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  1. Calculate the desired freezing concentration ensuring to set aside 100 μL of working sample for pre and post equilibration quality assessment.
  2. In a set of three Eppendorf tubes (set 1), adjust sperm samples to proper target concentrations by diluting with Hanks balanced salt solution (HBSS).
  3. After sperm samples are adjusted to target concentrations, add the Styrofoam float portion of the Cryokit to the liquid nitrogen to cool for ≥ 5 min.
  4. Set up the thermocouple straw by filling a 0.25mL straw with 1:1 ratio of HBSS and cryoprotectant (CPA). Insert the thermocouple wire into the tube so that the end of the wire is positioned in the center of straw. Plug the opening with the 3D printed straw sealers.
  5. In a separate set of three Eppendorf tubes (set 2), prepare different volumes of CPA that correspond to the target concentrations. CPA should be mixed with sperm sample 1:1, therefore the volume of CPA should be equal to the final volume of sperm sample based on the concentration. Adjust this based on the requirements of your experiment.
  6. Add the sperm samples from set 1 to the CPA in set 2, making sure to discard your pipette tip between target concentration samples. Gently flick and invert to mix.

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When finished with these steps move on to equilibration

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Pre and post equilibration quality assessment

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Preparation for quality assessment should be done at a convenient time during the previous steps so that your sample can be checked as soon as equilibration is done

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  1. Quality assessment setup-Set aside 5 microliters of sperm and dilute each at a 1:1 ratio with CPA. Sample should be equilibrated at room temperature. CPA addition should be staggered by 5 minutes so you have time to analyze each sample right after equilibration.

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Equilibration time for this sample should be exactly the same as the samples in your experiment above.

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  1. After waiting equilibration time, dilute with extender for analysis and do ****pre and post equilibration ****quality assessment (this quality assessment should ideally be done after freezing is complete).

Hanks Balanced Salt Solution (HBSS) recipe at 200 mOsm/kg