29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

Abstract

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although >80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200 µL of M199 medium supplemented with alanyl-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL⁻¹), epidermal growth factor (50 ng mL⁻¹), pLH (5 µg mL⁻¹), cysteamine (0.1 mM), and 10% FBS for 22 to 24 h in humidified air and 5% CO₂. Oocytes were divided into the following groups: 1× pFSH (2 µg mL⁻¹), 1× rhFSH (0.01 UI mL⁻¹), 1× pFSH + 1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24 h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1 × 10⁶ sperm mL⁻¹. The 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 under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos 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 development progress (24-48 h), and hatching of the zona pellucida (72 h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level = 0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors.

Table 1.  Cleavage, embryo development rate, and cryotolerance of FSH of various origins