This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

Transcription

1 This rticle ppered in journl pulished y Elsevier. The ttched copy is furnished to the uthor for internl non-commercil reserch nd eduction use, including for instruction t the uthors institution nd shring with collegues. Other uses, including reproduction nd distriution, or selling or licensing copies, or posting to personl, institutionl or third prty wesites re prohiited. In most cses uthors re permitted to post their version of the rticle (e.g. in Word or Tex form) to their personl wesite or institutionl repository. Authors requiring further informtion regrding Elsevier s rchiving nd mnuscript policies re encourged to visit:

2 Generl nd Comprtive Endocrinology 66 () 9 6 Contents lists ville t ScienceDirect Generl nd Comprtive Endocrinology journl homepge: Orl progestin induces rpid, reversile suppression of ovrin ctivity in the ct R.A. Stewrt,,, K.M. Pelicn,, J.L. Brown, D.E. Wildt,, M.A. Ottinger, J.G. Howrd Deprtment of Reproductive Sciences, Center for Species Survivl, Smithsonin s Ntionl Zoologicl Prk, Conservtion & Reserch Center, Front Royl, VA 6, USA Deprtment of Animl nd Avin Sciences, University of Mrylnd, College Prk, MD 7, USA rticle info strct Article history: Received 8 July 9 Revised 7 Decemer 9 Accepted 8 Decemer 9 Aville online Jnury Keywords: Ct Progestin Altrenogest Ovrin suppression Fecl hormone monitoring Estrous cycle The influence of orl progestin (ltrenogest; ALT) on ct ovrin ctivity ws studied using non-invsive fecl steroid monitoring. Queens were ssigned to vrious ALT dosges: () mg/kg (control; n = 5 cts); (). mg/kg (LOW; n = 5); ().88 mg/kg (MID; n = 6); or ().5 mg/kg (HIGH; n = 6). Fecl estrogen nd progestgen concentrtions were quntified using enzyme immunossys for 6 dys efore, 8 dys during nd 6 dys fter ALT tretment. Initition of folliculr ctivity ws suppressed in ll cts during progestin tretment, wheres controls continued to cycle normlly. Femles (n = 6) with elevted fecl estrogens t tretment onset completed norml folliculr phse efore returning to seline nd remined suppressed until tretment withdrwl. All cts receiving orl progestin re-initited folliculr ctivity fter tretment, lthough MID cts experienced the most synchronized return (within 6 dys). Men seline fecl estrogens nd progestgens were higher (P <.5) fter tretment in HIGH, ut not in LOW or MID cts compred to pre-tretment vlues. The results demonstrte tht: () orl progestin rpidly suppresses initition of folliculr ctivity in the ct, ut does not influence folliculr phse tht exists efore tretment initition; nd () queens return to norml folliculr ctivity fter progestin withdrwl. This study provides foundtionl informtion for reserch imed t using progestin priming to improve ovrin response in felids scheduled for ovultion induction nd ssisted reeding. Ó Elsevier Inc. All rights reserved.. Introduction Endngered felids hve enefited from ssisted reproduction strtegies designed to mintin or increse genetic diversity in smll popultions mnged ex situ (Wildt nd Roth, 997; Wildt et l., ). Approches such s rtificil insemintion (AI) nd in vitro fertiliztion/emryo trnsfer cn improve reproductive efficiency in rre ct species when nturl reeding fils (Howrd, 999; Swnson, ; Howrd nd Wildt, 9). A lproscopic intruterine AI technique hs een successful, producing offspring in the cheeth (Howrd et l., 99, 997), clouded leoprd (Howrd et l., 996, 997), tiger (Donoghue et l., 99), pum (Brone et l., 99), leoprd ct (Wildt et l., 99), snow leoprd (Roth et l., 997), tigrin (Swnson nd Brown, ) nd ocelot (Swnson et l., 996). However, pregnncy success is vrile cross species. AI efficiency is 5% in the cheeth (Howrd et l., 997), ut success remins low (<5%) in the clouded leoprd (Howrd et l., 997) nd tiger (Grhm et l., 6), nd multiple Corresponding uthor. Fx: E-mil ddress: wildtd@si.edu (D.E. Wildt). Present ddress: Center for the Integrtive Study of Animl Behvior, Indin University, Bloomington, IN, USA. Present ddress: Deprtment of Veterinry Popultion Medicine, College of Veterinry Medicine, University of Minnesot, St. Pul, MN, USA. ttempts hve filed in the fishing ct (Buer et l., ) nd Plls ct (Brown et l., ). Although cler etiology for pregnncy filure fter AI hs not een estlished, one contriutor ppers to e inconsistent ovrin response fter exogenous gondotropins re used to stimulte the ovry efore insemintion (Howrd nd Wildt, 9). Most felid ovultion induction protocols involve dministering speciesspecific dosge of equine chorionic gondotropin (ecg) to initite folliculr growth followed y humn chorionic gondotropin (hcg) to complete meiotic mturtion of the oocyte(s) nd ovultion (Howrd, 999). However, ecg cn stimulte ovultion if given during estrus, nd hcg hs folliculogenic properties in the ct (Swnson et l., 997). Thus, when ecg nd hcg re dministered in tndem t rndom intervls in the estrous cycle, hyper-folliculr stte cn occur (Swnson et l., 996), resulting in normlly high nd protrcted circulting estrogen profiles, reduced emryo qulity nd delyed emryo trnsport through the oviduct (Roth et l., 997; Grhm et l., ). Felids re lso notorious for exhiiting unusully high vrition in ovultion type. Trditionlly considered induced ovultors (Wildt et l., 98), more recent investigtions hve confirmed spontneous ovultion in lortory-housed domestic cts (Lwler et l., 99; Gudermuth et l., 997; Grhm et l., ; Pelicn et l., 5) nd certin non-domestic species, including the fishing ct, mrgy nd clouded leoprd (Brown et l., ; Moreir et l., ; Brown, 6). Spontneous ovultion further complictes 6-68/$ - see front mtter Ó Elsevier Inc. All rights reserved. doi:.6/j.ygcen.9..6

