Background
The ability to gain from the benefits of embryo transfer (ET) in commercial settings is predominantly due to the ease with which these procedures can be executed. Surgical approaches are not necessary to perform ET in most species of veterinary interest but rather the transcervical route is used. The ovine cervix, however, is a narrow and rigid structure with a complex, tortuous arrangement that precludes easy transcervical passage and intrauterine deposition of harvested embryos in recipient ewes using conventional ET catheters [
1,
2]. The main method used to circumnavigate this anatomical barrier involves surgical intervention; intrauterine ET in sheep is usually performed via mid-ventral laparoscopy [
3,
4]. Although considered moderately invasive, this procedure causes undue stress to animals, is time consuming, costly, labor intensive, and requires specialized equipment and qualified personnel. The repeated use of this technique is also a concern for animal welfare. As a result, the use of laparoscopic ET is not widespread in the sheep industry and is limited to small flocks of usually rare, expensive, heritage breeds. The cost to benefit ratio of surgical transfers is not favorable especially if the transfer to multiple recipients is required. Therefore, a reliable transcervical method of ET would be a valuable asset to the sheep breeders. Wulster-Radcliffe et al. [
5] were able to achieve cervical penetration and intrauterine embryo deposition in ewes. It was found that 53.5% of embryos were retained at day 12 of pregnancy. Although promising, these results were never repeated and the development of the embryos past day 12 in utero was not assessed.
Several modified catheters have been developed to traverse cervical canal in sheep but the penetration rates remain inconsistent and unpredictable [
1]. Furthermore, cervical trauma and local inflammation caused by repeated attempts to pass a rigid catheter through a highly constricted cervix may have a negative impact on the ensuing pregnancy and lambing rates [
1]. A plausible solution would be to devise a means of dilating the ovine cervix prior to manipulation to enlarge the cervical lumen and thus enable the passage of ET instruments; this would greatly decrease potential cervical injury and its associated detrimental effects. Cervical dilation could also increase the ease of transcervical ET making it less dependent on operators’ skills and decreasing the amount of time taken to perform the procedure. Dilation of merely the first two caudal folds of the cervix, which are the most constricted and misaligned, would be sufficient to significantly facilitate transcervical instrument passage [
1].
An ideal pharmacological agent to induce cervical dilation for the purpose of ET in ewes must be designed for easy delivery and cause minimal discomfort to the animal [
1]. Such an agent should not yield adverse residual effects, neither maternal nor embryonic. In particular, it should not induce uterine atony or hyperstimulation as both of these conditions could interfere with the ability of transferred embryos to successfully implant in the endometrial layer. Cervidil® is a vaginal insert containing synthetic analog of prostaglandin E
2 (PgE
2). It has been established as a safe agent to induce cervical dilation in periparturient women. Cervidil® releases PgE
2 at a constant rate to sustain physiological concentrations of the prostaglandin, similar to those observed at early stages of labor, and promoting cervical ripening without strong concomitant effects on uterine contractility. We proposed that the application of Cervidil® to cyclic sheep may facilitate transcervical embryo deposition.
Hence, the present study was undertaken to assess the utility of Cervidil® as an inducer of cervical dilation for transcervical ET in ewes. The emphasis was on the dilatory effects of Cervidil® inserts and so the embryos from only one source (frozen-thawed ovine embryos) were used and no comparisons between fresh or frozen/in vitro- or in vivo-produced embryos were attempted in the present trial.
Results
In terms of overall cervical penetrability, 32% (7/22) of ewes were successfully penetrated. Of those that were penetrated, 86% (6/7) had been treated with Cervidil® and 14% (1/7) was an untreated control. Within non-penetrated ewes, only 33% (5/15) had been treated whereas 67% (10/15) had not received treatment. Of the non-penetrated ewes primed with Cervidil®, 60% (3/5) had a 12-h Cervidil® treatment and 40% (2/5) a 24-h exposure. Within the treated ewes that had been penetrated, 67% (4/6) had been exposed to Cervidil® for 24 h and the remaining ewes had had a 12-h exposure. When comparing the treatment and control groups overall, the proportion of successfully penetrated ewes (55%, 6/11) was significantly greater in the Cervidil®-treated animals compared with controls (9%, 1/11). Within this subset of ewes, the 24-h treatment group exhibited significantly greater cervical penetration rate than respective controls, but the difference between 12-h treatment group and corresponding controls was not significant (Table
2). Serum E2 concentrations and E
2 : P
4 ratios declined (p < 0.05) from 24 h before to the time of TCET in both the 12-h Cervidil®-treated ewes and their respective controls (Table
2). A significant rise in circulating P
4 concentrations occurred over the 24 h before TCET in the 24-h treated ewes and between 24 and 12 h prior to TCET in the 12-h control group (Table
2).
