FULL PAPER Theriogenology

Effective Treatment on Placental Expulsion after Foaling in Heavy Draft Mares

Mitsuo ISHII1)*, Syuichi KOBAYASHI1), Tomas J. ACOSTA1), Wataru MIKI2), Motozumi MATSUI1), Takahiro YAMANOI3), Yoh-Ichi MIYAKE1) and Akio MIYAMOTO1)

1)Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-sen, Inada-cho, Obihiro, Hokkaido 080–8555, 2)Hokkaido NOSAI, 612 Motonopporo, Ebetsu-shi, Hokkaido 069–0867 and 3)Hokkai Gakuen University, Faculty of Engineering, Minami 26-jo, Nishi 11- chome, Chuo-ku, Sapporo, Hokkaido 064–0926, Japan

(Received 29 October 2007/Accepted 7 November 2008)

ABSTRACT. The aim of this study was to establish the effectiveness of administration of oxytocin (OT) on placental expulsion after foaling. Four foaling mares with the retained for up 1 hr after foaling received OT (50 IU) administration at 1 hr intervals before expul- sion of the . The changes in the plasma concentrations of OT and the PGF2alpha metabolite (PGFM) were investigated, and the influence of OT administration was considered. The results were as follows. The placenta was expelled after one to three OT admin- istrations in all four mares that received OT. In two mares, which expelled the placenta within 30 min after OT administration, the OT concentration increased and remained high. Expulsion of the placenta was delayed in two mares, and one of these mares, which received three doses of OT beginning 1 hr after foaling, showed only a small increase in the OT concentration after the first administration; the other mare did not receive OT until 3 hr after foaling. The OT concentration was increased before placental expulsion in all the mares, and the PGFM concentration also increased in the two mares with retained placentas. In conclusion, we suggest that intramuscular administration of 50 IU of OT at 1-hr intervals beginning 1 hr after foaling is effective for inducing placental expulsion. KEY WORDS: mare, oxytocin, PGFM, placental expulsion. J. Vet. Med. Sci. 71(3): 293–297, 2009

