Lab Animal Breeding

LABORATORY ANIMAL BREEDING
Presented By:
Dr. Dhaval Chaudhary
M.V.Sc Scholar(AGB)
College of Veterinary Science and Animal Husbandry, AAU, Anand
Content
General Consideration for Colony Management in Laboratory Animals
Reproductive Behaviour of Laboratory Animals

Introduction
• Colony management in its broadest sense involves animals, facility,
staff and administration.
• Colony management issues range in scope from simple tasks
performed day after day (checking water bottles and changing cages)
to complex tasks that you hope you never have to perform
(recovering from microbial contamination or a natural disaster).
• Colony management has one purpose: to produce and maintain
healthy, genetically consistent animals, suitable for biomedical
research.
(The Jackson Laboratory Handbook on Genetically Standardized Mice)

General Consideration for Colony Management
 Sources of experimental animals
 Animal Health
 Laboratory Animal Husbandry and Management
I. Housing and Environment
II. Nutrition and Feeding
III. Experimentation and Veterinary Care

Sources of Experimental Animals
• The only authentic source of getting right type of animals for research
should be from recognized scientific animal facilities where the
animal colonies of known genetic and health status are available.
Such animals only can provide reliable results.
• The scientists should therefore insist upon getting defined animals
through organized colonies eliminating unscrupulous traders, which
not only supply poor stock of animals but also maintain these animals
under most unethical and unhygienic conditions
(Guideline for Care and Use of Animals in Scientific Research, INSA, Delhi)

Animal Health
• If you manage a production or research colony, maintaining animal
health is one of your most critical responsibilities.
• Whether you purchase mice or acquire them from other research
colonies, your concern also includes the possibility of introducing
contaminated animals into your colony.
• This issue includes preventing and identifying microbial
contamination as well as ridding your colony of a contamination.
(The Jackson Laboratory Handbook on Genetically Standardized Mice)

Physiological Norms of Commonly Used Laboratory Animals
Mouse Rat Hamster Guinea Pig Rabbit
Weight at Birth(grams) 1-2 4-5 2-3 80-100 40-60
Age at Weaning(weeks) 3 3 3 3 8
Weight at Weaning(grams) 9-12 40-50 30-40 250-300 800-900
Age at Maturity(weeks) 6-8 10-12 6-8 16-20 24-32
Weight at Maturity 18-22 g 150-200 g 80-90 g 250-400 g 1.5-2.0 kg
Adult Weight 25-30 g 200-300 g 80-100 g 400-500 g 2.0-2.5 kg
Rectal Temp oC(Average) 37.4 37.5 37.6 38.6 38.7
Respiratory Rate/min 90-180 80-150 40-120 60-110 35-56
Pulse Rate/min (Average) 600 300 450 150 133
Life Span(years) 1.5-2.0 2.5-3.0 1.5-2.0 4-5 4-5
Diploid Chromosome number (2n) 40 42 44 64 44

Laboratory Animal Husbandry and Management
(i) Housing and Environment
• Laboratory animals are very sensitive to their living conditions. It is
important that they are housed in an isolated building located as far
away from human habitations as possible.
• They should not exposed to dust, smoke, noise, wild rodents, insects
and birds. The building, cages and environment of animal rooms are
the major factors which affect the quality of animals.

• The cages should be made of suitable metal (stainless steel,
galvanized iron sheet/rods) or synthetic material
(polypropylene/polycarbonate).
• They should be of suitable size for each species of animal and should
have adequate arrangement for feeding and watering. They must be
free from crevices, corners and sharp edges for easy cleaning and to
avoid injury.

• The bedding should be of right material and sterilized before use.
Common bedding materials used in India are paddy husk, saw dust,
paper cuttings, dry grass and crushed corn cobs.
• The environment of animal room (Macro-environment) and animal
cage (Micro-environment) is an important factor on which the
production and experimental efficiency of the animal depends. Since
animals are very sensitive to environmental changes, sharp
fluctuations in temperature, humidity, light, sound and ventilation
should be avoided.

Housing and Environmental Requirements
Types of Housing Cage Cage Cage Cage/Pan Cage
Floor Area/animal (sq. cm) 65-100 100-150 90-120 300-600 3700-4600
Cage Height Minimum(cm) 12 14 12 18 36
Room Temp oC 22-24 22-24 22-24 22-24 22-24
Relative Humidity(%) 45-60 50-60 45-60 45-60 45-60
Suitable Bedding Material
Paddy
Husk
Saw
Dust
Paddy
Husk saw
Dusk
Paddy
Husk
Shredded paper
Paddy
Husk saw
Dust
Shredded paper
Straw
Nesting Material Paper cutting Paper cutting Paper cutting
Wood shavings
Dry grass
Cotton paper
Cutting dry grass
Light Intensity(LUX) 300-400 300-400 300-400 300-400 300-400
Photocycle(Light:Dark) 12:12 12:12 12:12 12:12 12:12

• A constant room temperature is essential, because variation in room
temperature causes change in food and water intake. The
temperature also affects fertility and lactation.
• Coupled with high humidity the increase in temperature causes
ammonia built up. If the ventilation is not proper the high ammonia
concentration causes respiratory irritation to both animals and
attendants, predisposing them to infection by lowering their
resistance.
• An effective ventilation system with 10-12 air changes per hour of
100% fresh air must be provided for animal rooms.

