Shorter6 project protocol

Male reproduction in 12 Collaborative Cross strains of mice: Sperm motility (CASA)   (2020)

Shorter JR, O'Brien DA, Pardo-Manuel de Villena F
With: Pan W, Odet F, Aylor DL, Kao C-Y, Fu C-P, Greenstein S, Bell TA, Stevans AM, Patel S, Cates SE, Shaw GD, MIller DR, McMillian L

Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 Mice euthanized and weighed (see Shorter4) - -
2 Reproductive organs harvested and weighed (see Shorter4) - -
3 Sperm harvested from right cauda epididymis and counted (see Shorter4) - -
4 Sperm harvested from left cauda epididymis for in vitro capacitation study on sperm motility Microscope (Computer Assisted Sperm Analysis (CASA)) Motility analysis
5 Sperm from left cauda epididymis prepared to assess morphology (see Shorter4) - -


General Information for All Procedures: See Shorter4 for reproductive organ weights, sperm count, motility and morphology.

Procedure 1: Sperm motility

Definitions & Abbreviations: CASA: Computer Assisted Sperm Analysis

Equipment, software, and supplies

  • CEROS imaging system (Hamilton Thorne)
  • CEROS imaging system software (Hamilton Thorne, version 12.3 IVOS)
  • CASAnova, a support vector machines program based on CASA parameters of CD1 sperm
  • Leja chambers (Leja, Netherlands)
  • 5% CO2 tissue culture incubator

Reagents and solutions

  • Human tubal fluid medium (HTF), 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM KH2PO4, 0.2 mM MgSO4·7H2O, 2 mM CaCl2, 25 mM NaHCO3, 2.78 mM glucose, 0.33 mM pyruvate, 21.4 mM lactate, 5 mg/mL of bovine serum albumin, 100 U/mL of penicillin G, and 0.1 mg/mL of streptomycin
  • Bovine serum albumin (BSA)


  1. Mice are euthanized by CO2 asphyxiation followed by cervical dislocation.
  2. Reproductive organs are harvested and weighed (Shorter4).
  3. The left cauda epididymis is clipped with iris scissors and incubated in human tubal fluid medium (HTF) at 37°C (5% CO2) for 10 min.
  4. Sperm suspensions are diluted with HTF and incubated for 2 h at 37°C (5% CO2).
  5. Motility is assessed at 30 min intervals throughout the in vitro capacitation period by computer-assisted sperm analysis (CASA) using Leja chambers.
  6. Sperm tracks (90 frames, 1.5 s) and kinetic parameters for individual sperm are captured at 60 Hz using motility analysis parameters (mouse 2) recommended by Hamilton Thorne Biosciences, except that slow sperm are considered motile.
  7. Tracks in 10 fields are typically recorded for each mouse; motility measurements are captured.
  8. CASAnova is used to classify individual sperm into one of five motility groups. Classification is based on Support Vector Machines equations incorporating independent CASA parameters for each sperm track. See Goodson et al., 2011 for details.


Protocol References

Goodson SG, Zhang Z, Tsuruta JK, Wang W, O'Brien DA. Classification of mouse sperm motility patterns using an automated multiclass support vector machines model. Biol Reprod. 2011 Jun;84(6):1207-15. doi: 10.1095/biolreprod.110.088989. Epub 2011 Feb 23.   PubMed 21349820     FullText