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Characterization of Endocrine Events During the Periestrous Period in Sheep After Estrous Synchronization with Controlled Internal Drug Release (CIDR) Device
DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 15(1):23–34, 1998
CHARACTERIZATION OF ENDOCRINE EVENTS DURING THE PERIESTROUS PERIOD IN SHEEP AFTER ESTROUS SYNCHRONIZATION WITH CONTROLLED INTERNAL DRUG RELEASE (CIDR) DEVICE1 J. Van Cleeff,*2 F.J. Karsch,* ** and V. Padmanabhan* ***3 *Reproductive Sciences Program and the Departments of **Physiology and ***Pediatrics, University of Michigan, Ann Arbor, MI 48109-0404 Received March 11, 1997 Accepted August 11, 1997
The Controlled Internal Drug Releasing (CIDR) device is an intravaginal pessary containing progesterone (P4) designed for synchronizing estrus in ruminants. To date, there has been little information available on the timing, duration, and quality of the follicular phase after CIDR removal and how those characteristics compare with natural periovulatory endocrine events. The present communication relates the results of methods we used to characterize the endocrine events that followed CIDR synchronization. Breeding-season ewes were given an injection (10 mg) of Lutalyse (PGF2a), and then studied during three consecutive estrous cycles, beginning in the luteal phase after the estrus induced by PGF2a. Cycle 1 estrus was synchronized with 1 CIDR (Type G) inserted for 8 d beginning 10 d after PGF2a. Cycles 2 and 3 were synchronized with two CIDRs for 8 d beginning 10 d after previous CIDR removal. Cycle 1 estrous behavior and serum gonadotropins showed a follicular phase (the interval from CIDR withdrawal to gonadotropin surge [surge] peak) of 38.2 6 1.5 hr. Two CIDRs lengthened the interval to 46.2 6 1.5 hr (P , 0.0001). At CIDR removal, circulating P4 concentrations were higher in ewes treated with two CIDRs (5.1 6 0.3 and 6.4 6 0.4 ng/mL in Cycles 2 and 3 vs. 2.7 6 0.3 ng/mL in Cycle 1), whereas estradiol concentrations were higher in the 1 CIDR cycle (3.3 6 0.5 pg/mL in Cycle 1 vs. 0.5 6 0.1, and 0.7 6 0.2 pg/mL in Cycles 2 and 3), suggesting that the lower levels of P4 achieved with one CIDR was not sufficient to arrest follicular development. There were no differences in any other endocrine variable. Both one and two CIDR synchronization concentrated surges within a 24-hr period in 92% of the ewes in Cycles 1 and 2. Cycle 3 ewes were euthanized at estimated luteal, early follicular, late follicular, LH surge, and secondary FSH rise timepoints. Endocrine data and ovaries showed that 88% of the ewes synchronized with two CIDRs were in the predicted stage of the estrous cycle. These data demonstrate that the CIDR device applied during the luteal phase effectively synchronizes estrus and results in a CIDR removal-to-surge interval of similar length to a natural follicular phase. © Elsevier Science Inc. 1998
INTRODUCTION Synchronization of the estrous cycle has aided producers with reproductive management and facilitated scientific study of reproductive endocrine events. The Controlled Internal Drug Releasing (CIDR) device is an intravaginal pessary containing only the natural hormone progesterone (P4), which is used for synchronizing estrus in a variety of farmed and captive wild ruminants (1–12). The CIDR device was primarily designed for use in a farm setting. It was meant to be a tool for concentrating and scheduling breeding efforts to maximize efficiency in terms of ram (or inseminator), facility, and labor usage. Typical on-farm use for normal breeding in sheep consists of the application of one CIDR © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010
0739-7240/98/$19.00 PII S0739-7240(97)00059-3
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device for 12–14 d in randomly cycling ewes (4 – 6). In a farm setting, synchrony is normally defined as acceptable when a high percentage, typically 90% or better, of treated animals come into estrus within a 72-hr period. Further refinement into periods shorter than 2–3 d has not been necessary for the producer. In addition, the CIDR device has the advantage of being noninvasive and nonluteolytic, as well as being easy to use, and so resynchronization after breeding are possible (7,8). The CIDR device can clearly synchronize estrus, bringing groups of ewes into estrus over a short, predictable period. However, there has been little direct scrutiny of the effects of the device on the endocrinology of the follicular phase after CIDR removal in terms of such elements as follicular estradiol production, gonadotropin secretion patterns, or the interval to and duration of the preovulatory gonadotropin surge, particularly in sheep. As part of our ongoing investigation of the control of gonadal function, we needed to preserve as much as possible the timing, duration, and quality of natural periovulatory endocrine events. The present communication relates the results of methods we used to characterize the events after a synchronization regimen using the CIDR device. Although not derived from a controlled and balanced experiment, these data demonstrate the efficacy of the CIDR device, and provide useful information on which to base further experimentation. MATERIALS AND METHODS Twenty-six intact mature Suffolk ewes were given a pretreatment injection (10 mg) of Lutalyse (PGF2a; Upjohn, Kalamazoo, MI) to induce luteolysis. Ewes were then studied during three consecutive estrous cycles, beginning in the luteal phase after the estrus induced by PGF2a. Cycle 1 estrus was synchronized by treatment for 8 d with a CIDR device (Type G, 9% natural progesterone; InterAg, Hamilton, New Zealand) beginning 10 d after PGF2a. Estrous behavior was monitored using vasectomized rams. Briefly, two rams painted with stock marking paint on the brisket were introduced to the ewes 24 hr after one CIDR removal and, at 4-hr intervals after collection of blood samples (described below), ewes were inspected for rump markings and observed for 15 min. Marked and mounted ewes were removed from the group and tested with a third ram to confirm receptivity. Unconfirmed ewes were returned to the main group, whereas confirmed ewes were removed to a separate pen until all ewes were detected in estrus. Initial observations of estrous behavior after one CIDR removal indicated that onset of estrus occurred earlier than the expected time of approximately 40 hr (13,14). Subsequent analysis of blood samples for gonadotropins confirmed a follicular phase (the interval from CIDR removal to gonadotropin surge) shorter than is usually seen in naturally cycling ewes in our flock; ewes at our facility typically exhibit an interval of about 48 hr from onset of the fall in circulating progesterone to gonadotropin surge peak (13,14). Therefore, for Cycle 2 synchronization, 25 ewes from the same group were treated with two CIDR devices simultaneously 10 d after first CIDR removal, and treatment lasted 8 d (same period as one CIDR cycle). Because ewes were cannulated and sampling frequency was high, estrous detection was not used during Cycle 2. Cycle 3 was conducted exactly as the second cycle (2 CIDR devices were inserted 10 d after the previous CIDR removal and left in place for 8 d), except that rams were introduced within 4 hr of CIDR removal to monitor the onset of estrus. During Cycle 3, on the basis of hormonal data obtained from Cycle 2, ewes were killed and tissues were collected at five specific time points (luteal, early follicular, late follicular, surge, secondary FSH rise) during the periestrous period (five ewes per group). Luteal group ewes were killed 3 d before CIDR removal. Early follicular and late follicular groups were killed 12 and 32 hr after CIDR removal. Surge ewes and secondary FSH rise ewes were killed 8 and 32 hr after detection of estrus. Ovaries were examined
CIDR SYNCHRONIZATION AND THE PERIESTROUS PERIOD
25
at the time of euthanasia and gonadotropin and steroid concentrations measured to determine whether ewes were at the predicted endocrine stage. For determination of steroid concentrations at critical points during the study, single blood samples were collected via jugular venipuncture 2 d after PGF2a injection before the first cycle, and, for all cycles, samples were collected immediately before CIDR insertions and immediately before CIDR removals. Care was taken to prevent exposure of samples to any progesterone contamination from the CIDR. Intensive sampling was conducted during the periestrous periods of the three CIDR-synchronized cycles. During the one CIDR cycle, jugular venipuncture sampling at 4-hr intervals began 24 hr after CIDR removal, and continued through 120 hr after CIDR removal. In Cycle 2 (two CIDR), blood samples were collected more extensively and with decreasing intervals during the expected time of the gonadotropin surge to obtain better precision in the timing of events. Samples were collected every 6 hr at the beginning and end of the periestrous period (24 hr before CIDR removal until 12 hr after CIDR removal, and from 66 hr–96 hr after CIDR removal). Sampling interval was decreased to 4-hr intervals from 12 hr after CIDR removal, and then to 2-hr intervals from 24 – 66 h after CIDR removal, during the time when the gonadotropin surge was expected. During Cycle 3 (two CIDR), blood samples were collected infrequently until euthanasia from 3 d before through 12 hr after CIDR removal, and at 2-hr intervals beginning 12 hr after CIDR removal until euthanasia. Assays. Circulating progesterone (P4) concentrations were measured in two 100-mL aliquots using a standard commercial solid-phase radioimmunoassay kit (Coat-A-Count P4, Diagnostic Products Corp., Los Angeles, CA). This assay kit has been validated for use in sheep (15). Standard curves were modified to include a lower concentration and exclude the highest concentration of progesterone (range, 0.05–20 ng/mL). Intra-assay coefficients of variation (C.V.) at 80% and 20% binding were 13.5% and 6.8%, respectively. Median variance ratio was 0.04. Interassay C.V. (mean of five assays) was 10.4%. Detection limit of the assay, calculated as 2 SD of buffer control, averaged 0.04 6 0.01 ng/mL. Estradiol (E2) assays were performed on 200 ml duplicates of serum extracted with 2 mL anhydrous ethyl ether (Fisher Scientific), using a modification (16) of a commercially available radioimmunoassay kit that uses magnetic particle separation (Estradiol MAIA, Serono, Italy). For Cycles 1 and 2, E2 was measured in samples collected from CIDR removal through onset of estrus on a subset of 12 ewes that included those ewes that in Cycle 3 formed the surge group (n 5 5) and the secondary surge group (n 5 5), and two ewes from the late follicular group. In Cycle 3, samples from all ewes except those in the luteal phase group were assayed. Intra-assay C.V. for 80% and 20% binding were 20.3% and 10.2%, respectively. Median variance ratio was 0.10. Interassay C.V. (mean of five assays) based on three quality control sera was 23.4%. Detection limit (2 SD of buffer control) of the E2 assay was 0.29 6 0.09 pg/mL. Serum LH was measured by a previously validated RIA (17) in duplicate 100 –200 mL aliquots using a serum standard calibrated against NIH-LH-S12 (biopotency 9.82 3 S1) (18). Repeat assays of samples during the surge were performed as needed using duplicate 5–50 mL aliquots. The LH assay sensitivity (2 SD of buffer control) and ED50 (50% displacement point) averaged 0.17 and 0.65 ng, respectively (n 5 7 assays). The intraassay C.V. at the 80% and 20% displacements averaged 8.5 6 0.9% and 4.3 6 0.5%, respectively. The interassay C.V., based on two quality control pools measured at 22.1 6 0.6 and 16.1 6 0.4 ng/ml, averaged 12% and 10%, respectively. Serum FSH was measured in duplicate 50 –100 mL aliquots using NIDDK-oFSH-1 (AFP 5679C) as the standard and NIDDK-anti-oFSH-1 (AFP-C5288113) antiserum at a working dilution of 1:12K. The sensitivity and ED50 of the FSH assay averaged 0.04 and 0.42 ng, respectively (n 5 5 assays). The intra-assay C.V. at 80% and 20% displacement points averaged
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VAN CLEEFF ET AL.
