Investigation of chicken embryo metabolism and substrate utilization during later development
Bequette, Brian J
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The objective of this research was to determine metabolic adaptations and substrate utilization during chicken embryo development and to determine the influence of breeder age and egg size on embryonic growth and metabolism during development (embryonic day 11 to posthatch day 1). In Study One, for both small (n = 60, 53.2 ± 1.04 g) and large (n = 60, 69.0 ± 1.86 g) eggs from 26 versus 42 wk-old broiler breeders, glucose content in albumen decreased to negligible levels by embryonic day (e) 11 whereas mannose and fucose remained constant. Higher yolk glucose content was observed in small eggs from e17 onwards whereas proportions of yolk linoleic and linolenic acids were greater in larger eggs. Liver adenosine monophosphate protein kinase (AMPK), the central cellular energy-sensor, was higher in activity in embryos from large eggs, and AMPK activity was at its highest for both sizes of eggs on e14. These observations suggest that glucose was consumed in early development (before e11). Lower liver AMPK activity and higher yolk glucose at later stages in small eggs from young hens suggests that anaplerotic metabolism is enhanced to alleviate the relative nutrient deficiency. In Study Two, a gas chromatography-mass spectrometry-based metabolomic profiling approach was employed to investigate effects of hen age and egg size on embryo metabolism. Principal component analysis of liver and blood metabolites showed separate clusters on both e14 and e20 from 32 and 51 wk-old hens. The separate clusters featured branched-chain amino acid, glycine, serine and threonine metabolism. Clear separation of metabolites was not observed for embryos from small versus large eggs at any developmental age. Breeder age had a larger influence on embryo metabolism and growth. Three clusters corresponding to liver metabolites from e14, e17 and e20 embryos formed a sub-tree that merged with the cluster from posthatch day 1 chicks in the Hierarchical clustering analysis. This result confirmed that embryo metabolism adapted during later development. Embryos from 51 wk-old hens displayed predominant developmental changes in ketone, glycerolipid and glutathione metabolic pathways in the liver compared to 32 wk-old hens. Study Three aimed to quantify gluconeogenesis, and substrate utilization and partition in e14 and e19 embryos. A constant infusion protocol (8 h) was developed for delivery of [U-<super>13</super>C] glucose and [U-<super>13</super>C] glycerol into the chorio-allantoic fluid. Gluconeogenesis was higher in e19 compared to e14 embryos, consistent with the need for increased liver and muscle glycogen by e19 embryos in preparation for emergence. The contribution of glucose to non-essential amino acid (NEAA) synthesis was greater in e14 <italic>vs</italic> e19 embryos, indicating a higher demand for amino acids for tissue growth. Glycerol contributed very little (< 5%) to gluconeogenesis; thus the remainder must be ascribed to amino acids. Relatively more of acetyl-CoA flux was derived from fatty acid metabolism in e14 embryos compared to 3-carbon pool substrates. In summary, this thesis research established aspects of embryo metabolism and nutrient partition in developing embryos that previously were unknown. This included demonstration of a role of AMPK in development, the influence of breeder age on global embryo metabolism, and metabolic adaptations in gluconeogenesis, Krebs cycle activity and glycerol metabolism during the latter half of chicken embryo development.