Module 3 (cont.)

• Identification• Breeding and egg production• Disease• Anaesthesia• Minor procedures

Identification

• Use natural markings.• Zebra stripes v Leopard spots.• Photographs of Xenopus• Anaesthesia for

– Pigmented elastomer – Freeze branding

• Transponders• http://www.unobv.com/PICO-ID%20Transponder.html

Breeding / egg production –Zebra fish

• Zebra fish are egg layers – up to 300/clutch• Quality can vary - viability for micro-injection• The female Zebra fish - slightly larger, more

swollen abdomen than the smaller, slimmer and slightly longer male.

• Females more silvery in colour than yellowish coloured males.

Male and Female ?

Breeding (cont)

• Zebra fish sexually mature at > 4 months • Start of the light cycle stimulus to the fish to

breed if laying sites are available • Presence of male gonadal pheromones in water• Eggs scattered at random and zebra fish will

cannibalise their own eggs • Separate adults from eggs by means of a mesh

platforms or marbles on the base of the tank.

Adapted tank

Breeding (cont.)

• Rotate breeding tanks• 4males + 12 females for large scale production• E.g. Monday & Thursday: Tuesday & Friday• Wash eggs to remove debri• Incubated in petri dishes at around 28.5C• Eggs hatch in around 48-36hrs.• Embryos then placed into separate tanks

Breeding frequency

• Baseline of every 1 to 2 weeks• Avoids gravid females• Avoids stress of over production• Can lay daily!

Methylene Blue

• Can use Methylene Blue solution for fish up to 20 days of age.

• Stock solution made up using system water, then diluted to a light sky blue/aquamarine colour.

• Depth of only 1cm in the base of the tank.• Prevents fungus on fish eggs and embryos, decreases

the toxicity of nitrite and is effective against parasites.

Embryos (cont.)

• Survival criteria – 80-95% is good with a final size of 1.0-1.5cm at 21 days.

• However dependent upon the mutation, can be as low as 10%.

In Vitro Methods

• In Vitro fertilisation achieved by collecting eggs and sperm from anaesthetised fish.

• Gently squeezing the abdomen of the gravid females and the sides of the male – or dissecting testes.

• Eggs and sperm are mixed and water added. Activates the sperm and fertilisation should occur.

Breeding and egg production - Xenopus

• Natural mating – pairs. • Amplexus around 12 hrs – male releases sperm

as female lays

Induced breeding

• Induction – hCG treatment (500IU for females 36hrs before laying and 50IU for males – few days before mating – clicks signify readiness)

• Squeezing/milking – rubbing the belly of female

• Egg quality determines rest periods – 3 months?

• Common diseases and recognition

Ill Health – Zebrafish

• Average lifespan in the lab – 3.5 years• Any sign of ill health should be recorded and

investigated.• Spinal curvature observed in the lab.• Sick animals should be culled where possible or

placed into separate quarantine tanks.• Dead fish should be removed immediately.

Clinical Signs - Zebrafish• Changes in body colour / haemorrhage• Clamped fins and/or Improper buoyancy• Fin damage• Emaciation• Exopthalmos• Lethargy• Opercular flaring• Scale loss – mucus & ulcers• Surface breathing• Sudden death

Clinical signs - Xenopus

• Skin sloughing• Redleg & subcutaneous haemorrhaging• Dermal ulcers• Increased mucus production• Swelling of abdomen• Change in behaviour, response to handler

Possible Causes

• Bacterial & viral infections• Parasites• Chemical irritation or Toxicity• Environmental stress• Gas supersaturation• Oxygen depletion• Mechanical trauna• Starvation

Ill health

• Water quality should always be checked.• Water quality parameters and instrument

calibration should be checked.• Corrective action taken as necessary.

Ill Health (cont)

• Fish are subject to a range of bacterial, viral, fungal and parasitic diseases.

• Snail Vectors!!• The aim should be prevention and culling (if

necessary) rather than treatment. • All treatment must be under the supervision of

the named veterinary surgeon.

Treatment

• It should be noted that many agents are designed to be added to the water.

• Antibiotics and other chemical treatments could damage the biological filters.

• Therefore individual fish should be treated in isolation / quarantine tanks.

Treatment (cont)

• Doses should be first checked with a small number of fish to ensure that no adverse effects occur.

• The effect of the treatment agent to the ongoing research programme must be considered as some agents can be possible mutagens/carcinogens.

• e.g Malachite Green

Zebra fish - Common Symptoms & Causes

• Ulcers, white patches and/or white spots – Bacteria or virus infection– Protozoal disiease

• Fin damage – Infection– Fighting– Water quality poor

Common Symptoms & Causes

• Haemorrhage – Infection– Water quality poor

• Scale damage– Poor handling– Fighting– Rough objects in the tank– Nutritional problems

Common Symptoms & Causes

• Distended abdomen– Female with eggs– Worm burden– Dropsy

• Mucus trails – Infection– Water quality poor

Common Symptoms & Causes

• Bulging eyes (pop eye) exophthalmia.– Cloudy eyes – Infection– Water quality poor– Vitamin deficiency

• Stringy faeces – Nutritional problems– Infection

Common Symptoms & Causes

• Physical defects & deformities– Genetic defect– Environmental toxicity during development– Tumours – external and internal

• Spinal Curvature– Mycobacteriosis– Genetic defect

Common Diseases

• Microspoidiosis –(pseudoloma neurophilia)– Parasitic infection of larvae. Affects CNS & skeletal muscle.

Emaciation, ataxia and spinal malformations.– No treatment – UV light sterilisation of water helpful.