11.4 6 1.4% and 5.7 6 0.7% respectively. The interassay C.V. based on three quality control pools measuring 2.4 6 0.04, 8.7 6 0.2, and 16.6 6 0.4 ng/ml averaged 7%, 10%, and 9%, respectively. Statistical Analysis. The start of the LH surge was defined as the time at which LH exceeded baseline secretion by 2 3 SD and remained at or above this level for at least 4 hr. The end of the LH surge was defined as the time at which LH concentrations returned to baseline plus 2 3 SD. Differences in mean intervals (hr) from CIDR removal to various periestrous events after one and two CIDR synchronization (Cycles 1 and 2) were analyzed using analysis of variance. Statistical analyses are presented as a comparison of one versus two CIDR treatment for Cycles 1 and 2 only. All means of hormone concentrations and intervals are presented with standard errors. Additionally, a Spearman Rank Correlation Test was applied to assess repeatability between Cycle 1 and 2 of the temporal order in which LH peaks occurred after CIDR removal. RESULTS Estrous Behavior Following One CIDR Synchronization. Estrous detection was used in Cycle 1 after one CIDR removal and in Cycle 3 after two CIDR removal (estrus was not monitored in Cycle 2). Mean time from device removal to detected onset of estrus was 36.2 6 2 hr (n 5 26) during the one CIDR cycle. This calculation does not account for the exact onset of estrus of the earliest ewes, which may have occurred earlier than 24 hr after device removal. Of 26 ewes treated with one CIDR device, 6 ewes were already in estrus when vasectomized rams were introduced at 24 hr after CIDR removal, and another 5 ewes were detected in estrus at 28 hr. Overall, 24 of 26 treated ewes were detected in estrus between 24 and 48 hr after CIDR removal. One ewe (Ewe No. 12) was not synchronized (determined on the basis of progesterone measurements, see below). With the nonsynchronized ewe excluded (detection of estrus 60 hr after one CIDR removal), mean time to detected onset of estrus was 35.2 6 1.9 hr. Mean time from device removal to detection of estrus for Cycle 3 (two CIDR) was 42.4 6 3.4 hr (n 5 10; others were killed before detection of estrus). Circulating Sex Steroid Levels After One and Two CIDR Synchronization. Concentrations of P4 achieved before, during, and after one and two CIDR treatments are shown in Figures 1 and 2. Concentrations of P4 averaged 0.5 6 0.1 ng/mL (n 5 26) in samples collected 2 d after injection of PGF2a. P4 concentrations were greater than 1 ng/mL at this time in 6 of the 26 ewes. At time of 1 CIDR insertion (10 days after PGF2a), P4 concentrations averaged 3.3 6 0.2 ng/mL (range, 1.0 –5.9; n 5 26). Immediately before one CIDR device removal, circulating P4 concentration averaged 2.7 6 0.3 ng/mL (range, 1.4 – 8.5 ng/mL; n 5 26) and declined to 0.5 6 0.2 ng/ml (n 5 26) at 24 hr after device removal. Of the 26 ewes 1 (Ewe No. 12) had P4 concentration of 4.9 ng/mL at 24 hr after CIDR removal, suggesting the presence of functional corpus luteum. This nonsynchronized ewe was removed from the study after the first cycle. Progesterone concentrations in the other 25 synchronized ewes averaged 0.4 6 0.02 ng/mL 24 hr after CIDR removal. At the time of two CIDR insertion in Cycle 2, P4 concentrations averaged 5.0 6 0.3 ng/mL (range, 1.68 –7.76; n 5 25). At the time of two CIDR removal in Cycle 2, P4 concentrations averaged 5.1 6 0.3 ng/mL, but had declined to 0.8 6 0.1 ng/mL by 6 hr later, and to 0.4 6 0.04 by 20 hr (n 5 25; Figure 1). Progesterone concentrations before CIDR insertion, just prior to and 20 h after CIDR removal averaged 3.2 6 0.2, 6.4 6 0.4 and 0.34 6 0.1 ng/mL for Cycle 3 (2 CIDR; n 5 25). Mean E2 concentrations after CIDR removal from a subset of 12 ewes in Cycles 1 and 2 are shown in Figure 2 (top panel, open circles). Concentrations of E2 achieved during Cycle 3 by the seven ewes that were killed after the LH surge are also presented for
CIDR SYNCHRONIZATION AND THE PERIESTROUS PERIOD
27
Figure 1. Progesterone concentrations in ewes treated with 1 or 2 CIDR devices for 8 d beginning in the luteal phase. (A) Mean 6 SE, 1 CIDR, Cycle 1; (B) Mean 6 SE, 2 CIDR, Cycle 2; (C) Mean 6 SE, 2 CIDR, Cycle 3; (D) Individual ewes, 1 CIDR, Cycle 1 (n 5 25); 1 of the 26 ewes studied (Ewe No. 12) had P4 concentration of 4.95 ng/mL 24 hr after CIDR removal suggesting the presence of a functional corpus luteum and was considered unsynchronized (data from this ewe is not shown); (E) individual ewes, 2 CIDR, Cycle 2 (n 5 25), (F) individual ewes, 2 CIDR, Cycle 3 (n 5 25).
comparison (right panel). At one CIDR removal, the concentration of E2 was variable (mean 6 SE 5 3.3 6 0.5 pg/mL; range, 1.6 –7.5 pg/mL). It seemed that a single CIDR device, by the end of 8 d of use, failed to inhibit progression of follicular development. Two CIDR devices, however, successfully inhibited E2 production while the device was in place. Immediately before two CIDR removal in Cycle 2, mean E2 concentration was 0.5 6 0.1 pg/mL, significantly lower than that for 1 CIDR (P , 0.0001), and similar to previously reported luteal phase concentrations (13,14). The interval from CIDR removal to peak E2 for Cycles 1 and 2 were not different (Table 1), but maximum E2 was significantly lower after treatment with two CIDR devices (7.4 6 0.8 vs. 5.25 6 0.8 pg/mL; P , 0.01). Immediately before two CIDR removal in Cycle 3, mean E2 concentration was 0.7 6 0.1 pg/mL (n 5 20 ewes), similar to those seen in Cycle 2. For the seven Cycle 3 ewes that showed an LH peak before euthanasia, mean E2 concentrations immediately before CIDR removal and peak E2 concentrations were 1.4 6 0.5 pg/mL and 5.4 6 0.6 pg/mL, respectively. Timing and Nature of Gonadotropin Surges After One and Two CIDR Synchronization. Mean circulating patterns of LH after one and two CIDR removal are shown in Figure 2 (bottom panels) and timing relationships in Table 1 and Figure 3. Average interval from one CIDR device removal to gonadotropin surge peak was 38.2 6 1.5 hr (range, 24 –52 hr), excluding the unsynchronized ewe (ewe No. 12), and 39.4 6 1.9 hr (range, 24 – 68 hr) with all ewes. In 24 of 26 ewes, the LH peaks occurred over a 24-hr period, but no obvious clustering within a shorter time frame was notable (Figure 3). In five of the six ewes that were detected in estrus at 24 hr peak, LH concentrations occurred
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Figure 2. Mean (6SE) E2, P4, LH and FSH for the 12 ewes in which all hormones were measured after 1 CIDR synchronization (Cycle 1, left panel) and 2 CIDR synchronization (Cycle 2, middle panel). Mean (6SE) E2, P4, LH, and FSH for the seven ewes from Cycle 3 that were killed during or after the primary gonadotropin surge are shown for comparison (right panel). Two of the seven ewes were euthanized before onset of secondary FSH rise and five during the secondary FSH surge. All data are normalized to LH peak (time 0, vertical line). CIDR removal coincides with first data point.
