2008

Asiatic Herpetological Research, Vol. 11

pp.147–152

Survival and Metabolic Responses to Freezing Temperature in the
Northeast Forest Frog Rana dybowskii
XIANGHONG XIAO*, DONG ZHENG, CUIJUN YANG AND LONGHUI CHAI
Division of Physiology, Northeast Forestry University, Harbin 15004, China.
*Corresponding author E-mail: xiaoxh@vip.0451.com
Abstract.- Dynamic changes in water content, crude oil, general proteins, blood sugar and hepatic glycogen duringfreezing temperatures in the Northeast forest frog (Rana dybowskii Günther, 1876) were investigated by establishingfrog freeze-tolerant models. Chemical and biochemical analyses showed that a temperature drop from 4°C to -3°C
resulted in (1) increase in integrative water content and decrease in in vivo moisture and dissociative water contents;(2) decrease in hepatic glycogen and crude oil and significant increase in blood sugar; (3) significant increase(p > 0.05) in general protein content; (4) mortality below temperatures of -1°C; (5) and increase in blood sugar andglucose levels in skeletal muscle following injection of glucose at 4°C and -2°C (hepatic glycogen levels showed similar increases in test groups injected with 650 mmol/L and 1500 mmol/L glucose-PBS, but not in groups injected with
2000 mmol/L glucose-PBS). These physiological and metabolic responses suggest that the Northeast forest frog
adopts a positive freeze-tolerant strategy in which glucose serves as the primary mechanism by which damage due tofreezing is prevented.
Keywords.- Freezing tolerance, moisture contents, blood sugar, crude oil, general proteins.

Introduction
The Northeast forest frog (Ranidae: Raninae: Rana
dybowskii Günther, 1876), formerly classified as anortheastern population of Rana chensinensis David,
1875 (Xie et al., 1999), is found throughout eastern
Asia, with records from Heilongjiang Province, Jilin
Province, Liaoning Province, the northeast of the Inner
Mongolia Autonomous Region, as well as the Russian
Far East, eastern Mongolia, the Republic of Korea, andTsushima island (Japan) (Fei et al., 2005). The climate
in the northern province of Heilongjiang, the region
from which research material was collected, is typified
by intermediate and frigid-temperature zones with acontinental monsoon climate. From November toMarch, the average temperature is usually less than 0°C,
while in January, temperatures reach -15°C to -30°C.
Due to climatic factors such as west-wind circumfluence, Siberian air mass, Mongolia high pressure andBaikal cyclone, winters in Heilongjiang Province areoften dry and without snow.
As is typical of northern amphibians facing freezing temperatures, the Northeast forest frog hibernates tosubtly adjust its physiological functions and metabolism
to survive the winter. Although little physiological andbiochemical investigations have been made on thisspecies, various freeze-tolerant strategies have been
examined in species with similar biochemical metabolisms, including Rana sylvatica LeConte, 1825, Hylaversicolor LeConte, 1825, Hyla chrysoscelis and Rana

© 2008 by Asiatic Herpetological Researchridibunda Pallas, 1771 (Storey and Storey, 1986;
Voituron et al., 2000). These have included studies on
ecological behavior, genetic characteristics (Jiang andZhou, 2001; Yang et al., 2001; Xia et al., 2006), classification (Jiang and Zhou, 2001; Xie et al., 1999; Yang etal., 2001), artificial breeding and reproduction (Wei etal., 2005) and biochemical composition (Xiao et al.,2005). To further explore freeze-tolerant mechanisms
and cryobiology in the Amphibia, we here examine themoisture, crude oil, proteins, blood sugar and glycogencontents of the Northeast forest frog when subjected tofreezing temperatures.

Materials and Methods
Materials and freeze-tolerant models.- Adult malefrogs weighing 20 to 22 g were collected from the
Yichun area of Heilongjiang Province in September.
Following one week of acclimation to room temperature(25°C), ten randomly-selected frogs were placed into
separate glass boxes (40 × 40 × 40 cm) and transferred
to digitally-controlled refrigerators. Temperature
dropped at a rate of 1°C every 12 h and was held at 4°C
for up to 60 d. One third of the water in each box was
exchanged with pre-cooled fresh water (4°C) every twodays.
Freeze temperature impacting survival ratios.- To
investigate survival ratios at different temperatures, six
test groups (held at 2°C, 1°C, 0°C, -1°C, -2°C, and -3°C,

