Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Fertility assessment
• Procedure: Testis and semen preparation
• Procedure: Computer assisted sperm analysis (CASA)
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Breeding cages set up for 1 wk	-	Litter size
2	Testis and semen harvested	Dissecting kit	Testis weight
3	Computer assisted sperm analysis (CASA)	Microscope	Sperm count, percentage of sperm that are motile and percentage that are processive

Equipment and supplies

• IVOS automated microscopy
• Computer assisted sperm analysis (CASA) system (Hamilton Thorne CEROS imaging system with IVOS software (v. 12.3H))

Reagents and solutions

• Phosphate-buffered saline
• 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

Procedure: Fertility assessment

1. One male mouse is co-housed with two FVB/NJ female mice (at 8-12 wks of age) for 1 week.
2. Females are observed for at least 21 days following male removal.
3. Live born pups are counted.

Procedure: Testis and semen preparation

1. One testis is weighed per mouse.
2. Semen is harvested from one cauda epididymis per mouse: One epididymis is placed in 500 µL of phosphate-buffered saline and incubated for 20 min at 37°C. Each epididymis is dissected into 500 µL of HTF media which is then diluted 1:20 for subsequent analysis as previously described (Odet et al., 2015).

Procedure: Computer assisted sperm analysis (CASA)

1. The following settings are used for the imaging system to determine sperm count and the percentage of total sperm that are motile and processive (sperm analyses performed by the UNC Mutant Mouse Resource and Research Center):
   • Frame rate: 60 Hz
   • Minimum Contrast: 50 (the minimum acceptable brightness, above the brightness of the background, that must be shown by an object to be counted)
   • Minimum Cell Size: 5 pixels (minimum number of pixels an object must have to be counted)
   • Minimum Static Contrast: 15
   • Straightness (STR), Threshold: 50% (Straightness = average value of the ratio of VSL/VAP (measures the departure of the cell path from a straight line; minimum value a cell must possess to be considered progressively motile)
   • VAP Cutoff: 5.0 µm/s (VAP = path velocity; the average velocity of the smoothed cell path in µm/s that a cell must possess to be considered progressively motile)
   • Progressive Minimum VAP: 80 µm/s
   • VSL Cutoff: 0.0 µm/s (VSL = Progressive Velocity; the average velocity measured in a straight line from the beginning to the end of the track)
   • Cell Size: 8 pixels
   • Cell Intensity: 90
   • Static Head Size: 0.30 to 3.67
   • Static Head Intensity: 0.50 to 1.30
   • Static Elongation: 0 to 62
   • Slow Cells Motile: Yes
   • Magnification: 0.79
   • Video Frequency: 60
   • Bright Field: No
   • LED Illumination Intensity: 2338
   • IDENT Illumination Intensity: 3000
   • Temperature, Set: 37°C
   • Chamber Depth: 100 µm

Data collected by investigator

• Testis weight (1 testis)
• Number of sperm per mL semen
• Percentage of sperm that are motile
• Percentage of sperm that are processive
• Mean litter size

 

Reference

Odet F, Pan W, Bell TA, Goodson SG, Stevans AM, Yun Z, Aylor DL, Kao CY, McMillan L, Pardo-Manuel de Villena F, O'Brien DA. The founder strains of the Collaborative Cross express a complex combination of advantageous and deleterious traits for male reproduction. G3: GENES, GENOMES, GENETICS (2015) 5(12):2671-83.