Cuticle Secretion Duration in the Uterus Is One of the Major Factors Affecting Eggshell Cuticle Deposition in White Leghorn Laying Hens

Background: The cuticle formed in the uterus is the outermost layer as the rst defense line of eggshell against microbial invasions in most avian species, and analyzing its genetic regulation and inuencing factors are of great importance to egg biosecurity in poultry production. The current study compared the uterine transcriptome and proteome of laying hens producing eggs with good and poor cuticle deposition (GC and PC, the top and tail of the cuticle deposition distribution), and identied several genes involved with eggshell cuticle deposition. Results: Overall, transcriptomic analysis identied 53 differentially expressed genes (DEGs) between PC vs GC group hens, among which 25 were up-regulated and 28 were down-regulated. No differences were found in the uterine proteome. Several up-regulated DEGs of PC vs GC hen uterus, including PTGDS, PLCG2, ADM and PRLR related to uterine functions and reproductive hormones, were validated by qPCR analysis. Phenotypic measurements for verication between GC and PC hens showed GC hens had longer laying interval between two consecutive ovipositions (25.64 ± 1.23 vs 24.94 ± 1.12 h), thicker eggshell thickness (352.01 ± 23.04 vs 316.20 ± 30.58 μm) and more laying days (20.67 ± 2.58 vs 18.48 ± 2.46) within 27 laying days (P < 0.05). Apart from eggshell traits, other egg quality traits didn’t differ. The results above suggested that the expression patterns of PTGDS, PLCG2, ADM and PRLR genes in the uterus regulated the muscular activity rhythm and secretion function, which ensured the eggshell and cuticle formation duration, and lead to the good eggshell and cuticle quality of the GC hens. Conclusions: The present study demonstrated eggshell and cuticle secretion duration in the uterus is one of the major factors affecting eggshell cuticle deposition in laying hens. PTGDS, PLCG2, ADM and PRLR genes were discovered and might play crucial roles in cuticle deposition by regulating the uterine muscular activities and secretion function. Moreover, the cuticle is associated Functional enrichment of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the 53 DEGs showed that there was no signicant enriched category of GO functional annotation or KEGG pathway when subject to analysis by the DAVID Functional Annotation Tool. This may be due to the small amount of DEGs as only functions of 32 genes from the DEGs are known in the DAVID database.


Background
The annual Global Report on Food Crises 2020 by the Food Security Information Network (FSIN) indicates that the world is facing an unprecedented food crisis [1]. As a source of low-cost and highquality animal protein, poultry production not only meets the basic nutritional needs of humans, but also is an important contributor to the economies and cultures, and thus the demand for poultry egg and meat is increasing [2]. Egg safety is absolutely pivotal to the success of today's poultry industry, however, egg contamination with pathogenic bacteria is considered one of the leading causes of economic loss in the poultry industry worldwide and represents a threat to public health [3,4]. Furthermore, faced with current environmental pressures and animal welfare requirements, the modern poultry industry, which relies on the extensive use of arti cial incubation, is more dependent on strict biosecurity of the egg [5].
Avian eggshells have evolved multiple physical and chemical barriers in response to microbial challenges, and these barriers are essential for the successful reproduction of avian species as well as to maintain safe and nutritious table eggs for human consumption [6,7]. The eggshell is a complex with several highly ordered and distinct layers (i.e., mammillae, palisades, vertical crystal layer, and cuticle) and the cuticle is the outermost layer as the rst defense line of the eggshell against microbial invasions in most avian species [8][9][10]. The cuticle has been a relatively neglected structure while recent researches on the properties, composition, functions, physiology and genetics of the cuticle have highlighted its important role in ensuring egg biosecurity and quality in poultry production. The cuticle is deposited during the nal hr of egg formation in the uterus (shell gland pouch), covering the shell surface and lling the external openings of the gas pores so that effectively exerting waterproof and antibacterial effects [11,12]. The cuticle mainly consists of hydroxyapatite crystals, glycoprotein, polysaccharides, lipids, and pigment [13,14], and abundant antibacterial proteins (e.g., lysozyme C, ovotransferrin, ovocalyxin-32, ovocleidin-17) constitute the molecular basis for the antimicrobial function of the cuticle [15,16]. It has been demonstrated that good cuticle deposition of the eggshell can signi cantly reduce the opportunity of pathogen invasion [5,17,18], and the quantity of cuticle is a heritable trait that genetic selection to this trait can be an effective strategy to reduce transmission of microorganisms in poultry production [8,19].