148

Asiatic Herpetological Research, Vol. 11

respectively) and one control group (25°C) were established with ten frogs per group. For each test group,water temperature was lowered from 4°C to the target
temperatures specified above at a rate of 2°C per day.
Frogs in those test groups with target temperatures lessthan -1°C were placed on water-soaked sponges.
Survivorship was checked once daily and PT100 thermo-sensors were used to monitor temperatures.
Following 10 d of freezing stress, each test group wasreturned to room temperature (25°C) for 24 hrs and survivorship examined, followed by euthanasia and biochemical analysis. Each test was repeated three times.
Measurement of moisture content, crude oil, generalproteins, blood sugar and glycogen.- The drying
method outlined by Han et al. (2005) was used to measure dissociative and integrative water content: three 10 g
samples from each frog (recorded as Ww) were incubated at 70°C for 6 or 7 hrs until the semi-dry samples
reached a constant weight (recorded as W70). ; the semidried samples were incubated at 105°C for 5 or 6hrs
until the samples again reached a constant weight(recorded as W105). Calculated dissociative and integrative water percentages were calculated as follows:
Dissociative water (%) = 100 –Integrative water (%) =

W 70
WW

W 70 − W105
WW

× 100
× 100

The Soxhlet extraction method was used for measuring crude oil (Wei et al., 2004): for each group, 10 dry,1 g samples (recorded as Wfat) were placed into theSoxhlet extraction flask and degreased with low-meltpoint aether/petroleum; samples were packaged andincubated at 105°C for 1hr until the weight again
became constant (recorded as W′fat). Crude oil percentage was calculated using the following equation:
Crude oil (%) =Wfat − W'fat
× 100
Wfat

General protein content was determined by the
Kjeldahl nitrogen determination method: for each group,semi-dried 0.5 g samples were transferred into a digestion tube; 2.5 g Na2SO4, 0.13 g CuSO4, and 10 ml H2SO4
were added and digested at 400°C for 3 h until the solution color changed to pea green; the tubes were thenplaced on the Kjeldahl nitrogen determination apparatus
for distillation; 15 ml 1% H3BO3 were added to theErlenmeyer flask and connected to the condensator exit;
20 ml saturated NaOH was added to the reaction andmethyl red/bromocresol green indicator was added until
the solution turned grey; the solution was finally titrated
with HCl standard buffer until the solution color
changed from grey to blue. General proteins percentages

2008

were calculated with following equation:
Vs is the volume of HCl standard buffer added toadjust the distilled sample solution, V0 is the volume ofGeneral proteins (%) =(Vs–Vo)x 0.01 x 0.014 x 6.25 x 100
W

HCl standard buffer needed to adjust the control solution, and W is the dry weight of sample.
The ortho-toluidine o-toloidine colorimetry method
and the anthrone colorimetry method were used formeasuring blood sugar and glycogen, respectively (Heet al., 2004): after 24 h of freezing-temperature stress,
blood sugar was measured from heart tissue (immediately treated with heparin from phlebotomized specimens
within each group); 1 g of liver tissue was used to measure glycogen; tissue was homogenated and centrifuged,
the supernatant was collected and added to an equal volume of ethanol; the solution was centrifuged, and 100
ml of distilled water was added to the precipitatedglycogen, which was then measured.
Exogenous glucose intervention assay.- The method
described by Costanzo et al. (1991) was used to detect
the effects of exogenous glucose on glucose metabolism
in frog liver and muscle tissue, and to demonstrate
whether glucose was involved in any freeze-tolerance
mechanisms. For each target temperature (4°C or -2°C),
ten frogs in three test groups and one control group(25°C) were examined. For the control group, approximately 2.3–2.5 ml 115 mmol/L PBS was injected into
the dorsal lymph bursa, comprising approximately 6.7 %of total volume in the bursa. (For the three test groups,
different concentrations (650, 1500, 2000 mmol/L,
respectively) of glucose-PBS (pH 7.4) were injected toagain comprise approximately 6.7% of total bursa volume. All frogs were euthanized following 72 hrs offreezing temperatures. One gram of blood, liver andmuscle were immediately collected to analyze glucose
contents by the same methods described above.
Statistical analysis.- SPSS (ver.15.0) software was used
for statistic analysis. Confidence intervals were set to0.05. Data were presented as means±standard error (SE)and analyzed using a one-way ANOVA; these results
were subsequently analyzed using the Tukey test.

Results
Freezing temperatures and their impact on survival
ratios.- During the temperature decrease from 4°C to-1°C, ice crystals became visible on the skin of the frogs.
After ten days, mortality was observed only below -1°C
(see Fig. 1).

