Exploring swine oviduct anatomy through micro-computed tomography: a 3D modeling perspective

Abstract

The oviduct plays a crucial role in the reproductive process, serving as the stage for fertilization and the early stages of embryonic development. When the environment of this organ has been mimicked, it has been shown to enhance in vitro embryo epigenetic reprogramming and to improve the yield of the system. This study explores the anatomical intricacies of two oviduct regions, the uterotubal junction (UTJ) and the ampullary-isthmic junction (AIJ) by using micro-computed tomography (MicroCT). In this study, we have characterized and 3D-reconstructed the oviduct structure, by measuring height and width of the oviduct’s folds, along with the assessments of fractal dimension, lacunarity and shape factor. Results indicate distinct structural features in UTJ and AIJ, with UTJ displaying small, uniformly distributed folds and high lacunarity, while AIJ shows larger folds with lower lacunarity. Fractal dimension analysis reveals values for UTJ within 1.189–1.1779, while AIJ values range from 1.559–1.770, indicating differences in structural complexity between these regions. Additionally, blind sacs or crypts are observed, akin to those found in various species, suggesting potential roles in sperm sequestration or reservoir formation. These morphological differences align with functional variations and are essential for developing an accurate 3D model. In conclusion, this research provides information about the oviduct anatomy, leveraging MicroCT technology for detailed 3D reconstructions, which can significantly contribute to the understanding of geometric-morphological characteristics influencing functional traits, providing a foundation for a biomimetic oviduct-on-a-chip.

Full text

Frontiers in Veterinary Science 01 frontiersin.org
Exploring swine oviduct anatomy
through micro-computed
tomography: a 3D modeling
perspective
RamsesBelda-Perez
1,2, CostanzaCimini
1, LucaValbonetti
1,
TizianaOrsini
3, AnnunziataD’Elia
3, RobertoMassari
3,
CarloDiCarlo
1, AlessiaParadiso
1, SeeratMaqsood
1,
FerdinandoScavizzi
3, MarcelloRaspa
3, NicolaBernabò
1* and
BarbaraBarboni
1
1 Department of Biosciences and Technology for Food, Agriculture and Environment, University of
Teramo, Teramo, Italy, 2 Physiology of Reproduction Group, Department of Physiology, Faculty of
Veterinary Medicine, International Excellence Campus for Higher Education and Research (Campus
Mare Nostrum), University of Murcia, Murcia, Spain, 3 Institute of Biochemistry and Cell Biology (CNR-
IBBC/EMMA/Infrafrontier/IMPC), National Research Council, Rome, Italy
The oviduct plays a crucial role in the reproductive process, serving as the
stage for fertilization and the early stages of embryonic development. When the
environment of this organ has been mimicked, it has been shown to enhance
in vitro embryo epigenetic reprogramming and to improve the yield of the
system. This study explores the anatomical intricacies of two oviduct regions,
the uterotubal junction (UTJ) and the ampullary-isthmic junction (AIJ) by using
micro-computed tomography (MicroCT). In this study, wehave characterized
and 3D-reconstructed the oviduct structure, by measuring height and width of
the oviduct’s folds, along with the assessments of fractal dimension, lacunarity
and shape factor. Results indicate distinct structural features in UTJ and AIJ,
with UTJ displaying small, uniformly distributed folds and high lacunarity,
while AIJ shows larger folds with lower lacunarity. Fractal dimension analysis
reveals values for UTJ within 1.189–1.1779, while AIJ values range from
1.559–1.770, indicating dierences in structural complexity between these
regions. Additionally, blind sacs or crypts are observed, akin to those found in
various species, suggesting potential roles in sperm sequestration or reservoir
formation. These morphological dierences align with functional variations
and are essential for developing an accurate 3D model. In conclusion, this
research provides information about the oviduct anatomy, leveraging MicroCT
technology for detailed 3D reconstructions, which can significantly contribute
to the understanding of geometric-morphological characteristics influencing
functional traits, providing a foundation for a biomimetic oviduct-on-a-chip.
