Identification Of Hypoxic Adaptation Phenotypes And The Differential Expression Genes Of Myocardial Tissue In Chicken Embryo

The growth and development of animal embryos are affected by hypoxia and extreme low temperature in high-altitude environments. Birds’ embryos are particularly sensitive to environmental stress due to the development in vivo. In fact, the hatchability decreases with the increase of altitude. Hypoxia can lead to several problems, including the reduction of embryonic development, the extension of incubation period, and the decline of hatching rate. The adaptabilities to hypoxia are based on chicken embryo varieties. In this study, distinctions of chicken embryo mortalities, organ developments, and other phenotypic indexes under different hypobaric hypoxia and normobaric hypoxia respectively compared in different chicken breeds. Furthermore, transcriptome and proteomic sequencings were also conducted to identify candidate genes involved in biological pathways, which were responsible for the adaptabilities of chickens to hypoxia. Our research provides theoretical bases for further study to decode the mystery of genetic mechanisms of adaptabilities to hypoxia in chicken embryos.Tibet chicken (TC) and Chahua chicken (CH) eggs were collected and incubated at normoxia (21%O2) and hypoxia (13%Oa), respectively. Then the embryonic weight, each organ weight, hatchability, etc. phenotypic indexes were measured. The results showed that the mortality of CH embryos was much higher than that of TC under hypoxia incubation. The peak of mortality was come after D16in hypoxia. We also found that heart and brain were the most sensitive organs subject to low oxygen, analyzed the development of embryonic organ at normoxia and hypoxia respectively.Tibetan chicken (TC), White Leghorn E (WL-E), Chahua chicken (CH), White Leghorn D (WL-D), Lhasa White chicken (LWC) and Beijing-You chicken (BY) eggs were collected and incubated at normobaric hypoxia (13%O2) and hypobaric hypoxia (as3000m) separately. The embryonic weight, each organ weight, hatchability, etc. phenotypic indexes and embryonic blood hemoglobin concentration and other blood indexes were measured. Results showed that the adaptabilities to hypoxia in WL-E and TC were better compared with other breeds, while the mechanisms of adaptation to hypoxia might be different in the two breeds. In addition, embryonic organs in different chicken varieties had their own traits. The hemoglobin concentration, number of red blood cells and hematokrit in hypoxia adaptive breeds were higher than that in sensitive breeds in incubation time. Our research first time found that the WL-E had a high hatchability, which was very similar with TC under hypoxic incubation.Eggs from Tibet chicken (TC), Chahua chicken (CH), Tibet x Chahua (TC×CH) and Chahua x Tibet (CHxTC) chickens were incubated under hypoxic condition. The results indicated that the egg weight and the water loss during incubation were mainly determined by female parent. The intercrossing eggs between Tibet chicken and lowland chicken had obviously increased hatchability and had heterosis in traits of hypoxic hatchability. Although there was no difference influence on hypoxic hatchability of the eggs from Tibet chicken as female parent compared with the eggs from the Tibet chicken as male parent, the former had better developments in early stage and the latter had better developments in late stage of incubation when comparing the ratio of embryo to egg weight between the intercrossing groups.Embryonic heart issues of TC, CH, WL-E and WL-D were collected on the D16under hypoxia incubation, total RNA was then extracted, and RNA-seq was performed. Differentially expressed genes (DEGs) were identified according to P≤0.05and log2FC≥1or<-1. Go enrichment analyses of DEGs were performed on DAVID website, and pathway enrichment were conducted with GESA. Each sample of sequencing had more than4G dates with genome mapping rate more than83%. In total,18467positively expressed genes were identified in myocardial tissues of chicken embryos, including16340genes of four breeds shared expressed. There were354DEGs between TC and CH, including130up-regulated DEGs and224down-regulated DEGs in TC.516DEGs were identified between TC and WL-D, including292up-regulated DEGs and224down-regulated DEGs in TC. There were432DEGs between WL-E and CH, which had294up-regulated DEGs and138down-regulated DEGs in WL-E.384DEGs were identified between WL-E and WL-D, which had206up-regulated DEGs and178down-regulated DEGs in WL-E.62DEGs were identified in TC compared with CH and WL-D, which were enriched in several GO terms, including heart development, blood vessel development, etc.64DEGs were identified in WL-E compared with CH and WL-D, which were enriched in several GO terms, including neurotransmitter receptor activity, GTP binding, etc. Although both TC and WL-E exhibited excellent adaptabilities to hypoxia, only6DEGS were shared by them that were HSP90AA1, BEAN1, GBP and three novel genes. Pathways of TC were enriched in categories, such as mTOR signaling pathway, MAPK signaling pathway, etc., which were the upstream or downstream pathways of HIF signaling pathway. The particular enrichment pathways of WL-E trended to energy metabolic pathways, such as TCA cycle and so on.Embryonic heart issues of TC and CH were collected on the D16under hypoxia incubation, total protein was then extracted, and iTRAQ was performed. Differentially expressed proteins were screened with P<0.05and FC≥2or≤0.5. Analyzed the differentially expressed proteins enrichment GO, COG, pathway by PANTHER website. There were118differentially expressed proteins, including46up-regulated and72down-regulated in TC. The differentially expressed proteins screened from iTRAQ were enriched in HIF signaling pathway and some pathways related to angiogenesis, including EGLN1, MAP2K2, RAB5C, etc.In the present study, the most oxygen sensitive organs, DEGs, and proteins were identified by analyses of the different varieties of chicken embryos phenotypic under hypoxia, transcriptome and proteomic. This work provides new suggestion and genetic basis for future research in the molecular mechanism of chicken embryo in hypoxia adaptation.