RECOVERY CAPACITY ASSESSMENT IN VITRIFIED EMBRYOS OF SUPEROVULATED CF-1 MICE

MARURI, A.; LOMBARDO, DM.; MARURI, A.; LOMBARDO, DM.; TELLO, MF.; CRUZANS, PR.; TELLO, MF.; CRUZANS, PR.; LORENZO, MS.; GULÍN, E.; LORENZO, MS.; GULÍN, E.

Varadero

Taller; Taller internacional "Modelos animales e Investigación Preclínica 2019 (Animod 2019)"; 2019

Centro de Ingeniería Genética y Biotecnología de la Habana, la Sociedad Cubana de Farmacología y el Centro Nacional de Producción de Animales de Laboratorio (CENPALAB)

The vitrification of embryos is a useful tool for assisted reproductive techniques. It is an ultra-rapid cooling technique using a minimum volume with highly concentration of cryoprotectants avoiding ice crystals formation. The aim of this study was to obtain a high number of embryos using a superovulation (SOV) protocol and to evaluate its morphology and recovery capacity after vitrification. The embryos were retrieved from 6 to 8-week-old SOV CF-1 females (n=3). They were injected with 7.5 IU eCG and 7.5 IU of hCG (48 h later); immediately they were single mated overnight with 9-week-old CF1 males. The following morning the presence of a vaginal plug was checked. A total of 116 embryos (blastocyst=58; cleavage=58) were retrieved by uterine flushing. A group of blastocysts (n=37) were exposed to an equilibration solution (5 min), then to vitrification solution (1 min) and finally placed on a vitrification device with a minimum volume and plunged into liquid nitrogen. For warming, they were placed into a prewarmed solution at 37ºC (3 min) then diluted and kept in a washing solution. For recovery, they were incubated in 50-μL drops of an enriched blastocyst medium for 10 h. The morphology, re-expansion, andhatching were evaluated using a stereomicroscope, the number of cells withHoechst 33342 (n=21) and in average a 2,7% of blastomeres presented DNAfragmented ? TUNEL assay (n=15). The treated embryos presented a normalmorphology, mitotic figures, cells number and apoptosis rates according to their development stage. Embryos obtained with this SOV protocol maintain theircapacity to re-expand and hatching after vitrification protocol.