This study aimed to determine if levels of pancreatic lipase in nestling house sparrows increased when their diet changed from low-lipid to high-lipid. Pancreases from birds fed different diets were assayed to measure lipase activity. However, the assays did not yield reliable data as the lipase activity measurements decreased over time instead of increasing. Adjustments to the assay methods did not improve the results. As a reliable assay could not be established, no conclusions could be drawn about the effect of diet on lipase levels in house sparrows. Future research should test if freezing pancreas samples denatures lipase and establish an effective assay method.

1. Pancreatic Lipase Activity in Nestling House Sparrows Passer Domesticus
Fiona Rambo with Dr. William Karasov and Katie Rott
UW Department of Forest and Wildlife Ecology
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Abstract
One of the main goals of the Karasov Lab has been to study digestive enzyme
flexibility in the house sparrow Passer domesticus, essentially seeing how changes in diet
affect the levels of digestive enzymes produced by the birds. This research sought to
determine whether levels of pancreatic lipase increase in birds that transition from being
fed a low-lipid diet to a high-lipid diet. In order to conduct this research, wild house
sparrow Passer domesticus nestlings were collected and fed one of three diets: high-
carbohydrate; high-protein; or high-lipid. By switching the diets of some birds halfway
through the feeding trials and keeping other birds on a control diet, enzyme levels were
compared in relation to changes in diet. Pancreas samples from juvenile house sparrows
were assayed using the Sigma Aldrich Lipase Activity Assay Kit. Initially the samples
had absorbance measurements much higher than the highest glycerol standard, making it
impossible to determine how much lipase they contained. After diluting the samples to fit
the glycerol standard curve, a graph of the absorbance measurements versus time showed
a downward trend of lipase activity in each sample. In the presence of a substrate, lipase
is expected to release increasing amounts of glycerol over time. The downward trend that
was observed would seem to indicate that lipase was either being improperly extracted
from the tissue samples or that the glycerol was going undetected by the assay. In order
to correct for these possible faults related to the assay, two adjustments to the method
were tried. First, the pancreatic tissue was homogenized using a mortar and pestle with
liquid nitrogen to better separate the lipase from other components of the tissue. The

2. lipase activity of the samples continued to show a downward trend. Second, calcium
chloride was added to the assay buffer to test whether the lipase was calcium dependent.
There were no indications that the lipase was calcium dependent because the data
maintained the same pattern.
Since the lipase assay did not yield reliable data, no conclusions can be made
about the effect of high-lipid diets on the production of lipase in house sparrows using
these methods. However, future research should test whether the freezing of pancreas
samples denatures the lipase, thereby rendering the assay unviable. It should also
establish a reliable method for assaying the samples.
Introduction
Omnivorous birds consume a range of different foods based on their nutritional
needs and the availability of food throughout the seasons. It was predicted that digestive
enzyme activity would increase in tandem with the amount of substrate in the animal’s
diet. (Brzek et al. 2009). Evolutionarily, enzyme production flexibility would save an
organism energy by adjusting levels of enzymes based on the need to break down certain
substrates. The goal of this research is to contribute knowledge about and further the
understanding of the components of digestive enzyme flexibility, which have been
studied in a limited number of species. In order to conduct this research, wild house
sparrow Passer domesticus nestlings were collected and fed one of three diets. By
switching the diets of some birds half way through the feeding trials and keeping other
birds on a control diet, enzyme levels were compared in relation to changes in diet.
Overall, the goal of this research on digestive enzyme flexibility was to improve
predictions about birds’ abilities to adapt to changes in their environment, promote the

