Aquatic on Line Ver 1 1 of 102

Home , Fish slaughter

Aquatic on line ver 1 1 of 102

! Laboratory Animal Science & Training (Last-Ireland)

Training Course Materials

Aquatic Module

Tutor :Dr T.Murphy

Course web page http://www.LAST-Ireland.ie Email [email protected] Aquatic on line ver 1 2 of 102

LAST Ireland online aquatic module for captive fish and frogs

Aquatic Module Tutors: Drs T Murphy, Susan Mitchell , Aileen Cronin, Gerhard Schlosser & Peter Nowlan

EU Modules Schedule of work study Done Natural History of fish 4

Basic Anatomy and Physiology of fish TM 3.1; 4; 5;

Diseases and welfare of fish TM 3.1; 4;5;20;22

Biology of frogs GS 3.14

Biology and Husbandry of farmed fish SM 3.1;4;5

Biology and Husbandry of Zebra Fish

Welfare, stress and pain in fish. TM & Humane endpoints 4;5 Euthanasia and humane end-points TM 6.1 Quiz

13‐Jan‐20

Aquatic on line ver 1 3 of 102

Introduction to fish biology: A brief natural history Tom Murphy, MVB, MVSc, Ph.D., MRCVS

Introduction

(i) Natural history of fish

Vertebrates : Animals with a backbone or spinal column

Five categories of vertebrates: (i) Fish (Salmon, Plaice, Haddock etc) (ii) Amphibians (Frogs etc) (iii) Reptiles (Crocodiles, Snakes) (iv) Birds (Sparrows, Starlings, Hawks etc) (v) Mammals (Horse, Dog, Human etc)

Vertebrates Characterised by: (a) Being bilaterally symmetrical (e.g. muscular system consists of paired muscle masses) (b) Central nervous system partially enclosed within a backbone and bony skull

Fish: 1st vertebrates: To evolve from the chordates [Notochord evolved to become part of the vertebral column protecting the main nerve]

Ancestry: At least 500 million years Fish: Aquatic poikilothermic vertebrates with gills and limbs in the shape of fins and whose bodies are usually covered in scales

1 13‐Jan‐20

Aquatic on line ver 1 4 of 102

Why study fish?

Physiology Biochemistry For a fundamental understanding of Developmental Biology general processes Immunology Pathology

i) Economic Commercial Fisheries Sport Economic importance ii) Political Fish farming

Distribution of fishes: Occupy all aquatic environments (almost everywhere water occurs) Highest diversity : Tropics

Ecological Main factor Distribution due to Phylogenetic Factors Continental drift (180-90 millon yrs ago ) Geological

Various tectonic plates

Fish have successfully conquered almost all naturally occurring aquatic ecosystems: Distribution of fishes: 58% Marine: Ecological division of marine species: i) Epipelagic (surface dwelling (1%)) ii) Deep pelagic (5%) iii) Deep Benthic (6%) iv) Inshore, littoral, continental shelf (46%)

41% Freshwater: Freshwater regions i) Neoartic (N. America) 950 sp. ii) Neotropical (Middle and S. America), 450 sp. Iii) Paleartic (Europe, Asia( north of Himalayans), 500 sp. iv) Africa, 2,900 sp. v) Orient (India, S. China, S.E. Asia), 3,000sp vi) Australia (Australia, New Zealand), 2 sp.

1% Move between freshwater and the marine : 160 sp Anadromous species (Salmon) Northern hemisphere ( High latitudes).

2 13‐Jan‐20

Aquatic on line ver 1 5 of 102

Demonstrated:

Physiological In response to specific Anatomical Adaptations environmental Ecological pressures

Ocean:

Deeper one goes there is: i) Less light ii) Decreasing temperature iii) Less food iv) Increasing pressure Increase up to 20‐400 atmospheres. Fish in Epipelagic, Mesopelagic and Bathypelagic zones:

i) Proteins < sensitive to pressure ii) Modified gas bladder (negative buoyancy)/lost in some species iii) Modified musculature + skeleton (no heavy components in their bodies) (little movement) iv) Adaptations of eyes: Large, > sensitivity to light, wavelength 470nm v) Bioluminescence for attracting prey + sexual mates vi) Modified sexual behaviour vii) Metabolic activity: Low

Mesopelagic/bathypelagic deep sea fish

Anglerfish with two males attached

Other features: Large eyes Large expandable mouths Reduced musculature

3 13‐Jan‐20

Aquatic on line ver 1 6 of 102

Epipelagic zone: Numerous fish species: Herring, Sardines, Mackerel, Cod Very productive zone Tuna, Sword fish Fish abundant Fish undergo long migrations

Features: i) Counter shaded, silver undersurface ii) Very active: High % red muscle for prolonged swimming iii) Negatively buoyant, have to keep swimming iv) Ram gill ventilation: Mouth open water flows across gills v) Efficient circulatory system, large heart, large blood

volume, enhanced O2 to muscles vi) Respiration facilitated by numerous thin gill lamellae vii) Feed on plankton

Polar regions: 0 H2O: ‐2.1 C Zone is O2 & nutrient rich a) Blood contains anti freeze glycoproteins b) Kidneys lack glomeruli (stops filtration of glycoproteins) c) Neutral buoyancy: Reduced mineralisation of skeleton( cartilaginous skull and caudal bones) d) Increased lipid deposits throughout the body E) Loss of blood pigments (reduced Hb & myoglobin)

f) Increased vascularisation of skin (for O2 exchange Hillstream Loach Fast flowing rivers: [fish hide under rocks etc] a) Dorso ventrally compressed b) Sub‐terminal mouth for feeding on algae on rocks, stones c) Some species: Pelvic fins fused to form a suction disc

To be continued:

4 Aquatic on line ver 1 7 of 102 1/21/20

Basic anatomy and physiology of fish T.M. Murphy

Basic anatomy

Surface anatomy

Internal organs

Skin: Multipurpose tissue

Epidermis

Dermis Physical Protection Chemical

Metabolically active Epidermis: Stratified squamous epithelium A few cell layers (5-10) thick, non keratinised Covered by a layer of mucus secreted by goblet cells Mucus has antimicrobial properties (first line of defense). Dermis: Stratum spongiosum Also specialised Two layers Dense collagen fibres and scales Chromatophores Stratum compactum

Hypodermis: Closest to muscle, collagen fibres and blood vessels 3

1 Aquatic on line ver 1 8 of 102 1/21/20

Chromatophores [Melanophores Iridophores ] Specialised cells in the skin Mucous cells [ Antimicrobial activity] Keratocytes [Rapid wound healing] Ion regulated cells

Fish mucus: Antimicrobial peptides Still present in mammals including humans

Salmon: No. of mucus cells in skin affected by stress and also by gonad development

Zebra fish: Ion regulated cells in embryos: Analogous to mammalian kidney tubule cells

Types of muscle: Musculature and locomotion i) Skeletal: Striated most of fishes’ mass ii) Smooth: Non-striated, involuntary contractions associated with intestines and blood vessels iii) Cardiac: Non-skeletal, striated (only found in the heart)

Two types of skeletal muscle: White: i) Lies on either side of the dorsal column ii) Divided by a horizontal tissue septum ( Epaxial: Upper pair of myomeres) (Hypaxial : Lower pair of myomeres)

Red muscle: Wedge shaped under skin along horizontal septum [between the epaxial and hypaxial muscle on each side]

2 Aquatic on line ver 1 9 of 102 1/21/20

Skeletal muscle continued

Red Muscle White Muscle Aerobic Anaerobic Hard to fatigue Easily fatigued No build up of lactates High build up of lactate Recovery fast Recovery slow, up to 12 hrs Abundant myoglobin Little myoglobin Highly vascularised Little vascularisation Rich oxygen supply Poor oxygen supply Fibres rich in mitochondria Fibres poor in mitochondria

Blood circulation Heart: Two chambers: (i) Sinus venosus (ii) Atrium (iii) Ventricle (iv) Bulbus arteriosus Various valves between each chamber (to prevent backflow of blood) Bulbus arteriosus: Dampens pulsing (allows a continuous flow of blood) Nerve innervation: Vagus nerve

Blood circulation is continuous

Only passes through the heart once

Deoxygenated blood returns to the heart via afferent blood vessels Then is pumped by the heart through the gill where it’s oxygenated and then to the rest of the body( via Dorsal Aorta)

3 Aquatic on line ver 1 10 of 102 1/21/20

Respiration

i) Fish pumps H2O over gills ii) Ram ventilation

Anatomy:

4 gill arches on each side of the body Large Numerous gill filaments on each arch surface Secondary gill lamellae on the filaments area

H2O flows between gill filaments & over the 2ndary lamellae

Secondary lamellae : Site of O2 exchange Gas exchange enhanced by: a) Thin epithelial membrane on 2ndary lamellae b) Counter flow of blood [blood flows in opposite direction to water passing over lamellae] 10

Gills and osmoregulation: Process of maintaining an internal balance of salt & H2O in the body

Blood hyperosmotic to freshwater

Blood hypo-osmotic to seawater

Chloride cells: Site of active Na+, Ca++ and Cl- uptake (freshwater) and extrusion (marine)

Other cells: Neuro-endocrine cells[paraneurons] [ oxygen sensing cells l][signal transmitted to hypothalamus via glossopharyngeal (ix) and vagus (x) nerves]

Gas transport (Oxygen)

O2 is transported to the tissues in red blood cells attached to haemoglobin

Red blood cells in fish nucleated

Haemoglobin (Hb) Molecule : Transports 4 molecules of oxygen

Haemoglobin /oxygen transport system: Central to the evolutionary success of teleost fish

4 Aquatic on line ver 1 11 of 102 1/21/20

Cell metabolism

Basic reaction: CH2O + O2 CH2O3 CO2 + H2O

CO2 has to be transported from the tissues to the gills

O2 and CO2 transport (Bohr Effect)

The Bohr Effect describes how C02 produced in metabolising tissues acidifies the surrounding >pH7.4

CO2 C02 transported to the gills as: (i) (i) Bicarbonate (CHO3) in the plasma [70%] (ii) (ii) Attached to proteins in the plasma or attached to the amine group of Hb in the erythrocyte [23%] (iii) (iii) Dissolved in plasma [7%]

Bohr Effect versus Root Effect

Rete Mirabile: Looping bundle of blood vessels associated with gas bladder that functions as counter current multiplier

5 Aquatic on line ver 1 12 of 102 1/21/20

Digestive system Alimentary canal: Mouth Anterior foregut: Buccal cavity Pharynx

Oesphagus Posterior foregut: Stomach 1) Digestion and absorption of Midgut: Pyloric caeca nutrients 2)Role in salt and Hindgut: Intestine water balance 3) Immunity Rectum

Fish possess the main digestive enzymes present in all vertebrates: Trypsin, maltase, amylase, aminopepsidases, lipase & alkaline phosphatase Stomach: pH 1-4 [ Pepsin + HCl] Intestine: pH 6.8 – 7.8 Histology: Columnar epithelium + goblet cells 17

6 Aquatic on line ver 1 13 of 102 1/21/20

Digestive system continued Carnivore species: Intestine short Herbivore species : Intestine long

Absorption (nutrients) efficiency: Carnivores: 80% Herbivores: 40 -50%

Other aspects of digestive system:

Pancreas: Usually scattered diffusely amongst pyloric caeca Endocrine Islet cells: Insulin + glucagon secretions: Exocrine Pancreatic cells: Proteases, amylases , lipases

Liver: Nutrients transported from intestine via the hepatic-portal vein Assimilates nutrients, detoxifies toxins from endogenous and external sources, secretes bile Gall bladder: Stores bile and bile salts before releasing them back into the intestine for fat digestion.

