2008

Asiatic Herpetological Research, Vol. 11

pp.143–146

Effects of Starvation on Urinary Nitrogen Composition of Juvenile
Chinese Three-keeled Pond Turtles (Chinemys reevesii)
JIE WANG AND CUIJUAN NIU*
Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering, College of Life
Sciences, Beijing Normal University, Beijing, 100875, People’s Republic of China.
*Corresponding author E-mail: cjniu@bnu.edu.cn
Abstract.- We investigated the effect of starvation on urinary nitrogen composition in the juvenile Chinese threekeeled pond turtle (Chinemys reevesii). Under normal conditions, ammonia, urea, and uric acid constitute 2.3, 95.8
and 1.9% of total urinary nitrogen, respectively. During starvation periods of one to four weeks, the concentration ofurea changed little, while that of ammonia rose sharply and that of uric acid fell significantly. After feeding wasresumed for four weeks, the levels of ammonia and uric acid returned to control levels. Changes in urinary nitrogencomposition during starvation may be related to the anti-oxidative function of uric acid during periods of stress.
Keywords.- Pond turtle, Chinemys reevesii, bladder urine content, stress, uric acid.

Introduction
The nature of an animal’s nitrogenous waste, including
ammonia, urea and uric acid, is dependent on the environment in which it lives (Delaunay, 1931).
Furthermore, the spatial distribution of nitrogenous end
products in the body provides important data on howthat animal responds physiologically to that environment. The highly diverse excretory function of theReptilia has been well-documented (Campbell, 1995),
and although aquatic and semiaquatic turtles are primarily ureotelic, a number of exceptions are known where
the predominant form of nitrogenous waste is ammonia(Lee et al., 2007).
The first objective of this study was to determine
the composition of excretory nitrogen in the bladder of
the Chinese Three-keeled pond turtle Chinemys reevesii.
This research will add to the body of literature on nitrogen excretion in freshwater turtles, which is currently
very limited (Lee et al., 2007; Singer, 2003).
Nitrogenous end-products have diverse physiological functions in different animal groups, including acidbase regulation, osmoregulation, etc. (Wright, 1995). Inturtles, two of the major factors affecting nitrogen excretion are availability of water and amount of dietarynitrogen ingested (Singer, 2003), which are of course
highly influenced by starvation and dehydration, which
turtles have developed a magnificent physiologicalcapability to resist. The aquatic Sonoran mud turtle(Kinosternon sonoriense), for example, can aestivate for
11 weeks without food or water; during the initial period
of deprivation, urine in the bladder is apparently used
for osmoregulation (Peterson and Stone, 2000). Whileammonia is quite toxic, more inert forms of excretory© 2008 by Asiatic Herpetological Research

nitrogen (e.g. urea and uric acid) are more costly to synthesize (in ATP equivalents; Baze, 1970), making
ammonia the most frequently produced form of waste inaquatic reptiles because they can lose this waste rapidly
to the environment (Cragg et al., 1961). Uric acid is considered to be an endogenous antioxidant, and as such,might play a role in the clearance of free radicals that areproduced during starvation (Zhu et al., 2005). The second objective of the present work is to investigate how
Chinemys reevesii changes its nitrogen metabolism during starvation.

Materials and methods
Experimental animals and diet.- Juvenile turtles wereobtined from a turtle farm in Guangzhou, China andacclimated to a fixed photoperiod of 12L:12D at a temperature of 29±°C for three weeks in December 2004.
Each of the three turtles were placed in a glass tank withthe dimension of 19 × 23 × 27 cm. Turtles were fed toapparent satiation twice a day on a commercial formulated feed powder (composition as percentage of dry
matter: crude protein 41.66%, crude lipid 5.84%, ash
18.24%, and water 8.62% of total weight), which wasadded to water and extruded to strip pellets before usage.
Each turtle was marked by sawing one to three notchesat the edge of its carapace.
Experimental procedure.- Before the experiment, 108
healthy turtles with a mean body mass of 13.58±1.84 g(mean±SD) were chosen, randomly divided into fivegroups (C [control], S1, S2, S3 and S4), and placed into
a total of 36 tanks. This experiment lasted eight weeks,with a period of starvation interrupting periods when the7S4F