28 –36 hr after one CIDR removal, whereas the maximum LH concentration was found in one ewe at 24 hr after one CIDR removal. As in Cycle 1, all ewes treated with two CIDR devices in Cycle 2 (n 5 25) experienced a gonadotropin surge after two CIDR removal; all ewes could be considered synchronized (Figure 3). Mean interval from two CIDR device removal to surge onset and surge peak (range, 30 – 60 hr) in Cycle 2 was significantly longer than for 1 CIDR cycle (P , 0.0001; Table 1). Use of two CIDR devices delayed the LH peak by an average of 8 hr compared with one CIDR. Figure 4 shows the cumulative percentage of ewes reaching the LH peak TABLE 1. TIMING
OF
GONADOTROPIN SURGE EVENTS IN EWES TREATED WITH ONE HOURS (MEAN 6 SE; EWE NO. 12 EXCLUDED)
OR
TWO CIDR DEVICES,
IN
Treatment
Event Interval from CIDR removal to peak estradiol Interval from CIDR removal to onset of LH surge Interval from CIDR removal to LH surge peak Duration of LH surge Interval from surge peak to onset of secondary FSH rise
Cycle 1 1 CIDR (Estrus monitored)
Cycle 2 2 CIDR (Estrus monitored)
Cycle 3 2 CIDR (Estrus not monitored)
33.8 6 1.0 (n 5 9) 33.8 6 1.4 (n 5 25) 38.2 6 1.5 (n 5 25) 15.1 6 1.4 (n 5 25) 16.3 6 1.4 (n 5 25)
39.0 6 1.7 (n 5 12) 42.3 6 1.3a (n 5 25) 46.2 6 1.5a (n 5 25) 14.8 6 1.4 (n 5 25) 16.6 6 1.4 (n 5 25)
40.3 6 4.0 (n 5 7*) 40.3 6 3.5 (n 5 7*) 46.0 6 4.0 (n 5 7*) 15.6 6 1.0 (n 5 5**) 14.4 6 0.6 (n 5 5**)
2 CIDR (Cycle 2) . 1 CIDR, P , 0.0001. * Includes all ewes not euthanized before LH peak. ** Secondary FSH rise group only.
a
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29
Figure 3. LH surges in individual ewes treated with 1 CIDR and 2 CIDR devices (Cycles 1 and 2, respectively) for 8 d beginning in the luteal phase.
after device removal for Cycles 1 and 2. One-CIDR treatment concentrated LH peaks in 24 of 26 ewes within a 24 h period, whereas in Cycle 2 LH peaks occurred within an 18-hr period for 22 of 25 ewes (Figure 3), though the trend toward a smaller variation in timing of peaks was not statistically significant (P 5 0.10). Although there was a difference in the timing of the onset of the LH surge, there were no differences between Cycles 1 and 2 in the LH surge maximum (155.1 6 9.4 vs. 152.6 6 7.3 ng/mL, Figure 2) or duration (Table 1). Maximum surge FSH for the Cycles 1 and 2 averaged 8.9 6 0.5 and 7.5 6 0.5 ng/mL and were not different. The average interval from CIDR removal to peak of LH surge for the seven ewes in
Figure 4. Cumulative percent of ewes treated with one or two CIDR devices (Cycles 1 and 2, respectively) reaching peak of LH surge.
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TABLE 2. NUMBER OF EWES WITH PREDOMINANT OVARIAN STRUCTURES AND CORRESPONDING STEROID CONCENTRATIONS WHEN KILLED. EWES WERE SYNCHRONIZED WITH 2 CIDR DEVICES INSERTED ON DAY 10 CYCLE 2, AND SACRIFICED AT TIMES INDICATED BY GROUP NAMES. Luteal Structures (P4, ng/mL) Group (n 5 5) Luteal Early follicular Late follicular Surge Secondary FSH rise
Yellow, Vascular
Pale, Avascular
4 — (12.2 6 2.0) 1 4 (0.5) (0.6 6 0.1) — 5 (0.3 6 0.1) — 5 (0.2 6 0.04) — —
None 1 (3.2*) — — — 5 (0.1 6 0.03)
OF
Follicular Structures (E2, pg/mL) Large, Clear 1 (**) 5 (2.1 6 0.8a) 5 (2.1 6 0.5b) 1 (2.43a) —
Pre-Ovulatory, Corpus Hyperemic Hemorrhagicum None —
—
—
—
4 (**) —
—
—
—
4 (2.2 6 1.1c) —
—
—
5 (0.3 6 0.03d)
—
* P4 concentration is within what is expected from 2 CIDR devices after 5 d of use in the absence of a CL (unpublished data). ** Samples not assayed for E2. a Highest concentration in final sample. b Three ewes declining after peak concentration, two ewes highest concentration in final sample. c Two ewes declining after peak concentration, two ewes highest concentration in final sample. d Declining after peak concentration.
Cycle 3 that exhibited an LH peak before euthanasia was similar to that of Cycle 2. Concentrations averaged 148.5 6 9.6 and 7.6 6 0.8 ng/mL for LH and FSH, respectively. Secondary FSH rise was defined as beginning at the first sample after the primary surge ended, which exceeded the pre-surge mean FSH concentration by 2 3 SD and which was followed by at least 8 hr of sustained increase in FSH concentration. All but one of the periestrous periods examined in Cycles 1, 2, and 3 (n 5 56) exhibited clearly distinguishable secondary FSH rises. After one CIDR treatment, the secondary FSH rise was first detected at 16.3 hr after peak LH (Figure 2). Secondary FSH rise after synchronization with two CIDR devices began on average 16.6 hr after peak LH in Cycle 2, not significantly later than with one CIDR. There was some evidence for a relationship in the temporal order of LH peaks from the first cycle to the second. This effect does not seem to be related to the number of CIDR devices used to synchronize the peaks. For example, those ewes that were in estrus by 24 hr after one CIDR removal in the first cycle were the first to experience LH surges in the second cycle, with peaks occurring 30 –38 hr after two CIDR removal, earlier than the average interval. When a Spearman ranking correlation coefficient was calculated, the hour by which each ewe reached peak LH in the first cycle was significantly correlated to the hour by which she reached peak LH in the second cycle (r 5 0.74). This suggests that the timing of events after P4 withdrawal is consistent within ewe and is independent of the number of CIDR devices. Prediction Outcome. During Cycle 3, ewes were sacrificed (five per group) at five time points during the periestrous period (see Materials and Methods for details). Gross examination of the ovaries at slaughter and subsequent endocrine data revealed that 4/5 or 5/5 ewes in each group were in their expected phases at time of sacrifice (Table 2). Gonadotropins were at basal concentrations for both early and late follicular groups. In the Surge group, ewes were euthanized prior to the LH surge (n 5 1), on the ascending limb (n 5 2), or on the descending limb (n 5 2) of the LH surge. The two ewes killed on the descending limb of the surge had E2 profiles that had peaked and begun to decline, whereas for all others the final sample contained the highest E2 concentration. None of the
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Surge group ewes had ovulated, but four of five had begun the ovulatory process and showed markedly hyperemic preovulatory follicles, indicating that they had been exposed to the gonadotropin surge (19). Secondary FSH rise ewes had basal LH concentrations, and FSH concentrations indicative of being in the secondary FSH rise period. The combined success rate over all groups in achieving the intended stage at sacrifice was 88% (22/25), or 92% including the early follicular ewe with a CL of functional appearance but P4 below 0.5 ng/mL. DISCUSSION Temporal relationships among periestrous events in naturally cycling ewes and after synchronization with subcutaneous implants of P4 pockets have been well characterized (13,14,18,20,21). Where CIDR devices are used in sheep, P4 profiles, timing of estrus, and pregnancy and lambing rates are all well established (1– 6). However, the temporal relationships between endocrine events that follow the removal of CIDR devices have not been characterized in detail. Although the present study was not performed as a balanced crossover experiment, and comparisons between one and two CIDR synchronization must be made with caution, some general conclusions can be drawn as to the efficacy of the described synchronization system that may prove useful to researchers requiring precise control of periovulatory events. Our results do suggest that for an 8-d CIDR-treatment period beginning in the luteal phase, a single CIDR device will prevent onset of estrus and the surge for at least 8 d when applied during the luteal phase. However, the concentration of P4 delivered by a single CIDR device during the final days of treatment was not sufficient to prevent progression of follicular development to an estrogenic stage. The result was an early onset of estrus in many ewes and an unnaturally short follicular phase on average. Given the low concentrations of P4 achieved in our study at the end of 8 d, it does not seem that the traditional 12-d regimen using a single CIDR device in randomly cycling ewes would be adequate for achieving optimal synchronization of all ewes in a group. Treatment with two CIDR devices simultaneously for the same period did successfully inhibit E2 secretion and lengthen the interval from device removal to gonadotropin surge to a duration more closely resembling the naturally occurring follicular phase (13,14). The longer follicular phase in Cycle 2 may be attributable to more uniform follicular maturation, or at least synchronous initiation of the preovulatory estradiol rise. Thus, the secretory status of the preovulatory follicle(s) seemed to be the key factor in determining the difference between one and two CIDR treatments. This difference was confined to the timing of the surge relative to P4 withdrawal and to the length of the follicular phase. No other differences in surge parameters, such as amplitude or duration, or effects on the secondary FSH rise were detectable. The estrogenic status of the follicle seemed to be determined primarily by the concentration of P4 circulating at the time of CIDR withdrawal, which was lower for one CIDR compared with two CIDR (2.68 6 0.27 vs. 5.15 6 0.27 ng/mL). Johnson and coworkers (21) found that ewes treated to induce a peripheral concentration of P4 less than 1 ng/mL during the luteal phase had increased peripheral E2 when compared with ewes with circulating P4 greater than 1 ng/mL. Our ewes, regardless of treatment, had higher, CIDR-induced, concentrations of P4 compared to the Johnson study (21), but the difference in P4 concentrations between 1 and 2 CIDR treatment may have similarly led to the difference in E2 concentration. The suppressive effects of P4 on circulating E2 concentrations may be mediated via the proven negative feed back effects on pulsatile LH/GnRH secretion (23).
