7766

• UCREFRP Catalog Number: 7766
• Author: Scholz, A. T., et al.
• Title: Notes on the Development and Measurement of Egg and Larval Thyroxine Concentration in Flannelmouth Suckers ‎(
• USFW Year: 1992.
• Copyright Material: NO

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2.0 METHODS

Flannelmouthsucker eggs were collected from the Green River, UT
on May 28, 1991, and again on June 10, 1991. The eggs were fertilized and
immediately transported to a U.S. Fish and Wildlife Service operated
hatchery located on the Ouray National Wildlife Refuge, UT. The fish were
reared in vertical stacked Heath Tray incubators until the swim-up stage
and then transfered into rearing troughs. Water temperature was a
constant 21.1 :l:1.0oC throughout incubation and fry rearing. In the first
group, 25 eggs or larvae were collected at daily intervals from day 1 to
day 10 post-fertilization. In the second group, 15 eggs or larvae were
collected at daily intervals from day 1 to day 18 post-fertilization then
at 7 day intervals until 45 days post-fertilization. Each day fish were
sampled between 0830 and 0930 h Rocky Mountain Daylight Savings time,
so that hormone levels at a particular time of day could be established and
temporal trends determined indepentdent of dirunal fluctuations in
thyroxine (T4) levels. Sampled fish were weighed to the nearest 0.1 mg
using a Mettler AJ 100 analytical balance, flash frozen on dry ice and
stored at -80oC until T 4 was extracted and concentrations det'errnined by
radioummunassay.

2.1 T4 Extraction Procedures

Thyroxine (T4) was extracted from eggs and larvae using procedures
adapted from Parker (1988). Eggs or larvae were minced with scalpel on a
metal cutting block on ice and placed into a 15 x 85 mm test tube. Ice
cold 100% ethanol containing 1.0 mM 6-n-Propyl-2-thiouracil (ETOH-PTU)
was added to the tube at a J.l1 volume equal to 2 x's the mg weight of the
fish. The tissue sample was homogenized for 20 sec at 20,000 RPM using
a Polytron 3000 tissue grinder (Binkman Instruments, Inc.), then vortexed
for 5 seconds and decanted into a centrifuge tube. The test tube was then
washed with a J.l1 volume of ETOH-PTU equal to 1 x the mg weight of the
fish, homogenized for an additional 20 sec at 20,000 rpm, vortexed for 5
seconds, and then transfered into the centrifuge tube. The centrifuge tube
was vortexed for 10 seconds to thoroughly mix the two subsamples, then
centrifuged 10 min at 3000 RPM using a Dynac refrigerated centrifuge at
40C (Clay Adams, Inc.) The supernatent was decanted into a 10 ml drying
tube. The pellet was resuspended in 100% ETOH at a J.l1 volume equal to
1.5x's the mg weight of the fish, vortexed for 10 seconds. and centrifuged
again for 10 minutes at 3000 rpm at 40C. The supernatent was combined
with first supernatent in the drying tube. The drying tube, which
contained the extracted T 4, was dried overnight in vacuum .oven at 600C

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