Unravelling how in vitro capacitation alters ram sperm chromatin before and after cryopreservation

[Background]: Sperm chromatin structure provides valuable information for the prediction of male fertility and can be altered during different procedures. Previous studies have shown that sperm chromatin condensation decreased during in vitro capacitation. Moreover, cryopreservation can affect sperm DNA integrity and chromatin compaction.[Objectives]: This study aimed to investigate dynamic modifications produced in the chromatin structure of ram spermatozoa during in vitro capacitation before and after cryopreservation.[Materials and methods]: Chromatin decondensation (AB+), DNA methylation, DNA fragmentation index (%DFI) and high DNA stainability (HDS) were evaluated in fresh and frozen-thawed ram spermatozoa incubated under capacitating (CAP) conditions at 1, 5, 15, 30, 60, 120, 180 and 240 minutes and under non-capacitating (NC) conditions at 0, 15 and 240 minutes.[Results]: Incubation in NC conditions did not induce significant changes in chromatin condensation (P > .05; AB + and HDS). However, incubation of fresh and cryopreserved ram spermatozoa under CAP conditions significantly increased chromatin decondensation (P .05).[Discussion and conclusions]: Our results suggest that sperm chromatin condensation decreased progressively during in vitro capacitation of ram spermatozoa, while sperm DNA integrity remained intact. Such changes in chromatin condensation appeared faster after sperm cryopreservation.PFP and DAMC were supported by a University of Castilla-La Mancha scholarship. MIC and AMM were supported by a Ministry of Economy and Competitiveness scholarship. The study was supported by grants from ALF-Research (Region Östergötland, LIO-698951), FORSS (Forskningsrådet i Sydöstra Sverige, 473121 and 745971), Lions Forskningsfond (DNRLIU-2016-00641), Linköping and the Swedish Research Council FORMAS, Stockholm (Project 2017-00946 and Project 2019-00288) and by the MINECO (AGL2013-48421-R).Peer reviewe