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1/9 VOL. 55 2025: e55bc23384
http://dx.doi.org/10.1590/1809-4392202303841
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BIODIVERSITY AND CONSERVATION | ORIGINAL ARTICLE
CITE AS: Avelino-de-Souza, K.; Valdevino, G.C.M.; Melo-Santos, G.; Mota, B.; Da Silva, V.M.F. 2025. How to remove the brain of Amazonian manatee
(Trichechus inunguis) calves preserving the skull for morphological analysis. Acta Amazonica 55: e55bc23384.
How to remove the brain of Amazonian manatee
(Trichechus inunguis) calves preserving the skull for
morphological analysis
Kamilla AVELINO-DE-SOUZA1,2,3* , Gisele de Castro Maciel VALDEVINO3,8,9 ,
Gabriel MELO-SANTOS3,4,5,6,7, Bruno MOTA1,2,3, Vera Maria Ferreira DA SILVA3,8,9
1 Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Rio de Janeiro, RJ, Brazil
2 Universidade Federal do Rio de Janeiro, Instituto de Física, Laboratório de Biologia Teórica e Matemática Experimental (MetaBIO), Rio de Janeiro, RJ, Brazil
3 Rede Brasileira de Neurobiodiversidade (RBNB), Rio de Janeiro, RJ, Brazil
4 Universidade Federal Rural da Amazônia, Biologia e Conservação de Mamíferos Aquáticos da Amazônia (BioMa), Belém, PA, Brazil
5 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia, Rio de Janeiro, RJ, Brazil
6 Instituto de Desenvolvimento Sustentável Mamirauá, Centro de Estudos da Várzea, Rede de Pesquisa e Conservação de Sirênios no Estuário Amazônico (SEA), Tefé, AM, Brazil
7 Universidade Federal do Rio de Janeiro, Laboratório de Ecologia e Conservação Marinha (Ecomar), Rio de Janeiro, RJ, Brazil
8 Instituto Nacional de Pesquisas da Amazônia (INPA), Laboratório de Mamíferos Aquáticos, Manaus, AM, Brazil
9 Instituto Nacional de Pesquisas da Amazônia (INPA), Programa de Pós-Graduação em Biologia de Água Doce e Pesca Interior (PPG-BADPI). Manaus, AM, Brazil
*Corresponding author: kamilla_bio@ufrj.br
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. e 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. e
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.
KEYWORDS: Sirenia; neuroanatomy; dissection protocol; osteology, aquatic mammals
Como remover o cérebro de lhores de peixe-boi amazônico (Trichechus
inunguis) preservando o crânio para análises morfológicas
RESUMO
Embora existam diversos estudos em crânios de peixes-boi amazônicos (Trichechus inunguis) a fim de melhor compreender a anatomia,
fisiologia e comportamento desses animais, análises do cérebro tem sido negligenciadas. Tipicamente, em estudos osteológicos, o
cérebro é descartado para preservar a integridade do crânio. Um dos principais motivos para essa negligência com o cérebro é a falta
de protocolos adequados de dissecação que permitam a extração do cérebro intacto enquanto preserva-se a integridade do crânio.
Neste estudo, apresentamos um protocolo simples, passo a passo, para um procedimento de extração e fixação do cérebro em filhotes
de peixe-boi, garantindo a preservação da estrutura do crânio ao máximo possível para estudos futuros.. O protocolo é baseado
em uma incisão na parte posterior do crânio, estendendo-se lateralmente em direção ao osso parietal até alcançar o osso frontal,
seguido pela remoção da porção superior da calota craniana para extrair o cérebro. Ao final do procedimento, a porção removida
pode ser reconstituída de forma a manter a estrutura completa do crânio. O protocolo também oferece adaptações para simplificar
a metodologia de acordo com a realidade de locais com pouca estrutura laboratorial, permitindo a preservação de tecidos raros com
recursos limitados e/ou em áreas de difícil acesso. A metodologia proposta permite a máxima utilização do animal coletado, o que
não apenas está em conformidade com considerações éticas e práticas, mas também torna o material disponível para uma descrição
detalhada dos cérebros dos peixes-boi e, consequentemente, a melhor compreensão da neuroanatomia de mamíferos aquáticos em geral.
