Fish Physiol Biochem
https://doi.org/10.1007/s10695-019-00676-9
Effects of dietary hydroxyproline on collagen metabolism,
proline 4-hydroxylase activity, and expression of related
gene in swim bladder of juvenile Nibea diacanthus
Hua Rong & Yunlong Zhang & Meilin Hao &
Weiguang Zou & Jun Yu & Chuanqi Yu & Qinchao Shi &
Xiaobo Wen
Received: 11 January 2019 / Accepted: 18 June 2019
# Springer Nature B.V. 2019
Abstract This study was conducted to investigate the
effects of dietary hydroxyproline (Hyp) on tissue colla-
gen level, proline 4-hydroxylase (P4H) activity as well
as transcript levels of COL1As (COL1A1 and CO-
L1A2) and P4Hαs (P4Hα(I), P4Hα(II), and P4Hα(III))
in juvenile Nibea diacanthus. A total of 450 fishes were
randomized to six equal groups and fed the diet with
graded supplementary Hyp—0, 5, 10, 15, 20, and
25 g kg−1 of dry matter for 8 weeks. Results showed
that fish fed diets with 10 g kg−1 Hyp had significantly
higher acid-soluble collagen (ASC) and total collagen
(TC) concentrations in swim bladder than fish fed with
the other diets (P < 0.05). The activity of P4H in liver
and swim bladder showed a similar trend, showing first
increase and then decrease with increasing dietary Hyp
(P < 0.05). The mRNA expression of COL1As in swim
bladder and muscle were significantly higher than those
H. Rong : W. Zou : J. Yu : C. Yu : Q. Shi : X. Wen (*)
Guangdong Provincial Key Laboratory of Marine Biology,
Shantou University, Shantou 515063, People’s Republic of China
e-mail: wenxbo@stu.edu.cn
H. Rong
College of Animal Science and Technology, Yunnan Agricultural
University, Kunming 650201, People’s Republic of China
Y. Zhang
Department of Food Safety Technology, China National
Analytical Center, Guangzhou 510642, People’s Republic of
China
M. Hao
College of Marine Science, South China Agriculture University,
Guangzhou 510642, People’s Republic of China
in the liver and intestines. Meanwhile, with increasing
dietary Hyp, the relative expression of COL1As genes
in swim bladder showed a similar pattern with the TC
concentrations of swim bladder, increased significantly
initially followed by a decrease. Increased dietary Hyp
content corresponded with significant decrease in the
mRNA level of P4Hαs in swim bladder. These results
indicated that the dietary Hyp promotes the collagen
accumulation of swim bladder to some extent, and the
promoting action may be related to the expression of
COL1As. The optimum supplement of dietary Hyp was
estimated from TC of swim bladder with piecewise
regression analysis to be 9.66 g kg−1.
Keywords Hydroxyproline . Swim bladder . Collagen
metabolism . Proline 4-hydroxylase . Gene expression .
Nibea diacanthus
Introduction
The swim bladder is rich in collagen, and as a traditional
functional food, collagen is believed to improve brain
function, maintain a normal endocrine status, and mod-
ulate immune functions (Lu et al. 2010). In Chinese
cuisine, fish maw (the dried swim bladder) is a delicacy,
and regarded as one of the four sea treasures (abalone,
sea cucumber, shark fin, and fish maw). For many
centuries, Asians have come to believe that the swim
bladder of fish possess some traditional medicinal prop-
erties, especially, as a tonic for postpartum women, to
speed up their recovery (Clarke 2004). Major

commercial sources of collagen have recently been ex-
plored from the swim bladder of miiuy croaker (the
same genus as Nibea diacanthus) (Lu et al. 2010). Nibea
diacanthus is a large, fast-growing, and long-lived pred-
atory fish species, which is widely regarded as excellent
food fish, especially its fish maw, which is used as a
tonic, has high market value and huge trade volume in
Southeast Asia, especially in Hong Kong and Southern
China (Chen et al. 2011; Li et al. 2016, 2017). In fact, it
is very common for swim bladders produced using
N. diacanthus in China, especially swim bladder from
N. diacanthus is the most nutritious compared with that
from other fishes (Wen et al. 2016).
Fish collagen or collagen peptides are rich in type I
collagen which is the most important component of the
skin, swim bladder, and other connective tissues (Karim
and Bhat 2009; Ohara et al. 2007). Type I collagen
consists of two a1 (I) chains (COL1A1) and one a2 (I)
chain (COL1A2), regulated by COL1A1 and COL1A2
genes, respectively (Kimura et al. 1988; Sato et al. 1989;
Rest et al. 1993; Miller 1985). Collagen synthesis also
involves many post-translational modifications. For in-
stance, prolyl 4-hydroxylase (P4H) is the key collagen-
specific enzyme that catalyze the modifications or for-
mation of 4-Hyp from proline (Kivirikko et al. 1990). In
vertebrates, the P4H has at least three isoenzymes,
encoded by three genes, e.g., P4Hα(I), P4Hα(II), and
P4Hα(III) (Kukkola et al. 2003). Recent studies have
shown that total collagen level in muscle increased
significantly with significant decrease in P4Hα(I) ex-
pression in muscle of turbot fed on diets with increased
hydroxyproline (Hyp) (Zhang et al. 2013). There is
however no related research work on the other isoen-
zymes of prolyl 4-hydroxylase (i.e., P4Hα(II) and
P4Hα(III)), and no reports on the relationship between
swim bladder collagen accumulation and the expression
of metabolism-related genes (COL1A1, COL1A2,
P4Hα(I), P4Hα(II), and P4Hα(III)). Moreover, there
has always been an interest in the question of whether
the administration of certain nutrients could specifically
enhance collagen synthesis. Some recent studies have
reported that endogenous synthesis of hydroxyproline is
inadequate for maximum growth and feed efficiency in
pigs, chickens, and fish (Zhang et al. 2013; Li and Wu
2018; Wu et al. 2011; Zhang et al. 2015; Liu et al. 2014).
Hydroxyproline does not exist in free-form amino acids
in nature, and it cannot be used as substrate for the
synthesis of protein, since it is derived from proline in
collagen proteins by vitamin C-dependent prolyl
Fish Physiol Biochem
hydroxylase (Stanley 1983). Hydroxyproline is a major
AA in collagen proteins, the major extracellular compo-
nents of connective tissues such as skin, tendon, carti-
lage, vessels system, and bone (Phang et al. 2010; Krane
2008). In addition, hydroxyproline has important meta-
bolic and physiological roles in the structural functions
of collagens (Brinckmann et al. 2005). It is even be-
lieved that the effect of dietary Hyp on collagen metab-
olism goes beyond meeting the requirement for growth-
promoting effect in some fishes (Li et al. 2009; Wu et al.
2008).
The present study was conducted to test the hypoth-
esis that hydroxyproline as the main component of
collagen, may play an important role on collagen accu-
mulation. Our research team has for a long time, dedi-
cated our research to studying how to strengthen the
development and utilization of the swim bladder of
N. diacanthus for its special value in food nutrition. It
is currently unknown whether dietary Hyp supplemen-
tation has any effects on the collagen accumulation of
swim bladder as well as on metabolism-related genes
expression in N. diacanthus. The focus of the present
study was on adding Hyp as a nutrient to the diet of
N. diacanthus for 56 days so as to assess the level of
collagen in muscle and swim bladder, as well as the
activities of related enzymes, and expression of genes
involved in collagen biosynthesis. The main purpose is
to investigate the optimum nutritional level of hydroxy-
proline supplementation that could be used to obtain
maximum collagen synthesis in swim bladder. The re-
sults could lay a theoretical foundation for the develop-
ment and utilization of functional foods.
Materials and methods
Experimental diets and feeding trial
This study used the same experimental diets and feeding
trial as our previous study (unpublished result). Briefly,
six isonitrogenous (420 g kg−1) crude protein (CP) and
isolipidic (84 g kg−1) diets (I–VI) were formulated
(Table 1). As shown in Table 1, fish meal and mixed
amino acids were used as the main dietary protein
sources, and were found to be limiting in hydroxypro-
line, while fish oil was the main dietary lipid sources.
The experimental diets were supplemented with graded
levels of hydroxyproline (0, 5, 10, 15, 20, and 25 g kg−1
diet). The hydroxyproline powder was purchased from
Fish Physiol Biochem

