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Theriogenology 67 (2007) 767777

www.theriojournal.com

The effects of the prostaglandin E analogue Misoprostol


and follicle-stimulating hormone on cervical penetrability
in ewes during the peri-ovulatory period
Sukanya Leethongdee a,*, Muhammad Khalid b, Aleem Bhatti b,
Suppawiwat Ponglowhapan b, Claire M Kershaw b, Rex J Scaramuzzi a
a

Departments of Veterinary Basic Sciences, Royal Veterinary College, Hawkshead Lane, North Mimms, Hertfordshire, AL9 7TA, UK
b
Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Lane, North Mimms, Hertfordshire, AL9 7TA, UK
Received 9 May 2006; received in revised form 18 October 2006; accepted 19 October 2006

Abstract
Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxation over the peri-ovulatory
period and investigated FSH and Misoprostol as cervical relaxants to facilitate transcervical passage of an insemination pipette into
the uterine cavity. Following synchronisation of oestrus using progestagen sponges and PMSG (500 IU) the depth of cervical
penetration was determined using a modified cattle insemination pipette as a measuring device.
Penetration of the cervix was least at the time of sponge removal and increased to a maximum at 72 h after sponge removal and
then declined. Intra-cervical administrations of either ovine FSH (Ovagen; 2 mg) or Misoprostol (1 mg; a Prostaglandin E1
analogue) facilitated cervical penetration. Ovagen given 24 h after sponge removal allowed transcervical intrauterine penetration in
100% of ewes at 54 and 60 h after sponge removal while Misoprostol given 48 h after sponge removal allowed trans-cervical
penetration in 100% of ewes at 54 h. A combination of Ovagen and Misoprostol was as effective but not more so than Ovagen or
Misoprostol alone.
These results show that there is natural relaxation of the cervix at oestrus and that maximum relaxation occurs 72 h after sponge
removal, which is too late for the correct timing of insemination. The intra-cervical administration of FSH or Misoprostol enhanced
relaxation of the cervix and both were able to relax the cervix to allow intrauterine penetration 54 h after sponge removal, the
optimum time for insemination. The results also show that FSH is biologically active after intracervical, topical application.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Prostaglandin E; Misoprostol; FSH; Cervix; Sheep

1. Introduction
Genetic improvement has had a significant impact on
sheep breeding and, artificial insemination (AI) with
either fresh or frozen and thawed (F-T) semen has had an

* Corresponding author. Tel.: +44 1707 66 62 80;


fax: +44 1707 65 20 90.
E-mail address: sleethongdee@rvc.ac.uk (S. Leethongdee).
0093-691X/$ see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2006.10.012

important role in this. The role of artificial insemination


in sheep breeding is to increase the rate of genetic
improvement. It contributes to the following goals: (i) it
allows more extensive use of the best rams, therefore
increasing selection pressure and the rate of response to
selection; (ii) younger rams may be used more widely,
resulting in faster genetic progress; (iii) superior rams can
be identified more easily through progeny testing and;
(iv) AI reduces the risk of genital infection during natural
mating. Furthermore, it is associated with certain animal

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S. Leethongdee et al. / Theriogenology 67 (2007) 767777

