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Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares
Theriogenology 66 (2006) 1397–1404 www.journals.elsevierhealth.com/periodicals/the
Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares Johannes Handler a,b,*, Danielle Hoffmann a, Frank Weber c, Dieter Schams d, Christine Aurich b b
a Clinic for Obstetrics, Gynaecology and Andrology, Veterina¨rplatz 1, A-1210 Vienna, Austria Centre for Artificial Insemination and Embryo Transfer, Department of Animal Breeding and Reproduction, University of Veterinary Medicine Vienna, Veterina¨rplatz 1, A-1210 Vienna, Austria c Clinic for Ruminants, Ludwig-Maximilian-University, D-85764 Oberschleißheim, Germany d Research Centre for Milk and Food, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany
Received 25 November 2005; received in revised form 24 April 2006; accepted 26 April 2006
Abstract The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 mg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from 0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4 0.1 cm in control group, 1.5 0.1 in oxytocin and carbetocin group and 1.3 0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare. # 2006 Elsevier Inc. All rights reserved. Keywords: Mare; Early pregnancy; Embryonic growth; Oxytocin; Cervical dilation
1. Introduction * Corresponding author. Tel.: +43 1 25077 5410; fax: +43 1 25077 5490. E-mail address: [email protected] (J. Handler). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.04.032
Economic success of equine embryo transfer is limited by some species-related factors. Factors
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comprise the inability to sufficiently induce superovulation in mares, unsatisfactory synchronization protocols, poor viability of cryopreserved embryos and variable pregnancy rates. Earlier studies revealed lower pregnancy rates obtained by transcervical transfer when compared with surgical transfer (27–77% versus 53–80%) [1–5]. More recent studies yielded much better results by transcervical transfer technique up to pregnancy rates from 65% to 85% [6–10] and even exceeding 90% [11]. However, several factors are under discussion to contribute to variable pregnancy rates when this technique is used: poor uterine and cervical tone [12], bacterial contamination and subsequent inflammation of the endometrium [13,14] as well as the release of PGF2a due to manipulation of the cervix [15,16]. In a recent study, we could demonstrate that cervical manipulation induces significant secretion of oxytocin in cyclic mares [17]. For this purpose, we developed a method for standardized manipulation of the cervix (cervical dilation), because previous studies revealed conflicting results, probably due to different methods for stimulation of the cervix [15,16,18]. In our study, total length of the estrous cycle was significantly shortened by 2 days. Secretion of progesterone by the corpus luteum decreased after dilation, and basal concentration was reached 2 days earlier when compared with controls [17]. Further on, we investigated the effects of cervical dilation on early pregnancy [19]. Surprisingly, cervical dilation at Day 7 after ovulation did neither reduce pregnancy rates nor increase the incidence of early embryonic death. Moreover, embryonic growth was even enhanced when cervical dilation was performed in pregnant mares [19]. The fact that cervical dilation stimulates oxytocin release and embryonic development suggests a potential role of oxytocin in the control of progesterone secretion. This is supported by the important functions of oxytocin in the regulation of luteolysis and maternal recognition of pregnancy [20,21] and secretion of oxytocin by the endometrium [22,23]. Moreover, it has been shown that in human trophoblast cell lines a functional oxytocin receptor contributes to the regulation of cellular proliferation and, thus, to the growth of the conceptus [24]. In the horse, no studies have been performed to clarify the impact of oxytocin on embryonic growth. Therefore, in the present study, we investigated the potential role of oxytocin for the enhanced growth of conceptuses after cervical dilation [19]. The aim of the present study was, to analyze the effects of oxytocin administration on Day 7 post-
ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared with the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. 2. Materials and methods 2.1. Animals and study design Eight healthy Haflinger mares, 3–14 years of age, were checked for estrous cycle stage and breeding soundness by palpation and ultrasonography (5 MHz linear transducer; SV 600, Sonoace, Kretz, Austria) per rectum and vaginal inspection before the trial started. When estrus was detected, mares were mated to a fertile Haflinger stallion (one to three matings per estrus and mare) until ovulation was detected. The day of ovulation was defined as Day 0. Uterus and ovaries were examined by palpation and ultrasonography per rectum daily, from ovulation to Day 13 after ovulation. Diameters of corpora lutea and conceptuses were recorded. At Day 7 after ovulation, two mares at a time had to undergo one of four treatments in a crossover design: (a) non-treated control, (b) oxytocin (Synpitan 10 IU per mare, i.v.), (c) carbetocin (Depotocin 280 mg per mare, i.m.) and (d) cervical dilation. At Day 13, conceptuses were collected by uterine flushing with a phosphate buffered saline (2000 mL) and an embryo-flushing catheter (Cook, Queensland, Australia). The trial was approved by an animal utilization protocol (GZ 68.205/66-BrGT/2003). The mares were kept at the research facility ‘‘Rehgras’’ of the University of Veterinary Sciences, Vienna, from April to October on pasture and during the winter in a loosebox, where they were fed grass or hay and minerals without supplementation of grain. Water was freely available. 2.2. Cervical dilation At first, the mares tail was wrapped and the perineal region and vulva were thoroughly cleaned with soap and a disinfectant (polyvidon-iodine, 1.0%; BraunodermTM, Braun, Austria). Cervical dilation was performed as published recently [17]. Shortly, the cervix-dilator (modified balloon catheter) was inserted into the cervical lumen via a beak-formed vaginoscope and an insertion aid, and the balloon was inflated up to a diameter of 4.5 cm for 10 min. When dilation was completed, the air was withdrawn from the balloon and the catheter was removed.
