The effect of dose and route of oestradiol benzoate administration on plasma concentrations of oestradiol and FSH in long-term ovariectomised heifers

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1 Ž. Animl Reproduction Science The effect of dose nd route of oestrdiol enzote dministrtion on plsm concentrtions of oestrdiol nd FSH in long-term ovriectomised heifers M. O Rourke,, M.G. Diskin,), J.M. Sreenn, J.F. Roche Tegsc Agriculture nd Food DeÕelopment Authority, Athenry Reserch Centre, Athenry, Co. Glwy, Irelnd Fculty of Veterinry Medicine, UniÕersity College Dulin, Bllsridge, Dulin 4, Irelnd Received 5 June 1999; received in revised form 3 Novemer 1999; ccepted Decemer 1999 Astrct Oestrdiol Ž E. suppresses FSH nd ffects follicle wve dynmics in cttle. However, neither the optimum dose of ODB required to suppress FSH nor the effect of route of ODB dministrtion on lood concentrtions of E re known; hence, the im of this experiment ws to nswer these questions. Ovriectomised heifers received Progesterone Relesing Intrvginl Device Ž PRID. for 7 dys, nd 4 dys lter heifers received one of eight ODB tretments t second PRID insertion s follows; Ž mg Ž Control; ns3.ž., 0.5 mg Ž ns4.ž., mg Ž ns4.ž., 4.5 mg Ž ns6., Ž mg Ž ns4., Ž mg Ž ns4., Ž mg Ž ns4., nd Ž mg Ž ns5.. For tretments 6 inclusive, ODB ws dministered intrmusculrly in oil, while for tretments 7 nd 8, the ODB in powder form ws dministered topiclly in the vgin y geltine cpsule ttched to the PRID. Blood smples were collected every 6 h for the first 48 h, every 1 h for the next 48 h, nd twice dily for further 6 dys. The intervl from ODB dministrtion to pek E concentrtion ws similr Ž P) for tretments 6 where ODB ws dministered intrmusculrly Ž men 13.4" 1.4 h., nd ws longer Ž P for the intrvginl cpsule tretments Ž men 5.5".84 h.. Plsm concentrtions of E incresed with incresing intrmusculr dose of ODB injected, Žplsm E Ž. Ž. sy0.37q dose y0.74 dose, R s0.75; P Pek plsm concentrtions of E following the 5- nd 10-mg cpsules were similr to ech other nd to those following the 0.5-mg injection Ž P) 0.05., ut were lower thn concentrtions otined following injection of mg Ž P Across ll tretments, oth ) Corresponding uthor. Tel.: q ; Fx: q Ž. E-mil ddress: mdiskin@thenry.tegsc.ie M.G. Diskin r00r$ - see front mtter q 000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 M. O Rourke et l.raniml Reproduction Science the mximum percentge decline in FSH nd the intervl to FSH ndir were relted to the pek plsm concentrtions of E Žmximum % decline in FSHs11.17q1.564 Ž pek E. y0.009 Ž pek E., R s0.75; P , Žhours to FSH ndirs10.68q1.486ž hours to pek E. y 0.08Ž hours to pek E., R s0.; P Concentrtions of FSH incresed s E declined from its pek vlue, irrespective of mximum vlue chieved. It ws concluded tht the intrmusculr dministrtion of ODB in oil to ovriectomised heifers given PRID results in higher plsm concentrtions of E nd cuses greter reduction in FSH thn dministrtion topiclly y intrvginl geltine cpsule. E trnsiently suppresses FSH in ovriectomised heifers, nd the mgnitude of the suppression is dose-dependent; however FSH concentrtions egin to increse 1 dys fter ODB dministrtion while concentrtions of E were declining ut still high. q 000 Elsevier Science B.V. All rights reserved. Keywords: Cttle; Endocrinology; Ovriectomy; Oestrdiol; FSH 1. Introduction Follicle growth in cttle is highly regulted process under the control of FSH, LH nd locl intr-folliculr growth fctors. There re recurrent wves of folliculr growth throughout the oestrous cycle ŽSvio et l., 1988; Sirois nd Fortune, 1988; Ginther et l., 1989., most of pregnncy Ž Ginther et l., 1996., nd they resume erly in the postprtum period Ž Murphy et l., 1990; Svio et l., 1990; Stgg et l., Ech wve