1. biology rodentiaredkitevets.co.uk/staging/wp-content/uploads/small...5/24/2017 1 small laboratory...

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Small laboratory animals

1. Biology

• Rodents are successful order of placental mammals – 2277 spp. and counting….

• Colonise almost all habitats

• All members are small

• Open rooted sharp teeth (grow 2.2-2.8 mm/week, 0.4 mm per day in rat)

• Care – malocclusion leads to overgrowth of teeth

Classification of rodents

Rodentia

Myomorpha

Hystricomorpha

Sciuromorpha

Muridae

Cricetidae

Caviidae

Chinchillidae

Mouse, rat

Hamster, gerbil

Guinea pig

Chinchilla

Squirrel, beaver,

chipmunk

Small rodents

• Myomorpha

• Mouse Mus musculus

• Rat Rattus norvegicus (Brown or Norway rat)

• Most commonly used laboratory animals (85%)

• Inbred and outbred strains

• Balb/C, C57/Bl mice,

• Sprague Dawley, Wistar, Lewis rat

• Golden or Syrian Hamster Mesocricetus auratus

2. Nutrition

• Herbivores

• Large caecum = microbial fermentation chamber.

• Rats are cautious eaters and will reject strange food

Rat and mouse nutrition

• Rodents can eat a variety of foodstuffs, including seeds

and grains, roots, leaves, fruit, invertebrates. Cereal

based diet in laboratory.

• Simple stomach, glandular and non glandular parts

• Digestion takes place in caecum, between ileum and

colon….

• …but this is at the end of the digestive tract• coprophagy

• soft faeces ingested during dark period

• hard faeces deposited on the floor of the cage

http://images.google.com/imgres?imgurl=http://www.wildanimalsonline.com/mammals/capybara-hydrochoerushydrochaeris.jpg&imgrefurl=http://www.wildanimalsonline.com/mammals/capybara.php&usg=__HvN0ZIjL8eYf5PRwzLe1XwdNxBE=&h=480&w=640&sz=181&hl=en&start=12&itbs=1&tbnid=koBZBPgxTtJ8ZM:&tbnh=103&tbnw=137&prev=/images%3Fq%3Dcapybara%26gbv%3D2%26hl%3Den

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3. Biological data

• Small rodents – lifespan all 1-3 years

• Normal adult weights

• Mouse 20-40g

• Rat 250-500 (M>F)

• Hamster 85-150 (F>M)

4. Reproduction

• Generation time for small rodents is 9-10 weeks

Rodent Breeding

Oestrus, mating and ovulation occur at night

Mating results in a vaginal plug

Pups are altricial –naked, blind and deaf (except

guinea pigs)

If a female is disturbed in the immediate post-

partum period, she may destroy her young,

Hamsters are particularly immature (gestation only 15-16

days). Very hard to hand rear

Weaned at three weeks

Breed until 12-18 months old

Rodent Breeding Data

• Puberty (days)

• Age to breed (days)

• Gestation (days)

• Litter size

• Birth weight (grams)

• Weaning age (days)

• Oestrous cycle (days)

• Post partum oestrus

Mouse

28-49 (av 42)

F 60-84

M 70

19-21

4-12

1-1.5

18-21

4-5

Fertile

Rat

60-63

65-110

20-23

6-12

5-6

21

4-5

Fertile

Hamster

32-42

42-70

>70

15-16

5-9

2-3

21

4

Infertile

Rodent breeding systems

• Mice

• Harem - one male for two to six females, pregnant females

removed to give birth

• Monogamous pair – mate at post partum oestrus, litter every 3-4

weeks. Young are removed before next litter is born

• Mice will breed until they are 12–18 months old, although economic

breeding life of most strains is around 6 months

• Some breeders operate traffic light system – female has 3 litters

before being retired, or maximum breeding age of e.g. 200 days.

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• Rats

• Polygamous - one male with 2-6 females. Pregnant females

removed prior to parturition and returned after weaning. Females in

produce more milk and have larger litters. Females produce 1–12

litters per year, and may nurture young collectively.

• Monogamous systems - female is mated at the post-partum

oestrus, and young removed at weaning. Maximum number of

litters, but male may interfere with the young. He can be removed

at parturition, and returned after weaning.

• In a variation on this system, a single male is moved between

singly housed females, spending a week with each. One male is

used for every seven females.


