In-ovo imaging using ostrich eggs: Biomagnetism for detection of cardiac signals and embryonal motion

Abstract

In-ovo imaging using ostrich eggs has been described as a potential alternative to common animal testing. The main advantage is its independence from small animal imaging devices as ostrich eggs provide good image quality on regular CT, MRI, or PET used in examinations of humans. However, embryonal motion during dynamic imaging studies produce artifacts. The aims of this study were (1) to explore the feasibility of biomagnetism to detect cardiac signals and embryonal motion and to use these findings (2) to investigate the effect of isoflurane anesthesia on ostrich embryos. A standard magnetoencephalography developed for brain studies was used to detect embryonal signals of ostrich eggs on developmental day 34. Signals were instantly shown on a screen and data were also postprocessed. For assessing the effects of anesthesia, nine ostrich eggs were investigated using isoflurane 6% for 90 min. Biomagnetic signals were recorded simultaneously. A control group consisting of eight different ostrich eggs was also investigated. Cardiac signals similar to electrocardiography were observed in all eggs. Postprocessing revealed frequent motion of embryos without anesthesia. The exposure to isoflurane led to a significant decrease in motion signals in 9/9 ostrich embryos after 8 min. Motion was significantly reduced in the isoflurane group versus control group. There were no isoflurane-related deaths. This study shows that biomagnetism is feasible to detect cardiac signals and motion of ostrich embryos in-ovo. Application of isoflurane is safe and leads to a rapid decrease in embryonal motion, which is an important prerequisite for the implementation of in-ovo imaging using ostrich eggs.

Keywords: In-ovo imaging; alternative animal testing; biomagnetism; isoflurane anesthesia; ostrich eggs.

Figure 1.

A total of 36 channels (nine detectors with four channels each) of biomagnetic signals of an ostrich egg on DD 31 over 30 s (x-axis). Examples of periodically occurring spikes are depicted in Detectors 20 and 23. Detectors 18 and 22 show noisy signals without the detection of spikes. At 2377 s, an artifact produced a high amplitude signal in all channels. (A color version of this figure is available in the online journal.)

Figure 2.

MEG-system setup. (1) The helium-cooled MEG system detects magnetic field changes by multiple SQUID, arranged in a semi-concentric pattern similar to a helmet, so-called dewar (2). The gas-tight system (3) holding the ostrich egg is set on a stretcher (4) and is connected to four gas tubes: (a) an air supply, optionally equipped with a vaporizer (5) for narcotic gases (e.g. isoflurane). (b) Outflow and exhaustion of air and – if applied – narcotic gases is realized by a wall-mounted gas exhaustion system for general anesthesia. (c) Part of the gas in the gas-tight system holding the ostrich egg is analyzed by a gas monitoring system and subsequently re-transferred into the gas-tight system (d). Photography of the air-tight system holding the ostrich egg is seen in (6). (A color version of this figure is available in the online journal.)

Figure 3.

A total of 19 MOG channels over time of an ostrich egg on DD 31 identifying periodically occurring low-amplitude spikes (yellow arrows), representing cardiac signals. In addition, signals with larger amplitude and longer duration of signals are depicted (red arrows) representing embryonal motion.

Figure 4.

Time schedule for isoflurane anesthesia (Group A) and control group (Group B). (A color version of this figure is available in the online journal.)

Figure 5.

Heart rate over time of an ostrich egg on DD 34. The black arrow marks the beginning of isoflurane anesthesia at 15 min.

Figure 6.

Motion analysis of two ostrich eggs of Group A (intervention group) with isoflurane (a–c) and of Group B (control group) without exposure to isoflurane (d–f) on DD 34. (a) Time-Frequency-Spectrogram, representing power (color scale) of signals at different frequencies (y-axis) over time (x-axis). After 15 min, isoflurane 6% was started (gray dashed line) and after 90 s, signal intensity decreased and did not increase during constant isoflurane exposure. (b) Graph representing dichotom scale classification of either positive (colored box) or negative (no box/white) of different frequencies bands at 0.05 Hz intervals (range 0–3 Hz) over time. Each frequency band is represented by a different color. The threshold for differentiating positive from negative signals was defined at a power spectral density (PSD) of 200 pT2/Hz. Data were analyzed with a time resolution of 1 min. (c) Based on the sum of positive frequency bands, see (b), mean embryonal activity was calculated during resting phase (0–15 min) and interventional phase (15–90 min). Both values were graphically shown and connected using a sigmoidal curve fitting (red graph). The inflection point of this sigmoid curve function was determined as the time point of successful immobilization (black dashed line). (d) Analogous to (a): Ostrich egg (Group B; control group) without isoflurane exposure. Note the missing decline of signal intensity after 15 min. (e) Analogous to (b): Note the constant positive signals above 90 min without distinct discrimination of resting phase and interventional phase. Periodical phases of higher (around 250 s) and lower (1800–2100 s) activity. (f) Analogous to (c): Note the higher embryonal activity above 90 min with only slight decrease after 1300 s, attributable to continuous cooling. Comparing (c) and (f), the graph in (f) has much higher values on y-axis.

Figure 7.

Mean heart rate change of different time intervals during interventional phase of Group A (isoflurane exposure, black dots) and Group B (control group, white dots). Mean heart rate during time intervals of 40–50, 50–60, 60–70, 70–80, and 80–90 min is compared to the mean heart rate during resting phase of 0–15 min. **p < 0.01, ***p < 0.0001.

Figure 8.

Level of activity during resting phase and interventional phase of Group A (isoflurane exposure, black dots) and Group B (control group, white dots). **p < 0.01; n.s. not significant.