RESEARCHES REGARDING THE ULTRASTRUCTURAL MODIFICATIONS OF SPERMS CELLS BEFORE AND AFTER FREEZING IN DIFFERENT MEDIA
Stela Zamfirescu, Andreea Anghel
Sperm cryopreservation is a method of ex situ preservation of gametes in all domestic animals, which is extensively used in artificial insemination. Factors that depend on the success of freezing sperm and hence obtain the product live are very numerous. Seminal plasma composition, extenders used for dilution, cryoprotectant concentration, reactive oxygen species, and not finally, cooling rate, influence the quality of sperm cells frozen and thawed and viable product number obtained after insemination of cryopreserved sperm. Research for recent years confirms that in sheep and goat fertility outcome is still very variable, regardless of conditions and environments of freezing-thawing used. Research shows that, regardless of solvent used, motility and membrane integrity of sperm deteriorates during cooling and storage at low temperature. These degenerative changes may be the result of lipid peroxidation and excessive production of reactive oxygen species (ROS). Our study presents the results of changes in the integrity of Saanen goat cell sperm membranes after freezing-thawing, after with Tris base extender enriched with 10mm L-cysteine (Sigma), 5 mg/ml BSA (Sigma) and 1mm vitamin E (DL-a-Tocopherol, Merck). After thawing, the biggest motility was obtained in vitamin E and cysteine variants (between 51-55%). Goat semen samples diluted with the 3 antioxidants were processed for examination and analyzed in terms of electron microscopic cellular integrity at all levels (head, mid piece, principal piece and endpiece). Electron photography analysis (X10.000) shows that full membrane were observed in a proportion of 49% of cells diluted with Tris-vitamin E at all levels of the cell, and at a rate of 37% in sperm cells diluted with Tris-cysteine. Swollen membranes, but continuous, were considered to be normal. The cell sperm diluted with medium supplemented with BSA present protein deposits with acicular aspect and membrane which presents small and frequent interruptions. Future research aim in vivo testing of goat sperm cryopreserved with different antioxidants to determine its fecundity.
Key words: goat, sperm cell ultrastructure, cryopreservation, antioxidant additives