Therefore, how to improve cuticle deposition to ensure egg biosecurity and quality and further promote poultry production has drawn a worldwide interest.
Avian birds are oviparous and produce an egg at intervals precisely controlled by the hormonal secretion and gene expression of the hypothalamo-pituitary-gonadal-oviduct axis [10,12,[20][21][22]. Previous transcriptomic and proteomic studies of hen (Gallus gallus) reproductive tract have well pro led the temporal and spatial transcriptome landscapes and important genes that regulate egg formation [22][23][24][25][26][27][28]. Cuticle deposition is also a well-de ned and speci c process that occurs in the uterus rather than the extension of mineralized eggshell [12]. It's reported that there is a signi cant association between ovocleidin-116, ovocalyxin-32, ovalbumin, and estrogen receptor (ESR1) genes and cuticle deposition [8,29]. Transcriptome analysis of the uterus of hens laying eggs with good or poor cuticle deposition suggested that clock genes and immediate early genes are prime candidates for the control of cuticle deposition and the uterus transcriptome did not differ that the genetic effects of cuticle deposition might not be expressed in the uterus [30]. However, there is few duration for the secretion of the cuticle compared with other components of the egg, making it di cult to exactly obtain the uterine tissue and the transcriptomic and proteomic data during cuticle deposition. Thus, speci c pathways and genes that regulate cuticle deposition still remain unclear.
In present study, transcriptomic and proteomic status of uterus from White Leghorn laying hens that produced eggs with good and poor cuticle deposition (GC and PC, the top and tail of the cuticle deposition distribution) were analyzed to identify unknown candidate regulatory genes involved in eggshell cuticle deposition and provide deeper insights into the biological basis of cuticle deposition. This is also an accurate study to describe the uterus transcriptome and proteome during the process of chicken eggshell cuticle formation, providing a reference for further improving the eggshell cuticle deposition in laying hens.

Results
Cuticle opacity of eggs from GC and PC hens for transcriptomic and proteomic analysis Earlier observations of present study showed that the cuticle quality (α value) may have great variability in the same individual. Therefore, cuticle quality of eggs produced by the experimental ock was monitored for the 14 consecutive laying days to obtain individuals that stably produced eggs with good or poor cuticle deposition (GC or PC) for subsequent sampling and analysis. The cuticle deposition was more abundant in the GC group versus the PC group as indicated by a signi cant difference in α value (GC 30.18 ± 7.62 vs PC 10.34 ± 3.67 α value; n = 7 and 4, respectively; F1,9 < 0.001). In other words, the GC group hens had a strong ability for cuticle deposition whilst the PC groups hens had almost no secretion of cuticle. MST cuticle blue dye results of eggs taken from uterus of GC and PC hens during sampling demonstrated that the uterus of GC group had secreted a remarkable thicker cuticle layer than PC group (Additional le 1: Figure S1). This signi cant difference of the cuticle deposition phenotype was in line with expectations that the tissue samples obtained in present study could be used for subsequent analysis.

Analysis of uterus Transcriptomic data of GC and PC group
Seven and four biological replicate samples of uterus from GC and PC group hens during cuticle formation were analysed. A total of 590,111,516 clean reads were generated from the eleven libraries of GC and PC group (Additional le 2: Table S1). The reads feature summary indicated that the percentage of reads mapped to Gallus gallus genome was ≥ 85% (Additional le 3: Table S2). Differential gene expression analysis showed 53 differentially expressed genes (DEGs) (with 26 genes |log 2 fold change| >1) between the GC and PC group during cuticle formation. Among the 53 DEGs, there were 25 upregulated and 28 down-regulated genes at the PC group relative to the GC group. A full list of the 53 DEGs was shown in Table 1.
The heatmap of the 53 DEGs between GC and PC group was plotted and the pattern of expression for the 53 DEGs was visualised in Fig. 1, showing there were signi cant differences in the expression patterns of the 53 DEGs between GC and PC group. Veri cation of gene expression differences by quantitative real-time PCR (qPCR) analysis To validate the transcriptomics results, the relative expression of four genes [ADM (Adrenomedullin), PLCG2 (Phospholipase C gamma 2), PRLR (Prolactin receptor) and PTGDS (Prostaglandin D2 synthase)] to GAPDH were determined in GC and PC group by qPCR on the basis of their known or potential in uence on the hen uterus functions during cuticle secretion. The four genes were all up-regulated in PC group relative to GC group ( Table 1). The qPCR results showed a highly similar expression pattern compared with the RNA-Seq analysis for the genes being validated though there was slight difference in the magnitude of changes in gene expression analyzed by RNA-Seq and qPCR (Table 1; Fig. 2). The qPCR results suggested that the RNA-Seq data obtained in present study provided a good reference for the study of gene expression differences in the uterus of GC and PC group during cuticle formation.
