Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Sperm motility
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected*
1	Mice euthanized and weighed (see Odet1)	-	-
2	Reproductive organs harvested and weighed (see Odet1)	-	-
3	Testis fixed, prepared for histology, and imaged (see Odet1)	-	-
4	Sperm harvested from right cauda epididymis and counted (see Odet1)	-	-
5	Sperm harvested from left cauda epididymis for in vitro capcitation study on sperm motility	Microscope (Computer Assisted Sperm Analysis (CASA))	Motility analysis
6	Sperm from left cauda epididymis prepared to assess morphology (see Odet1)	-	-
7	Sperm from left cauda epididymis prepared to measure lactate production (see Odet1)	-	-

*Supplementary data are available for this project. See Odet2 downloads.

Equipment and supplies

• Hamilton Thorne CEROS imaging system with version 12.3 IVOS software (Goodson et al. 2011) for computer-assisted sperm analysis (CASA)
• CASAnova (a support vector machines program based on CASA parameters of CD1 sperm)
• Leja chambers (100 µm-depth) (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 (Goodson et al. 2011).
• Bovine serum albumin (BSA)

Procedure: Sperm motility

1. Mice are euthanized by CO₂ asphyxiation followed by cervical dislocation.
2. Reproductive organs are harvested and weighed (Odet1).
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.

Data collected by investigator

CASA parameters for individual sperm tracks and means for each sample:

• average path velocity (VAP)
• straight line velocity (VSL)
• curvilinear velocity (VCL)
• amplitude of lateral head displacement (ALH)
• beat cross frequency (BCF)
• straightness (STR) = VSL/VAP
• linearity (LIN) = VSL/VCL

Sperm motility groups classification:

• percentage of sperm defined as progressive
• percentage of sperm defined as intermediate
• percentage of sperm defined as hyperactivated
• percentage of sperm defined as slow
• percentage of sperm defined as weakly motile
• percentage of sperm considered vigorous = progressive + intermediate + hyperactivated

 

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 84(6): 1207-15.