Reinitiation of Ovulatory Cycles in Incubating Female Turkeys by an Inhibitor of Serotonin Synthesis, P-Chlorophenylalanine

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1 BIOLOGY OF REPRODCTION 28, (1983) Reinitition of Ovultory Cycles in Incubting Femle Turkeys by n Inhibitor of Serotonin Synthesis, P-Chlorophenyllnine M. E. EL HALAWANI,2 J. L. SILSBY, S. C. FEHRER nd E. J. BEHNKE Deprtment of Animl Science niversity of Minnesot St. Pul, Minnesot 5518 ABSTRACT Nest deprivtion of incubting turkeys cused decrese in serum prolctin (PrI) levels from ± ng/mi to ± 83. ng/mi 1 dy fter initition of deprivtion, with further decline to ± ng/mi t the end of the 22-dy experimentl period. Serum luteiniing hormone (L), progesterone nd estrdiol levels following nest deprivtion were similr to those in birds llowed to incubte (controls). Orl dministrtion of p-chlorophenyllnine (PCPA, 5 mg/kg) to incubting turkeys for 3 consecutive dys reduced nesting frequency (P<.5) on the 4th dy fter initition of tretment nd the nesting virtully cesed by the 9th dy. Pretretment PrI ws 1655 ± 21 ng/ml nd declined (P<.5) fter PCPA dministrtion to low of 28.6 ± 2.8 ng/mi. In ddition, PCPA cused sustined rise in serum L peking (5.59 ± 1.9 ng/ml) 3 dys fter tretment initition.contrry to nest deprivtion, serum levels of progesterone nd estrdiol incresed (P<.5) s consequence of PCPA tretment. Seven of 8 PCPA-treted birds lter cme into ly when their PrI levelsnd nesting frequency incresed gin. The resultssuggest role for serotonin (5-HT) in incubtion behvior, nd PrI nd LH secretion in turkeys. INTRODCTION Incubtion behvior in yes is chrcteried by decresing circulting levels of luteiniing hormone (LH), estrdiol nd progesterone, nd blood prolctin (Pri) levels re higher thn those found in lying hens (Cogger et l., 1979; Etches et l., 1979; Shrp et!., 1979; Burke nd Dennison, 198; El Hlwni et l., 198; Goldsmith nd Willims, 198, Bedrk et!., 1981; Proudmn nd Opel, 1981). Incubtion is lso ssocited with ggressive nest-protective behvior, cesstion of ovultion nd ovrin regression (Collis, 195; El Hlwni nd Burke, 1976). The mechnism(s) responsible for these chnges remins mtter for specultion nd the sequence of events nd the hormonl bsis tht led to initition nd mintennce of incubtion re equivocl (Nkjo nd Tnk, 1956; Lehrmn, 1963; Accepted August 3, Received June 21, This ppter is Scientific Journl Series pper number 12,293 of the Minnesot Agriculturl Experiment Sttion. 2Correspondence: M. E. El Hlwni, 495D An. Sci./Vet. Med. Building, Dept. of Animl Science, niversity of Minnesot, St. Pul, MN Riddle, 1963; Shni et l., 1973; Cmper nd Burke, 1977b; Burke nd Dennison, 198; El Hlwni et t, 198; Opel nd Proudmn, 198; Le et l., 1981; Proudmn nd Opel, 1981). The involvement of serotonin (5-HT) hs been suggested in the control of pituitry function, gondl ctivity nd incubtion behvior. The physiologicl relevnce of 5-HT involvement in incubtion behvior is supported by the incresed hypothlmic 5-HT turnover rte in incubting turkeys (El Hlwni nd Burke, 1976). Phrmcologiclly induced chnges in the metbolism of 5-HT in the centrl nervous system hve been shown to modify the relese of both Prl nd LH nd gondl development. Degenertion of serotonergic neurons s consequence of 5,7- dihydr.oxytryptmine tretment ugments testiculr growth in response to photostimultion in quil (Cls, 1975), s does p-chlorophenyllnine (PCPA) which inhibits 5-HT synthesis (El Hlwni et l., 1978). Augmenttion in LH relese is lso observed following PCPA tretment in the turkey (El Hlwni et t, 1982). Conversely, intrventriculr injection or implnttion of 5-HT reduces LH relese nd light-induced gondl growth (Rudnev, 1978; 221 on 1 April 218

