Reprod Dom Anim 45, 1059–1064 (2010); doi: 10.1111/j.1439-0531.2009.01493.
x
ISSN 0936-6768
Site of Intrauterine Artificial Insemination in the Bitch does not Affect Sperm
Distribution within the Uterus
FB Fukushima, C Malm, M Henry, VA Gheller, R Serakides, MM Neves, SP Macedo, MS Figueiredo, MEJ Andrade,
MS Chaves, MX Silva, CMF Rezende and EG Melo
Department of Veterinary Clinics and Surgery, Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Contents
The aim of this study was to evaluate the distribution of
frozen–thawed spermatozoa within the uterine lumen and
oviducts following intrauterine laparoscopic deposition at two
sites. Twelve bitches of unknown reproductive history were
randomly distributed into two groups. Semen (3 ml containing
300 · 106 frozen–thawed spermatozoa) was infused at the
uterine body (UB group) or at the cranial tip of the left uterine
horn. A 22-G catheter was used to access the uterine lumen.
Sperm cell distribution was evaluated after ovariohysterecto-
my performed 3 h after artificial insemination (AI). There was
no difference between groups in mean time to perform AI.
Spermatozoa were detected in all uterine segments, including
the tip of both horns, but none was detected in the oviduct.
The 22-G catheter facilitated deposition of semen in the uterine
lumen, particularly at the UB site. Sperm cell distribution
occurred evenly along both horns, independent of the site of
semen deposition.
Introduction
Artificial insemination (AI) of the bitch with frozen–
thawed semen requires intrauterine insemination,
because of reduced post-thaw viability. However, the
difficulty in passing through the cervix is a constraint to
obtain acceptable fertility and prolificacy. To overcome
this difficulty, several techniques have been developed,
including the use of laparoscopy (Andersen 1975; Wildt
1986; Fontbonne and Badinand 1993; Wilson 1993,
2003; Silva and Verstegen 1995; Silva et al. 1995, 1996;
Linde-Forsberg et al. 1999; Linde-Forsberg 2001; Tsut-
sui et al. 2003; Valocký et al. 2003). Laparoscopy is a
minimally invasive surgical approach with minor tissue
trauma, good visualization of intra-abdominal struc-
tures, magnification of images and fewer complications
than laparotomy (Frazee et al. 1991). Hence, this
technique remains as an option for AI of frozen–thawed
semen (Silva and Verstegen 1995; Silva et al. 1995, 1996)
and, particularly, when cranial intrauterine deposition
of semen is intended.
When semen is deposited into the uterus, spermatozoa
are transported both passively and actively whereas only
those with progressive motility will be able to traverse
the uterotubal junction (Scott 2000). Spermatozoa are
kept in sperm reservoirs to maintain their viability until
fertilization (England and Pacey 1998). Nevertheless, the
evaluation of sperm distribution in the uterus of the
bitch was only performed with fresh semen following
either natural mating (Doak et al. 1967; England and
Pacey 1998; England and Burgess 2003) or intravaginal
insemination (Rijsselaere et al. 2004). The evaluation
methods used in those studies to identify sperm distribution
 2009 Blackwell Verlag GmbH
were uterine and oviductal flushing, mucosa imprint or
histology.
It is well established that freezing and thawing sperm
decrease their viability, impairing the process of fertil-
ization. Therefore, intraluminal deposition of sperma-
tozoa closer to the apex of the horn can theoretically
increase the quantity of spermatozoa at the uterotubal
junction, enhancing the possibility of conception
(Tsutsui et al. 1989; Wilson 1993), particularly with
frozen semen. Hence, the primary aim of this study was
to evaluate uterine and oviductal distribution of frozen–
thawed semen following laparoscopic intraluminal
deposition into the uterine body (UB) or horn.
Material and Methods
Animals
Twelve healthy adult mixed-breed bitches weighing from
6 to 17 kg and with unknown reproductive history were
randomly distributed in two groups of six animals.
Bitches belonged to the town Zoonosis Control Division
and were added in the adoption programme, which
included a free ovariohysterectomy, as part of a popu-
lation control programme1.
