Acta Veterinaria Hungarica 56 (1), pp. 125–131 (2008) DOI: 10.1556/AVet.56.2008.1.13

MELENGESTROL ACETATE AS A TOOL FOR INDUCING EARLY OVULATION IN TRANSITIONAL MARES Consuelo LÓPEZ-BAYGHEN1, Heidy ZOZAYA1, Luis OCAMPO1, Gordon W. BRUMBAUGH2 and Héctor SUMANO1* 1

Department of Physiology and Pharmacology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico (UNAM), Av. Universidad 3000, Coyoacán, Mexico City 04510, Mexico; 2Pfizer Animal Health, Pfizer Inc., Wellborn, TX, USA (Received 26 January 2007; accepted 19 April 2007)

The efficacy of melengestrol acetate (MGA) to shorten the vernal transition of mares by synchronising and accelerating the first ovulation of the year after 60 days of phototherapy was determined by ultrasonographic monitoring. Sixteen mares in late transition were fed two doses of MGA (150 mg/mare/day and 100 mg/mare/day, respectively) for 10 days. A luteolytic dose of prostaglandin was administered to each mare one day after the end of MGA treatment. The presence and duration of oestrus, follicular growth, uterine oedema and presence of ovulation were monitored by ultrasonography and the cervical tone was evaluated by rectal palpation. Ovulation was detected in 87.5% of the mares treated with 150 mg MGA/mare/day for 10 days, and in 62.5% of the mares receiving 100 mg MGA/mare/day for 10 days. This was statistically different (P = 0.03) from the untreated control mares having an ovulation rate of 20%. Mares that received 150 mg MGA/day for 10 days had a mean treatment to ovulation interval of 13.1 ± 5.97 days after the end of treatment, while mares that received 100 mg MGA/day for 10 days had a mean of 25.6 ± 10.50 days (P = 0.01) to ovulation. These results suggest that MGA can be used for synchronising and hastening the first ovulation of the year in mares. Key words: Mare, transition, oestrus, ovulation

It is well known that the reproductive efficiency of mares during the vernal transitional period is low in terms of oestrus cycling. The use of progesterone *

Corresponding author; E-mail: [email protected]; Phone: 0052 (55) 56225980; Fax: 0052 (55) 56225980 0236-6290/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

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or synthetic progestins (i.e. progesterone in oil and altrenogest) is useful to control the irregular, often anovulatory, oestrous periods and to accelerate the date of the first ovulation of the year in mares (McKinnon et al., 1997). However, the authors found no reports about the oral administration of melengestrol acetate (pregnan-4,6-diene-3,20-dione, 17-hydroxy-6-methyl-16-methylene-acetate; or MGA) in mares for such purpose. Its ease of administration, low cost and potential to induce oestrus renders MGA a good choice for use in mares. In the bovine, the dose for suppressing and synchronising oestrus is 0.5 mg MGA/day/animal during a 7-day period (Perry, 2002). The synchronisation, conception and pregnancy rates are high with this dosage regimen in heifers. If administered for 14 days, oestrus synchrony is good but fertility decreases (Zimbelman et al., 1970). Considering the above, we hypothesised that MGA would have acceptable bioavailability in mares when administered in the feed; and therefore, would be a viable alternative to synchronise the first ovulation of the year during the late transitional period. The purpose of this study was to determine, by ultrasonographic monitoring, the efficacy of MGA in shortening the vernal transition while synchronising and hastening the first ovulation of the year in mares.

Materials and methods In order to avoid irregular oestrous cycles of younger or older mares, 21 Thoroughbred mares between 4 and 10 years of age were used. They were housed on the same premises in Northeast Mexico (25° latitude, 100° longitude) with an average annual temperature of 24 °C and under the same feeding regimen. None of the animals had a history of reproductive problems, as they were not usually used as breeders, and none had any important medical condition that could affect their reproductive performance. They were all examined in November 2004 to determine their ovarian activity. When they were confirmed to be anoestrous, they were randomly assigned into one of three groups as follows: Group A: Eight mares received 150 mg MGA (Pfizer de México)/mare/ day for 10 days orally with their morning feed. Group B: Eight mares received 100 mg MGA/mare/day for 10 days. Group C: Five mares served as nonmedicated control. The MGA used in this study was obtained from the manufacturer and it is the same product as that used for cattle. Phototherapy was started on 15 December 2004 for all the mares in the three groups, increasing the photoperiod to 16 hours light/day with a 200-Watt light-bulb for 60 days (Ginther, 1979; Samper, 2000). After the MGA treatment was completed, Dinoprost-tromethamine was administered at a dose of 5 mg/mare by deep intramuscular injection on day 71 (Meyer, 1997).