2008

149

Asiatic Herpetological Research, Vol. 11

increase with the addition of exogenous glucose.
Hepatic glycogen also increased with increasing concentrations of exogenous glucose in the test groups injectedwith 650 mmol/L and 1,500 mmol/L glucose-PBS, however, it was found to decrease in the test groups injectedwith 2,000 mmol/L, particularly at -2°C (p < 0.01) (seeTable 2–3).

Discussion
Figure 1. Impact of freeze temperatures and their duration on survival ratios of Rana dybowskii. Y-axis is survival ratios in percentage, X-axis is duration. The continuous line with empty triangles represents data from thetest groups at 4°C, 0°C and -1°C. The dotted line withcrosses is for the test groups at -2°C. The dashed linewith empty diamonds is for the test groups at -3°C.

Changes of moisture contents in vivo, crude oils, general proteins and glycogen.- Following the temperature
decrease from 4°C to -3°C, moisture contents in vivo
and dissociative water contents decreased gradually,
whereas integrative water was found to increase; furthermore, blood sugar was found to increase while
hepatic glycogen and crude oil decreased significantly.
During freezing-temperature stress, general proteins
were found to decrease slightly with decreases in temperature (p > 0.05) (see Table 1).
Changes in blood sugar and glycogen levels with addition of exogenous glucose under freezing-temperatures.- At 4°C and -2°C, blood sugar and glucose concentrations in skeletal muscle were always found to

Since the 1980s, investigators have studied the freezingtolerance mechanisms of amphibians, and have foundthat species such as Rana sylvatica can precisely regulate their metabolic levels in order to tolerate extracellular ice crystallization (Storey and Storey, 1988), whichplays as key role in survival and evolution.
Investigations on Rana sylvatica, Pseudacris triseriata
and Rana ridibunda (Churchill and Storey, 1995;
Costanzo et al., 1991; Edwards et al., 2004; Layne andJones, 2001; Storey and Storey, 1985) illustrate that in atleast some amphibians, endogenous glucose is used as aprotectant during hibernation.
In the present study, it was found that in Rana
dybowskii, freezing temperatures are associated withdehydration, an increase in blood sugar and a decrease inhepatic glycogen; temperatures below -1°C are also
associated with increased mortality. Some investigations
have proposed that endogenous water redistribution aidsin the tolerance of freezing temperatures by changing
dissociative water into integrative water, condensing
extra-cellular solutes and promoting intracellular water
trafficking out of cells. This prevents intracellular icing,
lowers the freezing point of the body, induces antifreeze

Table 1. Effects of lowering temperature on percentage of water, crude oil, general proteins and sugar in Rana
dybowskii.

Controlgroups
at 25°C
Test groups
at 4°C
Test groups
at 0°C
Test groups
at -1°C
Test groups
at -2°C
Test groups
at -3°C

Dissociative
water (%)

Integrative
water (%)

Moist contents (%)

Crude oil (%) General proteins (%)

81.25±0.70d

1.44±0.11a

82.68±0.73c

4.64±0.06b

79.69±0.47cd 1.79±0.06ab 81.49±0.50c

Blood sugar(mg%)

Hepatic
glycogen (%)

70.49±1.71b 152.38±6.57a

5.60±0.02e

4.63±0.03b 64.90±0.61ab 184.97± 13.18a 4.68±0.08d

77.67±1.44bcd 1.90±0.13ab 79.58±1.57bc 4.55±0.03ab 64.51±0.66a 196.98± 15.08a 4.12±0.05c
76.24±2.07bc

2.07±0.14b 78.32±2.21bc 4.49±0.03ab 63.33±1.50a 203.78± 1.84a

73.33±1.24ab

2.18±0.02b 75.52±1.23ab 4.41±0.05a

63.19±1.18a 266.98± 23.08b 3.21±0.02a

70.40±0.94a

2.26±0.04b

63.09±1.90a 305.26± 5.88b

72.66±0.91a

4.41±0.09a*Different letters in the same column represent significant differences (p < 0.05).

3.65±0.03b

3.10±0.01a

150

Asiatic Herpetological Research, Vol. 11

2008

Table 2. Effect of exogenous glucose on blood sugar andliver glycogen in Rana dybowskii at 4°C.

Table 3. Effect of exogenous glucose on blood sugar andliver glycogen in Rana dybowskii at -2°C.

