TMT-based quantitative proteomic analysis reveals eggshell matrix protein

changes correlated with eggshell quality in Jing Tint 6 laying hens of different
ages

Dan-rong Zhao,*,y Li-bing Gao ,y Fei Gong,* Jia Feng,* Hai-jun Zhang,y Shu-geng Wu,y
Jing Wang ,y and Yu-na Min *,1
*
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China; and yKey
Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Laboratory of Quality & Safety Risk
Assessment for Products on Feed-origin Risk Factor, Ministry of Agriculture and Rural Affairs, Institute of Feed
Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China

ABSTRACT The decline in eggshell quality resulting
from aging hens poses a threat to the financial benefits
of the egg industry. The deterioration of eggshell quality
with age can be attributed to changes in its ultrastruc-
ture and chemical composition. Specific matrix proteins
in eggshells have a role in controlling crystal growth
and regulating structural organization. However, the
variations in ultrastructure and organic matrix of egg-
shells in aging hens remain poorly understood. This
study assessed the physical traits, mechanical quality,
chemical content, as well as the microstructural and
nanostructural properties of eggs from Jing Tint 6 hens
at 38, 58, 78, and 108 wk of age. Subsequently, a quanti-
tative proteomic analysis was conducted to identify dif-
ferences in protein abundance in eggshells between the
ages of 38 and 108 wk. The results indicated a notable
decline in shell thickness, breaking strength, index, frac-
ture toughness, and stiffness in the 108-wk-age group
compared to the other groups (P < 0.05). The
Key words: eggshell quality, ultrastructure, matrix proteins, proteomics
ultrastructure variations primarily involved an
increased ratio of the mammillary layer and a reduced
thickness of the effective layer of eggshell in the 108-wk-
age group (P < 0.05). However, no significant differen-
ces in eggshell compositions were observed among the
various age groups (P > 0.05). Proteomic analysis
revealed the identification of 76 differentially expressed
proteins (DEPs) in the eggshells of the 38-wk-age
group and 108-wk-age group, which comprised proteins
associated with biomineralization, calcium ion binding,
immunity, as well as protein synthesis and folding. The
downregulation of ovocleidin-116, osteopontin, and cal-
cium-ion-related proteins, together with the upregula-
tion of ovalbumin, lysozyme C, and antimicrobial
proteins, has the potential to influence the structural
organization of the eggshell. Therefore, the deteriora-
tion of eggshell quality with age may be attributed to
the alterations in ultrastructure and the abundance of
matrix proteins.
2024 Poultry Science 103:103463
https://doi.org/10.1016/j.psj.2024.103463

INTRODUCTION quality eggshells throughout the egg production cycle.

Lower eggshell quality can lead to egg damage during
transportation and storage, which greatly impacts the
economic benefit of egg industry (Bain et al., 2016; Fathi
et al., 2019). The financial loss caused by broken and
cracked eggs at the end of the laying period accounts for
approximately 12 to 20% of the total loss (Travel et al.,
2011). Therefore, it is important to maintain high-
Ó 2024 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Received November 8, 2023.
Accepted January 10, 2024.
1
Corresponding author: minyuna2003@163.com
The shape, size, thickness, and material distribution of
the eggshell all contribute to its quality and mechanical
properties (Ma et al., 2020). As hens age, there is a
decline in eggshell quality, characterized by increased
egg weight but decreased shell thickness and index
(Rayan et al., 2010; Feng et al., 2020).
The physical properties of the eggshell result from its
unique structure, which is a bio-ceramic formed by the
highly ordered precipitation of minerals (mainly calcium
carbonate, CaCO3, » 95%) and organic matrix (»

3.5%) (Ketta and Tumov
a, 2016; Nys et al., 1999). The
calcified part of eggshell consists of the cuticle, effective
layer (palisade and vertical crystal layer), and mammil-
lary layer, with the effective layer being the main load-

