124 COMPARISON OF TWO EMBEDDING SYSTEMS FOR FOLLICULAR CULTURE

Abstract

Recent research has focused on developing secondary, preantral follicles as a new way to collect female gametes to develop in vitro or to study folliculogenesis. Secondary follicles are abundant in ovaries of almost all females, and can be isolated from the ovarian cortex of fresh or cryopreserved tissue. These follicles must be matured to obtain mature oocytes, but the mechanism for early follicle development is not well understood. Currently, culture techniques are being developed to promote the growth and quality of these follicles. The aim of this preliminary study was to compare 2 embedding systems for porcine follicles to improve preantral follicle culture. We collected secondary preantral follicles isolated from porcine ovaries recovered from the abattoir. Ovaries were transported to our laboratory in saline solution (0.9% NaCl) and cut into smaller fragments with a scalpel. Follicles were further mechanically isolated with a 23 1/2-gauge needle and placed into a dish with medium TCM199 plus Hepes (Lonza 12–117F) supplemented with 5% fetal bovine serum (FBS). Secondary follicles less than 300 µm were selected. Furthermore, only clear follicles with dark oocytes and no antrum were used. The selected follicles were randomly divided into 5 different treatment groups: 1) embedded in 0.5% alginate gel; 2) 1.0% alginate gel; 3) 1.0% agarose gel; 4) 2.0% agarose gel; and 5) control. Three follicles were placed in each well with 280 µL of α-minimal essential medium supplemented with 3.5 μg mL^–1 of insulin 10 μg mL^–1 of transferrin, 100 μg mL^–1 of l-ascorbic acid, and 7.5% porcine serum and cultured for 4 days at 39°C at 5% CO₂. Media (140 µL) from each well was changed on Day 2 and saved for metabolic and functional analysis. Initial diameters and measurements on Day 2 and 4 were taken to analyse dimensional growth. We evaluated 6 follicles per group per replicate, for a total of 3 replicates (18 per group). All recorded parameters were subjected to a 2-way ANOVA using the procedure of the Generalized Linear Model (SPSS, 18th version, SPSS Inc., Chicago, IL, USA). Independent variable was the result of the balance between the size on Day 2 and 0 and the size on Day 4 and 0 time of observation. Data were normally distributed. Least squares means tests were used to perform statistical multiple comparisons. The α level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results in the Table 1 show that at Day 2 there was no difference between groups. However, after 4 days of development, the group agar 2% and alginate 1% showed a statistical difference when compared with the control. These results suggest that there is a positive effect when the follicles are embedded during culture.

Table 1.  Growth of Follicles (in μm) since Day 0 in different embedding systems