Abrupt decrease in daylength and short-term changes in

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1 Abrupt decrease in daylength and shrt-term changes in the plasma cncentratins f FSH, LH and prlactin in anestrus ewes J. S. Waltn, Janet D. Evins, B. P. Fitzgerald and F. J. Cunningham Department fphysilgy & Bichemistry, The University, Whiteknights, Reading RG6 2AJ, Berkshire, U.K. Summary. The plasma cncentratins f FSH, LH and prlactin in ewes were measured at frequent intervals during 24-h perids in anestrus at different times frm the nset f the breeding seasn. Ewes kept under natural daylength cnditins were cmpared with ewes in which the nset f the breeding seasn was advanced by expsure t cnstant shrt days (8L:16D). The cncentratins f FSH during mid-anestrus did nt vary during the day and there was n effect f shrt days r any changes which culd be assciated with the nset f vulatin. In all f the ewes pulsatile releases f LH were bserved n each sampling ccasin. During mid-anestrus the ccurrence f LH pulses varied between 1 and 3/day and ne f the pulses appeared t be synchrnized with dawn. Expsure t shrt days did nt affect the frequency f LH release. In bth grups f ewes an increased frequency f LH pulses was bserved in the perid 12\p=n-\14days befre the first vulatin but this was assciated with a decrease in the magnitude f each pulse. Prlactin cncentratins were raised during anestrus and tended t be higher during the hurs f darkness and in the early mrning. Expsure t shrt days fr 3 weeks ablished these diurnal changes and reduced the cncentratins t nn-detectable amunts. Intrductin The seasnal pattern f reprductive activity f the ewe is largely determined by changes in daylength (Hafez, 1952). Attempts t assciate changes in the cncentratins f gnad trphins in plasma with the annual changes in varian activity have, hwever, been largely incnclusive. Fr example, plasma cncentratins f fllicle-stimulating hrmne (FSH) exhibit marked day-t-day fluctuatins thrughut anestrus and during the breeding seasn but n changes which culd accunt fr the cessatin r resumptin f vulatin can be detected (Waltn, McNeilly, McNeilly & Cunningham, 1977). Similarly, plasma cncentratins f luteinizing hrmne (LH) d nt crrespnd clsely with the changing varian status (Rche, Fster, Karsch, Ck & Dziuk, 1970; Yuthasastraksl, Palmer & Hwland, 1975; Waltn et al, 1977). In all f these experiments, hwever, bld was nt taken at intervals frequent enugh t characterize fully the pattern f hrmne release; this is particularly imprtant when measuring LH which is released in discrete pulses f shrt duratin (Scaramuzzi & Baird, 1977). In cntrast, changes in the plasma cncentratins f prlactin can be related t breeding * Present address: Department f Animal and Pultry Science, Ontari Agricultural Cllege, University f Guelph, Guelph, Ontari, Canada, NIG 2W /80/ S02.00/ Jurnals f Reprductin & Fertility Ltd

2 activity and have been shwn t be assciated with changes in daylength (Waltn et al, 1977; Thimnier, Ravault & Ortavant, 1978): values are high when the ewes are anvulatry and the days lng but the levels fall markedly when daylength decreases and vulatin ccurs. These changes in the release f prlactin mirrr the changing sensitivity f the hypthalamus t the negative feedback actin f estradil, which was suggested by the bservatin that the cncentratin f LH in the plasma f variectmized ewes bearing estradil-cntaining implants was high during the time f the nrmal breeding seasn and lw during the time f anestrus (Legan, Karsch & Fster, 1977). If hypthalamic activity is reduced during anestrus and prlactin release is nrmally regulated by hypthalamic inhibitin (MacLed, 1976), then the changing pattern f prlactin release may be a useful index f hypthalamic activity in the ewe. The high levels f prlactin during anestrus may als exert an actin which is antignadal since it has been demnstrated that the hyperprlactinaemia induced by suckling delays the resumptin f varian activity in ewes pst partum (Kann, Martinet & Schirar, 1978). The relatinship between the release f prlactin and the gnadtrphins is, therefre, f cnsiderable imprtance in understanding the regulatin f seasnal breeding in the ewe. The aim f the present study was t define shrt-term changes in the plasma cncentratins f FSH, LH and prlactin in ewes during anestrus and during the transitin frm anestrus t breeding activity under tw daylength envirnments. Materials and Methds Crss-bred ewes (25% Finnish Landrace: 25% Drset Hrn: 50% Scttish Blackface) that lambed in March 1978 and had nt suckled their lambs were available fr the experiment. On 18 June, the ewes were weighed and randmly divided int 2 grups f 5. One grup (S) was hused in a light-prfed cabin (fr details see Ducker, Thwaites & Bwman, 1970) in which the lights were adjusted t cincide with the prevailing natural phtperid, and the ther grup (N) was hused in an pen-frnted shed. A vasectmized ram equipped with a marking crayn and harness (Radfrd, Watsn & Wd, 1960) fr the detectin f estrus was included in each grup. On 27 June, the ewes in each grup were penned individually and a cannula was inserted in an external jugular vein and filled with heparin-saline slutin (100 i.u./ml). Frm 10:00 h n 28 June until 11:30 h n 29 June, bld samples (5 ml) were withdrawn at intervals f 45 min. Samples were taken during darkness with the aid f a dim red trch and care was taken t illuminate nly the area arund the end f the cannula n the back f the animal's head. Samples were immediately placed in heparinized (20 i.u.) tubes in ice and centrifuged within 30 min f cllectin at 1500 g fr 10 min. The plasma was separated and stred at 20 C until assayed. On 29 June the lighting schedule in the light-prf cabin was adjusted s that the ewes f Grup S were expsed t a phtperid f 8 h light (06:00-14:00 h) and 16 h darkness (8L: 16D). This phtperid was maintained fr the rest f the experiment. Ewes in Grup were kept under natural daylength cnditins thrughut the experiment. Bld was als btained, using an identical prtcl, frm each ewe at 1, 3, 6 and 8 weeks (5 July, 19 July, 9 August and 23 August) after the change f phtperid. Each ewe was checked daily fr evidence f estrus and additinal bld samples were btained by venepuncture twice weekly thrughut the experiment. The experiment ended when all ewes had exhibited the first estrus f the new breeding seasn. Plasma cncentratins f FSH, LH and prgesterne were measured using radiimmunassays described previusly (McNeilly, McNeilly, Waltn & Cunningham, 1976; Waltn et al, 1977). When tested using highly purified preparatins, the crss-reactin f LH in the assays fr FSH was 0-07% and the activity f FSH in the LH assay was 0-3%. Other

3 pituitary hrmnes r plasma did nt interfere in either assay. The least detectable amunts f hrmne were 0-5 ng NIH-LH-S16/ml and 25 ng NIH-FSH-S8/ml. The assay fr prgesterne was essentially specific (Furr, 1973) and culd detect 0-2 ng prgesterne/ml with a recvery f 75%. Plasma cncentratins f prlactin were measured using a specific, duble-antibdy radiimmunassay fr vine prlactin. The antiserum was raised in rabbits against NIHprlactin-S9, which was als used as the standard in the assay. NIH-prlactin-S6 was labelled with idine-125 by a mdificatin f the methd described by Gldfine, Amir, Petersen & Ingbar (1974). Prlactin (5 µg) was added t a mixture f 1 mci sdium 125idide (Radichemical Centre, Amersham, U.K.) and 800 ng chlramine in 0-5 M-phsphate buffer, ph 7-5. The ttal reactin vlume was 50 ul and the reactin was allwed t prceed fr min. T stp the reactin, 100 ul 0-05 M-phsphate buffer cntaining 2-5% bvine serum albumin and 0-005% ptassium idide were added and the idinated prlactin was separated immediately by gel filtratin n Sephadex G25. When the antiserum was used at a final dilutin f 1: the sensitivity f the assay was 3 ng NIH-prlactin-S9/ml using a prtcl similar t that used fr LH (Waltn et al, 1977), with the exceptin that the labelled hrmne was added n the same day as the antiserum. Crss-reactins with ther hrmnes (NIH-FSH-S8, NIH-LH-S16, NIH-TSH-S6, NIH-GH-S10) were all <0-01% and there were n bservable nn-specific effects caused by plasma. Fr any hrmne, all samples frm each animal and frm ne animal per treatment were assayed tgether in a single assay. All estimatins were carried ut in duplicate and the precisin f the assays, as reflected by the intra-assay cefficient f variatin, was always < 10%. The first vulatin f the breeding seasn in each case was presumed t precede the first nrmal luteal-phase prfile f prgesterne (Waltn et al, 1977). A pulse f LH was defined as the incidence f a significantly elevated level fllwed by at least tw prgressively lwer values which were still elevated abve basal cncentratins. On a small number f ccasins, when the ccurrence f these pulses was very frequent, this criterin culd nt be satisfied and a subjective assessment was made f the number f pulses ccurring during the cllectin perid. Statistical cmparisns were carried ut using Student's t test. Onset fvulatin and estrus Results The ewes in Grup weighed (s.e.m.) kg and thse in Grup S weighed 71-6 ± 1-6 kg. The first estrus f the breeding seasn was bserved 91-8 ± 0-5 (s.e.m.) days in Grup ewes and 74-2 ±3-2 days after the lngest day in Grup S ewes (P < 0-001, d.f. 8, t = 5-41). Measurements f plasma cncentratins f prgesterne indicated that each ewe vulated nce withut exhibiting estrus befre the first vert estrus. The estimated mean ± s.e.m. nset f vulatin was 74-8 ± 0-5 days and days after the lngest day in the ewes in Grup and S respectively. The endcrine respnses t the reduced daylength will be cnsidered nly fr the first 4 weeks f the experiment because changes after this may be a reflectin f the nset f vulatin rather than a respnse t phtperid. FSH Mean plasma cncentratins f FSH during mid-anestrus ranged frm 50 t 80 ng/ml with n significant changes ccurring during the 24-h perid f study (Text-fig. la). There was a trend twards increasing cncentratins in the early mrning but this was nt significant. The pattern 1 week and 3 weeks later was similar in bth grups f ewes (Text-figs lb and lc).

4 c \n <t c cm ^ CO CM -1-r CSI - 5 " -1- O O O Csl ( iu/6u) uiieijj ( LU/6U) U!lOB OJd ( W/6u) U!10B OJd I -r~ r cm -1-r- CO CM ( UJ/6u) HI ( LU/ßu) m ( UJ/6u) HI li : X i CM CO «r I 00 <* ( UJ/ßu) HSd ( W/ßu) HSd (iw/6u) HSd

5 LH Plasma cncentratins f LH exhibited fluctuatins indicative f episdic release in all f the ewes studied. Thrughut mid-anestrus, 1-3 shrt pulses f LH release were bserved per 24 h in all ewes. The magnitude f these pulses ranged frm 3 t 10 times the basal levels f LH when samples were taken every 45 min (Text-figs la, lb and lc). The pulses were f shrt duratin ranging frm 2-25 t 3-00 h. These data are presented in tw ways; as means (Text-fig. 1) and as changes in the frequency f the pulses (Text-fig. 2b). Pling the data gave an indicatin f the degree f synchrny f the pulses during the study perid. In mid-anestrus there was cnsistent release f LH sn after dawn and anther in the middle f the afternn (Text-figs la and lb). The early mrning surge was als bserved in Grup S ewes 1 week and 3 weeks after the light treatment was impsed (Text-figs lb and lc). This synchrny f the pulses was, hwever, almst ttally lst in the ewes in Grup by 3 weeks after the start f the experiment (Text-fig. lc). There was n suggestin, hwever, that mre LH release ccurred in the ewes in Grup S than in the ewes in Grup during this perid. There was als n indicatin that the pulsatile release f LH was restricted t different phases f light r darkness except fr the early mrning peak. A secnd surge f LH was bserved in the Grup S ewes at the time when it wuld nrmally be dusk (Text-figs lb and lc). Prlactin In all ewes n the first sampling ccasin the first plasma samples taken had variable and high levels f prlactin (Text-fig. la). This was als bserved t a lesser extent n the later ccasins. During husing under natural daylength cnditins in mid-anestrus, ewes had high mean levels f prlactin (15-45 ng/ml) which varied accrding t the time f day. Plasma cncentratins f prlactin were elevated during perids f darkness and int early mrning (Text-fig. 1). Hwever, expsure t shrt days fr nly 1 week (Grup S) almst cmpletely ablished the rise in prlactin that ccurred in the dark, and after 3 weeks n shrt days prlactin cncentratins were ften undetectable (Text-figs lb and lc). Changes related t the nset fvulatin In an attempt t islate changes in the cncentratins f these hrmnes that may be related t the nset f vulatin, the data have als been arranged accrding t the day f first vulatin (Text-fig. 2). Changes in the mean cncentratins f FSH were nt clsely related t the nset f vulatin in any f the ewes (Text-fig. 2a). Sme ewes in Grup S tended t have higher cncentratins f FSH than thse in Grup but this was nt a cnsistent finding. Ewes in bth grups exhibited a similar frequency f LH discharge until apprximately 20 days befre the first vulatin (Text-fig. 2b). There was a suggestin that the frequency f these pulses was increasing and that the magnitude f each pulse was decreasing as the first vulatin apprached. In ne ewe the pulses were very frequent and f lw magnitude 3 days befre the first vulatin, a pattern which is similar t that fund n Day 14 f the estrus cycle (Text-fig. 3). Plasma cncentratins f prlactin were dminated by the effect f the daylength envirnment and n changes in relatin t vulatin were detected (Text-fig. 2c). Text-fig. 1. Changes in the mean plasma cncentratins f FSH, LH and prlactin in (a) 9 ewes kept in natural daylength n June; (b) 4 f the 9 ewes in natural daylength ( ) 1 week later and the remaining ewes (O) which had been kept in shrt days (8L: 16D) fr 1 week; and (c) the same ewes as in (b) after 3 weeks in natural daylength (#) r shrt days (O). Vertical bars represent s.e.m. and the timing f the light and dark perids is indicated by the hrizntal bars.

