Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

The overall goal of this dissection and morphometric analysis is to illustrate the robustness of our morphometric methodology for studying structures with complex shapes like the mouse penis bone. We first had the idea for this study when we realized that existing methods of measuring baculum variation such as taking length and width dimensions lost all information about curvature and shape complexity, therefore we devised this method to quantify size and shape variation of any bone or structure of interest in three dimensions. The main advantage of this technique is that it applies modern morphometrics to structures without landmarks.

This should advance the field by providing improved repeatable measurements of complex structures. Position a euthanized, sexually mature male mouse supine and protract the penis by applying thumb pressure lateral to the preputial opening. Once the penis emerges, extend the tissue through the prepuce as far as possible.

Next, using scissors, cut the penile body proximal to the gland's penis where the baculum resides, detaching the penis. Then fully submerge the penis in about 200 microliters of tap water in a microcentrifuge tube. To soften the tissue, incubate it at about 50 degrees celsius for three to five days.

Tissue softening reduces the change of the bone breaking during extraction. After incubation, use a dissection microscope and forceps to remove most of the tissue surrounding the baculum. After the gross dissection, gently squirt 70 percent ethanol to push off the remaining tissue and clean the bone.

Then place the dissected baculum into a new microcentrifuge tube, and let the bone dry at room temperature with the cap left open. To begin, prepare the material in which the bone will be placed. Press a CT scan cylindrical holder into a brick of florist foam.

Then extract the resulting cylinder of foam and cut slices of foam that are between two and five centimeters thick. Now push 10 to 15 dried bacula into the florist foam around the periphery of an individual slice to minimize interference during scanning. Take note of the precise orientation of the bone for future identification.

Next, carefully place the bones in foam into the micro CT holder and load the holder into a micro CT 50 scanner. Set the micro CT 50 scanner as follows. Use 90 kilovolt pascals, a voxel size of 155 angstroms, a 5 milliliter aluminum filter, 750 projections per 180 degrees, and a 500 millisecond exposure time.

Then proceed to scan the specimen. Baculum bones were dissected, prepared, and scanned using the described protocol. Many bones can be packed into one CT scan.

The images were then processed using computational geometry as described in the text protocol. This imaging process should be applicable to the study of a wide array of bones and other subjects of interest. The analysis provides detailed information on the landmark features of the object and is highly automated.

This process only required about 10 manual adjustments when analyzing 369 bacula. The exciting thing is that our methods could be applied to a wide variety of complex structures. These methods allowed us to quantify the size and shape of the baculum in three dimensions.