Table 1 Formulation and proximate composition of the experimental diets

Ingredient composition (g kg−1) Diets

I II III IV V VI

1
Fish meal 345 345 345 345 345 345
Fish oil 56 56 56 56 56 56
Starch 222.5 222.5 222.5 222.5 222.5 222.5
2
Mixed amino acids 177 177 177 177 177 177
Cellulose microcrystalline 134.5 134.5 134.5 134.5 134.5 134.5
Choline chloride 10 10 10 10 10 10
Monocalcium phosphate 10 10 10 10 10 10
3
Vitamin premix 10 10 10 10 10 10
4
Mineral premix 10 10 10 10 10 10
Trans-4-hydroxy-L-proline 0 5 10 15 20 25
Glutamic 25 20 15 10 5 0
Proximate composition (%)
Crude protein 42.34 42.38 42.45 42.19 42.06 42.22
Crude lipid 8.45 8.39 8.40 8.38 8.47 8.41
Crude ash 10.94 10.87 10.93 12.00 12.27 11.35
Hydroxyproline (Hyp) 0.03 0.51 1.05 1.48 2.06 2.54
1
The white fish meal of the USA: crude protein content of 64.7%, crude fat content of 10%
2
Mixed crystalline amino acid (g/100 g diet): glycine, 13.29; phenylalanine, 0.31; arginine, 0.28; threonine, 0.18. alanine, 3.63
3
Vitamin premix (mg/kg diet): thiamin, 25; vitamin B12, 0.1; vitamin K3, 10; inositol, 800; pantothenic acid, 60; retinal acetate, 32; niacin
acid, 200; folic acid, 20; riboflavin, 45; cholecalciferol, l,5; biotin, 1.20; pyridoxine hydrochloride, 20; α-tocopherol, 120; ascorbic acid,
2000; ethoxyquin, 150
4
Mineral premix (mg/kg diet): NaF,1; KI,0.4; NaCl,50; CoCl2·6H2O,25; MgSO4·7H2O,600; FeSO4·H2O,40; Ca(H2PO4)2·H2O,1500;
ZnSO4·H2O,25; MnSO4·H2O,30; CuSO4·5H2O,5.0; Zeolite,7725

Hebei Runying Biotechnology (Hebei, China). The
quantity of hydroxyproline was increased at the expense
of glutamate so as to make the diets isonitrogenous, as
glutamate levels do not affect the parameters being
measured. The levels of diet amino acid are shown in
Table 2, as described using an HPLC-Ultimate 3000
(Thermo Scientific Dionex, USA). Feedstuffs were
sifted through a 2.5-mm diameter grinder, fan-dried at
room temperature, and stored at − 20 °C until use.
The experiments were carried out in floating pens at
Nan’Ao Marine Biology Station (NAMBS), Shantou
University, Shantou, China. The N. diacanthus were
provided by a local marine fish hatchery (Raoping,
Guangdong, China). A 56-day feeding trial was carried
out from June to Aug 2016. Similar size (mean 133 ±
2.14 g) fish were selected and individually weighed, and
randomly distributed into 18 pens (1 m × 1 m × 1.5 m,
L:W:H) with 25 fish per pen. Fish were hand-fed to
apparent satiation twice daily (07:00 and 16:30 h).
During the experiment, the temperature ranged from
23 to 30 °C, pH 7.8 to 8.1, ammonia nitrogen lower
than 0.05 mg/L, salinity 31 to 33 g/L, and dissolved
oxygen 5.2 to 6 mg/L.
Sample collection
At the end of the feeding trial, all fish were fasted for
24 h, and anesthetized with eugenol (1:10,000) (purity
99%, Shanghai Reagent, China) to avoid being stressed
before harvest. Plasma samples were collected from the
caudal vein of fish using heparinized syringes and serum
using nonheparinized syringes. Plasma was recovered
after centrifugation (3000g for 10 min at 4 °C) and
serum for (4000g for 10 min at 4 °C), and then imme-
diately stored at − 80 °C until analysis. Tissue sample
(liver, muscle, intestines, and swim bladder) were
dipped in liquid nitrogen first, and then stored at −
 Fish Physiol Biochem