Timing of insemination
The time of the insemination was determined by the use
of vaginal smears and progesterone assays. Ovulation
was confirmed by the presence of corpora lutea in the
ovaries. Vaginal smears were collected daily during pro-
oestrus and oestrus and progesterone assays were
performed every 2–3 days when serum progesterone
concentration was <2 ng ⁄ ml and every day until the
time of the insemination when concentrations were
>2 ng ⁄ ml.
Vaginal smears were collected with a wet cotton swab;
the secretion content of the swab was gently rolled over a
glass slide, dehydrated in absolute alcohol and stained
using Harris-Schorr technique (Luna 1968). Serum
progesterone concentrations were measured using chemi-
luminescence immunoassay (Chapwanya et al. 2008). A
single AI was performed when serum progesterone
concentrations were between 4 and 8 ng ⁄ ml, there was
‡80% superficial cells in the vaginal smear and bitches
displayed sexual receptivity.
1
This research was conducted in accordance with Federal
legislation and was approved by the Animal Ethics Committee
of Minas Gerais Federal University.
1060 FB Fukushima, C Malm, M Henry, VA Gheller, R Serakides, MM Neves, SP Macedo, MS Figueiredo, MEJ Andrade, MS
Chaves, MX Silva, CMF Rezende and EG Melo
Laparoscopic procedure
Bitches were fasted for 8 h before laparoscopy. Acepro-
mazine (0.05 mg ⁄ kg i.m.) and pethidine hydrochloride
(3 mg ⁄ kg i.m.) were used as a pre-medication. Anaes-
thesia was induced with 1% propofol (5 mg ⁄ kg i.v.) and
was maintained with isoflurane in a semi-closed circuit.
After induction of pneumoperitonium (8–10 mmHg),
using a veress needle, skin incisions were performed as
shown in Fig. 1. Two trocars (P1, 11 mm; P2, 6 mm)
and an endoscope (KARL STORZ, 43 cm, 30 and 10
mm diameter; Tuttligen, Germany). were used. The
omentum and uterus were repositioned using a claw
grasping forceps (KARL STORZ, 31431FMS model)
(Fig. 2a).
The UB and the left uterine horn (UH) tip, clamped
approximately 2 cm caudal to the uterine–tubal junc-
tion, were brought as close as possible to the abdominal
wall at the left paramedian retro-umbilical region. The
left UH was chosen randomly. Assessment of the lumen
was performed using a 22-G catheter (Insyte; Becton,
Dickinson and Company, São Paulo, Brazil). Sites of
uterine assessment are shown (Fig. 2b,d). Once within
the lumen, the mandrel was withdrawn and the catheter
was carefully advanced cranially closer to the UB
bifurcation or to the uterine–tubal junction (UH group).
When necessary, the spleen was first dislocated to the
right and then cranially (Fig. 2c). The seminal volume
was consistently as 3 ml. Any complications and
alterations observed during laparoscopic procedure as
Fig. 1. Positioning of the surgical team, laparoscopic equipment and
anaesthesiologist in relation to the animal in dorsal recumbence.
V – site of Veress needle introduction; P1 and P2 – trocar
positions; P1 – portal 1 for the endoscope; P2 – portal 2 for the
grasping forceps; C1 – introduction of the 22-G catheter for AI in to
the uterine body (GI); C2 – introduction of the 22-G catheter for AI in
the cranial edge of the left uterine horn (GII). Figure adapted from
Leonard (1968)
well as the number of follicles and corpora lutea were
recorded.
Following the laparoscopic AI procedure, the abdo-
men was closed, the bitches were allowed to recover
from anaesthesia and were maintained in a warm
kennel (30–32C) until ovariohysterectomy. To evalu-
ate the occurrence of sperm reflux through the cervix,
10 min after semen deposition, a vaginal swab was
collected and processed as described for pre-breeding
evaluation.
Semen preparation
A healthy mongrel dog was chosen as a semen donor,
based on the following seminal characteristics: >80%
and >50% progressive motility before and after freez-
ing ⁄ thawing, respectively and <25% abnormal sperm
morphology (CBRA 1998). The sperm-rich fraction was
frozen in egg-Tris extender with 5% glycerol (Rodrigues
1997). Total sperm concentration was adjusted to
100 · 106 sperm cells ⁄ ml for freezing. Each insemina-
tion was performed using a total sperm cell count of
300 · 106. Post-thaw sperm motility was always checked
before each insemination.