• Light and sound are other important factors. The light intensity, the
wave length and the photo cycle affect the health and behaviour of
the animals.
• Sudden and sharp sounds in the animal rooms disturb the health and
behaviour of animals and may give rise to ear damage, hypertension,
cannibalism, etc.

(ii)Nutrition and Feeding
• The results of an experiment are likely to be influenced by co-
existence of nutritional deficiencies and imbalance. It is, therefore,
essential that laboratory animals are maintained on a balanced diet
based on nutritional requirements of each species.
• Special care is needed on nutritional elements, ingredients used in
diet, and feeding practices.
• A balanced diet should contain protein, carbohydrates, fat, minerals,
vitamins, roughage and water in required proportions for each
species of animal.

Nutritional Requirements of Common Laboratory Animals

• Only quality ingredients should be used in a diet and they should be
free from dust, moulds, fungi and other contaminants. Each animal
must get required quantity of feed, based on animal maintenance and
production requirements.
• The feed should be palatable so that it is consumed in adequate
quantity by the animals. Any undesirable odour always causes under
consumption resulting in nutritional deficiency in the animals.
• No drug, hormone or antibiotic should be added in the feed as these
are likely to disturb the normal metabolism of the animals and
produce biased results.

• The ingredients and the prepared feed must be stored and handled
carefully so as to avoid any contamination.
• The food must be of right consistency and should be presented to
animals in proper type of hoppers to avoid wastage. In some cases
the feed may be divided in 2-3 meals during the day.
• Pelleted feeds balanced for different species of animals are now
available commercially. These are easy to procure and use without
wastage. However one has to be careful on quality of the feed from
batch to batch.

• It should be obligatory on manufacturer to mark each bag with the
type of food, date of manufacture, the batch number, the ingredients
used and chemical composition.
• Random chemical analysis must be carried to for major nutrients to
monitor the quality of food from time to time.
• Clean, chlorinated water should be available to the animals ad lib.

(iii) Experimentation and Veterinary Care
• The experimental animal units should generally be looked after by
qualified investigators. These units must have adequate housing and
technical facilities for experiment and post-operative care.
• The equipment provided in the experimental unit should be
appropriate for the needs of the experiments. No technique should
be used which may cause avoidable discomfort to the animals. The
post-operative holding rooms and cages should be comfortable and
such animals should remain under the care and supervision of an
experienced scientist or a qualified veterinarian.

Reproductive Data of Commonly used Laboratory Animals
Oestrus Cycle(days) 4-5 4-5 4-5 16 -
Duration of Oestrus(hours) 10 13-15 20 6-11 -
Time of Ovulation
2-3 h after
Est. (spont.)
8-10 h
(spont.)
8-10 h
(spont.)
10 h
(spont.)
Induced 10-11 h
(After mating)
Gestation Period (days) 21 21 16 68 30
Litter size 6-10 8-12 5-8 1-4 4-6
Oestrus after parturition Post partum Post partum 1-8 d Post partum 35 d
Reproductive Life span (years) 1 1 1 3-4 2-3
Max. number
of females per male
5 5 5 6 10
Mammary Glands (T.A.P.)
no. of pairs
3,1,1
(Five)
3,1,2
(Six)
-,5,1
(Six)
-,-,1
(One)
1,2,1
(Four)
A=Abdominal, P=Pelvic, T=Thoracic

Reproductive Behaviour- Mice
• Sexual maturity in mice occurs very early in life.
• Vaginal opening may occur as early as 24–28 days, but sexual
maturation varies with strain and environmental influences. Ovarian
follicle development begins at 3 weeks of age and matures by 30
days. Puberty in males occurs up to 2 weeks later.
• The cyclicity of oestrus and ovulation are controlled by the diurnal
rhythm of the photoperiod, and oestrus and ovulation most often
occur during the dark phase (Fox et al. 2002).
(THE UFAW HANDBOOK ON The Care and Management of Laboratory and Other Research Animals)

• Light cycles of 12–14 h light and 10–12 h dark are necessary to
maintain regular oestrous cycles.
• Light intensities in the range commonly encountered in animal rooms
can affect the oestrous cycle (Clough 1984), e.g., too much light or a
too long period of dark will cause irregularities in the oestrous cycle.

Estrus Detection
A B
A) Estrus Swollen open moist appearance of vaginal opening
B) Non Estrus Closed vaginal opening
(University of Kentucky, U.S.)

