A precise evaluation of embryo quality is important to estimate the suitability of embryo transfer to recipient animal. Recently, an objective evaluation method was reported for bovine embryos, in which the oxygen consumption of embryos can be noninvasively determined by scanning electrochemical microscopy (SECM) (Shiku et al. 2001 Anal. Chem. 73, 3751–3758). Trimarchi et al. (2000 Biol. Reprod. 62, 1866–1874) suggested that the oxygen consumption reflects the cell number and mitochondrial activity of embryos. The objectives of this study were (1) to examine the oxygen consumption of in vivo-derived embryos by SECM, (2) to investigate the relationship between oxygen consumption and morphological estimation of embryos, and (3) to assess the correlation among the oxygen consumption, embryo viability, and pregnancy rates. Fifty-six embryos were collected from Japanese Black cattle, which were superovulated with a total dose of 20 mg porcine FSH (FSH-R; Kawasaki Pharmaceutical Co., Ltd., Tokyo, Japan) followed by AI. The qualities of collected embryos at the stage of compacted morulae (CM), early blastocysts (EB), and blastocysts (BL) on Day 7 after AI were categorized as grade 1 and grade 2, according to the IETS manual (2002). The oxygen consumption rates of embryos were evaluated by SECM, as previously described by Abe et al. (2004 J. Mamm. Ova Res. 21). Embryos were frozen by programmable freezer in Dulbecco's PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20% calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30°C water bath for 15 s. After thawing, the embryos were examined for oxygen consumption. Twenty-eight embryos were then cultured in TCM-199 supplemented with 20% fetal bovine serum and 0.1 mM β-mercaptoethanol for 24 h to assess the viability of embryos by re-expansion of blastocole. The remaining 28 embryos were transferred to recipients. The pregnancy rates were determined by rectal palpation on Day 70. Data were analyzed by ANOVA. The consumption rates of BL embryos on Day 7 were significantly higher (P < 0.05) than those of CM collected on the same day (0.84 vs. 1.29 × 10−14 mol s−1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P < 0.05). After freezing–thawing, the average oxygen consumption rates of embryos were 0.52 × 10−14 mol s−1 for CM (n = 9), 0.67 × 10−14 mol s−1 for EB (n = 8), and 0.96 × 10−14 mol s−1 for BL (n = 11). The CM embryos with rates of < 0.5 × 10−14 mol s−1 and the EB and BL embryos with those < 0.6 × 10−14 mol s−1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates <0.5 × 10−14 mol s−1 for CM (n = 5) and <0.7 × 10−14 mol s−1 for EB (n = 9). A high pregnancy rate (67%) was obtained from embryos with rates >1.0 × 10−14 mol s−1 for BL (n = 14). These results suggest that the measurement of oxygen consumption of embryos after embryo freezing and prior to embryo transfer may be useful for estimating embryo quality and suitability of embryo transfer.
Studies on factors affecting superovulation and embryo transfer in Hungarian Merino ewes
