Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for for matings and pregnancy monitoring
Step Procedure performed Equipment Data collected 1 Nulliparous females allowed to bear one litter before testing - - 2 After first litter, candidate breeding females placed into separate rooms to acclimate to testing conditions monitoring equipment and infrared lighting - 3 Females and males pair mated - 4 Females checked early mornings for the presence of vaginal plug blunt forceps presence of copulatory plug 5 Presumptive pregnant females weighed and separated from males scale baseline body weight 6 At E14.5 females weighed and housed singly scale, monitoring equipment and infrared lighting pregnant body weight 7 Pregnant females monitored continuously from E14.5 till appearance of first pup; immediately after giving birth, number of pups born counted gestation duration, number of pups born 8 At postnatal day 3, nursing females and pups moved to a new pen, live pups counted and weighed scale number pups alive, body weight of pups
- balance scale
- infrared lighting and video cameras (Inter-Pacific, Wheeling, IL)
- blunt probe or forceps (for copulatory plug detection)
- bed-o-cob bedding
- white pine shavings
- disinfectant or cleaning solution
Acclimation to test conditions
Mice are transported from the mouse room into the monitoring room and are allowed to acclimate for at least 1 wk before test mating.
Procedure for matings and pregnancy monitoring
a. To eliminate variability that might arise from first litters, female mice are mated and allowed to deliver one litter before being placed into the queue for gestation time monitoring.
b. Following first litters, females from each strain are separated from males, moved simultaneously into a different room outfitted with monitoring equipment and infrared lighting, and allowed to acclimate for at least 1 wk before test mating.
c. After 1 wk males and females are pair-mated at the end of the day.
d. Females are checked for the presence of a vaginal plug early the next morning as an indication of a successful mating.
e. Fertilization (0 h) is calculated to be the midpoint of the dark cycle prior to the appearance of the copulation plug.
f. Pregnant females are weighed and housed separately from the male until monitoring.
g. At E14.5 pregnant females are again weighed and housed singly in pens containing bed-o-cob bedding (chosen because it cannot be piled and obscure the view of the cameras).
h. Gestating females are monitored continuously from E14.5, using infrared lighting and video cameras until the appearance of the first pup.
i. Following delivery of the last pup, nursing females and pups are moved to a new pen containing white pine shavings, and the litter is weighed as a group.
j. Each video is examined for the precise time of birth, determined by the appearance of the first pup (varies from 451 h to 493 h).
Notes by submitting PI: "Variability in the total number of females measured is due to differences in reproductive performance or morbidity associated with characteristics of the individual strain, such as lymphoma in the AKR/J strain and diabetes in NOD/ShiLtJ."
E14.5: embryonic day 14.5, beginning at fertilization and estimated base on the presence of a vaginal or copulatory plug
gestation length: duration time from conception to birth
pregnancy load: [(pregnant (E14.5) body weight - non-pregnant body weight) / non-pregnant body weight] X 100
Data collected by investigator
- percent increase in maternal body weight by E14.5 (pregnancy load)
- gestation duration
- number of pups born per litter
- percent of pups that survived to day 3
- pup body weight at day 3