• Fish tuberculosis (mycobacteriosis)– Lethargic, open sores, raised scales & emaciated.– No treatment – UV light sterilisation of water helpful.

• Velvet Disease (oodinium pillularis) parasitic algae– Rubbing behaviour, fins close to the body, lethargy– Treat with Atabrine

Diseases of Xenopus

• Red leg – bacterial septicaemia– Treatment by NVS – antibiotics, potassium

permanganate, malachite green• Nematode Infection (Pseudocapillaroides

xenopdis)– Contagious - infection from eggs or larvae

through the skin– Skin sloughing– Treatment by NVS - ivermectin

Bd

• Batachochytrium dendrobatidis• Chytrid fungus – carried by Xenopus• Asymptomatic – zoospores released• Massed amphibian ‘die offs’• Health screening?

Health Screening and Diagnosis

• Sentinel programme by holding a small group of fish in a separate tank. The water in this tank is only changed using water from the tank drain before it is filtered.  

• Health screens may include:– Bacteriology– Virology– Check for fungal infection– Skin scrapings.– Fin and gill biopsy– Histopathology– Necropsy

Post Mortem

• Examination of gills. • Fin examination.• Removal of the GI tract and microscopic

examination. • Bacteria for culture obtained from the kidney,

liver, spleen and swim bladder • Lab Animal Europe (March 2002) Vol 2. No. 3

Pain and Distress

• Fish can experience pain and distress. • Nociceptive pathways and neuro-physiological

mechanisms are in place.• Fish avoid adverse stimuli e.g. electric shock,

show signs of fear and stress and respond to analgesia (affect of morphine in goldfish). (UFAW).

Anaesthesia & Analgesia

• Sedation may be used to reduce stress when animals are transported, handled and mixed between tanks.

• Anaesthesia may be used as part of an experimental procedure.

• Overdose for S1 culling.

Immersion Anaesthesia

• This must be conducted in a separate tank. • The dose is calculated according to the volume

of water in the tank.• The fish is introduced into the anaesthetic

solution and takes up the agent via the gills.

Anaesthesia (cont)

• Agents used:• Tricaine methanesulphonate (MS222). (200mg/L

fish and 300-50mg/L Xenopus)– Dissolves in water and reduces the pH therefore

buffer with NaHCO3 (200mg/L) – Increased up to 500mg/litre for killing by overdose. – Fat soluble anaesthetic, – Difficult to maintain stable anaesthetic, fish progress

to deep anaesthesia and death, therefore of benefit for short procedures only.

Anaesthetics (cont.)

• Benzocaine.  • 2-phenoxyethanol (2-PE) • Metomidate

Seek veterinary advice before use.

Anaesthetic Planes

• Light Sedation – Responsive to stimuli but activity reduced

• Deep sedation – No response to all but major stimuli. Some analgesia

• Light anaesthesia – Partial loss of equilibrium– Good Analgesia

• Deep anaesthesia – Total loss of muscle tone and equilibrium.– Slow ventilation rate

Anaesthetic Planes (cont)

• Surgical Anaesthesia– Total loss of reaction to stimuli– Very slow ventilation rate

• Medullary Collapse• Required for S1 by overdose of anaesthetic

– Ventilation ceases– Cardiac arrest– Death

Recovery

• This should be conducted in a recovery tank.• The system water should be free of anaesthetic

and aerated.• pH and temperature should be consistent with

the system and anaesthetic chambers.• The anaesthetic is cleared from the circulation

by the gills.• Full recovery (normal swimming behaviour)

before the fish is returned to the main system.

Analgesia

• No clinical trials have been undertaken to evaluate analgesic use in the fish.

• However the NVS should attempt to provide analgesia in situations that would cause pain in mammals. (Flecknell)

Experimental Procedures

• Subject to legislation under 2010/63/EU • Record keeping

– waterproof tank identification cross-linked to main record files. 

• Handling should be avoided.– external mucous layer of the fish is essential

in maintaining fluid balance and protecting against infection

Minor Procedures

• Creation of transgenic and mutagenic  • Caudal Fin Clip – to obtain tissue for DNA

isolation and PCR analysis.  • Blood sampling – in small Zebra fish, the fish is

first killed and then the caudal peduncle is cut through completely.

• Cryopreservation – recent success

Xenopus surgery

• Oocyte collection from ovarian tissue• Laparotomy (multiple surgeries)• Anaesthesia, analgesia (lidocaine?)and

veterinary fitness to proceed.

Websites

• Zfish book• http://zfin.org/zf_info/zfbook/cont.html#cont1• ZFIN• http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB_home.apg

• RSPCA  http://www.rspca.org.uk/sciencegroup/researchanimals/reportsandresources/housingandcare

• Directive 2010/63/EUhttp://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm

References

Sprague,J. Doerry,e. Douglas,S. and Westerfield, M. (2001) The Zebrafish Information Network (ZFIN) : A resource for genetic, genomic and developmental research. Nucleic Acids Res.29, 87-90.

Schlofeldt,H and Aldermann, D.J. (1995) A practical guide for the fresh water fish farmer.

Kestin,S.C. (1993) Pain and Stress in fish. RSPCA Report.

Astrfsky,K.M. et.al. Diagnostic techniques for clinical investigation of laboratory zebra fish in Lab Animal Europe (March 2002) Vol 2. No. 3

The UFAW Handbook on the Care and Management of Laboratory Animals 7 th Ed. Vol.2 Amphibious & Aquatic Vertebrates & Advances Invertebrates (1999) Blackwell Science Ltd.

Flecknell, p and Waterman-Pearson, A. Pain Management in Animals (2000)

Lab Animal Europe (March 2002) Vol 2. No. 3