292 OOCYTES FROM FOLLICLES OF DIFFERENT DIAMETERS: EMBRYO IN VITRO DEVELOPMENTAL POTENTIAL AND QUALITY

The present study aimed to assess the developmental potential and quality of embryos produced from oocytes originating from follicles of different sizes. Ovaries were collected at a slaughterhouse and follicles of <3, 3 to 6, and >6 mm were aspirated. Follicle diameters were estimated based on the size of their exposed surface on the ovarian cortex using a ruler as reference for the first aspirations and were then based on visual evaluation. Aspirated oocytes, separated by their follicular origin, were matured in vitro in TCM-199 supplemented with 10% FCS, 5.0 �g mL-1 of LH, 0.5 �g mL-1 of FSH, 0.2 mM pyruvate, and 10 �g mL-1 of gentamicin for 22 h. After in vitro maturation, oocytes were in vitro-fertilized (IVF) using frozen–thawed semen prepared by Percoll gradient. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 of heparin. After 20 h, presumptive zygotes were partially denuded and transferred to in vitro culture (IVC) medium (TCM-199 supplemented with 10% FCS, 2.0 mM pyruvate, and 10 mg mL-1 of gentamicin). All cultures were incubated at 38.5�C under 5% CO2 in air and maximum humidity. The cleavage rate was assessed after 48 h of IVC, and blastocyst development was assessed on Day 7 (D7). On Day 9 (D9), the hatching rate was assessed and the hatched embryos were fixed for 1 h (3% paraformaldehyde in PBS), permeabilized for another h (0.5% Triton-X, 0.1% sodium citrate, and 0.1% plyvinyl alcohol in PBS), and evaluated by the TUNEL technique (in situ cell death detection kit). Total cell number and TUNEL-positive cells in embryos were counted under an epifluorescence microcope. Data of 4 replicates were analyzed by ANOVA and comparisons among groups were made by the Tukey test. The level of significance used was 5%. From the oocytes used (<3 mm = 100; 3–6 mm = 99; >6 mm = 88), cleavage rates increased with increasing follicular diameter. Oocytes originating from <3-, 3- to 6-, and >6-mm follicles resulted in 75, 93.6, and 95.5% cleavage rates, respectively (P > 0.05). For blastocyst rates on D7, <3-mm follicles showed 21.5% blastocyt development, which was lower (P < 0.05) than for the other follicle diameter groups (3–6 mm = 35.4% and >6 mm = 41.1%; P > 0.05). Regarding the hatching rate, total cell numbers, and TUNEL-positive cells on D9, <3 mm (20.4%, 268, and 0.37%, respectively), 3–6 mm (29.1%, 248, and 0.32%, respectively), and >6 mm (32.3%, 237, and 0.23%, respectively) were not different (P > 0.05). The results suggest that oocytes from larger follicles are more competent for cleavage and blastocyst development on D7; however, when oocytes reach the blastocyst stage, hatching, total cell numbers, and apoptotic cell numbers are not influenced by follicle diameter. Financial suport for this work was provided by the FAPESP, Brazil.