Download Breeding / egg production

Module 3 (cont.)
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Identification
Breeding and egg production
Disease
Anaesthesia
Minor procedures
Identification
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Use natural markings.
Zebra stripes v Leopard spots.
Photographs of Xenopus
Anaesthesia for
– Pigmented elastomer
– Freeze branding
• Transponders
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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.)
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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
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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
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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
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Skin sloughing
Redleg & subcutaneous haemorrhaging
Dermal ulcers
Increased mucus production
Swelling of abdomen
Change in behaviour, response to handler
Possible Causes
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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
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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
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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
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