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PLoS ONEAnimal Behavior & CognitionPeer-reviewed journal article

Zebrafish facility report on implementation of artificial plants as structural enrichment

Aymene Youcef Krachni, Richard Busch, Indigo Brakus, Angelina Schumann, Jenny WilzopolskiiD, Nils OhnesorgeiD

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Abstract

There is a broad consensus that husbandry conditions of laboratory animals need constant improvement to guarantee optimal animal welfare and research data quality. Zebrafish (Danio rerio) as one of the main animal models in biomedicine and toxicology are currently kept in barren tanks in most experimental setups, as well as in animal husbandry. Structural enrichment with artificial plants is currently discussed as a potential refinement measure to provide a more diverse environment. It was shown, that this can reduce stress and improve cognitive abilities, survival rate and fertility in these animals. Still, concerns remain regarding its long-term benefits and drawbacks. Therefore, we introduced artificial plants in our husbandry tanks, and evaluated over a one-year period if in our specific system, the benefits would outweigh the risks. Comparing six different lines of zebrafish raised in enriched tanks or under our standard conditions, we saw no significant difference in terms of sex ratio, fertility or pathogen burden. Enrichment increased the survival rate of 4 % during upbringing, albeit not statistically significant due to high variation. When analyzing zebrafish behavior in their 8 L home-tanks, we found less zebrafish in areas close to the plants but rather a preference for the open water in the middle or opposite side of the tank. This effect was even more pronounced at lower holding densities of less than 4 fish per liter. In summary, we found that introducing structural enrichment to our zebrafish facility carried low cost and no detrimental effects for the animals but at the same time benefits were difficult to determine in our readouts as our survival rates already were very high without these measures. We like to encourage other animal facilities to evaluate enrichment measures to ensure a broader discussion on long-term benefits of structural enrichment in zebrafish husbandry systems.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (24 Feb 2026) · CC BY 4.0

Across six zebrafish lines over one year, artificial home-tank plants left sex ratio, fertility and microbial counts unchanged, while fish avoided plant areas.

Few zebrafish facilities adopt structural enrichment, citing workload, cost, pathogen risk and doubts that short studies on single or paired fish apply to group-housed husbandry. This facility report follows artificial plants for one year in standard home tanks, using only routine, non-invasive readouts: survival, sex ratio, mating, water microbial load and place preference.

Key results

  1. ShownArtificial plants on the left wall of 8 L home tanks did not draw shoaling fish in. Instead, the fish moved toward open water and the opposite wall. The shift was larger during human activity than when fish were undisturbed. The plants did not reduce how many fish could be seen.

    Human present: left:right 49:51 standard vs 36:64 enriched; left:middle:right 24:48:28 vs 14:45:41. Undisturbed: 50:50 vs 44:56 and 27:43:30 vs 20:47:33 (n = 36). Fish detected: 83 +/-9 % in both tank types under direct observation; 74 +/-12 % vs 75 +/-9 % on video. · Observation of fish/plant interaction; Results; Figure legends

  2. SuggestedIn enriched tanks of lines 3 to 6, fish avoided the plant side more at lower holding densities, where more of them gathered at the opposite wall. The plant area held a larger share of fish at the two higher densities than at the two lower ones.

    With humans present, 14% and 7% of fish were in the plant area at 2.5 and 3.5 fish/L vs 28% and 23% at 4.1 and 5.5 fish/L. Over 50% were near the right wall at low density vs 30-35% at higher density. Undisturbed: 43% at 2.5 fish/L stayed near the right wall vs 31-34% in other lines (n=9). · Observation of fish/plant interaction; Results; Figure legends

  3. SuggestedRearing with plants gave a small survival gain that was not statistically significant. Survival in the standard tanks matched the facility's historical baseline.

    Mean survival 74 % standard vs 77 % enriched (historical mean 73 %). The enriched tank did better in 6 of 8 pairs; paired differences ranged from -16 % to +20 % (mean 4 %, p = 0.353). 16 tanks from 6 lines. · Results; Discussion; Figure legends

  4. ShownSex ratio, mating success and clutch size did not change, either with plants in the home tank during rearing or with a plant added to the mating tank.

    Females 60 % standard vs 66 % enriched. First and second matings: success 64 +/-20 % and 75 +/-20 % (standard) vs 61 +/-24 % and 82 +/-19 % (enriched); eggs 72 +/-43 and 127 +/-56 vs 68 +/-55 and 122 +/-58 (n = 7-8). Plant in Tübingen wildtype mating tanks: 135 vs 149 (plain) and 124 (gravel picture) eggs, p = 0.564, n = 25 per group. · Mating; Results; Figure legends

  5. SuggestedThe plants did not raise the microbial load of tank water. Biofilm grew on the suction cup rather than on the plants. The plants were cheap and needed little extra work to maintain.

