Optimizing Mouse Breeding

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Optimizing Mouse Breeding

• Assisted Reproduction Technologies Laboratory
• UCLA Division of Laboratory Animal Medicine
• March 1, 2006

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Reproduction - Estrous Cycle

• Annual Polyestrous (cycle throughout the year)
• Cycle duration 4-5 days
• Phases of the cycle have characteristic duration,
  vaginal appearance cytology changes

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Stages of the Estrous CycleVaginal Appearance
Proestrus (Early 11 hours late -21 hours)
lip gaping, pink, moist, striation. Ovarian
follicular development phase
Estrus (21 hours) gaping, pale pink, less
moist, striation on lips. Sexual receptivity
ovulation
Metestrus (22 hours) closing, pale, dry CL
formation eggs in oviduct
Diestrus (22 hours) closed or very small
opening, blue lips, moist If no pregnancy, new
wave of follicles develops
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Stages of the Estrous CycleVaginal Cytology
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Reproductive Behavior

• Social animals
• Considerable variation between outbred and inbred
  across inbred strains
• Fecundity extremes (FVB vs. Balb/c) see next
  slide
• Light cycle is critical for normal cycling,
  mating and pup rearing (seasonal variations)
• Olfactory cues important (pheromones)
• Dominance structure (aggression, territory)
• Mating cues

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Female

• Puberty occurs around 4-5 weeks old
• Begin breeding at 6 weeks old
• Always take the female to the males cage for
  mating
• Females of most inbred strains will breed well
  until 7-9 months old outbreds, longer

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Male

• Puberty usually occurs by 5-6 weeks of age
• House the male singly for 1 week prior to mating
• Make sure the males cage has been undisturbed
  for 1-2 days prior to mating
• Males usually remain fertile for life (except
  obese mice)

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Pheromones

• Three effects observed in female mice
• Lee-Boot effect
• Whitten effect
• Bruce effect
• These can be used to our advantage for breeding
  mice

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Pheromones in action

• Lee-Boot Effect Estrus suppression (anestrus) in
  females housed together for about 2 weeks
• Whitten Effect Induction of estrus in females by
  the presence of the male or males urine
• ?Best observed when housing one male and one
  female ? gt50 of females will conceive on the
  3rd evening after pairing? Most effective when
  used together with the Lee-Boot effect
• Bruce Effect Pheromones from a strange male may
  prevent implantation of the embryo in recently
  (4-5 d) bred females
• ? Most commonly a problem with males from a
  different strain
• ? Change gloves after handling males

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Mating (1)

• Select your breeders carefully (correct sex!,
  age, physical condition and size compatibility)
• Select breeding pairs or breeding trios
• Mating occurs during late proestrus to early
  estrus phase of the cycle
• Set up matings in the evening (record the setup
  date/time and who was set up with whom)
• Ovulation usually occurs 4-5 hr after onset of
  the dark cycle (around midnight on a 1410 LD
  light cycle 5AM-7PM)

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Mating (2)

• Check females the morning after mating for
  presence of a copulatory or vaginal plug (waxy,
  tan to off-white material found in the vagina
  may need probe check as early in the morning as
  feasible) see next slide
• The vaginal plug indicates that mating has
  occurred (but it does not guarantee pregnancy)
• Up to 80-90 of females with a vaginal plug will
  become pregnant (very strain dependent)

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Examples of copulatory plugs
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Fertilization Gestation

• Fertilization occurs in the proximal oviduct
  ampulla shortly after ovulation
• Implantation of the embryos occurs 4-5 days after
  conception (do not introduce strange male odors
  during this time Bruce effect can abort)
• Gestation 19-21 days (strain- and
  mutant-dependent)

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Pregnancy Detection (1)

• Visually pregnant by 12-13 days (pear-shaped
  abdomen) first-timers even earlier
• Weigh the female before placing her with the male
  and again at 17 days (weight increase of 10 g is
  generally indicative of pregnancy)
• Do not pick up late-pregnant mice by the tail
  (cup instead)
• Palpation of the abdomen not recommended
  trauma, stress may cause abortion

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Pregnancy Detection (2)
Pregnant vs. non (at 12 days after detecting
vaginal plug)

• Pregnant

• Non-pregnant

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Birth

• Birth can be predicted by knowing when mating
  occurred (plug date) and the expected gestation
  of the strain
• You must know how other researchers are referring
  to the plug date, e.g., Day 0, 0.5, 1
• Do not disturb the female between about 3-4 days
  before and 3-4 days after delivery (place DND
  sign on the cage)
• Change into a clean cage at day 17 (_at_ weight
  check) and do not touch again until the pups are
  3-4 days old

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Birth Postpartum

• Birth most often occurs between 12-4 AM
• Litter size 1-12 pups
• Following birth, females undergo a Postpartum
  Estrus
• Postpartum Estrus
• Occurs 12-28 hr following birth
• At least 50 fertile
• Pregnancy subsequent to the postpartum estrus may
  be longer than usual

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Postpartum

• Do not disturb the female for about 3-4 days
  following the birth of her pups
• Pups are born hairless
• External ears open about day 3
• Eyes open around day 12-14
• Hair coat is well developed by day 14

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Postpartum

• Pups start to eat solid food around day 11 or
  when eyes open
• Females lactate for about 4 weeks
• Wean pups at 21 days of age by sex
• Males have a longer anogenital distance than
  females
• Only females have nipples
• Remember that weaning pups beyond 21 days
  requires ARC approval, e.g., 28-day weaning age

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Determining Age in Mouse Pups (1)
Day 1
Day 2
Day 4
Day 3
Day 5
Day 9
Day 6
Day 8
Day 12
Day 10
Day 11
Day 14
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Determining Age in Mouse Pups (2)
Like mother, like daughter
3 Weeks
4 weeks Adult
4 weeks Adult
21 days old
17 days old
21 days old
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Determining Sex in Mouse Pups
male
female
female
male
male
female
male
(Notice nipples)
(Notice nipples)
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Birth

• Some strains experience problems around the time
  of birth (pup neglect, cannibalism)
• To reduce the problems try the following
• Place Do Not Disturb signs on the cage
• Cover the cages bottom with bonnets or other
  solid material
• Place cotton Nestlets (Ancare) in the cage
• To get Nestlets, please contact your local DLAM
  facility supervisor

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Birth Problems

• Dystocia The outcome depends on
• Health status of the female when first detected
• Availability of early treatment
• Availability of foster mothers

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Continuing Reproduction

• If you take advantage of the first estrus
  postpartum, give the females a break about every
  other birth
• Avoid using aged females (over 7-9 months) or
  males (over 1 year of age)
• Avoid using overweight breeders
• Avoid using mice that appear sick

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Records

• Keep good breeding records (see Lee Silvers
  book www.informatics.jax.org/silver/
• Birth date
• Pairings
• Offspring
• Number of Males
• Number of Females
• Number born vs. Number weaned
• Quality
• Common heritable health problem Malocclusion

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Records

• Dont use records to judge animals prematurely
• Determine from your records when these animals
  should be culled from the colony
• Poor quality young
• Undesirable traits (malocclusion, cannibalism)
• Poor weaning percentage

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Fertility problems