Plasma nentratins f teststerne in the develping ram in different sial envirnments A. W. Illius, N. B. Haynes, K. Purvis and G. E. Lamming Department fphysilgy and Envirnmental Studies, University f Nttingham Shl f Agriulture, Suttn Bningtn, Lughbrugh, Leiestershire LE12 5RD, U.K. Summary. Plasma teststerne prfiles were determined by taking frequent bld samples at intervals frm birth t 21 mnths f age frm rams reared in islatin, in an all-male grup and in a mixed-sex grup. The teststerne pattern was nt mdified by these different sial envirnments. The ability f the ram t pulate when first expsed t an estrus ewe, apparently a maturatinal press, was als independent f sial rearing. At birth plasma teststerne levels were detetable but lw, they were higher at 10 and 16 weeks f age and shwed a marked rise by 26 weeks, inident with the time f puberty. Depressive effets f seasn n test\x=req-\ sterne prfiles urred during the 2nd year f life but nt the 1st year. Intrdutin Reently it has been demnstrated fr several speies that peripheral plasma levels f teststerne shw marked flutuatins ver relatively shrt perids. In the adult ram, up t 10 peaks f testster ne have been bserved in 24 hr, with levels flutuating frm <1 ng t 25 ng/ml plasma, and seasnal variability in teststerne levels has been reprted (Attal, 1970 ; Katngle, Naftlin & Shrt, 1974 ; Purvis, Illius & Haynes, 1974; Sanfrd, Winter, Palmer & Hwland, 1974). It has been suggested that individual rams may be haraterized by differenes in their frequeny and level f teststerne peaks sine these tended t be nsistent within animals when teststerne prfiles were measured n different asins (Purvis et al., 1974). Plasma teststerne levels are altered by pulatin in males f a number f speies, e.g. rabbit, bull, man, rhesus mnkey and rat (Saginr & Hrtn, 1968; Katngle et al., 1971 ; Fx, Ismail, Lve, Kirkham & Lraine, 1972; Rse, Grdn & Bernstein, 1972; Purvis & Haynes, 1974). In bulls, the hanges are marked in 3-6-year-ld animals but less evident frm 1-5 t 2-5 years f age (Smith, Mngknpunya, Hafs, Cnvey & Oxender, 1973). In the adult ram, teststerne levels were unaffeted by pulatin (Purvis et al., 1974), but the experiments were nduted in Marh when plasma teststerne levels were seasnally lw. The intentin f the present study was t establish when episdi seretin f teststerne first beame evident in the ram and t examine the develpment f teststerne prfiles frm an early age until maturity, thus prviding a basis fr the investigatin f the rle f teststerne in the develp ment f male sial behaviur. Further, the experiments were designed t test the hypthesis that the bserved differenes in teststerne prfiles between adult rams uld result frm different sial envirnments during develpment. Finally, the animals were used t investigate in mre detail the effet f pulatin n plasma teststerne prfiles. Animals and rearing predure Materials and Methds Twenty Hampshire Clun rams brn during February 1973 were used. Fur treatments were designed t prvide extremes f sial envirnment. Five rams were reared artifiially frm 2 until Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess
40 days f age using milk substitute and subsequently maintained in individual islatin (Grup I, Rams 1-5). Five rams were reared naturally until 6 weeks f age, then kept in islatin after weaning (Grup II, Rams 6-10). After weaning at 6 weeks f age, 5 rams were kept tgether in an all-male grup (Grup III, Rams 11-15) and 5 were plaed with 3 female lambs f the same age t make a mixed-sex grup (Grup IV, Rams 16-20). At 9 mnths, the females were separated frm the rams by a hurdle. All animals were kept in ntrlled-envirnment rms with the temperature and light adjusted weekly t rrespnd t external seasnal nditins. A nentrate diet was fed