2 222 HALAWANI ET AL. Rudnev et!., 1978), s does tretment with monomine oxidse inhibitor resulting in elevted 5-HT (Forbes, 197; El Hlwni et!., 1982). The serotonin ntgonist, methysergide, decreses plsm Pr! levels in the chicken (Buonomo et!., 198) nd PCPA prevents the nesting induced PrI relese in the turkey (El Hlwni et!., 198b). The evidence tht 5-HT inhibits the relese of LH, inhibits gondl ctivity nd stimultes the relese of Pr! combined with the observtion tht the incubting turkey hs n incresed hypothlmic 5-HT turnover rte, elevted Prl nd depressed LH levels, led us to hypothesie tht interference with 5-HT synthesis in incubting turkeys should restore the endocrine condition to tht of lying hens. The present study reports the effect of dministrtion of PCPA ( reltively specific inhibitor of tryptophn hydroxylse, the rte limiting enyme in 5-HT synthesis) to incubting turkeys on the circulting levels of LH, Pr!, progesterone nd estrdiol. MATERIALS AND METHODS Nichols lrge white strin femle turkeys (3 weeks old) were used in this study. The birds were housed in floor pens, 6-8 birds per pen, with trpnests. They were exposed to 54 lux light intensity, with lights on constntly from 4 to 19 h. This ws the first stimultory photoperiod the birds received s dults. Feed nd wter were constntly vilble. Trpnests were inspected 6 times dily from 8 to 17 h. Birds nd ny ccompnying eggs were removed from the nests t ech nest check. The number of times ech bird entered the nest per dy nd the dily egg production ws tbulted. The birds entered the study when they hd nested continuously for 4-6 dys fter the lst egg ws lid. Blood smples were collected for estrdiol nd progesterone nlysis 6 dys before initition of tretment nd immeditely before decpittion. The birds in ll groups were killed by decpittion 44 dys fter the initition of tretment. At this time the ovries, oviducts nd pituitry glnds were excised nd weighed. Experiment 1. Effect of Nest Deprivtion on Serum Prl nd LH Levels of Incubting Turkeys Incubting turkeys were rndomly divided into two groups (6 birds/group). One group remined undisturbed, while the birds in the second group were nest-deprived by plcing ech bird into n individul wire cge. Serum LH nd PrI concentrtions were determined in blood smples collected from both groups before (3 nd dys) nd fter (1, 2, 3, 4, 7, 9 nd 22 dys) nest deprivtion. Experiment 2. Effect of PCPA on Nesting Frequency nd Serum Hormone Levels in Incubting Turkeys Incubting turkeys were rndomied between two groups consisting of 7 birds in the control group nd 8 birds in the tretment group. The birds in the tretment group received orlly dministered PCPA (DLp-chlorophenyllnine, crystlline, Sigm St. Louis, MO; 5 mg/kg in geltin cpsules) for 3 consecutive dys. The birds in the control group received empty geltin cpsules. Blood smples for subsequent Prl nd L nlysis were collected from both groups 6, 3 nd dys before drug tretment nd 1, 2, 3, 4, 7, 9, 22, 31 nd 42 dys fter tretment initition. Rdioimmunossy Blood (4 ml) ws obtined from the brchil vein, llowed to clot overnight t 4#{176}Cnd centrifuged. Serum ws poured off nd stored t -2#{176}C until ssyed for LH, Prl, estrdiol nd progesterone. LH nd PrI rdioimmunossys were conducted ccording to the methods of Burke et l. (1979) nd Burke nd Ppkoff (198), respectively. Estrdiol concentrtions were mesured by rdioimmunossy using the method of Etches et l. (1981). Progesterone rdioimmunossy ws conducted utiliing commercil ntiserum (Arnel). The procedure yielded estimtes of progesterone not significntlydifferent from estimtes derived from the sme extrcts using the method described by Cmper nd Burke (1977). Serum smples for ech experiment were run in duplicte in single ssy. The within ssy coefficients of vrition were 5.9% for Pr!, 3.2% for L, 11.7% for estrdiol nd 16.