2008

Asiatic Herpetological Research, Vol. 11

pp.45–49

Effect of Stocking Density on the Energy Budget of Juvenile
Soft-Shelled Turtles (Pelodiscus sinensis)
RUNZHEN JING1 AND CUIJUAN NIU1,*
1

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.- The present work investigates the effect of stocking density on the energy budget of juvenile soft-shelledturtles (Pelodiscus sinensis). Turtles (body weight: 16.22±0.28 g) were stocked at densities of SD1 (8 animals/m2,
0.14 kg/m2), SD2 (48 animals/m2, 0.81 kg/m2) and SD3 (96 animals/m2, 1.62 kg/m2) in aquaria in triplicate for eachtreatment. The experiment lasted for 35 days. Survival rate, coefficient of size variation, productivity, and apparent
digestibility coefficient were not significantly different at the three stocking densities. While there were no significant
differences between treatments SD2 and SD3, turtles in group SD1 showed a lower excretion rate and significantly
higher food intake and growth rate. Turtles in group SD1 also showed higher crude lipid content and lower crude ash
content. No significant differences were found among the treatments in body moisture and crude protein.
Keywords.- Survival, growth, food consumption, stress, body composition.

Introduction
Stocking density is one of the most important biotic factors in aquaculture because it directly influences survival, growth, behavior, health, feeding, and production.
High densities may interfere with intra-population interactions and eventually affect biomass gain. The relationship between stocking density and growth for fish has
been shown to be positive (Papst et al., 1992), negative(Hengsawat et al., 1997; Irwin et al., 1999) or densityindependent (Fairchild and Howell, 2001; Rowland etal., 2004; Rowland et al., 2006), depending on different
experimental density ranges. In the fish farming industry, it is very important for the farmer to know the optimum stocking density of the animals being reared tomaximize production and profitability.
The soft-shelled turtle (Pelodiscus sinensis) is acommonly cultured aquatic reptile species in China witha yield of more than 140,000 tons in 2004 (Shen et al.,2006; Zhang, 2005). Despite the fact that the aquaculture of this species is widespread, scientific studies concerning the effects of stocking density on biologicalcharacters are limited (Mayeaux et al., 1996). The objective of the present study is to evaluate the effect of stocking density on the energy budget of juvenile P. sinensis.

Materials and Methods
Turtles and rearing conditions.- Juvenile Pelodiscus
sinensis (body weight: 16.22±0.28 g) were obtained
from a commercial turtle farm in Beijing. Turtles were

© 2008 by Asiatic Herpetological Researchreared in rectangular aquaria (80 length [L] × 35 width[W] × 30 cm height [H]), with 11 individuals per aquarium, at a water depth of 15 cm. Water temperature wasmaintained at 29.5±0.5ºC by a thermo-controlled heater.
Aquaria were supplied with dechlorinated water. The
dissolved oxygen level was over 5 mg/L and the pH was
7.95. Natural photoperiod was followed. Turtles were
fed to satiation once daily at 1500 h. Commercial turtle
food was used with 0.5% Cr2O3 added for the apparent
digestibility coefficient assay. Proximate dry mattercomposition of the diet was as follows: moisture 3.97%;
crude protein 40.27%; crude lipid 7.04%; and crude ash
15.73%. Energy content was 16.06 kJ/g. Turtles wereallowed to acclimate to the laboratory conditions forthree weeks before the experiments began.
Experimental process.- Healthy turtles were randomly
stocked at initial densities of SD1 (8 animals/m2, 0.14
kg/m2), SD2 (48 animals/m2, 0.81kg/m2) and SD3 (96animals/m2, 1.62 kg/m2) in aquaria (40 L ×30 W ×30 cm
H) in triplicate for each treatment. There were no significant differences in initial average body weight or coefficient of size variation within each aquarium among thetreatments. The experiment lasted for 35 days. The final
densities were 0.34 kg/m2, 1.20 kg/m2 and 2.26 kg/m2,
respectively. To maintain a constant numbers of animals,
an alternative turtle with approximately the same bodyweight was added when an initial turtle died. All thewater in the tanks was replaced by an equal amount offresh water daily after surplus food was removed. The
aquaria were inspected once daily for mortalities anddead turtles were removed immediately after detection.

