226 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING CHEMICAL PACKETS THAT REGULATE CO₂ AND O₂

Abstract

Bovine embryos are now routinely produced with oocytes collected from slaughterhouse ovaries or by transvaginal ovum pickup. The oocytes are matured, fertilized, and cultured in a water-jacketed CO₂/O₂ incubator. Gas phase in incubators is usually maintained at 5% CO₂ in air for in vitro maturation (IVM) and IVF of oocytes and at 5% CO₂, 5% O₂, and 90% N₂ for in vitro culture (IVC) of embryos. Here we investigated whether two chemical packets that regulate CO₂ and O₂ for culturing bacteria (Mitsubishi Gas Chemical, Tokyo, Japan) could be used to control the gas phase in vitro production (IVP) of cattle embryos. One packet (Anaero Pack-CO₂) was maintained at a CO₂ level of ~5% in a 2.5-L container and the other (Anaero Pack-MicroAnaero) was maintained at a CO₂ level of 5–8% and an O₂ level of 6 to 12%. Bovine cumulus-oocyte complexes (COC, n = 970) were collected from slaughterhouse ovaries, matured in HEPES-buffered TCM-199 (catalog number 12340–030, Invitrogen) supplemented with 10% FCS, 0.02 Armour unit mL^–1 FSH and 1 µg mL^–1 E₂ for 22 h, and fertilized in medium IVF100 [Research Institute for the Functional Peptides Co. Ltd. (IFP), Yamagata, Japan] with frozen-thawed sperm (4 × 10⁶ cells mL^–1) for 6 h. Sperm and cumulus cells were removed from the oocytes. The denuded oocytes were cultured in IVD101 (IFP, 20 to 30 embryos/50 μL) for 8 days (Day 0 = IVF). Culture was carried out at 39°C with maximum humidity. Five different combinations of gas conditions were used for incubation (Table 1). Experiments were repeated 3 times. Cleavage and blastocyst rates were assessed on Day 8. Data were analysed by ANOVA followed by Fisher's PLSD test. In the five conditions, rates of matured oocytes (oocytes at MII, n = 210) were 70 to 73% and rates of normal fertilized oocytes (oocytes with 2 pronuclei, n = 310) were 67 to 75%. Cleavage rates of embryos after 8 days of culture (n = 450) were 68 to 75%, and rates of blastocysts from cleaved embryos were 25 to 40%. None of the above measures were significantly different among the 5 conditions (P > 0.05). These results indicate that gas phase control is not needed for IVM and IVF of bovine oocytes for their subsequent development. Anaero Pack-MicroAnaero (5–8% CO₂, 6–12% O₂) can be used for IVC of bovine embryos. The CO₂-generating and deoxidizing packets can be successfully used to control the gas phase during bovine embryo production.

Table 1.  Five different combinations of gas conditions used for incubation