Influence of hydroxytyrosol in semen extenders on fertilizing capacity and embryo development using cooled and cryopreserved bull sperm

This study investigated the effects of hydroxytyrosol (HT), a potent antioxidant polyphenol, on sperm functionality, fertilizing ability, and embryo development when added to semen extenders using cooled and cryopreserved bull semen. In a preliminary trial, 1 and 25 μM HT were identified as the most effective concentrations and subsequently used in the main experiments. Semen from 12 Angus bulls was divided into aliquots and stored either refrigerated at 4 °C for 24 h or cryopreserved. Sperm parameters (motility, viability, membrane and acrosomal integrity), malondialdehyde (MDA) levels, total antioxidant capacity (TAC), zona pellucida (ZP) binding, and in vitro fertilization outcomes were assessed. In cooled semen, HT enhanced functional membrane integrity and acrosomal status, and increased sperm–ZP binding. Supplementation with 1 μM HT improved embryo quality, increasing hatching rate and blastocyst cell number. In cryopreserved semen, 25 μM HT improved ZP-binding capacity, blastocyst rates, and blastocyst cell numbers. These findings suggest that HT supplementation improves sperm functionality and embryo development under both cooled and cryopreserved storage conditions, highlighting its potential to enhance assisted reproductive technologies in cattle.