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BOR - Papers in Press, published online ahead of print October 31, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007278
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BIOLOGY OF REPRODUCTION 68, 1403–1412 (2003)
DOI: 10.1095/biolreprod.102.007278
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Effects of a 6-Day Treatment with Medroxyprogesterone Acetate after Prostaglandin F2{alpha}-Induced Luteolysis at Midcycle on Antral Follicular Development and Ovulation Rate in Nonprolific Western White-Faced Ewes1

Pawel M. Bartlewski3,a, Rajesha Duggavathia, Jayaprakash Aravindakshan4,a, David M.W. Barretta, Susan J. Cooka, and Norman C. Rawlings2,a

a Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Medroxyprogesterone acetate (MAP) from intravaginal sponges prolongs the lifespan of large ovarian follicles when administered after prostaglandin F2{alpha} (PGF2{alpha})-induced luteolysis early in the luteal phase of ewes. The present study was designed to determine whether a PGF2{alpha}/MAP treatment applied at midcycle would alter the pattern of antral follicle growth and increase ovulation rate in nonprolific ewes. A single injection of PGF2{alpha} (15 mg, i.m.) was given, and an intravaginal MAP (60 mg) sponge was inserted for 6 days, on ~Day 8 after ovulation, in 7 (experiment 1), 8 (experiment 2) or 11 (experiment 3) ultrasonographically monitored, cycling Western white-faced ewes; seven ewes (experiment 1) served as untreated controls. Blood samples were collected each day and also every 12 min for 6 h, halfway through the period of treatment with MAP (experiment 1), or every 4 h, from 1 day before to 1 day after sponging (experiment 2). Seventeen of 26 treated ewes (experiment 1, n = 6; experiment 2, n = 5; experiment 3, n = 6) ovulated 1 to 6 days after PGF2{alpha}, but this did not affect the emergence of ensuing follicular waves (experiments 1 and 2). These ovulations, confirmed by laparotomy and histological examinations of the ovaries (experiment 3), were not preceded by an increase in LH/FSH secretion and did not result in corpora lutea, as evidenced by transrectal ultrasonography and RIA of serum progesterone (experiments 1 and 2). Following the removal of MAP sponges, the mean ovulation rate was 3.1 ± 0.4 in treated ewes and 2.0 ± 0.3 in control ewes (experiment 1; P < 0.05). In experiments 1 and 2, the ovulation rate after treatment (3.1 ± 0.4 and 2.8 ± 0.4) was also greater than the pretreatment rate (1.9 ± 0.3 and 1.9 ± 0.1, respectively). Ovulations of follicles from two consecutive waves before ovulation were seen in five treated but only in two control ewes (experiment 1), and in seven ewes in experiment 2. There were no significant differences between the MAP-treated and control ewes in mean daily serum concentrations of FSH and estradiol, and no differences in the parameters of LH/FSH secretion, based on frequent blood sampling. Treatment of nonprolific Western white-faced ewes with PGF2{alpha} and MAP at midcycle changed follicular dynamics and increased ovulation rate by approximately 50%. These effects of MAP, in the absence of luteal progesterone, may not be mediated by changes in gonadotropin secretion.

follicular development, ovary, ovulatory cycle, pituitary hormones, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic ewes have regular, emerging waves of antral follicles that grow to ovulatory diameters (>=5 mm) before regression or ovulation [1–4]. The interovulatory interval typically consists of three or four follicular waves. In nonprolific breeds such as the Western white-face [3–4], Suffolk, Texel, and Ile-de-France [5], ovulatory follicles originate mostly in the last wave of the cycle, but in prolific Finn ewes, ovulatory follicles are recruited also from the penultimate wave of the estrous cycle, which emerges during the midluteal phase [3].

It was shown that lower than normal luteal phase concentrations of progesterone prolonged follicular lifespan in ewes [6, 7]. Similarly, the lifespan of large antral follicles was prolonged in ewes injected with PGF2{alpha} on Day 6 and treated with vaginal sponges soaked with medroxyprogesterone acetate (MAP), from Days 5 to 19 after ovulation [8]. Therefore, the MAP treatment appeared to mimic the effects of a low progesterone regimen in ewes. Interestingly, in the breeding season, mean serum concentrations of progesterone are higher in nonprolific Western white-faced ewes compared to prolific Finn sheep [9]. Lower circulating concentrations of progesterone in Finn sheep may facilitate the prolongation of the lifespan of follicles in the penultimate wave so that they can ovulate with follicles from the final wave of the cycle.

The aim of this study was to determine whether a short treatment with MAP would increase the ovulation rate by causing ovulations from two consecutive antral follicle waves. Luteal and endocrine function after treatment (i.e., corpus luteum [CL] formation, progesterone secretion, and changes in circulating concentrations of FSH and estradiol) were also examined. The study was replicated to confirm the findings.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Procedures: Experiment 1