KEYWORDS
oviduct, microCT, 3D-reconstruction, swine model, utero tubal junction, ampullary-
isthmic junction
OPEN ACCESS
EDITED BY
Stefan Gregore Ciornei,
University of Life Science (IULS), Romania
REVIEWED BY
Carlos Eduardo Ambrósio,
University of São Paulo, Brazil
Mihaela Spataru,
Ion Ionescu de la Brad University of
Agricultural Sciences and Veterinary Medicine
of Iași, Romania
*CORRESPONDENCE
Nicola Bernabò
nbernabo@unite.it
RECEIVED 28 June 2024
ACCEPTED 14 August 2024
PUBLISHED 03 September 2024
CITATION
Belda-Perez R, Cimini C, Valbonetti L,
Orsini T, D’Elia A, Massari R, Di Carlo C,
Paradiso A, Maqsood S, Scavizzi F, Raspa M,
Bernabò N and Barboni B (2024) Exploring
swine oviduct anatomy through
micro-computed tomography: a 3D
modeling perspective.
Front. Vet. Sci. 11:1456524.
doi: 10.3389/fvets.2024.1456524
COPYRIGHT
© 2024 Belda-Perez, Cimini, Valbonetti,
Orsini, D’Elia, Massari, Di Carlo, Paradiso,
Maqsood, Scavizzi, Raspa, Bernabò and
Barboni. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 03 September 2024
DOI 10.3389/fvets.2024.1456524

Belda-Perez et al. 10.3389/fvets.2024.1456524
Frontiers in Veterinary Science 02 frontiersin.org
1 Introduction
e oviduct is the organ where the journey of life begins for all
mammals. It is divided into 4 regions, the infundibulum, the ampulla,
the isthmus, and the uterotubal junction. Aer ovulation, the
infundibulum is responsible for collecting the cumulus-oocyte complex,
and on the isthmus, the sperm will form the spermatic reservoir
attaching to the epithelial cells, suering changes in its membrane, and
prolonging their viability. Before ovulation, spermatozoa will bereleased
from the reservoir and they reach the ampulla, where fertilization takes
place. On the other hand, the uterine-tubal junction could beconsidered
one of the main selection barriers (1).
The epithelium of the oviduct is composed of two major types
of cells: secretory and ciliated. The secretory cells are responsible
for the formation of oviductal fluid (2), which plays an important
role in creating an appropriate environment for the transport and
nourishment of gametes, as well as in protecting the fertilized egg
during its journey through the oviduct (3). Conversely, the ciliated
cells possess carbohydrate residues that are recognized by lectin-
like proteins on the head of spermatozoa, leading to their binding
and the creation of the previously mentioned sperm reservoir (4).
The oviduct offers a dynamic environment, since its cell
proportion and functionality change in response to the hormonal
swings that take place throughout the cycle (5, 6). For instance,
during the follicular phase, ciliated cells prevail in the ampulla of
the oviduct while during the luteal phase, secretory cells take the
forefront in this region (7). By contrast, in other segments of the
oviduct, like the isthmus, the proportion of these cell types
remains relatively stable with minimal variations throughout the
estrus cycle (7).
Due to the relevance of oviduct in fertilization, some authors
have tried to mimic its effect in in vitro fertilization platforms
used in artificial reproductive techniques (8, 9). Indeed, the
addition to artificial environment designed to allow the
fertilization could improve the system’s out, mainly reducing the
epigenetic differences between the in vivo derived and in vitro
produced embryos.
