Intraruminal soluble glass boluses containing melatonin

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1 Intraruminal sluble glass bluses cntaining melatnin can induce early nset f varian activity in ewes A. L. Pultn, A. M. Symns, M. I. Kelly and J. Arendt Department f Bichemistry, University f Surrey, Guildfrd, Surrey GU2 5XH, and * Research and Develpment Labratries, Pilkingtn Brthers p.i.e., Lathm, Ormskirk, Lancashire L40 5UF, U.K. Summary. Sluble phsphate glass bluses (preparatins A, B, and C) have been develped t release melatnin int the reticul-rumen f ewes at relatively different (fast, medium r slw) rates. A furth type (preparatin D) cntaining n melatnin was used as a sham cntrl. Fur grups f seasnally anestrus Sufflk-crss ewes were respectively dsed with preparatins A, B, C r D n 4 July. Plasma samples were cllected twice weekly fr melatnin and prgesterne assay. In Grups A, B and C, elevated daytime plasma cncentratins f melatnin culd be detected fr abut 5 weeks after blus administratin. Hwever, the pattern f hrmne release was variable between grups, with Grup C animals maintaining higher plasma melatnin cncentratins fr a lnger perid. The cntrl animals had undetectable daytime melatnin levels. The nset f cyclic varian activity in the animals treated with the 'slw' release blus (Grup C) was significantly (P < 0\m=.\05) advanced cmpared t the cntrl grup. The 'fast' and 'medium' release treatments (Grups A and B) did nt significantly alter the nset f varian activity. The results indicate the ptential f a nvel and cnvenient methd f melatnin delivery fr inductin f early breeding activity in ewes. Intrductin Secretin f the pineal hrmne melatnin mediates the effect f changing phtperid upn seasnal reprductive functin in the ewe (see reviews by Bittman, 1984; Kennaway, 1984). Timed administratin f supplementary melatnin by feeding (Kennaway et al, 1982; Arendt et al, 1983), injectin (Nett & Niswender, 1982) r infusin (Bittman & Karsch, 1984) can stimulate an early nset f breeding activity by pharmaclgically mimicking the nset f shrt phtperidic cn ditins. Cntinuus melatnin administratin via subcutaneus (English et al, 1986) r vaginal implants (Nwak & Rdway, 1985) is prbably perceived as a 'very shrt-day' respnse (Lincln & Ebling, 1985) and can als induce early breeding activity. In breeds such as the Sufflk-crss and Mule, bth timed and cntinuus melatnin treatments are nly effective arund mid-summer, prbably fllwing expsure t a critical interval f lng phtperid (Nwak & Rdway, 1985; English et al, 1986). In the less seasnal Merin crss breeds, melatnin treatment can be effective befre the summer slstice (Kennaway et al, 1982) and can induce a relative increase in vulatin rate (Kennaway et al, 1984). It is clear that any cnvenient and inexpensive methd f melatnin administratin nt nly prvides a useful research tl but wuld be f practical benefit t the cmmercial sheep industry. The ruminai stmach prvides an ideal site fr cntrlled delivery preparatins (Telfer, 1984; Cardinal, 1985) which, by virtue f their size, shape and density are nrmally retained in the reticu lum. This study describes perfrmance data f a bidegradable sluble glass/melatnin device develped fr ral administratin in the ewe. Cntaining the same amunt f melatnin, 3 types f

