205 HOLDING PIG OOCYTES AT 24°C PRIOR TO IN VITRO MATURATION ALTERS THE DEVELOPMENTAL CAPACITY AFTER IN VITRO FERTILISATION BUT NOT PARTHENOGENETIC ACTIVATION
Authors: Quadalti C. , Lagutina I. , Lazzari G. , Galli C. 
Published: December 2015

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.