USGS Forest and Rangeland Ecosystem Science Center

3200 SW Jefferson Way

Corvallis, OR 97331

Phone: 541-750-7482; FAX: 541-758-8806; Email: Susan_Haig@usgs.gov

**[GENETIC SAMPLE COLLECTION]{.underline}**

Thanks for offering to help collect tissue samples for our conservation
genetics project. Detailed below is a brief outline of how to collect
and store samples. If you have problems or questions, please call me
(Susan Haig; see \# above) or my lab manager, Tom Mullins (541-750-7389,
tom_mullins@usgs.gov).

[Blood Samples]{.underline}

1\. Hold bird upright (i.e., not on its back in your lap). Extend wing
out *slightly* so you can expose the brachial vein (vein crossing
joint). Use a q-tip dipped in rubbing alcohol to part

feathers and expose vein. This clears, cleanses, and somewhat
anesthetizes the area.

2\. Use the needle (26 g or smaller) to poke a small hole in the vein. I
usually hold the needle parallel to the vein and approach in a direction
from the wing towards the heart. Remove the needle immediately as blood
will begin to flow.

If this doesn\'t work, you can try one or two more times on that wing
before switching to the other. If a hematoma forms, apply pressure until
swelling stops. Normally, you cannot get blood

from a hematoma as it is clotted. Change wings and try again.

3\. Use a heparinized tube to collect blood as it drips *down* from the
vein\--do not touch the vein

with the tube, just collect drops as they drip onto the feathers. The
tube should be held

underneath the flow and in such a way that the blood flows down into the
tube. We are *not*

depending on capillary action to fill the tube\--just gravity! If the
flow stops, you can

carefully try to open the wound again, but chances are it won\'t start
again. We are trying to get

as full a tube as possible, but if you have at least several good drops,
don\'t worry about it. If it

stops right away, try the other wing. We are hoping to get 0.2-0.5 ml of
blood per sample.

Note: the amount of blood you draw is well below the limits recommended
by the Veterinary Handbook of Surgery and Medicine and the American
Ornithologists Union Guidelines on Care of Wild Birds in Research (Oring
et al. 1998). A bird\'s blood volume is 10 % of it\'s body

weight. Thus, the guidelines recommend drawing 10-15% of a bird\'s
blood. By taking even 0.5 ml of blood, you will sample less than the
most conservative allowable sample.

4\. Usually blood stops flowing sooner than you want it to, but if it
doesn\'t, apply pressure (but

don\'t break their wing!) until it does. You can use cotton to help
apply pressure or clean up the blood. Bleeding profusely is very
uncommon, but you should know what to do if it happens.

5\. Blood should be gently blown from the tube into the vial. Seal vial
tightly and store in an

*upright* position so the blood does not get caught up in the screw part
of the top. Keep

vials out of the sun and cool in the field, store them in freezer until
shipping, or you can

leave vials at room temperature (if they contain buffer)\--*do not
refrigerate* as buffer will come

out of solution and not preserve the blood.

[Eggs and Embryos]{.underline}

Embryos from abandoned eggs can be a great source of DNA. The easiest
way to deal with eggs would be for you to crack open each egg and
carefully look for an embryo. Place the embryo in the vial with buffer.
If there is no embryo, it probably will not be of use to us, so please
don\'t send the yolk or shells.

[Muscle Tissue]{.underline}

Heart or pectoralis (breast) tissue can be very helpful in genetic
analyses. Cut the heart or pectoralis out of the body with a clean
(sterile if possible---we can provide) scissors or scalpel. Place the
entire heart or pectoralis in the buffer vial and label accordingly. If
the tissue needs to be saved for other purposes, cut a piece that is
approximately 1 cm^3^. See directions below for storing and shipping.

[Other Body Parts]{.underline}

We can use almost anything---bones, organs, hair, etc. Just place it in
the buffer vial as above.

[Feathers]{.underline}

Feathers can also be a handy source of DNA and are preferable tissue to
blood in mtDNA analyses.

1\. Clip 2-3 secondary feathers from the base of the feather-do not
pluck the feather as it is painful for the bird and will bleed
unnecessarily.

or\...

If feathers are just growing in and have not attached (i.e. blood
feathers), you can wipe

several of them off the wing or tail and place them in the vial. They
will grow back quickly. Do

not pull these feathers out as it will be painful to the bird.

2\. If using dry feathers (not blood feathers), place feathers from each
bird in a single plastic bag or

vial and label with species, bird id# , location, etc. (see #6. above)

3\. Keep feathers as clean as possible to reduce contamination- be
especially careful not to touch

the base (quill) with your hands.

4\. Keep feathers in cool dry place until mailing to us.

[Labeling Vials]{.underline}

**Please be sure each vial is labeled** with: species, bird id#,
location (including state and site). Other pertinent information which
can be noted on a separate sheet (attached) is the county of collection,
collectors name, and tissue type i.e. blood, muscle, feather, etc.
**Also let us know if you have sampled siblings or parents and
offspring.**

[Sample Storage]{.underline}

We have or will supply you with vials containing a buffer solution.
Samples can be kept in these vials at room temperature for short periods
of time (a week or so) and then mailed via regular mail w/o ice or they
can be frozen and mailed on dry ice. **DO NOT REFRIGERATE SAMPLES** or
the buffer will come out of solution.

\*\*\*Please label sample vial with species, USFWS #, location, sex

**SAMPLE LOG:**

Species USFWS \# Color bands Sex State/Province County Location Date
Tissue Type Collector (email address) Comments

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

**Thanks again for your help-this will be a great contribution to the
project.**