Care and treatment for experimental animals was carried out according to guidelines issued by the Canadian Council on Animal Care. Fourteen sexually mature, clinically healthy, cyclic Western white-faced ewes were used in the study, which was conducted from September to November. Estrus was initially synchronized in 24 ewes by a 14-day treatment with progestogen-releasing intravaginal sponges (MAP, 60 mg; Veramix, Upjohn, ON, Canada). Ewes were examined for estrus with two vasectomized crayon-harnessed rams, and an electronic estrous detector for measuring changes in vaginal mucous impedance [10–11]. The study began at the second estrus after the synchronization treatment. The 14 ewes were in estrus within a 24-h period, and ovulated between 24 to 48 h after the onset of estrus. Days of ovulation were regarded as the days on which large (>=5 mm in diameter) ovarian follicles that had been identified by ultrasonography were no longer detected. Nine days after estrus, or on about Day 8 after ovulation (7.7 ± 0.2 days; at 0800 h), seven ewes received a single injection of PGF2{alpha} (15 mg i.m.; Lutalyse, Upjohn, Orangeville, ON, Canada) and an intravaginal MAP sponge, which remained in place for 6 days. Treatment with progestogen was designed to encapsulate the growth of follicles in the last two waves of the cycle [3]. The remaining seven ewes served as untreated controls. Daily transrectal ultrasonography of ovaries (performed between 1300 and 1500 h) started at ~Day 5 after ovulation. The size and position of CL and ovarian antral follicles >=3 mm in size were then recorded for 19 days. Ovarian ultrasonography was performed using a real-time, B-mode echo camera (Aloka SSD-500; Overseas Monitors Ltd., Richmond, BC, Canada) equipped with a stiffened, 7.5-MHz transducer. That this technique can accurately quantify ovarian antral follicles >=3 mm in diameter [2, 8, 12–19] and to detect CL [9, 20–22] have been shown previously. Ultrasonographic detection of ovulations has also been recently verified in our laboratory (unpublished) via laparotomy performed 1, 2, and 3 days after ovulation had been determined with transrectal ultrasonography. Twenty-three of 24 ovulations (96%) identified with ultrasonography were confirmed by laparotomy, and the single questionable ovulation was that of a 4-mm follicle.

In the present study, blood samples (10 ml) were collected each day before ultrasonographic examination by jugular venipuncture using vacutainers (Becton Dickinson, Rutherford, NJ). Three days after PGF2{alpha} injection and MAP sponge insertion to treated ewes, all ewes were bled every 12 min for 6 h via indwelling jugular catheters (4 ml/sample; vinyl tubing, 1.00 mm inside diameter x 1.50 mm outside diameter; SV70, Critchley Electrical Products Pty Ltd., Auburn, NSW, Australia).

Ovarian data summary and analyses A follicular wave was defined as one or more antral follicles that grew from 3 to >=5 mm in diameter; the day the follicles were detected at 3 mm was the day of emergence. Groups of follicles emerging within 48 h were included in a wave. The characteristics of ovulatory antral follicles [3] in the last and penultimate follicular waves before ovulation detected after the end of MAP treatment were compared within and between the treatment and control groups by ANOVA (SigmaStat 2.0 for Windows; SPSS Inc., Chicago, IL). Comparisons were not made for the ovulating follicles in preceding waves because none of the control ewes and only three of the seven treated animals had ovulatory follicles that emerged prior to the penultimate wave. The proportions of ovulatory follicles emerging in the penultimate and final waves before ovulation (follicles growing to >=5 mm in diameter) were compared between treated and control ewes by the {chi}2 test (SigmaStat 2.0). The number of ovulations and time at which they occurred, as well as mean numbers of luteal structures per ewe (i.e., CL and luteinized unovulated follicles), were compared between the two groups of ewes by Student t-test (SigmaStat 2.0). Ovulation rates were also compared for the first (immediately before the PGF2{alpha}/MAP treatment) and last (after sponge removal in treated ewes) ovulation of the study period, within each group of ewes. Ovulations were observed in MAP-treated ewes during the period of sponging. Therefore, the characteristics of follicles ovulating during MAP treatment in the six treated ewes were noted (i.e., percentage of follicles >=5 mm in diameter on the day of PGF2{alpha} that ovulated, day of emergence, maximum follicular diameter, diameter on the day before ovulation, and interval between the attainment of maximum size and ovulation).

Hormone analyses Serum samples were analyzed by RIA for concentrations of LH [23], FSH [24], estradiol [25], and progesterone [26]. Earlier studies in our laboratory (unpublished) showed that the antiserum to progesterone did not cross-react with MAP, which permitted the measurement of endogenous progesterone in MAP-treated ewes. Concentrations of LH and FSH are given in terms of NIAMDD-oLH-24 and NIAMDD-oFSH-1, respectively. The sensitivities of assays were as follows: 0.10 ng/ml (LH and FSH), 1.0 pg/ml (estradiol), and 0.03 ng/ml (progesterone). The range of standard curves was from 0.06 to 8.0 ng/ml, 0.12 to 16.0 ng/ml, 1.0 to 50 pg/ml, and 0.1 to 10 ng/ml, in the LH, FSH, estradiol, and progesterone assays, respectively. All LH analyses were conducted in a single assay; the intraassay coefficients of variation (CVs) for mean LH concentration of 0.19 or 0.97 ng/ml were 12.3% or 8.9%, respectively. For FSH, the intraassay and interassay CVs were 6.4% and 2.9% or 7.1% and 5.9%, for reference sera with mean concentrations of 1.28 or 2.41 ng/ml, respectively. The intraassay and interassay CVs for estradiol were 14.0% and 12.2%, or 10.3% and 7.2%, for reference sera with mean concentrations of 5.1 or 13.9 pg/ml, respectively. Intraassay and interassay CVs for reference sera with mean progesterone concentrations of 0.29 or 0.69 ng/ml were 13.2% and 9.4% or 18.5% and 7.3%, respectively.

Daily serum concentrations of FSH, estradiol, and progesterone for all ewes were aligned with the day of PGF2{alpha} treatment for the treated ewes (Day 0), and analyzed for the period from Day -1 to Day 7. Daily concentrations of the hormones were also aligned with the day of ovulation after the MAP treatment period (Day 0) and analyzed for the period from Day -3 to Day 5 in all ewes (the period when sponges were withdrawn in the treated ewes to 1 day after CL detection in all ewes studied). Main effects of group, day, and group-by-day interaction were determined by repeated measures ANOVA (SigmaStat 2.0).