Table 2 Amino acid contents of experimental diets (g kg−1 protein)

Amino acid contents Diets

I II III IV V VI

EAA
Arginine, Arg 68.99 67.09 67.14 71.13 71.75 67.82
Threonine, Thr 23.35 22.50 22.87 28.37 23.36 26.67
Phenylalanine, Phe 25.87 24.61 23.50 23.99 22.32 23.94
Histidine, His 5.05 5.26 5.04 5.01 5.63 5.25
Methionine, Met 11.78 11.99 13.22 12.10 12.72 12.39
Lysine, Lys 24.40 26.50 25.18 25.66 25.86 24.15
Isoleucine, Ile 57.84 59.31 58.96 60.91 59.03 59.84
Leucine, Leu 14.93 13.88 13.64 13.77 13.98 13.65
Valine, Val 23.56 23.55 23.50 23.36 23.36 23.31
NEAA
Aspartic, Asp 35.34 34.91 35.04 34.62 34.42 34.02
Serine, Ser 24.61 24.40 24.13 24.61 23.36 24.99
Glutamic, Glu 68.78 63.51 57.07 54.02 51.10 45.98
Alanine, Ala 85.61 79.29 77.42 76.34 84.27 73.91
Glycine, Gly 273.66 259.31 252.63 246.54 255.31 254.70
Tyrosine, Tyr 43.75 47.32 45.74 44.84 45.47 44.72
Proline, Pro 33.66 31.55 31.05 29.62 36.29 35.28

EAA essential amino acid, NEAA nonessential amino acid

80 °C for gene expression analysis, Hyp content, and
collagen content analysis.
Biochemical analysis
Hyp content determination Samples were processed
using Diagnostic Reagent Kits (Art. No. A030-3 and
Art. No. A030-2; Nanjing Jiancheng Bioengineering
Institute, Nanjing, China) according to the manufac-
turer’s instructions with some modifications. The Hyp
content of the samples was analyzed on the Infinite®
Pro 200 multi-functional micro porous plate detector
(Tecan, Switzerland). Briefly, approximately
30~100 mg tissue sample was hydrolyzed with 1 mL
6 M hydrochloric acid under 95 °C for 5 h (500 μl
plasma samples for 20 min). The liquid pH was adjusted
by adding liquid A and liquid B according to the in-
structions, after which the samples were diluted, mixed,
and filtered through 0.20 μm filter. Aliquots of 1-ml
diluted samples, 1-ml ultra-pure water, or 1-ml standard
Hyp (5 μg/ml; prepared from stock solution of Hyp)
were mixed with 500 μl reagent A and incubated for
10 min at room temperature. Reagent B (500 μl) was
added and the mixture was incubated for further 5 min at
room temperature before adding 500 μl of reagent C.
The mixture was then heated for 20 min at 60 °C,
cooled, centrifuged at 3500 rpm at room temperature
for 10 min, and the absorbance determined at 550 nm.
The Hyp concentration was determined from a standard
curve.
Calculation of collagen content Hydroxyproline is an
amino acid that is unique to collagen and is traditionally
used to quantify this protein. The content of total colla-
gen is the sum of soluble collagen and insoluble colla-
gen, the content of Hyp is 12.5% among the collagen
protein of connective tissue (AOAC 1995). To convert
the quantity of hydroxyproline into collagen, a factor of
12.5% was used and expressed as mg g−1 protein.
Activity of proline 4-hydroxylase determination Total
soluble protein of the homogenate was measured using
Folin phenol reagent by casein digestion method of
Lowry et al. (1951). All measurements were done using
a Diagnostic Reagent Kit (Jiancheng Bioengineering
Institute, Nanjing, China) according to the
Fish Physiol Biochem

manufacturer’s instructions, and analyzed on a multi-
functional microporous plate detector (Infinite_200 Pro,
Tecan, Switzerland). The levels of proline 4-
hydroxylase (P4H) in tissues and serum were deter-
mined by double antibody sandwich method. First, solid
phase antibody is made using PH antibody of fish pack-
aged with microporous plate, after which P4H was
added into the micropore packaged with monoclonal
antibody which comes from tilapia. This was then com-
bined with HRP-labeled P4H antibodies, to form the
antibody-antigen-enzyme-labeled antibody complex.
Finally, thorough TMB coloring was performed after
washing thoroughly, the color depth was positively cor-
related with concentrations of PH in the sample. The
absorbance (OD value) was determined by ELISA at
450 nm, and the concentrations of P4H was calculated
using a standard curve.
Analysis of metabolism-related genes expression
Total RNA was extracted from a section of each tissue
(liver, muscle, intestines, and swim bladder) using
Trizol (Invitrogen™) according to the manufacturer’s
instructions. Tissues were first homogenized with 1 ml
of Trizol, and then 300 μl chloroform was added with
vigorous shaking before centrifuging at 12,000 rpm at
4 °C. Supernatant was transferred to a new tube and
precipitated with isopropanol. The RNA pellet was
washed with 75% ethanol and dissolved in diethyl
pyrocarbonate (DEPC) water. Contaminating genomic
DNA were eliminated by DNase I digestion (Takara,
Dalian, China). The final concentration and quality of
RNA were determined by measuring the absorbance at
260 nm/280 nm using a Thermo Scientific NanoDrop
2000 spectrophotometer (Thermo Scientific Nanodrop,
USA), while the integrity was confirmed through anal-
ysis on 1.5% (w/v) agarose gel.
First strand cDNA was synthesized from 1 μg of
mRNA using the TransScript® One-Step gDNA Re-
moval and cDNA Synthesis SuperMix Kit (TransGen
Biotech, Beijing, China). The following gene-specific
primer pairs were designed using the program, Primer
Premier 5.0 software (Premier Biosoft International,
Palo Alto, CA, USA), COL1A1 and COL1A2 accord-
ing to sequences of Danio rerio (Genbank accession no.
NM_199214 and NM_182968, respectively) and
P4Hα(I), P4Hα(II), and P4Hα(III) according to se-
quences of Larimichthys crocea (Genbank accession
no. XM_010731575, XM_010729350 and
XM_010739670, respectively) (Table 3). The PCR cy-
cling conditions were 95 °C for 5 min and 35 cycles of
amplification at 95 °C for 15 s, 58 °C for 1 min, exten-
sion 72 °C for 1 min, and finally, extended at 72 °C for
10 min. The PCR fragments were subjected to electro-
phoresis on a 1.2% agarose gel to ascertain product size
and sequenced by BGI Tech (The Beijing Genomics
Institute, Shenzhen City). The resulting sequences were
verified and subjected to cluster analysis in NCBI.
Real-time fluorescent Quantitative Polymerase chain
reaction (qRT-PCR) was performed with on the Roche
Light Cycler® 480 System. Two microliters of cDNA
were used in each PCR reaction, and analyzed in tripli-
cates. The housekeeping gene beta-actin rRNA (Ap-
plied Biosystems) was used for normalization and water
was used as a no-template control. The relative quanti-
fication was calculated using the ΔΔCT method. The

