125 FOLLICULAR FLUID AND GRANULOSA CELL RECOVERY FROM BOVINE FOLLICLES OF DIFFERENT DIAMETERS USING AN ADAPTED TRANSVAGINAL-GUIDED FOLLICULAR ASPIRATION SYSTEM

Abstract

Most studies of granulosa cell (GC) function have been performed in vitro, using follicular fluid (FF) and GC recovered from slaughterhouse ovaries. This approach does not consider the reproductive status and follicular developmental stage of the donor, limiting data interpretation and usefulness. The aim of this study was to evaluate the efficiency of an adapted ultrasound-guided transvaginal follicular aspiration (TVFA) procedure to recover FF and mural GC from live cows. A preliminary in vitro trial was performed to calculate fluid losses using a conventional TVFA circuit. A known volume of PBS, expected to be the volume of FF in follicles ranging from 4 to 12 mm in diameter, was aspirated using a 20 G needle connected to a Teflon circuit 80 cm long, with a 1.0-mm internal diameter, connected to a 1.5-mL tube. Losses of the expected volume of FF were 12.7 ± 1.1, 19.9 ± 2.5, 54.4 ± 4.0, 87.6 ± 4.3 and 100%, for follicles of 12, 10, 8, 6 and 4 mm in diameter (0.90, 0.52, 0.27, 0.11 and 0.03 mL, respectively). When an adapted system for small-volume recoveries was used, there was a decrease (P < 0.05) in fluid losses for follicles of 8, 6 and 4 mm. An experiment was performed in vivo, using the adapted system, to evaluate FF and GC recovery from follicles of 4 and 5 (n = 16), 6 (n = 19), 7 (n = 7), 8 (n = 13), 9 (n = 15), 10 (n = 24), 11 (n = 11), 12 (n = 15), 13 (n = 13), 14 (n = 6) and 16 (n = 9) mm in diameter. Follicular wave emergence was synchronized with 2 mg of oestradiol benzoate and an intravaginal progesterone device and follicles that reached the desired diameter were aspirated using an ultrasound machine equipped with a 7.5-MHz probe and disposable 20 G needle. The recovered FF volume was measured and centrifuged at 600 × g for 10 min. The GC pellet was vortexed with 0.1% hyaluronidase (5 min) and washed twice in PBS and the number of cells was determined using a Neubauer chamber. Ribonucleic acid was extracted using an RNAeasy Microkit and quantified in NanoDrop. The efficiency of FF recovery was estimated by the difference between the recovered and expected volumes for each follicle diameter (4/3πr³), which were compared by ANOVA. From all the follicles aspirated, the recovery of FF and GC was not successful in 2 (1.3%). Overall, FF recovery efficiency was 84.7%. The recovered volume ranged from 0.03 to 3.80 mL and increased with follicular diameter (y = 0.011x² – 0.012x + 0.043; R² = 0.99). Losses of FF were significant (P < 0.05) for follicles larger than 12 mm. The mean (±s.e.m.) number of mural GC recovered was 716,708 ± 68,536, providing 6.8 ± 0.7 samples of 100 000 cells with 14.8 ± 0.7 ng of RNA μL^–1 for each punctured follicle. A high coefficient of variation (57.4%) was observed in cell recovery. There was no difference in the number of cells recovered from follicles of different diameters, but sample contamination with blood was more frequent (75%) in follicles larger than 10 mm. In conclusion, an adapted TVFA system can be used successfully on an individual basis and from follicles of different diameters for in vivo recovery of FF and GC for further endocrine and gene expression analyses.

This research was supported by CNPq and Fapemig (CVZ APQ 02863/09).