Table 2
The cervical penetrability, total time taken to perform the procedure (TT), time of the final attempt (FT) and estradiol/progesterone (E
2
: P
4
) concentrations/ratios for the Cervidil®-treated cyclic Rideau Arcott x Polled Dorset ewes and their respective controls
Cervical penetrability
| 2/5 (40%) | 4/6 (67%)* | 1/6 (16%) | 0/5 (0%)* |
TT (sec) | 168 ± 22 | 110 ± 29 | 170 ± 28 | 180 ± 26 |
FT (sec) | 38 ± 23 | 27 ± 5 | 154 | - |
E
2
– 24 h prior (pg/ml) | 2.4 ± 0.4† | 1.8 ± 0.4 | 2.9 ± 0.4† | 1.5 ± 0.2 |
E
2
– 12 h prior (pg/ml) | 1.9 ± 0.3 | 1.7 ± 0.4 | 2.6 ± 0.5 | 1.6 ± 0.5 |
E
2
– TCET (pg/ml) | 1.7 ± 0.3† | 1.7 ± 0.4 | 1.9 ± 0.5† | 1.2 ± 0.2 |
P
4
– 24 h prior (ng/ml) | 3.4 ± 0.6 | 2.6 ± 0.4§ | 2.7 ± 0.3§‡ | 2.9 ± 0.6 |
P
4
– 12 h prior (ng/ml) | 3.8 ± 0.4 | 4.0 ± 0.7 | 3.5 ± 0.5§ | 3.4 ± 0.6 |
P
4
– TCET (ng/ml) | 4.1 ± 0.5 | 3.8 ± 0.6§ | 3.7 ± 0.6‡ | 3.6 ± 0.4 |
E
2
: P
4
– 24 h prior
| 0.9 ± 0.3‡ | 0.8 ± 0.3 | 0.9 ± 0.2• | 0.6 ± 0.1 |
E
2
: P
4
– 12 h prior
| 0.5 ± 0.1 | 0.5 ± 0.07 | 0.8 ± 0.3 | 0.6 ± 0.3 |
E
2
: P
4
– TCET
| 0.4 ± 0.08‡ | 0.5 ± 0.2 | 0.6 ± 0.2• | 0.4 ± 0.07 |
The variation in the age, weight, breed, parity, lifetime lamb production and post-partum interval among the penetrated and non-penetrated ewes in this experiment was not significant (p > 0.05, Table
3). Similarly, serum P
4 and E
2 concentrations and E
2 : P
4 ratios at 24 and 12 h prior to TCET and at the time of TCET did not vary between penetrated and non-penetrated ewes (p > 0.05, Table
3). Serum E
2 concentrations and E
2 : P
4 ratio declined (p < 0.05) from 24 h before to just prior to TCET, whereas P
4 levels increased from 24 to 12 h before TCET in non-penetrated ewes (Table
3). In the successfully penetrated ewes, serum concentrations of P4 increased and values for E
2 : P
4 ratio declined from 24 h before to the time of TCET (p < 0.05, Table
3).
Table 3
A comparison of ewe-related parameters, and estradiol/progesterone concentrations/ratios (at 24 h prior to transcervical embryo transfer (TCET) and at the time of TCET) between the penetrated and non-penetrated Rideau Arcott x Polled Dorset ewes
Age (days) | 1524 ± 155 | 1391 ± 88 |
Weight (kg) | 80.6 ± 3.1 | 80.0 ± 2.9 |
Breed (% Rideau Arcott) | 58.1 ± 8.7 | 63.8 ± 4.1 |
Parity
| 2.3 ± 0.5 | 2.1 ± 0.3 |
Lifetime lamb production
| 5.0 ± 1.2 | 4.2 ± 0.7 |
Post-partum interval (d) | 261 ± 20 | 354 ± 70 |
E
2
– 24 h prior (pg/ml) | 2.0 ± 0.4 | 2.0 ± 0.2† |
E
2
– 12 h prior (pg/ml) | 1.6 ± 0.2 | 2.1 ± 0.3 |
E
2
– TCET (pg/ml) | 1.6 ± 0.3 | 1.7 ± 0.2† |
P
4
– 24 h prior (ng/ml) | 2.8 ± 0.4§ | 3.0 ± 0.3§‡ |
P
4
– 12 h prior (ng/ml) | 3.3 ± 0.4 | 3.9 ± 0.3§ |
P
4
– TCET (ng/ml) | 3.8 ± 0.5§ | 3.8 ± 0.3‡ |
E
2
: P
4
– 24 h prior
| 0.9 ± 0.3† | 0.8 ± 0.1• |
E
2
: P
4
– 12 h prior
| 0.6 ± 0.1 | 0.6 ± 0.1 |
E
2
: P
4
– TCET
| 0.5 ± 0.09† | 0.5 ± 0.1• |
The total time (TT) required to perform the procedure and recorded in all ewes or in the penetrated animals only was not correlated with the ewe-related and endocrine parameters or with the VMI values determined in the present experiment (p > 0.05, Table
4). However, the duration of the final attempt (FT) preceding successful cervical penetration and uterine deposition of embryos was negatively correlated (p ≤ 0.05) with the ewes’ age, parity, lifetime lamb productivity and the duration of the post-partum interval (Table
4).
