biostudies-literatureFerrara MAItalian Ministry for University and ResearchItalian Ministry for University and Research (MUR)43190https://www.ebi.ac.uk/biostudies/studies/S-EPMC12680650biostudies-literatureUnknown15(1)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 difScientific reportsQuantitative holographic analysis in stallion spermatozoa following cryopreservation.PMC12680650PRIN EVIDENCE - code number: 2022ERKW4P - CUP MASTER: C53D23005260006Preziosi GBoni RRuggiero RFerrara MAGualandi SCfalseQuantitative 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 dif2025-01-01T00:00:00Z2025 Dec2026-06-09T04:49:27.251Z2026-06-09T03:07:32.476ZS-EPMC126806504134514010.1038/s41598-025-24885-w