Effect of mitochondrial uncoupling and glycolysis stimulation on the functionality, bioenergetics and oxidative homeostasis of bovine spermatozoa subjected to cryopreservation

Abstract

Sperm cryopreservation is considered a key process for the use of reproductive biotechnologies, such as artificial insemination and in vitro fertilization. However, it is known that this technique causes a decrease in sperm quality. Among the factors involved (e.g., ice crystal formation, increased osmolarity) an aggravating mechanism directly or indirectly involved in damage during cryopreservation would be oxidative stress. Since mitochondria represent the main source of pro-oxidant agents, it is believed that this organelle plays a central role in the oxidative imbalance of spermatozoa. Therefore, mitochondrial dysfunctions during sperm cryopreservation, especially related to intracellular damage in organelles, are possibly the origin of the excessive release of reactive oxygen species (ROS). This event characterizes oxidative stress, which can be an important source of damage to the post-thawing spermatozoa. In addition, a reduction in the antioxidant capacity of spermatozoa after cryopreservation was verified, further predisposing these cells to oxidative stress.In this way, several studies have been conducted aiming to develop antioxidant therapies for sperm samples submitted to cryopreservation, aiming at the prevention of oxidative imbalance. However, for an antioxidant therapy to be efficient an ideal concentration of these antioxidants is necessary to maintain a balance of oxidation, since the ROS have a physiological role in the processes of sperm capacitation and fertilization. In addition, each antioxidant acts by preferentially eliminating a specific ROS. In addition, each sperm structure may be more susceptible to a particular ROS. Therefore, for these treatments to be effective the ideal would be to perform a therapy composed of an association between these antioxidants with precise and specific concentrations, which may make this treatment impracticable.Thus, the use of a specific mitochondrial protector during cryopreservation could improve sperm quality after cryopreservation. Such approach would be interesting since it would reduce oxidative stress by avoiding the excessive formation of ROS and not combating the already formed free radicals. In this context, a possible alternative would be a mild mitochondrial uncoupling during the process of sperm cryopreservation, so that possible deleterious effects of mitochondrial dysfunctions be softened by the lower release of reactive oxygen species. In fact, the activity of some uncouplers were identified in physiological processes of somatic cells, acting in the prevention of oxidative stress.The protective effect of mitochondrial uncoupling during sperm cryopreservation was verified in fish and primate spermatozoa. However, there is a need for more detailed studies to verify the role of this molecule in sperm metabolism and oxidative homeostasis. In this context, studies involving bovine spermatozoa would be extremely important since this species is widely used in reproductive biotechnologies, of extreme importance for the increase in the production of high quality protein and in the increase of the national livestock productivity.Therefore, the objective of this study is to promote a moderate mitochondrial uncoupling of bovine spermatozoa during the sperm cryopreservation process and to verify the effect of this therapy on sperm functionality, bioenergetics and oxidative homeostasis, aiming at an improvement both in the quality of post-thaw bovine spermatozoa and in of in vitro fertilization processes and embryonic development.