The PC-PULSAR program [27] was used to estimate LH/FSH pulse frequency, amplitude, and duration as well as mean and basal serum concentrations of LH and FSH obtained by frequent blood sampling. The basal serum level ("smoothed series") was generated after the removal of short-term variations in hormone concentrations, including possible pulses. Standard deviation criteria (G and Baxter parameters) were used for pulse detection [28]. These characteristics of FSH/LH secretion were then compared between the treatment and control groups by ANOVA (SigmaStat 2.0).

Additional analyses Daily serum concentrations of FSH, estradiol, and progesterone in the six treated ewes that ovulated after PGF2{alpha} injection and before the removal of progestogen sponges were aligned with the day of such ovulations in each ewe (Day 0) and analyzed for the period from Day -2 to Day 2 by one-way repeated measures ANOVA (SigmaStat 2.0).

Following MAP sponge removal in treated ewes, some ovulated follicles in three treated and three control sheep failed to form detectable CL (i.e., inadequate CL). In order to assess differences between the ewes with normal CL only (n = 8) and those that had both normal and inadequate CL (n = 6), hormonal data were compared between the two groups in question, after alignment with the day of ovulation, as described above for endocrine data.

Experiment 2

The first experiment was repeated to confirm the ovulations that occurred during sponging and the effects of MAP treatment on ovulation rate after sponge withdrawal. In addition, we examined the gonadotropin dependency of the ovulations that occurred during MAP treatment. Ewes were treated later in the breeding season (December–January) than they were in experiment 1, but general animal husbandry, handling of the ewes, and data analyses were similar in both experiments.

Ovarian ultrasonography was performed for 30 days from the beginning of the second estrus after the synchronization treatment using a real-time, high-resolution ultrasound scanner (Aloka SSD-900; Aloka Inc., Japan) connected to a stiffened, 7.5-MHz transducer. From 1 day before MAP treatment to 1 day after sponge removal, ultrasonography was performed every 12 h. The ability to detect and enumerate ovarian antral follicles >=1 mm in diameter using the Aloka SSD-900 scanner has been verified in our laboratory [4]. Ultrasonographic detection of ovulations and corpora hemorrhagica (CH) has also been recently validated in our laboratory by laparotomy and postoperative examinations of dissected ovaries (unpublished observations). Eight ewes were treated with PGF2{alpha}/MAP on Day 8 after ovulation. Blood samples (10 ml) were collected during ultrasonographic examinations, and also every 4 h from 1 day before to 1 day after the 6-day treatment with MAP sponges (5 ml), in order to determine serum concentrations of LH and FSH.

Using the higher resolution echo camera (Aloka SSD-900), ovarian follicles smaller than 1 mm (>=0.4 mm) in size could be visualized, and 2-mm follicles were accurately assigned at the origin and end of sequential follicular waves [4]. Therefore, a follicular wave was defined as one or more antral follicles that grew from the pool of 2 to >=3-mm follicles to >=5 mm in diameter (emergent follicles) within 48 h.

Analyses for each hormone were performed in single assays. The intraassay CVs for mean LH concentrations of 0.49 or 0.98 ng/ml were 7.6% or 5.9%, respectively. For FSH reference sera with mean concentrations of 0.42 or 2.32 ng/ml, the intraassay CVs were 5.6% or 1.6%, respectively. Intraassay CVs for reference sera with mean progesterone concentrations of 0.45 or 1.22 ng/ml were 14.4% or 4.6%, respectively. Serum concentrations of LH and FSH in blood samples taken every 4 h were analyzed for the period from 1 day before to 1 day after MAP treatment in all ewes, and also for the period from 48 h before to 12 h after ovulations detected during the period of treatment with MAP sponges (breakthrough ovulations), in five of eight ewes studied.

Experiment 3

The third experiment was undertaken to confirm and document the ovulations that occurred during sponging and to remove the ovaries for histology. Ewes were treated in September and October, as described for the first two experiments. Ovarian ultrasonography was performed every 12 h, starting 1 day before PGF2{alpha} injections and insertion of MAP sponges (8 days after ovulation), using an Aloka SSD-900 echo camera connected to a 7.5-MHz transducer. When ovulations were detected with ultrasonography, the ewes were transferred indoors and deprived of food for 12–18 h before surgery the following day. General anesthesia was induced with sodium pentobarbital (Somnotol, MTC Pharmaceutical, Cambridge, ON, Canada; 65 mg/ml; 20–30 ml/ewe, i.v.) and maintained with 2.5% halothane/oxygen (Halocarbon Laboratories, River Edge, NJ) administered by an endotracheal tube at an average flow rate of 0.75 L/min. Ovaries were exposed, photographed, and then dissected, and small tissue blocks containing ruptured follicles were fixed in Bouins solution. Paraffin wax sections were prepared and stained with hematoxylin and eosin. The histology of the ruptured follicles/CH was studied via light microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Follicular dynamics and ovulations Individual follicle profiles (follicles that grew from 3 to >=5 mm in diameter) are shown for two different animals for each group in order to illustrate the pattern of follicular wave development and the number and time of emergence of ovulatory follicles during the study period (Fig. 1).



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  		FIG. 1. The diameter profiles of individual antral follicles growing from 3 to >=5 mm in two Western white-faced ewes that received PGF2{alpha} and a progestogen (MAP) sponge on Day 7 (A) or Day 8 (B) after ovulation, and two control ewes (C, D; experiment 1). Day 0 = day of treatment with PGF2{alpha}, Day 6 = day of MAP sponge removal (treated ewes). The arrows along the X-axis (top panels; {uparrow}) denote the days of follicle wave emergence, and the arrows within the upper chart area (<-OV) indicate the ovulatory follicles on the day before ovulation

Within 2 to 5 days of the injection of PGF2{alpha} and insertion of progestogen sponges (Day 0), 6 of 7 treated ewes ovulated, giving a total of 11 ovulations (Table 1). Inspection of growth profiles for individual antral follicles revealed that ovulation occurred in 50% (8/16) of all follicles >=5 mm in diameter detected on the day of PGF2{alpha} injection in the treatment group. In addition, in one treated ewe, two follicles emerged on Day 2 and ovulated on Day 5, and in one other ewe, a follicle that emerged on Day 1 ovulated on Day 4 after PGF2{alpha}. No ewes were marked by rams and none of the follicles that ovulated after PGF2{alpha} and before the withdrawal of sponges formed a CL.