Table 3 Real-time PCR primer sequences

Name Sequences of primers Product length

COL1A1 Forward 5′-AGACCTGCGTGACTCCCA-3′ 138

Reverse 5′-AGCCCTCGCTGCCATACT-3′
COL1A2 Forward 5′-CAAGAACAGCGTTGCCTACAT-3′ 120
Reverse 5′-ACGGAGAAGGTGAAGCGG-3′
P4Ha1 Forward 5′-GTGCTTGGCTCACTGGCTAC-3′ 212
Reverse 5′-CCACGTTGCTATGCGATTG-3′
P4Ha2 Forward 5′-ACCAGGTGTTCACTCCAATGC-3′ 243
Reverse 5′-ATAGCCACAAGTCGGCGTGT-3′
P4Ha3 Forward 5′-CTGAGAATGAAGGGACTTTGGA-3′ 134
Reverse 5′-CAAAACTCTTCCATTCATCGG-3′
β-actin Forward 5′-GGTTACTCCTTCACCACCACAG-3′ 147
Reverse 5′-TCCGTCGGGCAGCTCATA-3′
 Fish Physiol Biochem

mean CT of triplicate samples was used to calculate the
ΔCT as the difference in CT between the target and
housekeeping gene. The ΔΔCT was expressed by
ΔCT for each sample minus the experimental reference
ΔCT control. The relative quantification was expressed
as the fold expression of the target gene compared with
the reference control expression according to the formu-
la 2-ΔΔCT and expressed in relative expression units.
Calculation and statistical analysis
The Software SPSS, 13.0 (SPSS, Inc., Chicago, IL,
USA) was used for all statistical evaluations. All data
were analyzed by one-way ANOVA followed by
Tukey’s test to inspect differences among all the treat-
ments. Differences were regarded as significant when
P < 0.05. Data are presented as mean ± standard error.
dietary Hyp levels (Table 4), and with significant differ-
ence between any two groups (P < 0.05). There was no
significant difference in the collagen contents of muscle
including acid-soluble collagen (ASC), the insoluble
collagen (ISC) and total collagen (TC) among the die-
tary treatments (P > 0.05). On the contrary, the collagen
(ASC, ISC and TC) of different dietary treatments in the
swim bladder showed significant difference with the
different dietary Hyp levels (P < 0.05). Maximum TC
was observed in fish fed diet III, and significantly de-
creased as dietary Hyp levels increased. The piecewise
regression analysis (y1 = 5990x + 203.4 R2 = 0.9851 and
y2 = −3852.6x + 298.45 R2 = 0.9954) of total collagen in
swim bladder against different dietary Hyp levels in
diets of juvenile Nibea diacanthus, which is presented
in Fig. 1, shows that maximum total collagen in swim
bladder could be possibly obtained with diets to which
9.66 g kg−1 Hyp is added.

Results Activity of proline 4-hydroxylase

Free hydroxyproline in plasma, total hydroxyproline
contents in liver, and the collagen of muscle and swim
bladder
Free Hyp levels in plasma and total Hyp levels in liver
showed a gradual increase in accordance with increasing
The activity of prolyl 4-hydroxylase (P4H) in tissues of
juvenile Nibea diacanthus fed the experimental diets is
shown in Table 5. The activity of serum and muscles
P4H exhibited a similar pattern, i.e., significant increase
with increasing dietary Hyp levels (P < 0.05). However,
the activity of P4H in liver and swim bladder were

Table 4 Plasma-free hydroxyproline, liver total hydroxyproline, acid-soluble collagen (ASC), insoluble collagen (ISC), and total collagen
(TC) contents in muscle and swim bladder of juvenile Nibea diacanthus fed the experimental diets with different levels of hydroxyproline

Biochemical indexes Diets

I II III IV V VI

Hyp
Liver (g kg−1 wet basis) 0.28 ± 0.00a 0.33 ± 0.00b 0.38 ± 0.00c 0.48 ± 0.01d 0.62 ± 0.00e 0.68 ± 0.00f
−1 a b c d e
Plasma (μg ml ) 33.83 ± 0.16 43.07 ± 0.93 48.77 ± 0.51 52.57 ± 0.14 57.03 ± 0.24 62.69 ± 0.87f
−1
ASC (g kg wet basis)
Muscle 13.69 ± 0.38 13.22 ± 1.75 14.28 ± 1.81 14.88 ± 1.42 13.85 ± 0.51 13.63 ± 1.18
Swim bladder 187.07 ± 2.71a 217.23 ± 3.29bc 242.49 ± 1.75d 221.01 ± 2.83c 208.25 ± 5.36ab 187.08 ± 3.65a
ISC (g kg−1 wet basis)
Muscle 0.81 ± 0.15 1.01 ± 0.10 0.69 ± 0.07 0.91 ± 0.04 0.72 ± 0.01 0.88 ± 0.35
Swim bladder 14.20 ± 2.25a 20.40 ± 1.38c 18.69 ± 1.26c 17.25 ± 0.48bc 14.22 ± 0.38ab 15.14 ± 0.34ab
TC (g kg−1 wet basis)
Muscle 14.51 ± 1.05 14.22 ± 1.76 14.97 ± 1.53 15.80 ± 1.48 14.56 ± 0.85 14.52 ± 1.83
a bc d c b
Swim bladder 201.27 ± 4.26 237.60 ± 3.43 261.17 ± 3.44 238.25 ± 1.03 222.47 ± 2.21 202.22 ± 4.09a

