Estresse controlado induzido por pressão negativa na criopreservação de sêmen equino

Abstract

Brazil is a vast and highly productive country, with an expressive and growing number of production animals. Horse agribusiness has followed this growth, driven largely by the use of animals in sport, leisure and work activities. This growth is due, in most cases, to improvements in production and reproduction techniques. In this sense, reproduction biotechnologies are important tools that help breeders in the genetic improvement of animals. Artificial insemination has been gaining more and more space in the segment as an alternative to facilitate the exchange of genetic material (semen) between properties, optimizing and preserving the reproductive life of stallions. It also allows animals with acquired reproductive problems to be multiplied. One of the emerging techniques in equine reproduction is semen cryopreservation. Although cryopreservation protocols have advanced a lot in recent years and have numerous advantages (conservation of genetic material, possibility of storage, ease of transport) they still need to be revised and improved due to the great variability in results. Thus, several studies have been carried out in an attempt to improve the quality of cryopreserved semen by improving freezing techniques. One of the causes cited in the literature related to decreased fertility of equine semen is the damage caused by the accumulation of reactive oxygen species (ROS). In this sense, some authors have proposed that the submission of gametes to some type of controlled stress can improve the cryotolerance of cells. Thus, the objective of this work was to verify whether the controlled stress provided by different intensities of negative pressure (200, 500 and 800 mbar) in equine semen improves post-thawing seminal reproductive parameters. Thirteen ejaculates from 4 stallions were used. Each ejaculate was divided equally into 4 groups (control, P200, P500 and P800). The treatments were submitted to different pressures for 15 minutes. After that, the freezing protocol was performed for the 4 groups. The samples were analyzed for their sperm kinetics using CASA (Computer Assisted Sperm Analysis). Oxidative stress was analyzed by quantifying ROS and total antioxidant potential. Membrane functionality was performed using the hypoosmotic test. Results were expressed as mean and standard deviation, being considered significant when p<0.05. In the hyposmotic test, the control and the P500 group showed similar results (17.6±4.1 and 18.0±4.1 respectively), showing a significant difference from the P200 group (11.5±1.8) and greater difference from the P800 group (7.6±2.0). Regarding sperm kinetics, there was no statistically significant difference between the groups, remaining similar to the control in the parameters total motility , circular motility , immobile , slow motility , local motility , progressive motility . There were no statistical differences between the groups regarding the production of reactive species, nor regarding the total antioxidant potential. We can observe that there was an influence of the use of negative pressure as a form of controlled stress, against the membrane functionality of equine sperm cells, however, it is necessary to carry out more studies to obtain a better understanding on the subject.