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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

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Module 3 (cont.)

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Module 3 cont

Module 3 (cont.)

  • Identification

  • Breeding and egg production

  • Disease

  • Anaesthesia

  • Minor procedures


Identification

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

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

Male and Female ?


Breeding cont

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

Adapted tank


Breeding cont1

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

Breeding frequency

  • Baseline of every 1 to 2 weeks

  • Avoids gravid females

  • Avoids stress of over production

  • Can lay daily!


Methylene blue

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

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 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

Breeding and egg production - Xenopus

  • Natural mating – pairs.

  • Amplexus around 12 hrs – male releases sperm as female lays


Induced breeding

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?


Module 3 cont

  • Common diseases and recognition


Ill health zebrafish

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

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

Clinical signs - Xenopus

  • Skin sloughing

  • Redleg & subcutaneous haemorrhaging

  • Dermal ulcers

  • Increased mucus production

  • Swelling of abdomen

  • Change in behaviour, response to handler


Possible causes

Possible Causes

  • Bacterial & viral infections

  • Parasites

  • Chemical irritation or Toxicity

  • Environmental stress

  • Gas supersaturation

  • Oxygen depletion

  • Mechanical trauna

  • Starvation


Ill health

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

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

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

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

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

Common Symptoms & Causes

  • Haemorrhage

    • Infection

    • Water quality poor

  • Scale damage

    • Poor handling

    • Fighting

    • Rough objects in the tank

    • Nutritional problems


Common symptoms causes1

Common Symptoms & Causes

  • Distended abdomen

    • Female with eggs

    • Worm burden

    • Dropsy

  • Mucus trails

    • Infection

    • Water quality poor


Common symptoms causes2

Common Symptoms & Causes

  • Bulging eyes (pop eye) exophthalmia.

    • Cloudy eyes

    • Infection

    • Water quality poor

    • Vitamin deficiency

  • Stringy faeces

    • Nutritional problems

    • Infection


Common symptoms causes3

Common Symptoms & Causes

  • Physical defects & deformities

    • Genetic defect

    • Environmental toxicity during development

    • Tumours – external and internal

  • Spinal Curvature

    • Mycobacteriosis

    • Genetic defect


Common diseases

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

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


Module 3 cont

Bd

  • Batachochytrium dendrobatidis

  • Chytrid fungus – carried by Xenopus

  • Asymptomatic – zoospores released

  • Massed amphibian ‘die offs’

  • Health screening?


Health screening and diagnosis

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

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

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

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

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

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

Anaesthetics (cont.)

  • Benzocaine.  

  • 2-phenoxyethanol (2-PE) 

  • Metomidate

    Seek veterinary advice before use.


Anaesthetic planes

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

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

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

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

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

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

Xenopus surgery

  • Oocyte collection from ovarian tissue

  • Laparotomy (multiple surgeries)

  • Anaesthesia, analgesia (lidocaine?)and veterinary fitness to proceed.


Websites

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/EU

    http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm


References

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 7th 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


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