Aquaculture Reports 13 (2019) 100174

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

A study of the digestive enzyme activities in scaleless carp (Gymnocypris T

przewalskii) on the Qinghai-Tibetan Plateau
Haining Tianb,1, Yuqiong Menga,b,1, Changzhong Lib, Lunxiang Zhangb, Guoqian Xub, Yuan Shib,
⁎
Jianquan Shic, Hongfang Qic, Rui Maa,
a
State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, China
b
College of Eco-Environmental Engineering, Qinghai University, Xining, China
c
Emergency Centre of Gymnocypris Przewalskii, Xining, China

A R T I C LE I N FO A B S T R A C T

Keywords: Two experiments were conducted to study the digestive enzyme activities in scaleless carp (Gymnocypris prze-
Gymnocypris przewalskii walskii) on the Qinghai-Tibetan Plateau. In experiment 1, the distribution of digestive enzymes in various di-
Digestive enzyme gestive organs of G. przewalskii (body weight: 118.24 ± 9.48 g) was studied. Results showed that digestive
Distribution enzyme specific activities in intestine were significantly higher than those in hepatopancreas (P < 0.05). In
Temporal changes
intestine, the highest levels of trypsin and chymotrypsin specific activities were found in foregut and hindgut, the
highest levels of lipase and amylase specific activities were observed in foregut (P < 0.05). In experiment 2,
temporal changes of the digestive enzyme specific activities after feeding were investigated in G. przewalskii
(body weight: 305.32 ± 28.78 g). Results showed that lipase and amylase specific activities in foregut ap-
proached maximum levels at 2 h after feeding and then decreased to the basic level at 6 h after feeding. The
trypsin specific activities in foregut and hindgut reached the maximum levels at 2 h after feeding and then to the
basic level at 8 h after feeding. In conclusion, as stomach-less fish, foregut of G. przewalskii contributed more to
the nutrients digestion. The fish could digest nutrients (protein, fat and carbohydrate) efficiently. Thus, in-
creasing in the amount of G. przewalskii resources could be achieved by improving nutritional state in artificial
culture systems.

1. Introduction
Scaleless carp (Gymnocypris przewalskii) is the dominant cold-water
species in Lake Qinghai on the Qinghai-Tibetan plateau with the alti-
tude of 3200 m. Due to its fishery importance and ecological sig-
nificance in the fish-bird-grassland system around Lake Qinghai (Wei
et al., 2014), G. przewalskii is one of the main endemic cultured fish.
Normally it takes 7 years to attain marketable size of 300 g under
natural condition (Walker et al., 1995). Nowadays, it is listed in China
Red Data Book of Endangered Animals as the result of overfishing and
habitat destruction (Xiong et al., 2010). In recent years, local govern-
ment and researchers focused on the artificial propagation and re-
leasing juvenile G. przewalskii to Lake Qinghai. However, lacking nu-
trition research and specific diets severely limited the sustainable
development of fish resources protection.
Utilization of nutrients in fish depend on the activities of various
digestive enzymes present in digestive organs (Natalia et al., 2004).
Studies on digestive secretions in fish can elucidate certain aspects of its
nutritive physiology and help resolve nutritional problems, such as
artificial diets (Xiong et al., 2011). Hence, lots of studies focused on the
definition of digestive enzyme activity profiles (Sabapathy and Teo,
1993; Natalia et al., 2004; Tramati et al., 2005; Xiong et al., 2011) and
the temporal changes in the enzyme expression in response to feeding
(Uys et al., 1987; Caruso et al., 2008; Rodiles et al., 2012; Castro et al.,
2016) in other fish species. To our knowledges, there were very few
studies on the above fields for G. przewalskii.
The present studies were conducted to determine the distribution of
digestive enzymes in various digestive organs of G. przewalskii
(Experiment 1), and then investigate the temporal changes in digestive
enzyme activities in different digestive parts following feeding
(Experiment 2). Such information may contribute to improve nutri-
tional protocols currently adopted for the farming of G. przewalskii.