In that context, for instance an oviduct-on-a-chip has been
recently created (9). In one hand it represents a very interesting
device, but in the other one hand it ignores the architectural
features of the organ. Since it has been demonstrated that there is
a correlation between oviduct architecture and function (10) here
wecarried out a set of measures to lay the foundation to a sort of
reverse engineering work. In fact, in a recent work weidentified,
from a selection of 3D-printing-biocompatible materials
previously used in cell cultures, the one suitable for construction
the model of the oviduct, evaluating its toxic effects by mean of
embryo development (11). Consequently, the present work
emerges as a pilot study aimed at creating an anatomically
accurate-3D model of the oviduct to furtherly design a biomimetic
oviduct-on-a-chip. This approach will consider the organ’s
morphology, then weadopted an approach based on the use of
microcomputed tomography (MicroCT) as a reliable tool for the
anatomical study of oviduct: it is non-destructive technology
characterized by high resolution and three-dimensional
visualization capabilities, and it is able to allow sophisticated
quantitative analysis and detailed 3D reconstructions.
2 Results
2.1 Utero tubal junction
In this segment of the oviduct, it is noteworthy that there are small
folds present, and their dimensions typically fall within the range
between 144–988 μm length and 70–366 μm width
(Supplementary Table S1; Figure1). ese folds do not reach a great
percentage of the lumen, an observation that is supported by the high
lacunarity value (Supplementary Table S1).
On the contrary, the distribution of values for fractal dimension
and lacunarity in UTJ is concentrated around a dierent subpopulation
(Figure2). e fractal dimension encompasses values within the range
1.189–1.1779, while the lacunarity values range within 0.901–2.701
(Supplementary Table S1). On the other hand, the shape factor values
of the UTJ external and internal regions are provided (Table1).
2.2 Ampullary-isthmic junction
In this section of the oviduct, we not only observe a higher
prevalence of folds but also an increase in their individual sizes,
ranging between 126–1,446μm length (Supplementary Table S2) and
40–512 μm width (Supplementary Table S2), resulting in a more
substantial occupancy within the lumen, reducing the lacunarity
(Supplementary Table S2). In AIJ, distinct subpopulations of folds are
discernible, with one set characterized by larger dimensions and
another set exhibiting smaller dimensions. By contrast to the width
measurements, where the data distribution is not centered around
specic values, but rather displays a more homogeneous spread
(Figure3).
Similar to what weobserved with the length of the folds, the fractal
dimension and lacunarity are also divided into dierent subpopulation
(Figure4). e fractal dimension encompasses values within the range
1.559–1.770, while the lacunarity values ranges within 0.577–1.544
(Supplementary Table S2). On the other hand, the shape factor values
of the AIJ external and internal region is provided (Table2).
2.3 3-D reconstruction
In the 3D reconstruction, the tortuous structure of the oviduct is
represented oering a detailed view of the high tortuosity it possesses
(Supplementary Movie 1). e visualization highlights the spatial
arrangement of the folds of the AIJ section and small blind-ended sacs
(Figures5A–D). On the other hand, in the UTJ of the oviduct, the
lumen is narrower and exhibits a less tortuous structure compared to
the other sections (Figures5E–H).
3 Discussion
In this study, wehave delved into the architectural examination of
dierent segments of swine oviduct by using a medical imaging
approach, reporting for the rst-time parameters such as the shape
factor, fractal dimension and lacunarity of the oviduct through
microCT imaging. ese parameters will bethe basis for a more

Belda-Perez et al. 10.3389/fvets.2024.1456524
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accurate representation of reality in an oviduct-on-a-chip, enabling
improved IVF and embryo culture performance, presumably
enhancing epigenetic embryo reprogramming (9).