1
2 ZHAO ET AL.
bearing part that prevents crack propagation (Radwan,
2016). The composition and distribution of these layers
are closely related to the mechanical performance of the
eggshell. It was reported that the eggshell strength was
positively correlated with the ratio of the effective layer
and mammillae density but was negatively correlated
with the ratio of the mammillary layer and width of the
mammillary knob (Carnarius et al., 1996; Hincke et al.,
2011). Furthermore, the eggshell structure at the nano-
meter level has been observed and it occurs through the
interaction between inorganic and organic components,
which affects the eggshell mechanical properties (Lam-
mie et al., 2005; Rodríguez-Navarro et al., 2015). In
nanomaterials, hardness tended to increase as the sub-
unit size decreases (Naik and Walley, 2020; Wu et al.,
2020). The eggshell distinct regions displayed varying
nanostructures, with the smallest nanoparticles found in
the outermost vertical crystal layer and upper palisade
layer. This reduction in particle size was inversely associ-
ated with the increase in eggshell hardness and elastic
modulus (Athanasiadou et al., 2018).
Additionally, the composition of the eggshell, including
inorganic calcium (Ca), phosphorus (P), and organic
matter, plays a crucial role in determining the material
performance of the eggshell. Various components of the
organic matrix, including proteins, polysaccharides, and
proteoglycans, are secreted into the uterine fluid to
accomplish the eggshell biomineralization (Gautron et
al., 1997; Brionne et al., 2014). The organic constituents
in eggshells play a vital role in controlling crystal growth,
shaping, and determining the mechanical performance of
the material (Addadi and Weiner, 1992; Hern
andez-
Hern
andez et al., 2008; Le Roy et al., 2021). Proteomic
analysis has identified over 900 proteins in the eggshell
matrix, prompting investigations into their distribution
across different structural layers and their related func-
tions (Mann et al., 2006; Marie et al., 2015a; Gautron,
2019; Yang et al., 2020). The variation in matrix proteins
in eggshells, which exhibit different mechanical perform-
ances, has become the focus of exploring the mechanisms
underlying these proteins. Previous studies have demon-
strated an increase in the amount of matrix proteins,
such as ovalbumin (OVA), ovotransferrin, and ovoclei-
din-17, in weakened eggshells from aging hens (Panh
eleux
et al., 2000). After molting, there was observed improve-
ment in eggshell quality and a reduction in crystal size,
which was attributed to an increase in ovocleidin-116
(OC-116) content in the eggshell gland (Ahmed et al.,
2005). However, comprehensive information on the differ-
ences in eggshell matrix proteins from hens of different
ages is still limited.
This study was performed to explore the change in the
eggshell quality and hierarchical ultrastructure with
increasing age and then we employed a TMT-based com-
parative quantitative proteomics analysis to reveal the
key differential proteins in the eggshell of peak and late
laying hens. Consequently, the study aimed to gain a
better understanding of the underlying mechanisms
involving crucial matrix proteins and to reveal the rea-
sons behind poor egg quality in aged laying hens.
MATERIALS AND METHODS
Animals and Samples
The animal protocol for this study was conducted
under the management of the Animal Care and Use
Committee of the Institute of Feed Research of the Chi-
nese Academy of Agricultural Sciences. A total of 90
healthy Jing Tint 6 (38 wk of age) laying hens were
divided into 6 replicates of 15 hens in a randomized com-
plete block design, and three birds were assigned to 1
cage. All the laying hens were fed with a compound basal
diet and were given water ad libitum. The room temper-
ature was controlled, and artificial light consisted of 16
h of light and 8 h of dark. All laying hens remained in
good health during the feeding period. The 54 egg sam-
ples (3 eggs/replicate/d £ 3 d) were collected from hens
at 38, 58, 78, and 108 wk old respectively. Eggshell sam-
ples were carefully washed and dried at room tempera-
ture for ultrastructural and component analysis.
Egg and Eggshell Quality Assessment
Each egg was weighted and measurements of egg
length (L) and width (W) were taken with an egg shape
index analyzer (FHK, Fujiping Company, Tokyo,
Japan) on the day of sampling. Eggshell thickness was
measured by Egg Shell Thickness Gauge (Israel Orka
Food Technology Ltd., Ramat Hasharon, Israel) and
breaking strength was determined by Egg Force Reader
(Israel Orka Food Technology Ltd., Ramat
Hasharon, Israel). Shell stiffness was measured by a
physical property structure analyzer (TMS-Pro, Ensoul
Technology Ltd., VA) and the fracture toughness of
each egg was calculated according to the formulas devel-
oped by Mabe (Mabe et al., 2003). After removing egg
contents, eggshell was cleaned, dried at room tempera-
ture for 48 h, and weighed. Shell percentage [(eggshell
weight/egg weight) £ 100], egg surface area
(S = 4.68 £ W2/3, where W = egg weight), and eggshell
index [(shell weight/egg surface area) £ 100] were also
calculated.
Analysis of Eggshell Components
The determination of organic matter in the eggshell
was taken by the ignition loss method. The method
involves heating the sample to a high temperature, typi-
cally around 550 to 600°C, which causes the organic
matter to combust and volatilize, leaving behind a resi-
due of inorganic material (Pliya and Cree, 2015).
Approximately 1.0 g eggshell powder from each replicate
was weighed in the crucibles, dried at 110°C for 5 h,
weighed as W1, and then ignition to constant weight by
muffle furnace at 550°C, weighed as W2. The percentage
of organic matter content was calculated as (W1-W2)/
W1 £ 100.
The method used to determine Ca and P contents of
eggshells was based on a previous study (Feng et al.,
2020). The eggshell samples were dissolved in the 3 mL
 EGGSHELL QUALITY-RELATED MATRIX PROTEINS
nitric acid and 3 mL H2O2, and then digested by the
microwave dissolution instrument (MDS-10, Shanghai
Xinyi Instrument Technology Co., Ltd., Shanghai,
China). The Ca and P content of eggshells were tested
with an atomic absorption spectrometer (Zeenit700 P,
Analytik Jena) and a spectrophotometer (UV-2000, Shi-
madzu).
Eggshell proteins were extracted and determined as
previously described (Ahmed et al., 2005). A total of
250 mg of the dried sample per replicate was demineral-
ized with 20% acetic acid. Thereafter, the samples were
mixed with ultrapure water (v:v = 1:1) and lyophilized.
The lyophilized powders were dissolved lasted 12 h at 4°
C in an extraction milieu (containing 4 M guanidine
hydrochloride, protease inhibitors, 0.05 M pH 5.8
sodium acetate, 10 mM EDTA, and 0.5% Tween 20) as
the previous study demonstrated (Gautron et al., 2001)
and the extracts were dialyzed (dialysis bag cut off 3,500
Da). Then, the solutions were centrifugated (2,500 g, 20
min), and the supernatant was collected. Finally, the
total protein content in the eggshell was determined
using the Bradford Protein Assay Kit (PC0010; Beijing
Solarbio Science & Technology Co., Ltd., Beijing,
China).
Eggshell cracking typically originates from a single
point on the surface. The chemical composition per unit
area may be associated with crack propagation. To pro-
vide a more precise representation of the variations in
eggshell components, the content of organic matter, egg-
shell protein, Ca, and P per eggshell area [the content/
egg surface area] were calculated.
Eggshell Ultrastructure
The pieces of shell (about 1 cm2) from the equatorial
region of each egg were evaluated for the ultrastructure
of the internal shell surface and cross-section by scan-
ning electronic microscopy (SEM, SU8020, Hitachi,
Japan). The preparative treatments of eggshell speci-
mens referred to the previous method (Bain, 1990). The
cross-sectional lengths (total thickness of palisade, verti-
cal crystal layer and cuticle) of effective and mammillary
layers and the width of the mammillary knobs were
directly measured in the images photo’d by the SEM at
a magnification of £ 180. Mammillae density was
counted and observed as the number of knobs per unit
by the SEM at a magnification of £ 150.
Eggshell Nanostructure
The pieces of shell samples from the equatorial section
were embedded in epoxy resin vertically, and the top
cross sections were successively ground with 150, 1,500,
4,000, and 8,000 mesh sandpaper, then polished with
0.06 silica suspension. Height and amplitude images of
palisade and mammillary layers were taken using an
atomic force microscopy (AFM, Dimension Icon,
Bruker AXS) operating in tapping mode at room tem-
perature in air with a scanning area of 800
3
nm £ 800 nm, referring to the method of Athanasiadou
(Athanasiadou et al., 2018). The Feret diameter meas-
urements of the nanoparticles observed by AFM were
calculated using Image-J software and a total of 210
Feret diameter measurements of each sample. The Feret
diameter is a descriptor for evaluation of particle size,
which is defined as the longest distance between the two
furthest points of the shape (Drazi
c et al., 2016).
Proteomic Analysis
Every group consisted of 6 biological replicates (n = 6)
in order to minimize individual level variation. Each egg-
shell sample from 38wk and 108 wk groups was ground
in liquid nitrogen, and SDT (4% SDS, 100 mM Tris-
HCl, pH 7.6) buffer was used for sample lysis and protein
extraction. The 20 mg of extracted protein was taken
and mixed with 5 times loading buffer and boiled for
5 min. The proteins were separated on 4%-20% SDS-
PAGE gel (180V, 45 min). Protein bands were visual-
ized by Coomassie Blue R-250 staining. Protein diges-
tion by trypsin was performed according to a filter-aided
sample preparation procedure. The digest peptides of
each sample were desalted on MCX, concentrated by
vacuum centrifugation, and reconstituted in 40 mL of
0.1% formic acid, the quantitative peptide (OD280) was
obtained.
The peptide (100 mg) mixture of each sample was
labeled using TMT reagent according to the instructions
(Thermo Scientific, MA). Labeled peptides were mixed
equally and then fractionated by High pH Reversed-
Phase Peptide Fractionation Kit (Thermo Scientific,
MA). The peptide mixture was reconstituted and acidi-
fied with a 0.1% TFA solution. Following this, it was
loaded onto an equilibrated, high-pH, reversed-phase
fractionation spin column, where the peptides were
bound to the hydrophobic resin under aqueous condi-
tions and desalted through low-speed centrifugation
with water washing. Subsequently, a step gradient of
increasing acetonitrile concentrations in a volatile, high-
pH elution solution was applied to the columns to elute
the bound peptides, resulting in the collection of 10 dif-
ferent fractions by centrifugation. Finally, the collected
fractions were vacuum dried and lyophilized using 12
mL 0.1% FA.
LC-MS/MS analysis was performed on a Q Exactive
mass spectrometer (Thermo Scientific, MA). The pepti-
des were loaded onto a reverse phase trap column
(Thermo Scientific Acclaim PepMap100, 100
mm £ 2 cm, nanoViper C18) connected to the C18-
reversed phase analytical column (Thermo Scientific
Easy Column, length 10 cm, 75 mm inner diameter, 3mm
resin) in buffer A (0.1% Formic acid in water) and sepa-
rated with a linear gradient of buffer B (84% acetonitrile
and 0.1% Formic acid) at a flow rate of 300 nL/min.
The mass spectrometer was operated in positive ion
mode. MS data was acquired using a data-dependent
top20 method dynamically choosing the most abundant
precursor ions from the survey scan (300−1800 m/z) for
4 ZHAO ET AL.

