Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

Summary

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

Abstract

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male's cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 μL capillary pipette was placed into the tube at a 180° angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 μL line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 μL capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Introduction

The female reproductive tract is comprised of the upper and lower tracts. The upper reproductive tract includes Fallopian tubes, the uterus, and the endocervix. The lower reproductive tract includes the ectocervix and the vagina. In humans, the ejaculated semen is deposited at the anterior wall of the vagina, adjacent to the ectocervix¹. In mice, however, the semen is swept into the uterus within minutes after mating².

Semen contains seminal gel that is solidified within seconds of ejaculation, causing the sperm to be entrapped and immobilized³. Liquefaction releases th....

Protocol

All animals and procedures used in this study were handled according to Washington State University (WSU) Animal Care and Use Committee guidelines and in compliance with WSU-approved animal protocols #4702 and #4735.

1. Mice

1. Use standard C57BL/6J adult male mice or other strains of interest. Maintain animals under a temperature- and humidity-controlled environment, with ad libitum access of water and food.
   1. Use male breeders ranging from 8 weeks old to 10 months old. Use only proven breeders in the experiments.
2. Use adult female mice with a tissue selective dele....

Results

Semen was collected from CTRL and KO females approximately 8 h after mating with fertile CTRL males. Liquefaction (or semen viscosity) is quantified by the time taken to fill 25 μL capillary tube with semen. Semen collected from CTRL uteri took 2.06 ±0.12 min (mean±S.E.M, n = 5) to fill the capillary tube (Figure 1). However, the semen collected from KO uteri took longer than 60 min (60.0 ±0.0, mean±S.E.M, n = 5) of experimental time. These data suggest that the semen is si.......

Discussion

Collecting semen from mouse uteri can provide advantages over semen analysis in vitro as the former can illustrate the physiological interactions between the semen and the secretions from the female reproductive tract. The techniques presented in this study allow researchers to determine semen quality as well as sperm viability and motility in ideal physiological conditions. Moreover, there are various downstream analyses that can be performed using the semen collected from the uteri, for example, the s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Toothpick	Diamond	750-Flat	Autoclaved sterile, use blunted toothpick. Do not use point-ended
Dissecting scissors	Fisher	08-940
Spring scissors	Roboz	RS-5600
Fine forceps	Roboz	RS-5045
25-μL glass capillary tube	VWR	53432-761
Leibovitz’s L-15 media	Life Technologies	11415064	Supplement with 1% FBS
Fetal bovine serum (FBS)	Gemini Bio-Products	100-106
1.5-mL microcentrifuge tube	Axygen	MCT-150-C
2-mL microcentrifuge tube	Axygen	MCT-200-A
Digital dry block heater	VWR	13259-052	For warming the microcentrifuge tube prior to semen collection
Minicentrifuge	Corning	LSE 6765
35-mm culture dish	VWR	10861-656
15-mL polypropylene centrifuge tube	VWR	10025-686
Waterbath	Precision	TSGP05
Heated stage stereomicroscope	Leica Microsystems	MZ10F	To keep the tissue warm prior to semen collection
Brightfield microscope	Leica Microsystems	DMi8	Optional. For video imaging of the sperm motility
Camera	Leica Microsystems	DMC2900	Optional. For video imaging of the sperm motility
Glass slides	Fisher	12-552-3	Optional. For video imaging of the sperm motility
Coverslip	VWR	48393-059	Optional. For video imaging of the sperm motility
Bleach			Dilute in dH2O to 10% concentration