Peripartal endocrine changes associated with retained placenta in dairy cows

THERIOGENOLOGY

PERIPARTAL ENDOCRINE CHANGES ASSOCIATED WITH RETAINED PLACENTA IN DAIRY COWS A.T. Peter and W.T.K. Bosu Department of Medical Sciences School of Veterinary Medicine University of Wisconsin Madison, WI 53706 Received for publication: November 23, 1986 Accepted: May 15, 1987 ABSTRACT Peripheral s e r u m concentrations of prostaglandln F2a metabollte (PGFM), c o r t l s o l , and progesterone ( P ~ ) w e r e determined in cows that retained placentae (RP; n = IB) and cows that did not retain placentae after p a r t u r i t i o n (NRP; n = 2 B ) . Blood samples collected from Day 15 before until Day 15 after p a r t u r i t i o n were assayed. Significant increases (P < 0.05) in PGFM concentrations occurred in RP cows between Days 7 and 6 before p a r t u r i t i o n compared with NRP cows. There were no differences in the interval required to attain peak concentrations of PGFM and the interval required to reach basal concentrations after p a r t u r i t i o n in both groups. However, the amount of PGFM secreted during the perlpartum period was significantly higher in RP cows (P < O.O1). Starting from Day 6 before p a r t u r i t i o n , serum cortlsol concentrations in RP cows increased significantly to peak levels 3 d before parturition (P < 0.05). Cortlsol levels decreased to basal concentrations in RP cows on Day 1 postpartum. The perlpartal serum P4 concentrations in RP and NRP cows did not d i f f e r . The results indicate that prepartal PGFM and cortlsol increases may constitute r e l i a b l e indicators of retained placenta in dairy cows. Key words: retained placenta, dairy cow, prostaglandln F2~ metabollte, progesterone, cortlsol

Acknowledgments We thank MS. Kandl Rutledge for permission to use cows at the State Correctional Center at Oregon, Wisconsin. We gratefully acknowledge the assistance by the staff of the Correctional Center during the study and thank Randy DeDecker and Kevln Butler for their excellent technical assistance. This research was supported in "part by funds from University of Wisconsin, College of Agriculture and Life Sciences Pro~ect No. 2927, and in part by funds from UW Graduate School.

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INTRODUCTION The s c i e n t i f i c l i t e r a t u r e contains extensive information regarding the various factors associated with retained placenta in dairy cows. For instance, a relationship between retained placenta and length of gestation, prepartum p l a c e n t l t l s , dystocla, twinning, and various other stressors has been established (1). However, l i t t l e information regarding the exact mechanisms of placental separation postpartum is available, nor are the details of biochemical and hormonal changes associated with retained placenta in dairy cows. T h i s dearth of information on the basic mechanism leading to retained placenta is the major reason for lack of effective methods to prevent the condition. Identifying prepartum biochemical or hormonal indices or markers for retained placenta are therefore important, since this w i l l lead to the formulation of effective methods for treatment and prevention. Various altered prepartum hormonal patterns have been associated with retained placenta (2-4). Interpretatlng the data from these and other studies is d i f f i c u l t because of the wide differences in the experimental designs used. In a previous study, we reported early prepartal increases in concentrations of peripheral PGFM in dairy cows that retained fetal membranes postpartum (5). We suggested that early prepartal increases in concentrations of PGFM could constitute a prepartal indicator of retained placenta. Thus, one of the aims of the study reported here was to characterize further the perlpartal changes in PGFM concentrations in cows that did or did not retain fetal membranes postpartum by using a larger number of experlmental animals to extend and confirm our e a r l i e r observations. Results were analyzed to determine i f prepartal increases in PGFM would constitute a sensitive and accurate indicator of retained placenta foIIowlng parturition. The results of a study by BJorkman and Sollen (6) indicated that prepartum hlstopathologlcal changes were present in the uterus of cows that retained fetal membranes postpartum. I t was hypothesized that the hlstopathologica] prepartal changes may be associated with the early prepartal increases in PGFM concentrations described in cows with retained placenta (5) and may also constitute stressors to the animal. Because prepartal inflammatory conditions of the uterus may constitute a significant stressor and because cortlsol concentrations increase as a result of stress (?), this study also examined the changes in the prepartal cortlsol concentrations in cows that did or did not retain fetal membranesafter calving. MATERIALS AND METHODS Holstein cows aged 3 to 7 yr were selected from a large herd maintained at the State Correctional Center, Oregon, Wisconsin, for the study. Cows that produced singletons on the farm between July IgB4 and June IgB5 were included in the study. Three weeks befbre the expected day of calving, cows were separated from the herd and housed in individual calvlng pens. After calving, the cows were closely observed to determine the interval from calving to shedding of placenta. Cows were classlfled as NRP (n = 28) I f the placenta was shed within 24 h after calvlng or RP (n = IB) i f the placenta was retained for 24 h or more after calving. No treatment to increase the rate of placental expulsion was given and no attempts to remove the placenta manually were made. The 18 cows in the RP group were peer-matched with the IB cows in