Values are presented as means ± SEM, n = 3. Means in the same row with different letters are significantly different from each other
(P < 0.05)
Fish Physiol Biochem

Fig. 1 Piecewise regression analysis of total collagen in swim hydroxyproline estimated from total collagen contents of swim
bladder against different dietary Hyp levels in diets of juvenile bladder was 9.66 g kg−1. Each point represents the mean of three
Nibea diacanthus. (y1 = 5990x + 203.4 R2 = 0.9851 and y2 = groups of Nibea diacanthus with 25 fish per group
−3852.6x + 298.45 R2 = 0.9954). Addition amount of dietary

significantly increased initially and were followed by a
decrease as Hyp content in diets (P < 0.05) increases.
The maximum activity of P4H appeared in diets III
(10 g kg−1 Hyp) and diets II (5 g kg−1 Hyp).
Expression of genes related to collagen metabolism
To determine the potential involvement of the hydroxy-
proline in the regulation of collagen metabolism-related
genes expression, we performed qRT-PCR analysis
(Fig. 2 and Fig. 3). Relative gene expressions of CO-
L1A1 and COL1A2 of juvenile N. diacanthus fed with
graded levels of hydroxyproline are presented in Fig. 2.
The expression of COL1A1 and COL1A2 genes in
swim bladder were significantly higher than that in
muscle, liver and intestines, and showed a similar trend
with COL1A1 gene expression in muscle and liver
when increasing dietary hydroxyproline. The expression

Table 5 Content of prolyl 4-hydroxylase (P4H) in serum, swim bladders, muscles, and livers of the juvenile Nibea diacanthus fed the
experimental diets

P4H (pg/g wet basis) Diets

I II III IV V VI

Serum 81.31 ± 5.82a 86.39 ± 5.03b 92.64 ± 4.72c 98.035 ± 3.62d 101.82 ± 5.32e 123.12 ± 6.23f
cd e bc bc b
Liver 198.96 ± 19.31 232.67 ± 8.73 185.47 ± 21.80 188.10 ± 20.18 175.41 ± 19.68 140.24 ± 18.40a
ab a a ab b
Muscle 112.97 ± 9.26 105.32 ± 8.67 105.38 ± 7.34 116.73 ± 7.56 126.57 ± 8.96 143.93 ± 7.31b
Swim bladder 376.29 ± 21.42a 428.53 ± 19.37bc 490.81 ± 23.56e 442.37 ± 18.97cd 406.45 ± 24.57abc 390.48 ± 17.86ab

Values are presented as means ± SEM, n = 3. Means in the same row with different letters are significantly different from each other
(P < 0.05)

