Theriogenology 127 (2019) 120e129

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Theriogenology
journal homepage: www.theriojournal.com

Morphological changes in the porcine cervix: A comparison between

nulliparous and multiparous sows with regard to post-cervical
artificial insemination
F.A. García-Va
zquez a, b, *, P.J. Llamas-Lo

pez a, M.A. Jacome a, L. Sarrias-Gil c,
, **
O. Lo
pez Albors c

a
Department of Physiology, Faculty of Veterinary Science, University of Murcia, International Excellence Campus for Higher Education and Research,
Campus Mare Nostrum, Murcia, Spain
b
Institute for Biomedical Research of Murcia, 30100, Murcia, Spain
c
Department of Anatomy and Comparative Pathology, University of Murcia, International Excellence Campus for Higher Education and Research, Campus
Mare Nostrum, 30100, Murcia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In recent decades, new artificial insemination (AI) methods, such as post-cervical AI (PCAI), have been
Received 7 November 2018 developed in pig. PCAI involves crossing the cervix to deposit the sperm in the uterine body. Although
Received in revised form PCAI application in sows is frequent, its application in nulliparous (gilts) females it is still limited due to
23 December 2018
the difficulty of passing through the cranial part of the cervical lumen. We hypothesized that ageing and
Accepted 9 January 2019
parity would modify the cervical canal, facilitating the introduction of AI devices through the cervix. The
Available online 10 January 2019
aim was to compare the morphology of the uterus at different levels between multiparous and nullip-
arous females. Morphological analysis of the uterus pointed to a longer cervix (25.9 ± 4.6 vs.
Keywords:
Assisted reproductive technology
21.6 ± 3.3 cm, p < 0.001) and greater length of the part of the reproductive tract involved in PCAI (from
Collagen rima vulvae to the last cervical cushion) (56.2 ± 6.0 vs. 50.3 ± 5.2 cm, p < 0.001) in multiparous sows
Endometrium compared with nulliparous animals. As regards the structure of the vaginal and uterine parts of the
Morphometry cervix (the part in contact with the vagina and uterine body, respectively), the cross-sectional area,
Myometrium perimeter and total thickness were greater in the uterine part of multiparous than of nulliparous animals
Uterus (area: 4.07 ± 1.46 vs. 2.46 ± 0.56 cm2, p < 0.01; perimeter: 8.50 ± 1.44 cm vs. 6.28 ± 0.92 cm, p < 0.001;
thickness: 10.79 ± 0.96 vs. 8.35 ± 0.62 mm, p < 0.05), but not in the vaginal part. The tissue content
analysed in histological cross-sections also showed differences between female groups, a greater content
of connective tissue (58.86 ± 10.78 vs. 67.60 ± 13.38%, p < 0.001) and a lower amount of muscle fibres
(39.79 ± 10.24 vs. 30.66 ± 13.69%, p < 0.001) being observed in multiparous sows. Finally, silicone casts of
the cervical lumen revealed differences between the two groups in the size and shape of the ridges in the
lumen trajectory. Parity, which is also influenced by ageing, determines important changes in the size,
structure and tissue content of the cervix wall, as well as in the morphology of the cervical canal, which
may be responsible for the different levels of performance of PCAI in the female populations. Therefore,
the future design of AI strategies and catheters should take into consideration the morphological vari-
ations of the cervix lumen, which will depend on age and parity of the females.
© 2019 Published by Elsevier Inc.

1. Introduction

* Corresponding author. Department of Physiology, Faculty of Veterinary, Uni-
versity of Murcia, Campus de Espinardo, Murcia, 30100, Spain.
** Corresponding author. Department of Anatomy and Comparative Pathology,
Faculty of Veterinary, University of Murcia, Campus de Espinardo, Murcia, 30100,
Spain.

zquez), albors@um.es (O. Lo

pez
E-mail addresses: fagarcia@um.es (F.A. García-Va
Albors).
A characteristic feature of the mammalian uterus is its inter-
specific variable morphology. Differences are visible to the naked
eye in the external morphology of its three main parts e neck
(cervix), body and horns -, as well as in the appearance of the
endometrium and uterine lumen [1,2]. Unlike the uterus body and
horns, the cervix plays no part in placentation, but is responsible for

https://doi.org/10.1016/j.theriogenology.2019.01.004
0093-691X/© 2019 Published by Elsevier Inc.