HCD fragmentation. The Automatic Gain Control annotated using the software program Blast2GO (Ash-
(AGC) target was set to 1e6, with a maximum injection burner et al., 2000; G€otz et al., 2008). Following annota-
time of 50 ms. Dynamic exclusion duration was set to tion steps, the studied proteins were blasted against the
30.0 s. Survey scans were acquired at a resolution of online Kyoto Encyclopedia of Genes and Genomes
60,000 at 200 m/z, while the resolution for HCD spectra (KEGG) database (http://geneontology.org/) and
was established at 15,000 at 200 m/z, with an isolation were mapped to pathways in KEGG (Kanehisa et al.,
width of 1.5 m/z. The normalized collision energy was 2012). Enrichment analysis of all data was applied based
maintained at 30 eV, and the underfill ratio, defining on the Fisher exact test and Benjamini- Hochberg cor-
the minimum percentage of the target value reached at rection for multiple testing was further applied to adjust
maximum fill time, was specified as 0.1%. Peptide recog- derived p-values. The protein-protein interaction (PPI)
nition mode was enabled during instrument operation. information of the studied proteins was retrieved from
The raw data for each sample were searched using the the IntAct molecular interaction database (http://
MASCOT engine (Matrix Science, London, UK; version www.ebi.ac.uk/intact/) by the STRING software
2.2) embedded into Proteome Discoverer 2.4 software, (http://string-db.org/) (Szklarczyk et al., 2019). And
and the database search analysis was carried out using the results were visualized by the Cytoscape software
the Gallus gallus database (uniprot_Gallus_gal- (http://www.cytoscape.org/, version 3.10.0).
lus_51560_20230524.fasta). The MS tolerance was set
at § 20 ppm, with a fragment ion tolerance of 0.1 Da.
Trypsin was employed as the digestive enzyme, allowing RESULTS
for up to 2 missed cleavages. All proteins with at least Egg and Eggshell Quality Assessment
one unique peptide and false discovery rate (FDR) ≤
0.01 were qualified for further quantification data analy- The change in egg properties and eggshell parameters
sis. Protein ratios are calculated as the median of only from different aged hen groups (38, 58, 78, and 108 wk
unique peptides of the protein. of age) is presented in Table 1. Compared with the egg
from 38-wk-old hens, egg weight and egg surface area of
78-wk-old hens increased significantly (P < 0.05),
Correlation Analysis Between Proteomic and whereas there were no significant changes in shell per-
Eggshell Traits centage, thickness, breaking strength, and fracture
toughness (P > 0.05). As for the egg of 108-wk-old hens,
The abundance of DEPs in this study and related lit- egg weight, and egg surface area raised considerably (P
erature for functional description provided a basis for < 0.05) and shell thickness, breaking strength, index,
selecting putative proteins. These putative proteins fracture toughness, and stiffness decreased vastly (P <
were subjected to Pearson correlation analysis to deter- 0.05) than those of other groups.
mine their potential relationship with the thickness of
effective and mammillary layers, the calcium contents of
eggshells, and the Feret diameters of nanoparticles in Analysis of Eggshell Components
palisade layers. No significant changes were observed between differ-
ent age groups in the contents of organic matter, Ca, P,
Statistical and Bioinformatics Analyses
Table 1. Comparison of egg properties and eggshell parameters
All analyses were performed using SPSS 20.0 (SPSS of laying hens with different weeks of age (n = 6).
Inc., Chicago, IL). Data were analyzed using one-way Hens age (wk of age)
analysis of variance (ANOVA), and the means were
Items 38 58 78 108 SEM1 P-value
compared using Duncan’s multiple range test among the
Egg weight (g) 54.12 c
58.12 b
58.31 63.06b a
0.390 <0.001
groups. Data were shown as mean with pooled standard Shape index 1.34b 1.37a 1.34b 1.37a 0.004 0.001
error of the mean (SEM). P-values < 0.05 were consid- Surface area 66.93c
70.20b
70.35b
74.12a 0.316 <0.001
ered statistically significant. (cm2)
5.02c 5.51a 5.24b 5.20bc <0.001
Proteins with fold change (FC) in a comparison > 1.2 Shell weight (g)
Shell percentage 9.28ab 9.48a 9.00b 8.24c
0.038
0.064 <0.001
(or < 0.83) and P < 0.05 were considered to be signifi- (%)
cantly different (differentially expressed proteins, Thickness (*102 44.25a 43.59a 43.10a 41.36b 0.285 0.004
mm)
DEPs). Protein domains were identified using the Inter- Breaking 38.68a 37.55a 35.63a 30.30b 0.817 0.002
ProScan software from the InterPro member database strength (N)
Pfam (Finn et al., 2016). Complexheatmap R (R Version Eggshell index 7.49b 7.84a 7.45b 7.01c 0.048 <0.001
2
(g/100 cm )
3.4) was used to perform hierarchical clustering analysis Fracture tough- 321.20a 332.85a 324.13a 272.50b 7.074 0.006
(based on the Euclidean distance algorithm for similar- ness (N/mm3/
2
ity measure and average linkage clustering algorithm). )
Stiffness (N/mm) 71.18a 72.07a 66.94b 60.65c 0.833 <0.001
The protein sequences of the selected DEPs were locally
a,b,c
searched using the NCBI BLAST+ client software and Means within a row with no common superscripts differ signifi-
cantly (P < 0.05).
InterProScan to find homolog sequences, then gene 1
SEM: standard error of means.
ontology (GO) terms were mapped and sequences were
 EGGSHELL QUALITY-RELATED MATRIX PROTEINS 5
Table 2. Comparison of eggshell chemical composition of laying
hens with different weeks of age (n = 6).

