Best Practices: Husbandry

Handling Mice

Pick up mouse from base of tail and hold in your hand

• Always have a grip on the tail so the animal is secure.
• A transfer cup or cage can be used if your taking it to a new location.
• Some new data suggests picking up the mouse with a tube or cup reduces handling-related stress that is caused by the experimenter picking up the mouse from behind. The data are mixed on this so consider this in light of the study focus and the strain.

Handling Habituation

• Especially important for behavior
• If you are stressed the mouse will be stressed
• Handling habituation removes the effects of handling stress from the data collection.
• We suggest at least three sessions, more if the assay requires more stressful handling like injections.

Stay in control of the mouse at all times

• Do not let go of the tail when they are outside of a cage

Do not walk away from an open cage. Open cages should be handled in the hood with blower and lights on. Mice between P14 – P25 can “popcorn” or jump unexpectedly right out of the cage – so be extra vigilant at these ages.

Breeding

Breeding Ages/Timelines

Optimal Breeding Age Range:

• Females: 8 weeks (P56) – 8 months. Females start going through menopause at 8 months, so she will stop cycling regularly and giving regularly timed litters.
• Males: 8 weeks (P56) – ~1 year

Gestation is 3 weeks (21 days)

Refresh your breeders at ~8 months for a female and ~1 year for a male.

Types of Lines

Maintenance

• 2 breeding cages
• 2 backup cages at breeding age (1 cage of males, 1 cage of females)

Replenish breeders ~4-6months and replenish backup cages regularly

Experimental/Cohort

• No cage limit (try to wean into as few cages as possible)
• As many breeders as needed for experimental numbers (determine ahead of time)

Organization is key

Types of Breedings

Single Pair Breedings

• Best option especially for maintenance lines.
• Continuous breeding
• Requires 1 cage card

Harem Breeding

• 1 male with 2 females
• Must separate multiple litters
• Requires a breeding card for each pair/female (2/cage)
• Checking which mother gave birth
• Difficult for continuous breeding
• Not recommended for maintenance lines.

If Harem breedings, remember cage policies:

• 5 post-weaning mice per cage
• 3 adults and 1 litter only up to 14 days old
• 2 dams with 2 young litters only <7 days of age
  • Unless C57 strain, then our IACUC protocol has a special approval for two dams and two litters until P21.
• DCM will mark these cages that are out of the policy and contact or charge us to fix it.

Steps for Successful Breedings

• Females start puberty ~P35, Males ~P42 (strain dependent)
• Sexually mature at P56 (early adulthood).
• Breedings are most successful if start at P56 for both males and females.
  • Put male in cage for 3-7 days by himself to create a territory – this increases success of breeding right away (important for timed pregnancies)
  • You can induce estrus in the female by adding soiled male bedding to her cage for 4 days prior to breeding (and 4 hours before adding her to the male’s cage on day of breeding).
  • Leave breeding pair together for 4 days before separating (this will ensure the estrus phase occurred).
  • If you want to continually produce litters from a pair, best to leave them together – mice have a fertile postpartum estrus that occurs 14-24 hours following parturition.

For Timed Pregnancies

• Put male in cage for 3-7 days by himself to create a territory – this increases success of breeding right away (important for timed pregnancies). Use yellow cage cards to indicate to DCM not to change the cage (make sure to check food and water)
• You an induce estrus in the female by adding soiled male bedding to her cage for 4 days prior to breeding (and 4 hours before adding her to the male’s cage on day of breeding).
• weigh females before breeding
• Place female into male’s cage late in the afternoon. They should breed overnight.
• Check for Vaginal Plugs in the morning (usually you can see if visually, but can also use a small probe to feel it)
  • A plug usually means `~5% of the time you’ll get a pregnancy.
  • If you don’t see a plug, that does not mean the breeding was not successful.
• Physically see female pregnant @ E14 – E21
  • Take weight to confirm pregnancy around E16.

Tips

• Increased stress can increase cannibalism of pups
• Avoid handling female mouse close to due date.
• Avoid disturbing a cage with new pups
• DCM protocol states: pups <P7 are left undisturbed (even during cage change)
• Don’t use wt littermates. Breed to wt C57Bl/6j animals
  • Suggested to replenish from JAX every two generations.
• Plenty of Nestlets
• Tubes for difficult breeders
  • Exception: behavior animals that didn’t have it before first behavior testing.
  • Bio-Serv Tunnels Order the Sterile tubes so we do not have to autoclave them ourselves.
• If you suspect your dams are stressed, and/or observe high rates of cannibalization, you can supplement with Black Oil Sunflower Seeds.
  • Sprinkle a handful of seeds at the bottom of the cage 3 days prior to breeding up to 3 days after pups are born. Distributing them widely allows a foraging opportunity, which also lowers stress. (Lecker, & Froberg-Fejko, Lab Anim (2016))
  • Bio-Serv Black Oil Sunflower Seeds Order sterile from Bio-Serv so you don’t have to autoclave. Product No: S5137-1.
• Love Mash is a high fat supplement, with high omega-3 fatty acids, made specifically for breeding mice.
  • Put 2 pellets/breeder on the floor of the cage. You can also use the dust at the bottom of the package. Scattering Love Mash pellets or dust also provides a foraging opportunity, which can decrease stress in both males and females (Lecker, & Froberg-Fejko, Lab Anim (2016))
  • Beware of over-supplementing Love Mash because mice love eating it and can become obese! Obese mice are not good breeders.
  • Bio-Serv Love Mash Order from Bio-Serv, product No: S3823P.

