Saddleback Journal of Biology - Saddleback College

Spring 2010 Biology 3B Paper
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.
It is hypothesized that Hyla regilla,
collected from more temperate climates, will not
exhibit significant increases in plasma glucose
associated with cold temperature.
Materials and Methods
Participants
Hyla regilla were collected from a pond in
Irvine, California on February 20, 2010 (n=20).
Investigators purchased a One-day Sport Fishing
License from the California Fish and Game
(License#: 20018254). The frogs were observed for
two weeks prior to determining their blood glucose
levels, 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 TRUE2go TM glucometer and
GoldSensor TM Laser Accuracy blood glucose strips
(HOMEdiagnostics TM, 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 SafetyGlide TM
Insulin needles (1mL 29G x ½ inch) were used to
draw blood from the ventral, pelvic region of the
frogs. A Kenmore TM (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.
Cooling/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 (Excel TM 2008).
Results
The blood glucose levels of the control and
experimental groups of Hyla regilla were measured
immediately prior to and after protocol. Prior to
protocol, the mean blood glucose level was 33.40
±3.45 mg • dL -1 (±SEM, n=10), and the mean blood
glucose level after protocol was 31.70 ± 2.29 mg •
dL -1 (±SEM, n=10). The mean blood glucose level of
the experimental group prior to protocol was 32.80
±2.93 mg • dL -1 (±SEM, n=10), and the mean blood
glucose level after experimental protocol was 50.20
±3.10 mg • dL -1 (±SEM, n=10).
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). 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
illustrates the results of the control group, and figure
2 illustrates the results of the experimental group.
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Saddleback Journal of Biology
Spring 2010