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In addition to qualitative differences in secretory activity of the follicle, a role for P4 in controlling the timing of periovulatory events has also been suggested. Using P4-impregnated sponges, Robinson and Smith (24) and Pearce and Robinson (25) showed a direct relationship between the dose of P4 and the timing of estrus, with the larger dose of P4 resulting in a longer latency. Although the differences in latency between Cycle 1 and 2 in our studies may be directly related to the P4 concentrations achieved, a concern that needs to be addressed is the possibility of a ram effect (26) in the 1 CIDR cycle, which would have been absent in Cycle 2. However, this does not seem to be the case because the latency from P4 removal to peak LH surge after 2 CIDR removal were similar, averaging 46 6 1.5 and 46 6 4 hr for Cycle 2 (n 5 25, estrus not monitored) and Cycle 3 (n 5 7; estrus monitored), respectively. Comparative studies performed in the presence or absence of rams by Hauger and coworkers (18) have also shown that the interval between P4 removal and the onset of the gonadotropin surge and gonadotropin surge characteristics are not affected by the presence of a ram. From a synchronization standpoint, the application of the CIDR device during the luteal phase of the estrous cycle allows for more precise control over the resulting synchronized estrus. While both one and two CIDR treatments concentrated LH peaks within a 24-hr period, some surges occurred earlier than desired, especially with one CIDR treatment. The precise control afforded by the high P4 at the end of treatment with two CIDR devices resulted in the desired timing of gonadotropin surge peak, at about 48 hr after CIDR removal, and the desired concentration of gonadotropin surges to within 24 h of each other. Studies conducted in cycling ewes by Wheaton and coworkers (2) and our preliminary studies [unpublished data] in ovariectomized ewes show that circulating P4 concentrations remain above 2 ng/ml for the first 5 d of CIDR insertion and decline steadily thereafter. Given the relatively low P4 output from a single CIDR device by 8 d, a single CIDR device inserted too early in the luteal phase would not suffice in maintaining the levels of P4 required for precise synchronization regimens. It is a possibility that the mere repetition of the synchronization regimen may have affected the quality of the synchronization response (i.e., concentration and/or timing of LH surge peaks). Information on repeated synchronization in sheep is lacking, especially repeated synchronization of unbred animals. Inseminations that fail are still likely to affect resynchronization responses (7). In heifers first synchronized using PGF2a only, then resynchronized using CIDR devices for Days 17–22 after estrus, estrous responses were 78% and 94% in inseminated-nonpregnant and non-inseminated heifers, respectively (8). Our non-inseminated ewes achieved a resynchronization rate of 100% for the second cycle, but seemed to be slightly less closely synchronized in the third. It is more likely that repeated synchronizations would lead to a gradual decline in the precision of the synchrony, rather than an increase. Therefore, the improved response seen in the second cycle compared to the first is more likely due to the increased P4 concentration than to the effects of repeated synchronization. In summary, application of two CIDR devices for 8 d beginning in the luteal phase resulted in a CIDR removal-to-surge interval of similar length to a natural follicular phase, and effectively synchronized and concentrated the timing of gonadotropin surges. Data derived from Cycles 2 and 3 also suggest that this synchronization regimen is capable of producing repeated, well-synchronized cycles. ACKNOWLEDGMENTS/FOOTNOTES We are grateful to Mr. Douglas D. Doop and Mr. Gary McCalla for help with the animal maintenance; Dr. Gordon D. Niswender and Leo E. Reichert, Jr. for supplying assay reagents for the LH assay, and National Hormone and Pituitary Program for generous supply of FSH reagents used in the FSH assay. Support was also provided by the Sheep Research, Standards and Reagents, Data Analysis, and Administrative Core Facilities of
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the Center for the Study of Reproduction. All procedures were performed within the guidelines of the University’s Committee for the Use and Care of Animals at the University of Michigan. 1 Published as part of the National Cooperative Program for Infertility Research. Supported by NIH grants U54 HD-29184 (UM), and P30 HD-18258 (UM). Portions of this work were presented at the 29th Annual meeting of the Society for the Study of Reproduction, London, Ontario, Canada. 2 Current address: Animal Science Group, Faculty of Agriculture, University of Western Australia, Nedlands, WA 6907, Australia. 3 Address reprint requests to Vasantha Padmanabhan, Ph.D., Reproductive Sciences Program, University of Michigan, 300 N. Ingalls Bldg., Room 1110, Ann Arbor, MI 48109-0404.
REFERENCES 1. Welch RAS. Development of CIDR dispensers for use in nulliparous ewes. New Zealand Ministry of Agriculture and Fisheries, Agricultural Research Division Annual Report, 1983/84, p. 58, 1985. 2. Wheaton JE, Carlson KM, Windels HF, Johnston LJ. CIDR. A new progesterone-releasing intravaginal device for induction of estrus and cycle control in sheep and goats. Anim Rep Sci 33:127–141, 1993. 3. Smith JF, Konlechner JA, Parr J. The efficacy of used CIDR devices for synchronization of oestrus and post-mating treatment. Proceedings of the New Zealand Society of Animal Production 5:111–115, 1991. 4. Carlson KM, Pohl HA, Marcek JM, Muser RK, Wheaton JE. Evaluation of progesterone Controlled Internal Drug Release dispensers for synchronization of estrus in sheep. Anim Reprod Sci 18:205–218, 1989. 5. Rhodes L, Nathanielsz PW. Comparison of a Controlled Internal Drug Release device containing progesterone with intravaginal medroxyprogesterone sponges for estrous synchronization in ewes. Theriogenology 30:831– 836, 1988. 6. Thompson JG, Simpson AC, James RW, Tervit HR, Asher GW, Peterson AJ. Timing of the LH peak and ovulations in superovulated Coopworth ewes synchronized with progesterone containing CIDR devices. Proc NZ Soc Anim Prod 52:171–174, 1992. 7. Van Cleeff J, Drost M, Thatcher WW. Effects of postinsemination progesterone supplementation on fertility and subsequent estrous responses of dairy heifers. Theriogenology 36:795– 807, 1991. 8. Van Cleeff J, Macmillan KL, Drost M, Lucy MC, Thatcher WW. Effects of administering progesterone at selected intervals after insemination of synchronized heifers on pregnancy rates and resynchronization of returns to service. Theriogenology 46:1117–1130, 1996. 9. Asher GW, Fisher MW, Jabbour HN, Smith JF, Mulley RC, Morrow CJ, Veldhuizen FA, Langridge M. Relationship between the onset of oestrus, the preovulatory surge in luteinizing hormone and ovulation following oestrous synchronization and superovulation of farmed red deer (Cervus elaphus). J Reprod Fertil 96:261–273, 1992. 10. Bowen JM, Barrell GK. Duration of the oestrous cycle and changes in plasma hormone concentrations measured after an induced ovulation in scimitar-horned oryx (Oryx dammah). J Zoology 238:137–148, 1996. 11. Jabbour HN, Asher GW, Smith JF, Morrow CJ. Effect of progesterone and oestradiol benzoate on oestrous behaviour and secretion of luteinizing hormone in ovariectomized fallow deer (Dama dama). J Reprod Fertil 94:353–361, 1992. 12. Jabbour HN, Veldhuizen FA, Mulley RC, Asher GW. Effect of exogenous gonadotrophins on oestrus, the LH surge and the timing and rate of ovulation in red deer (Cervus elaphus). J Reprod Fertil 100:533–539, 1994. 13. Karsch FJ, Foster DL, Legan SJ, Ryan KD, Peter GK. Control of the preovulatory endocrine events in the ewe: Interrelationship of estradiol, progesterone and luteinizing hormone. Endocrinology 105:421– 426, 1979. 14. Karsch FJ, Legan SJ, Ryan KD, Foster DL. Importance of estradiol and progesterone in regulating LH secretion and estrous behaviour during the sheep estrous cycle. Biol Rep 23:404 – 413, 1980. 15. Padmanabhan V, Evans NP, Dahl GE, McFadden KL, Mauger DT, Karsch FJ. Evidence for short or ultrashort loop negative feedback of GnRH secretion. Neuroendocrinology 62:248 –258, 1995. 16. Evans NP, Dahl GE, Glover BH, Karsch FJ. Central regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 134:1806 –1811, 1994. 17. Niswender GD, Reichert LE Jr, Midgley AR Jr, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166 –1173, 1969. 18. Hauger RL, Karsch FJ, Foster DL. A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101:807– 817, 1977. 19. Murdoch WJ. Endothelial cell death in preovulatory ovine follicles: Possible implication in the biomechanics of rupture. J Reprod Fert 105:161–164, 1995.
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20. Goodman RL. Neuroendocrine control of the ovine estrous cycle. In: The Physiology of Reproduction, 2nd Ed., E. Knobil, JD Neill (eds.), Raven Press, Ltd., New York, pp. 659 –709, 1994. 21. Karsch FJ, Bittman EL, Foster DL, Goodman RL, Legan SJ, Robinson JE. Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 40:185–232, 1984. 22. Johnson SK, Daily RA, Inskeep EK, Lewis PE. Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes. Domest Anim Endocrinol 13:69 –79, 1996. 23. Karsch FJ. Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing hormone. Annu Rev Physiol 49:365–382, 1987. 24. Robinson TJ, Smith JF. The time of ovulation after withdrawal of SC-9880 impregnated intravaginal sponges from cyclic Merino ewes. In: The Control of the Ovarian Cycle in Sheep, TJ Robinson (ed.), Sydney University Press, Sydney, Australia, pp. 158 –168, 1967. 25. Pearce DT, Robinson TJ. Plasma progesterone concentrations, ovarian and endocrinological responses and sperm transport in ewes with synchronized oestrus. J Reprod Fertil 75:49 – 62, 1985. 26. Parsons SD, Hunter GL, Rayner AA. Use of probit analysis in a study of the effect of the ram on time of ovulation in the ewe. J Reprod Fertil 14:71– 80, 1967.
