No abstract

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs.

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.

The growth hormone (GH) and growth insulin-like factor-1 (IGF-1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF-1 in the long-duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1-7), second half (days 7-14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF-1; IGF-1 → GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO . The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF-1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF-1 → GH and GH + IGF-1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF-1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.

El objetivo del estudio fue evaluar el efecto de la adición de ácido ascórbico (AA) al medio de vitrificación de folículos preantrales (FPs) bovinos previamente enfriados a 4 °C durante 4 o 24 h. Se recolectaron ovarios de novillas Nelore a los 14 meses de edad. En el laboratorio se extrajeron fragmentos de ovario de la región cortical y se distribuyeron a fragmentos para control fresco (C0h), y fragmentos para refrigeración a 4 ºC por 4 y 24 horas en TCM-199 más HEPES y antibióticos. De los fragmentos enfriados, dos se fijaron como controles para cada tiempo de enfriamiento (C4h, C24h), y los restantes se distribuyeron en cuatro tratamientos de vitrificación, utilizando el medio TCM-199 asociado con etilenglicol y dimetilsulfóxido (V), sacarosa (VSUC) o ácido ascórbico (VAA), y el tratamiento con etilenglicol, dimetilsulfóxido, sacarosa y AA (VSUC+AA). Después de 72 h, los fragmentos se calentaron y fijaron para el análisis histológico y el aislamiento folicular mecánico. No se detectó diferencia (p>0.05) entre C0h y C4h para FPs morfológicamente normales (99.3 y 96.0%, respectivamente). La vitrificación redujo la integridad morfológica y la viabilidad folicular en todos los tratamientos en comparación con C0h; sin embargo, el tratamiento VAA mantuvo la viabilidad folicular similar a C24h (p>0.05). Se concluye que los FAs bovinos se conservaron eficientemente a 4 °C durante 4 h, y la adición de ácido ascórbico al medio de vitrificación mejoró las tasas de supervivencia y mantuvo la integridad morfológica folicular.