Download Key: The Effect of Near Freezing Temperatures on Blood Glucose

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The Effect of Near Freezing Temperatures on Blood Glucose Concentrations
in the Pacific Tree Frog Collected in Coastal Southern California
Brian W Capen and Paige H Taylor
Department of Biological Sciences
Saddleback College
Mission Viejo, California 92692
Abstract
The adaptation of freeze tolerance (or winter cold hardiness) in ectothermic
vertebrates, such as amphibians, is an important acclimatization that ultimately allows
survival when temperatures approach freezing. Freeze tolerance associated with
distribution of cryoprotective agents to cells throughout the body is well documented in
several species of vertebrates. The pacific tree frog Pacific Tree Frog, Hyla regilla,
possesses this ability. The cryogenic mechanism is promoted by an increase in blood
glucose levels as the environmental temperature approaches 0˚C. This increase in blood
glucose concentration helps prevent tissue damage during a temporary freezing or near
freezing episode. A control group (n=10) (N=10) and an experimental group (n=10)
(N=10) of Hyla regilla were used in this experiment to investigate this relationship. The
experimental frogs were cooled to an average temperature (1.13˚C) for five hours. The
blood glucose levels were measured prior to and after the control protocol or experimental
protocol. The mean glucose concentration (prior to freezing) prior to freezing was 32.80 mg
• dL -1 ±2.93 (±SEM, n=10) 32.80 ± 2.93 mg • dL -1 (±SEM, N=10), and the mean glucose
concentration after freezing was 50.20 mg • dL -1 ±3.10 (±SEM, n=10) 50.20 ± 3.10 mg • dL 1 (±SEM, N=10). A significant difference was found between the groups (p=0.0001, one
tailed, paired t-test), indicating an increase in blood glucose levels as the experimental
temperature decreased.
Introduction
The adaptation of freeze tolerance (or winter cold hardiness) in ectothermic vertebrates,
such as amphibians and reptiles, is an important acclimatization that ultimately promotes
survival when temperatures approach freezing. “Cold-blooded” animals can be active only
within ranges of environmentally induced body temperatures to which they are specifically
adapted (Cunningham and Mullally, 1956). Freeze tolerance is a biophysical and physiological
response to ice formation within the tissues of ectothermic vertebrates whose body temperature
equalizes to the surrounding environments. This mechanism refers to an organism’s ability to
survive an extensive freezing of body fluids under thermal and temporal conditions of ecological
significance to the species (Costanzo, et al, 1993).
Freeze tolerance may be promoted by the rapid synthesis of glucose from liver glycogen
and the distribution of this cryoprotective agent to cells throughout the body. The accumulated
glucose apparently enhances the survival of cells, tissues, and organs because experimentally
administering additional glucose to the frog increases its tolerance to freezing (Costanzo et al.
1993). Chemicals such as glycerol are cryoprotective agents that help protect against protein
denaturation. The glycerol component can be converted to glucose by the liver and provides
energy for cellular metabolism.
Previous studies such as Croes and Thomas (2000), propose that the rise in plasma
glucose, along with increased levels of liver glucose and glycerol in response to freezing,
suggests that these compounds are being used as cryoprotectants. Storey and Storey (1984)
determined that the onset of freezing triggers a mobilization of glucose from liver glycogen; the
glucose becomes distributed throughout the body. Based on this correlation, it was suggested
that glucose protected the animal from cryoinjury.
Croes and Thomas (2000) demonstrated that the specimens, Hyla regilla, frozen at -2˚ C
for six and 12 hours had a survival rate of 10% and 80%, respectively, in the spring and the fall.
Freezing caused a fivefold increase in plasma glucose levels in the spring and a 14-fold increase
in the fall. Steiner, et al. (2000) demonstrated that blood glucose concentration increased from
40.35 ± 7.25 to 131.87 ± 20.72 mg/dL (P < 0.01) when the frogs, Rana catesbeiana, were
transferred from 20 to –2ºC.
In all of the previous studies, the frogs were collected at either a high altitude, or in a
region where the climate commonly approached near freezing or freezing temperatures. Under
these conditions it necessary for frogs to have the freeze tolerance adaptation in order to survive.
However, in more temperate climates, the freeze adaptation is not essential for their survival. To
date there has been no investigation of temperature-induced changes in plasma glucose in Hyla
regilla from more temperate climates in the southern portion of their range.
Application of the physiological response of freeze tolerance has become an interest in
future medicine and furthered research for cryopreserving mammalian organs. The
determination of how long tissues can be kept at near freezing temperatures without irreversible
damage taking place is of clinical and surgical interest. It is known that lowering body
temperature decreases the metabolic rate of cellular respiration. However, in addition to time
limits, there is a limit to how low the temperature can go before tissue damage occurs.