MicroCT has already been used to characterize the architecture of
organs such as lungs (12), tendons or arteries (13), or even to recreate
3D images of breast cancer specimens (14). It generates 3D models
that can then beconverted into les suitable for 3D printing, thus
enabling the creation of three-dimensional replicas of those organs
(15). is technology has been used to create custom prostheses (16),
and could beused to create models for surgeon planning (17) or for
educational purposes (18). Recently, it has been used to create 3D bone
phantoms through 3D printing, using CT generated images (19). In
this context, this preliminary study means a progress into the creation
of a novel oviduct 3D model considering the architecture of the organ,
in the light to design a biomimetic 3D scaold suitable for IVP.
e classical method for reconstructing the oviduct has
traditionally involved using histology slides. However, it has been
shown that this method may not bethe most suitable, as it could
result in gaps (20). Additionally, when using histology, it can
be reconstruction inaccuracies ranging due to deformation of
misalignment (21), due to the diculty of managing three-
dimensional orientation. Multiple manual artifacts aect the perfect
alignment of the sample in all directions of space. In fact, the
positioning procedures of the sample inside the embedding medium,
the mounting of the histological specimen on the microtome, the
simultaneous three-dimensional angulation of the specimen and the
instrument are evident causes of problematic management of the
spatial orientation of the specimens. is type of problem is partially
overcome in the case of virtual histology that can beperformed by
microCT, which also becomes the basis of the three-dimensional
model perfectly tting the original sample. MicroCT technique has
proven to bea valuable tool for studying mineralized biologic tissues
(22), although its utility in the investigation of so tissues has been
somewhat constrained due to the limited contrast these tissues
typically exhibit (23). Some tries have been made previously to study
the internal structure of the oviduct through microCT (24). With the
oviduct unmodied and without contrast, and other ones ll the
oviduct with a strong contrast agent (24). e initial attempt proved
FIGURE1
Violin plot representing the dierent measurement of length (left) and width (right) of the folds from the UTJ.
FIGURE2
Violin plot representing the dierent measurement of fractal dimension (left) and lacunarity (right) of the folds from the UTJ.

Belda-Perez et al. 10.3389/fvets.2024.1456524
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to beunsuccessful as it yielded no discernible internal structures. In
contrast, the subsequent approaches provided what could belikened
to a photographic “negative” of the oviduct. is eect was achieved
due to the signicant disparity in clarity between the contrast agent
and the surrounding tissue. However, when compared with histology
slides, the structure observed was not very detailed. In our work,
we employed the paran embedding method, a technique that
enables us to visualize the high-resolution structure of the oviduct
by leveraging its endogenous contrast, as previously described for
murine embryos (25). Although in human the use of MicroCT to
study the oviduct has been already achieved successfully (26), to our
knowledge, those represent the rst data obtained from swine
oviducts through microCT.
Like what has been observed in humans (26, 27) or sheep (28), our
results show throughout all phases of the cycle, at the UTJ level,
mucosal folds are sparse and smaller in size, occupying limited space
within the lumen. However, as wemove away from the uterus and
progress toward the oviduct until the ampulla, a signicant increase
in the size of these folds and the proportion of space they occupy
within the lumen becomes evident. is increase in lumen occupancy
is not only apparent from the measurements of the folds in the images
but is also corroborated by the decrease in lacunarity. In addition, even
TABLE1 Shape factor of the oviduct in UTJ region.
External shape factor
Late follicular Early follicular Late luteal Early luteal
Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2
Volume
(mm3)
66.31 127.71 31.53 26.64 41.55 43.64 84.22 39.80
Surface
(mm2)
234.11 353.68 124.53 109.56 170.13 130.69 268.53 143.59
Height
(mm)
4.46 4.46 4.46 4.46 4.46 4.46 4.46 4.46
Shape index 15.75 12.35 17.62 18.34 18.26 13.36 14.22 16.09
shape index
(1/mm)
3.53 2.77 3.95 4.11 4.09 2.99 3.19 3.61
Internal shape factor
Volume
(mm3)
3.95 3.2 0.52 0.80 1.66 1.58 7.78 2.87
Surface
(mm2)
77.88 50.2 13.83 16.31 30.20 23.92 106.27 30.19
Height
(mm)
4.46 4.46 4.46 4.46 4.46 4.46 4.46 4.46
Shape index 87.94 69.97 118.62 90.93 81.14 67.52 60.92 46.92
shape index
(1/mm)
19.72 15.69 26.60 20.39 18.19 15.14 13.66 10.52
FIGURE3
Violin plot representing the dierent measurement of length (left) and widths (right) of the folds from the AIJ.

