47 Phenformin can reduce lipid peroxidation and improve the mitochondrial activity of post-thaw stallion semen

Cryopreserved stallion spermatozoa show impaired mitochondrial activity due to oxidative stress and the osmotic damage that occurs during freezing-thawing. This compromises the capability of thawed semen to produce ATP through oxidative phosphorylation. Thus, cryopreserved spermatozoa have lower motility and lower ATP content, compromising their functionality and, finally, their fertilising ability. Some molecules, such as phenformin, can modulate the cell metabolism through the mitochondria activity, suggesting phenformin as a substance with antioxidant capacity. The objective of this study was to evaluate the effect of phenformin on cryopreserved stallion semen quality, oxidative status, and mitochondrial activity. Five horses were used for the collection of 15 ejaculates. Each ejaculate was extended in Equiplus^® (Minitube) at a concentration of 100 × 10⁶ cells/mL and supplemented before its freezing with phenformin at 1,000 (P1000), 2,000 (P2000), and 3,000 (P3000) µM and a control group without supplementation. A slow-freezing protocol was performed using a programmable freezer. Post-thawing, sperm motility and kinetics were evaluated using a computerised system; sperm vitality and morphology, through supravital staining and plasma membrane integrity through the hyposmotic test. Lipid peroxidation, mitochondria membrane potential (ΔΨM), and reactive oxygen species (ROS) production were evaluated by spectrofluorometry through the fluorescent probes BODIPY™, JC-1™ (ThermoFisher), and 2′,7′-dichlorofluorescein, respectively. These parameters were measured as relative fluorescence units (RFU). For the statistical analysis, a mixed model was used and means between treatments were compared using the Tukey test. The percentage variables were transformed through the Arcsen Öx method. All data were analysed using SAS version 9.2 software. For post-thaw semen, P3000 reduced progressive motility (12.44 ± 1.81%) compared with control (17.65 ± 2.08%) (P < 0.05). Average speed and rectilinear speed of spermatozoa were reduced for all treatments. Curvilinear speed decreased for P2000 (49.12 ± 2.19) and P3000 (48.50 ± 1.64). Linearity index of spermatozoa only decreased for P3000 (37.16 ± 1.94) (P < 0.05). Lipid peroxidation was lower for P3000 than for the control (4.77 ± 0.35 RFU vs 4.02 ± 0.29 RFU, respectively) evaluated in terms of a higher red/green ratio (P < 0.05). The ΔΨM^high for P2000 and P1000 was higher compared with the control, with values of 58.64 ± 3.46, 55.11 ± 2.27, and 51.10 ± 3.04 RFU, respectively (P < 0.05). Likewise, ROS production was higher for P3000 and P2000 with respect to the control, with values of 91.11 ± 3.2, 97.1 ± 3.75, and 81.90 ± 2.82 RFU, respectively (P < 0.05). It is concluded that phenformin can reduce lipid peroxidation and improve the mitochondrial activity of post-thaw stallion semen, despite reducing some parameters of sperm kinetics and increase reactive oxygen species.

The authors thank CES University for its financial support and the Food Science Laboratory of Universidad Nacional de Colombia, sede Medellín, for its technical support.