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2.3. Blood sampling and hormone assays Daily blood samples (Day 0–13) for determination of progesterone were obtained from the jugular vein by use of vacutainer vials. Serial samples (every 10 min for 90 min) for determination of oxytocin were collected by intravenous cannulation of one jugular vein from all mares at the same time (9 a.m.). Treatments started 10 min after the first sampling (Fig. 3). Blood samples were centrifuged immediately after collection for 10 min at 2000 g. Plasma was decanted and stored at 20 8C until assayed. Plasma concentrations of progesterone were determined with an enzyme immunoassay described by Bollwein et al. [25]. Measurements were performed with 5 mL plasma by use of a monoclonal antibody (progesterone-7a-BSA; enzyme: progesterone-3-HRP). The antibody showed 50% cross-reaction with dihydroprogesterone, the biological active progestin in the mare and 100% with progesterone. The intra-assay coefficient of variation was 10% and the inter-assay coefficients of variation were 10% for low (1.5 ng mL 1) and 6% for higher (4 ng mL 1) progesterone concentrations. The radioimmunoassay for oxytocin was performed by a method developed for the use in cattle [26,27] and subsequently validated for equine plasma oxytocin [28]. The lower detection limit ranged between 0.25 and 1 pg mL 1. The intra-assay coefficient of variation was, on average, 6% and the inter-assay coefficients of variation were 9.2 and 14.5% in samples with low and high oxytocin concentrations, respectively. 2.4. Statistical analysis The relationship between hormone (progesterone, oxytocin) and clinical data (diameters of corpora lutea and conceptuses) were tested by ANOVA for repeated measurements and post hoc Fisher’s PLSD test. Results were considered to be significant at P < 0.05. A
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software program (StatViewTM, Abacus, USA) was used for all calculations, e.g. descriptive analyses (mean S.E.M.), area under curve (AUC), ANOVA and Fisher’s PLSD test. Ultrasonographic measurements of the conceptuses diameters were performed daily at a given time (8:00–9:00 a.m.) by only one investigator. Images showing the maximum crosssectional area of the conceptus were frozen at the screen and the longest and perpendicular diameters measured by use of electronic calipers. To increase accuracy, each diameter was calculated as the mean of three different measurements. For better comparison, the size of conceptuses (spherical and elliptic) was demonstrated as maximum cross-sectional area (A = abp; A – area; a,b – radius b perpendicular to maximum radius a). For calculation of growth rates of the conceptuses, a hypothetical diameter was calculated from the crosssectional area of the conceptuses (d = 2 times the square root of the quotient A/p; d – diameter of spherical conceptus). The AUC values for progesterone (ng mL 1 h 1) and oxytocin (pg mL 1 h 1) were calculated for the total sampling periods (daily samples: Day 0–13; serial samples: 90 min). Hormone concentrations exceeding at least two-fold standard deviation of basal values were defined as peaks. Data given are means S.E.M. 3. Results 3.1. Pregnancy rate and embryonic growth Almost all mares were pregnant on Day 13 irrespective of treatment: control – 8/8 mares, oxytocin – 8/8, carbetocin – 8/8 and dilation – 6/8 mares. In one mare transient fluid accumulation at Day 4 of control treatment was detected. Uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death (EED) by Day 11 (conceptus diameter at Day 10: 0.7 cm and Day 13: 0.7 cm) occurred during dilation treatment in the same mare. Another mare, which did
Table 1 Conceptuses growth rates (means S.E.M.) from Day 9 to 13 during treatments (control, oxytocin, carbetocin, dilation) in mares Treatment
Control Oxytocin Carbetocin Dilation
Conceptus growth rate (cm per day) n
Day 9–10
n
Day 10–11
n
Day 11–12
n
Day 12–13
0 1 0 1
– 0.20 – 0.30
1 4 2 5
0.30 0.33 0.02 0.17 0.03 0.32 0.06
6 7 7 6a
0.41 0.04 0.36 0.10 0.34 0.05 0.35 0.10
8 8 8 6a
0.39 0.04 0.40 0.07 0.40 0.04 0.23 0.09
No significant differences could be detected among groups. a One mare did not become pregnant and a second mare developed early embryonic death.
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not become pregnant during dilation treatment, developed signs of endometrial inflammation such as uterine fluid accumulation, uterine edema and reddening of the external cervical os from Day 10 to 14. In 10 out of 30 pregnancies transient mild uterine fluid accumulation (smaller than 0.5 cm in uterine corpus) without uterine edema was noticed irrespective of treatment. In these cases, fluid accumulation seemed not to be caused by inflammation and did not interfere with pregnancy. Daily mean growth rates of conceptuses are shown in Table 1 and daily mean maximum cross-sectional areas in Fig. 1, respectively. Mean growth rates did not differ among treatment groups; growth rates of individual conceptuses varied in a wide range from 0.1 to 0.8 cm per day. In the one case of EED, growth rate was between 0.1 and 0 cm. Early detection of conceptuses (diameters: 0.3 and 0.4 cm) was possible in two mares on Day 9 post-ovulation. At Day 13, conceptuses gained similar mean diameters among groups (1.4 0.1 cm in control group, 1.5 0.1 in oxytocin and carbetocin group and 1.3 0.2 cm in dilation group). 3.2. Corpus luteum At Day 0, the mean maximum cross-sectional area of corpora hemorrhagia did not vary among treatment groups (8.7 1.2 cm2 in control group, 7.4 1.1 in oxytocin, 9.5 0.9 in carbetocin and 9.1 1.5 cm2 in dilation group; Fig. 2a). Mean maximum cross-
Fig. 2. (a) Mean maximum cross-sectional area (means S.E.M.) of corpora lutea during treatments (control, oxytocin, carbetocin, dilation) in mares. No significant differences could be detected among treatment groups; (b) daily plasma progesterone concentrations (means S.E.M.) during treatments (control, oxytocin, carbetocin, dilation) in mares. Progesterone pattern of two mares (dilation-EED), which developed uterine inflammation and early embryonic death after dilation, is separately shown. No significant differences among treatment groups. In two mares (*dilation-EED) progesterone concentrations dropped to basal values within 4 days after dilation.
