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Embryo temperature was measured with a thermometer inside the egg and maintained at 37��C �� 1��C with the help of a heated egg holder on a heated plate and a heat lamp. Ultrasound biomicroscopy measurements were performed with a high frequency ultrasound system: the Vevo 770 (VisualSonics, Toronto, Ontario, Canada) equipped with a 55-MHz transducer (RMV 708) exhibiting an axial and lateral resolution of ?30 and 75 ��m, respectively (Zhou et al., 2002; McQuinn et al., 2007). The embryonic heart was imaged at a focal depth of 4.5 mm. The pulsed Doppler sample volume varied between 0.15 and 0.17 mm and the scanning depth ranged between 4.15 and 5.15 mm. The measurements were http://www.selleckchem.com/products/DAPT-GSI-IX.html performed and described in detail previously by our research group (Oosterbaan et al., 2009). From each embryo velocity signals were recorded at three cardiac sites, in the primitive left ventricle (PLV), the primitive right ventricle (PRV), and the OFT (Fig. 1a). To acquire signals from these sites the transducer was outlined along the cross section from the apex to the OFT (Fig. 1b). Because http://www.selleckchem.com/products/Gefitinib.html chicken embryos are generally floating on their left side, these embryos were included to increase group homogeneity and limit the variation in the angle of insonation. To ensure accurate probe positioning B-mode ultrasound was used (Fig. 1c). Power Doppler mode was used to visualize the blood flow in the cross section through the heart (Fig. 1d). After identifying these flows, the Doppler sample volume was placed in the centre of the blood stream to obtain flow velocity waveform recordings. From the obtained velocity waveforms we determined the heart rate (bpm; beats http://en.wikipedia.org/wiki/NK_cells per minute), peak velocities (cm s?1), velocities integrated over time (VTI; cm) and wave times (ms) (Fig. 2a). The Doppler angle was arbitrarily set to zero as flow direction could not be assessed accurately. Therefore, the Doppler velocities should be interpreted as relative velocities, which more precisely are the vectorial velocities in the direction of the ultrasound beam. From the cardiac cycle the ��Passive�� (P) wave, Phase I, and the ��Active�� (A) wave, Phase II of ventricular filling, and the ejection wave, Phase III of the cardiac cycle, were determined (Fig. 2a). Wave times were additionally calculated in percentages of total cycle time to find proportional changes of the three phases of the cardiac cycle between homocysteine and control embryos. For each embryo we analyzed three consecutive cardiac cycles. Embryo temperature was monitored and collected simultaneously with the Doppler data. The data are presented as mean (standard deviation; SD). To determine heart rate and temperature variance within embryos during the experimental time span, analysis of variance (One-Way ANOVA) was performed. All other statistics were carried out using the independent-samples t test. Statistical significance was defined as a P value of