The effects of perphenazine and bromocriptine on follicular dynamics and endocrine profiles in anestrous pony mares

ELSEVIER

THE EFFECTS OF PERPHENAZINE AND BROMOCRIPTINE ON FOLLICULAR DYNAMICS AND ENDOCRINE PROFILES IN ANESTROUS PONY MARES K. Bennett-Wimbush,’

W.E Loch,’ H. Plata-Madrid2

and T. Evans 3

‘Ohio State University Agricultural Technical Institute, Wooster, OH 44691 * Department of Animal Sciences, University of Missouri, Columbia, MO 652 11 3 College of Veterinary Medicine, University of Missouri, Columbia, MO 65211 Received for publication: Accepted:

August September

15,

1996 19,

1997

ABSTRACT Nineteen anestrous pony mares were used m a project designed to determine the effects of altered prolactin concentrations on follicular dynamics and endocrine profiles during spring transition, The dopamine antagonist, perphenazine, was administered daily to mares (0.375 mg/kg body weight) in Group A (n=6), while Group B mares (n=7) received 0.08 mg/kg metabolic weight (kg”) dopamine agonist, Z-bromo-ergocriptine, intramuscularly twice daily. Mares in Group C (n=6) received 0.08 mg/kg”, im., saline twice daily. Treatment began January 20, 1994, and continued until ovulation occurred. Mares were teased 3 times weekly with an intact stallion. The ovaries of the ponies were palpated and imaged weekly using an ultrasonic Bmode unit with a 5 Mhz intrarectal transducer until they either exhibited estrual behavior and had at least a ZO-mm follicle, or had at least a 25-mm follicle with no signs of estrus. At this time, ovaries were palpated and imaged 4 times weekly. Blood samples were obtained immediately prior to ultrasonic imaging for measurement of prolactin, FSH and estradiol-17P. Perphenazine treatment advanced the spring transitional period and subsequent ovulation by approximately 30d. Group A exhibited the onset of estrual behavior earlier (P < 0.01) than control mares. In addition, Group A mares developed large follicles (> 30 mm) earlier (P < 0.01) than Group B mares, with least square means for Groups A and 13 of 47.0 i 8.8 vs 88.1 i 8.2 d, respectively. Control mares developed 30-mm follicles intermediate to Groups A and B at 67.3 & X.8 d. Bromocriptine decreased (I’ < 0.05) plasma prolaclnn levels throughout the study, while perphenazine had no significant overall effect. However, perphenazine treatment did increase (P < 0.05) mean plasma prolactin concentrations from Day 3 1 to 60 of treatment. There were no differences in mean plasma FSH or estradiol- 176 between treatment groups, We concluded that daily perphenazine treatment hastened the growth of follicles and subsequent ovulation while bromocriptine treatment appeared to delay the growth of preovulatory size follicles without affecting the time of ovulation. 0 19% by Elswer

Science

Inc

Key words: mare. transitional estrus, follicular development, perphenazine. bromocriptine Acknowledgments This paper is a contribution from the Missouri Agricultural Series 12,455. Themgenology 49.717-733. 0 1998 by Elsevier Science

1998 Inc.

Experiment Station Journal 0093-691x/98/$19.00 PIISOO93-691X(98)00021-1

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718

INTRODUCTION The mare is a seasonal breeder, exhibiting nonovulatory anestrus during the winter months and ovulatory estrous periods during the spring and summer months. Transition between the physiological breeding season and the period of quiescence is characterized by erratic estrual behavior and follicular dynamics as well as atypical endocrine profiles. This is an important time period for horse breeders since it is when horses are bred to produce early foals. Many methods have been developed to hasten the first ovulatory estrous cycle of the spring. An extended photoperiod can initiate ovarian activity and lead to ovulation within 60 to 90 d (11,3 1, 33,43). Both GnRH and GnRH agonists have been used to induce ovulation in anestrous mares (35), with success rates of 69.2% (14) and 35% (39). However, the frequency of administration and the degree of ovarian activity appear to be correlated with the success of the treatment ( 39). Progestin therapy also appears to have limited success in inducing follicular development and ovulation in deeply anestrous mares (50). Human chorionic gonadotropin (hCG) has been used to hasten the time to ovulation in transitional mares without any apparent adverse effects on the formation or function of the corpus luteum (CL; 5) but hCG requires at least a 40-mm follicle in order to consistently induce ovulation (9). Finally a PGF,a analog, iupristiol, has been shown to cause a sustained release of both FSH and LH in both jugular ptasma and intercavernous sinus samples ofStransitional mares (28); however, it does not appear to induce estrual behavior. Although many of the above methods are effective in inducing ovulation in late transitional mares, they are not always effective in stimulating follicular development from deeply anestrous mares. It has recently been suggested that prolactin may be involved in follicular growth in other species (36). Prolactin receptors have been localized on granulosa cells late in follicular development (24), and it has been shown to increase follicular steroidogenesis (18). In the mare. prolactin exhibits a seasonal increase during sprmg transition (29, 5 1, 55). Both in vitro and in vivo work demonstrates a significant correlation between prolactin concentrations and day length in mares (13). Recent studies have shown that follicular growth in anestrous mares was accelerated by increased exposure to daylight and the use of fluphenazine (a D, dopamine receptor antagonist). which was correlated with a rise in serum prolactin (I 9,40). Additionally administration of’ ovine proiactin to anestrous mares stimulated ovarian activity and was also correlated with an endogenous rise in prolactin (40). However, neither treatment resulted in hastened ovulation. These previous studies suggest that prolactin may play a direct I ale in the initiation of early transitional events in the mare (19,40). Prolactin secretion is controlled by tonic inhibition via the hypothalamus. Most evidence indicates that dopamine is the inhibitory factor (2, 15). It has been amply shown that D, dopamine receptor agonists such as bromocriptine inhibit prolactin secretion in vivo (20,25. 30, 38,4 I, 46,49), while D, dopamine receptor antagonists such as the phenothiazine derivatives increase prolactin secretion (25). The purpose of the present study was to tdeterminc the effects of both perphenazine (dopamine antagonist) and 2-bromo-ergocriptine (dopamine agonist) on follicular dynamics,