⁎
Corresponding author.
E-mail address: myrui713@163.com (R. Ma).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.aqrep.2018.10.002
Received 3 July 2018; Received in revised form 19 September 2018; Accepted 27 October 2018
2352-5134/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
H. Tian et al.
2. Materials and methods
The present study was performed in strict accordance with the
Standard Operation Procedures (SOPs) of the Guide for the Use of
Experimental Animals of Qinghai University. All animal care and use
procedures were approved by the Institutional Animal Care and Use
Committee of Qinghai University.
2.1. Experiment 1, experimental animals and rearing condition
G. przewalskii were supplied by the Emergency Centre of G. prze-
walskii in Xining, China. All fish were reared in the intensive re-
circulating aquaculture system. Fish were fed commercial feed of
common carp (32% crude protein, 7% crude lipid) twice daily. Water
temperature ranged from 10 to 18 °C, dissolved oxygen was higher than
5 mg/L.
2.2. Experiment 1, sample collection and chemical analysis
Nine fish with body weight of 118.24 ± 9.48 g were anesthetized
with eugenol (1: 10,000) (Shanghai Reagent Corp, China) and weighed.
The hepatopancreas and intestine were immediately dissected. Then
intestine was divided into foregut, midgut and hindgut according to the
method of Shi et al (2004). All the samples were kept in liquid nitrogen
and then stored in −80 °C for further analysis.
Samples of liver, foregut, midgut and hindgut were homogenized by
an electric homogenizer (XHF-D, Xinzhi, China) on ice. For the homo-
genization, the special buffer was used for each enzyme according to
the manufacturer’s instruction of commercial kit. The homogenate was
then centrifuged at 4 °C at 10,000× g for 15 min. The supernatant was
then stored at −80 °C prior to analysis. The enzyme specific activities in
different digestive parts were assayed by different test kits as listed in
following.
Trypsin specific activity was assayed by using trypsin kit (Nanjing
Jiancheng Bioengineering Institute, No. A080-2). Unit definition: the
trypsin in 1 mg protein, which make absorbance change 0.003 in 1 min
at 37 °C, pH 8.0, is considered as 1 activity unit.
Chymotrypsin specific activity was assayed by using chymotrypsin
kit (Nanjing Jiancheng Bioengineering Institute, No. A080-3). Unit
definition: the chymotrypsin in 1 mg protein, which hydrolyze protein
to 1 μg amino acid at 37 °C, is considered as 1 activity unit.
Lipase specific activity was assayed by using lipase kit (Nanjing
Jiancheng Bioengineering Institute, No. A054-1). Unit definition: the
lipase in 1 g protein, which hydrolyze 1 μmol substrate (triglyceride) at
37 °C, is considered as 1 activity unit.
Amylase specific activity was assayed by using amylase kit (Nanjing
Jiancheng Bioengineering Institute, No. C016-1). Unit definition: the
amylase in 1 mg protein, which hydrolyze 10 mg substrate (starch) in
30 min at 37 °C, is considered as 1 activity unit.
Tissue protein content was determined by coomassie brilliant blue
method by using commercial kit (Nanjing Jiancheng Bioengineering
Institute, No. A045-2).
Table 1
Special activities of trypsin, chymotrypsin, lipase and α-amylase in various digestive organs in Gymnocypris przewalskii.
Digestive tract Trypsin
(U/mgprot)