Proteomic analysis of uterus of GC and PC group TMT labeling based proteomic analysis of uterus samples of GC and PC group hens (n = 4 and 3, respectively) during cuticle formation was further performed to identify differentially expressed proteins (DEPs). The peptide detection results of the TMT sequencing showed the length of the peptides is in a partial normal distribution with a mean value of 11, and the main length is between 8-13, which is reasonable for subsquent proteomic analysis. The proteomic analysis showed no DEPs were detected between the uterus samples from GC and PC group hens.
Laying interval, laying performance and serum hormone levels of GC and PC group hens In order to further verify the results above and explore factors that affect cuticle deposition, laying interval between two consecutive ovipositions, laying performance during the twenty-seven days and concentrations of two serum hormones related to the RNA-Seq and qPCR results were determined in another larger White Leghorn laying hen ock. Signi cant differences were found in the laying interval and laying performance between GC and PC group that the GC group hens had a longer laying interval and better laying performance compared to those of PC group (P < 0.05) (Fig. 3, Table 2). The concentrations of prostaglandin D2 and oxytocin of PC group hen serum at 1 h before oviposition were higher than those of GC group, which were consistent with the RNA-Seq and qPCR results, and then the concentrations of the two hormones decreased after oviposition, but the statistical differences were not signi cant (P > 0.05) (Fig. 4).

Egg quality between GC and PC group hens
Egg quality between GC and PC group hens were further measured to investigate possible reasons for the differences in laying interval and laying performance between the two groups. The results of egg quality indicated that there were signi cant differences in eggshell thickness (EST), eggshell strength (ESS) and eggshell weight between GC and PC group hens (P < 0.05), and other egg quality traits were not signi cantly different, suggesting the differences between GC and PC eggs were mainly re ected in the eggshell quality (Table 3). Observations and measurements of the eggshell ultrastructure by scanning electron microscope (SEM) also visually showed the huge difference in the cuticle phenotype of GC and PC group hens (Fig. 5). Further, the thicker EST of GC group was largely derived from the increase in thickness of the effective layer (including the cuticle layer) (P < 0.05) ( Fig. 5; Table 4).   3 Effective layer thickness is the combined thickness of the palisade, vertical crystal and cuticle layer.

Discussion
Egg formation in avian reproductive tract is strictly regulated by the hormons and gene expression [20,22], and the cuticle layer is the last process of egg formation in most avian species [12], however, the genetic regulation of cuticle deposition is still poorly understood. Considering the importance of eggshell cuticle and the genetic improvement of this trait in poultry production, present study were conducted by analyzing the DEGs and DEPs of the uterus between GC and PC group hens to elucidate potential genes and networks that regulate cuticle deposition. Besides, results of qPCR and a series of phenotypic measurements validated the RNA-Seq data. The current work not only picturised the differential expression pro le of the uterus of GC vs PC group hens during eggshell cuticle formation, but also revealed important genes affecting cuticle deposition.
DEGs obtained in the transcriptome analysis of the uterus of GC and PC group hens during cuticle formation (1 h before oviposition) are very likely to be genes that regulate cuticle formation. Based on the results of DEGs, multiple genes (such as PTGDS, PLCG2, ADM and PRLR) related to uterine functions and reproductive hormones were found. The qPCR results further validated the DEGs that the gene expression levels of PTGDS, PLCG2, ADM and PRLR in the uterus of PC group hens were all higher than those of GC group, suggesting their vital roles during cuticle formation.
PTGDS is one of the prostaglandin synthases [31], and it's also an important catalytic enzyme for the synthesis of prostaglandins [32,33]. Studies on humans and mice have demonstrated that prostaglandins are involved in sleep induction [34,35], intake behavior [36] and reproduction processes [37]. Prostaglandins are mostly used to induce parturition in clinical practice. In poultry, injection of prostaglandins can be used to control the uterine contractility and then induce premature oviposition [12,38,39].