Hens age (wk of age)

Items 38 58 78 108 SEM1 P-value
Organic matter 3.46 3.43 3.33 2.97 0.092 0.325
(%)
Organic content 2.60 2.69 2.48 2.08 0.090 0.087
per eggshell
area (mg/cm2)
Eggshell protein 13.66 13.15 13.63 13.54 0.098 0.250
content (mg/g)
Protein content 1.02a 1.03a 1.02a 0.95b 0.013 0.027
per eggshell
area (mg/cm2)
Calcium content 422.03 416.56 418.44 416.55 1.046 0.210
(mg/g)
Calcium content 31.64 b
32.39 a
31.19 c
29.23 d
0.357 <0.001 Figure 1. Eggshell ultrastructure of cross section of laying hens at
per eggshell different weeks of age (scanning electron microscope images, scale bar,
area (mg/cm2) 300 mm, magnification, 180 £). wk, weeks of age; MT, mammillary
Phosphorus con- 1.13 1.13 1.15 1.15 0.004 0.070 layer thickness; ET, effective layer thickness; TT, total thickness.
tent (mg/g)
Phosphorus con- 0.08 0.09 0.09 0.08 0.002 0.121
tent per egg-
shell area (mg/
cm2)
a,b,c
Means within a row with no common superscripts differ signifi-
cantly (P < 0.05).
1
SEM: standard error of means.

and total matrix protein in eggshells (P > 0.05)

(Table 2). However, the total protein per eggshell area
and Ca content per eggshell area of 108-wk-old groups
declined substantially compared with other age groups
(P < 0.05).

Eggshell Ultrastructure
The age-related deterioration in eggshell ultrastruc-
ture is presented in Table 3. The thickness of the total
calcified and effective layer of eggshell decreased signifi-
cantly in 78-wk-age and 108-wk-age groups than egg-
shells of other groups (P < 0.05). Especially, the
proportion of effective layer declined and the proportion
of mammillary layer increased greatly in the 108-wk-age
group compared with others (P < 0.05). Whereas there
was no difference (P > 0.05) in mammillary knob width
among various age groups (Figure 1). Moreover, com-
pared with the 38-wk-old group, the decreased mammil-
lae density of eggshell was found in the 108-wk-age
group (P < 0.05) (Figure 2).
Figure 2. Eggshell ultrastructure of the inner surface of laying hens
at different weeks of age (scanning electron microscope images, scale
bar, 300 mm, magnification, 150 £). wk, weeks of age.
Eggshell Nanostructure
The nanostructure size analysis of different regions in
eggshell is revealed in Table 4, and associated AFM
images are observed in Figure 3. For the nanoparticle
size of palisade layer, the Feret diameter of 108-wk-age
group expanded significantly than those of 78-wk-age
group (P < 0.05). However, no significant difference was
observed among all groups in nanogranules size of the
mammillary layer (P > 0.05).

