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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
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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.
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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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)
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
<0.001
<0.001
0.010
0.095
0.006
0.002
<0.001
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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.
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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
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.
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[7] More J. Anatomy and histology of the cervix uteri of the ewe:
new insights. Acta Anatom 1984;120:1569.
[8] Kershaw CM, Khalid M, McGowan MR, Ingram K, Leethongdee S, Wax G, et al. The anatomy of the sheep cervix and its
influence on the trans-cervical passage of an inseminating pipette into the uterine lumen. Theriogenology 2005;64:122535.
[9] Kershaw CM. Mechanisms of cervical relaxation in the ewe.
Ph.D. thesis. University of London; 2006.
[10] Naqvi SM, Pandey GK, Gautam KK, Geethalakshmi V, Mittal
JP. Evaluation of gross anatomical features of cervix of tropical
sheep using cervical silicone moulds. Anim Reprod Sci
2005;85:33744.
[11] Halbert GW, Dobson H, Walton JS, Buckrell BC. The structure
of the cervical canal of the ewe. Theriogenology 1990;33:977
92.
[12] Ayad VJ, Leung ST, Parkinson TJ, Wathes DC. Coincident
increases in Oxytocin receptor expression and EMG responsiveness to Oxytocin in the ovine cervix at oestrus. Anim Reprod Sci
2004;80:23750.
[13] Shemesh M, Dommbrovski L, Gurevich M, Shore LS, Fuchs AR,
Fields MJ. Regulation of bovine cervical secretion of prostaglandins and synthesis of cyclooxygenase by oxytocin. Reprod
Fertil Dev 1997;9:52530.
[14] Mizrachi D, Shemesh M. Follicle-stimulating hormone receptor
and its messenger ribonucleic acid are present in the bovine
cervix and can regulate cervical prostanoid synthesis. Biol
Reprod 1999;66:77684.
[15] Shemesh M, Mizrachi D, Gurevich M, Stram Y, Shore LS, Fields
MJ. Functional importance of bovine myometrial and vascular
LH receptors and cervical FSH receptors. Seminars Reprod Med
2001;19:8796.
[16] Mizrachi D, Shemesh M. Expression of functional luteinizing
hormone receptor and its messenger ribonucleic acid in bovine
cervix: luteinizing hormone augmentation of intracellular cyclic
AMP, phosphate inositol and cyclooxygenase. Mol Cell Endocrinol 1999;157:191200.
[17] Derecka SA, Gawronska B, Bodek G, Zwierzchowski L, Shemesh, Ziecik AJ. LH/hCG receptors in the porcine uterusa
new evidence of their presence in the cervix. J Physiol Pharmacol 2000;51:91731.
[18] Fields MJ, Shemesh M. Extragonadal luteinizing hormone
receptors in the reproductive tract of domestic animals. Biol
Reprod 2004;71:14128.
[19] Buckrell BC, Buschbeck C, Gartley CJ, Kroetsch T, McCutcheon W, Martin J, et al. Further development of a transcervical
technique for artificial insemination in sheep using previously
frozen semen. Theriogenology 1994;42:60111.
[20] Anel L, Alvarez M, Martinez-Pastor F, Garcia-Macias V, Anel E,
dePaz P. Improvement strategies in ovine artificial insemination.
Reprod Domest Anim 2006;41(Suppl. 2):3042.
[21] Stys SJ, Dresser BL, Otte TE, Clark LE. Effect of prostaglandin
E2 on cervical compliance in pregnant ewes. Am J Obstetr
Gynaecol 1981;140:4159.
[22] Ledger WL, Ellwood DL, Taylor MJ. Cervical softening in late
pregnant sheep by infusion of prostaglandin E-2 into a cervical
artery. J Reprod Fertil 1983;69:5115.
[23] Menarguez M, Pastor LM, Odeblad E. Morphological characterization of different human cervical mucus types using light and
scanning electron microscopy. Hum Reprod 2003;18:17829.
[24] Honmode J, Pachlag SV. Time of ovulation and chloride content
of vaginal mucus during oestrus in sheep. Ind J Anim Sci
1973;43:13640.
[25] Kopito LE, Kosasky HK, Sturgis SH, Leiberman BL, Shwachman H. Water and electrolytes in human cervical mucus. Fertil
Steril 1973;24:499506.
