Sperm cryopreservation is considered a key process when using reproductive biotechniques such as artificial insemination, embryo transfer and in vitro fertilization. However, significant reduction on sperm quality is often observed when using such biotechnique. Among the factors related to impaired sperm quality in thawed semen samples (e.g., intracellular ice crystals, increased extracellular osmolarity), a mechanism directly or indirectly involved is the oxidative stress. Because mitochondria represent the main source of pro-oxidative agents, this organelle may play a central role on cases of disrupted oxidative homeostasis in sperm. Therefore, mitochondrial disfunctions during sperm cryopreservation, especially those related to intracellular damages to organelles, are possibly the origin of the excessive reactive oxygen species (ROS) formation. Such picture characterizes a situation of oxidative stress, which may be harmful to different sperm structures during cryopreservation and thawing. Therefore, the use of a specific mitochondrial mediator during cryopreservation may improve post-thaw sperm quality. Such approach would be interesting due to a decrease on oxidative stress by avoiding the production of excessive ROS and not by scavenging already formed free radicals, strategy with inconsistent results. In this context, a possible alternative would be a controlled mitochondrial uncoupling during sperm cryopreservation. Also, to avoid a reduction on ATP production, vital to sperm kinetics, an alternative energetic metabolic pathway (i.e. glycolysis) would be stimulated by the association with glucose. Therefore, the objective of the present study is to evaluate the levels of ATP, mitochondrial and oxidative status and sperm kinetics on ram sperm submitted to both mitochondrial uncoupling and glycolysis stimulation during the process of cryopreservation. Towards this aim, ejaculates of 7 healthy and sexually mature rams will be collected using artificial vagina. Samples will be diluted to a final concentration of 100 x 106 sperm/mL with Botubov® semen extender (Botupharma, Brasil) supplemented with increasing concentrations of oxidative phosphorylation uncoupler CCCP (0, 1, 10 e 20µM) with or without glucose (0 e 5mM), in a 4x2 factorial arrangement. Samples will then be cryopreserved. After thawing, samples will be evaluated for ATP levels (luminescence; ATP Determination kit A22066), mitochondrial status (3'3 diaminobenzidine / DAB and JC1), oxidative status by Cell-Rox probe and TBARS assay, and sperm kinetics by Computer Assisted Sperm Analysis (CASA; Hamilton-Thorne®, Ivos 12.3, USA). Results will be submitted to statistical analysis according to the 4x2 factorial design, using parametric or non-parametric tests according to residue normality and variance homogeneity. A probability value of p<0.05 will be considered significant. We expect, with the use of a mitochondrial uncoupler associated to the stimulation of the glycolytic pathway during sperm cryopreservation, the maintenance on ATP levels, the improvement on both mitochondrial and oxidative status and enhanced sperm kinetics may be observed in thawed samples.
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