Embryo Collection and Vitrification Protocol

The collection of embryos was performed in a specifically designed surgical room located on the farm. The donors were subjected to a mid-ventral laparotomy on Day 6 of the estrous cycle (Day 0: onset of estrus). The donors were sedated with azaperone (2 mg/kg body weight, intramuscular). General anesthesia was induced using sodium thiopental (7 mg/kg body weight, intravenous) and was maintained with isoflurane (3.5-5%). After exposure of the genital tract, the corpora lutea on the ovaries were counted. The embryos were collected by flushing the tip of each uterine horn with 30 mL of Tyrode’s lactate (TL)-HEPES-polyvinyl alcohol (PVA)15 (link) with some modifications4 . The recovered embryos were evaluated under a stereomicroscope at a magnification of 60× to grade the developmental stage and quality. One-cell eggs and poorly developed embryos were classified as unfertilized oocytes and degenerated embryos, respectively. The remaining embryos with the appropriate morphology according to the criteria determined by the International Embryo Transfer Society16 were considered viable. Vitrification was only performed on compacted morulae and unhatched blastocysts with morphology graded as excellent or good.
The ovulatory response of the donors was determined by counting the number of corpora lutea on both ovaries. Recovery rate was defined as the ratio of the number of embryos and oocytes and degenerated embryos recovered to the number of corpora lutea present. Fertilization rate was defined as the ratio of the number of viable embryos at collection to the total number of embryos and oocytes and degenerated embryos collected.