How to remove the brain of Amazonian manatee (Trichechus inunguis) calves preserving the skull for morphological analysis

ABSTRACT Although there are several studies on the skull of Amazonian manatees (Trichechus inunguis) to better understand the anatomy, physiology, and behavior of these animals, the analysis of the brain has been often neglected. Typically, in osteological studies, the brain is discarded to preserve the integrity of the skull. One of the main reasons for this neglect of the brain is the lack of adequate dissection protocols to allow the extraction of the intact brain while preserving the integrity of the skull. In this study, we present a simple step-by-step protocol for a comprehensive procedure of brain extraction and fixation in manatee calves while ensuring the preservation of the skull structure to the best possible extent for further studies. The protocol is based on an incision at the posterior part of the skull, extending laterally toward the parietal bone until reaching the frontal bone, followed by removing the upper portion of the skullcap to extract the brain. After the procedure, the removed skull portion can be reconstituted to preserve the entire skull structure. The protocol also offers adaptations to simplify the methodology according to the reality of places with little laboratory structure, allowing the preservation of rare tissues with limited resources and/or in areas of difficult access. Our proposed methodology enables maximum utilization of the collected animal, which not only aligns with ethical and practical considerations, but also makes material available for a detailed description of the manatee brain, and a better understanding of the neuroanatomy of aquatic mammals in general.