| Hatching is the prerequisite and foundation during the poultry breeding process.In commercial hatching,poultry eggs are commonly incubated in completely dark environment,while eggs exposed to the light environment sometimes in the natural hatching process.Exploring lighting effects on hatching and post-hatching performance have important application value for improving the hatching efficiency.Pineal gland is the pacemaker of poultry circadian clock,which plays an important role in the transformation of light energy into biological signals,and may be an important response organ in the light-hatching process.Therefore,this study analyzed the lighting effects on hatching characteristics and explored the possible mechanism of light mediated chicken embryo development with the involvement of pineal gland.High-throughput sequencing technology and bioinformatics methods were used to identify and analyze the characteristics and functions of circadian elements in chicken pineal gland.The results will enrich the research of poultry biorhythm,and will be of great significance for probing the adaptation mechanism of poultry to the external environment.The main research contents are as follows:1.Lighting effects on hatching and post-hatching characteristics.In this study,4 strains of layer breeder eggs,namely White Leghorn(W),Rhode Island Red(R),Columbia Rock(C),and Barred Rock(B)were used to assess the effects of 12L:12D green light during embryogenesis on hatching time,hatching performance,and pubertal growth.The results showed hatching time were significantly shorter among the 4 strains in the light group(P<0.01),while hatch window and peak hatching period were not extended(P>0.05).There was no significant difference in hatchability,chick weight/egg weight,or chick quality among the 4-strain eggs between the light group and dark group(P>0.05).There was no difference(P>0.05)in posthatch BW between different light treatments of the 3 strains(W,C,and B),whereas the BW of R was higher in light group than that of the dark group at 8 to12 wk of age(P<0.05)and the difference disappeared from week 14.The results demonstrate that 12L:12D monochromatic green light stimulation during embryogenesis shortens the hatching time with no negative effects on hatching and posthatch performance.2.Exploration of the possible molecular mechanism of light-regulated chicken embryo development.The liver of embryos incubated in the 12L:12D light condition was heavier than those of the 0L:24D group on d 21 post incubation which may be linked to the observed increase in the serum concentration of IGF-1;primary secretion of the liver.Histological structure analysis of pineal gland demonstrated that light stimulation increased follicle area,wall thickness and lumen area on d 10 and d 12 post incubation.Rhythmic function analysis demonstrated that three CLOCK related genes(BMAL1,CLOCK,CRY1)and a melatonin rate-limiting enzyme related gene(AANAT)were rhythmically expressed in the pineal gland of the light group,but not in the dark group.Simultaneously,the light stimulation also altered the concentration of MT,which was linked to hepatocyte proliferation and IGF-1 secretion in previous studies.Further studies showed that pineal gland’s early histological development and maturation of its rhythmic function were accelerated by the light stimulation.This organ further promotes the secretion of MT with consequent increase in IGF-1 secretion in livers,which promotes subsequent embryonic development and shortening of the hatching time.3.Identification and analysis of chicken pineal circadian genes.Eggs were incubated for 17 days in a 12L:12D light environment to establish biorhythm in advance.On d 18 post incubation,eggs were incubated in 12L:12D(LD group)and 24L:0D(LL group)light conditions respectively.High-throughput sequencing technology and bioinformatics methods were used to screen pineal circadian genes in LD and LL groups.Totals of 1299 genes in LD group and 1358 in LL group were significantly oscillating in a rhythmic manner in the chick pineal,and the intersection circadian genes of the two groups were identified as endogenous circadian genes,with a total of 400.The proportion of endogenous circadian genes with a ± 4 h shifted phase in the two light environments reached 97.5%.It was verified that 6 randomly selected endogenous circadian genes were still rhythmically expressed in constant dark condition,suggesting that the method of identifying endogenous circadian genes in this study is highly accurate.The endogenous circadian genes are more likely to be distributed in transcripts with high expression abundance.A compilation of peak phases of expression across all endogenous circadian genes revealed two major peaks,morning(CT 0-CT 4)and early-night(CT 12-CT 16).GO analysis revealed that genes peaking at the daytime were significantly annotated to the cellular components of myofibril and perform DNA binding molecular functions,whereas annotation to membrane components and perform transmembrane transport molecular functions for the genes peaking at the nighttime.It is speculated that genes in the above 2 clusters play an important role in starting and shutting down the body’s working mechanisms.In addition,this is the first report to found an interesting phenomenon that serveal significant enrichment pathway of endogenous circadian genes were time-specific expression,such as adipocytokine signaling pathway,Erb B signaling pathway,glycerolipid metabolism,steroid biosynthesis,and terpenoid backbone biosynthesis.Genes with a rhythmic expression pattern only in one group were considered as light-sensitive genes,and its distribution is independent of expression abundance.The compositional genes of the chicken pineal oscillator were predicted using bioinformatics methods,including CLOCK,ARNTL,BHLHE40,PRKAG2,CRY1,CRY2,PER2,PER3 and HLF.The output of the oscillator includes signal transduction,metabolic pathways dominated by tryptophan and lipid metabolism,and endocrine systems dominated by thyroid hormone and relaxin.4.Exploration of the possible molecular mechanism of pineal biorhythm with the involvement of miRNA.A total of 120 and 76 rhythmic miRNAs in LD group and LL group.A compilation of peak phases of expression across all circadian miRNA in LD cycles showed two clusters,one cluster at the daytime and another cluster at the nighttime.When light condition switched to constant lighting,the rhythm of the nighttime cluster genes disappeared.Analyzing the lighting effects on its expression,we found that a cluster of miRNA expression was regulated by light,named light-sensitive miRNAs.Cell assay identified two possible pathways for light-regulated light-sensitive miRNAs.Pathway 1:pineal cells directly sensing the lighting changes through their own opsin,regulate the expression of light-sensitive miRNA by changing the level of c AMP and c GMP.Pathway 2:retina transmits light information to SCN,which releases NE to the pineal gland through sympathetic nerve.NE regulates the expression of light-sensitive miRNA through adrenergic receptor/c AMP mechanism.Light-sensitive miRNAs directly regulate the oscillator through CRY2 gene,and directly regulate metabolism and endocrine system through INPP4 A and PFKP genes.In summary,the 12L:12D monochromatic green light stimulation during incubation shortened hatching time among the 4 layer strains.Pineal gland may be the key organ in the photo-endocrine axis that regulates embryo development.During embryogenesis,large number of genes and some miRNAs in pineal gland showed rhythmic expression patterns under the 12L:12D light stimulation,which played an important role in regulating the biological rhythm process. |
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