Table 4
Summary of correlations between the ewe-related characteristics and estradiol/progesterone concentrations/ratios at 24 h prior to transcervical embryo transfer (TCET) and at the time of TCET with the total time taken to perform the procedure (TT) and the time of the final attempt (FT) (the latter for penetrated ewes only) determined in 22 Rideau Arcott x Polled Dorset ewes
Age (days) | r = −0.26, p = 0.25 | r = −0.21, p = 0.29 |
r = −0.59, p < 0.05
|
Weight (kg) | r = 0.0007, p = 0.98 | r = 0.01, p = 0.96 | r = 0.02, p = 0.95 |
Breed (% Rideau Arcott) | r = −0.16, p = 0.49 | r = −0.02, p = 0.93 | r = −0.11, p = 0.71 |
Parity
| r = −0.12, p = 0.56 | r = −0.12, p = 0.56 |
r = −0.52, p = 0.05
|
Lifetime lamb production
| r = −0.07, p = 0.76 | r = 0.05, p = 0.81 |
r = −0.59, p < 0.05
|
Post-partum interval (days) | r = −0.27, p = 0.23 |
r = −0.35, p = 0.08
|
r = −0.60, p < 0.05
|
E
2
– 24 h prior (pg/ml) | r = −0.31, p = 0.18 | r = −0.22, p = 0.29 | r = −0.31, p = 0.28 |
E
2
– 12 h prior (pg/ml) | r = −0.03, p = 0.90 | r = −0.07, p = 0.72 | r = −0.36, p = 0.20 |
E
2
– TCET (pg/ml) | r = 0.08, p = 0.72 | r = 0.02, p = 0.93 | r = −0.16, p = 0.59 |
P
4
– 24 h prior (ng/ml) | r = −0.16, p = 0.51 | r = −0.22, p = 0.30 | r = 0.19, p = 0.51 |
P
4
– 12 h prior (ng/ml) | r = −0.15, p = 0.51 | r = −0.15, p = 0.45 | r = 0.44, p = 0.11 |
P
4
– TCET (ng/ml) | r = −0.31, p = 0.16 | r = −0.34, p = 0.08 | r = 0.22, p = 0.45 |
E
2
: P
4
– 24 h prior
| r = −0.12, p = 0.61 | r = −0.04, p = 0.84 | r = −0.39, p = 0.16 |
E
2
: P
4
– 12 h prior
| r = 0.06, p = 0.79 | r = 0.04, p = 0.84 |
r = −0.52, p = 0.06
|
E
2
: P
4
– TCET
| r = 0.22, p = 0.31 | r = 0.21, p = 0.29 | r = −0.40, p = 0.15 |
Serum P4 concentrations on days 29 and 30 of the experiment (9 and 10 days post-TCET, respectively) declined to basal levels in all but two animals. Ultrasonographic pregnancy detection performed 25 days post-TCET confirmed that the two ewes (one from the 12-h and one from the 24-h treatment group) were pregnant; however, a subsequent ultrasonographic scan on day 55 post-TCET revealed that these pregnancies had not been sustained.
Discussion
The extremely low proportion of control ewes successfully penetrated during the present TCET trial indicates that the ovine cervix is highly constricted in the luteal phase of the estrous cycle. The application of Cervidil® has been met with a sizeable dilation of the cervix, especially after the 24-h exposure. The average total time taken to penetrate the cervix was 1–2 min and this is within the range of TT observed in Cervidil®-treated ewes from the transcervical artificial insemination (TCAI) trials in the breeding (unpublished results) and non-breeding seasons [
13].
Kershaw-Young et al. [
14] have shown that PgE
2 mediates its effects on the cervix through four receptors, EP
1 through EP
4; EP
2 and EP
4 especially have been found to be involved in cervical dilation. EP
2 and EP
4 mRNA is expressed in the sheep cervix throughout the estrous cycle. In fact, Kershaw-Young et al. [
14] revealed that a gradient of EP
2 and EP
4 mRNA expression existed along the cervix such that the greatest expression was found in the vaginal and mid-cervical region and the lowest in the uterine region. This indicates that the vaginal region may actually be more responsive to the effects of PgE
2, and considering that dilation of merely the first two caudal folds is thought to produce sufficient cervical dilation for the purpose of TCAI/ET, the local application of Cervidil® may be beneficial. Although the EP
2 mRNA has been shown to be highest prior to the LH surge, the EP
4 mRNA expression remains constant over the estrous cycle. The direct binding of locally applied PgE
2 to constitutively expressed receptors may lead to PgE
2-induced cervical dilation during diestrus.