and hay and water were always available. Bdy weight was rerded weekly. Rams were examined at intervals fr penis develpment using the riterin f Wiggins & Terrill (1953) f the separatin f the glans penis and pressus urethrae frm the prepue. Bld sampling Bld was taken frm lambs up t 8 weeks f age by jugular venepunture. All ther samples (2-3 ml) were taken frm indwelling plythene annulae (Prtex Ltd) intrdued int the jugular vein under lal anaesthesia, and rams were allwed an vernight alimatizatin perid befre bld samples were taken. After eah lletin the annula was flushed with 5 % aqueus sdium itrate slutin. Bld was transferred int heparinized tubes and entrifuged at 1500 g fr 20 min immediately after lletin. The plasma was remved and stred at 20 C until required fr assay. Samples were taken hurly fr 6 hr at varius ages frm birth t 8 weeks. At apprximately 10, 16, 26 and 38 weeks and 12 and 15 mnths f age, bld samples were taken every 30 min fr 12-, 18- r 24-hr perids mmening at 08.00 hurs. Samples were als taken at 18 and 21 mnths f age after the animals had been regruped. At the latter age, sampling mmened between 16.00 and 19.00 hurs. During pulatin studies, bld was lleted at 30-min intervals frm 2 hr befre t 6 hr after expsure t the ewe exept fr the 1st hr after expsure when samples were lle ted every 10 min. Cpulatin studies On the basis f teststerne prfiles btained (belw) it was deided that Grups I and II shuld be mbined fr this study. Nine rams (3 frm Grup IV, 3 frm Grup III and 3 frm Grups I and II) were eah allwed ntat fr 30 min with an varietmized ewe brught int estrus by injetin f 100 µg estradil benzate fllwing a 10-day treatment with a prgesterne pessary (Synhrmate: G. D. Searle & C., Buks). Eah animal was tested n 5 asins between Deember 1973 and February 1974. They were again tested n tw asins in May 1974, at a time when teststerne levels were assumed t be seasnally lw (Katngle et al., 1974 ; Purvis et al., 1974). Five ther rams (2 frm Grup IV and 3 frm Grups I and II) were first expsed t an adult estrus female at this time. Cpulatin was rerded as suessful if the ram ahieved intrmissin and ejaulatin. Assay f teststerne This was arried ut as desribed previusly fr rams by Purvis et al. (1974). Results Statistial evaluatins were by analysis f variane r t test unless therwise stated. Plasma teststerne prfiles fr 4 lambs sampled at varius times between birth and 8 weeks f age are shwn in Table 1. It is apparent that lw, but flutuating, levels f teststerne ur at an early age. Seventeen prfiles were btained in ttal ; flutuatins were present in all, with levels ranging frm t 4 ng teststerne/ml plasma. The develpment f teststerne prfiles fr 3 animals up t 38 weeks f age (in Otber) is illustrated in Text-fig. 1. Peak levels f teststerne inreased up t 38 weeks. Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess
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Table 1. The nentratins f peripheral plasma teststerne (ng/ml) ver 6 hr in fur ram lambs at different ages Age 0 Time (hr) Birth (Grup III; Ram 13) 6 weeks (Grup III; Ram 11) 6 weeks (Grup IV; Ram 19) 8 weeks (Grup III; Ram 15) 0-4 1-6 20 4-4 0-7 2-2 1-5 1-8 1-3 0-4 10 0-3 0-3 0-3 10 0-3 2-6 1-4 2-8 0-5 1-1 2-1 0-8 Mean height f teststerne peaks, mean teststerne peak intervals and mean teststerne prfile areas (adjusted t equalize the length f the sampling perid) fr all grups at sampling perids between 10 weeks and 21 mnths f age are shwn in Table 2. There were n signifiant differenes between any f these parameters at any given time as a result f the sial grupings. Cmbining the data fr all grups, the mean teststerne prfile areas fr May, August and Nvem ber 1974 were signifiantly higher (P < 001, < 005 and < 001 respetively) than the rrespnd