3 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 rtificil control of the ovry ecuse corpor lute presence nd prllel elevtions in circulting progesterone reduce (or prevent) the effective use of exogenous gondotropins (Pelicn et l., 6, 8). For ssisted reproduction to e mximlly efficient in felids, quiescent ovry t the time of ovultion induction is required. This environment increses the chnce for consistent nd uniform folliculogenic response to ovrin stimultion while gretly diminishing the risk of n ltered endocrine milieu. This concept ppers especilly vlid in felids s illustrted y the cheeth, consistent induced ovultor with frequent periods of cyclicity (Brown et l., 996). Both these trits no dout hve contriuted to norml ovultion numers post-ecg/hcg nd comprtively high incidence of AI success in the cheeth (Howrd et l., 99, 997) s well s in nother induced ovultor, the ocelot (Swnson et l., 996). In non-felids tht spontneously ovulte, including domestic livestock (Lofstedt, 988; Wood et l., 99; Deliginnis et l., 5) nd wild ungultes (Monfort et l., 99; Morrow et l., ), progestins re routinely dministered prior to ssisted reeding to temporrily suppress folliculr ctivity nd improve ovrin response. Although the progestins melengestrol cette (MGA), megestrol cette nd medroxyprogesterone cette re well-known contrceptive gents in felids (Romtowski, 989; Munson, 6), their ility to temporrily suppress ovrin ctivity for the purpose of ssisted reproduction hs not een investigted. Our lortory confirmed the efficcy of the progestin levonorgestrel (Norplnt Ò ) delivered s n implnt for short-term suppression of ovrin ctivity in the ct (Pelicn et l., 5). However, this pproch requires nesthesi to insert nd remove the implnts nd, more importntly, results in vrile returns to ovrin cyclicity fter implnt removl (rnging from weeks to > months). The purpose of the present study ws to ssess the impct of n orl progestin on ovrin function in the ct. We selected ltrenogest (ALT), which hs een used to synchronize folliculr ctivity efore ssisted reeding in the horse (Lofstedt nd Ptel, 989), pig (Wood et l., 99), killer whle (Roeck et l., ) nd ottlenose dolphin (Roeck et l., 5). The efficcy of three ALT dosges for short-term inhiition of ovrin ctivity ws ssessed y non-invsively evluting fecl steroid profiles. We hypothesized tht orl progestin would provide rpid, reversile inhiition in dose-dependent fshion.. Mterils nd methods.. Animls Thirteen dult ( 5 yer old) femle domestic cts were housed t the Conservtion & Reserch Center of the Smithsonin s Ntionl Zoologicl Prk. Ech queen ws mintined individully in stinless steel cge (.5 m ) under rtificil fluorescent illumintion ( h light: h drk) during the -month study. Cts were provided dry commercil diet (Purin ONE Ò, Nestlé Purin PetCre Co., St. Louis, MO) nd hd continul ccess to wter, perch ords, edding nd enrichment items. Ech ct ws weighed on the dy efore tretment onset using digitl veterinry scle, nd weights (in kg) were used to clculte ALT dosge. All reserch ctivities were pproved y Institutionl Animl Cre nd Use Committees t Smithsonin s Ntionl Zoologicl Prk (5-5) nd the University of Mrylnd, College Prk (R-6-7)... Altrenogest dministrtion The gol ws to chieve n ovrin inhiition intervl tht coincided with the durtion of norml lutel phse (6 8 dys) in domestic ct (Brown et l., 99; Pelicn et l., 5). We presumed tht suppression for this intervl would ensure lysis of existing corpor lute (if present) nd promote sufficient ovrin quiescence y the end of tretment. Ech ct received two different dosges (in Tril versus Tril ) seprted y n 8-month intervl. Resumption of norml folliculr ctivity fter Tril ws confirmed y fecl hormone nlyses efore eginning Tril. Cts were rndomly ssigned to the two tretments in lnced order to void crry-over effects etween the consecutive trils. Altrenogest orl suspension (Regu-Mte Ò ;. mg/ml; Intervet Inc., Millsoro, DE) ws stored t room temperture ( C) in n opque continer. During ech tril, the progestin ws mixed into 5 g of wet food (Friskies Ò ; Nestlé Purin PetCre Co.) nd dministered dily for 8 consecutive dys. In Tril, ech ct ws ssigned to one of four ALT tretments: () mg/kg (control; wet food only; n = cts); ().88 mg/kg (n = ); ().76 mg/ kg (n = ); or ().5 mg/kg (n = ). Bsed on Tril results, the.76 mg/kg tretment ws removed from the experimentl design nd replced with new test dosge (. mg/kg) to etter refine the lowest effective dosge. In Tril, ech ct ws ssigned to one of four tretments: () mg/kg (n = ); (). mg/kg (n = 5); ().88 mg/kg (n = ); or ().5 mg/kg (n = )... Fecl hormone extrction Over the course of the study, fecl smples were collected dily, seled in plstic gs leled with the individul s nme/dte nd stored t C. Fecl liquots from 6 dys efore, 8 dys during nd 6 dys fter ech tretment period were extrcted to quntify estrogen nd progestgen concentrtions using ssys vlidted for the domestic ct (Brown et l., 99). Briefly, ech fecl smple ws lyophilized, pulverized, nd.8. g of dry fecl powder ws oiled in 5 ml of 9% ethnol for min. During oiling, % ethnol ws dded, s needed, to mintin pproximte pre-oil