ZEBRAFISH, DANIO RERIO AS A

REPLACEMENT ALTERNATIVE

MANGALA GUNATILAKE

Dept. of Physiology

Faculty of Medicine

University of Colombo

Overview:

Introduction Concept of Replacement

Alternatives and Relative

replacement

Objective Sharing information on

scientific attributes of ZF model

and the plan under the Sri

Lankan context

Methods ZF model in CKDu - with ISO

and OECD

Acknowledgements

2. Refinement Alternatives

1. Reduction Alternatives

3. Replacement Alternatives

“Alternative” Any change in an animal test that achieves one or more of the 3Rs Russell &

Burch

in 1959

As per Russell & Burch, REPLACEMENT alternative technique

Any scientific method employing

non-sentient material replacing methods which use

conscious living vertebrates

• Invertebrates and

• Non-mammalian animal models

ABSOLUTE RELATIVE

Abolitionists Animal researchers

Zebrafish, Danio rerio

Relative Replacement

• Fresh water fish

• Cyprinidae family

• India Pakistan Nepal Myanmar Bangladesh • Most popular

vertebrate model organism in ecotoxicology

Introduced to Sri Lankan Researchers at the pre & post Conference workshops of the

Inaugural Scientific Sessions of the SLALAS : 24-26 January 2014

Dr Francois Busquet, PhD

Center for Alternatives to

Animal Testing (CAAT)

Europe Policy Coordinator

• Native fish

• Ornamental purposes only

• Unaware of its scientific importance as a

research model

Zebrafish & fertilized embryo

Genetics

Neuroscience Vertebrate developmental biology Physiology

Toxicology

Biomedicine

Human disease model

Developmental toxicity (teratogenecity)

Mammalian organotoxicity

Wide spectrum of scientific applicability

- 1930s

• Genetic similarity to humans • Small size - Large numbers in a small space • Easy maintenance & economic benefits • Rapid embryonic development

• Life span 2–4 years ………up to 6 years

Attributes for scientific importance

• Embryos are permeable to drugs - easy manipulation

• Development of embryo and larvae – Transparent • External fertilization • High spawning rate

Copyright: R. A. McKee and R. A. Wingert. Zebrafish Renal Pathology: Emerging Models of Acute Kidney Injury. Curr Pathobiol Rep (2015) 3:171–181

Fish Husbandry in a laboratory setting

• Housing

• Water quality

• Purification of water - filters

• Feeding

• Health monitoring and diseases

• Reproduction – conditions required

16

Development Stages of Embryos Period hpf Description

Zygote 0 Newly fertilized egg

Cleavage 0.75 - 2 Beginning of Cleavage cycles

Blastula 2.25 - 4.7 Formation of yolk syncytial layer forms Beginning of Epiboly formation

Gastrula 5.25 - 10 Formation of Epiboly

Segmentation 10 - 22 Somite formation, Organogenensis, Beginning of Movement

Pharyngula 24 - 42 Beginning of Circulation & Pigmentation, Development of Fins

Hatching 48 - 72 Completion of primary organs, Hatching begins

Early larva 72 Inflation of Swim bladder & further development, Capable in swimming in shallow water Digestive tract development

Mid larval Completion of Fins and pigmentation, swim bladder, GIT formation

Four-cell-stage approximately 1 hpf

Segmentation phase approximately 12 hpf

Head

Tail

somites

17

Stages of Embryonic Development of the Zebrafish CHARLES B. KIMMEL, WILLIAM W. BALLARD, SETH R. KIMMEL, BONNIE ULLMANN, AND THOMAS F. SCHILLING. DEVELOPMENTAL DYNAMICS 203: 255-310 (1995)

After 24 h Tail is detached from the yolk and spontaneous movement starts

After 48 h well structured spine, blood circulation and regular heart beats

18

Dr Francois Busquet. Development of a new screening assay to identify proteratogenic compounds using Zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT). PhD Thesis. Toxicology institute of Merck KGaA in Darmstadt, Germany, 2008.

All major organs appear within 36 hpf and hatching takes place 12–36 h later 19

E,F: Protruding-mouth stage (72 h). Progressive increase dorsally in yellow pigmentation owing to xanthophore development, and the progressive filling of melanophores into the lateral stripe. G,H:The early larva (120 h) – Inflation of swim bladder 20

Tail tip Tail

Chorda/Spinal cord Sacculi/ otoliths

Heart

Yolk sac & extension

Eye

Brain

Anus

Zebrafish (Danio rerio) embryo at 72 hpf

Plan in SL – ZF in CKDu

CKD is uniformly distributed in all provinces while CKDu is restricted to a few provinces.

Clinical Kidney Journal, 2015, 1–7

CKDu • First patient in 1994

• Multifactorial

• Over 1,100 CKDu patients are hospitalized per month

in Sri Lanka and 300 deaths recorded per year

• > 70,000 patients in SL

• 50- 60 yrs affected……………..trend is changing

…very young

• Prevalence among farming communities

• Slowly progressing and irreversible

• Asymptomatic until late stages

• Tubulointerstitial fibrosis and tubular atrophy – in renal

biopsies

International Standardization Organization : ISO

15088:2007(E) – Determination of the acute toxicity

of waste water to zebrafish eggs (Danio rerio)

Organization of Economic Cooperation and

Development : OECD TG 236 - Fish embryo acute

toxicity test (FET) with the ZF for the testing of

chemicals

No ethics approval needed

For water quality and toxicity:

Scheme of the zebrafish embryo acute toxicity test procedure – TG 236

Spawning unit

Glass vessel with respective test con- centrations/controls at volumes to fully cover eggs

Selection of fertilized eggs and fertilizing rate

Fertilized eggs

0 hpf ≤ 1.5 hpf ≤ 3 hpf

Removal of coagulated eggs

Duration of the

test : 96 h

Based on the 4 End points, toxicity of substances will be determined.

Coagulated embryo

No somite formation

somites

Non-detachment of the tail

Lack of heart beat

Other tests:

Fish Acute Toxicity Test (OECD 203) with whole

fish – 96 h

Fish Early-life Stage Toxicity Test (OECD 210) –

30 days post hatch

- cumulative mortality

- numbers of healthy fish at end of test

- time to start of hatching and end of hatching

- numbers of larvae hatching each day

- length and weight of surviving animals

- numbers of deformed larvae

- numbers of fish exhibiting abnormal

behaviour

Fish Juvenile Growth Test (OECD 215) – 28 days

Plan: 1. Collection of water samples from CKDu endemic and non-endemic areas 2. Performance of tests with

• Collected water samples • Filtered water samples using special filters • Suspected heavy metals in different concentrations

3. Histopathology with H & E for renal effects

• Whole embryo • Harvested kidneys of adult fish

• Dr Hajime Kojima - co-chairman of the Congress

and members of the organizing committee

• Prof M A Akbarsha – Director, Mahatma Gandhi–

Doerenkamp Center for Alternatives to Use of

Animals in Life Science Education, India

• Distinguished Audience