Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Summary

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Abstract

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Introduction

The transition through puberty is required to attain sexual maturity and reproductive competence. The pubertal transition and the maintenance of fertility in adulthood is regulated by the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis (Figure 1). The timing of pubertal onset and maintenance of fertility is tightly regulated by internal as well as environmental factors to increase the chances of survival of offspring and parents^1,2. This protocol provides a non-invasive approach to determine pubertal onset in mice and rats to confirm sexual maturity prior to....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee of Michigan State University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

1. Determine Pubertal Onset

1. Follow institutional guidelines for clothing, at a minimum, it is necessary to wear a clean lab coat and clean gloves. Always handle mice wearing clean gloves.
2. Prepare the work area by placing a pad on the table.
   1. Place a cl.......

Discussion

The overall wellbeing of the mice is critical for a successful fertility assay²¹. When performing a fertility assay, it is important to not physically check on the mice every day as this can cause stress. Further avoid frequent cage changes, as these are also stressful. Ideally cage changes will be done no more than 1-2 times per week. Light exposure during the dark phase negatively impacts breeding in nocturnal rodents. Do not turn on lights in the breeding room during the dark hours. If entry to.......

Materials

Name	Company	Catalog Number	Comments
Sterile Cotton Balls	Fisher	22456885
Surface protector	Fisher	1420637
Light meter	VWR	21800-014
Methylene blue	Sigma-Aldrich	M9140
Microscope Slides	Genesee Scientific	29-101
Optimouse rack with cages	AnimalCare systems	C89100
Water Bottle Basket	AnimalCare systems	C61011
Filtered Cage Tops	AnimalCare systems	C78210
Optimice Standard Feeder	AnimalCare systems	C40100SG
Cage Card Holder	AnimalCare systems	C43251
Cage Cards	AnimalCare systems	M52010
Bottle Assambley	AnimalCare systems	C79122P
Bed R'Nest Nesting	The Andersons	BRN4WSR
1/8" Corn Cob bedding	The Andersons	8B
Standard mouse chow	Teklad	7904 (7004)
Scale	VWR	10205-004
Polypropylene Beaker	Fisher	14-955-111F