Zebrafish as a research model

Transcription

1 March , Bergen-Norway Lab Animal Science: Special Focus on Fish Zebrafish as a research model Tommaso Sala Customer care Manager Aquatic Line Tecniplast - Italy

2 Contents Laboratory aquatic species Some numbers Why Zebrafish? Applications in research Zebrafish and the 3Rs

3 Laboratory aquatic species Environment: FRESH vs SALTY

4 Laboratory aquatic species Phylum: INVERTEBRATE vs VERTEBRATE Atlantic Lobster Homarus americanus Oyster Crassostrea virginica

5 Laboratory aquatic species Zebrafish Brachydanio rerio Oncorhynchus mykiss(rainbow Trout) Salmo salar (Salmon)

6 Laboratory aquatic species Class amphibia Hello! Order ANURA URODELES APODA

7 Laboratory aquatic species Plurodeles Waltl Axolotol Xenopus Spp.

8 Laboratory aquatic species Class Chondrichthyes Teleostei (Actinopterygii) Order Cypriniformes Family: cyprinidae Salmoniformes Family: salmonidae

9 Some numbers

10 Some numbers Figure 1.1 Classes of animals Guinea-Pigs 2,11% Rats 21,5% Other rode nts 1,04% Rabbits 2,49% Cold-blooded animals 15,4% Birds 4,99% Year 02 / 99 Rodents: -3,26% Cold blooded animals: + 2,5% Mice 50,9% Artio+perissodactyla 1,18% Carnivores 0,28% Prosimians+Monkeys +Apes 0,10% Other mammals 0,03%

11 Some numbers Numberofanimalsusedforscientificpurposesin 1975 and 2002 (% oftotal animalused) SPECIES % change Mice 1,174,350(43,51%) 759,790 (36,1%) -35% Rats 594,678 (22,03%) 332,065(15,78 %) -44% Birds 293,563 (10,88%) 117,958 (5,60 %) - 60 % Fish 130,984 (4,85%) 607,367 (28,87%) %

12 Some numbers

13 Some numbers Zebrafish Researcher Worldwide Researcher US Labs worldwide Labs US

14 Why Zebrafish?

15 Why Zebrafish? Taxonomy - Kingdom: Animalia - Phylum: Chordata - Subphylum: Vertebrata - Class: Actinopterygii - Subclass: Neopterygii - Superorder: Ostariophysi - Order: Cypriniformes - Family: Cyprinidae - Subfamily: Rasborinae - Genus: Danio - Species: rerio Hamilton-Buchanan, 1822

16 Why Zebrafish? Genus & species albolineatus choprae dangila feegradei Danio kerri kyathit muongthanhensis nigrofasciatus rerio roseus

17 Why Zebrafish? Characteristics of the species Females Generally more rounded abdomen Less of a color variation Bigger size between the two Males More torpedo shaped body Anal and Caudal fin tend to have a yellow tint

18 Why Zebrafish? Characteristics of the species Upward mouth

19 Why Zebrafish? Native countries

20 Why Zebrafish? Habitat preferences Rivers Swamps Ponds Rice paddies

21 Why Zebrafish? Habitat preferences Shallow water

22 Why Zebrafish? Advantages relatively simple vertebrate -CNS less complex easy to maintain; low costs each female produces >200 eggs fertilisation is external; fast development ex-utero embryos are transparent genome project almost finished

23 Why Zebrafish? Advantages Extremely high housing density

24 Why Zebrafish? Advantages Embryos transparency

25 Why Zebrafish? Advantages Zebrafish dimensions Adult =maximum 4/5 cm Fry =about 1 mm after hatching

26 Why Zebrafish? Advantages Zebrafish embryogenesis

27 Why Zebrafish? Advantages Periods of early development -Zygote -Cleavage -Blastula -Gastrula - Segmentation - Pharyngula -Hatching -Early larva

