Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined treatment with IL-7 (5 ng/ml) and Tempol (10 and 25 mM) were comparable with that of the control. The proportion of NBT stained area did not differ significantly between the 8-cell embryos and morula, but was found significantly greater in the degenerating embryos. In conclusion, the developed NBT staining method was found effective for detecting and interpreting the level of intracellular ROS in oocytes, cumulus cells and embryos. This method can be used as an alternative to the DCFH-DA staining method.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4–8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3β, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

The aim of the study was to explore the anti-methanogenic potential of phyto-sources from Northeast region of the country and assess the effect on rumen fermentation characteristics and protozoa for their likely inclusion in animal diet to reduce methane emission