health benefits because it may be possible to move semen


where disease risk prevents ram movement [1].
However, the use of artificial insemination in sheep
has been not been widely adopted because deep vaginal
insemination and cervical insemination give poor
fertility when used with F-T semen [2,3]. A more
successful alternative is laparoscopic intrauterine
insemination which produces good fertility with F-T
semen but the technique is costly and not welfare
friendly [1,4]. Trans-cervical artificial insemination
(TCAI) is a potential alternative to laparoscopic
insemination [5,6]. This involves passing an AI pipette
through the cervix to deposit semen within the uterus.
This technique is used in other species but the unusual
anatomy of the ovine cervix is a major limitation to the
use of this technique in sheep.
The ovine cervix is a long, fibrous, tubular convoluted
organ [7]. The average length of the cervical canal is
6.7 cm and contains about six ventral facing, funnelshaped annular rings [7]. Other authors have reported
similar findings [5,8,9]. There are among and within
species differences in the complexity of the cervical
rings, organization of the inner and outer orifices, length
and complexity of the cervical lumen and anatomical
relationships with the uterine body and vagina. The
internal morphology of the cervix is characterized by
about, six annular rings [8]. In the ewe, Silastic casts
show the tortuous, corkscrew-like nature of the cervical
lumen [10]. Successful passage of a cervical insemination instrument is difficult because of failure to easily
identify the cervical opening and because of the restricted
diameter (2.7  1.1 mm) of the cervical rings [10]. In
addition, cervical rings are not always concentrically
aligned [11]. The second ring is consistently out of
alignment with the first ring. Consequently, passage of an
artificial insemination pipette beyond the second ring is
difficult and rarely achieved.
There is a degree of cervical relaxation at naturally
occurring oestrus, enabling trans-cervical penetration in
a small proportion of multiparous ewes. This relaxation
is the result of the peri-ovulatory reproductive
hormones, progesterone, oestradiol and oxytocin acting
on the cervix. Increases in oxytocin receptor expression
during oestrus are detectable in luminal epithelial cells
of the cervix [12]. The potential effect of oxytocin on
cervical relaxation in cows is mediated by a local
increase in cyclooxygenase-2 (COX-2) and a subsequent increase in prostaglandin E2 (PGE2) synthesis
[13]. The bovine cervix, at the time of peak periovulatory concentrations of follicle stimulating hormone (FSH), has high levels of follicle stimulating
hormone receptor (FSH-R) and responds to FSH by

increasing PGE2 synthesis that may lead to cervical


relaxation at oestrus [14,15]. Luteinizing hormone
receptor (LH-R) mRNA and LH receptor protein are
also present in the cervix of cattle [1618] and sheep
Leethongdee, Scaramuzzi, Kershaw and Khalid; unpublished data) and in the ewe they reach their maximum
concentration at oestrus. However, the potential role of
LH in cervical relaxation at oestrus has not been
investigated.
This objective of these investigations were: (i) to
determine the pattern of natural cervical relaxation at
oestrus in the ewe and (ii) to examine the use of intracervical deposition of FSH and the prostaglandin E1
analogue, Misoprostol as cervical relaxants.
2. Materials and methods
2.1. Animals and management
Two experiments were carried out using parous Welsh
Mountain ewes. The first experiment in October
November 2004 used 24 ewes, randomly assigned to
six groups of four. Ewes were used in 3 replicates allowing
12 observations per treatment. After the ewes were used in
each replicate they were allowed to rest for 5 days before
commencement of the next cycle of treatment. The
second experiment, AprilJune 2005, used 18 ewes
randomly assigned to four groups of four or five ewes.
This experiment had two replicates so that each treatment
had nine observations. In all cases, the ewes were rerandomised between replicates and the data treated as
independent observations. The ewes were kept in groups
of six to eight indoors on straw. All ewes were fed with a
commercial concentrate diet with hay and water provided
ad libitum. All procedures were conducted under Home
Office authorization in compliance with the Animal
(Scientific Procedures) Act, 1986.
2.2. Synchronisation of the oestrous cycle
The oestrous cycles of the ewes were synchronized
to a common day of oestrus using intravaginal sponges
(Chronogest: Intervet UK Ltd., Milton Keynes, Northamptonshire, UK) for 12 days followed by PMSG
(Folligon: Intervet UK Ltd.) at sponge removal. In
experiment 1, the dose used was 250 IU PMSG while in
experiment 2, 500 IU was used.
2.3. Measurement of cervical penetration
The ewes were restrained in a yoke fitted with
sidebars to minimise lateral and forward movements

S. Leethongdee et al. / Theriogenology 67 (2007) 767777

769

Fig. 1. Cervical penetration was measured using a sounding device made by shortening a standard cattle insemination pipette. The stainless steel
outer pipette sheath was reduced in length making it 10 cm shorter than the solid metal pipette plunger. The metal tip of the plunger was rounded. The
outer 10 cm of the plunger was etched with 0.5 cm graduations with the zero point aligned at the outer end of the pipette sheath when the pipette tip
was aligned with the inner end of the pipette sheath.