Evaluation of sperm distribution
Uterine sperm distribution was evaluated after ovario-
hysterectomy (Stone et al. 1993), which was performed
3 h after laparoscopic intrauterine deposition of fro-
zen–thawed semen. Bitches were pre-medicated and
anaesthetized as described above. To avoid random
sperm migration by manipulation, seven uterine liga-
tures (cotton suture A–0) were placed as shown in
(Fig. 3). Post-operative analgesia was provided by
ketoprofen (Ketofen 10%, Merial, Brazil; 1 mg ⁄ kg
once daily for 3 days) and tramadol (2 mg ⁄ kg twice
daily for 3 days).
After ovariohysterectomy, uteri were kept on a flat
surface and the lumen was exposed through a dorsal
incision along the longitudinal axis of each segment.
Three glass slide imprints of the endometrium were
prepared for each segment (UB, left and right UHs and
uterotubal junctions) and for both oviductal lumina
(Lovell and Getty 1968; Hunter et al. 1980). Imprints
were immediately dehydrated in absolute alcohol and
stained using the Harris-Schorr technique (Luna 1968).
Sperm enumeration was performed using a bright-field
microscope at ·400 magnification. Spermatozoa were
counted in the entire imprinted surface of the slides.
Total sperm count per segment was the sum of the
sperm cells found in each of the three imprints. All slides
were evaluated within a 7-day period.
Statistical analyses
Mean length of time for each laparoscopic seminal
deposition procedure was compared using a Student’s
t-test. Occurrence of seminal reflux and specific surgical
complications were analysed with Fisher’s exact test.
Mean sperm numbers per segment were compared using
Mann–Whitney U-test. Means were considered different
when p £ 0.05.
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Site of Intrauterine AI in Bitch 1061

(a) (b)

(c) (d)

Fig. 2. (a) Dislocation of the omentum (O) with grasping forceps and visualization of the urinary bladder (B) and uterine bifurcation (UB). (b)
Uterine body (U) and uterine bifurcation (UB) elevated near the abdominal wall, with the support of the claw grasping forceps and perforation
with the 22-G catheter (C). (c) Retracted left uterine horn, allowing visualization of the ovarian bursa (OB), uterotubal junction (UTJ), cranial
edge of the left uterine horn (LUH) and spleen, which was previously dislocated. (d) Cranial portion of the left uterine horn (LUH) elevated near
the abdominal wall, with the support of the claw grasping forceps and perforation of its wall with the 22-G catheter (C)

Results
Timing of insemination
There was no difference between groups for serum
progesterone concentration (overall mean ± SD
6.0 ± 1.52 ng ⁄ ml, range 3.95–8.55 ng ⁄ ml) and corpora
lutea were found in the ovaries of all bitches.

Fig. 3. Scheme of the bitch’s uterus, illustrating the ligatures per-
formed in situ before removal of the uterus. The ligatures were placed
cranially the cervix, on the uterus bifurcation, in the middle of the
uterine horns and 0.5 cm caudally the oviduct
Laparoscopic procedure
The average time necessary to complete laparoscopic
intrauterine AI from initiation of pneumoperitoneum to
semen deposition was 25.5 ± 7.53 and 26.67 ± 2.94
min for UB and UH groups respectively (p = 0.06). At
first visualization, the uterus was partially covered by
the omentum, but was easily identified in the pelvic
region, near the bladder. Moving the omentum cranially
with a grasping forceps allowed visualization of the UB,
uterine bifurcation and caudal portion of the horns. In
all bitches, the uterus was turgid, with prominent blood
vessels in the serosa as well as in the broad ligament, as
expected for this phase of the oestrous cycle (Feldman
and Nelson 1996).