CHARACTERIZATION OF ENDOCRINE EVENTS DURING THE PERIESTROUS PERIOD IN SHEEP AFTER ESTROUS SYNCHRONIZATION WITH CONTROLLED INTERNAL DRUG RELEASE (CIDR) DEVICE1 J. Van Cleeff,*2 F.J. Karsch,* ** and V. Padmanabhan* ***3 *Reproductive Sciences Program and the Departments of **Physiology and ***Pediatrics, University of Michigan, Ann Arbor, MI 48109-0404 Received March 11, 1997 Accepted August 11, 1997
The Controlled Internal Drug Releasing (CIDR) device is an intravaginal pessary containing progesterone (P4) designed for synchronizing estrus in ruminants. To date, there has been little information available on the timing, duration, and quality of the follicular phase after CIDR removal and how those characteristics compare with natural periovulatory endocrine events. The present communication relates the results of methods we used to characterize the endocrine events that followed CIDR synchronization. Breeding-season ewes were given an injection (10 mg) of Lutalyse (PGF2a), and then studied during three consecutive estrous cycles, beginning in the luteal phase after the estrus induced by PGF2a. Cycle 1 estrus was synchronized with 1 CIDR (Type G) inserted for 8 d beginning 10 d after PGF2a. Cycles 2 and 3 were synchronized with two CIDRs for 8 d beginning 10 d after previous CIDR removal. Cycle 1 estrous behavior and serum gonadotropins showed a follicular phase (the interval from CIDR withdrawal to gonadotropin surge [surge] peak) of 38.2 6 1.5 hr. Two CIDRs lengthened the interval to 46.2 6 1.5 hr (P , 0.0001). At CIDR removal, circulating P4 concentrations were higher in ewes treated with two CIDRs (5.1 6 0.3 and 6.4 6 0.4 ng/mL in Cycles 2 and 3 vs. 2.7 6 0.3 ng/mL in Cycle 1), whereas estradiol concentrations were higher in the 1 CIDR cycle (3.3 6 0.5 pg/mL in Cycle 1 vs. 0.5 6 0.1, and 0.7 6 0.2 pg/mL in Cycles 2 and 3), suggesting that the lower levels of P4 achieved with one CIDR was not sufficient to arrest follicular development. There were no differences in any other endocrine variable. Both one and two CIDR synchronization concentrated surges within a 24-hr period in 92% of the ewes in Cycles 1 and 2. Cycle 3 ewes were euthanized at estimated luteal, early follicular, late follicular, LH surge, and secondary FSH rise timepoints. Endocrine data and ovaries showed that 88% of the ewes synchronized with two CIDRs were in the predicted stage of the estrous cycle. These data demonstrate that the CIDR device applied during the luteal phase effectively synchronizes estrus and results in a CIDR removal-to-surge interval of similar length to a natural follicular phase. © Elsevier Science Inc. 1998
INTRODUCTION Synchronization of the estrous cycle has aided producers with reproductive management and facilitated scientific study of reproductive endocrine events. The Controlled Internal Drug Releasing (CIDR) device is an intravaginal pessary containing only the natural hormone progesterone (P4), which is used for synchronizing estrus in a variety of farmed and captive wild ruminants (1–12). The CIDR device was primarily designed for use in a farm setting. It was meant to be a tool for concentrating and scheduling breeding efforts to maximize efficiency in terms of ram (or inseminator), facility, and labor usage. Typical on-farm use for normal breeding in sheep consists of the application of one CIDR © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010
0739-7240/98/$19.00 PII S0739-7240(97)00059-3
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device for 12–14 d in randomly cycling ewes (4 – 6). In a farm setting, synchrony is normally defined as acceptable when a high percentage, typically 90% or better, of treated animals come into estrus within a 72-hr period. Further refinement into periods shorter than 2–3 d has not been necessary for the producer. In addition, the CIDR device has the advantage of being noninvasive and nonluteolytic, as well as being easy to use, and so resynchronization after breeding are possible (7,8). The CIDR device can clearly synchronize estrus, bringing groups of ewes into estrus over a short, predictable period. However, there has been little direct scrutiny of the effects of the device on the endocrinology of the follicular phase after CIDR removal in terms of such elements as follicular estradiol production, gonadotropin secretion patterns, or the interval to and duration of the preovulatory gonadotropin surge, particularly in sheep. As part of our ongoing investigation of the control of gonadal function, we needed to preserve as much as possible the timing, duration, and quality of natural periovulatory endocrine events. The present communication relates the results of methods we used to characterize the events after a synchronization regimen using the CIDR device. Although not derived from a controlled and balanced experiment, these data demonstrate the efficacy of the CIDR device, and provide useful information on which to base further experimentation. MATERIALS AND METHODS Twenty-six intact mature Suffolk ewes were given a pretreatment injection (10 mg) of Lutalyse (PGF2a; Upjohn, Kalamazoo, MI) to induce luteolysis. Ewes were then studied during three consecutive estrous cycles, beginning in the luteal phase after the estrus induced by PGF2a. Cycle 1 estrus was synchronized by treatment for 8 d with a CIDR device (Type G, 9% natural progesterone; InterAg, Hamilton, New Zealand) beginning 10 d after PGF2a. Estrous behavior was monitored using vasectomized rams. Briefly, two rams painted with stock marking paint on the brisket were introduced to the ewes 24 hr after one CIDR removal and, at 4-hr intervals after collection of blood samples (described below), ewes were inspected for rump markings and observed for 15 min. Marked and mounted ewes were removed from the group and tested with a third ram to confirm receptivity. Unconfirmed ewes were returned to the main group, whereas confirmed ewes were removed to a separate pen until all ewes were detected in estrus. Initial observations of estrous behavior after one CIDR removal indicated that onset of estrus occurred earlier than the expected time of approximately 40 hr (13,14). Subsequent analysis of blood samples for gonadotropins confirmed a follicular phase (the interval from CIDR removal to gonadotropin surge) shorter than is usually seen in naturally cycling ewes in our flock; ewes at our facility typically exhibit an interval of about 48 hr from onset of the fall in circulating progesterone to gonadotropin surge peak (13,14). Therefore, for Cycle 2 synchronization, 25 ewes from the same group were treated with two CIDR devices simultaneously 10 d after first CIDR removal, and treatment lasted 8 d (same period as one CIDR cycle). Because ewes were cannulated and sampling frequency was high, estrous detection was not used during Cycle 2. Cycle 3 was conducted exactly as the second cycle (2 CIDR devices were inserted 10 d after the previous CIDR removal and left in place for 8 d), except that rams were introduced within 4 hr of CIDR removal to monitor the onset of estrus. During Cycle 3, on the basis of hormonal data obtained from Cycle 2, ewes were killed and tissues were collected at five specific time points (luteal, early follicular, late follicular, surge, secondary FSH rise) during the periestrous period (five ewes per group). Luteal group ewes were killed 3 d before CIDR removal. Early follicular and late follicular groups were killed 12 and 32 hr after CIDR removal. Surge ewes and secondary FSH rise ewes were killed 8 and 32 hr after detection of estrus. Ovaries were examined
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at the time of euthanasia and gonadotropin and steroid concentrations measured to determine whether ewes were at the predicted endocrine stage. For determination of steroid concentrations at critical points during the study, single blood samples were collected via jugular venipuncture 2 d after PGF2a injection before the first cycle, and, for all cycles, samples were collected immediately before CIDR insertions and immediately before CIDR removals. Care was taken to prevent exposure of samples to any progesterone contamination from the CIDR. Intensive sampling was conducted during the periestrous periods of the three CIDR-synchronized cycles. During the one CIDR cycle, jugular venipuncture sampling at 4-hr intervals began 24 hr after CIDR removal, and continued through 120 hr after CIDR removal. In Cycle 2 (two CIDR), blood samples were collected more extensively and with decreasing intervals during the expected time of the gonadotropin surge to obtain better precision in the timing of events. Samples were collected every 6 hr at the beginning and end of the periestrous period (24 hr before CIDR removal until 12 hr after CIDR removal, and from 66 hr–96 hr after CIDR removal). Sampling interval was decreased to 4-hr intervals from 12 hr after CIDR removal, and then to 2-hr intervals from 24 – 66 h after CIDR removal, during the time when the gonadotropin surge was expected. During Cycle 3 (two CIDR), blood samples were collected infrequently until euthanasia from 3 d before through 12 hr after CIDR removal, and at 2-hr intervals beginning 12 hr after CIDR removal until euthanasia. Assays. Circulating progesterone (P4) concentrations were measured in two 100-mL aliquots using a standard commercial solid-phase radioimmunoassay kit (Coat-A-Count P4, Diagnostic Products Corp., Los Angeles, CA). This