Nodestructive Sexing Of Chicken Eggs Based On Odor And Matrix Differences Analyzed By Multi-Omicmulti-Omics

In laying hens industry,non-destructive sexing of chicken eggs during the early stage of incubation could not only meet the actual production needs,but also avoid a lot of waste of egg resources.The odor diffence of different breeds,different fertilization statuses,male and female fertitle eggs were analyzed by non-destructive were used to screen for potential markers.Furthermore,high-throughput omics and bioinformatics analysis were used to analyze the mechanism of difference between unhatched male and female eggs.Finally,two non-destructive identification technique or model for male and female eggs baesd on odor was preliminarily established.The main results are as follows:?1?The optimal conditions for non-destructive identification of odor from eggs by SMPE-GC-MS were as follows:DVB/CAR/PDMS fiber was extracted at 40°C for 60min and a total of 17/18 volatiles were identified in fertilized eggs.The VOCs from three breeds of fertilized eggs well differentiated in discriminant models,and the accuracy of RFC and CD model were 100%,100%?initial?and 83.3%?cross-validation?.Further,2-ethyl-1-hexanol and 2-nonen-1-ol were only identified in W eggs and3-?bromomethyl?-piperidine and 2-cyano-Acetamide were only identified in J eggs.Heptanal,hexanal,decanal,6-methyl-5-hepten-2-one,2-ethyl-1-hexanol,etc.have greatly contributed to the distinction of W,H and J fertilized eggs.These differential VOCs may become potential marker for non-destructive identification breeds of egg.?2?SPME/GC-MS combined with E-nose were successfully used to non-destructively discriminate the unfertilized,fertile and infertile eggs.The accuracy of CDA model for nondestructive discrimination of egg fertilization?status?by GC-MS were 100%and the accuracy of CDA model for nondestructive discrimination of UF-Fd and F'-IF eggs were 100%and 89.5%,respectively.Cedrene,decanal,hexanal,2-ethyl-1-hexanol and undecanal etc.were greatly contributed to the classification of unfertilized and fertilized eggs.Cedrene and decanal etc.contributed significantly to the differentiation between unfertilized and infertile eggs.Heptanal and undecanal etc.were greatly contributed to the classification of fertile and infertile eggs.And these compounds may become potential markers for identifying infertile eggs.Sensors P40/1,P10/2,P10/1,TA/2,T40/2 were mostly contributed to the distinction of unfertilized and fertilized eggs,and sensors T30/1,P30/1,P40/2,PA/2,T40/2 were mostly contributed to the distinction of fertile and infertile.?3?There were certain differences between male and female eggs with different breeds?W,H,J?,and during E₀-E₉ of incubation for W eggs.The accuracy of CDA model for nondestructive sexing of?embryo?chicken eggs during early stage of incubation were almost 100%?abundance,52/52;percentage 51/52?;the accuracy of CDA model for nondestructive sexing of unhatched?chicken?eggs for W,H and J breed were 100%?abundance?,90%?percentage?;68.8%?abundance?,76.6%?percentage?;90.4%?abundance&percentage?.Male and female eggs have a good separation trend in canonical discriminant model.Furthermore,the abundance of VOCs in female eggs was higher than that in male eggs and there were certain correlation between2-ethyl-1-hexanol?E₀?,cedarene,heptanal,decanal?E₁?,carbon dioxide?E₅?,octanal?E₇&E₉?with the sex of W hatched egg during early of incubation.In addition,2-nonen-1-ol,2-ethyl-1-hexanol,6-methyl-5-hepten-2-one and hexane etc.were contributed greatly to the gender discrimination of W eggs;nonanal,2-nonen-1-ol,1-octanol,1-Heptadecanamine and 6-methyl-5-hepten-2-one etc.were contributed greatly to the gender discrimination of H eggs;cedrene,decanal and undecanal were contributed mostly to the gender discrimination of J eggs.?4?A total of 900 species of lipids were identified in?male and female?unhatched egg yolk,76 species were significantly different,including 3 species were up-regulated?F>M?,73 species were down-regulated.Namely,differentially expressed lipids,acylcarnitine,triglyceride?TG,12:0/22:1/22:3?,lysophosphatidylserine?LPS,16:0/0:0?,ceramide?Cer,d18:0/18:0?,cholesterol lipid?CE,16:1?,phosphatidylcholine?PC,O-18:3/20:4?etc.lipids have greater contribution to the discrimination of male and female?unhatched?eggs.A total of 582 metabolites and 72 differential metabolites were identified in?male and female?unhatched egg yolk,including 46 species were up-regulated?F>M?and 26 species were down-regulated?F<M?.Namely,differentially expressed metabolites,?-valerolactam,11,12-EET[?±?11,?12?-epoxy-5Z,8Z,14Z-eicosatrienoic acid,docosahexaenoi cacid,eicosapentaenoic acid,2-pyrrolidinone,9-oxo-10E,12Z-octadecadienoic acid,cis-7-hexadecenoic acid,isonicotinamide and 10-Formyl-Thf etc.have greater contribution to the discrimination of male and female?unhatched?eggs.Many differential metabolites are involved in following pathways:tyrosine,purine metabolism,unsaturated fatty acid synthesis,metabolism,and vitamin degradation and absorption.It is noteworthy that estrone 3-sulfate involved in the steroid hormones biosynthesis pathways and cuminaldehyde involed in the xylene degradation pathways,which participates in the formation of aldehydes the main VOCs emitted from fertilized eggs,may have certain relationship with gender of fertilized eggs.?5?The amino acids composition between male and female egg yolks is similar;except for small significant difference in glutamic acid,glycine,cysteine and arginine.411 proteins were identified in?male and female?egg yolks.Among them,276 species have been reported in chickens?gallus gallus?and the remaining 135 proteins may be hypothetical"new proteins"for chicken.35 differential proteins were screened from male and female egg yolk,including 18 proteins up-regulated and 17 proteins?M>18 of F?down-regulated in male eggs,17 of which are down-regulated?M<F?.Among them,37533205,1015809562,763550308,973583714,1741943,etc.contributed significantly to the discrimination of male and female?unhatched?eggs.Differential proteins are mainly distributed in extracellularl?component?and response to stimulus and immune system process,with binding and chemoattractant activity.Many differential proteins are involved in following pathways:response to stimulus,immune response,immune system process,immunoglobulin production and production of molecular mediator of immune response.Down-regulated proteins are involved in the folding,sorting and degradation,signaling molecules and interaction,cell groth and death,transport and catabolism and infectious diseases:Viral.Whlie the up-regulated differential proteins were less annotated in KEGG database.?6?The fitting effects of MLP model for non-destructive detection of eggs gender based on VOCs performed by GC-MS and PTR-MS were better than CDA and RFC modes.The discriminative accuracy of MLP models?traning set?for non-destructive detection of eggs gender based on VOCs performed by GC-MS and PTR-MS were 100%and 91%,respectively.