Head Kidney: Haematopoietic tissue

Kidney: Excretion of metabolic wastes and osmoregulation

Glomerulus filters out: a) H2O, sugars, salts and nitrogenous wastes b) Filtrate collected in Bowman’s capsule c) Travels along the mesonephron tubule d) H2O , sugars and other substances are selectively reabsorbed

Important aspects of osmoregulation:

i) Freshwater fish: Higher conc. of ions in body than water (hyper-osmotic) ii) Marine fish: Lower conc. of ions in body than in the sea (hypo-osmotic ) iii) H2O & ions (NaCl, MgSO4, K+) absorbed and lost via skin, gills, mucous membranes, intestine, iv) Freshwater fish: dilute copious urine v) Marine fish: Little but highly concentrated urine vi) Gills very important for osmoregulation in marine fish [Cl- excreted by numerous Chloride cells in secondary lamellae]

7 Aquatic on line ver 1 14 of 102 1/21/20

Differences in kidney function between freshwater and marine fish

Freshwater fish Marine fish (small amounts of highly concentrated (copious amounts of dilute urine) urine)

Glomerular filtration High Low or non-existent (b) Glomerulus rate

Divalent ions reabsorbed from the Divalent ions pumped into the glomerular Proximal segment I glomerular filtrate filtrate

Distal segment and collection duct Impermeable to water Permeable to water

Central and peripheral nervous system: Comprises the brain, spinal cord and afferent and efferent nerves of the somatic and autonomic nervous system

Fish brain Fore-brain (Telecephalon): Olfaction & possibly nociception

Diencephalon: Between fore & mid brain, acts as a centre for controlling the endocrine system (Pineal Body is in this part of the brain)[ Thalmus-Hypothalmus-Pituitary axis]

Mid-brain (Mesencephalon): Important for vision

Hind-brain( Metencephalon) [includes the cerebellum), Functions in maintaining muscular tone and equilibrium in swimming. The cerebellum is the reception site for all the electrical impulses from the peripheral nervous system

Myelencephalon (brain stem) Has centres which control somatic and visceral functions as well as respiratory and osmoregulation centres

8 Aquatic on line ver 1 15 of 102 1/21/20

Functional division of the CNS and peripheral nervous system

Incoming stimuli (somatic and visceral) Output pathways Motor innervation to Motor innervation to smooth & cardiac muscle muscles & glands & some endocrine glands

Fight or flight system Rest & digestive system

Effects of these two systems are antagonistic 25

Transmission of nerve impulses:

Transmission occurs as a result of changes in electrical potential across the membrane of the neuron:

This is due to changes in the balance of Na+ and K+ inside and outside the axon

When the nerve is resting there is more Na+ outside the cell than inside (cell is polarised ) Passage of nerve impulse is due to movement of Na+ into the cytoplasm and movement of K+ out of the cell

Myelinated v unmyelinated nerves

BASIS OF COMPARISON MYELINATED NERVE FIBER UNMYELINATED NERVE FIBER

Myelinated Nerve Fibers are nerve Unmyelinated Nerve fibers are nerve Description fibers that are insulated by a myelin fibers that do not have a myelin sheath. sheath.

The myelinated nerve fibers are white The unmyelinated nerve fibers are gray Color in color. in color.

The myelinated nerve fibers have nodes Unmyelinated nerve fibers do not have Nodes of Ranvier of Ranvier. nodes of Ranvier.

Due to presence of nodes of Ranvier on Unmyelinated nerve fibers do not have myelinated nerve fibers, the speed of myelin insulations, and therefore, the Speed of Transmission transmission of nerve impulses is high speed of the transmission of the nerve in myelinated nerve fibers. impulses is low.

Most neurons in the central and Unmyelinated neurons can be found in peripheral nervous system are both the peripheral and central nervous Location myelinated because they require fast system in the group c nerve fibers, conduction speed such as neuron responsible for transmission of involved in spinal reflexes. secondary pain

Due to presence of myelin sheath, Unmyelinated nerve fibers can lose the Impulse Conduction myelinated nerves do not lose the impulse during conduction nerve impulse during conduction.

Axons The nerve fibers with long axons are The short axon nerve fibers are myelinated. unmyelinated.

9 Aquatic on line ver 1 16 of 102 1/21/20

Synapses and conduction of nerve impulses

Synapse: Permits a neuron to pass on an electrical impulse to another cell or to a target organ

When the nerve impulse reaches the end of the axon I) It stimulates the release of neurotransmitters. ii) These diffuse across the synaptic space and bind onto receptors iii) Nerve impulse is then transmitted along the next nerve cell to the target tissue

Endocrine system Complex network of chemical signals that control many immediate and life-long functions and responses and is very sensitive to stress

Three parts i) Production glands to the ii) Hormones (messengers) system iii) Target cells/tissue

Main endocrine glands in fish Hormones regulate a) Sexual activity Controlled b) Reproduction in part c) Growth by the d) Osmotic pressure CNS e) Metabolic activity(liposis, gluconogenesis) f) Blood pressure g) Smoltification h) Aspects of skin (colour) i) Ionic regulation

Endocrine glands and tissues in teleost fish 1) Pineal gland: Photosensory organ, secretes melatonin 2) Hypohysis (Pituitary): Central role in endocrine signalling 3) Saccus vasculosus: Photosensory , has a role in seasonal reproduction 4) Thymus: Role in adaptive immunity, T-cells develop from thymocytes 5) Pseudobranch: Implicated in O2 and CO2 sensing 6) Ultimobranchial Bodies: Calcium metabolism, secretes calcitonin 7) Thyroid: Role in early development and metamorphosis, influences growth , nitrogen metabolism, osmoregulation, prepares fish for migration and smoltification in Atlantic salmon 8) Interrenal tissues: Homologous to adrenal cortex in mammals, secretes cortisol & mineralocorticoid 9) Chromaffin tissue: Homologous to adrenal medulla, secretes catecholamines hormones: Adrenaline (epinephrine ) & noradrenaline (norepinephrine) 10) Renal Juxtaglomerular cells : Renin-angiotensin-aldosterone system? 11) Corpuscles of Stannous: Involved in calcium metabolism 12) Gonads: Testes and ovaries secrete testosterone and oestrogen respectively 13) Islets of Langerhans: Present in pancreas , secretes insulin 14) Stomach: Secretes gastrin and other peptide hormones which control appetite and digestion 15) Enterochromaffin cells in the intestine: Involved with endocrine control of appetite and digestion 16) Urophysis: Neurosecretory system situated at the caudal end of the spinal column, may play a role in ion regulation particularly Na+

10 Aquatic on line ver 1 17 of 102 1/21/20

Various divisions of the pituitary, cell types, secretions and actions of hormones produced in the proximal and rostral Pars distalis, Pars intermedia and pars nervosa

Part of Pituitary Division Cell types Secretions Functions Adenohypophysis Proximal pars distalis Thyrotrophs Thyrotropins e.g TSH Regulates the growth and secretion from thyroid

Gonadotrophs Gonadotropin e.g. FSH Regulates secretion of (follicular stimulating gonadal hormone, Meso-adenohypophysis hormone) and LH spermatogenesis and (leutinizing hormone) oogenesis

Somatotrophs Somatotropins e.g.GH Increase growth and (Growth hormone) BMR of the fish body

Rostral pars distalis Lactotrophs Prolactin Regulation of osmoregulation and Pro-adenohypophysis melanogenesis Corticotrophs Corticotropin viz. ACTH Regulates secretion of corticotropins from interrenal. Pars intermedia MSH and MCH Regulates the (melanophore dispersing concentration and Meta-adenohypophysis and melanophore dispersion of pigments contracting hormone) within melanophores.

Neurohypophysis Pers-nervosa Vasopression and Regulates oxytocin osmoregulation, saltwater balance, mating and egg laying

Immune system of teleost fish

Cytokines: Transferrin: Binds iron Interleukin 1: Enhances the complement cascade Interferon: Reduces the replication rate of viruses in infected cells 32

11 Aquatic on line ver 1 18 of 102 1/21/20

12 Aquatic on line ver 1 19 of 102 1/21/20

13 Aquatic on line ver 1 20 of 10213‐Jan‐20

Basic disease epidemiology and welfare in a fish research facility

T.M. Murphy

Disease outbreaks in fish holding facilities: (a) Can have disastrous effects on research programmes [due to disruption of the normal physiology and biochemistry of diseased fish]

Disease especially chronic/”clinically in‐apparent” infection can confound experiments, (i) adversely affect the validity of results (ii) reduce reproducibility (/repeatability) of expt. protocols

Also following infection (b) There can be an extensive work effort involved and a high cost in eradicating the pathogen as well as disinfecting and decontaminating ponds, tanks, feeding and fish handling (including surgical) equipment etc and buildings (c) Valuable (genetically important) fish stocks may have to destroyed.

Thus it behoves everyone who work with experimental fish to understand how diseases may be introduced into and spread within a fish holding facility

Objectives

At the end of this lecture the student will be able to

(a) Understand how fish become infected

(b) How diseases can spread within a fish holding facility

(d) The precautions that can be taken to prevent entry of disease

(e) Understand how diseases can be contained if they are introduced into a facility

(f) Understand the basics of a health monitoring programme

1 Aquatic on line ver 1 21 of 10213‐Jan‐20

Good welfare = Good science

Welfare in fish (especially fish in research facilities) : Not well defined

Fish medicine: Health and welfare: general terms that are often used synonymously (both terms are used interchangeably)

Health & Welfare Monitoring can be performed: (a) Directly by clinical observation of the fish population (b) Indirectly by monitoring the fishes’ environment

Thus the tac of health monitoring is considered an integral part of the process for ensuring good fish welfare

Good welfare continued

For fish welfare to be acceptable: A number of conditions must be fulfilled

(a) Fish should be free of disease, injury and functional impairment (b) Should have all their biological systems functioning appropriately

However, the link between health and welfare is complex

Although a reasonable assumption is: Fish showing signs of disease are in a poor state of welfare

Which is indicative of : (i) An underlying problem with the environment (ii) Stressful conditions (experimental or husbandry) to which the fish are being subjected to

Pathogenic micro‐organisms are ubiquitous: Disease precipitated by stressful environmental conditions

Transmission of infectious disease in a fish holding facility

Chain of infection:

No. of links

Each link : Considered a critical disease control point (infection cycle can be broken)

2 Aquatic on line ver 1 22 of 10213‐Jan‐20

Infectious diseases can be caused by: Microorganisms Pathogens:

Viruses

Bacteria

Fungi

Parasites (i) protozoa (ii) helminths (nematodes, cestodes, trematodes) (iii) arthropods

Microorganisms are ubiquitous in most water bodies and fish are frequently exposed to potential pathogens

Occurrence of disease depends on: (a) Inherent virulence of the microorganism (b) Number of infectious particles the fish are exposed to (c) Immune status of the fish. (d) Level and etyp of stress fish are being subjected to

Sources and Reservoirs of infection

During an outbreak: Main Source of infection for healthy fish: Clinically ill fish

Asymptomatic carriers: Very important alternative source of infection [Latent or silent infections] [Clinically in‐apparent infected fish]

(i) During an outbreak newly infected/sub‐clinically infected fish (ii) Recovered fish (can often carry and shed pathogens that caused the initial disease)

Best way of preventing the transmission of infectious diseases: Avoid their introduction Greatest danger of introducing disease into a facility: Buying in or transferring fish from populations of unknown health status

Portal of exit from diseased fish

i) Leakage from skin wounds ii) Faeces iii) Urine iv) Leakage from autolysing fish carcasses

3 Aquatic on line ver 1 23 of 10213‐Jan‐20

Transmission of disease within a fish research facility

a) Direct: When healthy and infected fish are in the same tank or pond

b) Indirect

i) Mechanically: Organism suspended in the water body or attached to sediment particles and/or other fomites(farm and pond utensils, fish handlers hands and clothes)

ii) Feed: Contaminated feed especially if live feed is being used

iii) Gametes : Some viruses and bacteria transmitted vertically through fertilised eggs (furunculosis)

Portal of Entry of pathogenic microorganisms

(1)Passive absorption of pathogens in the water body and feed via: a) Skin (wounds) b) Gills [disrupted gill lamellae & epithelium] c) Mucous membranes of intestine, urinary system

(3) Inoculation by biting vectors etc [special cases, should not occur in fish research facilities]

Infectious disease: Three‐way interaction requiring (a) Host (b) Pathogen (c ) Environment