Fig. 1. Embryonic growth (mean maximum cross-sectional area of conceptuses S.E.M.) during treatments (control, oxytocin, carbetocin, dilation) in mares. No significant differences could be detected among groups.
sectional area of corpora lutea slightly decreased by Day 13 (control: 7.2 1.4 cm2, oxytocin: 6.0 0.4, carbetocin: 6.4 0.6 and dilation: 5.6 0.8 cm2). No statistically significant differences were obtained among treatment groups. From the two mares with early luteolysis in dilation group, the one mare that did
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not conceive showed a markedly reduced size of the corpus luteum by Day 12, while in the mare with EED, the corpus luteum size did not differ from other mares until Day 13. 3.3. Progesterone concentrations Secretion of progesterone was not affected by treatments (Fig. 2b). Progesterone concentrations increased from basal values on Day 0 to maximum concentrations between Days 6 and 9. After that, progesterone concentrations slightly decreased until Day 13. In two mares which showed clinical signs of inflammation and early embryonic death plasma concentrations of progesterone dropped rapidly after cervical dilation at Day 7 to basal values on Days 10 and 11, respectively (Fig. 2b). Total progesterone secretion before (AUCd0–7) and after treatments (AUCd7–13) did not differ among treatments—with the exception of the two mares, which underwent premature luteolysis (control: 569.7 38.7 and 1008.2 72.3 ng mL 1 h 1; oxytocin: 557.1 29.6 and 893.9 58.1; carbetocin: 601.2 48.0 and 910.2 73.7; dilation: 615.2 37.2 and 826.5 139.5 ng mL 1 h 1; P > 0.05).
of oxytocin resulted in three oxytocin peaks in plasma (at 20 min: 125.6, 50 min: 494.8 and 80 min: 85.6 pg mL 1) within 90 min.
3.4. Oxytocin
4. Discussion
Concentrations of oxytocin varied significantly among treatments as demonstrated by AUC values: control 275.9 87.7 pg mL 1 h 1, oxytocin: 3549.7 872.5, carbetocin: 11861.5 2633.2, and dilation: 1105.0 364.8 pg mL 1 h 1 (P < 0.05). Mean plasma oxytocin concentrations are shown in Fig. 3. The fastest increase of oxytocin was obtained after intravenous administration, while intramuscular application of carbetocin resulted in slightly delayed peaks of longer duration. Cervical dilation revealed smallest oxytocin peaks when compared with oxytocin and carbetocin treatment. In individual mares, maximum oxytocin concentrations varied from 2.5 to 17.2 pg mL 1 in controls, 120.4 to 941.8 during oxytocin, 112.6 to 828.3 during carbetocin and 7.2 to 244.4 pg mL 1 during dilation treatment. Maximum peak concentrations differed significantly among treatment groups (control: 6.2 1.7, oxytocin: 362.8 95.2, carbetocin: 450.3 104.6 and dilation: 63.2 26.6 pg mL 1; P < 0.05), with the exception of oxytocin and carbetocin (P > 0.05). One mare showed a small rise of oxytocin concentration (17.2 pg mL 1) during control treatment, while another mare did not respond to dilation with oxytocin release. In one mare, a single intravenous dose
An increase in the knowledge on the effects of cervical manipulation on hormone secretion patterns and embryonic development after transcervical transfer of equine embryos is an important point as its impact on success rates of embryo transfer is still under discussion. The basis for appropriate investigation of the effects of cervical manipulation was the development of a standardized method for cervical dilation [17]. Consequently, we investigated the effects of oxytocin on embryonic development and secretion of progesterone in pregnant mares in the present study. Oxytocin has been shown to directly stimulate pituitary secretion of LH without the involvement of GnRH in estrous mares [29]. So far, no studies have been published to investigate the impact of oxytocin on LH secretion and luteal function in diestrous and early pregnant mares. However, in the present study, cervical dilation and single doses of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect embryonic growth or progesterone secretion. This is in contrast to the results of recent studies where growth of conceptuses was enhanced [19] or duration of luteal phase and progesterone release in non-pregnant mares were adversely affected [17] by cervical dilation.
Fig. 3. Plasma concentrations of oxytocin (means S.E.M.; serial sampling) after treatments (control, oxytocin, carbetocin, dilation). a– d values with different superscripts differ significantly among treatment groups (P < 0.05).
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In that study, increased embryonic growth [19] might be influenced by the administration of xylazine hydrochloride to the mares before cervical dilation, in contrast to the present study. However, that observation needs further investigation, because the impact of the small numbers of mares and the large variability of embryonic growth have to be ruled out as well as potential mechanisms of xylazine hydrochloride have to be identified. Different progesterone patterns after cervical dilation in pregnant mares and cyclic mares [17] give evidence for possible changes of regulation of progesterone secretion during early pregnancy. Therefore, it can be concluded that oxytocin release caused by cervical manipulation seems not to be involved in the variation of pregnancy results after non-surgical embryo transfer. However, the results of the present and recent studies of our group show that even a controlled dilation of the cervix results in very inconsistent effects on progesterone secretion. Two mares showed clinical signs of uterine inflammation such as uterine fluid accumulation and reddening of the external os of cervix uteri. Moreover, plasma progesterone concentrations dropped to basal values within 4 days after cervical dilation. Subsequently, early embryonic death occurred in one mare within a few days, suggesting a contribution of ascending bacterial contamination to EED in this case. These data support earlier findings in the mare [14] and in humans [30,31] that uterine bacterial contamination and successive endometrial inflammation is one of numerous causes for embryonic loss after transcervical transfer of embryos. Thus, Lagneaux et al. [14] observed a pronounced improvement of pregnancy rates after transcervical transfer by changing embryo handling procedures and transfer technique diminishing the frequency of uterine inflammation. Similarly, Wilsher and Allen [11] were able to improve pregnancy rates dramatically after transcervical transfer of equine embryos by inserting the transfer catheter more aseptically via a vaginoscope and fixation of the external cervical os by forceps. In the present study, different routes to increase plasma oxytocin concentrations in mares at Day 7 after ovulation resulted in different patterns of oxytocin concentrations. Cervical dilation induced only a small peak when compared with exogenous oxytocin and carbetocin treatments. The oxytocin profile in response to cervical dilation in pregnant mares was less pronounced with almost one-third lower maximum values when compared with previous data in cyclic mares obtained by frequent sampling (every 30 s) during dilation [17]. This is most likely caused by longer sampling intervals (10 min) in the present study.