PLCG2, which is a type of the phospholipase C (PLC), is an important mediator of oxytocin to regulate uterine contractions [40]. One of the important functions of oxytocin is to stimulate the contractions of myometrium during parturition in mammals and birds [41][42][43]. Oxytocin directly binds to the oxytocin receptor, which is subsquent activated and coupled to the G protein, to activate the PLC on the intracellular membranes, activating the in ux of calcium and nally regulating contractions of the myometrium by voltage [40,[43][44][45][46]. In addition, another function of oxytocin is to increase the synthesis of prostaglandin by stimulating the activity of endometrial prostaglandin synthetase, and then the synergetic effect of prostaglandin and oxytocin enhances the activity of myometrium, thereby inducing labor or oviposition [44][45][46][47][48][49][50][51].
ADM is a peptide with several functions, including vasodilation, regulation of hormone secretion, promotion of angiogenesis, regulation of follicular growth and luteinization of ovarian follicles [52]. In the labor activities of human and mice, ADM in the uterus binds to the receptor and enters the cell to participate in the relaxation of myometrium [43]. Though the relaxation function of ADM is opposite to the contractile effects of oxytocin and prostaglandin in myometrium, ADM could cooperate with oxytocin and prostaglandin to participate in the rhythmical contraction and relaxation of the myometrium [43,48,53,54].
Prolactin (PRL), secreted from the anterior pituitary, plays a series of roles in osmoregulation, corpus luteum formation and maintenance of broody behaviour in laying hens, and its receptor, PRLR, plays an important role in the PRL signal transduction cascade and cell growth and differentiation [55]. The PLR and PRLR genes are expressed in many tissues including the hypothalamus, ovary and oviduct [56,57], and mediate the formation of egg quality [58]. PRL is generally considered to be a key factor in the onset and maintenance of broody behaviour in birds, and it has been well established that the elevated plasma PRL inhibits gonadotropin release, ovum development and ovulation, resulting poor laying performance and even complete cessation of egg production in laying hens [59][60][61][62]. Transcriptome studies have also revealed PRL is associated with egg production performance in chicken hypothalamic-pituitary-ovarian axis [63,64]. Therefore, the signi cant difference in eggshell quality (EST, ESS, eggshell weight, and cuticle opacity) and egg production performance between GC and PC group hens may be related to the different expression patterns of PRLR gene in the uterus. Previous study has shown the inhibitory effects of excessive PRL on eggshell formation [62]. Gonadotropin-releasing hormone (GnRH) is considered to be an important factor in promoting the cuticle deposition [12], however, the elevation of PRL can signi cantly inhibit the secretion and function of GnRH [55]. Besides, environmental stressors would negatively affect the cuticle deposition with an increased PRL secretion [12,65]. Follicle maturation, ovulation and egg formation are physiological events involving multiple organizations and processes, thus, the physiological difference of the hen uterus alone seems to be insu cient to explain its impact on egg production performance. Considering the inhibitory effects of PRL on eggshell formation and egg production, it's speculated that the PRL level and PRLR gene expression level in the hypothalamopituitary-gonadal-oviduct axis are higher in the PC group hens. However, the effects of PRL and PRLR on hen uterine function are still unclear.
Summarizing the functions of PLCG2, PTGDS, ADM and PRLR expressed in the uterus, their express patterns in the uterus regulate the muscular activities and secretion function, which may lead to the difference in egg-laying rhythm between GC and PC group hens. The relatively high expressions of PLCG2, PTGDS and ADM genes could increase the frequency and intensity of contraction and relaxation activities of the myometrium that might negatively affect uterine functions and eventually facilitate oviposition. Combined the results of RNA-seq and qPCR analysis, in other words, hens with good cuticle deposition are more likely to oviposition accompanied by a extended duration for the egg staying in the uterus. This point of view was con rmed by the observations during the experiment, that is, in order to more accurately collect the uterine tissue samples during the cuticle secretion period, in the process of estimating the oviposition time of the hens, it was found that the laying interval between two consecutive ovipositions of the GC group was longer. The difference in laying interval between GC and PC group hens was further con rmed by the larger experimental ock, namely the laying interval between two consecutive ovipositions of GC group hens was signi cantly longer than that of PC group by 0.7 h (Fig. 3). At the same time, the concentrations of prostaglandin D2 and oxytocin of PC hen serums at 1 h before oviposition were higher than those of GC group hens though the statistical differences were not signi cant (P > 0.05). There was also a tendency that the concentrations of prostaglandin D2 and oxytocin of hen serum after oviposition were decreased, implying their important role in regulating uterine functions. The statistically insigni cant differences may be due to the large difference in physiological status between individuals and the short biological half-life of oxytocin and prostaglandin [66,67], however, the serum hormone levels was consistent with the RNA-Seq and qPCR results. The cuticle only begins to secrete 2-1.5 h before oviposition, and the secretion stops after the egg enters the vagina, so the secretion duration is relatively short [12]. The results above indicated that during the cuticle secretion period in the uterus, the frequency and intensity of myometrium contraction and relaxation in GC group hens might be lower than that of PC group hens, which might extend the duration of the cuticle formation, and then lead to the good cuticle quality.
