on a scale of 0–5, where 0 is no motility and 5 is vigorous progressive movement.
Acrosomal integrity after cryopreservation is a well-accepted parameter for estimating sperm function and response to cryopreservation , , especially after relatively long incubation periods to record changes in plasma membrane sensitively . For the assessment of the percentage of spermatozoa with intact acrosomes, 10 µl of sperm suspension in PBS were diluted with 50 µl of a 2% glutaraldehyde in 0.165 M cacodylate/HCl buffer (pH 7.3), and examined by phase-contrast microscopy. A total of 200 spermatozoa were acquired per ejaculate. Percentages of acrosome integrity were recorded.
Sperm viability was assessed in sperm smears stained with eosin-nigrosin. Thus, 5 µl of sperm suspension with PBS and 10 µl eosin-nigrosin solution were mixed on a glass slide placed on a stage at 37°C and 20 s later the mix was smeared and allowed to air-dry. Smears were examined at ×1000 under bright field and 200 spermatozoa per male were examined to evaluate sperm viability. Live spermatozoa were those excluding eosin (from the eosin-nigrosin stain).
Only those sperm samples with a minimum quality (percentage of motile sperm above 80% and quality of movement above 3.5) were cryopreserved, as a way to assure that all sperm samples showed good quality before freezing. If an ejaculate did not meet the requirements of quality was discarded and the male was recollected again to complete the three semen collections. Cryopreservation was performed as described by García-Alvarez et al. . Briefly, fresh semen was diluted with freezing extender Biladyl® (Minitüb, Tiefenbach, Germany) with 20% egg yolk and an osmolarity balanced at 310 mOsm/kg for the Fraction A (non-glycerol fraction). Semen was diluted to 400×106 spermatozoa/ml with Biladyl®, Fraction A, at 30°C. Diluted semen was cooled to 5°C for 2 h and then was further diluted with the same volume of Biladyl®, Fraction B. Sperm samples were allowed to equilibrate at 5°C for 2 h and packed in 0.25 ml plastic 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 occu