90 OPEN PULLED STRAW VITRIFICATION OF IN VITRO PORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM

Abstract

The aim of this study was to design a chemically defined medium for the vitrification of in vitro produced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitro in NCSU-23 for 44 h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess in vitro fertilization (IVF) parameters (N = 200) or for 6 days (N = 600) in order to obtain blastocysts. For chemically delipidation, 10 μM forskolin was added to the culture medium on Day 5 of in vitro culture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n = 65), with 0.1% of polyvinyl alcohol (PVA; n = 64) or without additives (WA; n = 65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al. 2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitro for 24 h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitro culture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. The maturation, penetration, and monospermy rates were 98.5 ± 1.2%, 85.3 ± 3.6%, and 48.8 ± 5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0 ± 4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8 ± 1.4% and 37.3 ± 1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8 ± 5.9) PVA (40.6 ± 6.0), and WA (30.8 ± 5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4 ± 6.8 to 50.8 ± 8.3) among vitrification groups. In conclusion, in vitro derived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitro produced pig blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07).