The overall goal of this procedure is to enable non-invasive, economic and efficient sexing and marking of altricial chicks as early as possible after hatching. This is accomplished by first taking buccal samples from newly hatched chicks. The second step is to trim the down feathers in a specific pattern which stays recognizable until 10 days after hatching.
Next, the DNA is extracted from the buccal samples using the kele DNA extraction method. The final step is to perform PCR using primers that amplify a sexually dimorphic region on the z and w chromosome of birds and load the samples on a gel to visualize the band pattern. Ultimately on day 10, after hatching, each chick in a nest is identified by its unique haircut and a leg band is applied.
The main advantage of this technique over existing ones is that it is non-invasive and much faster than sexing from blood samples using conventional DNA extraction methods In general, people new to this method will struggle because they are not thorough enough in taking the buccal swap, which is key for a successful result. To begin, prepare 50 milliliters of 5%kelex 100 solution. In molecular grade water, constantly re homogenize the suspension with a magnetic stir to prevent the kele resin from precipitating while Ali Watting 200 microliters of the solution In standard 1.5 milliliter reaction tubes Next cut Watman paper into pieces smaller than the width of the bird's beak.
The piece should be long enough to enable sample collection without the forceps being inserted into the beak of the bird. Prepare the PCR premix as described in the accompanying text protocol. Capture the bird of interest and gently hold it in one hand using the ringer's grip.
Do not squeeze the bird. Next, hold a piece of watman paper with forceps and swipe the paper several times across the inside of the bird's cheeks. Sampling from hatchlings or nestlings is especially easy using this technique as their begging behavior provides easy access to the inside of the beak.
Immediately store the paper in a reaction tube containing 200 microliters of previously prepared kele solution. If you also intend to or need to mark chicks individually for later recognition, do so now. As described in a later section, store samples at minus 20 degrees Celsius samples can be stored for more than three years.
To begin DNA extraction, remove the stored samples from the freezer and thaw the samples at room temperature. Ensure that the Watman paper is submerged in the kelex 100 solution on the bottom of the tube. Incubate the samples for 15 minutes at 56 degrees Celsius.
Then briefly vortex the sample and spin the tube. Next, incubate the samples at 100 degrees Celsius for eight minutes. Then spin the samples at 15, 000 times G for three minutes.
Then store the sample for subsequent genotyping at minus 20 degrees Celsius. Prepare one PCR tube with six microliters of pre-mix per sample, and two additional tubes for the negative and positive control. Next, add 19 microliters of the supernatant from the DNA extraction to the PCR premix.
Make sure to pipette from the surface of the solution to avoid carryover of kele beads. Then run the PCR program. Next, prepare a 2%standard TBE AROS gel to separate the PCR products.
Then load the gel with the PCR sample and run the aros gel with appropriate voltage settings. Once the gel has finished running, visualize the bands under UV light and take a picture. Make sure the two bands in the samples originating from females are well separated.
Distinguish male from female samples by their band pattern. One band for males, two bands for females check ness in the morning daily for newly hatched chicks in zebra finches. Tufts of down grow at four characteristic and distinct areas across the nestlings body.
Cut the down feathers of each chick within a nest. For each chick cut one area. If there are more than four chicks within one nest, the unique four haircuts can be combined.
10 days post hatching inspect the haircuts to identify chicks. The down feathers are now sitting on the tips of the developing feathers. At that age, nestling size allows you to apply leg bands and note down who is who.
Sexing results from buccal swabs were obtained from various avian species. One band appears in male samples and two bands appear in female samples. Similar amounts of DNA bearing material were gained from all ages.
Kele carryover into the PCR reaction inhibits the amplification of DNA as seen in the column where a small amount of kele beads were added to the PCR reaction. The presence of beads can be readily identified as dark staining in the pocket of the gel, and an absence of primer dimer formation in the contaminated sample. Successful extraction can be performed after long storage due to the use of kele.
Samples of two animals were stored under different conditions as indicated in the table. If performed properly, this technique can be done in about three hours. After watching this video, you should have a good understanding of how to mark and sex altru chicks noninvasively as early as the day of hatching.
By combining the down feather haircuts and the fast and easy to perform buccal swabs in combination with a helix based DNA extraction.