Hepatopancreas 1466.54 ± 365.51c
Foregut 34575.51 ± 6027.89a
Midgut 13709.65 ± 3195.12b
Hindgut 25348.31 ± 3825.22a
Values (mean ± standard error) in the same column with different superscripts are significantly different (P < 0.05).
Aquaculture Reports 13 (2019) 100174
2.3. Experiment 1, statistical analysis
Data were assessed by one-way ANOVA by using SPSS 19.0 for
windows and then were expressed as mean ± standard error (S.E.).
When differences were significant (P < 0.05), Duncan’s test was used
to compare the means among individual treatments.
2.4. Experiment 2
One hundred of G. przewalskii were kept in net cage (6 m × 6 m ×
6 m) in Li Jiaxia reservoir, Qinghai province, China. The fish were fed
the commercial feed of common carp (32% crude protein, 7% crude
lipid) for 15 days before sampling. During the feeding trial, the water
temperature ranged from 10 to 12 °C, dissolved oxygen was higher than
7 mg/L.
Then, all the fish were starved for 24 h. Five fish were collected as
pre-feeding treatment which were named as T0. Other fish were fed to
satiation. Five fish per group were randomly taken at 2, 4, 6 and 8 h
after feeding and they were named as T2, T4, T6 and T8, respectively.
All the fish taken form the cage was firstly anesthetized with eugenol
(1: 10,000) (Shanghai Reagent Corp, China) and weighed (body weight:
305.32 ± 28.78 g). The hepatopancreas and intestine with contents
were immediately isolated. The intestine was divided into foregut,
midgut and hindgut separately as pervious discussed. All the samples
were kept in liquid nitrogen, and then stored in −80 °C for further
analysis.
Trypsin, lipase and amylase specific activities in different digestive
parts were assayed by the same method in experiment 1. All the data in
experiment 2 were analyzed using one-way ANOVA.
3. Results
3.1. Experiment 1
The specific activities of trypsin, chymotrypsin, lipase and amylase
in various digestive organs of G. przewalskii are shown in Table 1.
Generally, the digestive enzyme specific activities in intestine were
significantly higher than those in hepatopancreas (P < 0.05).
The trypsin and chymotrypsin specific activities in foregut and
hindgut were significantly higher than those in midgut (P < 0.05), no
difference was observed between foregut and hindgut (P > 0.05). The
highest lipase specific activity was found in foregut, which was sig-
nificantly higher than that in midgut (P < 0.05). The significant
descending order of amylase specific activity was listed as follows:
foregut, hindgut and midgut (P < 0.05).
3.2. Experiment 2
Time changes of trypsin specific activities in different digestive parts
after feeding are presented in Fig. 1. The specific activity of trypsin in
hepatopancreas increased to the maximum value (15,627 U/mg pro-
tein) at 2 h after feeding (T2) and then underwent a sharp decrease to
pre-feeding level at 4 h (Fig.1A). The trend of trypsin specific activity in
foregut increased first and then decreased with highest level observed
at 4 h after feeding (T4, 346,011 U/mg protein) (P < 0.05; Fig. 1B).
Chymotrypsin Lipase A-amylase
(U/mgprot) (U/gprot) (U/mgprot)
3.59 ± 0.33b 38.84 ± 7.55c 0.23 ± 0.01d
6.62 ± 0.57a 206.31 ± 52.34a 1.58 ± 0.09a
3.13 ± 0.45b 126.76 ± 31.99b 0.61 ± 0.05c
8.10 ± 1.37a 143.16 ± 14.61ab 1.10 ± 0.10b
2
H. Tian et al. Aquaculture Reports 13 (2019) 100174

