University of Wisconsin, Madison, WI 53706, USA. 2Departamento ... This research was supported by the College of Agricultural and Life Sciences, University of.
ELSEVIER
EFFECT OF OXYTOCIN, PROSTAGLAND1N F2~, AND CLENBUTEROL ON UTERINE DYNAMICS IN MARES M.O. Gastal, 1'2 E.L. Gastal, 1 C.A.A.Torres 2 and O.J. Ginther I 1Animal Health and Biomedical Sciences University of Wisconsin, Madison, WI 53706, USA 2Departamento de Zootecnia Federal University of Vi~osa, Vi~osa, MG 31570, Brazil Received for publication: Harch 18, 1998 Accepted: June 25, 1998 ABSTRACT The effects of oxytocin, prostaglandin F2~ (PGF2~), and clenbuterol on uterine contractility and tone during anestrus and diestrus, and during mobility and postfixation of the embryonic vesicle were studied in 51 pony mares. Contractility was assessed by scoring real-time ultrasound images, and tone was assessed by transrectal digital compression. Scoring was done by an operator who had no knowledge of treatment assigments. In anovulatory mares primed with progesterone for 16 d, oxytocin did not significantly alter contractility but did stimulate an increase in tone, whereas clenbuterol depressed both contractility and tone. The PGF2a given on Days 12, 15, and 18 did not significantly alter uterine contractility in pregnant mares, but it increased contractility on all days in nonpregnant mares. Clenbuterol decreased both tone and contractility when given to pregnant mares on the day of embryonic-vesicle fixation, while it decreased tone but not contractility when given on Day 19. Clenbuterol treatment was associated with dislodgment of the fixed embryo in only 1 of 5 mares. However, on Day 19, clenbuterol treatment was associated with a change in shape of the conceptus when viewed in a cross section of the uterine horn. The conceptus shape became more circular rather than irregular or triangular, as indicated by a significant decrease in the variation in the distances between adjacent walls measured in 4 different directions. Results indicated that: 1) oxytocin increased uterine tone but did not alter contractility in progesterone-primed anestrous mares; 2) on Days 12, 15 and 18, PGF2~ increased uterine contractility in nonpregnant mares but not in pregnant mares; 3) clenbuterol decreased both tone and contractility at all reproductive states except for a lack of a decrease in contractility on Day 19 of pregnancy; and 4) reduction in uterine tone from clenbuterol treatment on Day 19 was associated with a change in the two-dimensional shape of the in situ conceptus from irregular to a more circular form. © 1998 by ElsevierScience Inc.
Key words: uterine contractility, uterine tone, oxytocin, PGF2a, clenbuterol, mare
Acknowledgments This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by EquiCulture, Inc., Cross Plains, Wisconsin. The authors thank Pharmacia & Upjohn Co. for the donation of Lutalyse. M.O.G. and E.L.G. were supported by the Federal University of Vi~osa and by a CAPES scholarship from Brazil. Correspondence and reprint requests to O.J. Ginther. Theriogenology 50:521-534, 1998 © 1998 by Elsevier Science Inc.
0093-691X/98/$19.00 PII S0093-691X(98)00158-7
Theriogenology
522
INTRODUCTION The early equine conceptus is mobile, traveling to all parts of the uterus 10 to 20 times a day, especially on Days 12 to 14 (ovulation=Day 0) in ponies (reviewed in 16,17). This mobility is temporally associated with increased ultrasonically-assessed uterine contractility (11). Clenbuterol, a selective 152 adrenergic agonist, interfered with the mobility of the embryonic vesicle (29), presumably by diminishing uterine contractions. During embryo mobility, the uterus increases in tone and decreases in diameter (reviewed in 16,17). Apparently, the increasing uterine tone together with increasing growth of the embryonic vesicle leads to cessation of mobility (fixation) on mean Day 15 in ponies. Fixation occurs at a flexure in the caudal portion of one of the uterine horns. By 2 d after fixation, the expanding and spherical embryonic vesicle assumes an irregular or triangular shape when viewed by ultrasound in a cross section of the turgid uterine horn. Progesterone is needed for mobility, fixation, and postfixation shape changes (15,27). Ultrasonography has been used to study uterine contractility by evaluating real-time images of wave-like movements or the to-and-fro movements of endometrial reflectors in mares (10,11,18,19), cattle (6), and women (1,25,26). In nonpregnant mares, maximum uterine contractility occurs during luteolysis (Days 14 to 18), whereas in early pregnant mares, maximum uterine contractility occurs during maximum mobility of the embryonic vesicle (11). Studies involving trapping of the embryo by surgical ligation of a uterine horn have indicated that the embryo locally stimulates