PALAVRAS-CHAVE: sirênios; neuroanatomia; protocolo de dissecção, osteologia, mamíferos aquáticos
Avelino-de-Souza et al. Approach to remove brain of Amazonian manatees
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INTRODUCTION
e Amazonian manatee (Trichechus inunguis Natterer, 1883)
belongs to the Sirenia order and is endemic to the Amazon
Basin in South America (Rosas 1994; Amaral et al. 2023). It is
an herbivorous, fully aquatic mammal and, uniquely, the only
sirenian that inhabits exclusively freshwater environments.
Despite being the smallest member of its group, calves are born
weighing 10-15 kg and measuring 85-105 cm and can grow
up to 2.75 meters long and weigh as much as 450 kilograms
(Best 1983; Best 1984; Amaral et al. 2010).
e skull is one of the most studied structures in the
Amazonian manatee. Analyses of its components, such as
the tympanic bulla and the occipital condyles, have helped to
shed light on ecological, behavioral, and morphological issues
(Domning 1978; Domning and Hayek 1986; Castelblanco-
Martínez et al. 2014; Barros et al. 2017; Valdevino et al. 2021).
Additionally, cranial endocasts have been widely used as a
tool in neuroanatomy to provide information about the brain
anatomy of diverse fossil taxa, including sirenians (O’Shea
and Reep 1990; Macrini 2023). Also, the nervous system in
manatees has several peculiarities that are of great interest to
researchers, particularly from evolutionary-developmental
biology and comparative neuroanatomical perspectives
(O’Shea and Reep 1990; Popov and Supin 1990; Kelava et
al. 2013; Charvet et al. 2016). Manatees are known for their
distinctive smooth cerebral cortex and thick gray matter, which
may be of relevance to better understand the mechanism that
generates cortical gyrification (Mota and Herculano-Houzel
2015). ey are also noted for their small brain-to-body size
ratio and low encephalization quotients, which have been
the subject of discussions regarding their cognitive abilities
(O’Shea and Reep 1990; Reep and Bonde 2006). In this sense,
studying the brain morphology of manatees is not only of
interest to address the structure and function of their unique
neuroanatomical features, but also provide valuable data to
broaden comparative studies of mammalian brains in general.
However, despite their considerable potential, studies
focused on the nervous system of manatees are notably
sparse. Published neuroanatomical descriptions of the West
Indian manatee (Trichecus manatus, Linnaeus, 1758) (Reep
and O’Shea 1990; Marshall and Reep 1995) are decades
old and thus did not incorporate modern techniques not
available at that time. Furthermore, detailed information
regarding the dissection and fixation procedures used in these
studies is no longer accessible, hindering further analysis.
For the Amazonian manatee specifically, to the best of our
knowledge, there are no published detailed descriptions of its
brain anatomy. is likely results from the logistic challenges
associated with collecting the brains of aquatic mammals,
especially in the challenging climatic and logistic conditions
of the Amazon, combined with a lack of scientific funding for
studying the neuroanatomy of these animals in South America.
Another factor that contributes to the scarcity of
neuroanatomical studies of manatees is the invasive
nature of the usual approaches for brain extraction, which
typically damage the skull, making it unsuitable for future
morphological studies. ere is thus a trade-off between
extracting the intact brain and preserving the integrity of the
brain case. As a result, and given the usual lack of the necessary
materials and protocols, in most cases, the brain is discarded
to preserve the bone structure for osteological investigations.
A methodology for obtaining material that simultaneously
enables both osteological and neuroanatomical research would
therefore be highly valuable.
To address this challenge, we have developed and proposed
in here a simple and cost-effective protocol to remove
the brain from manatee calves while preserving the main
components of the skull. It includes a detailed description
of a removal and fixation procedure compatible with non-
invasive approaches, such as magnetic resonance imaging
(MRI), as well as histologically invasive approaches, such as
immunohistochemistry (Herculano-Houzel and Lent 2005).
Our main objective was to pave the way for expanding the
diversity of materials deposited in anatomical collections
for future research on manatee brains and to maximize the
utilization of collected specimens, enabling investigations
through diverse modern neuroanatomical methodologies.
