White paper – Is the EggSorter biocompatible with Zebrafish embryos?

White paper – Is the EggSorter biocompatible with Zebrafish embryos?

Have you ever thought of the impact of the EggSorter in the development of your zebrafish embryos?

This white paper presents a set of trials evaluating the safety and efficacy of the EggSorter in standardising the sorting process of zebrafish embryos.

Results demonstrate that the use of the EggSorter does not induce adverse effects on the development of zebrafish embryos.

This white paper presents a set of trials evaluating the safety and efficacy of the EggSorter in standardising the sorting process of zebrafish embryos.

Results demonstrate that the use of the EggSorter does not induce adverse effects on the development of zebrafish embryos.

      ABSTRACT

      In the past decade, the zebrafish has become an increasingly powerful experimental model in basic biomedical research. These fish represent a viable and cost-effective alternative to mammalian animal models as they possess many favourable qualities, including optical transparency, the ability to be produced on demand in large quantities, and their early developmental stages are generally not subject to regulations for animal study in most locations. Together, these and other features make the zebrafish model one that is uniquely suitable for high throughput studies.

      In an effort to capitalise on the potential of this system, Bionomous has developed the EggSorter, a device that automates sorting and dispensing of the zebrafish embryos prior to hatching. The automation of these processes saves labour and improves reproducibility compared to traditional manual techniques.

      In this white paper, we describe a set of trials that evaluated the performance of the EggSorter and its impact on the health and well-being of zebrafish embryos run through the device. Specifically we examined whether or not 1) manipulation by the device would influence the rate of development, 2) contact and exposure within the device would result in any toxic side effects on the embryos, and 3) repeat runs of the same embryos through the device would have any deleterious impacts. We observed no significant effect on any of the three measured conditions on zebrafish embryos up to 120 hours post-fertilisation.

      These results suggest that the EggSorter can be used safely as a tool to increase the efficiency and standardise the sorting of zebrafish embryos, as it does not induce any negative effects on their development. 

      MAIN FIGURES

      Scroll lateraly below to explore the main figures of the white paper.

      Figure 2: Comparison of health status of zebrafish embryos at 120hpf. Each replicate has an EggSorter group (embryos handled by the device) and a control group (embryos handled manually with a transfer pipette). Significant changes were calculated between percentages of healthy embryos within replicates using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 2: Comparison of health status of zebrafish embryos at 120hpf. Each replicate has an EggSorter group (embryos handled by the device) and a control group (embryos handled manually with a transfer pipette). Significant changes were calculated between percentages of healthy embryos within replicates using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 3: Comparison of the health status at 120 hpf of all conditions among the three replicates. Significant differences were calculated between each condition and its corresponding Control condition (negative control) using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant differences are to be noted.
      Figure 3: Comparison of the health status at 120 hpf of all conditions among the three replicates. Significant differences were calculated between each condition and its corresponding Control condition (negative control) using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant differences are to be noted.
      Figure 5: Comparison of swim bladder inflation in zebrafish embryos at 120 hpf. Significant changes were calculated between percentages of healthy embryos within replicates using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 5: Comparison of swim bladder inflation in zebrafish embryos at 120 hpf. Significant changes were calculated between percentages of healthy embryos within replicates using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 6: Comparison of health status of zebrafish embryos at 120 hpf for multiple runs in the EggSorter. “EggSorter 1x” means that embryos were sorted and dispensed once in the device; “EggSorter 2x” means that embryos were sorted and dispensed twice in a row; and “EggSorter 3x” means that embryos were sorted and dispensed three times in a row in the EggSorter. Significant changes were calculated between percentages of healthy embryos of the control group against the other conditions using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 6: Comparison of health status of zebrafish embryos at 120 hpf for multiple runs in the EggSorter. “EggSorter 1x” means that embryos were sorted and dispensed once in the device; “EggSorter 2x” means that embryos were sorted and dispensed twice in a row; and “EggSorter 3x” means that embryos were sorted and dispensed three times in a row in the EggSorter. Significant changes were calculated between percentages of healthy embryos of the control group against the other conditions using Fisher’s exact test, * (p≤0.05), ** (p≤0.01), *** (p≤000.1), with a significance level of α=0.05. No significant changes are to be noted.
      Figure 1: Conditions of the experiment. For the viability experiment, only the conditions “EggSorter” and “Control” were used. For the toxicity experiment, all four conditions (“EggSorter”, “EggMech”, “Control” and “EggTox”) were used. The “EggSorter” condition is defined by the embryos being handled by the EggSorter and the water coming out of the EggSorter is kept in the wells. The “EggMech” condition is with system water and embryos handled by the EggSorter. The “Control” condition is defined by embryos handled by hand and the water in the wells is system water. Finally, the “EggTox” condition is given by embryos being handled manually and water from the EggSorter is placed in the wells.
      Figure 1: Conditions of the experiment. For the viability experiment, only the conditions “EggSorter” and “Control” were used. For the toxicity experiment, all four conditions (“EggSorter”, “EggMech”, “Control” and “EggTox”) were used. The “EggSorter” condition is defined by the embryos being handled by the EggSorter and the water coming out of the EggSorter is kept in the wells. The “EggMech” condition is with system water and embryos handled by the EggSorter. The “Control” condition is defined by embryos handled by hand and the water in the wells is system water. Finally, the “EggTox” condition is given by embryos being handled manually and water from the EggSorter is placed in the wells.

      CONCLUSION

      Bionomous has developed a device, the EggSorter, to automate and standardise the sorting process of zebrafish embryos. The aim of this white paper is to certify that this device does not influence the viability and development of zebrafish embryos.

      Three experiments were conducted: a viability test of zebrafish embryos at 120 hpf, a toxicity test from 5 to 120 hpf, and an additional viability test at 120 hpf with embryos running up to three times in the device.

      The first viability experiment showed that there was no measurable difference in the survival of zebrafish embryos subjected to manual handling vs. handled by the EggSorter. The toxicity test demonstrated again no significant differences between the four tested conditions, meaning that the contact and exposure to the device does not have an effect on the development of the embryos. Moreover, results showed that even when a batch of embryos is more fragile, the EggSorter does not induce more stress to their development. There were also no differences in the occurrence or frequencies of malformations between embryos sorted manually or with the EggSorter. A delay in swim bladder inflation (7% observations in total) was observed, but it was distributed equally across the conditions. The last experiment testing multiple runs in the EggSorter did not result in any significant differences compared to the control group.

      In conclusion, these results show that the EggSorter does not induce any negative effects on embryonic development, compared to manual sorting and pipetting methods. Thus, the Eggsorter from Bionomous can be used as a tool to safely accelerate and standardise the screening, sorting and dispensing of zebrafish embryos. 

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