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  		TABLE 1. Percentage of ewes ovulating (treated ewes), mean ovulation rates and characteristics of ovarian antral follicles

In the six ewes that ovulated 2 to 5 days after PGF2{alpha} treatment, there were no significant changes (P > 0.05) in serum concentrations of FSH in daily blood samples, from 2 days before to 2 days after ovulation, but mean serum concentrations of estradiol declined from 4.1 ± 0.2 pg/ml on Day -1 before ovulation to 1.5 ± 0.5 pg/ml on the day of ovulation (P < 0.001), and subsequently increased (P < 0.05) to 3.4 ± 0.7 pg/ml on Day 2 after ovulation.

The last follicular wave detected before the PGF2{alpha}/MAP treatment of treated ewes emerged, on the average, 1.9 ± 0.2 days before the day of PGF2{alpha} injection (range, 1 to 3 days), in both treatment and control groups (P > 0.05). During the 6-day period in which treated ewes had MAP sponges in place, follicles from at least two waves were present in all ewes studied. The mean number of emerging follicular waves during MAP treatment in treated ewes did not differ (P > 0.05) from that observed during the same period in control ewes (1.7 ± 0.1/ewe; range, 1 to 2).

Following the removal of sponges, the mean ovulation rate was 3.1 ± 0.4 (range, 2 to 5) in treated ewes vs. 2.0 ± 0.3 (range, 1 to 3) in control ewes (P < 0.05; Table 2). The mean ovulation rate after treatment (3.1 ± 0.4) was also greater (P < 0.05) than the pretreatment ovulation rate (1.9 ± 0.3) for ewes given PGF2{alpha} and MAP sponges. In control ewes, there was no significant difference (P > 0.05) in the mean ovulation rate at the beginning (1.4 ± 0.2) and end (2.0 ± 0.3) of the interovulatory interval studied. All but one ovulation were detected after the onset of estrus. A single treated ewe ovulated one follicle 24 h before estrus (left ovary) and then two follicles 24 h after the onset of estrus (both in right ovary); the ovulation prior to estrus did not result in formation of a CL.


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  		TABLE 2. Ovulatory responses in ewes that received treatment and in control (untreated) ewes.*

At the time of sponge removal in treated ewes, the last follicular wave before ovulation contained ovulatory follicles in all ewes studied. Of all follicles reaching >=5 mm in diameter in this wave, 85% (11/13) and 73% (11/15) ovulated in the treatment and control groups, respectively (P > 0.05). The penultimate wave contained ovulatory follicles in five treated ewes but in only two control sheep. In the penultimate follicular wave, 70% (7/10) and 21% (3/14) of follicles >=5 mm in size were ovulatory in the treated and control ewes, respectively (P < 0.05). In addition, one treated ewe had a follicle that emerged in the follicular wave before the penultimate wave (Day -2 before PGF2{alpha}) and it ovulated 11 days after emergence. One treated ewe ovulated a follicle from a wave that emerged >3 days before PGF2{alpha} and the time elapsed from emergence to ovulation was >12 days. Finally, one treated ewe had an ovulatory follicle that emerged in each of the two waves before the penultimate wave.

None of the characteristics of follicle growth for ovulatory follicles in the last wave before ovulation detected after MAP treatment of treated ewes differed between the treated and control ewes (Table 3). In both groups of ewes, ovulatory follicles in the penultimate follicular wave emerged approximately 3 days earlier than ovulatory follicles in the final wave before ovulation. Ovulatory follicles in the penultimate wave in the treated ewes had a significantly longer growing phase (5.1 ± 0.5 vs. 3.0 ± 0.0 days) but a shorter static phase (2.7 ± 0.6 vs. 5.0 ± 1.0 days) compared with the control ewes (P < 0.05).


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  		TABLE 3. Characteristics of ovulatory antral follicles (growing to >=5 mm diameter; means ± SEM) originating from the final or penultimate follicular wave before ovulation (posttreatment).*

Progesterone concentrations and CL/luteal structures Mean daily serum concentrations of progesterone for the ewes given PGF2{alpha} and MAP and for control ewes are shown in Figure 2. All treated ewes responded to PGF2{alpha} as evidenced by an abrupt decline in circulating concentrations of progesterone and luteal regression observed with ultrasonography. Circulating concentrations of luteal progesterone declined (P < 0.05) to a basal level 24 h after injection of PGF2{alpha} and remained low (P > 0.05) during the entire period of treatment with progestogen sponges. Mean serum concentrations of progesterone were significantly higher in control ewes than in treated ewes for 6 consecutive days, from Day 1 to Day 6 after PGF2{alpha}.



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  		FIG. 2. Daily serum concentrations of progesterone (mean ± SEM) during the 19-day study period in seven Western white-faced ewes that received PGF2{alpha} injection on ~Day 8 and a progestogen (MAP) sponge from ~Days 8 to 14 after ovulation (treated ewes, filled symbols), and seven untreated white-faced ewes (control ewes, hollow symbols; experiment 1). Data were aligned to the day of treatment with PGF2{alpha} (Day 0) of the treated ewes. *P < 0.05 (data analyzed for the period from 1 day before to 1 day after MAP treatment). See text for additional statistical descriptions

All CL were detected by Day 4 after ovulation following sponge withdrawal in the treatment group (both groups, 3.3 ± 0.2 days after ovulation; P > 0.05). Three treated ewes and three control animals had fewer CL than ovulated follicles. A short-lived CL (observed once only on Day 3 after ovulation) was observed in one control ewe, and in this same ewe, a luteinized unovulated follicle was detected. The mean number of all luteal structures per ewe (2.4 ± 0.3 and 1.4 ± 0.2 for treated and control ewes, respectively) differed from the mean ovulation rates (3.1 ± 0.4 and 2.0 ± 0.3 for treated and control ewes, respectively; P < 0.05).

