Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production.

The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse-derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 µM), cysteamine (100 µM), carnitine (0.5 mg/ml), vitamin C (50 µg/ml) or resveratrol (2 µM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2',7'-dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p < .05) in the groups with quercetin (27.5 ± 3.4), vitamin C (27.1 ± 3.0) or resveratrol (28.1 ± 4.7), in comparison with those with cysteamine (34.9 ± 4.5), carnitine (34.6 ± 3.8) or to the control group (36.5 ± 5.2). GSH levels increased (p < .05) in cysteamine (63.5 ± 5.5) or carnitine (60.8 ± 4.4) groups in comparison with quercetin (52.7 ± 5.1), vitamin C (53.0 ± 3.8), resveratrol (53.1 ± 4.4) or to the control (49.6 ± 4.5). Nuclear maturation cleavage and hatched blastocysts rates did not differ (p > .05) between groups. However, blastocyst rates after in vitro fertilization in quercetin (53.5 ± 3.9%), vitamin C (52.1 ± 3.1%) resveratrol (54.2 ± 4.0%), cysteamine (52.4 ± 2.7%) or carnitine (54.2 ± 3.1%) groups were higher (p < .05) than in the control (47.2 ± 2.7%). Total cell numbers in embryos from the vitamin C, resveratrol, cysteamine or carnitine groups were higher than in quercetin and control groups, which were similar to each other. The results suggest that using antioxidants during IVM may reduce oxidative stress either by decreasing ROS levels directly or by increasing GSH levels in oocytes, depending on the type of antioxidant used. Overall, oxidative stress control during IVM with the antioxidants examined here improved blastocyst development with similar efficacy.

The influence of morphology, follicle size and Bcl-2 and Bax transcripts on the developmental competence of bovine oocytes.

This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1-3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6-9 mm (95.6; 41.1%), but similar (p > 0.05) to 3-6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1-3 mm (Bax; 1.0 and Bcl-2; 1.0), 3-6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6-9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.