ing values fr June, August and Otber 1973, respetively. Furthermre, the mean teststerne prfile area fr February 1974 was signifiantly lwer ( < 005) than the values fr Otber 1973 and May 1974, but that fr August, 1974 tended t be lwer than that fr May 1974 and was signifi antly lwer (P < 001) than that fr Nvember 1974. Hwever, the prfile area fr August 1973 was signifiantly higher than that fr June 1973 ( < 005). The mean teststerne peak interval was signifiantly greater (P< 005) fr February 1974 than at any ther time. The teststerne prfile areas fr individual animals at partiular liveweights up t 38 weeks f age are illustrated in Text-fig. 2. These results shw that a marked inrease in plasma teststerne nentratin takes plae between 30 and 40 kg liveweight, whih is reahed at abut 20 weeks f age. 200 s 1 150 140 120 A 100-80 T = 3-23L-72-4 r =0-63 60 ".0 20 20 30 40 Liveweight (kg),t = 0-634L + 2-05 r = 0-54 50 60 Text-fig. 2. A plt f the areas under the teststerne prfiles fr individual animals up t 38 weeks f age against liveweight. Sine sampling perids varied frm 12 t 24 hr, all values have been adjusted t a 24-hr perid. A, Grups I + II (islatin);, Grup III (all male);, Grup IV (mixed sex). Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess
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Table 3. The pulatin (C) rerd findividual rams during 7 perids f expsure (see text fr details) t an estrus ewe Expsure perid 1 (De) 2 (Jan) 3 (Jan) 4 (Feb) 5 (Feb) 6 (May) 7 (June) Grup I/II Ram 1 Ram 4 Ram 10 Ram 6 Ram 7 Ram 3 Grup III Ram 11 Ram 12 Ram 13 Grup IV Ram 16 Ram 17 Ram 18 Ram 19 Ram 20 C C The utline f the pulatin experiene f the animals in eah grup is shwn in Table 3. Of 9 animals tested n 5 asins between Deember and Marh, nly 4 pulated, but eah grup was represented. The lak f effet f sial gruping was maintained during the later expsure perids, in whih mst f the rams pulated. Frty-five teststerne prfiles were btained during the exp sures. Typial examples are shwn in Text-fig. 3. The prfile shwn in Text-fig. 3(a) shws n apparent effet f pulatin n teststerne levels; 17 prfiles were in this ategry, but nly 3 were btained when animals pulated. There were 12 prfiles f the type represented in Text-fig. 3(b), 4 frm pu lating animals. The high teststerne levels befre expsure t a female may have preluded the pssibility f an immediate respnse. In the 7 animals, 2 pulating, in whih the prfiles resembled Text-fig. 3. Typial teststerne prfiles f individual rams expsed tan estrus ewe fr 30 min (hathed area). Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess
that shwn in Text-fig. 3(), teststerne levels were rising befre expsure and any effet f expsure uld have been masked. There were 4 pulating rams in the 9 that exhibited a teststerne prfile like that in Text-fig. 3(d), whih shws a pssible inrease in teststerne levels as a result f expsure t females. 2 analysis shwed that the number f prfiles falling int these ategries did nt differ signifiantly frm the expeted randm distributin f teststerne peaks. In sme ases, an animal whih had pulated at a previus expsure had high teststerne levels at the time f a subsequent expsure t a female but did nt pulate. Disussin The differenes in sial envirnment impsed in these studies did nt appear t affet the develping patterns f peripheral plasma teststerne in the yung ram sine statistial analysis failed t shw differenes in frequeny f plasma teststerne peaks, peak heights r prfile areas at ages frm 10 weeks t 15 mnths as a result f sial envirnment. Furthermre, the develpment f sexual be haviur did nt appear t be mdified by sial gruping; few animals pulated at 10 mnths, whilst the majrity did s at 15 mnths, even thse given their first expsure t a female at this age. Libid