Effects of melatonin implants in ram lambs D. J. Kennaway and T. A. Gilmore Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia 5011, Australia Summary. Sixteen pinealectomized and 19 unoperated ewes were exposed to constant light for about 4 weeks before and 4 weeks after lambing. Six ram lambs born to unoperated ewes were implanted s.c. with melatonin sachets while 8 ram lambs were implanted with empty sachets. The 8 ram lambs born to pinealectomized dams also received empty implants. Ewes and lambs were then returned to the field. Analysis of weekly blood samples indicated that prolactin secretion was significantly decreased in the ram lambs with empty implants between 44\p=n-\51weeks of age whereas lambs treated with melatonin failed to show a significant change during development. All 3 groups of rams had elevated LH levels between 7 and 17 weeks of age, and a second period of high LH between 7 and 40 weeks. There were no significant differences between groups in the patterns of FSH secretion; FSH was highest between 7 and 17 weeks of age, and again between 7 and 40 weeks of age. Plasma testosterone levels in all groups increased gradually between 4 and 35 weeks. Between 38 and 48 weeks of age testosterone concentrations were markedly elevated in all groups. Growth was not affected by melatonin treatment. These results indicate that neonatal melatonin treatment has subtle endocrine effects; whether these effects are sufficient to compromise fertility in the ram, however, remains to be established. Introduction The impact of daylength upon gonadotrophin secretion and ram fertility has been shown by Lincoln & Short (1980). Alternating periods of short and long daylength can induce cycles of testicular development and regression, presumably in response to changes in the frequency of GnRH release from the hypothalamus. When adult rams are superior cervical ganglionectomized to inhibit pineal gland function, the rams cannot entrain to photoperiod challenge (Lincoln & Short, 1980). It has therefore been proposed that a seasonal variation in pineal gland activity is responsible for changes in ram fertility (Lincoln & Short, 1980). The effects of daylength on the sexual development of ram lambs is far less clear. There have been no studies on the effects of photoperiod manipulation on sexual development in rams. However, superior cervical ganglionectomy has been used to determine the influence of the daylength/pineal gland axis: in ganglionectomized Romney ram lambs testosterone secretion is reduced between 6 and 36 weeks of age and the seasonal pattern of prolactin secretion is disrupted (Fisher & Lapwood, 1981). The nature of sexual development in rams is such that it is difficult to determine exactly when puberty occurs, but longitudinal studies of some of the hormones involved in the establishment of spermatogenesis may give some insight into the factors involved in sexual maturation. We therefore studied the effects of melatonin treatment upon the patterns of hormone secretion in ram lambs. Pinealectomized and unoperated dams were used to determine whether the hormone patterns were influenced by the maternal pineal gland.
Materials and Methods The experiment was conducted at the Mortlock Experiment Station, Mintaro, South Australia, between January 1981 and June 198. Sixteen 5-year-old Saxon Merino Border Leicester ewes that had been pinealectomized previously (at 6 months of age) according to the procedure of Kennaway, Obst, Dunstan & Friesen (1981) and 19 unoperated ewes were mated to Dorset rams in early January 1981. At 4 weeks before lambing, the ewes were moved from the field to an animal house where they were kept in groups of 8-9 ewes per pen. The lights (G.E.C.-Multivapor, providing in excess of 1500 lux at sheep's eye level) operated continuously until the lambs were 1-4 weeks old to suppress melatonin production before any treatments were imposed (Rollag & Niswender, 1976). Fourteen ram lambs were born to operated (Group C) ewes and 8 ram lambs were born to pinealectomized dams (Group P). Six ram lambs born to Group C dams were implanted with one melatonin sachet (Kennaway, Gilmore & Seamark, 198a) subcutaneously on the back at 3-4 weeks of age, followed by a second sachet at 13-14 weeks (Group Cm). Empty implants were placed in the remaining 16 lambs (Groups Ce and Pe). Every Tuesday for 5 weeks a single blood sample was taken by venepuncture (10:00-11:00 h). The rams were weaned at 16 weeks, vasectomized at 18 weeks and shorn at 0 and 7 weeks. At 35-36 weeks of age the rams were placed in single pens in an open shed and hourly blood samples were taken during the late afternoon and throughout the night. Dim red lighting was used when necessary. Melatonin was assayed by RIA (Kennaway et ai, 198b). Sensitivity was < 86 pmol/1 and intraand inter-assay coefficients of variation at 0-63 nmol/1 were 1% and 17% respectively. Luteinizing hormone (LH) was assayed by a modification of the RIA method of Oldham, Martin & Knight (1978/79). A liquid assay system was used (rather than coated tube) with the UWA-3B antiserum at a final dilution of 1:400 000 and a pre-precipitated second antibody (goat