3. gastrointestinal tract as an model of phenotypic flexibility, and enhance the limited,
current knowledge about Passerine birds and the regulation of digestive enzymes.
Hypotheses
In this study, I explored the relationship between the levels of substrate in the diet
of juvenile house sparrows Passer domesticus and the amount of pancreatic lipase
activity they exhibited. I hypothesized that the amount of pancreatic lipase activity would
increase in tandem with the amount of lipid substrate in the bird’s diet. This is a
reasonable hypothesis to make because evolutionarily, it would be more efficient for
levels of enzymes to fluctuate in response to availability of substrates, eliminating the
wasteful production of enzymes that are not being used. This hypothesis is also supported
by a study on dietary enzyme modulation in Pine Warblers that found that birds fed a
high-lipid insect and seed diets displayed higher levels of lipase activity than birds fed the
high-carb fruit diet (Levey et al. 1999).
However, a research project conducted in the Karasov lab by Pawel Brzek in 2012
concluded that pancreatic enzyme activity is not strongly correlated with the activity of
intestinal enzymes (Paweł Brzek. 2012). This information implies that pancreatic enzyme
production is under genetic control and may not fluctuate in response to diet.
Methods
Collection
House Sparrow nestlings Passer domesticus were collected three days post-hatch
from an array of natural and man-made nests located in the UW Dairy Cattle Instruction
and Research center and the parking lot to the rear of Russell Labs. Nests were numbered
and checked daily in order to monitor the number of eggs and nestlings. Nestlings were

4. given a number and marked with permanent markers of varying colors. New hatchlings
were labeled as “day zero” and marked. Each day, nests known to contain nestlings were
checked and the birds were re-marked so as to insure that they would be discernable from
their siblings. On day three, birds were retrieved from their nests and transported to the
Karasov Lab in Tupperware containers lined with scrunched up paper towel. The day-
three birds were weighed, assigned one of three diets, and added (in their nest container)
to the humidified, incubated tub with the other subjects. Once in the lab, bird’s body
temperatures were recorded once a day using a thermometer inserted into the cloaca,
birds were weighed three times a day before feedings, and the tarsus bone was measured
at the end of the day to indicate growth. The birds were contained in a room that was kept
at 35 degrees Celsius with 29% humidity in order to simulate their natural environment.
Every hour from 6:30am until 8:30pm, the birds were fed one of the three assigned diets
with a syringe. After being fed for three days on one diet, some birds were switched to a
different diet for three days, while other birds were fed the same diet for the whole
treatment period.
Below is a table of the three diets used in this study.
Diets designed to have adequate essential nutrients (“e”), similar energy contents (kj/g).
Diet Carb. Protein. Lipid. e i kj/g
C 50 15 8 10 17 14.8
P 5 60 8 10 17 14.8
L 5 15 25 10 45 13.4
C = carbohydrate – starch; P = protein – casein (essential amino acids are part of “e”) L =
lipid – corn oil. “i” is inert ingredient – grit and alphacel-cellulose; all % dry mass.

5. Euthanasia and tissue collection
Once birds had been fed in the lab for a total of six days and had reached an age
of nine days, they were weighed and then euthanized in a chamber attached to a carbon
monoxide tank (per animal protocol A1570). Upon euthanasia, birds were immediately
decapitated and blood samples were collected using capillary tubes. During the
dissection, a sample of the pancreas was removed, weighed, and frozen in liquid nitrogen
to be preserved for use in the lipase assays. Additionally, samples of intestine and liver
were harvested and stored for use in future studies. After dissection, birds were placed in
labeled plastic bags and stored in the freezer at -20 degrees Celsius.
Pancreatic Lipase Assay
All assays were conducted using materials from the Sigma Aldrich Lipase
Activity Assay Kit MAK046. Frozen pancreas samples were removed from the -80
degree freezer and set on ice. Pancreases were cut in half, halves were weighed, and one
half was returned to the freezer for use in future studies. The remaining halves which had
masses all of about 0.050 g were placed in Axygen microtubes on ice. 60µL of lipase
assay buffer was added to each microtube and samples were homogenized for 45 seconds
using an OMNI International homogenizer probe. Homogenized samples were then
transferred to a centrifuge and centrifuged at four degrees Celsius for 10 minutes at a
speed of 12,000 RPM. Supernatant from the centrifuged homogenates was pipetted into a
96-well plate-- 3 wells per sample--along with enough buffer to bring the volume up to
50µL. A glycerol calibration curve was created on the plate by pipetting increasing
volumes of a 1 mM glycerol standard solution over 6 wells. Buffer was added to the
glycerol calibration curve to bring each well up to 50µL volume. One well of the plate

