Molecular, enzymatic responses and in vitro embryonic developmental competency of heat-shocked buffalo embryos co-cultured with granulosa cells monolayer.

The present study was designed to establish a suitable alternative approach to mitigate the adverse effect of high culture temperature on in vitro embryo development and the related molecular response in buffalo. Pre-cultured granulosa cells (GCs) were used as a monolayer during in vitro embryo culture until day 8 (day of fertilization = D0). Post fertilization, presumptive embryos were randomly assigned into two culture conditions: embryos cultured in the presence of GCs monolayer under normal culture temperature (N: 38.5 °C; GEN group) or heat shock (H: 40.5 °C; GEH group) and their counterpart groups of embryos cultured without GCs (EN and EH groups). Additionally, two groups of GCs monolayer were cultured without embryos up to day 8 under 38.5 °C (GN) or 40.5 °C (GH) for further spent culture media enzymatic analyses. Heat shock was administered for the first 2 h of culture then continued at 38.5 °C until day 8. The results indicated that under heat treatment, GCs enhanced (P ≤ 0.05) embryo cleavage and development (day 8) rates, which were comparable to the embryos cultured at 38.5 °C. On the molecular level, blastocysts of the GEH group showed similar expressions of metabolism-regulating genes (CPT2 and SlC2A1/GLUT1) and an antioxidant gene (SOD2) when compared to the blastocysts of the EN group. The relative expression of HSP90 was significantly up-regulated under heat shock and/or co-culture conditions. However, HSF1 expression was increased (P ≤ 0.05) in the GEH group. No statistical differences were observed among the study groups for the pluripotency gene NANOG, and stress resistance transcript NFE2L2. Regarding the enzymatic profile, the concentrations of SOD, total protein, and MDA were decreased (P ≤ 0.05) in the GEH group compared to the cultured GCs without embryos (GH group). In conclusion, GCs as a monolayer have a beneficial impact on alleviating heat stress at the zygote stage through the regulatory mechanisms of metabolic activity, defense system, and heat shock response genes.

Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation.

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus-oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB- and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB- group. BCB- oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB- under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB-) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB- oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.