6 160' la) \ i. i 12-, (b) I 2 itt t i t. (c) l ó 8 % Days Text-fig. 2. Summary f the hrmnal prfiles fr each 24-h study perid in relatin t the presumed day f first vulatin (Day 0) in ewes kept under natural daylength cnditins ( ) and shrt days (8L: 16D) (O). 10:00 16:00 22:00 04:00 10:00 10:00 16:00 22:00 04:00 Hurs 10:00 Text-fig. 3. Plasma cncentratins f LH in individual ewes frm Grup S n Days 9 (Ewe 3), 3 (Ewe 4) and +14 (Ewe 6) relative t the presumed day f first vulatin (Day 0). The hrizntal bars indicate perids f light and darkness.

7 Discussin The nset f the breeding seasn was advanced by subjecting anestrus ewes t a cnstant, shrt phtperid frm the lngest day nwards. This is cnsistent with earlier studies (Ducker et al, 1970; Waltn et al, 1977), althugh a small part f the effect may have been due t the heavier bdyweight f the ewes n shrt days. There was an unusual degree f synchrny in the nset f estrus in the ewes kept under natural daylength. Hwever, these ewes, unlike thse in Grup S, were expsed t a cntinually changing farm-yard envirnment, and this synchrnizatin may have been caused by anther envirnmental stimulus, e.g. by inadvertent expsure t strange rams (Hunter & Lishman, 1967), in additin t the effect f declining daylength. There was little suggestin that plasma cncentratins f FSH fluctuated in accrdance with the lighting envirnment and n changes which culd be related either t the fighting treatment r t the nset f vulatin were detected. On ccasins, increases in the cncentratins f FSH were assciated with pulsatile releases f LH but these were f lw magnitude and were nt cnsistently bserved. The trend twards increasing cncentratins f FSH in the early mrning during mid-anestrus is prbably assciated with the surge f LH that ccurs at this time. These results cntrast sharply with the effect f shrt phtperids n plasma cncentratins f FSH in the ram. A 3 t 4-fld increase in the cncentratin f FSH was bserved in Say rams after expsure t shrt days (8L: 16D) fr 5 weeks after expsure t lng days (16L:8D) (Lincln & Peet, 1977). Since these shrt phtperids stimulate gnadal functin in ewes and rams the mechanisms which cntrl the release f FSH in each sex must be different. Pulsatile release f LH was bserved in all animals n all ccasins studied. During the majrity f the anvulatry perid between 1 and 3 pulses were bserved in 24 h. The peridicity and magnitude f these pulses agree with thse described by Scaramuzzi & Baird (1977) when differences between the tw experiments in the timing and duratin f bld sampling are accunted- fr. During mid-anestrus there was an indicatin that ne f these pulses f LH was clsely assciated with the end f the dark perid and this assciatin was maintained in a large prprtin f the animals in bth grups during subsequent sampling perids. There was als a suggestin that there was anther pulse in the middle f the afternn and that this was delayed by extending the perid f darkness. Apart frm changes in the timing f these pulses the data indicated that similar amunts f LH were released in bth phtperids. There appeared t be an increase in the number f pulsatile releases as the first vulatin apprached and just befre the first vulatin there were many pulses f lw magnitude. This pattern resembles that fund during the later stages f the estrus cycle (Baird, Swanstn & Scaramuzzi, 1976). At apprximately 10 days befre the first vulatin, therefre, there was an increase in the frequency f pulses and a reductin in the magnitude f each pulse. It is uncertain whether these changes in LH release are the cause r a reflectin f the changing varian status. In additin t these pulsatile fluctatins ther releases f LH were bserved. One ewe was discarded because a surge f LH f prevulatry prprtins was bserved n the very first sampling ccasin. This was prbably induced by the intrductin f the rams used t detect estrus (Hunter & Lishman, 1967) just befre the experiment started. A similar surge was als bserved in 1 ewe 5 days befre the first vulatin, which was assessed by subsequent changes in the plasma cncentratins f prgesterne. This supprts the view that there are tw surges f LH f prevulatry dimensins separated by 5 days befre the first nrmal prfile f prgesterne secretin after anvulatin (Waltn et al, 1977). Plasma cncentratins f prlactin were high during mid-anestrus althugh nt as high as recrded in earlier experiments in which bld samples were taken by venepuncture (Waltn et al, 1977; Thimnier et al, 1978). The release f prlactin bserved at the start f the cllectin perid in mst f the ewes which had high prlactin levels was prbably an initial respnse t stress (Lamming, Mseley & McNeilly, 1974), and was much less nticeable in the ewes n