Theriogenology

719

estrual behavior, endocrine profiles and ovulation in anestrous pony mares. Bromocriptine has been used as a model for fescue toxicosis in late gestation mares. Therefore, we also wanted to study the effect consumption of endophyte-infected tall fescue may have on transitional characteristics in mares. MATERIALS

AND METHODS

Animals Nineteen anestrous pony mares (ages 3 to 20 yr) were blocked by age and randomly assigned to 1 of 3 treatment groups. Mares were determined to be anestrous if follicles > 20 mm were not detected on either ovary by palpation per rectum and ultrasonic imaging of the ovaries for 3 consecutive weeks prior to the initiation of treatment and if progesterone levels were lower than I ngiml on January 20. Group A mares (n=6) received 0.375 mgikg body weight oral perphenazine twice daily; Group B (n=7) received 0.08 mgikg metabolic body weight (kg”) 2alpha-ergo-bromocriptine (Sigma Co., St. Louis, MO), administered twice daily by intramuscular injection, and Group C (n=6) received 0.08 mg/kg/body weight ‘I of saline administered twice daily by intramuscular injection. Treatment was administered at 0730 and I700 h daily. Treatment began January 20, 1994, and continued until ovulation occurred. Management Techniques All ponies were maintained in dry lots and fed ad libitum hay and water. Ponies were teased 3 times a week with an intact stallion. True behavioral estrus, versus the occasional signs of estrus sporadically observed in anestrous mares, was indicated when a mare displayed signs of tail raising, winking, urination and copulatory stance upon exposure to a stallion. When a mare displayed at least 3 of the 4 criteria she was determined to be in estrus. Reproductive organs were imaged weekly with a B-mode ultrasound unit equipped with a 5 Mhz transrectal transducer. Reproductive organs were imaged 4 times a week after a mare exhibited estrual behavior accompanied by at least a 20-mm follicle on either ovary or exhibited ovarian activity of at least a 25-mm follicle with no accompanying estms being observed. Ovulation was determined as described by Ginther (17). The size of all individual follicles was recorded. If there was a discrepancy between the height and width of a follicle, the 2 values were averaged to obtain the recorded diameter of the follicle. Blood samples were collected in sodium heparinized Vacu-tainer tubes by jugular venipuncture immediately prior to each recta1 ultrasound exam. Blood was centrifuged at 1500 g for 20 min at 5°C. The plasma was decanted and stored at 20°C in duplicate aliquots. Prolactin Assay Prolactin concentrations were measured using a homologous equine prolactin radioimmunoassay as described by Roser (47) with moditications to the method by Worthy (54).

Theriogenology

720

Pituitary equine prolactin (AFP-7730B), rat antisera (AFP-361687) and equine prolactin reference preparation (AFP-7730B) were obtained from Dr. A.F. Parlow, National Hormone and Pituitary Program. This technique gave rise to a stable tracer with total binding of 28% and nonspecific binding of 1.9%. All samples were analyzed using tracer obtained from a single iodination, and all samples from one animal were analyzed in the same assay. Intra-assay and inter-assay CV were 13.6 and 15.8%, respectively (n=7).

FSH Assay Plasma FSH was measured by a heterologous system described by Hines (23) and modified as described. Ovine FSH (ovine FSH- 19-SIAFP, lot AFP-4 I 17A), ovine FSH reference preparation (ovine FSH-19-SIAFP-RP-2, lot AFP4117A) and human antiserum (antihuman FSH-6, lot AFP-005) were obtained from the National Hormone and Pituitary Program. Unlabeled FSH (loo-u1 sample) or ovine reference standard (0.3 1 to 100 @ml) was added to protein-assay buffer followed by 100 ul of rabbit-human FSH antisera at an initial dilution of 1: 10,000. This was allowed to incubate for 24 h at 4°C. The lzJI-oFSH was added and allowed to incubate 48 h at 4°C. Separation of the free and bound was achieved by a second antibody technique. This gave rise to specific binding of 22% and nonspecific binding of 2%. All samples were analyzed using tracer obtained from a single iodination, and all samples from one animal w-ere analyzed in the same assay. The ovine reference preparation (0.31 to 100 ngiml) ran parallel with both an equine nonreferenced FSH preparation (1 to 1000 rig/ml) and a cyclic mare pool. The intra-assay CV and inter-assay CV were 13 and 13.5%, respectively (n=8).

