“Nuevas estrategias heteroespecíficas en la reproducción 2 asistida...
The development of assisted reproductive technologies (ART) in species of the Felidae family has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Sperm cryopreservation research and IVF have been studied in several Felidae species, but the lack of viab...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7527 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7527.dir/7527.PDF |
| Aporte de: |
| Sumario: | The development of assisted reproductive technologies (ART) in species of the Felidae family has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Sperm cryopreservation research and IVF have been studied in several Felidae species, but the lack of viable homologous oocytes makes it difficult to assess the fertile capacity of sperm after cryopreservation. We propose in chapter 2 to evaluate the ability of cryopreserved sperm from Puma concolor (Pc), Leopardus geoffroyi (Lg) and Panthera onca (Po) to induce fertilization of domestic cat oocytes and subsequent embryonic development prior to implantation. In addition, embryo quality was analyzed based on the number of total cells, the expression of pluripotent (Oct4) and early differentiation (Cdx2) genes, DNA fragmentation and the distribution of the OCT4 protein. After ovariectomy, domestic cat (Felis catus, Fc) oocyte cumulus complexes were matured in vitro in 5% CO2 in humidified air at 38.5°C for 22 h and cryopreserved Pc, Lg, and Po spermatozoa were used for heterologous IVF. The straws were thawed by exposing them to air for 10 s and then immersing them in a 37°C water bath for 30 s. The content of the straws was poured into a sterile 1.5 ml microtube preheated to 37°C. The sperm suspension was diluted (1:3 v/v) by slow enhancement (dropwise) of modified Tyrode's solution. For IVF, in vitro matured oocytes were co-incubated with 2×105 366 motile spermatozoa/mL under 5% CO2 at 38.5°C for 18-20 h. After gamete incubation, putative zygotes were cultured in vitro in 50 µl drops of modified Tyrode's medium in 5% CO2, 5% O2, 90% N2. Cleavage was prolonged 48 h after fertilization and the blastocyst stage was evaluated on day 7. The blastocyst rate was found to be higher using Lg sperm compared to the others. No differences were found in the expression of the pluripotent gene; however, the hPc and hLg hybrids show a higher expression of Cdx2 compared to the Fc embryos. No differences were found between the hPo and Fc hybrids in relation to the expression of the Cdx2 gene. Additionally, hPc blastocysts show less apoptosis and positive cells with OCT4 protein than domestic cat and hLg blastocysts. Hybridization in felines is known, however, no report on the characterization of hybrid preimplantation embryos in vitro has yet been published, and it could be a powerful tool to generate knowledge about sperm inheritance in early embryonic development. Heterospecific embryo transfer of an endangered species has been carried out using recipients of related domestic females. The aggregation of an embryo of an endangered species with a tetraploid embryo of the species to be transferred could improve pregnancy development to term. The main objective of Chapter 3 was to analyze embryo aggregation in a domestic cat model using hybrid embryos. For this purpose, we compared the in vitro development of synchronous (Sync) or asynchronous (Async) and asynchronous with tetraploid (Async4n) aggregation of domestic cat IVF embryos. In addition, the quality of the aggregated blastocyst was analyzed by evaluating the total number of cells, the location of cells by mitotracker staining, the expression of the Oct4, Nanog, Sox2, Cdx2 genes, the number of OCT4+ nuclei, and the presence of DNA fragmentation. In addition, developmental rates of Async4n aggregation of domestic cats with hybrid embryos of Leopardus geoffroyi (hLg) were evaluated. Asynchronous aggregation increased the number of blastocyst cells and the number of OCT4+ nuclei compared to nonaggregated diploid (2n) and tetraploid (4n) embryos. Furthermore, blastocysts produced by Async4n aggregation showed reduced rates of DNA fragmentation. No differences were found in the expression of pluripotent genes, except for the expression of Cdx2, which was higher in 4n and aggregated embryos compared to the control group. Interestingly, hybrid embryos derived from Async4n aggregation with domestic cat embryos have similar rates of development to blastocyst compared with the control. Taken together, the results support the use of two-cell fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos |
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