anti-rabbit gamma globulin). The cross-reactivities of the primary antibody have been reported previously (Oldham et al., 1978/79). Sensitivity, using NIH-LH-S19 as standard, was 0-3 pg/1, with intra- and inter-assay coefficients of variation of 10% and 15% respectively at 5 pg/1. Follicle-stimulating hormone (FSH) was assayed by the RIA method of Cheng, Simaraks & Palmer (1981). Sensitivity was < µg/l using NIH-FSH-S15 as standard. Intra- and inter-assay coefficients of variation were < 15% over the range -00 µg/l. Prolactin was assayed by RIA (Kennaway et al., 1981) using NIH-P-S13 as standard. Sensitivity was <3 µg/l and intra- and inter-assay coefficients of variation through the range 10-300 µ /1 were < 10%. Testosterone was assayed by RIA. This assay uses a goat antibody (0 II, 19/6/79) raised against testosterone-15-(3-thiopropionic acid)-bovine serum albumin. Plasma (00 µ ) was extracted with 1 ml ethyl acetate/hexane. (3 :, v/v). Cross-reactivity was 1% for dihydrotestosterone and < 0-3% for androstenedione, dehydroepiandrosterone, oestradiol-17ß, progesterone and pregnenolone. Sensitivity was <0-6 nmol/1 and intra- and inter-assay coefficients of variation were < 14% at 6-9 and nmol/1. All samples from an individual animal were assayed for LH, FSH and prolactin in single assays. One animal from each of Groups Ce and Pe was excluded from statistical analysis because of very low body weight (<47 kg at 5 weeks). The mean hormone levels for 4-5-week periods of development were calculated and analysed by Friedman's non-parametric two-way analysis of variance and Wilcoxon's critical range test (Colquhoun, 1971). Melatonin Results The implanting of melatonin sachets at 3/4 weeks of age resulted in blood levels of melatonin of 0-77 ± 013 nmol/1 at 1 week, decreasing to 0-8 + 0-06 nmol/1 at 13 weeks, reflecting a previously
observed change in melatonin metabolism during early neonatal development (Kennaway & Gilmore, 1984). After insertion of a second sachet, levels reached 0-4 + 0-05 nmol/1 and were maintained for the duration of the study. Daytime melatonin levels in Group Ce and Group Pe remained low (86 pmol/1) throughout the experiment. At 36 weeks of age all 3 groups of lambs showed a nocturnal increment of melatonin in excess of 0- nmol/1 (data not shown). Neither melatonin treatment nor the pineal status of the lamb's dam affected growth rate (Textfigs 1-3). The fleece weights at 0 and 7 weeks were not significantly different between groups (data not shown). 40 Group Ce -a o m 0 8 _ 61 ( 5 u. 3-4 4 3 3-1 m 1 300 100 10 10 15 0 5 30 35 40 Age (weeks) -Winter I!-Spring-11 Summer 11 1981 45 50 -Autumn- 198 Text-fig. 1. Bodyweights, plasma FSH, LH, prolactin and testosterone concentrations in ram lambs in Group Ce (empty Silastic sachet and intact dam). Bodyweights are shown as mean and range. The weekly hormone levels are represented as the mean ± s.e.m. for 6-7 animals. 55
40 Group Pe 01 "C o m 10 I' 8 61 4 4 5> 3 1 = _ 300 J tu CD 100 i O =r 30 ] ( ) - 10 10 15 5 30 35 Age (weeks) -Spring " Summer " 1981 -Winter- -Autumn- 198 Text-fig.. Bodyweights, plasma FSH, LH, prolactin and testosterone concentrations in ram lambs in Groups Pe (empty Silastic sachet and pinealectomized dam). Bodyweights are shown as mean and range. The weekly hormone levels are represented as the mean + s.e.m. for 6-7 animals. Prolactin The prolactin concentrations in the weekly blood samples showed large variations between animals and between groups (Text-figs 1-3). Groups Pe and Ce had significant time interactions in prolactin concentrations (P < 0-001) with the highest values occurring at -5 weeks (09 ± 33
Group Cm 5 30 35 Age (weeks) -II Spring II Summer IL 1981 -Winter- -Autumn- 198 Text-fig. 3. Bodyweights, plasma FSH, LH, prolactin and testosterone concentrations in ram lambs in Group Cm (melatonin-containing sachet and intact dam). Bodyweights are shown as mean and range. The weekly hormone levels are represented as the mean ± s.e.m. for 6-7 animals. and 174 ± 30 pg/1, respectively) and lowest levels between 44 and 51 weeks (107 ± 33 and 7 + 9 pg/1, respectively). In contrast, the melatonin-treated rams (Groups CM) showed no change during development (171 ± 8 pg/1 at -5 weeks and 163 ± 3 µg/l at 44-51 weeks). LH Plasma concentrations of LH were similarly characterized by marked weekly variation (Textfigs 1-3). There were significant changes in concentrations during development in all three groups
