مستخلص: |
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 38.5C 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. [ABSTRACT FROM AUTHOR] |