with the hindquarters of the ewe raised about 4 in. The


sounding device used in this experiment was modified
from a stainless steel, cattle AI pipette. The stainless steel
outer pipette sheath was 52 cm in length but the pipette
sheath was reduced to 42 cm in length, making it 10 cm
shorter than the solid metal pipette plunger. The metal tip
of the plunger had a blunt rounded end. The outer 10 cm
of the plunger was etched with 0.5 cm graduations with
the zero point aligned at the outer end of the pipette
sheath when the pipette tip was aligned with the inner end
of the pipette sheath (Fig. 1). The plunger had a diameter
of 1.5 mm and the pipette sheath had an external diameter
or 3.0 mm and an internal diameter of 1.6 mm. The
perineum was cleaned with a disinfectant wipe and a
speculum introduced into the vagina so that the external
os cervix could be seen in the light of the speculum lamp.
The pipette tip was placed at the external os of the cervix
(to become the zero point and the plunger was passed
into the cervix and advanced as far as possible using
gentle manipulation but without force. The plunger was
then clamped to the pipette and the apparatus withdrawn
and the distance the plunger had advanced beyond the tip
of the pipette was recorded as the depth of cervical
penetration. A depth of penetration of greater than 8 cm
was regarded as intra uterine penetration [8]. Measurements were made in duplicate.
2.4. Experiment 1
For five of the groups (groups 15), penetration of
the cervix was determined only once at 0, 24, 48, 72 and
96 h after sponge removal (the once only groups).
For group 6 penetration of the cervix was determined on
five occasions at 0, 24, 48, 72 and 96 h (the repeated
group) after sponge removal.

2.5. Experiment 2
Cervical penetration was measured at 24, 48, 54, 60,
66 and 72 h after sponge removal in groups of ewes
treated intra-cervically with (i) Ovine FSH (2 mg;
Ovagen; ICPbio (UK) Limited, Wiltshire, UK) and/or
(ii) a prostaglandin E1 analogue (1 mg; Misoprostol;
SigmaAldrich Co., Dorset, England) as follows:
Group 1: Ovagen; treated with intra-cervical FSH at
24 h after sponge removal followed by intracervical gelatine vehicle at 48 h after sponge
removal.
Group 2: Misoprostol; treated with intra-cervical gum
acacia vehicle at 24 h after sponge removal
followed by intra-cervical Misoprostol at 48 h
after sponge removal.
Group 3: Ovagen plus Misoprostol; treated with intracervical FSH at 24 h and intra-cervical
Misoprostol at 48 h after sponge removal.
Group 4: Control; treated with intra-cervical gum
acacia vehicle at 24 h and intra-cervical
gelatine vehicle at 48 h after sponge removal.
Ovagen was administered at a dose of 2 mg
dissolved in 0.5 ml of 50% Gum acacia (Sigma
Aldrich Co.). Misoprostol was administered at a dose
of 1 mg dissolved in 0.5 ml of 30% gelatine (Sigma
Aldrich Co.). For treatment, the ewes were restrained
as described above for the cervical penetrability tests.
A 1 ml Eppendorf (Eppendorf AG, Hamburg,
Germany) pipette fitted with a 10 cm extension
consisting of 3  1000 ml tips glued together was
used for intra-cervical administration. The tip of the
extension pipette was blunted, by cutting off about

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S. Leethongdee et al. / Theriogenology 67 (2007) 767777

0.2 mm of the tip, the extension pipette was then


sterilised. The perineum was wiped clean with a
disinfectant wipe and a speculum introduced into the
vagina so that the external os cervix could be seen in
the light of the speculum lamp. The pipette tip was
inserted about 24 cm into the cervix and the 0.5 ml
bolus deposited in 80% of ewes, when this was not
possible and the FSH was placed at the os cervix. In a
series of preliminary tests, we established the
maximum volume (0.5 ml) and viscosity of vehicle
required to ensure that the bolus did not leak from the
cervical canal.

2.8. Statistical analysis


The data for depth of cervical penetration at different
times after sponge removal was analysed by a mixed
model ANOVA using ewe as a random factor and time
(experiment 1) and time and treatment (experiment 2) as
fixed factors. Additional post hoc comparisons, between
time and between treatment comparisons were made
using Sidaks test. Vaginal and cervical changes were
analysed using the Chi square test for proportions. The
tests were carried out using SPSS 14.0 for Windows.
3. Results

2.6. Vaginal and cervical morphology


3.1. Experiment 1
Vaginal and cervical morphology and the characteristics of cervical mucus were also recorded prior to the
penetration tests. These were not recorded at sponge
removal.
(i) Vaginal epithelial colour was recorded and
categorized as white, pink, red and deep red.
(ii) Cervical mucous production was recorded. Cervical mucous production was categorized as none (no
cervical discharge flowing from the cervical
opening), some (some cervical discharge around
the area of the cervical opening) and much (copious
cervical discharge at the cervical opening, cervical
discharge running out of the cervical opening into
the vagina and an accumulation of cervical
discharge in the vagina).
(iii) The colour of cervical mucus was categorized as
clear, yellow and pink.
(iv) Spontaneous movement of the cervical opening
was recorded and categorized as still (no spontaneous movement of the cervical opening before
manipulation of the cervical opening and moving
(spontaneous movement of the cervical opening
seen before manipulation of the cervical
opening).