2 blus designed t release the hrmne within the rumen at different rates, were administered t seasnally anestrus Sufflk-crss ewes in mid-summer. Daytime plasma melatnin cncen tratins prvided an index f the release pattern f melatnin frm the ruminai bluses whilst cyclic varian activity was indicated by plasma prgesterne prfiles. Materials and Methds Melatninjsluble glass bluses. Plymer-bnded sluble phsphate glass bluses incrprating pwdered melatnin (150 mg) (Sigma Chemical Crpratin, Ple, Drset, U.K.) were prepared at the Research and Develpment Labratries, Pilkingtn Brthers p.l.c. in cllabratin with the Department f Bichemistry, University f Surrey. Each blus was cylindrical in shape (diameter 1-8cm, length 30cm) with an average mass f 23g, resulting in an average specific gravity f 30. Variatin in the plymer-bnded sluble glass frmulatin alters the aqueus slubility f the blus which in turn determines the release-rate f melatnin. Preliminary in-vitr studies (data nt published) indicated 3 frmulatins (preparatins A, and C) that wuld disslve within the rumen at relatively different (fast, medium and slw) rates. A furth type f blus (preparatin D) cntaining n melatnin was develped as a sham-cntrl treatment. Experimental animals. Sixteen maiden Sufflk-crss ewes, brn in spring 1984, were maintained under natural phtperid at the MAFF Central Veterinary Labratries (CVL), Weybridge, Surrey (latitude 51 41'N). The animals were penned tgether in a large pen shed within an area measuring 10 5 metres and were fed a ratin f sheep rearer nuts (Suthern Cunties Agricultural Trading Sciety, Gdalming, Surrey, U.K.) at 230 g/ewe/day and allwed free access t hay and water. The ewes were randmly divided int fur grups f fur and n 4 July 1985 (Day 0) were respectively dsed (esphageal balling gun n. V687A, Arnlds Veterinary Prducts, Reading, Berkshire, U.K.) with preparatins A, B, C r D. The animals were weighed 2 days befre and 116 days after treatment. Bld sampling. Frm 28 June t 31 Octber 1985, bld samples (10 ml) were cllected between 09:00 and 11:00 h twice weekly by jugular venepuncture using heparin-cated vacutainers (Bectn Dickinsn Ltd, Cwley, Oxfrdshire, U.K.) and the plasma stred at 20 C until hrmne assay. Melatnin assay. Plasma melatnin was estimated by a direct radiimmunassay (RIA) (Fraser et al, 1983) described previusly (Pultn as et al, 1986). Plasma samples f 25, 100 and 478 pg melatnin/ml had inter-assay cefficients f variatin f 21, 19 and 13% respectively and intra-assay cefficients f variatin f 12, 12 and 7% respectively. The limit f assay sensitivity, as expressed as the value f tw standard deviatins belw maximum (zer melatnin standard) binding, was 4 pg/ml fr 500 µ plasma assayed. Prgesterne assay. Plasma prgesterne was estimated by a direct enzyme-linked immunabsrbent assay (ELISA) (Sauer et al, 1986) as described previusly (English et al, 1986). Plasma samples f 0-2 and l-5ng pr gesterne/ml had inter-assay cefficients f variatin f 23 and 13% respectively and intra-assay cefficients f variatin f 3 and 4% respectively. The limit f assay sensitivity, expressed as the value f tw standard deviatins belw maximum (zer prgesterne standard) binding, was 0-1 ng/ml fr 20 pi plasma assayed. Reprductive activity. T distinguish between 'nrmal' and 'shrt luteal phase' estrus cycles, the frmer were defined as a series f at least 3 plasma samples with detectable prgesterne cncentratins, prviding at least ne f these samples had a prgesterne cncentratin > 1-0 ng/ml. The nset f cyclic varian (breeding) activity was defined as the ccurrence f at least 2 cnsecutive 'nrmal' cycles. Statistical analysis. Values are given as mean + s.e.m. Daytime plasma melatnin cncentratins in Grups A, and C, during the perid 1-33 days after blus administratin, were analysed using tw-way analysis f variance and tested at each bld sampling episde using ne-way analysis f variance. Onset f varian activity between all grups was assessed by ne-way analysis f variance. Differences between treatment grups were tested using Duncan's new multiple range test at 2 levels f significance (P < 005 and < 001). Results Pled plasma melatnin and individual plasma prgesterne cncentratins are presented in Fig. 1. In Grups A, and C, daytime plasma melatnin cncentratins > 10 pg/ml culd be detected until days after treatment. Grup D ewes had undetectable levels thrughut the study. During Days 1-33 (Day 0 day f treatment) there = was n difference in verall melatnin cncentratins between Grups A, and C althugh there was variatin ver time (P < 0002).