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the NRP group by parity, sex of the c a l f , month of calving, and gestation length (~ 2 d). Daily blood samples were collected by venlpuncture of coccygeal vessels from 15 d before calving until 15 d after calvlng. From 7 d before until ? d after calving, the sampling frequency was increased to four times per day. The blood samples were centrifuged Immedlately and the sera harvested and stored at -20°C u n t i l assay. Serum concentrations of P4 were estimated by a radlolmmunoassay (RIA) method (B). The Intraassay coefficient of variation (CV) was 1.6% (n = 12) and Interassay CV was 5.3% (n = 5) (g). The concentrations of PGFM in the samples were determined by a RIA method already characterized and validated (8). The Intraassay CV was 4.2% (n = 7) and interassay CV was 7.9% (n = B) (g). Serum cortlsol concentrations were determined by a RIA method (lO). A solld-phase RIA klta with I125 as the tracer was used without extraction. The assay procedure of the k i t was adopted with minor modifications. The lowest dose of the standards (43 ng/ml) provided in the k i t was s e r i a l l y diluted with charcoal-strlpped bovine serum to arrive at the following standards: 0.08, 0.17, 0.34, 0.67, 1.34, 2.69, 5.38, 10.75, and 21.50 ng/ml. Due to the low levels of bovine serum c o r t l s o l , lO0 ~l of standard and test samples were used instead of 25 ~l. The s e n s i t i v i t y of the assay was O.OB ng/ml. Specificity data of the antiserum listed by the manufacturer indicates a nondetectable or low (1.2B%) cross-reactlvlty with steroid hormones or structural analogues which might be present in the serum samples (aldosterone O.OOg%, cortlcosterone 1.40%, cortisone 0.60%, ll-deoxycortlcosterone l.SO%, ll-deoxycortlsol 1.28%, estradlol 0.05%, estrone 0.025%, pregnanedlol 0.001%, and progesterone 0.15%). Validation of the assay included quantitative recovery of unlabeled cortlsol from bovine serum. With known concentrations of cortlsol (lO ng/ml) added to cortlsol-depleted bovine serum, the recovery was 94.3% (n = lO). Serum (lO0 ~l) was dispensed in duplicate with 1 ml of i125 cortlsol buffer solution into antlbody-coated polypropylene tubes and incubated for 3 h. After incubation, the l i q u i d phase was decanted and the tubes were l e f t to dry at room temperature. The radioactivity of the precipitate was measured and the concentrations of the hormone were read off the standard curve by the programmable counter. The Intraassay CV was 4.3% (n = lO) and Interassay CV was 8.4% (n = 5) (g). S t a t i s t i c a l Analysis The PGFMdata were smoothed by using running medians as described by Tukey ( l l ) to identify d i s t i n c t l y the time of onset of prepartum increases in PGFM, time of peak concentrations of" PGFM, and interval required to reach basal concentrations of PGFM after p a r t u r i t i o n . The probability of significant differences in these three parameters, between RP and NRP cows, was analyzed by a General Linear Model procedure of the S t a t i s t i c a l Analysis System (12). The t r a i t s , parity, and duration of retention of placentae were treated as a Coat-A-Count, Diagnostic Products Co., Los Angeles, CA.

