Marco Brocca Market Manager Aquatic Line Tecniplst Spa Italy

1. Zebrafish as a research model: latest in housing technologies and husbandry procedures Marco Brocca Market Manager Aquatic Line Tecniplst Spa Italy

10. рерио

13. THE ZEBRAFISH BOOKA guide for the laboratory use of zebrafish-Danio (Brachydanio) rerio

15. 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 19th century already a popular aquarium fish ~ 1960’ – potential as a vertebrate model organism was first realized (Stresinger, Kimmel and co,) Courtesy of Gregory Paull

16. Zebrafish Taxonomy • 9 described Danio species • All relatively small • Adult typically 40mm - 50mm • 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

19. 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

22. 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

24. 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

25. 1968 1993 1990

29. Housing tanks Pipes Drain Primary solid removal Chemical filtration Reservoir Pump Biofilter Water Disinfection Secondary solids removal Water source Schematic representation of a recirculating system for zebrafish

30. “RAS” Stand-Alone systems

33. 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

34. 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

37. Grouping Stocking Isolation 1 Isolation 2

38. Zebrafish prefers group housing If housed in pairs, aggressive behaviours can occur

39. Sometimes it happens!

41. 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

42. 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

43. BREEDING TANKS PC UP to 121 °C PSU UP to 134°C

44. The Zebrafish as a Biomedical Model Organism Attractive Features • External fertilization • Transparent embryo • Rapid development • Vertebrate • Relative small size • High Fecundity

45. Zebrafish embryogenesis

46. Fertilization of zebrafish eggs +

47. 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

48. 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.

49. 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

50. Breeding: two major aspects for success • Shallow water/gradient • Physical separation