3.1.1. The depth of cervical penetration


There was significant variation in the depth of cervical
penetration at different times after sponge removal
(Fig. 2). The mean  S.E.M. of the depth of penetration
in the once only groups increased significantly
(P < 0.001) from 0.73  0.28 cm at sponge removal
to a maximum of 5.0  1.2 cm 72 h after sponge removal
before decline to 1.6  0.84 cm at 96 h. The depth of
penetration was significantly (P = 0.019) reduced at 96 h
compared to 72 h (Fig. 2). In the repeated group, the
mean  S.E.M. depths of penetration also increased
significantly (P < 0.001) from sponge removal
(0.56  0.15 cm) to a peak at 72 h (6.2  1.2 cm) before
declining. At sponge removal the depth of penetration

2.7. Smears of cervical mucus


Cervical mucus was collected from the anterior
vagina or fornix by gentle suction with a syringe that
had a long, soft plastic extension tube attached. Cervical
mucus was smeared onto slides and the slides left to air
dry. The air-dried smears were examined by microscopy
and photographed. Different ferning patterns were
observed and these were classified as: (i) incomplete or
atypical ferning, (ii) complete or typical ferning and (iii)
degraded ferning.

Fig. 2. The depth of penetration of the cervix in Welsh Mountain ewes


at different times after sponge removal and during the peri-ovulatory
phase of the oestrous cycle. The ewes in the once only groups were
tested at only one time point while the ewes in the repeated group
were tested at each time point. Within the once only groups and the
repeated group columns with different superscripts are significantly
different (P < 0.05). Asterisk * indicates a significant difference
(P < 0.05) in the depth of penetration between ewes in the once only
and repeated groups.

S. Leethongdee et al. / Theriogenology 67 (2007) 767777

771

Table 1
The changing patterns, expressed as percentages, of epithelial colour and some characteristics of cervical mucus in Welsh Mountain ewes at different
times after sponge removal
Time relative to sponge removal (h)

Pink vaginal epithelium


Pale vaginal epithelium
Spontaneous movement of the os cervix
Clear cervical mucus
Yellow cervical mucus
Copious cervical mucus discharge
Ferning of cervical mucus

P-value

24

48

72

96

0
100
N/A
N/A
N/A
N/A
N/A

100
0
62.5
25
75
0
All incomplete

100
0
62.5
87.5
12.5
12.5
All complete

75
0
37.5
62.5
25
50
Some degraded

0
100
0
50
50
0
All degraded

was significantly lower than at all other times (P < 0.05).


The depth of penetration increased gradually between 24
and 72 h and then decreased at 96 h but these differences
were not significant. When the depth of penetration in the
once only was compared to the repeated groups
(Fig. 2), cervical penetration was not different at 0, 24, 48
and 72 h. However, in the repeated group 96 h after
sponge removal the depth of penetration was greater than
in the once only group (P < 0.05).
3.1.2. Vaginal and cervical morphology
Vaginal and cervical morphology was recorded only
in once only group, immediately before the penetration test thus, avoiding potential changes induced by
prior manipulation of the cervical passage.
3.1.2.1. Vaginal epithelial colour. At 0 h all ewes had
a pale vaginal epithelium. At 24 and 48 h after sponge
removal the vaginal epithelial colour changed to pink in
all ewes. At 72 h, 25% of ewes had a red and 75% of
ewes had a pink vaginal epithelial colour. At 96 h after
sponge removal all ewes once again had a pale vaginal
epithelial colour (Table 1).
3.1.2.2. Spontaneous movement of the cervical opening. Spontaneous movement of the cervical opening
was observed and the pattern was different at different
times after sponge removal. Spontaneous movements
were not recorded at time 0 h (sponge removal). At 24
and 48 h, 62.5% of ewes had spontaneous movement of
the cervical opening, by 72 h this had decreased to
37.5% and at 96 h no ewes had spontaneous movements
of the cervical opening (Table 1).
3.1.3. Cervical mucus
3.1.3.1. Discharge of cervical mucus. There are no
data at 0 h because cervical discharge was mixed with
accumulated fluid and detritus associated with the
progestagen sponge. Some cervical discharge was

