Effect of Freezing Rates and Supplementation of α-Tocopherol in the Freezing Extender in Equine Sperm Cryosurvival

This study was conducted to determine the impact of antioxidant (α-tocopherol) supplementation in the cryopreservation extender and three different freezing rates (FRs) on quality of post-thaw semen to elaborate a new protocol for stallion semen cryopreservation. Six ejaculates from each of four stallions were subjected to cryopreservation with a commercial extender (Gent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2 mM α-tocopherol. The semen was exposed to three different freezing rates between 5°C and −15°C: slow (5°C/min), moderate (10°C/min), and fast (20°C/min). After thawing, the sperm viability (Sybr-14 and propidium iodide [PI]), mitochondrial membrane potential (MMP) (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine), lipid membrane peroxidation (LPO; C11-BODIPY581/591), and apoptosis status (fluorescein isothiocyanate-conjugated annexin V and PI) of the plasmatic membrane of each sample were determined by flow cytometry. For both extenders, the percentage of viable cells was higher for spermatozoa cooled at 5°C/min than at 10°C/min and 20°C/min (P ≤ .05). The FR of 20°C/min demonstrated a lower value of MMP than the FR of 5°C/min and 10°C/min (P ≤ .05). The α-tocopherol extender improved (P ≤ .05) post-thaw membrane LPO; however, it did not improved viability and the apoptosis status of the sperm plasmatic membrane after thawing. In conclusion, results clearly indicate that the cryosurvival of stallion spermatozoa is improved when FR of 5°C/min is used from 5°C to −15°C.