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Sexing rodents


• Hamsters

• Female Syrian hamsters are aggressive, and may not tolerate

unfamiliar males unless in oestrus

• Males and females kept separately, and put together for a short

period after dark. If the female does not accept the male he should

be removed at once. Otherwise remove male before lights on

• Females can be rotated through the male’s cage at weekly

intervals, one male being used for seven females

• *Monogamous pairs - male and female are put together at weaning

and kept permanently together

• *Harem - several males and females together. Females are

removed prior to parturition and returned after weaning

• *These systems can lead to fighting.

• Hamsters commonly abandon or kill their young - triggered by

environmental disturbances, inadequate nesting material, handling.

Disturbed females may hide their young in their cheek pouches.

This can protect young but may also suffocate them

• Nursing mothers should not be disturbed for 7 days post-partum


Development of rodent pups


5. Nutrition

• Rodents feed on a variety of foodstuffs• seeds and grains, roots, leaves or fruits, invertebrates and even

small fish.

• Laboratory species eat mostly vegetable matter and cereals.

• Rodents have a simple stomach, with glandular and non glandular parts, and cannot vomit.

• Mice need water to lubricate food and may not eat if deprived of water

Rodent diets

• Nutrient requirements vary

• strain

• maintenance vs production

• environment and microbiological status (different

intestinal flora influence nutrient requirements

• Pelleted, (compressed or expanded), powdered

or in gel form

• Energy requirements:

• obese mice < lean mice

• females < males

• Mice

• Daily metabolisable energy (ME) requirement

maintenance 160 kcal (670 kJ)/BWkg0.75/day

• Maintenance: 4–5% lipid and 12–14% protein

• Growth and reproduction: 7–11% lipid and 17–19%

protein

• 3.5–6.2 g diet daily

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• Rats

• Omnivorous: seeds, grains and other plant matter,

invertebrates and small vertebrates

• Rats do not have a gall bladder

• Maintenance 20–27% protein and 5% fat. Higher

protein levels may reduce reproduction

• Rats are cautious eaters and will reject strange

food.

• Eat 5–10 g/100 g body weight daily

• 15 g/day for maintenance, 15–20 g/day in pregnancy,

30–40 g in lactation

• As rats age, beneficial to restrict food intake to 80% of ad

lib: increases life span and reduces neoplasia

6. Housing

• Depends on species and strain

• Can keep together if sufficient space and plentiful resources

• Males are most aggressive when competing for food, territory or females and are usually separated

Housing for laboratory animals

• Laboratory animal housing needs to provide for both

physiological and ethological needs

• Food, water, environmental parameters, bedding,

cleaning

• Must also provide for behavioural needs of animals

• Opportunities for animals to perform natural behaviours

• Failure to provide these opportunities can lead to abnormal

behaviours, called stereotypies

Stereotypic behaviour

• Persistent repetitive behaviour, with no obvious purpose

• - pacing

• - flipping

• - overgrooming/ barbering

• - diet crunching

• - bar biting


• Rats, mice and guinea pigs are social animals and can be held in compatible groups (some strain differences)

• Hamsters are solitary and both sexes are generally kept singly to avoid aggression

Mice in the laboratory

• Housing

• Social groups of

compatible animals

(males may fight)

• Strain differences

• BALB/c aggressive

• Bedding and nesting

material,

environmental

enrichment

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Rats in the laboratory

• Rats like to stand erect, need cages with high lids

• Rats are intelligent and inquisitive and need an

enriched environment to provide variety and

prevent boredom

• Cages with solid floors, with bedding

• Rats are usually friendly and amenable and will

become friendlier with more frequent handling

• Beware strain differences

• Males >females

• www.ratlife.org

7. Behaviour

• Crepuscular – active dawn and dusk. Feeding, drinking

and mating all usually occur at night. Their eyesight is

poor, and blind rats behave as if perfectly normal

• Thigmotactic – ‘wall hugging’, dislike open spaces

• Need tubes and bedding so they can feel secure

• Wild rabbits are also crepuscular or nocturnal, but in the

laboratory can become diurnal

8. Communication

• Pheromones most important

• Mice

• Remote sensing via volatile compounds

• Contact sensing of territory marks

• removing pheromones causes increased territory marking and

fighting, e.g. during cage cleaning

• Subordinate animals may be barbered

9. Environment

• Environmental changes can impact negatively on animals and science, e.g. • Low humidity predisposes to ring-tail in rats

• High light levels cause retinal degeneration in albino animals

• Sudden changes can precipitate respiratory disease in rabbits

10. Rodent diseases

• Zoonoses: • Hantaan virus, LCMV

Leptospirosis,

• Viruses: • M, R, H: Sendai virus,

pneumonia virus of mice

• R: rat coronavirus, Kilham rat virus, rat parvovirus

• M: mouse hepatitis virus (MHV), mouse rotavirus (EDIM), mouse norovirus, Theiler`s murine encephalomyelitis virus (TMEV), minute virus of mice (MVM)…….