<0.001
<0.001
0.010
0.095
0.006
0.002
<0.001

observed in all ewes at 24 h; by 48 h, 75% of ewes


had some cervical discharge, while 12.5% had none and
12.5% had a copious discharge. At 72 h, ewes with a
copious discharge had increased to 50% while 37.5%
had some vaginal discharge and the remainder none. At
96 h, 75% of ewes had some cervical discharge and 25%
had none (Table 1).
3.1.3.2. Colour of cervical mucus. There are no data at
0 h because cervical discharge was mixed with accumulated fluid and detritus associated with the progestagen sponge. At 24 h, cervical mucus was predominantly
yellow although it was clear in 25% of ewes. At 48 h this
pattern had reversed and 85% had clear cervical mucus
and the remainder yellow. At 72 h, 25% of ewes had
yellow, 62.5% had clear and 12.5% had clear mucus
tinged with pink. By 96 h 50% of ewes had yellow
cervical mucus that had a thicker consistency and 50%
had clear cervical mucus (Table 1).
3.1.3.3. Smears of cervical mucus. The different
ferning patterns observed in smears of cervical mucus
collected at different times in the peri-ovulatory period
are illustrated in Fig. 3. At 24 h after sponge removal
some ewes had incomplete ferning and some had
complete ferning. Maximum ferning was observed at
48 h, some degradation of the ferning was seen at 72 h;
and by 96 h the degradation of ferning was completed
and no ferning was seen at this time (Fig. 3).
3.2. Experiment 2
3.2.1. Cervical penetrability
Cervical penetrability (Table 2) was significantly
affected by both time (P < 0.001) and treatment
(P = 0.002) but the time by treatment interaction was
not significant (P = 0.201). In the control group, the
mean  S.E.M. of the depth of penetration was least at
24 h and deepest at 72 h (Table 2). When ewes received

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S. Leethongdee et al. / Theriogenology 67 (2007) 767777

Fig. 3. Ferning patterns in cervical mucus collected at different times during the peri-ovulatory period in Welsh Mountain ewes, (top left) no obvious
ferning in mucus collected 24 h after sponge removal, (top right) complete ferning in mucus collected 48 h after sponge removal, (bottom left) partially
degraded ferning in mucus collected 72 h after sponge removal, (bottom right) degraded ferning in mucus collected 96 h after sponge removal.

Ovagen alone the depth of penetration was least at 24 h.


At 54 h and 60 h, penetration was at a maximum and
into the uterus in all ewes. Penetration was reduced at 66
and 72 h (Table 2). In the group that received
Misoprostol the depth of penetration was least at
24 h and increased to maximum penetration at 54 h
before declining (Table 2). In the combination group
penetration was lowest at 24 h and increased to a
maximum at 54 h before declining (Table 2).
3.2.2. Other changes
Other qualitative and quantitative differences were
noted. In control ewes, maximum penetration was later

at 72 h compared to other treatments where maximum


penetration was observed at 54 h (Table 2). Ewes
treated with Ovagen appeared to produce a longer
period of intrauterine penetration compared to the
Misoprostol alone or in combination with Ovagen
(Table 2). There were also noticeable qualitative
differences that were not quantified in this experiment
but none-the-less they are worth noting. The external os
cervix was noticeably dilated particularly in the Ovagen
and Misoprostol treated ewes and penetration of the
cervix was technically much easier in these ewes. The
order of ease of penetration the easiest to the most
difficult was the combination followed by Ovagen, the

S. Leethongdee et al. / Theriogenology 67 (2007) 767777

773

Table 2
The depth of penetration of the cervix (mean  S.E.M.) during the peri-ovulatory period in Welsh Mountain ewes treated intra-cervically with
vehicle (Control), 2 mg of ovine FSH (Ovagen), 1 mg of the prostaglandin E analogue (Misoprostol) or a combination of Ovagen and Misoprostol
Treatment

Time relative to the removal of progestagen sponges (h)