    Mean CFU/mL with vs without plants: 4633 vs 5673 (line 1a) and 2387 vs 14620 (line 1b), over three occasions; repeated-measures one-way ANOVA p = 0.446. Sump mean 8843 CFU/mL. Cost 3-5 Euro per tank; about one person-hour per month for 50 enriched tanks. · Total microbial count; Results; Discussion; Figure legends

Methods, data and reproducibility

Fish from six lines were reared in adjacent tanks with or without bundles of non-leaching polyethylene plants. Survival, sex ratio, the first two matings, plate-count microbial load and home-tank place preference (fish counted per tank zone in photos and time-lapse frames) were compared with t-tests and ANOVA.

  • Six zebrafish lines (elavl3/HuC GCaMP and Gal4/UAS:RFP transgenics, mitfa^w2, Tübingen and Tüpfel-Longfin wildtypes) reared in paired enriched vs standard tanks · zebrafish (Danio rerio) · n = 16 tanks (6 lines)
  • Home-tank photos of lines 1 and 2 in 8 L tanks, scored by left/right and left/middle/right zones, with human activity or undisturbed (time-lapse) · zebrafish (Danio rerio) · n = 36 per condition; 4 tanks per condition
  • Enriched 8 L tanks of lines 3, 4, 5 and 6 at different holding densities, scored for place preference · zebrafish (Danio rerio) · n = n=9 images per tank; 2.5 to 5.5 fish/L
  • First two matings of fish reared with vs without plants (mating success, egg counts) · zebrafish (Danio rerio) · n = n = 7-8
  • Tübingen wildtype matings in tanks with a plain beige base, a gravel picture, or a gravel picture plus a plant · zebrafish (Danio rerio) · n = 25 per group
  • Total microbial counts of tank water (lines 1a and 1b, with vs without plants) and of the facility sump · n = lines 1a and 1b, with and without plants, three occasions
  • PubMed sample of the most recent zebrafish research articles, checked for any mention of enrichment · n = 100 articles
Methods & tools
GraphPad Prism 10.1.2, NC3Rs online power calculator (R 3.5.2, power.t.test), Shapiro-Wilk test and QQ plots, ROUT outlier detection (FDR 1%), Paired/unpaired t-tests, one- and two-way ANOVA, Tukey's test, Plate count agar total microbial count (Mast Group DM 195), ZIRC Embryo Surface Sanitation bleaching protocol, Olympus SZX16 stereo microscope, Tecniplast Active Blue standalone husbandry rack, Dohse Aquaristik polyethylene artificial plants (Hygrophila, Vallisneria, Rotala, Bacopa), Printed gravel pictures under tanks (Supplement 3)
Reagents & environment
GraphPad Prism 10.1.2; sample sizes from the NC3Rs online tool using R 3.5.2 and power.t.test (alpha 0.05, power 0.90).
Reproducibility gaps
No code or data repository is given. Raw rearing and mating tables are only in Supplement 2, the place-preference images and videos are not stated as shared, and the clutch size vs age analysis is reported as data not shown.
Limitations (6)
  • Survival varied much more than planned, mostly because of a poor-quality line 6 clutch that was kept in the data. The study therefore lacked the samples to test whether the 4 % mean survival gain is real; a larger dataset is needed. · stated by the authors
  • Aggression and territorial behaviour were not measured systematically. Single fish were occasionally seen guarding the plants and chasing others away. · stated by the authors
  • The facility's strong baseline (high hygiene, monitored water quality, gravel pictures, likely low stress) may have left little room for artificial plants to show measurable benefit. · stated by the authors
  • Cognitive effects were not tested, and older fish (> 15 mpf) were not studied. It is unresolved whether the remaining shift in undisturbed fish reflects the physical volume of the plants or a real change in behaviour. · stated by the authors
  • The density analysis uses one enriched tank per line, so holding density is confounded with genotype and rearing history (fish were merged from two tanks). Each estimate rests on 9 images. · noted in this summary
  • Microbial load was compared in only two tank pairs over three occasions. Place preference was scored from fish positions in still frames rather than tracked movement, which limits sensitivity. · noted in this summary

Read this if you run a zebrafish facility and want long-term, home-tank data on the costs, hygiene risks and behavioural effects of adding artificial plants.

Citations
1
FWCI
3.38
Field percentile
top 9%

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