• Bacteria: • Helicobacter spp. Pasteurella

pneumotropica, Staphylococcus spp, Clostridium piliformis (Tyzzer’sdisease), streptococci and Mycoplasma spp

• Parasites: • pinworms, protozoa, mites,

dwarf tapeworm (R only)

Recognition of PSD and LH

• Mice

• Increase in sleep time

• Weight loss

• Piloerection and hunched

• Isolated

• Persistent ‘dormouse’ posture

• More docile/ aggressive

• Squeals on handling or pressure on affected area

• May eat bedding or neonates

• Abdominal writhing

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• Rat

• Reluctance to move or explore

• Failure to stand erect

• More aggressive, and resist handling

• Vocalization and struggling

• Licking or guarding a painful area

• Crouched appearance

• Disturbed sleep patterns

• Chromodacryorrhoea

• Abdominal pain – back arching, twitching and a cat-likestretch

• www.digires.co.uk/product/pain-assessment-in-the-rat

Hamster

• Few clinical diseases. May develop non specific enteritis,

and can carry LCM (zoonosis)

• Weight loss, extended sleep period and increased

aggression or depression.

• Ocular discharge and diarrhoea may be associated with

stress.


Anaesthesia


Anaesthesia of small animals

• Particular issues:

• Significant risk due to small size

• access to blood vessels difficult

• increased heat loss

• fasting may result in hypoglycemia (but cannot vomit and

coprophagic)

• Ensure healthy, stress free animals

• Keep warm during and after anaesthesia

• Give supplementary fluids

Inhalation anaesthesia

• Isoflurane

• high safety margin

• rapidly excreted

• no biotransformation

• Induction concentration 3-4%

• Anaesthetic chamber for mice, rats and guinea pigs (not rabbits), or

via a facemask (all species)

• Maintenance concentration 1-3%

• Endotracheal tube, intranasal tube, facemask

• Can supplement with additional analgesics

Injectable anaesthesia

• Intra-venous route is to be preferred:

immediate effect, safer

• BUT...venous access difficult in

rodents

• So most agents are administered i.m,

i.p, or s.c

• Larger dose, slow absorption, slow

recovery, risk of mis-dosing

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Anaesthetic management

• Monitoring

• Physiological stability: airway, breathing, circulation

• heart rate, colour, pulse oximeter, ECG, apnoea alarm

• Depth of anaesthesia: narcosis, relaxation, analgesia

• breathing, reflexes, muscle tone

• Body temperature

Maintenance

• Provide supplemental heat

• Eye care: corneal drying

• Oxygen essential

• Airway blockage

• Fluid needed - but avoid

overload

• Posture –prolonged

recumbency in unnatural

position

Schedule 1 methods for small

mammals


Schedule 1 methods

• Overdose of anaesthetic – all animals

• Rising concentration of CO2 – rodents >10 days,

• Concussion – rodents & rabbits up to 1kg

• Cervical dislocation - rodents

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Standardisation

• Genetic characteristics

• Breeding systems, genetic monitoring

• Environment

• Code of practice

• Nutrition

• Standardised lab diets

• Microbiological status

• Health monitoring and disease prevention

Breeding Systems

• Aims:

• Selective breeding to produce animals with desired

characteristics

Different methods employed depending on what is to be

achieved, e.g. inbreeding, outbreeding, backcrossing

Laboratory Animals - Genetic Characteristics

• Rat and mouse - wide range of inbred strains and outbred

stocks available

• Rabbits and guinea pigs - mainly outbred, some inbred

lines

• Dog and cat – outbred

• Farm animals – mostly outbred

• Birds – some inbred lines of chickens

• Fish - depends on species and purpose

Genetics - Definitions

• Inbred

• Outbred

• Backcross

• Congenic

• Coisogenic

• F1, F2 hybrid

• Genetically altered animals

• Transgenic

• Mutant

Inbred v outbred?

• Inbred

• 20 generations of brother x sister matings

• Almost completely homozygous

• Genetically stable

• Mice: Jackson Lab www.jax.org

• Outbred

• Random bred, or mated to maximise outbreeding

• Heterozygous

• Animals not uniform

• Genetically unstable

• Closer to ‘natural’ population?