egins with the emergence of cohort of oestrogen ctive follicles of 3 5 mm in dimeter, preceded y n increse in concentrtions of FSH ŽAdms et l., 199; Bo et l., 1993; Sunderlnd et l., 1994; Stgg et l., This is followed y the selection of generlly one dominnt follicle Ž DF., which occurs in ssocition with declining FSH concentrtions Ž Sunderlnd et l., 1994; Mihm et l., Finlly, the fte of the DF is determined y LH pulse frequency, where infrequent pulses Ž 1 every 3 4 h. led to DF tresi while more frequent pulses Ž 1 per hour. leds to ovultion. Thus, ech wve hs n inherent life spn, determined y the intervl from wve emergence to dominnce nd the durtion of dominnce. The durtion of dominnce is dependent on the numer of wves Ž or 3. during the oestrous cycle, nd the influence of nutrition, lcttion nd suckling during the postprtum period Ž Roche et l., on wve dynmics. The fctors tht regulte the recurrent FSH increses re minly of ovrin origin, prticulrly from the DF Ž Ginther et l., with oestrdiol Ž E. one of the key intr-folliculr components involved. The reltionship etween systemic E nd FSH concentrtions however, is uncler nd the reported effects of E on FSH re inconsistent. Bolt et l., Ž reported tht 10 mg of oestrdiol 17 in 5 ml of sterile corn oil dministered intrmusculrly, either lone or in comintion with norgestomet, suppressed plsm FSH in ovriectomised heifers for t lest 64 h. Kesner nd Convey Ž 198. lso reported suppressive effect of 1 mg of E 17 in 3 ml sfflower seed oil injected intrmusculrly on FSH s did Price nd We Ž nd Brnes et l. Ž 1990., where E ws dministered in crystlline form in sucutneous implnt to intct heifers. Wolfe et l., Ž 199. found decrese in FSH when high doses Žsucutneous implnts. of E17 were dministered to ovriectomised heifers ut no effect on FSH following lower doses. However, Schoenemnn et l., Ž found little or no effect of E Ž 1 mg E17 in ml sfflower oil. on FSH in ovriectomised nimls.

3 M. O Rourke et l.raniml Reproduction Science The ility to exogenously regulte FSH concentrtions in cttle is importnt to synchronise follicle wve development in oth cyclic nd noestrous cttle. Although progress hs een mde in recent yers on improving the efficiency of oestrous synchronistion regimens, vriility in oestrous response nd pregnncy rtes fter tretment re still mjor limittions preventing its greter use in cttle. It is necessry to control follicle wve dynmics s well s lutel function in order to hve helthy DF present t the end of tretment Ž Mihm et l., When used together, E nd progesterone cn synchronise follicle wves, with vrile success Ž Bo et l., However, the optimum dose of E necessry to suppress FSH is not cler, neither is the effect of route of dministrtion of E on lood concentrtions of E known. Hence, the im of this experiment ws to determine the effect of dose nd route of E dministrtion t time of progesterone dministrtion on circulting concentrtions of FSH, in order to select optimum dose to use in susequent studies to mnipulte follicle wves. In this experiment, ovriectomised nimls given progesterone were used to prevent confounding effects of endogenous E production.. Mterils nd methods.1. Animls nd tretments Thirty-four long-term ovriectomised crossred eef heifers, 18 4 months of ge nd weighing kg were used. All heifers received Progesterone Relesing Intrvginl Device Ž PRID, Snofi Frnce. for 7 dys in order to simulte lutel phse concentrtions of progesterone efore tretment. Four dys fter removl of the first PRID, new PRID ws inserted for further 7 dys. Animls were rndomly llocted to receive one of eight oestrdiol enzote Ž ODB. tretments t this time; Ž mg Ž Control; ns3., Ž. 0.5 mg Ž ns4., Ž. 3 1mgŽ ns4., Ž. 4.5 mg Ž ns6., Ž. 5 5mg Ž ns4., Ž 6. 