Animal Reproduction Science 59 Ž2000. 87–97 www.elsevier.comrlocateranireprosci Levels of progesterone and changes in prostaglandin F2 a release during luteolysis and early pregnancy in llamas and the effect of treatment with flunixin meglumine M.A. Aba a,b,d,) , H. Kindahl c,d , M. Forsberg a,d , M. Quiroga b, N. Auza b a Department of Clinical Chemistry, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7038, S-750 07Uppsala, Sweden b Department of Physiopathology Campus UniÕersitario, Faculty of Veterinary Sciences, UNCPBA, Paraje Arroyo Seco sr n, Tandil, 7000Buenos Aires, Argentina c Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7039, S-750 07Uppsala, Sweden d Centre for ReproductiÕe Biology, SLU, Uppsala, Sweden Received 19 March 1999; received in revised form 7 December 1999; accepted 7 December 1999 Abstract The secretory patterns of progesterone in relation to concentrations of 15-ketodihydro-PGF2 a ŽPGFM. during the period of luteolysis or of maternal recognition of pregnancy were determined in the blood of llamas mated either with an intact or a vasectomized male. The ability of flunixin meglumine ŽFM. to postpone luteolysis in non-pregnant llamas was investigated by injecting the drug intravenously every 6 h at a dose of 2.2 mgrkg from days 6 to 12 post-copulation into a group of non-pregnant llamas. A pulsatile pattern of prostaglandin release was recorded during luteolysis in non-pregnant llamas, giving further support to the hypothesis that PGF2 a is the luteolytic agent in llamas. The mean number of peaks per animal rose from 0.3 on day 7 to 3.8 on day 10 and then declined to 1.1 on day 12 with corresponding mean peak amplitude changing from 465 to 1234 and 566 pmol ly1, respectively. In pregnant llamas, prostaglandin pulsatile release also occurred. The mean number of peaks per animal rose from 0.4 on day 7 to 0.8 on day 10 and then declined to 0.2 on day 11 and 0.6 on day 12, with corresponding mean peak ) Corresponding author. UNCPBA Faculty of Veterinary Sci, Dept. of Physiopathology, Campus Universitario, Paraje Arroyo Seco, 7000 Tandil Buenos Aires, Argentina, Tel.: q54-293-26667; fax: q54-293-28485. E-mail address: [email protected] ŽM.A. Aba.. 0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 6 8 - 3 88 M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 amplitude changing from 494 to 676, 388 and 547 pmol ly1, respectively. The transient decrease and subsequent recovery in progesterone concentrations was observed to occur in connection with prostaglandin release during early pregnancy. Oestradiol-17b plasma peak concentrations attained after luteolysis were significantly higher than those recorded in early pregnant animals Žaround 30 pmol ly1 and ll pmol ly1 .. Concentrations of PGFM decreased rapidly after the first administration of FM and remained low throughout the first 2 days of treatment. Thereafter, pulsatile release of prostaglandins started, and luteolysis proceeded; but a delay of 1–1.5 days in the progesterone decline was observed. Thus, it might be suggested that a higher dose andror a more intensive injection schedule is required in llamas than in other ruminants to prevent luteolysis. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Llama; Progesterone; Prostaglandins; Flunixin meglumine; Corpus luteum; Pregnancy 1. Introduction In non-pregnant llamas Ž Lama glama. a dramatic fall in progesterone concentrations is observed between days 9 and 11 post-mating, in connection with repeated surge release of PGF2 a from the uterus ŽSumar et al., 1988; Aba et al., 1995.. In pregnant animals, a transient decrease and subsequent recovery in progesterone concentrations has been reported during the period when maternal recognition of pregnancy is expected to occur ŽAdams et al., 1991; Aba et al., 1995.. In these studies, no pulsatile release of PGF2 a has been reported in association with the transitory decline in progesterone concentrations in llamas, but in the closely related alpaca Ž L. pacos ., prostaglandin metabolite peaks have been detected from days 8 to 13 of pregnancy, indicating a temporal relationship between the progesterone decline and PGF2 a pulsatile release ŽAba et al., 1997.. Flunixin