has been reprted t be sub-ptimal in rams at this seasn (see Yeates, 1954 ; Pepelk & Clegg, 1965). Our results supprt the bservatins f Katngle et al. (1974) and Purvis et al. (1974) that adult rams will shw apparently nrmal pulatin at times when teststerne levels are knwn t be lw. There is n evidene frm the present study that teststerne prfiles f yung rams are affeted by pulatin, irrespetive f sial rearing, but it is intended t repeat the bservatins n these animals when they are lder beause f the differenes reprted between yung and ld bulls (Smith et al., 1973). T assess teststerne hanges during develpment, these have been redued t a single parameter, namely the area under the prfile. Althugh this value is a resultant f a mplex f metabli vari ables whih annt be separated, it gives a useful mparative indiatin fthe amunt fteststerne sereted by the animal ver a given perid. Plasma teststerne levels were lw but detetable befre 8 weeks f age and flutuatins in teststerne levels were apparent at this time. Fster (1974) has bserved flutuatins in plasma teststerne fllwing peaks f endgenus plasma LH in rams f this age. The levels then rse, in nn-linear fashin, twards adult values by 38 weeks f age. This pattern ards with that fund fr testis grwth in similar ram lambs by Illius, Haynes & Lamming (1976). The marked hange in the rate f inrease in teststerne levels urring as the animals reahed 30-40 kg liveweight, arund 20 weeks f age, agrees well with the average liveweights and ages at puberty reprted by Dyrmundssn & Lees (1972) fr Clun Frest rams. The riterin f penis develpment used by Wiggins & Terrill (1953) was fulfilled by 16 weeks f age in the present study. These authrs shwed that teststerne was invlved in the breakdwn f preputial adhesins and the lw levels f irulating teststerne befre 16 weeks f age are bviusly suffiient fr mpletin f this maturatin. The hange f teststerne levels in plasma as rams mature apparently represents an interatin between maturatinal and seasnal variables. The signifiantly higher levels f teststerne fund in August and Nvember 1974 mpared t similar perids in 1973 shw that teststerne levels inrease until at least 21 mnths f age. The seasnal mdifiatin f teststerne patterns is affeted by the maturatinal trend as shwn by the different patterns f teststerne seretin in the 1st and 2nd years f life; in 1974 teststerne levels were lw in February, inreased in May and lw again in August, but in 1973 there were n suh hanges and the levels ntinued t inrease frm April t Otber. It uld be argued that immature rams are insensitive t seasnal effets, but this is unlikely sine Curt, de Reviers & Pelletier (1972) have shwn differenes in LH seretin frm birth up t 80 days in lambs brn in September r February/Marh, and Attal (1970) has nted that the seasnal yle in plasma teststerne is partly masked by testiular grwth during the 1st year f a ram's life. The depressin f plasma teststerne levels in February 1974 is partly a result f teststerne peaks urring at less frequent intervals than at ther times f the year; Katngle et al. (1974) have Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess
reprted that LH disharge is less frequent at times when teststerne levels in the ram are seasnally lw. The reasn fr an elevatin f teststerne levels in May and August 1974 mpared t February 1974 in the present study is nt lear, espeially sine Katngle et al. (1974) reprt that values fr teststerne in adult rams are nsistently lw between January and September and high frm Ot ber t Deember as a result f hanging phtperid. It may be that maturatinal effets during the spring and summer in the 2nd year f the animal's life are still interating with seasnal depressive effets. Hwever, in ne 4-year-ld ram a seasnal depressin in