Fig. 2 Relative expression of collagen type 1 α1 (COL1A1, A)
and collagen type 1 α2 (COL1A2, B) mRNA in juvenile Nibea
diacanthus fed with diets by adding graded levels of hydroxypro-
line. Relative mRNA expression was evaluated by real-time quan-
titative PCR. Data are expressed as means with S.E.M. (N = 3).
of COL1A1 and COL1A2 were initially increased and
then decreased, with the highest COL1A1 gene expres-
sion observed in muscle and liver of the groups fed with
20 g kg−1 hydroxyproline diet and 5 g kg−1 hydroxy-
proline diet, respectively (P < 0.05). The highest CO-
L1A1 and COL1A2 gene expressions in the swim blad-
der were found with 10 g kg−1 hydroxyproline diet
(P < 0.05). The pattern of COL1A2 mRNA in the intes-
tines was the opposite compared with that in the swim
bladder (P < 0.05). The lowest COL1A2 gene expres-
sion in the intestines was found with 10 g kg−1 hydroxy-
proline diet (P < 0.05). The COL1A2 mRNA expression
in muscle was significantly decreased with increasing
dietary hydroxyproline, while the trend of COL1A1
mRNA expression in the intestines was different from
trend of the COL1A2 mRNA expression in muscle.
As shown in Fig. 3, the levels of P4Hα(I) and
P4Hα(II) mRNA in the liver were significantly higher
(P < 0.05) than in other tissues. On the contrary, the
P4Hα(III) mRNA abundance in liver and intestines
were significantly lower than that found in swim bladder
and muscle, with the highest found in swim bladder
(P < 0.05). The relative expression of P4Hαs
(P4Hα(I), P4Hα(II), and P4Hα(III)) mRNA in the liver,
swim bladder, and muscle significantly decreased as
levels of dietary hydroxyproline (P < 0.05) increases,
with the exception of the expression of P4Hα(III) in
muscle, which increased initially before decreasing with
increased dietary hydroxyproline. The highest
P4Hα(III) gene expressions in muscle was found in
Fish Physiol Biochem
Value with different capital letters is significantly different among
the dietary treatments in the same tissues (P < 0.05). Values with
unlike lowercase letters were significantly different among the
different tissues in the group of basal diet (diets I) (P < 0.05)
the group fed with 10 g kg−1 hydroxyproline diet
(P < 0.05). Furthermore, the P4Hα(I) gene expression
in intestines was also found to be significantly decreased
with increasing levels of dietary hydroxyproline
(P < 0.05).
Discussion
This study is part of the growth trial (unpublished
result), which showed that dietary Hyp supplementa-
tion could significantly stimulate growth in juvenile
Nibea diacanthus, with the estimated optimum
amount of dietary hydroxyproline from SGR found
to be 16.6 g kg−1. Unfortunately, in that study, we did
not study the effect of dietary Hyp on collagen syn-
thesis, hence, the effect of dietary Hyp on collagen
biosynthesis in juvenile N. diacanthus is unknown. In
this study, we showed that the addition of Hyp to
diets significantly increased Hyp levels in plasma and
liver. This observation is consistent with previous
findings by Pinilla-Tenas et al. (2003), where dietary
Hyp supplementation significantly increased Hyp
levels in plasma and muscle, while low levels of
Hyp was found in the serum of Atlantic salmon fed
on diets with low levels of Hyp compared with fish
fed with high dietary Hyp (Aksnes et al. 2006).
Similarly, Zhang et al. (2013) found that the content
of Hyp in muscle, liver, and serum increased signif-
icantly with increase in dietary Hyp. It is believed
Fish Physiol Biochem
Fig. 3 Relative expression of prolyl 4-hydroxylase ((P4Hα(I), a),
(P4Hα(II), b), and (P4Hα(III), c)) mRNA in juvenile Nibea
diacanthus fed with diets by adding graded levels of hydroxypro-
line. Relative mRNA expression was evaluated by real-time quan-
titative PCR. Data are expressed as means with S.E.M. (N = 3).
that Hyp could be effectively absorbed and
transported in tissues without dehydroxylation. The
collagen level of swim bladder was significantly dif-
ferent among different dietary Hyp supplementation.
Some studies (Aksnes et al. 2008; Kousoulaki et al.
2012) have reported similar results that dietary Hyp
significantly increases collagen and crosslink con-
centrations, which could improve tissue firmness
and flesh quality in aquaculture. However, the colla-
gen level of muscle was not affected by the different
levels of dietary Hyp supplements. The results here
were different from the previous findings in turbot by
Liu et al. (2014), where the addition of dietary Hyp
significantly increased muscle collagen content. So,
we consider that the different results indicated that
the effect of dietary Hyp on collagen biosynthesis
could be tissue-specific, also depending on the dif-
ferent composition of the diet and animal species.
Value with different capital letters is significantly different among
the dietary treatments in the same tissues (P < 0.05). Values with
unlike lowercase letters were significantly different among the
different tissues in the group of basal diet (diets I) (P < 0.05)
Collagen biosynthesis can be influenced by the
activity of proline 4-hydroxylase (P4H), the enzyme
that catalyzes the post-translational formation of 4-
hydroxyproline from proline (Karpakka et al. 1991;
Kivirikko et al. 1989; Hyvarinen et al. 2010; Gelse
et al. 2003), this process is essential for the folding
of newly synthesized procollagen polypeptide chains
into stable triple-helical molecules (Myllyharju and
Kivirikko 2001). We usually use the activity of P4H
to measure the synthesis potential of collagen. In the
present study, P4H was affected by the dietary sup-
plementation of Hyp, as significant increase in P4H
was observed in serum and muscles as dietary Hyp
levels increase. This suggests that the hydroxylation
capacity of proline in muscle increased with the
increase of hydroxyproline supplementation in feed,
and muscle P4H activity might depend on that in
serum, all of which also depend on dietary Hyp

