Springer et al. • CHANGES IN WHITE-THROATED SPARROW MUSCLE 
117 
standard bird seed and then weaned onto a ground 
commercial diet (Mazuri small bird maintenance, 
PMI Nutrition International, Brentwood, MO, 
USA) over 4 weeks. Food and water were 
changed on a daily basis and provided ad libitum. 
Room temperature ranged between 21 and 24 C. 
Five sparrows, after short day exposure, were 
switched to long days (16L:8D) for 28 days to 
stimulate migratory behavior (Agatsuma and 
Ramenofsky 2006), while the remaining birds 
were kept on short days. Hyperphagia and nightly 
hopping (Zugunruhe; characteristic of captive 
birds in migratory condition) were demonstrated 
by long day animals (D. J. Cerasale, D. M. Zajac, 
andC. G. Guglielmo, unpubl. data). Short day and 
long day birds were euthanized by decapitation 
following anesthesia with isofluorane at the end of 
this period. Right pectoralis muscle samples were 
rapidly removed and flash frozen in liquid 
nitrogen prior to storage (—80" C). Nearly all 
birds could be classified as juvenile or adult by the 
extent of skull ossification (DeHaven et al. 1974). 
Protocols were approved by the University of 
Western Ontario Council on Animal Care Animal 
Use Subcommittee (protocol #2005-060-08) and 
appropriate collection permits were obtained from 
the Canadian Wildlife Service (CA 0170, CA 
0230) and the U.S. Fish and Wildlife Service 
(MB758364-1 issued to F. R. Moore). 
Wild Sparrows. —Migratory White-throated 
Sparrows were captured using mist nets in April 
bpring, n = 8) and October (fall, n = 26) near 
Uong Point, Ontario. Eleven wintering sparrows 
were captured during January near Stoneville, 
Mississippi at the Delta Experimental Forest. 
Birds were anesthetized with isofluorane and 
euthanized by cervical dislocation. The right 
pectoralis muscle was removed and immediately 
frozen in liquid nitrogen. Samples were stored at 
C until analysis. Age was classified as 
juvenile or adult (DeHaven et al. 1974). 
Lipid Extraction and Analysis. —Lipids were 
extracted following a modified Folch procedure 
1 Folch et al. 1957). Pectoralis samples (100 mg) 
Were homogenized in 2.5 mL chloroform/methanol 
( 2 ; 1 v/v) containing 25 mg/L butylated hydro- 
x ytoluene as an antioxidant. Total lipid extracts 
were centrifuged (2,056 X g) for 15 min, 1 mL of 
- % KC1 so | ut j on was added, an d samples were 
le ated in a water bath for 10 min at 70° C to 
^eparate the aqueous and lipid-soluble components. 
e resulting organic layer was removed and dried 
Lincl er a stream of nitrogen, followed by re¬ 
suspension in 1 mL chloroform/methanol (2:1 v/ 
v) containing 25 mg/L butylated hydroxytoluene. 
Extracts were stored at -20° C prior to analysis. 
Sphingomyelin from bovine brain (Sigma, Oak¬ 
ville, ON, Canada) was used as an internal standard 
in wild sparrow samples and L-oc-phosphatidyl-L- 
serine (PS) from Glycine max (Sigma) was used as 
the internal standard in captive sparrow samples to 
correct for sample loss during extraction. Both 
internal standards were chosen because sphingo¬ 
myelin and PS were below detection threshold in 
preliminary analyses of our tissues. 
We used thin layer chromatography coupled to 
a flame ionization detector (TLC-FID) to separate 
and quantify phospholipids (De Schrijver and 
Vermeulen 1991). Samples were spotted (1.5 pL) 
using a syringe (Pressure Lok VICI Precision 
Sampling, VICI Valeo Canada, Brockville, ON, 
Canada) and developed on a quartz rod coated 
with a thin layer of silica oxide (Chromarod, 
Shell-USA, Fredericksburg, VA, USA). The 
spotted samples were developed in a solvent 
system consisting of chloroform:methanol:water 
(20:12:1) (Sherma and Fried 2003). Chromarods 
were analyzed with an Iatroscan MK-6 instrument 
(Mitsubishi Kagaku Iatron, Tokyo, Japan) using 
the following settings: flow rate of 2 L/min air, 
160 mL/min hydrogen, and scanning speed of 
3.0 s/cm. FID results were analyzed with 
PeakSimple 3.29 software (SRI instruments, 
Torrance, CA, USA), which generated area counts 
(AC) for each separated lipid component. Phos¬ 
pholipid classes were identified by comparison to 
a mix of polar standards containing PC (L-ot- 
phosphatidylcholine, Type XVI-E —99%, Sigma), 
PE (L-a-phosphatidylethanolamine, Type III: 
from egg yolk -98%, Sigma), sphingomyelin, 
and PS. Standard curves were developed using 
phospholipid standards (PC and PE). Area count 
data for PC and PE were normalized based on the 
recovered internal standard, and were used for the 
final calculation of PC/PE ratios. 
Statistical Analysis .—The statistical signifi¬ 
cance level was set at oc = 0.05 for all statistical 
tests. SPSS software (Version 17, SPSS Inc., 
Chicago, IL, USA) was used for all analyses. 
Student’s /-tests were used to examine the effect 
of season on the PC/PE ratio in captive sparrows. 
A general linear model was used to test for effects 
of season and age, and their interaction on PC/PE 
ratios in wild birds. Student-Newman-Keuls test 
was used to examine differences among groups in 
wild birds. 