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Serum samples were obtained for determination of progesterone by microplate ELISA as suggested by Nagy et al. (1998) on the day prior to starting treatment with MGA, on the last day of MGA treatment (before administering prostaglandin), and on day 5 postovulation (if ovulation occurred). Rectal ultrasonographic evaluations were performed with an Aloka 500 SD ultrasound machine with a 5 mHz lineal transducer (Fraser et al., 1973) and all images were printed according to the following time schedule: during phototherapy (from day 1 to day 60), twice weekly, making a total of 18 exams per mare; one evaluation on day 61 (prior to MGA treatment), and daily, starting from day 72 until ovulation. Mares that did not ovulate were examined up to 48 days after finishing treatment. Uterine oedema was graded from 0 to 3 and the ovarian follicular diameter measured in mm according to Ginther and Pierson (1983) and McKinnon (1987). Data recorded included the presence and duration (days) of oestrus as estimated by follicular growth, cervical tone, uterine oedema and ovulation. The SAS System for Windows (The SAS Institute, 9.0) was used to perform an analysis of variance to determine the difference in mean follicular size before and after treatment with MGA. An exact Fisher test was used to determine differences in the percentage of ovulated mares according to Kuehl (1994).

Results A total of 21 mares were checked by means of 1109 ultrasound exams during a period of 5 months, from 12 November 2004 to 31 March 2005. The mean ± SD values for days to ovulation, percentage of ovulated mares, mean uterine oedema grade and cervical tone of each group, as well as the statistically significant differences for the three groups are shown in Table 1. Serum progesterone concentrations are summarised in Table 2. Table 1 Days to ovulation after treatment (mean), percentage of ovulated mares, grade of oedema (mean) and cervical tone in mares in Group A (150 mg/mare/day MGA), Group B (100 mg/mare/day MGA) and Group C (control)

Treatment group

A (150 mg/mare/day) B (100 mg/mare/day C (untreated control)

Mean ± SD Treatment to ovulation interval

Per cent of mares that ovulated

Mean ± SD grade of uterine oedema at ovulation*

13.1 ± 5.97a 25.6 ± 10.50b 44c

87.5%a 62.5%a 25%b

1.71 ± 1.25 1.2 ± 1.09 3

Cervical tone Closed (% of mares)

Relaxed (% of mares)

42.85% 20%

57.14% 80% 100%

a, b, c Within columns, different letters mean statistically significant means (P < 0.05); *0 = none; 1 = low; 2 = moderate; 3 = high

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Table 2 Mean serum progesterone concentrations (ng/ml) of the mares in the three groups before and after treatment with MGA and at day 5 after ovulation Progesterone (ng/ml) Day 60 (before MGA treatment)

Group

A B C

Progesterone (ng/ml) Day 72 (after MGA treatment)

0.3 ± 0.5 0.2 ± 0.9 0.3 ± 0.7

Progesterone (ng/ml) Day 5 after ovulation

1.6 ± 1.1 1.4 ± 1.4 0.8 ± 1.7

8.4 ± 8.1 7.6 ± 10.2 6

After treatment with MGA an increase in follicular activity could be observed, mainly in the mares which ovulated. The mean follicular diameters per day and per group are shown in Fig. 1 and they were fitted into Gaussian regression curves. In most mares, the dominant follicle started to grow and reached ovulation at different days after finishing treatment, while the rest of the follicles remained practically the same size. Ovulation group A

55

Group A Group B Group C

50

Follicular diameter (mm)

45 Ovulation group B Only on day 87

40 35 30 25 20 15 10 60

65

70

75

80

85

90

Time (days) Fig. 1. Gaussian regression curves for mean follicular diameter size (mm) of mares treated from day 60 to 70, as follows: Group A, 150 mg/mare/day, Group B, 100 mg/mare/day and Group C, untreated control group. Dotted lines mark ovulation period in Group A and ovulation day in Group B. Untreated mares in Group C did not ovulate during this period