volumes. After centrifugtion (g, min), the superntnt ws recovered, nd the pellet ws resuspended in 5 ml of 9% ethnol, vortexed for s nd re-centrifuged (g, 5 min). The first nd second superntnts were comined, dried under ir nd reconstituted in ml methnol. Methnol extrcts were vortexed riefly nd plced in sonictor for 5 min to free prticles dhering to the glss tue. Ech extrct ws diluted : in steroid dilution uffer (. M NH PO,. M N HPO,.5 M NCl, ph 7.) nd ws stored in polypropylene tues t C until enzyme immunossy (EIA) nlyses... Estrone sulfte EIA A single ntiody estrone sulfte (ES) EIA ws used to quntify estrogen metolites in ll fecl extrcts (Stenfeldt et l., 99). This ssy cross-rected with rod rnge of estrogen metolites previously identified in domestic ct feces y high-performnce liquid chromtogrphy (Brown et l., 99). The ssy relied on polyclonl ntiody (R58; :; supplied y C.J. Munro, University of Cliforni, Dvis, CA) produced ginst estrone--glucuronide in coting uffer (.5 M N CO,.5 M NHCO, ph 9.6) dded to 96-well, flt-ottomed microtiter pltes (Nunc-Immuno, Fisher Scientific Inc., Pittsurgh, PA) nd incuted overnight t C. Pltes were wshed (.5% Tween in.5 M NCl solution) to remove undsored ntiody, nd.5 ml steroid ssy uffer (. M NH PO,. M N HPO,.5 M NCl,. g/l BSA, ph 7.) ws dded to ech well nd mintined t room temperture for 5 h. Next,.5 ml of diluted smple (rnge, : to :) or E SO stndrd (rnge,.95 pg; Sigm Aldrich Chemicl Co., St. Louis, MO) ws dded to wells in duplicte immeditely followed y.5 ml estrone sulfte horserdish peroxidse (:,; supplied y C.J. Munro).

4 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 Following -h incution t room temperture, pltes were wshed nd. ml sustrte (. M ABTS,.5 M H O in.5 M citric cid solution) ws dded to ech well. Opticl densities (ODs) were red using microplte reder (MRX, Dynex Technologies, Chntilly, VA) t 5 nm when pg stndrd wells reched n OD of.9. Seril dilutions of ct feces yielded displcement curve tht ws prllel to the stndrd curve (R =.99). Recovery of known mounts of E SO stndrds dded to pool of fecl extrcts (:) ws 7. ±.% (y =.8x.; R =.99). Intr-ssy vrition ws <%, nd inter-ssy vrition ws.% nd.% t % nd 7% inding, respectively (n = pltes)..5. Pregnne EIA A single ntiody progesterone EIA ws used to quntify progestgen metolites in every other fecl smple (Schwrzenerger et l., 99; Grhm et l., ). The EIA relied upon monoclonl ntiody (CL5; :,; supplied y C.J. Munro) tht cross-rected with rod rnge of progestgen metolites previously identified in domestic ct feces y high-performnce liquid chromtogrphy (Brown et l., 99). Procedures nd ssy regents were the sme s descried ove for the ES ssy unless otherwise noted. After overnight ntiody incution, pltes were wshed, nd.5 ml of diluted smple (rnge, : to :) or progesterone stndrd (rnge,.78 pg; Sigm Aldrich) ws dded to wells in duplicte immeditely followed y.5 ml enzyme conjugte (progesterone-cmo horserdish peroxidse; :,; supplied y C.J. Munro). Following -h incution, pltes were wshed,. ml sustrte ws dded to ech well, nd OD ws red. Seril dilutions of ct feces yielded displcement curve tht ws prllel to the stndrd curve (R =.99). Recovery of known mounts of progesterone stndrd dded to pool of fecl extrcts (:) ws 65.9 ±.9% (y =.x 5.; R =.99). Intr-ssy vrition ws <%, nd inter-ssy vrition ws.8% nd 6.8% t % nd 7% inding, respectively (n = 68 pltes)..6. Sttisticl nlyses For ech individul, seline fecl estrogen concentrtions were determined using n itertive process wherey ll vlues exceeding the men plus stndrd devitions (SDs) were deleted from the dt set. The verge ws then reclculted, nd the elimintion process ws repeted until no vlues exceeded the men plus SD(Brown et l., 99; Pelicn et l., 5). The finl verge generted using this process ws considered the seline men for tht niml, nd ll vlues removed from the dt set during the itertive process were clssified s elevted. Durtion of folliculr phse ws defined s the numer of consecutive dys during which estrogens were elevted (minimum dys), nd the highest fecl estrogen vlue within n rry of elevtions ws the pek for tht folliculr phse. Estrous cycle length ws clculted s the numer of dys etween fecl estrogen peks with no susequent elevtion in fecl progestgens. Bseline progestgen concentrtions were determined using similr itertive process, except the men plus.5 SD ws used. Vlues greter thn twice the progestgen seline were considered elevted for tht individul. A metric of.5 SD ws chosen for progestgens ecuse SD ws too sensitive to dequtely seprte seline concentrtions from elevtions. A lutel phse ws defined when progestgen levels rose ove seline nd remined elevted for t lest consecutive weeks. Lutel phse length ws the totl numer of dys where progestgens remined ove seline. Following tretment, return to folliculr ctivity ws clculted s the numer of dys from progestin removl until the first dy fecl estrogens rose ove seline for tht individul. Dt from Trils nd were not different (P >.5) nd, thus, were comined for further nlyses to compre: () mg/kg ALT dily (n = 5 cts, control); (). mg/kg (n = 5, LOW); ().88 mg/kg (n = 6, MID); nd ().5 mg/kg (n = 6, HIGH). Differences were evluted for the 6 dys efore, 8 dys during nd 6 dys fter tretment. Men estrous cycle trits (durtion of folliculr phse, men estrogens/folliculr phse, pek estrogens/folliculr phse, estrous cycle length, seline nd men estrogens, seline nd men