8 shrt daylengths which had lw levels f prlactin. In ewes n lng days, prlactin levels exhibited a marked diurnal variatin: the cncentratins increased during the dark perid and fell during the beginning f the light perid. A similar pattern has als been reprted in Say rams n lng days (Lincln, McNeilly & Camern, 1978). This pattern was, hwever, almst cmpletely ablished by nly 1 week n shrt days and a lw level was fund in all the ewes kept n shrt days fr the rest f the experiment. These data are cnsistent with earlier wrk (Waltn et al, 1977; Thimnier et al, 1978) which demnstrated that prlactin levels are reduced in ewes kept in shrt days. Expsure t shrt days, therefre, des nt apparently increase the release f FSH r LH in the ewe as has been reprted fr the ram (Lincln & Peet, 1977), at least in the shrt term. This des nt, hwever, preclude the pssibility f daylength cntrlling gnadtrphin release. This must ccur if the hypthesis f the changing hypthalamic sensitivity t estradil during the year hlds true (Legan et al, 1977). The pulsatile release f gnadtrphin was reasnably well synchrnized by the change frm darkness t light, suggesting a daylength-mediated hypthalamic cntrl ver gnadtrphin release. Althugh increased hypthalamic activity, as reflected by the frequency f LH release, was bserved just befre the nset f vulatin, it is uncertain whether this is causing, r is a cnsequence f, the changing varian status. There was, hwever, n suggestin f a perid f increased LH release due t mre pulses f increased magnitude, as has been shwn in the ewe (Kann et al, 1978) and cw (Lamming, 1978) psi partum. The majr effect f decreased daylength was t reduce the circulating cncentratin f prlactin. This decline was nt, hwever, immediately fllwed by a return t vulatin. It might be expected that, if inhibitin by prlactin was the nly factr restraining varian activity, then ewes wuld have respnded mre quickly t shrt daylengths than they did. The administratin f 2-brm-a-ergcryptine (CB154, Sandz: 2 mg/day) t anestrus ewes frm 14 July t 14 August reduced prlactin cncentratins t an undetectable level (<3 ng/ml) but the nset f estrus was nly marginally advanced (B. P. Fitzgerald, J. S. Waltn & F. J. Cunningham, unpublished bservatins). It is unlikely, therefre, that the seasnal hyperprlactinaemia per se is slely respnsible fr anvulatin. Nevertheless, part f the respnse t shrtened daylength may invlve reducing prlactin levels and s relieve a suppressin n the events which nrmally initiate the new breeding seasn. We thank Dr I. T. Kechik fr his assistance and the staff f the University farm, Snning, Berkshire, fr care f the animals; Dr S. S. Lynch and Dr. J. A. Furr fr the antisera t human FSH and prgesterne; and NIAMDD, NIH, fr purified hrmnes. This study was financed by a grant (A.G. 45/157) frm the Agricultural Research Cuncil t F.J.C. References Baird, D.T., Swanstn, I. & Scaramuzzi, RJ. (1976) Pulsatile release f LH and secretin f varian sterids in sheep during the luteal phase f the estrus cycle. Endcrinlgy 98, Ducker, M.J., Thwaites, CJ. & Bwman, J.C. (1970) Phtperidism in the ewe. 2. The effects f varius patterns f decreasing daylength n the nset f estrus in Clun Frest ewes. Anim. Prd. 12, Furr, J.A. (1973) Radiimmunassay f prgesterne in peripheral plasma f the dmestic fwl in varius physilgical states and in fllicular venus plasma. Acta endcr., Cpenh. 73, Gldfine. I.D., Amir, S.M., Petersen, A.W. & Ingbar, S.H. (1974) Preparatin f bilgically active 125I- TSH. Endcrinlgy 95, Hafez, E.S.E. (1952) Studies n the breeding seasn and reprductin f the ewe. J. agrie. Sci., Camb. 42, Hunter, G.L. & Lishman, A.W. (1967) Effect f the ram early in the breeding seasn n the incidence f vulatin and estrus in sheep. Prc. S. Afr. Sc. Anim. Prd, ß, Kann, G., Martinet, J. & Schirar, A. (1978) Hypthalamic-pituitary cntrl during lactatin in sheep. In Cntrl f Ovulatin, pp Eds. D. B.