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v a r i a b l e s and t h e i r e f f e c t s on the PGFM concentrations were analyzed tn the same a n a l y s i s . Student's t - t e s t was a p p l i e d to t e s t the s i g n i f i c a n c e in the amount of PGFN produced by RP and NRP cows. Differences in serum c o r t t s o l and P4 concentrations In RP and NRP cows were analyzed by a s p l i t - p l o t a n a l y s i s of variance f o r repeated measures (13). RESULTS The g e s t a t i o n lengths were s i m i l a r tn RP (280 ± 5 d, mean ± SER) and NRP cows (278 ± 7 d, mean ± SEM). In the peer-matched cows, the g e s t a t i o n lengths were also s i m i l a r (278 ± 2 d ) . The placenta was shed w i t h i n 3 h a f t e r p a r t u r i t i o n In NRP cows, and tn RP cows the placenta was r e t a i n e d from 24 to 72 h a f t e r p a r t u r i t i o n . None of the cows developed systemic t ] ] n e s s during the p e r t p a r t a l p e r i o d . In both groups, the mean P4 c o n c e n t r a t i o n s were high (7 ± 2.5 ng/ml) from Day 15 before p a r t u r i t i o n unt11 Day 3 before p a r t u r i t i o n , but a tendency to a slow d e c l i n e was c ] e a r ( F i g . 1). S i g n i f i c a n t and p r e c i p i t o u s declines in P4 concentrations occurred in both groups between 72 and 48 h before parturition. T h e r e a f t e r , the Pa c o n c e n t r a t i o n s decreased to 1 ng/ml or less on the day of c a l v i n g . The mean P4 values postpartum never exceeded I ng/m] in e i t h e r group than tn NRP cows ( F i g . 2). Increases In PGFR concentrations occurred e a r l i e r (P < 0.05) in RP cows than In NRP cows ( F i g . 2). The f i r s t significant increases tn PGFR concentrations in RP cows occurred between Days 7 and 6 before p a r t u r i t i o n . The PGFR concentrations were s i g n i f i c a n t l y higher in RP cows on Days 6 to 2 before c a l v i n g . In both groups a dramatic increase in PGFR concentrations occurred 48 to 24 h before c a l v i n g . PGFR concentrations remained elevated postpartum but g r a d u a l l y decreased to basal concentrations by Day 13 a f t e r c a l v i n g in both groups. There was no d i f f e r e n c e in the i n t e r v a l required to a t t a i n peak concentrations of PGFM and the i n t e r v a l required to reach basal c o n c e n t r a t i o n s of PGFM in RP and NRP cows. However, the amount of PGFfl secreted during the perlpartum period was s i g n i f i c a n t l y higher in RP cows than tn NRP cows (P < 0 . 0 1 ) . P a r i t y and d u r a t i o n of placenta r e t e n t i o n a f t e r p a r t u r i t i o n had no i n f l u e n c e on the onset of p r e p a r t a l increases, i n t e r v a l to peak c o n c e n t r a t i o n s , or i n t e r v a l required to reach basal concentrations of PGFN in both groups.

In both groups, mean c o r t t s o l concentrations ranged between 3 and 5 ng/ml u n t i l Day 6 before p a r t u r i t i o n ( F i g . 3). S t a r t i n g from Day 6 prepartum, the corttsol concentrations in RP cows increased g r a d u a l l y to m e a n peak c o n c e n t r a t i o n s of 28 ng/ml on Day Z before p a r t u r i t i o n , f o l l o w e d by a p r e c i p i t o u s f a l l between Day 2 before p a r t u r i t i o n and Oay 1 a f t e r p a r t u r i t i o n w i t h a subsequent gradual d e c l i n e to low c o n c e n t r a t i o n s postpartum. In the NRP cows, c o r t l s o t concentrations were about 5 ng/ml and increased to 7 ng/ml during the l a s t 3 d before p a r t u r i t i o n , f o l ] o w e d by a d e c l i n e to low concentrations postpartum.