Belda-Perez et al. 10.3389/fvets.2024.1456524
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though our sample size is small, it is worth noting that the maximum
fold amplitude is consistently observed during the follicular phase, as
previously described using electronic microscopy (29). Cyclic
variations during the estrous cycle have been even described in the
serosal part of the oviduct where cells of the tubal epithelium are also
present, being the ciliated cells predominant during the follicular
phase and the secretory cells during the luteal phase (30). It has been
suggested that the folds in the oviduct could play a crucial role in the
transport of oocytes and embryos during the fertilization process.
ese oviductal epithelial folds serve to signicantly increase the
surface area of the epithelium, thereby enhancing the likelihood of
contact between the oocyte/embryo and the ciliated cells within the
oviduct (31). Moreover, the specic structure of these folds appears to
play a key role in alleviating the pressure dierence in the oviductal
uid before and aer the passage of the oocyte/embryo (31). It has
been observed that the Celsr1 gene controls the proper formation of
these oviductal folds, and Celsr1-decient mice present an altered
ciliary beating coordination, and aberrant fold orientation distribution,
even leading to infertility (32), presumably due to diculties in the
eective transport of oocytes and embryos through the oviduct.
FIGURE4
Violin plot representing the dierent measurement of fractal dimension (left) and lacunarity (right) of the folds from the UTJ.
TABLE2 Shape factor of the oviduct in AIJ region.
External shape factor
Late follicular Early follicular Late luteal Early luteal
Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2 Oviduct 1 Oviduct 2
Volume
(mm3)
40.79 44 23.6 26.51 29.83 23.79 35.10 48.41
Surface
(mm2)
226.97 225.73 234.99 286.61 155.14 84.67 166.89 323.40
Height
(mm)
4.46 4.46 4.46 4.46 4.46 4.46 4.46 4.46
shape index 24.82 22.88 44.41 48.21 23.20 15.87 21.21 29.79
shape index
(1/mm)
5.56 5.13 9.96 10.81 5.20 3.56 4.75 6.68
Shape factor
Volume
(mm3)
4.87 4.32 6.36 5.88 1.99 0.40 0.81 8.34
Surface
(mm2)
66.76 73.22 103.86 141.18 41.52 8.96 18.91 195.45
Height
(mm)
4.46 4.46 4.46 4.46 4.46 4.46 4.46 4.46
shape index 61.15 75.59 72.83 107.16 92.96 99.90 104.12 104.56
shape index
(1/mm)
13.71 16.95 16.33 24.03 20.84 22.40 23.35 23.44

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In the 3D reconstruction of the oviduct, wecan observe numerous
blind sacs or crypts, which are small, pouch-like structures within the
oviduct. ese anatomical features have been described in pigs (29)
and in other animal species, including humans (26), ovine (28),
marsupials (33, 34), hamster (35), bovine (6), and moles (36). In some
animals, such as shrews, it has been suggested that the crypts in the
oviduct mucosa may serve the function of “sequestering” sperm,
thereby preventing polyspermy (37). Meanwhile, in other animals like
cows (6) or sows (29), it has been proposed that these structures could
also collaborate to form the sperm reservoir.
e data gleaned from this study possesses signicant potential to
lay the groundwork for the development of a sophisticated 3D model.
Advanced printing techniques, including additive manufacturing
processes, can then beemployed to fabricate the physical device layer
by layer, resulting in a tangible replica of the organ. is model would
bemeticulously craed to encompass the intricate architecture of the
oviduct. By meticulously incorporating the detailed structural nuances
revealed by the microCT imaging, this 3D printing le would serve as
a blueprint for creating a physical device that faithfully replicates the
natural features of the organ. is innovative device, when combined
with microuidic systems, could have the potential to replicate the
physiology of organs, oering a valuable tool for enhanced studies of
organ function and disease (38). Notably, in recent years, research has
shown that conducting in vitro fertilization processes within devices
mimicking the oviduct can yield superior outcomes (9, 39).