Current investigators hypothesize that Pacific Tree Frogs collected from more temperate
climates will not exhibit significant increases in plasma glucose associated with cold
temperature.
Materials and Methods
Participants
Hyla regilla, pacific tree frogs, were collected from a pond in Irvine, California on
February 20, 2010 20 February 2010 (n=20) (N=20). Investigators purchased a One-day Sport
Fishing License from the California Fish and Game (License#: 19017106). The frogs were
observed for two weeks prior to determining their blood glucose concentration, and they were
stored in a habitat outdoors to maintain their natural climate fluctuation. The investigators
monitored the water and food intake of the species.
Materials
A TRUE2goTM glucometer and GoldSensorTM Laser Accuracy blood glucose strips
(HOMEdiagnosticsTM, E3HDI04 Rev.5; LOT TJ1083) were obtained to measure the blood
glucose concentrations of the frogs prior to and after the control or freezing protocol. BD
SafetyGlideTM Insulin needles (1mL 29G x ½ inch) were used to draw blood from the ventral,
pelvic region of the frogs. A KenmoreTM (2.5 CU FT, model: 564.94256400, serial#:
060308018) refrigerator was calibrated to 1-2˚C for the freezing protocol, recording a mean
temperature of 1.13˚C over a five hour time period.
Freezing/Control Protocol
The frogs did not receive food or water 12 hours prior to experimentation because the
presence of food in the gut of freeze-tolerant animals is believed to cause uncontrolled ice
nucleation and thus reduce their survivorship during freezing (Storey and Storey, 1987). The
cooling protocol was as follows: The incubator was adjusted to reach an average temperature
between 1 and 2˚C. Baseline blood glucose levels were obtained at room temperature prior to the
cooling protocol. The frogs were transferred to plastic containers containing a damp paper towel
to encourage ice nucleation. After five hours of cooling, the blood glucose levels were measured
again from the pelvic region of the frogs. The control protocol was as follows: Baseline blood
glucose levels were obtained from the control frogs at room temperature. They were transferred
to plastic containers containing a damp paper towel, similar to the experimental group. The
frogs were placed in a dark room at room temperature for five hours, and then their blood
glucose levels were measured.
Statistical Analysis
The blood glucose levels of the experimental and control groups were measured before
and after protocol, and they were compared using a one-tailed, paired t-test (ExcelTM 2008).
Results
The blood glucose levels of the control and experimental groups of Hyla regilla were
measured immediately prior to and after protocol, as shown in Table 1 Table 1and Table 2
Table 2. Table 1 Table 1 shows the blood glucose levels of the control group. Prior to protocol,
the mean blood glucose level was 33.40 mg • dL -1 ±3.45 (±SEM, n=10) 33.40 ± 3.45 mg • dL -1
(±SEM, N=10), and the mean blood glucose level after protocol was 31.70 mg • dL -1 ±2.29
(±SEM, n=10) 31.70 ± 2.29 mg • dL -1 (±SEM, N=10). Table 2 Table 2 shows the blood
glucose levels of the experimental group. The mean blood glucose level of the experimental
group prior to protocol was 32.80 mg • dL -1 ±2.93 (±SEM, n=10) 32.80 ±2 .93 mg • dL -1
(±SEM, N=10), and the mean blood glucose level after experimental protocol was 50.20 mg • dL
-1
±3.10 (±SEM, n=10) 50.20 ± 3.10 mg • dL -1 (±SEM, N=10).
Table 1.This table is not necessary - don’t report individual data points, just the mean for each
group Blood glucose levels (mg/dL) of the control group of Hyla regilla recorded prior to and
after protocol at 23˚C.
Glucose Concentration (mg/dL)
Frog (n) (N)
Before (23˚C)
After (23˚C)
1
42
34
2
24
27
3
25
28
4
49
37
5
26
23
6
23
27
7
47
37
8
25
26
9
33
34
10
40
44
33.40
Mean
±3.45 (±SEM)
31.70
±2.29 (±SEM)
Table 2. This table is not necessary - don’t report individual data points, just the mean for each
group Blood glucose levels (mg/dL) of the experimental group of Hyla regilla recorded prior to
(23˚C) and after protocol (1.13˚C).
Glucose Concentration (mg/dL)
Frog (n) (N)
Before (23˚C)
After (1.13˚C)
1
40
67
2
25
39
3
29
45
4
36
40
5
25
47
6
23
42
7
47
67
8
24
53
9
42
58
10
37
44
Mean
32.8
±2.93 (±SEM)
50.2
±3.10 (±SEM)
A one-tailed, paired t-test was utilized to determine whether or not there was a significant
difference between the control and cooling-exposed groups (p ≤ 0.05) (p<0.05). A significant
difference was not determined between the control group prior to and after protocol (p=0.3176),
but a significant difference was determined between the experimental group prior to and after
protocol (p=0.0001) indicating a respectable increase in blood glucose levels as the experimental
temperature decreased. Figure 1 Figure 1 illustrates the results of the control group and figure 2
Figure 2 illustrates the results of the experimental group.
