Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro.

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1ß on primordial follicle activation and survival in vitro.

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.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles.

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Muscle delta13C change in Nile tilapia (Oreochromis niloticus) fingerlings fed on C3-or C4-cycle plants grain-based diets.

The effects of grain-based diets from C3 or C4-cycle plants on muscle delta(13)C change process in Nile tilapia (Oreochromis niloticus) fingerlings were investigated. Two groups of sex reversal males Nile tilapia fingerlings were fed with isoproteic (32.0% DP) and isocaloric (3200 kcal DE/kg) diets, differing from each other by their delta(13)C. Muscle samples were collected and the carbon isotopic composition was measured. For C4 diet, the formula for the muscle delta(13)C change related to the intake time of a new diet was delta(13)C=-14.88-9.21e(-0.0209t) and the half-life (T) of the muscle carbon was 33.2 days. For C3 diet, the formula was delta(13)C=-25.43+8.59e(-0.0533t) with T=13 days. The C3 diet was considered more appropriate based on its palatability and consequent larger food intake than the C4 diet, resulting in an increased muscle delta(13)C change rate. However, for future studies, would be necessary to mix both the C3 and C4 feedstuffs to formulate diets nutritionally appropriated, with contrasting stable isotopes signatures. Tissue delta(13)C change rate is therefore indicated as a promising tool to better understand the biotic and abiotic factors that influence nutrients utilization from the diet and animal growth.

Muscle delta13C change in Nile tilapia (Oreochromis niloticus): effects of growth and carbon turnover.

The contribution of growth and turnover to the muscle delta(13)C change process was investigated using mathematical models which associate delta(13)C change to time of intake of a new diet or increase in body mass. Two groups of Nile tilapia (Oreochromis niloticus) were fed on diets based on C3 (delta(13)C=-25.64+/-0.06 per thousand) or C4 (delta(13)C=-16.01+/-0.06 per thousand) photosynthetic cycle plants to standardize the muscle delta(13)C. After establishing the carbon isotopic equilibrium, fish (mean mass 24.12+/-6.79 g) then received the other treatment diet until a new carbon isotopic equilibrium could be established, characterizing T1 (C3-C4) and T2 (C4-C3) treatments. No significant differences were observed in fish productive performance. Good fits were obtained for the models that associated the delta(13)C change to time, resulting in carbon half-life values of 23.33 days for T1 and 25.96 days for T2. Based on values found for the muscle delta(13)C change rate from growth (0.0263 day(-1) and 0.0254 day(-1)) and turnover (0.0034 day(-1) and 0.0013 day(-1)), our results indicate that most of the delta(13)C change could be attributed to growth. The application of model that associated the delta(13)C change to body mass increase seems to produce results with no apparent biological explanation. The delta(13)C change rate could directly reflect the daily ration and growth rate, and consequently the isotopic change rates of carbon and other tissue elements can be properly used to assess different factors that may interfere in nutrient utilization and growth.