الفهرس 
In vitro buffalo embryo production under heat stress conditions : role of granulosa cells as a mono-layer
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Abstract
This study was performed to alleviate the effect of heat shock during pre-implantation stage (zygote) using co-culture system by granulosa cells (GCs) mono-layer. In addition to, study the effect of heat stress and co-culture system on granulosa cells viability, mitochondrial activity and gene expression. Cumulus-oocyte complexes (COCs) were collected from ovaries (n= 448) of slaughtered buffalo. Good quality immature oocytes (n=1512) were subjected to in vitro maturation and fertilization. Post in vitro fertilization (18-22 h), presumptive zygotes were randomly assigned into four groups: (G1) No heat shock (38.5oC), (G2) Heat shock (40.5°C), (G3) Co-culture with GCs monolayer and heat shock and (G4) Co-culture with GCs monolayer and no heat shock.Heat-shocked groups were exposed to temperature of 40.5°C for the first two hours of culture then continued at 38.5°C up to day 8 Of fertilization. Embryo development (cleavage rate at D3and blastocyst rate at D8) was monitored throughout pre-implantation period. The results indicated that COCs expansion rate was 90.8±1.1% and nuclear maturation rate (telophase + metaphase II) was 73.8%. Cleavage rate as recorded at day 3 was significantly higher (p≤0.05) for G1 (71.1±10.5%), G3 (80.2±7.0%) and G4 (70.5±7.9%) than G2 (43.7±7.0%). In addition, embryos of G3 showedapproximately the same rate of developed transferable embryos (Morula and blastocyst stages at D 8 of culture) as of G1 (50.9±5.3 and 51.7±7.9, respectively). The expression profile of genes CPT2 and GLUT1 was increased (P≤0.05) in G1 and G3 compared to G2 and G4 groups. SOD2 had similar patternin G1 and G3 being however higher than G2 group. HSF1 and HSP90 were significantly up-regulated in G2 and G3 compared to G1 group.While no statistical differences were observed for NANOG and NFE2L2 among the study groups. Granulosa cells viability rate significantly decreased in heat stress group (25.1±3.7) compared to control group (36.6±5.3) at day 3. The viability rate at day 7 decreased in heat stress compared to control (83.7±4.5 and 97.4±0.4, respectively). The result showed that there was a significant increase in mitochondria activity at day 7 in G5 compared to G6 and non-significant difference in ROS concentration between groups but graphically increase in G5. There was a non significant difference in ROS accumulation between G3 and G4. On the other hand, the mitochondrial activity was significantly increased in G4 compared to G3. The expression of heat shock factor 1 (HSF1) and apoptosis-inducing gene (P53) was significantly up-regulated in granulosa cells exposed to heat shock compared to control group. StAR gene was down-regulated (P ≤ 0.05) in granulosa cells cultured under heat shock compared to control group