Considering this, the reconstructions obtained in this study hold the
promise of serving as the blueprint for a 3D-printed device that
accurately reects this complex architecture. anks to its accuracy
anatomy reconstruction, together with a microuidic system, could
even allow the study of the biophysics of the organ and a more reliable
representation of the processes that’s occurs in the oviduct, as sperm
capacitation or the “taxis” (chemotaxis, thermotaxis and rheotaxis)
that guides sperm cells within the oviduct (40).
In this work, we conducted a comprehensive assessment to
determine the most suitable segmentation techniques for accurately
representing the morphological traits of interest. is evaluation
encompassed various segmentation methods, including automatic,
interactive, and manual approaches. e challenge of segmentation in
biomedical imaging, as highlighted in both the existing literature and
this experimental investigation, predominantly arises from the lack of
ecient automated tools.
In summary, the pursuit of optimal segmentation techniques in
biomedical imaging is crucial for advancing both research and clinical
practice. By addressing the challenges inherent in segmentation, such
as automation and accuracy, researchers and clinicians can harness the
full potential of biomedical imaging. Additionally, the integration of
3D printing technology further amplies the impact of biomedical
imaging by that facilitating the conversion of virtual models into
physical prototypes capable of faithfully reproducing even the most
intricate shapes and geometric features. For that reason and
considering that our group have previously identied a suitable
material together with the suitable appropriate 3D printing technology,
the data presented in this work will allow the engineer of a new 3D
scaold as closely as possible to the oviduct in order to increase the
quality of embryos produced with ARTs.
4 Materials and methods
4.1 Oviduct selection and collection
Genital tracts from sows and gilts were obtained at the local
slaughterhouse and transported into the lab within 2 h of slaughter.
Once in the lab, the cycle stage of the tracts was determined based
on the ovarian morphology as described previously (41) and
classied into early follicular (n = 2), late follicular (n = 2), early
FIGURE5
3D reconstruction of AIJ (A–D) and UTJ (E–H) regions in dierent phases of the cycle late follicular (A,E), early follicular (B,F), late luteal (C,G), and early
luteal (D,H).

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luteal (n = 2), or late luteal phase (n = 2). Two oviduct from 4
animals were selected for each phase of the reproductive cycle,
dissected and divided into segments. e portions corresponding
to the isthmus and the uterine-tubal junction were washed in PBS
and xed in 4% paraformaldehyde for 1 h. Subsequently, the
samples underwent dehydration through a series of alcoholic
solutions (ranging from 50 to 100%) and soaked in xylene 3 times
for 15 min each (45 min tot). An incubation step with xylene
paran (1:1) was carried out for 45 min at 56°C before embedding
in paran wax.
4.2 MicroCT and image acquisition
MicroCT datasets of swine oviducts at dierent stages of estrous
cycle were acquired by using the high-resolution 3D-imaging
system Skyscan 1172G (Bruker, Kontich – Belgium), using an
L7901-20 Microfocus X-ray Source (Hamamatsu), with image pixel/
size of 7.4 μm, camera binning 2×2, source voltage of 39 kV, source
current of 240 μA, exposure time of 500 ms. e reconstructed
tomographic volumes of the acquired images were performed using
built-in NRecon Skyscan reconstruction soware (Version: 1.6.6.0;
Skyscan Bruker). 3D-images were generated using 3D-Visualization
Soware CTvox v. 2.5, while the volume rendering and virtual
sectioning views using DataViewer v. 1.4.4 (Skyscan Bruker) and
the analysis of the sample was performed using CT-Analyser
soware version 1.13.
