226 Effect of culture time on maturation of oocytes obtained by ovum pickup of alpacas (Vicugna pacos)

Development of an in vitro fertilisation (IVF) technique requires oocyte maturation as a phase essential for the transition from a gamete to an embryo competent. In alpacas, studies on IVF have been reported but there are controversies about the maturation times required to obtain competent cumulus-oocyte complexes (COCs). The study was carried out to evaluate the effect of culture time on maturation of oocytes obtained by the ovum pickup technique. Adult nonpregnant alpacas (n = 6) of 6–8 years of age and weighing 55–65 kg, were used as oocyte donors. Animals were evaluated by transrectal ultrasonography (EXAPAD MINI) with a 7.5 MHz linear array transducer to determine a follicle ≥ 7 mm; the dominant follicle was then ablated. Animals were induced to hormonal stimulation with 150 mg of FSH (FOLLTROPIN-V) applied IM in a decreasing manner for three days from 36 h post-follicular ablation. Animals were subjected and anaesthesia caudal epidural was applied (2.0 mL lidocaine) to facilitate the transvaginal ultrasound-guided follicle aspiration using a 5.0 MHz convex-array ultrasound transducer coupled to an 18-gauge needle. Follicles ≥ 5 mm were aspirated into a 50 mL conical tube containing a commercial medium aspiration at 37°C, supplemented with heparin. Follicular content aspirates were transferred to Petri dishes for COCs examination using a NIKON stereomicroscope. COCs were distributed to the culture times under evaluation (28, 32, and 34 h). COCs were classified as grade I (≥4 layers of compacted granulosa and homogeneous ooplasm), grade II (partial layers of granulosa cells and homogeneous ooplasm), grade III (denuded), and grade IV (expanded/degenerated, pyknotic). Only COCs I and II were matured in groups of 20 COCs in 40-μL drops of TCM-199 supplemented with 10% heat-treated fetal calf serum, 0.2 mM sodium pyruvate, 0.5 μg/mL FSH, 1 μg/mL oestradiol-17β, and 25 µg/mL gentamycin. Microdrops were covered with mineral oil and cultured for the time in study at 38.5°C in a humidified atmosphere of 5% CO₂. Maturation rate was assessed after denuding the COCs and fixation in 1:3 acetic acid/ethanol by 36 h and after staining with orcein, and evaluated using phase-contrast microscopy and classified as GV, GVBD, MI, and MII. Maturation rate between culture times was analysed using the Wilcoxon-Mann-Whitney test with a significance level of ≤0.05. Results indicate that recuperation rate was 37.5% (36/96) and maturation rate was 16.7%; 60.0% and 66.6% of oocytes in MII to 28, 32, and 34 h. Results suggest that COCs recovered by OPU require more than 32 h of maturation time.

This research was supported by Project 149-2017, FONDECYT-UNMSM.