zquez et al. / Theriogenology 127 (2019) 120e129
F.A. García-Va
other, equally important functions in reproduction, such as
participating in the microbiological isolation of the uterine cavity,
and acting as a filter-barrier from the external environment [3]
and as a selective barrier for spermatozoa (reviewed by Ref. [4]).
During pregnancy it maintains the foetus in the uterus and then
opens sufficiently to allow the passage of the foetus at term
(reviewed by Ref. [5]). In sows, the cervix is divided into a cranial or
uterine part and a caudal or vaginal part [6], and its lumen (cervical
canal) is characterized by the presence of numerous cushion-
shaped prominences, which define a narrow “cork-screw” trajec-
tory within this organ [7]. Such a distinctive feature is essential for
facilitating attachment of the penis during the lengthy copulation
process but establishes a physical barrier for artificial insemination
(AI), which has conditioned the design of the insemination cathe-
ters used.
Indeed, pig AI procedures have reached high levels of efficiency,
such as 80e90% for farrowing rates, and producing between 11 and
14 total born pigs (reviewed by Ref. [8]). In addition to productive
and economic efficiency, a minimal semen dose is desirable to
optimize the ejaculate of boars of high biological and productive
value. In this respect, the number of spermatozoa required per
seminal dose decreases with the depth of semen deposition in the
uterus. Therefore, insemination catheters designed for conventional
deposition (cervical artificial insemination, CAI) have gradually
been modified to aid deposition of the spermatozoa in the uterus
body (post-cervical insemination, PCAI, or intrauterine insemina-
tion) [9,10], or even for deposition within the uterine horns (deep
intrauterine insemination) [11]. During the last decade, CAI has
been progressively replaced by PCAI worldwide, because it provides
similar or improved fertility and prolificacy rates, while substan-
tially reducing costs since the number of spermatozoa per dose is
decreased [10]. However, the success of PCAI varies significantly,
depending on the age and number of parturitions. In multiparous
sows (more than one farrowing) there is no restriction to the use of
PCAI and it can be applied in virtually 100% of cases. In primiparous
sows (only one farrowing), although its application is more limited,
the success rates achieved are close to 86% [12]. However, in
nulliparous gilts PCAI rates are only around 20% [13] mainly due to
the difficulties encountered while inserting the inner cannula of the
insemination device, especially through the deepest (cranial) part of
the cervix. Whether these difficulties are related to the morpho-
logical and structural changes of the cervix associated with parity or
ageing is a topic which deserves particular further attention [7].
The female genital tract of mammals undergoes important
physiological changes from birth to adulthood. In different species,
including porcine, morphological and functional changes in the
reproductive organs have been related to the onset of puberty
[14e18], pregnancy and ageing [19e25]. In the cervix, the
morphological, structural and compositional changes that take
place are relevant for understanding its mechanical properties, e.g.
elastic response, distension capacity, stiffness, etc., during preg-
nancy or through the effect of ageing [26e33]. However, due to
interspecific anatomical differences, the mono- or polytocous
condition, and the complexity of the different tissue components of
the cervix, the changes are neither unique nor conclusive. Thus, the
amount of collagen (the main protein in connective tissue, and
which is responsible for the mechanical strength of the extracel-
lular matrix) was found to decrease in pregnant women and mice
[27,32,34], whereas it gradually increased in rats [35]. On the other
hand, in both human and mouse, ageing has been related with
controversial effects such as increased collagen and greater
distension capacity of the cervix [27,32].
As regards the pig species, the morphological and compositional
changes of the cervix during a sow's reproductive life are still
poorly understood. Only a few studies have characterized the
121
physical properties, composition and structure of the cervix to-
wards the end of pregnancy [5,36e38] or in multiparous sows [7],
but there is a complete lack of information about the precise
changes that take place in the morphology of the cervical canal and
the structure of the tissue due to ageing or comparing nulliparous
and multiparous animals. Such knowledge would be important
because it would help understand the physiological reasons for the
restrictions in the use of PCAI in nulliparous sows. For that reason,
the present work was aimed at evaluating the morphology and
structure of the uterine cervix in two groups of sows - nulliparous
and multiparous - with different physiological backgrounds (parity
and age) and opposite success rates in PCAI procedures. More
precisely, this work focuses on evaluating the cervix at three levels:
i) the gross morphology of the part of the female genital tract of
interest for PCAI; ii) the morphology and structure of both of the
cervix (vaginal and uterine); and finally, iii) the inner morphology
of the cervical canal.
2. Material and methods
2.1. Ethics
All the procedures carried out in this work were approved by the
Ethical Committee of Animal Experimentation of the University of
Murcia and by the Animal Production Service of the Agriculture
Department of the Region of Murcia (Spain) (Ref. Nº. A13160609).
2.2. Animals
Reproductive tracts from hybrid females, a product of the
crossbreeding of the species Landrace x Large White and raised in
the same productive and treatment conditions, were obtained from
a local abattoir. Nulliparous females (cycling gilts) were of a ho-
mogenous age (~220 days; ~0.6 years of age) and weight
(165.76 ± 8.5 kg). Multiparous females had the reproductive fea-
tures of animals that had experienced at least 5 farrowings
(7.26 ± 1.22 on average), were ~2.5e4.0 years of age and weighed
234.05 ± 11.71 kg. Multiparous sows were intended for slaughter
after a minimum period of 28 days since the last parturition.
2.3. Study design
A total of 79 reproductive tracts were used in the present study,
41 corresponding to nulliparous females and 38 to multiparous.
According to the goals of the study the reproductive tracts were
used to analyse (Fig. 1): i) the gross morphology of the part of the
female genital tract involved in PCAI e the length of the vaginal
vestibule plus vagina (pre-uterine reproductive tract), cervix and
body of uterus; ii) the morphology and structure of the vaginal and
uterine parts of the cervix: perimeter and cross-sectional area,
endometrium and myometrium thickness, and tissue components
(connective tissue, muscle fibres and capillaries), and iii) the inner
morphology of the cervical canal.
2.4. Gross morphology of the part of the genital tract involved in
PCAI
Once in the dissection room, the reproductive tracts were
cleaned, and the rectum, bladder and fat tissue were removed. The
length (cm) of the pre-uterine reproductive tract (from rima vulvae
to the first cervical cushion, the pulvini cervicalis), the cervix (from
the first to the last cervical cushion) and the body of the uterus
(from the last cervical cushion to the beginning of the horns)
(Fig. 2a) were measured with a tape measure (scale in millimetres-
mm).
122
F.A. García-Va
Fig. 1. Flow chart of the study design.
2.5. Morphology and structure of the vaginal and uterine parts of
the cervix
The cervix was isolated and divided into three equal parts with
the help of a scalpel. The separated parts were classified as vaginal
section (caudal third, section of the cervix in contact with vagina);
middle section (middle third, intermediate part of the cervix) and
uterine section (cranial third, related with the uterine body)
(Fig. 3a). The vaginal and uterine sections of each cervix were
covered with talcum powder, immersed in liquid nitrogen for 1 min
and then stored at
20  C until further processing. The middle
section was discarded because the middle third of the cervix is a
transition part with a trunk-cone shape, which makes any stan-
dardization of the measurements inconsistent.
From the middle point of the frozen parts (vaginal and uterine),
a whole cross-section of approximately 0.5 cm thickness was ob-
tained with a butcher band saw. Cross sections were immediately
photographed with a digital camera (Nikon D3200/D40, Tokyo,
Fig. 2. Gross morphology of the female genital tract of interest to PCAI. (a) Schematic representation of the parts measured in nulliparous (n ¼ 41) and multiparous (n ¼ 38) females.
The letter A in the image represents the vaginal section of the cervix, B the middle section and C the uterine section of the cervix. (b) Length (cm) of the pre-uterine reproductive
tract (V), cervix (Cx), V þ Cx, and uterus body (UB). Data of mean ± SD. *** indicates p < 0.001.