Table 3. Comparison of eggshell ultrastructure of laying hens with different weeks of age (n = 6).

Hens age (wk of age)

Items 38 58 78 108 SEM1 P-value
Total thickness (mm) 339.30 a
337.07 a
317.64 b
314.96 b
2.086 <0.001
Thickness of mammillary layer (mm) 71.16bc 68.65c 72.61b 79.06a 0.638 <0.001
Thickness of effective layer (mm) 268.95a 267.05a 246.57b 235.55b 2.357 <0.001
Mammillary knobs width (mm) 67.17 67.85 68.06 67.08 0.295 0.563
Mammillary layer (%) 20.79c 20.75c 22.72b 25.25a 0.291 <0.001
Effective layer (%) 79.21a 79.25a 77.28b 74.75c 0.291 <0.001
Mammillae density (1 mm2) 250.13a 244.33ab 241.40bc 235.20c 1.334 0.001
a,b,c
Means within a row with no common superscripts differ significantly (P < 0.05).
1
SEM: standard error of means.
6 ZHAO ET AL.

Table 4. Comparison of the nanostructure size in eggshell varies Functional Analysis of DEPs
layers (palisade and mammillary layers) of laying hens with differ-
ent weeks of age (n = 6). To better realize the functional roles of proteins, the
GO enrichment, KEGG pathway, and protein-protein
Hens age (wk of age)
interaction network analyses were conducted. Gene
38 58 78 108 SEM1
Items P-value Ontology functional enrichment analysis (P < 0.05) was
ab ab b a
Feret diameter of 41.82 41.92 41.26 42.66 0.174 0.026 performed based on biological process (BP), molecular
palisade layer
(nm) function (MF), and cellular component (CC) modules,
Feret diameter of 50.92 50.78 50.30 50.05 0.236 0.541 and all functional classes of DEPs were found in
mammillary Figure 5A. The GO enrichment in BP mainly included
layer (nm)
complement activation, classical pathway
a,b,c
Means within a row with no common superscripts differ signifi- (GO:0006958), humoral immune response mediated by
cantly (P < 0.05).
1
SEM, standard error of means. circulating immunoglobulin (GO:0002455), protein acti-
vation cascade (GO:0072376) and B cell mediated
immunity (GO:0019724), which were primarily related
Proteomic Analysis of Eggshell From 108 wk to immunity. The MF classes were mostly enriched in
misfolded protein binding (GO:0051787), lipoprotein
and 38 wk Groups particle receptor binding (GO:0070325), and molecular
TMT-based quantitative proteomics of eggshells was function regulator (GO:0098772) which was composed
performed to analyze the different matrix proteins of 12 proteins. The key CC was the apical junction com-
between hens at two wk of age. In the LC-MS/MS analy- plex (GO:0043296).
sis, we observed the average peptides mass error was < The pathways of KEGG enrichment analysis per-
10 ppm, which is reasonable for subsequent proteomic formed for upregulated and down-regulated proteins
analysis. Overall, 9808 peptide spectrum matches respectively were further demonstrated (Figure 5B).
(PSMs) were matched to 2,516 peptides, of which 2,131 The upregulated protein annotation was enriched in oxi-
were unique. A total of 655 proteins were identified from dative phosphorylation, adherens junction, cytokine-
the two groups, and there were 43 significant upregu- cytokine receptor interaction, and proteasome. Whereas,
lated and 33 significant down regulated proteins (P < the ECM-receptor interaction, focal adhesion, MAPK
0.05, FC > 1.2) in the 108wk group compared to the 38 signaling pathway, and lysosome were downregulated.
wk group (Figure 4A). The detailed information about Using the STRING database and Cytoscape software,
these DEPs is provided in Table S1, and all identified the protein interaction networks were constructed for
and quantified proteins are listed in Table S2. The VH1, the DEPs (Figure 6). The PPI networks indicated 41
protein TENP, avidin, lysozyme C, ovalbumin, Immu- proteins, of which 20 were up-regulated and 21 down-
noglobulin lambda-like polypeptide 1, ovodefensin A1 regulated. The crucial proteins that closely interacted
and avian beta-defensin 11 were regarded as high abun- with other DEPs had greater connectivity and impor-
dance proteins within the differentially expressed pro- tance, like osteopontin (OPN) (14 interactions), apoli-
teins (FC > 1.5). To analyze the change in protein poprotein AI (11 interactions), OC-116, lysozyme C
abundance of inter-group and intra-group samples, hier- (LYZ) and OVA (all had 4 interactions).
archical cluster analysis was used to classify the DEPs of To speculate on all proteins that might be associated
the comparison groups. In the result, the expression pat- with the decline of eggshell quality, every DEPs was
terns of DEPs were similar within the intra-group sam- searched and analyzed from related databases and
ples, suggesting that a significant difference in protein research. Among all the DEPs, 15 proteins were found
abundance was only present between the two groups to be more essential for the information of eggshell struc-
(Figure 4B). ture and thus affected the eggshell quality. The

Figure 3. Eggshell nanostructure in palisade (A) and mammillary (B) layer of laying hens with different weeks of age (atomic force microscopy
amplitude images, scanning area, 800 nm £ 800 nm). wk, weeks of age.
 EGGSHELL QUALITY-RELATED MATRIX PROTEINS 7