2.7±0.27p2.8±0.3

6S3

1.261
0.313

2.1±0.3

2.1±0.5

S20.475Fp

2.4±0.20.915

65

3.1±0.5

6S3S463.2±0.6

5S2C

1.178

4.0±1.2

2S1

S1

235.7±32.3

2.1±0.5

5C

107.3±21.6 b
4.077
0.012

119.8±10.1 b

150.0±26.7 ab242.4±40.5 a

165.7±24.9 ab

0.352

139.4±41.9

136.1±45.6

166.3±38.5

172.5±33.0

2.1±0.6

NGroupUrea

Percentage
of urine inbody weight

0.304

1.311

0.776
0.5521.21±0.17

4.81±0.83

1.37±0.33

1.08±0.32

0.96±0.17

224.1±43.8 b
4.064
0.012

251.1±21.0 b

312.8±53.8 ab496.6±80.9 a

341.3±49.7 ab

After feeding for four weeks

0.000

25.5590.22±0.11 b
496.6±66.8

267.4±104.1

0.36±0.13

284.5±94.2

b352.0±82.0

363.0±66.5

0.71±0.14 b

0.44±0.42b

2.19±0.20 a

1.45±0.19

1.138
0.362

Total NAt the end of fasting

Uric acid

5.59±0.88

7.40±1.58

7.43±1.1

6.10±0.56

0.040

3.103

24.25±2.61a

13.24±5.44ab

9.37±3.21 b

17.45±3.64

ab9.21±0.44 b

Ammonia

Concentration: mmol•L-1

1.947
0.135

94.7±1.2

95.4±0.595.1±0.997.5±0.5

96.8±0.5

0.794

0.417

94.8±0.3

95.6±0.4

94.7±0.994.6±0.3

94.5±0.8

Urea

1.010
0.422

2.6±0.6

2.2±0.3

2.6±0.5

1.6±0.3

1.9±0.2

0.007

5.015

5.0±0.2 a

3.6±0.5ab

3.4±0.3 ab

5.0±0.1ab

2.9±0.6 b

Ammonia

2.010
0.125

2.7±0.6

2.3±0.3

2.3±0.7

1.0±0.4

1.3±0.3

0.008

4.789

0.2±0.1 b

0.9±0.3 b

1.9±0.8 ab

0.4±0.4ab

2.6±0.3 a

Uric acid

Partition of total Nitrogen

Table 1. Percentage of urine in body weight, nitrogen concentration (mmol•L-1) and partition (percent) formed as ammonia, urea and uric acid in the bladder of freshwater turtles (Chinemys reevesii) at the end of fasting and after feeding for four weeks. Data expressed as Mean ± SE. Different letters denote significant differences
within the same column, p <0.05.
Group C was the control group. Group S1 was starved during the fourth week; group S2 was starved during the third and fourth weeks; group S3 was starved fromthe second to the fourth week; group S4 was starved for the first four weeks.

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turtles were fully fed. The C group included eight tanks
where the turtles were always fed to satiation. The S1,S2, S3 and S4 groups included seven tanks each. The S4group was starved in the first four weeks of the experiment. The S3 group was fed in the first week and thenstarved for three weeks. The S2 group was fed for twoweeks and then starved for two weeks. The S1 group
was fed for three weeks and then starved for one week.
All groups were fed to satiation from the fifth to eighthweeks.
Analytical methods.- One turtle was sampled randomly
from every tank at the end of the fourth and eighthweeks, starved for 24 hours to empty the gut, toweledoff, weighed to within 0.01 g with an electronic balance,
placed in a plastic bag and euthanized at a temperatureof -80°C.
The turtles were dissected and the urinary bladders,which contained frozen urine, were extracted andweighed. Urine samples were centrifuged and diluted
twenty times with 0.9% NaCl. Urea and uric acid concentrations were determined using a Roche Diagnostics
Cobas INTEGRA 400. The concentration of ammonia
was measured using Roche Modular-P and Integra systems.
Data processing.- All statistical analyses were performed using the SPSS13.0 software package. The
Kolmogorov-Smirnov test revealed that the data (including percentages) followed a normal distribution. A oneway ANOVA was employed to assess the effects of starvation. The Tukey HSD or Games-Howell test was used
for making multiple comparisons between the means ofdifferent groups; p < 0.05 was taken as the level of significance.