zquez et al. / Theriogenology 127 (2019) 120e129
Japan), and fixed in 10% formalin. Due to a mistake during the
processing 8 samples from nulliparous group were processed for
histological analysis before being photographed. The digital images
allowed the perimeter and cross-sectional area of the cervix to be
accurately measured with the Sigma Scan Pro 5 image analysis
software (scale in micrometres-mm). The data were then exported
to an Excel spreadsheet.
The formalin fixed cross-sections were trimmed into four
to five quadrants, which were further processed for routine
paraffin embedding, microtome sectioning (5 mm thickness) and
Masson trichrome staining (Bio-Optica Milano spa, Milano, Italy)
(Fig. 4a). Endometrium and myometrium thickness were evaluated
separately in the stained histological cross sections. Three mea-
surements per quadrant were taken with a digital camera
(Leica MC170 HD/Leica Microsystems, Wetzlar and Mannheim,
Germany) coupled to a binocular magnifier (Wild Heerbrugg/Leica
transmitted-light stand EB, Switzerland) and the Leica Application
Suite 4.7 Software (Leica Microsystems, Switzerland 2003e20015).
The Masson trichrome-stained histological sections were also
photographed with a light microscope (Zeiss Axioskop 40,
Go€ttingen, Germany), coupled to a digital camera (Zeiss Axiocam
105 color, Go €ttingen, Germany), and digital software (Zen 2012
€ttingen, Germany). Four pictures of both the endometrium
Zeiss, Go
and myometrium were randomly taken from each histological
section, and then analysed by means of digital morphometry soft-
ware (SigmaScan Pro 5, Systat Software Inc, California, USA). The
percentage of stained area corresponding to connective tissue
(blue) and muscle fibres (red) was estimated after conversion to a
grey scale (Fig. 5a) and segmentation according to the following
threshold levels: hue for connective tissue, 130e185; hue for
muscle fibres, 220e254. Also, the area of the capillaries was
quantified by drawing directly on the digital images.
2.6. Inner morphology of the cervical canal
Reproductive tracts were dissected, and the vagina, cervix and
uterine body were isolated. Then, an insemination catheter
(Import-Vet S.A., Barcelona, Spain) was placed at the entrance to
the vaginal cervix. A mixture of pure silicone (ERTV Base; Dow
Corning Corporation, Midland, Michigan, USA) and 10% catalyst
(ERTV Curing Agent) was prepared and transferred to a syringe for
injection through the catheter. The silicone was slowly injected into
the cervical canal while gently massaging the sample to help the
polymer progress up to the uterus body. The injected samples were
then placed in a tray, covered with wet foil and left at room