Blood sugar
Hepatic(mg %)
glycogen (%)

Skeletalmuscle
glucose (%)

SkeletalBlood sugar
Hepatic
muscle(mg %)
glycogen (%) glucose (%)

124.20±0.69a 5.15±0.45a

0.10±0.01a

ControlgroupsTest groups 475.50±1.27b 28.68±0.55b
injected with
650 mmol/L

2.03±0.07b

Test groups 539.07±1.24c 47.65±2.16d
injected with1,500 mmol/L

2.57±0.06c

Test groups
651.69±0.28b 18.43±1.13b 2.54±0.07b
injected with
650 mmol/L
Test groups
715.67±2.16c 33.92±1.66c 5.66±0.02c
injected with1,500 mmol/L

Test groups 945.07±1.65d 37.94±0.89c
injected with2,000 mmol/L

4.59±0.13d

Controlgroups131.18±0.17a

4.19±0.59a 1.63±0.03a

Test groups 1,012.58±3.90d 20.99±1.67b 7.33±0.24d
injected with2,000 mmol/L*Different letters in the same column represent significant differences(p < 0.05).

*Different letters in the same column represent significant differences(p < 0.05).

synthesis, and prevents damage to critical organs fromintracellular ice crystallization (Churchill and Storey,
1995; Hermes-Lima and Storey, 1996; Horton, 1996).
Below a certain temperature, however, small ice crystals
enlarge to a point that causes damage to cell membranes
and cellular substructure, thereby causing death.
Stability of the cell membrane would also be compromised, resulting in plasma-membrane fusion, phasetransformation from liquid crystal to gels, membrane
lipids deficiencies, phospholipid separation, etc., andwould destabilize membrane structure (Tong and Nie,
1996). In amphibians glucose is known to function as
antifreeze, lowering the freezing-point of the body,maintaining cell membranes and stablizing internal
environments (Costanzo and Lee, 1994; Katz, 1989;
King et al., 1995). In situations involving dehydration,such as those observed here, hydrogen bond formation
between glucose hydroxyls and the heads of membrane
phospholipids have been found to stabilize lipid bilayers
at the liquid crystalline state, preventing membranefusion, phase change, side phase separation, cell leakage
and membrane protein displacement (Tong and Nie,
1996). Simultaneously, freezing temperatures stimulate
the catabolism of hepatic glycogen and crude oils,
increasing blood sugar concentrations, initiating the glucose antifreeze system, stabilize cell membranes, andenhancing physiological cold-tolerance. These factors
combined provide a physiological and biochemical strategy that the Northeast forest frog uses to survive freezing annual temperatures.
In the present study, it has been illustrated that the
injection of exogenous glucose into the lymph bursa
results in increased levels of blood sugar and skeletal
muscle glucose levels, increasing as higher concentrations of glucose solution are injected, particularly in thetest groups kept at -2°C. In comparison, hepatic glycogen levels also showed an increase with higher concentration of the injected glucose solution, although the test
groups injected with 2000 mmol/L glucose-PBS showeda significant decrease. A possible explanation for thisunexpected result is that extremely high concentrationsof glucose induce diabetes-like symptoms, causing glycol-metabolism disorders and decreased glycogen synthesis (Wang and Zhuang, 2001).

Acknowledgments
We thank Liu W. S., Zou Q. and Zeng K.W. for assistantship in samples collection. This study was granted by
the Nature Science Fund (C9726) and the Key Projects
of Heilongjiang Province (ZJN0604-02).

Literature Cited
Churchill, T. A. and K. B. Storey. 1995. Metabolic
effects of dehydration on an aquatic frog Rana pipiens. The Journal of Experimental Biology 198:
147–154.
Costanzo, J. P. and R. E. Lee. 1994. Biophysical andphysiological responses promoting freeze tolerance
in vertebrates. News in Physiological Sciences 9:252–256.
Costanzo, J. P., R. E. Lee and M. F. Wright. 1991. Effectof cooling rate on the survival of frozen wood frogs,
Rana sylvatica. Journal of Comparative Physiology
B 161: 225–229.