progestgens) were clculted for ech individul nd then the mens verged within ech tretment. To normlize the itertive process, only the 8 dys efore, during nd fter tretment were used when determining seline fecl steroid concentrtions. Vlues within tretment cross time were evluted using mixed model repeted mesures ANOVA followed y lest significnt difference (LSD) men comprisons. Dt mong tretments within single time intervl were nlyzed using mixed model one-wy ANOVA followed y LSD men comprisons. When necessry, vlues were corrected for non-norml distriution efore ANOVA using log trnsformtions (Sokl nd Rohlf, 99). Differences in the rnge of return to folliculr ctivity mong tretments were compred using F-test for vrince in Excel (Microsoft Corportion, Redmond, WA). All other nlyses were performed using SAS 9.. (SAS Institute Inc., Cry, NC). Dt re presented s men ± SEM.. Results.. Estrous cycle chrcteristics efore tretment For the 6 dys efore tretment onset, the durtion of the folliculr phse verged 5 dys (rnge, 7 dys) with wide vrition (rnge, 7 dys) in estrous cycle length (i.e., intervl etween consecutive estrogen peks; Tle ). In compring metrics mong groups prior to tretment, seline estrogen metolite concentrtions were higher in LOW (P <.5) thn in HIGH cts, nd folliculr phse durtion ws longer (P <.5) in LOW nd HIGH cts thn in controls. All other metrics, including frequency of the folliculr phse, estrous cycle length, men estrogens/folliculr phse, pek estrogens/folliculr phse nd seline progestgens, were similr (P >.5) mong groups. During this pre-tretment monitoring phse, two of (5.%) cts ovulted spontneously. Lutel phse length, pek progestgen concentrtion nd men progestgen concentrtion/lutel phse could not e chrcterized ecuse progestgens were lredy elevted on the first dy of smple collection... Influence of ltrenogest dosge on estrous cycle chrcteristics Control cts rndomly cycled t lest twice during the experimentl intervl (sed on temporl rise nd fll in fecl estrogens; Fig. A). In contrst, no femle receiving orl progestin (t ny dosge) initited folliculr ctivity (Fig. B D). Consequently, the numer of estrogen peks did not chnge (P >.5) during or Tle Reproductive trits (efore orl progestin dministrtion) in cts ssessed y longitudinl fecl steroid metolite nlyses (n = femles). Durtion of folliculr phse (dys) 5. ±. Estrous cycle length (dys).6 ±.8 Numer of folliculr phses/6 dys. ±. Bseline estrogen concentrtion (ng/g feces) 9.8 ±.5 Pek estrogen concentrtion (ng/g feces).8 ± 6. Men estrogen concentrtion/folliculr phse (ng/g feces).5 ± 6.5 Bseline progestgen concentrtion (lg/g feces).8 ±. Vlues re mens ± SEM.

5 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 fter tretment in controls, wheres fewer (P <.5) peks were oserved during orl progestin tretment t ll ssessed dosges (Fig. ). Cts treted with the HIGH dosge produced fewer (P <.5) estrogen peks efore (.5 ±. peks/6 dys) compred to fter (.7 ±. peks/6 dys) tretment. Bseline estrogens were elevted (P <.5) in HIGH cts fter tretment (95.7 ± 5. ng/g dry feces) compred to efore tretment (6. ± 6.9 ng/g; Fig. A). Bseline progestgens were lso elevted (P <.5) in HIGH cts fter tretment (. ±. lg/g dry feces) compred to efore tretment (.6 ±. lg/g; Fig. B). In contrst, seline estrogen nd progestgen concentrtions were similr (P >.5) cross time in LOW nd MID cts. An increse (P <.5) in seline estrogens ws oserved in control cts during plceo tretment (Fig. A). Femles demonstrting folliculr ctivity efore tretment, ut with seline estrogens t the onset of exogenous progestin, remined t seline (see Fig. A for representtive profile). Cts exhiiting folliculr ctivity on Dy of progestin tretment (n = 6) consistently returned to seline within 6 dys nd remined inhiited for the durtion of tretment (Fig. B for representtive profile). In the six femles displying folliculr ctivity t tretment initition, durtion of tht folliculr phse (7.7 ±. dys), men fecl estrogens (5. ±.9 ng/g feces) nd pek fecl estrogens (95. ± 7. ng/g feces) were similr (P >.5) to estrous cycle chrcteristics preceding tretment. No femles exhiited lutel ctivity on Dy of progestin tretment. After tretment, ll cts returned to norml folliculr ctivity in the LOW (8. ±.8 dys post-alt withdrwl), MID (.5 ±.9 dys) nd HIGH (6. ±. dys) groups (Fig. 5). However, more synchronized (P <.5) return to folliculr ctivity ws oserved in A C 9 9 B D Numer of fecl estrogen peks MID (rnge, 6 dys) thn in HIGH (rnge, 9 5 dys) cts, with intermedite vrition (P >.5) in the LOW tretment group (rnge, dys). Two of (5.%) cts ovulted spontneously fter tretment.. Discussion Results demonstrted the efficcy of orl progestin for shortterm, reversile inhiition of ovrin ctivity in the domestic ct with no oserved side effects. Consistent with previous studies 9 9 Before During After Control LOW MID HIGH Fig.. Influence of orl progestin dosge on numer of fecl estrogen peks per femle (men ± SEM) efore (solid), during (open) nd fter (htched) progestin tretment. Within tretment, mens with different superscripts differ (P <.5). c Dy from tretment initition Dy from tretment initition Fig.. Influence of orl progestin on longitudinl fecl estrogens. Queens were ssigned to (A) mg/kg ltrenogest (control), (B). mg/kg (LOW), (C).88 mg/kg (MID) or (D).5 mg/kg (HIGH). Ech lck r indictes the 8-dy tretment period. Individul nimls within ech tretment re represented y different line mrkers.