zquez et al. / Theriogenology 127 (2019) 120e129
F.A. García-Va 123

Fig. 3. Sampling strategy and overall dimensions of the cervix at its cranial and caudal parts. (a) Representation of the strategy followed to sample the vaginal and uterine parts of
the cervix. (b) Perimeter and (c) cross-sectional area in multiparous (n ¼ 11) and nulliparous (n ¼ 10) females. Mean ± SD. ** indicates p < 0.01; *** indicates p < 0.001.

Fig. 4. Evaluation of the total thickness of the cervical wall in histological sections stained for Masson trichrome in nulliparous (n ¼ 18) and multiparous (n ¼ 11) females. (a) Picture
showing how the thickness of the endometrium and myometrium was evaluated. (b) and (c), Myometrium and endometrium thickness (and the sum of both represented as Total)
at the vaginal part (b) and uterine part (c) of the cervix. Mean ± SEM. * indicates p < 0.05.
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F.A. García-Va

Fig. 5. Tissue content in the cervix of nulliparous and multiparous sows. (a) Analysis of tissue content by SigmaScan Pro 5 software: i) representative histological image of Masson's
trichrome staining (vaginal section of the cervix of a multiparous sow); ii) green and yellow represent connective tissue and muscle fibres respectively, as detected by the software;
iii) capillaries are labelled in dark blue (indicated by white arrows head in the image). Bar scale ¼ 500 mm. (b) Global content (%) of connective tissue, muscle fibres and capillaries in
cervical tissues of nulliparous and multiparous females. (c) Distribution of tissue content in the cervix by sections (vaginal and uterine sections of the cervix), and (d) by sections and
cervical layers (endometrium-upper graphs and myometrium-lower graphs). Nulliparous animals are represented by dark grey bars and multiparous by light grey bars. A total of 28
animals, 17 corresponding to nulliparous and 11 to multiparous females, were used. Four images of each sample were analysed. Data are expressed as mean ± SD. *, **, *** indicate
p < 0.05, p < 0.01 and p < 0.001, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

