Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Summary

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Abstract

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Introduction

The field of morphometrics includes a diversity of methods to quantify the size and shape of the biological form, a fundamental step in scientific inquiry^1,2,3,4,5,6. Traditionally, the statistical analysis of size and shape begins by identifying landmarks on a biological structure, and then measuring linear distances, angles and ratios, which could be analyzed in a multivariate framework. Landmark-based Geometric Morphometrics is an approach that retains the spatial posit....

Protocol

All procedures and personnel were approved by the University of Southern California's Institute for Animal Care and Use Committee (IACUC), protocol #11394.

1. Baculum Dissection and Preparation

1. Euthanize a sexually mature male mouse via carbon dioxide over-exposure, according to protocols set forth by the relevant Institutional Animal Care and Use Committee (IACUC).
2. Lay the animal in the supine position, and protract the penis by applying pressure with thumbs lateral to the preputial opening.
3. Once the penis is protracted, extend the tissue through the prepuce as far as possible.
4. With scissors, c....

Results

The x-y-z coordinates of the semi-landmarks produced in Protocol 6 can be directly imported into any landmark-based geometric morphometrics analysis¹⁷. The computational pipeline above has been applied to study mouse bacula¹⁴, as well as whale pelvic and rib bones¹⁶. More details on the computational definition of semi-landmarks are presented here, in an attempt to help researchers visualize steps that might be modifi.......

Discussion

The critical steps in the above protocol are 1) dissecting the bacula, 2) gathering the microCT images, 3) converting the microCT output to a flat file of x-y-z coordinates, 4) segmenting out each specimen's point cloud, 5) transforming each specimen to a standardized coordinate system, and 6) defining semi-landmarks. These steps are easily modified to accommodate different objects.

These methods can likely be applied to any object that is essentially "rod-shaped", or at least not .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissecting scissors	VWR	470106-338	Most sizes should work
Dissecting Forceps, Fine Tip, Curved	VWR	82027-406
1.7 mL microcentrifuge tube	VWR	87003-294
Absolute Ethanol	Fisher Scientific	CAS 64-17-5	To be diluted to 70% for dissections
Floral Foam	Wholesale Floral	6002-48-07
uCT50 scanner	Scanco Medical AG, Bruttisellen, Switzerland