There was a significant difference between the treated and control ewes in terms of progesterone concentrations after ovulation following MAP treatment in treated ewes. The treated ewes exceeded control ewes in mean serum concentrations of progesterone on Day 4 (1.49 ± 0.33 vs. 1.03 ± 0.22 ng/ml) and Day 5 (2.41 ± 0.29 vs. 1.32 ± 0.25 ng/ml) after ovulation (treated vs. control ewes, respectively, P < 0.05).

Serum concentrations of gonadotropins and estradiol The main effect of group (P = 0.68), day (P = 0.89), and interaction (P = 0.33) were not significant for daily serum concentrations of FSH for the period from 1 day before to 1 day after MAP treatment of treated ewes. Circulating concentrations of estradiol in the ewes of the present study differed by day (P <= 0.001), but there was no significant effect of treatment (P = 0.60) and interaction of the main effects was not significant (P = 0.29). Mean daily concentrations of FSH and estradiol did not vary between the two groups during the period from 3 days before to 5 days after ovulation following MAP treatment (P > 0.05).

The secretory parameters of FSH and LH in the present ewes, based on the intensive blood sampling, were not affected by the treatment with PGF2{alpha} and MAP. The mean serum concentrations of FSH did not differ between groups (1.32 ± 0.16 and 1.53 ± 0.26 ng/ml for treated and control ewes, respectively; P > 0.05). Mean and basal levels of LH, LH pulse frequency, amplitude, and duration were 0.20 ± 0.02 and 0.20 ± 0.03 ng/ml, 0.13 ± 0.02 and 0.15 ± 0.01 ng/ml, 0.29 ± 0.09 and 0.27 ± 0.07 pulse/h (range, zero to three pulses per 6 h in both treated and control ewes); 0.47 ± 0.10 and 0.47 ± 0.12 ng/ml; and 54 ± 15 and 48 ± 11 min; for treated and control ewes, respectively (P > 0.05).

Comparisons between ewes with normal and inadequate CL after the final ovulation of the study period After the ovulation following MAP treatment of treated ewes, eight ewes formed as many CL as ovulated follicles (ewes with normal CL), but six ewes had fewer CL than ovulations (three treated and three control ewes; ewes with inadequate CL). The mean ovulation rate was 2.1 ± 0.3 and 3.2 ± 0.4, for ewes with normal and inadequate CL, respectively (P < 0.05). The mean number of detected luteal structures per ewe was 2.1 ± 0.3 and 1.7 ± 0.2 (P > 0.05) for ewes with normal and inadequate CL, respectively. The time from ovulation to detection of CL averaged 3.1 ± 0.2 days for both groups of ewes in question (P > 0.05). When data were normalized to the day of ovulation, mean serum concentrations of estradiol were significantly higher in ewes with normal CL on Day 1 (4.2 ± 0.4 vs. 2.5 ± 0.4 pg/ml) and Day 2 (4.6 ± 0.3 vs. 2.8 ± 0.4 pg/ml), and daily serum concentrations of progesterone were higher (P < 0.05) on Day 3 (1.03 ± 0.20 vs. 0.50 ± 0.14 ng/ml) and Day 4 (1.60 ± 0.28 vs. 0.81 ± 0.18 ng/ml) after ovulation (ewes with normal CL vs. ewes with inadequate CL, respectively).

Experiment 2

Seven ovulations were detected in five of eight ewes between 1 and 6 days after the injection of PGF2{alpha} and insertion of MAP sponges (Table 1). Six of 19 follicles (32%) that were >=5 mm in diameter on the day of PGF2{alpha} injection ovulated during treatment with MAP. In addition, one ewe had a follicle that emerged on Day 3 and ovulated on Day 6 after PGF2{alpha}. The mean day of emergence of the follicles that ovulated during sponging was -3.9 ± 1.5 days before PGF2{alpha} (range, from 8 days before to 3 days after PGF2{alpha} injection). No ewes were marked by rams and none of the follicles that ovulated during the treatment with MAP formed a CL; however, CH were detected after ovulations occurred (Fig. 3, A–D) for ~4 days (Table 1). For comparison, a CL detected with ultrasonography 48 h after ovulation posttreatment is shown in Figure 3E. In the five ewes that ovulated 1 to 6 days after PGF2{alpha}, there were no changes (P > 0.05) in serum concentrations of FSH or LH in blood samples taken every 4 h, from 48 h before to 12 h after ovulations (Fig. 4), and serum concentrations of LH remained basal (time effect, P > 0.05) from 1 day before to 1 day after treatment with MAP sponges.