More frequent blood sampling [17] revealed a distinct increase of oxytocin to maximum concentrations within 3 min after the beginning of dilation followed by a rapid decrease within 10 min and return to basal concentrations within an hour. Thus, we possibly truncated the oxytocin peak by choosing a longer sampling interval. However, pregnancy is unlikely to cause the smaller peaks during cervical dilation, because Sharp et al. [32] showed similar secretion patterns of oxytocin in pregnant and non-pregnant mares, respectively. The long-acting analogue carbetocin has been used to facilitate milk let down in pigs and cows [33,34], to accelerate uterine involution in cows [35,36] and to synchronize parturition in sows [37]. Reports concerning its use in mares are not available. However, carbetocin also would be beneficial for the treatment of uterine fluid accumulation because of its longer action when compared with oxytocin as shown in the present study. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations, but onset and duration of peaks differed. As expected, maximum concentrations were obtained almost 20 min later after intramuscular application of carbetocin when compared to intravenous administration of oxytocin. However, duration of peaks after injection of the long-acting oxytocin analogue was more than threefold longer than after administration of native oxytocin. Interestingly, one mare in the oxytocin group showed two – one higher and one smaller – oxytocin peaks after the initial one within the sampling period, resembling a wave-like pattern. This observation raises the question of the mechanisms involved. This mare remained pregnant until Day 13 when conceptuses were flushed from the uterus. Thus, there was no evidence of luteolysis and successive possible positive feedback mechanism involving PGF2a, which has been shown in a previous study to induce the secretion of oxytocin in individual mares [38]. The fact that additional peaks were not observed in other mares may be due to the sampling technique used as sampling from the jugular vein reveals much lower concentrations of oxytocin when compared to sampling from the intercavernous sinus [39]. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin on Day 7 after ovulation did not affect progesterone secretion nor area of corpora lutea, pregnancy rate or embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: in the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm did not have negative consequences on progesterone secretion and pregnancy outcome. However,
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cervical dilation caused uterine inflammation, subsequent luteolysis and early embryonic death in one mare. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare. The critical factor seems to be ascending bacterial contamination and direct intrauterine inoculation of bacteria during transfer. Acknowledgements We greatly acknowledge the technical assistance of Mrs. Heidrun Mayrhofer (Oberschleißheim) and Mrs. Gabi Schwentker (Freising-Weihenstephan). References [1] Squires EL, Imel KJ, Juliano MF, Shideler RK. Factors affecting reproductive efficiency in an equine embryo transfer program. J Reprod Fertil Suppl 1982;32:409–14. [2] Juliano MF, Squires ED, Cook VM. Effect of age of equine embryos and method of transfer on pregnancy rate. J Anim Sci 1983;60:258–63. [3] McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer: a review. Comp Cont Educ Pract Vet 1988;10:343–55. [4] Wilson JM, Rowley MB, Rowley WK, Smith HA, Webb RL, Tolleson DR. Successful non-surgical transfer of equine embryos to post-partum lactating mares. Theriogenology 1987;27:295. [5] Sertich PL. Transcervical embryo transfer in performance mares. JAVMA 1989;195:940–4. [6] Pashen RL, Lascombres FA, Darrow MD. The application of embryo transfer to polo ponies in Argentina. Equine Vet J Suppl 1993;15:119–21. [7] Riera FL, McDonough J. Commercial embryo transfer in polo ponies in Argentina. Equine Vet J Suppl 1993;15:116–8. [8] Squires EL, Seidel Jr GE. Collection and transfer of equine embryos. Fort Collins, CO: Colorado State University, Anim Reprod Biotechnol Lab Bull 1995; 11:7–9,11–5,27–32. [9] Meadows S, Lisa H, Welsh C. Factors affecting embryo recovery, embryo development and pregnancy rate in a commercial embryo transfer programme. In: Allen, WR, Wade, JF (Eds.), R&W Publications Ltd, Newmarket, vol. 1. Havemeyer Foundation Monograph Series; 2000, p. 61–6. [10] Jasko DJ. Comparison of pregnancy rates following non-surgical transfer of day 8 equine embryos using various transfer devices. Theriogenology 2002;58:713–6. [11] Wilsher S, Allen WR. An improved method for non-surgical embryo transfer in the mare. Equine Vet Educ 2004;16:39–44. [12] Carnevale EM, Ramirez RJ, Alvarenga MA, McCue PM. Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology 2000;54:965–79. [13] Squires ED, Garcia RH, Ginther OJ. Factors affecting success of equine embryo transfer. Equine Vet J Suppl 1985;3:92–5. [14] Lagneaux P, Tainturier D, Palmer E. Luteolysis and bacterial contamination associated with unsuccessful cervical embryo transfer in the mare. Theriogenology 1988;29:285. [15] Hurtgen JP, Ganjam VK. The effect of intrauterine and cervical manipulation on the equine oestrous cycle and hormone profiles. J Reprod Fertil Suppl 1979;27:191–7.
