Potencial do extrato da folha de Morus nigra L. componente de diluidor para criopreservação de espermatozoides

Abstract

The objective of this work was to evaluate different concentrations of Morus nigra L. leaf extract, in fruiting stages, as an additive to a dilute for sperm cryopreservation. The leaves collected from mulberry trees presented on Campus I (7° 05' S 34° 50' W) of the Federal University of Paraíba. The amount of 297 g of leaves were dried in an oven at 55 0C for 48 h; then, they were macerated and immersed in one liter of 95% ethanol for 72 h, protected from light, so that there was no change in the composition of photosensitive metabolites. After this process, filtration was carried out and the filtrate was dried in a vacuum rotary evaporator, with a water bath temperature of 55 0C, to obtain the crude extract. In qualitative phytochemical analysis, the presence of steroid and flavonoid compounds is obtained, and the absence of saponin, terpenoid, tannin and glycoside compounds. No Fourier Transform Infrared Spectroscopy (FTIR) test confirmed the presence of steroids and flavonoids in the extract. In the Quantification of Reducing Sugars (DNS Method), we obtained the presence of 34% of reducing sugars in the extract. In the Determination of Antioxidant Potential by the DPPH Method, the result was IC50=0.5848 mg/mL, which is favorable for a diluter. Next, the crude extract was diluted in four possible solvents: TRIS, physiological solution (NaCl 0.09%) and distilled water. The solvent chosen was TRIS to provide the best dilution of the extract. Then, the crude extract was diluted 4 mg/mL in TRIS, forming the mother solution, to later form the experimental groups: 1) Control Group (CG), composed of the Tris Gema dilutor (3.605 g of tris-hydroxymethylaminomethane; 2,024 g of citric acid 1,488 g of fructose 100 mL of double-distilled water; 3) Test Group 2 (GT2), Tris Yolk and 5% blackberry extract; 4) Test Group 3 (GT3), Tris Yolk and 10% blackberry extract. The extenders were subjected to the Hydrogenion Potential (pH) test with the following results: The mother solution had pH 6.0 and the others pH 7.0. Considering the above parameters, the extract has potential for sperm cryopreservation due to the presence of flavonoids, which promote the reduction of oxidative stress, a satisfactory amount of sugars that can protect and provide energy for the cell, and also has considerable value as an antioxidant. As a final step, tests were carried out on equine sperm. The spermatozoa were subjected to refrigeration and freezing/thawing in the aforementioned experimental groups and subsequent analyzes (sperm motility and vigor, integrity and functionality of plasma membranes and sperm mitochondrial activity. The experiment was carried out with ejaculate from eight individuals and the evaluations carried out in duplicate and subsequently the averages were generated. Results were presented within acceptable post-thawing standards for analyzes regarding the integrity and functionality of plasma membranes.