There were no DEPs between the uterus samples of GC vs PC group hens analyzed by the proteomic analysis, implying there was no signi cant difference in the protein composition and content to a large extent. Alternative hypothesis is that undetected DEPs may be due to the extensive posttranslational modi cation regulation in the biological process [68,69]. On the other hand, the extremely short half-life of the hormones (e.g. oxytocin) may be the reason why the DEPs were not found [66,67,70]. The expression characteristics of the transcriptome and proteome between GC and PC group are highly similar (53 DEGs and no DEPs), suggesting that the biological processes of cuticle formation of the two groups may be the same, but mainly the difference in the duration of cuticle secretion.
Therefore, the duration of cuticle secretion in the uterus may be a major factor affecting cuticle quality. However, from ovulation to oviposition, it takes about 24 hours for the formation of a complete egg in chicken [10,20]. A complete egg includes egg yolk, egg white, eggshell membranes, calci ed eggshell and cuticle formation. The forming egg stays in different segments of the oviduct (i.e., infundibulum, magnum, isthmus and uterus) for different duration, and the egg remains in the uterus for the longest period during shell and cuticle formation, for a duration over 18 h in laying hens [10,20,71]. Thus, it is not clear whether the longer laying interval in GC group hens is entirely derived from the extended cuticle secretion time. The egg quality measurement results showed that there were no signi cantly differences in the egg weight, yolk weight, yolk color, albumen height, haugh unit and egg shape index between GC and PC group. Surprisingly, the eggshell quality (EST, ESS, eggshell weight, and cuticle opacity) of GC group were signi cantly increased compared with PC group, further suggesting that the the longer laying interval in GC group was mainly due to the extended formation duration of the eggshell in the uterus. The eggshell comprises four morphologically distinct layers that are formed sequentially, starting with the innermost mammillary layer, followed by the palisade, the vertical crystal layer and the cuticle [10].
Eggshell ultrastructure of GC and PC group eggs by SEM shows the signi cantly increased EST of GC group is largely due to the increase in the thickness of the effective layer (palisade, vertical crystal layer and cuticle), demonstrating the extended laying interval of GC group hens both positively affects the calci ed shell and cuticle formation. EST and ESS traits are highly correlated in phenotype and genetic [58,72]. The effective layer thickness is the major ultrastructural characteristic compromising ESS [73].
Eggshell formation takes place in the uterus over an 18 h period and the laying interval between ovipositions were positively correlated with the duration of eggshell formation, eggshell deposition rate and eggshell quality, and in detail, a difference of 0.7 hours between egg laying intervals could signi cantly affect eggshell weight and eggshell deposition rate by about 30% [71]. Consequently, in present study, the prolonged laying interval by 0.7 h of GC group hens could explain the signi cant increase in the effective layer and cuticle layer thickness.
It is hypothesized that extended duration of eggshell formation and better secretion function of the uterus simultaneously promote the formation of good eggshell and cuticle quality. The heritability of cuticle trait is 0.27-0.40 [8,19,74]. Previous studies have also shown the cuticle quality is weakly positive correlated with the EST/ESS (< 0.2 in phenotypic and genetic correlation) in several commercial laying hen ocks (brown-shelled, white-shelled and tinted-shelled) [17,74]. The results of present study directly prove that eggs with good or poor cuticle generally embody thicker or thinner EST that the top (good) and tail (poor) of the cuticle deposition distribution ampli ed the difference in eggshell quality traits. It's suggested that eggshell calci cation is precisely regulated by the interaction processes between mineral and organic precursors under gene expressions, including transcellular and paracellular transport of minerals and the secretion of different matrix proteins [28,[75][76][77][78]. The processes establish the eggshell texture and ultrastructure, and then in uence eggshell quality traits [78][79][80]. Eggshell matrix proteins (e.g., ovocleidin-17, ovocleidin-116, ovocalyxin-32, ovocalyxin-21 and ovalbumin) present in both the calci ed eggshell and the cuticle, and eggshell matrix proteins constitutes the main components of the cuticle [78,81,82].