In heavy draft mares, occurs frequently PG during delivery are mutually and closely related [1, 15]. and can have serious consequences, such as puerperal fever The aim of present study was to establish an effective and laminitis. Extension of the placental retention time method to administer OT after foaling in heavy draft mares. reduce the subsequent conception rate in heavy draft mares For this purpose, we conducted tested several methods of [5]. Although the conception rate of foal heat in mares with administering OT after foaling and monitored the time of a placental retention time of less than 1 hr is 66%, it falls placental expulsion. Changes in the OT and PGF2alpha significantly to 51.7% in mares with a placental retention metabolite (PGFM) concentrations were also analyzed to time that exceeds 4 hr [5]. To promote expulsion of the pla- assist in evaluation of these methods. centa, oxytocin (OT) administration is usually used in mares after foaling. There are some reports that describe effective MATERIALS AND METHODS OT treatments for retained placenta, such as an intravenous drip infusion containing 30–100 IU of OT [3, 14] and Animals: This study was conducted in Kushiro, Hok- repeated subcutaneous or intramuscular administration of kaido, Japan. It involved a total of 4 heavy draft mares from 20–120 IU of OT [4, 7, 16]. Ishii et al. [5] reported that the 4 farms that were mixed breeds belonging to Breton, conception rate at foal heat of heavy draft mares whose pla- Percheron or Belgian Draft hoses with an estimated average centas were expelled after intramuscular administration of body weight of 900 kg after foaling and varying from 6 to 50 IU of OT up to 4 hr after foaling was more than 70%. In 16 years in age. Foaling occurred from March to June, and addition, the conception rate at foal heat of mares that pregnancy ranged from 331 to 340 days in length. All foal- received OT was higher than that of non-OT treated mares ing appeared to be normal, and all foals lived. The owner of among the mares that expelled the placenta 1–4 hr after foal- the mares provided minimal help during delivery of the ing [5]. However, whether this is a more effective method foals. of treatment to not only induce expulsion of the placenta, OT administration: All four mares received OT after but also to improve subsequent reproductive performance foaling. Two mares whose placentas were retained for up to after foaling, has not yet been clarified. (PG) 1 hr after foaling received 50 IU of OT intramuscularly 1 hr F2α might play a role in the contraction stimulus of the myo- after foaling (Nos. 1 & 2; Figs. 1 & 2), and one mare metrium during delivery. The action and release of OT and received three doses of OT at 1 hr intervals for up to 3 hr after foaling because the placenta had not been expelled *CORRESPONDENCE TO: ISHII, M., Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and (No. 3; Fig. 3). The other mare that had a retained placenta Veterinary Medicine, Nishi 2-sen, Inada-cho, Obihiro, Hokkaido received one dose of OT 3 hr after foaling, and OT was 080–8555, Japan. administered once again at 1-hr intervals (No. 4; Fig. 4). e-mail: [email protected] Data and investigation: The placental retention time of 294 M. ISHII ET AL. each mare and whether the foal stood, sucked or was hand- to three OT administrations in all four mares that received milked were recorded. OT administration and changes in OT before placental expulsion (Nos. 1–4). In two (Nos. 1 & circulating OT and PGFM were investigated by comparing 2) of the three mares that received OT 1 hr after foaling, the these data. placenta was expelled around 30 min after administration Blood sampling: Blood samples were collected from 20 (Figs. 1 & 2). In mare No. 3, which received three doses of min before foaling for one mare (No. 4) and immediately OT (50 IU) at 1-hr intervals, the placenta was expelled about after foaling for the other 3 mares; blood sampling contin- 1 hr after the third OT administration (Fig. 3). In the other ued until 2 hr after expulsion of the placenta in all mares. mare, No. 4, which received the first dose of OT 3 hr after Blood samples were collected every 5 min from immedi- foaling, the placenta was expelled 30 min after the second ately after foaling to 1 hr after foaling in all mares. When OT administration at 1-hr intervals (Fig. 4). the placenta was retained, sampling was continued at 5-min Changes in the plasma OT concentration: The fluctua- intervals until expulsion of the placenta, and blood samples were collected 1 and 2 hr after placental expulsion. A 160 mm long 14-G catheter with a 2-m long tube was kept in the jugular vein during the experiment and filled up 10 ml of physiologic saline including 200 IU of heparin sodium after collection of each sample. The blood samplings were repeated carefully with as little stress as possible to the mare. After collection, the blood was placed in a chilled 10 ml glass tube containing ethylenediaminetetraacetic acid (EDTA). The blood was centrifuged immediately after col- lection. The harvested plasma was frozen at –20°C until assayed. OT extraction: The plasma samples (5 ml) were diluted with 5 ml of distilled water, and the pH was adjusted to 2.5. All samples were then applied to a Sep-Pak C18 Cartridge (Waters, Millford, MA, U.S.A.) as described previously [10]. The residue was evaporated and then dissolved in 200 µl assay buffer (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl, 4.8 mM EDTA, 0.05% bovine serum albumin [BSA], Fig. 1. Transition of the OT and PGFM concentrations in the blood of mare No. 1, which received OT 1 hr pH 7.5) for a peptide enzyme immunoassay (EIA). Thus, the after foaling and the placenta was expelled. P: parturi- samples were concentrated 25-fold as a result of this pro- tion M: hand-milking E: expulsion of the placenta cess, which enabled us to determine peptide concentrations OT50: intramuscular administration of 50 IU of OT. in EIA within the range of a standard curve. The recovery rate of OT added to the plasma was 70%. OT determination: The EIA for OT, which has been pre- viously described [12], was based on the second antibody method using the biotin-streptavidin-peroxidase technique [11]. The standard curve for OT ranged from 1.9 to 2000 pg/ml, and the ED50 of the assay was 80 pg/ml. The intra- and interassay CVs were 5.8 and 6.4%, respectively. PGFM extraction: The plasma was extracted by diethyl ether after adjusting the pH of each plasma sample (2 ml) to 3.5 using HCl [10]. The residue was evaporated and then dissolved in 200 µl assay buffer (40 mM PBS, 0.1% BSA, pH 7.2). The samples were concentrated 10-fold as a result of this process. The recovery rate of PGFM added to the plasma was 70%. PGFM determination: PGFM concentrations were deter- mined by a second antibody EIA as reported previously [9]. The standard curve for PGFM ranged from 4 to 1,000 pg/ml, and the ED50 of the assay was 48 pg/ml. The intra- and inter-assay CVs were 7.7 and 13.0%, respectively. Fig. 2. Transition of the OT and PGFM concentrations RESULTS in the blood of mare No. 2, which received OT 1 hr after foaling and the placenta was expelled. P: parturi- Placental expulsion: The placenta was expelled after one tion S: suckling E: expulsion of the placenta OT50: intramuscular administration of 50 IU of OT. EFFECTIVE OXYTOCIN TREATMENT ON PLACENTAL EXPULSION IN HEAVY DRAFT MARES 295

Fig. 3. Transition of the OT and PGFM concentrations in the blood for mare No. 3, which had a retained placenta (exceeding 4 hr after foaling). P: parturition M: hand-milking S: suckling C: Colic SW: sweating E: expulsion of the placenta OT50: intramuscular administration of 50 IU of OT.

