Embryos from 30 superovulated Santa Ines ewes were collected 5–7 days after laparoscopic artificial insemination. Embryos were recovered by surgical procedure (laparotomy followed by flushing of the uterus horns). The obtained morulae and blastocysts were selected and classified according to the International Society of Embryo Transfer (IETS) [32]. Grade I and II embryos were washed in Phosphate Buffered Saline (PBS) plus 20% fetal calf serum (PBSS), maintained

in holding medium (Holding Plus®, Vitrocell, PI3K Inhibitor Library São Paulo, Brazil) at 36 °C and protected from light until cryopreservation or fixation. Grade I and II embryos were divided into three groups: slow freezing (n = 22), vitrification (n = 24) and control (n = 33). Embryos were randomly

distributed, but always maintaining similar Trametinib cell line numbers of blastocysts and morulae, and Grade I and II embryos in every group. Fresh embryos (control group) were immediately evaluated for mitochondrial activity and cytoskeleton structure by confocal microscopy and for ultrastructure by transmission electron microscopy (TEM). Grade III embryos were not cryopreserved. Some were processed only as controls, both for mitochondrial activity and cytoskeleton structure (n = 3) and for transmission electron microscopy (n = 2). Slow freezing was performed using the protocol of Garcia-Garcia et al., [19] with a slight modification on the freezing program, which missed the third cooling ramp (0.1 °C/min from −30 to −35 °C). All cryoprotectant solutions were prepared in PBSS. Initially, embryos were equilibrated in 0.75 M EG for 10 min and then placed for a further 10 min in 1.5 M EG at 32 °C. One to four embryos were loaded into each 0.25 mL straw. Afterwards, the straws were placed in a controlled-rate freezer (Dominium K, Biocom, MG, Brazil) at 10 °C and immediately cooled

at 1 °C/min to −7 °C and then manually seeded. After 5 min at −7 °C, embryos were cooled at 0.3 °C/min to −35 °C. After Branched chain aminotransferase 10 min at −35 °C, the straws were immersed into liquid nitrogen and stored for 2–9 months. Straws were thawed by immersion in distilled water at 32 °C for 30 s. Embryos were then transferred to a 0.25 M sucrose solution in PBSS for 10 min, and washed three times in PBSS for 5 min each. Vitrification was performed using the protocol of Dattena et al. [9] with the equilibrium time modified (1.5 min instead of 3 min). All vitrification solutions were prepared using PBSS. Embryos were exposed to 10% EG and 10% DMSO for 1 min and 30 s, and then to 20% EG, 20% DMSO and 0.5 M sucrose for 30 s, always at room temperature. The embryos were loaded into OPS according to Vajta et al. [38], by capillarity together with ∼2 μl of medium and directly immersed into liquid nitrogen and stored for 2–9 months. Embryos were warmed according to Vajta et al.