Fig. 1. Time changes of trypsin special activity in hepatopancreas (A), foregut (B), midgut (C) and hindgut (D) of Gymnocypris przewalskii after feeding. Reported are
the mean ± standard error, n = 5. Within each experimental group, different letters indicate significant differences (P < 0.05).

Trypsin specific activity in hindgut showed similar trend with that in
foregut. Compared with T0 and T8, significantly higher values of
trypsin specific activity in hindgut were found in T4 and T6 (394,027
and 431,846 U/mg protein, respectively) (P < 0.05; Fig. 1D). At 8 h
after feeding, trypsin specific activities in foregut and hindgut re-
covered to the pre-feeding level. There was no significant difference in
trypsin specific activity in midgut of fish after feeding (P > 0.05;
Fig. 1C).
Time changes of lipase specific activity in different digestive parts
after feeding are shown in Fig. 2. Lipase specific activity in
hepatopancreas (38.47–40.99 U/g protein) and hindgut
(348.17–439.13 U/g protein) remained stable after feeding (P > 0.05;
Fig. 2A, 2D). Lipase specific activity in foregut significantly increased in
the first two hours and reached its maximum in T2 (550.49 U/g pro-
tein), then deceased thereafter (P < 0.05; Fig. 2B). In midgut, the
highest level of lipase specific activity was found at 4 h after feeding
(282.46 U/g protein) and then extremely remarkable deceased (P <
0.05; Fig. 2C). Lipase specific activity in foregut and midgut recovered
to the pre-feeding level at 6 h and 8 h after feeding, respectively.
Time changes of amylase specific activities in different digestive

Fig. 2. Time changes of lipase special activity in hepatopancreas (A), foregut (B), midgut (C) and hindgut (D) of Gymnocypris przewalskii after feeding. Reported are
the mean ± standard error, n = 5. Within each experimental group, different letters indicate significant differences (P < 0.05).

