Detailed Sperm Morphometry Protocol

Sperm samples were directly recovered from the cauda of both epididymides. Spermatozoa were fixed in glutaraldehyde solution. A sub-sample of 2 µl was used to prepare the smears. Semen smears were air-dried for one day, then immersed in the glutaraldehyde fixative solution for 5 min and immediately mounted, sealing the edges with dibutyl phthalate xylene (DPX). This method avoids floating cells on the slide (Figure 1A), which greatly helps sperm morphometry analysis. Sperm samples were photographed using a high-resolution camera DXM1200 (Nikon, Tokyo, Japan) under a phase-contrast microscopy using an Eclipse E600 microscope (Nikon, Tokyo, Japan), and a 40X objective (Nikon, Tokyo, Japan). The resolution of the pictures was 3840×3072 pixels (TIFF format). A scale of 10 µm (181 pixels) was used for the measurements. The pixel size was 0.055 µm in the horizontal and vertical axes. Sperm lengths were assessed using ImageJ software (National Institutes of Health, USA). The main structures of red deer spermatozoon are shown in Figure 1B. The following sperm morphometry parameters were determined: head width, head length, proximal midpiece width, distal midpiece width, midpiece length, flagellum length, and terminal piece length (Table S1). From these measurements, we calculated other morphometric parameters such as total sperm and principal piece lengths. The head area was calculated using the formula for the area of an ellipse [31] (link), [32] :
Head perimeter was calculated using the Ramanujan's formula for calculating the perimeter of an ellipse [33] :
In both formulae, L and W are the semi-major and semi-minor axis of the sperm head, respectively. Twenty-five representative sperm were measured for each male as described by Malo et al. [16] (link).