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[37] Leike J, Huhn U. The synchronization of sow parturition using a combined treatment regimen of cloprostenol Jenapharm and depotocin Spofa. Berl Mu¨nch Tiera¨rztl Wochenschr 1992;105: 345–9. [38] Handler J, Wu¨stenhagen A, Schams D, Kindahl H, Aurich C. Effect of administration of subluteolytic doses PGF2a at Day 7 on estrous cycle and progesterone secretion in Pony mares. Theriogenology 2004;61:1573–82. [39] Vanderwall DK, Silvia WJ, Fitzgerald BP. Concentrations of oxytocin in the intercavernous sinus of mares during luteolysis: temporal relationship with concentrations of 13,14dihydro-15-keto-prostaglandin F2a. J Reprod Fertil 1998; 112:337–46.
Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares Johannes Handler a,b,*, Danielle Hoffmann a, Frank Weber c, Dieter Schams d, Christine Aurich b b
a Clinic for Obstetrics, Gynaecology and Andrology, Veterina¨rplatz 1, A-1210 Vienna, Austria Centre for Artificial Insemination and Embryo Transfer, Department of Animal Breeding and Reproduction, University of Veterinary Medicine Vienna, Veterina¨rplatz 1, A-1210 Vienna, Austria c Clinic for Ruminants, Ludwig-Maximilian-University, D-85764 Oberschleißheim, Germany d Research Centre for Milk and Food, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany
Received 25 November 2005; received in revised form 24 April 2006; accepted 26 April 2006
Abstract The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 mg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from 0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4 0.1 cm in control group, 1.5 0.1 in oxytocin and carbetocin group and 1.3 0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare. # 2006 Elsevier Inc. All rights reserved. Keywords: Mare; Early pregnancy; Embryonic growth; Oxytocin; Cervical dilation
1. Introduction * Corresponding author. Tel.: +43 1 25077 5410; fax: +43 1 25077 5490. E-mail address: [email protected] (J. Handler). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.04.032
Economic success of equine embryo transfer is limited by some species-related factors. Factors
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comprise the inability to sufficiently induce superovulation in mares, unsatisfactory synchronization protocols, poor viability of cryopreserved embryos and variable pregnancy rates. Earlier studies revealed lower pregnancy rates obtained by transcervical transfer when compared with surgical transfer (27–77% versus 53–80%) [1–5]. More recent studies yielded much better results by transcervical transfer technique up to pregnancy rates from 65% to 85% [6–10] and even exceeding 90% [11]. However, several factors are under discussion to contribute to variable pregnancy rates when this technique is used: poor uterine and cervical tone [12], bacterial contamination and subsequent inflammation of the endometrium [13,14] as well as the release of PGF2a due to manipulation of the cervix [15,16]. In a recent study, we could demonstrate that cervical manipulation induces significant secretion of oxytocin in cyclic mares [17]. For this purpose, we developed a method for standardized manipulation of the cervix (cervical dilation), because previous studies revealed conflicting results, probably due to different methods for stimulation of the cervix [15,16,18]. In our study, total length of the estrous cycle was significantly shortened by 2 days. Secretion of progesterone by the corpus luteum decreased after dilation, and basal concentration was reached 2 days earlier when compared with controls [17]. Further on, we investigated the effects of cervical dilation on early pregnancy [19]. Surprisingly, cervical dilation at Day 7 after ovulation did neither reduce pregnancy rates nor increase the incidence of early embryonic death. Moreover, embryonic growth was even enhanced when cervical dilation was performed in pregnant mares [19]. The fact that cervical dilation stimulates oxytocin release and embryonic development suggests a potential role of oxytocin in the control of progesterone secretion. This is supported by the important functions of oxytocin in the regulation of luteolysis and maternal recognition of pregnancy [20,21] and secretion of oxytocin by the endometrium [22,23]. Moreover, it has been shown that in human trophoblast cell lines a functional oxytocin receptor contributes to the regulation of cellular proliferation and, thus, to the growth of the conceptus [24]. In the horse, no studies have been performed to clarify the impact of oxytocin on embryonic growth. Therefore, in the present study, we investigated the potential role of oxytocin for the enhanced growth of conceptuses after cervical dilation [19]. The aim of the present study was, to analyze the effects of oxytocin administration on Day 7 post-
ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared with the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. 2. Materials and methods 2.1. Animals and study design Eight healthy Haflinger mares, 3–14 years of age, were checked for estrous cycle stage and breeding soundness by palpation and ultrasonography (5 MHz linear transducer; SV 600, Sonoace, Kretz, Austria) per rectum and vaginal inspection before the trial started. When estrus was detected, mares were mated to a fertile Haflinger stallion (one to three matings per estrus and mare) until ovulation was detected. The day of ovulation was defined as Day 0. Uterus and ovaries were examined by palpation and ultrasonography per rectum daily, from ovulation to Day 13 after ovulation. Diameters of corpora lutea and conceptuses were recorded. At Day 7 after ovulation, two mares at a time had to undergo one of four treatments in a crossover design: (a) non-treated control, (b) oxytocin (Synpitan 10 IU per mare, i.v.), (c) carbetocin (Depotocin 280 mg per mare, i.m.) and (d) cervical dilation. At Day 13, conceptuses were collected by uterine flushing with a phosphate buffered saline (2000 mL) and an embryo-flushing catheter (Cook, Queensland, Australia). The trial was approved by an animal utilization protocol (GZ 68.205/66-BrGT/2003). The mares were kept at the research facility ‘‘Rehgras’’ of the University of Veterinary Sciences, Vienna, from April to October on pasture and during the winter in a loosebox, where they were fed grass or hay and minerals without supplementation of grain. Water was freely available. 2.2. Cervical dilation At first, the mares tail was wrapped and the perineal region and vulva were thoroughly cleaned with soap and a disinfectant (polyvidon-iodine, 1.0%; BraunodermTM, Braun, Austria). Cervical dilation was performed as published recently [17]. Shortly, the cervix-dilator (modified balloon catheter) was inserted into the cervical lumen via a beak-formed vaginoscope and an insertion aid, and the balloon was inflated up to a diameter of 4.5 cm for 10 min. When dilation was completed, the air was withdrawn from the balloon and the catheter was removed.