% for progesterone. Sensitivities were.69 ng for PrI,.6 ng for L,.76 pg for estrdiol nd.19 ng for progesterone. Sttisticl Anlysis Dt were nlyed by nlysis of vrince using ANOVA procedure in the Sttisticl Anlysis System (Helwing nd Council, 1979). Duncn s multiple rnge test ws used to compre mens t the.5 level.the estrdiol nd progesterone levels nd the weights of the ovries, oviducts nd pituitries were nlyed using the two-wy Student s t test. RESLTS Experiment 1. Effect of Nest Deprivtion on Serum Pri nd LHLevels of Incubting Turkeys The results re presented in Fig. 1. During pretretment while the birds were still in their home pens, serum Pr! levels of nest-deprived nd control groups were similr. A prompt decrese in serum Pr! levels ws observed 1 dy fter nest deprivtion with further depression the following 2-9 dys. The low serum Pr! levels persisted throughout the experiment. Control serum Pri levels remined elevted nd stble. Men serum LH levels were not significndy ltered in either the control or the nest-deprived groups. on 1 April 218

3 PCPA AND INCBATION-INDCED GONADAL REGRESSION E C 16 CONTROL S--i -J cr 3-8 w ) 4 NEST- DEPRIVED 4 NEST- DEPRIVED E c :i: -J 3 2 CONTROL w (1) I I I I I DAYS FIG. 1. Men prolctin nd luteiniing hormone responses to nest deprivtion of incubting femle turkeys. Nest deprivtion ws ccomplished on Dy by plcing the birds in individul cges. Control birds remined on the nests during the experimentl period. The verticl line bove or below ech point represents the mgnitude of the stndrd error of the men vlue. There were 6 birds/group. Experiment 2. Effect of PCPA on Nesting Frequency nd Serum Hormone Levels in Incubting Turkeys Seven of 8 incubting turkeys strted to ly 24.7 ± 1.7 (X ± SEM) dys following PCPA dministrtion. The one bird tht did not ly showed serum Pr! levels nd nesting frequency similr to the lying turkeys. Her serum LH, however, remined t pretretment level nd the ovry nd oviduct were regressed. Nesting frequency nd seurm Pr! nd LH levels of the 7 lying hens re presented in Fig. 2. The PCPAtreted birds were found on the nests 4.75 ± on 1 April 218

4 - 224 HALAWANI ET AL. TREATMENT > 6 4 cx L& I- ) 2 CONTROL #{149} 1. 4Th - - r T r IT PCPA, TREATMENT ; 144 I.- 1-T 1 CONTROL 1 -J cx - 6kcx f_/) 2k 1 PC PA OVIPOSITION (PCP e 8 TREATMENT 111 S.s 6 I -J 4 cx 2 NTRO OVIPOSITION CPA DAYS FIG. 2. Men nesting frequency, prolctin nd luteiniing hormone responses to orl dministrtion of PCPA in incubting femle turkeys. The dministrtion of PCPA is indicted by rrows. The verticl line bove or below ech point represents the mgnitude of the stndrd error of the men vlue. The boriontl line with verticl rrow beds indictes the period during which the birds first ovulted following PCPA tretment. Asterisk () represents the dy on which the first significnt (P<.5) chnge ws observed. There were 7 birds/ group. on 1 April 218

5 PCPA AND INCBATION-INDCED GONADAL REGRESSION 225 u.14 to 5.25 ±.37 times/dy during the 6. r. pretretment dys. On Dy 4 following the o o o initition of PCPA tretment, nesting frequency I (1 eqe#{216}f. I < VVV fell (P<.5) to 3.88 ±.72. Therefter, o nest-occuption behvior virtully cesed nd El there ws no sign of nesting interest for dys. Nest visits incresed gin when the birds ER I u oooso strted to ly. Control birds persisted in their i I T I N.. nesting behvior for the durtion of the cx- I VVVV periment. The level of Pr! ws high before tretment #{149}, (1655 ± 21 ng/ml; time), but fell steeply fter PCPA dministrtion. On Dy 2 of tret- n r-.,.