3 -. * S ( UI/6u) 0U0J9}S0ß0Jd - '> fai "* 3 ) Il 2 g *^» t a. u QQ O - > S u ( C >. «ß ** "ß vi Û12 s.s [ aü..2 «J < ce C «< c a ; c O C /-S C«P., C ß te ^ "03 > ( uj/6d) u]uib 0 /\j ce g c vi O 03 ai. E 5 * S jj D. tu C ce -> S I w 6 ni O &û --, t-., g S C Vi E ce

The plasma melatnin prfile was similar within grups and biphasic in nature. In Grups A and B, melatnin peaks f abut 300 and 150 pg/ml ccurred at Days 1 and 7.

4 The plasma melatnin prfile was similar within grups and biphasic in nature. In Grups A and B, melatnin peaks f abut 300 and 150 pg/ml ccurred at Days 1 and 7. In Grup C, peaks f abut 180 and 200 pg/ml ccurred at Days 1 and 18. At Days 14, 18 and 21, plasma melatnin cncentratins were significantly greater in Grup C (Fig. 1). There was a significant grup effect in the nset f cyclic varian activity (P < 003) with the mean date f nset in Grup C (9 August, days) significantly advanced cmpared t that f the cntrl grup (14 September, days) (P < 005). The start f varian cyclicity was nt advanced in Grups A (3 September, days) r (21 August, days). There were n ther significant interactins. All grups displayed a similar weight increase ver the 4-mnth experimental perid. Weights (verall mean + s.e.m. values) f the ewes, 2 days befre and 116 days after treatment, were and kg, respectively. Discussin All animals in Grups A, and C displayed elevated daytime plasma cncentratins f melatnin fr apprximately 5 weeks, indicating that, while disslving in the rumen, these preparatins released (immunlgically active) hrmne which was taken up int the peripheral bld system ver this perid. As endgenus melatnin is mainly secreted ncturnally, it was assumed that daytime plasma levels culd be attributed t the treatments. It wuld appear that n bluses were regurgitated, indicating that the blus mass was sufficient t be retained in the ruminai system and the cnsistent plasma melatnin prfile within each treat ment grup suggests that the bluses were unifrmly and nt prematurely degraded. All animals gained weight ver the experimental perid suggesting an absence f any adverse treatment effects. Plasma cncentratins in Grup C ewes were markedly elevated ( > 50 pg/ml) days after blus administratin cmpared t thse in the ther grups. It wuld appear that this extended perid f elevated plasma hrmne cncentratin has cntributed t the cnsistent early nset f varian activity in these animals. The critical (daytime) plasma melatnin cncentratin and the duratin f such treatment required t mimic effectively a shrt phtperidic respnse in ewes remain t be accurately defined. This study suggests that at least ncturnal cncentratins (i.e pg/ml) fr apprximately 5 weeks are needed, althugh supraphysilgical levels ver a lnger perid are als effective (Hsieh & Chien, 1985). The sham-cntrl ewes exhibited the start f varian activity earlier than expected f Sufflkcrss ewes in this lcality (Arendt et al, 1983; English et al, 1986). Within an enclsed area, ewes displaying early cyclic activity may (via phermnal cues?) stimulate a similar but premature respnse in ther (untreated) animals. In an 'early breeding' flck, this is actually a desirable phenmenn but may cnfund experimental treatment versus cntrl cmparisns. This issue shuld be cnsidered in further studies f this nature. Cautin shuld als be exercised in drawing cnclusins frm a study that was limited in grup size and restricted t ne breed. Hwever, these preliminary results prvide a psitive indicatin f the efficacy f this technique. Field trials t establish the perfrmance f the sluble glass/ melatnin blus with respect t inductin f early estrus and subsequent lambing rates are currently underway. Similar trials have been extended t the gat and deer. A bidegradable intraruminal device capable f sustaining elevated plasma melatnin cncen tratins wuld prvide a nvel and inexpensive methd f inducing thse seasnal respnses in ruminants assciated with shrt phtperid. We thank Mr Phil Irlam, Mr Ted White and Dr Brian Algar f Pilkingtn Brthers pic fr their participatin in the develpment f the melatnin/sluble-glass blus; Mr Ben Parker and Mr Tny Quinn f MAFF, CVL, Weybridge, fr their management f the experimental animals; and

Dr Jhn Fulkes f MAFF, Cattle Breeding Centre, Shinfield, fr reagents and assistance with the prgesterne assay. This wrk was spnsred by Pilkingtn Brthers pic, U.K.

