270 HIGH GASEOUS PRESSURE PRETREATMENT IN IN VITRO MATURATION OF CANINE OOCYTES

Abstract

Stress processes, such as hydrostatic pressure treatment of oocytes in different species, have been reported to increased embryo rate following in vitro maturation (IVM). However, studies on high gaseous pressure (HGP) pretreatment in IVM of oocytes from domestic animals are lacking in the literature. This experiment aimed to test HGP pretreatment of canine oocytes to increase meiosis achievement (metaphase II) after IVM. A total of 502 canine oocytes (6 replicates) were used in this study. Ovaries from 15 bitches were obtained from local shelters or rescue organizations after ovariohysterectomy. Sample collection was blind as to reproductive stage and dog age. The ovaries were transported to the laboratory in 0.9% NaCl and were processed within 3 h of collection. The ovarian cortex was sliced and washed in PBS with 1% FCS to release cumulus–oocyte complexes. Grade 1 and 2 cumulus–oocyte complexes were selected for IVM and randomly distributed into 3 treatment groups: HGP (oocytes placed in PBS and subjected to pressure chamber; 206 oocytes), ambient control (oocytes maintained in TCM-HEPES at room temperature for 60 min; 130 oocytes), and laboratory protocol (oocytes IVM after morphologic selection; 166 oocytes). The average pressure, initial and final temperature, and duration of oocytes in the HGP pretreatment were 76.19 atm (±0.92), 32.20°C (±5.17) and 27.71°C (±3.17), and 60 min, respectively. In vitro maturation was carried out for 72 h at 37°C in a high-glucose medium, consisting of TCM-199 with 2.2 mg mL^–1 of sodium bicarbonate (11150, Gibco, Grand Island, NY, USA), and supplemented with 0.1% polyvinyl alcohol (P-8136, Sigma, St. Louis, MO, USA), 0.991 mg mL^–1 of glucose (108337, Merck, Darmstadt, Germany), 50 µg mL^–1 of gentamicin, 22 µg mL^–1 of pyruvic acid, 20 µg mL^–1 of oestradiol (E-8875, Sigma), 0.5 µg mL^–1 of FSH (Folltropin-V, Vetepharm Inca), 0.03 IU mL^–1 of hCG (Chorulon^®, Intervet, Kenilworth, NJ, USA), under 20% oxygen tension. The number of oocytes at each stage (prophase to metaphase II) was recorded according to the morphology of nuclear content after staining with Hoechst 33342. For comparison purposes of nuclear maturation in oocytes, data were analysed by Fisher’s exact test. Differences at a P-value ≤0.05 were considered significant. Oocytes from the HGP, ambient control, and laboratory protocol groups had similar meiotic progression to the metaphase II stage (metaphase I–anaphase I–telophase I–metaphase II), and were 35.4% (73/206), 30.8% (40/130), and 34.9% (58/166), respectively (P ≥ 0.05). The proportion of oocytes without chromatin or having an irregular organisation was not different among groups. In conclusion, results indicate that HGP pretreatment as used in this experiment did not improve meiosis rates in IVM canine oocytes. Further investigations to understand the significance of HGP pretreatment in IVM and in vitro production of canine embryos are ongoing in our laboratory.