6 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 A 5 5 Before During After, A Estrogens Progestgens Progestgens (µg/g feces) 5 B Progestgens (µg/g feces) 5 Control LOW MID HIGH Fig.. Influence of orl progestin on (A) seline fecl estrogens nd (B) seline fecl progestgens efore (solid), during (open) nd fter (htched) tretment (men ± SEM). Within tretment, mens with different superscripts differ (P <.5). B Dy from tretment initition Fig.. Representtive fecl steroid profiles efore, during nd fter orl progestin tretment. Profiles include femle: (A) exhiiting seline fecl estrogens t the time of progestin tretment initition; nd (B) experiencing folliculr ctivity t the time of progestin tretment initition. Asterisks indicte pek estrogens nd the solid r represents the 8-dy ltrenogest tretment intervl Progestgens (µg/g feces) using progestin implnts for ovrin suppression (Pelicn et l., 5, 8), orl dministrtion of this steroid did not lter folliculr ctivity lredy in progress. Apprently, once threshold in folliculogenesis ws reched, even the highest dosge of orl progestin tested here ws incple of cutely perturing finl folliculr dvncement in the ct (lthough the viility of this structure nd its enclosed oocyte remins to e studied). We lso demonstrted tht orl progestin exerted dose-dependent effect tht ws quntifile vi fecl hormone metolite monitoring. Present results ffirmed the dvntge of using fecl steroid hormones for detecting even sutle differences mong tretments. Most importntly, our oservtions reveled tht, lthough the ovry ws highly sensitive to progestin chllenge, re-initition of folliculr development ws resilient nd occurred consistently even t the highest progestin dosge tested. Given the sustntil ody of literture on progestin therpy for controlling the ovrin cycle in diverse species, it ws not surprising tht orl ltrenogest inhiited folliculr ctivity in our test species. However, it ws cler tht oth progestin dosge nd stge of ovrin ctivity t tretment onset were mjor regultors of tretment success. Pelicn et l. (5) chrcterized the effects of nother progestin (levonorgestrel; dministered vi n implnt) in the ct nd lso oserved rpid cesstion of ovrin steroidogenesis, ut gin no impct on the course of n estrogen surge tht ws initited efore tretment onset. This hs een lso oserved in the mre, where ltrenogest is unle to relily suppress ovultion if given during estrus (Lofstedt nd Ptel, 989). Thus, the ct shows consistent ovrin sensitivity (regrdless of progestin source) t preventing initil recruitment of smll ntrl follicles, ut n inility to override selection nd dominnce of mture follicles. There is remrkly little informtion ville out the interction etween progesterone nd gondotropins nd susequent effects on folliculogenesis in the ct. In other mmmls, folliculr recruitment is primrily under the influence of high FSH nd low LH, wheres folliculr selection is regulted y low FSH nd incresing LH (Roche, 996; Monniux et l., 997; McGee nd Hsueh, ). While endogenous progesterone ctions re more dominnt during the lutel phse nd pregnncy, progesterone receptors hve een loclized to the follicle, nd exogenous progestins regulte grnulos cell function in multiple wys (Drummond, 6). It is likely tht, in this cse, the exogenous progestin ws simultneously cting t the pituitry to influence FSH nd LH relese nd t the ovry to lter steroidogenesis nd cell remodeling in grnulos cells. Potentil ctions on ctivin, inhiin nd other regultory fctors must lso e considered nd wrrnt future investigtion. Although ovrin suppression with orl progestin ws predicted to e dose-dependent, folliculr ctivity ws olished even t the lowest dosge, suggesting tht the minimum effective ltrenogest dosge for ovrin suppression ws t or elow. mg/kg. Other studies hve reveled tht dosge is not ody mss-dependent s lower dose is required in the horse (. mg/kg) thn in the dog (.88 mg/kg; Lofstedt nd Ptel, 989; Root Kustritz, ). Interestingly, n evlution of individul fecl hormone profiles in the ct receiving the lowest dosge (dt not shown) reveled modest elevtions in estrogens pproching the delinetion etween seline nd elevted vlues.