Estradiol-

17

p Assay

Estradiol- 17 p was analyzed from ether extracted plasma samples using a radioimmunoassay procedure. Samples (500 ul) and standards (0.5 to 20 pgiml) were extracted using methyl-text-butyl ether (Sigma Chemical Co., St. Louis, MO). Following extraction, the assay reagents were added in the following order: 100 ul 1% BSA buffer, 100 ul estradiol antibody (ICN Biomedicals, Costa Mesa, CA) to all tubes except total count and nonspecific binding tubes; and then 100 ul 3-ido-estradiol-17p (ICN Biomedical) diluted in PAB to yield 1750 uciiug. This was incubated for 30 min at 37°C. Separation of the free and bound 3-idoestradiol was achieved by the charcoal extraction method. All samples from one animal were analyzed in the same assay. Intra-assay and inter-assay CVs were 7 and 10.9%, respectively (n=lO).

Statistical Analysis The follicular data were analyzed using General Linear Model (SAS; 48) for the following parameters: 1) days from initiation of treatment until signs of estrus, 2) days from initiation of treatment until appearance of a 30-mm follicle, 3) days from initiation of treatment until ovulation, 4) days in estrus and 5) size of the largest follicle at the onset of behavioral

Theriogenology

721

estrus. Differences between groups were tested using least squares means methods. Withintreatment variation was used as the error term, and hypothesis tests were performed at the P < .OS significance level. Endocrine data were analyzed both by day relative to ovulation and by day of treatment, The categories used for day relative to ovulation included Days 0 to -7, -8 to - 14, -15 to -30, -3 1 to -60 and -61 to -115 (Day O=ovulation). In addition, the categories used for day of treatment included Day 0 to 30,3 1 to 60, 6 1 to 90 and 9 1 to 115. The endocrine data (prl, FSH and estradiol-17P) and the number of small follicles were analyzed as a repeated measurement design as outlined by Gil and Hafs (1971). The split plot ANOVA contained the main effect of treatment, day and the interaction of treatment by day. The main effect of treatment was tested using individual within treatment as the denominator of F. Mean differences were ascertained using Fisher’s least significant difference test for split-plot designs (8), and hypotheses tests were performed at the P < .05 significance level. RESULTS Daily perphenazine treatment appeared to advance the spring transitional period and subsequent ovulation. Group A mares exhibited the onset of estrus behavior 27.5 2 10.4 d from the initiation of treatment, which was earlier (P < 0.01) than Group C (70.7 k 10.4) but not Group B (42.1 + 9.6 d). In addition, Group A developed preovulatory size follicles (> 30 mm) earlier (P < 0.01 j than Group B and ovulated earlier (P < .05) than either Group B or Group C. Control mares developed preovulatory size follicles in 67.3 d, which was intermediate to Groups A and B. A difference in the duration of estrual behavior and the size of the largest follicle at the onset of behavioral estrus was also observed. Bromocriptine-treated mares exhibited behavioral estrus for 52.3 t 7.5 d compared with 35.5 2 8.1 and 21.8 + 8.1 d for Groups A and C, respectively. Furthermore. mares in both Groups A (16.2 mm) and B (18.4 mm) exhibited the onset of behavioral estrus with smaller follicles (P c: 0.01) on the ovaries than the control mares (30.5 mm). Mean plasma prolactin was significantly decreased in Group B throughout the experimental period both relative to day of treatment (P < 0.01) and relative to day of ovulation (P c 0.05). Groups A, B and C had mean plasma prolactin concentrations of’7.5,4.1 and 8.9 rig/ml, respectively. Perphenazine treatment did not significantly alter mean prolactin concentrations either relative to day of treatment or day of ovulation. However, a treatment-bytime interaction relative to day of treatment was observed. Group A mares exhibited higher (P < 0.01) mean plasma prolactin concentrations than control mares from Days 30 to 60 of treatment. Mean plasma prolactin increased significantly (P < 0.05) in control mares 2 wk prior to ovulation, coinciding with a difference in prolactin concentrations between perphenazine-treated and control mares for Days - 14 to -8 and Days -7 to 0 observed in the analysis of variance. Table 2 summarizes mean plasma prolactin concentrations relative to day of treatment and Table 3 summarizes plasma prolactin relative to the day of ovulation. Both FSH and estradiol-17p as well as the number of small follicles (10 to 19 mm) were unaffected by either bromocriptine or perphenazine treatment.

722

Theriogenology

Table 1. Effects of daily perphenazine (Group A), bromocriptine (Group B) and saline (Group C) treatment on the interval from the initiation of treatment to estrus, preovulatory size follicle (> 30 mm), appearance and ovulation as well as the duration of behavioral e&us in anestrous pony mares. Perphenazine treatment significantly advanced ovulation while bromocriptine delayed the development of preovulatory size follicles and prolonged the duration of behavioral estrus in seasonally transitional mares.