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DISCUSSION All cows included in the experiment were exposed to the same management procedures; no prepartum diseases or calving problems that have been associated with retained placenta in dairy cows occurred in any anlmal (1). Furthermore, the gestation lengths were similar in RP and NRP cows. Thus, the qualitative and quantitative differences in hormonal patterns observed between RP and NRP cows cannot be attributed to differences in the above factors. The pattern of prepartal decline in serum P4 concentrations found in this study is similar to those reported prevlously (5,14). However, the elevated prepartal P4 concentrations recorded on Day 7 (5) or Day 6 (3,4) In RP cows were not evident in this study. The significance of changes in prepartal P4 concentrations in cows that retain fetal membranes remains to be elucidated. The early prepartal increases in PGFM In RP cows confirms and extends our previous observation (5). These results indicate that prepartum prostaglandin F2= (PGF2=) synthesis and/or release o c c u r s e a r l i e r in cows that retain fetal membranes postpartum. The increase in PGF2~ represents increased synthesis since, according to Poyser (15), prostaglandlns are not stored in tissues. The source of PGF2= is believed to be the uterus and i t s contents (16,17). In one study PGF2= concentrations were measured in fetal cotyledons and maternal caruncle collected through cesarean section. PGF2= concentrations were found to be higher in fetal cotyledons compared to maternal caruncle (16). However, recent studies (17,18) identified caruncular tissue as the major source of PGF2= in postpartum cows. Our results would lead us to suggest that PGF2a synthesis In these tissues was activated e a r l i e r prepartum in RP cows and that the mechanisms leading to retained placenta may involve early onset of and increased secretion of prostaglandlns before p a r t u r i t i o n . The slgnal that provokes the accelerated synthesis of prostaglandtns prepartum in RP cows remains to be determined. Shemesh et a l . (19) demonstrated the presence of a h e a t - l a b i l e c y t o s o l t c f a c t o r in bovine caruncles that i n h i b i t e d the conversion of arachtdonlc acid to prostanotds and proposed that a decline in the level of t h i s i n h i b i t o r at term may be one f a c t o r t r i g g e r i n g increases in prostanotd synthesis p r i o r to p a r t u r i t i o n . The same workers also demonstrated stlmulatory a c t i v i t y toward prostanotd synthesis of cotyledon extracts and suggested that t h i s tissue may contain a separate f a c t o r that enhances production of prostanolds. Therefore, our data raises the p o s s i b i l i t y that the prepartal events associated with retained placenta may e i t h e r induce early removal of t n h t b t t o r s of prostaglandtn synthesis from the caruncle or promote early a c t i v a t i o n of the prostanotd s t i m u l a t i o n f a c t o r in the cotyledons. Further work is needed to determine the mechanisms involved In e a r l l e r " prepartal onset of PGF2= synthesis in RP cows. Existing data regarding PGF concentrations and r e t e n t i o n of f e t a l membranes are c o n t r a d i c t o r y . Results of previous studies (16,20) i n d i c a t e decreased prepartal concentrations of PGF in placental retention and conversely increased prepartal concentrations of PGF in cows which did not r e t a i n placenta a f t e r p a r t u r i t i o n .

