Different narcotic gases and concentrations for immobilization of ostrich embryos for in-ovo imaging

Abstract

In-ovo imaging using avian eggs has been described as a potential alternative to animal testing using rodents. However, imaging studies are hampered by embryonal motion producing artifacts. This study aims at systematically comparing isoflurane, desflurane and sevoflurane in three different concentrations in ostrich embryos. Biomagnetic signals of ostrich embryos were recorded analyzing cardiac action and motion. Ten groups comprising eight ostrich embryos each were investigated: Control, isoflurane (2%, 4%, and 6%), desflurane (6%, 12%, and 18%) and sevoflurane (3%, 5%, and 8%). Each ostrich egg was exposed to the same narcotic gas and concentration on development day (DD) 31 and 34. Narcotic gas exposure was upheld for 90 min and embryos were monitored for additional 75 min. Toxicity was evaluated by verifying embryo viability 24 h after the experiments. Initial heart rate of mean 148 beats/min (DD 31) and 136 beats/min (DD 34) decreased over time by 44-48 beats/minute. No significant differences were observed between groups. All narcotic gases led to distinct movement reduction after mean 8 min. Embryos exposed to desflurane 6% showed residual movements. Isoflurane 6% and sevoflurane 8% produced motion-free time intervals of mean 70 min after discontinuation of narcotic gas exposure. Only one embryo death occurred after narcotic gas exposure with desflurane 6%. This study shows that isoflurane, desflurane and sevoflurane are suitable for ostrich embryo immobilization, which is a prerequisite for motion-artifact free imaging. Application of isoflurane 6% and sevoflurane 8% is a) safe as no embryonal deaths occurred after exposure and b) effective as immobilization was observed for approx. 70 min after the end of narcotic gas exposure. These results should be interpreted with caution regarding transferability to other avian species as differences in embryo size and incubation duration exist.

Keywords: alternative animal testing; animal model; biomagnetism; in-ovo imaging; magnet-ovography; narcotic gases; ostrich eggs.

FIGURE 1

Time schedule for narcotic gas exposure. Phase “rest” (15 min) allows for reduction of embryonal arousal after egg transport to MEG-facility. This phase is followed by “narcotic gas exposure” (90 min) and a “follow-up phase” (75 min). During rest and follow-up phase, ambient air is inflated into the chamber holding the ostrich eggs. During “narcotic gas exposure,” respective narcotic gas is applied. In control group, during this phase ambient air is inflated. During 180 min MOG, the ostrich egg is held by a container which is located at the head rest (a) of the MEG system. Narcotic gas inflow is provided by specific vapor (b) and narcotic gas outflow (c) is provided by exhaustion system. During the whole experiment, narcotic gas concentration was continuously measured using a gas measurement module (d) for anesthetic care (Scio Four Oxi, Draeger, Luebeck, Germany), ensuring adequate concentration of narcotic gas within the chamber holding the ostrich egg.

FIGURE 2

Embryonal movement using advanced automated movement detection according to [26] in a data set derived from [1]. Left: Boxplot diagram showing level of activity during resting phase (0–15 min; grey) and isoflurane/ambient air (15–105 min; red) in two groups (isoflurane 6% and control) on DD 34. Right: Four Bland-Altmann plots showing adequate agreement of advanced automated movement detection and manual movement detection. The four plots refer to the four boxes in the boxplot diagram. The Bland-Altmann plots compare each value between advanced automated movement detection and manual movement detection. The dots indicate the difference between both methods (y-axis) over absolute value (embryonal movement; x-axis) and the solid line represents the mean value of all dots. Dashed lines indicate 2x standard deviation of all differences. All values are in the range of 2x standard deviation, indicating no significant underestimation or overestimation.

FIGURE 3

Boxplot diagram of heart rate during different time intervals of all 80 ostrich embryos investigated on DD 31 and 34. Significant differences between DD 31 and DD 34 occur within the first 5 min after initiation of narcotic gas exposure, in the middle of narcotic gas exposure and at the end of follow-up. Aggregated data of all groups including control group was deemed appropriate in view of Figures 4, 5, excluding significant differences between control group and narcotic gases. ***p < 0.001.

FIGURE 4

Mean heart rate over time for each narcotic gas and concentration (green: isoflurane, blue: desflurane; orange: sevoflurane). Top row: DD 31; bottom row: DD 34. In each graph, control group is depicted in grey. Dashed lines represent start and end of narcotic gas exposure. In each group, eight ostrich embryos on DD 31 and 34 were investigated, respectively.

FIGURE 5

Boxplot diagram of mean heart rate reduction (difference) between 0–15 min and 151–180 min for each narcotic gas and concentration. Considering DD 31 and DD 34 separately, comparison between control group and each narcotic gas and concentration revealed no significant differences.

FIGURE 6

Mean embryonal movement over time for each narcotic gas and concentration (green: isoflurane, blue: desflurane; orange: sevoflurane). Top row: DD 31; bottom row: DD 34. In both rows, results of control groups are depicted in grey. Dashed lines represent start and end of narcotic gas exposure. Note the light blue graph on DD 31 and DD 34 (desflurane 6%) during narcotic gas exposure (15–105 min) which indicates residual movement whereas the other graphs (desflurane 12%, 18%; sevoflurane, isoflurane) show almost constant 0 values. This figure mainly focuses on visualization of group differences. For quantification of effectiveness of immobilization, see Figure 7.

FIGURE 7

Boxplot diagram of duration between start of narcotic gas exposure and reduction of movements (“time point sleep”; left diagrams) as well as duration between end of narcotic gas exposure and re-appearance of movements (“time point awake”; right diagrams) for ostrich embryos on DD 31 (top row) and DD 34 (bottom row). In order to compare concentration levels of narcotic gases, categories “low,” “medium” and “high” were used as provided by dedicated vaporizers. Note the shorter time until re-appearance of movements for desflurane [as compared to sevoflurane and isoflurane (highest concentrations)] on DD 34. *p < 0.05; **p < 0.01; ***p < 0.001.