assay kit has been validated for use in sheep (15). Standard curves were modified to include a lower concentration and exclude the highest concentration of progesterone (range, 0.05–20 ng/mL). Intra-assay coefficients of variation (C.V.) at 80% and 20% binding were 13.5% and 6.8%, respectively. Median variance ratio was 0.04. Interassay C.V. (mean of five assays) was 10.4%. Detection limit of the assay, calculated as 2 SD of buffer control, averaged 0.04 6 0.01 ng/mL. Estradiol (E2) assays were performed on 200 ml duplicates of serum extracted with 2 mL anhydrous ethyl ether (Fisher Scientific), using a modification (16) of a commercially available radioimmunoassay kit that uses magnetic particle separation (Estradiol MAIA, Serono, Italy). For Cycles 1 and 2, E2 was measured in samples collected from CIDR removal through onset of estrus on a subset of 12 ewes that included those ewes that in Cycle 3 formed the surge group (n 5 5) and the secondary surge group (n 5 5), and two ewes from the late follicular group. In Cycle 3, samples from all ewes except those in the luteal phase group were assayed. Intra-assay C.V. for 80% and 20% binding were 20.3% and 10.2%, respectively. Median variance ratio was 0.10. Interassay C.V. (mean of five assays) based on three quality control sera was 23.4%. Detection limit (2 SD of buffer control) of the E2 assay was 0.29 6 0.09 pg/mL. Serum LH was measured by a previously validated RIA (17) in duplicate 100 –200 mL aliquots using a serum standard calibrated against NIH-LH-S12 (biopotency 9.82 3 S1) (18). Repeat assays of samples during the surge were performed as needed using duplicate 5–50 mL aliquots. The LH assay sensitivity (2 SD of buffer control) and ED50 (50% displacement point) averaged 0.17 and 0.65 ng, respectively (n 5 7 assays). The intraassay C.V. at the 80% and 20% displacements averaged 8.5 6 0.9% and 4.3 6 0.5%, respectively. The interassay C.V., based on two quality control pools measured at 22.1 6 0.6 and 16.1 6 0.4 ng/ml, averaged 12% and 10%, respectively. Serum FSH was measured in duplicate 50 –100 mL aliquots using NIDDK-oFSH-1 (AFP 5679C) as the standard and NIDDK-anti-oFSH-1 (AFP-C5288113) antiserum at a working dilution of 1:12K. The sensitivity and ED50 of the FSH assay averaged 0.04 and 0.42 ng, respectively (n 5 5 assays). The intra-assay C.V. at 80% and 20% displacement points averaged
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11.4 6 1.4% and 5.7 6 0.7% respectively. The interassay C.V. based on three quality control pools measuring 2.4 6 0.04, 8.7 6 0.2, and 16.6 6 0.4 ng/ml averaged 7%, 10%, and 9%, respectively. Statistical Analysis. The start of the LH surge was defined as the time at which LH exceeded baseline secretion by 2 3 SD and remained at or above this level for at least 4 hr. The end of the LH surge was defined as the time at which LH concentrations returned to baseline plus 2 3 SD. Differences in mean intervals (hr) from CIDR removal to various periestrous events after one and two CIDR synchronization (Cycles 1 and 2) were analyzed using analysis of variance. Statistical analyses are presented as a comparison of one versus two CIDR treatment for Cycles 1 and 2 only. All means of hormone concentrations and intervals are presented with standard errors. Additionally, a Spearman Rank Correlation Test was applied to assess repeatability between Cycle 1 and 2 of the temporal order in which LH peaks occurred after CIDR removal. RESULTS Estrous Behavior Following One CIDR Synchronization. Estrous detection was used in Cycle 1 after one CIDR removal and in Cycle 3 after two CIDR removal (estrus was not monitored in Cycle 2). Mean time from device removal to detected onset of estrus was 36.2 6 2 hr (n 5 26) during the one CIDR cycle. This calculation does not account for the exact onset of estrus of the earliest ewes, which may have occurred earlier than 24 hr after device removal. Of 26 ewes treated with one CIDR device, 6 ewes were already in estrus when vasectomized rams were introduced at 24 hr after CIDR removal, and another 5 ewes were detected in estrus at 28 hr. Overall, 24 of 26 treated ewes were detected in estrus between 24 and 48 hr after CIDR removal. One ewe (Ewe No. 12) was not synchronized (determined on the basis of progesterone measurements, see below). With the nonsynchronized ewe excluded (detection of estrus 60 hr after one CIDR removal), mean time to detected onset of estrus was 35.2 6 1.9 hr. Mean time from device removal to detection of estrus for Cycle 3 (two CIDR) was 42.4 6 3.4 hr (n 5 10; others were killed before detection of estrus). Circulating Sex Steroid Levels After One and Two CIDR Synchronization. Concentrations of P4 achieved before, during, and after one and two CIDR treatments are shown in Figures 1 and 2. Concentrations of P4 averaged 0.5 6 0.1 ng/mL (n 5 26) in samples collected 2 d after injection of PGF2a. P4 concentrations were greater than 1 ng/mL at this time in 6 of the 26 ewes. At time of 1 CIDR insertion (10 days after PGF2a), P4 concentrations averaged 3.3 6 0.2 ng/mL (range, 1.0 –5.9; n 5 26). Immediately before one CIDR device removal, circulating P4 concentration averaged 2.7 6 0.3 ng/mL (range, 1.4 – 8.5 ng/mL; n 5 26) and declined to 0.5 6 0.2 ng/ml (n 5 26) at 24 hr after device removal. Of the 26 ewes 1 (Ewe No. 12) had P4 concentration of 4.9 ng/mL at 24 hr after CIDR removal, suggesting the presence of functional corpus luteum. This nonsynchronized ewe was removed from the study after the first cycle. Progesterone concentrations in the other 25 synchronized ewes averaged 0.4 6 0.02 ng/mL 24 hr after CIDR removal. At the time of two CIDR insertion in Cycle 2, P4 concentrations averaged 5.0 6 0.3 ng/mL (range, 1.68 –7.76; n 5 25). At the time of two CIDR removal in Cycle 2, P4 concentrations averaged 5.1 6 0.3 ng/mL, but had declined to 0.8 6 0.1 ng/mL by 6 hr later, and to 0.4 6 0.04 by 20 hr (n 5 25; Figure 1). Progesterone concentrations before CIDR insertion, just prior to and 20 h after CIDR removal averaged 3.2 6 0.2, 6.4 6 0.4 and 0.34 6 0.1 ng/mL for Cycle 3 (2 CIDR; n 5 25). Mean E2 concentrations after CIDR removal from a subset of 12 ewes in Cycles 1 and 2 are shown in Figure 2 (top panel, open circles). Concentrations of E2 achieved during Cycle 3 by the seven ewes that were killed after the LH surge are also presented for
CIDR SYNCHRONIZATION AND THE PERIESTROUS PERIOD
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Figure 1. Progesterone concentrations in ewes treated with 1 or 2 CIDR devices for 8 d beginning in the luteal phase. (A) Mean 6 SE, 1 CIDR, Cycle 1; (B) Mean 6 SE, 2 CIDR, Cycle 2; (C) Mean 6 SE, 2 CIDR, Cycle 3; (D) Individual ewes, 1 CIDR, Cycle 1 (n 5 25); 1 of the 26 ewes studied (Ewe No. 12) had P4 concentration of 4.95 ng/mL 24 hr after CIDR removal suggesting the presence of a functional corpus luteum and was considered unsynchronized (data from this ewe is not shown); (E) individual ewes, 2 CIDR, Cycle 2 (n 5 25), (F) individual ewes, 2 CIDR, Cycle 3 (n 5 25).
comparison (right panel). At one CIDR removal, the concentration of E2 was variable (mean 6 SE 5 3.3 6 0.5 pg/mL; range, 1.6 –7.5 pg/mL). It seemed that a single CIDR device, by the end of 8 d of use, failed to inhibit progression of follicular development. Two CIDR devices, however, successfully inhibited E2 production while the device was in place. Immediately before two CIDR removal in Cycle 2, mean E2 concentration was 0.5 6 0.1 pg/mL, significantly lower than that for 1 CIDR (P , 0.0001), and similar to previously reported luteal phase concentrations (13,14). The interval from CIDR removal to peak E2 for Cycles 1 and 2 were not different (Table 1), but maximum E2 was significantly lower after treatment with two CIDR devices (7.4 6 0.8 vs. 5.25 6 0.8 pg/mL; P , 0.01). Immediately before two CIDR removal in Cycle 3, mean E2 concentration was 0.7 6 0.1 pg/mL (n 5 20 ewes), similar to those seen in Cycle 2. For the seven Cycle 3 ewes that showed an LH peak before euthanasia, mean E2 concentrations immediately before CIDR removal and peak E2 concentrations were 1.4 6 0.5 pg/mL and 5.4 6 0.6 pg/mL, respectively. Timing and Nature of Gonadotropin Surges After One and Two CIDR Synchronization. Mean circulating patterns of LH after one and two CIDR removal are shown in Figure 2 (bottom panels) and timing relationships in Table 1 and Figure 3. Average interval from one CIDR device removal to gonadotropin surge peak was 38.2 6 1.5 hr (range, 24 –52 hr), excluding the unsynchronized ewe (ewe No. 12), and 39.4 6 1.9 hr (range, 24 – 68 hr) with all ewes. In 24 of 26 ewes, the LH peaks occurred over a 24-hr period, but no obvious clustering within a shorter time frame was notable (Figure 3). In five of the six ewes that were detected in estrus at 24 hr peak, LH concentrations occurred
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Figure 2. Mean (6SE) E2, P4, LH and FSH for the 12 ewes in which all hormones were measured after 1 CIDR synchronization (Cycle 1, left panel) and 2 CIDR synchronization (Cycle 2, middle panel). Mean (6SE) E2, P4, LH, and FSH for the seven ewes from Cycle 3 that were killed during or after the primary gonadotropin surge are shown for comparison (right panel). Two of the seven ewes were euthanized before onset of secondary FSH rise and five during the secondary FSH surge. All data are normalized to LH peak (time 0, vertical line). CIDR removal coincides with first data point.