4.3 Histology and light microscopy
Paran embedded oviducts were microtome-sectioned at 10 μm
and stained with Hematoxylin (Sigma-Aldrich, cat. MHS16) – Eosin
(Sigma-Aldrich, cat. 109,844) standard protocol to perform
histological assay. Images were obtained with the stereomicroscope
MZ12 (Leica) equipped with a color camera. e histological analysis
highlighted the accuracy and delity of the two-dimensional and
three-dimensional images obtained from microtomography, which
has the further advantage of guaranteeing the structural integrity of
the sample and avoiding distortions and artifacts resulting from the
sectioning procedures (Figures2, 5, 6).
4.4 Image analysis
Using microCT images, measurements of the length and width of
the oviduct folds were conducted, as illustrated in Figure6. e FIJI
program (ImageJ 2.0.0-rc-43/1.50e) was employed for this purpose,
utilizing its built-in measurement tool to ensure accuracy in
anatomical dimensions. Similarly, to calculate the fractal dimension
and lacunarity values, weapplied the box-counting method using the
FracLac plugin in Fiji.
4.5 Two-dimensional image processing
For the processing of two-dimensional images, the soware Mimics
from the Belgian company Materialise (Materialise, Leuven, Belgium)
and 3Matic (Materialise, Leuven, Belgium) were used. Mimics is the
primary soware for processing biomedical images in the rapid
prototyping sector. Initially, the DICOM les were loaded, and the
correct orientation of the images (Top-Bottom, Anterior–Posterior,
Right–Le) was set. Once the images were loaded, the soware provided
their visualization of the three anatomical planes: transverse/axial,
coronal/frontal, and sagittal. It was decided to analyze, in particular, the
series of images from the ampullary-isthmus junction (AIJ) and of the
utero-tubal junction (UTJ). is approach ensured that images from all
phases acquired were available, regardless of their original format. Each
image series generated a project within Mimics, i.e., a le in .mcs format.
At this point, an initial segmentation was performed on each of the four
les by selecting the region of interest through the crop project
operation. Specically, various slices along all three directions (x, y, z)
were excluded from the project, retaining only the structures of interest
and reducing the number of slices to beanalyzed. is helped eliminate
the inuence of regions not relevant to the study. To achieve this, the
position and size of a rectangle were set in all three views (axial, coronal,
and sagittal) to delimit the volume of interest in the resulting
parallelepiped in the three-dimensional view. Aer a careful analysis of
FIGURE6
Comparison between virtual and histological oviduct sectioning, with a schematic representation of the measurement method performed.

Belda-Perez et al. 10.3389/fvets.2024.1456524
Frontiers in Veterinary Science 08 frontiersin.org
the images from various contrast phases, the dierent anatomical
structures that would beincluded in the overall three-dimensional
virtual model have been segmented separately: both the external and
internal structures of the reference tract (UTJ and AIJ). For each of these
structures, it was assessed which image series was most suitable for their
segmentation. is decision was based on the presence of enhanced
visibility of the mentioned structures in those specic images. Regarding
the improvement of image quality, especially in terms of contrast, a
point-wise enhancement technique was employed. is involves
applying a transformation that maps a small range of grey levels onto
the entire possible range, aiming to achieve visual enhancement. Finally,
it is specied that the term “mask” refers to a set of pixels that have been
grouped together as a result of various operations performed on the
three two-dimensional views. Each mask is associated with a specic
color, a minimum HU value, and a maximum HU value.
