Porcine Embryo Collection and Evaluation

Embryo collection was performed in a surgical room located on the farm. The donors were subjected to a midventral laparotomy on Days 5 and 6 of the estrous cycle (Day 0: onset of estrus) to obtain morulae and unhatched blastocysts, respectively. The donors were sedated by the administration of azaperone (2 mg/kg body weight, intramuscular). General anesthesia was induced using sodium thiopental (7 mg/kg body weight, intravenous) and maintained with isoflurane (3.5–5%). After exposure of the genital tract, the corpora lutea on the ovaries were counted. Embryos were collected by flushing the tip of each uterine horn with 30 mL of a chemically defined medium consisting of Tyrode's lactate (TL)-HEPES-polyvinyl alcohol (PVA) [17] (link) with some modifications. This medium (TL-PVA) was composed of 124.3 mM NaCl, 3.2 mM KCl, 2 mM NaHCO₃, 0.34 mM KH₂PO₄, 10 mM Na-lactate, 0.5 mM MgCl₂·6H₂O, 2 mM CaCl₂·2H₂O, 10 mM HEPES, 0.2 mM Na-pyruvate, 12 mM sorbitol, 0.1% (w/v) PVA, 75 µg/ml potassium penicillin G and 50 µg/mL streptomycin sulfate. The collected embryos were evaluated to verify their developmental stage and quality grade. One-cell eggs and poorly developed embryos were classified as oocytes and degenerate embryos, respectively. The remaining embryos that exhibited appropriate morphology according to the criteria determined by the International Embryo Transfer Society [18] were considered viable. Only compacted morulae and unhatched blastocysts graded as excellent or good based on morphological appearance were used in the experiments according to the specific experimental design.
The ovulatory response of the donors was determined by counting the number of corpora lutea in both ovaries. To evaluate the effectiveness of the superovulation treatment, the numbers of viable embryos and oocytes and degenerate embryos were counted in each donor. The recovery rate was defined as the ratio of the number of embryos and oocytes and degenerate embryos recovered to the number of corpora lutea present. The fertilization rate was defined as the ratio of the number of viable embryos to the total number of embryos and oocytes and degenerate embryos collected.