Recovery capacity assessment in vitrified embryos of superovulated CF-1 mice

TELLO MF; LORENZO MS; MARURI A; CRUZANS PR; GULIN JEN; LOMBARDO DM

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

The vitrification of embryos is a useful tool for assisted reproductive techniques. It is anultra-rapid cooling technique using a minimum volume with highly concentration ofcryoprotectants avoiding ice crystals formation. The aim of this study was to obtain ahigh number of embryos using a superovulation (SOV) protocol and to evaluate itsmorphology and recovery capacity after vitrification. The embryos were retrieved from 6to 8-week-old SOV CF-1 females (n=3). They were injected with 7.5 IU eCG and 7.5 IUof hCG (48 h later); immediately they were single mated overnight with 9-week-old CF-1 males. The following morning the presence of a vaginal plug was checked. A total of116 embryos (blastocyst=58; cleavage=58) were retrieved by uterine flushing. A groupof blastocysts (n=37) were exposed to an equilibration solution (5 min), then tovitrification solution (1 min) and finally placed on a vitrification device with a minimumvolume and plunged into liquid nitrogen. For warming, they were placed into a pre-warmed solution at 37ºC (3 min) then diluted and kept in a washing solution. Forrecovery, they were incubated in 50-µL drops of an enriched blastocyst medium for 10h. The morphology, re-expansion, and hatching were evaluated using astereomicroscope, the number of cells with Hoechst 33342 (n=21) and in average a2,7% of blastomeres presented DNA fragmented ? TUNEL assay (n=15). The treatedembryos presented a normal morphology, mitotic figures, cells number and apoptosisrates according to their development stage. Embryos obtained with this SOV protocolmaintain their capacity to re-expand and hatching after vitrification protocol.