Shorter5 project protocol

Male reproduction in 28 F1 hybrids of Collaborative Cross strains of mice (RIX): Organ weights and sperm count, motility, and morphology   (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 Scales Body weight
2 Reproductive organs harvested and weighed Scales Testis, epididymis with attached vas deferens, and seminal vesicle weights
3 Sperm harvested from right cauda epididymis and counted Hemocytometer Sperm counts
4 Sperm harvested from left cauda epididymis for in vitro capacitation study on sperm motility (see Shorter7) - -
5 Sperm from left cauda epididymis prepared to assess morphology Microscope Sperm morphology


General Information for All Procedures: See Shorter7 for assessing sperm motility

Procedure 1: Body weight and organ weights

Equipment, software, and supplies

  • Scales


  1. Mice are euthanized by CO2 asphyxiation followed by cervical dislocation.
  2. Carcasses are weighed
  3. Reproductive organs are harvested and weighed

Procedure 2: Sperm count and morphology

Equipment, software, and supplies

  • Microscope (Olympus, BX51)
  • Digital camera (Olympus, DP72)
  • Image analysis software (Molecular Devices, Sunnyvale, CA)
  • Iris scissors
  • Fine forceps
  • Microfuge tubes
  • Microscope slides
  • Hemocytometer

Reagents and solutions

  • Phosphate buffered saline (PBS)
  • 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)
  • Peanut agglutinin (PNA), conjugated to a fluorescent tag (Invitrogen, Alexa Fluor 488)
  • Methanol


  1. The right cauda epididymis is clipped with iris scissors in 500 µL of PBS and incubated for 10 min at 37°C.
  2. Sperm are extruded with fine forceps and the suspension is transferred to a microfuge tube; the collection well is rinsed with an additional 500 µL of PBS.
  3. Sperm are diluted appropriately and counted using a hemocytometer.
  4. The left cauda epididymis is clipped with iris scissors and incubated in HTF at 37°C (5% CO2) for 10 min, allowing sperm to swim into the medium.
  5. Sperm motility is determined (see Shorter7).
  6. Aliquots (10 µL) of the HTF sperm suspension are spread onto positively charged microscope slides and allowed to air dry briefly until moisture just evaporates.
  7. Samples are fixed with -20°C methanol for 10 min, air dried and stored at -20°C.
  8. Acrosomes are stained with PNA conjugated to a fluorescent tag.
  9. Sperm are scored using phase contrast optics (normal morphology, abnormal head shape, abnormal tail bending (≥ 90°), or broken tails (severed at head/neck junction or at more distal locations along the length of the flagellum).