Treatment grow - .

Interval to estrus

Interval to formation of 30mm follicle

Interval to ovulation

Length of estrus

A

27.5 & 10.4a**

47.0 .+ 8.8a

63.3 + 9.4a

35.5 & 8.1a’b

B

42.1 & 9.6a’b

88.1 t 8.2b**

97.3 + 8.7b**

52.3 2 7sa+*

C

70.7 + 10.4b**

67.3 i 8.8a

93.5 + 9.4b*

21.8+8.1b**

Values in the same column with different superscripts* are different (P < 0.05) Values in the same column with different superscripts ** are different (I’ < 0.0 1).

Table 2. Mean plasma prolactin concentrations (ngiml) relative to the day of treatment in perphenazine-treated mares (Group A).. bromocriptine-treated mares (Group B) and the saline treated control mares (Group C). Bromocriptine decreased plasma prolactin throughout the experiment, while perphenazine treatment increased mean plasma prolactin only during the second time period (Days 3 1 to 60). Day of treatment

Group A

Group B

Group C

O-30

6.5 & .8a

4.9 + .8”

6.6 f .8”

31-60

9.9 2 1.oa

3.42 .gb

5.9 2 .8’

61-90

8.2 + 1.4a

3.6 ;t .gb

7.8 + .8a

91-115

8.6 + 1.7”

5.01 l.ob

12.5 + I .Oa

Values in the same row with different superscripts are different (P < 0.05).

Theriogenology

723

Table 3. Mean plasma prolactin concentrations (ngiml) in perphenazine treated mares (Group A), bromocriptine treated mares (Group B) and saline-treated, control mares (Group C) relative to ovulation. Bromocriptine decreased plasma prolactin concentrations throughout the experiment. Plasma prolactin increased in control mares 2 weeks prior to ovulation (Day -14 to -8 and Day -7 to 0, with ovulation = 0). Day Relative to Ovulation

Group A

Group B

Group C

-115to-61

5.2 2 1.3a

4.3 + .Ya

5.3 2 .ga

-60 to -3 1

8.1 2 l.Oa

3.6 2 .Yb

6.9 k .8a

-30 to -15

7.8 i 1 .Oa

3.6 & .gb

8.9 2 .ga

-14 to -8

7.9 2 .9a

3.8 k .5Jb

12.3 ;t .Y’

-7 to 0

8.6 &.9a

5.4 + .!lb

11.2+.8’

Values in the same row with different superscripts are different (P < 0.05)

The authors observed changes in endocrine profiles relative to season as well as to treatment. Since there were differences between treatment groups. data from Group C was used to describe changes in plasma prolactin over time. Prolactin concentrations iu Group C mares were lowest during winter anestrous. Mean plasma prolactin concentrations increased from 6.6 5 0.8 ngiml during Days 0 to 30 of the study (.lanuary 2 I to February 20) to 12.5 ” I .O ngiml during Days 90 to 115 (April 23 to May 18). In addition, plasma prolactin increased significantly (P < 0.01) two weeks prior to ovulation from baseline levels of 58.3i 0.8 ngiml that were observed during Days -115 to -90 (ovulation = Day 0) to 12.3 k 0.9 and 11.2 i 0.Y on Days -14 to -8 and Days -7 to 0; respectively. Data on FSl-I and estradiol-17p were pooled across treatment groups since no treatment effect was observed. Mean plasma FSH concentrations were consistent throughout the study until they decreased (P c 0.05) from 7.1 + 0.7 rig/ml one month prior to ovulation to 3.9 5 0.7 ngiml one week prior to ovulation. Baseline levels ofestradiol17 p averaged 1.2, 1.4 and 1.3 pg/ml prior to Day -7 in Groups A, B and respectively increasing significantly (P < 0.01) to 7.4 + 0.2 pg/ml from Day -7 until ovulation. DISCUSSI(>N The daily treatment of anestrous mares with the dopamine antagonist perphenazine clearly hastened both the growth of preovulatory size follicles and ovulation compared with that of the control mares or of mares treated with the dopamine agonist bromocriptine. However, the effects may not have been mediated through an increase in prolactin. as expected. Plasma prolactin was not significantly increased throughout the experiment. The effects of a dopamine antagonist on follicular development were similar, in part, to work by other researchers (1 Y. 40).

Theriogenology

724

Legend -@-+--+-

Group A Groupi GroupC

Figure 1. Effect of treatment on the mean number of small follicles (10 to 19 mm) and weekly mean prolactin concentrations for Weeks 1 through 12 of treatment. Bromocriptine decreased (P < 0.05) mean plasma prolactin concentrations throughout the experiment while perphenazine increased them from Days 30 to 60, as is readily observed during Weeks 5 to 8.

Theriogenology

725

Legend +

Group

A

-*-

Group Group

B c

- -),-

Day relative to ovulation

Figure 2. Plasma prolactin concentrations (nghl) in mares 30 days prior to ovulation. Bromocriptine decreased plasma prolactm throughout the study while perphenazine had no overall effect. A rise in prolactin was observed in control mares at Days - 14 to -8 and Days -7 to 0, which was not observed in Groups A and B. Note: The values reported for each even day relative to ovulation are the prolactin levels for either the Day 0 or - 1, Day -2 or -3, Day -4 or -5, Day -6 or -7, Day -8 or -9, Day -10 or -11. Day -12 or -13, Day -14 or -15, Day -16 or -17, Day -18 or -19, Day -20 or -21, Day -22 or -23, Day -24 or -25, Day -26 or -27, Day -28 or -29, Day -30 or -3 1. depending on when the mare ovulated relative to ultrasonic examination of the reproductive tract.

726

Theriogenology

Legend ?

,’ i

! +

Group

A

2

0

--r--r--30

-28

-26

--24

-22

/. -20

-16

~--r -16

~-~~.~ -14

-12

i -10

-6

-6

-4

-2

0

Day relative to ovulation

Figure 3. Mean plasma FSH concentrations @g/ml) 30 days prior to ovulation. There were no differences in mean plasma FSH between treatment groups. F‘SH concentrations decreased one week prior to ovulation in all mares. Note: The values reported for each even day relative to ovulation are for levels for either the Day 0 or - 1, Day -2 or -3, Day -4 or -5, Day -6 or -7, Day -8 or -9, Day - 10 or-11,Day-12or-13,Day-14or-15,Day-16or-17,Day-18or-19,Day-20or -21, Day -22 or -23, Day -24 or -25, Day -26 or -27, Day -28 or -29, Day -30 or -3 1, depending on when the mare ovulated relative to ultrasonic examination of the reproductive tract.

727

Theriogenology 14

Legend ”

2E iz-

IJA-30

~

+ -+I(--

Group A GroupB

-+-

GroupC

~

~j. -26

-26

-24

-22

-20

-16

-16

..34

-12

-10

-8

-6

4

-2

0

Day relative to ovulation

Figure 4. Mean plasma estradiol-17P concentrations (pgiml) for 30 days prior to ovulation. There was no difference in estradiol- 17 p concentrations between treatment groups. Estradiol increased in all the mares 1 week prior to ovulation. Note: The values reported for each even day relative to ovulation are for levels for either the Day 0 or -1, Day -2 or -3, Day -4 or -5. Day -6 or -7, Day -8 or -9, Day - 10 or -11, Day -12 or -13, Day -14 or -15, Day -16 or -17, Day -18 or -19, Day -20 or -2 1, Day -22 or -23, Day -24 or -25, Day -26 or -27, Day -28 or -29, Day -30 or -3 1, depending on when the mare ovulated relative to ultrasonic examination of the reproductive tract.

728

Theriogenology

In our present study, both follicular development and ovulation were advanced while in earlier studies only follicular development was affected by the use of a dopamine antagonist (fluphenazine). The treatment regimen of twice-daily administration of oral perphenazine may have had a more consistent dopaminergic effect than a single dose of the long-acting fluphenazine administered every 21 d. Fluphenazine (178.6 ug/kg, im) significantly increased prolactin 3 to 8 h post administration, with a return to baseline values after this time (40). Perphenazine and other dopamine antagonists have been shown to increase prolactin concentrations in pregnant mares (25,44, 56), anestrous mares (19, 32,40), cyclic mares (I) and in stallions (lo), In our present study, perphenazine only increased prolactin levels from Day 3 1 to 60 of treatment. The absence of a continual and significant prolactin rise may be due to the frequency of sampling or to pituitary stores. Prolactin concentrations have been shown to be highly correlated with photoperiod in mares (13, 30). In vitro studies have indicated that the releasable pool of pituitary prolactin varies with the season (13). It has also been hypothesized that the releasable pituitary prolactin or a decrease in the sensitivity of a prolactin releasing factor accounts for the seasonal difference (1). In our current study, perphenazine treatment may not have increased prolactin from Days 0 to 30 of treatment because pituitary stores were low. As synthesis increased, so did the releasable prolactin and, consequently, the mean plasma levels. As mares ovulated, they were removed from the study. Thus the mean prolactin concentrations reported from Days 60 to 115 of the study represented only one-half of the mares, so individual variation may have played a role in the failure to detect a significant difference in prolactin Bromocriptine treatment significantly decreased mean plasma prolactin levels in anestrous pony mares, as reported in an earlier study (30), in which a decrease in serum prolactin in response to 10 mg bromocriptine was observed in May but not November. Additionally, bromocriptine has been shown to decrease serum prolactin in lactating rnares (41) and in periparturient mares (25, 56). There were no significant differences m plasma FSH between the groups or over time. Little information is available on the effects of dopamine agonists and antagonists on gonadotropin response. Long-term administration of sulpiride (dopamine antagonist) decreased daily FSH levels in stallions (10). as has also been shown by other researchers (3,7) suggesting that prolactin might reduce the responsiveness of the pituitary gland to GnRH. Marchetti and Labrie (34) showed that hyperprolactinaemia exerts an inhibitory effect on pituitary GnRH receptors and basal gonadotropin secretion in rats. However, our data do not support this since perphenazine-treated mares developed preovulatory size follicles earlier than Group B or C mares without a rise in FSH. The pattern of estradiol-I 7p observed during our study was consistent with the observations of Mienecke et al. (37) and Oxender et al. (43). In our study, estradiol increased in all mares 1 wk prior to ovulation. Mienecke et al. (37) report that estradiol increases 6 to 8 d before ovulation, peaks on Day -3 or -4 and then decreases on Day -1 during the normal estrous cycle, while Oxender et al. (43) reported a longer, but lower estradiol surge in association with the first ovulation following anestrus. WC did not analyze for differences in hormone concentrations by day; however, Figure 4 clearly shows a peak in estradiol on Day -4 in all groups, with a decline in estradiol concentrations on Day -2.

