Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA)

The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.

Obtenção e conservação de espermatozoides de cutia (Dasyprocta leporina Linneaus, 1753) do semiárido brasileiro

The general objective of this thesis was to stablish protocols to obtain and conserve agouti (Dasyprocta leporina) sperm bred in captivity in the Brazilian semi-arid, aiming its sustainable production. The thesis was divided in three experiments. In the first one, we studied the influence of the interaction between two probes (quadratics and sine waves) and two stimulation protocols (continuous and in series) on the agouti sperm collection by electroejaculation efficiency. The most efficient interaction on this obtainment was the one with probes with rings associated with stimuli in series (4/7; 57%, P<0.05). In the second experiment we compared the cryoprotectant effects of different substances (glycerol, ethyleneglycol, dimethylsulfoxide, dimethylformamide) on epididymis sperm cryopreservation. The highest values on motility (39.5±4.6%), vigor (2.9±0.2) and membrane integrity (30.6±4.5%) were observed on the samples cryopreserved using glycerol when compared to those with ethyleneglycol and dimethylformamide, but there was no difference (P>0.05) when compared to the samples cryopreserved with dimethylsulfoxide. At last, we studied the effects of the methods to obtain sperm (electroejaculation vs epididymal collection) on post thawing sperm quality. The samples obtained by epididymal retrograde flushing showed values for motility of 25.0±10.9% and vigor 2.4±0.8, and those obtained by electroejaculation had 31.2±14.2% of motility and vigor of 2.2±0.7, however, without statistical difference (P>0.05), which shows the possibility to successfully use the epididymal sperm cryopreservation protocol on agouti ejaculated sperm. In conclusion, significant advances on obtainment and processing of agouti sperm were made, allowing the establishment of germplasm banks from sperm samples obtained from the epididymis or by electroejaculation.