teststerne fund in Marh was nt evident by June (Purvis et al., 1974). Als, there are indiatins frm the data presented by Katngle et al. (1974) that the seasnal fall in plasma teststerne levels in the adult ram between January and September is less prnuned in April-June. Pelletier (1971) reprts a seasnal inrease in ram testiular size in June, and a diphasi inrease in teststerne levels in re deer ming int the breeding seasn, very similar t that bserved fr rams in the present paper, has been reprted by Giménez, Brth, Hffman & Karg (1975). The subjet f phtperidi ntrl f testiular fun tin in the ram is under further investigatin. This wrk was arried ut during tenure f a Ministry f Agriulture, Fisheries and Fd Pst graduate Shlarship (A.W.I.). Referenes Attal, J. (1970) Measure des estrgènes et des andrgènes testiulaires et plasmatiques dans Vespèe vine par des mirmethdes de Chrmatgraphie en phase gazeuse: influene de l'âge de la saisn et du yle diurne. Thèse de Dtrat D'Etat Es-Sienes Naturelles. Curt, M., de Reviers, M.M. & Pelletier, J. (1972) Pituitary LH and bld LH in the male lamb. /. Reprd. Fert. 31, 497^198. Dyrmundssn, O.R. & Lees, J.L. (1972) Pubertal develpment f Clun Frest ram lambs in relatin t time f birth. J. agrie. Si., Camb. 79, 83-89. Fster, D.L. (1974) Regulatin f gnadtrphins during fetal and early pst-natal develpment in the sheep. INSERM 32, 143-156. Fx, CA., Ismail, A.A.., Lve, D.N., Kirkham, K.E. & Lraine, J.A. (1972) Studies n the relatinship between plasma teststerne levels and human sexual ativity. J. Endr. 52, 51-58. Giménez, T., Brth, D., Hffmann, B. & Karg, H. (1975) Bld levels f teststerne in the re deer, Caprelus aprelus, in relatinship t the seasn. Ata endr., Cpenh., Suppl. 193, 59, Abstr. Illius, A.W., Haynes, N.B. & Lamming, G.E. (1976) Effets fewe prximity n plasma teststerne levels and behaviur in the ram. J. Reprd. Fert. 48,25-32. Katngle, C.B., Naftlin, F. & Shrt, R.V. (1971) Relatinship between bld levels f luteinizing hrmne and teststerne in bulls and the effets f sial stimulatin. /. Endr. 50, 457-466. Katngle, C.B., Naftlin, F. & Shrt, R.V. (1974) Seasnal variatins in bld luteinizing hrmne and teststerne levels in rams. /. Endr. 60, 101-106. Pelletier, J. (1971) Influene du phtpérìdìsme et des andrgènes sur la synthèse et la libératin de LH hez le Bélier. Thèse de Dtrat D'Etat Es-Sienes Naturelles. Pepelk, W.E. & Clegg, M.T. (1965) Influene f seasn f the year upn patterns f sexual behaviur in male sheep. /. Anim. Si. 24, 633-637. Purvis, K. & Haynes, N. B. (1974) Shrt-term effets f pulatin, human hrini gnadtrphin injetin and nn-tatile assiatin with a female n teststerne levels in the male rat. /. Endr. 60, 429^139. Purvis, K., Illius, A. W. & Haynes, N.B. (1974) Plasma teststerne nentratins in the ram. /. Endr. 61, 241-253. Rse, R.M., Grdn, T.P. & Bernstein, I.S. (1972) Plasma teststerne levels in the male rhesus: influenes f sexual and sial stimuli. Siene, N. Y. 178, 643-645. Saginr, M. & Hrtn, R. (1968) Reflex release f gnadtrphin and inreased plasma teststerne nentratin in male rabbits during pulatin. Endrinlgy 82, 627-630. Sanfrd, L.M., Winter, J.S.D., Palmer, W.M. & Hwland,B.E. (1974) The prfile f LH and testst erne seretin in the ram. Endrinlgy 95, 627-631. Smith, O.W., Mngknpunya, K., Hafs, H.D., Cnvey, E.M. &Oxender, W.D. (1973) Bld serum teststerne after sexual preparatin r ejaulatin, r after injetins flhrprlatin in bulls./. Anim. Si. 37, 979-984. Wiggins, E.L. & Terrill, CE. (1953) Variatin in penis develpment in ram lambs. J. Anim. Si. 12, 524. Yeates, N.T.M. (1954) Daylight hanges. In Prgress in the Physilgy f Farm Animals, vl. 1, pp. 362-390. Ed. J. Hammnd. Butterwrths, Lndn. Reeived 10 Deember 1975 Dwnladed frm Bisientifia.m at 01/16/2019 05:22:32PM via free aess