46

2008

Asiatic Herpetological Research, Vol. 11

Table 1. Survival, specific growth rate, food consumption, apparent digestibility coefficient, and excretion of juvenile
soft-shelled turtles (Pelodiscus sinensis) held at different stocking densities (Mean ± S. E.)1.
Treatment
Initial average weight (g)SD1
16.90±0.45

SD216.27±0.23

SD316.15±0.16

P-Value
0.294

Final average weight3 (g)40.30±1.96a

24.09±0.38b

22.49±0.70b

0Survival rate (%)

100±0.00

91.67±8.33

77.78±10.02

0.1

Coefficient of size variation (%)—

30.82±5.24

26.84±2.72

0.504

Specific growth rate2 (%/day)

2.48±0.22a

1.12±0.09b

0.94±0.11b

0.002

5.57±0.57

7.08±4.96

7.60±7.98

0.963

2

Productivity (g/day/m )3Food consumption (mg/day/g)

40.54±0.46

a25.76±0.43b24.34±0.62b0Apparent digestibility coefficient (%)

74.29±2.1275.60±0.10

78.14±1.44

0.319

Excretion (mg N/day/kg)

60.44±10.64b

104.36±5.75a

108.59±3.54a

0.012

1

Values in each row with different superscript letters are significantly different (p < 0.05).

2

Means are significantly different among treatments (p < 0.01).

3

Means are extremely different among treatments (p < 0.001).

Turtles were weighed to an accuracy of 0.1 g before andafter the experiment following three days starvation. Six
turtles at the beginning of the experiment and all turtles
remaining at the end of the experiment were sacrificed
and dried at 65ºC to constant weight for analysis of bodybiochemical composition. Crude protein was determinedby the Kjeldahl method, crude lipid was extracted byether, and crude ash was determined after 12 h of burning at 550º in a muffle furnace. Energy contents were
measured using a calorimeter (CA-4P, Shimadzu,
Japan). All samples were analyzed in triplicate.

productivity were calculated by the following formulae:
CV (%) = 100 × Standard deviation/Average bodyweightSGR (%/day) = 100×(lnW2-lnW1)/(t2- t1)
Productivity (g/day/m2) = (Wt2-Wt1)/S/( t2- t1)

ADC (%) = 100 × (1- Cr2O3 content in diet/Cr2O3
content in feces)where W2 and W1 express final average weight at time
t2 and initial average weight at time t1 in days, respectively. Wt2, Wt1 and S express biomass at day t2, biomass
at day t1, and aquarium surface area, respectively.
Energy allocated to growth was calculated from weightgain (g) and energy content (kJ/g) of the whole body.
Energy lost via excretion was calculated from theammonia and urea excreted using energy equivalents forammonia (24.83 J/mg N) and urea (23.03 J/mg N)(Elliott, 1976). Ammonia and urea concentrations inside
the water were measured by firstly catalyzing urea toammonia using urease, and then assaying the totalammonia via standard Nessler’s colorimetric technique.
The turtles were kept in a given amount of renewed
experimental water for 48 h and fasted during the measurement. Water samples were taken before and after thisperiod.
The energy budget for juvenile animals can be
described as:

F = I × (100-ADC) × Ef/100

C=F+U+R+G

where I and Ef are food consumption in dry weight andfeces energy content, respectively.
The coefficient of variation in body weight (CV)within each aquarium, specific growth rate (SGR), andwhere C is the energy in the food consumed, F is theenergy lost in fecal production, U is the energy lost in
nitrogenous excretory products, R is the energy spent inmetabolism, and G is growth energy. In this study, C, F,

Measurements of various components of the energy
budget.- A weighed excess of feed pellets was fed to theturtles once daily (at 1500 h) with a fraction of feed
retained for determination of dry matter content.
Uneaten food was collected an hour later and dried.
Food intake was determined as the difference between
the food supplied and the food left uneaten.
Fresh complete feces were collected once daily.
Cr2O3 content in the diet and feces were determined by
the method described in detail by Bolin et al. (1952).
The apparent digestibility coefficient (ADC) and theenergy lost via feces (F) were calculated by the following expressions:

2008

47

Asiatic Herpetological Research, Vol. 11

Table 2. Chemical composition of juvenile soft-shelled turtles (Pelodiscus sinensis) held at different stocking densitiesin 35-day experimental period (Mean ± S. E.)1.
Treatment

Moisture (%)

Crude protein*(%)

Crude lipid* (%)

Crude ash*,2 (%)

SD175.86±0.28

63.58±0.65

13.46±0.62a

19.16±0.15bSD2

76.27±1.45

65.05±0.13

8.31±1.01b

22.39±0.54a

61.89±1.04b23.11±0.22a

SD3

76.46±0.83

10.13±0.73*Crude protein, crude lipid, and crude ash based on the contents in the dry matter.
1
Values in each column with different superscript letters are significantly different p < 0.05).
2
Means were extremely different among treatments (p < 0.001).

U, and G were determined directly, and R was calculatedby the equation:
R = C - F - U - G.
Statistical analysis.- All data were analyzed with SPSS
for Windows, Version11.0. A one-way ANOVA was used
to test the differences among treatment means whenassumptions of normality and homogeneity were met.
When a significant treatment effect was found, theLeast-Significant-Difference (LSD) test was applied todetermine which specific pairs differed. The nonparametric Kruskal-Wallis test was applied when therequired homogeneity of variance and normality were
not satisfied. A regression analysis was carried out toestimate the relationship between stocking density andgrowth rate. The significant level was set at p < 0.05.

Results
All turtles in one aquarium of treatment SD2 died due toa malfunction of the thermo-controlled heater. Thisreplicate was not taken into account for any statisticalcomparisons.
The effects of stocking density on survival, specificgrowth rate, food consumption, apparent digestibility
coefficient, and excretion.- Survival rates were not significantly different among the three treatments (Table 1),
but all showed a negative relationship with increased
stocking density (r = -0.708, p = 0.050). Stocking density showed a clear influence on final body weight, specific growth rate, food consumption, and excretion, as
identified by the statistical significance (Table 1).
Turtles in group SD1 showed significantly higher foodintake and growth rate than those held at the other two
densities, which did not differ significantly from eachother. Lower excretion rate was observed in group SD1
compared to groups SD2 and SD3. The apparent
digestibility coefficients of juvenile turtles ranged from74.29% to 78.14%. The relationship of SGR and stocking density (SD, animals/m2) can be described as the lin-

ear model or the quadratic model:
SGR = -0.0171× SD + 2.4360(p < 0.01, R2 = 0.769)
SGR = 0.0003 × SD2 - 0.0531× SD + 2.8805(p < 0.01, R2 = 0.915).
The effect of stocking density on body composition.Body composition for each treatment group is shown in
Table 2. There were significant differences in lipid andash contents between treatments (F2,5 = 11.520,
p = 0.013; F2,5 = 64.577, p = 0.000). Crude lipid contents of group SD1 were much higher than those ofgroups SD2 and SD3 while crude ash contents werelower. No significant differences were found among
treatments in body moisture and crude protein(F2,5 = 0.155, p = 0.860; F2,5 = 3.412, p = 0.116).
The effect of stocking density on energy budget.- No
marked differences were found among treatments in F/C
and R/C (Table 3; F2,5 = 0.058, p = 0.945; F2,5 = 2.561,
p = 0.171). Stocking density significantly influenced
U/C and G/C (F2,5 = 27.151, p = 0.002; F2,5 = 6.243,
p = 0.044). Energy budgets for the different treatments
can be described as:
100C = 10.0F + 0.3U + 73.3R + 16.5G; SD1
100C = 10.3F + 0.7U + 78.3R + 10.8G; SD2
100C = 10.0F + 0.8U + 79.1R + 10.2G; SD3

Discussion
Stocking density has been considered to be chronicallystressful to reared animals (Vijayan and Leatherland,1988). Several studies have also demonstrated that
increased stocking density has a negative effect on survival and growth (Penha-Lopes et al., 2006; Schram etal., 2006), except in some fish species that exhibit
schooling behavior (Jørgensen et al., 1993;
Papoutsoglou et al., 1998). This impaired growth bystocking density may be attributed to reduced food con-

