Evaluation of arachidonic acid supplementation in the extender on the integrity of the acrosome membrane in post-freezing goat semen (Atena Editora)

Abstract

Seminal cryopreservation causes damage to the integrity of sperm membranes, caused by oxidative stress, impairing the functional capacity of sperm cells. However, maintaining the integrity of the acrosomal membrane after cryopreservation is essential for fertilization to occur. Therefore, it is necessary to add substances that can improve the antioxidant defenses of biological systems, combating the high production of ROS. In this sense, the addition of arachidonic acid, a polyunsaturated fatty acid, was able to improve membrane fluidity, reduce the action of free radicals and maintain the integrity of sperm membranes in bovine and swine species. Thus, the objective was to evaluate the supplementation of arachidonic acid in the TRIS-yolk extender on the acrosome integrity during the cryopreservation of goat spermatozoa. Six ejaculates/animal were collected from four Anglo-Nubian goats, using artificial vagina. After immediate analysis, they were pooled, diluted in TRIS-Gema and divided into treatments: control and supplementation with 0.5μM, 5 μM and 50μM of arachidonic acid. Then the semen was cryopreserved. After thawing, the acrosomal membrane integrity analysis was performed using the FITC-PNA dye. 20μL aliquots of FITC-PNA were placed on smears of slides containing sperm and incubated for 20 minutes in a humid chamber at 4°C, in the absence of light. Then slides were rinsed twice in refrigerated PBS and dried in the dark. Immediately prior to evaluation, 5μL of UCD mounting medium was placed on the slide and covered with a coverslip. 200 spermatozoa/slide were evaluated in an epifluorescence microscope (400x), using an emission filter LP 515nm and BP 450-490nm for excitation. After the analyses, it was found that the supplementation of 50μM of arachidonic acid in the extender significantly differed from the other treatments in relation to the acrosomal integrity. A possible explanation for the lower values ​​after the addition of 50μM arachidonic acid would be its excessive concentration, considering that arachidonic acid, through the action of cyclooxygenase, is converted into prostaglandin E2, inducing the influx of Ca2+ through the membrane. sperm, leading to membrane fusion. It is concluded that the addition of 0.5μM and 5μM of arachidonic acid to the extender maintained the integrity of the acrosome membrane in post-cryopreservation goat semen, being beneficial for the process of sperm capacitation and fertilization, suggesting further research aiming at a greater accuracy of the concentrations of arachidonic acid as an antioxidant.