28 –36 hr after one CIDR removal, whereas the maximum LH concentration was found in one ewe at 24 hr after one CIDR removal. As in Cycle 1, all ewes treated with two CIDR devices in Cycle 2 (n 5 25) experienced a gonadotropin surge after two CIDR removal; all ewes could be considered synchronized (Figure 3). Mean interval from two CIDR device removal to surge onset and surge peak (range, 30 – 60 hr) in Cycle 2 was significantly longer than for 1 CIDR cycle (P , 0.0001; Table 1). Use of two CIDR devices delayed the LH peak by an average of 8 hr compared with one CIDR. Figure 4 shows the cumulative percentage of ewes reaching the LH peak TABLE 1. TIMING
OF
GONADOTROPIN SURGE EVENTS IN EWES TREATED WITH ONE HOURS (MEAN 6 SE; EWE NO. 12 EXCLUDED)
OR
TWO CIDR DEVICES,
IN
Treatment
Event Interval from CIDR removal to peak estradiol Interval from CIDR removal to onset of LH surge Interval from CIDR removal to LH surge peak Duration of LH surge Interval from surge peak to onset of secondary FSH rise
Cycle 1 1 CIDR (Estrus monitored)
Cycle 2 2 CIDR (Estrus monitored)
Cycle 3 2 CIDR (Estrus not monitored)
33.8 6 1.0 (n 5 9) 33.8 6 1.4 (n 5 25) 38.2 6 1.5 (n 5 25) 15.1 6 1.4 (n 5 25) 16.3 6 1.4 (n 5 25)
39.0 6 1.7 (n 5 12) 42.3 6 1.3a (n 5 25) 46.2 6 1.5a (n 5 25) 14.8 6 1.4 (n 5 25) 16.6 6 1.4 (n 5 25)
40.3 6 4.0 (n 5 7*) 40.3 6 3.5 (n 5 7*) 46.0 6 4.0 (n 5 7*) 15.6 6 1.0 (n 5 5**) 14.4 6 0.6 (n 5 5**)
2 CIDR (Cycle 2) . 1 CIDR, P , 0.0001. * Includes all ewes not euthanized before LH peak. ** Secondary FSH rise group only.
a
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Figure 3. LH surges in individual ewes treated with 1 CIDR and 2 CIDR devices (Cycles 1 and 2, respectively) for 8 d beginning in the luteal phase.
after device removal for Cycles 1 and 2. One-CIDR treatment concentrated LH peaks in 24 of 26 ewes within a 24 h period, whereas in Cycle 2 LH peaks occurred within an 18-hr period for 22 of 25 ewes (Figure 3), though the trend toward a smaller variation in timing of peaks was not statistically significant (P 5 0.10). Although there was a difference in the timing of the onset of the LH surge, there were no differences between Cycles 1 and 2 in the LH surge maximum (155.1 6 9.4 vs. 152.6 6 7.3 ng/mL, Figure 2) or duration (Table 1). Maximum surge FSH for the Cycles 1 and 2 averaged 8.9 6 0.5 and 7.5 6 0.5 ng/mL and were not different. The average interval from CIDR removal to peak of LH surge for the seven ewes in
Figure 4. Cumulative percent of ewes treated with one or two CIDR devices (Cycles 1 and 2, respectively) reaching peak of LH surge.
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TABLE 2. NUMBER OF EWES WITH PREDOMINANT OVARIAN STRUCTURES AND CORRESPONDING STEROID CONCENTRATIONS WHEN KILLED. EWES WERE SYNCHRONIZED WITH 2 CIDR DEVICES INSERTED ON DAY 10 CYCLE 2, AND SACRIFICED AT TIMES INDICATED BY GROUP NAMES. Luteal Structures (P4, ng/mL) Group (n 5 5) Luteal Early follicular Late follicular Surge Secondary FSH rise
Yellow, Vascular
Pale, Avascular
4 — (12.2 6 2.0) 1 4 (0.5) (0.6 6 0.1) — 5 (0.3 6 0.1) — 5 (0.2 6 0.04) — —
None 1 (3.2*) — — — 5 (0.1 6 0.03)
OF
Follicular Structures (E2, pg/mL) Large, Clear 1 (**) 5 (2.1 6 0.8a) 5 (2.1 6 0.5b) 1 (2.43a) —
Pre-Ovulatory, Corpus Hyperemic Hemorrhagicum None —
—
—
—
4 (**) —
—
—
—
4 (2.2 6 1.1c) —
—
—
5 (0.3 6 0.03d)
—
* P4 concentration is within what is expected from 2 CIDR devices after 5 d of use in the absence of a CL (unpublished data). ** Samples not assayed for E2. a Highest concentration in final sample. b Three ewes declining after peak concentration, two ewes highest concentration in final sample. c Two ewes declining after peak concentration, two ewes highest concentration in final sample. d Declining after peak concentration.
Cycle 3 that exhibited an LH peak before euthanasia was similar to that of Cycle 2. Concentrations averaged 148.5 6 9.6 and 7.6 6 0.8 ng/mL for LH and FSH, respectively. Secondary FSH rise was defined as beginning at the first sample after the primary surge ended, which exceeded the pre-surge mean FSH concentration by 2 3 SD and which was followed by at least 8 hr of sustained increase in FSH concentration. All but one of the periestrous periods examined in Cycles 1, 2, and 3 (n 5 56) exhibited clearly distinguishable secondary FSH rises. After one CIDR treatment, the secondary FSH rise was first detected at 16.3 hr after peak LH (Figure 2). Secondary FSH rise after synchronization with two CIDR devices began on average 16.6 hr after peak LH in Cycle 2, not significantly later than with one CIDR. There was some evidence for a relationship in the temporal order of LH peaks from the first cycle to the second. This effect does not seem to be related to the number of CIDR devices used to synchronize the peaks. For example, those ewes that were in estrus by 24 hr after one CIDR removal in the first cycle were the first to experience LH surges in the second cycle, with peaks occurring 30 –38 hr after two CIDR removal, earlier than the average interval. When a Spearman ranking correlation coefficient was calculated, the hour by which each ewe reached peak LH in the first cycle was significantly correlated to the hour by which she reached peak LH in the second cycle (r 5 0.74). This suggests that the timing of events after P4 withdrawal is consistent within ewe and is independent of the number of CIDR devices. Prediction Outcome. During Cycle 3, ewes were sacrificed (five per group) at five time points during the periestrous period (see Materials and Methods for details). Gross examination of the ovaries at slaughter and subsequent endocrine data revealed that 4/5 or 5/5 ewes in each group were in their expected phases at time of sacrifice (Table 2). Gonadotropins were at basal concentrations for both early and late follicular groups. In the Surge group, ewes were euthanized prior to the LH surge (n 5 1), on the ascending limb (n 5 2), or on the descending limb (n 5 2) of the LH surge. The two ewes killed on the descending limb of the surge had E2 profiles that had peaked and begun to decline, whereas for all others the final sample contained the highest E2 concentration. None of the
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Surge group ewes had ovulated, but four of five had begun the ovulatory process and showed markedly hyperemic preovulatory follicles, indicating that they had been exposed to the gonadotropin surge (19). Secondary FSH rise ewes had basal LH concentrations, and FSH concentrations indicative of being in the secondary FSH rise period. The combined success rate over all groups in achieving the intended stage at sacrifice was 88% (22/25), or 92% including the early follicular ewe with a CL of functional appearance but P4 below 0.5 ng/mL. DISCUSSION Temporal relationships among periestrous events in naturally cycling ewes and after synchronization with subcutaneous implants of P4 pockets have been well characterized (13,14,18,20,21). Where CIDR devices are used in sheep, P4 profiles, timing of estrus, and pregnancy and lambing rates are all well established (1– 6). However, the temporal relationships between endocrine events that follow the removal of CIDR devices have not been characterized in detail. Although the present study was not performed as a balanced crossover experiment, and comparisons between one and two CIDR synchronization must be made with caution, some general conclusions can be drawn as to the efficacy of the described synchronization system that may prove useful to researchers requiring precise control of periovulatory events. Our results do suggest that for an 8-d CIDR-treatment period beginning in the luteal phase, a single CIDR device will prevent onset of estrus and the surge for at least 8 d when applied during the luteal phase. However, the concentration of P4 delivered by a single CIDR device during the final days of treatment was not sufficient to prevent progression of follicular development to an estrogenic stage. The result was an early onset of estrus in many ewes and an unnaturally short follicular phase on average. Given the low concentrations of P4 achieved in our study at the end of 8 d, it does not seem that the traditional 12-d regimen using a single CIDR device in randomly cycling ewes would be adequate for achieving optimal synchronization of all ewes in a group. Treatment with two CIDR devices simultaneously for the same period did successfully inhibit E2 secretion and lengthen the interval from device removal to gonadotropin surge to a duration more closely resembling the naturally occurring follicular phase (13,14). The longer follicular phase in Cycle 2 may be attributable to more uniform follicular maturation, or at least synchronous initiation of the preovulatory estradiol rise. Thus, the secretory status of the preovulatory follicle(s) seemed to be the key factor in determining the difference between one and two CIDR treatments. This difference was confined to the timing of the surge relative to P4 withdrawal and to the length of the follicular phase. No other differences in surge parameters, such as amplitude or duration, or effects on the secondary FSH rise were detectable. The estrogenic status of the follicle seemed to be determined primarily by the concentration of P4 circulating at the time of CIDR withdrawal, which was lower for one CIDR compared with two CIDR (2.68 6 0.27 vs. 5.15 6 0.27 ng/mL). Johnson and coworkers (21) found that ewes treated to induce a peripheral concentration of P4 less than 1 ng/mL during the luteal phase had increased peripheral E2 when compared with ewes with circulating P4 greater than 1 ng/mL. Our ewes, regardless of treatment, had higher, CIDR-induced, concentrations of P4 compared to the Johnson study (21), but the difference in P4 concentrations between 1 and 2 CIDR treatment may have similarly led to the difference in E2 concentration. The suppressive effects of P4 on circulating E2 concentrations may be mediated via the proven negative feed back effects on pulsatile LH/GnRH secretion (23).