10mgŽ ns4. ll dministered y intrmusculr injection in ml of corn oil, Ž. 7 5 mg Ž ns4., nd Ž mg Ž ns5. ODB in powder form dministered y geltine cpsule ttched to the PRID. The heifers were kept indoors for the durtion of the experiment nd fed kg of concentrtes per dy with d liitum ccess to grss silge... Blood smpling nd hormone ssys Blood smples were collected y jugulr venipuncture into 10 ml vcutiner tues contining EDTA K 3. The first smple ws tken just efore tretment dministrtion, nd then t 6-h intervls for the first 48 h fter ODB dministrtion, t 1-h intervls for the second 48 h nd twice dily Ž 0900 nd 1500 h. for further 6 dys, which covered the period up to 3 dys fter PRID removl. Blood smples were plced in iced wter nd then centrifuged immeditely t 1000 = g t 48C for 15 min. Following seprtion, the plsm ws stored t y08c until ssyed for E nd FSH. A previously vlidted rdioimmunossy Ž Prendiville et l., ws used to mesure E. The sensitivity of the E ssy ws 0. pg ml y1 nd the inter- nd

4 4 M. O Rourke et l.raniml Reproduction Science intr-ssy coefficients of vrition were 10.5% Ž n s 9. nd 8.7% Ž n s 4., respectively. The FSH ssy used USDA FSH B1 s the stndrd preprtion Ž Crowe et l., The sensitivity of the ssy ws 1 ng ml y1 nd the inter- nd intr-ssy coefficients of vrition were 14.7% Ž N s 5. nd 13.7% Ž n s 6., respectively..3. Sttisticl nlyses One heifer, receiving 10.0 mg ODB y intrmusculr injection, filed to show ny increse in plsm concentrtions of E until 4 dys following tretment nd her hormonl dt ws excluded from nlyses. The E, nd FSH dt were sujected to repeted mesures nlyses of vrince Ž PROC GLM, SAS, followed y regression nlyses Ž SAS, Becuse of the etween niml vriility, concentrtions of FSH were nlysed s % chnges from pre-tretment concentrtions. 3. Results 3.1. Plsm concentrtions of E All nimls hd similr nd non-detectle concentrtions of E pre-tretment. Pek concentrtions of E following ll injection nd intrvginl cpsule tretments Ž Tle 1. were significntly higher thn pre-tretment concentrtions nd those in untreted control nimls Ž Fig. 1.. The intervl Ž h. from dministrtion of ODB to pek E concentrtion Ž Tle 1. ws similr Ž P) 0.1. in heifers given intrmusculr injections Tle 1 Effect of ODB dministrtion using different doses nd routes of dministrtion on chrcteristics of plsm concentrtions of oestrdiol Ž Men"S.E.. in ovriectomised heifers given progesterone intrvginl device Ž PRID. ODB No. Of Pre-tretment Pek No. of hours to No. of hours No. of hours tretment heifers Concentrtions Concentrtion rech mximum to rech -3 to rech -1 Ž mg. Ž n. of E of E Ž pgrml. concentrtion of E pgrml of E pgrml of E Controls "0.05 Topicl dministrtion y intrõginl cpsule "7.1 d 5.5" " " "6.3 d 7.6" " "17.3 Intrmusculr injection in ml corn oil c 11.9"7.1 c 13.5" " "19.4 c c c " " " "19.4 c c c " " " "15.8 d c d " " " " d 84.6"8. c 18.0" "19.4 d 188.0".4,,c,d Ž. Within column, tretments with different superscripts re significntly different P

5 M. O Rourke et l.raniml Reproduction Science Fig. 1. Effect of different doses Ž 0, 0.5, 1,.5, 5, nd 10 mg. of ODB dissolved in ml of corn oil nd dministered y intrmusculr injection to ovriectomised heifers t the time of PRID insertion on plsm concentrtions of oestrdiol Ž pgrml. Ž ns3 6 heifers per dose.. Ž men 13.4"1.4 h., nd ws longer Ž P in heifers given the 5 nd 10 mg ODB y intrvginl cpsules Ž men 5.5".84 h.. Pek plsm concentrtion of E Fig.. Effect of different doses of ODB dministered either y powdered form in geltine cpsule Ž5- nd 10-mg doses. ttched to PRID nd two doses Ž 0 nd 0.5 mg. dissolved in ml of corn oil nd dministered y intrmusculr injection to ovriectomised heifers t the time of PRID insertion on plsm concentrtions of oestrdiol Ž pgrml. in ovriectomised heifers Ž ns3 4 heifers per dose..

