1. I ntroduction Sex identification in birds allows scientists to track population trends as well as monitor fitness and sex ratios in species of concern. Sexing birds is often difficult due to the fact more than 50% of bird species are sexually monomorphic. Physical characteristics can lead to sex determination but are not always accurate and require extensive handling of birds which is often undesirable (Jensen et al. 2003). Without external features, scientists are left with dissection and molecular techniques to sex many birds. Sexing using DNA samples offers the easiest and least invasive way to accomplish this task (Wang et al. 2006). However, it is difficult to determine the sex of some bird species using molecular techniques. Owls in general and barn owls in particular, are difficult to sex because the replicated regions on the sex chromosome are very similar in length. It is hard to distinguish between the ZW double band of a female from the ZZ single band of a male when compared on agarose gels (Griffiths et al. 1998). Polyacrilamide gel electrophoresis (PAGE) can accomplish this but is expensive, time consuming, and difficult to use. The purpose of this experiment was to refine the methodology of using agarose gels for the molecular sexing of barn owls. Sexing Barn Owls Using Molecular Techniques Ben Heyler Extract DNA from feather sample PCR Agarose Gel Electrophoresis Image gel to determine sex Methods The procedure for PCR reaction using primer P2 and P8 was outlined by Griffiths et al. (1998). These methods for agarose gel electrophoresis were modified. A 4.1% agar gel was used to distinguish between fragments with as little as 20 bp difference. 1.45g agar was dissolved in 35 ml 1xLB buffer. To reduce air bubbles, the agar and buffer were swirled in the flask while hot. The gel was cast after adding 2 µl gelstar and allowed to cool at room temperature until hard. Electrophoresis was performed at 4 o C in a refrigerator using pre- cooled 1xLB buffer. Gels were run initially for 30 minutes at 250 V and run for additional 10 minute increments if necessary. Results Objective: The purpose of this experiment was to refine the methodology of using agarose gels for the molecular sexing of barn owls. Polyacrimamide Gel Agarose Gel Matching samples were run on both 10% PAGE and 4.1% agarose gels. Both PAGE and agarose gels yielded similar results. On PAGE, samples 1,2,3,4,5,6, and 7 were sexed as female. On agarose, samples 4,5,6, and 7 were all sexed as female. Sample 8 on PAGE and samples 1,2, and 3 on agarose could not be read. There were no males sexed out of the samples. Agar Gel 1Agar Gel 2 Samples 6, 9 and 11 from Gel 1 (4.1%) were found to be male while samples 7,8,and 10 were found to be female. Sample 18 on Gel 2 (4.1%) was found to be male while samples 17, 19, and 30 were found to be female. Conclusion: 4.1% Agarose gels can be used with methods modified from Griffiths et al. (1998) to successfully sex barn owls. Not every owl could be sexed on the first try and some samples need to be run additional times. Acknowledgments: I would like to thank Dr. Karl Kleiner for all the help he provided for my project. Literature Cited Griffiths, R., Double, M.C., Orr, K. and Dawson, R.J.G. 1998. A DNA test to sex most birds. Molecular Ecology 7:1071- 1075. Jensen, T., Pernasetti, F.M. and Durrant, B. 2003. Conditions for rapid sex determination in 47 avian species by PCR of genomic DNA from blood, shell-membrane blood vessels, and feathers. Zoo Biology 22:561-571. Wang, L., Chen, C., Lee, H., Li, S., Lir, J., Chin, S., Pu, C. and Wang, C. 2006. Cut feather containing rachis as a sampling way for avian sexing. Zoo Biology 25:279-283. F F F F F F F F F F F M F F M F M F M F F