MATERIAL AND METHODS
We dissected five Amazonian manatee calves from the Aquatic
Mammals Laboratory at the National Institute of Amazonian
Research (Instituto Nacional de Pesquisas da Amazônia -
INPA), Manaus, Brazil. is research facility serves as a hub
for receiving, rehabilitating, and facilitating scientific studies
of endangered orphaned animals. The procedures were
authorized by the Scientific Breeding Facility of Instituto
Brasileiro do Meio Ambiente e dos Recursos Naturais
Renováveis (IBAMA) under process # 21105.004653/1984-
60. e skulls were obtained from animals that did not survive
the rehabilitation process and were collected during necropsy
procedures. e study was conducted under the aegis of the
recently established ‘Rede Brasileira de Neurobiodiversidade’
(neurobiodiversidade.org), created specifically to enable the
collection and analysis of aquatic mammal brains in Brazil.
e age of the calves ranged from 0.5 to 1 year. eir sex,
body size, and weight are informed in Table 1. Two whole
individuals were frozen immediately after death and kept in
a freezer for no more than three months before the procedure,
while the other three had only the skull cleaned (with no
skin and muscles) and placed in formalin immediately after
death for at least 24 hours before dissection (Table 1). For
brain extraction, we developed a protocol based on prior
examinations of the cranial anatomy and position of the
brain within the skull of the Amazonian manatee, using
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the aquatic mammal collection at INPA. Our goal was to
establish a cutting plan that would enable the extraction of the
whole brain while preserving the integrity of the skull to the
greatest extent possible. To carry it out, the following tools are
required: scalpel, microsurgery scissors, dental forceps pliers,
hacksaw blade, spatula, metal spoon, tweezers, and epidural
needles (Figure 1). e following reagents are required for the
fixation and storage steps: 10% formalin or 4% formaldehyde
(PFA), phosphate buffer, sodium azide, and deionized water
(see composition in the Supplementary Material, Tables S1-
S4). All the reagents and equipment must be made available
beforehand. In institutions that receive stranded manatees
and manatee deaths are deemed likely, it is recommended
that a staff veterinarian or resident researcher be trained in
the procedure.
e anatomical nomenclature of bone structures follows
Domning and Hayek (1986), and Hoson et al. (2009) for
suture terminology. e protocol described in here can be
applied to Amazonian manatees of any developmental stage
but is particularly recommended for the extraction of the
brains of calves, which are more susceptible to damage than
those of adults.
RESULTS
e brains of the two individuals that were frozen postmortem
were apparently well preserved externally but not internally,
and disintegrated during the removal process. erefore the
protocol described below is based on the dissection of the
three individuals that had their skulls cleaned and immediately
immersed in formalin after death.
Dissection protocol
1 - Separate the head from the rest of the body using a scalpel
or sharp dissecting knife through the junction between the
occipital condyle and the first cervical vertebra (Figure 2a,b).
2 - Remove the skin and muscles from the head for a clear
visualization of the frontal, parietal, and occipital bones using
the tweezers, scalpel and surgical spatula (Figure 2c, 3). As the
cranial sutures are not totally closed in calves (Figure 3c), the
brain can be seen through the bones. e removal of the brain
immediately after death is not indicated in the case of calves,
as their brains are exceedingly fragile and may not withstand
the forces applied during removal.
3 - After the removal of the skin and muscles, inject fixative
into the most internal regions of the brain through the foramen
magnum and nasal bone openings using an epidural needle. is
will aid in the fixation of the inner parts of the brain (Figure
3c) as the fixative does not reach all regions of the tissue at the
same rate (Gage et al. 2012; Latini et al. 2015; Loomis 2016).
en immerse the whole skull in 4% paraformaldehyde or 4%
formalin for at least 24 hours before initiating the brain dissection
process, thus avoiding the potential leakage of sensitive structures,
such as the cerebellum, through the foramen magnum during
the subsequent skull-cutting process. Use a volume of fixative 10
to 20 times greater than the volume of the skull being preserved,
covering the whole structure, as a low liquid volume often results
in poor fixation (Everitt and Gross 2006; Loomis 2016).
e status of the fixation process can be assessed by
observing the color of the brain tissue. Fresh tissue is soft, rich
in blood, and exhibits a reddish appearance. Upon fixation, the
tissue becomes harder and takes on a slightly pale brownish
hue (Figure 4). Additionally, the consistency of the brain
tissue can be inspected by gently palpating it with the fingers.