meglumine ŽFM., a non-steroidal anti-inflammatory drug, has the ability to reduce prostaglandin biosynthesis by inhibiting the enzyme cyclo-oxygenase. In consequence, when used intensively, the drug has shown the ability to inhibit the synthesis of PGF2 a to the extent that luteolysis is prevented in cattle, without interfering irreversibly with the pulsatile mechanism of prostaglandin ŽAiumlamai et al., 1990.. The objective of the present study was twofold: first, to investigate the plasma concentrations of progesterone in relation to concentrations of 15-ketodihydro-PGF2 a ŽPGFM. during the period of luteolysis and maternal recognition of pregnancy in llamas and, second, to evaluate the ability of FM to inhibit luteolysis in non-pregnant llamas. 2. Materials and methods 2.1. Animals Eleven sexually mature female llamas Ž L. glama., 3–5 years old with an average weight of 105 kg Žrange 95–120., were used. The animals were kept in a natural pasture at the Faculty of Veterinary Sciences, UNCPBA, Tandil, Argentina, Ž37817X S.L., sea level. and were supplemented with hay twice a day. Since several animals were assigned M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 89 to successive trials, a complete rest period of 2 months was provided to the animals between treatments in order to circumvent possible carry-over effects of the previous treatment to the outcome of the following. 2.2. Treatments Intra-vaginal sponges containing medroxyprogesterone acetate ŽMPA. Ž120 mg, Syntex, Buenos Aires, Argentina. were inserted and left in the vagina for 9 days. Six days after removal of the sponges Žday 15., 10 animals were bred with an intact male, while 11 llamas were mated with a vasectomized male. Five of the animals mated with the vasectomized male were treated with FM ŽFinadyne w vet., Schering-Plough, Union, NJ, USA.. FM was injected intravenously every 6 h Ž06:00, 12:00, 18:00 and 24:00 h. at a dose of 2.2 mgrkg from days 6 to 12 post-copulation. 2.3. Blood sampling Blood samples were collected daily from days 3 to 6 post-mating. Thereafter, samples were obtained every second hour during the day Ž08:00–20:00 h. and every fourth hour during the night Ž20:00–08:00 h. until day 16 post-mating. In animals treated with FM, the first sample was collected on day 5 post-mating, and the following samples were collected at 2-h intervals from 06:00 to 24:00 h from days 6 to 12 after mating. All blood samples were collected by venipuncture and immediately drawn into heparinized tubes. Plasma was separated by centrifugation and stored at y208C until analysed. In order to minimise possible damage to the jugular veins from the sampling protocol, puncture was performed according to a schedule at high, medium and low positions, on both the left and right sides of the neck. 2.4. Hormone assays Progesterone was assayed with an enhanced luminescence immunoassay technique ŽAmerlite; Kodak Clinical Diagnostics, England. previously validated for llama plasma ŽAba et al., 1995.. The intra-assay coefficient of variation calculated from the precision profiles was below 8% for concentrations between 2 and 160 nmol ly1 . The inter-assay coefficients of variation, for three quality-control samples were 7% Ž2 nmol ly1 ., 8% Ž18 nmol ly1 . and 6% Ž54 nmol ly1 .. The sensitivity of the assay was 0.2 nmol ly1 . The plasma metabolite of PGF2 a , PGFM, was analysed by RIA according to Kindahl et al. Ž1976. and Granstrom ¨ and Kindahl Ž1982.. The intra-assay coefficients of variation were below 8% for samples containing 240 and 485 pmol ly1 . The corresponding inter-assay coefficients of variation were 5.5% and 8.4%, respectively. The practical detection limit of the assay was 30 pmol ly1 . Oestradiol-17b concentration was determined using RIA kit ŽDiagnostic Products, Los Angeles, CA, USA., reported for use with bovine plasma ŽSirois and Fortune, 1990., and validated for use with llama plasma after minor modifications ŽAba et al., 1995.. The intra-assay coefficients of variation calculated from the precision profiles were 18% at 6 pmol ly1 , and below 11% for concentrations between 19 and 180 pmol M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 90 ly1 . The inter-assay coefficients of variation for three control samples were 22% Ž13 pmol ly1 ., 6% Ž39 pmol ly1 . and 10% Ž84 pmol ly1 .. The lowest amount of oestradiol-17b detectable Ždefined as the intercept of maximal binding y2 SD. was 4 pmol ly1 . Hormone concentrations are expressed in SI units. To convert from pmol ly1 to pg y1 ml and from nmol ly1 to ng mly1 the following factors should be used: PGF2 a metabolite: 2.8; oestradiol-17b: 3.7 and progesterone: 3.2. 2.5. Analysis of data Basal concentrations of PGF2 a metabolite were calculated by averaging the values obtained on each particular day and removing values higher than two standard deviations from the mean value, until the baseline remained unchanged. Concentrations that exceeded the baseline by more than two standard deviations between days 7 and 12 were defined as peaks. The amount of PGF2 a released during each peak was estimated by calculating the areas under the release curve according to the formula: PGF2 a release s Ý Ž Ž PGFM i q PGFM iq120 . r2 . = 120 min, where i s 0, 120, 240, . . . , min and where PGFM i s basal concentration was considered equal to 0. Daily progesterone concentrations were estimated by averaging the concentrations measured in the sample collected at 08:00, 16:00 and 24:00 h for each individual animal. Analysis of variance, using a repeated measures Žwithin-SS. design, was applied to detect differences in hormone concentrations. In all cases, a least-significant difference test ŽLSD. was used to determine differences between means. Because of variations in the time required to attain peak progesterone concentrations in pregnant animals, values were normalised against the highest concentration during days 7, 8 and 9. The mean peak value was further compared with mean concentrations attained during the following days. All statistical analyses were carried out using the StatisticarW, release 4.0, software package Statsoft, USA ŽStatistica for Windows, 1993.. Results are expressed as mean " SEM. 3. Results The progesterone profiles showed that all animals ovulated in response to copulation. Increasing concentrations of progesterone were recorded by day 4 post-mating. Five out of 10 llamas mated with an intact male became pregnant Ž50%.. By day 10–11 after mating, all non-pregnant llamas showed progesterone concentrations close to the detection limit of the assay. Visual examination of the results for each individual confirmed that a pulsatile pattern of prostaglandin release occurred during luteolysis in non-pregnant llamas. Fig. 1Ža. shows the mean Ž"SEM. plasma progesterone concentrations in non-pregnant animals mated with an intact male and the PGFM secretory pattern in a representative llama from days 3 to 16 after mating. Although only statistically significant in three animals, slight increases in plasma PGFM were observed as early as day 7 after mating M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 91 Fig. 1. Plasma concentrations of progesterone Žmean, dotted line, "SEM, shaded area. in non-pregnant Ža. and pregnant Žb. llamas and a prostaglandin metabolite secretory profile Žsolid line. in a representative animal from days 3 to 16 post-mating. Values identified as significant pulses of PGF2 a metabolite are indicated by asterisks. Note the logarithmic scale for the prostaglandin metabolite values. in most of the animals. PGFM pulses were registered between days 8 and 9 and day 13 after mating in non-pregnant animals. Since no significant differences in the hormonal secretory patterns were registered between non-pregnant llamas, whether mated with an intact or a vasectomized male, all animals were considered as one group for further analysis of PGFM profiles. Table 1 shows the characteristics of luteolytic pulses of Table 1 Characterisation of normal luteolytic release of PGF2 a as determined by PGFM analysis in llamas Ž ns11.. Numbers within parentheses in the second column represent the mean number of peaks detected on each particular day. M s 08:00 h, As16:00 h, N s 24:00 h Days after mating Cumulative number of peaksr animal Area under curve Žpeaks. Žpmol ly1 . Ž%. Peak-to-peak interval Žh. 7 8 9 10 11 12 0.3 1.4 Ž1.1. 4.3 Ž2.9. 8.2 Ž3.8. 10.5 Ž2.3. 11.6 Ž1.1. 2.6 49.7 358.9 520.8 227.2 23.3 – 19.6 7.5 7.1 9.1 11.7 0.1 3.6 28.6 45.7 19.9 2.1 Mean peak amplitude Žpmol ly1 . Progesterone Žpmol ly1 . M A N 465 748 1112 1234 779 566 7.9 8.9 4.9 1.0 0.5 0.5 9.3 6.5 1.9 0.6 0.5 0.5 8.3 7.6 3.3 0.7 0.7 0.5 M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 92 Table 2 Mean Ž"SEM. plasma progesterone concentrations from days 10 to 16 after mating in pregnant llamas compared with the mean maximum concentration between days 7 and 9 Mean days 7–9 13.00 "1.90 Plasma progesterone concentrations Žnmol ly1 . Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 9.00UU "0.72 8.93UU "0.61 9.26UU "0.70 8.83UU "0.63 8.38UU "0.64 9.66 "0.94 9.15U "0.83 U P - 0.05 P - 0.01 UU PGFM during luteolysis Ždays 7–12 after mating. in non-pregnant animals Ž n s 11.. In all cases, the initial pulses were lower in amplitude Ž525.9 " 36.7 pmol ly1 . than those recorded later Ž P - 0.01.. The mean peak concentration of the luteolytic pulses was 1019 " 48.5 pmol ly1 . On average, 11.6 Žrange 10–14. peaks per animal were recorded between days 7 and 12 post-mating. Decreasing concentrations of progesterone were already registered by the afternoon of day 8, when only one or two prostaglandin metabolite peaks were recorded in each individual animal. Although all animals had reached basal plasma concentrations of progesterone on day 10 after mating, PGFM peaks were recorded during the following 2 days. In pregnant animals, plasma concentrations of progesterone remained high until the end of the experiment ŽFig. 1Žb... Peak plasma progesterone concentrations were attained between days 7 and 9. When the mean progesterone concentrations registered on those days were compared with the mean concentrations registered from days 10 to 16, a significant drop in progesterone was seen after day 9 ŽTable 2.. In connection with the progesterone decline, PGF2 a pulsatile release was observed in all pregnant animals from days 7 to 15 after mating. Fig. 1Žb. shows the PGFM concentrations in a representative llama. The characteristics of prostaglandin release during early pregnancy Ždays 7–12 after mating. are shown in Table 3. The overall mean frequency of PGF2 a Table 3 Characterisation of PGF2 a release during early pregnancy in llamas as determined by PGFM analysis Ž ns 5.. Numbers between parentheses in the second column represent the mean number of peaks detected on each particular day. M s 08:00 h, As16:00 h, N s 24:00 h Days after mating Cumulative number of peaksr animal Area under curveŽpeaks. Žpmol ly1 . Peak-to-peak interval Žh. Ž%. 7 8 9 10 11 12 0.4 1.0 Ž0.6. 1.4 Ž0.6. 2.2 Ž0.8. 2.4 Ž0.2. 3.0 Ž0.6. 11.1 6.0 11.0 69.3 7.3 28.8 0.1 3.6 28.6 45.7 19.9 2.1 – – 28.0 35.3 38.0 99.0 Mean peak amplitude Žpmol ly1 . Progesterone Žnmol ly1 . M A N 494 443 488 676 388 547 7.6 9.1 10.0 10.0 8.7 8.0 8.7 8.2 7.3 8.0 8.4 9.4 6.2 11.3 8.0 9.0 9.8 10.4 M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 93 Fig. 2. Plasma concentrations of oestradiol-17b Žmean"SEM, shaded area. in non-pregnant Ža. and pregnant Žb. llamas from days 3 to 16 post-mating. metabolite peaks detected in pregnant animals was 0.64 peaksranimalrday, while the mean peak amplitude was 503.8 " 49.1 pmol ly1 . Oestradiol-17b plasma concentrations increased slowly from days 3 to 8 after mating in all lamas. Thereafter, concentrations sharply increased in non-pregnant llamas, until peak concentrations Ž30.9 " 3.8 pmol ly1 . were attained on day 15. Conversely, a slow oestradiol-17b rise was recorded in pregnant animals in which the highest concentrations Ž11.2 " 4.9 pmol ly1 . were observed 12 days after mating ŽFig. 2Ža. and Žb. respectively.. Fig. 3. Plasma concentrations of progesterone Žmean, dotted line, "SEM, shaded area. in non-pregnant llamas mated with a vasectomized male Ža. and in non-pregnant llamas treated with FM Žblack bar. four times a day Žb., and a prostaglandin metabolite secretory profile Žsolid line. in a representative animal from days 5 to 12 post-mating. Values identified as significant pulses of PGF2 a metabolite are indicated by asterisks. 94 M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 Prostaglandin metabolite concentrations decreased rapidly in all animals after the first administration of FM from about 450 pmol ly1 to concentrations of around 180 pmol ly1 Ž P - 0.01.. Concentrations of the metabolite remained low throughout the first 2 days of FM treatment Ždays 6 and 7 post-mating.. Thereafter, pulsatile release of PGF2 a started, and concentrations similar to those recorded during the first 2 days of treatment were recorded between peaks. Fig. 3 shows the changes in hormone concentrations in non-pregnant llamas mated with a vasectomized male during normal luteolysis Ža. and during FM treatment Žb.. In llamas treated with FM, the cumulative number of peaksranimal were 1 Žday 8., 2.2 Žday 9., 4.4 Žday 10., 5.8 Žday 11. and 6 Žday 12.. The mean peak prostaglandin metabolite amplitudes were 261 pmol ly1 Žday 8., 547 pmol ly1 Žday 9., 561 pmol ly1 Žday 10., 993 pmol ly1 Žday 11. and 864 pmol ly1 Žday 12.. Increasing concentrations of progesterone were recorded from days 5 to 7 post-mating in both groups. Thereafter, progesterone concentrations declined and were close to the detection limit of the assay by day 9 post-copulation in untreated animals. Progesterone concentrations remained high until day 8 post-mating, after which, they started to decrease until basal concentrations were attained by day 10 or 11 post-mating. 4. Discussion The PGFM, pattern registered during luteolysis, correlates well with previous reports showing the close temporal association between pulsatile release of PGF2 a and termination of the corpus luteum function in llamas ŽSumar et al., 1988; Aba et al., 1995.. The sampling schedule used in this study, allowed a more detailed study of the luteolytic pulses of PGF2 a in llamas, although some peaks could have been missed. As has been shown in cattle, sheep and goat ŽFredriksson et al., 1984; Basu and Kindahl, 1987; Zarco et al., 1988b., the first peaks Žregistered on days 7 and 8. had consistently lower amplitudes than those recorded during the following days. Calculating the area under the peaks revealed that about 35% of the total release of PGF2 a occurred during days 8 and 9, when luteolysis was at its highest, while about 65% of the total release was seen after progesterone levels had decreased below 1 nmol ly1 . Sampling was not frequent enough to permit an accurate estimation of the duration of pulses. However, based on the observation that increased PGFM concentrations were registered in two successive samples in most cases during the period of maximum secretion, an estimate of 4–5 h for peak duration may be suggested. Moreover, it was observed during this period that in several cases, concentrations did not reach basal values between two consecutive peaks, indicating that the interval between the end of one peak and the beginning of the next could be shorter than 2 hours. This suggestion is supported by the observation that most of the animals had at least four peaks during 1 day Žday 10.. The overall secretory pattern of PGF2 a appears to be similar to that observed in other ruminants. However, PGF2 a pulses during the period of luteolysis seemed to be somewhat shorter in duration and to be produced at a higher frequency in llamas than those reported in cattle ŽBasu and Kindahl, 1987.. M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 95 The luteolytic release of PGF2 a starts on day 7 or 8 post-mating Žapproximately day 5–6 after ovulation. and is completed by day 9 or 10 after mating in non-pregnant llamas. It has also been shown that in South American camelids, the first significant increase in progesterone concentrations is observed 3–4 days after mating ŽSumar et al., 1988; Bravo et al., 1996.. Exposure to an environment characterised by high progesterone concentrations is claimed to play a key role in the regulation of uterine oxytocin receptors and, consequently, of prostaglandin pulsatile release in ruminants ŽSilvia et al., 1991; Lamming and Mann, 1995.. Such a period lasts only 3–4 days in llamas, while it lasts around 3 and 5 times longer in the sheep and in the cow, respectively. In addition, it is apparent that luteal activity after a sterile mating in llamas, is considerably shorter than that observed in other induced ovulators. Thus, using