The apposition of the uterine site of seminal deposi-
tion close to the abdominal wall was slightly more
laborious for the UH group, as the tip of the left horn

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1062 FB Fukushima, C Malm, M Henry, VA Gheller, R Serakides, MM Neves, SP Macedo, MS Figueiredo, MEJ Andrade, MS
Chaves, MX Silva, CMF Rezende and EG Melo

was usually located under the spleen and the intestinal Table 1. Number of sperm cells counted in endometrium imprints of
loops. Otherwise, relocation of uterine segments to- several uterine segments and in the oviducts of bitches after laparo-
scopic AI in the uterine body (UB) and in the cranial portion of the left
wards the abdominal wall was not difficult in any uterine horn (UH)
bitches. AI was more laborious in one bitch from the
UB and in two from the UH group, because of UB UH z (Mann–
splenomegaly. Segment Mean ± SD Mean ± SD Whitney)
Based on preliminary trials (data not shown), the
Body 6.2 ± 2.6a 1.5 ± 1.0b 0
22-G peripheral venous access catheter was used instead Right horn – segment 1 17.8 ± 10.0a 23.3 ± 13.8a 0.42
of the 18-G described by Silva et al. (1995). Its diameter Right horn – segment 2 19.3 ± 9.0a 34.7 ± 22.9a 0.17
seemed to be more appropriate to reach the lumen with Right UTJ 7.0 ± 4.7a 17.2 ± 16.7a 0.23
less trauma and its length facilitated advancing the Right oviduct 0a 0a 0.32
catheter within the uterine lumen. The catheter was Left horn – segment 1 18.0 ± 6.9a 24.3 ± 14.0a 0.38
Left horn – segment 2 22.5 ± 11.1a 30.7 ± 20.6a 0.63
introduced smoothly and without resistance and the Left UTJ 7.8 ± 3.1a 19.0 ± 12.9a 0.09
retraction of the mandrel tip before further intraluminal Left oviduct 0a 0a 0.32
introduction of the plastic tube may have reduced the
risk of dorsal uterine wall perforation. UTJ, uterotubal junction. Means in the same line followed by the same letter are
not significantly different.
Intraluminal introduction of the catheter was suc- Time interval between AI and hysterectomy = 3 h.
cessful at first attempt in nine of the 12 bitches; the other
three required a second attempt. Resistance to semen
introduction occurred only in the UH group. To avoid the UB segment. In this area, less sperm cells were
reflux through the puncture site, the grasping forceps detected in UB group than in UH. In both UHs, sperm
was then slightly loosened. However, there was no counting showed a reduction at the uterotubal junction.
resistance when semen was deposited in the UB.
Using laparoscopy, visible visceral lesions seldom
occurred and were usually small (<0.5 cm). In this Discussion
study, there was minor haemorrhage from one spleen in The hypothesis that cranial intrauterine deposition of
one case, which resolved spontaneously after 5 min. On frozen–thawed semen influences sperm counting along
two occasions, small blood vessels on the serosal surface the uterus in the bitch was tested in this study.
of the uterus were perforated while introducing the Laparoscopic AI in the bitch with fresh semen was first
catheter in the uterine wall and the claw grasping described by Silva et al. (1995) and was reported to
forceps damaged a vessel within the broad ligament. In result in pregnancy. However, influence of the site of
all cases, the haemorrhage was focal, discrete and ceased semen deposition and sperm distribution along the
spontaneously after a few minutes. uterus was studied only in other species and revealed in
Reflux of sperm cells through the cervical os occurred sows that deep intrauterine deposition of semen allowed
in all bitches. On vaginal cytology performed 10 min reduction in the number of spermatozoa inseminated
after AI, numerous spermatozoa were apparent and, on and dose volume without decreasing fertility (Martinez
one occasion, a small drop of semen was detected in the et al. 2002).
ventral vulvar commissure. Sperm cell distribution in the uterine lumen of bitches
is influenced by several factors including phase of the
oestrous cycle, method of seminal deposition (natural
Sperm preparation mating, intravaginal or intrauterine AI), type of semen
There was no difference between groups for post-thaw (fresh, chilled or frozen), sperm quality (total and
total motility (overall mean ± SD 57.08 ± 4.98, range progressive motility and sperm speed) and variations
50–70%), progressive motility (overall mean ± SD because of animal individualities (Rijsselaere et al. 2004;
50.83 ± 2.87, range 45–55%) and sperm speed (overall England et al. 2006). Aiming to reduce the factors
mean ± SD 3.08 ± 0.29, range 3–4). affecting the responses; AI time, seminal characteristics
and volume and animal size were standardized in this
study and were not different between groups, indicating
Evaluation of sperm distribution that the likelihood of those variables influencing the
In this study, using endometrial imprints, spermatozoa results was low. Additionally, the corpora lutea found in
were found in all segments of the uterine mucosa. the ovaries and the serum progesterone concentrations of
However, there was no difference (p = 0.41) between all bitches indicated that the AI was performed during
groups for total number of spermatozoa in the uterus the ovulatory phase, as it would occur in natural mating.
