Zebrafish as a research model latest in housing
Zebrafish as a research model: latest in housing technologies and husbandry procedures Marco Brocca Market Manager Aquatic Line Tecniplst Spa Italy
THE ZEBRAFISH BOOK A guide for the laboratory use of zebrafish-Danio (Brachydanio) rerio
Members of the carp family Cyprinidae Danio rerio first described to science in 1822 ~ Francis Hamilton indentified 10 Danio species in the River Ganges catchment ~ Late 19 th century already a popular aquarium fish ~ 1960’ – potential as a vertebrate model organism was first realized (Stresinger, Kimmel and co, ) Courtesy of Gregory Paull
Zebrafish Taxonomy • 9 described Danio species • All relatively small • Adult typically 40 mm - 50 mm • Larvae approx 2 mm • Common body shape, fusiform & laterally compressed, terminal oblique mouth pointing upwards D. coprae Adapted from: Parichy, D. M. Current Biology, 2003. Modified from Gregory Paull
Males – More torpedo shaped body – Anal and Caudal fin tend to have a yellow tint Females – Generally more rounded abdomen – Less of a color variation – Bigger size between the two
Zebrafish habitat • Zebrafish typically found inhabiting relatively shallow slow-moving or standing water bodies which may have seasonal connections to the main rivers as well as man-made lakes, ponds, and irrigation channels, constructed for fish and rice cultivation • Name Danio derives from the Bengali name ‘dhani’ meaning ‘of the rice field’ Courtesy of Gregory Paull
Why using zebrafish as a lab model • relatively simple vertebrate • easy to maintain; low costs • each female produces hundreds of eggs • fertilisation is external; fast development • embryos are transparent • Sequenced genome
1968 1993 1990
Housing tanks Drain Pipes Primary solid removal Chemical filtration Reservoir Pump Biofilter Water source Secondary solids removal Water Disinfection Schematic representation of a recirculating system for zebrafish
“RAS” Stand-Alone systems
DIMENSIONS Big enough in order not to stress the animals housed in and dedicated to proper experimental needs Which is the best density value? Are there any indications? More than one size, different needs
Density Management • 5 – 14 Days = 25 Fry per liter • 14 – 30 Days = 12 Fry per liter • 30 + Days = 6 - 10 Fish per liter • This is optimum – This number varies dramatically depending on space and research requirements – This will allow for quick growth and reproduction of the lines – Systems can handle much higher numbers
Grouping Stocking Isolation 2 Isolation 1
Zebrafish prefers group housing If housed in pairs, aggressive behaviours can occur
Sometimes it happens!
The effect of fish density on reproductive performance in laboratory fish 8 labs in the World, more than 2. 000 single pair crosses Zebrafish, 2011; Sept 8(3); 141 - 146
Water quality From the APPENDIX A of ETS 123: “All the enclosure should be kept free of fish waste products or uneaten food. If these are allowed to accumulate, water quality and thus fish health will be adversely affected” At least in Europe they mention it even at tank level
BREEDING TANKS PC PSU UP to 121 °C UP to 134°C
The Zebrafish as a Biomedical Model Organism Attractive Features • External fertilization • Transparent embryo • Rapid development • Vertebrate • Relative small size • High Fecundity
Zebrafish embryogenesis
Fertilization of zebrafish eggs +
Collecting fertilized eggs Breeding: • 1 female + 2 males • refresh 50% water with RO • reduced conductivity Light-dark cycle 14 hrs light / 10 hrs dark within ± 15 min fertilized eggs
Breeding • Can be bred weekly • Breed every 2 – 3 weeks for longer lasting fish • Fish bred every couple of weeks should produce good viable eggs for 18 – 24 months.
Olfactory control of zebrafish spawning. 1. Male zebrafish (orange) releases pheromone that triggers ovulation in the female (blue). 2. The “ripe” female (green) then releases a pheromone that triggers courtship and chasing behavior in male. 3. The “activated” male (green) then courts and chases the ripe female. 4. Oviposition – female (red) releases eggs and male (red) fertilizes them. Courtesy of Chris Lawrence
Breeding: two major aspects for success • Shallow water/gradient • Physical separation
UNTILTED (Sessa et al, . 2008)
Fish enjoying the “beach” Fish in the deep area
Temperature Poikiloterm Body T varies according to the environmental changes The methabolism is function of the temperature
Temperature T T time 25 – 28, 5 °C
p. H more acidic 0 more basic 7 Sudden changes are much more stressful and dangerous than gradual smaller changes (both in quantity and time) 6, 5 - 8, 5 In recirculating system, both nitrification and respiration (of animals and nitrifying bacteria) reduce p. H => it is necessary to buffer the water to prevent p. H from falling too much 14
Conductivity Is the ability of water to carry an electrical charge and is indicative of the amount of electolytes in solution Indicator of salinity and can be used as an efficiency test for R. O. systems In fresh water it is measured in micro. Siemens (�S)
Conductivity 250 - 1900 µS The electrolytes associated to conductivity promote the osmoregulatory functions in aquatic animals In fresh water, conductivity can reach very different values
Dissolved Oxygen 6 - 8 ppm Measure of the gaseous oxigen (O 2) dissolved into a solution Oxigen can mix with water by means of diffusion from the surrounding air, areation (mechanical agitation) and also as a product of the photosyntesis Water temperature can influence the DO levels
Best water quality conditions = best fish health p. H: 7. 0 – 7, 5 Temp: 27 – 28, 5 °C Cond: 600 – 1600 µS Ammonia: < 0, 1 ppm Nitrites: < 0, 2 ppm Nitrates: < 25 ppm
Thanks a lot! Marco Brocca marco. brocca@tecniplast. it
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