated for mammals, although they could also be adapted to fish spermatozoa. The present study aimed to characterise the European eel spermatozoa morphometry, comparing the measurements (area and perimeter) obtained by computer-assisted spermatozoa between pre-treated or non-treated eel sperm samples (n = 5) with fixation solution. In both protocols, the TruMorph® tool was used to apply a constant force to extend the cells in a thin layer. Images of spermatozoa were captured using a 40× negative phase contrast objective and analysed with the ISASv1 CASA-Morph system. Sperm head morphometry showed significant differences in area and perimeter comparing both protocols. Besides, the head size analysis using TruMorph® without fixation along time showed that the sperm membrane remains intact with the use of this technique, preserving the semipermeable condition. Considering the fact that fixation solution produces dehydration/hydration that could affect the real spermatozoa size, the simple use of TruMorph® without fixation combined with CASA-Morph analysis could allow more realistic measurements of eel spermatozoa head.