RESUMO
The aims were to identify the effects of growth differentiation factor 9 (GDF-9) on the in vitro development of ovarian preantral follicles (PAFs) of collared peccaries. Ovarian fragments were in vitro cultured for 1 or 7 days without or with inclusion of GDF-9 in the medium (0, 50, 100, or 200 ng/mL). The non-cultured (control) and cultured fragments were evaluated for PAF viability, activation, and cell proliferation. Although there were no differences in the percentage of morphologically normal follicles, the percentage of growing follicles was greater compared to the control in all treatment groups, especially those cultured with 200 ng/mL GDF-9 for 7 days (P < 0.05). The inclusion of GDF-9 in the medium did not interfere with PAF viability (P> 0.05); however, treatment with 200 ng/mL GDF-9 resulted in greater (P < 0.05) cell proliferation in PAFs cultured for 1 or 7 days (â¼2.5 nucleolar organizing regions - NORs) compared to the follicles of the control group (2.0 NORs). In addition, peccary ovarian cortexes were subjected to PCR analysis and there was detection of the mRNA GDF-9 receptor transcripts of the BMPR2 (type I receptor) and ALK-5 (type II receptor) types. In conclusion, GDF-9, especially at a 200 ng/mL inclusion in the culture medium, was actively involved in the in vitro development of collared peccary PAFs.
Assuntos
Artiodáctilos/fisiologia , Fator 9 de Diferenciação de Crescimento/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Técnicas de Cultura de TecidosRESUMO
Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.
Assuntos
Artiodáctilos/fisiologia , Gonadotropina Coriônica/farmacologia , Ovário/efeitos dos fármacos , Animais , Peso Corporal , Gonadotropina Coriônica/administração & dosagem , Feminino , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Receptores da Gonadotropina/genética , Receptores da Gonadotropina/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismoRESUMO
The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species.
Assuntos
Cervos/embriologia , Cervos/genética , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Genes Mitocondriais , Animais , Blastocisto/metabolismo , Bovinos , Células Cultivadas , Clonagem de Organismos/veterinária , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Fertilização in vitro , Cabras , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mórula/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Técnicas de Transferência Nuclear/veterinária , Oócitos/metabolismo , Regulação para CimaRESUMO
A resposta ovariana de catetos tratados com eCG/hCG foi analisada pelo desenvolvimento folicular e pela expressão gênica. O tratamento DS (dose utilizada em suínos) e DA (dose ajustada alometricamente) foram comparados. Todas as fêmeas apresentaram corpos hemorrágicos (CHs) e as taxas de ovulação foram semelhantes entre DS (4,00±1,17) e DA (2,50±0,43). O número de folículos por animal e as quantidades de folículos P (≤3mm), M (3-5mm) e G (≥5mm) foram similares entre os grupos. Os folículos ovarianos do mesmo tamanho expressaram FSHR e LHCGR em níveis similares entre os grupos. FSHR foi mais expresso na fase inicial, e LHCGR, na fase tardia da foliculogênese ovariana de cateto. A expressão de LHCGR nos CHs foi igual entre os tratamentos e entre os ovários direito e esquerdo.
The ovarian response of catches treated with eCG/hCG was analyzed by follicular development and gene expression. The treatment DS (dose used in pigs) and AD (dose adjusted alometrically) were compared. All females showed hemorrhagic bodies (CHs) and ovulation rates were similar between DS (4.00±1.17) and AD (2.50±0.43). The number of follicles per animal and the numbers of follicles P (≤3mm), M (3-5mm) and G (≥5mm) were similar between the groups. Ovarian follicles of the same size expressed FSHR and LHCGR at similar levels between groups. FSHR was more expressed in the early phase, and LHCGR, in the late phase of ovarian catheter folliculogenesis. LHCGR expression in CHs was equal between treatments and between right and left ovary.
Assuntos
Feminino , Animais , Artiodáctilos/fisiologia , Folículo Ovariano/fisiologia , Gonadotropina Coriônica/uso terapêutico , Hormônios , Sincronização do Estro/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
A resposta ovariana de catetos tratados com eCG/hCG foi analisada pelo desenvolvimento folicular e pela expressão gênica. O tratamento DS (dose utilizada em suínos) e DA (dose ajustada alometricamente) foram comparados. Todas as fêmeas apresentaram corpos hemorrágicos (CHs) e as taxas de ovulação foram semelhantes entre DS (4,00±1,17) e DA (2,50±0,43). O número de folículos por animal e as quantidades de folículos P (≤3mm), M (3-5mm) e G (≥5mm) foram similares entre os grupos. Os folículos ovarianos do mesmo tamanho expressaram FSHR e LHCGR em níveis similares entre os grupos. FSHR foi mais expresso na fase inicial, e LHCGR, na fase tardia da foliculogênese ovariana de cateto. A expressão de LHCGR nos CHs foi igual entre os tratamentos e entre os ovários direito e esquerdo.(AU)
The ovarian response of catches treated with eCG/hCG was analyzed by follicular development and gene expression. The treatment DS (dose used in pigs) and AD (dose adjusted alometrically) were compared. All females showed hemorrhagic bodies (CHs) and ovulation rates were similar between DS (4.00±1.17) and AD (2.50±0.43). The number of follicles per animal and the numbers of follicles P (≤3mm), M (3-5mm) and G (≥5mm) were similar between the groups. Ovarian follicles of the same size expressed FSHR and LHCGR at similar levels between groups. FSHR was more expressed in the early phase, and LHCGR, in the late phase of ovarian catheter folliculogenesis. LHCGR expression in CHs was equal between treatments and between right and left ovary.(AU)
Assuntos
Animais , Feminino , Artiodáctilos/fisiologia , Hormônios , Sincronização do Estro/métodos , Folículo Ovariano/fisiologia , Gonadotropina Coriônica/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
The brown brocket deer Mazama gouazoubira is 1 of the 10 recognized brocket deer of the Neotropical region. Recently, this species has suffered a population decline due to current threats, mainly poaching and habitat loss. Several studies have shown that some endangered species can benefit from interspecies somatic cell nuclear transfer technology through the use of their somatic cells, such as the fibroblasts. Thus, the aim of this study was to verify the viability and the effect of cryopreservation on fibroblasts after several passages. For this purpose, fibroblast cells were cultured until passages 4, 7, and 10 (cultured control groups) and cryopreserved in cryotubes (frozen/warmed groups). The cellular viability, functionality, and percentage of cells undergoing necrosis and apoptosis were evaluated. The survival rates were always higher than 80% irrespective of the tested group, except for passage 10 in the frozen/warmed group. Population doubling time of cultured cells from passage 10 was significantly higher than that of passages 4 and 7, exhibiting low metabolic activity and a higher percentage of cells in initial apoptosis. In conclusion, the M. gouazoubira fibroblast-derived cell line provides an essential resource for further studies regarding reproductive biotechniques and is likely to be useful as an ex situ conservation strategy.