https://repositorio.ufu.br/handle/123456789/32762 Wed, 22 Apr 2026 22:12:19 GMT 2026-04-22T22:12:19Z https://repositorio.ufu.br/handle/123456789/36304 Title: 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. Thu, 06 Oct 2022 00:00:00 GMT https://repositorio.ufu.br/handle/123456789/36304 2022-10-06T00:00:00Z https://repositorio.ufu.br/handle/123456789/31880 Title: Risco de exposição a carrapatos Amblyomma sculptum e Amblyomma dubitatum e à bactéria Rickettsia bellii em área urbana indene para Febre Maculosa Brasileira Abstract: Diseases caused/transmitted by ticks presuppose physical contact and trophic exchange between the host, vector, and pathogen. In this context, we can establish three levels/sequential stages of relationship among these beings and that are essential for the epidemiology of diseases associated with ticks: 1- hazardous area, 2- exposure and 3- parasitic interaction. The hazardous area refers to the occurrence, within a certain area, of the tick and in this, when applicable, of the pathogenic microorganism. The second level, exposure, refers to physical contact with the tick and that will occur in a hazardous area. The third level, parasitic interaction, refers, after physical contact, to the host’s attractiveness to the tick and its parasitic capacity towards this specific host. Facing the unlikelihood of controlling/eliminating ticks from a hazardous area and/or impossibility to avoid the parasitic interaction for disease prevention, it is necessary to lessen the risk of host exposure to ticks. To this end, it is essential to know the bioecology, especially host-seeking behavior of tick species that are associated with host disease in a hazardous area. Two species of ticks in Brazil, Amblyomma dubitatum and Amblyomma sculptum, occur in environmental sympatry when maintained by capybaras. The number of these parasites is particularly high in areas under anthropic impact in southeastern Brazil and, in some places, they are associated with Brazilian Spotted Fever (BSF), a disease with extremely high lethality caused by the bacterium Rickettsia rickettsii. Although the tick nymph A. sculptum is considered the main vector of the pathogen, the proportions between the two species and the behavior of each stage of the ticks influence the risk of human exposure to ticks and the bacteria. In addition, Rickettsia bellii, a non-pathogenic bacterium and that frequently infects A. dubitatum interferes with the transmission of the lethal Rickettsia by mechanisms that are still unclear. Therefore, this work aimed to contribute with information for protocols that reduce the risk of human exposure to ticks at areas hazardous for the transmission of BSF or other diseases associated with these two tick species. Among others, the density, behavior, and proportional relationship between these two tick species in the urban area of an BSF-free municipality and related to the risk of human exposure to ticks were evaluated. For this purpose, ticks were collected in ten locations with capybaras and bordering water bodies in the three seasons of the year characterized by peaks of activity of the parasitic stages of the A. sculptum tick. The collections of ticks in each location were made exhaustively in a standardized collection unit of 400 m2 and the coordinated application of four techniques. These techniques also allowed the evaluation of the host-seeking behavior of ticks according to the species and stage. Finally, the tick infection rate with R. bellii was correlated to the relative proportion of A. sculptum/A. dubitatum at each location. During the study period, 52,953 ticks were collected exclusively from two species, A. sculptum and A. dubitatum. Of these ticks, 83.4% were A. sculptum (1,523 adults, 10,545 nymphs and 32,104 larvae) and 16.6% A. dubitatum (464 adults, 2,153 nymphs and 6,164 larvae). Although the largest number of ticks collected was of the A. sculptum species, the prevalence of species varied among the ten study sites. During the study period, an average annual density of 4.4 ticks/m2 was found, varying between 0.03 and 31.8 ticks/m2 according to the location, season, species, and stage of ticks. As for the host-seeking behavior of ticks, ambush predominated in larvae and hunter in nymphs and mainly in adults, for both tick species. However, it was noted that the ambush host-seeking occurred in a significantly greater proportion of larvae and nymphs of A. sculptum in relation to A. dubitatum ticks and the latter remained more restricted to the ground. Adults and nymphs of A. sculptum were significantly more attracted to humans in relation to the same stages of A. dubitatum. Conversely, larvae of A. dubitatum were significantly more attracted to humans than those of A. sculptum. Taken together, these observations indicate that a human being in movement along A. sculptum and A. dubitatum hazardous area will have an almost exclusive exposure to A. sculptum larvae and/or nymphs, particularly in autumn and winter. When in a stationary position, humans are more attractive to adults and especially nymphs of A. sculptum, but also to larvae of A. dubitatum. This unexpected observation needs additional studies within the epidemiology of tick-borne diseases. Finally, it was observed that the higher the proportion of A. sculptum in the tick community, the lower the infection rate of A. dubitatum by R. bellii and that indicates a refractory effect of A. sculptum on the diffusion of the bacteria. Based on these observations, preventive measures to avoid exposure to the two species of ticks in hazardous areas are proposed. Notes: Tese para promoção à classe de Professor Titular Mon, 10 May 2021 00:00:00 GMT https://repositorio.ufu.br/handle/123456789/31880 2021-05-10T00:00:00Z