the length of the normal pregnancy for each specie as 100%, the lifespan of the CL in non-pregnant animals varies from 100% in mink and ferret ŽMøller, 1973; Heap and Hammond, 1974., to approximately 55–60% in rabbit and cat ŽPaape et al., 1975, Rowlands and Weir, 1984. and to about 3% in llamas. The mechanism behind this rapid increase in sensitivity and the responsiveness of the developing CL to prostaglandins remains to be determined in llamas. In cows, no prostaglandin peaks are seen during maternal recognition of pregnancy ŽBasu and Kindahl, 1987.. Similarly, there is no pulsatile pattern in pregnant goats during this period, but PGFM concentrations in the peripheral circulation start to increase on day 14 or 15 ŽFredriksson et al., 1984.. In pregnant sheep, although no pulsatile pattern has been observed, two or three PGF2 a pulses of low amplitude, separated by long intervals, can be distinguished during maternal recognition of pregnancy. However, no changes in plasma progesterone concentrations have been reported in connection with these pulses ŽZarco et al., 1988a.. In the present study, prostaglandin peaks were recorded in all pregnant llamas during the period when maternal recognition of pregnancy was expected to occur. The magnitude and the frequency of the pulses registered during days 7–12 of pregnancy were not comparable to those observed in non-pregnant animals. The amount of PGFM recorded as peaks during early pregnancy averaged about 3% of the total amount of prostaglandin released during luteolysis; nevertheless, this prostaglandin production proved to be effective in inducing a decline in progesterone concentrations. A similar transient decrease in progesterone concentrations during maternal recognition of pregnancy has been reported previously in llamas and alpacas ŽAdams et al., 1990; Aba et al., 1995,1997.. Prostaglandin metabolite peaks with relatively low amplitude have been observed in connection with the decline in progesterone concentrations in pregnant alpacas ŽAba et al., 1997.. The mechanism by which the pulsatile release of PGF2 a from the uterus is depressed Žbut not suppressed. in the pregnant llama is not known. Attempts to identify any substance similar to ovine or bovine interferons in camel Žthe related Old World camelids. conceptus incubates from days 10 to 33 after ovulation have been unsuccessful ŽSkidmore et al., 1994.. Moreover, no increase in oestrone sulphate concentrations in urine or oestradiol-17b in plasma has been recorded during early pregnancy in South American camelids ŽBravo et al., 1991; Aba et al., 1995.. The divergent pattern of oestradiol-17b plasma concentrations registered in pregnant and non-pregnant llamas after day 9 post-mating further confirms the hypothesis that 96 M.A. Aba et al.r Animal Reproduction Science 59 (2000) 87–97 progesterone from the corpus luteum exerts a negative influence on follicular activity during early pregnancy in llamas ŽAdams et al., 1990; Aba et al., 1995.. The dose of FM used here, proved to be effective in depressing prostaglandin synthesis. This effect was clearly seen during the first 2 days of the treatment and during the periods between peaks on the successive days. A negative influence on the pulsatile pattern of PGF2 a was also observed. Thus, both the amplitude of the PGFM peaks and the cumulative number of peaks per animal were significantly lower in llamas treated with FM than in untreated animals; but this negative effect was not strong enough to completely inhibit the occurrence of luteolytic pulses of PGF2 a , and luteolysis proceeded in these animals. However, the progesterone patterns indicated that luteolysis was delayed for 1–1.5 days in treated llamas, as compared to untreated animals. A similar dose of FM has been previously proven to be effective in other ruminants to prevent luteolysis; however, earlier reports indicate that the FM half-life is shorter in the related camel than it is in the cow. The authors concluded that, using the same dose regimen in camels as has been used in cattle, would result in lower pharmacological effectiveness ŽOukessou, 1994; Ali et al., 1996.. 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