Gizele Augusta Lemos Silva

The present study aimed to evaluate the effect of N-acetylcysteine (NAC) on in vitro maturation (IVM) and in vitro embryo production in sheep. Cumulus–oocyte complexes (COCs) were collected and matured in vitro in a control medium or supplemented with different concentrations of NAC (1, 1.5 and 2.0 mM). After maturation, a subset of oocytes was used for morphological assessment and chromatin configuration analysis, as well as for evaluating intracellular levels of reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and DNA fragmentation using the TUNEL assay. Another subgroup of oocytes was subjected to in vitro fertilization (IVF), and the presumed zygotes were cultured in in vitro culture (IVC) medium to assess cleavage rates. Data were statistically analysed, considering significance at P < 0.05. The concentration of 1 mM NAC resulted in lower ROS levels compared to the control and 1.5 mM groups, in addition to reducing DNA fragmentation. No significant differences ( P > 0.05) were observed in GSH levels or mitochondrial activity between treated and control groups. Furthermore, oocytes treated with 1 mM NAC showed higher cleavage rates compared to the other experimental groups. In conclusion, supplementation with 1 mM NAC improves oocyte quality and early embryonic development, as demonstrated by reduced oxidative stress and DNA fragmentation, resulting in increased cleavage rates.

Oxidative stress resulting from the inherent conditions of in vitro culture can compromise oocyte quality. In this regard, there is a growing interest in the use of natural products to prevent oxidative damage and to promote in vitro follicular development. This study was conducted to evaluate the effects of the addition of acidified extract of grape industrial residue (Vitis vinifera cv. Syrah) as a supplement to the base medium for in vitro culture of isolated sheep secondary follicles. Secondary follicles were isolated and cultured for 12 days in α-MEM+ (control medium) or in α-MEM+ supplemented with different grape residue extract concentrations (0.1, 0.2, or 0.4 mg/ml). Follicular morphology, antrum formation, follicular and oocyte diameter, glutathione (GSH) levels, mitochondrial activity, DNA fragmentation and meiotic resumption were evaluated. After 12 days of culture, there was no difference (P >0.05) among the treatments in relation to morphology, antral cavity formation, follicular diameter, DNA fragmentation and the percentage of fully grown oocytes (≥ 110 µm). Furthermore, the GSH levels were similar (P >0.05) among the α-MEM+, 0.2, and 0.4 mg/ml grape residue extract groups at the end of culture. Nevertheless, oocytes from secondary follicles cultured in 0.4 mg/ml grape residue extract showed higher (P <0.05) mitochondrial activity and greater meiotic resumption than oocytes cultured in the control medium. In conclusion, supplementation with 0.4 mg/ml grape residue extract maintained survival, improved mitochondrial activity, and promoted the meiotic resumption of oocytes from sheep secondary follicles cultured in vitro.

Summary: The aims of this study were to evaluate the doxorubicin concentration that induces toxic effects on in vitro culture of isolated mouse secondary follicles and to investigate whether resveratrol can inhibit or reduce this toxicity. Secondary follicles were isolated and cultured for 12 days in control medium (α-MEM⁺) or in α-MEM⁺ supplemented with doxorubicin (0.1 µg/ml) or different concentrations of resveratrol (0.5, 2, or 5 µM) associated with doxorubicin (0.1 µg/ml) (experiment 1). For experiment 2, follicles were cultured in α-MEM⁺ alone or supplemented with doxorubicin (0.3 µg/ml) or different concentrations of resveratrol (5 or 10 µM) associated or not with doxorubicin (0.3 µg/ml) (experiment 2). The endpoints analyzed were morphology (survival), antrum formation, follicular diameter, mitochondrial activity, glutathione (GSH) levels and DNA fragmentation. In the first experiment, doxorubicin (0.1 µg/ml) maintained survival and antrum formation similar to the control, while 5 µM resveratrol showed increased parameters, maintained mitochondrial activity and increased GSH levels compared to the control. In the second experiment, doxorubicin (0.3 µg/ml) reduced survival, antrum formation and follicular diameter compared to the control. Resveratrol at a concentration of 10 µM attenuated the damage caused by doxorubicin by improving follicular survival and did not present DNA fragmentation. In conclusion, supplementation of the in vitro culture medium with 0.3 µg/ml doxorubicin reduced the survival and impaired the development of mouse-isolated preantral follicles. Resveratrol at 10 µM reduced doxorubicin-induced follicular atresia, without DNA fragmentation in the follicles.