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  		FIG. 3. Photographic reproductions of ultrasonograms of ovaries (left panels) with accompanying diagrams depicting the outlines of the ovary and intraovarian structures (right diagrams). Images obtained using an Aloka SSD-900 echo camera equipped with a 7.5-MHz transducer (experiment 2) were recorded on S-VHS, digitized, and then converted into gray scale bit-map images in order to delineate ovaries, antral follicles, CH, and CL. For illustrative purposes, ovarian stroma is shown in light gray, follicles in black, and CH/CL in dark gray. Sequential ultrasonograms (A–D) show an ovary recorded in a treated ewe at 0, 12, 24, and 48 h relative to ovulation, which was detected on Day 1 after PGF2{alpha} injection and insertion of a MAP sponge, and E depicts an ovary of a ewe 48 h after ovulation that was detected 3 days after the removal of MAP sponges. All ewes underwent transrectal ultrasonography of ovaries every 12 h, from 1 day before PGF2{alpha}/MAP treatment until 1 day after sponge withdrawal, and then daily for 8 days. Each scale bar represents 10 mm. Note the difference in the size and echogenicity (brightness) between demising CH (D) that did not form a CL, and a growing (full lifespan) CL (E) at 48 h postovulation



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  		FIG. 4. Mean (± SEM) concentrations of FSH (•) and LH ({circ}) in serum samples collected every 4 h, and analyzed for the period from 48 h before to 12 h after breakthrough ovulations (n = 7) detected with ultrasonography in five of eight Western white-faced ewes, between Days 1 and 6 after PGF2{alpha}/MAP treatment (experiment 2)

The mean number of emerging follicular waves during MAP treatment was 1.7 ± 0.2 per ewe (range, 1 to 2), and the last wave before sponging emerged 1.9 ± 0.3 days before treatment (range, 1 to 3 days). After the sponges were withdrawn, the mean ovulation rate was 2.8 ± 0.4 (range, 2 to 5). The mean ovulation rate after treatment was greater (P < 0.05) than the pretreatment ovulation rate (1.9 ± 0.1; Table 2). All but one ewe were well marked by rams; one ewe was not marked. In four ewes, a total of nine ovulations were observed before estrus, and five ovulations were detected after the onset of behavioral estrus; in the remaining four ewes all ovulations occurred after estrus. However, in all but one ewe, ovulations occurred within a 48-h period, between 2 and 5 days after sponge withdrawal. A single ewe ovulated on Days 2, 3, 4 (before estrus), and again on Day 6 after MAP treatment (after the onset of estrus).

The last follicular wave before ovulation following MAP treatment contained ovulatory follicles in all ewes; of all follicles reaching >=5 mm in diameter in this wave, 86% (12/14) ovulated (Table 2). The penultimate wave before ovulation contained ovulatory follicles in seven of eight ewes. In the penultimate follicular waves, 60% (9/15) of follicles >=5 mm in size were ovulatory. None of the follicles that emerged before the penultimate wave ovulated. Ovulatory follicles in the penultimate wave emerged approximately 3 days earlier and had a significantly longer static phase (4.5 ± 0.9 vs. 1.7 ± 0.4 days; P < 0.05) compared with ovulatory follicles in the last follicular wave.

All but one treated ewe responded to PGF2{alpha} with a decline in circulating concentrations of progesterone within 24 h after injections. In one ewe, CL could be observed for 7 days after PGF2{alpha} until regression just before estrus after MAP treatment, and serum concentrations of progesterone declined to 0.50 ng/ml 24 h after treatment (Day 0), then rose to 1.23 ng/ml on Day 4, and finally declined to 0.10 ng/ml on Day 7 after PGF2{alpha}. In this same ewe, a breakthrough ovulation occurred on Day 4 after PGF2{alpha} and ovulations of follicles from the last two waves of the cycle were seen.

Following the ovulation after sponge withdrawal, four of eight animals had fewer CL than ovulated follicles. One ewe ovulated two follicles but no CL was detected by Day 5 after ovulation, and serum progesterone concentrations were ± 0.10 ng/ml. In the remaining three ewes, some ovulated follicles failed to form observable CL. One ewe with two CL formed a luteinized, unovulated follicle that was first detected 3 days after ovulation. The mean number of CL per ewe (1.6 ± 0.2) was less than the mean ovulation rate (2.8 ± 0.4; P < 0.05). The total number of all luteal structures per ewe (1.8 ± 0.2) was also less (P < 0.05) than the ovulation rate above.

Experiment 3

Within 3 to 6 days of the injection of PGF2{alpha} and insertion of MAP sponges, 6 of 11 ewes ovulated, giving a total of 11 ovulations (Table 1). No ewes were marked by rams. All of the ovulations detected with ultrasonography were subsequently confirmed by laparotomy (Fig. 5). On the basis of ultrasonographic records, 28% (9/32) of all follicles >=5 mm in diameter present on the day of PGF2{alpha} injection ovulated during MAP treatment. In addition, one ewe had a follicle that emerged on Day 1 and ovulated on Day 4, and in one other ewe a follicle that emerged on Day 2 ovulated on Day 5 after PGF2{alpha}. Ruptured ovarian follicles with distinct blood-stained ovulatory sites, from 1 to 5 mm in diameter, protruding above the surface of the ovary, were observed at laparotomy (Fig. 5, A and C). Histological micrographs of the dissected CH are shown in Figure 5, B and D.



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  		FIG. 5. Photographs of ovaries at laparotomy conducted in two Western white-faced ewes (A and C; experiment 3) 1 day after follicular rupture detected with ultrasonography 5 and 3 days after PGF2{alpha} injection and insertion of MAP impregnated sponges, respectively. The ewes received PGF2{alpha} and a MAP sponge on Day 8 after ovulation. Arrows indicate ovulatory sites observed during the inspection of ovaries just before dissection. B and D depict histological sections through the CH illustrated in (A) and (C) (x40). Arrowheads delineate outlines of the CH/ruptured follicles. For comparison, a section through the CL of a ewe 24 h after normal ovulation (Duggavathi et al., unpublished data) showing darkly stained thecal cells, the hypertrophied granulosa cells, and the innermost connective tissue with the reminder of a blood clot, is shown in (E) (x40). Note differences in diameter and cell differentiation (luteinization) between the growing CL (E) and transient CH in the present study (B and D). Scale bars in (B), (D), and (E) represent 1 mm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, in nonprolific Western white-faced ewes, a 6-day treatment with MAP from midcycle in the absence of luteal progesterone resulted in a significant increase in ovulation rate by approximately 50% when compared with control ewes or the pretreatment cycle of treated ewes. This increase was mainly the result of the ovulation of follicles from the penultimate follicular wave before ovulation, and some follicles from earlier waves.

The high ovulation rate in prolific Finn sheep is achieved by ovulation of ~50% of follicles attaining >=5 mm in diameter in the penultimate follicular wave, which ovulate along with the follicles from the final wave of the estrous cycle [3]. The occurrence of two ovulatory waves has also recently been shown in 3/4 Rambouillet ;ts 1/4 Booroola ewes [29]. This contrasts with nonprolific Western white-faced ewes, in which very few ovulatory-sized follicles ovulate from the penultimate wave of the cycle [3]. In the present study (experiments 1 and 2), the percentage of follicles that ovulated from the penultimate wave before ovulation in MAP-treated ewes was 70% and 60%, respectively. There is, therefore, a striking similarity in the pattern of ovulatory follicle recruitment between the Western white-faced ewes that received PGF2{alpha} and MAP, and that of normally cycling Finn sheep.

Submaximal concentrations of progesterone prolonged the lifespan of large antral follicles in cyclic ewes [6, 7]. A similar effect was observed when luteolysis was induced with PGF2{alpha} and progesterone was replaced by MAP being released from intravaginal sponges [8], or when MAP-impregnated sponges were inserted on Day 12 after ovulation and ovaries were exposed to MAP in the absence of functional CL [18]. This suggests that MAP treatment mimics a low progesterone regimen in sheep. During the midbreeding season, mean serum concentrations of progesterone are higher in nonprolific Western white-faced than in prolific Finn sheep [9]. We speculate that treatment of nonprolific Western white-faced ewes with MAP after PGF2{alpha}-induced luteolysis created the equivalent of a low progesterone environment, and hence promoted an increase in ovulation rate to resemble that of the Finn sheep [3].

There is marked variation in prolificacy among breeds of sheep [30–34]. Earlier studies failed to produce convincing evidence that differences in circulating concentrations of gonadotropic hormones were solely responsible for differences in ovulation rate in ewes [34]. It has been suggested that the high ovulation rate in prolific sheep is due to intraovarian rather than pituitary factors [34–36]. We propose that the difference in the pattern of follicle development and ovulation rate between breeds of sheep differing in prolificacy may be partly due to a difference in circulating concentrations of progesterone.

In earlier studies, prolongation of follicle lifespan was consistently observed in ewes given PGF2{alpha} early in the estrous cycle (Days 5 or 6 after ovulation), with a subsequent treatment to create low serum concentrations of progesterone [6, 7] or with MAP [8] for 10 or 14 days, respectively. The proposed mechanism when the treatments began early in the luteal phase was an increase in LH pulse frequency [8], similar to the explanation given for cattle [37]; however, LH pulse frequency was measured only once, after 13 days of MAP treatment in sheep [8]. In the present study, a shorter treatment with MAP was applied at midcycle, and no increase in LH pulse frequency was noted as determined 3 days after sponging. In a previous study in which intravaginal MAP sponges were inserted for 12 days on Day 12 after ovulation (i.e., just before natural luteolysis), prolonged follicle lifespan was also observed, but LH pulse frequency did not change over the first 6 days of treatment, and only a numerical but not significant increase was seen after 12 days [18]. Therefore, the extended follicular lifespan and increased ovulation rate after PGF2{alpha}/MAP treatment commenced late in the sheep estrous cycle, and applied for a short period of time, may not be dependent on changes in LH pulse frequency.

It is possible that creation of lower than normal luteal phase concentrations of progesterone or progestogen treatment could have direct effects on the ovary. Certainly, the CL can exert local effects on developing follicles in sheep [22, 38–42] in spite of morphological barriers between the luteal and follicular compartments, and the high concentrations of progesterone in ovine follicular fluid (10-fold to 40-fold serum concentrations; [19]). Progesterone may modify follicular responsiveness to gonadotropins in both CL-bearing and non-CL-bearing ovaries [41, 43–45]. It is also feasible that circulating concentrations of progesterone or MAP could affect ovarian follicles indirectly by extraovarian mechanisms. Luteal progesterone may alter the countercurrent transfer of hormones in the subovarian vascular plexus, probably via changes in estrogen:progesterone ratios and their effects on the constriction of blood vessels [46].

In the ewes of the present study, even though MAP sponges were inserted on the day of PGF2{alpha} treatment, injection of a luteolytic dose of PGF2{alpha} on ~Day 8 after ovulation was followed by ovulation of some large follicles, between Days 1 and 6 after injection. There was no change in circulating concentrations of FSH in samples taken daily during that period, but a rapid decline in daily serum concentrations of estradiol to basal concentrations in the six ovulating ewes after PGF2{alpha}/MAP treatment coincided with the day of ovulation (experiment 1). Such a decline in serum concentrations of estradiol is typically seen around ovulation during the normal estrous cycle [3]. We initially suspected that PGF2{alpha}-induced luteolysis in the presence of MAP resulted in aberrant or truncated preovulatory changes in gonadotropin release. Obviously, in cyclic ewes, there is tremendous variation in the magnitude and duration of the preovulatory LH surge [47]. However, frequent blood sampling conducted on Day 3 after PGF2{alpha} did not indicate the occurrence of phasic secretion of LH in the treated ewes. Moreover, based on blood sampling every 4 h throughout the period of treatment with MAP (experiment 2), there was no evidence of a preovulatory mode of gonadotropin secretion.

We have now confirmed that ovulations detected in the ewes of the present study during MAP treatment (Figs. 3 and 5) were not preceded by an increase in gonadotropin secretion. The underlying mechanism of these ovulations that occurred during administration of the synthetic progestogen remains to be elucidated.

