The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400 µL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL−1), EGF (50 ng mL−1), ovine FSH (50 ng mL−1), bLH (3 µg mL−1), cysteamine (0.1 mM), and 10% fetal bovine serum (FBS) for 22 to 24 h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90 µg mL−1), penicillamine (3 µg mL−1), hypotaurine (11 µg mL−1), and heparin (20 µg mL−1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5–6, blastocysts at Day 6–6.5, and expanded blastocysts at Day 6.5–7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45 min. The slow freezing protocol consisted of 1.5 M ethylene glycol (EG) + 20% FBS + 0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing the 0.25-mL straws to air (23°C) for 10 s and then underwater at 35°C for 1 min. The vitrification (Cryo-Top) medium was 15% (vol/vol) EG + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos from both groups were cultured in SOF-BSA + 10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0 h), re-expansion, and hatching of the zona pellucida (72 h). Three replicates were performed for each treatment level. Fisher’s l.s.d. test with Bonferroni correction was used to determine treatment differences (P < 0.05). The post-thaw survival, re-expansion, and hatching results showed that either expanded blastocysts (84.7 ± 3.2%, 74.1 ± 3.9%, and 60.9 ± 4.4%) or blastocysts (81.7 ± 3.5%, 69.6 ± 4.2%, and 55 ± 4.6%) were preferred (P < 0.05) embryo stages for cryopreservation compared with morulae (67.6 ± 4.4%, 52.5 ± 4.6%, and 33.2 ± 4.3%). Vitrification and cytochalasin-B pre-freezing exposure (61.3 ± 3.6% and 56.6 ± 3.8%) provided better (P < 0.05) hatching results compared with slow-freezing and without cytochalasin-B (37.8 ± 3.6% and 42.5 ± 3.7%).
In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification
