Gonadotropin Response to Naloxone in the Mare: Effect of Time of Year and Reproductive Status¹

^a Department of Veterinary Science, The Maxwell Gluck Equine Research Center, University of Kentucky, Lexington, Kentucky 40546–0099

	ABSTRACT

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In the mare, endogenous opioids have been implicated in the suppression of gonadotropin secretion during seasonal anestrus (AN). The present study tested whether continuation of reproductive activity during the nonbreeding season (NBS) reflects the absence of a seasonal shift in opioid tone compared to what occurs in AN mares. During the NBS, 11 AN and 8 luteal-phase mares received 0.1, 0.05, 0.025 mg/kg naloxone (NAL) or vehicle on alternate days. Whereas cycling mares responded to all dosages of NAL, AN mares responded only to the higher dosages for FSH, and LH failed to increase at any dosage employed. During the breeding season (BS), the response to these dosages of NAL was reevaluated in 12 mares in the luteal phase of a synchronized estrous cycle. Although there was no difference between cycling mares during the breeding and nonbreeding seasons in FSH response, those mares that had cycled during the NBS showed a greater LH response to 0.05 mg/kg NAL than mares during the BS. From these data, we conclude that opioid tone is lower during the BS than during AN and that this shift in inhibitory tone does not occur in mares that cycle during the NBS. Thus, reduced opioid tone may play a role in the mechanisms controlling the nonseasonal exhibition of estrous cycles in the mare.

	INTRODUCTION

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Although horses are seasonal breeders, a small percentage of mares continue to exhibit ovarian activity throughout the nonbreeding season [1–3]. Among these mares, it has been shown [2] that there is no discernible pattern in the occurrence or absence of unseasonal cyclicity from one year to the next. That is, the occurrence of estrous cycles during the nonbreeding season cannot be attributed to a failure to respond to photoperiodic cues, an individual propensity to cycle unseasonally, or to the occurrence of cyclicity during the preceding year. The mechanisms that may lead to this phenomenon, thus far, remain unknown. In anestrous mares, current evidence suggests that the absence of reproductive activity reflects a suppression of gonadotropin secretion due to inhibition of GnRH secretion resulting from increased dopaminergic [4–6] and opioidergic activity [7–10]. Thus, the continuation of estrous cycles during the nonbreeding season may result from a reduced inhibitory action (i.e., reduced "tone") by one or both of the aforementioned neural systems. In the mare, a role for continued glutamatergic stimulation of GnRH secretion as a possible cause for continuation of estrous cycles has also been suggested; however, no differences were observed between anestrous and cycling mares in gonadotropin response to the glutamatergic agonist N-methyl-D,L-aspartic acid during the nonbreeding season [3]. In view of this consideration, the present study investigated the hypotheses that the occurrence of estrous cycles during the nonbreeding season reflects reduced opioidergic activity on GnRH/gonadotropin secretion compared with observations in anestrous mares and that opioid tone among these mares would resemble that of mares during the breeding season. To examine this proposal, two related experiments were performed. The first experiment determined whether the occurrence of reproductive activity during the nonbreeding season was associated with a difference in the gonadotropin response to administration of the opioid antagonist naloxone in comparison to that in anestrous mares. The second experiment reevaluated the gonadotropin response to naloxone administration in the same mares during the breeding season. In both studies, a range of doses of naloxone was employed among luteal phase mares in an attempt to determine whether the dose response is different with respect to physiological state and time of year. This experimental approach mirrored an earlier study [8], which suggested that a change in opioidergic tone plays a role in the seasonal cessation of reproductive activity in the mare. This conclusion was reached based on the observation that larger dosages of naloxone were required to induce increased gonadotropin secretion during anestrus than during the breeding season.

	MATERIALS AND METHODS

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Animals

All animals used in this study were mature mares (> 5 yr of age) of mixed, predominantly Thoroughbred breeding maintained at the University of Kentucky research farm (lat 38°2'N). Prior to the experiment the mares were kept together on pasture in the absence of a stallion. Their diet was supplemented with hay, and they had free access to water and trace-mineral salt blocks. The body weights of the mares were recorded on the day preceding treatment.

Experiment 1

During the nonbreeding season (NBS; March 1995), 6 anestrous (AN) mares and 6 mares that continued to demonstrate estrous cycles were selected from a larger herd in which reproductive status had been continuously monitored for 5 mo via rectal palpation and ultrasound of the ovaries. Reproductive status of the selected mares was monitored throughout the experimental period by palpation and ultrasound of the ovaries per rectum. The occurrence or absence of ovulation was confirmed by determination of serum progesterone (P₄) concentrations in blood samples collected daily. Only mares verified to be diestrous were included in the statistical analysis. The 6 mares that continued to demonstrate cyclic activity were selected from the larger herd based upon the likelihood that they would ovulate in relative synchrony due either to a recently confirmed ovulation (n = 3) or to the presence of a large (> 45 mm) preovulatory follicle 3 days prior to the initial sampling day (n = 3). Blood samples taken during the experiment were subsequently assayed for P₄ to confirm that the mares had ovulated and were luteal throughout each period of sample collection. To verify that mares deemed to be cyclic remained cyclic throughout the NBS, blood samples were collected from all mares 2–3 times per week after completion of the experiment until all AN mares ovulated, indicative of the onset of the breeding season. None of the mares designated cyclic became AN during this time.

Each mare received (i.v.), on separate days, 0.1, 0.05, and 0.025 mg/kg of the opiate antagonist naloxone (NAL; naloxone hydrochloride; Sigma Chemical Co., St. Louis, MO), dissolved in 10 ml physiological saline (0.9% w:v NaCl), and 10 ml saline vehicle. In order to minimize the possibility that the day of the luteal phase might affect the response to NAL, all 12 mares were randomly assigned to one of 4 treatment groups (n = 3 per group), and the doses were administered in a staggered 4 x 4 Latin square design [11]. Blood samples were collected at 12-min intervals for 2 h before and 5 h after administration of NAL or vehicle. A day was allowed between each collection period to rest the mares and to reduce the risk of an accumulated effect of the drug.

Though those cycling mares verified to have ovulated by the first day of the experiment were presumed to be luteal throughout, retrospective analysis of P₄ in serum from each sampling day revealed that one of the mares had entered the follicular phase during the experiment. In the 3 mares with large, preovulatory follicles, two of these follicles unexpectedly failed to ovulate and became atretic, while the third (51 mm) resisted ovulation until the second sampling day and P₄ levels were luteal only during the last 2 days. Samples taken during the follicular phase or during follicular atresia were removed from inclusion in the statistical analysis, which resulted in a low sample size at each dosage (n = 4: saline; n = 3: 0.025 and 0.1 mg/kg NAL; n = 2: 0.05 mg/kg NAL). Therefore, this experiment was repeated during the following winter (February 1996) using an additional 5 AN and 6 cycling mares, and the resultant data were pooled with those of the first experiment for statistical analysis.

During this second NBS, only 6 of the mares available for study continued to demonstrate cyclic activity. Of these, 2 had been designated cyclic and had been utilized in the previous breeding season experiment. None of the AN mares had been previously used. Five of the cycling mares were verified to have ovulated, and one mare possessed a large preovulatory follicle prior to the experiment.

Again, irregularities in the estrous cycles common to the NBS (unpublished results) created difficulties in the sample size for luteal mares. Of the 6, only 1 was luteal for all 4 sampling days, and the mare that was preovulatory was determined to have undergone follicular atresia and had to be removed from inclusion in the statistical analysis. For this experiment, therefore, the sample sizes for each dosage were n = 4, saline; n = 5, 0.025 mg/kg NAL; n = 3: 0.05 mg/kg NAL; n = 2: 0.1 mg/kg NAL. When these data were pooled with those of the first experiment, the final sample size for AN mares was n = 11 mares per dosage. In the case of the cycling mares, however, the final sample size for each dosage was n = 8 for both saline and 0.025 mg/kg NAL and was n = 5 for both 0.05 and 0.1 mg/kg NAL.

The second NBS experiment (Year 2) followed the same protocol as the first except that 100 µg GnRH (Sigma) was administered i.v. to saline controls (n = 4 cycling and 5 AN mares) 2 h after treatment with vehicle to exclude the possibility that differential pituitary stores of the gonadotropins between the groups may account for variation in the response to NAL. This dosage was selected based upon previous studies [12, 13] in which it had been determined to be maximally stimulating to pituitary secretion of both gonadotropins.

Only cycling mares that were verified to have P₄ levels higher than 1 ng/ml throughout each period of blood sample collection were considered to be luteal and included in the statistical analysis.

Experiment 2

During the breeding season (BS; July 1995), the same 12 mares that had been selected for use in the initial NBS experiment (March 1995; both cycling and AN) were used with an identical experimental procedure. These mares were intentionally selected in order to investigate the possibility that prior reproductive status during the NBS might affect the response to NAL during the succeeding BS. The low sample size for the initial NBS experiment, however, made this comparison impossible. All mares were tested during the luteal phase of the estrous cycle, and the timing of ovulation for each mare was synchronized by treatment with daily i.m. injections of P₄ plus estradiol-17ß (150 mg and 9.99 mg/mare, respectively) in 3 ml cottonseed oil for 10 days. Prostaglandin F₂ (Lutalyse; Upjohn Co., Kalamazoo, MI) was administered (10 mg; i.m.) on the first and last days of treatment to lyse any residual luteal structure. Reproductive status was monitored by measurement of P₄ concentration in blood samples collected daily, together with rectal palpation and ultrasound of the ovaries throughout the study. All mares ovulated within 10–13 days after the final hormonal treatment and all remained luteal throughout the experiment.

Collection of Blood Samples

Blood samples (6 ml) were collected via an indwelling catheter (Angiocath; Becton Dickinson, Sandy, UT) inserted into the jugular vein on the day prior to the initial period of blood collection. Patency of the cannula was maintained using a removable stylet.

All blood samples were allowed to clot and were kept overnight at 4°C. The next day, samples were centrifuged (15 min at 1500 rpm), and the serum was harvested and stored at -20°C until assayed for LH, FSH, and P₄.

RIA

Concentrations of LH were measured in serum by a previously described RIA [14] using ovine LH antiserum (CSU120; Dr. Terry M. Nett, Colorado State University, Fort Collins, CO) and ¹²⁵I-ovine LH (LER1374A; Dr. L.E. Reichert Jr., Albany Medical College, Albany, NY). Sensitivity of the assay averaged 0.22 ng/ml (reference standard E98A; Dr. Harold Papkoff, University of California, Davis, CA). Inter- and intraassay coefficients of variation were 18% and 15%, respectively. Cross-reactivity of the purified equine FSH standard (E99B; see below) with CSU120 anti-LH was 1.1%.

FSH was measured in serum by RIA [15]. Human FSH antiserum (#6) and ovine FSH for iodination were supplied by the National Hormone and Pituitary Program (Rockville, MD). Sensitivity of the assay averaged 1.19 ng/ml (reference standard E99B; Dr. Harold Papkoff), and inter- and intraassay coefficients of variation were 19% and 10%, respectively. Cross-reactivity of the purified equine LH standard (E98A; see above) with human anti-FSH was 0.2%.

Serum concentrations of P₄ were determined by an RIA described previously [16]. Sensitivity of the assay averaged 35.89 pg/ml, and inter- and intraassay coefficients of variation were 17% and 15%, respectively.

Analysis of Data

The mean gonadotropin response to NAL and GnRH was compared among groups using an ANOVA procedure for repeated measures design in the Statistical Analysis System [17] using PROC MIXED. Effects of season, status, dose, and time were examined. To minimize the possibility that mean increases in gonadotropin levels over time reflected changes in endogenous secretion, rather than a true response to the drug, only hormone values during the first hour following treatment were used in the analysis.

This experiment was originally designed such that each mare would be represented in each treatment group, as well as in the saline control group. Because of the irregularities in the lengths of the luteal phase for cycling NBS (C-NBS) mares, however, all mares were not equally represented in each group. For this reason, changes in gonadotropin secretion in response to each dosage of NAL were compared with the pretreatment means for each status (AN, C-NBS, or BS). The pretreatment means for each status were derived using all samples taken prior to NAL or saline injection on all four sampling days. Using a one-way ANOVA for repeated measures, comparisons were made within status to determine whether there were any differences in pretreatment values between mares when grouped by dose. Pretreatment means were compared between status using a Student's t-test.

Because of the irregularities known to exist in cycle lengths of C-NBS mares, this study was designed intentionally in such a way that day of the estrous cycle would not be a significant factor. Because each mare did not contribute equally to data for each dosage, the mean (± SEM) day of the cycle (ovulation = Day 0) during which each dose was administered was calculated for both C-NBS and BS mares to determine whether data for any dosage might be inadvertently biased for the day of the estrous cycle.

In order to examine the hypothesis that the C-NBS response to NAL resembled that of the BS, the incremental change in gonadotropin secretion (ng/ml) from the pretreatment mean was calculated for each dosage and these values were examined as before using PROC MIXED.

	RESULTS

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Analysis of the data revealed a significant effect of time on mean response to NAL (p < 0.0001 for each analysis performed). For this reason, the gonadotropin response in each experiment was examined at fixed time points: 12, 24, 36, 48, and 60 min after administration of the drug. Data are expressed as the mean ± SEM of all samples during the entire first hour following treatment and are graphically represented in Figures 1 and 2 (bottom panels). For the sake of simplicity, the p values reported here represent the time point at which the maximal significant response was obtained. Unless otherwise stated, where there was a significant response, it was significant (p < 0.05) at each time point.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Changes in serum concentrations of LH in response to graded doses of NAL during the first hour following treatment. Data are expressed as the mean (± SEM; top) and as the overall mean (± SEM) incremental increase from pretreatment values (bottom) for luteal phase mares during the BS (n = 12 all dosages) and NBS (0.025 mg/kg: n = 8; 0.05 and 0.1 mg/kg: n = 5), and for AN mares (n = 11 all dosages).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Changes in serum concentrations of FSH in response to graded doses of NAL during the first hour following treatment. Data are expressed as the mean (± SEM; top) and as the overall mean (± SEM) incremental increase from pretreatment values (bottom) for luteal phase mares during the BS (n = 12 all dosages) and NBS (0.025 mg/kg: n = 8; 0.05 and 0.1 mg/kg: n = 5), and for AN mares (n = 11 all dosages).

The luteal phases for C-NBS mares used in the data analysis ranged from 9 to 19 days in length. The mean (± SEM) days postovulation for these mares at each dose level were as follows: saline = 6.88 ± 1.87 (n = 8); 0.1 mg/kg NAL = 6.6 ± 1.81 (n = 5); 0.05 mg/kg = 5.0 ± 1.05 (n = 5); and 0.025 mg/kg = 7.25 ± 1.26 (n = 8) days. During the BS, in which the mares were synchronized, the days postovulation at each dosage were saline = 9.08 ± 1.10; 0.1 mg/kg NAL = 9.08 ± 1.03; 0.05 mg/kg = 9.08 ± 0.87; and 0.025 mg/kg = 9.08 ± 0.98 (n = 12 each dosage).

Pretreatment gonadotropin levels (LH and FSH) for independent sampling days did not differ within any group of mares (p > 0.05 for each status). Neither was there a gonadotropin response to saline among any group. Pretreatment vs. saline FSH concentrations were 22.89 ± 0.69 vs. 16.89 ± 0.87 for C-NBS mares (n = 8), 23.63 ± 0.63 vs. 19.97 ± 1.54 for AN mares (n = 11), and 30.26 ± 2.47 vs. 30.39 ± 0.85 for BS mares (n = 12), respectively (p > 0.05 each status). Pretreatment vs. saline LH concentrations were 2.16 ± 0.27 vs. 2.57 ± 0.09 for C-NBS mares, 1.91 ± 0.08 vs. 2.10 ± 0.03 for AN mares, and 5.65 ± 0.29 vs. 4.53 ± 0.17 for BS mares, respectively (p > 0.05 each status). There was no difference in pretreatment LH or FSH concentrations between AN and C-NBS mares (p > 0.05). As expected, pretreatment LH levels in BS mares were significantly greater (p < 0.05) than those of both AN and C-NBS mares. Although mean FSH levels were higher during the BS than in either C-NBS or AN mares, this difference approached but did not reach significance.

During the NBS, administration of NAL evoked increased FSH at each dose level in C-NBS mares (NAL dose 0.025 mg/kg: 38.35 ± 3.76 ng/ml, p < 0.05; 0.05 mg/kg: 66.20 ± 5.48 ng/ml, p < 0.001; 0.1 mg/kg: 42.05 ± 3.18 ng/ml, p < 0.001). In AN mares, the lowest dosage of NAL was unaccompanied by increased FSH values (NAL dose 0.025 mg/kg: 25.98 ± 1.58 ng/ml, p > 0.05); however, the highest dosages evoked a significant increase in FSH (NAL dose 0.05 mg/kg: 31.24 ± 2.60 ng/ml, p < 0.05; 0.1 mg/kg: 42.26 ± 3.35 ng/ml, p < 0.001). Although NAL evoked increased FSH in AN mares, circulating concentrations of LH did not change significantly in response to NAL (NAL dose 0.025 mg/kg: 2.18 ± 0.08 ng/ml; 0.05 mg/kg: 2.68 ± 0.14 ng/ml; 0.1 mg/kg: 3.03 ± 0.17 ng/ml, p > 0.05 each). In contrast, all dosages of NAL induced a significant increase in LH in C-NBS mares (NAL dose 0.025 mg/kg: 5.03 ± 0.56 ng/ml, n = 8, p < 0.01; 0.05 mg/kg: 9.87 ± 1.36 ng/ml, n = 5, p < 0.001; 0.1 mg/kg: 4.46 ± 0.58 ng/ml, n = 5, p < 0.01).

When challenged with GnRH, the LH response was greatest among C-NBS mares (11.49 ± 0.87 vs. 3.42 ± 0.20 ng/ml; p < 0.001). Though the FSH response to GnRH was also greater among C-NBS mares, this difference approached, but did not reach, significance (96.13 ± 5.78 vs. 71.62 ± 6.27 ng/ml, respectively; p > 0.05).

During the BS, NAL increased FSH secretion at all dose levels (NAL dose 0.025 mg/kg: 48.75 ± 3.86 ng/ml, p < 0.05; 0.05 mg/kg: 61.62 ± 6.15 ng/ml, p < 0.01; 0.1 mg/kg: 58.11 ± 3.96 ng/ml, p < 0.01). Likewise, LH secretion increased in response to all dosages of NAL (NAL dose 0.025 mg/kg: 8.81 ± 0.82 ng/ml, p < 0.05; 0.05 mg/kg: 8.93 ± 0.83 ng/ml, p < 0.01; 0.1 mg/kg: 9.76 ± 0.67 ng/ml, p < 0.001).

When the incremental change in response from pretreatment between C-NBS and BS mares was compared at each dosage, it was found that whereas the FSH response to NAL was similar (p > 0.05; Fig. 2, top) in cycling mares at both times of year, C-NBS mares exhibited a greater incremental increase in LH than did BS mares at a dosage of 0.05 mg/kg (p < 0.001), but a similar response at the lowest and highest dosages, respectively (Fig. 1, top).

	DISCUSSION

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In the present study, we reexamined the hypothesis that a change in opioidergic tone between the breeding and nonbreeding seasons plays a role in the inhibition of gonadotropin secretion during AN. The results of previous research [7–10] in which luteal phase mares during the BS demonstrated greater gonadotropin secretion in response to an opioid antagonist than did AN mares suggested that opioid tone, or inhibition, was greater among AN mares. It is currently thought that this increase in opioidergic inhibition of gonadotropin secretion may be partially responsible for the occurrence of AN in the mare. Our results confirm these earlier findings. During AN, the LH response to NAL was insignificant at all dosages tested, whereas FSH increased at the two higher dosages. Conversely, BS mares responded to all dosages of NAL for both gonadotropins, suggesting that the opioid inhibition of gonadotropin secretion is less during the BS. The present study, however, extends these earlier observations by demonstrating that for a subpopulation of mares, the occurrence of estrous cycles during AN may reflect reduced opioidergic inhibition of the hypothalamic-pituitary axis. In this regard, for both gonadotropins, the response to NAL was greater among these cycling mares than among AN mares. Furthermore, not only did the incremental increase from pretreatment values appear to be greater in magnitude among cycling mares, particularly at the 0.05 mg/kg dose, but the dose-response curves for FSH demonstrated a shift in sensitivity to NAL between the two groups (Fig. 2). Cycling mares did not simply release more of the hormone; the minimal effective dosage was less than that required to induce increased gonadotropin secretion in AN mares.

As expected, when the response of cycling mares to administration of NAL was compared during the BS and AN, the FSH response was similar. Both groups responded to all dosages, with the greatest FSH response observed at the moderate dosage. In the case of LH, however, the two groups responded similarly at the lowest and highest dosages of NAL, but the moderate dosage induced a greater incremental increase in C-NBS than in BS cycling mares. The significance of this observation to the mechanisms controlling the continuation of estrous cycles during the NBS is unclear. It is also unclear why pretreatment concentrations of LH were lower in mares that cycled during the NBS than during the BS. Conceivably, mares that exhibit estrous cycles during the NBS may exhibit subtle variation in the activity of inhibitory neural systems that is sufficient to result in the suppression of LH secretion, but not in the resultant cessation of reproductive activity. In support of this suggestion, although we selected mares that exhibited estrous cycles during the NBS, we observed variation in the length of estrous cycles as compared to the regularity of cycles during the BS (unpublished results). Indeed, as a result of this phenomenon, a low sample size for observations during Year 1 resulted in the collection of further data in a second year.

Studies in the mare have shown both a reduced hypothalamic content of GnRH and pituitary concentrations of LH, and thus the amount of hormone that may be released by GnRH between the BS and AN [12–16, 18, 19]. The pituitary content of FSH, however, does not vary with season. The possibility of differential pituitary responsiveness between cycling and AN mares during the NBS, however, has not previously been addressed. In the present study, no difference was observed between groups in the FSH response to GnRH. However, for LH the maximally stimulating dosage of GnRH [12, 13] employed in this study induced a greater response in cycling than in AN mares during the NBS. This observation might suggest that the greater responsiveness to NAL found in C-NBS mares is simply indicative of greater stores of LH in the pituitary. However, it is notable that a difference was observed between the groups in both opioid tone and sensitivity to NAL, with respect to secretion of FSH, that cannot be explained by a difference in pituitary responsiveness or stores. This observation, therefore, supports the suggestion that during the NBS, opioid tone is greater among AN mares than in mares that continue to exhibit estrous cycles.

Current evidence suggests that the occurrence of AN in the mare, as in other seasonal breeders, reflects an active suppression of GnRH secretion [20]. Two neural systems have been proposed as potential inhibitors of GnRH secretion during the NBS, namely opioidergic [7–10] and dopaminergic systems [4–6]. This study focused on changes in opioid inhibition of gonadotropin secretion; however, in sheep, interaction between the dopaminergic and opioidergic systems has been demonstrated [21, 22]. The possibility that similar interaction may occur in the mare remains to be investigated, particularly in view of recent studies demonstrating that prolonged treatment of AN mares with a dopamine antagonist results in increased LH and FSH secretion and advances mean time of first ovulation [5, 6].

In conclusion, while the cessation of reproductive activity during AN reflects an increase in opioid tone, the occurrence of estrous cycles during the NBS in a subpopulation of mares may reflect, in part, reduced opioidergic inhibition of the hypothalamic-pituitary axis. The role and possible interaction of additional inhibitory neural systems, notably dopaminergic inhibition, remains to be explored.

	ACKNOWLEDGMENTS

 
We thank Sarah Wheeler for assistance in sample collection, Lelia Garrison for performance of RIAs, Steven Grambow for statistical consultation and assistance with SAS, and Barry Curd and the staff of the University of Kentucky research farm for care and handling of the animals.

	FOOTNOTES

 
¹ This work was supported by funds from the Kentucky Equine Research Foundation. An abstract containing some of these data was presented at the 29th annual meeting of the Society for the Study of Reproduction, London, Ontario, Canada, 1996. The research reported in this article (No. 97–14–162) is published in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of its director.

² Correspondence. FAX: 606 257 8542; ldavison{at}pop.uky.edu

Accepted: July 9, 1998.

Received: October 27, 1997.

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