Video acquisition and CASA analysis of mouse sperm

Video acquisition of sperm

Reagents

1. EmbryoMax human tubal fluid 1×: Cat# MR-070-D, Sigma-Aldrich, St. Louis, U.S.
2. 12 well tissue culture plate: Cat# 229111, CELLTREAT, Pepperell, U.S.
3. Leja sperm counting chamber slides, 100 micron 2 chamber: Cat# 025998, IMV Technologies, Saint-Ouen-sur-Iton, France

Instrument, software, and tools

1. Nikon Diaphot 200 microscope: Nikon, Tokyo, Japan
2. ZWO ASI 174mm Monochrome CMOS Imaging camera: ZWO, SuZhou, China
3. SharpCap: https://docs.sharpcap.co.uk/2.9/
4. Fiji (Image J): https://imagej.net/software/fiji/

Procedure

Collection of cauda epidydimal sperm was performed as previously described (Goodson et al., 2011). Epidydimal sperm were collected from each mouse and immediately placed in 1mL pre-warmed 37ºC human tubal fluid media (HTF) containing 4mg/ml bovine serum albumin (EmbryoMax human tubal fluid 1×, Sigma-Aldrich) in a 12 well tissue culture plate with a lid (CELLTREAT). The plate containing sperm samples was then transferred to an incubator with 5% CO₂. At indicated time points, the tissue culture plate was briefly removed from the incubator, and 25µL sperm was gently pipetted from the suspension into a chamber on the pre-warmed 100µm sperm counting glass chamber slide (IMV Technologies). The sperm sample was first visually inspected under the microscope and further diluted with warm HTF if the imaging field was over-crowded. For video acquisition, a Nikon Diaphot 200 microscope with darkfield optics equipped with Nikon E Plan 10×/0.25 160/- Ph1 DL objective (Nikon) and ZWO ASI 174mm Monochrome CMOS Imaging camera (ZWO) were set up in a 37ºC chamber. The SharpCap software was used to record sperm movement using darkfield at 10× magnification. Video acquisition was performed for at least five fields for each biological replicate. Video lengths were edited for publication using Fiji (Image J). 

Sperm imaging for CASA analysis

Reagents

1. EmbryoMax human tubal fluid 1×: Cat# MR-070-D, Sigma-Aldrich, St. Louis, U.S.
2. 12 well tissue culture plate: Cat# 229111, CELLTREAT, Pepperell, U.S.
3. Leja sperm counting chamber slides, 20 micron 4 chamber: Cat# 025107, IMV Technologies, Saint-Ouen-sur-Iton, France

Instrument, software, and tools

1. IVOS II: Hamilton Thorne, Beverly, MA
2. CASAnova: http://csbio.unc.edu/CASAnova/index.py (Goodson et al., 2011)
3. RStudio: https://www.rstudio.com/
4. ggplot2: https://ggplot2.tidyverse.org/

Procedure

Sperm collection and preparation procedures are as described in “Video acquisition of sperm” except for the use of 20µm chamber slides (IMV Technologies). Imaging was conducted using an IVOS II instrument (Hamilton Thorne) pre-warmed to 37°C with the following settings: 100 frames acquired at 60 Hz; minimal contrast = 50; 4 pixel minimal cell size; minimal static contrast = 5; 0%straightness (STR) threshold; 10 μm/s VAP Cutoff; prog. min VAP, 20 μm/s; 10 μm/s VSL Cutoff; 5 pixel cell size; cell intensity = 90; static head size = 0.30–2.69; static head intensity = 0.10–1.75; static elongation = 10–94; slow cells motile = yes; 0.68 magnification; LED illumination intensity = 3000; IDENT illumination intensity = 3603; 37°C. The raw data files (i.e., .dbt files for motile sperm and .dbx files for static sperm) were used for sperm motility analysis with CASAnova (Goodson et al., 2011). For each sample, at least five fields were imaged to reach 500–1000 total sperm cells. Data points acquired for each field of the same sample were combined either directly on IVOS II or manually after exporting .dbt files to Microsoft Excel. For the motile sperm, only those whose movement was captured with ≥45 consecutive frames were analyzed to remove less reliable data points while retaining sufficient data points for analysis. Accordingly, the number of static sperm was re-scaled for each mouse based on the percentage of motile sperm with ≥45 frames. For example, if 90% of all motile sperm cells from an animal have ≥45 frames in the .dbt file, the number of static sperm in the corresponding .dbx file would be re-scaled by a factor of 0.9. For hyperactivated and progressive motility analysis, .dbt files of motile sperm were used as input for CASAnova as described by the tool developers (Goodson et al., 2011). Boxplots were generated using RStudio and ggplot2. 

References

1. Goodson, S. G., Zhang, Z., Tsuruta, J. K., Wang, W., and O’Brien, D. A. (2011). Classification of mouse sperm motility patterns using an automated multiclass support vector machines model. Biol Reprod 84, 1207–1215.

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