Gamete buffer

20 MII oocytes from each experimental group were stained with 0.2 mM of Mito Tracker Green fluorochrome for 5 min at 37°C (42 (link)). After washing them three times by gamete buffer (Cook, Bloomington, Indiana‎, USA), the oocytes were placed in glass bottom cell culture dishes and were observed under an inverted fluorescence microscope with 490 nm filters. After 3 replications, the fluorescence intensity of the 40 oocytes was quantified and analyzed by Image J software.

After collection by masturbation, sperm samples were liquefied at room temperature for 30–60 min. Semen volume, sperm concentration and motility were evaluated following the WHO guidelines [1 ]. The SpermFreeze™ medium (FertiPro NV, Beernem, Belgium) was added to the sperm in drops while gently swirling (0.7 mL of medium per ml of sperm). During the 10 min incubation for equilibration at room temperature, the mixture was sucked into the CBS High Security sperm straws (Cryo Bio System, L’Aigle, France) that were placed in liquid nitrogen vapor phase for 15 min. Then the straws were quickly transferred to liquid nitrogen and stored at −196 °C for one week.
To thaw, the straws were removed from the liquid nitrogen and warmed at room temperature until the sample thawed. Then the end of the straw was cut off and the semen–medium mixture was put into a tube containing 1 mL of Gamete buffer (Cook Medical) prewarmed at 37 °C. After centrifugation at 1400 rpm for 10 min the pellet was transferred into the QIAzol^® Lysis Reagent (Qiagen) and processed as described below.
We checked the percentage of round cells in our samples, and it was very low, with an average 5% (range 0–20%) of the sperm count.

Oocytes were vitrified using commercial kits (Irvine Scientific CA) and closed vitrification devices (CryoTip). The oocytes were washed in a drop of hepes-buffered medium (gamete buffer Cook) and, subsequently in three drops of equilibration solution containing ethylene glycol (EG) (7.5% vol/vol) and dimethylsulphoxide (DMSO) (7.5% vol/vol). After 8 minutes, eggs were transferred into a vitrification solution drop containing 15% vol/vol EG, 15% vol/vol DMSO, and 0.5 mol/L sucrose, for a total of 20 seconds before being loaded into a Cryotip and sealed properly at both ends. The device was directly plunged into liquid nitrogen and stored.
Oocytes were rapidly warmed by transferring the Cryotip directly from liquid nitrogen to a 37°C water bath for 3 seconds. The device was cut at the end and the oocyte/s released in a thawing solution (1.0 mol/L sucrose) for a minute then moved to a dilution media (0.5 mol/L sucrose) for 4 minutes and finally washed twice in a washing solution (6 minutes). Survived oocytes were cultured in Cleavage medium (Cook) before the fixation.