28 Why Zebrafish? Advantages Comparison embryonic development zebrafish vs mouse 1 dpf 21 dpf Karlstrom RO, Kane DA (1996). A flipbook of zebrafish embryogenesis. Development 123:461 made by Brad Smith

29 Why Zebrafish? Advantages Easy to breed in lab +

30 Why Zebrafish? Advantages Easy to breed in lab Photo induced breeding cycle 14 hrs light / 10 hrs dark Light on: ±30 min later

31 Why Zebrafish? Advantages Easy to breed in lab

32 Applications in research Who had the idea? George Streisinger

33 Applications in research Fields of research Cell Biology Molecular Biology Developmental Biology Embriology Genetics Toxicology/ EcoToxicology

34 Applications in research Example Morpholino technology Sintetic oligonucleotide that works through antisense technology: BindmRNAtarget Stop the translation at the beginning High specif sequence specificity Wellsolublein water Resistant to nucleasi AAAAAAA AUG No protein (gene knockdown)

35 Applications in research Example Morpholino technology: micro-injection in fertilized zf eggs

36 Applications in research Example Morpholino technology: micro-injection in fertilized zf eggs 24 hrs later

37 Applications in research Example Morpholino technology: Heart development (72hpf) after fxr1 MO injection Speed 1/6 Normal 190 beats/ min fxr1 MO 70 beats/ min

38 Zebrafish and the 3Rs Replacement Russell & Burch Whenever possible, animals should be replaced by non animal alternatives(such as in vitro toxicology tests). It also includes the possibility to choose an animal with a less complex nervous system. Reduction The number of animals used in research should be reduced through careful planning and proper experimental design, which allows investigators to use the smallest number of animals possible to obtain statistical significance. Refinement When there is no alternative to the use of live animals in research, the procedures and housing conditions they are subjected to should be refied to minimize pain and distress.

39 Zebrafish and the 3Rs: Replacement Mammals A licence is required to conduct regulated procedures from half-way through the gestation period. Fish A licence is required from the time they become capable of independent feeding (rather than from yolk reserve), which is in zebrafish accepted to be at5d.p.f. Within 72 hours fry attain an adult-like stage. By4d.p.f.theyarealreadyabletoseeandswim. At unlicensed stages, non neuronal organs(heart) are well developed. The CNS remains relatively primitive: visual and auditory stimuli are only apparent from 5 d.p.f. onwards: higher brain development is delayed.

40 National Animal Ethics Advisory Committee (2003 Annual report)

41 Zebrafish and the 3Rs Refinement Advantage of size and transparency of zebrafish larvae (adults) to perform similar procedures as those performed in mammals at licensed stages but using less invasive methods.

42 Zebrafish and the 3Rs Refinement Animal welfare improvement Environmental enrichment Rodents Fish?

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44 Zebrafish and the 3Rs Refinement FOCUS on: Water quality Water quality is the most important factor in maintaining the well-being of fish and in reducing stress and the risk of disease. Water-quality parameters should at all times be within the acceptable range that sustains normal activity and physiology for a given species. Housing conditions Fish behaviour will influence stocking density and schooling or territorial behaviour should be considered. Measures should be taken to avoid or minimise conspecific aggression without otherwise compromising animal Welfare.. In principle, groups should consist of fish of the same size to minimize the risk of injuries or cannibalism.

45 Zebrafish and the 3Rs Reduction Large number of animals are used in the drug discovery process. Compounds having an activity in vitro must be tested in animal models. There is often a failure to reproduce in vitro results in vivo due to problems with adsorption, distribution, metabolism and excretion that cannot be predicted in cell-based models. Zebrafish can provide a cost-effective model to bridge the gap between in vitroandinvivowork.

46 Zebrafish and the 3Rs Reduction Normal 190 beats/ min fxr1 MO 70 beats/ min Speed 1/6

47 Zebrafish and the 3Rs Reduction Angiogenesis studies Inflammatory process Toxicology assays

48 Conclusions Zebrafish is now a validated research model Zebrafish will become an important tox model From base research into Pharma

49 Thank you very much for your attention!