Oxidative assessment of thawed donkey semen with seminal plasma, permeable and non-permeable cryoprotectants

Abstract

During cryopreservation, donkey semen is exposed to oxidative and osmotic damage that alters the plasma membrane and causes alteration of sperm metabolism. In equids, the elimination of seminal plasma (SP) is a necessary process for the conservation of sperm in artificial conditions such as freezing, however, it is known that SP has an important role in physiological processes for sperm functions such as protection, transport, antioxidant defense, regulation of the inflammatory response in the uterus and interaction with the oocyte. The objective of this study was to evaluate total antioxidant capacity (TAC), reactive oxygen species (ROS) production and mitochondrial membrane potential of sperm, with different alternatives for donkey semen cryopreservation with permeable, non-permeable cryoprotectants and SP. Samples of semen were collected from ten Colombian Creole donkeys (Equus asinus), three ejaculates were collected from each donkey, for a total of 30 ejaculates. Eight treatments (T) were used with different combinations of 5% of dimethylformamide (DMF), 200 mM of sucrose (SUC), 1% of bovine serum albumin (BSA) and 10% of homologous seminal plasma (SP), as following: T1: DMF, T2: DMF/SP, T3: SAC/BSA, T4: SAC/BSA/SP, T5: DMF/SAC/BSA, T6: DMF/SAC/BSA/SP, T7: DMF/BSA, T8: DMF/BSA/SP. Besides the treatment, sperm was extended in EquiPlus® and 5% of clarified egg yolk, until a sperm concentration of 100 × 106 spermatozoa/mL. Posthawing, TAC was evaluated by the cationic radicalmethod (ABTS) and by the method of oxygen trapping capacity (ORAC) methodologies, total reactive oxygen species (ROS) production, superoxide anion (O2-) and hydroxyl radical (OH-) content were evaluated by spectrofluorimetry; sperm viability and mitochondrial membrane potential (ΔΨM) were measured by flow cytometry. For statistical analysis, a general linear model was fitted for each dependent variable. Normal distribution of variables was validated with the Kolmogorov–Smirnov test. Means were compared by the Tukey's test. Treatments T3 and T4 presented higher TAC (p<0.05) (ABTS and ORAC) compared to T1. For ΔψM, the lowest mean value was 9.1% for T4 (p<0.05), corresponding to one of the treatments without permeable cryoprotectant and SP. For sperm viability, the lowest values were found in both treatments with non-permeable cryoprotectant. Moreover, the treatments that were supplemented with SP showed higher production of ROS (p<0.05). In conclusion, supplementation of donkey semen with seminal plasma for cryopreservation does not alter the total antioxidant capacity; additionally, freezing semen with non-permeable cryoprotectants generates low sperm viability, low mitochondrial membrane potential and a high amount of reactive oxygen species and the composition of the freezing extender for donkey semen with seminal plasma influences the generation of total reactive oxygen species.