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Since the concentrations of PGFM reflect the status of PGF2~ synthesis in the pregnant uterus and i t s contents (17,1g) we can assume that differences in prepartal concentrations of PGFM between RP and NRP cows indicate differences in PGF2s synthesis in the two groups. T h i s theory supports the hypothesis that the occurrence of retained placenta is already determined some time before p a r t u r i t i o n (5). The elevated PGFMconcentrations in RP cows suggest that events determining whether or not retained placenta w i l l occur probably take place at least 5 d before p a r t u r i t i o n . Indeed, BJorkman and Sollen (6) demonstrated pathological conditions of the placenta which were presumed to be present prior to p a r t u r i t i o n . The lesions included bleeding between chorlonlc v i l l i and cryptal walls and varying degrees of necrosis of tips of the v i l l i , trophoblast, and cryptal epithelium. The extent of the necrotic lesions varied from small spots in restricted areas to large areas intervening between the v i l l i and cryptal epithelium; the extent of necrosis varied with the degree of placental attachment postpartum. Since increased PGF2~ synthesis is associated with inflammation (21), prepartal inflammatory lesions in the myometrlum, cotyledon, caruncle, or endometrlum probably stimulate e a r l i e r synthesis and release of PGF2= in RP cows. The f a i l u r e of P4 concentrations to decrease in the face of increasing prepartal PGFM concentrations observed in this study can be explained by the fact that the levels were not high enough to induce complete "luteolysls" or were due to "slow luteolysls," as suggested for cows in which premature parturition was induced by dexamethasone (22). On the other hand, extragonadal sources, p r i n c i p a l l y the adrenal gland, might have contributed to maintenance of normal P4 levels under stimulation of PGF2a. In this regard, prostaglandln injections have been shown to stimulate adrenocortlcal function in the heifer, thus resulting in increases in cortlcosterold levels (23). The increases in the maternal peripheral cortlsol concentrations in RP cows has not, to our knowledge, been previously reported. Prior to parturition in the bovine, maternal cortlsol concentrations do not increase, although fetal cortlsol concentrations increase slx-fold (24). Results of a recent study suggest that glucocortlcoids can cross from the fetal to the maternal circulation (25). However, the prepartal increases of cortlsol in RP cows may also be attributed to inflammatory conditions in the pregnant uterus or may be a response to increased prostaglandln production associated with inflammation. Indeed, a closer examination of the PGFM and cortlsol patterns reveals that increases in PGFM concentrations occurred a day e a r l i e r than increases in c o r t l s o l . The physiologic role of the elevated prepartal cortlsol concentrations in RP cows remains to be determined. During p a r t u r i t i o n the blood supply to the placenta is interrupted and thus the placental tissue becomes a foreign tissue to which the animal mounts a rejection process, involving leucocytlc a c t i v i t y . Results of a recent study indicate that reduced prepartu~ uterine leucocytlc a c t i v i t y against the cotyledon was associated with retained placenta (26). Since cortlsol inhibits leucocytlc a c t i v i t y in the bovine (27,28), the increased cortlsol levels in the RP cows may suppress leucocytlc a c t i v i t y , resulting in retained placenta. Further studies are needed to confirm the cortlsol patterns in RP cows. In addition, studies are needed to determine the mechanisms leading to increases in cortlsol concentrations and to elucidate the role of cortlsol in the pathophyslology of placental retention in dairy cows. The relationship between cortlsol levels and leucocytlc a c t i v i t y in RP cows also warrants further investigation.

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Diseases. Edwards

2. Agthe, O. and Kolm, H.P. Oestrogen and progesterone levels in the blood plasma of cows with normal parturition or with retained placenta. J. Reprod. F e r t i l . 43:163-166 (1975). 3. Chew, B.P., Keller, R.E., Erb, R.E. and Malven, P. V. Perlparturlent concentrations of prolactin, progesterone and the estrogens in blood plasma of cows retaining and not retaining fetal membranes. J. Anim. Sci. 44:1055-I066 (1977). 4. Chew, B.P., Erb. R.E., Zamet, C.N., Colebrander, V.F., Malven, P.V. and D'Amlco, M.F. Variables associated with perlpartum t r a i t s in dairy cows. V. Hormonal proflles associated with retained fetal membranes. Theriogenology 12:245-253 (19?g). 5. Bosu, W.T.K., Llptrap, R.M. and Leslie, K . E . Peripheral changes in plasma progesterone and 15-keto-13,14-dihydro-prostaglandln F2~ concentrations in cows with or without retained foetal membranes. Anlm. Reprod. Sci. ~:497-510 (lgB5). 6. BJorkman, N. and Sollen, P. A morphologlcal study on secundinarum in cattle. Acta Vet. Scan. 2:165-16g (Ig61).

retention

7. Alam, M.G.S. and Dobson, H. Effect of various veterinary procedures on plasma concentrations of cortlsol, lutelnlzing hormone and prostaglandln F2~ metabolites in the cow. V e t . Rec. II___88:7-I0 (1986). 8. Peter, A.T. and Bosu, W.T.K. Effects of intrauterine infection on the function of the corpora lutea formed after f i r s t postpartum ovulations In dairy cows. Therlogenology. 9. Rodbard, D. Statistical quality control and routine data processing for radioimmunoassays and Immunoradiometric assays. Clinical Chemistry 20:1255-1270 (1974). lO. Hasler, M.J., Painter, K. and Niswender, G.D. An 1251-labeled cortisol radlolmmunoassay in which serum binding proteins are enzymatically denatured. Clln. Chem. 22:1850-1854 (1976). I I . Tukey, J.W. Exploratory Data Analysis. pp. 205-211. 12. SAS User's Guide: Statistics, Institute, Inc.~ lgB5,pp. 956.