Unsuccessful fixation will result in a pinkish color due to the
presence of blood. If the brain does not achieve the consistency
and color typical of fixed material after 24 hours, the fixative
needs to be replaced.
4 – To separate the top of the skull, use a hacksaw blade and
start the cut in the posterior part of the skull, at the level of
the supraoccipital-exoccipital synchondrosis, which is not
yet fused in calves (Figure 3c). en continue on both sides
towards the parietal bone until the frontal bone, after the
coronal suture. Laterally, the cut plane goes along the suture
between the parietal and squamosal bones (Figure 3a), where
Figure 1. Dissection tools used in the process of brain removal of Amazonian
manatee (Trichechus inunguis) calves.
Table 1. Information about the specimens of Amazonian manatee (Trichechus
inunguis) used in this study. NA = Not available information.
Animal identication Sex Standard length*
(cm)
Body weight
(kg)
Pb #291 Male 84 10
Pb #293 Male 83 11
Pb #294 Male 92 14
Frozen head 1 NA NA NA
Frozen head 2 NA NA NA
*Standard length = length in straight line from the tip of its snout to the base of
its tail.
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Figure 2. Line of incision (dashed line) behind the occipital condyle to separate the head from the body of Amazonian manatees, showing the position of the brain
inside the skull in lateral (A) and dorsal (B) view; C – Sketch showing the removal of skin and muscles from the head to allow the visualization of the skull bones.
Illustrations by Gabriel Melo-Santos.
the bone is less dense, reducing the risk of accidental damage
from applying too much pressure to harder skull regions. e
supraoccipital-exoccipital synchondrosis, is the last region on
the skull to begin the ossification process (Valdevino 2016).
is cutting line also protects delicate structures like the
tympanic bulla and other important bone regions.
5 - After completing the cut all around the skull, use dental
forceps pliers to carefully remove the skullcap and expose the
dorsal part of the brain (Figure 3d). e brain is enveloped
by thin membranes called meninges, which must be removed
carefully to avoid cutting into the brain during the extraction.
In these animals, the meninges were strongly adhered to both
Figure 3. Delimitation of the cutting area (dashed line) of the head of Amazonian manatees in lateral (A), dorsal (B, D), and posterior (C) view. The supraoccipital-exoccipital
synchondrosis is the ossication area between the supraoccipital bone and exoccipital bones shown in the red dotted line. The syringe in (C) indicates the injection
point for xative into the brain through the foramen magnum. The spatula in (D) indicates the insertion movement to release the brain from the skull after xation. Once
brain extraction is complete, the skullcap can be returned to its original position, maintaining the complete structure of the skull. Illustrations by Gabriel Melo-Santos.
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Figure 4. Brain of an Amazonian manatee calf in dorsal (left) and lateral (right) views after its removal from the skull. The right hemisphere in the dorsal view still
conserves the meninges. Abbreviations: bs: brainstem; cb: cerebellum; cc: cerebral cortex; rh: right hemisphere; lh: left hemisphere. Scale bar = 1cm.
the brain and the braincase, so use a blunt object without
sharp tips, such as a spatula or metal spoon, to very gently
and carefully separate the membrane from the brain tissue.
Microsurgery scissors can also be used to cut the meninges
and facilitate their separation from the skull. is precaution
is necessary to avoid potential damage to the tissue while
separating the brain from the skull.
6 - Keep the skull in dorsal orientation (Figure 3b) and, with
the aid of the surgical spatula and metal spoon, release the rest
of the brain from the skull to allow its removal without any
damage to the tissue (Figure 3d). Carefully slide your fingers
between the bone-brain boundary in the laterals, and also
underneath the brainstem and cerebellum, and gently lift the
posterior part of the brain. Be careful not to apply excessive
pressure to the brain tissue.