4.6 Processing of the three-dimensional
model
Once the segmentation of the dierent structures has been carried
out, it is necessary to process the three-dimensional model by performing
a smoothing operation with the appropriate number of iterations. is
is done to achieve a virtual model suitable for both three-dimensional
visualization and the subsequent potential 3D printing phase. e three-
dimensional object calculated from the mask inevitably exhibits a step
eect due to the spatial resolution of the images, which is evidently
insucient for our purposes. e edges of the three-dimensional object,
reconstructed from the mask, tend to follow individual pixels that have
dimensions of approximately 1 mm × 1 mm. is scaling eect can
beobserved both in the three-dimensional view and on the individual
slices for each anatomical structure. e smoothing operation was
performed in the Mimics soware rather than the modelling soware
3-Matic by Materialise, used in the later phase of processing the three-
dimensional model and optimizing the mesh. is choice is because,
once the smoothing operation with a certain number of iterations is
completed in Mimics, it is possible to compare the result with CT images
on individual sections (axial, coronal, and sagittal) by visualizing the
contours of the obtained three-dimensional object. In general,
smoothing operations contribute signicantly to enhancing the surface
quality of a model. Nevertheless, it is crucial to exercise caution, as an
excessively high number of smoothing iterations can lead to alterations
in the model, resulting in unexpected outcomes. It is important to keep
in mind that real-life organs do not possess perfectly smooth surfaces.
e complete virtual model obtained in this way enabled the three-
dimensional visualization of various anatomical structures. In this
visualization, appropriate degrees of transparency were set for dierent
structures, allowing for the visualization of outer and inner structures
as well.
4.7 Processing of the three-dimensional
model
e calculation of the shape factor is a procedure used in various
contexts, such as physics, engineering, or thermodynamics. e shape
factor is a quantity that expresses the geometry of one body in relation
to another, oen concerning thermal radiation exchange or uid
dynamics. Its mathematical expression can vary depending on the
specic context.
In general terms, the shape factor (F) that we used can
becalculated using the following simplied formula:
FSh
V
=
∗
where S and V are, respectively, the surfaces and volumes of the
two bodies under consideration, the UTJ and AIJ, and h is the height
of the traits. However, in more complex situations, such as radiation
exchange between non-ideal surfaces, the formula can be more
intricate and involve angles, distances, and emissivity properties of
the surfaces.
Data availability statement
e original contributions presented in the study are included in
the article/Supplementary material, further inquiries can bedirected
to the corresponding author.
Ethics statement
Ethical approval was not required for the study involving animals
in accordance with the local legislation and institutional requirements
because wecollected samples at local slaughterhouse without any
contact with live animals, and without interacting with them.
Author contributions
RB-P: Data curation, Formal analysis, Writing – original dra. CC:
Data curation, Formal analysis, Writing – original dra, Writing –
review & editing. LV: Conceptualization, Data curation, Formal analysis,
Methodology, Visualization, Writing – original dra. TO: Data curation,
Formal analysis, Writing – review & editing. AD'E: Data curation,
Formal analysis, Methodology, Writing – review & editing. RM: Data
curation, Formal analysis, Writing – review & editing. CDC: Data
curation, Formal analysis, Writing – review & editing. AP: Data curation,
Formal analysis, Writing – review & editing. SM: Data curation, Formal
analysis, Writing – review & editing. FS: Data curation, Formal analysis,
Writing – review & editing. MR: Data curation, Formal analysis, Writing –
review & editing. NB: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Supervision, Validation, Writing – original dra,
Writing – review & editing. BB: Data curation, Formal analysis, Funding
acquisition, Supervision, Writing – review & editing.
Funding
e author(s) declare that nancial support was received for the
research, authorship, and/or publication of this article. is research
was funded by the European Union-Next Generation EU. Project
Code: 427 ECS00000041; Project CUP: C43C22000380007; Project
Title: Innovation, digitalization and 428 sustainability for the diused
economy in Central Italy-VITALITY.

Belda-Perez et al. 10.3389/fvets.2024.1456524
Frontiers in Veterinary Science 09 frontiersin.org
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated organizations,
or those of the publisher, the editors and the reviewers. Any
product that may beevaluated in this article, or claim that may
bemade by its manufacturer, is not guaranteed or endorsed by the
publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fvets.2024.1456524/
full#supplementary-material
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