temperature for ~24e36 h to allow complete hardening of the
mixture. Casts were obtained by trimming the cervix wall with
scissors and the volume (vol) was estimated according to Archi-
medes' principle by immersing each cast in a 1 L graduated test
tube filled with 900 ml of doubly distilled water.
Casts were then photographed with a digital camera (Nikon
D3200/D40, Tokyo, Japan) to estimate the distance between
consecutive ridges (Dis), the depth of each ridge (Dp), the thickness
(Th), cross-sectional area (A) and perimeter (P) of the casts, and the
total number of ridges with the SigmaScan Pro 5 software (Systat
Software Inc, California, USA) program (Fig. 6a).
To make a digital reconstruction of the cervical lumen, the casts
were scanned with a NextEngine Desktop 3D Scanner (Model
2020i) and the images were managed with Scan Studio V2.0.2
software. The scanner has an accuracy of 0.13 mm resolution
(Macro mode), providing a maximum point density of 400 dots per
inch. These images acted as negative mould for digital cervix
reconstruction (Fig. 7).
2.7. Statistical analysis
The IBM SPSS 21 Statistics package (SPSS, Chicago, IL, USA) was
used. Data from experimental variables were compared for statis-
tically significant differences between the nulliparous and multip-
arous groups. The assumption of normality and homogeneity of
variances were checked by Shapiro-Wilk and Levene tests, respec-
tively. After both tests, a Student's t-test was applied (gross
morphology; area, perimeter and thickness of the vaginal part;
perimeter of the uterine part; tissue content; inner morphology and
cast volume). When either of the tests was not fulfilled the non-
parametric Mann-Whitney test was used (area and thickness in
uterine part). Differences were considered statistically significant at
p < 0.05. Thickness (endometrium, myometrium and total thick-
ness) and luminal casts (Dis, Dp, Th, A, P and total number of ridges)
data were expressed as the mean ± SEM. The gross morphology of
uterus, cross-sectional area, perimeter of cervix, tissue content
(connective tissue, muscle fibres, and capillaries) and cast volume
were expressed as the mean ± SD.
3. Results
3.1. Gross morphology of the female genital tract interested in PCAI
The measurements made of the reproductive tracts [pre-uterine
reproductive tract (V), cervix (Cx) and uterine body (UB)] of
nulliparous and multiparous sows are depicted in Fig. 2b. The re-
sults showed that Cx was significantly longer in multiparous than
in nulliparous females (25.93 ± 4.64 vs. 21.65 ± 3.39 cm, p < 0.001).
The other morphological parameters, V (28.92 ± 4.59 vs.
30.43 ± 3.32 cm) and UB (4.71 ± 2.14 vs. 5.07 ± 1.56 cm), were not

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F.A. García-Va 125

Fig. 6. Cast of the cervical lumen in nulliparous and multiparous females. (a) Image showing how the depth (Dp, cm), thickness (Th, cm), distance (Dis, cm), perimeter (P, cm) and
area (A, cm2) of the ridges, as well as the number of ridges per 10 cm (density) were measured. (b) Comparison of the cast of a nulliparous (upper panel) vs. multiparous sow (lower
panel). (c) Results observed in the different sections (vaginal and uterine parts of the cervix) of the luminal casts. (d) Comparison of cast volume (mL). Nulliparous animals are
represented by dark grey bars and multiparous by light grey bars. A total of 33 uteri were used, in which cervical insemination using silicone was simulated in 19 nulliparous and 14
multiparous sows. Data are expressed as mean ± SEM, except cast volume that is expressed as mean ± SD. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

significantly different. The average total length of the reproductive

tract which must be passed by the cannula in PCAI (obtained by
adding V and Cx) was longer in multiparous (56.23 ± 6.01 cm) than
in nulliparous females (50.39 ± 5.25 cm) (p < 0.001).