• These effects are mediated by pheromones in the urine of the males (Cunliffe -
Beamer & Les 1987).
The Lee - Boot effect
It is a phenomenon concerning the suppression or prolongation of oestrous cycles
of mature female mice (and other rodents), when females are housed in groups
and isolated from males.
The Whitten effect
When the pheromones contained in the urine of male mice stimulate synchronous
estrus in a population of female mice.
The Bruce effect
If the female is housed with a second male within 24 h after a successful mating,
implantation of fertile egg cells will be prevented and the female will return to
oestrus in 4–5 days.

• Mating also often occurs during the dark phase (Fox et al.2002), and
can be detected within 24 h after copulation by the formation of a
waxy vaginal plug (a mixture of sperm and secretions from the
seminal vesicles and the coagulating glands of the male). As this
detection method is not always reliable it is often combined with
detection of spermatozoa in the vaginal fluid after flushing.

Reproductive Behaviour- Rat
• The rat has an exceptionally high reproductive capacity.
• In the wild, a receptive female will be followed and mounted by the
dominant male and often by a number of other male colony members
as well.
• After several successful copulations, the female’s willingness starts to
decline; this is indicated by aggressive actions and escape behaviour
by the female(McClintock & Adler 1978).

• Gestation is 20–21 days, and during the few days prior to delivery of
the pups, the pregnant female starts to build a nest and will defend
her nest against both male and female colony members.
• This maternal aggression will continue throughout the ﬁrst week of
nursing, after which it gradually declines and another reproductive
cycle may start (Rosenblatt et al. 1994).

• Copulation may occur for 2-
24 hours depending on how
long the estrus cycle lasts.
The male will mount the
female while she is standing
causing her to arch her back
and present herself for
insertion (lordosis).
• Copulation is short and ends
in the female darting off as
the male bends down and
cleans his penis. Copulation is
generally repeated many
times.
(The Care and Breeding of Laboratory Animals by Edmond J. Farris)

Reproductive Behaviour- The Syrian Hamster
• Female hamsters are relatively aggressive in the presence of
unfamiliar males and generally, unless sexually receptive, will not
tolerate their presence.
• Using a breeding system in which females are selected in this way,
one male can serve a harem of 12 females. This is a common method
of breeding Syrian hamsters but is very labour intensive.

• The optimal reproductive life of a Syrian hamster is considered to be
around 10 months and a significant reduction in reproductive
capacity occurs from 1 year of age onwards.
• The hamsters are mated soon after dark, when they are naturally
most active. The female is placed in the same cage as the male and
the pair observed to ensure mating occurs. Because when ﬁghting
start, the pair must be immediately separated.

Reproductive Behaviour- Guinea Pig
• Female guinea pigs exhibit a post- partum oestrus which means they
become receptive immediately after giving birth. If they do not become
pregnant at this time, females show periodic oestrous cycles of about 16
days (Shi et al. 1999).
• Guinea pigs do not show a day–night cycle of activity. Rather, they are
characterised by an ultradian rhythm, that is, alternating phases of activity
and rest last for about 2–3 h.
• Thus, the activity is not dependent on the light – dark regime (Sachser et
al. 1992).
• The olfactory sense is most important in social behaviour (Beauchamp et
al. 1979, 1980, 1982 ; Martin & Beauchamp 1982). Male guinea pigs mark
individual females with their anal glands.

Reproductive Behaviour- Rabbit
• The European wild rabbit is a seasonal breeder, with seasonality
determined by the interaction of day length, climate, nutrition,
population density, social status and other factors (Bell & Webb 1991;
Bell 1999).
• Before mating an elaborate courtship takes place– the rabbits circle
around each other, parade side by side, jump over each other and
sniff the genital region (Lehmann 1991).
• Hafez (1960) described seven degrees of sex drive, which ranged from
aggressive with immediate mounting and ejaculation to offensive
reaction with general smelling of the skin, biting and no ejaculation.

• When the doe allows mating she raises the hindquarters (Bennett
2001).
• Mating takes only a few seconds, after which the buck falls to one
side or backwards (Bennett 2001).
• Ovulation is induced by the act of coitus and conception occurs 8–10
h after mating (Bennett 2001).

Breeding of Laboratory Animals
Sr.
No.
Animal Scientific Name Preferred phase of Mating Remarks
1. Mice Mus musculus Dark Phase Immediate removal of male during the late
gestation to avoid growth interference of
young ones.2. Rat Rattus norvegicus Dark Phase
3. Hamster Mesocricetus auratus Dark phase
One male and one female are put together
for a short time till the mating is over and
then separate them.
4. Guinea pig Cavia porcellus Dark and Light phase
5 to 10 sows with one boar in community
living.
5. Rabbit Oryctolagus cuniculus Dark and Light phase
Bucks are sensitive to familiar surroundings.
So doe is placed in cage of bucks. Reversal of
which will leads to savage attack and possible
injuries to buck.
(Laboratory Animal Science, Dr. S. Ratnam;
The Care and Breeding of Laboratory Animals Edmond J. Farris;
THE UFAW HANDBOOK ON The Care and Management of Laboratory and Other Research Animals)

Lab Animal Breeding

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