9 Crightn,. B. Haynes, G. R. Fxcrft & G. E. Lamming. Butterwrths, Lndn. Lamming, G.E. (1978) Reprductin during lactatin. In Cntrl f Ovulatin, pp Eds D. B. Crightn, N. B. Haynes, G. R. Fxcrft & G. E. Lamming. Butterwrths, Lndn. Lamming, G. E., Mseley, S.R. & McNeilly, J.R. (1974) Prlactin release in the sheep. /. Reprd. Fert. 40, Legan, SJ., Karsch, FJ. & Fster, D.L. (1977) The endcrine cntrl f seasnal reprductive functin in the ewe. A marked change in respnse t the negative feedback actin f estradil n luteinizing hrmne secretin. Endcrinlgy 101, Lincln, G.A. & Peet, MJ. (1977) Phtperidic cntrl f gnadtrphin secretin in the ram: a detailed study f tempral changes in plasma levels f fllicle-stimulating hrmne, luteinizing hrmne and teststerne fllwing an abrupt switch frm lng t shrt days. /. Endcr. 74, Lincln, G.A., McNeilly, A.S. & Camern, CL. (1978) The effects f sudden decrease r increase in daylength n prlactin secretin in the ram. /. Reprd. Feri. 52, MacLed, R.W. (1976) Regulatin f prlactin secre tin. In Frntiers in Neurendcrinlgy, Vl. 4, pp Eds L. Martini & W. F. Ganng. Raven Press, New Yrk. McNeilly, J.R., McNeilly, A.S., Waltn, J.S. & Cun ningham, FJ. (1976) Develpment and applicatin f a heterlgus assay fr vine fllicle-stimulating hrmne. /. Endcr. 70, Radfrd, H.M., Watsn, R.H. & Wd, G.F. (1960) A crayn and assciated harness fr the detectin f mating under field cnditins. A ust. vet. J. 36, Rche, J.F., Fster, D.L., Karsch, FJ., Ck, B. & Dziuk, PJ. (1970) Levels f luteinizing hrmne in sera and pituitaries f ewes during the estrus cycle and anestrus. Endcrinlgy 86, Scaramuzzi, RJ. & Baird, D.T. (1977) Pulsatile release f luteinizing hrmne and the secretin f varian sterids in sheep during anestrus. Endcrinlgy 101, Thimnier, J., Ravault, J.P. & Ortavant, R. (1978) Plasma prlactin variatins and cyclic varian activity in ewes submitted t different light regimens. Annls Bil. anim. Bichim. Biphys. 18, Waltn, J.S., McNeilly, J.R., McNeilly, A.S. & Cun ningham, FJ. (1977) Changes in the cncentratins f fllicle-stimulating hrmne, luteinizing hrmne, prlactin and prgesterne in the plasma f ewes during the transitin frm anestrus t breeding activity. J. Endcr. 75, Yuthasastraksl, P., Palmer, W.M. & Hwland, B.E. (1975) Luteinizing hrmne, estrgen and pr gesterne levels in peripheral serum f anestrus ewes as determined by radiimmunassay. J. Reprd. Feri. 43, Received 12 Octber 1979