Previous transcriptomic study in the uterus of laying hens that produce eggs with good or poor eggshell quality (EST/ESS) demonstrated the most of identi ed DEGs were involved in eggshell calci cation (ion transport) and cuticularization (matrix proteins) pathways [83]. It was found that the expression of FGF13 ( broblast growth factor 13) is up-regulated in the uterus of PC group as compared to GC group (Table 1).
FGF13 is a well-known growth factor belonging to the FGF family and is the modulator voltage-gated Na + , Ca 2+ and K + channels [84,85]. FGF13 could increase voltage-gated Na + and Ca 2+ channel availability [84,86], and it's suspect that the up-regulated FGF13 also increased the uterine contractility. Simultaneously, there is also a signi cant association between ovocleidin-116, ovocalyxin-32 and ovalbumin genes with EST and cuticle deposition [8,29]. Thus, the poor eggshell and cuticle qualities are likely mediated by dysregulation of both ion channels and matrix proteins during eggshell formation, which may also caused by the high expressions of PTGDS, PLCG2, ADM and PRLR genes that the uterine functions are negatively affected by the muscular activities based on the voltage-gated ion channels. To put it simply, during the eggshell formation period, the uterine secretion environment of GC hens is more stable, which ensures the duration for eggshell formation and thus the eggshell and cuticle quality.
The difference in eggshell quality and laying interval between GC and GC group may result from the difference in the uterine biological clock rhythm. Generally, hens lay eggs at intervals precisely controlled by the hormonal secretion and gene expression of the hypothalamo-pituitary-gonadal-oviduct axis. In laying hens, during the maturation of follicular, ovulation, and formation of the egg, the coincidence of the different physiological processes in time demonstrates that the laying rhythm is regulated by the endogenous circadian clock [21]. The circadian clock is a major regulator of a wide range of behavioral and physiological processes including metabolism, food intake, sleep, body temperature, endocrine, immune, reproductive systems and orchestrates rhythmic gene expression in diverse organisms to maintain consistency between body metabolism and the external environment [87,88]. In chickens, clock genes include PER, CRY, BMAL and CLOCK genes, which are expressed in several organisms including the central circadian clock (the pineal, the retina and the hypothalamus) and the peripheral circadian clock (almost all tissues throughout the body), is essential for synchronizing gene expression of key metabolic pathways to coordinate the time course of physiological and behavioral processes [89,90]. In present study, the difference in laying interval of GC and PC group hens is mainly due to the difference in the calci ed eggshell and cuticle formation period in the uterus, showing the uterine clock may be a main factor affecting the cuticle deposition as the eggshell formation is directly regulated by the uterine clock [91]. This inference is supported by earlier research, that is, clock gene expression in the uterus during shell formation may be responsible for controlling the cuticle deposition, and PER2, CRY2, CRY1, CLOCK and BMAL1 genes were differentially expressed when cuticle deposition was prevented by arginine vasotocin [30]. The contractile responses of the uterus to prostaglandins and arginine vasotocin play an important role in inducing oviposition [92]. Arginine vasotocin and prostaglandin is thought to mediate the brain to ovary signalling of oviposition timing being involved in cuticle deposition, and the premature oviposition induced by arginine vasotocin and prostaglandin signi cantly reduce the EST and cuticle deposition [12], which supports the results of present study. The transcriptome results of present study also captured genes that regulate uterine muscular activities such as PLCG2, PTGDS, and ADM, however, clock genes and proteins were not detected in the transcriptome and proteome analysis, which could be due to the half-life of clock genes and proteins that would not be apparent in the transcriptome and proteome [30,93]. Therefore, genes related to uterine timing mechanism and muscular events constitute the components of cuticle deposition regulation, but the regulatory networks need further research.