Backcrossing

• To transfer a desired genetic characteristic from one

background strain to another

• Strain 1 carries mutation/genetic alteration of interest,

which is to be transferred to animal of strain 2

• Mate strain 1 animal with strain 2 animal

• Select offspring carrying gene (strain 1/2 hybrids)

• Mate these back to strain 2 animals

• Repeat selection and cross back to desired strain 5-10

times


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Definitions ctd

• Congenic strains

• Strains differ in a single length of chromosome

• Coisogenic strains

• Two strains differing at a single locus (often arise by mutation in an

inbred strain)

• F1, F2

• F1 – first generation offspring from a cross between two strains

• F2 – offspring produced by mating F1 animals

• Genetically Altered Animals

• GAA have an (artificially) induced change in their DNA

F1, F2

• F1 – first generation offspring

• F2 – offspring produced by mating F1 animals

AA BB

AB ABAB AB

ABAA AB BB

F1

F2

Genetically Altered Animals

• Genetically altered animals have an (artificially) induced

change in their DNA

• Spontaneous mutants: selective breeding of animals with

particular characteristics. May be harmful.

• Mutagenesis: rate of mutation of DNA increased by

exposure to chemicals or radiation.

• Transgenesis: Addition or deletion of genetic material into

nuclei.

Uses of GAA

• Use of genetically altered animals is increasing• Understanding genetic basis for disease

• To generate models of particular diseases

• Current level of use likely to be sustained, and the range

and sophistication of experimental procedures will

increase

• Creation and use of Genetically Modified Organisms

covered by additional legislation

• GMO (Contained Use) Regulations 2005

How are GAA Produced?

• Pronuclear microinjection

• Embryonic stem (ES) cell manipulation

• Viral vectors

• Cytoplasmic injection

• Primordial germ cells

• Nuclear transfer

• Spermatogonial manipulation

Pronuclear Microinjection

Oocytes isolated from donor

mouse immediately after

fertilisation (mouse

killed and eggs removed)

Foreign DNA is injected into

the (larger) male pronucleus.

The egg is implanted in a foster

mother and develops to term.

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Pronuclear Injection

• The DNA integrates many copies at random

positions in the genome. The resulting mouse

may be only partially transgenic.

• If the transgenic cells contribute to the germ line,

then some transgenic eggs or sperm will be

produced

• These founder animals are bred and offspring

carrying the gene selected

Cross with wild type

Genotype offspring, mate

hemizygotes

25% Homozygous transgenics produced

Embryonic Stem Cell

Collect embryos, culture in-

vitro and isolate stem cells

Introduce DNA into stem

cells in culture

Collect embryos (from mouse of

different colour), inject stem

cells - stem cells contribute to

development of embryo

Implant embryo into recipient

mouse - resulting offspring are

chimeras (mixture of two

genotypes). If ES cells

contributed to germ line, some

offspring will be transgenic.

Welfare Implications?

• Effects of genetic modification

• gain in function (eg producing a new protein)

• loss of function, if the alteration affects or ‘knocks out’ another

gene

• Effects of procedures involved in production and

maintenance of GAA animals

• Protection under ASPA from the moment of conception

• Effects of Genetic Alteration

• DNA is inserted randomly. May be no clinical effects, or

adverse effects only in particular circumstances or if on a

different background strain

• May cause major alterations in embryonic development and

can be lethal

Production of Transgenics

• Superovulation of donor female

• Hormone injection, often very young

• Mating - fertilised eggs produced

• Female killed and eggs harvested

• Recipient female made pseudopregnant by mating with

vasectomised male

• Surgery to vasectomise male

• Reimplantation of embryos

• Surgical implantation into oviduct of recipient

• i.e. Many surgical procedures

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Advances in Technology

• Advances in technology

enable more control of

phenotype

• E.g. Targeted gene

insertion – only ES cells

with gene in correct

location implanted into

blastocysts

Genotyping

• A variety of techniques available

• Choice depends on quantity and quality of material

required

• Tail-tipping was commonly used

• Causes pain, neuroma formation and allodynia/

hyperalgesia

• Can use PCR on very small sample – blood, saliva

• Should use least severity procedure

Identification methods

• Appearance/coat colour

• Ear notching

• Microchips

• Fur & tail marking

• Tattooing

• Ear tags

• Use least severity method

Mouse Passports for GAA

• GAA should ideally come with a ‘passport’, giving details of the effects of the modification

and any special care needed

• Comparison of their welfare with that of non-GA mice, but very little data on non-GA animals!

• NC3Rs working party recommended structured welfare assessments (www.nc3rs.org.uk/GAmice)

• Assess neonates, at weaning, and adults• Use a simple check sheet, record proportion with

compromised welfare

• Develop a welfare profile to aid in management

Guinea pigs


Guinea pig biology

• Guinea pig Cavia porcellus

• Hystricomorph rodent

• (NB it was proposed in 1990s that caviomorphs should

be reclassified as a separate order. Subsequent research

supported current classification).

