Male fertility assessment in 347 pre-CC lines of the Collaborative Cross that are now extinct (2017)
Shorter JR, O'Brien DA, Pardo-Manuel de Villena F
With: Odet F, Aylor DL, Pan W, Kao CY, Fu CP, Morgan AP, Greenstein S, Bell TA, Stevans AM, Feathers RW, Patel S, Cates SE, Shaw GD, Miller DR, Chesler EJ, McMillian L
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
Morphological assessment of individual sperm
Seminiferous tubule morphology
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
Phosphate-buffered saline (PBS)
Human tubule fluid (HTF)
Bovine serum albumin (BSA)
Peanut agglutinin conjugated to a fluorescent tag (Alexa Fluor 488; Invitrogen, Carlsbad CA)
Periodic acid-Schiff reagent
Procedure: Fertility testing and harvesting organs
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.
Pairs are set up for a minimum of 7 weeks after a line is declared extinct.
Males that are unproductive in all matings are considered infertile.
Each male is euthanized using CO2 asphyxiation followed by cervical dislocation.
The carcass is weighed and reproductive organs harvested and weighed.
Procedure: Sperm counts
Sperm counts are determined after collecting sperm from the right cauda epididymis after being stored at 4° overnight in PBS.
The epididymis is clipped with iris scissors in 500 µL of PBS and incubated for 10 min in a 37° C incubator.
Sperm are extruded from the cauda with fine forceps.
The sperm suspension is transferred to a microfuge tube and the collection well rinsed with an additional 500 µL of PBS.
Sperm are counted with a hemocytometer.
Procedure: Sperm motility
Motility is measured from sperm collected from the left cauda epididymis.
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).
After appropriate dilution with the same medium, sperm are transferred to Leja chambers.
Motility is assessed by computer-assisted sperm analysis (CASA) using CEROS imaging system.
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.
Tracks in 10 fields are typically recorded for each mouse.
Procedure: Sperm morphology
The HTF sperm suspension (above) is also used to assess sperm morphology.
Ten-microliter aliquots of this suspension are spread onto positively charged microscope slides and allowed to air dry briefly until moisture has just evaporated.
Slides are fixed in -20° C methanol for 10 min, air dried, and stored at -20° C.
Acrosomes are stained with peanut agglutinin conjugated to a fluorescent tag before microscopic analysis.
Procedure: Testis histology
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).
Testes are fixed in Bouin's solution, cut in half horizontally, and embedded in paraffin.
Testes sections (8 µm) are stained with periodic acid-Schiff reagent and counterstained with hematoxylin.
Multiple images of each section are recorded using an Olympus microscope equipped with a digital camera.
A composite image of each transverse section (20x) is generated using MetaMorph automation and image analysis software.
Testis composite images are annotated using a custom interactive image analysis package.
For each testis, the tubule centers are automatically found and refined by a trained observer.
The mean tubule radius and the number of tubules are used to estimate the length of the seminiferous epithelium.
An interactive tool is used to denote tubules with few or many vacuoles.
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).
Fertility testing and harvesting organs
Weight for each testis
Weight for each epididymis with attached vas deferens
Weight for seminal vesicles
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
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
Mean seminiferous tubule radius
Number of seminiferous tubules per transverse section
Seminiferous tubule epithelium length per transverse section