48

2008

Asiatic Herpetological Research, Vol. 11

Table 3. Energy budget of juvenile soft-shelled turtles (Pelodiscus sinensis) held at different stocking densities in 35day experimental period (Mean ± S. E.)1.
Treatment

C3 (J/day/g)

SD1

651.21±7.33

aSD2413.83±6.89bSD3b390.96±9.94

F/C (%)

U/C2 (%)

R/C (%)G/C (%)

10.04±0.83

0.31±0.06

b73.29±2.61

16.45±1.77a

10.31±0.04

0.74±0.06a

78.32±0.63

10.75±0.63b

9.96±0.65

a79.12±1.56

10.23±1.13b

0.79±0.03

C: food intake in energy; F: energy lost in fecal production; U: energy lost in nitrogenous excretion; R: energy lost in metabolism; G: energy allocated to
growth.
1
Values in each column with different superscript letters are significantly different (p < 0.05).
2
Means are significantly different among treatments (p < 0.01).
3
Means are extremely different among treatments (p < 0.001).

sumption, lowed food conversion rate or increased
metabolic cost (Jørgensen et al., 1993; Li and Brocksen,
1977; Vijayan and Leatherland, 1988).
In the present study, an obvious trend of decreased
survivorship of juvenile soft-shelled turtle with elevatedstocking density was observed. This agrees with theresults of Mayeaux et al. (1996), who reported that common snapping turtles (Chelydra serpentine) stocked at
58 animals/m2 exhibited greater mortality, lower weight
gain, and higher food consumption compared to thosestocked at 29 animals/m2. Food consumption also
reduced with increasing stocking density in the present
study, however, conflicting with the above snapping turtle results. This discrepancy may be caused by differences in life habit or varying experimental conditions.
Knights (1985) observed that more aggressive (and usually larger) eels of Anguilla anguilla ate more, while thefeeding of smaller subordinates was inhibited, evenwhen food was offered to excess. In the present experiment, despite food being divided between at several
spots in each tank, a similar phenomenon was observed
during the feeding process. It is likely that the appetiteof the subordinate turtles was suppressed and their
growth inhibited when the turtles were grouped at highdensity.
The lower feed intake in treatments SD2 and SD3
appear to explain the lower growth in the same treatments, because reduced food ingestion reduces theamount of energy available for growth (Table 1).
Moreover, the proportions of food energy spent ingrowth (indicated by the gross energy efficiency, Table3) in treatments SD2 and SD3 were obviously lower in
relation to that in treatment SD1 (10.8% and 10.2%,
compared to 16.5%). These results may suggest that theingested energy was not efficiently converted to bodyreserves, especially at high stocking density.
Juvenile soft-shelled turtles tend to grab each otherwith their sharp claws, and grabbing activities often
result in injuries of the toes and neck, and may evenresult in death. Agonistic interactions among individuals
and elevated swimming activity also lead to increased
metabolic expenditure. In the present experiment, turtlesof groups SD2 and SD3 exhibited hyperactivity compared to group SD1, and had reduced lipid and higher
ash contents. The difference in chemical composition inthese turtles suggest that elevated stocking density may
induce extra energy expenditure, subsequently allocating less energy to storage.
In conclusion, the pattern of energy allocation of theturtles in the present experiment was significantly influenced by different stocking densities. Turtles cultured atlower density had a relatively higher survival rate, distinctly higher growth rate and transfer more consumed
energy to growth. The lower energy input and lower
gross energy efficiency in treatments SD2 and SD3 may
have contributed to their reduced growth rate.
Furthermore, the excretion of nitrogenous wastes to theenvironment was relatively lower with reduced stocking
density. Conversely, higher stocking density could resultin higher productivity to some degree, since there wereno significant differences in productivity among thetreatments. We suggest that the turtle farmer pursue an
optimal stocking density based on profitability, considering that lower stock densities are shown to be related
to increased survivorship, growth rate and feed utilization, while also being associated with a reduction innitrogenous wastes.

Acknowledgments
We are thankful to Ms. C. Huang and our other colleagues at the animal physiological ecology lab, collegeof life science, Beijing normal university, for their valuable advice and assistance to the experiment. This work
was funded by the National Natural Science Foundationof China (No. 30271014, No. 30671598).