levels. However, the collagen content of muscle was
not significantly affected by P4H activity of muscle
in this study, suggesting the collagen content of
muscles could be influenced by the hydroxylation
capacity of proline in a limited way. It was similar
with the study of Wei et al. (2016) which indicated
that dietary-free Hyp resulted in significantly in-
creased collagen content in muscle of Larimichthys
crocea which is inconsistent with the hydroxylation
capacity of proline in muscle. On the other hand, the
activity of P4H in swim bladder and liver initially
increased significantly followed by a decrease with
incremental dietary Hyp levels, suggesting that there
is might a same mechanism about the P4H activity
in swim bladder and liver affected by dietary hy-
droxyproline. However, the possible reason needs to
be further investigated. Furthermore, the collagen
content of swim bladder was in accordance with
the activity of P4H in swim bladder, initially in-
creased significantly followed by a decrease with
incremental dietary Hyp levels. To sum up, the col-
lagen metabolism could be partially influenced by
P4H activity, which may be depend on the different
tissues, and/or the species and diet composition.
The COL1A1 gene is widely expressed in skate
tissues (muscle, skin, cartilage, gill, orbit, brain, liv-
er, intestine, heart, stomach, spleen, pancreas, egg,
ovary, uterine tube, shell gland, and spiral valve,
except for whole blood) (Hwang et al. 2006). Al-
though dietary Hyp could affect collagen synthesis
(Zhang et al. 2013, 2015), there are limited studies on
the effect of Hyp on the mRNA expression of CO-
L1As, e.g., COL1A1 and COL1A2 in fish species.
The findings here have shown significant difference
in the expression of COL1As in different tissues. For
instance, the expression of COL1As in swim bladder
and muscle were significantly higher than that in
liver and intestines. These differences might be due
to tissue-specific expression patterns of the gene and
different biological effects. For instance, the collagen
content in swim bladder was significantly higher than
that in muscle. With increasing dietary Hyp, the
mRNA level of COL1As in swim bladder initially
increased followed by a decrease, which is consistent
with the varying trend of collagen contents in swim
bladder (P < 0.05). These results indicated that the
effect of dietary Hyp on the metabolism of collagen
in swim bladder might be mediated by regulating the
transcription of the COL1As gene. However, the
Fish Physiol Biochem
mRNA expressions of COL1A1 and COL1A2 in
muscle did not show similar patterns, the reasons
for which require further studies.
Collagen synthesis involves a large number of post-
translational modifications in the polypeptide chains,
with P4H, being the main enzyme that catalyzes specific
intracellular collagen modification. An increase in P4H
activity are controlled mainly by regulating the expres-
sion of subunits of the P4Hα gene (P4Hα (I), P4Hα(II),
and P4Hα(III)) (Kivirikko et al. 1990; Myllyharju
2005). In the current study, the expression levels of
P4Hα(II) and P4Hα(III) in intestines were not signifi-
cantly different with increasing levels of dietary Hyp
(P > 0.05), while the levels of P4Hαs expression in
liver, muscle, and swim bladder were significantly in-
fluenced by the increasing dietary Hyp (P < 0.05). The
P4Hα(I) and P4Hα(II) expression in muscle as well as
the P4Hαs expression in swim bladder were significant-
ly reduced with increasing dietary Hyp (P < 0.05).
These observations are in agreement with recent studies
(Zhang et al. 2013, 2015), where increasing dietary Hyp
content was reported to decrease the mRNA level of
P4Hα (I) in muscle of turbot. However, in muscle, the
expression of P4Hα(III) showed an inconsistent pattern
compared with P4Hα(I) and P4Hα(II), which increased
initially and then decreased with increasing dietary hy-
droxyproline. Up to date, there are limited referable
information about the comparison among the P4Hα(I),
P4Hα(II), and P4Hα(III) expression in the same time
study. Therefore, the biological significance of the sub-
tle differences among the relative expression patterns of
P4Hα(I), P4H α(II), and P4Hα (III) needs to be further
explored and analyzed. The α subunits of P4H contain
the main catalytic sites, and are the rate-limiting steps
for the formation of active P4H, these can be used as
catalysts for P4H synthesis (Kivirikko et al. 1990). In
this study, the expression of P4Hαs in muscle showed
an opposite pattern compare with the activity of P4H in
muscle. To explore the possible causes, we speculate
that the influence of dietary Hyp supplementation on
fish collagen metabolism might be to help reduce pro-
hydroxylation to some extent, which was confirmed by
Li et al. (2009). Therefore, it can save Pro and P4H with
increasing dietary Hyp, leading to release P4H in the
proline-binding state and increase the activity of free
P4H. Meanwhile, for the need of maintaining homeo-
stasis, the negative feedback of P4H gene transcription
level is enhanced, and finally the transcription level of
P4H gene decreases. Because there is limited referable
Fish Physiol Biochem
information, the exact cause is needed to be further
investigated and confirmed. On the other hand, the
collagen level of swim bladder was however found to
increase as dietary Hyp level increases. We boldly spec-
ulated that the improved collagen level in swim bladder
of N. diacanthus fed on optimum Hyp might not be due
to increased ability to synthesize collagen, but suppres-
sion of collagen degradation in fish, since the accumu-
lation of collagen depends not only on the posttransla-
tional modification steps but also on the fractional deg-
radation of collagen (Laurent 1987; Zhang et al. 2015).
However, the role of dietary Hyp in inhibiting collagen
degradation and the relevant molecular mechanisms
involved require further exploration and confirmation.
In conclusion, the results from the present study
revealed that dietary Hyp levels significantly affect the
level of free Hyp in plasma and liver as well as collagen
contents in swim bladder but not in muscle. The opti-
mum amount of Hyp added to the basal diet was esti-
mated to be 9.66 g kg−1 based on the collagen content in
swim bladder. Moreover, the activity of P4H and the
mRNA levels of COL1A1, COL1A2, P4Hα (I), P4Hα
(II), and P4Hα (III) in different tissues were significant-
ly affected by the increasing dietary Hyp level to some
extent, thus partially influencing the collagen metabo-
lism in the N. diacanthus.
Acknowledgements We are grateful to all laboratory members
for technical advice and valuable help during the feeding trial and
sample analysis. Gratitude is extended to Dr. Jude Juventus Aweya
for English editing.
Funding information The present study was financially sup-
ported by grant no. 2016KQNCX058 from young creative talents,
major scientific research projects of Guangdong University Foun-
dation and grant no. A201005D06-1 from the China Guangdong
Oceanic and Fishery Science and Technology Foundation.
References
Aksnes A, Hope B, Jönsson E, Björnsson BT, Albrektsen S (2006)
Size-fractionated fish hydrolysate as feed ingredient for rain-
bow trout (Oncorhynchus mykiss) fed high plant protein
diets. I: growth, growth regulation and feed efficiency.
Aquaculture 261:305–317
Aksnes A, Mundheim H, Toppe J, Albrektsen S (2008) The effect
of dietary hydroxyproline supplementation on salmon
(Salmo salar L.) fed high plant protein diets. Aquaculture
275:242–249
Association of Official Analytical Chemists (AOAC) (1995)
Official methods of analysis of official analytical chemists
international, 16th ed. Association of Official Analytical
Chemists, Arlington, VA
Brinckmann J, Notbohm H, Müller PK (2005) Collagen, topics in
current chemistry. Springer, Berlin, p 247
Chen HD, Zheng JH, Shen ZK (2011) Technique for artificial
breeding of spotted drum N. diacanthus. Fish Sci 30:501–
504 (in Chinese)
Clarke S (2004) Understanding pressures on fishery resources
through trade statistics: a pilot study of four products in the
Chinese dried seafood market. Fish Fish 5:53–74
Gelse K, Poschi E, Aigner T (2003) Collagens-structure, function,
and biosynthesis. Adv Drug Deliv Rev 55:1531–1546
Hwang JH, Yokoyama Y, Mizuta S, Yoshinaka R (2006) cDNA
cloning and characterization of type I procollagen α1 chain in
the skate Raja kenojei. Comp Biochem Phys B. 144:1–10
Hyvarinen J, Parikka M, Sormunen R, Ramet M, Tryggvason K,
Kivirikko KI, Myllyharju J, Koivunen P (2010) Deficiency
of a transmembrane prolyl 4-hydroxylase in the zebrafish
leads to basement membrane defects and compromised kid-
ney function. J Biol Chem 285:42023–42032
Karim AA, Bhat R (2009) Fish gelatin: properties, challenges, and
prospects as an alternative to mammalian gelatins. Food
Hydrocoll 23:563–576
Karpakka J, Virtanen P, Väänänen K, Orava S, Takala TE (1991)
Collagen synthesis in rat skeletal muscle during immobiliza-
tion and remobilization. J Appl Physiol 70:1775–1780
Kimura S, Zhu XP, Matsui R, Shijoh M, Takamizawa S (1988)
Characterization of fish muscle type I collagen. Food Sci 53:
1315–1318
Kivirikko KI, Myllylä R, Pihlajaniemi T (1989) Protein hydrox-
ylation: prolyl 4-hydroxylase, an enzyme with four
cosubstrates and a multifunctional subunit. FASEB J 3:
1609–1617
Kivirikko KI, Helaakoski T, Tasanen K, Vuori K, Myllyla R,
Parkkonen T, Pihlajaniemi T (1990) Molecular biology of
prolyl 4-hydroxylase. Ann N Y Acad Sci 580:132–142
Kousoulaki K, Olsen HJ, Albrektsen S, Langmyhr E, Mjøs SA,
Campbell P, Aksnes A (2012) High growth rates in Atlantic
salmon (Salmo salar L.) fed 7.5% fish meal in the diet.
Micro-, ultra-and nano-filtration of stickwater and effects of
different fractions and compounds on pellet quality and fish
performance. Aquaculture 338:134–146
Krane SM (2008) The importance of proline residues in the
structure, stability and susceptibility to proteolytic degrada-
tion of collagens. Amino Acids 35:703–710
Kukkola L, Hieta R, Kivirikko KI, Myllyharju J (2003)
Identification and characterization of a third human, rat, and
mouse collagen prolyl 4-hydroxylase isoenzyme. J Biol
Chem 278:47685–47693
Laurent GJ (1987) Dynamic state of collagen: pathways of colla-
gen degradation in vivo and their possible role in regulation
of collagen mass. Am J Phys 1:C1–C9
Li P, Wu GY (2018) Roles of dietary glycine, proline, and hy-
droxyproline in collagen synthesis and animal growth.
Amino Acids 50:29–38
Li P, Mai KS, Trushenski J, Wu GY (2009) New developments in
fish amino acid nutrition: towards functional and environ-
mentally oriented aquafeeds. Amino Acids 37:43–53
Li WJ, Wen XB, Zhao J, Li SK, Zhu DS (2016) Effects of dietary
protein levels on growth, feed utilization, body composition

