Spatial distribution of preantral follicles in ovarian parenchyma of bovine species.

The goal of this study was to determine the distribution of preantral follicles in bovine ovaries. Follicular distribution in the ovaries (n = 12) was evaluated in the region of the greater curvature of the ovary (GCO) and the region close to the ovarian pedicle (OP) of Bos taurus indicus heifers of the Nelore breed. Two fragments were obtained from each region of the ovary (GCO and OP). The mean weight of the ovaries was 4.04 ± 0.32 g. The mean antral follicle count (AFC) was 54.58 ± 3.55 follicles (minimum and maximum variation of 30 and 71 follicles, respectively). In total, 1123 follicles were visualized in the region of the GCO; 949 (84.5%) of them were primordial follicles and 174 (15.5%) were developing follicles. The region close to the OP contained 1454 follicles, of which 1266 (87%) were primordial follicles and 44 (12.9%) were developing follicles. The OP region showed a higher proportion of intact follicles in the primordial (P < 0.0001) and primary (P = 0.042) stages compared with the GCO region. The proportion of secondary follicles was similar in the OP and GCO regions. The ovaries of two bovine females (16%; 2/12) contained multi-oocytes follicles, which were characterized as primary follicles. Therefore, the distribution of preantral follicles in the bovine ovary was heterogeneous, with the region close to the OP containing a greater number of preantral follicles compared with the GCO region (P < 0.05).

Culture of preantral ovarian follicles of Bos taurus indicus with alpha-lipoic acid.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at -80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

Impact of the antioxidant quercetin on morphological integrity and follicular development in the in vitro culture of Bos indicus female ovarian fragments.

We evaluated the effect of quercetin on the in vitro culture of bovine ovarian fragments in relation to morphology, development, and oxidative stress. Ovaries (n = 12) from Nelore heifers (n = 6) were used. Each pair of ovaries was divided into nine fragments, and one fragment from each animal was fixed in Bouin solution for 24 h (histology control) or frozen (- 80°C; control for oxidative stress). Other ovarian fragments (n = 8) were distributed into concentrations of 0, 10, 25, and 50 µg/mL of quercetin added to the culture medium for 5 or 10 d. Data were analyzed by chi-square test or ANOVA followed by Tukey's test (P < 0.05). Treatment with 25 µg/mL quercetin resulted in the highest proportion of total intact follicles for 5 (67.3%) and 10 d (57.1%); the concentration of 25 µg/mL also presented the best proportion of developing follicles for 5 d (68.7%) and 10 d (62.8%). Treatment with 25 µg/mL quercetin resulted in significant ferric reduction for 10 d of culture, but not for 5 d. No difference (P > 0.1) was observed in the production of reactive oxygen species or in the oxidative degradation of lipids between treatments and non-cultivated controls. Treatment with 25 µg/mL quercetin preserved the morphological integrity of the developing follicles for 5 and 10 d of culture, in addition to promoting the best antioxidant potential after 10 d of culture in bovine ovarian fragments.

In vitro culture supplementation of EGF for improving the survival of equine preantral follicles.

Folliculogenesis is a process of development and maturation of the ovarian follicles, being essential for the maintenance of fertility. In in vivo conditions, 99.9% of the follicles of an ovary do not ovulate and undergo atresia. In order to minimize this loss and to clarify the existing mechanisms, a technique was developed that allows for the in vitro follicular development. The objective of this study was to evaluate the effects of different epidermal growth factor (EGF) concentrations on the in vitro culturing of equine preantral follicles. Ovaries (n = 10) were collected from a local slaughterhouse of mares in seasonal anestrus, washed with 70% alcohol and PBS, and transported. The inner portion of the ovary was divided into 11 fragments of approximately 3 × 3 × 1 mm. A fragment of each ovary was immediately fixed in Bouin (control group). The remaining 10 fragments were individually cultured for 2 and 6 d. The medium was supplemented with different concentrations of EGF (0, 10, 50, 100, and 200 ng/mL). After cultivation, the fragments were processed and classified according to the developmental stage and morphology. In total, 1065 slides containing 6105 tissue sections were evaluated. Within 2 d of culture, there was a higher proportion of intact follicles at the EGF concentrations of 0 and 100 ng/mL (p > 0.05). After 6 d of culture, only the EGF concentration of 100 ng/mL demonstrated a difference when compared to the other treatments (0, 10, 50 and 200 ng/mL of EGF, p > 0.05). There was follicular development after 2 d at all EGF concentrations. Thus, we suggest that EGF promotes follicular survival in equines at a concentration of 100 ng/mL in in vitro cultures of ovarian fragments for 2 d. In addition, we suggest that EGF promotes follicular survival in equines at a concentration of 100 ng/mL in situ cultivation.