3.2. Morphology and structure of the vaginal and uterine parts of

the cervix

Fig. 3 (b, c) shows the metrical analysis (perimeter and area) of

Fig. 7. Digital 3D models of the cervix (mid longitudinal section) of nulliparous (upper
panel) and multiparous (lower panel) females obtained from the scanning (NextEngine
Desktop 3D Scanner, Model 2020i) of the luminal casts.
the vaginal and uterine parts of the cervix. The perimeter and cross-
sectional area of the cervix was always significantly higher in the
vaginal than in the uterine part, both in nulliparous (perimeter:
10.36 ± 0.95 vs. 6.28 ± 0.92 cm, p < 0.001; area: 6.36 ± 1.11 vs.
2.46 ± 0.56 cm2, p < 0.001) and multiparous groups (perimeter:
10.44 ± 1.98 vs. 8.50 ± 1.44 cm, p ¼ 0.016; area: 6.44 ± 1.22 vs.
4.07 ± 1.46 cm2, p ¼ 0.001). The comparison between nulliparous
and multiparous sows only yielded significant differences for these
parameters in the uterine part of the cervix, in favour of the
multiparous sows (perimeter: 8.50 ± 1.44 vs. 6.28 ± 0.92 cm,
p < 0.001; area: 4.07 ± 1.46 vs. 2.46 ± 0.56 cm2, p < 0.01).
Masson trichrome staining of the cervix showed two well
differentiated layers in the cervix cross-sections (Fig. 4a), an
external layer of myometrium mainly occupied by smooth muscle
fibres and an inner layer (endometrium) with abundant connective
tissue. In the myometrium, an outer and rather oblique layer of
smooth muscle fibres was separated from the inner circular layer by
a transitional area with abundant connective tissue and vessels.
Data of the thickness of the endometrium and myometrium in the
vaginal and uterine parts of the cervix are displayed in Fig. 4 (b, c).
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F.A. García-Va
In the vaginal part there were no differences between the groups of
sows in the endometrium or the myometrium, whereas in the
uterine part the myometrium was thicker in multiparous than in
nulliparous tracts. A comparison between vaginal and uterine
parts revealed the significantly higher thickness of the cervical
wall in nulliparous (vaginal part: 11.50 ± 0.81 vs. uterine part:
8.35 ± 0.62 mm, p ¼ 0.002) but not in multiparous females (vaginal
part: 12.37 ± 0.81 vs. uterine part: 10.79 ± 0.96 mm, p > 0.05).
Regarding the tissue components of the cervix, multiparous
sows had more connective tissue (58.86 ± 10.78 vs. 67.60 ± 13.38%,
p < 0.001) and less muscle fibres (39.79 ± 10.24 vs. 30.66 ± 13.69%,
p < 0.001) than nulliparous animals (Fig. 5b), with no statistical
differences between the capillaries (1.34 ± 1.00 vs. 1.73 ± 1.00%, for
nulliparous and multiparous respectively). The tissue content was
further evaluated in terms of the cervix part (vaginal vs. uterine)
and tissue layers (endometrium vs. myometrium). With regards to
the parts of the cervix (Fig. 5c), the vaginal part showed more
abundant connective tissue and fewer muscle fibres than the
uterine part (p < 0.05), whereas multiparous sows showed more
abundant connective tissue and fewer muscle fibres than nullipa-
rous females in both parts (p < 0.01 for the vaginal part, p < 0.001
for the uterine part). Further information was obtained by evalu-
ating the tissue components separately in the endometrium and
myometrium (Fig. 5d). In multiparous sows the connective tissue
was most abundant in the myometrium of the vaginal part
(p < 0.001) and in the endometrium and myometrium of the uter-
ine part (p < 0.01 and p < 0.001, respectively). Conversely, a lower
content of muscle fibres in these sows was observed in the myo-
metrium of the vaginal part (p < 0.05) and the endometrium of the
uterine part (p < 0.01). No changes in the capillary content were
observed among any of the compared parts or tissue layers
(p > 0.05).
3.3. Inner morphology of the cervical canal
The casts depicted the inner morphology of the cervical canal in
a very realistic and comprehensive way (Fig. 6b). Surprisingly,
instead of the expected curled or “cork-screw” trajectory, the cer-
vical canal always described a “wavy” shape in the vertical plane.
Only at the two extremes of the cervical canal was a sort of curled
shape observed. The caudal and cranial parts, separated by a short
transitional area, are clearly differentiated in the casts. The wavy
pattern was morphologically different in the two groups of females,
particularly in the cranial part of the cervix (Fig. 6b). The statistical
analysis between the vaginal and uterine parts is represented in
Fig. 6c. While no morphological differences between nulliparous
and multiparous sows were found in the vaginal part, the thickness
(Th) of the cast (0.40 ± 0.01 vs. 0.56 ± 0.05 cm, p < 0.01), the dis-
tance (Dis) between two consecutive ridges (1.26 ± 0.04 vs.
1.72 ± 0.13 cm, p < 0.001), and area (A) of the ridges (0.55 ± 0.04 vs.
0.72 ± 0.07 cm2, p < 0.05) were higher in the uterine part of
