RESUMO

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

Assuntos

RESUMO

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1ß on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1ß (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1ß, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1ß (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Assuntos

RESUMO

The residual carbon content of a variety of bovine-derived samples and forage was determined by inductively coupled plasma optical emission spectrometry with radial view configuration (ICP-OES) after microwave-assisted digestion under high pressure in a closed vessel. The original carbon concentration in the samples was determined by elemental analysis. The highest amount of original carbon content (64%) was found in viscera. After digestion, up to 75% of it was destroyed. Viscera presented the highest ether extract and blood exhibited a high crude protein content of up to 99%. The efficiency in destroying the organic matter in biological materials seemed to be related to their fat content and showed no significant difficulty for protein-rich samples. The correlation coefficient between the fat content of the samples and the residual carbon after acid decomposition was 0.9173 indicating a fair fit. However, no correlation was observed between % RC and the protein content.

Assuntos

RESUMO

A polyvalent flow injection system was proposed for spectrophotometric determination of calcium, magnesium and phosphorus in acid plant digests. O-cresolphtalein complexone (CPC) was the chromogenic reagent for calcium and magnesium determination, and ammonium molybdate/vanadate for phosphorus determination. Ammonium-ammonia was elected as buffer system, and EGTA or 8-hydroxyquinoline as the masking agents for calcium or magnesium. Linearity of the calibration equations was observed up to 40.0 mg Ca L-1, 10.0 mg Mg L-1 and 16.0 mg P L-1. Slight variations in the coefficients of the calibration equations (usually 3 %) were found after 4-h working periods. Precise results (r.s.d. 0.02), in agreement with alternative method (flame atomic absorption spectrometry) were obtained. When the system was applied to large scale analysis, good repeatability was observed, and a sample throughput, of about 107 h-1 for magnesium and 75 h-1 for calcium and phosphorus was achieved, emphasizing the simplicity, robustness and versatility inherent to flow analysis.

Um sistema polivalente de análises químicas por injeção em fluxo foi proposto para a determinação de cálcio, magnésio e fósforo em digeridos ácidos de plantas. O-cresolphtaleina complexona (CPC) foi selecionado como reagente colorimétrico para a determinação de cálcio e magnésio e molibdato de amônio/vanadato de amônio para a determinação de fósforo. O sistema empregou tampão amônio-amônia (pH 10,5) e EGTA ou 8-hidroxiquinolina como agentes mascarantes para cálcio ou magnésio, respectivamente. Equações de calibração lineares foram obtidas, sendo que para facilitar a utilização do sistema proposto, foi utilizada curva analítica mista (até 40,0 mg Ca L-1, 10,0 mg Mg L-1 e 16,0 mg P L-1), não sendo observadas interferências entre os elementos. Os limites de detecção foram: 0,10; 0,05 e 0,004 FONT FACE="Symbol">m /font>g L-1 respectivamente para Ca, Mg e P. Pequenas modificações no coeficiente das equações de calibração (normalmente 3%) foram observadas após períodos de 4 h de trabalho. Os resultados apresentaram-se precisos (d.p.r. 0,02), e em concordância com método alternativo (espectrometria de absorção atômica). Quando o sistema foi aplicado a análises em rotina, boa repetibilidade foi observada, sendo obtidas velocidade de amostragem de 107 amostras h-1 para magnésio e 75 amostras h-1 para cálcio e fósforo, enfatizando a simplicidade, robustez e versatilidade, inerentes aos sistemas de análises por injeção em fluxo.

RESUMO

A polyvalent flow injection system was proposed for spectrophotometric determination of calcium, magnesium and phosphorus in acid plant digests. O-cresolphtalein complexone (CPC) was the chromogenic reagent for calcium and magnesium determination, and ammonium molybdate/vanadate for phosphorus determination. Ammonium-ammonia was elected as buffer system, and EGTA or 8-hydroxyquinoline as the masking agents for calcium or magnesium. Linearity of the calibration equations was observed up to 40.0 mg Ca L-1, 10.0 mg Mg L-1 and 16.0 mg P L-1. Slight variations in the coefficients of the calibration equations (usually 3 %) were found after 4-h working periods. Precise results (r.s.d. 0.02), in agreement with alternative method (flame atomic absorption spectrometry) were obtained. When the system was applied to large scale analysis, good repeatability was observed, and a sample throughput, of about 107 h-1 for magnesium and 75 h-1 for calcium and phosphorus was achieved, emphasizing the simplicity, robustness and versatility inherent to flow analysis.

Um sistema polivalente de análises químicas por injeção em fluxo foi proposto para a determinação de cálcio, magnésio e fósforo em digeridos ácidos de plantas. O-cresolphtaleina complexona (CPC) foi selecionado como reagente colorimétrico para a determinação de cálcio e magnésio e molibdato de amônio/vanadato de amônio para a determinação de fósforo. O sistema empregou tampão amônio-amônia (pH 10,5) e EGTA ou 8-hidroxiquinolina como agentes mascarantes para cálcio ou magnésio, respectivamente. Equações de calibração lineares foram obtidas, sendo que para facilitar a utilização do sistema proposto, foi utilizada curva analítica mista (até 40,0 mg Ca L-1, 10,0 mg Mg L-1 e 16,0 mg P L-1), não sendo observadas interferências entre os elementos. Os limites de detecção foram: 0,10; 0,05 e 0,004 FONT FACE="Symbol">m /font>g L-1 respectivamente para Ca, Mg e P. Pequenas modificações no coeficiente das equações de calibração (normalmente 3%) foram observadas após períodos de 4 h de trabalho. Os resultados apresentaram-se precisos (d.p.r. 0,02), e em concordância com método alternativo (espectrometria de absorção atômica). Quando o sistema foi aplicado a análises em rotina, boa repetibilidade foi observada, sendo obtidas velocidade de amostragem de 107 amostras h-1 para magnésio e 75 amostras h-1 para cálcio e fósforo, enfatizando a simplicidade, robustez e versatilidade, inerentes aos sistemas de análises por injeção em fluxo.