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The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey''s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P< 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P< 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P< 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1of a-tocopherol after 6 d. After 8 days of in vitroculture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitroculture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships. [ABSTRACT FROM AUTHOR]

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitroculture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin''s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey''s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P≤ 0.05) and the percentage of growing follicles was increased (P≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitrofor 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship. [ABSTRACT FROM AUTHOR]