Results
Table 1 shows the changes in percentage of urine, total
excretory nitrogen concentration, and the proportion ofammonia, urea, and uric acid among the different groups
at the end of the fasting and refeeding periods.
After fasting, significant differences between thecontrol and deprived groups were found in the concentrations of ammonia (F4,19 = 3.103, p = 0.040)and uricacid (F4,18 = 25.559, p = 0.000), and in the composition oftotal excretory nitrogen (F4,18 = 5.015, p = 0.007;
F4,18 = 4.789, p = 0.008). Ammonia concentration and its
relative concentration showed a positive relationshipwith increading periods of starvation, while those of uricacid showed the reverse trend. Excretory urea did notappear to be significantly affected by starvation(p > 0.05).

145

After feeding for four weeks, urea concentrationand total excretory nitrogen of S1 were found to be significantly higher in groups S3 and S4, while there wereno clear differences between the other groups for thesetwo parameters. Furthermore, all groups did not differ
clearly in other parameters measured (p > 0.05). Urinary
nitrogen composition was approximately 2.3% ammonia, 95.8% urea, and 1.9% uric acid in the control group.

Discussion
In our study, Chinemys reevesii was found to be primarily ureogenic like other freshwater turtles, but it exhibited a relatively higher proportion of urea (about 95.8%)
in excretory nitrogen compared to that of other freshwater turtles such as Trachemys scripta (about 70%, urinein ureter; Dantzler and Schmidt-Nielson, 1966) and
Pelodiscus sinensis (54%, water samples; Lee et al.,2007). Schmidt-Nielsen and Skadhauge (1967) reported
that ureotelic fresh water turtles excreted 45–95% of
their waste nitrogen in the form of urea, comparable to
the results found here.
During food deprivation, the concentration of uricacid in the turtle’s bladder fell significantly (p = 0.000)
while that of ammonia clearly rose (p = 0.040), eventhough they constitute only a small proportion of total
nitrogenous end products. Rapatz and Musacchia (1957)
found that the fasted fresh water turtle Chrysemys picta(fasted for 6–8 weeks at 22°C) showed characteristic
biochemical properties in decreased liver total fatty
acids, decreased blood glucose and increased urine uricacid levels. Meanwhile, specimens in cold torpor (4–8weeks at 4°C) had increased liver total fatty acids, a significant increase in liver glycogenolysis and increased
urine uric acid levels. Zhu et al. (2005) found that
Chinemys reevesii retained higher uric acid in its bladderduring cold torpor, but these contents rapidly decreased
when exposed to air because of oxidation. They presumed that retention of uric acid in the bladder duringcold torpor (they induced hibernation for about oneyear) might have a benificial function during long periods of food deprivation, as uric acid or urate is known tohave antioxidative properties similar to those of vitamin
C and E. This may be the cause of the observed decreaseof uric acid in the bladder during starvation in the present study. Conflicting results observed between this andprevious studies may be due to differences in rearing
conditions, the use of a different species (Chinemys
reevesii vs. Chrysemys picta) or ontogenetic differences(juvenile vs. adult). The increase of ammonia in thebladder of the turtles may be the result of increased
activity in innate protein catabolism during starvation.
Further research on nitrogen metabolism in stressful

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environments should be conducted.
The concentration of total urinary nitrogen and urea
in our experiment did not significantly change with starvation, conflicting with prior experiments (Zhu et al.,2005). This conflict may be due to differences in nitrogen metabolism when hibernation is not induced.

Acknowledgments
We thank our colleague Miss Chen-Xi Huang and Dr.
Zhi-Gang Xie for their valuable comments. We are also
grateful to Professor Quan-zhong Shan and Miss Ou Liu
for their help in nitrogen analysis. This research wassupported by the National Natural Science Foundationof China (No. 30271014, No. 30671598).

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Submitted: 01 November 2006
Accepted: 22 September 20072008

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