Conclusions
By analyzing the transcriptome and proteome of the uterus from hens that produced eggs with good or poor cuticle deposition, PTGDS, PLCG2, ADM and PRLR genes were discovered and might play crucial roles in cuticle deposition by affecting uterine secretion rhythm and function. Compared with the PC hens, the relatively low expression of these genes in the uterus ensured the eggshell and cuticle formation duration, and lead to the good eggshell and cuticle quality of the GC hens. Moreover, eggshell quality and laying performance of hens that produce eggs with good cuticle are better, implying the importance of genetic selection to the cuticle trait in laying hens. This study also hints at the relationship between the cuticle formation and the endogenous circadian clock, which might provide a basis for subsequent poultry production improving the cuticle quality by improving exogenous daily inputs (e.g., photoperiod, nutrition).

Ethical statement
All the experimental procedures were conducted in accordance with the Guidelines for Experimental Animals established by the Animal Care and Use Committee of China Agricultural University (permit number: AW08059102-1). Animal studies were reported in compliance with the ARRIVE guidelines [94].

Experimental design
One 28-week-old White Leghorn laying hen ocks (n=208) were used in present study for omics analysis and then another 28-week-old White Leghorn laying hen ocks (n=574) were choosed for further veri cation tests. There was no genetic selection for the cuticle trait of the two ocks. Hens that stably produced eggs with good and poor cuticle (GC and PC, the top and tail of the cuticle deposition distribution) were obtained by evaluating the cuticle quality. According to the ARRIVE guidelines [94], in the preliminary experiment of present study, the differences of the top and tail of the cuticle deposition distribution of the two ocks were signi cant large to obtain enough GC and PC hens for the experiments (GC group hens had a strong ability for cuticle deposition whilst the PC groups hens had almost no secretion of cuticle). Transcriptomic, proteomic and qPCR analysis of GC and PC hen uterus tissues during cuticle deposition were designed for identifying genes involved with eggshell cuticle deposition.
Subsequently, phenotypic measurements (cuticle opacity, laying interval, laying performance, serum hormone level, egg quality) of GC and PC group hens were carried out to further verify the results above.
Animals and uterine tissue collection Two hundred and eight White Leghorn hens at 28-week-old were selected form the National Poultry Testing Center, China Agricultural University. All hens were caged individually under standard conditions with a photoperiod of 16L:8D, and the diet and water were provided ad libitum.
To obtain individuals that stably produced eggs with good or poor cuticle (GC or PC) for subsequent sampling, the eggs were collected for 2 weeks to measure the cuticle quality within 24 h after oviposition. The cuticle quality evaluation was conducted according to the staining method proposed by Chen et al.
previously [17]. Brie y, the cuticle opacity (α value) was measured based on differences in cuticle staining before and after staining with a dye solution of MST cuticle blue (MS Technologies Ltd, UK) using a spectrophotometer (CM-2600d; Konica Minolta, Japan) with the XYZ color space system. A higher α value represents to more cuticle deposition, that is better cuticle quality. Each egg was measured at 3 points: the large end, equator, and small end. Cuticle quality per egg was determined from the mean value of these points and at least 8 eggs were collected for cuticle quality measurement per hen.
Oviposition Raw data was quality controlled using the FastQC package (Babraham bioinformatics, Cambridgeshire, England). Clean reads were obtained by removing reads containing adapter, reads containing ploy-N, empty reads and low quality reads from raw data. Trimmed reads were mapped on the reference chicken genome Gallus_gallus_ncbi_GCF_000002315.6_GRCg6a (https://www.ncbi.nlm.nih.gov/assembly/GCF_000002315.6) using HiSAT2.0 [95,96]. The clean reads of each sample were assembled and nally merged to a transcriptome using Stringtie (http://ccb.jhu.edu/software/stringtie/) [95] with Ensembl Gallus gallus.v92 as the reference. Expression levels of the transcripts were quanti ed as RPKM (reads per kilobase per million) for gene expression analysis.
DEGs were identi ed using DESeq2 [97] according to the cretiera of adjusted P-value < 0.05. Ensembl gene IDs from each group were uploaded to the DAVID Functional Annotation Tool and analysed for gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment [98,99]. The transcriptome data (raw mRNA-seq reads) have been deposited with the National Center for Biotechnology  Finally, fractionated peptides were exposed to an NSI source followed by tandem mass spectrometry (MS/MS) with a Q Exactive HF-X spectrometer (Thermo Fischer Scienti c, MA, USA) coupled online to the UPLC system.
