Dynamic Metabolic Process Of Yolk Nutrients In Chicken Embryos And Effect Of Iron Homeostasis On Incubation Efficiency

The purpose of this study was to investigate the dynamic changes of yolk metabolites in chicken embryo stage and the metabolic regulation mechanism of iron homeostasis on nutrients utilized by yolk sac.By comparing the effects of yolk metabolites,in vitro cell culture and egg injection of different iron sources on the hatching efficiency of chicken embryos,the yolk sac withering and regulation mechanism were explored in order to improve the oxidative stress caused by yolk sac metabolism in the late stage of chicken embryos,improve their utilization efficiency of yolk nutrients,and achieve the purpose of improving the hatching efficiency of chicks.This study consists of three parts,experiment I:the study of dynamic metabolic changes of yolk in broilers during the embryonic period.100 Ross 308 fertilized eggs were selected and yolk samples were collected on embryonic days（E）7,9,11,15,17,and 19,and 10samples were collected repeatedly every day for non-targeted metabolomics analysis.Experiment Ⅱ:Study of the molecular mechanism of yolk sac withering by iron homeostasis.A total of 100 Ross 308 fertilized eggs were selected,and yolk and yolk sac samples were collected at E13,15,17,19 and on the day of shelling（D0）,and 10 replicates were collected daily for combined metabolic transcriptomic analysis of the yolk sac as well as iron response element-binding protein（IREB2）,lysophosphatidylcholine acyltransferase 3（LPCAT3）,mevalonate kinase（MVK）,nuclear receptor coactivator 4（NCOA4）,HMG-Co A reductase（HMGCR）,nuclear factor erythrocyte 2-related factor 2（NRF2）,glutathione peroxidase 4（GPX4）,acyl-Co A synthase long chain family member 4（ACSL4）,divalent metal transporter 1（DMT1）,transferrin（TF）,transferrin receptor（TFRC）,prostaglandin endoperoxidase 2（PTGS-2/COX-2）,and lysozyme carrier member 7 11（SLC7A11/Xc-）expression.ExperimentⅢ:The effect of egg injection of different binding forms of iron on yolk sac ferroptosis.500 Ross 308 fertilized eggs were selected and randomly divided into five groups of 100 eggs each.They were the control group（no injection,CON group）;ferrous sulfate（Fe SO₄）group（2,4 mg/m L,0.2 m L）;ferrous glycine（Fe-Bisgly）group（4 mg/m L,0.2 m L）;and desferrioxamine（DFO）group（5 mg/m L,0.2 m L）,which were injected into the yolk area at E15 to observe the effects of different iron sources on the shelling efficiency and early growth and development of chicks.The results showed that:1)The results of principal component analysis（PCA）of yolk metabolism showed that E07 and E09 metabolites were approximately similar,but E09,E11,E15,E17 and E19 were different from each other,indicating that yolk nutrients were drastically changed at E9-E19;yolk metabolites were enriched in amino acid metabolic pathways,such as arginine,lysine,cysteine and histidine metabolism,in E11-E15 and E17-E19.The increase of amino acid metabolism may be involved in the process of embryo growth and oxidative stress improvement.In addition,the ferroptosis pathway was found to be one of the major metabolic processes of yolk metabolites E15-E17 and E17-19 in this study.2)The yolk sac is the main site of yolk nutrient absorption,but the yolk sac weight then decreases after peaking at about E17.Total iron in the yolk decreased significantly after E13（P<0.05）,and absorbed iron may accumulate in the yolk sac.The metabolic pathways enriched in yolk sac metabolism E13-19 were mainly the metabolism of sulfur-containing amino acids and their derivatives,including cysteine,methionine and taurine metabolism.Sulfur could form iron-sulfur coenzyme factors or iron-sulfur clusters with iron to act on the redox reaction in cells,indicating that the yolk sac may be one of the main pathways to inhibit oxidative stress and regulate litter using iron and sulfur metabolism in the late incubation period.3)The results of RT-q PCR of the yolk sac showed that the expression of transferrin receptor（TFRC）in the yolk sac was significantly higher at E13 than at E19（P<0.05）,while the expression of genes such as E19 divalent metal ion transporter（DMT1）was not significantly different from that at E13（P>0.05）,while the key inhibitory receptors of the ferroptosis pathway and enzymes GPX4,HMGCR,MVK,and LPCAT3 were significantly decreased at E13-19（P<0.05）,but the expression of enzyme genes such as COX-2 and ACSL4 that promote the activation of the ferroptosis pathway was significantly increased at E19（P<0.05）.This suggests that the mode of transport of iron by the yolk sac is changed from metaphase（E13）mainly by transferrin（TF）transport uptake to anaphase（E19）mainly by DMT1 in the form of divalent metal ions（NTBI）.It is further suggested that the activation of ferroptosis pathway in this study is caused by the increase of NTBI and the peroxidation of polyunsaturated fatty acids（PUFAs）,and mediates the withering process of yolk sac.4)Fe SO₄ supplementation by egg injection could advance the shelling time by 3–4 h,while Fe-Bisgly or DFO was delayed by 4–8 h,and significantly increased yolk sac weight（P<0.05）and birth weight（P<0.05）and reduced the remaining yolk（P<0.05）compared with the CON group.Compared with the control group,each test significantly increased body weight by 1（P=0.002）.Therefore,injection of Fe-Bisgly or DFO at E15 can regulate yolk sac withering,increase yolk utilization rate,and improve hatching efficiency.In summary,yolk nutrition and function change dynamically in the process of chicken embryo development to meet the development and nutritional needs of different stages.The change of iron transport and absorption mode in the yolk sac at the late incubation stage triggers the activation of ferroptosis pathway and mediates the withering of the yolk sac.Injecting Fe-Bisgly or DFO into the yolk area at E15 can regulate the withering of the yolk sac,and then improve the hatching efficiency of chicks.