Obligate pathogens Pathogen Host Opportunistic pathogens

Environment

4 Aquatic on line ver 1 24 of 10213‐Jan‐20

Signs of disease Behavioural signs: i) Failure to feed properly ii) Flashing (turning on their sides) iii) Rubbing against the walls and the bottom of the tank iv) Gathering around the water inflow v) Reduced vitality vi) Gasping at the surface

Physical signs: a) Blisters b) Swollen bellies c) Pop‐eyes d) Haemorrhages on the fins and cloaca (vent) e) Erosion of fins and other body parts f) Growths on the body g) Excessive mucus production on the skin h) Stringy white faeces hanging from the vent

In the event of a disease outbreak

Initial sign: Anorexia Staff responsible for feeding Behavioural changes should be trained to observe changes in the feeding pattern

(a) Moribund and dead fish should be removed [when detected first] & sent for microbiological and pathological examination (b) Affected fish, tank/tanks should be isolated (c) All equipment (nets, feeding utensils etc) should be adequately disinfected and /or autoclaved. (d) Laboratory and research staff and animal technicians should observe good personal hygiene

(e) Appropriate control and treatment regimes instituted once the pathogen is identified (f) These include: (i) Isolation affected fish (and tanks etc) (ii) Medication of the affected groups (iii) Euthanasia [may be necessary to maintain the integrity of the experimental protocols ] (as recovered and in contact fish may not be suitable experimental subjects)

Routine monitoring of fish research facilities for disease

A major inhibitory factor for efficient screening is that there is a dearth of non‐invasive tests for the common fish diseases Health monitoring usually carried out on a population basis

Fish can only be observed for clinical signs

(a) Staff with responsibility for feeding fish should be trained to observe changes in feeding patterns and swimming behaviour (b) Fish exhibiting unusual feeding and swimming behaviour should be removed immediately and subjected to microbiological and histological examination [by a veterinarian or other fish health expert] (c) All incidental moribund and dead fish should be removed on first detection and subjected to a microbiological examination (d) If the population size allows it, a representative number of the fish should be examined at regular intervals to provide background information on the pathogens circulating in the group (e) If population size is small, sentinel fish held in the effluent water can be used to demonstrate eth presence or absence of disease in the main facility.

5 Aquatic on line ver 1 25 of 10213‐Jan‐20

Preventing the introduction of disease into a fish research facility

The best way to control infectious disease: Avoid their introduction

(a) Only use ova from SPF adults [disinfect the surface of the eggs with iodophors or sodium hypochlorite solutions depending on the fish species] (b) Use fish of known health status, preferably from specific pathogen free suppliers (c) Hold all newly introduced fish in a quarantine unit for several weeks before introducing them into the research facility Period of quarantine can vary depending on pathogens concerned and the rate of their detection [Standard quarantine periods can vary from: 14 days up to 90 days] Prophylactic treatment for some common problems such as external parasites may be allowed in a quarantine unit (c) Avoid the introduction of contaminated water with the new fish into the main system (d) Use pathogen free food if using live food (e) Filter and disinfect (UV sterilisation, ozone treatment) all incoming water to the main unit

Synopsis:

(1) Avoid the introduction of fish with asymptomatic disease

(2) Maintain a stress free environment Optimum: (a) Stocking density (b) Water quality and temperature (c ) Provide some level of environment enhancement

6 Aquatic on line ver 1 26 of 102

Xenopus – biology,

Gerhard Schlosser NUI Galway

Amphibians • Three lineages of amphibians – Urodeles: salamanders, newts – Anurans: frogs, toads – Caecilians

Frogs (Anurans)

• Approx. 5400 species • Highly modified adult body plan with specializiations for new type of jumping locomotion: – elongated hindlimbs – shortened vertebral column – tail reduction • Highly peculiar, light-weight skull, large frontal eyes and protrusible tongue for prey capture Triadobatrachus Modern frog (Fossil from Triassic) • Carnivorous as adults

1 Aquatic on line ver 1 27 of 102

Frogs (Anurans)

• Larvae also adopt new specialized body plan (tadpole), specialized for aquatic filter feeding • Metamorphosis of tadpoles into adult frogs involves dramatic reorganization of all body parts

Frogs (Anurans)

• Anurans have adopted a diversity of life styles – semiaquatic pond dwellers with webbed toes and long legs for jumping and swimming ("frogs", e.g. Rana) – more terrestrial anurans with short legs for hopping or walking ("toads"; sometimes associated with warty skin: e.g. Bufo) – arboreal frogs with toe pads assisting in climbing (e.g. Hyla, Agalychnis)

Frogs (Anurans)

Rana • Anurans have adopted a diversity of life styles – semiaquatic pond dwellers with webbed toes and long legs for Bufo jumping and swimming ("frogs", e.g. Rana) – more terrestrial anurans with short legs for hopping or walking ("toads"; sometimes associated with warty skin: e.g. Bufo) – arboreal frogs with toe pads assisting in climbing (e.g. Hyla, Agalychnis Agalychnis)

2 Aquatic on line ver 1 28 of 102

Frogs (Anurans)

Xenopus • Anurans have adopted a diversity of life styles – desert dwellers (e.g the spadefoot toad Scaphiopus) – fully aquatic frogs/toads Frogs (Anurans) (e.g. Xenopus)

Scaphiopus

Xenopus as a model organism

• Initially used in pregnancy test (1930s-1950s) • Hormone induced egg laying • Many embryos • Easily manipulated by microinjection and transplantation

Xenopus as a model organism

3 Aquatic on line ver 1 29 of 102

Biology of Xenopus

• Xenopus laevis, X. tropicalis and X. borealis

• Mainly aquatic & nocturnal • Lungs for gaseous exchange • Flattish antero-dorsal shape • Skin smooth and glandular. • Mucous glands secrete slimy protective layer (antimicrobial component magainin). • Serous glands (around head/shoulders)

Xenopus laevis

• South African clawed toad • Family Pipidae • Can live up to 25 yrs • Tetraploid – 26 chromosomes • Females >150mm, 300g body weight • Males >60-100mm, 100g body weight • Change colour according to surroundings • In the wild live in stagnant water – mud substrate

Xenopus laevis

• Underwater acoustic communication – clicks. • Pheromone communication possible • Detect food odours and eat non living food • Lateral line present

4 Aquatic on line ver 1 30 of 102

Xenopus tropicalis

• Diploid – used for genetic studies • Smaller size 40-60mm • Shorter generation time – 5 months

• Found in Ghana and the Ivory Coast • Warmer environment required • 24-27C

Housing.

• Traditional holding for research Xenopus used individual ‘stand alone’ tanks. • A ‘fill and dump’ systems OR independently filtered and serviced. • Labour intensive • Frequent water changes • Water quality varied markedly over time

Large Scale Holding • Purpose built system is recommended.

5 Aquatic on line ver 1 31 of 102

System requirements

• Maintain healthy Aquatic species. • Keep them free of environmental stress • Keep them free of disease. • Containment • Reliable & easy to service. • Labour saving & cost effective. • Meet research requirements. • Allow flexibility for breeding • Meet European directive requirements!!

What is involved

• Battery system of tanks – polycarbonate construction with mesh filters and lids plus non- corrosive support racking. • Flow through or Re-circulating system of water. • Series of filters to maintain water purity: • Biological, Mechanical, Chemical • U/V sterilisation.

Polycarbonate tank

http://thenode.biologists.com/a-day-in-the-life-ofa-xenopus-lab/lablife/

6 Aquatic on line ver 1 32 of 102

Xenopus – Housing and diseases

Gerhard Schlosser NUI Galway

Housing.

Large Scale Holding • Purpose built system is recommended.

1 Aquatic on line ver 1 33 of 102

System requirements

What is involved

Polycarbonate tank

http://thenode.biologists.com/a-day-in-the-life-ofa-xenopus-lab/lablife/

2 Aquatic on line ver 1 34 of 102

Tanks

• Transparent tanks – polycarbonate • Able to observe inhabitants from the side • Animals to be able to turn around easily

Battery of Tanks

What is involved

• Source of pure water (RO). This is obtained from a Reverse Osmosis unit.(Purite) • Header tank for conditioning of water. • Automatic monitoring of water quality. • Semi-automated water quality maintenance. • Temperature control and monitoring. • Flow control of water. • Electrical pumps and valves.

3 Aquatic on line ver 1 35 of 102

Header tank

Pumps & Filters

What is involved

• A flow system that allows some tanks to be decoupled from other tanks for quarantine of sick frogs • Alarm systems and back up systems in place.

4 Aquatic on line ver 1 36 of 102

Emergency

• Emergency alarms and stand-by systems. • A technologically-dependent animal facility is a vulnerable entity. • It is strongly recommended that such facilities are appropriately protected to detect the breakdown of essential equipment • Emergency call out and weekend cover. • Animal care staff should be on a call out list

Monitors

Requirements

• A room with a strong enough floor! • 1000 Litres of water = • 1000 kg

• A room with enough space. • Strong floor. • Waterproof non-slip floor. • Drainage & sink. • Hot & Cold water.

5 Aquatic on line ver 1 37 of 102

Requirements (cont)

• Electrical supply • Waterproof sockets, switches and electrical connections • Circuit breakers and earth wires • Installed by professional electricians

Requirements (cont)

• Room temperature control. • Helps control water temp without overloading system heaters. • Provides a safeguard if system fails. • Light Control • Light cycle for breeding • Noise & vibration – habituation to vibration of system?

Environmental Parameters

• Water Quality • Temperature •pH • Ammonia (NH3 /NH4) •Nitrite (NO2) •Nitrate (NO3) • Conductivity

6 Aquatic on line ver 1 38 of 102

Water Quality

• Vital – essential to get right • Aquatic species are ectothermic animals. • Completely dependent upon the quality and stability of their immediate environment – the water.

Water Quality

• Achieved by filtration • Aim: To remove the uneaten food, skin debris and solid waste products of digestion, plus dissolved faeces and ammonia. • Amphibians are susceptible to a build up of ammonia, toxic effects are reported at 0.2 – 0.5 mg/L of free ammonia (NH3) (Andrews et al, 1998).

Mechanical filters

• Pre-filters of mesh, or rough filter material e.g. white polyester pad. • These are used for the removal of gross high density and suspended solid debris including food and faeces from the water before it enters the biological filters

7 Aquatic on line ver 1 39 of 102

Pre-filters

• The pads require regular cleaning to remove the rapid build up of soiled on their surface • Can block the filter with consequent backflow of water. • 2 times per week to prevent contamination and maintain efficiency of the biological filters.

Biological Filter

• Fluidized bed biofilters – a highly efficient nitrification process takes place in such filters • Biological filters remove ammonia and nitrite using biological filters • Seeded with beneficial nitrifying bacteria (Nitrosomonas and Nitrobacter species) • Convert the ammonia into nitrite and then nitrate via the nitrogen cycle. • Pure RO water protects the biological filters from chlorine present tap water.

Biological Filter

8 Aquatic on line ver 1 40 of 102

Biological Filters

• Newly installed or cleaned biological filters need time to mature. • Build up of a nitrifying bacterial population required to become fully effective in removing ammonia and nitrite.

Biological Filters

• Mature in around 2 weeks. • During this time monitoring of ammonia levels is crucial. • Reduce toxic levels by removing quantities of contaminated water and adding more clean system water.

Chemical Filters

• Chemical filters - activated Charcoal or Carbon. • Remove organic compounds (including ammonia) from the system water. • Remove discoloration and odour by binding organic chemicals in suspension.

9 Aquatic on line ver 1 41 of 102

Ultraviolet (U/V) Filters

• Sterilising/disinfecting filter that will destroy bacteria and other micro-organisms • Recommended dosage of 30,000-60,000 2 mwattsec/cm or 240-280nm wavelength (UFAW). • Protozoa may not be affected. • Change at appropriate intervals (approx. once per year)

U/V Filters

Water testing & recording.

• Electronic meters allowing continuous monitoring of system conditions. • Aquanodes that can be linked to alarms • Ensure that the system is correctly calibrated and that probes are maintained in optimum condition. • The ’aquanode’ does not give levels of ammonia, nitrite or nitrate. Use the appropriate test kit. • Monitor twice per week and record details.

