Identification of Avian Gender Using Raman Spectroscopy

1
Identification of Avian Gender Using Raman
Spectroscopy

• Thaddaeus Hannel
• ARN Seminar
• Spring 2008

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Introduction

• Significance
• Current methods
• Commercial Chick Sexing
• Raman Spectroscopy
• Background
• Advantages
• Minimally Invasive Option
• Sample Prep
• UV Resonance Raman (UVRR)
• Experimental Results
• Data Analysis
• Chemometrics
• Conclusions

http//www.americanbirdcenter.com/abc-birdseyeview
.htm, 1/29/08
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Significance

• Aviculturists
• Establishment of breeding pairs
• Transfer endangered species to new breeding
  grounds
• 235 endangered species of bird
• Poultry Industry
• Economics
• Gender determines feeding conditions
• Feed is 65 percent of the cost of raising a
  chicken

http//www.precast.org/publications/solutions/2006
_ spring/images/images/parrot_parrot.jpg, 1/29/08
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Avian Physiology

• Cloaca
• Single opening in which contains the urinary,
  intestinal and genital tracts.

http//www.moyerschicks.com/MC-Web /DesktopDefault
.aspx, 1/29/08

• Physical
• Feather color and shape

http//www.charliesbirdblog.com/charlie/eclipse/
eclipsebills.jpg, 1/29/08
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Current Methods for Determining Gender

• Commercial Chick Sexing
• Manual sorting of poults based on cloaca
  morphology.
• Surgical Sexing
• The bird must be anesthetized.
• Polymerase chain reaction (PCR)
• Compare gene expression in embryos.

http//www.upc-online.org/pics/washingtonian/67.jp
g, 2/7/08
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Proposed Method for Gender Determination

• Sample prep
• Feathers are plucked from the bird
• Causes minimal duress to the bird
• UV Resonance Raman (UVRR) Spectroscopy
• Analysis of feather pulp to reveal Raman
  fingerprint

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Raman Spectroscopy
http//www.sunbeamtech.com/PRODUCTS/images/laser_b
eam_led_r_550.jpg, 1/29/08
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Raman Spectroscopy

• Discovered in 1928 by C.V. Raman
• Awarded the Nobel prize in 1931
• Measures the difference in the incident radiation
  and the radiation scattered by certain molecules.
  
• The wavelength shift is dependent on the chemical
  structure of the molecule.

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Raman Spectroscopy
Excited electronic states
2 1 0
Rayleigh Scattering E hv
Raman Scattering E hv ?E
E hv
Virtual states
?E
E hv
Stokes, E hv - ?E
Anti-Stokes, E hv ?E
2 1 0
Ground Electronic state
?E
Adapted from Skoog, Holler, Nieman, Principles
of Instrumental Analysis, Brooks/Cole, 1998, 431
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Raman Spectroscopy

• Identical Raman shifts can be found on both side
  of the Rayleigh peak.
• The Rayleigh line is of identical wavelength to
  excitation source.

Rayleigh scattering
Stokes lines
Anti-Stokes lines
Intensity
400 300 100 0
-100 -300 -400
? cm-1
Adapted from Skoog, Holler, Nieman, Principles
of Instrumental Analysis, Brooks/Cole, 1998, 431
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Raman Spectroscopy

• Advantages
• Raman Shifts are independent of excitation
  wavelength
• Small sample requirement
• No Sample manipulation is required
• Minimal water interference

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Raman Spectroscopy

• Disadvantages
• Rayleigh line
• Signal to Noise ratio (S/N lt 10-9)
• Limited scanning area
• Destructive
• Florescence

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Raman Instrumentation
Typical Continuous Wave (CW) Raman layout Andor
Technology, http//www.andor.com/chemistry/?app64
, 3/20/2007
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Minimally Invasive Option

• Sample prep
• Feather samples are picked from the skin and
  stored at -20 C.
• Thawed feathers are pressed from tip to base.
• The liquid sample is dried on a fused-silica
  surface
• UV Resonance Raman (UVRR) Spectroscopy
• Analysis of the dried sample to reveal unique
  Raman fingerprint

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Minimally Invasive Option
Harz, M. Krause, M. Bartels, T. Cramer, K.
Rosch, P. Popp, J. Anal. Chem. 2008 ASAP
Article
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Minimally Invasive Option

• UVRR Spectroscopy
• Analysis of complex biological samples can be
  performed.
• Selective enhancement of different sample
  constituents (i.e. aromatic amino acids, DNA
  bases) is realized.
• Genome size is larger in male birds.

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UVRR Spectroscopy

• Raman line intensities are enhanced be excitation
  wavelengths that closely approach that of an
  electronic absorption peak of an analyte
• Excitation energy can be targeted for specific
  absorption bands

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UVRR Spectroscopy
Excited electronic states
2 1 0
Rayleigh Scattering E hv
Raman Scattering E hv ?E
E hv
Virtual states
?E
E hv
Stokes, E hv - ?E
Anti-Stokes, E hv ?E
2 1 0
Ground Electronic state
?E
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Data Analysis
Harz, M. Krause, M. Bartels, T. Cramer, K.
Rosch, P. Popp, J. Anal. Chem. 2008 ASAP
Article
20
Data Analysis
Harz, M. Krause, M. Bartels, T. Cramer, K.
Rosch, P. Popp, J. Anal. Chem. 2008 ASAP
Article
21
Chemometric Analysis

• Hierarchical Cluster Analysis (HCA)
• Unsupervised data analysis

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Chemometric Analysis

• Support Vector Machine (SVM)
• Supervised data analysis

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Chemometric Analysis
Redrawn from Harz, M. Krause, M. Bartels, T.
Cramer, K. Rosch, P. Popp, J. Anal. Chem.
2008 ASAP Article
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Chemometric Analysis
Harz, M. Krause, M. Bartels, T. Cramer, K.
Rosch, P. Popp, J. Anal. Chem. 2008 ASAP
Article
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Conclusion

• UVRR Spectroscopy can be used for biological
  samples.
• Each spectrum can be compared to a database via
  chemometric methods.
• The SVM model is a practical solution for correct
  classification.
• Why pay for this?

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References

• Harz, M. Krause, M. Bartels, T. Cramer, K.
  Rosch, P. Popp, J. Anal. Chem. 2008 ASAP
  Article
• Skoog, Holler, Nieman, Principles of Instrumental
  Analysis, Brooks/Cole, 1998, 431
• Rosch, P. Harz, M. Schmitt, M. Popp, J. J.
  Raman Spectrosc. 2005 36 377379
• Schmitt, M. Popp, J. J. Raman Spectrosc. 2006
  37 2028

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• Thanks!