2008

Asiatic Herpetological Research, Vol. 11

Edwards, J. R., J. L. Jenkins and D.L. Swanson. 2004.
Seasonal effects of dehydration on glucose mobilization in freeze-tolerant chorus frogs (Pseudacris
triseriata) and freeze-intolerant toads (Bufo woodhousii and B. cognatus). Journal of Experimental
Zoology Part A: Comparative Experimental
Biology 301(6): 521–531.
Fei, L., C. Y. Ye, J. P. Jiang, F. Xie and Y. Z. Huang.
2005. An illustrated key to Chinese
amphibians.Chongdu: Sichuan publishing house ofscience and technology. 108 pp. (In Chinese).
Han, R. D., X. G. Sun, Y. Y. Xu and W. G. Zhang. 2005.
The biochemical mechanism of cold-hardiness inover wintering larva of Dendrolimus spectabilis
Butler (Lepidoptera: Lasiocampidae). Acta
Ecologica Sinica 25(6): 1352–1356. (In Chinese).
He, X. Z. 2004. Pp 60–64. In Biochemical experiment
technology. Beijing: Chemical Industry Press. (In
Chinese).
Hermes-Lima, M. and K. B. Storey. 1996. Relationshipbetween anoxia exposure and antioxidant status in
the frog Rana pipiens. The American Journal of
Physiology 271: 918–925.
Horton, D. R. 1996. Reduced cold-hardiness of pear
psylla caused by exposure to external water andsurfactants. The Canadian Entomologist 128(5):825–830.
Jiang, J. P. and K. Y. Zhou. 2001. Phylogenetic relationships of Chinese Brown Frogs inferred from mitochondrial DNA sequence of 12 S ribosome RNA
Gene. Zoological Research 22(1): 27–32. (In
Chinese).
Katz, U. 1989. Strategies of adaptation to osmotic stressin anuran Amphibia under salt and burrowing conditions. Comparative Biochemistry and Physiology
93(A): 499–503.
King, P. A., M. N. Rosholt and K. B. Storey. 1995.
Seasonal changes in plasma membrane glucose
transport in freeze tolerant wood frogs. Canadian
Journal of Zoology 73: 1–9.
Layne, J. R. Jr. and A. L. Jones. 2001. Freeze tolerance
in the gray tree frog: cryoprotectant mobilizationand organ dehydration. Journal of Experimental
Zoology 290(1): 1–5.

151

Storey, J. M. and K. B. Storey. 1985. Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog, Rana sylvatica.
Comp. Physiological Society, B 156: 191–195.
Storey, K. B. and J. M. Storey. 1986. Freeze tolerance
and intolerance as strategies of winter survival interrestrially-hibernating amphibians. Comparative
Biochemistry and Physiology A 83(4): 613–617.
Storey, K. B. and J. M. Storey. 1988. Freeze Tolerancein Animals. Physiological reviews 68(1): 27–84.
Tong, Q. and S. Q. Nie. 1996. Interaction of sugar withphosphalipid bilayer-study on the stabiliztion ofliposomes. Progress in Physiological Sciences
27(22): 123–128. (In Chinese).
Voituron, Y., M. Eugene and H. Barre. 2000. Survival
and metabolic responses to freezing by the water
frog (Rana ridibunda). Journal of Experimental
Zoology Part A: Comparative Experimental
Biology 299(2): 118–126.
Wang, C. J. and L. M. Zhuang. 2001. Pp. 16–21. In
Diabetes. Anhui science & Technology Publishing
House. (In Chinese).
Wei, G. Q., T. F. Wen, F. K. Cai and X. H. Zou. 2005.
Effects of pen rearing condition on morphologicalcharacteristics,body proportion and yield of
Oviducts ranae in the adult of Rana dybowskii.
Journal of Northwest Sci-Tech University ofAgriculture and Forestry 33(12): 17–20. (In
Chinese).
Wei, H., H. J. Zhong and H. Wang. 2004. Improvement
on determination of crude oil by Soxhlet extraction.
China Oils and Fats 29(6): 52–54. (In Chinese).
Xia, Y. G., W. G. Zhao, P. Liu, L. L. Liu, J. Y. Li and Y.
Q. Liu. 2006. The Karyotype of Rana huanrenesis
and R. dybowskii from Huanren, Liaoning
Province. Chinese Journal of Zoology 41(5): 103–
106. (In Chinese).
Xiao, X. H., Y. G. Xu and L. H. Chai. 2005. The
Isolation and Extraction of Active Peptides from theSkin of Rana chensinensis. Journal of Northeast
Forestry University 33(1): 44–46. (In Chinese).

152

Asiatic Herpetological Research, Vol. 11

Xie, F., C. Y. Ye, L. Fei, J. P. Jiang, X. M. Zeng and
Masafumi Matsu. 1999. Taxonomical Studies on
Brown Frogs (Rana) From Northeastern China(Amphbia:Ranidae). Acta Zootaxonomica Sinica.
24(2): 224–231. (In Chinese).
Yang, X. G., Y. Q. Wang, K. Y. Zhou and Z. Q. Liu.
2001. Phylogenetic Relationships of Chinese
Brown Frogs (Rana) Based on Sequence ofMitochondrial Cytochrome b Gene. ZoologicalResearch 22(5): 345–350. (In Chinese).

Submitted: 22 November 2006
Accepted: 22 September 20072008