Extract of Amburana cearensis maintains the survival of ovine preantral follicles during long-term ovarian tissue transport and promotes primordial follicle activation after in vitro culture

Abstract

This study evaluated the effect of Amburana cearensis extract as a preservation or culture medium for ovine ovarian tissue. Ovarian fragments were fixed in 4% buffered formaldehyde for 18 h (fresh control), stored in Minimal Essential Medium (MEM) or in A. cearensis extract (0.1; 0.2 or 0.4 mg/mL) at a temperature of 4ºC for 6, 12 or 24 h (preservation - experiment 1) or cultured for 7 days in ?-MEM+ or in A. cearensis extract without (0.1; 0.2 or 0.4 mg/mL) or with supplements (0.1+ ; 0.2+ or 0.4+ mg/ mL; experiment 2). The percentages of morphologically normal follicles and follicular activation were submitted to analysis of variance (ANOVA) and Tukey´s test. The values of TUNEL-positive cells were submitted to Chi-square test (P < 0.05). The storage of fragments for 6 h in MEM showed higher percentages of normal follicles (62%) and a lower rate of TUNEL positive cells (36.17%) compared to other treatments (normal follicles: 46%; 43% and 52%; TUNEL positive cells: 58.57%; 55.30% and 55.63% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, respectively). However, after 12 or 24 h, MEM (12 h: 48%; 24 h: 45%) and Amb 0.2 mg/mL (12 h: 37%; 24 h: 39%) showed similar percentages of normal follicles and TUNEL positive cells (MEM - 12 h: 43.26%; 24 h: 58%; Amb 0.2 mg/mL - 12 h: 50%; 24 h: 61%). After culture, ?-MEM+ recorded a higher percentage of normal follicles (58.25%) than A. cearensis treatments (32.8%; 25.4% and 34.2% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, and 22.25%; 20.0% and 36.6% for Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) (P < 0.05). Follicular activation increased in all treatments (52.5%; 36.73%; 54.05%; 47.5% and 58.19% for ?-MEM+ ; Amb 0.1; Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) compared to the fresh control (11.65%), except for Amb 0.2 mg/mL (23.69%) and Amb 0.4 mg/mL (28.85%) (P > 0.05). Moreover, after in vitro culture, A. cearensis at a concentration of 0.1 mg/mL maintained the percentage of TUNEL positive cells (30.0%) in a way that is similar to that observed in the fresh control (22%) (P > 0.05). In conclusion, ovine preantral follicles can be preserved at 4°C in MEM for 6 h. For longer periods of preservation (24 h), MEM and 0.2 mg/mL A. cearensis are recommended. Moreover, after in vitro culture, A. cearensis extract (0.1 mg/mL) showed higher activation and lower DNA fragmentation in ovine preantral follicles.