6 6 Tle Effect of ODB dministrtion using different doses nd routes of dministrtion on chrcteristics of FSH concentrtions Ž Men"S.E.. in ovriectomised heifers given PRID ODB No. of Pre-tretment Men concentrtion No. of hours Mximum decline in No. of hours to No. of hours for FSH to tretment heifers concentrtions of FSH t ndir to rech FSH expressed s % of rech first FSH to return to pre-tretment Ž mg. Ž n. of FSH Ž ngrml. Ž ngrml. FSH ndir pre-tretment vlues rise following ndir concentrtions Controls "16.96 Ž. 10.9" Topicl dministrtion y intrõginl cpsule " "11.3 d 4.0"4. 6.6" " " " " " " " "15.13 Intrmusculr injection in ml corn oil " " "4. 4.3" " "19.54 c " " " " " "16.9 c d " "9. 4.0" " " "13.8 c c " " " " " " "14.11 c.3"13.1 cd 34.0"4.9 c 69.5"8.1 cd 104.0" "19.54,,c,d Ž. Within column, tretments with different superscripts re significntly different P M. O Rourke et l.raniml Reproduction Science 59 ( 000 ) 1 1

7 M. O Rourke et l.raniml Reproduction Science incresed with higher dose of ODB injected, Žplsm E sy0.37 q Ž dose. y 0.74 Ž dose., R s 0.75; P ut not when given topiclly y the intrvginl route using the cpsule. The pek plsm concentrtions of E in heifers given 5- nd 10-mg injections were similr to ech other, ut were higher thn in heifers ssigned to ll other tretments Ž P Pek concentrtions of E following use of the 5- nd 10-mg intrvginl cpsules were similr to ech other nd to those following the 0.5 mg injection Ž P)0.05., ut were lower thn concentrtions in heifers given 1,.5, 5, nd 10 mg ODB intrmusculr Ž P Ž Fig... The increse in plsm concentrtions of E in nimls following the 5- nd 10-mg injections were more prolonged nd were slower to return to pre-tretment concentrtions thn in nimls following ll other tretments Ž Tle Plsm concentrtion of FSH The effects of ODB dministrtion on plsm concentrtions of FSH re presented in Tle. There ws significnt decline in FSH concentrtions following ll ODB tretments compred with pre-tretment concentrtions Ž P The mgnitude of the decline in FSH concentrtion ws greter s lood concentrtion of E incresed in treted nimls Ž Figs. 3 nd 4.. The mximum decline in FSH concentrtion expressed s % of pre-tretment concentrtions, ws significntly greter following the 5- nd 10-mg injections thn following ll other ODB tretments. Across ll tretments, oth the mximum percentge decline in FSH nd the intervl Ž. h to rech tht decline were relted to the pek plsm concentrtions of E Ž mximum% decline in FSH s q Ž pek E. y Ž pek E., R s 0.75; P , Žhours to FSH ndir s Fig. 3. Effect of different doses Ž 0, 0.5, 1,.5, 5, nd 10 mg. of ODB dissolved in ml of corn oil nd dministered y intrmusculr injection to ovriectomised heifers t the time of PRID insertion on % chnges in FSH Ž ns3 4 heifers per dose..

