Justicia insularis Improves the in vitro Survival and Development of Ovine Preantral Follicles Enclosed in Ovarian Tissue

Objectives： Evaluating the addition effect of./. insularis extract and FSH on the survival, activation and ROS production after in vitro culture of ovine preantral follicles enclosed in ovarian tissue. Methods： In the first experiment, ovarian fragments were fixed （non-cultured control） or in vitro cultured in α-MEM＋ （cultured control）, α-MEM＋ supplemented with FSH 50 ng/mL, or in α-MEM＋supplemented with J. insularis （JUS0.3; 1.25 or 5 mg/mL） for 1 or 7 days, at 39℃, 5% CO2. In the second experiment, fragments were fixed or cultured in α-MEM＋ supplemented with anethole 300 μg/mL ＋ FSH 50 ng/mL or in α-MEM＋ supplemented with anethole 300μg/mL ＋ 0.3 mg/mL JUS. Key findings： JUS0.3 was the only treatment that maintained the percentage of morphologically normal follicles similar to non-cultured control even after 7 days of culture. After 7 days of culture, a higher （p 〈 0.05） percentage of developing follicles was observed in JUS5 treatment compared with the other treatments except JUS 1.25. In the second experiment, FSH maintained the percentage of normal follicles and promoted activation of primordial follicles. A reduction （p 〈 0.05） of stromal cell density was observed in MEM＋＋ANE supplemented with JUS or FSH. Conclusions： J. insularis in a concentration-dependent manner maintained the levels of ROS and improved in vitro follicular survival and activation of ovine primordial follicles.

Effects of Nitric Oxide on the Growth and Development of Porcine Preantral Follicles Cultured in Vitro

In order to investigate the effects of nitric oxide(NO) on the growth and development of porcine preantral follicles,we treated the follicles with different concentrations of sodium nitroprusside(SNP,0, 0.001,0.01,0.1 and 1 mmol/L),a NO donor.The results showed that the follicle diameter increased during in vitro culture,but there were no significant differences between the treatments(P】0.05);the survival rate in the 1 mmol/L SNP group was significantly lower than that in the 1μmol/L SNP group(61.61% vs 81.52%,P【0.05),but no significant differences were found between other treatments(P】0.05);the rate of antrum formation in the 1μmol/L SNP group peaked at 50%on day 4,and the rate in the 1μmol/L SNP group on day 6 was higher than that in the 0.01 mmol/L SNP group;in addition,the rate of antrum formation in the 1μmol/L SNP group was significantly higher than that in the 0.1 and 1 mmol/L SNP groups (Day 6:73.07%vs 50%,47.62%,P【0.05).After 6 days of culture,the proportion of normal oocytes in the1 mmol/L SNP group was significantly lower than that in the 1μmol/L SNP group(71.21%vs 48.18%, P【0.05),with no significant differences between other treatments(P】0.05).The recovery rate of cumulus cells oocyte complexes(COCs) in the 1μmol/L SNP group was significantly higher than that in the controls and all other treatments(37.27%vs 22.88%,25.59%,20.74%and 19.39%,P【0.05).The results indicate that during the in vitro culture of porcine preantral follicles,low concentration of NO released from SNP improves growth and development of oocytes and follicular antrum formation while high levels of NO are toxic to follicular survival.