Figure 4. Identification and comparison of differential expressed proteins. (A) Volcano plots. The fold change (FC) criteria is > 1.2 (or < 0.83)
and p < 0.05. X-axis displays the fold change of differential proteins (value of log2), and Y-axis corresponds to the P value (value of -log10). The red
dots express the up-regulated significant differentially expressed protein, the blue indicate the down-regulated, and the gray are the proteins with no
difference change. (B) Hierarchical cluster heatmap. X-axis manifests groups of samples, and Y-axis demonstrates the differential proteins. The red
and blue signify the high and lower expressed levels respectively. And gray represents no quantitative protein information.

functional description and regulated type for these pro-
teins are listed in Table 5. To sum up, the results of func-
tional analysis suggested that different eggshell quality
in the aged hens (108 wk of age) and the young hens (38
wk of age) was regulated by proteins involved in biomin-
eral proteins, Ca ion-related proteins, immune-related
proteins, synthesis and folding of proteins, and other
proteins. Specifically, it was verified the concentrations
of two key proteins, LYZ and OVA, from the proteomic
data by Elisa, and the results indicated that the expres-
sion levels of these proteins were consistent with the pro-
teomics.

Correlation Analysis Between Proteomic and

Eggshell Traits
A Pearson correlation analysis was utilized to deter-
mine whether there was any association among the
thickness of effective and mammillary layers, the Ca
contents of eggshells, the size of nanoparticles in pali-
sades layers, and the quantification of main eggshell
matrix proteins (Figure 7). The results revealed that the
thickness of mammillary layer was negatively correlated
with the quantification of OPN, nephronectin

Figure 5. Gene ontology (GO) and KEGG pathway enrichment

analysis of up and down-regulated differential proteins. (A) GO func-
tional enrichment analysis. The abscissa presents enriched GO terms,
and ordinate represents the number of proteins. The color of column
corresponds to the P value, color gradient indicates significance of the
enriched GO terms and the number at the top of the column are rich
factor (≤ 1). BP, biological process; MF, molecular function; CC, cellu-
lar component. (B) Butterfly plot of KEGG enrichment pathway. X- Figure 6. Protein-protein interaction (PPI) networks. The color of
axis displays the P value (value of -log10) and the pathways involved in the circle indicates the difference in protein expression (blue for down-
up-regulated and down-regulated proteins are represented by red regulation and red for up-regulation), and the size of the circle indicates
(right) and blue (left) bars. the number of proteins that interact directly.
8 ZHAO ET AL.

Table 5. The vital matrix proteins contributing to decreased mechanical properties of eggshell in aged laying hens.

Functional class Protein name (Gene symbol) Annotation Regulated type

Biomineralization
Calcium ion related
Immune-related
Protein synthesis and folding
Lysozyme C (LYZ) Hydrolytic antimicrobials; a modulator for Up
CaCO3 crystallization
Ovalbumin (OVA) Regulate shell structure and present in eggshell Up
Ovocleidin-116 (OC-116) Major component of the eggshell matrix; Play an Down
important role in the regulation of calcite
growth during eggshell calcification.
Osteopontin (SPP1) Binds tightly to hydroxyapatite; Appears to form Down
an integral part of the mineralized matrix.
Annexin (ANXA11) Calcium ion binding Down
Nephronectin (NPNT) Cell-substrate adhesion Down
EF-hand domain-containing protein Calcium ion binding Down
Fibronectin (FN1) molecular adhesion; related to mechanical Down
toughness
Nucleobindin 2 (NUCB2) Contained EF-folding domains; Calcium ion Down
binding
Syndecan (SDC1) Cell surface proteoglycan. Down
Alpha 1 type IIA collagen (COL2A1) Extracellular matrix structural constituent Down
Ovodefensin A1 (OvoDA1) Defense response to bacterium Up
Avidin (AVD) Biotin binding; antibacterial humoral response Up
Avian beta-defensin 11 (AvBD11) Defense response to bacterium Up
DnaJ heat shock protein family (Hsp40) member Proteolysis involved in cellular protein catabolic Down
C3 (DNAJC3) process