(overall mean ± SD were 113.5 ± 29.5 and 150.7 ± Several methods to evaluate sperm distribution in the
43.2 for AI in the UB and in the UH respectively; range uterine lumen were described in different species (ovi-
from 76 to 198). The number of spermatozoa per uterine ductal and uterine washing, mucosal cytology and
and oviduct segments 3 h after insemination is shown in histology) (Doak et al. 1967; Rijsselaere et al. 2004;
Table 1. Sperm cell number was distributed throughout England et al. 2006; Sumransap et al. 2007), however,
the total extension of the uterus either using the UB or most of them used fresh semen and an inseminating dose
the cranial portion of the UH as deposition sites. equivalent to one ejaculate. The technique of cytology
Sperm counts were similar between groups and sperm imprints of the uterine mucosa was successfully used in
distribution occurred similarly in most of the evaluated swine (Lovell and Getty 1968) and in sheep (Hunter
uterine segments (Table 1). An exception occurred for et al. 1980) to evaluate sperm transport.

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Site of Intrauterine AI in Bitch
Table 2. Imprinted area (cm2) of each segment of the genital tract of the bitches (mean ± SD)
Left horn 1 Left horn 2
Groups Body (cm2) (cm2) (cm2)
UB 3.56 ± 1.31 8.12 ± 4.06 7.07 ± 2.35
UH 3.58 ± 0.74 9.55 ± 5.47 9.81 ± 3.12
Groups: sperm deposition in the uterine body (UB) and cranial portion of the left uterine horn (UH).
Although the technique used detected spermatozoa in
all segments, it was inefficient in estimating real sperm
count per uterine unity, considering that total number of
sperm deposited into the uterine lumen was 300 · 106
and the total number counted was considerably less.
However, other causes of sperm number decrease need
to be considered. Reflux of sperm cells through the
cervical os occurred in all bitches, as demonstrated by
vaginal cytology 10 min after AI. Additionally, one of
the most important sperm reservoirs in bitches, the
crypts of the endometrial glands (Rijsselaere et al. 2004;
England et al. 2006), might have contributed to reduce
sperm numbers. Perhaps, the 3-h interval between sperm
deposition and ovariohysterectomy enabled mobile
spermatozoa to enter reservoir, making them inaccessi-
ble to the imprints. Spermatozoa were detected in
endometrial crypts 24 h after insemination with fresh
semen (Rijsselaere et al. 2004), but apparently no exam-
ination have been conducted at shorter intervals after
insemination. Physiological removal of intrauterine sper-
matozoa also could have reduced sperm number. In the
bitch, seminal plasma presents immunomodulating fac-
tors, which control the uterine inflammatory response
(Ribeiro et al. 2006). Furthermore, the seminal extenders
induce a local inflammatory response and the presence of
spermatozoa in the uterus induces a chemotaxic stimulus
of polymorphonuclear cells, yielding to phagocytosis,
membrane lipases activation and sperm lysis. In this
study, leucocytes were observed in all imprints, indicating
that the presence of spermatozoa, extender or both
exerted a chemotaxic effect upon those cells.
Based on the presence of spermatozoa in the contra-
lateral UH tip of the UH group, insemination does not
need to be performed in both horns; however, further
studies are required to confirm whether the laparoscopic
AI-technique in the tip of one horn will compromise
fertility and prolificacy. Spermatozoa were observed in
the tip of the UH 30–60 s after natural mating in the
bitch, indicating the efficiency of sperm cell transport
(Tsutsui et al. 1989). Conversely, regardless of the effects
of uterine contractions, the insemination volume (3 ml)
may have promoted the distribution of semen along the
narrow uterine lumen. That insemination in the UH
group, as a result of the small intraluminal space left in
the tip of the horn to accommodate the seminal volume,
required loosening the forceps, which facilitated move-
ment of the semen in the uterine lumen. However, there
was no resistance when semen was deposited in the UB.
