142 MORPHOLOGICAL EVALUATION OF CANINE EMBRYOS OBTAINED IN VIVO

Abstract

The aim of this study was to evaluate re-expansion and ultrastructure of in vivo-produced canine embryos immediately after collection (Co; n = 6) and 24 h after in vitro culture (Co24; n = 6). For embryo collection, two bitches in pro-oestrous were monitored every 48 h by vaginal cytology up to the detection of 80 to 90% of superficial cells. Then, they were inseminated 3 times on alternate days with fresh semen from 1 fertile male. Embryo collections were performed after ovariohysterectomies and uterine flushing 12 days after the first artificial insemination. Each uterine horn was flushed 3 times with 60 mL of DPBS at 36 to 37°C and collecting embyos in Petri dishes. Embryos culture were performed at 38.5°C under an atmosphere with 5% CO₂ and maximum humidity, using SOFaa media added with 20% of fetal bovine serum (FBS). Embryo reexpansion was evaluated after 24 h of culture and the embryos were classified as re-expanded or not re-expanded, according the appearance of the blastocoele by stereomicroscopy (Leica MZ 12.5, Leica Microsystems, Wetzlar, Germany). Twelve embryos were collected, 5 from bitch A and 7 from bitch B; 100% of embryos (6/6) showed re-expansion after 24 h of culture. Blastocoele reexpansion was used as an embryo viability marker. The group Co showed perivitelline space and apical surface of blastomeres covered with microvilli, elongated mitochondria, rough endoplasmic reticulum (RER), tight junctions, and a large amount of lipid droplets that was similar to the results previously described in others mammals species. The Co24 group showed the same characteristcs of the group Co at the time of collection, however with a reduction of lipid droplets and the presence of myelinic structures. In conclusion, the lipid droplet reduction and presence of myelinic structures after 24 h of in vitro culture may indicate lipid consumption associated with embryo expansion.