Oxygen Consumption of Single Bovine Embryos Probed by Scanning Electrochemical Microscopy

Abstract

Oxygen consumption of individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). A probe microelectrode was used to scan near a single embryo surface in a culture medium to monitor the oxygen reduction current at 37 degrees C, under a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profiles near the embryos were in good agreement with the theoretical spherical diffusion. When an embryo reached the stage of a morula with a 74-microm radius on day 6 after in vitro fertilization, the oxygen concentration difference (deltaC) between the bulk solution and the morula surface was 6.90 +/- 1.35 microM. The oxygen consumption rate (F) of the single morula was estimated to be (1.40 +/- 0.27) x 10(-14) mol s(-1). After the SECM measurement, the embryo was continuously cultured for another 2 days and grew to the stage of a blastocyst with a 100-microm radius. For the blastocyst, the deltaC values for the inner cell mass side and the trophoblast side were 16.40 +/- 1.83 and 9.14 +/- 1.68 microM, respectively. The oxygen consumption rate of the blastocyst was found to be in the range of (2.50 +/- 0.46) x 10(-14) mol s(-1) < F < (4.49 +/- 0.50) x 10(-14) mol s(-1). We have carried out SECM measurements for 19 embryos, and the results were compared in detail with these from an optical microscopic observation. The deltaC values for the morulae on day 6 after in vitro fertilization were strongly related to the morphological embryo quality. The morulae showing a larger deltaC value developed into blastocysts of a larger size, and the deltaC value after the subsequent 2 days of cultivation was found to be increased.

Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development

“…The rate of oxygen consumption in embryos at 38.5 8C was , 2.8 £ 10 213 moles/s. This value is closer to the value of 0.14 £ 10 213 moles/s for bovine morula reported by Shiku et al (2001) than for values reported by Thompson et al (1996) of , 9 £ 10 211 moles/s for bovine embryos from day 0 -4 of culture. Reported values for bovine blastocysts range from 0.4 -1.5 £ 10 29 moles/s , Kaidi et al 2001, Donnay et al 2002.…”

“…Reported values for bovine blastocysts range from 0.4 -1.5 £ 10 29 moles/s , Kaidi et al 2001, Donnay et al 2002. Reasons for the differences in absolute amounts are not known but could include methodology since the lower values in the present study and by Shiku et al (2001) are based on electrochemical measurements of oxygen, while other papers cited above rely on quenching of pyrene fluorescence by oxygen. The electrochemical method employed here has been used extensively to characterize oxygen consumption in cultured islets of Langerhans (Jung et al 1999a(Jung et al ,b, 2000.…”

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

“…This overcomes the problem of the inhomogeneity of the blastocyst surface, encountered by Shiku et al (2001) when using the scanning electrochemical microscopy (SCEM) technique. In that paper, the authors reported that the oxygen consumption rate of a blastocyst varied, with higher oxygen consumption rates being observed in the inner cell mass side when compared with the trophoblast side.…”

“…When the oxygen-consuming embryo was placed at the bottom of the capillary, the cylindrical shape of the diffusion space resulted in a largely linear concentration gradient when the system reached steady state. Using oxygen respiration values reported in the literature (Shiku et al 2001) and the dimensions of the capillaries, it could be calculated that a typical embryo at the bottom of the capillary should reduce the oxygen concentration by about 25 -50% according to Ficks first law of diffusion (see section below). These predictions were subsequently verified by experimental measurement of the oxygen concentration near the embryo inside the glass capillary.…”

“…More recently, oxygen consumption of single bovine morulae and blastocysts was determined using scanning electrochemical microscopy (Shiku et al 2001). The measurements required constant microscopic observation, and each embryo was held with holding pipettes in order to perform oxygen measurements at several defined positions because of the inhomogeneity observed at the blastocyst surface.…”