Cervical dilation prior to parturition is initially induced by a decline in progesterone with a concomitant increase in estradiol [
1]. However, the notion that steroid hormone milieu may influence the effect of Cervidil® on the dilation of ovine cervix in diestrous ewes was not supported in this study. Serum concentrations of E
2, P
4 and E
2 : P
4 ratios generally did not vary between penetrated and non-penetrated ewes, and negative correlations between TT and P
4 levels at TCET and between FT and E
2 : P
4 ratios at 12 h before TCET in successfully penetrated ewes only approached to significance.
Windsor et al. [
15] found a significant effect of ewe parity on cervical penetration rates in synchronized ewes during seasonal anestrus but not in normally cycling, non-synchronized ewes in the breeding season. Some studies have reported a correlation between the breed and cervical penetrability [
16], which may be due to the morphological differences in cervical structure between various genotypes of ewes; in general, the breeds with higher cervical complexity exhibited a lower degree of cervical penetration. There was an apparent effect of breed (specifically, the % of Rideau Arcott genotype) on the penetration rates in the present diestrous ewes. Kaabi et al. [
16] also described a significant correlation of ewe age with cervical penetrability in a group of ewes within a similar age range to that in the present study; however, this may be confounded by factors such as parity. Significant negative correlations between FT and the ewes’ age, parity and lifetime lamb production were found in this study. Buckrell et al. [
17] suggested that penetration success in the ewes undergoing TCAI was affected by the post-partum interval; a shorter interval was shown to significantly increase penetration rate. However, Windsor et al. [
15] reported that cervical penetration rate was not affected by an increase in post-partum interval at TCAI, from 12 to 26 wks after lambing. The results from the present study have indicated that the duration of the post-partum interval from 225 to 1001 days (32 to 143 wks) was inversely related to the ease of cervical penetration or the time taken to traverse the cervix and deposit embryos.
The ultimate testament to the success of Cervidil®-facilitated TCET would be corroboration with sound fertility data comparable to laparoscopic ET or natural mating. In the ewes of the present study, serum P
4 measurements and ultrasonographic imaging enabled the detection of two pregnancies, which were not subsequently maintained until day 55 post-TCET. There are many factors that may have contributed to the low fertility observed in this experiment. The transfer of frozen-thawed ovine embryos is usually associated with lower pregnancy rates compared with the transfer of freshly obtained embryos [
3]. An additional factor that may have contributed to low fertility could have been embryo handling. Considering that the straw containing embryos is loaded into the insemination gun before cervical penetration is attempted, there is a considerable amount of time in which the embryos are exposed to external environment before they are deposited. Minimizing this exposure time would be beneficial since embryonic cells can be very sensitive to even minor fluctuations in temperature or air pressure or to possible contact with pathogens.
Sayre and Lewis [
18], and Campbell et al. [
19] have suggested that the transcervical method itself may have detrimental effects on fertility. Mechanical stimulation during manipulation of the cervix may damage the epithelial lining and result in the activation of sensitive areas triggering the release of various inflammatory mediators that affect uterine tone and create inhospitable environment for embryos [
5,
20]; such molecules may have direct embryocidal effects.
It is unlikely that dinoprostone, Cervidil® inserts and/or cervical plugs had a direct detrimental effect on fertility after TCET. Mammalian embryos secrete large quantities of PgE
2 to facilitate their entry into the uterus and PgE
2 also has a significant involvement in embryo implantation [
21]. It was shown that estradiol upregulates PgE
2 production by the endometrium in sheep and inhibition of PgE
2 synthesis prevents implantation, which can be restored by the administration of endogenous PgE
2[
22].
Acknowledgements
The authors would like to acknowledge the financial support and in-kind contributions from the Department of Biomedical Sciences at the University of Guelph, Ferring Pharmaceuticals Inc., and the Ontario Ministry of Agriculture Food and Rural Affairs. These results were presented, in the preliminary form, at the Annual Meeting of the Polish Society for Biology of Reproduction (February 27-March 1, 2013 in Gdańsk, Poland; Reproductive Biology (Elsevier), Volume 13, Supplement 2, pp. 1–70 (February 2013); Reproductive Biology, Biotechnology, Animal Welfare and Biodiversity, Abstract #6.4).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Both authors contributed equally to the present work, read and approved the final manuscript.