The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

Investigou-se a eficiência da solução salina 0,9% (SS) e TCM 199 na conservação de folículos pré-antrais (FOPA) bovinos in situ em diferentes temperaturas e tempos de incubação. Cada par ovariano foi dividido em 25 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado imediatamente após a coleta (controle). Os demais foram distribuídos em tubos contendo SS ou TCM 199 a 4, 20 ou 39ºC por 2, 4, 12 ou 24h. A análise histológica mostrou que a conservação a 4ºC em ambas as soluções manteve a porcentagem de FOPA normais similar ao controle. A conservação em SS a 20ºC por 12 ou 24h, TCM 199 a 20ºC por 24h e em ambas as soluções a 39ºC a partir de 2h aumentou (P<0,05) a porcentagem de FOPA degenerados comparada à porcentagem de folículos-controle. Em ambas as soluções, independente do tempo de incubação, a porcentagem de folículos normais, após conservação a 39ºC, foi (P<0,05) menor que a obtida com 4 e 20ºC. FOPA bovinos podem ser conservados eficientemente a 4ºC por até 24h em ambas as soluções, e a 20ºC por 4 e 12h em SS e TCM 199, respectivamente.

The efficiency of 0.9% saline solution (SS) and TCM 199 on the preservation of bovine preantral follicles (PF) in situ at different temperatures and incubation times was investigated. Each ovarian pair was divided into 25 fragments. One fragment was taken randomly and immediately fixed (control). The other fragments were distributed in tubes containing SS or TCM 199 at 4, 20 or 39ºC for 2, 4, 12 or 24h. The histological analysis showed that the storage at 4ºC in both solutions kept the percentage of normal follicles similar to control values. Preservation in SS at 20ºC for 12 or 24h, TCM 199 at 20ºC for 24h and in both solutions at 39ºC from 2 h onward (P<0.05) increased the percentage of degenerated follicles when compared with control. In both solutions, independent of incubation time, the percentage of normal follicles observed at 39ºC was (P<0.05) lower them those observed at 4 and 20ºC. Bovine PF can be preserved efficiently at 4ºC for up to 24h in both solutions, at 20ºC for 4 and 12h in SS and TCM 199, respectively.