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14. Edqvlst, L. E., Kindahl, H. and Stabenfeldt, G. Release of prostaglandln F2~ during the bovine perlpartal period. Prostaglandlns 1 6 : l l l - l l 9 (1978). 15. Poyser, N.L. Uterine prostaglandln production: arachldonic acid cascade, fact or fiction? I_En: Samuelsson, B., Paolettl, R. and Ramwell, P., (eds). Advances in Prostaglandln and Thromboxane and Leukotrlene Res. Raven Press, NY. 1983, Vol. 12, pp. 443-447. 16. Leidl, W., Hegner, D. and Rockel, P. Investigations on the PGF2~ concentrations of cows with and without retained fetal membranes. Zentrabl. Veterinarmed. Relhe. 27:6gl-696 (IgBO). 17. Guilbault, L.A., Thatcher, W.W., Foster, D.B. and Caton, D. Relationship of 15-keto-13,14-dlhydro-prostaglandln F2~ concentrations in peripheral plasma with local uterine production of F series prostaglandins and changes in uterine b l o o d flow during the early postpartum period of cattle. Biol. Reprod. 31:870-B78 (lgB4). lB. Guilbault, L.A., Thatcher, W.W., Drost, M. and Hopkins, S.M. Source of F series prostaglandins during the early postpartum period in cattle. Biol. Reprod. 3j_l:B79-B87 (19B4). 19. Shemesh, M.M., Hansel, W. and Strauss I l l , J.F. Modulation of bovine prostaglandin synthesis by an endogenous inhibitor. Endocrinology II__55:1401-1405 (1984). 20. Gross, T.S., Williams, W.F. and Manspeaker, J.E. In vitro placental prostaglandln synthesis in the late pregnant and perlpartum cow. Biol. Reprod. 32:154 abstr. (1985). 21. Vane, J.R. Prostaglandlnsas mediators of inflammation. In: Samuelsson, B. and Paolettl, R., (eds). Advances in Prostaglandln and Thrombaxane Res. Raven Press, NY. 1976, Vol. 2, pp. 7gl-BOl. 22. Lindell, J.O., Kindahl, H. and Edqvist, L.E. Prostaglandln release at dexamethasone induced parturition in cows. Acta Vet. Scan. I B:257-265

(1977). 23. Louis, T . M . , Stellflug, J.N., Tucker, H.A, and Hafs, H . D . Plasma prolactln, growth hormone, lutelnlzlng hormone and glucocortlcolds after prostaglandln F2~ In heifers. P r o c . Soc. Exptl. Biol. and Med. 14___77:128-135 (1974). 24. Comllne, R.S., Hall, L.W., Lavelee, L.W., Nathanielz, P.W. and Silver, M. Parturition in the cow: endocrine changes in animals wlth chronically implanted catheters In the foetal and maternal circulations. 3. Endocrlnol. 6_33:451-472 (1974). 25. Williams, W.F., Gross, T.S., Larson, G., Moreland, T.W. and Manspeaker, J.E. Maternal response to Intra-amltotlc hormonal In~ectlons of cattle. J. Anlm. Scl. 61:379 abstr. (1985).

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26. Gunnlnk, J.W. Retained placenta and leucocyttc a c t i v i t y . 6:49-104 (1984).

Vet. Quarterly

27. Muscoplat, C.C., Shope, R.E., Chen, A.W. and Johnson, O.W. Effects of cort~costero%ds on responses of bovtne perlpheral blood lymphocytes cultured w%th phytohemagglut~n%n. Am. J. Vet. Res. 36:1243-1244 (1975). 28. Roth, J.A. and Kaeberle, M.L. Effects of tn vtvo dexamethasone administration on %n v~tro bovine polymorphonuclear leukocyte functlon. Infect. Immun. 33:434-44] ( ] 9 8 ] ) .

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