Fig. 4. Transition of the OT and PGFM concentrations in the blood for mare No. 4, which had a retained placenta (exceeding 4 hr after foaling). P: parturition M: hand-milking S: suckling C: Colic E: expulsion of the placenta OT50: intramuscular administration of 50 IU of OT. tion range of circulating OT after administration differed two mares that had a retained placenta for over 4 hr after between the individual mares. In the two mares (Nos. 1 & foaling showed that the increase in the range was small after 2) in which the placenta was expelled 30 min after OT the first and second OT administration in one of the mare administration, the OT concentration in the blood increased (No. 3), which received OT starting 1 hr after foaling. After by about 15 pg/ml from the level before administration and the third OT administration, the mare exhibited symptoms remained high until 30 min after OT administration, which of a light colic condition, such as lying down and sweating, is when the placenta was expelled (Figs. 1 & 2). and the maximum increased range of the OT concentration Analysis of the changes in the OT concentration in the (43 pg/ml) was recorded at 1 hr after administration, at 296 M. ISHII ET AL. which time the placenta was expelled (Fig. 3). draft mares, the rate of incidence of retained placenta is high In mare No. 4, which did not receive OT until 3 hr after and is a cause of puerperal fever and laminitis. In the mare, foaling, the OT and PGFM concentrations immediately after the retained placenta needs to be expelled as soon as possi- foaling were observed to be low, 22.1 pg/ml and 2.3 ng/ml, ble. Medical treatments such as intravenous drip infusion or respectively. In this mare, the OT concentration rose gradu- intravenous administration into the umbilical vein [8] are ally following the stimulus of hand-milking or suckling not realistic and are not a choice for owners of foaling mares before OT administration. After receiving two OT adminis- late at night. Therefore, intramuscular administration of 50 trations biginning 3 hr after foaling, the OT concentration IU of OT was considered to be an adequate method after increased rapidly after each injection (Fig. 4). foaling in the present study. Changes in the plasma PGFM concentration: In two The dose of 50 IU of OT was chosen based on the values mares (Nos. 1 & 2), no change in the PGFM concentration normally used by the authors usually used for the purpose of accompanying the rise in the OT concentration of the blood placental expulsion [5]. We have previously reported that was observed (Figs. 1 & 2). the placenta was expelled a short time after foaling in mares In mare No. 3, no increase in the PGFM concentration with a high OT concentration, more than 100 pg/ml, imme- was observed after two OT administrations up to 3 hr after diately after foaling [6]. On the other hand, the OT concen- foaling. However, the PGFM concentration increased after tration immediately after foaling in a mare that had a the third OT administration, and the peak PGFM concentra- retained placenta was noticeably low (22.1 pg/ml) [6]. In tion was recorded 30 min after the third OT administration, order to increase the OT concentration sufficiently in mares or 30 min before the maximum elevation of OT (Fig. 3). after foaling, a fixed, adequate quantity of OT must be pre- In the other mare (No. 4), the PGFM concentration scribed immediately after foaling. However, it is expected increased when the foal suckled before OT administration. that use of increased doses can also produce undesirable Although OT was administered after the first PGFM eleva- side effects resulting from an excessively high concentra- tion, the PGFM concentration in the blood increased again tion of OT. Therefore, we recommend administration of a after OT administration, and the highest PGFM concentra- fixed quantity of OT (50 IU) to mares that do not expel their tion was recorded 40 min after OT administration. placenta during a certain fixed amount of time (1 hr after Although the second OT was injected 20 min after the sec- foaling). ond peak of PGFM, the PGFM concentration did not The OT concentration immediately after foaling of the increase, but the placenta was expelled 30 min after the sec- mare (No. 4) that retained its placenta was low (20 pg/ml), ond