7 - Immediately after removing the brain, place it in a
container filled with PBS in sodium azide (PbAz), and sealed
with a hermetic cover, for storage until analysis. Alternatively,
a 30% sucrose solution at 4 °C can be used to dehydrate the
tissue for cryoprotection. After dehydration, the brain sinks
to the bottom and can then be stored in an antifreeze solution
at -20 °C. is step is beneficial for immunohistochemical
analyses, as it gives greater flexibility for conducting
experiments (Corthell 2014).
8 – After the brain is removed, the skull can undergo further
cleaning and preparation for inclusion in an osteological
collection. If the upper part of the skull was removed with a
clean cut (Figure 3d), it can be easily fitted back into place,
preserving the original skull morphology for anatomical,
imaging and/or histological studies.
DISCUSSION
Our dissection protocol for brain extraction in Amazonian
manatees represents a novel methodology that allows for the
removal of the brain in good condition while preserving the
skull for a variety of modern morphological studies. It enables
analyses of brain tissue using both invasive and non-invasive
techniques, such as immunohistochemistry and magnetic
resonance imaging, respectively.
e brain tissue is soft, delicate, and prone to degradation.
It is surrounded by the meninges, which, among several other
functions, provide a protective covering for the underlying
neural tissue of the brain and the spinal cord (Decimo et al.
2012; Dasgupta and Jeong 2019). Based on previous necropsies
in the context of the Rede Brasileira de Neurobiodiversidade,
we have observed that in other aquatic mammals, such as
dolphins, the meninges surrounding the brain are not as tightly
attached to the brain and skull as in the Amazonian manatee
calves. is observation reinforces the recommendation of
immersing the head for brain removal in a bucket filled with
4% paraformaldehyde (PFA) or 10% formalin (preferentially
PFA, if planning to perform immunohistochemistry analyses
(Corthell 2014; Loomis 2016) to allow the brain to gain
consistency and better withstand handling during the
procedure. Further research is necessary to elucidate if this is a
general species-specific feature or just related to the calf stage.
One of the main concerns when fixating neural tissue is
selecting an appropriate fixative that preserves the brain tissue
as closely as possible to its living state while maintaining its
suitability for the intended analysis. Also, reducing the time
interval between death and fixation is crucial for maintaining
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tissue integrity (Insausti et al. 2023). While freezing is often more
practical than other preservation methods and is commonly
chosen when available, our experiments revealed that, in the
case of brain tissue, it caused significant degradation of the
sample even before the extraction procedure was completed. e
tissue loses its consistency and fails to fixate internally, resulting
in the complete loss of the sample. Besides that, freezing the
sample even after fixation in formalin is not a good choice since
it leads to the formation of ice crystals in the brain sample and
consequent damage to the tissue, rendering the organ unsuitable
for further procedures (Rosene and Rhodes 1990).
e use of 4% PFA is generally considered the gold
standard approach to preserve cellular morphology and
antigenicity (Li et al. 2017; Konno et al. 2023). However, due
to the logistical challenges involved in preparing this solution
— since 4% PFA cannot be ordered as a working solution,
requiring prior preparation, and must be kept cold—previous
tests showed that, alternatively, the use of formalin at low
concentrations (4%) as fixative also has provided satisfactory
results in immunohistochemistry and neuroimaging studies.
e time required for fixation is another concern and
depends on the size of the brain and the fixative used (Carter
and Shieh 2015; Loomis 2016). It is crucial to ensure that the
brain tissue becomes fixed enough to allow for manipulation
without disintegration. However, avoiding over-fixing is also
important to preserve the tissue for histological analysis,
such as immunohistochemical staining (Corthell 2014).
is timing must be adjusted to adapt to specific needs and
circumstances. Previous studies on humans have shown that
extending fixation time to 72h still allows techniques like
Nissl-staining (Insausti et al. 2023). However, smaller brains,
such as those of manatees, tend to overfix more quickly.
Further tests are necessary to determine the appropriate
fixation time for different morphological techniques.
