42 Vitrification of in vitro-produced feline embryos

Abstract

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of in vitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n = 32), vitrification with EG (n = 31), or control (n = 47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5 mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5 M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5 min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5 M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30 s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5 mL of 1 M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1 min. Next, embryos were moved to 0.5 mL of 0.5 M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3 min. Finally, embryos were transferred to 0.5 mL of FOCMH for 5 min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48 h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24 h after warming (84%) than embryos vitrified in PrOH (59%; P < 0.05). The continued embryonic growth of viable embryos after culture for 48 h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P > 0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24 h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.