3
H. Tian et al.
Fig. 3. Time changes of amylase special activity in hepatopancreas (A), foregut (B), midgut (C) and hindgut (D) of Gymnocypris przewalskii after feeding. Reported are
the mean ± standard error, n = 5. Within each experimental group, different letters indicate significant differences (P < 0.05).
parts after feeding are shown in Fig. 3. Similar trends of amylase spe-
cific activities in both hepatopancreas and foregut observed in the
present study. In both parts, the amylase specific activities increased
first and then decreased with highest level observed at 2 h after feeding
(T2, 0.28 and 15.52 U/mg protein, respectively) (Fig. 3A and B). At 8 h
after feeding, the specific activities of amylase in hepatopancreas and
foregut recovered to the pre-feeding level. Amylase specific activity in
midgut was unchanged after feeding (P > 0.05; Fig. 3C), while it in
hindgut reached the highest level (12.53 U/mg protein) at 6 h after
feeding (Fig. 3D).
4. Discussion
This study was first undertaken to determine the distribution of the
major digestive enzymes such as trypsin, chymotrypsin, lipase and
amylase in G. przewalskii. As the absence of stomach and pyloric caeca,
the main digestive organs of G. przewalskii were intestine and hepato-
pancreas (Shi et al., 2004). In the present study (both experiment 1 and
2), the digestive enzyme activity profile suggested that G. przewalskii
had the ability to hydrolyze proteins, carbohydrates and lipids. Mean-
while, the trypsin, chymotrypsin, lipase and amylase activity in intes-
tine were significantly higher than those in hepatopancreas. Higher
digestive enzyme activities in digestive tract than those in liver or
pancreas were also found in goldfish, tench, rainbow trout, European
eel and paddlefish (Hidalgo et al., 1999; Huang et al., 2013). The reason
could be that those digestive enzymes were secreted by hepatopancreas
and then activated in intestine of G. przewalskii.
In the intestine of G. przewalskii, higher values of trypsin, chymo-
trypsin, lipase and amylase activities were generally observed in the
foregut (experiment 1). Similar results were also found in sharptooth
catfish, gilthead sea bream and Glyptosternum maculatum for alkaline
protease (Uys and Hecht, 1987; Deguara et al., 2003; Xiong et al.,
2011), in Nile tilapia, Diplodus puntazzo and Glyptosternum maculatum
for lipase (Tengjaroenkul et al., 2000; Tramati et al., 2005; Xiong et al.,
2011), in sharptooth catfish, Pseudoplatystoma corruscans and Glyptos-
ternum maculatum for amylase (Uys and Hecht, 1987; Lundstedt et al.,
2004; Xiong et al., 2011). Digestive enzymes were secreted from exo-
crine pancreas into the anterior section of the digestive tract (Bakke
4
Aquaculture Reports 13 (2019) 100174
et al., 2010). Accordingly, this section seemed to contribute more for
nutrient digestion and absorption than posterior sections (Castro et al.,
2016). Especially in stomach-less fish, the foregut had similar capability
to store and digest food with stomach in stomach-containing fish
(Stroband and Kroon, 1981). The interesting result was observed in the
present study that hindgut of G. przewalskii also had higher activities of
trypsin and chymotrypsin. Similarly, higher alkaline protease activity in
posterior intestine was also found in pintado (Lundstedt et al., 2004)
and Oxyeleotris marmoratus (Ouyang et al., 2016). The reason needs
further investigation.
Knowledge of temporal changes in digestive enzyme activities could
provide a useful background on the time evolution of the digestive
process, which may contribute to improve nutritional protocols adopted
for both fish farming and research. Hence, the second experiment was
conducted to study time changes of the digestive enzyme activities in
different digestive parts after feeding. The high levels of enzyme ac-
tivities recorded at the pre-feeding phase in experiment 2 indicated that
those enzymes still held high activity levels in the digestive tract despite
the absence of food. It was in agreement with European eel (Caruso
et al., 2008). The activities of trypsin, lipase and amylase generally
displayed significantly increasing trend after feeding, and then de-
creased in the present study. Similar trends were also found in other
researches (Fountoulaki et al., 2005; Santigosa et al., 2008; Rodiles
et al., 2012). This indicated that the digestive enzymes secretion and
digestion were stimulated by food ingestion through the digestive tract.
In the present study, trypsin activity in hepatopancreas approached
the maximum level at 2 h after feeding. Then the activity in foregut and
hindgut reached the maximum level at 4 h and 4–6 h after feeding,
respectively. The response of protease activity to feeding was similar
with carp (5 h) (Onishi et al., 1976), Clarias gariepinus (4.5 h) (Uys et al.,
1987), rainbow trout (3 h) (Santigosa et al., 2008) and Senegalese sole
(3 h) (Rodiles et al., 2012). Amylase activities in hepatopancreas and
foregut synchronously approached its maximum levels at 2 h after
feeding, while in hindgut they reached their highest level at 6 h after
feeding. The response of amylase activity in intestine to feeding was
faster than that in carp (5–7.5 h) (Onishi et al., 1976) and gilthead sea
bream (5 h) (Fountoulaki et al., 2005). There was limited study to in-
vestigate time changes of lipase activity after feeding. In the present
H. Tian et al.
study, lipase activity in hepatopancreas remained stable after feeding.
However, it reached maximum level in foregut and midgut at 2 h and
4 h after feeding, respectively. The response of lipase activity in intes-
tine to feeding was faster than it in European eel (8 h) (Caruso et al.,
2008). From above, G. przewalskii could digest nutrients (protein, fat
and carbohydrate) efficiently. The digestive enzymes generally ap-
proached those maximum levels at 2–4 h after feeding, and then de-
creased to the pre-feeding level at 8 h after feeding.
Overall, as stomach-less fish, foregut of G. przewalskii contributes
more to nutrient digestion. G. przewalskii could digest nutrients (pro-
tein, fat and carbohydrate) efficiently. The digestive enzymes generally
approached maximum levels at 2–4 h after feeding and then decreased
to the pre-feeding level at 8 h after feeding. The slow growth of this fish
under nature condition could be related with limited food resources.
Thus, increasing in the amount of G. przewalskii resources could be
achieved by improving nutritional state in artificial culture systems.
Acknowledgements
This research was financially supported by grants from Provincial
Natural Science Foundation of Qinghai, China (2015-ZJ-921Q). We
would like to thank Zhanfang Xue, Xiaoping Xie, Yanlin Tong, Ruman
Zhu for their support and help during this study.
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