uterine contractility (22) which, presumably, results in embryo mobility. The substance produced by the embryo that stimulates uterine contractility is not known. The maximum uterine contractility during the time of luteolysis at the end of diestrus suggests that prostaglandin F2et (PGF2ct) may be involved in uterine contractility since PGF2~ is released at that time (16). Anovulatory mares treated daily with either estradiol or progesterone developed increased uterine contractility in 4 and 14 d, respectively (10), indicating that the steroidal environment is involved. In the same study, a PGF2~ injection after 10 d of progesterone treatment resulted in additional contractility in 30 min. A gradual increase in uterine thickness or tone occurs in both nonpregnant and pregnant mares between Days 0 to 6, followed by a subsequent decrease between Days 6 to 10 (3,5,21). After Day 10 in nonpregnant mares, a transient tone increase occurs for a few days (5), but in pregnant mares the increase continues, reaching a plateau on Days 25 to 30 (3,5,22,24,38). In anovulatory and ovulatory mares, progesterone stimulated uterine tone equivalent to the tone of diestrus (2,24). When the progesterone was given for a prolonged time, the tone continued to increase, mimicking the turgidity of pregnancy by Day 30 (5). Estradiol apparently also plays a role since the uterine tone of early pregnancy was best simulated by low doses of estradiol after priming for 2 wk with progesterone (24). The conceptus may be the estradiol source. Estrogens are produced by the conceptus (39) at the time when uterine turgidity is increasing, and isolation of the conceptus by uterine surgical ligation has demonstrated that the conceptus stimulates tone locally (22). In this regard, exogenous estradiol hastened the day of fixation of the embryonic vesicle and tended to increase uterine tone (4). Changes in intrauterine pressure, electrical activity, and palpable uterine tone occur after administration of uterokinetic agents (7,8,23,28,34,37). These studies indicated that intrauterine pressure and electrical activity increased after PGF2ot or oxytocin injection. A rapid rise in
Theriogenology
523
intrauterine pressure occurred after oxytocin treatment in ovulatory and anovulatory mares (23,32). Clenbuterol decreased palpable uterine tone during the first third of pregnancy (8) but seldom caused a reduction in electrical activity (28). In addition, clenbuterol decreased the prevalence of apparent segmental constrictions in pregnant mares on Days 67 to 72 (20). The present study had 2 objectives: 1) to measure the response of uterine contractility and tone to oxytocin, PGF2ct and clenbuterol during anovulatory, cyclic, and early pregnancy phases and 2) to study the effect of uterine contractility and tone on location of the fixed embryonic-vesicle and the postfixation shape of the vesicle. MATERIALS AND METHODS Pony mares weighing 174 to 376 kg and 2 to 20 yr of age were used in 4 experiments. The experiments were conducted from February through November, Northern Hemisphere. Within each experiment, mares were randomized into treatment groups. End point values were established by an operator who had no knowledge of treatment assignments. Uterine contractility was scored from 1 to 4 (minimal to maximal) in all experiments by continuous 1-min ultrasound scanning of a longitudinal section of the uterine body and scoring the extent of to-and-fro movements of endometrial reflectors as described (11). The ultrasound scanner (Tokyo Keiki, LS300, Products Group International, Lyons, CO) was equipped with a 5.0 MHz linear-array intrarectal transducer. The extent of uterine tone was scored before the ultrasound examinations from 1 to 4 (minimal to maximal) in Experiments 1, 3 and 4 by transrectal digital assessment as described (24). Uterine contractility and tone scores were made periodically during a 2-, 3- or 8-h trial as described for the experiments. The first score was made imrr~diately before treatment (Time 0). Time 0 was 16 d after beginning of progesterone priming for anovulatory mares (Experiment 1), Days 12, 15 or 18/19 for cyclic and pregnant mares (Experiments 2 and 4), and the day of the embryonic-vesicle fixation (Experiment 3). Contractility and tone scores at Time 0 were examined for differences among groups by a oneway ANOVA. The scores or values were not necessarily the same for the various groups at Time 0. Therefore the scores and values were converted to a percentage change from Time 0 in the analyses involving > 1 group; an arcsin transformation was used. Main effects of group and time and the group-by-time interaction were examined by a split-plot ANOVA for sequential data. Duncan's multiple range test was used to compare means representing significant effects of group, time or the group-by-time interaction. When the interaction was significant, the main effects were not further evaluated, but the interaction was considered by analyzing for differences among groups within each time. The effect of time within each group was also evaluated with one-way ANOVA using scores or values, rather than percentages; when significance was obtained, means were further examined by the multiple range test. Experiment 1 Seasonally anovulatory mares (n=15), based on the absence of a corpus luteum and absence of follicles >20 mm, were used in February. Mares were primed with 16 consecutive dally injections of progesterone (100 mg, sc) in corn oil (10). Mares were examined every other day to
