Sequential medium with GH and IGF-1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue.

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 â†’ IGF-1; IGF-1 â†’ GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO2 . 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 â†’ 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 â†’ 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 â†’ 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.

The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro.

The aim of this study was to determine the effectiveness of minimum essential medium alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's medium (McCoy's) on IVC of preantral follicles included in the bovine ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199, or McCoy supplemented ((+)) with glutamine, insulin, transferrin, selenium, ascorbic acid, BSA, penicillin, streptomycin, and HEPES buffer in 24-well plates, at 37 °C and 5% CO2 for 1 or 7 days. The morphology of follicles, normal, primordial and development (primary and secondary), as well as viability and morphometric variables of follicles and oocytes were assessed. The morphology and morphometry of preantral follicles were analyzed by ANOVA followed by the Tukey and Dunnett tests, and viability variables were determined by the chi-square test. The results showed that TCM-199(+) reduced significantly (P < 0.05) the percentage of morphologically normal and viable follicles after 7 days of culture compared to the control. Similar results were observed in McCoy(+), in which the percentage of viable follicles after 7 days of culture was significantly lower (P < 0.05) than in the control. However, it was similar (P > 0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P < 0.05) than in control and α-MEM(+) after 7 days of culture. In addition, the ultrastructure of preantral follicles was similar between the control and α-MEM(+) after 7 days of culture. In conclusion, α-MEM(+) showed to be the most effective medium to preserve morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring their viability and growth after in situ culture.

Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes.

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.