[26] Gould KG, Ansari AH. Electrolyte interactions in cervical
mucus and their relationship to circulating hormone levels.
Contraception 1981;23:50716.
[27] Nasir-ud-Din, Hoessli DC, Runnger-Brandl E, Hussain SA,
Walker-Nasir E. Role of sialic acid and sulfate groups in cervical
mucous physiological functions: study of Macaca radiata glycoproteins. Biochem Biophys Acta 2003;1623:5361.
[28] Rexroad Jr CE, Barb CR. Cervical mucus in estrous ewes after
treatment with estrogen, progesterone and intrauterine devices. J
Anim Sci 1977;44:1025.
[29] Wolf DP, Blasco L, Khan MA, Litt M. Human cervical mucus II
Changes in viscoelasticity during the ovulatory menstrual cycle.
Fertil Steril 1977;28:4752.
[30] Moor RM, Bruce NM. The distribution of blood flow to the
reproductive tract of anaesthetized ewes near oestrus. Acta
Endocrinol (Copenhagen) 1976;83:7949.
[31] Silva LD, Onclin K, Verstegen JP. Cervical opening in relation to
progesterone and oestradiol during heat in beagle bitches. J
Reprod Fertil 1995;104:8590.
[32] Khalifa RME, Sayre BL, Lewis GS. Exogenous oxytocin dilates
the cervix in ewes. J Anim Sci 1992;70:3842.
[33] Wulster-Radcliffe MC, Costine BA, Lewis GS. Estradiol-17boxytocin-induced cervical dilatation in sheep: application to
trans-cervical embryo transfer. J Anim Sci 1999;77:258793.
[34] Weiss G, Goldsmith LT. Relaxin and the cervix. Frontiers Horm
Res 2001;27:10512.
[35] Lee HY, Zhao S, Fields PA, Sherwood OD. Clinical use of
relaxin to facilitate birth: reasons for investigating the premise.
Ann NY Acad Sci 2005;1041:35166.
[36] Brown SE, Toner JP, Schnorr JA, Williams SC, Gibbons WE, de
Ziegler D, et al. Vaginal Misoprostol enhances intrauterine
insemination. Hum Reprod 2001;16:96101.
[37] Goldberg AB, Greenberg MB, Darney PD. Misoprostol and
pregnancy. N Engl J Med 2001;344:3847.
[38] Li YT, Kuo TC, Kuan LC, Chu YC. Cervical softening with
vaginal Misoprostol before intrauterine device insertion. Int J
Obstetr Gynaecol 2005;83:678.
[39] Fletcher H, Mitchell S, Frederick J, Simeon D, Brown D.
Intravaginal Misoprostol as cervical ripening agents. Br J Obstetr
Gynaecol 1993;100:6414.
[40] Srisomboon J, Tongsong T, Tosiri V. Pre-induction cervical
ripening with intravaginal prostaglandin E1 methyl analogue
Misoprostol: a randomised controlled trial. J Obstetr Gynaecol
Res 1996;22:11924.
[41] Singh K, Fong YF. Preparation of the cervix for surgical
termination of pregnancy in the first trimester. Hum Reprod
Update 2000;6:4428.
[42] Ataman M, Kaya A, Yildiz C, Duzgun H. The effect of misoprostol and valethamate bromide administered before insemination with frozen thawed semen on cervix dilatation and
fertility in sheep. Ind Vet J 2000;77:6002.
[43] Fuchs AR, Graddy LG, Kowalski AA, Fields MJ. Oxytocin
induces PGE2 release from bovine cervical mucosa in vivo.
Prostaglandins Other Lipid Mediat 2002;70:11929.
[44] Kershaw CM, Scaramuzzi RJ, McGowan MR, Wheeler-Jones
CPD, Khalid M. The expression of prostaglandin endoperoxide
synthase 2 messenger RNA and the proportion of smooth muscle
and collagen in the sheep cervix during the estrous cycle. Biol
Reprod 2007;76:1249.
777
[46] Kershaw CM, Scaramuzzi RJ, Pitsillides AA, Wheeler Jones CPD,
McGowan MR, Khalid M. Changes in the cervical extracellular
matrix of the sheep during the oestrous cycle. In: Proceedings of
the biennial joint meeting for the UK fertility societies; 2005. p. 45.