Separação de espermatozoides de alta qualidade por um método nanotecnológico de ativação magnética em animais selvagens e domésticos

Abstract

l of any species. Male fertility requires a lot of attention because sperm has to be able to fertilize oocyte and ensure the embryo’s development. The ejaculates’ quality is highly variable in both domestic and wild species, since it is composed of several spermatic subpopulations, bringing a variety of structural and functional modifications, and some of those abnormalities translate cellular apoptosis and are directly correlated with fertility failures in vivo and in vitro. This study aimed at improving selection of high-quality sperm through sperm separation by magnetic activation in fresh semen of cattle, deer, horses, and domestic cats by evaluating the cells’ quality after the sperm selection process. For this, semen was collected from animals with a high number of spermatic abnormalities (> 30%), being: 1) Nellore bulls (n=21), using the artificial vagina restraint; 2) six Mazama deer, using the electroejaculation technique and after chemical containment; 3) ten horses Mangalarga and American Quarter horse, using the artificial vagina technique, and 4) ten domestic cats without defined breed, using pharmacological ejaculation technique (dexmedetomidine) and urethral catheterization. Semen was analyzed in five statuses: 1) fresh semen; 2) post separation by density gradient centrifugation (DGC); 3) post separation by Magnetic-activated cell sorting (MACS) or Magnetic-activated sperm sorting (MASS) (non-apoptotic part); 4) post separation by MACS or MASS (apoptotic part) and 5) post separation by MACS followed by DGC. Motility was measured using either Computer Assisted Sperm Analysis (CASA) or optical microscopy, concentration by Neubauer chamber, sperm morphology by phase contrast, and supravital test was completed with eosin/nigrosin staining. In the sperm separation by DGC, 20 x 106 cells were used in a gradient of two layers of 400 μl each of Percoll at 90% and 45%; after centrifugation at 900 G for 5 minutes the pellet was diluted in HEPES. Sperm separation by MACS or MASS used 10 x 106 cells diluted in 1.5 ml of HEPES, followed by centrifugation at 300 G for 10 min. The pellet was resuspended in 150 μl HEPES and 20 μl of nanoparticles bound to anexin V were added; after incubation for 15 minutes at room temperature, filtration was carried out in the MiniMacs™ or MASS. The non-apoptotic fraction was collected in a 2 mL tube and the apoptotic fraction connected to the microspheres was collected by removing the magnet column and adding 300 μl of HEPES. The spermatic separation by MACS followed by DGC was a combination of the two methodologies. The evaluation of cell membrane quality and mitochondrial potential after selection was made using fluorescent probes with FITC/IP and JC-1, respectively, in flow cytometry. In cattle, the combination of the two sperm processing methods (DGC and MACS) was significantly more effective in the production of high-quality sperm samples than using them separately (70% more reduction in abnormalities). The subpopulation of non-apoptotic spermatozoa had morphologically superior sperm quality compared to the apoptotic fraction, as well as higher number of cells with intact membrane and better mitochondrial potential. In cervids, horses, and domestic cats, we found that DGC and MASS techniques were efficient in reducing sperm abnormalities, mainly removing tail and head defects, and the subpopulation of non-apoptotic sperm had morphologically superior sperm quality compared to the apoptotic fraction; the same was observed for the number of cells with intact membrane. In those three species, the motility of the post MASS samples was extremely low, perhaps due to the greater sensitivity of the spermatic cell in the species, the number of procedures to which the cell was submitted, and the large time spent in the analyses. Those findings suggest that that nanotechnological method is efficient in the production of high-quality semen samples for assisted reproduction procedures in both12 domestic and wild animals, but further studies are still needed to better understand the particular characteristics of the spermatic cells of each species, in order to improve the results of the technique.