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2.3. Blood sampling and hormone assays Daily blood samples (Day 0–13) for determination of progesterone were obtained from the jugular vein by use of vacutainer vials. Serial samples (every 10 min for 90 min) for determination of oxytocin were collected by intravenous cannulation of one jugular vein from all mares at the same time (9 a.m.). Treatments started 10 min after the first sampling (Fig. 3). Blood samples were centrifuged immediately after collection for 10 min at 2000 g. Plasma was decanted and stored at 20 8C until assayed. Plasma concentrations of progesterone were determined with an enzyme immunoassay described by Bollwein et al. [25]. Measurements were performed with 5 mL plasma by use of a monoclonal antibody (progesterone-7a-BSA; enzyme: progesterone-3-HRP). The antibody showed 50% cross-reaction with dihydroprogesterone, the biological active progestin in the mare and 100% with progesterone. The intra-assay coefficient of variation was 10% and the inter-assay coefficients of variation were 10% for low (1.5 ng mL 1) and 6% for higher (4 ng mL 1) progesterone concentrations. The radioimmunoassay for oxytocin was performed by a method developed for the use in cattle [26,27] and subsequently validated for equine plasma oxytocin [28]. The lower detection limit ranged between 0.25 and 1 pg mL 1. The intra-assay coefficient of variation was, on average, 6% and the inter-assay coefficients of variation were 9.2 and 14.5% in samples with low and high oxytocin concentrations, respectively. 2.4. Statistical analysis The relationship between hormone (progesterone, oxytocin) and clinical data (diameters of corpora lutea and conceptuses) were tested by ANOVA for repeated measurements and post hoc Fisher’s PLSD test. Results were considered to be significant at P < 0.05. A
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software program (StatViewTM, Abacus, USA) was used for all calculations, e.g. descriptive analyses (mean S.E.M.), area under curve (AUC), ANOVA and Fisher’s PLSD test. Ultrasonographic measurements of the conceptuses diameters were performed daily at a given time (8:00–9:00 a.m.) by only one investigator. Images showing the maximum crosssectional area of the conceptus were frozen at the screen and the longest and perpendicular diameters measured by use of electronic calipers. To increase accuracy, each diameter was calculated as the mean of three different measurements. For better comparison, the size of conceptuses (spherical and elliptic) was demonstrated as maximum cross-sectional area (A = abp; A – area; a,b – radius b perpendicular to maximum radius a). For calculation of growth rates of the conceptuses, a hypothetical diameter was calculated from the crosssectional area of the conceptuses (d = 2 times the square root of the quotient A/p; d – diameter of spherical conceptus). The AUC values for progesterone (ng mL 1 h 1) and oxytocin (pg mL 1 h 1) were calculated for the total sampling periods (daily samples: Day 0–13; serial samples: 90 min). Hormone concentrations exceeding at least two-fold standard deviation of basal values were defined as peaks. Data given are means S.E.M. 3. Results 3.1. Pregnancy rate and embryonic growth Almost all mares were pregnant on Day 13 irrespective of treatment: control – 8/8 mares, oxytocin – 8/8, carbetocin – 8/8 and dilation – 6/8 mares. In one mare transient fluid accumulation at Day 4 of control treatment was detected. Uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death (EED) by Day 11 (conceptus diameter at Day 10: 0.7 cm and Day 13: 0.7 cm) occurred during dilation treatment in the same mare. Another mare, which did
Table 1 Conceptuses growth rates (means S.E.M.) from Day 9 to 13 during treatments (control, oxytocin, carbetocin, dilation) in mares Treatment
Control Oxytocin Carbetocin Dilation
Conceptus growth rate (cm per day) n
Day 9–10
n
Day 10–11
n
Day 11–12
n
Day 12–13
0 1 0 1
– 0.20 – 0.30
1 4 2 5
0.30 0.33 0.02 0.17 0.03 0.32 0.06
6 7 7 6a
0.41 0.04 0.36 0.10 0.34 0.05 0.35 0.10
8 8 8 6a
0.39 0.04 0.40 0.07 0.40 0.04 0.23 0.09
No significant differences could be detected among groups. a One mare did not become pregnant and a second mare developed early embryonic death.
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not become pregnant during dilation treatment, developed signs of endometrial inflammation such as uterine fluid accumulation, uterine edema and reddening of the external cervical os from Day 10 to 14. In 10 out of 30 pregnancies transient mild uterine fluid accumulation (smaller than 0.5 cm in uterine corpus) without uterine edema was noticed irrespective of treatment. In these cases, fluid accumulation seemed not to be caused by inflammation and did not interfere with pregnancy. Daily mean growth rates of conceptuses are shown in Table 1 and daily mean maximum cross-sectional areas in Fig. 1, respectively. Mean growth rates did not differ among treatment groups; growth rates of individual conceptuses varied in a wide range from 0.1 to 0.8 cm per day. In the one case of EED, growth rate was between 0.1 and 0 cm. Early detection of conceptuses (diameters: 0.3 and 0.4 cm) was possible in two mares on Day 9 post-ovulation. At Day 13, conceptuses gained similar mean diameters among groups (1.4 0.1 cm in control group, 1.5 0.1 in oxytocin and carbetocin group and 1.3 0.2 cm in dilation group). 3.2. Corpus luteum At Day 0, the mean maximum cross-sectional area of corpora hemorrhagia did not vary among treatment groups (8.7 1.2 cm2 in control group, 7.4 1.1 in oxytocin, 9.5 0.9 in carbetocin and 9.1 1.5 cm2 in dilation group; Fig. 2a). Mean maximum cross-
Fig. 2. (a) Mean maximum cross-sectional area (means S.E.M.) of corpora lutea during treatments (control, oxytocin, carbetocin, dilation) in mares. No significant differences could be detected among treatment groups; (b) daily plasma progesterone concentrations (means S.E.M.) during treatments (control, oxytocin, carbetocin, dilation) in mares. Progesterone pattern of two mares (dilation-EED), which developed uterine inflammation and early embryonic death after dilation, is separately shown. No significant differences among treatment groups. In two mares (*dilation-EED) progesterone concentrations dropped to basal values within 4 days after dilation.
