Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Fertility testing and harvesting organs
• Procedure: Sperm counts
• Procedure: Sperm motility
• Procedure: Sperm morphology
• Procedure: Testis histology
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Fertility testing and harvesting organs	Dissection kit	Body and organ weights
2	Sperm count	Hemocytometer	Sperm count
3	Sperm motility	Microscope and Computer-assisted sperm analysis (CASA)	Count and percentage of motile sperm, velocities, amplitude of head displacement, beat cross frequency, and classification of individual sperm
4	Sperm morphology	Microscope	Morphological assessment of individual sperm
5	Testis histology	Microscope	Seminiferous tubule morphology

Equipment and supplies

• Dissection kit
• Fine forceps
• Iris scissors
• Microfuge tubes
• Hemocytometer
• Leja chambers (Leja Products BV, Nieuw-Vennep, The Netherlands)
• Computer-assisted sperm analysis (CASA) system
• CEROS imaging system (Hamilton Thorne Biosciences; version 12.3 IVOS software)
• Positively charged microscope slides
• Microscope (Olympus BX51 equipped with a motorized stage and an Olympus DP72 digital camera)
• MetaMorph automation and image analysis software (Molecular Devices, Sunnyvale CA)
• Custom interactive image analysis package

Reagents and solutions

• Phosphate-buffered saline (PBS)
• Human tubule fluid (HTF)
• Bovine serum albumin (BSA)
• Methanol
• Peanut agglutinin conjugated to a fluorescent tag (Alexa Fluor 488; Invitrogen, Carlsbad CA)
• Liquid nitrogen
• Bouin's solution
• Paraffin
• Periodic acid-Schiff reagent
• Hematoxylin

Procedure: Fertility testing and harvesting organs

1. Breeding pairs are set up for each previously declared infertile male plus females from the same funnel, as well as to unrelated females, such as Swiss Webster and FVB/NJ.
2. Pairs are set up for a minimum of 7 weeks after a line is declared extinct.
3. Males that are unproductive in all matings are considered infertile.
4. Each male is euthanized using CO₂ asphyxiation followed by cervical dislocation.
5. The carcass is weighed and reproductive organs harvested and weighed.

Procedure: Sperm counts

1. Sperm counts are determined after collecting sperm from the right cauda epididymis after being stored at 4° overnight in PBS.
2. The epididymis is clipped with iris scissors in 500 µL of PBS and incubated for 10 min in a 37° C incubator.
3. Sperm are extruded from the cauda with fine forceps.
4. The sperm suspension is transferred to a microfuge tube and the collection well rinsed with an additional 500 µL of PBS.
5. Sperm are counted with a hemocytometer.

Procedure: Sperm motility

1. Motility is measured from sperm collected from the left cauda epididymis.
2. The cauda is clipped with iris scissors and transferred to a 37° C incubator (5% CO₂ in air), allowing sperm to swim out for 10 min into 1 mL of human tubule fluid (HTF) medium + 5 mg/mL bovine serum albumin (HTF medium typically supports the development of hyperactivated motility over a 90-min period).
3. After appropriate dilution with the same medium, sperm are transferred to Leja chambers.
4. Motility is assessed by computer-assisted sperm analysis (CASA) using CEROS imaging system.
5. Sperm tracks (90 frames, 1.5 s) and kinetic parameters for individual sperm are captured at 60 Hz using motility analysis parameters recommended by Hamilton Thorne Biosciences, except that slow cells were counted as motile.
6. Tracks in 10 fields are typically recorded for each mouse.

Procedure: Sperm morphology

1. The HTF sperm suspension (above) is also used to assess sperm morphology.
2. Ten-microliter aliquots of this suspension are spread onto positively charged microscope slides and allowed to air dry briefly until moisture has just evaporated.
3. Slides are fixed in -20° C methanol for 10 min, air dried, and stored at -20° C.
4. Acrosomes are stained with peanut agglutinin conjugated to a fluorescent tag before microscopic analysis.

Procedure: Testis histology

1. After wet weights are determined (above), the left testis is flash frozen in liquid nitrogen and preserved for future use while the right testis is prepared for histological examination (if the left testis showed anomalies, it is used for histology instead of the right testis).
2. Testes are fixed in Bouin's solution, cut in half horizontally, and embedded in paraffin.
3. Testes sections (8 µm) are stained with periodic acid-Schiff reagent and counterstained with hematoxylin.
4. Multiple images of each section are recorded using an Olympus microscope equipped with a digital camera.
5. A composite image of each transverse section (20x) is generated using MetaMorph automation and image analysis software.
6. Testis composite images are annotated using a custom interactive image analysis package.
7. For each testis, the tubule centers are automatically found and refined by a trained observer.
8. The mean tubule radius and the number of tubules are used to estimate the length of the seminiferous epithelium.
9. An interactive tool is used to denote tubules with few or many vacuoles.
10. Vacuolization is classified in six categories ranging from no vacuoles to many vacuoles in > 20 tubules, and the six categories further classified into two that generalized few to no vacuoles (categories 1-4) and many vacuoles (categories 5-6).

Data collected by investigator

• Fertility testing and harvesting organs
  
  • Body weight
  • Weight for each testis
  • Weight for each epididymis with attached vas deferens
  • Weight for seminal vesicles
• Sperm counts
  • Sperm count
• Sperm motility
  • Number of motile sperm
  • Percentage of sperm that are motile
  • Average path velocity (VAP)
  • Straight-line velocity (VSL)
  • Curvilinear velocity (VCL)
  • Amplitude of lateral head displacement (AHL)
  • Beat cross frequency (BCF)
  • Percentage of sperm classified as progressive
  • Percentage of sperm classified as intermediate
  • Percentage of sperm classified as hyperactivated
  • Percentage of sperm classified as slow
  • Percentage of sperm classified as weakly motile
• Sperm morphology
  • Percentage of sperm having normal morphology
  • Percentage of sperm having abnormal head shape
  • Percentage of sperm having abnormal tail bending
  • Percentage of sperm having broken tails
• Testis histology
  • Mean seminiferous tubule radius
  • Number of seminiferous tubules per transverse section
  • Seminiferous tubule epithelium length per transverse section
  • Vacuole classification