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In addition to qualitative differences in secretory activity of the follicle, a role for P4 in controlling the timing of periovulatory events has also been suggested. Using P4-impregnated sponges, Robinson and Smith (24) and Pearce and Robinson (25) showed a direct relationship between the dose of P4 and the timing of estrus, with the larger dose of P4 resulting in a longer latency. Although the differences in latency between Cycle 1 and 2 in our studies may be directly related to the P4 concentrations achieved, a concern that needs to be addressed is the possibility of a ram effect (26) in the 1 CIDR cycle, which would have been absent in Cycle 2. However, this does not seem to be the case because the latency from P4 removal to peak LH surge after 2 CIDR removal were similar, averaging 46 6 1.5 and 46 6 4 hr for Cycle 2 (n 5 25, estrus not monitored) and Cycle 3 (n 5 7; estrus monitored), respectively. Comparative studies performed in the presence or absence of rams by Hauger and coworkers (18) have also shown that the interval between P4 removal and the onset of the gonadotropin surge and gonadotropin surge characteristics are not affected by the presence of a ram. From a synchronization standpoint, the application of the CIDR device during the luteal phase of the estrous cycle allows for more precise control over the resulting synchronized estrus. While both one and two CIDR treatments concentrated LH peaks within a 24-hr period, some surges occurred earlier than desired, especially with one CIDR treatment. The precise control afforded by the high P4 at the end of treatment with two CIDR devices resulted in the desired timing of gonadotropin surge peak, at about 48 hr after CIDR removal, and the desired concentration of gonadotropin surges to within 24 h of each other. Studies conducted in cycling ewes by Wheaton and coworkers (2) and our preliminary studies [unpublished data] in ovariectomized ewes show that circulating P4 concentrations remain above 2 ng/ml for the first 5 d of CIDR insertion and decline steadily thereafter. Given the relatively low P4 output from a single CIDR device by 8 d, a single CIDR device inserted too early in the luteal phase would not suffice in maintaining the levels of P4 required for precise synchronization regimens. It is a possibility that the mere repetition of the synchronization regimen may have affected the quality of the synchronization response (i.e., concentration and/or timing of LH surge peaks). Information on repeated synchronization in sheep is lacking, especially repeated synchronization of unbred animals. Inseminations that fail are still likely to affect resynchronization responses (7). In heifers first synchronized using PGF2a only, then resynchronized using CIDR devices for Days 17–22 after estrus, estrous responses were 78% and 94% in inseminated-nonpregnant and non-inseminated heifers, respectively (8). Our non-inseminated ewes achieved a resynchronization rate of 100% for the second cycle, but seemed to be slightly less closely synchronized in the third. It is more likely that repeated synchronizations would lead to a gradual decline in the precision of the synchrony, rather than an increase. Therefore, the improved response seen in the second cycle compared to the first is more likely due to the increased P4 concentration than to the effects of repeated synchronization. In summary, application of two CIDR devices for 8 d beginning in the luteal phase resulted in a CIDR removal-to-surge interval of similar length to a natural follicular phase, and effectively synchronized and concentrated the timing of gonadotropin surges. Data derived from Cycles 2 and 3 also suggest that this synchronization regimen is capable of producing repeated, well-synchronized cycles. ACKNOWLEDGMENTS/FOOTNOTES We are grateful to Mr. Douglas D. Doop and Mr. Gary McCalla for help with the animal maintenance; Dr. Gordon D. Niswender and Leo E. Reichert, Jr. for supplying assay reagents for the LH assay, and National Hormone and Pituitary Program for generous supply of FSH reagents used in the FSH assay. Support was also provided by the Sheep Research, Standards and Reagents, Data Analysis, and Administrative Core Facilities of
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the Center for the Study of Reproduction. All procedures were performed within the guidelines of the University’s Committee for the Use and Care of Animals at the University of Michigan. 1 Published as part of the National Cooperative Program for Infertility Research. Supported by NIH grants U54 HD-29184 (UM), and P30 HD-18258 (UM). Portions of this work were presented at the 29th Annual meeting of the Society for the Study of Reproduction, London, Ontario, Canada. 2 Current address: Animal Science Group, Faculty of Agriculture, University of Western Australia, Nedlands, WA 6907, Australia. 3 Address reprint requests to Vasantha Padmanabhan, Ph.D., Reproductive Sciences Program, University of Michigan, 300 N. Ingalls Bldg., Room 1110, Ann Arbor, MI 48109-0404.
REFERENCES 1. Welch RAS. Development of CIDR dispensers for use in nulliparous ewes. New Zealand Ministry of Agriculture and Fisheries, Agricultural Research Division Annual Report, 1983/84, p. 58, 1985. 2. Wheaton JE, Carlson KM, Windels HF, Johnston LJ. CIDR. A new progesterone-releasing intravaginal device for induction of estrus and cycle control in sheep and goats. Anim Rep Sci 33:127–141, 1993. 3. Smith JF, Konlechner JA, Parr J. The efficacy of used CIDR devices for synchronization of oestrus and post-mating treatment. Proceedings of the New Zealand Society of Animal Production 5:111–115, 1991. 4. Carlson KM, Pohl HA, Marcek JM, Muser RK, Wheaton JE. Evaluation of progesterone Controlled Internal Drug Release dispensers for synchronization of estrus in sheep. Anim Reprod Sci 18:205–218, 1989. 5. Rhodes L, Nathanielsz PW. Comparison of a Controlled Internal Drug Release device containing progesterone with intravaginal medroxyprogesterone sponges for estrous synchronization in ewes. Theriogenology 30:831– 836, 1988. 6. Thompson JG, Simpson AC, James RW, Tervit HR, Asher GW, Peterson AJ. Timing of the LH peak and ovulations in superovulated Coopworth ewes synchronized with progesterone containing CIDR devices. Proc NZ Soc Anim Prod 52:171–174, 1992. 7. Van Cleeff J, Drost M, Thatcher WW. Effects of postinsemination progesterone supplementation on fertility and subsequent estrous responses of dairy heifers. Theriogenology 36:795– 807, 1991. 8. Van Cleeff J, Macmillan KL, Drost M, Lucy MC, Thatcher WW. Effects of administering progesterone at selected intervals after insemination of synchronized heifers on pregnancy rates and resynchronization of returns to service. Theriogenology 46:1117–1130, 1996. 9. Asher GW, Fisher MW, Jabbour HN, Smith JF, Mulley RC, Morrow CJ, Veldhuizen FA, Langridge M. Relationship between the onset of oestrus, the preovulatory surge in luteinizing hormone and ovulation following oestrous synchronization and superovulation of farmed red deer (Cervus elaphus). J Reprod Fertil 96:261–273, 1992. 10. Bowen JM, Barrell GK. Duration of the oestrous cycle and changes in plasma hormone concentrations measured after an induced ovulation in scimitar-horned oryx (Oryx dammah). J Zoology 238:137–148, 1996. 11. Jabbour HN, Asher GW, Smith JF, Morrow CJ. Effect of progesterone and oestradiol benzoate on oestrous behaviour and secretion of luteinizing hormone in ovariectomized fallow deer (Dama dama). J Reprod Fertil 94:353–361, 1992. 12. Jabbour HN, Veldhuizen FA, Mulley RC, Asher GW. Effect of exogenous gonadotrophins on oestrus, the LH surge and the timing and rate of ovulation in red deer (Cervus elaphus). J Reprod Fertil 100:533–539, 1994. 13. Karsch FJ, Foster DL, Legan SJ, Ryan KD, Peter GK. Control of the preovulatory endocrine events in the ewe: Interrelationship of estradiol, progesterone and luteinizing hormone. Endocrinology 105:421– 426, 1979. 14. Karsch FJ, Legan SJ, Ryan KD, Foster DL. Importance of estradiol and progesterone in regulating LH secretion and estrous behaviour during the sheep estrous cycle. Biol Rep 23:404 – 413, 1980. 15. Padmanabhan V, Evans NP, Dahl GE, McFadden KL, Mauger DT, Karsch FJ. Evidence for short or ultrashort loop negative feedback of GnRH secretion. Neuroendocrinology 62:248 –258, 1995. 16. Evans NP, Dahl GE, Glover BH, Karsch FJ. Central regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 134:1806 –1811, 1994. 17. Niswender GD, Reichert LE Jr, Midgley AR Jr, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166 –1173, 1969. 18. Hauger RL, Karsch FJ, Foster DL. A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101:807– 817, 1977. 19. Murdoch WJ. Endothelial cell death in preovulatory ovine follicles: Possible implication in the biomechanics of rupture. J Reprod Fert 105:161–164, 1995.
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20. Goodman RL. Neuroendocrine control of the ovine estrous cycle. In: The Physiology of Reproduction, 2nd Ed., E. Knobil, JD Neill (eds.), Raven Press, Ltd., New York, pp. 659 –709, 1994. 21. Karsch FJ, Bittman EL, Foster DL, Goodman RL, Legan SJ, Robinson JE. Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 40:185–232, 1984. 22. Johnson SK, Daily RA, Inskeep EK, Lewis PE. Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes. Domest Anim Endocrinol 13:69 –79, 1996. 23. Karsch FJ. Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing hormone. Annu Rev Physiol 49:365–382, 1987. 24. Robinson TJ, Smith JF. The time of ovulation after withdrawal of SC-9880 impregnated intravaginal sponges from cyclic Merino ewes. In: The Control of the Ovarian Cycle in Sheep, TJ Robinson (ed.), Sydney University Press, Sydney, Australia, pp. 158 –168, 1967. 25. Pearce DT, Robinson TJ. Plasma progesterone concentrations, ovarian and endocrinological responses and sperm transport in ewes with synchronized oestrus. J Reprod Fertil 75:49 – 62, 1985. 26. Parsons SD, Hunter GL, Rayner AA. Use of probit analysis in a study of the effect of the ram on time of ovulation in the ewe. J Reprod Fertil 14:71– 80, 1967.