My sincerest thanks go to Dr. William Karasov for welcoming me into the Karasov lab and supporting me in all aspects of my research. I also thank Dr. Enrique Caviedes-Vidal, Katie Rott, and Cherry Brown for their constant support and guidance throughout the summer. I am grateful to Lisa Wachtel and Carmen Lombard, the leaders of the MMSD Science Research Internship program, as well as Rachel Eagan, for organizing this wonderful opportunity for students with an interest in the sciences.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.

Acknowledgements

Pancreatic Lipase Activity in Nestling House Sparrows Passer domesticusFiona Rambo with Dr. William Karasov and Katie Rott

University of Wisconsin-Madison Department of Forest and Wildlife Ecology

Results

Conclusion

Introduction Materials & Methods

Omnivorous birds consume a range of different foods based on their nutritional needs and the availability of food throughout the seasons. This study aimed to explore how changes in the diet of house sparrows Passer domesticus affect the production of pancreatic lipase. It was hypothesized that the amount of pancreatic lipase activity would increase in tandem with the amount of lipid substrate in the bird’s diet (Brzek et al. 2009), eliminating the wasteful production of enzymes that are not being used. This hypothesis is supported by a study on dietary enzyme modulation in Pine Warblers that found that birds fed a high-lipid insect and seed diet displayed higher levels of lipase activity than birds fed the high-carbohydrate fruit diet (Levey et al. 1999).

Overall, the goal of this research was to improve predictions about birds’ abilities to adapt to changes in their environment, promote the gastrointestinal tract as an archetype of phenotypic flexibility, and to enhance the limited, current knowledge about passerine birds and the regulation of digestive enzymes. However, conclusions cannot be made about the relationship between diet and pancreatic lipase activity because the assays did not yield reliable or valid data.

Body temperatures of the birds were recorded daily, birds were weighed three times a day before feedings, and the tarsus bone was measured at the end of the day to track growth. 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 the next three days, while other birds were fed the same diet for the whole treatment period.

All % dry massC=carbohydrate(starch), P=protein(casein), L=lipid(corn oil), i= inert ingredient (grit & cellulose)AssayAll assays were conducted using materials from the Sigma Aldrich Lipase Activity Assay Kit MAK046. Frozen pancreas samples were homogenized and transferred to a centrifuge. Supernatant from the centrifuged homogenates was pipetted into a 96-well plate along with buffer. 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 was dedicated to a lipase positive control consisting of a sample of lipase provided in the kit and buffer. A master solution of buffer, peroxidase, and enzyme mix was added to all wells in order to bring the volume up to 97μL. Finally, additional buffer was added to the wells containing sample blanks, and lipase substrate was added to all other samples and glycerol standards. Plates were mixed using a horizontal shaker and incubated at 40°C for 3 minutes. The absorbance measurements were read every 5 minutes at 570nm using a Wallace Victor 1420 multi-label plate reader and incubated at 40°C between reads.

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 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.

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 lipase is destroyed by freezing, 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.

Treatment

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

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. Below is a 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, the absorbance measurement of the real pancreas samples decreased. These results show that the activity of the lipase in the pancreas samples was not properly being detected.

0 10 20 30 40 50 60 70-0.060

-0.040

-0.020

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

Pancreas Sample

Lipase Pos. Control

Time (m)

Abso

rban

ce M

easu

rem

ent –

Pan

crea

s Sa

mpl

e(5

70nm

)

Materials & Methods

TreatmentHouse Sparrow nestlings Passer domesticus were collected three days post-hatch from locations on the UW campus. Nests were numbered and checked daily in order to monitor the number of eggs and nestlings. At three days post-hatch, birds were retrieved from their nests and transported to the Karasov Lab. The birds were weighed, assigned one of three diets, and added (in their nest container) to the humidified, incubated tub with the other subjects

In the first assay trials, 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 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.

Results

Time v. Absorbance measurement