A Method to Quantify Mouse Coat-Color Proportions

Coat-color proportions and patterns in mice are used as assays for many processes such as transgene expression, chimerism, and epigenetics. In many studies, coat-color readouts are estimated from subjective scoring of individual mice. Here we show a method by which mouse coat color is quantified as the proportion of coat shown in one or more digital images. We use the yellow-agouti mouse model of epigenetic variegation to demonstrate this method. We apply this method to live mice using a conventional digital camera for data collection. We use a raster graphics editing program to convert agouti regions of the coat to a standard, uniform, brown color and the yellow regions of the coat to a standard, uniform, yellow color. We use a second program to quantify the proportions of these standard colors. This method provides quantification that relates directly to the visual appearance of the live animal. It also provides an objective analysis with a traceable record, and it should allow for precise comparisons of mouse coats and mouse cohorts within and between studies

Means and SDs of coat percent yellow, as measured from the top and sides at different times by different raters.

A<p>Coat colors were measured as percentage yellow by two different raters each at three specific times within a one-week period. Measurements were also averaged by rater, time, or both.</p>B<p>Sides were calculated as the average of the measurements from the left side and right side.</p>C<p>Difference was calculated as Sides–Top. For “averaged” measures, the difference was calculated between the averaged Top and Sides measures.</p>D<p>Two One-Sided 95% Confidence Limits on the mean difference between Top and Sides. Under the Two One-Sided Tests (TOST) procedure, the Top and Sides are equivalent (at 5% alpha) to within ±10 percentage points if the two one-sided 95% confidence limits both lie inside equivalence-limit boundaries located at +10 and −10 percentage points.</p

Intra-rater reliability of each rater measuring the percentage of yellow coat color in each mouse on three different days from the sides^A and from the top.

A<p>Sides: Average measure from left- and right-side photos per mouse.</p>B<p>Intraclass Correlation Coefficient, type (2,1) in Shrout and Fleiss <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005414#pone.0005414-Shrout1" target="_blank">[25]</a> notation.</p

RGB proportions for the red, brown, and yellow colors.

<p>RGB proportions for the red, brown, and yellow colors.</p

Inter-rater reliability between the two raters on the individual days, evaluated using the average of measurements from photos taken from the left and right sides.

A<p>Intraclass Correlation Coefficient, type (2,1) in Shrout and Fleiss <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005414#pone.0005414-Shrout1" target="_blank">[25]</a> notation.</p

Photographs, colorings, and pie charts of mice.

<p>Photographs of mice were processed as described in the text to produce yellow areas of uniform color and agouti areas of uniform color that closely matched yellow and agouti areas on the live mouse. A script was used to generate a pie chart from these areas of uniform color to determine the percentage of yellow and agouti in the mouse. Two examples are shown here.</p

Inter-rater reliability estimate between the two raters for time-averaged measurement derived from the indicated photographic angle.

A<p>Intraclass Correlation Coefficient, type (2,1) in Shrout and Fleiss <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005414#pone.0005414-Shrout1" target="_blank">[25]</a> notation.</p>B<p>Sides: Average of two measurements per mouse, each derived from photos taken on the left and right sides of the mouse.</p>C<p>Top: Single measurement per mouse, derived from photo taken from vertical (top) angle.</p

A plot of the percent yellow in the coats of mice evaluated from their sides.

<p>Two raters matched colorings on 12 mice on three occasions by examination of each mouse from its left and right sides. Vertical and horizontal axes denote the average percent yellow of the mouse's left and right sides. Letters denote the identity of the mouse being rated, while the red diagonal line denotes the line of equality between percentages assigned by the two raters.</p

Inter-rater reliability between the two raters on the individual days, evaluated using measurements from photos taken from the top angle only.

A<p>Intraclass Correlation Coefficient, type (2,1) in Shrout and Fleiss <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005414#pone.0005414-Shrout1" target="_blank">[25]</a> notation.</p

A plot of the percent yellow in the coats of mice evaluated from the top.

<p>Two raters matched colorings on 12 mice on three occasions by examination of each mouse from the top. Vertical and horizontal axes denote the percent yellow assigned by each rater. Letters denote the identity of the mouse being rated, while the red diagonal line denotes the line of equality between percentages assigned by the two raters.</p