Theriogenology
524
determine uterine contractility and tone during progesterone treatment. Time 0 was on the 16th day of progesterone treatment. Three treatment groups were used: 1) control (saline vehicle, 1 mL, iv), 2) oxytocin treatment (20 IU, iv), and 3) clenbuterol treatment (0.8 I.tg/kg, iv). One replicate (1 mare/group) was initiated on each of 5 consecutive days, resulting in 5 mares per group. Beginning at Time 0, uterine tone and contractility examinations were performed every 20 rain during the first hour and every 1 h during the next 7 h. Experiment 2 Pregnant and nonpregnant mares (8 mares/group) were used. All mares received PGF2t~ (5 mg, im; Lutalyse, Pharmacia & Upjohn Co., Kalamazoo, MI, USA) on Days 12, 15, and 18. The eircu3ating progesterone concentrations and duration of exposure were standardized (27) by giving daily exogenous progesterone (100 mg/day, sc) on Days 12 to 17. On each day of PGF2~ treatment, uterine contractility was evaluated every 10 rain in a 2-h trial. Experiment 3 Pregnant mares (n=5/group) were used. On the day of embryonic-vesicle fixation mares were assigned to 2 groups: 1) control (saline vehicle, 6 mL, iv) and 2) clenbuterol treatment (600 gg/mare, iv). Time 0 was on the day of fixation of the embryonic vesicle. A 2-h trial with examinations every 10 rain was done after treatment to determine uterine contractility and tone. Fixation was defined as no location changes during a 2-h mobility trial (14); trials began on Day 13 and continued daily until fixation occurred. The location of fixation of the embryonic vesicle was established by dividing each horn and the uterine body into 3 segments of approximately equal length as described (14). Location was recorded at each examination during the trial and thereafter each day for 3 d. Experiment 4 Pregnant mares (n=5/group) were assigned on Day 19 to 2 groups: 1) control (saline vehicle, 6 mL, iv) and 2) clenbuterol treatment (600 I.tg/mare, iv). A 3-h trial with examinations every 30 min was performed after treatment to determine contractility and tone of the uterus and shape of the embryonic-vesicle. The ultrasound images of the embryonic vesicles were videotaped for later measurements. As an indicator of shape, wall-to-wall linear dimensions were measured, using a computer-driven image analysis program (NIH Image, NIH, Bethesda, MD), in 4 directions as on the face of a clock as follows: 12:00 to 6:00 o'clock, 1:30 to 7:30 o'clock, 3:00 to 9:00 o'clock, and 4:30 to 10:30 o'clock. The variance for the 4 dimensions was calculated and used as the end point for shape similar to a previously described technique for quantitating shape of follicles (35). Shape variance was statistically evaluated as for contractility and tone. RESULTS In Experiment 1, administration of progesterone in anovulatory mares for 16 d increased both uterine contractility and tone. Contractility scores increased between the first day of progesterone treatment (1.3 _+0.0) and 4 d after the beginning of treatment (1.9 _+0.2) and remained high until the end of the experiment. Uterine tone scores increased between the beginning of progesterone
Thefiogeno~gy
525
treatment (0.8 4-0.1) and 2 d later (1.1 4-0.1) and further increased 10 d after the beginning of treatment (1.5 4-0.1). In Experiment 2, uterine contractility and tone scores at Time 0, including the results of the analyses, are shown for nonpregnant and pregnant mares on Days 12, 15, and 18 (Table 1). Data for percentage changes in scores and values, including the results of the statistical analyses, are shown for Experiments 1 to 4 (Figures 1-4). Data for scores and values of individual groups, including the analyses over time, are shown for all experiments (Table 2).
Table 1. Uterine contractility scores at Time 0 before treatment with PGF2~ on Days 12, 15 or 18 postovulation in pregnant and nonpregnant mares (Experiment 2) Day
Number of mares
Nonpregnant
Pregnant
12
8
1.7 + 0.1Aa
2.5 + 0.1Ab
15 18
8 8
2.0 + 0.1Ba 2.1 + 0.1Ba
2.2 + 0.1Ba 1.6 + 0.1Cb
A, B, CValues with different capital letters in the same column are different (P