As a post-fixation step, freezing the tissue for slicing
techniques or sample storage can be an alternative option
after the PFA/formalin fixation process rather than PbAz. To
cryoprotect the tissue in this case, sucrose can be used to push the
water molecules out of the tissue, preventing ice crystal formation
and preserving the tissue available for further morphological
analysis (Corthell 2014). However, for neuroimaging studies,
preliminary results have demonstrated that sucrose-infiltrated
brain tissue preparation may alter the water diffusion properties
of the tissue (Mullins et al. 2013). is may prevent imaging
methodologies such as diffusion tensor MRI, which relies on
the anisotropy of the diffusion of water molecules to investigate
connectivity in the brain. In this context, preserving the brain
in PBS or PbAz after the fixation process is preferable. PbAz
not only acts as an antimicrobial preservative (de Prisco et al.
2022) but also enhances the contrast between tissues of their
nuclear spin relaxation times, resulting in more clearly defined
images (Wielenga et al. 2023). Although the post-fixation time
and reagents were adapted to immunohistochemistry studies
mentioned here, post-fixation time for other techniques can
be up to five years stored simply in paraformaldehyde/formalin
(Insausti et al. 2023; Nardi et al. 2023). However, it should be
noted that such prolonged exposure may lead to deformation
and shrinkage of the tissue which may, for example, confound
volumetric measurement studies (Su et al. 2014; de Guzman
et al. 2016; Insausti et al. 2023; McKenzie et al. 2024). ese
effects are still under debate but must be considered when
determining the appropriate post-fixation duration to ensure
the integrity of morphological data in long-term storage.
In rare instances, neuroimaging studies can be performed
in situ, right after the animal’s death. This allows the
immediate analysis of the brain and has the advantage of
investigating the organ in its correct position within the
skull, with no distortions related to the brain removal and
fixation (Montie et al. 2007). However, given the challenges
of carcass transportation, preparing the brain for ex-situ
analysis is more frequently the best choice. An alternative
approach is to perform in situ brain imaging in materials that
are preserved in formalin for a long time, as in the case of
museums or research collections. However, for these materials,
the immunohistological analysis may be unsuitable. In light
of this, our approach provides researchers with the necessary
time, equipment, and resources to perform comprehensive
imaging, histological, and osteological analyses, yielding a
wide range of valuable data for further research.
e acquisition of high-quality brain tissue, along with the
preservation of the skull, is a significant advancement in the
field. Both elements are crucial for comprehending the evolution
of mammalian brains and the potential cognitive capacities of
the species (Domning and Hayek 1986; Bauer and Reep 2022;
Henaut et al. 2022), but in many cases, one must be chosen at
the expense of the other. Our protocol introduces a standardized
method for whole brain extraction, increasing the availability of
this organ in zoological collections, and potentially minimizing
methodological discrepancies among comparative anatomical
descriptions. is standardization is crucial as varying protocols
can affect the accuracy, reproducibility, and comparison across
different neuromorphological studies (G.Vonsattel et al. 1995;
Ioannidis 2011; Klapwijk et al. 2021).
We expect this work will open the door to various
neuroanatomical studies on Amazonian manatees including
the potential analysis of adult specimens. e extracted manatee
brains can be used to produce detailed neuroanatomical
descriptions, high-fidelity cortical surface reconstructions from
structural MRI, white matter connectivity mapping through
imaging techniques such as diffusion tensor imaging (DTI),
and precise cellular counting for the various brain regions
using techniques such as the isotropic fractionator (Herculano-
Houzel and Lent 2005). In terms of comparative neuroanatomy,
studying Amazonian manatee brains allows for comparative
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analyses within the Sirenia group (Chapman 1875; Marshall
and Reep 1995; Reep and O’Shea 1990; Reep et al. 1989;
Sarko and Reep 2007; Bauer and Reep 2022; Macrini 2023)
as well as other mammals (Manger et al. 2012), providing
valuable insights into the relationship between evolutionary
and neuromorphological aspects of this group.
CONCLUSIONS
is study offers the first detailed method for extracting and
fixing manatee brains while preserving the skull for future
morphometrical investigations. Our research addresses the
scarcity of data on aquatic mammal brains, particularly
in Brazil, and provides the means to increase the material
available for multivariate studies aimed at advancing our
understanding of manatee neuroanatomy, contributing to
the broader field of mammalian neuroanatomy.