8 8 M. O Rourke et l.raniml Reproduction Science Fig. 4. Effect of different doses of ODB dministered either y powdered form in geltine cpsule Ž5- nd 10-mg doses. ttched to PRID nd two doses Ž 0 nd 0.5 mg. dissolved in ml of corn oil nd dministered y intrmusculr injection to ovriectomised heifers t the time of PRID insertion on the % chnge in FSH in ovriectomised heifers Ž ns3 4 heifers per dose q1.486 Ž hours to pek E. y0.08 Ž hours to pek E., R s0.; P Following ndir concentrtions, FSH concentrtion incresed s E concentrtion de- Fig. 5. Effect of different doses of ODB dministered either y powdered form in geltine cpsule Ž10 mg doses. ttched to PRID nd two doses Ž 1.0 nd dissolved in ml of corn oil nd dministered y intrmusculr injection to ovriectomised heifers t the time of PRID insertion on men plsm concentrtions of oestrdiol Ž E dotted lines. nd FSH Ž solid lines. in ovriectomised heifers Ž ns4 heifers per dose..

9 M. O Rourke et l.raniml Reproduction Science clined from pek vlue, irrespective of tretment. However, t the higher injected doses, concentrtions of FSH egin to rise gin in the fce of declining ut still elevted plsm concentrtions of E Ž Fig. 5.. Concentrtions of FSH following the 5- nd 10-mg injections took significntly longer to return to pre-tretment concentrtions thn in nimls ssigned to ll other tretments Ž P Discussion This experiment clerly shows tht the route of dministrtion of ODB nd the dose injected ffect susequent concentrtions of E nd FSH in lood. The concentrtion of E in lood ws elevted fster following ODB dministered vi the intrmusculr route when given in corn oil compred with topicl vginl ppliction using geltine cpsule ttched to the PRID. When pooled, concentrtions of E in nimls given ODB topiclly on the vginl epithelium y cpsule, took over 10 h longer to rech mximum concentrtion thn in heifers given similr dose y intrmusculr injection in oil. Pek concentrtions in heifers were 0 times greter for the 5- nd 10-mg injections thn similr doses dministered in powder form using the intrvginl cpsule. While pek plsm concentrtion of E ws not reched for further 10 h in these heifers, some of the E my hve een lost efore it would hve eing sored, given tht the geltine cpsule dissolves within 6 h. However, in the humn, vginl sorption of E in powder form hd reched mximum 1 h following dministrtion nd overll declines in plsm levels of FSH nd LH were oserved 5 h following tretment ŽSchiff et l., This experiment demonstrtes tht there is more efficient sorption following intrmusculr injection in oil thn following intrvginl cpsule dministrtion of ODB in powder form. Smith nd Tervit Ž nd Smith nd McGown Ž 198. hve shown tht to give similr oestrus response, geltine cpsule contining four times more ODB thn injection form is required. The rte of sorption of ODB from n oily solvent is slower nd less cute thn from drugs dministered in queous solvents, ut the rte of sorption is still much fster thn tht following topicl dministrtion y the intrvginl route. Hormones in solutions re sored more rpidly thn when given in solid form, which explins prt of the slower uptke following use of the intrvginl cpsule, even though sorption occurs redily nd rpidly when the intrvginl route is used. Retrogrde loss of powdered hormones in vginl secretions my lso e significnt source of loss from the vgin. The doses of ODB dministered resulted in pek E concentrtions spnning the rnge from lutel phse Ž pgrml., folliculr phse Ž 5 7 pgrml. to the phrmcologicl rnge Ž ) 10 pgrml. Ž Cooke et l., Pek E incresed in dose-dependent mnner with dose injected ut not following intrvginl route used in this experiment. The rte of sorption following injection ws similr to tht reported y Bo et l., Ž where pek concentrtions were chieved within 1 dy of dministrtion. Once pek concentrtions were reched, the rte of decline of E ws similr for ll tretments. This is the first experiment to show the difference etween pek plsm concentrtions of E following dministrtion of ODB y intrmusculr injection nd intrvginl geltine cpsule. Some synchronistion regimens hve in-