24

Control (Vehicle)
Ovagen
Misoprostol
Ovagen + Misoprostol

48
a,x

2.2  1.1
4.3  1.2a,y
3.5  1.1a,x
0.33  0.12a,x

54
b,x

5.3  1.3
7.4  0.6b,x
4.3  1.2a,x
6.2  1.9b,x

60
b,x

5.3  1.3
8.0  0.0b,y
8.0  0.0b,y
7.1  2.1b,y

66
b,x

4.3  1.2
8.0  0.0b,y
6.7  0.9b,y
6.2  1.7c,x

72
b,c,x

4.3  1.2
7.1  0.9b,x
6.3  1.1a,b,x
4.4  1.6b,c,x

7.1  0.9c,x
6.4  1.1b,x
3.9  1.3a,y
4.0  1.1b,c,y

Ovagen was administered at 24 h in a gum acacia vehicle and Misoprostol was administered at 48 h in a gelatine vehicle; within rows values with
different superscripts (a, b, c) are significantly different at P < 0.05; within columns values with different superscripts (x, y) are significantly
different from the vehicle control at P < 0.05.

Misoprostol and finally no treatment (control ewes).


There was also noticeably more, thin watery mucus
produced in Ovagen treated ewes compared to other
treatments.
4. Discussion
The success of TCAI requires the ability to pass an
insemination pipette through the cervix to deposit
semen directly into the uterus in a high proportion of
ewes. The unusual anatomy of the ovine cervix is a
major factor limiting the use of this technique. The
ovine cervix is a long and fibrous, tubular convoluted
organ with a number of features that effectively impedes
TCAI [711]. First, the anatomy of the os cervix is
variable and it is often difficult to locate when using a
speculum and light. Second, the presence of several
intra-cervical annular rings [8] with small openings [11]
that can impede the passage of an insemination pipette
and finally the first and second annular rings are
consistently mis-aligned effectively obstructing the
central lumen of the cervical canal [7,11]. The
arrangement of the internal cervical rings is eccentric
in the native Malpura and Kheri breeds [10] and in a
study in Welsh Mountain ewes cervical penetration was
affected by the anatomy of the cervical lumen [8]. A
cervical lumen with a simple arrangement of the
internal rings allowed the passage of pipette easier than
a cervix with a complex arrangement of internal rings.
Third, non-luteal (follicular) cervices could be penetrated further than luteal cervices [8].
The shape and type of pipette affects its passage
through the cervical lumen. The Guelph system for
trans-cervical AI allowed successful cervical penetration in most ewes [19]. However, others have not had
similar success with the Guelph system [4] or with other
mechanical methods [20]. Penetration success was
affected by the period since the last lambing and

inseminator experience [19] and lambing rate was


higher for ewes bred during the breeding season than
during other times [19].
The position of ewe during penetration was important
for the accuracy and success of passing a pipette through
the cervix. In our own experience the standing ewe was
more readily inseminated into the uterus than were ewes
in the over-the-rail position. We have found that the
most satisfactory position was with the ewe standing with
slightly elevated (approximately 15 cm) hindquarters. In
this position, uterine penetration was achieved in 82% of
the ewes. Invariably, if the external os could be penetrated
intrauterine insemination was achieved. The most
frequent problem was penetration of the external os
especially in those ewes with a flap type of external os
[8].
We were able to confirm that there is a degree of
cervical relaxation at naturally occurring oestrus that
enables trans-cervical penetration in a small proportion
of multiparous ewes. In our two experiments we used
parous Welsh Mountain ewes and penetration of the
cervix increased from 1 cm or less at the time of sponge
removal to a maximum depth of 67 cm 72 h after
sponge removal before declining again at 96 h after
sponge removal. Unfortunately, the optimum time for
artificial insemination of ewes is not at 72 h after sponge
removal. Highest fertility is achieved when ewes are
inseminated 54 h after sponge removal [1] and at this
time cervical penetration averaged only 45 cm in our
experiments.
Natural cervical relaxation at the time of oestrus and
ovulation is probably the result of the peri-ovulatory
changes in reproductive hormones that occur at this
time. The increases in oestradiol and oxytocin receptor
concentrations during the peri-ovulatory period are
thought to increase prostaglandin E2 synthesis [13]
leading to remodelling of cervical extracellular matrix
[21,22] to relax the cervix.