10 Aquatic on line ver 1 42 of 102

Aquanode monitor

Temperature

• Aquatic species keep as close to natural environmental temperature as practicable. • X. laevis require a water temperature of 18-20C • Room temperature of 20C • X. tropicalis water temp of 24-27C

Temperature (cont)

• It is important to maintain a constant temperature as changes greater than 2C in 24 hours are not recommended. • Water temperature affects the oxygen carrying capacity of the water.

11 Aquatic on line ver 1 43 of 102

Effect of temperature change.

• Low temp – stop eating. • Depressed metabolism and immune system

• Levels recommended for X.laevis are 6.5-8.5 • pH should be monitored daily, kept constant and buffering salts added as required to maintain the desired levels. • Sodium bicarbonate may be added to raise the pH as the nitrification cycle gradually reduces pH. • High pH and increased toxicity of NH3

pH (cont)

• Levels maintained at around pH 7 • Note: pH is measured on a logarithmic scale • Each unit change in pH value is equivalent to a 10 fold change in hydrogen ion concentration.

12 Aquatic on line ver 1 44 of 102

Ammonia levels

+ •(NH3/NH4 ): Either dissolved as ammonium ions or more dangerously as free ammonia. • Levels of free ammonia increase with increased pH or temperature. • Level of free ammonia that can be tolerated without toxic effects is 0.01-0.03mg/L • In a well established system, levels should be at zero or at least below 0.01ppm

- Nitrite levels (NO2 )

• Nitrite is less toxic than ammonia. • Toxicity occurring from 0.5-1.0mg/L with death at levels in excess of 10mg/L. • In a well established system, levels should be at zero.

- Nitrate levels (NO3 ):

• Affected by the level of bacterial nitrification occurring, as it is the final product in the nitrogen cycle. • Recommended maximum nitrate level is 50mg/L (Schlotfeldt and Alderman).

13 Aquatic on line ver 1 45 of 102

Test kits

Conductivity

• This determines the levels of dissolved solids in the system – measures ion content. • Indication of the general water quality. • RO water is free of all contaminants. • Synthetic Sea Salts (Instant Ocean) are added to the purified water to maintain conductivity. • Recommended level of 300-2000 µs • Adjusted by addition of salts.

Other Toxins

• If not using RO – obtain analysis of water. • Chlorine & chloramines in the supply. • Requires aeration before use.

14 Aquatic on line ver 1 46 of 102

Water changes

• Required to maintain water quality. • Recommended 3 changes per hour in tanks. • Re-circulating system = 10% change per day.

• Xenopus – if ‘fill & dump’ system. Change water after feeding at least 2 times per week.

Light Levels

• Strong circadian pattern – daytime activity • The photoperiod will influence the physiological regulation, affecting the activity, breeding behaviour and feeding pattern of the frogs. • Light cycle of 12L:12D • Xenopus – poss. require UV light for Vit D: – 100 lux at bench height – 40 lux at the top of the tanks – avoid retinal damage

Frog density

• Maximum animal housing density for 50 liter tanks (56x42.5x25 cm) (Appendix A, ETS123)

Animal Body Length Animals /Tanks Minimum Water (cm) Depth (cm) <6 56 6 6-9 28 8 9-12 12 10 >12 7 12.5 • Maximum animal housing density for 21 liter tanks (42.5x27x25 cm) (Appendix A, ETS 123)

Animal Body Length Animals /Tanks Minimum Water (cm) Depth (cm) <6 26 6 6-9 12 8 9-12 410 >12 2 12.5

15 Aquatic on line ver 1 47 of 102

Environmental enrichment

• Animals will be group housed • Each tank is supplied with a number of plastic or ceramic tubes/half tubes, stones or broken flower pots with smooth edges, or floating plastic bin liners to allow frogs to hide

http://www.xenopus.com/husbandry.htm http://aquaticfrogs.tripod.com/id1.html

Introducing frogs

• New system – Test before use. • Leave to run and stabilise for 2 weeks. • Check for leaks. • Check RO water supply. • Check environmental parameters.

Introducing frogs

• Seed the biological filters • Add culture of bacteria: • Nitrosomonas & Nitrobacter spp. • Introduce sentinels

16 Aquatic on line ver 1 48 of 102

Feeding

• Only use approved food that will not introduce pathogens to the system. • SDS aquatic diets • Do not overfeed OR underfeed. • Will affect the health of the frogs and the quality of the water. • Uneaten food will start to decay and also block up the filters. • Frogs should be eager to feed and consume the food quickly. • Too little food and egg production decreases

Xenopus feeding

Blades biological Xenopus diet- Size number 3 • The Xenopus are fed twice or three times weekly • Adult Xenopus should be given enough food so that there is some remaining in the tank after two hours. • Any remaining food should be removed after the three - five hours. • Beef liver – as part of the diet up to twice per week

Cleaning and disinfection:

• The hard way by hand! • Any debris or uneaten food should be removed from the holding tanks daily. • Achieved by means of a siphon – adapted squeezee bottle or by netting. • Small particles are flushed into the pre filters by the circulating water. • Pre filters are washed 3 times per week.

17 Aquatic on line ver 1 49 of 102

Cleaning (cont)

• Full clean of adult tanks takes place about every 6 wks. • Gross debris removed when emptying the tank of it’s contents. • Wipe away most of the algal staining • Rinse with tap water and finally add system water before replacing frogs.

Handling of frogs

• Goals: - Support and comfort - Avoid kicking out

www.uwm.edu/Dept/EHSRM/ACP/MANUAL/Frog2.jpg

Diseases of frogs

• Red leg – often stress/IC; control with proper sanitation and environmental quality – Treatment: (Tetracycline oral: 1mg/5g body weight for 5 days)

http://www.xenopus.com/disease.htm

18 Aquatic on line ver 1 50 of 102

Diseases of frogs

• Bloated frog – due to bacterial infections or metabolic problems (edema due to kidney and liver malfunction) – treatment: quarantine; treatment depends on cause

http://www.xenopus.com/disease.htm

Diseases of frogs

• Fungal or nematode infections – treatment: antifungal solutions; injections with invermectin against nematodes

http://www.xenopus.com/disease.htm

19 Aquatic on line ver 1 51 of13/01/2020 102

LAST course

Susie Mitchell Fish Vet Group Ireland

Outline

• Focus on farmed species • Farming systems / experimental units • Fish welfare & implications of using cultured fish as experimental models • Stress and disease interactions in fish • Signs of disease & principles of disease transmission

Husbandry

• Types of rearing systems • Freshwater • Marine • Aquaria

1 Aquatic on line ver 1 52 of13/01/2020 102

Freshwater Farming

Marine Farm

Aquaria

2 Aquatic on line ver 1 53 of13/01/2020 102

Marine & FW Experimental units

Water parameters

• Key parameters dependant on species • Example – Atlantic salmon • Oxygen > 7mg /L • Aerators / injection / flow • Stocking density • Temperature <18 Degrees • pH 6 ‐8.5 • Stability of temperature NB –Temperature shock! ‐> No more then 2 Degree change advised

Water parameters

• Contrast – Channel catfish • Oxygen > 4mg /L • Stocking density • Temperature 28‐30 Degrees • pH 6.5 ‐ 9

3 Aquatic on line ver 1 54 of13/01/2020 102

Fish Welfare

They are only fish!

Fish Welfare

• Importance of welfare: Industry / Science Consumer / Public

• Research ‐ poor welfare compromises experimental results

• Definitions of welfare: Function Feeling Nature

Definitions

• Function‐based ‐ how the animal copes with the environment

Disease Growth status

Reproduction

4 Aquatic on line ver 1 55 of13/01/2020 102

Definitions

• Feeling‐based ‐ subjective, mental state of the animal

Definitions

• Nature‐based ‐ animal is given the possibility to perform normal and natural behaviour.

Do fish feel pain?

• Pain in humans always has a psychological component • The neocortex absent in fish • Mounting evidence they can feel pain

5 Aquatic on line ver 1 56 of13/01/2020 102

Pain Check List

• Perception X • Emotional responses X

• “Nociceptors” • CNS & PNS • Physiological and behavioural responses

Fish can perceive pain and to suffer

Current Welfare Issues when using farmed fish in experimental units

Use of farmed species in experiments

• Used extensively for challenge trials, particularly Atlantic salmon • MANY POTENTIAL WELFARE ISSUES • Usually disease models and experimental treatment trials • Chance for things to go wrong are high!

6 Aquatic on line ver 1 57 of13/01/2020 102

Potential challenges

• Disease models ‐> sick fish, mortality • Treatment trials ‐> Adverse effects • Feed trials ‐> Malnutrition • Handling fish / procedures‐> stress

• Need to optimise controllable factors

Feeding

• Stress post transfer affects feeding • Size of tank / stocking density • Small aquaria without water exchange –easier not to feed fish.. • Food deprivation –how long is OK without food? • Malnutrition during feed trial

Poor Water Quality

Environmental Parameters

Oxygen Turbidity

7 Aquatic on line ver 1 58 of13/01/2020 102

Environmental Parameters

• Fish are in intimate contact with environment • Good water quality NB • Oxygen levels • FW ‐ Nitrates, ammonia etc. • Marine – salinity, phytoplankton / zooplankton

Stress in Fish Reducing stress = optimising welfare

Two Types of Stress

Acute stress ‐ caused by short term stressors

Chronic stress ‐ caused by long term stressors

8 Aquatic on line ver 1 59 of13/01/2020 102

Effects of Stress

Unavoidable! Acute stress .Transfer .Predators . Planning and Stress Effects Management

Time

Effects of Cumulative Stress

1st 2nd 3rd Acute Acute Acute stress stress stress

Stress Effects

Time

Chronic Stress

.Parasites Chronic stress .Low oxygen begins .Poor water quality .Reduces appetence & Immunity

Stress Effects

Time

9 Aquatic on line ver 1 60 of13/01/2020 102

Cortisol = The Stress Hormone

Effects of cortisol

 Increased metabolic rate Increase in muscle energy levels  Reduced appetence Immune response decreases

 COMPROMISED EXPERIMENTAL RESULTS

Effects of repeated stress (capture & 90 sec air exposure) at 0, 1 & 2hrs

repeat stress 100.0 single stress control

50.0 Water cortisol (ng/l) cortisol Water

0.0 02468 Time (h)

A non-invasive stress assay based on measurement of free cortisol released into the water by rainbow trout T Ellis, J D James, C Stewart & A P Scott

10 Aquatic on line ver 1 61 of13/01/2020 102

Other effects of stress

• Fright/escape response may lead to physical damage • Abnormal blood parameters / experimental results • Consequences of biochemical reactions inside fish can affect flesh quality / experimental results

Stress and disease interactions

• Fish Immune system • Fight infection, stay healthy • Stress can lower immunity

Challenge Threshold to disease

Disease High challenge

Low challenge

Challenge Threshold

Time

11 Aquatic on line ver 1 62 of13/01/2020 102

Challenge Threshold

Disease

Challenge Threshold Stress!

Time

Negative effects of stress

• Depressed appetite • Reduced growth • Increased conversion • Abnormal behaviour • Reduced immunity • Disease • Death

On a positive note…

• Allostasis • Relationship with stress is U‐shaped • No stress is as bad as too much stress • Stress enables learning and adaptive responses

12 Aquatic on line ver 1 63 of13/01/2020 102

How can we optimise welfare in farmed & aquarium species in experimental challenge models?

Optimise welfare ‐

• Healthy source of fish • Good biosecurity • High standards of hygiene and cleanliness • Good husbandry is key • Appropriate SD, water quality, oxygen, etc • Settling in period prior to experiment • Appropriate diet • Environmental enrichment?