#{176} 1IsoOSN ment, Pr! levels were lredy declining, nd I.i they reched their lowest vlue (28.6 ± 2.8 so ng/ml) by the 7th dy of tretment. The levels ; n 11 I.i - remined!ow until the birds cme bck into ly, when their serum levels of Pr! incresed to 4.. men vlue of 97.1 ± 24. ng/ml. The men I serum Pr! level ws not significntly ltered in n. - o so I the control group during the first 22 dys of the experimentl period. By Dy 31 the men so Pr! level of the controls ws reduced to 72% of I the plteu level observed t Dy 22, but * so4.. - remined significntly higher thn tht of the PCPA-treted birds (872.8 ± ng/ml vs. 6.3 ± 16.9 ng/ml). The decline in serum Pr! T levels in untreted birds ws ssocited with the. O N O bsence of ggressive nest-protective behvior H H H H nd reduced resistnce to dislodging from the.. -,-.,- nests, lthough nesting frequency ws not ii N - N N ltered (Fig. 2).. Pretretment serum LH levels were low..5 There ws n increse in the serum level of LH. on Dy 3 of PCPA tretment from 2.53 ±.33. o so ng/ml to 4.26 ±.54 ng/m!. A pek vlue of N 44HHH 5.58 ± 1.9 ng/ml ws observed 4 dys fter co tn.n soti tretment initition. The level dropped to 3.66 * m,,iv,i:s#{225} m V ±.61 ng/ml 2 dys fter tretment but con- -,,, tinued to remin significntly bove the level in B control birds. Serum LH level of the incubting. T untreted group remined low nd stble. b V P..H 44H+IH throughout the experiment. v tititir T I4HHII -4 Effect of PCPA nd Nest Deprivtion on so * - Gondl nd Pituitry Glnd Weight nd - Cs.5 on Serum Progesterone nd Estrdiol Levels. PN. The results re presented in Tble 1. Post-.41- mortem exmintion reveled tht the ovries X 41 nd oviducts of incubting control turkeys in the PCPA nd the nest deprivtion experiments were completely regressed. In PCPA-treted u. turkeys the men ovrin weight ws ±. so g nd men oviductl weight ws T. I- I- ZQO.,nIr;fn p * TP on 1 April 218

6 226 HALAWANI ET AL. ± 7.33 g. The ovries nd oviducts of nestdeprived turkeys remined regressed nd were not dissimilr from controls. The pituitry glnds of the nest-deprived turkeys were significntly smller thn those of incubting control birds (17.63 ±.77 mg vs ± 1.95 rng). However, the pituitry glnds of PCPA-treted birds weighed pproximtely the sme s those of incubting control birds. Serum progesterone ws nondetectbie in both incubting control nd nest-deprived turkeys. However, PCPA-treted birds exhibited elevted serum progesterone (6.19 ± 2.37 ng/ml) t the termintion of the experiment. Serum estrdiol levels were low in control birds nd remined low following nest deprivtion (4.72 ± 1.2 pg/rn! vs ± 8.88 pg/mi). In contrst, PCPA-treted birds hd significntly incresed serum estrdio! (43.69 ± 6.75 pg/mi) s compred to the pretretment level (8.7 ± 18 pg/mi) nd the control group (7.55 ±.95 pg/rn!). DISCSSION The results of the present study demonstrte tht PCPA suppresses rised serum Pr! levels, decreses nesting frequency nd increses LH levels in incubting turkeys. This effect is ssocited with return to functionl hypothlmic-pituitry-ovrin xis chrcteristic of photostimulted turkeys. The precise mechnism(s) of reinitition of ovrin ctivity hs not been elucidted. The Pr!