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In a few mares, some follicles larger than 40 mm became atretic and did not ovulate, as detailed below: (1) Group A. Mare 2: The largest follicle measured 40 mm thirty days after finishing treatment and decreased to 28 mm 17 days afterwards. (2) Group B. Mare 1: The largest follicle measured 43 mm 26 days after finishing treatment and decreased to 30 mm 18 days afterwards. Mare 4: The largest follicle measured 55 mm 28 days after finishing treatment and decreased to 40 mm 19 days afterwards. Mare 8: The largest follicle measured 50 mm 27 days after finishing treatment and decreased to 30 mm 20 days afterwards. (3) Group C. Mare 1: The largest follicle measured 40 mm 36 days after the other two groups finished treatment and decreased to 22 mm 11 days afterwards. Mare 3: The largest follicle measured 52 mm 12 days after the other two groups finished treatment and decreased to 18 mm 21 days afterwards. A statistical analysis of variance for non-normally distributed data (Kuehl, 1994) using the SAS System revealed that there was no statistical difference (P = 0.92) in the follicular size of the twenty-one mares before starting treatment with MGA. The same method was used to determine that the mares in Group A (150 mg/kg/day) had larger follicle sizes than mares in treatment group B (P = 0.048) and control group C (P = 0.04) one day after the end of treatment (day 72), but no statistical difference could be found (P = 0.96) when comparing mares from treatment group B and control group C. The follicular growth rate was determined by estimating the difference in follicular diameter on days 1 and 8 post-treatment divided by the number of days, considering only the follicles that ovulated. The daily follicular growth rate was statistically different when comparing all groups (P = 0.27). The preovulatory follicular sizes observed were relatively homogeneous in all groups. That is, all ovulated follicles had a diameter of 40 to 51 mm. A Student’s t-test (Kuehl, 1994) was used to compare the mean time to ovulation. The ovulations in the treated mares in Group A occurred earlier (P = 0.01) after finishing treatment (13.1 ± 5.97 days) compared to that for mares in Group B (25.6 ± 10.5 days). Also, the ovulation rate was higher (P = 0.03) in the treated mares in Group A (87.5%) and B (62.5%) as compared to that in Group C (20%). However, no statistical difference was found (P = 0.36) between mares in Group A and mares in Group B. When considering the mean time to ovulation, three mares in Group A (mare 4, mare 7 and mare 8) ovulated two dominant follicles. The ovulation registered was the one that happened first, except for mare 4, which ovulated both follicles on the same day (day 9 after finishing treatment). In the treatment to ovulation interval a statistical difference (P = 0.01) was found between mares in Group A (13.1 ± 5.97 days) and mares in Group B (25.6 ± 10.50 days). The mare in the untreated control group C ovulated on day 44. A statistically significant difference (P = 0.001) was found between the mean time to ovulation of the treated mares in Group A and the untreated mare in Acta Veterinaria Hungarica 56, 2008

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Group C, as well as between the treated mares in Group B and the untreated mare in Group C (P = 0.010). The duration of oestrus in mares of Group A and Group B was 8.6 days and 19.5 days, respectively. Mares in Group C had a mean oestrus length of 15 days, with only one mare ovulating.