Astra Zeneca

13 Aquatic on line ver 1 64 of13/01/2020 102

Environmental enrichment

• Group schooling species –house together • Dawn / dusk photoperiod • Appropriate flow rate for species • Interaction with aerator / filter • Different food to encourage a range of behaviours • Environmental enrichment promotes neural plasticity and cognitive ability in fish(Salvanes et al 2013) • Raising fish in an environment with "furniture" improved their learning ability

Optimise Welfare ‐

• Learn to recognise signs of disease • Close monitoring for adverse effects during treatments • Experienced person in charge of fish • Know when to terminate experiment and have plan in place on how to do so (e.g. total water change, euthanasia)

Examples –what can go wrong

1. Hydroid challenge (Controls compromised) 2. Clavochlamydia challenge (lack of feed, toxicity) 3. AGD challenge – compromised respiratory function, moribund fish, mortality 4. Amoebic gill disease (AGD) challenge – adverse reactions to novel bath treatments

14 Aquatic on line ver 1 65 of13/01/2020 102

In disease challenge models when is a humane endpoint?

Disease –Why worry?

• Disease can compromise experiment and skew results • Disease can compromise welfare • Always risk of disease introduction when using farmed stock –no “SPF” fish! • Vigilance for signs of disease NB • Disease can be sub‐clinical & covert

Viruses

Parasites

Types of disease causing organisms

Bacteria

Fungi

15 Aquatic on line ver 1 66 of13/01/2020 102

General signs of disease

• Behavioural signs: • Lethargy, anorexia, abnormal position in water column, seclusion, abnormal swimming behaviour, flashing, rubbing • Physical signs: • Lesions on body surface, pop‐eye, frayed fins, change in body colour, increased mucus, reddening around mouth or fin bases, • Presence of fungal plaques

Whats normal?

16 Aquatic on line ver 1 67 of13/01/2020 102

Non‐infectious disease

• Spinal deformities (zebrafish > 2yr) • Eye abnormalities • Fin erosion • Opercular shortening

Zebrafish Diseases

• Mycobacteriosis • Filamentous bacteria • Edwardsiella • Velvet disease • Microsporidian “skinny disease” • Scoliosis

What to do if something goes wrong

1. Establish the history of the fish – supplier, quarantine, etc. 2. General observations

17 Aquatic on line ver 1 68 of13/01/2020 102

General observations

NB: Person responsible for daily husbandry is best placed for this • What is level of mortality (high / low)? • Sudden or gradual (acute or grumbling)? • What pens / tanks affected –one vs all?‐> • One pen often indicates something infectious, all pens affected together, most likely environmental (eg. Compromised water quality)

General observations –behaviour • Feeding (increased or decreased)? • Unusual swimming behaviour (fish near surface / gathering at water inlet)? • Clinical signs –fish gasping (increased Respiratory rate) or keeling over in water column

What to do if something goes wrong

1. Establish the history of the fish – supplier, quarantine, etc. 2. General observations 3. Clinical examination of sick fish 4. Examine recent mortalities 5. Take clinical samples if required 6. Initiate treatment / euthanasia of affected stock as appropriate

18 Aquatic on line ver 1 69 of13/01/2020 102

PREVENT STRESS + OPTIMISE WELFARE = HAPPY FISH + SOLID EXPERIMENTAL RESULTS!

RSPCA

• Welfare\RSPCA salmon standards 2012.pdf

19 Aquatic on line ver 1 70 of 10213‐Jan‐20

Zebrafish

LAST Aquatics Course Ali Cronin

Content

• Background • Development of Zebrafish • Biology of Zebrafish • Husbandry • Aquaria systems • Transport • Anaesthesia • Surgery • Signs of distress • Common disease • Euthanasia • Authorisation • Record keeping

Zebrafish

• Freshwater tropical fish native to streams of the southeast Himalayan region. • Found in slow moving rivers and floodplains. • Temperatures ranging from 17‐30°C. • In natural habitat spawn annually, large clutch numbers. • No parental care high mortality at juvenile stages.

1 Aquatic on line ver 1 71 of 10213‐Jan‐20

Phylogeny

• Teleost fish of the cyprinid family • Lineage leading to the cyprinids and mammals split 450 million years ago

Behaviour

• Shoaling species with dominance hierarchies. • At low densities they show aggression however if densities are too high it will result in stress in the fish. • Introducing objects into tank can reduce aggression if fish need to be kept in small numbers.

Gender

• Females tend to be larger than males and require more food. • Grey/Silver in colour with a round abdomen. • Males are more slender and sometimes appear more purple/pink than females. • Life span is up to 6 years. • After 2 years egg quality deteriorates.

2 Aquatic on line ver 1 72 of 10213‐Jan‐20

Advantages • Development ex utero. • Large clutch numbers (100‐300 eggs). • Embryos are completely transparent for initial 24 hours post fertilisation (hpf). • Rapid development. • Up to 5 days post fertilisation (dpf) not covered by legislative requirements of EU Directive 63 of 2010. • Genome has been fully sequenced. • Relatively low cost when compared to upkeep of rodent facilities (over 15 times less cost than rodents)

Genome

• Zebrafish genome 1.7 GB in size • 25 chromosomes • 2 homologues of the mammalian equivalent gene (genome duplication event) • Function of ancestral genes may be divided between two genes

Development

3 Aquatic on line ver 1 73 of 10213‐Jan‐20

Early development

Early development • Fertilised eggs are protected by the chorion. Development begins with one cell on top of a yolk sac. • By 1 dpf clear anatomical structures can be seen. • Pigment begins to develop at 2 dpf. • 2‐3 dpf the embryo will hatch out of the chorion (larvae once hatched). • Feeding will be required from 5 dpf. • At 5 dpf the swim bladder will develop and inflate. • The first 4‐6 weeks embryos and larvae will be kept at 28°C.

Zebrafish development

4 Aquatic on line ver 1 74 of 10213‐Jan‐20

Biology

Husbandry

Cleaning

• Cleanliness is the most important feature of keeping zebrafish in healthy breeding conditions. • Cleaning will also reduce the risk of spread of disease. • Filters will need to be checked for blockages daily. • Tanks not on a recirculation system will need to be cleaned weekly.

5 Aquatic on line ver 1 75 of 10213‐Jan‐20

Room conditions

• A suitable zebrafish housing room will have water supply, ideally one with reverse osmosis water, a sink, floor drain, power lines, and a programmable room light. • All furniture and equipment in the room will need to be fit to tolerate high humidity and frequent exposure to water. • Zebrafish require a temperature between 25°C and 28°C.

Circadian Rhythm

• Fish are kept on a 14 hour light –10 hour dark cycle using automatic timers. • Zebrafish spawn when the light is turned on in the morning.

Fish Densities

• Depends on the efficiency of the filter, age of the fish and the amount of feeding. • Lower densities are required for spawning fish. • 1‐3 fish in quarantine do not need filters but need daily cleaning (max 3 fish per litre). • Large scale recirculation systems 5 fish per litre (60 fish in 12 litre tanks).

6 Aquatic on line ver 1 76 of 10213‐Jan‐20

Aquaria systems

Systems without filtering • Individual Glass tanks • Flow through systems Advantages – Cheaper to set up Disadvantages – Require far more space – Labour intensive – Not suitable for large number of fish

Aquaria systems

Systems with filtering • Glass tanks with individual filter. Recirculation systems • Serial tanks • Overflow system Advantages – Can stock higher densities of fish – Requires far less space – Less labour intensive Disadvantages – Expensive to set up

Aquaria systems

• Water needs to be kept clean to avoid toxicity. – Recirculation systems water change‐every 6 weeks – Systems without filtering water change a min of once a week. • Low density of fish – Recirculation system (5 fish/litre) – System without filtering (1‐2 fish/litre) • In filtered systems bacteria in filter sponge degrade toxic ammonia and less toxic nitrates. • In large recirculation systems the water from tanks is recycled through one common filter.

7 Aquatic on line ver 1 77 of 10213‐Jan‐20

Systems without filtering

Single Glass tanks (no filter) • Cheap to set up • Very space demanding as fish need to be kept at low densities • 1‐2 fish per litre needs water exchanged every week.

Flow through systems (20‐200 litres) • 1‐2 fish per litre • Tap water: charcoal filtered and run through UV lamps before entering the aquaria • Aerate tanks using air stones • Requires daily cleaning

System with filtering • Single glass tanks with individual filters. • Lids to stop evaporation and fish escaping from tank. • Three sizes – Adult tanks usually 10 L capacity – Juvenile tanks 3 L – Spawning tanks 1 L

Recirculation systems

• With biological filters the water is purified, cleaned and reused. • High quality water and a high water exchange rate. • Water in tanks is cleaned and regenerated through a common filter unit. • Allows for very large fish numbers in a smaller space. • Disadvantage is disease can easily spread but this risk can be reduced by sterilising water using UV irradiation.

8 Aquatic on line ver 1 78 of 10213‐Jan‐20

Recirculation systems

• Two types of recirculation systems: • Serial tanks: Long glass tanks divided by partitions. • Compartment of about 12 litres allows for 60‐ 70 fish.

Recirculation systems

• Overflow container: • Individual containers placed in a row on a glass shelf. • Water enters each tank through an inlet and exits through an overflow onto the shelf and drained into a communal filter.

Recirculation systems • Biological filtration – Trickle filters, efficient nitrification, needs separate SS filtration and prone to stopping – Submerged filters, fluidised sand filters • Suspended solids filtration – Silica sand filters – In‐line sock filters, pad filters – Drum filters • UV irradiation – Kills most bacteria and viruses – Requires cleaning quartz sleeve and bulb replacement – Suspended solids affect efficacy • Chemical filters – Activated charcoal or carbon – Removes organic compounds including ammonia

9 Aquatic on line ver 1 79 of 10213‐Jan‐20

Feeding

• Feeding routine is important to maintain healthy fish especially for spawning fish. • Overfeeding or underfeeding will have detrimental effects on fish health. • Most will be fed twice daily. – Dry feed in the morning – Live feed in the evening (Artemia or Paramecia). • Will have effect on quality and quantity of eggs. • ZM systems recognised as a good quality supplier of special diet services (SDS) feeds for zebrafish.

Feeding

• Fish should be monitored for health status during feeding. • All food should be eaten within ten minutes. • Overfeeding will block filters and encourage bacteria growth quickly reducing water quality. • Live feed will be needed along with dry food to maintain good breeding conditions.

Feeding

Paramecia

Dry food

Artemia

Fertilisation 5dpf 10d 12d 15d 20d 28d

10 Aquatic on line ver 1 80 of 10213‐Jan‐20

Paramecia • Unicellular ciliated protozoan. • Widespread in freshwater and abundant in stagnant ponds. • Cells are ovoid in shape and covered with cilia. • Reproduce asexually by binary fission. • Cultures are kept to feed to zebrafish larvae.

Artemia • Genus of aquatic crustaceans known as brine shrimp. • Artemia are a source of omega 3 & 6 oils. • Daily feeding with Artemia continues for adult fish, good nutritional source and behavioural stimulant. – Important for environmental enrichment • Found worldwide in inland saltwater lakes can tolerate very high salinity (up to 250g/l). • Produce dormant eggs (cysts) which can be hatched on demand.

Artemia

• Cysts are placed into artemia hatcher containing artificial sea water. • Also require light source, aeration and 28°C for hatching.

11 Aquatic on line ver 1 81 of 10213‐Jan‐20

Spawning

• Zebrafish sexually mature at 4 months. • Typically are spawned every week. • Spawning pairs are placed into a 1L tray with a grid bottom. Eggs will drop through the grid and into an outer tray where they can be easily collected. • Healthy spawning pairs will produce 100‐300 eggs. • Fertilisation rate should be >95%.

Water

• Mineral content and buffering capacity of the water is important for fish health. • Elevated salt concentrations have a positive effect on embryo and larvae health (bacterial growth and pH). • However lower salt concentrations promote fertility of the adult fish. • Embryos are kept in water with slightly elevated salt content (1g/l) to help prevent bacteria growth. • To remove possible organic contaminants water can be passed through a charcoal filter.