Fig. 1. Embryonic growth (mean maximum cross-sectional area of conceptuses S.E.M.) during treatments (control, oxytocin, carbetocin, dilation) in mares. No significant differences could be detected among groups.
sectional area of corpora lutea slightly decreased by Day 13 (control: 7.2 1.4 cm2, oxytocin: 6.0 0.4, carbetocin: 6.4 0.6 and dilation: 5.6 0.8 cm2). No statistically significant differences were obtained among treatment groups. From the two mares with early luteolysis in dilation group, the one mare that did
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not conceive showed a markedly reduced size of the corpus luteum by Day 12, while in the mare with EED, the corpus luteum size did not differ from other mares until Day 13. 3.3. Progesterone concentrations Secretion of progesterone was not affected by treatments (Fig. 2b). Progesterone concentrations increased from basal values on Day 0 to maximum concentrations between Days 6 and 9. After that, progesterone concentrations slightly decreased until Day 13. In two mares which showed clinical signs of inflammation and early embryonic death plasma concentrations of progesterone dropped rapidly after cervical dilation at Day 7 to basal values on Days 10 and 11, respectively (Fig. 2b). Total progesterone secretion before (AUCd0–7) and after treatments (AUCd7–13) did not differ among treatments—with the exception of the two mares, which underwent premature luteolysis (control: 569.7 38.7 and 1008.2 72.3 ng mL 1 h 1; oxytocin: 557.1 29.6 and 893.9 58.1; carbetocin: 601.2 48.0 and 910.2 73.7; dilation: 615.2 37.2 and 826.5 139.5 ng mL 1 h 1; P > 0.05).
of oxytocin resulted in three oxytocin peaks in plasma (at 20 min: 125.6, 50 min: 494.8 and 80 min: 85.6 pg mL 1) within 90 min.
3.4. Oxytocin
4. Discussion
Concentrations of oxytocin varied significantly among treatments as demonstrated by AUC values: control 275.9 87.7 pg mL 1 h 1, oxytocin: 3549.7 872.5, carbetocin: 11861.5 2633.2, and dilation: 1105.0 364.8 pg mL 1 h 1 (P < 0.05). Mean plasma oxytocin concentrations are shown in Fig. 3. The fastest increase of oxytocin was obtained after intravenous administration, while intramuscular application of carbetocin resulted in slightly delayed peaks of longer duration. Cervical dilation revealed smallest oxytocin peaks when compared with oxytocin and carbetocin treatment. In individual mares, maximum oxytocin concentrations varied from 2.5 to 17.2 pg mL 1 in controls, 120.4 to 941.8 during oxytocin, 112.6 to 828.3 during carbetocin and 7.2 to 244.4 pg mL 1 during dilation treatment. Maximum peak concentrations differed significantly among treatment groups (control: 6.2 1.7, oxytocin: 362.8 95.2, carbetocin: 450.3 104.6 and dilation: 63.2 26.6 pg mL 1; P < 0.05), with the exception of oxytocin and carbetocin (P > 0.05). One mare showed a small rise of oxytocin concentration (17.2 pg mL 1) during control treatment, while another mare did not respond to dilation with oxytocin release. In one mare, a single intravenous dose
An increase in the knowledge on the effects of cervical manipulation on hormone secretion patterns and embryonic development after transcervical transfer of equine embryos is an important point as its impact on success rates of embryo transfer is still under discussion. The basis for appropriate investigation of the effects of cervical manipulation was the development of a standardized method for cervical dilation [17]. Consequently, we investigated the effects of oxytocin on embryonic development and secretion of progesterone in pregnant mares in the present study. Oxytocin has been shown to directly stimulate pituitary secretion of LH without the involvement of GnRH in estrous mares [29]. So far, no studies have been published to investigate the impact of oxytocin on LH secretion and luteal function in diestrous and early pregnant mares. However, in the present study, cervical dilation and single doses of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect embryonic growth or progesterone secretion. This is in contrast to the results of recent studies where growth of conceptuses was enhanced [19] or duration of luteal phase and progesterone release in non-pregnant mares were adversely affected [17] by cervical dilation.
Fig. 3. Plasma concentrations of oxytocin (means S.E.M.; serial sampling) after treatments (control, oxytocin, carbetocin, dilation). a– d values with different superscripts differ significantly among treatment groups (P < 0.05).
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In that study, increased embryonic growth [19] might be influenced by the administration of xylazine hydrochloride to the mares before cervical dilation, in contrast to the present study. However, that observation needs further investigation, because the impact of the small numbers of mares and the large variability of embryonic growth have to be ruled out as well as potential mechanisms of xylazine hydrochloride have to be identified. Different progesterone patterns after cervical dilation in pregnant mares and cyclic mares [17] give evidence for possible changes of regulation of progesterone secretion during early pregnancy. Therefore, it can be concluded that oxytocin release caused by cervical manipulation seems not to be involved in the variation of pregnancy results after non-surgical embryo transfer. However, the results of the present and recent studies of our group show that even a controlled dilation of the cervix results in very inconsistent effects on progesterone secretion. Two mares showed clinical signs of uterine inflammation such as uterine fluid accumulation and reddening of the external os of cervix uteri. Moreover, plasma progesterone concentrations dropped to basal values within 4 days after cervical dilation. Subsequently, early embryonic death occurred in one mare within a few days, suggesting a contribution of ascending bacterial contamination to EED in this case. These data support earlier findings in the mare [14] and in humans [30,31] that uterine bacterial contamination and successive endometrial inflammation is one of numerous causes for embryonic loss after transcervical transfer of embryos. Thus, Lagneaux et al. [14] observed a pronounced improvement of pregnancy rates after transcervical transfer by changing embryo handling procedures and transfer technique diminishing the frequency of uterine inflammation. Similarly, Wilsher and Allen [11] were able to improve pregnancy rates dramatically after transcervical transfer of equine embryos by inserting the transfer catheter more aseptically via a vaginoscope and fixation of the external cervical os by forceps. In the present study, different routes to increase plasma oxytocin concentrations in mares at Day 7 after ovulation resulted in different patterns of oxytocin concentrations. Cervical dilation induced only a small peak when compared with exogenous oxytocin and carbetocin treatments. The oxytocin profile in response to cervical dilation in pregnant mares was less pronounced with almost one-third lower maximum values when compared with previous data in cyclic mares obtained by frequent sampling (every 30 s) during dilation [17]. This is most likely caused by longer sampling intervals (10 min) in the present study.