Literature Cited
Bolin, D. W., R. P. King and E. W. Klosterman. 1952. A
simplified method for the determination of chromicoxide (Cr2O3) when used as an index substance.
Science 116: 634–635.

2008

Asiatic Herpetological Research, Vol. 11

Elliott, J. M. 1976. Energy losses in the waste productsof brown trout (Salmo trutta L.). Journal of Animal
Ecology 45(2): 561–580.
Fairchild, E. A. and W. H. Howell. 2001. Optimal stocking density for juvenile winter flounder
Pseudopleuronectes americanus. Journal of theWorld Aquaculture Society 32(3): 300–308.
Hengsawat, K., F. J. Ward and P. Jaruratjamorn. 1997.
The effect of stocking density on yield, growth andmortality of African catfish (Clarias gariepinus
Burchell 1822) cultured in cages. Aquaculture152(1–4): 67–76.
Irwin, S., J. O'Halloran and R. D. FitzGerald. 1999.
Stocking density, growth and growth variation injuvenileturbot,Scophthalmus
maximus(Rafinesque). Aquaculture 178(1–2): 77–88.
Jørgensen, E. H., J. S. Christiansen and M. Jobling.
1993. Effects of stocking density on food intake,
growth performance and oxygen consumption in
Arctic charr (Salvelinus alpinus). Aquaculture110(2): 191–204.
Knights, B. 1985. Energetics and fish farming. Pp. 309340. In: P. Tytler and P. Calow (eds.), Fish
Energetics: New Perspectives. Croom Helm,
London. Total # pages??
Li, H. W. and R. W. Brocksen. 1977. Approaches to theanalysis of energetic costs of intraspecific competition for space by rainbow trout (Salmo gairdneri).
Journal of Fish Biology 11(4): 329–341.

49

Penha-Lopes, G., A. L. Rhyne, J. Lin and L. Narciso.
2006. Effects of temperature, stocking density anddiet on the growth and survival of juvenile
Mithraculus forceps (A. Milne Edwards, 1875)(Decapoda: Brachyura: Majidae). Aquaculture
Research 37(4): 398–408.
Rowland, S. J., G. L. Allan, M. Hollis and T. Pontifex.
2004. Production of silver perch (Bidyanus
bidyanus) fingerlings at three stocking densities incages and tanks. Aquaculture 229(1–4): 193–202.
Rowland, S. J., C. Mifsud, M. Nixon and P. boyd. 2006.
Effects of stocking density on the performance of
the Australian freshwater silver perch (Bidyanus
bidyanus) in cages. Aquaculture 253(1–4):
301–308.
Schram, E., J. W. Van der Heul, A. Kamstra and M. C. J.
Verdegem. 2006. Stocking density-dependent
growth of Dover Sole (Solea solea). Aquaculture252(2–4): 339–347.
Shen, X., Q. Zhang and W. Zhu. 2006. [Status quo anddevelopmental strategy of turtle industry in China].
Gansu Agriculture (1): 48. (In Chinese)
Vijayan, M. M. and J. F. Leatherland. 1988. Effect ofstocking density on the growth and stress-response
in brook charr, Salvelinus fontinalis. Aquaculture75(1–2): 159–170.
Zhang, J. 2005. [Economic Analysis and Forecast of
Tortoise and Turtle Industry in 2005]. Chinese
Fisheries Economics (3): 49–50. (In Chinese)

Mayeaux, M. H., D. D. Culley Jr. and R. C. Reigh. 1996.
Effects of dietary energy : Protein ratio and stocking density on growth and survival of the common
snapping turtle Chelydra serpentina. Journal of theWorld Aquaculture Society 27(1): 64–73.
Papoutsoglou, S. E., G. Tziha, X. Vrettos and A.
Athanasiou. 1998. Effects of stocking density on
behavior and growth rate of European sea bass
(Dicentrarchus labrax) juveniles reared in a closed
circulated system. Aquacultural Engineering 18(2):135–144.
Papst, M. H., T. A. Dick, A. N. Arnason and C. E. Engel.
1992. Effect of rearing density on the early growthand variation in growth of juvenile Arctic charr,
Salvelinus alpinus (L.). Aquaculture and Fisheries
Management 23: 41–47.
Submitted: 26 October 2006
Accepted: 29 May 2007