304 EFFECT OF SYNTHETIC OVIDUCT FLUID MEDIUM (SOF) v. OPTIMIZED SIMPLE POTASSIUM MEDIUM (KSOM) ON EMBRYO DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE OOCYTES

Abstract

The objective of the study was to compare the effect of 2 free serum chemically defined media, synthetic oviduct fluid medium (SOF) and optimized simple potassium medium (KSOM) on embryo development of in vitro-produced embryos. Bovine ovaries were collected from a local abattoir. Cumulus oocyte complexes (COC) were obtained by follicular aspiration of 3- to 6-mm follicles and in vitro-matured in TCM-199 for 24 h at 39°C, 5% CO₂, and high humidity. After IVM, COC were co-incubated with sperm at a concentration of 1.5 × 10⁶ spermatozoa per mL in TALP medium for 18 h at 39°C, 5% CO₂, and high humidity. Presumptive zygotes were randomly assigned to SOF (n = 312) or KSOM (n = 290) media, both supplemented with essential and nonessential amino acids plus 0.6% BSA. Zygotes were cultured at 39°C, 5% CO₂, 5% O₂, 90% N₂, and high humidity for 9 days. Cleavage rate was evaluated on Day 2 (Day 0 = day of insemination) and the number of blastocysts and hatched blastocysts were recorded on Days 8 and 9. Expanded blastocysts from Day 8 (n = 45/per culture medium) were fixed and stained with bisbenzimide (Hoechst 33242), and the total number of embryonic nuclei was counted by fluorescence microscopy (Optiphot, Nikon, Tokyo, Japan). The study was conducted in 6 replicates for embryo development assessment and 3 replicates for embryonic nuclei counting. Data were analyzed using Fisher’s exact test and Student’s t-test. The percentage of presumptive zygotes to develop into 2 to 4 cells (22.4 v. 24.2%) or > 4 cells (47.7 v. 53.4%) did not differ (P > 0.05) between SOF and KSOM on Day 2, but total embryo cleavage ≥ 2 cells was greater (P < 0.05) in KSOM (76.8%) than in SOF (68.5%). However, the percentage of blastocysts developed on Day 8 and 9 was greater (P < 0.05) for zygotes cultured in SOF (22.1 and 25.6%) than for those cultured in KSOM (15.1 and 18.9%). Furthermore, the percentages of hatched blastocysts on Day 8 and 9 were also greater (P < 0.05) for zygotes cultured in SOF (18.8 and 36.2%) than for those cultured in KSOM (4.7 and 12.5%). In addition, expanded blastocysts developed in SOF had higher total mean number of embryonic nuclei than those developed in KSOM (P < 0.05; 128.8 ± 7.5 v. 84.3 ± 8.9, respectively). In conclusion, the use of SOF resulted in significantly greater percentage of blastocysts and hatched blastocysts compared with KSOM. Additionally, expanded blastocysts developed in SOF displayed a higher number of cells.

This study was supported by Convenio Desempeño en Investigacion (2007-DGI-CDA-04), Universidad Catolica de Temuco and DiD-UAcH: S-2009-26.