Water • Water needs to be at a pH of 7‐7.5. • If it drops below 6‐toxic effects. Can be solved by changing the water or adding bicarbonate to the water in a recirculation system. • If filters are not functioning correctly the water may rise to above pH 8 (stops growth of denitrifying bacteria) pH should be measured weekly. • Water that is not replaced frequently enough will lead to the accumulation of toxic ammonium compounds in the water. • Any water used to replace in tanks should be left overnight in the fish room to allow chlorine to evaporate and also for the temperature to rise. Water that is too cold can shock the fish and result in death.

12 Aquatic on line ver 1 82 of 10213‐Jan‐20

Water Parameters

Parameter Zebrafish Temperature (°C) 25‐28 Oxygen >6mg/ml Ammonia <0.02mg/ml Nitrite <1mg/ml Carbon dioxide <5 Chlorine 0 pH 7.0‐7.5 Salinity 0.5‐1g/l

Biological filters

• Ammonia is the primary nitrogenous waste product excreted by fish. • Primary function of biological filters is to convert this ammonia to less toxic form. • Two step biological process – Nitrosomonas bacteria convert ammonia to nitrite – Nitrobacter bacteria convert nitrite to less toxic nitrate • Be careful using disinfectants on culture tanks/equipment • All filters will require a start‐up phase until denitrifying bacteria have colonised the filter sponges (several weeks). • Water will need to be checked daily during this phase. If nitrite levels rise water will need to be changed immediately. • Aeration is important for culture survival.

Conditioning a filter

• Incubate biological filter sponge with nitrifying bacteria. – Need ammonia for Nitrosomonas bacteria – Need nitrite for Nitrobacter bacteria • Sponges not in use are left in container with bacteria. – Aeration is required – Ammonia added weekly to keep culture going • Sponges are rinsed out with water before use. • Nitrite declines to <1mg/ml, nitrate rises: culture is mature.

13 Aquatic on line ver 1 83 of 10213‐Jan‐20

Supply and transport

• Directive EU 63 of 2010 states zebrafish used for research must be from purpose bred stock. • Source from universities or licenced centres (European Zebrafish Resource Centre) – Currently has over 12,400 different strains of zebrafish. Large number of wildtype and mutant lines.

Transportation

• To import, must be registered with the regulatory authority (Marine Institute) as an importer of live fish • Must enter through an approved border inspection point (Dublin Airport, Shannon Airport or Dublin Port) • From outside EU, require health certification from supplier • Better to transport as embryos or early larvae

Transportation

• Egg transport – Chorion disinfection – reduces risk of disease transmission – Low oxygen demand – Better protected from physical trauma – Can be shipped in 50 ml tissue culture flasks containing about 20 eggs filled to the top with water • Transport as Adults – Double walled container, inner plastic fish bag and outer polystyrene insulation box – Approximately 10 fish in 2 L – Fill 2/3rd with water and 1/3rd oxygen or air – Withhold food for one day prior to shipment

14 Aquatic on line ver 1 84 of 10213‐Jan‐20

Transportation

• Upon arrival – Check water temperature, pH and ammonia – Avoid temperature shock • Place fish bag into a tank of water to allow gradual acclimation – Feed adult fish on day of arrival (useful initial indicator of stress level) • Maintain in quarantine for 3‐4 weeks

Anaesthesia

• Tricaine methane sulphonate (MS222) used to anaesthetise zebrafish. • Is acidic, must be buffered before use and water pH should be measured before and after adding MS222. • Use either Phosphate buffer or Sodium hydrogen carbonate (bicarbonate) to raise the pH to the desired range (7‐7.5). • Light sedation in zebrafish (30‐50mg/l) • Deep anaesthesia (100‐200mg/l)

Surgery on zebrafish

• Do not feed for at least 12 hours prior to surgery. • Usually will not require any sutures for incision to heal. • Restrained by being placed into a sponge. • Will need quarantine and close observation post surgery.

15 Aquatic on line ver 1 85 of 10213‐Jan‐20

Recognition of pain/distress

• Reluctance to eat • Unusual behaviour • Discolouration • Reddening around mouth, gills, abdomen. • Swimming at surface • Abnormal body shape • Scales standing • Any fish showing illness should be removed and isolated immediately

Fish health

16 Aquatic on line ver 1 86 of 10213‐Jan‐20

Common diseases

• Pseudoloma neurophilia (microsporidiosis) – Parasite transmitted from parents to offspring. – It infects the CNS and skeletal muscle of zebrafish causing chronic emaciation (or ‘skinny disease’), reduced growth, ataxia and spinal malformations. – No known effective treatment, UV light sterilisation of the water has proven to help in reducing its incidence (Kent 2007). – PCR‐based tests can be used to screen for carriers (such that Pseudoloma‐free facilities may be established and maintained).

Common diseases • Fish tuberculosis (or mycobacteriosis) – Bacteria frequently present in aquaria but by keeping a clean, well‐watered system and the fish healthy, this infection should not pose a problem (Vargesson 2007). – There is a high risk of infection between fish. – Some evidence it can be spread to humans: gloves must be worn. – Fish may look unwell e.g. they may have open sores, be lethargic, have raised scales or appear emaciated (Vargesson 2007). – Sterilising UV lamps can be incorporated into the circulation system, which kills 99% of all Mycobacterium tuberculosis. – There is currently no known successful treatment for this disease.

Common disease • Velvet disease – Zebrafish are highly susceptible to the very contagious 'velvet disease' caused by Oodinium pillularis. Parasite attaches to the fish near the fins, and around the gills (Westerfield 2006). – Rubbing behaviour, lethargy, fins held close to the body. – This disease can be cured using a 3‐day treatment of Atabrine (Quinacrine hydrochloride) (10mg/ml stock )Westerfield (2006): – Day 1: Slowly drip 2 litres of sea salts solution (140g/l) into an infected 10‐gallon (38 litre) tank. Add 3.3 ml of the Atabrine stock solution – Day 2: Add 3.3 ml Atabrine stock. – Day 3: Add 3.3 ml Atabrine stock for a total of 9.9 .ml At the end of the day clean the bottom of the tank thoroughly and slowly dilute out the salt and the Atabrine with fresh water. Continue cleaning the bottom of the tank daily for several days.

17 Aquatic on line ver 1 87 of 10213‐Jan‐20

Euthanasia • Limit the pain, suffering and distress felt by the animal. • Carried out by authorised personnel only. • EU Directive 2010/63 permits euthanasia by overdose of anaesthetic (buffered MS222 250mg/l). • Death by MS222 usually occurs within a minute but fish must be left for 10 minutes after opercular movements cease. • All methods of euthanasia must be followed by pithing of the brain.

Authorisation

• Work with zebrafish older than 5 dpf requires authorization and ethical approval • First need ethical approval from the institution (e.g. University) • Then apply for authorisation to the HPRA (Health Products Regulatory Authority) • Individual licence • Project licence • Establishment licence

Record keeping

• All tanks need to be clearly labelled with the tank number and date of birth. • Records must be kept of the fish number in each tank. • Daily records – Fish health – Room temperature – Feeding checklist – Filters functioning • Weekly records – Spawned tanks – Cleaned tanks • Any ill or dead fish must be recorded and fish tank numbers updated immediately. • A record needs to be kept of the amount of culled fish. Date of euthanasia, amount of fish, reason for euthanasia and method need to be recorded as well as who it was carried out by. • Regular inspections to ensure compliance.

18 Aquatic on line ver 1 88 of 10213‐Jan‐20

References

• Statutory Instrument 543 of 2012 • Directive EU 63 of 2010 • Wilson et al 2013, Animal Technology and Welfare. • Kent, M.L. (2007) Oral presentation ‘Overview of diseases of zebrafish, with emphasis on mycobacteriosis and microsporidiosis’ 22 November 2007, Brighton, UK • Astrofsky, K.M., Harper, C.M., Rogers, A.B. & Fox, J.G. (2002) 'Diagnostic techniques for clinical investigation of laboratory zebrafish' Lab Animal 31 (3), p41‐45 • Vargesson, N.A. (2007) ‘Zebrafish’ in Manual of Animal Technology (ed. S. Barnett) Blackwell Publishing Ltd: Oxford, UK. Westerfield, M. (2000) 'The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)' ‐ 4th edition, University of Oregon Press, Eugene • Zebrafish by Volhard & Dahm (2002). • RSPCA Guidance on the housing and care of Zebrafish, Reed and Jennings (2010) • Directive EU 63 of 2010

19 Aquatic on line ver 1 89 of 10213‐Jan‐20

Basic disease epidemiology and welfare in a fish research facility

T.M. Murphy

Disease outbreaks in fish holding facilities: (a) Can have disastrous effects on research programmes [due to disruption of the normal physiology and biochemistry of diseased fish]

Thus it behoves everyone who work with experimental fish to understand how diseases may be introduced into and spread within a fish holding facility

Objectives

At the end of this lecture the student will be able to

(a) Understand how fish become infected

(b) How diseases can spread within a fish holding facility

(d) The precautions that can be taken to prevent entry of disease

(e) Understand how diseases can be contained if they are introduced into a facility

(f) Understand the basics of a health monitoring programme

1 Aquatic on line ver 1 90 of 10213‐Jan‐20

Good welfare = Good science

Welfare in fish (especially fish in research facilities) : Not well defined

Fish medicine: Health and welfare: general terms that are often used synonymously (both terms are used interchangeably)

Health & Welfare Monitoring can be performed: (a) Directly by clinical observation of the fish population (b) Indirectly by monitoring the fishes’ environment

Thus the tac of health monitoring is considered an integral part of the process for ensuring good fish welfare

Good welfare continued

For fish welfare to be acceptable: A number of conditions must be fulfilled

(a) Fish should be free of disease, injury and functional impairment (b) Should have all their biological systems functioning appropriately

However, the link between health and welfare is complex

Although a reasonable assumption is: Fish showing signs of disease are in a poor state of welfare

Which is indicative of : (i) An underlying problem with the environment (ii) Stressful conditions (experimental or husbandry) to which the fish are being subjected to

Pathogenic micro‐organisms are ubiquitous: Disease precipitated by stressful environmental conditions

Transmission of infectious disease in a fish holding facility

Chain of infection:

No. of links

Each link : Considered a critical disease control point (infection cycle can be broken)

2 Aquatic on line ver 1 91 of 10213‐Jan‐20

Infectious diseases can be caused by: Microorganisms Pathogens:

Viruses

Bacteria

Fungi

Parasites (i) protozoa (ii) helminths (nematodes, cestodes, trematodes) (iii) arthropods

Microorganisms are ubiquitous in most water bodies and fish are frequently exposed to potential pathogens

Sources and Reservoirs of infection

During an outbreak: Main Source of infection for healthy fish: Clinically ill fish

Asymptomatic carriers: Very important alternative source of infection [Latent or silent infections] [Clinically in‐apparent infected fish]

(i) During an outbreak newly infected/sub‐clinically infected fish (ii) Recovered fish (can often carry and shed pathogens that caused the initial disease)

Portal of exit from diseased fish

i) Leakage from skin wounds ii) Faeces iii) Urine iv) Leakage from autolysing fish carcasses

3 Aquatic on line ver 1 92 of 10213‐Jan‐20

Transmission of disease within a fish research facility

a) Direct: When healthy and infected fish are in the same tank or pond

b) Indirect

i) Mechanically: Organism suspended in the water body or attached to sediment particles and/or other fomites(farm and pond utensils, fish handlers hands and clothes)

ii) Feed: Contaminated feed especially if live feed is being used

iii) Gametes : Some viruses and bacteria transmitted vertically through fertilised eggs (furunculosis)

Portal of Entry of pathogenic microorganisms

(1)Passive absorption of pathogens in the water body and feed via: a) Skin (wounds) b) Gills [disrupted gill lamellae & epithelium] c) Mucous membranes of intestine, urinary system

(3) Inoculation by biting vectors etc [special cases, should not occur in fish research facilities]

Infectious disease: Three‐way interaction requiring (a) Host (b) Pathogen (c ) Environment

Obligate pathogens Pathogen Host Opportunistic pathogens

Environment

4 Aquatic on line ver 1 93 of 10213‐Jan‐20

In the event of a disease outbreak

Initial sign: Anorexia Staff responsible for feeding Behavioural changes should be trained to observe changes in the feeding pattern

Routine monitoring of fish research facilities for disease

A major inhibitory factor for efficient screening is that there is a dearth of non‐invasive tests for the common fish diseases Health monitoring usually carried out on a population basis

Fish can only be observed for clinical signs

5 Aquatic on line ver 1 94 of 10213‐Jan‐20

Preventing the introduction of disease into a fish research facility

The best way to control infectious disease: Avoid their introduction

Synopsis:

(1) Avoid the introduction of fish with asymptomatic disease

(2) Maintain a stress free environment Optimum: (a) Stocking density (b) Water quality and temperature (c ) Provide some level of environment enhancement

6 Aquatic on line ver 1 95 of 102

L 276/72 EN Official Journal of the European Union 20.10.2010

ANNEX IV

METHODS OF KILLING ANIMALS

1. In the process of killing animals, methods listed in the table below shall be used.

Methods other than those listed in the table may be used:

(a) on unconscious animals, providing the animal does not regain consciousness before death;

(b) on animals used in agricultural research, when the aim of the project requires that the animals are kept under similar conditions to those under which commercial farm animals are kept; these animals may be killed in accordance with the requirements laid down in Annex I to Council Regulation (EC) No 1099/2009 of 24 September 2009 on the protection of animals at the time of killing ( 1 ).