None of the ovulations above were followed by the formation of CL, only transient CH (Table 1), as evidenced by both ultrasonographic examinations (experiment 2; Fig. 3) and radioimmunoassay of serum progesterone (experiment 1, Fig. 2; and experiment 2). The failure of normal luteogenesis has previously been reported in ewes induced to ovulate during the luteal phase [48]. This may be attributed to low LH concentrations during an established luteal phase [48] or to the presence of a fresh MAP sponge (present study). Histological micrographs of the dissected CH (Fig. 5, B and D) revealed their relatively small sizes, suggesting limited hypertrophy of granulosa cells. In addition, the pattern of staining of the thecal and granulosa lutein cells as seen in the forming ovine CL (Fig. 5E) was not observed in the present study (experiment 3).

Impaired luteogenesis of some ovulated follicles was observed in both treated and control ewes after the final ovulation in this study. Luteal inadequacy in ewes may be due to a lack of luteinization of some ovulated follicles [20, 48, 49] or premature regression of CL [20, 49]. A report by Mann and Lamming [49] showed that in cattle, low serum concentrations of estradiol around the time of ovulation were associated with an earlier increase in an oxytocin receptor level and augmented PGF2{alpha} response to oxytocin stimulation (i.e., stronger luteolytic signal). In the present study, mean serum concentrations of estradiol were significantly higher in ewes that produced as many CL as ovulations compared with animals that had inadequate CL, on Days 1 and 2 after ovulation, but normal CL were observed in both groups of ewes in question. This suggests that luteal insufficiency may be a product of inadequate developmental competence of preovulatory follicles; this would explain the coexistence of apparently normal and inadequate CL in a ewe, or even in an ovary of a ewe [3, 20].

The first experiment was replicated to confirm its findings. There was close agreement between the first two experiments in terms of the incidence of breakthrough ovulations (i.e., ovulations detected during MAP treatment), and the number of ovulations and time at which they occurred after sponge withdrawal in MAP-treated ewes. However, the ewes studied late in the breeding season (experiment 2) appeared to have a delayed onset of estrus, by approximately 1.5 days, and a somewhat greater number of inadequate CL compared with the ewes in the middle portion of the breeding season (experiment 1). These differences could be due to decreased estrogenicity of antral follicles [50, 51], or to diminished ovarian responsiveness to gonadotropic stimuli [52], or both, in ewes approaching seasonal anestrus (experiment 2). The important point, however, is that nonprolific ewes at different stages of the breeding season exhibited similar patterns of change in follicular development and ovulation rate in response to a PGF2{alpha} /MAP treatment applied at midcycle.

Induction of multiple births has been identified as one of the primary goals in controlled sheep breeding [53]. The results of the present study may provide a basis for devising practical follicle manipulation techniques to increase the ovulation rate in commercial flocks of sheep. However, in cattle, there is evidence [54, 55], albeit not unequivocal [56], that oocyte quality is compromised in follicles with an extended lifespan or dominance, resulting in low fertility. Recent studies suggest that this is not the case in sheep [57], which means that the addition of prolonged lifespan follicles to the ovulatory cohort could effectively increase fertility. In the study by Johnson et al. [6], fertility was depressed by a PGF2{alpha}/low progesterone treatment in ewes; however, in that study, treatment was applied from early in the cycle and for a longer duration (Days 5 to 15 after ovulation). In a recent study by Vinoles et al. [58], the pregnancy rate was significantly higher in cyclic Polwarth sheep that received MAP sponges for 6 days (87%) rather than 12 days (63%).

In conclusion, a PGF2{alpha}/MAP regimen applied late in the cycle of nonprolific Western white-faced ewes increased the ovulation rate, mainly through maintenance of ovulatory-sized follicles in the penultimate and earlier waves, and their addition to ovulatory follicles from the final follicular wave before ovulation. Breakthrough ovulations were observed in 65% (17/26) of treated ewes between 1 and 6 days after PGF2{alpha} treatment and insertion of MAP sponges. These ovulations did not affect the emergence of successive follicular waves, were not preceded by a preovulatory discharge in gonadotropin secretion, and did not result in CL, but rather, only in transient CH. In the present study, we were unable to show a dependency of the effects of MAP treatment on serum concentrations of FSH/LH or LH pulse frequency, suggesting that the altered follicular dynamics and increased ovulation rate may have been related to other direct or indirect effects of MAP, independent of changes in gonadotropin secretion.


   ACKNOWLEDGMENTS
 
We thank Drs. B. Singh and J. Singh for help with the histology of ovaries, and Mr. Edward Bagu, Ms. Kate Davies, Mr. K. Janardhan, and Ms. Christine Smetschka for their excellent technical assistance. We also thank the National Institute for Diabetes and Digestive and Kidney Diseases and the U.S. Department of Agriculture for providing reagents for the gonadotropin assays.


   FOOTNOTES
 
1 This study was funded by the Alberta Agricultural Research Institute (AARI) in the form of a Farming for the Future grant to N.C.R. and by the Natural Sciences and Engineering Council of Canada (NSERC). P.M.B. was supported by a University of Saskatchewan Graduate Scholarship and Saskatchewan Health Services Utilization and Research Commission postdoctoral fellowship. Back

2 Correspondence: N.C. Rawlings, Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, Canada S7N 5B4. FAX: 306 966 7376; norman.rawlings{at}usask.ca Back

3 Current address: Department of Obstetrics, Gynecology and Reproductive Sciences, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4 Back

4 Current address: Human Health Research Center, INRS-Institut Armand Frappier, Quebec City, QC, Canada H7V 1B7 Back

Received: 9 May 2002.

First decision: 19 June 2002.

Accepted: 30 October 2002.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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