A new system for the rapid collection of large numbers of developmentally staged zebrafish embryos

Abstract

The zebrafish is an excellent genetic and developmental model system used to study biology and disease. While the zebrafish model is associated with high fecundity, its reproductive potential has not been completely realized by scientists. One major issue is that embryo collection is inefficient. Here, we have developed an innovative breeding vessel designed to stimulate the natural reproductive behavior of the fish. This novel apparatus allows us to collect large numbers of developmentally synchronized embryos in brief and defined windows of time, and with minimal investments in labor and space. To demonstrate the efficacy of this approach, we placed three separate groups (n = 180) of fish in the vessel and allowed them to spawn for 10-minute intervals. During these trials, which were repeated three times, the fish produced 8600±917, 8400±794, and 6800±1997 embryos, respectively. This level of embryo production is nearly twice what we were able to achieve when using conventional crossing equipment with some of the same fish, and it required significantly less room and time to set up and break down. This system overcomes major space and labor restrictions inherent in spawning equipment currently used in the field, and will greatly accelerate efforts to improve the scale and throughput of experiments.

Figure 1. Architecture of the zebrafish breeding vessel.

(A) The three primary components of the breeding vessel. (B) Framework of the bottom or “floor” of the spawning platform, showing variation in topography. (C) The breeding vessel, with all three primary components engaged and ready for operation.

Figure 2. Schema depicting the operation of the breeding vessel, cross-sectional view.

(A) The breeding vessel is filled with conditioned water, and fish are added to it so that female fish and male fish are contained within the spawning platform, below and above the separator, respectively. (B) The separator is removed and the male and female fish swim together in deep water. (C) The platform is raised within the outer chamber so that the male and female fish swim together and spawn in shallow water. The fertilized embryos fall through the floor of the spawning platform. (D) After the fish are removed from the breeding vessel, the fertilized embryos that have settled at the bottom of the outer chamber are collected.

Figure 3. Quantitative assessment of embryo production and viability.

(A) The average number of embryos produced in the breeding vessel during a 10 minute interval in separate populations of three zebrafish strains; (error bars, s.d.; n = 3). (B) The average viability of embryos produced in the breeding vessel during a 10 minute interval in separate populations of three zebrafish strains: (100 embryos sampled for each event; error bars, s.d.; n = 3).

Figure 4. Embryo production over sequential shallow and deep water intervals.

(A) The average number of embryos produced in the breeding vessel during five sequential intervals in one population of zebrafish; (error bars, s.d.: n = 3). s1 = 0–10 minutes post-release, shallow water, d1 = 10–70 minutes post-release, deep water, s2 = 70–80 minutes post-release, shallow water, d2 = 80–140 minutes post-release, deep water, s3 = 140–150 minutes post-release, shallow water. (B) The average rate of embryos produced per minute during five sequential intervals in one population of zebrafish; (error bars, s.d.: n = 3).