2. The killing of animals shall be completed by one of the following methods:

(a) confirmation of permanent cessation of the circulation;

(b) destruction of the brain;

(d) exsanguination; or

(e) confirmation of the onset of rigor mortis.

3. Table

Dogs, cats, Animals-remarks/ Large Non-human Fish Amphibians Reptiles Birds Rodents Rabbits ferrets and methods mammals primates foxes

Anaesthetic (1) (1) (1) (1) (1) (1) (1) (1) (1) overdose

Captive bolt (2)

Carbon dioxide (3)

Cervical (4) (5) (6) dislocation

Concussion/ (7) (8) (9) (10) percussive blow to the head

Decapitation (11) (12)

Electrical stunning (13) (13) (13) (13) (13) (13)

Inert gases (14) (Ar, N 2 )

Shooting with a (15) (16) (15) free bullet with appropriate rifles, guns and ammunition

( 1) OJ L 303, 18.11.2009, p. 1. Aquatic on line ver 1 96 of 102 20.10.2010 EN Official Journal of the European Union L 276/73

Requirements 1. Shall, where appropriate, be used with prior sedation.

2. Only to be used on large reptiles.

3. Only to be used in gradual fill. Not to be used for foetal and neonate rodents.

4. Only to be used for birds under 1 kg. Birds over 250 g shall be sedated.

5. Only to be used for rodents under 1 kg. Rodents over 150 g shall be sedated.

6. Only to be used for rabbits under 1 kg. Rabbits over 150 g shall be sedated.

7. Only to be used for birds under 5 kg.

8. Only to be used for rodents under 1 kg.

9. Only to be used for rabbits under 5 kg.

10. Only to be used on neonates.

11. Only to be used for birds under 250 g.

12. Only to be used if other methods are not possible.

13. Specialised equipment required.

14. Only to be used on pigs.

15. Only to be used in field conditions by experienced marksmen.

16. Only to be used in field conditions by experienced marksmen when other methods are not possible. Aquatic on line ver 1 97 of 102 CIR1525

Fish Slaughter, Killing, and Euthanasia: A Review of Major Published U.S. Guidance Documents and General Considerations of Methods1 Roy P.E. Yanong, Kathleen H. Hartman, Craig A. Watson, Jeffrey E. Hill, B. Denise Petty, and Ruth Francis-Floyd2

Introduction The terminology used to describe the deliberate ending of the life of a fish is largely based on the contextual applica- Fish are important to society for a number of different tion of the activity (Table 1). Three main categories used positive uses: for food, for recreation, for research, and as to describe ending the life of a fish are slaughter, killing, pets. However, non-native fish illegally released into the and euthanasia. The term slaughter is used primarily by environment pose a nuisance. In each case, death of fish agricultural producers and commercial fishers for products may be necessary or desirable. Cultural and ethical norms intended for human consumption (e.g., agricultural harvest, for causing death of fish reflect differing human percep- commercial fisheries). The term killing is used to describe tions according to the circumstances. There is an ongoing some recreational fishing practices and may also include scientific debate regarding how fish process negative activities such as fish sampling, depopulation or eradication, stimuli, specifically, whether they feel pain in the same or and control. These words are all used to describe activities an equivalent manner as do mammals. This debate will by field personnel, resource managers, and veterinarians not be resolved easily. Ultimately, science will only provide or fish health specialists for ecological research, fish health part of the answer. Differing circumstances and societal inspections, or to eliminate unwanted fish (including values will also be important in determining how fish are diseased or non-native fish) from a water body. The term handled in each situation. Parallels can be drawn with harvest specifically refers to the act or process of gathering societal considerations of terrestrial species. For example, a crop, for example, as in aquaculture and commercial in research, mice and rats are important laboratory animals; fishing; however, harvest may also be used to describe fish to some, they are valuable pets; however, in other situations, removed from a water body by anglers. As described above, they are unwanted pests, for which a variety of legal control harvested fish may be slaughtered or killed, depending upon methods are available. Methods used to cause death of fish the context of the activity. The term euthanasia (meaning should, as much as practical, seek to “1) minimize stress an easy or good death) is typically used by veterinarians and 2) minimize time to death” (Hartman 2006). and laboratory research scientists to describe ending the

1. This document is CIR1525, one of a series of the Fisheries and Aquatic Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Original publication date June 2007. Revised December 2007. Reviewed March 2012. Visit the EDIS website at http://edis.ifas.ufl.edu.

2. Roy P.E. Yanong is associate professor and extension veterinarian, Kathleen H. Hartman is courtesy assistant professor, Craig A. Watson is assistant director of aquaculture programs, and Jeffrey E. Hill is assistant professor, Tropical Aquaculture Laboratory-Ruskin, Department of Fisheries and Aquatic Sciences; B. Denise Petty is clinical assistant professor and extension veterinarian, and Ruth Francis-Floyd is professor and director of aquatic animal health, Department of Fisheries and Aquatic Sciences, and Large Animal Clinical Sciences; Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences.

The Institute of Food and Agricultural Sciences (IFAS) is an Equal Opportunity Institution authorized to provide research, educational information and other services only to individuals and institutions that function with non-discrimination with respect to race, creed, color, religion, age, disability, sex, sexual orientation, marital status, national origin, political opinions or affiliations. U.S. Department of Agriculture, Cooperative Extension Service, University of Florida, IFAS, Florida A&M University Cooperative Extension Program, and Boards of County Commissioners Cooperating. Millie Ferrer-Chancy, Interim Dean Aquatic on line ver 1 98 of 102 Table 1. Terminology used to describe the deliberate ending of the life of fish Term Possible Applications Examples Slaughter o Agricultural harvest o Catfish, salmon, and tilapia o Commercial fisheries o Wild-caught grouper and snapper catches Killing o Recreational fisheries o Largemouth bass and red drum o Depopulation or eradication o Non-native species eradication (e.g., walking catfish) o Population disease control or testing [e.g., outbreaks of spring viremia of carp (SVC1) or viral hemorrhagic septicemia (VHS1)] o Ecological research

Euthanasia o Pets o Hobbyist koi and goldfish o High-value wildlife or zoo animalso o Public aquarium sharko Small scale toxicology work in zebra danios Laboratory research 1Spring viremia of carp (SVC) and viral hemorrhagic septicemia (VHS) are viral diseases, that are under international and national regulatory control and for which depopulation of infected populations is warranted. life of a pet, research animal, or other high-value (financial by relevant legalities and restrictions when using or adding or sentimental) fish (e.g., public aquarium fish) (Hartman compounds/chemicals to water bodies or for treating fish. 2006). One group of chemicals, listed in the JSA Guide, includes EPA-registered and labeled fish toxicants/piscicides such Available Guidance Documents as chlorine (sodium hypochlorite, calcium hypochlorite), antimycin A, and rotenone. from Different Organizations and Agencies Limitations of the JSA Guide to Drug, Vaccine, and Pesticide Use in Aquaculture Four major guidance documents currently available in the U.S. will be discussed in this publication: 1) JSA Guide to Although the JSA Guide provides excellent information and Drug, Vaccine, and Pesticide Use in Aquaculture; 2) Use linkages to relevant agencies as well as specific information of Fishes in Research Committee’s Guidelines for the Use on drugs and chemicals available for use in aquaculture, of Fishes in Research; 3) Guide to the 2000 Report of the the document does not specifically address fish slaughter, AVMA Panel on Euthanasia; and 4) AAZV’s Guidelines for killing, or euthanasia as topic areas. Rather, it lists specific Euthanasia of Nondomestic Animals. Each document has a compounds that are approved for use (or considered low specific target audience, and contains useful information, regulatory priority) by FDA as anesthetic agents or labeled but also has limitations. by EPA as fish toxicants.

1. JSA Guide to Drug, Vaccine, and Pesticide 2. UFR-C Guidelines for the Use of Fishes in Use in Aquaculture Research The Federal Joint Subcommittee on Aquaculture (JSA) The American Fisheries Society (AFS), American Institute Working Group on Quality Assurance in Aquaculture of Fisheries Research Biologists (AIFRB), and the American Production’s Guide to Drug, Vaccine, and Pesticide Use in Society of Ichthyologists and Herpetologists (ASIH) also Aquaculture (April 2007 revision) is an important guidance provide guidance, specifically for research purposes, in document for aquaculture and for use of specific aquatic- their jointly developed Use of Fishes in Research Committee’s site labeled products. The entire document is available on (UFR-C) Guidelines for the Use of Fishes in Research http:// the Web as an html Web page at http://aquanic.org/jsa/ www.fisheries.org/afs/publicpolicy/guidelines2004.pdf. wgqaap/drugguide/drugguide.htm. This document was These guidelines are intended to provide general recom- produced collaboratively by the US Department of Agricul- mendations to assist researchers as well as state and federal ture (USDA-APHIS and USDA-CSREES), US Department agencies, with regards to experimental design and handling of Health and Human Services (FDA-CVM and FDA- of fish species. CFSAN), and the US EPA (Office of Pesticide Programs). The JSA Guide emphasizes the importance of knowing who the regulatory agencies are and understanding and abiding

