 straws. At this point, sperm quality was evaluated again. Finally, they were frozen in a programmable biofreezer (IceCube 14S ver. 1.30; SYLAB Geräte GmbH, Neupurkdersdof, Austria) at –20°C/min to –100°C, and at –10°C/min from –100°C to –140°C. Frozen semen was stored in liquid nitrogen (–196°C) for a minimum period of 6 months before thawing. Thawing was performing by dropping the straws in a water bath with saline serum solution at 37°C for 20 s.
After thawing, sperm quality was assessed as for the two previous stages. The assessment took place just after thawing and after 2 hours of incubation at 37°C. The assessment of sperm viability of thawed semen was performed by evaluating sperm membrane stability by flow cytometry using the combination of YO-PRO-1 and propidium iodide .
Assessment of Sperm Head Morphometry
For each step of the cryopreservation process, sperm head dimensions were assessed objectively by using the morphometric module of the Sperm-Class Analyzer® (SCA) (Microptic, Barcelona, Spain). Sperm morphometry analysis was performed as described by Maroto-Morales et al. . Briefly, microscope slides were prepared by placing 5 µl of sperm diluted in PBS on the clear end of a frosted slide and dragging the drop across the slide. Semen smears were air-dried and stained using the commercial kit Hemacolor™ (Merk, cat. No. 11661, Darmstadt, Germany). Then, stained samples were permanently mounted to the slide with a coverslip and dibutyl phthalate xylene (DPX). A total of 200 images were acquired per ejaculate to ensure a minimum of 125 properly measured sperm heads after improperly measured heads were removed from the analyses.
Morphometry traits assessed were: sperm head length (L, µm), width (W, µm), area (A, µm2), perimeter (P, µm), and p2a (perimeter to area; Perimeter2/4·π·Area) shape factor. The latter compares the perimeter of an object with its area. The p2a takes a minimum value of 1 when the object is a circle and increases its values for more elongated objects.
To examine the changes that occur on sperm head dimension throughout the cryopreservation process, the five sperm head morphometry parameters described above were used. However, we focused mainly on three sperm head measures, the sperm head length, sperm head area, and the p2a sperm head factor when displaying these differences. These three parameters have been shown to be sufficient to describe the dimensions of the sperm head in a precise way . Coefficients of variation (CV) among ejaculates were calculated to evaluated the within-male (between ejaculate of the same male) variability.
For the recognition of the different patterns of response, an initial graphical exploration of the sperm population head dimensions throughout the cryopreservation process were performed. Scatter plots of the sperm head area against the sperm head length and p2a were drawn for each male and each stage of the cryopreservation process (see Figures S1 and S2). Three different patterns of response were observed. Males were assigned to one of these patterns based on the following rules: if no significant differences were observed among the stages of the cryopreservation process (fresh, prefrezee and post-thaw) for the 3 sperm head morphometry parameters listed above males were assigned to response pattern 1; if sperm morphometry significantly differed for the three stages, males were assigned to response pattern 2; and if head morphometry in fresh semen was significant different from the other to stages, being the latter similar, males were assigned to response pattern 3. For the particular case in which a male did not meet these requirements for the three morphometric parameters, it was assigned to the closest group. For that, graphical visualization of data was considered as well (see Figures S1 and S2).
Once males were assigned to one of the different pattern of response, multiple comparisons between different stages of the cryopreservation process within each response pattern, and between different responses patterns within each cryopreservation stage were carrie