205 HOLDING PIG OOCYTES AT 24°C PRIOR TO IN VITRO MATURATION ALTERS THE DEVELOPMENTAL CAPACITY AFTER IN VITRO FERTILISATION BUT NOT PARTHENOGENETIC ACTIVATION

Abstract

The in vitro production of porcine embryos is of great interest because of the increasing importance of the swine as an animal model and a tissue donor for biomedical or biotechnological applications. Availability of ovaries at selected time of the day can be a limitation; therefore, the possibility to maintain immature oocytes for some hours can be very useful. The aim of this study is to determine whether holding recovered oocytes at 24°C for 24 h alters the maturation process and/or the developmental capacity. Immature sow oocytes were either matured in vitro for 42 h at 38.5°C (control group; CTR) or kept in 2 mL of HEPES-SOF in the dark at 24°C for 24 h before maturation (experimental group; +24 h). After maturation, cumulus cells were removed, and the number at metaphase II were recorded. For parthenogenetic activation (PGA), oocytes with a visible polar body were activated at 48 h of maturation as previously described (Lagutina et al. 2006). For IVF experiments frozen-thawed boar semen was prepared through a discontinuous density gradient, washed in TALP Ca²⁺-free, diluted in TALP : SOF = 1 : 1 supplemented with 6 mg mL^–1 of fatty acid-free BSA, hypotaurine and epinephrine, mixed with oocytes after partial removal of the cumulus cells, at 43 h of maturation and cultured in 5% CO₂ in humidified air at 38.5°C. After 24 h of IVF, oocytes were denuded and cultured in mSOF-1 in atmosphere of 5% O₂ and 5% CO₂. The same culture conditions were used after parthenogenetic activation. Half of the medium was changed with mSOF-1 at Day 3 and with mSOF-2 at Day 5. The cleavage and the cumulative Day 7 blastocyst (BLD7) rates and cell number of IVF BLD6 were recorded. For each group, blastocysts on Day 6 were fixed and cell number counted, whereas the other embryos were left in culture until Day 7 (cumulative D7 = BLD6 fixed + BLD7). All experiments were done in 3 replicates. The data were compared by Student’s t-test and chi-square test. Maturation rates as recorded for the presence of the polar body did not differ (CTR: 255/312, 82%; +24 h: 208/256, 81%). There was no significant difference (P < 0.05, chi-squared test) between CTR and +24 h group cleavage (144/165: 87% and 127/138: 92%, respectively) and BLD7 rate (47/165: 28% and 34/138: 25%, respectively) in the PGA. Whereas no difference (P < 0.05, chi-squared test) was observed between CTR and +24 h group cleavage (111/180: 62% and 99/186: 53%, respectively) in the IVF, but the BLD7 rate in +24 h group was significantly lower (48/180: 27% in the CTR group, 27/186: 15% in the +24 h group). However, the cell number of IVF BLD6 was not altered by holding at 24°C (n = 22: 25 ± 10 cells in the CTR, n = 8: 22 ± 13 cells in the +24 h) (P < 0.05, 2-tailed Student t-test). These experiments show that holding at 24°C for 24 h before maturation can alter the developmental capacity of IVF-produced embryos but not that of parthenogenetically activated ones. More replicates are needed to study the kinetics of maturation and to confirm our results.

MitCare project (ERC n 322424) is acknowledged for support of this project.