Quantitative holographic analysis in stallion spermatozoa following cryopreservation

: This study employs Holographic tomography (HT) to examine structural and biophysical changes occurring during the cryopreservation of stallion sperm. HT is an advanced imaging technique that integrates digital holography with tomography to achieve three-dimensional, quantitative reconstructions of objects without the need for treatment or reporter dyes. By using refractive index (RI) intervals to represent specific structural regions of sperm cells, variations in optical density, surface area, volume, and dry mass across different cryopreservation extenders and donors have been quantified. Three main sperm components, (i) nuclear region, (ii) post-acrosomal region and midpiece and (iii) whole cell were identified and discriminated based on different RI. Our results revealed significant differences in volume of post-acrosomal region and midpiece among stallions as well as between fresh and frozen/thawed sperm, whereas no significant differences were observed between freezing extenders, aligning with our findings on sperm kinetics. A significant stallion-extender interaction underscores the need to personalize the sperm freezing process.