ACKNOWLEDGMENTS
We thank Instituto Serrapilheira and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq). GCMV
was supported by Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior - Brasil (CAPES) Financing Code 001,
Fundação de Amparo à Pesquisa do Estado do Amazonas –
FAPEAM/POSGRAD.
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RECEIVED: 21/11/2023
ACCEPTED: 14/10/2024
ASSOCIATE EDITOR: Paulo D. Bobrowiec
DATA AVAILABILITY: The original datasets produced and examined
during this study can be obtained, upon reasonable request, from the
corresponding author [Kamilla Avelino de Souza].
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Avelino-de-Souza et al. Approach to remove brain of Amazonian manatees
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SUPPLEMENTARY MATERIAL
Avelino-de-Souza et al. How to remove the brain of Amazonian manatee (Trichechus inunguis) calves preserving the skull for
morphological analysis
Table S1. Composition of the phosphate buer (PB) 0.4M used in the protocol for
skull-preserving removal of the whole brain of Amazonian manatees (Trichechus
inunguis).
Ingredient Total volume (L)
1 5 10
Distilled water (L) 0.5 2.5 5
Anhydrous di-potassium
hydrogen phosphate [K2HPO4] (g) 56 280 560
Sodium di-hydrogen phosphate
[NaH2PO4 H2O] (g) 10.6 53 106
Distilled water adjust to 1L adjust to 5L adjust to 10L
Check pH to be between 7.2-7.4.
The solution can be stored at 4°C for up to 6 months.
To prepare the working solution of 0.1 M, dilute 1:4 with distilled water, i.e., 250 ml
of PB 0.4 M plus 750 ml of distilled water generates 1 liter of PB 0.1 M.
To prepare the solution, the following equipment is necessary: magnetic stirrer to
dissolve the components, pH-meter, precision balance, and a glass Pyrex bottle.
Table S2. Composition of the 0.1 M sodium azide (NaAz) solution used in the
protocol for skull-preserving removal of the whole brain of Amazonian manatees
(Trichechus inunguis).
Ingredient Total volume (L)
1 5 10
0.1 M PB (L) 1 5 10
Sodium azide (g) 1 5 10
SAFETY NOTE: Sodium azide must be handled and prepared carefully due to its
toxic and carcinogenic nature. It is crucial to avoid inhaling its dust and avoid
using metal utensils during its manipulation. Safety guidelines for this substance
are available at <https://www.ehs.harvard.edu/sites/default/les/lab_safety_
guideline_sodium_azide.pdf>.
The solution can be stored in the refrigerator or at room temperature for up to one
year.
To prepare the solution, the following equipment is necessary: magnetic stirrer to
dissolve the components, precision balance, and a glass Pyrex bottle.
Table S3. Composition of the 4% paraformaldehyde (PFA) in 0.1M PB solution
used in the protocol for skull-preserving removal of the whole brain of Amazonian
manatees (Trichechus inunguis).
Ingredient Total volume (L)
1 5 10
Distilled water 0.75 3.75 7.5
PFA powder stir (g) 40 200 400
NaOH 10M drops drops drops
0.4M PB (L) 0.25 1.25 2.5
PREPARATION: Heat distilled water to 60 °C, add PFA and stir. Neutralize pH with
the NaOH 10M drop by drop until the solution becomes clear, then add 0.4 M PB.
The output solution should equal pH = 7.2. Filter the solution using lter paper and
store at -20 °C up to 1 month. If kept in the refrigerator (at 4 °C) it must be used in
up to one week.
To prepare the solution, the following equipment and material is necessary: fume
hood, precision balance, heated stir plate, Erlenmeyer ask (500 ml), magnetic
stir bar, pH meter, lter paper, glass Pyrex bottle. The following EPIs are necessary:
gloves, lab coat, mask, eye protection.
Table S4. Composition of the 30% sucrose solution that can be used in the
protocol for skull-preserving removal of the whole brain of Amazonian manatees
(Trichechus inunguis).
Ingredient Total volume (L)
1 5 10
0.1 M PB (L) 1 5 10
Sucrose (kg) 0.3 1.5 3
Complete with 0.1PB to the nal volume.
The solution can be stored at 4°C for up to 1 month.
The following equipment will be needed: stirring plate, magnetic stirrer, glass Pyrex
bottle.