(NPNT), alpha 1 type IIA collagen (COL2A1), fibro- DISCUSSION

nectin (FN1) and nucleobindin 2 (NUCB2) (P <
0.05). The thickness of effective layer had a positive cor-
relation (P < 0.05) with the quantification of OPN, OC-
116, DnaJ heat shock protein family (Hsp40) member
C3 (DNAJC3), annexin (ANXA11), EF-hand
domain-containing protein, epithelial cell adhesion mole-
cule, insulin-like growth factor II and syndecan (SDC1).
Whereas, this was negatively correlated with the concen-
tration of LYZ, OVA, ApoA-I, FAM20C golgi associ-
ated secretory pathway kinase, ovodefensin A1
(OvoDA1), and Avian beta-defensin 11 (AvBD11) (P
< 0.05). There was no significant correlation between
the Ca contents of eggshells, and the nanoparticle size in
palisades layers with the quantification of these eggshell
matrix proteins (P > 0.05).
Figure 7. Corrplot based on Pearson’s correlation analysis. Red
represents a significant positive correlation (P < 0.05), blue indicates
significantly negative correlation (P < 0.05), and white shows that the
correlation was not significant (P > 0.05). MT, mammillary layer thick-
ness; ET, effective layer thickness; CC, calcium content; NP, nanostruc-
ture size in palisade layer.
The age of the hen is one of the main factors that
affects eggshell quality, with eggs laid by older hens more
likely to have poorer mechanical performance (Kemps et
al., 2006; Sirri et al., 2018). Our study found that the
breaking strength of eggshells significantly decreased in
older hens, particularly at 108 wk of age. Compared to
eggshell samples from hens at 38 wk of age, the strength
declined by 3.05N (7.89%) at 78 wk and by 8.38N
(21.66%) at 108 wk. In addition to breaking strength,
stiffness, and fracture toughness are commonly used to
assess the physical properties and material strength of
eggshells (Bain, 1990; Bain, 2005). The significantly lower
values of stiffness and elastic modulus of eggshells with
hen age were observed in the previous study (Mabe et al.,
2003). It was consistent with our results that a significant
decline in the 108-wk-age group. Furthermore, eggshell
characteristics, such as thickness and surface area, were
found to vary during the production cycle of laying hens,
reflecting changes in shell material properties (Fathi et
al., 2019). In our study, the decrease in eggshell thickness
and increase in surface area were likely attributed to the
lower mechanical quality observed in the 108-wk-age
group. Previous studies have indicated that the age-
related changes in egg and eggshell traits contribute to
decreased eggshell quality, with increases in egg weight
and surface area but decreases in shell percentage and
index (eggshell weight per area) (Roberts et al., 2013;
Kim et al., 2014; Moln
ar et al., 2016). Although shell
weight did not show a significant change, the shell per-
centage declined significantly with age (Nys, 1986). Simi-
larly, our study found that eggshells at 108 wk of age
exhibited increased surface area and altered shape, but
the shell weight did not proportionally increase.
The chemical composition and ultrastructure play
crucial roles in determining the mechanical properties of
eggshells. The eggshell consists of CaCO3 crystals and
the organic matrix as well as magnesium, P, and a
 EGGSHELL QUALITY-RELATED MATRIX PROTEINS
variety of trace elements (Hincke et al., 2012). The
decline in Ca deposition in aging laying hens may con-
tribute to poor eggshell quality. A recent study by Park
and Sohn, 2018 found that although the eggshell weight
increased with hen age, the proportionate rise in the Ca
content of eggshell deposition was not observed, which
is consistent with our findings. Our study did not
observe a change in the total Ca content in eggshells
with age but a decrease in distribution (Ca content per
eggshell area), which aligns with our results indicating
decreased thickness. Previous research has demon-
strated that P influences CaCO3 deposition in the shell
gland (Cusack et al., 2003). However, our results indi-
cate no change in the P content and distribution of egg-
shells with age. Further investigation is needed to
explore the impact of inorganic minerals on eggshell
mechanical performance. Regarding eggshell protein
content, our study also did not observe a significant
change with age, consistent with the findings of Ahmed
(Ahmed et al., 2005). It suggested that the total amount
of protein may have a lesser effect on eggshell quality.
The structural organization of the eggshell at various
levels significantly influences its mechanical properties