7 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 A Animl numer B Animl numer C Animl numer Cycling Tretment Inhiition Cycle Return Therefore, the. mg/kg tretment my e close to the minimum dosge necessry to suppress folliculr ctivity in this species. The highest dosge ssessed (while effective) incresed post-withdrwl seline estrogen nd progestgen concentrtions. A similr phenomenon for elevted seline estrogen hs een oserved in cts fter tretment with the progestin implnt levonorgestrel (Pelicn et l., 5). The highest dosge lso led to n increse in the numer of estrogen peks following tretment. These ltertions t the highest dosge could e due to chnges in the responsiveness of the rin to endogenous gondotropins tht, in turn, could increse sl estrdiol nd Dys from tretment initition Fig. 5. Comprison of individul inhiition durtion following orl progestin tretment. Cts received (A). mg/kg ltrenogest (LOW), (B).88 mg/kg (MID) or (C).5 mg/kg (HIGH). Brs represent dys of inhiition during the tretment period (htched), inhiition following the removl of orl progestin (solid), nd folliculr ctivity (open). Numers within rs represent intervl (dys) from progestin removl to first elevtion in fecl estrogens progesterone production nd lso stimulte utocrine regultion of steroidogenesis through negtive feedck on the hypothlmic pituitry ovrin xis. Although the rmifictions for elevted gondl steroids re not definitively known, it is possile tht persistent elevtions could influence the peri-implnttion period in femles undergoing ssisted reproduction. Thus,.88 mg/kg ltrenogest ppers to e optiml in the ct, llowing no rekthrough ovrin ctivity while producing normtive seline estrogen nd progestgen concentrtions nd resumptive ovrin folliculogenesis. The sme dosge is used to support pregnncy in the dog (Root Kustritz, ). This study demonstrted two interesting ssocitions etween orl progestin dosge nd return to folliculr ctivity post-drug withdrwl. First, there ws positive reltionship etween dosge nd men durtion of suppression where the intervl from end of tretment to onset of the folliculr phse incresed with dosge. A similr reltionship hs een oserved in pigs receiving orl ltrenogest to synchronize estrus (Kreling et l., 98). Although phrmcokinetics of ltrenogest hve not een studied in the ct, this finding proly cn e explined y dose-dependent differences in drug persistence in circultion. Second, we hypothesized tht n orl progestin would lwys provide more consistent return to ovrin cyclicity compred to previous efforts tht relied on n implnt formultion (Pelicn et l., 5). Insted, the present results indicted tht dosge plyed significnt role in determining the vriility of estrous cycle return. For exmple, the high dosge yielded fr greter vriility in return to cyclicity (9 5 dys) thn the mid-rnge counterprt ( 6 dys). Synchroniztion with the orl progestin proved superior to levonorgestrel implnts in the ct, prticulrly when compring return to folliculr ctivity in the mid-rnge dosge ( 6 dys) to six levonorgestrel implnts ( 79 dys; Pelicn et l., 5). Our findings hve prcticl implictions ecuse of the comprtively high incidence of spontneous ovultion in the domestic ct nd certin popultions of wild felids (Brown, 6) tht, in turn, contriutes to ovultion induction inefficiency (Pelicn et l., 6). Interestingly, the prticulr group of cts studied here hd n unusully low prevlence of spontneous ovultion (5.%) compred to lortory-housed queens in erlier studies where 5 87% of cts ovulte without mting stimulus (Lwler et l., 99; Gudermuth et l., 997; Pelicn et l., 5). An incresed incidence of this phenomenon hs een ttriuted, in prt, to stressful events, interctions with cge mtes nd visul nd olfctory cues from djcent mles (Concnnon, 99; Gudermuth et l., 997). Cts in the current study did receive olfctory nd uditory cues from mles, ut were housed individully with no visul mle contct. An dditionl hypothesis is tht spontneous ovultion is more prevlent in older femles (Lwler et l., 99). Although not the focus of our investigtion nd ecuse our study popultion ws too smll to evlute this fctor, we nonetheless oserved tht the two spontneous ovultors were in the oldest (5 yers) ge clss (dt not shown). In conclusion, short-term tretment with three dosges of the orl progestin ltrenogest induced rpid nd reversile inhiition of folliculr ctivity in the ct. These findings provide the necessry foundtion for ongoing studies evluting if n ovry primed with orl progestin: () expresses ltered sensitivity to exogenous gondotropins; nd () produces more norml endocrine milieu tht mitigtes disruption of ovum trnsport, fertiliztion nd implnttion. Finlly, it will lso e necessry to ensure tht even this short-term progestin tretment does not contriute to other helth risks. This is especilly importnt in tht the commonly used steroid melengestrol cette (MGA) hs een ssocited with n incresed incidence of endometril hyperplsi in felids (Munson et l., ). Although the ltter were ssocited with long-term (>6 yers) dministrtion, it nonetheless is criticl to