Figure 1. The mean blood glucose levels of the control group, prior to and after protocol.
Figure 2. The mean blood glucose levels of the experimental group, prior to and after
experimental protocol.
Discussion
The freeze tolerance mechanism of Hyla regilla allows the species to survive episodes of
freezing and near freezing temperatures. Environmental temperature determines the body
temperature of ectotherms and thereby affects many of their biological processes (Voituron,
2002). Glucose, a primary cryoprotectant, increases prior to hibernation. This may limit
intracellular dehydration via osmotic and water-binding effects. The function of glucose helps
stabilize proteins and maintain membrane structure.
All participants survived in the control and the experimental groups. The frogs that
survived the cooling protocol demonstrated their ability to tolerate near freezing temperatures for
a short period of time. For the experimental group, a significant increase in the blood glucose
concentration was observed (p=0.0001), even though they were not previously adapted to
temperatures that approach freezing. The Pacific Tree Frogs used in this investigation were
acclimated to temperatures averaging about 20 ˚C ± 5 ˚C 20 ± 5°C; therefore, investigators
hypothesized that the Pacific Tree Frogs collected from more temperate climates would not
exhibit significant increases in plasma glucose associated with cold temperature.
Blood glucose levels significantly increased as the temperature decreased (see Figure 2)
(Figure 2). The mean glucose level of the control group after protocol was 31.70 mg • dL -1
±2.29 (±SEM, n=10) 31.70 ± 2.29 mg • dL -1 (±SEM, N=10), and the mean glucose level of the
near freezing group was 50.20 mg • dL -1 ±3.10 (±SEM, n=10) 50.20 ± 3.10 mg • dL -1 (±SEM,
N=10).
The significant findings were unexpected for the Hyla regilla collected at a temperate
climate and did not support our hypothesis. Our preliminary results may suggest a widespread
distribution of the freeze tolerance adaptation in this species. Further research is necessary in
order to fully understand other factors that may have an effect on this mechanism.
Literature Cited
Costanzo et al. (1993). Glucose Concentration Regulates Freeze Tolerance in the Wood Frog
Rana sylvatica. Physiological and Biochemical Zoology, 76(3), 331-338. Retrieved from
JSTOR.
Croes, S.A. & Thomas, R.E. (2000). Freeze Tolerance and Cryoprotectant Synthesis of the
Pacific Tree Frog, Hyla regilla. Copeia, 2000(3), 863-868. Retrieved from JSTOR.
Cunningham, J.D. & Mullally, D.P. (1956). Thermal Factors in the Ecology of the Pacific
Treefrog. Herpetologica, 12(1), 68-79. Retrieved from JSTOR.
Storey, K.B. & Storey, J.M. (1987). Persistence of Freeze Tolerance in Terrestrially Hibernating
Frogs after Spring Emergence. Copeia, 1987(3), 720-726. Retrieved from JSTOR.
Voituron, et al. (2002). To Freeze or Not to Freeze? An Evolutionary Perspective on the ColdHardiness Strategies of Overwintering Ectotherms. The American Naturalist, 160(2),
255-270. Retrieved from JSTOR.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Brian W Capen and Paige H Taylor
Title: The Effect of Near Freezing Temperatures on Blood Glucose Concentrations in the
Pacific Tree Frog Collected in Coastal Southern California
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
Investigators hypothesized that the Pacific Tree Frog would not exhibit significant
increase in plasma glucose associated with cold temperature (1-2°C). Twenty frogs were
collected and categorized into two groups consisting of the experimental group and the
control group. Results indicated a significant difference (one-tailed paired t-test, p=0.0001)
between plasma glucose levels prior to protocol and after protocol for experimental group.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our
current state of knowledge.
The paper was coherent and easy to follow. The introduction was well structured and well
referenced with important information of past studies. Hypothesis was well stated and
tested. Materials and methods were well organized and structured from the beginning of
the experiment to the end of the experiment. The titles in material and methods made it
easy to follow what they did throughout the experiment. Materials and methods could have
used more specification on the population used in the studied, such as, sex and rough
estimate of age/size. Results section needed minor revisions to graphs. For example, the
graphs were presented without error bars. Also, the graphs need y-axis values to be set to
the same range or both graphs could have been fused together as one graph for better
comparison of data. Discussion needs more citations that support or contradict the
conclusions of their study. This is important because in this section you tie your project in
with other research projects/papers. Future studies should be described for improvement
or clarification of doubts.
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