(Ketta and Tumov
a, 2016). The age-related deteriora-
tion in ultrastructure may contribute to weakened egg-
shell quality in older laying hens. Previous studies have
suggested that the reduction in thickness and proportion
of the effective layer is the main factor affecting shell
strength (Bozkurt et al., 2009; Dunn et al., 2012). Addi-
tionally, the increased height of mammillary columns
and irregular arrangement of mammillary cores may fur-
ther negatively impact the quality of formed eggshells
(Feng et al., 2020; Radwan, 2016). In our study, we
observed a decreased ratio of the effective layer and an
increased ratio of the mammillary layer in eggshells laid
at 78 wk of age, which continued to decline in the 108-
wk-age group. Besides, a decline in mammillae density
was observed in 108-wk-age eggshells, which has been
reported to compromise the attachment points of the
eggshell mineral to the membranes and negatively affect
eggshell mechanical properties (Benavides-Reyes et al.,
2021). The eggshell nanostructure arises from the inter-
action between organic components and CaCO3 crys-
tals, forming "nanosphere particles" (Dauphin et al.,
2018). This unique nanoscopic organization plays a role
in the macroscopic properties and functionality of the
material. The size of eggshell nanometer-scale granules
in the palisades layer and mammillary layer, as demon-
strated by AFM, is approximately 30-70 nm, consistent
with our observations (P
erez-Huerta and Dauphin,
2016; Athanasiadou et al., 2018). An inversely propor-
tional relationship has been found between the size of
nanogranules and the mechanical characteristics of the
material (Wolf et al., 2016). However, in our study, the
size of nanogranules in the palisade layer appeared to
gradually increase with age, although not significantly.
Further investigation is needed to understand the rela-
tionship between nanostructure and age-related decline
in eggshell mechanical performance. Thus, the varia-
tions in eggshell structural organization observed in the
9
108-wk-aged hens contributed to impaired eggshell qual-
ity. Although there were no considerable changes in
composition among different age groups, the abundance
of different types of proteins was suggested to play
important roles in influencing eggshell quality.
The matrix proteins play a key role in regulating egg-
shell formation and controlling structural organization
(Gautron and Nys, 2007). In total, the present study
identified 43 upregulated and 33 downregulated proteins
in the 108-wk-age group, primarily related to four func-
tional classes: biomineralization, calcium-ion-related,
immune-related, and protein synthesis and folding. The
proteins with a key role in the control of eggshell biomin-
eralization process show the different regulated types in
this study. OC-116 is one of the most crucial proteins in
the eggshell, related to mineralization. It is predomi-
nantly located in the palisade region of the shell and
found an overabundance in the uterine fluid at the active
growth phase (Hincke et al., 1999; Marie et al., 2015b).
A candidate gene association analysis demonstrated
that single nucleotide polymorphisms in the OC-116
gene show an association with elastic modulus, shell
thickness, and egg shape (Dunn et al., 2009). In our
study, OC-116 was found downregulated in the eggshell
from aged hens and had a positive correlation with the
thickness of the effective layer. Meanwhile, OPN is a
highly concentrated phosphoprotein found in the pali-
sade layer (Pines et al., 1995; Hincke et al., 2012). Previ-
ous research has highlighted associations of OPN gene
expression with abnormalities, fracture toughness, and
cracks in the eggshell (Arazi et al., 2009). Lower abun-
dance of osteopontin has been identified in weak egg-
shells compared to strong eggshells (Sun et al., 2013),
which aligns with our findings of downregulated OPN in
the 108-wk-age group. Our results also showed a close
positive relationship between the effective layer and
OPN quantification. Therefore, the decreased levels of
OC-116 and OPN in the eggshell may contribute to the
decline in eggshell quality. The quantification of OVA
and LYZ increased in the eggshells from the 108-wk-age
group. OVA, a widespread egg white protein, could be
entrapped within the mineral layer during eggshell for-
mation and was localized in the mammillary knobs
(Hincke, 1995). The presence of OVA in eggshells sug-
gests its influence on the initiation of eggshell formation.
Similarly, previous studies have demonstrated higher
concentrations of OVA in eggshell extracts from older
hens compared to young hens (Panh
eleux et al., 2000).
The increase in OVA may induce added thickness to the
mammillary layer and abnormal formation of the mam-
millary core. Moreover, LYZ is another egg white pro-
tein abundant in the early stage of eggshell formation.
(Marie et al., 2015b). Lysozyme C, in addition to its
antibacterial defense role, has been suggested to play a
role in eggshell biomineralization (Moreau et al., 2022).
Under in vitro conditions, high concentrations of LYZ
have been shown to alter crystal morphology (Hincke et
al., 2000). The close positive relationship between LYZ
and the mammillary layer may explain the variations in
eggshell structure observed in the aged hens.
10 ZHAO ET AL.
Several proteins involved in Ca binding and regula-
tion participate in the eggshell formation process. In the
108-wk-age eggshell, ANXA11, NPNT, EF-hand
domain-containing protein, NUCB2, FN1, and proteo-
glycans (SDC1 and COL2A1) were downregulated com-
pared to the 38-wk-age group. ANXA11, proposed to be
present in vesicle membranes, can promote Ca ion entry,
and annexins serve as nucleation sites for mineral forma-
tion due to their Ca ion-binding properties (Wu et al.,
2008; Stapane et al., 2020a; Stapane et al., 2020b).
NUCB2, identified as an abundant noncollagenous bone
protein associated with the mammillary cones, has an
EF-hand Ca ion-binding domain and is involved in
maintaining Ca levels (D’Ambrosio et al., 2008; Rose-
Martel et al., 2015). The downregulation of these pro-
teins with hen aging may compromise the mammillary
knob and consequently impact eggshell structure and
quality. Similarly, NPNT is a Ca-binding protein identi-
fied in the unique organization responsible for the
mechanical properties of the Guinea fowl eggshell (Le
Roy et al., 2019). FN1, a glycoprotein found in the min-
eralized layer, interacts with the extracellular matrix
and facilitates molecular adhesion by binding proteogly-
cans (Zeng et al., 2023). These proteins are likely to play
a crucial role in eggshell structure, and their downregu-
lation may contribute to decreased eggshell quality in
the 108-wk-age group. Meanwhile, NUCB2, NPNT, and
FN1 exhibited closely positive correlations, may suggest-
ing their co-participation in biological pathways perti-
nent to the eggshell calcification process. Furthermore,
COL2A1 and SDC1, as the extracellular matrix pro-
teins, also participate in eggshell mineralization by inter-
acting with Ca (Arias et al., 2004; Brionne et al., 2014).
In total, the declining abundance of these proteins may
indicate impaired eggshell calcification processes in aged
hens, subsequently resulting in poor eggshell quality.
The decline in immunity is a notable characteristic of
aged laying hens. Our previous findings demonstrated
that impaired reproductive function and inflammation
in aged laying hens led to increased expression of anti-
bacterial proteins in the uterus (Feng et al., 2020). How-
ever, proteins involved in defense against bacteria have
been negatively correlated with strong eggshell forma-
tion (Sun et al., 2013). In the present study, plenty of
proteins in the 108-wk-age weak eggshells were enriched
in the GO function of the immune response. Avian
b-defensins (AvBDs) are major antimicrobial peptides,
and the expression of uterine AvBD11 increased with
aging of hens compared to young hens (Elhamouly et
al., 2019). OvoDA1 is also a b-defensin-related antimi-
crobial peptide that appears to be specifically expressed
in the avian oviduct. Avidin is secreted in the magnum
with antimicrobial activity, and its expression is higher
in adult hens compared to younger ones (Jung et al.,
2011). Meanwhile, significant correlations between the
immune-related OvoDA1, avidin, and AvBD11, have
been exhibited notably positive relationships, which
may co-influence eggshell structure. In addition,
DNAJC3, annotated in the function of misfolded protein
binding, was found to be downregulated in the 108-wk-
age group, indicating an abnormal state of the uterus.
Therefore, our present study suggests that the abnormal
deposition of immune-related proteins in eggshells from
aged hens can be attributed to uterine senescence and
subsequent damage to eggshell quality.
CONCLUSIONS
In summary, the eggshell thickness and mechanical
properties of Jing Tint 6 at 108 wk of age exhibited a sig-
nificant decrease compared to the peak laying period.
This decline could primarily be attributed to changes in
the layer proportion of eggshell ultrastructure. However,
no significant variations were observed in eggshell com-
positions. The DEPs in the eggshells from 108-wk-age
hens may be associated with the formation of an altered
structure. The decreased levels of OC-116 and OPN,
along with the increased abundance of OVA and LYZ,
are considered to be the main factors contributing to the
structural changes.
ACKNOWLEDGMENTS
This work was financially supported by the earmarked
fund for China Agriculture Research System (CARS-40-
K12; CARS-40-S20), the National Natural Science
Foundation of China (32302776) and the Agricultural
Science and Technology Innovation Program (ASTIP)
of CAAS.
DISCLOSURES
The authors have declared that they have no conflicts
of interest.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article
can be found in the online version at doi:10.1016/j.
psj.2024.103463.
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