8 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () conduct pproprite sfety ssessments with ltrenogest, especilly s most eventul trget popultions of felids re rre if not endngered. Acknowledgments R.A.S. ws supported y Predoctorl Reserch Fellowship from the Smithsonin s Ntionl Zoologicl Prk nd Mtching Tuition Scholrship from the University of Mrylnd Grdute School. Reserch ws supported y Ntionl Institutes of Helth SERCA grnt to K.M.P. (KO--RR7-). We re grteful to Michele Sommers, Cthi Morrison nd Stcey Wise for providing excellent niml cre. We lso thnk Ryn Berger, Dvid Kersey, Kte McKinnon, Bernrdo Mes nd Nicole Presley for technicl ssistnce. Dry ct food ws donted y the Purin Nestlé PetCre Compny. References Brone, M.A., Wildt, D.E., Byers, A.P., Roelke, M.E., Glss, C.M., Howrd, J.G., 99. Gondotrophin dose nd timing of nesthesi for lproscopic rtificil insemintion in the pum (Felis concolor). J. Reprod. Fertil., 8. Buer, R.A., Ottinger, M.A., Pelicn, K.M., Wildt, D.E., Howrd, J.G.,. Chllenges to developing n ovultion induction protocol in the fishing ct (Prionilurus viverrinus), felid with high incidence of spontneous ovultion. In: Proc. 5th Int. Symp. Cnine Feline Reprod., So Pulo, Brzil, pp Brown, J.L., Wsser, S.K., Wildt, D.E., Grhm, L.H., 99. Comprtive spects of steroid hormone metolism nd ovrin ctivity in felids, mesured noninvsively in feces. Biol. Reprod. 5, Brown, J.L., Wildt, D.E., Wielenowski, N., Goodrowe, K.L., Grhm, L.H., Wells, S., Howrd, J.G., 996. Reproductive ctivity in cptive femle cheeths (Acinonyx jutus) ssessed y fecl steroids. J. Reprod. Fertil. 6, 7 6. Brown, J.L., Grhm, L.H., Wielenowski, N., Swnson, W.F., Wildt, D.E., Howrd, J.G.,. Understnding the sic reproductive iology of wild felids y monitoring of fecl steroids. J. Reprod. Fertil. Suppl. 57, 7 8. Brown, J.L., Grhm, L.H., Wu, J.M., Collins, D., Swnson, W.F.,. Reproductive endocrine responses to photoperiod nd exogenous gondotropins in the Plls ct (Otocolous mnul). Zoo Biol., 7 6. Brown, J.L., 6. Comprtive endocrinology of domestic nd nondomestic felids. Theriogenology 66, 5 6. Concnnon, P., 99. Reproduction in the dog nd ct. In: Cupps, P.T. (Ed.), Reproduction in Domestic Animls. Acdemic Press, Inc., New York, pp Deliginnis, C., Vlsi, I., Rekks, C.A., Gouls, P., Theodosidou, E., Lins, T., Amiridis, G.S., 5. Synchroniztion of ovultion nd fixed time intruterine insemintion in ewes. Reprod. Domest. Anim., 6. Donoghue, A., Johnston, L.A., Armstrong, D.L., Simmons, L.G., Wildt, D.E., 99. Birth of Sierin tiger cu (Pnther tigris ltic) following lproscopic intruterine insemintion. J. Zoo Wildl. Med., Drummond, A.E., 6. The role of steroids in folliculr growth. Reprod. Biol. Endocrinol., 6. Grhm, L.H., Swnson, W.F., Brown, J.L.,. Chorionic gondotropin dministrtion in domestic cts cuses n norml endocrine environment tht disrupts oviductl emryo trnsport. Theriogenology 5, 7. Grhm, L.H., Schwrtzenerger, F., Mostl, E., Glm, W., Svge, A.,. A verstile enzyme immunossy for the determintion of progestogens in the feces nd serum. Zoo Biol., 7 6. Grhm, L.H., Byers, A.P., Armstrong, D.L., Loskutoff, N.M., Swnson, W.F., Wildt, D.E., Brown, J.L., 6. Nturl nd gondotropin-induced ovrin ctivity in tigers (Pnther tigris) ssessed y fecl steroid nlyses. Gen. Comp. Endocrinol. 7, 6 7. Gudermuth, D.F., Newton, L., Dels, P., Concnnon, P., 997. Incidence of spontneous ovultion in young, group-housed cts sed on serum nd fecl concentrtions of progesterone. J. Reprod. Fertil. Suppl. 5, Howrd, J.G., Donoghue, A.M., Brone, M.A., Goodrowe, K.L., Blumer, E.S., Snodgrss, K., Strnes, D., Tucker, M., Bush, M., Wildt, D.E., 99. Successful induction of ovrin ctivity nd lproscopic intruterine rtificil insemintion in the cheeth (Acinonyx jutus). J. Zoo Wildl. Med., 88. Howrd, J.G., Byers, A.P., Brown, J.L., Schwrtz, R.J., Evns, M.Z., Brrett, S.J., Wildt, D.E., 996. Successful ovultion induction nd lproscopic intruterine rtificil insemintion in the clouded leoprd (Neofelis neulos). Zoo Biol. 5, Howrd, J.G., Roth, T.L., Byers, A.P., Swnson, W.F., Wildt, D.E., 997. Sensitivity to exogenous gondotropins for ovultion induction nd lproscopic rtificil insemintion in the cheeth nd clouded leoprd. Biol. Reprod. 56, Howrd, J.G., 999. Assisted reproductive techniques in nondomestic crnivores. In: Fowler, M.E., Miller, R.E. (Eds.), Zoo nd Wild Animl Medicine: Current Therpy IV. W.B. Sunders Co., Phildelphi, pp Howrd, J.G., Wildt, D.E., 9. Approches nd efficcy of rtificil insemintion in felids nd mustelids. Theriogenology 7, 8. Kreling, R.R., Dziuk, P.J., Pursel, V.G., Rmpcek, G.B., Weel, S.K., 98. Synchroniztion of estrus in swine with llyl trenolone (RU-67). J. Anim. Sci. 5, Lwler, D.F., Johnston, S.D., Hegstd, R.L., Keltner, D.G., Owens, S.F., 99. Ovultion without cervicl stimultion in domestic cts. J. Reprod. Fertil. Suppl. 7, Lofstedt, R.M., 988. Control of the estrous cycle in the mre. Vet. Clin. North Am. Equine Prct., Lofstedt, R.M., Ptel, J.H., 989. Evlution of the ility of ltrenogest to control the equine estrous cycle. J. Am. Vet. Med. Assoc. 9, 6 6. McGee, E.A., Hsueh, A.J.,. Initil nd cyclic recruitment of ovrin follicles. Endocr. Rev.,. Monfort, S.L., Asher, G.W., Wildt, D.E., Wood, T.C., Schiewe, M.C., Willimson, L.R., Bush, M., Rll, W.F., 99. Successful intruterine insemintion of Eld s deer (Cervus eldi thmin) with frozen thwed spermtozo. J. Reprod. Fertil. 99, Monniux, D., Huet, C., Besnrd, N., Clement, F., Bosc, M., Pisselet, C., Monget, P., Mrin, J.C., 997. Folliculr growth nd ovrin dynmics in mmmls. J. Reprod. Fertil. Suppl. 