2 Aquatic on line ver 1 99 of 102 Limitations of the UFR-C Guidelines for Limitations of the Guide to the 2000 the Use of Fishes in Research Report of the AVMA Panel on Euthanasia This document provides useful guidance. However, the Because of the pivotal role that veterinarians play in the “Euthanasia” section does not clearly differentiate between arenas of animal health and welfare, the AVMA Guide the activities of fish slaughter, killing and euthanasia. has become an important and foundational reference for Within this section, methods for fish slaughter by fish many different species and scenarios requiring euthanasia. processors are described and include cold or electrical The AVMA has taken the lead in developing a thoughtful, shock. Pithing, spinal cord dislocation, and decapitation, scientifically based, and comprehensive approach to animal if done quickly and accurately, are considered acceptable euthanasia, and the AVMA Guide has undergone revisions methods for euthanasia of research fish, as is use of as new information emerges. The AVMA Guide, therefore, is tricaine methanesulfonate. Although these practices often assumed to be the only acceptable source for methods may be feasible for small numbers of research fish, they for the death of animals. However, the AVMA Guide is not are often not practical for large scale field research, and intended for all persons, agencies, or organizations, and these differences should be taken into consideration. In is applicable and practical under a limited set of circum- addition, tricaine methanesulfonate is FDA-approved stances. In fact, the AVMA’s recommendations are intended for temporary immobilization only. Currently, there are for “use by members of the veterinary profession” and the no FDA-approved drugs for euthanasia of fish. UFR-C “failure to list or recommend a means of euthanasia in this Guidelines suggest stunning with an electroshock followed report does not categorically condemn its use.” While use of by rapid decapitation or cold shock if large numbers of fish the AVMA Guide is appropriate for veterinarians working are collected. However, UFR-C Guidelines also provide the with fish species in many controlled settings (e.g., clinical caveat that these methods may be impossible to implement and laboratory environments), there are conflicts with these in commonly used fish sampling with nets that collect recommendations for veterinarians and non-veterinarians thousands of individuals (see General Considerations and a working in the field with fish (e.g., agriculture and natural Review of Methods Discussed). resources). Many agencies attempt to use or to apply the AVMA euthanasia recommendations for fish but cannot because the recommendations are limited in scope (Hart- 3. Guide to the 2000 Report of the AVMA man 2006). Panel on Euthanasia As described earlier in the UFR-C Guidelines section, The American Veterinary Medical Association’s (AVMA’s) tricaine methanesulfonate is labeled and approved only Guide to the 2000 Report of the AVMA Panel on Euthanasia for “temporary immobilization” and not for euthanasia. (hereafter, AVMA Guide) http://www.avma.org/issues/ It also carries a 21-day withdrawal time which should be animal_welfare/euthanasia.pdf, now also known as the considered when the final disposition of the euthanized fish AVMA Guidelines for Euthanasia http://www.avma.org/ may result in products for human or animal consumption. products/animal_welfare/euthanasia.asp provides recom- Likewise, the conditions of use for drugs considered to be mendations specifically for veterinarians involved with pets, low regulatory priority (LRP) by the FDA should also be research animals in controlled laboratory settings, and in observed. The use of the unapproved drug carbon dioxide other situations where euthanasia specifically, as opposed is typically “for anesthetic purposes in cold, cool, and to fish slaughter or killing, is under consideration. The warmwater fish” rather than for euthanasia. FDA will only AVMA Guide, developed over many years and revisions, consider their use to be a low enforcement priority when has provided a good, scientific foundation for discussions the conditions of use are observed. The list of LRP drugs and considerations in many situations where euthanasia is and their conditions of use are described on the FDA/CVM necessary. The AVMA Guide is not intended for use by the Web site http://www.fda.gov/cvm/Documents/LRPDrugs. general public or other persons without consultation with pdf. Furthermore, many of the other non-LRP drugs listed a veterinarian, who is then also tasked to use the document in the AVMA Guide are unapproved for use as euthanasia only as a guide, and to rely on his or her clinical experience agents for fish. In addition, the AVMA Guide does not and judgment to apply methods described in the AVMA specifically address slaughter or killing issues. Guide where appropriate. A number of acceptable methods are described in the AVMA Guide, including both chemical and physical means.

3 Aquatic on line ver 1 100 of 102 4. Guidelines for Euthanasia of carbon dioxide is considered a low regulatory priority drug Nondomestic Animals (AAZV Guidelines) but only if used for anesthetic purposes in fish, not for euthanasia. Similarly, methods discussed for pet fish owners The American Association of Zoo Veterinarians (AAZV) include unapproved uses for all of the chemicals listed. recently published their Guidelines for Euthanasia of Nondomestic Animals (AAZV Guidelines) which focuses on euthanasia of captive nondomestic species and free- General Considerations and a ranging wildlife. This report attempts to summarize current Review of Methods Discussed thinking and standards and to act as a starting point for There are a number of insightful guidance documents further consideration and improvement of knowledge and available to help determine the best mechanism of fish methods. The authors acknowledge that more research is slaughter, killing, or euthanasia. These and other documents necessary for non-mammalian vertebrates. are intended to serve only as guidelines, and are not the The AAZV Guidelines recognize that factors including fish result of regulatory authority. None of these guidance species, numbers and size, ultimate use, environmental documents currently addresses all imaginable situations factors, and personnel experience and skill should be con- that require fish death. Further clouding the issue, the sidered when determining the best method of euthanasia. existing patchwork of regulations and regulatory agencies The AAZV Guidelines distinguishes the differences between that have jurisdiction in these differing situations has use of the terms slaughter, killing, and euthanasia and also caused confusion regarding outcomes and intentions of fish discusses limitations of the AVMA Guide. slaughter, killing, or euthanasia among many professionals working with fish. The AAZV Guidelines include a combination of standard practices used in laboratory research, aquaculture, and in Because there are many different circumstances under fisheries work in their methods of euthanasia section, and which the death of fish may be warranted, the following provides a summary for each method discussed. practical approach is suggested. Whatever method is used, fish stress and time to death must be minimized as much Limitations of the Guidelines for as practical, and death ensured. At the same time, human Euthanasia of Nondomestic Animals safety (with regard to chemical usage and food safety) and environmental safety should be taken into consideration, (AAZV Guidelines) as well as all local, state, and federal regulations. These Although differences between underlying implications for suggestions are based on those described guidance docu- the terms slaughter, killing and euthanasia are discussed, ments and current information (May 2007), and changes to there is overlap of the use of these terms throughout these suggestions may be required over time. Appropriate the text, which may be confusing for some. The AAZV personnel and experts intimately involved with the specific Guidelines author(s) have chosen to expand the definition situation and current regulations should be part of the of euthanasia to include these other terms. Rotenone decision-making process. and antimycin are registered by the EPA as fish toxicants (pesticides), and are not officially labeled for euthanasia. In all situations, it is important to realize that within Chlorine products are also EPA-registered as fish toxicants, society, interpretative differences exist between the termi- but these are not listed in these guidelines. nology and actions involved with fish harvest/slaughter, killing, or euthanasia, and their synonyms. In addition, There are other interpretative issues with regard to use of decision-makers must be aware of practical differences certain products. For example, as discussed previously, between small-scale laboratory (small closed systems, although use of tricaine methanesulfonate is FDA approved simple manipulation, controlled environmental condi- for use in temporary immobilization, the AAZV Guidelines tions) and large-scale field scenarios (large water bodies, state that other uses are allowable according to the Animal hundreds or thousands of fish, large fish sizes, weather and Medicinal Drug Use Clarification Act (AMDUCA). How- other uncontrollable elements), and must understand the ever, extra-label drug use under AMDUCA is only allow- different perceptions and practices associated with each able for therapeutic uses and under those conditions listed scenario. In many instances, the method will be based on in the Act (21 CFR 530.2). Technically, slaughter, killing, accepted standard practices, and decisions should include and euthanasia are not considered therapeutic uses ac- the input of those with experience in the specific field or cording to AMDUCA. In reality, all of the drugs listed also circumstance. are unapproved for use as fish euthanasia drugs. Likewise,

4 Aquatic on line ver 1 101 of 102 Use either the AVMA Guide or the AAZV Guidelines if the Another important consideration is final disposition of methods discussed are applicable to the specific situation, euthanized animals. Carbon dioxide is currently the only e.g., for small scale laboratory studies or for veterinary eu- chemical described above for which there are no food safety thanasia of pet fish. Work with a veterinarian, if possible, in concerns with regard to tissue residues. Use of any of the these cases. In field situations, including research, where the other chemicals for euthanasia or depopulation therefore recommendations of the AVMA Guide may prove impracti- automatically prohibits entry of the fish into the food chain, cal or do not otherwise lend themselves to the particular either by rendering, as fish meal, or as directly consumed situation at hand (e.g., chemical residues in food/game fish; product. large numbers of fish or logistical considerations), consider options discussed under the AAZV Guidelines. In addition Other institutions have developed and provided rational, to the AVMA Guide and the AAZV Guidelines, the UFR-C alternative methods for euthanasia of small numbers of Guide and the JSA Guide should be consulted, along with fish used in research, because of difficulties with using one discussions with relevant experts. Agencies and other or another guidance document alone. For example, one non-veterinary fish professionals should develop their own university recommends the use of a saturated solution of protocols for fish euthanasia, harvest/slaughter, or killing carbon dioxide using chemical means for release, or cooling using the best available and legal methods. Other groups of tropical species to 4 °C, and chilling of non-tropical and documents may also provide reasonable approaches species in chilled salt water (at temperatures significantly and methods for consideration. Many of the methods will less than 0 °C) (University of Washington, 2002). require a certain level of expertise and training, and so may not be suitable for situations lacking appropriate expertise. In summary, fish slaughter, killing, and euthanasia differ in their context, societal value, and methods. Available Physical methods of causing death in fish include decapita- guidance documents are helpful but are limited in scope. tion, pithing, or stunning followed by decapitation and There is no single document or list of protocols that will pithing (AVMA Guide; AAZV Guidelines); cold shock, encompass all potential circumstances for which ending electrical shock, stunning with electroshock followed the life of fish will be necessary. Careful consideration of by rapid decapitation or cold shock (UFR-C Guidelines); methodologies among stakeholders and relevant experts oxygen deprivation (de-watering) may be required for should include discussion of pertinent science, generally ac- mandatory depopulation of production level-facilities cepted practices for a given activity, specific circumstances, (UFR-C Guide) but the UFR-C Guide does not recommend and applicable regulations. this particular method (de-watering) for research. Acknowledgements The JSA Guide lists the EPA-labeled fish toxicants chlorine, The authors thank Donald Prater, Aquaculture Drugs antimycin A, and rotenone, as described previously, for Team Leader, FDA-CVM; Jennifer Matysczak, Aquaculture use in aquaculture for depopulation/eradication, and two Drugs Team, FDA-CVM; and Scott Hardin, Exotic Species of these, rotenone and antimycin A are also listed in the Coordinator, Florida Fish and Wildlife Conservation AAZV Guidelines. Under certain situations, if label require- Commission for their assistance with this document. ments can be met for aquatic environments, use of these legal, EPA-labeled fish toxicants should be considered (see JSA Guide), and these products may be the most logical References options under a variety of circumstances, including for Animal Medicinal Drug Use Clarification Act of 1994 research. (AMDUCA). Accessed August 20, 2007. http://www.fda. gov/cvm/amducatoc.htm Additional chemical methods for causing death in fish include use of tricaine methanesulfonate (AVMA Guide; AVMA Animal Welfare Principles, Approved November UFR-C Guidelines), barbiturates, inhalant anesthetics, 2006. Accessed August 20, 2007. http://www.avma.org/ carbon monoxide, benzocaine hydrochloride, 2-phenoxy- issues/policy/animal_welfare/principles.asp ethanol, or carbon dioxide. However, as discussed previously, neither tricaine methanesulfonate, carbon dioxide, FDA-CVM. Drugs Approved for Use in Aquaculture. nor any of the other fish anesthetics mentioned in any Accessed August 20, 2007. http://www.fda.gov/cvm/ of the documents are currently approved by the FDA for drugsapprovedaqua.htm euthanasia or by the EPA for depopulation of fish.

5 Aquatic on line ver 1 102 of 102 FDA Guidance Document 1240.4200, Low Regulatory Priority Aquaculture Drugs. http://www.fda.gov/cvm/ Documents/LRPDrugs.pdf http://www.fda.gov/cvm/hydro- genperoxide.htm Both sites accessed August 20, 2007.

Guidelines for the Use of Fishes in Research, 2004. American Fisheries Society, American Institute of Fisheries Research Biologists, and American Society of Ichthyologists and Herpetologists. American Fisheries Society. Accessed August 20, 2007. http://www.fisheries.org/afs/publicpolicy/ guidelines2004.pdf

Guide to Drug, Vaccine, and Pesticide Use in Aquaculture (April, 2007 revision). Joint Subcommittee on Aquaculture Working Group on Quality Assurance in Aquaculture Production. Both sites accessed August 20, 2007. http:// aquanic.org/jsa/wgqaap/drugguide/drugguide.htm

Guide to the 2000 Report of the AVMA Panel on Euthanasia. American Veterinary Medical Association (AVMA). 2001. JAVMA 281(5): 669-696. Web version: AVMA Guidelines on Euthanasia. June 2007. Accessed August 20, 2007. http:// www.avma.org/issues/animal_welfare/euthanasia.pdf

Hartman, K.H. 2006. Fish. In: Guidelines for Euthanasia of Nondomestic Animals. American Association of Zoo Veterinarians (AAZV). 111 pp.

University of Washington Policy for the Euthanasia of Fish Species, 2002. Institutional Animal Care and Use Commit- tee, University of Washington. Accessed August 20, 2007. http://depts.washington.edu/iacuc/policies/fish_euthanasia. html