Background:

       As a group of more than 10,000 species, birds are the most diverse among the terrestrial vertebrates.  They are of considerable economic, ecological, and social importance, and represent some of the most conspicuous members of most faunae.  Nevertheless, with respect to the sizes of their genomes (i.e., the amount of DNA per haploid chromosome set), they are virtually unknown.  After more than 50 years of continuous research into genome size variation, only about 200 avian genome sizes have been published.  Genome size data are important not only for determining the future of complete sequencing projects, but also as fundamental biological characters with important consequences for organismal phenotypes.  Thus, despite the chronic lack of data for this group, preliminary analyses suggest that genome size is positively associated with cell size and negavtively correlated with metabolic rate in birds.  There are also intriguing indications that flight ability is linked to genome size, and it remains an ongoing question whether flight or small genome sizes came first (or indeed, if they co-evolved in tandem).  In order to investigate these issues, genome size data are needed from a wide diversity of bird species.
       A project underway in the Gregory Lab at the University of Guelph, led by graduate student Chandler Andrews, will seek to greatly expand the avian genome size dataset and to elucidate the patterns and consequences of variation in DNA content in this group.  To accomplish this, Chandler will use both flow cytometry (e.g., on frozen tissues) and Feulgen image analysis densitometry.  In terms of the latter, all that is needed are air-dried blood smears, which are easily prepared and shipped.  It is therefore possible for members of the ornithological community to make a significant contribution to the study, and at the same time to obtain genome size data for their species of interest free of charge.

Data use policy:

• Collaborators who provide samples will be free to use the genome size data as they see fit, so long as we are cited as having performed the measurement(s).  We will only ask for coauthorship on papers using the data if the genome size estimates form a major component of the study in question.  We reserve the right to publish the data as part of the larger avian genome size project or subsets thereof, and will offer coauthorship as appropriate in cases where collaboration is extensive.  All genome size data will ultimately be deposited in the Animal Genome Size Database, which is freely accessible.

Sampling blood by toenail clip:

• In many birds, it is possible to obtain a small volume of blood simply by clipping the toenail high enough to sever the vein.  This can be done with veterinary clippers sterilized with alcohol poses little to no risk to the bird if done properly.  Be sure to wipe the cut nail with alcohol when finished to prevent infection.

• You can touch the droplet of blood directly to a microscope slide and then smear it as described below.

Sampling blood by needle:

• Another effective way to get blood from birds is with a needle and syringe.  The brachial and jugular veins are considered the best sites for sampling in birds.  A guide including photographs by Ohio State University is available here.  Be sure to choose a needle guage that is appropriate for the size of the bird you are sampling.  A small (1mL or 3mL) syringe is sufficient – only a drop of blood is required.

• You may treat the syringes with heparin before use if clotting is a concern.  To do so, suck some heparin in and out of the syringe a few times to coat the inside.  Without treatment with an anticoagulant, the blood will clot quickly and will be very difficult to smear, but this is not a problem if you are preparing the smear immediately after sampling.

• Be sure to apply pressure to the needle site when removing the needle in order to prevent hemorrhaging below the skin.
  

• See below for details on preparing smears from the blood in the syringe.

Preparing blood smears:

• One individual per species is infinitetly better than none, but we will seek to analyze up to five individuals per species whenever possible.
  

• We use Fisherbrand Superfrost Plus slides (25 x 75 x 1.0mm, Fisher catalogue #12-550-15), but any standard microscope slide will suffice.   Slides are available in individual packages of ½ gross, or by the case of 10 gross.  Slide boxes are also needed for storage and shipping.  Plastic 100 slide capacity boxes are available from Fisher (and others) and cost on the order of $5 each.  Smaller slide boxes of 5, 25, and other capacities are also available (see below).

• Label the slide, in pencil, with the frosted side up.  Do not label in pen or marker (this will wash off during staining).  If you are providing samples from more than one individual, please take note of the individual number on each slide.

• Place a drop of blood on the slide near the label edge.

• Place a second slide at a roughly 45-degree angle to the first.  Gently back the angled slide into the blood drop.  When you touch the drop, the blood will expand to fill the space between the first and second slide.  Do not push the second slide through the droplet – you may damage the cells that way.  Instead, you will “pull” the cells along to smear them into a thin film.

• Gently move the second slide along toward the end of the first slide.  The blood will be pulled with it and will leave a thin film along the way.

• Allow the smear to air-dry, and store the slides in a slide box at room temperature before shipping.

Shipping slides:

• Please keep in mind that microscope slides are small pieces of glass and are very fragile.  Slide shipping containers are available in capacities of 2, 5, 25, 50, or 100 slides.  These are available from major suppliers such as Fisher at low cost and will ensure that your samples arrive in good shape.

• Pack the slides carefully in the slide box (put tissues in between them if you want), then pack the slide box in a cardboard box with some packing material (e.g., styrofoam “peanuts” or something of that nature). 
  

• If you have to fill out a customs form, just call the contents “microscope slides -- non-hazardous” or something of that sort.

• Air-dried slides will keep for a long time, so they do not need to be shipped by express courier.

• Unfortunately, we are not able to cover the cost of shipping for small numbers of slides.  However, if financial constraints prevent you from sending material or if you intend to send a large number of samples, we will attempt to work something out.

Permits and animal care:

• Please take all steps to minimize the stress experienced by the animals and make sure that all sampling takes place in accordance with your institution's animal care regulations.  Refer to the Guidelines to the Use of Wild Birds in Research (also available in PDF format) for detailed guidelines.

• Sampling of birds in the wild requires permits, often both state/provincial and federal.  Please consult with officials in your area and at your institution before collecting any specimens as only legally acquired material will be used in the study. 

• Shipping samples from some species out of your home country and/or into Canada may require special permits.  This will be particularly true for species that are listed in the International Convention on Trade in Endangered Species of Wild Fauna and Flora (CITES).  We can receive material from CITES-listed species through our direct affiliation with the Biodiversity Institute of Ontario, but you may also require export permits for these.

• Shipping bird material may be complicated by issues surrounding avian flu, especially if the species in question is a known carrier.  Please be sure that the material you wish to send is not restricted before shipping.
  

Links:

• The Gregory Lab, University of Guelph, Canada
  

• Chandler Andrews -- M.Sc. Candidate in charge of the bird project

• Animal Genome Size Database

• Biodiversity Institute of Ontario

• Reprint of Hardie et al. (2002) with details of the image analysis methodology

• Mammalian Genome Size Project
  

© T. Ryan Gregory, 2006.  Last updated Dec. 20, 2006.