10 10 M. O Rourke et l.raniml Reproduction Science cluded the dministrtion of 10 mg ODB in oil y intrmusculr injection, ŽMcmilln nd Peterson, 1993; Glvin, 1995., which would elevte concentrtions of E to phrmcologicl levels nd result in slow return of E to sl cyclicl vlues. While there is dose-dependent elevtion of E following intrmusculr injection, the sme is not true following dministrtion y intrvginl cpsule. Concentrtions of E chieved lso depend on the derivtive of E dministered. E17 is the most potent nturlly occurring form of E. On sorption, ODB is immeditely converted to E17 Ž Hoffmnn, nd therefore is similr in potency. E vlerte is long cting form, which is slowly metolised nd hence is more potent thn ODB. The pek concentrtions of E chieved using ODB intrmusculrly in oil re lower thn those chieved y Bo et l., Ž using E vlerte in oil. However, it must e noted tht Bo et l., Ž used intct nimls, which would hve higher E concentrtions thn ovriectomised nimls. The dt presented here clerly show tht vrying concentrtions of E in lood trnsiently suppress FSH concentrtion in ovriectomised heifers given PRID, nd tht the mgnitude of the decline in FSH ws ffected y the pek concentrtions of E chieved. This grees with other studies Ž Price nd We, 1988; Bo et l., in cttle, where E ws dministered in crystlline form in sucutneous implnt to intct heifers nd in sesme oil to intct heifers, respectively. Wolfe et l., Ž 199. reported tht concentrtions of FSH were suppressed in cuoidl mnner following dministrtion of E y sucutneous implnt to ovriectomised heifers. However, unlike this study, lower doses Ž mg. of E did not effect men FSH concentrtions. Bolt et l., Ž hs shown tht 10 mg E17 dministered intrmusculrly in 5 ml corn oil to norgestomet-treted ovriectomised heifers suppressed FSH for the entire smpling durtion of 64 h. Similrly, Burke et l., Ž 1996., using ovriectomised cows, recorded mximl suppression of FSH within 4 h of the dministrtion of ODB y intrvginl cpsule with concentrtions of FSH remining suppressed for l lest further 4 dys. However, this experiment shows tht the durtion of the suppression is much more trnsient nd lso dose-dependent nd with E lood concentrtion of 80 pgrml in nimls following 10 mg ODB intrmusculrly in oil, the FSH suppression lsts considerly longer, viz. up to 150 h. This trnsient suppression contrsts with the findings of Schoenemnn et l., Ž where no suppression of FSH ws found following dministrtion of 17-oestrdiol intrmusculrly in ml enzyl enzote to ovriectomised progesterone-treted zeu cows. Di Gregorio nd Nett Ž ltered the synthesis of gondotrophins in the nterior pituitry y dministering E nd P4 to ewes. This is the first report to show n increse in FSH concentrtions while concentrtions of E re still very high Ž 45 pgrml., s shown in heifers given 10 mg ODB in oil Ž Fig. 5.. This demonstrtes tht E is only one prt of the complex negtive feedck mechnism involved in the control of FSH. It is cler tht E is only one of the components produced y DFs tht mintins FSH suppressed etween successive new wve emergence Ž Cooke et l., 1997; Stgg et l., However, the reltive roles of other folliculr products such s inhiins, ctivins nd follisttin, nd tht of locl nterior pituitry products in the regultion of FSH re yet well delineted. The results of this work show tht there is only trnsient suppression of FSH fter E dministrtion nd, whether this trnsient suppression lone is dequte to control

