A New System for the Rapid Collection of Large Numbers of Developmentally Staged Zebrafish Embryos

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 86006917, 84006794, and 680061997 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.Stem Cell and Regenerative Biolog

A novel method for rearing first-feeding larval zebrafish: Polyculture with Type L saltwater rotifers (Brachionus plicatilis)

Promoting high rates of growth and survival can be a major challenge in zebrafish culture, especially during the first-feeding stage. Here we describe a new rearing technique in which zebrafish larvae are polycultured in static tanks with Type “L” saltwater rotifers (Brachionus plicatilis) for the first 5 days of feeding (days 5–9 postfertilization). To demonstrate the effectiveness of this technique, we conducted rearing trials using fish from two different strains: AB and nacre. Growth, survival, water quality, and rotifer density were assayed daily through the polyculture phase (days 5–9), and during the transition to standard rearing conditions (days 10–12). After that point, once the fish were fully integrated onto recirculating systems, parameters were measured once per week out to day 30. In all trials, the fish displayed high rates of growth and survival throughout the three phases (polyculture, transition, and recirculating flow), indicating that this method may be employed during the critical first-feeding stage to help improve rearing performance in zebrafish facilities. Additionally, water quality parameters observed during the polyculture phase of the trials reveal that early zebrafish larvae are much more tolerant of elevated levels of ammonia and salinity than previously believed.Jason Best, Isaac Adatto, Jason Cockington, Althea James, Christian Lawrenc

Measurement of reproductive performance in Tü wild-type fish, F₁ generation.

Reproductive performance for the F1 (A, B) AB wild-type strain and (C, D) Tü wild-type strain. (A) Fecundity defined as the total number of live embryos spawned and (B) viability defined as the percentage of live embryos at 24-hours post fertilization. Data shown as the mean ± standard deviation. No significant differences were measured in either fecundity or viability, n = 3 represented by 30 fish from each growth treatment.</p

Growth curves of two F₀ wild-type strains from 5 to 60 DPF.

Mean total body length for two F0 generation wild-type strains, (A) AB and (B) Tü. Growth curves are represented by mean ± standard deviation. Open circles = LF, closed squares = HF.</p

Initial reproductive performance of fish from LF and HF.

Values recorded at first successful group spawn of 35 fish. Fish were allowed to spawn for a total of 30 minutes.</p

Growth Curves of two F₁ wild-type lines from 5 to 90 DPF.

Growth curves for two F1 generation wildtype lines (offspring of F0), (A) AB and (B) Tü from 5 to 90 DPF. Slow growth curves are represented by mean ± standard deviation. Open circles = LF from F0 LF parents, closed squares = LF from F0 HF parents.</p

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Post spawn total fish weight (mg).

Total fish weight after each spawning trial is represented by • for fish in the HF group and □ for fish in the LF group. A = AB–F0, B = Tü–F0, C = AB–F1 and D = Tü–F1. No significant differences are reported in weight over the 8 weeks fish were spawned. (TIF)</p

Rotifer & <i>Artemia</i> delivery per tank per day.

(A) Daily rotifer concentration delivered per growth treatment, excluding weekends (day 8 & 9). Open circles = LF, closed squares = HF. (B) Daily Artemia nauplii delivered per growth treatment, excluding weekends. Open circles = LF, closed squares = HF.</p

Measurement of reproductive performance in AB wild-type fish, F₀ generation.

Reproductive performance for the F0 (A, B) AB wild-type strain and (C, D) Tü wild-type line. (A) Fecundity is defined as the total number of live embryos spawned and (B) viability is defined as the percentage of live embryos at 24-hours post fertilization. Data shown as the mean ± standard deviation. Significant differences at each week are noted with * indicating p ≤ 0.05, ** indicating p ≤ 0.01, and *** indicating p ≤ 0.001 by successive two-tailed T-Test, n = 3 represented by 30 fish from each growth treatment.</p