multiparous sows. Conversely, the number of cervical ridges per
10 cm (density of ridges) was significantly higher in the uterine part
of nulliparous females (11.41 ± 2.72 vs. 8.51 ± 2.02, p < 0.01). The
estimated volume of the cervical canal (Fig. 6d) was greater in
multiparous than in nulliparous sows (p < 0.01).
4. Discussion
AI in porcine species is considered one of the most successful
animal reproductive techniques in the world. Nowadays, up to
90e95% of farms in developed countries use this technology [39].
Although AI has been successfully used for decades, many studies
have tried to improve its efficiency. One result of this has been the
development of PCAI, which allows the number of spermatozoa
used per insemination to be reduced, while maintaining repro-
duction performance. However, and in contrast to traditional cer-
vical AI, the use of PCAI in nulliparous females is restricted due to
the difficulty involved in passing through the cervical canal. By
comparing the macro- and microscopic morphology of the cervix in
nulliparous and multiparous females, the present work contributes
to our understanding of such limitations. Also, the information
should help in the design of insemination catheters that match the
unique morphology of the cervical canal of nulliparous females.
Several previous works have described the morphological fea-
tures of the female genital tract in pre-pubertal gilts [40e42],
cycling gilts [41] and sows [7]. However, to the best of our knowl-
edge, a comparison of the morphology of the cervix between
nulliparous and multiparous females has not been made. Two main
processes differentiate the biological condition of the cervix in
these two groups of sows - ageing and parity. Both processes, which
are simultaneous in multiparous sows, determine changes in the
size and tissues of the cervix [27,32]. The slow and gradual growth
of the reproductive organs after birth is intensified at puberty [17],
and is then strongly influenced by parity. In our study, multiparous
sows showed longer and thicker cervices than nulliparous females.
The greatest thickness was mainly in the uterine part of the cervix,
particularly in the myometrium. In agreement with our results,
some authors have observed an increase in uterine size associated
to parity in women [24,43], although this is not always the case
[44,45]. With regard to ageing, there is also some controversy be-
tween our results and those relating to humans. In women, age-
correlated differences were not observed in uterus size [24,46],
but have been observed in rodent and livestock species such as
ewes [21,47]. It seems that the anatomy and pregnancy character-
istics of each particular species strongly determine the effect of
ageing and parity on the morphology of the cervix. Thus, although
human and porcine uterus show some similarities [48], their sub-
stantial differences in size and morphology make such a compari-
son of limited value. For instance, in women the length of the cervix
is around 3e4 cm long whereas in the porcine species is up to
20e30 cm.
However, our research was restricted to the study of only one
cross-breed (Landrace x Large White), although it is probably the
most common in commercial practice. It is plausible that the size of
the uterus/cervix could be different in other breeds of interest for
porcine industry, such as the Meishan (one of the most prolific
porcine breeds) or the Iberian breed (considered as a very low-
prolific breed) [49]. In fact, other authors [40] have observed
some differences in the reproductive system when two different
porcine breeds (Polish Large White vs. Polish Landrance) were
compared. Further studies to evaluate the breed effect would in-
crease the knowledge in this field.
The differences observed in the gross morphology of the cervix
between nulliparous and multiparous females may be attributable
to changes in the structure (tissues) of the cervix [27,50,51]. The
results of the present study showed differences mainly in the
content of connective tissue and muscle fibres. The most noticeable
difference in tissue composition was found in the uterine part
where, in both the myometrium and endometrium, the content of
connective tissue was higher in multiparous than in nulliparous
animals. Connective tissue is mostly composed of collagen, which is
the most abundant protein in the cervix and responsible for its
tensile properties [52]. In women, the cervix collagen content was
seen to increase by 0.5% per year of age, but to decreased by 1.7%
per birth [27]. The connective tissue also determines the flexibility
of the cervix, which could influence the morphology of the cervical
cushions in sows [7]. Moreover, parity was associated in women
with a weaker collagen network [34,53], which may contribute to
an increase in cervix distension. Taken together, such previous