The MS/MS data were searched against the Gallus gallus database (http://www.uniprot.org/proteomes/UP000000539, chicken proteome ID: UP000000539) for peptide identi cation and quanti cation using the Maxquant search engine (v.1.6.6.0). All proteins with at least one unique peptide and false discovery rate (FDR) < 0.01 were quali ed for further quanti cation data analysis. Protein abundance was quanti ed by hi-yer through loading (SL), internal reference scaling (IRS) and trimmed mean of M values (TMM) corrections in the Maxquant search engine system. DEPs between GC and PC group were identi ed using limma [100] and P < 0.05 was used as the cut-off for signi cance by FDR correction.
Quantitative real-time PCR (qPCR) Expression of mRNA was veri ed by qPCR with cDNA from 11 uterine tissue samples. GAPDH gene served as a housekeeping gene. Primer Primer (v. 5.0) was used with default parameters to design primers on exon-exon spans for selected genes (Additional le 4: All eggs produced by this ock were collected, and cuticle opacity measurement and oviposition time recording were in accordance with the rst experiment for seven successive laying days. On the sixth day, hens that stably produced eggs with good and poor cuticle quality (GC and PC) were selected and their oviposition time on the seventh day were estimated. On the seventh day, whole blood samples were collected from the brachial veins of the GC and PC group hens at 1 h before oviposition, and the blood samples were also collected from the GC hens at 0.5 h after oviposition (laid) (n=7). All the eggs were also collected after oviposition for validation by cuticle opacity measurement. The blood sample was collected only once per chicken and the cuticle quality and oviposition time were in line with expectations.
Laying interval between two consecutive ovipositions was calculated (at least 4 ovipositions per hen). Laying recording sheet during the 27 consecutive laying days from 28-week-old age was obtained to evaluate laying performance (i.e., total laying days, total sequences, mean sequence length and intersequence pause length). Concentrations of prostaglandin D2 and oxytocin of the blood serum were subsequently determined by chicken prostaglandin D2 (PGD2) and oxytocin (OT) ELISA kits (mlbio, Shanghai, China) according to the manufacturer's protocols.
After cuticle opacity measurement, other egg quality traits of GC and PC group hens (n=30) including egg weight, eggshell thickness (EST, mean of the large end, equator, and small end without eggshell membranes), eggshell strength (ESS), yolk weight, albumen height, yolk colour, haugh unit and egg shape index were determined using an egg multitester (EMT-5200, robotmation, Japan), an Eggshell Strength Tester (EFG-0503, robotmation, Japan) and a digital display micrometer gauge (Mitutoyo, Kawasaki, Japan) following the manufacturer's instructions. Eggshell pieces (about 1 cm 2 ) of the GC and PC group hens (n=30) were taken around the equator of the egg and the pieces were mounted on an aluminum stub and gold sputter-coated using an EIKO IB-3 (EIKO Engineering CO., Ltd, Japan) for about 15 min.
Thereafter, they were subjected to the SEM (JSM-7401, JEOL Ltd., Japan) for observations, photographing, and measurements of the eggshell cross-section ultrastructure. The eggshell crosssection ultrastructure in present study refers to eggshell thickness (EST), effective layer thickness (combined thickness of the palisade, vertical crystal and cuticle layer) and mammillary layer thickness, and the determination were consistent with previous studies [29,102].

Statistical analysis
Statistically signi cant differences among descriptive statistics were determined by the Duncan's test. All statistical analysis and gure plotting were performed with the statistical software RStudio (IRT 15R62). The funders did not involve in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials
The transcriptome data (raw mRNA-seq reads) of present article data are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number PRJNA664894 (https://www.ncbi.nlm.nih.gov/sra/PRJNA664894).

Ethics approval and consent to participate
All the experimental procedures were conducted in accordance with the Guidelines for Experimental Animals established by the Animal Care and Use Committee of China Agricultural University (permit number: AW08059102-1).

Consent for publication
No applicable.

Competing interests
The authors declare that they have no competing interests.   Validation of the DEGs of GC vs PC hen uterus during cuticle deposition from the transcriptomic analysis.
The qPCR was performed to quantify the the relative gene expression level based on the 2−ΔΔCT method. For gene expression data normalization, GAPDH was used as a reference gene. Fold changes between the GC (good cuticle) and PC (poor cuticle) group were calculated for the genes ADM, PLCG2, PRLR and PTGDS. The Y axis shows the fold changes.
Page 30/32 The concentrations of prostaglandin D2 and oxytocin in hen serum. GC, good cuticle quality hen 1 h before oviposition; PC, pore cuticle quality hen 1 h before oviposition; Laid, good cuticle quality hen 0.5 h after oviposition.