Sperm counting showed a reduction at the uterotubal
junction, in both sides. This can be partially explained
by the fact that this fragment surface was smaller
than the other uterine segments. The wide variation
in spermatozoa numbers among uterine segments
(Table 1) may have been caused by differences in the
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1063
Left UTJ Left tube Right horn Right horn Right UTJ Right tube
(cm2) (cm2) 1 (cm2) 2 (cm2) (cm2) (cm2)
1.99 ± 0.81 3.25 ± 1.02 8.22 ± 3.40 7.27 ± 2.45 2.18 ± 0.41 2.92 ± 0.88
3.58 ± 1.01 5.02 ± 3.16 15.01 ± 15.82 10.97 ± 3.80 3.28 ± 0.89 3.28 ± 0.74
examined area (Table 2). Despite attempts at standard-
ization, it was difficult to apply consistent pressure,
leading to variation in smear thickness. Furthermore,
areas of imprints with high cellularity may have hidden
sperm cells. This technique was chosen because during
the preliminary trials using uterine and oviductal flush-
ing and histology we could not detect sperm cells in the
uterus and oviducts (data not shown).
It was not possible to detect sperm cells in the
oviductal mucosa by the imprint method. In a study
concerning oviductal flushing and cytology, only a few
bitches had a small number of spermatozoa in the
oviduct after natural breeding (Doak et al. 1967).
Rijsselaere et al. (2004) found 12.1 ± 26.4 · 104 sperm
cell in canine oviducts, by washing them 24 h after AI
with 500 · 106 sperm cells of fresh semen. However, as
they concomitantly washed the uterotubal junction, it
was not clear how many sperm cells were in fact derived
from the oviduct. In cows, the presence of spermatozoa
was observed at the ampulla and at the infundibulum 2 h
after AI with frozen–thawed semen (Larsson 1988). In
addition, spermatozoa were observed at the oviduct of
hamsters 1 h after natural mating (Smith et al. 1987).
However, considering that the oviduct is not a major
sperm reservoir in the bitch (Rijsselaere et al. 2004),
difficulty was expected in finding spermatozoa in this site.
In conclusion, laparoscopic uterine intraluminal
deposition of a 3-ml inseminate containing 300 · 106
fronzen–thawed sperm cells resulted in sperm cell
distribution through the entire uterine lumen, indepen-
dent of the site of deposition (tip of one horn or UB).
Acknowledgements
The authors thank Ethicon Inc. for supplying Caprofyl and Nylon
sutures and Becton, Dickinson and Company (BD) for supplying the
Insyte catheters.
Author contributions
Fabiola Bono Fukushima is the Postgraduate student, who wrote the
project and developed all the phases of the study. Christina Malm is
the main Professor, who supported the whole study. Marc Henry is the
Professor, who helped freezing and thawing the semen and who also
helped evaluating the results and reviewing the manuscript. Valentim
Arabicano Gheller is the Professor, who assisted the surgical
technique. Rogéria Serakides is the Professor, who helped standard-
izing the sperm cell counting evaluation. Mariana Machado Neves is
the PhD student, who froze and thawed the semen and helped to
review the manuscript. Sabrina Pereira Macedo is the Postgraduate
student, who participated in all surgical procedures and helped to
review the manuscript. Mariana da Silva Figueiredo, Maria Elisa
Jatobá Andrade and Marcela Silva Chaves are the graduate students,
who took part in all surgical procedures. Marcos Xavier Silva is the
Professor, who analysed all statistical data. Cleuza Maria de Faria
Rezende and Eliane Gonçalves de Melo are the Professors, who
collaborated in the laparoscopic procedure.
1064 FB Fukushima, C Malm, M Henry, VA Gheller, R Serakides, MM Neves, SP Macedo, MS Figueiredo, MEJ Andrade, MS
Chaves, MX Silva, CMF Rezende and EG Melo

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