zquez et al. / Theriogenology 127 (2019) 120e129
F.A. García-Va
findings enhance the importance of the connective tissue of the
cervix with regard to its mechanical properties and suggest that the
higher content of connective tissue observed in the uterine part of
the cervix in multiparous sows determines the higher distension
capacity of the cervix than in nulliparous females. This would partly
explain the easier progress of the PCAI cannula through the cervix
in multiparous sows. However, to further progress in this line of
reasoning and better understand the ageing and parity effects on
the morphology and tissue composition of the cervix, a group of
nulliparous females with a similar age to that of the multiparous
animals used in this study is necessary. This has been evaluated in
mice where an effect of ageing on cervix distension was demon-
strated in both non-pregnant and pregnant animals, so that, inde-
pendently of parity, the cervix of older mice showed a greater
distension capacity [32].
The porcine cervix has a thick muscular wall [54] and the
amount of smooth muscle fibres is an important feature [5].
Changes in the structural organization of these smooth muscle fi-
bres related with pregnancy have been described [5], so, multiple
parturitions could involve variations in the content and contract-
ibility of the cervix wall. In fact, the rate of stillbirths is higher in
older sows, which may be related to changes in the tone of the
reproductive tract during parturition [55e59]. Also, impaired
sperm transport has been found to increase as the number of par-
turitions increases [10,60]. Moreover, as described in other species,
ageing also affects the contractibility of the myometrium [20,22]. In
the present work, multiparous sows showed a reduced amount of
muscle tissue in the uterine part of the endometrium and in the
vaginal part of the myometrium. This reduction in the amount of
muscular tissue was accompanied by an increase in connective
tissue, and both situations are likely to contribute to a loss of
stiffness in the cervix of multiparous sows. In the uterine part of the
cervix of multiparous females connective tissue increases as the
muscular component decreases, while the opposite is true for
nulliparous animals, which may be related with the limitations
experienced with PCAI in the nulliparous females. To further
illustrate the relationship between the variable amount of con-
nective and muscular tissues in the organs, changes in the pro-
portions of these components were also observed in the urethra in
rats [61] and the vaginal wall of ewes [25]. In both cases, the in-
crease in connective tissue may be considered as a compensatory
mechanism for the gradual loss of muscle tissue.
As previously mentioned, the morphological and structural
changes observed in the cervix of multiparous sows, particularly
in the uterine part, may contribute to explaining the success of
PCAI in these pigs. Conversely, the lower size and proportion of
connective tissue and higher amount of muscle fibres in this part
of the cervix could partly explain the difficulties experienced with
cannula insertion during PCAI of nulliparous females. Further
morphological evidence for this reasoning is presented by the
silicone casts of the cervical lumen. In all the casts the density of
the ridges (specular image of the cervical cushions), as well as
their thickness and area, were higher in the most cranial part of
the cervix (uterine part), and in nulliparous females. Likewise, in
ewes, the number of cervical folds was seen to decrease with
ageing [47] and parturitions [62], which was related with
morphometric changes and probably the rupture or modifications
in shape of the cervical ridges. Moreover, as in ewes, the shorter
distance between the cervical ridges not only reduces the lumen
but makes it more difficult to locate the orifice of the cervical
canal when using the insemination device [47]. Therefore, the
higher number of cervical cushions, their greater height and the
consequent reduction in the diameter of the cervical canal in the
uterine part of the cervix of nulliparous females (Figs. 6 and 7)
make cervical penetration by the PCAI catheter extremely
127
complicated compared with multiparous females, as previously
demonstrated [13].
So, the deep deposition of spermatozoa in nulliparous females
must take into account the morphology and shape of the repro-
ductive tract when new AI devices are being designed, with
particular attention paid to the length and diameter of the inner
cannula. Moreover, the AI devices could be combined with previous
successfully tested strategies [13] for a greater success.
5. Conclusions
Ageing and parity determine changes in the morphology of the
porcine cervix, which is longer and wider in multiparous sows than
in nulliparous females. Also, changes in tissue composition prob-
ably determine differences in the tensile properties of the cervix,
especially its uterine part. Moreover, in this part of the cervix the
lumen gradually becomes wider and less wavy. Together, these
features are of special relevance from a practical point of view for
the porcine industry. The findings of the present study suggest that
future efforts concerning the design of AI catheters for deep sperm
deposition should be based on the morphological characteristics of
the cervix and consider the parity condition of the sows.
Author contributions
FAGV and OLA acted as the advisors of the project, conceived,
designed and performed the experiments, analysed the data and
wrote the manuscript. PJLL contributed to the design of the study,
performed the experiments and wrote the draft of the paper. MAJ
and LSG performed the experiments. All the authors have revised
the final version of the manuscript.
Declaration of interest
None of the authors has any conflicts of interest to declare.
Acknowledgments
The authors would like to thank the Department of Research and
Development of CEFUSA (especially to Mateo Martínez-Espí), El

nchez) for providing
Pozo S.A. and Agropor S.L. (especially Paula Sa
~ arro
the biological material. Thanks, too, to Juan Francisco Min

nez from the Mechanical Unit (University of Murcia) for 3D
Jime
cervical images.
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