Theriogenology There is considerable evidence to support the role of prolactin in the return to normal cyclicity following winter anestrus in mares. There is also widespread consensus that the pineal gland is involved in the interaction between adenohypophyseal hormones and the neuronal activity of the hypothalamus (6,26, 27,45). Specific prolactin receptors have been localized in the pineal gland of rats (4) and sheep (42). Moreover, administration of prolactin to rats can increase pineal hydroxyindole-0-methyl-transferase and tyrosine hydroxylase activity (4,21). Since prolactin secretion is increased during increased photoperiod in horses, it is probable that the pineal gland has an effect on prolactin levels. An alternate explanation for the hastened development of follicles and ovulation observed in anestrous pony mares during this study may be that dopamine agonists and antagonists alter other pituitary hormones as well as prolactin. A likely candidate would be the growth hormone. Growth hormone has been shown to enhance follicular development in many species. However, there is no conclusive evidence that indicates that dopamine agonists or antagonists affect growth hormone concentrations in livestock. Neither the dopamine receptor blockers haloperidol in ewes (12) or sulpiride in mares (52) affected growth hormone secretion in any way despite the increases in prolactin that were observed. Luteinizing hormone (LH) was not measured in this study. Most of the literature indicates that the dopaminergic system is not involved with the secretion of LH. Metoclopramide (dopamine antagonist) had no effect on mean LH, or on LH pulse frequency or amplitude in cows until a GnRH challenge was given. Following administration of 7 mg/kg GnRH, metoclopramide-treated cows exhibited an increase in mean LH (53). However, metoclopramide-induced hyperprolactinemia failed to alter FSH, LH or the time of ovulation in cyclic light horse mares (1). Additionally, sulpiride (dopamine antagonist) failed to alter FSH or LH in breeding stallions (10). However, a recent study in anestrous ewes indicates that 2 dopaminergic cell groups (A-14 and A-l 5) may be involved in the seasonal shift in the response of LH to estradiol negative feedback. Lesions in either of these areas decreased estradiol inhibition of LH pulse frequency during anestrus. Additionally, apomorphine (dopamine agonist) decreased LH pulse frequency, while pimozide (dopamine antagonist) increased pulse frequency (22). Thus, the possibility that perphenzaine altered LH secretion throughout the dopaminergic system can not be ruled out. We conclude that perphenazine hastened the development of preovulatory size follicles compared with that of bromocriptine-treated mares and advanced the subsequent ovulation when compared with both bromocriptine-treated mares and control mares. The administration of bromocriptine appeared to lengthen the time period that mares demonstrated estrual behavior during the spring transitional period. Moreover, the observed estrual behavior did not coincide with observed ovarian structures. However, it is not clear whether the hastened development of follicles and ovulation in the perphenazine-treated mares were due to altered prolactin concentration or to some other mechanism.