Discussion Because no previous studies with MGA in mares were known to the authors, it was decided to use the dosing schedule of 100 or 150 mg/mare/day for 10 days, considering that the oral bioavailability of this drug in cattle ranges between 10 to 20% (Zimbelman et al., 1970). Toxicity with such doses is unlikely, as the intravenous dose of MGA of 125 mg/horse/day did not have any toxic effects (Zimbelman et al., 1970). To avoid behavioural or pheromonal influences on oestrus presentation, the use of a teaser stallion was avoided (Oxender et al., 1978; McKinnon and Voss, 1993; Nagy et al., 2000). Evaluation criteria were limited to internal morphological changes on uterine oedema and follicular growth as observed through ultrasonography, and to cervical relaxation and tone judged by rectal palpations. Treatment with prostaglandins to eliminate all progesterone of ovarian origin, as used in this trial, is widely accepted when exogenous progesterone or progestins are utilised to induce oestrus in mares (Asbury, 1991; McKinnon et al., 1997; Barrier-Battut et al., 2001). There appeared to be no correlation between treatment and length of oestrus. However, no evaluation was done to determine if these cycles were fertile. The mares used in this study were not intended for reproduction. Yet, one mare from Group A was inseminated shortly afterwards and became pregnant. The other mares treated were followed until their subsequent oestrus and it was concluded that they had started to cycle regularly. It is possible to suggest that MGA can be a viable alternative to shorten the transitional period from vernal anoestrus and hasten the first ovulation of the year in mares. However, further studies are required to assess if such induced oestrous cycles are fertile and to define oral bioavailability and pharmacokinetics of MGA, as well as its potential toxicity. References Asbury, A. C. (1991): Examination of the mare. In: Mansmann, R. A. (ed.) Equine Medicine & Surgery. American Veterinary Publishers, Inc., Santa Barbara, CA. pp. 954–955. Barrier-Battut, I., Poutre, N. T. E., Hechst, S., Raux, A. G. N., Bertrand, J., Fieni, F., Hoier, R., Renault, A., Egron, L., Tainturier, D. and Bruyas, J. F. (2001): Use of buserelin to induce ovulation in the cyclic mare. Theriogenology 55, 1679–1695. Acta Veterinaria Hungarica 56, 2008

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Fraser, A. F., Keith, N. W. and Hastie, H. (1973): Summarized observations on the ultrasonic: Detection of pregnancy and foetal life in the mare. Vet. Rec. 92, 20–21. Ginther, O. J. (1979): Reproductive Biology of the Mare. Basic and Applied Aspects. Equiservices. pp. 83–108, 233–290. Ginther, O. J. and Pierson, R. A. (1983): Ultrasonic evaluation of the reproductive tract of the mare: Principles, equipment and techniques. J. Eq. Vet. Sci. 3, 195–201. Kuehl, R. O. (1994): Statistical Principles of Research Design and Analysis. Sci. 28, 72–92. Duxbury Press, Belmont, CA, USA. McKinnon, A. O. (1987): Diagnostic ultrasonography of uterine pathology in the mare. Proceedings of the American Association of Equine Practitioners. pp. 605–622. McKinnon, A. O. and Voss, J. L. (1993): Equine Reproduction. Lea & Febiger, Philadelphia, USA. McKinnon, A. O., Vasey, J. R., Lescun, T. B. and Trigg, T. E. (1997): Repeated use of the GnRH analogue deslorelin (Ovuplant) for hastening ovulation in the transitional mare. Equine Vet. J. 29, 153–155. Meyer, J. P. (1997): Control and synchronization of the estrus cycle and ovulation. In: Morrow, E. P. (ed.) Current Therapy in Large Animal Theriogenology. W. B. Saunders, Philadelphia. pp. 166–171. Nagy, P., Guillaume, D. and Dales, P. (2000): Seasonality in mares. Anim. Reprod. Sci. 60–61, 245–262. Nagy, P., Solti, L., Kulcsár, M., Reiczigel, J., Huszenica, G. and Abaváry, K. (1998): Progesterone determination in equine plasma using different immunoassays. Acta Vet. Hung. 46, 501–513. Oxender, W. D., Noden, P. A. and Hafs, H. D. (1978): Oestrus, ovulation and serum progesterone, estradiol and LH concentrations in mares after an increased photoperiod during winter. Am. J. Vet. Res. 38, 203–207. Perry, G. A., Kojima, F. N., Salfen, B. E., Bader, J. F., Patterson, D. J. and Smith, M. F. (2002): Effect of an orally active progestin on follicular dynamics in cycling and anestrous postpartum beef cows. J. Anim. Sci. 80, 1932–1938. Samper, J. (2000): Equine Breeding Management and Artificial Insemination. W. B.Saunders Co., Philadelphia. Zimbelman, R. G., Lauderdale, J. W., Sokolowsky, J. H. and Schalk, T.G. (1970): Safety and pharmacologic evaluations of melengestrol acetate in cattle and other animals: a review. J. Am. Vet. Med. Assoc. 157, 1528–1536.

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