5 Dr Jhn Fulkes f MAFF, Cattle Breeding Centre, Shinfield, fr reagents and assistance with the prgesterne assay. This wrk was spnsred by Pilkingtn Brthers pic, U.K. and is published with the permissin f the Directrs and Mr A. S. Rbinsn, Directr f Grup Research and Develpment. References Arendt, J., Symns, A.M., Laud, CA. & Pryde, S.J. (1983) Melatnin can induce the early nset f the breeding seasn in ewes. J. Endcr. 97, Bittman, E.L. (1984) Melatnin and phtperidic time measurement: Evidence frm rdents and ruminants. In The Pineal Gland, pp Ed. R. J. Reiter. Raven Press, New Yrk. Bittman, E.L. & Karsch, F.J. (1984) Nightly duratin f pineal melatnin secretin determines the repr ductive respnse in inhibitry daylength in the ewe. Bil. Reprd. 30, Cardinal, J.R. (1985) Cntrlled drug delivery: veter inary applicatins. J. cntr. Release 2, English, J., Pultn, A.L., Arendt, J. & Symns, A.M. (1986) A cmparisn f the efficiency f melatnin treatments in advancing estrus in ewes. J. Reprd. Fert. 11, Fraser, S., Cwen, P., Franklin, M., Franey, C. & Arendt, J. (1983) Direct radiimmunassay fr melatnin in plasma. Clin. Chem. 29, Hsieh, D.S.T. & Chien, Y.W. (1985) Enhanced release f drugs frm silicne elastmers (III). Subcutaneus cntrlled administratin f melatnin fr early nset f estrus cycles in ewes. Drug Develpment and Industrial Pharmacy 11, Kennaway, D.J. (1984) Pineal functin in ungulates. Pineal Research Reviews 1, Kennaway, D.J., Gilmre, T.A. & Seamark, R.E. (1982) Effect f melatnin feeding n serum prlactin and gnadtrpin and the nset f seasnal estrus cyclicity in sheep. Endcrinlgy 110, Kennaway, D.J., Dunstan, E.A., Gilmre, T.A. & Seamark, R.F. (1984) Melatnin feeding increases vulatin rate in ewes. Prc. 7th Int. Cngr. Endcrinlgy, Quebec City, Abstract n. 21. Lincln, G.A. & Ebling, F.J.P. (1985) Effect f cnstantrelease implants f melatnin n seasnal cycles in reprductin, prlactin secretin and multing in rams. J. Reprd. Fert. 73, Nett, T.M. & Niswender, G.D. (1982) Influence f exgenus melatnin n seasnality f reprductin in sheep. Therigenlgy 17, Nwak, R. & Rdway, R.G. (1985) Effect f intravaginal implants f melatnin n the nset f varian activity in adult and prepubertal ewes. /. Reprd. Fert. 74, Pultn, A.L., English, J., Symns, A.M. & Arendt, J. (1986) Effects f varius melatnin treatments n plasma prlactin cncentratins in the ewe. J. Endcr. 108, Sauer, M.J., Fulkes, J.A., Wrsfld, A. & Mrris, B.A. (1986) Use f prgesterne 11-glucurnide alkaline phsphatase cnjugate in a sensitive micrtitre-plate enzymeimmunassay f prgesterne in milk and its applicatin t pregnancy testing in dairy cattle. J. Reprd. Fert. 76, Telfer, S.B. (1984) Cntrlled release glass (CRG) its actin and applicatin in sheep. Prc. Sheep Veter inary Sciety 8, Received 16 September 1986

Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams

Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams Effect f cnstant-release implants f melatnin n seasnal cycles in reprductin, prlactin secretin and multing in rams G. A. Lincln and F. J. P. Ebling MRC Reprductive Bilgy Unit, Centre fr Reprductive Bilgy,