Theriogenology

730 REFERENCES

1. Becker SE, Johnson AL. Failure of metoclopramide induced acute hyperprolactinemia to affect time of ovulation or spontaneous luteolysis in the cycling mare. Equine Vet J 1990; 10:275-279. 2 Birge CA, Jacobs LS, Hammer CT, Daughaday WH. Catecholamine inhibition ofprolactin secretion by isolated adenohypophyses. Endocrinology 1979; 86: 120- 130. 3. Brar AK, McNeilly AS, Fink G. Effects of hyperprolactinaemia and testosterone on the release of Lh releasing hormone and the gonadotrophins in intact and castrated rats. J Endocrin 1985; 104:35-43. 4. Cardinali DP. Hormone effects on the pineal gland. In: Reiter JR (ed.) The Pineal Gland Vol. 1. Anatomy and Biochemistry. 5. Carnevale EM, Squires EL, McKinnon AO, Harrison LA. Effect of human chorionic gonadotropin on time to ovulation and luteal function in transtional mares. Equine Vet Sci 1989; 9127-29. 6. Casson VM, Chesworth MJ, Armstrong SM. Entrainment of rat circadian rhythms by daily injections of melatonin depends upon the hypothalamic suprachiasmatic nuclei. Physiol Behav 1986; 36:1111-1121. 7. Cheung CJ. Prolactin suppresses lutenizing hormone secretion and pituitary responsiveness to lutenizing hormone-releasing hormone by a direct action at the anterior pituitary, Endocrmology 1983; 113:632-636. 8. Cochran W!G, C’ox, GM. Experimental Designs. New York: John Wiley and Sons Inc, 1992. 9. Colburn DR, Squires EL, Voss JL. Use of altrenogest and human chorionic gonadotropin to induce normal ovarian cyclicity in transitional mares. Equine Vet Sci 1987; 769-72. 10. Colburn DR, Thompson DL, Rahmanian MS, Roth TL. Plasma concentrations of cortisol, prolactin, LH and FSH in stallions after physical exercise and injection of secretagogue before and after sulpiride treatment in winter. J Anim Sci 1991; 69:3724-3732. 11. Cooper WL, Nert NE. Wintertime breeding of mares using artificial light and insemination. Proc. 21st Am Assoc Equine Prac 1975; 245-253. 12. Elasasser TH, Bolt DJ. Dopaminergic-like activity in toxic fescue alters prolactin but not growth hormone or thyroid stimulating hormone in ewes. Dom Anim Endocrin 1987; 4: 259-269. 13. Evans MJ, Alexander SL, Irvine CHG, Levesey JH, Donald RA. In vitro and in vivo studies of equine prolactin secretion throughout the year. J Reprod Fertil 1991; 44(Suppl):27-35. 14. Fitzgerald BP, Affleck KJ, Pemstein R, Lay RG. Investigation of the potential of LHRM or an agonist to induce ovulation in seasonal1 y anoestrous mares with observations on the use of the agonist in problem acyclic mares. J Reprod Fertil 1987; 35(Suppl):683-84, 15. Gibbs DM, Neil1 JD. Dopamine levels in hypophyseal stalk blood in the rat are sufficient to inhibit prolactin secretion in vivo. Endocrinology 1978; 102: 18958-1900. 16. Gil SL. Hafs I-ID. Analysis of repeated measurements of animals. J Anim Sci 1971; 33: 331-336. 17. Ginther OJ. Reproductive Biology of the Mare. La Cross WI: Equiservices 1992. 18. Gore-Langton RE, Armstrong DT. Follicular steroidogenesis and its control. In: Knobil E, Neil1 JD (eds), Physiology of Reproduction. New York: Ravenwood Press 35 1.

Theriogenology

731

19. Gow GM, King SS, Nequin LG, Ferreira-Davis GM, Johnson AL. Differential effects of dopamine inhibition and photo-stimulation on ovarian recrudescence and ovulation following anestnrs. 13th Symp Equine Nutr Phys Sot 1993; 356-357. 20. Graf K, Neumann F, Horowski. Effect of ergot derivative lisuride hydrogen maleate on serum prolactin concentrations in female rats, Endocrinology 1976; 98598-605. 2 1. Haldar-Misra C, Pevet P. Influence of prolactin on the processes of protein and/or peptide secretion in mouse and rat pinealocytes. An in vitro study. J Neural Tran 1983;58:245-259. 22. Havem RL, Whisnant CS, Goodman RL. Dopaminergic structures in the ovine hypothalamus mediating estradiol negative feedback in anestrous ewes. Endocrinology 1994; 134: 19051914. 23. Hines KK, Fitzgerald BP, Loy RG. Effect of pulsatile gonadotrophin release on mean serum LH and FSH in peri-parturient mares, J Reprod Fertil 1987; 35(Suppl):635-640. 24. Hughes JP, Elsholtz HP, Friesen HG. Growth hormone and prolactin receptors, In: Posner BI, Dekker M (eds), Polypeptide Hormone Receptors. New York: 1988; 157-159. 25. Ireland FA, Loch WE, Worthy K, Anthony RV. Effects of bromocriptine and perphenazine on prolactin and progesterone concentrations in pregnant pony mares during late gestation. J Reprod Fertil 1990; 92:179-186. 26. Iwantani N. Kodama M, Seto H. A child with pituitary gigantism and precocious adrenarchi: does GH and/or prolactin advance the onset of adrenarch. Endocrinology (Japan) 1992; 39:234-57. 27. Juszczak M, Cuzek JW. Hypothalamic and neurohypophyseal vasopressin and oxytocin in melatonin treated pinealectomized male rats. J Pineal Res 1988; 5:545-552. 28. Jochle W, Irvine CHG, Alexander SL, Newby TJ. Release of LH, FSH and GnRH into pituitrary venous blood in mares treated with a PGF analogue, luprostiol during the transitional period. .JReprod Fertil 1987; 35(Suppl):261. 29. Johnson AL. Serum concentrations of prolactin, thyroxine and triiodothyronine relative to season and the estrous cycle in the mare. J Anim Sci 1986; 62: 10 12- 1020. 30. Johnson AL, Becker SE. Effects of physiological and pharmacological affects on serum prolactin concentrations in the non-pregnant mare. J Anim Sci 1987; 65: 1292- 1297. 3 1. Kooistra LH, Ginther OJ. Effect of photoperiod on reproductive activity and hair in mares. Am J Vet Res 1975; 36:1413-1419. 32. Loch WE, Worthy D, Ireland F. The effect of phenothiazine on plasma prolactin in nonpregnant mares. Equine Vet J 1990; 22:30-32. 33. Loy RG. Effects of artificial lighting regimes on reproductive patterns in mares. Am Assoc Equine Prac 1968; 159-l 69. 34. Marchetti B, Labrie F. Prolactin inhibits pituitary Lhreleasing hormone receptors in the rat. Endocrinology 1982; 111:1209-1216. 35. McCue PM, Troedsson MHT, Liu IKM, Stabenfeldt GH, Hughes JP. Lasley BL. Follicular and endocrine responses of anestrousmares to administration of native GnRH agonist. J Reprod Fern1 1991; 44(Suppl):227-233. 36. McNeilly AS, Glasier A, Janassen J, Howie PW. Evidence for direct inhibition of ovarian function by prolactin. J Reprod Fertil 1982; 65:559-569. 37. Meinecke B, Gips H, Meinecke-Tillman S. Progestagen, androgen and estrogen levels in plasma and ovarian follicular fluid during the estrous cycle of the mare. Anim Repod Sci 1987; 121255265.

