RESUMEN
Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.
RESUMEN
Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.