11 M. O Rourke et l.raniml Reproduction Science follicle wve dynmics in cttle t different physiologicl stges remins to e estlished. It ws concluded tht dministrtion of ODB in oil y intrmusculr injection Ž ut not y dministrtion topiclly in powder form. results in dose-dependent increse in pek E concentrtions. Incresing concentrtions of E in plsm cuse dose-dependent trnsient suppression of FSH. Concentrtions of FSH egin to increse 1 dys fter ODB dministrtion while concentrtions of E re declining ut still elevted. In the ovriectomised heifer model given progesterone, E hs only trnsient suppressive effect on FSH concentrtions. Acknowledgements The uthors re grteful to Dr. M. Grely, A. Glynn nd N. Hynes for ssistnce with the hormone ssys, J. Nlly, P. Joyce, G. Morris nd W. Connolly for technicl support, G. Burke, G. McHugh, P. Crevn nd P. Reilly for cre of the nimls, Snofi Frnce for providing the PRIDs. We cknowledge NIDDK Ž Bltimore, MD. for providing ofsh. This study ws conducted y Tegsc ŽAgriculture nd Food Development Authority. nd under the Opertionl Progrmme for Agriculture, Rurl Development nd Forestry, Mesure 5 Ž., Reserch Stimulus Fund. MO R ws recipient of Tegsc Wlsh Fellowship. References Adms, G.P., Mtteri, R.L., Kstelic, J.P., Ko, J.C.H., Ginther, O.J., 199. Assocition etween surges of follicle-stimulting hormone nd the emergence of folliculr wves in heifers. J. Reprod. Fertil. 94, Brnes, M.A., Kzmer, G.W., Bierley, S.T., Gondotropic nd ovrin response in diry cows treted with norgestomet nd estrdiol vlerte. Theriogenology 16, Bo, G.A., Adms, G.P., Ccci, M., Mrtinez, M., Pierson, R.A., Mpletoft, R.J., Ovrin folliculr wve emergence fter tretment with progestogen nd estrdiol in cttle. Anim. Reprod. Sci. 39, Bo, G.A., Adms, G.P., Nsser, L.F., Pierson, R.A., Mpletoft, R.J., Effect of estrdiol vlerte on ovrin folliculr wves nd circulting gondotrophins in heifers. Theriogenology 40, Bo, G.A., Adms, G.P., Pierson, R.A., Triulo, H.E., Ccci, M., Mpletoft, R.J., Folliculr wve dynmics fter estrdiol-17 tretment of heifers with or without progestogen implnt. Theriogenology 41, Bolt, D.J., Scott, V., Kircofe, G.H., Plsm LH nd LH fter estrdiol, norgestomet nd Gn-RH tretment in ovriectomized eef heifers. Anim. Reprod. Sci. 3, Burke, C.R., Mcmilln, K.L., Bolnd, M.P., Oestrdiol potentites prolonged progesterone-induced suppression of LH relese in ovriectomised cows. Anim. Reprod. Sci. 45, Cooke, D.J., Crowe, M.A., Roche, J.F., Circulting FSH isoform ptterns during recurrent increses in FSH throughout the oestrous cycle of heifers. J. Reprod. Fertil. 110, Crowe, M.A., Pdmnhn, V., Hynes, N., Sunderlnd, S.J., Enright, W.J., Beitins, I.Z., Roche, J.F., Vlidtion of sensitive rdioimmunossy to mesure serum follicle-stimulting hormone in cttle: correltion with iologicl ctivity. Anim. Reprod. Sci. 48, Di Gregorio, G.B., Nett, T.M., Estrdiol nd progesterone influence the synthesis of gondotropins in the sence of gondotropin-relesing hormone in the ewe. Biol. Reprod. 53, Glvin, J., Ovrin folliculr development following oestrus synchronistion progrmmes t different stges of the oestrous cycle in lctting diry cows. M. Agr. Sc. Thesis NUI, Dulin, p. 41.

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