732

Theriogenology

38. Muller EE, Panerai AE, Cocchi D, Mantegazza P, Endocrine profile of ergot alkaloids. Life Sci 1977; 21:1545-1558. 39. Mumford EL, Squires EL, Peterson KD, Nett TM, Jasko DJ. Effect of various doses of a gonadotropin-releasing hormone analogue on induction of’ ovulation in anestrous mares. J Anim Sci 1994; 72:178-183. 40. Nequin LG, King SS, Ferreira-Davis G, Gow 6. Possible role for prolactin during the equine spring reproductive transition. 13th Symp Equine Nutr Phys Sot 1993; 358-359. 41. Neuschaefer A, Bracher V, Allen WR. Prolactin secretion in lactating mares before and after treatment with bromocriptine. J Reprod Fertil 1991; 44(Suppl):551-559. 42. Notebum HP, van Balan PP. van der Gugten AA, Hart IC, Ebels I, Salamink CA. Presence of immunoreactive growth hormone and prolactin in ovine pineal gland. J Pineal Res 1993; 14: 1 l-22. 43. Oxender WD, Norden PA, Hafs HD. Estrus, ovulation and serum progesterone, estradiol and LH concentrations in mares after an increased photoperiod during winter. Am J Vet Res 1977; 44.

45. 46. 47 48. 49. 50. 5 1.

52.

53.

54.

55.

38:203-

207.

Redmond LM. Cross DL. Stricland JR, Kennedy SW. Efficacy of domperidone and sulpiride as treatments for fescue toxicosis in horses. Am J Vet Res 1994; 55:722-729. Reiter RJ. The pineal and its hormones in the control of reproduction in mammals. Endocrin Rev 1980; 1:109-131. Rolland R, Schelleken LA. Inhibition of puerperal lactation by bromocriptine. Acta Endocrinol 1978; 216(Suppl):119-130. Roser JF. Chang YS, Papkoff H, Li CH. Development and characterization of a homologous radioimmunoassay for equine prolactin. Proc. Sot. Exper. Biol. Med. 1984. 175:5 1O-5 17. SAS User’s Guide. Cary NC: SAS Institute Inc, 1985. Schams D, Reinhardt V. Karg Il. Effects of:,-Br-ergokryptine on plasma prolactin levels during parturition and onset of lactation in cc’ws. Expcrientia 1972; 28:697-699. Squires EL. Heeseman CP, Webel SK. Shidelcr RK, Voss JL. Relationship of altrenogest to ovarian activity, hormone concentration and fertility of mares. 1983; 56:901-906. Thompson DL, Johnson L, St. George RL, Garza F. Concentrations of prolactin, LH and FSH in pituitary and serum of horses: effect:; of sex. season and reproductive state. J Anim Sci 1986; 63:854-860. Thompson DL Jr, Rahmanian MS, DePew CL, Burleigh DW, DeSoLrza CJ, Colbom DR. Growth hormone in mares and stallions: pulsatile secretion. response togrowth hormonereleasing hormone and effects of exercise, sexual stimulation and ph;armacological agents. J Anim Sci 1992; 70:1201-1207. Thompson DL, Jones RD, Studemann JA, Mizinga KM, Smith CK. Effect of metoclopramide on luteinizing hormone secretion in postpartum and anestrous cows. Am J Vet Res 531727-733. Worthy K. Escreet R, Renton JP, Eckersall I’D, Douglas TA, Flint DJ. Plasma prolactin concentrations and cyclic activity in pony mares during parturition and early lactation. J Reprod Fertil 1986; 771569-574. Worthy K. Colquhoun K. Escreet R, Dunlop M, Renton JP, Douglas TA. Plasma prolactin concentrations in non-pregnant mares at different times of the year and in relation to events in they cycle, J Reprod Fertil 1987: 35(Suppl):269-276.

Theriogenology 56. Zimmer RL, Loch WE, Bennett-Wimbush K, Anthony RV. Effects of 2-bromo-crergocriptine on pregnant pony mares during late gestation. Proc 13th Symp Equine Nutr Phys Sot 1993; p. 326-327.

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