and ammonia–nitrogen excretion in juvenile Nibea
diacanthus. Fish Sci 82:137–146
Li WJ, Wen XB, Huang YS, Zhao J, Li SK, Zhu DS (2017) Effects
of varying protein and lipid levels and protein-to-energy
ratios on growth, feed utilization and body composition in
juvenile Nibea diacanthus. Aquac Nutr 23:1035–1047
Liu YZ, He G, Wang QC, Mai KS, Xu W, Zhou HH (2014)
Hydroxyproline supplementation on the performances of
high plant protein source-based diets in turbot
(Scophthalmus maximus L.). Aquaculture 433:476–480
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the Folin-phenol reagent. J Biol Chem
193:265–275
Lu XJ, Chen J, Chen MZ, Lü JN, Shi YH, Li HY (2010)
Hydrolysates of swim bladder collagen from miiuy croaker,
miichthys miiuy, enhances learning and memory in mice.
Curr Top Nutraceut R. 8:149–156
Miller EJ (1985) The structure of fibril-forming collagens. Ann N
Y Acad Sci 460:1–13
Myllyharju J (2005) Intracellular post-translational modifications
of collagens. Top Curr Chem 247:115–147
Myllyharju J, Kivirikko KI (2001) Collagens and collagen-related
diseases. Ann Med 33:7–21
Ohara H, Matsumoto H, Ito K, Iwai K, Sato K (2007) Comparison
of quantity and structures of hydroxyproline-containing pep-
tides in human blood after oral ingestion of gelatin hydroly-
sates from different sources. J Agric Food Chem 55:1532–
1535
Phang JM, Liu W, Zabirnyk O (2010) Proline metabolism and
microenvironmental stress. Annu Rev Nutr 30:441–463
Pinilla-Tenas J, Barber AM, Lostao P (2003) Transport of proline
and hydroxyproline by the neutral amino-acid exchanger
ASCT1. J Membr Biol 195:27–32
Rest M, Garrone R, Herbage D (1993) Collagen: a family of
proteins with many facets. Adv Mol Cell Biol 6:1–67
Sato K, Yoshinaka R, Itoh Y, Sato M (1989) Molecular species of
collagen in the intramuscular connective tissue of fish. Comp
Biochem Phys B. 92:87–91
Fish Physiol Biochem
Stanley DW (1983) Relation of structure to physical properties of
animal materials. In: Peleg M, Bagley EB (eds) Physical
properties of foods. Avi Publish. Comp., Inc., Westport
Wei ZH, Ma J, Pan XY, Mu H, Li J, Shentu J, Zhang WB, Mai KS
(2016) Dietary hydroxyproline improves the growth and
muscle quality of large yellow croaker Larimichthys crocea.
Aquaculture 464:497–504
Wen J, Zeng L, Chen ZM, Xu YH (2016) Comparison of nutri-
tional quality in fish maw product of croaker Protonibea
diacanthus and perch Lates niloticus. J Ocean Univ China
15:726–730
Wu G, Bazer FW, Datta S, Johnson GA, Li P, Satterfield MC,
Spencer TE (2008) Proline metabolism in the conceptus:
implications for fetal growth and development. Amino
Acids 35:691–702
Wu GY, Bazer FW, Burghardt RC, Johnson GA, Kim SW, Knabe
DA, Li P, Li XL, McKnight JR, Carey-Satterfield M, Spencer
TE (2011) Proline and hydroxyproline metabolism: implica-
tions for animal and human nutrition. Amino Acids 40:1053–
1063
Zhang KK, Ai QH, Mai KS, Xu W, Liufu ZG, Zhang YJ, Peng M
(2013) Effects of dietary hydroxyproline on growth perfor-
mance, body composition, hydroxyproline and collagen con-
centrations in tissues in relation to prolyl 4-hydroxylase α(I)
gene expression of juvenile turbot, Scophthalmus maximus L.
fed high plant protein diets. Aquaculture 404-405:77–84
Zhang KK, Mai KS, Xu W, Zhou HH, Liufu ZG, Zhang YJ, Peng
M, Ai QH (2015) Proline with or without hydroxyproline
influences collagen concentration and regulates prolyl 4-
hydroxylase α (I) gene expression in juvenile turbot
(Scophthalmus maximus L.). J Ocean Univ China 14:541–
548
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional
affiliations.