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When cervical mucus from oestrous ewes is air-dried


on a glass slide it forms a characteristic crystallized
pattern known as ferning [23] because it has a fern leaflike appearance (Fig. 3). We found that a complete
ferning pattern was present at 48 h after sponge removal
and that by 72 h the ferning pattern had degraded and
had disappeared by 96 h. Thus, ferning was most clearly
observed 48 h after sponge removal just before
ovulation and at a time of maximum oestradiol
secretion, oestrus, mating and sperm transport. Ferning
is associated with an increase in water content, the
concentration of certain electrolytes particularly chloride [2426] and alterations in the relative quantities of
glycoproteins [27,28]. The formation of fern-like
structures bears a close relationship to spinbarkeit, a
measure of the viscosity and elasticity of cervical
mucous fluid [29].
Oestradiol has a major influence on blood flow to the
reproductive tract in sheep. Blood flow to the
myometrium, endometrium and oviducts increases 2fold and blood flow to vagina and cervix increases over
10-fold from day 14 to 15 of the oestrous cycle [30].
This increase is reflected in the colour of the vaginal
epithelium and we observed that the colour of the
vaginal epithelium changed from pale (indicating low
blood flow) to pink and eventually to red indicative of
high blood flow. Our data suggest that blood flow
through the vaginal and cervical tissues increased from
time 0 when all ewes had a pale, whitish vaginal
epithelium to 72 h when all ewes had a red or pink
vaginal epithelium and then decreased at 96 h all ewes
again had a pale whitish vaginal epithelium. Similar
patterns of change were seen in amount, consistency
and colour of cervical mucus and in the apparent
spontaneous movements of the os cervix. These are all
presumed to be responses to the high circulating levels
of oestradiol present at this time [31] although the role
of other hormones present in elevated concentrations at
this time cannot be discounted.
When designing experiment 1 we were concerned
that (i) repeated penetration of the cervix might initiate
some local reactions, possibly involving prostaglandin
production, in response to repeated penetration, (ii) we
may inadvertently puncture the cervical wall and record
this as intra-uterine penetration and (iii) that the
intracervical hormonal bolus may not be retained with
the intracervical lumen. We found that a frequency of
penetration up to four times over 72 h, did not affect the
relaxation of the cervical canal. However, penetration
for the fifth time did produce a significant relaxation of
the cervix that allowed deeper penetration at 96 h
(Fig. 2). The reasons for this effect are not clear and

require further investigation, but may as we suggest,


involve mechanical stimulation of prostaglandin production. In a preliminary test involving 48 Welsh
Mountain ewes during the breeding season, intrauterine penetration was attempted either during the late
luteal phase (n = 24) or the late follicular phase (n = 24)
of the oestrous cycle, one h before the animals were due
to be killed. After death the reproductive tracts were
recovered and examined for inadvertent puncturing of
the cervical wall. The data show that intrauterine
penetration was possible in 23/48 ewes (7 luteal phase
and 16 follicular phase; P = 0.009) and the post-mortem
examination confirmed that in all 23 ewes, there was no
evidence of the pipette tip having penetrated the wall of
the cervix and entered the pelvic cavity and we
conclude that penetration was intra-uterine. This study
also showed that the cervical canal could not be entered
in 20% of ewes (9/48; six luteal phase and three
follicular phase; P = 0.267). The principal impediment
to successful cervical penetration is the nature of the os
Cervix [8] and this is not influenced by the stage of the
oestrous cycle. The flap type cervix [8] is the most
difficult if not impossible, to penetrate past. In other
preliminary studies using a variety of viscous media
coloured with blue dye we were able to establish that a
hormonal bolus in 0.5 ml of a viscous medium such as
glycerol and gum acacia was retained within the
cervical lumen for several hours.
Investigation of the hormonal control of cervical
function has in the past concentrated on oestradiol,
progesterone,
prostaglandins
and
oxytocin
[12,13,22,32,33] with some interest in relaxin
[34,35]. However in recent years numerous reports
have appeared describing the presence of both LH-R
([1618], Leethongdee, Scaramuzzi, Kershaw and
Khalid; unpublished data) and FSH-R in the cervix
([14,15], Leethongdee, Scaramuzzi, Kershaw and
Khalid; unpublished data). From in vitro tests, these
receptors appear functional [14,16] and this prompted
us to investigate the in vivo action of FSH as a
cervical relaxant. The prostaglandin E1 analogue,
Misoprostol is a well-known cervical relaxant and is
used clinically for this purpose [36,37]. Our results
show that both ovine FSH and Misoprostol cause
relaxation of the ovine cervix to such an extent that
cervical penetration was possible in 27 out of 28
treated ewes 54 h after sponge removal. Our finding
that intracervical FSH allows easier penetration of
the cervix between 54 and 60 h after sponge removal
is undoubtedly of potential significance for sheep
artificial insemination. However, some caution
should be exercised at this time and our findings