-lowering effect of PCPA confirms our previous report tht tretment with the drug prevents the nesting-stimulting Pr! rise (El H!wni et!., 198b) nd increses serum LH (El Hlwni et!., 1982). The concept of serotonergic stimultion of Pr! relese is further supported by the findings tht quipine, 5-HT gonist, increses serum Pr! levels in the turkey (Fehrer et i., 1982), s does intrventncuir!y dministered 5-HT (Hrgis nd Burke, 1982). The findings of ugmenttive effects of 5,7-dihydroxytryptmine nd PCPA on LH levels nd gond! recrudescence (Cls, 1975; El H!wni et l., 1976) nd their inhibition by intrventriculrly dministered 5-T (Rednev, 1978) re consistent with the inhibitory role of 5-T on LH relese nd gondl growth. There is thus considerble evidence suggesting role for 5-HT in incubtion behvior nd L nd Pr! relese. However, the involvement of dopmine is possible since PCPA hs been shown to depress brin dopmine slightly (El Hlwni et!., 1978). Pro!ctin ppers to hve role in the regultion of ovrin function. The ntigondi effect of elevted blood Pr! levels hs been suggested by studies demonstrting tht gondotropin-induced ovrin steroidogenesis (Cmper nd Burke, 1977b) nd LH-induced premture ovultion (Tnk et!., 1971) re blocked by exogenous mmmlin Pr!. Moreover, high circulting PrI during incubtion is ssocited with gond! regression (El Hlwni nd Burke, 1976). Administrtion of mmmlin Prl termintes egg lying nd induces ovrin regression (Opel nd Proudmn, 1981), probbly by interfering with ovrin gondotropin receptors. In the rt, Pr! prticiptes in the regultion of LH receptors in the ovry (Holt et!., 1976). Elevted levels of Pr! pprently interfere with gondotropin secretion, since diminished LH levels re ccompnied by incresed levels of circulting Pr! in incubting birds, nd tretment with Pr! ntiser increses LH levels (Le et!., 1981). The results of PCPA dministrtion in the present study re consistent with the possibility tht Pr! my be ntigond! since reduction in circulting Pr! levels is ccompnied by elevted serum L levels nd ovrin development. In humns, 5-T ntgonists effectively lower serum Pr! levels nd restore spontneous ovultory cycles in hyperprolctinemic infertile women (D Agt et!., 1977; D Alberton et l., 1977). While Pr! is clerly implicted in the events leding to incubtion behvior nd/or ovrin regression, the finding of the present study, tht PCPA increses serum LH levels nd reinitites ovrin ctivity, implies tht this effect my be medited through suppression of serotonergic mechnisms. This ide is supported by the observtion in the present study tht the PCPA-induced increses in serum LH levels occurs before significnt reduction in circulting Pr! is detected (Fig. 2). Furthermore, Go!dsmith et l. (1981) nd Goldsmith nd Willims (198) presented dt indicting tht the decline in circulting LH levels in incubting doves nd mllrd ducks is not contingent upon n increse in Pr! levels. It is conceivble tht the low LH levels ssocited with incubtion is consequence of the elevted hypothlmic 5-HT turnover rte (El Hlwni nd Burke, 1976). While it would be premture to imply tht Pr! hs no inhibitory effect on LH relese, the dt presented here from the nest deprivtion experiment do not show n increse on 1 April 218

7 PCPA AND INCBATION-INDCED GONADAL REGRESSION 227 in LH levels ssocited with the Pr!-lowering effect of nest deprivtion. It hs not been estblished, however, tht the lck of LH rise in nest-deprived turkeys ws not due to the stress of cging, lthough the sme cges re commonly used for housing lying turkeys. Also, the suppressive effect of PCPA on Pr! levels ws more mrked thn tht of nest deprivtion. The results further demonstrte n ssocition between Pr! levels nd nesting frequency (Fig. 2). It is not cler, however, whether the decline in serum Pr! levels is responsible for the termintion of incubtion behvior or whether the reduction in nesting frequency cuses circulting levels of Pr! to decline. Our erlier findings (El Hlwni et!., 198) showed tht nest-deprived incubting turkeys resume nesting when returned to their home pens in spite of low levels of serum Pr!. REFERENCES Bedrk, E., Hrvey, S. nd Chdwick, A. (1981). 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