• Originated in the Andes - domesticated descendants of a

closely related species.

• do not exist naturally in the wild.

• Important in folk culture in South America

• food source, in folk medicine and in religious ceremonies.

• Introduction to Europe by traders in the 16th century

• Widespread popularity as a household pet since.


http://www.nc3rs.org.uk/GAmice

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• Docile, responsive to handling, easy to care for

• Specialized breeds, with varying coat colours

• Research on guinea pigs carried out since the 17th

century. Model organism in the 19th and 20th centuries

(hence "guinea pig" for a test subject)

• Largely replaced by other rodents now

• Still used for research into asthma, juvenile

diabetes, tuberculosis, scurvy, and pregnancy

complications.

• Lifespan 4-8 years

• Adult weight 700-1200g (M>F)


Guinea pig breeding data

• Puberty (days)

• Age to breed (months)


• Litter size


• Weaning age (days)


• Post partum oestrus

60 (M) 30 (F)

F 2-3

M 3-4

59-72

2-5

70-100

21-28

15-17

Fertile

Guinea pig breeding

• Vagina covered by an epithelial membrane - opens during

oestrus and parturition

• In the last week of gestation, the pubic symphysis

separates: females should breed before 7-8 months of

age, or the symphysis cannot separate (dystocia)

• Neonates are precocious: eat solid food within a few

days, wean at 180g (15-28 days), or 21 days (165-240g)

Guinea pig nutrition

• Guinea pigs are messy and fastidious eaters - much food is wasted so excess should be supplied

• Guinea pigs need vitamin C (5mg/kg daily, or up to 30mg/kg if pregnant)• In food or water, or cabbage, kale or oranges

• vitamin C is labile – food must be in date

• DO NOT USE FEED FORMULATED FOR OTHER SPECIES

• Give fresh vegetation, but take care – some plants are poisonous to guinea pigs

• Coprophagy does occur, but may not be essential

Guinea pigs in the laboratory

• Group housing ideally

• Bedding materials provide comfort and a substrate for

rooting behaviour

• Guinea pigs are messy animals and will disperse opaque,

creamy coloured urine and faecal pellets throughout the

pen

• They should be provided with bolt holes and barriers

within the pen, handled frequently to habituate the

animals and reduce stress.

Guinea pig health and disease

• Relatively few infectious diseases

• Vitamin C deficiency

• Most infectious diseases seen in guinea pigs are bacterial

(abscesses and non-specific infections)

• Can carry lymphocytic choriomeningitis virus and

respiratory viruses e.g. Sendai virus

• Large animals kept on grid floors or in unsanitary

conditions may develop foot infections (bumble foot)

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• Guinea pigs

• Alert, apprehensive animals who will try to avoid

capture and restraint

• Any unusual sign of acceptance indicates the animal is

unwell

• Loud vocalization common

• May appear sleepy - rarely show aggression

• Stoic - can be difficult to assess pain

Guinea pig - summary

• Rodent of South American origin

• Social animals, docile and easy to care for

• Long gestation – young precocious

• Breed females before 7 mo

• Dietary requirement for vitamin C

• Group house with bolt holes

• Few infectious diseases

• Stoic – sick guinea pigs are very sick!


Rabbits


Rabbit

• Lagomorph (‘hairy ears’) • originally classified as rodents but have 6 incisors and rodents

have only 4

• Oryctolagus cuniculus

• Lifespan 6-12 years

• Adult weight 900-6000g

• Breeds - New Zealand White, Dutch

• Originate from Asia and southern Europe – arrived in UK with the Normans?

• Open rooted teeth• Grow 2-2.4mm/week

Rabbit nutrition

• Rabbits have evolved to eat a low quality, high fibre diet (12-22%)

• The main site for microbial digestion is the caecum

• Coprophagy is essential

• Excess readily digestible carbohydrate leads to disruption of the microbial flora in the caecum, and leads to diarrhoea, fur chewing and hairballs

• Overeating may be a stereotypy: ad lib feeding may result in obesity • Provide environmental enrichment and a variety of foodstuffs

• Hay can be given

• Fibre increases bulk and reduces the incidence of

hairballs

• Important to feed diet appropriate for the species and

physiological status of animals

• Changes in diet should be gradual to avoid diarrhoea

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Rabbit breeding data

• Puberty (days)

• Age to breed (months)


• Litter size


• Weaning age (weeks)


90-120 (f

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