Supplementation of the BIOXcell extender with the antioxidants crocin, curcumin and GSH for freezing bull semen

Semen cryopreservation is routine in cattle, but the results of artificial insemination need improvement. A strategy to these aims is the supplementation of the freezing extender with novel antioxidants. This study aimed at testing the natural antioxidants curcumin and crocin as supplements to the commercial extender BIOXcell for freezing semen from 8 Holstein bulls. We tested curcumin at 0.05 and 0.1 mM (CU0.05, CU0.1) and crocin at 0.5 and 1.5 mM (CR0.5, CR1.5), with 0.5 mM reduced glutathione (GSH0.5) as reference, and a control (CTL, without supplementation). The samples were evaluated post-thawing and after 5 h at 38 °C by CASA for motility and flow cytometry for viability, apoptotic, capacitation, acrosomal status, cytoplasmic and mitochondrial reactive oxygen species (ROS) production, and chromatin status (SCSA). Control and GSH0.5 showed similar results, possibly because of the good protection from BIOXcell. CU0.05 and CU0.1 showed little effects but increased cytoplasmic ROS production and motility ALH. CR0.5 and CR1.5 decreased viability and increased apoptotic features significantly post-thawing and after the incubation, resulting in lower motility (significant after the incubation) but decreasing SCSA %HDS (loose chromatin). Whereas crocin at these concentrations seems incompatible with BIOXcell, maybe because of a prooxidant activity, curcumin use merits further research, considering the elevation of ROS with no significant negative effects. •The antioxidants crocin, curcumin, and GSH were tested as supplements for freezing bull semen, using the BIOXCell extender.•None of the antioxidant formulations clearly benefit sperm quality after thawing or incubation.•Crocin (0.5 and 1.5 mM) exerted mostly a negative effect on sperm physiology.•Curcumin (0.05 and 0.1 mM) showed little effects but stimulated the production of cytoplasmic ROS.