EP055

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

Ca²⁺ staining is an experimental technique that utilizes specific fluorescent probes (such as Fluo-4 AM, Fura-2, etc.) to qualitatively or quantitatively detect dynamic changes in intracellular Ca²⁺ concentrations; this is achieved by monitoring the changes in fluorescent signals generated when these probes bind to free intracellular calcium ions. The underlying principle relies primarily on the presence of chelating groups within the probe's molecular structure that possess high affinity for calcium ions.

Sperm thawing for functional recovery is a post-cryopreservation procedure designed to restore and measure sperm motility, viability, membrane/acrosome integrity, DNA integrity, and usable motile sperm yield after freezing-induced injury; published human studies show that cryopreservation reduces motility and viability and can damage the plasmalemma, acrosome, tail, and DNA integrity. The experimental readout is generated by comparing post-thaw motility recovery, viability, acrosomal status, DNA integrity, and recovered motile sperm after thawing and optional sperm-selection steps; thawing at 40°C improved motility recovery compared with 20-37°C without significant differences in viability, ATP content, acrosomal status, or DNA integrity in one human donor-semen study.