Breeding Strategy

Work backwards form the final number of mice you need to work out how many litters and breeding pairs you will need.

1. Determine how many mice you need of each experimental group (genotype, drug Tx, etc) including males and females.
2. What genotypes you need determines your breeding pair.
   • E.g Het x Het gives you 25% WT, 50% Het and 25% KO littermates
3. Figure out the average mice you will get in one litter based on genotypes and sex:
   • C57 gives on average 6 mice
   • FVB on average gives 9 mice
   • CD1 on average gives 11 mice.
     • All half males and half females
   • If you are breeding Het x Hets, one litter provides (on average for C57 mice):
     • 4 Hets (2 males and 2 females)
     • 1 WT (0.5 male and 0.5 female)
     • 1 KO (0.5 male and 0.5 female)
4. Divide the number of mice you need by how many you get in one litter based on the lowest number to determine how many litters total you will need.
   • Example:
   • If you need 60 mice total (10M, 10F of each of WT HET and KO genotypes) on C57 background:
     • 4 Hets (2 males and 2 females)
     • 1 WT (0.5 male and 0.5 female)
     • 1 KO (0.5 male and 0.5 female)
   • To get 10 M KOs, Need 20 litters. So, 20 female and 20 male hets to breed all at once, or some fraction of that and multiple rounds of breeding.
   • Try to batch your mice as infrequently as possible – batch effects are real!!

If you want to have your mice all within a close age range for a study, like behavior, try this strategy for your breeding cages:

1. Put male in cage for 4 days by himself to create a territory – this increases success of breeding right away (suggest a Fri to a Mon)
2. Put a DO NOT CHANGE card on the cages!
3. Induce estrus in the female by adding soiled male bedding to her cage for 4 days prior to breeding (and 4 hours before adding her to the male’s cage on day of breeding).
4. Leave breeding pair together for 4 days before separating (this will ensure the estrus phase occurred).

The less the cages are handled the higher the chance of a successful breeding.

Be generous with your DO NOT CHANGE cards!!!

Here’s how to think about breeding a cohort:

Example: two genotypes, Hets & WTs

If you want 40, thats 20 het and 20 WT. We get about an even split with this line. So, on average, you get 6 pups in a C57 line. So that’s 3 Hets and 3 WTs. 20/3=6.667. So that’s at least 7 litters to get what you need. But, I always add an extra 20% breeding pairs to cover the inevitable pair that doesn’t drop. So 7 x .2 = 1.4, so I would add another 2 breeding pairs to that. 9 breeding pairs should ensure that you get the 40 mice that you need to drop at the same time (within 4 days of each other, given estrous cycle spread)

Example: three genotypes, Hets & WTs & KOs

If you want 60, thats 20 KOs, 20 hets and 20 WTs. We get 25% KO, 25% WT, and 50% het per litter. So, on average, you get 6 pups in a C57 line. So that’s 3 Hets, 1.5 WTs, 1.5 KOs. 20/1.5=13.3333 litters to get all the WTs and KOs needed. So that’s at least 14 litters to get what you need. But, I always add an extra 20% breeding pairs to cover the inevitable pair that doesn’t drop. So 14 x .2 = 2.8 2.8, so I would add another 3 breeding pairs to that. 17 breeding pairs should ensure that you get the 60 mice that you need to drop at the same time (within 4 days of each other, given estrous cycle spread). – Batch that with two rounds of 9 pairs.

Pups

DCM will mark cages when pups are seen in cage.

• This marker is related to the date when pups were seen not always date of birth.
• Use the JAX Mice Pup Appearance by Age schematic to help you determine the correct DOB.
  • Smaller litters may appear older than they are due to more access to maternal care and nutrition. Similarly, larger litters (>8) may appear younger than they actually.

Date of Birth = postnatal day (P)0, P0 Average litter size

• C57Bl/6j: 6 pups
• FVBant/j: 9 pups

Weaning Mice

• Wean mice between P21–P28
  • DCM will mark with the date of weaning. The day before P29 Amanda will contact you as a reminder.
  • On P29, DCM will wean the cage and charge the lab. This should be avoided.
  • Wean as close to P21 as possible because another litter may be about to drop.
    • unless your mice are runty and need another day or two with the dam.
• When weaning:
  • Use clean food, water and cage parts when creating a new cage.
  • Put food pellets on the floor of the cage for weanlings
  • Use Nestlets and a cardboard tube

Wean mice into as FEW cages as possible. Mice from different litters can be housed together.

• Do not add young males to existing male cages that are aged P40 or older (they will attack the new guy).
• Females can be combined at any age – however, a P21 female added to a cage with all much older (into adulthood) females may not survive, like a dominance issue/lack of access to food/water.

When weaning, house mice by:

1. experimental line – NEVER co-house mice from separate lines (unless crossing two lines to create a new line, like a double transgenic for example).
2. sex – mice will start breeding if left together after P35.
3. genotype/experimental condition – applies to most behavioral studies, and should be determined on a study by study basis.

Housing Mice

• 5 mice per cage
  • You can house 6 per cage until 6 weeks old.
• 3 adults and 1 litter (up to 14 days old)
• 2 dams with 2 young litters (<7 days of age)
  • However, 2 dams with 2 litters until P21 for C57BL/6J strains. This is a special housing condition approved on the Dougherty IACUC protocol, initiated for drug studies.

DCM will mark these cages that are out of the policy and contact. If DCM has to split the cage, the lab will be charged.