OT administration (Fig. 4). and the rise in OT resulted from the stimulus of hand-milk- ing or suckling. This was reflected by an increase in the OT DISCUSSION range of about 20 pg/ml, which was mostly comparable with the increase in the range after administration of 50 IU of OT In the present study, 50 IU of OT was administred to four to mares in the present study. A dose of 50 IU of OT was mares to induce proper placental expulsion. In all the mares, considered to be physiologically proper for mares that had the placenta was expelled within one to three OT (50 IU) retained their placenta and that therefore showed a defi- administrations, and this was accompanied by an increase in ciency of OT in the circulating blood. In order to raise and the circulating OT concentration. This result supports pre- maintain the OT concentration in the blood, the interval for vious studies with various OT treatments, such as an intra- OT administration considered to be most effective was venous drip infusion containing 30–100 IU of OT [3, 14] about 1 hr, which would be in synch with the timing of the and repeated subcutaneous or intramuscular administration next administration before the OT concentration decreased. of 20–120 IU of OT [4, 5, 7, 13, 16], that have been shown Two mares that retained their placentas for more than 4 hr to be effective for discharging the retained placenta in after foaling were administered OT in the present study. In mares. It has been reported that the pregnancy rate also one mare (No. 3), the OT concentration was slightly lower decreases in subsequent breeding of mares with a placental (49.0 pg/ml), and the PGFM concentration was as high as retention time greater than 1 hr [7]. In addition, the concep- 43.9 ng/ml and differed clearly from the level in mare No. 4. tion rate at foal heat of mares that receive OT is significantly The cause of the retained placenta in mare No. 3 is higher (72.4%) than non-OT treated mares (54.0%) among unknown. The OT and PGFM concentrations rose again mares that expel the placenta 1–4 hr after foaling [5]. There- after OT was administered three times, and the placenta was fore, administration of OT 1 hr after foaling to mares that expelled more than 4 hr after foaling. However, the range of have a retained placenta is considered to be the most suitable the increased OT concentration after the first OT adminis- timing. tration was very low (2 pg/ml), but it gradually increased Various treatment methods for the purpose of placental after the second (22 pg/ml) and third (43 pg/ml) administra- expulsion have been reported previously. An intravenous tions. This gradual increase in the OT concentration might low dose or drip infusion of OT for mares that have a have been caused by accumulation of OT after repeated retained placenta has previously recommended [2, 3, 14]. administrations. The OT concentration was remarkably However, most mare deliveries occur during the night, and high (78.3 pg/ml) at the time of placental expulsion follow- it is difficult for veterinarians to attend all foalings. In heavy ing the rise in the PGFM concentration (12.8 ng/ml). Colic EFFECTIVE OXYTOCIN TREATMENT ON PLACENTAL EXPULSION IN HEAVY DRAFT MARES 297 symptoms were observed with the rise in PGFM, and we receptors and prostaglandin in the parturition process in the rat. believe that a strong probably occurred Biol. Reprod. 46: 58-64. at this time. However, the colic symptoms around the time 2. Cox, J. E. 1971. Excessive retainment of the placenta in a of expulsion of the placenta recovered within a short period mare. Vet. Rec. 89: 252-253. of time without treatment, which is required to consider a 3. Held, J. P. 1987. pp. 547-550. Retained Placenta: Current Ther- apy in Equine Medicine, 2nd ed., (Robinson, N. E. ed.), WB treatment method safe and without any side effects. We Saunders, Philadelphia. believed that the uterine contraction accompanied by the 4. Hillman, R. B. 1983. pp. 80-90. Equine Reproduction (Neely rise in the PGFM concentration at this time was due to D. L., Liu I, K. M. and Hillman, R. B. eds.), Hoffmann-La release of endogenous OT and that placental expulsion was Roche Inc, New Jersey. promoted by the interaction of both. The action and release 5. Ishii, M., Jitsukawa, T., Shimamura, T., Utsumi, A., Endo, S. of OT