6. was dedicated to a lipase positive control consisting of a sample of lipase provided in the
kit and buffer. Next, a master solution of buffer, peroxidase, and enzyme mix was added
to all wells in order to bring the volume up to 97µL. Finally, 3µL of buffer was added to
the wells containing sample blanks and 3µL of lipase substrate was added to all other
samples and glycerol standards. The plate was then mixed for 2 minutes using a
horizontal shaker and incubated at forty degrees Celsius for 3 minutes (T-initial). The
absorbance measurements were read at 570 nm every five minutes using a Wallace
Victor² 1420 multilabel counter and incubated in between reads. Once the absorbance
measurement of the most active sample had exceeded the highest glycerol standard, the
reaction was terminated. Absorbance measurements were converted to milliunits/mL
using the following formula:
G*sample dilution factor/(reaction time) *sample volume
Results
Lipase activity in the pancreas samples was to be quantified by analyzing the
absorbance measurements of the samples over a period of time. As time passes, the
amount of glycerol freed by the lipase enzyme should increase in the presence of the
substrate, therefore increasing the absorbance measurement of the samples. The Assays
for pancreatic lipase did not perform in the expected manner. At first, the sample wells
had absorbance measurements much higher than the highest glycerol standard. I adjusted
this by adding four sample-volumes worth of buffer. As a result of diluting the samples
more, the initial absorbance measurements of the sample wells were much lower than the
highest glycerol standard and therefore could be compared to the glycerol standard curve
in order to compute the amount of glycerol they contained. Though the samples were

7. diluted sufficiently, when I graphed the absorbance measurements versus time, I saw a
downward trend. In other words, as time went by, the absorbance measurements of the
samples decreased. The absorbance measurements of the samples would sometimes
increase by a little each time, but the sample blanks were increasing at a faster rate so that
when the blanks were subtracted from the samples each time, the real absorbance
measurements were becoming lower with time. These results showed that lipase was
either being improperly extracted from the tissue samples or undetected by the assay.
Below is a typical graph of time versus the absorbance measurement of a pancreas
sample.
As with all of the assays, the absorbance measurement of the lipase positive
control, provided by the assay kit, increased over time. However, over time, the
absorbance measurement of the real pancreas samples decreased. The downward trend
that was observed would seem to indicate that lipase was either being improperly
extracted from the tissue samples or that the glycerol was going undetected by the assay.
In order to correct for these possible faults related to the assay, two adjustments to
the method were tried. First, the pancreatic tissue was homogenized using a mortar and
pestle with liquid nitrogen to better separate the lipase from other components of the

8. tissue. The absorbance signal of the samples continued to show a downward trend,
showing that the mortar and pestle method did not improve lipase extraction. Second,
calcium chloride was added to the assay buffer to test whether the assay reaction was
calcium dependent. There were no indications that this was the case, because the data
maintained the same pattern.
Conclusion
Because the lipase assays did not yield reliable data, no conclusions can be made
about the effect of high-lipid diets on the production of lipase in Passer domesticus using
these methods. However, future research should test whether the freezing of pancreas
samples denatures the lipase, thereby rendering the assay unviable. To test whether
freezing temperatures destroy lipase, an assay should be performed using fresh pancreas
tissue that has never been frozen. Additionally, need remains to establish a reliable
method for assaying pancreatic lipase.
Sources Cited
Pawel Brzek, Kevin Kohl, Enrique Caviedes-Vidal, and William H. Karasov,
(2009).
Douglas J. Levey, Allen R.Place, Pedro J.Rey, Carlos Martinez del Rio, (1999). An
Experimental test of dietary enzyme modulation in Pine Warblers Dendroica Pinus
Brzęk, Paweł. "Effect of Age and Diet Composition on Activity of Pancreatic Enzymes in
Birds." National Center for Biotechnology Information. PubMed Central, 27 Dec. 2012.
Web. 6 Jan. 2015.