More frequent blood sampling [17] revealed a distinct increase of oxytocin to maximum concentrations within 3 min after the beginning of dilation followed by a rapid decrease within 10 min and return to basal concentrations within an hour. Thus, we possibly truncated the oxytocin peak by choosing a longer sampling interval. However, pregnancy is unlikely to cause the smaller peaks during cervical dilation, because Sharp et al. [32] showed similar secretion patterns of oxytocin in pregnant and non-pregnant mares, respectively. The long-acting analogue carbetocin has been used to facilitate milk let down in pigs and cows [33,34], to accelerate uterine involution in cows [35,36] and to synchronize parturition in sows [37]. Reports concerning its use in mares are not available. However, carbetocin also would be beneficial for the treatment of uterine fluid accumulation because of its longer action when compared with oxytocin as shown in the present study. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations, but onset and duration of peaks differed. As expected, maximum concentrations were obtained almost 20 min later after intramuscular application of carbetocin when compared to intravenous administration of oxytocin. However, duration of peaks after injection of the long-acting oxytocin analogue was more than threefold longer than after administration of native oxytocin. Interestingly, one mare in the oxytocin group showed two – one higher and one smaller – oxytocin peaks after the initial one within the sampling period, resembling a wave-like pattern. This observation raises the question of the mechanisms involved. This mare remained pregnant until Day 13 when conceptuses were flushed from the uterus. Thus, there was no evidence of luteolysis and successive possible positive feedback mechanism involving PGF2a, which has been shown in a previous study to induce the secretion of oxytocin in individual mares [38]. The fact that additional peaks were not observed in other mares may be due to the sampling technique used as sampling from the jugular vein reveals much lower concentrations of oxytocin when compared to sampling from the intercavernous sinus [39]. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin on Day 7 after ovulation did not affect progesterone secretion nor area of corpora lutea, pregnancy rate or embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: in the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm did not have negative consequences on progesterone secretion and pregnancy outcome. However,
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cervical dilation caused uterine inflammation, subsequent luteolysis and early embryonic death in one mare. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare. The critical factor seems to be ascending bacterial contamination and direct intrauterine inoculation of bacteria during transfer. Acknowledgements We greatly acknowledge the technical assistance of Mrs. Heidrun Mayrhofer (Oberschleißheim) and Mrs. Gabi Schwentker (Freising-Weihenstephan). References [1] Squires EL, Imel KJ, Juliano MF, Shideler RK. Factors affecting reproductive efficiency in an equine embryo transfer program. J Reprod Fertil Suppl 1982;32:409–14. [2] Juliano MF, Squires ED, Cook VM. Effect of age of equine embryos and method of transfer on pregnancy rate. J Anim Sci 1983;60:258–63. [3] McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer: a review. Comp Cont Educ Pract Vet 1988;10:343–55. [4] Wilson JM, Rowley MB, Rowley WK, Smith HA, Webb RL, Tolleson DR. Successful non-surgical transfer of equine embryos to post-partum lactating mares. Theriogenology 1987;27:295. [5] Sertich PL. Transcervical embryo transfer in performance mares. JAVMA 1989;195:940–4. [6] Pashen RL, Lascombres FA, Darrow MD. The application of embryo transfer to polo ponies in Argentina. Equine Vet J Suppl 1993;15:119–21. [7] Riera FL, McDonough J. Commercial embryo transfer in polo ponies in Argentina. Equine Vet J Suppl 1993;15:116–8. [8] Squires EL, Seidel Jr GE. Collection and transfer of equine embryos. Fort Collins, CO: Colorado State University, Anim Reprod Biotechnol Lab Bull 1995; 11:7–9,11–5,27–32. [9] Meadows S, Lisa H, Welsh C. Factors affecting embryo recovery, embryo development and pregnancy rate in a commercial embryo transfer programme. In: Allen, WR, Wade, JF (Eds.), R&W Publications Ltd, Newmarket, vol. 1. Havemeyer Foundation Monograph Series; 2000, p. 61–6. [10] Jasko DJ. Comparison of pregnancy rates following non-surgical transfer of day 8 equine embryos using various transfer devices. Theriogenology 2002;58:713–6. [11] Wilsher S, Allen WR. An improved method for non-surgical embryo transfer in the mare. Equine Vet Educ 2004;16:39–44. [12] Carnevale EM, Ramirez RJ, Alvarenga MA, McCue PM. Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology 2000;54:965–79. [13] Squires ED, Garcia RH, Ginther OJ. Factors affecting success of equine embryo transfer. Equine Vet J Suppl 1985;3:92–5. [14] Lagneaux P, Tainturier D, Palmer E. Luteolysis and bacterial contamination associated with unsuccessful cervical embryo transfer in the mare. Theriogenology 1988;29:285. [15] Hurtgen JP, Ganjam VK. The effect of intrauterine and cervical manipulation on the equine oestrous cycle and hormone profiles. J Reprod Fertil Suppl 1979;27:191–7.
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