5,. Moreir, N., Monteiro-Filho, E.L., Mores, W., Swnson, W.F., Grhm, L.H., Psquli, O.L., Gomes, M.L., Moris, R.N., Wildt, D.E., Brown, J.L.,. Reproductive steroid hormones nd ovrin ctivity in felids of the Leoprdus genus. Zoo Biol., 6. Morrow, C.J., Wolfe, B.A., Roth, T.L., Wildt, D.E., Bush, M., Blumer, E.S., Atkinson, M.W., Monfort, S.L.,. Compring ovultion synchroniztion protocols for rtificil insemintion in the scimitr-horned oryx (Oryx dmmh). Anim. Reprod. Sci. 59, Munson, L., Grdner, A., Mson, R.J., Chssy, L.M., Sel, U.S.,. Endometril hyperplsi nd minerliztion in zoo felids treted with melengestrol cette contrceptives. Vet. Pthol. 9, 9 7. Munson, L., 6. Contrception in felids. Theriogenology 66 (), 6. Pelicn, K.M., Brown, J.L., Wildt, D.E., Ottinger, M.A., Howrd, J.G., 5. Short term suppression of folliculr recruitment nd spontneous ovultion in the ct using levonorgestrel versus GnRH ntgonist. Gen. Comp. Endocrinol.,. Pelicn, K.M., Wildt, D.E., Pukzhenthi, B., Howrd, J.G., 6. Ovrin control for ssisted reproduction in the domestic ct nd wild felids. Theriogenology 66 (), 7 8. Pelicn, K.M., Wildt, D.E., Ottinger, M.A., Howrd, J.G., 8. Priming with progestin, ut not GnRH ntgonist, induces consistent endocrine response to exogenous gondotropins in induced nd spontneously ovulting cts. Domest. Anim. Endocrinol., Roeck, T.R., Steinmn, K.J., Gerhrt, S., Reidrson, T.R., McBin, J.F., Monfort, S.L.,. Reproductive physiology nd development of rtificil insemintion technology in killer whles (Orcinus orc). Biol. Reprod. 7, Roeck, T.R., Steinmn, K.J., Yoshiok, M., Jensen, E., O Brien, J.K., Ktsumt, E., Gili, C., McBin, J.F., Sweeney, J., Monfort, S.L., 5. Estrous cycle chrcteristion nd rtificil insemintion using frozen thwed spermtozo in the ottlenose dolphin (Tursiops trunctus). Reproduction 9, Roche, J.F., 996. Control nd regultion of folliculogenesis symposium in perspective. Rev. Reprod., 9 7. Romtowski, J., 989. Use of megestrol cette in cts. J. Am. Vet. Med. Assoc. 9 (5), 7. Root Kustritz, M.V.,. Use of supplementl progesterone in mngement of cnine pregnncy. In: Concnnon, P., Englnd, G., Verstegen, J.P., Linde-Forserg, C. (Eds.), Recent Advnces in Smll Animl Reproduction. Interntionl Veterinry Informtion Service, Ithc ( document # A.). Roth, T.L., Armstrong, D.L., Brrie, M.T., Wildt, D.E., 997. Sesonl effects on ovrin responsiveness to exogenous gondotrophins nd successful rtificil insemintion in the snow leoprd (Unci unci). Reprod. Fertil. Dev. 9, Roth, T.L., Wolfe, B.A., Long, J.A., Howrd, J.G., Wildt, D.E., 997. Effects of equine chorionic gondotropin, humn chorionic gondotropin, nd lproscopic rtificil insemintion on emryo, endocrine, nd lutel chrcteristics in the domestic ct. Biol. Reprod. 57, Schwrzenerger, F., Mostl, E., Bmerg, E., Pmmer, J., Schmehlik, O., 99. Concentrtions of progestgens nd oestrogens in the feces of pregnnt Lipizzn, trotter nd thoroughred mres. J. Reprod. Fertil. Suppl., Sokl, R.R., Rohlf, F.J., 99. Biometry: The Principles nd Prctices of Sttistics in Biologicl Reserch, third ed. W.H. Freemn nd Compny, New York. Stenfeldt, G.H., Dels, P.F., Munro, C.J., Kindhl, H., Hughes, J.P., Lsley, B., 99. An oestrogen conjugte enzyme immunossy for monitoring pregnncy in the mre: limittions of the ssy etween dys nd 7 of gesttion. J. Reprod. Fertil. Suppl., 7. Swnson, W.F., Grhm, K., Horohov, D.W., Thompson, D.L., Godke, R.A., 996. Ancillry follicle nd secondry corpor lute formtion following exogenous gondotropin tretment in the domestic ct nd effect of pssive trnsfer of gondotropin-neutrlizing ntiser. Theriogenology 5, Swnson, W.F., Howrd, J.G., Roth, T.L., Brown, J.L., Alvrdo, T., Burton, M., Strnes, D., Wildt, D.E., 996. Responsiveness of ovries to exogenous gondotrophins nd lproscopic rtificil insemintion with frozen thwed spermtozo in ocelots (Felis prdlis). J. Reprod. Fertil. 6, Swnson, W.F., Wolfe, B.A., Brown, J.L., Mrtin-Jimenez, T., Riviere, J.E., Roth, T.L., Wildt, D.E., 997. Phrmcokinetics nd ovrin-stimultory effects of equine nd humn chorionic gondotropins dministered singly nd in comintion in the domestic ct. Biol. Reprod. 57, 95.

9 6 R.A. Stewrt et l. / Generl nd Comprtive Endocrinology 66 () 9 6 Swnson, W.F.,. Reserch in nondomestic species: experiences in reproductive physiology reserch for conservtion of endngered felids. ILAR J., 7 6. Swnson, W.F., Brown, J.L.,. Interntionl trining progrms in reproductive sciences for conservtion of Ltin Americn felids. Anim. Reprod. Sci. 8 8,. Wildt, D.E., Seger, S.W., Chkrorty, P.K., 98. Effect of copultory stimuli on incidence of ovultion nd on serum luteinizing hormone in the ct. Endocrinology 7, 7. Wildt, D.E., Monfort, S., Donoghue, A.M., Johnston, L.A., Howrd, J.G., 99. Emryogenesis in conservtion iology or, how to mke n endngered species emryo. Theriogenology 7, 6 8. Wildt, D.E., Roth, T.L., 997. Assisted reproduction for mnging nd conserving thretened felids. Int. Zoo Yer. 5, 6 7. Wildt, D.E., Swnson, W.F., Brown, J.L., Sliw, A., Vrgs, A.,. Felids ex situ: mnged progrms, reserch nd species recovery. In: Mcdonld, D., Loveridge, A. (Eds.), Biology nd Conservtion of Wild Felids. Oxford University Press, Oxford. Wood, C.M., Kornegy, E.T., Shipley, C.F., 99. Efficcy of ltrenogest in synchronizing estrus in two swine reeding progrms nd effects on susequent reproductive performnce of sows. J. Anim. Sci. 7, 57 6.