and PG during foaling are mutually and closely and Yamanoi, T. 1999. Effect of placental retention time and related [1, 15]. The relationship between the circulating OT associated treatments on reproductive performance in heavy and PGFM concentrations were clarified not only in the draft horses. J. Equine Vet. Sci. 19: 117-121. stage of expulsion of the foal but also in the stage of expul- 6. Ishii, M., Kobayasi, S., Acosta, J. T., Ogata, H., Shimizu, H., sion of the placenta in the present study. Takeuchi, Y., Kajiya, R., Yamamoto Y., Okamoto, T., Nozawa, T., Kukino, T., Yamanoi, T. and Miyamoto, A. 2003. In mare No. 4, which had also retained its placenta, the Relationship between plasma oxytocin and placental retention circulating OT (22.1 pg/ml) and PGFM (2.3 ng/ml) concen- time immediately after foaling in heavy draft mares. J. Equine trations immediately after foaling were clearly low com- Sci. 13: 101-107. pared with the results of a previous study in which the 7. Jeffcott, L. B. and Rossdale, P. D. 1977. A critical review of adequate level of OT was more than 100 pg/ml [6]; on the current methods for induction of parturition in the mare. other hand, the mean PGFM concentration of the other three Equine Vet. J. 9: 208-215. mares was 34.5 ng/ml in the present study. The deficiency 8. Kovavisarach, E. and Rojsangruang, S. 1998. Effect of umbili- of OT or PGFM immediately after foaling was considered to cal vein oxytocin injection on the third stage of labor: a ran- be caused by the retained placenta. Furthermore, in this domized controlled study. J. Med. Assoc. Thailand. 81: 693- mare, after the OT concentration rose gradually following 697. 9. Meyer, H. H. D., Eisele, K. and Osaso, J. 1989. A biotin- the stimulus of hand-milking or suckling and OT adminis- streptavidin amplified enzymeimmunoassay for 13, 14-dihy- tration (which started 3 hr after foaling and resulted in a rise dro-15-keto-PGF2α. 38: 375-383. in the PGFM concentration), the placenta was expelled. We 10. Miyamoto, A., Tashiro, Y., Nakatsuka, T., Meyer, H. H. D., felt that the OT administration in this case was effective. If Taguchi, K., Abe, N. and Fukui, Y. 1995. Effect of tumor the OT administration had started sooner and was repeated necrosis factor-α and interleukin-1 on local release of progest- earlier after foaling, early uterine contraction and placental erone, prostaglandin F2α and oxytocin in microdialyzed ovine expulsion might be occurred. corpus luteum in vitro. Assist. Reprod. Tec., Androl. 8: 21-32. 11. Miyamoto, A., Kobayashi, S., Arata, S., Ohtani, M., Fukui, Y. CONCLUSION and Schams, D. 1997. Prostaglandin F2α promotes the inhibi- tory action of endothelin-1 on the bovine luteal function in vitro. J. Endocrinol. 152: 7-11. Based on the profiles of the changes in plasma OT and 12. Mutayoba, B., Meyer, H. H. D., Schams, D. and Schallen- PGFM, we suggest that intramuscular administration of 50 berger, E. 1990. Development of a sensitive enzyme immu- IU of OT at 1-hr intervals beginning 1-hr after foaling is noassay for LH determination in bovine plasma using the effective for inducing placental expulsion. streptavidin-biotin technique. Acta Endocrinol. 122: 227-232. 13. Threlfall, W. R. 1993. pp. 614-621. Retained Placenta. Equine ACKNOWLEDGMENTS. The authors wish to thank all of Reproduction. pp. Lea & Febiger, Philadelphia. the owners of the mares in the present study for their gener- 14. Vandeplassche, M., Spincemaille, J. and Bouters, R. 1971. ous offer of samples, their colleagues (Hiroshi Ogata, Hiro- Aetiology, pathogenesis and treatment of retained placenta in michi Shimizu, Yoshizumi Takeuchi, Toshinori Nozawa, the mare. Equine Vet. J. 3: 144-147. Yasuyuki Yamamoto, Taku Okamoto, Tsutomu Shimam- 15. Vivrette, S. L., Kindahl, H., Munro, C. J., Roser, J. F. and Sta- benfeldt, G. H. 2000. Oxytocin release and its relationship to ura, Akihiko Utsumi, Takeshi Fukuda and Tsuyoshi Jit- dihydro-15-keto PGF2alpha and arginine vasopressin release sukawa) at the Kushiro District Agricultural Mutual Aid during parturition and to suckling in postpartum mares. J. Association, and Mr. Jay Andrus for technical assistance. Reprod. Fertil. 119: 347-357. 16. White, T. E. 1980. Retained placenta. Wod. Vet. Pract. 61: 87- REFERENCES 88.

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