S. Leethongdee et al. / Theriogenology 67 (2007) 767777

should be considered preliminary and subject to


further experimental verification.
Ewes were given local intra-cervical FSH 24 h after
sponge removal and 24 h later that is, 48 h after sponge
removal the cervix had responded and penetration was
increased, the cervix was still relaxed at 72 h. This
result is explainable because it occurred at the same
time as the peripheral plasma peak of FSH and at a time
when FSH-R levels would also be elevated. The intracervical administration of FSH was 24 h after sponge
removal. This time was selected because of the
difference between the time of the normal ovulatory
surge of FSH in the oestrous cycle and the time of
maximum penetration of the cervix observed in the
control group from experiment 1. We reasoned that if
endogenous FSH were causing cervical relaxation at
72 h then a period of at least 24 h would be required to
elicit an effect so that the maximum effect would be
observed at 4854 h after sponge removal.
Misoprostol a prostaglandin PGE1 analogue is used
commonly and effectively to induce cervical softening
[38] and cervical relaxation at parturition in women [37]
and vaginal application of Misoprostol enhanced
intrauterine insemination in women [4]. Cervical
ripening at parturition can be induced with 100 mg of
intravaginal Misoprostol [39,40]. The recommended
dose for cervical ripening in women during the first
trimester of pregnancy is 400 mg [41]. Cervical
relaxation in sheep was not observed 45 min after
intra-cervical administration of 400 mg [42] and in
preliminary tests Kershaw, Scaramuzzi and Khalid;
unpublished data) we noted that 200 mg of intracervical Misoprostol did not enhance cervical relaxation 2472 h after administration. The effect of
Misoprostol is short lived and Goldberg et al. [37]
suggested that the length of time for cervical ripening
after Misoprostol is 46 h. For this reason Misoprostol
was administered 48 h after sponge removal and 24 h
later than FSH so that the maximum effect of
Misoprostol would also be observed at 54 h after
sponge removal. In our experiment, ewes were
administered intra-cervical Misoprostol 48 h after
sponge removal and the maximum depth of penetration
was observed at 54 h after which penetration declined
(Table 2). The effect of Misoprostol was more rapid and
of a shorter duration compared to the effect of FSH.
The prostaglandin synthetic system is active in the
cervix and PGE2 is secreted from cervical mucosa
during the LH surge [43]. In a recent series of
experiments increased expression of COX-2 mRNA
has been reported in the ovine cervix during the
follicular phase of the oestrous cycle [9,44] and

775

abundant mRNA concentrations of the prostaglandin


E2 receptors, EP2 and EP4 have been reported in the
ovine cervix [9,45]. We suggest that relaxation of the
cervix at oestrus could involve FSH-mediated stimulation of PGE2 and glycosoaminoglycans [46].
These results show that there is natural relaxation of
the cervix at oestrus and that maximum relaxation is
72 h after sponge removal, which is too late for the
correct timing of TCAI. The intra-cervical administration of Ovine FSH (2 mg) or Misoprostol (1 mg) was
able to enhance the natural relaxation of the cervix at
oestrus and if given at an appropriate time both were
able to relax the cervix to allow effective intrauterine
penetration at 54 h after sponge removal the optimum
time for TCAI [1]. It is somewhat surprising that a large
glycoprotein such as FSH (MW approximately 20,000)
is biologically active after topical application but, FSHR is present in all tissue layers of the cervix except the
external serosa and the highest concentrations are in
the luminal epithelium and the inner band irregular
smooth muscle (Leethongdee, Scaramuzzi, Kershaw
and Khalid; unpublished data).
Acknowledgements
The research was supported by a grant from The
Royal Veterinary College, Internal Grant Scheme and a
research scholarship awarded to Ms Sukanya Leethongdee by the Royal Thai government. The authors wish
to thank Ms. Anongnart Somchit and Mr. Waliul
Chowdhury for assistance with the animal experiments.
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