of rams. The highest values were observed between 7 and 17 weeks of age, particularly in Group Pe, decreasing to low levels for the next 10 weeks. A second period of high LH concentrations occurred between 7 and 40 weeks of age in all groups. FSH Plasma FSH concentrations exhibited a statistically significant (P < 0-001) biphasic pattern during development, being highest between 7 and 17 weeks and decreasing over the next 10 weeks before rising again between 7 and 40 weeks (Text-figs 1-3). Lambs in all three groups exhibited similar patterns and there were no statistically significant differences between any of the groups. Testosterone Plasma testosterone concentrations gradually increased between 4 and 35 weeks. Between 38 and 48 weeks of age, Groups Ce and Pe had markedly elevated values followed by a decline after this time. Group Cm exhibited a statistically significant time interaction in testosterone levels; the highest values occurred at the same age as in the other two groups, but the pattern of secretion differed from that of the other groups (Text-figs 1-3). Discussion Ram lambs treated with subcutaneous melatonin implants were subtly affected, which is in contrast to the profound, delayed puberty observed in the simultaneous studies of ewe lambs (Kennaway & Gilmore, 1984). Rams treated with empty sachets exhibited the expected age/seasonal decrease in prolactin in autumn, which was at the same time and age as in the ewe lambs (Kennaway & Gilmore, 1984) and confirms the observations of Ravault (1976), Ravault, Courot, Gamier, Pelletier & Terqui (1977) and Barenton & Pelletier (1980). Rams treated with melatonin, however, showed no significant temporal changes in prolactin during the study and the plasma concentrations were not depressed. The significance of the lower prolactin levels during the breeding season of the control rams is unclear, particularly since chronic bromocriptine treatment to lower circulating prolactin concentrations has no effect on numerous indicators of testis function (Ravault et al., 1977; Barenton & Pelletier, 1980). The patterns of gonadotrophins in the ram are similar to those reported previously, particularly the post-natal elevation (Foster et al., 197; Lee et al., 1976). The subsequent nadir and a second period of high LH values just before full sexual maturity were not observed by Fisher & Lapwood (1981). Lee et al. (1976) similarly did not observe the secondary FSH rise, presumably due to assay differences (their basal FSH levels were 35 pg/1 compared with pg/1 in this study). The period of high testosterone secretion coincided with the period of cyclic ovarian activity in the developing ewe lambs run in the same flock (Kennaway & Gilmore, 1984), as well as with the period of low prolactin levels. Although the pattern of testosterone secretion in melatonin-treated rams appeared different, the statistical tests used still indicated a highly significant increase in testosterone in this group. Unfortunately the ram lambs had to be vasectomized for practical reasons of flock management and so it is not known whether lambs in any of the 3 groups had impaired testicular function. The presence or absence of a maternal pineal gland had no effect on the hormonal characteristics of the lambs studied in this experiment, which confirms the findings obtained with the ewe lambs born to the same dams (Kennaway & Gilmore, 1984). The exposure to constant light before lambing and during early neonatal life similarly is unlikely to have been of consequence in determining the onset of puberty. Exposure of ewe lambs to up to 10 weeks of constant light has no effect on puberty onset (D. J. Kennaway, T. A. Gilmore & F. A. Dunstan, unpublished results).
On the basis of these results it may be concluded that there is a difference in sensitivity to melatonin between rams and ewes or that there are different mechanisms involved in sexual maturation between the sexes. The present study also indicates that a seasonal fall in prolactin levels is not required for normal sexual maturation in rams. Neonatal melatonin treatment of ewe lambs results in delayed onset of puberty by more than 0 weeks, whereas in rams there were only minimal hormonal changes. Part of the reason for this may be the lower melatonin levels achieved by the same implants in rams, which may have been due to differences in metabolism (Kennaway & Gilmore, 1984). If the dosage of melatonin is not the reason, the results are consistent with previous findings which have indicated that ram puberty is only minimally affected by photoperiod (Dyrmundsson, 1973). Different approaches are clearly required to uncover the factors responsible for the timing of sexual maturation in rams. We thank Dr R. F. Seamark for his enthusiastic support during this study; Mr P. Geytenbeek, Mr P. Van Beusichem, Mr P. Attick and Mr P. Steele of the Waite Institute and Mortlock Experiment Station for excellent care of the animals; Miss A. Diamanti, Miss T. Lloyd, Mrs M. Kolo, Miss W. Jones, Miss L. Hourigan and Miss S. Hodson for technical assistance; Dr H. G. Friesen for anti-prolactin serum; Dr G. Martin for anti-lh serum; Dr K. W. Cheng for anti-fsh serum; Dr L. E. Reichert for LH for iodination; NIH for supply of prolactin and LH and FSH standards; and Mr P. Turner, Osram-GEC (Australia) Ltd, for the generous loan of the multivapour lamps. This work was supported by a grant from the National Health and Medical Research Council of Australia. References Barenton,. & Pelletier, J. (1980) Prolactin, testicular growth and LH receptors in the ram following light and -Br-a-ergocryptine (CB-154) treatments. Biol. Reprod., 781-790. Cheng, K.W., Simaraks. S. & Palmer, W.M. (1981) Characterization of a radioimmunoassay for ovine FSH utilizing an anti-bovine FSH serum. J. Reprod. Fert. 61, 115-11. Colquhoun, D. (1971) Lectures on Biostatistics. Oxford University Press, London. Dyrmundsson, O.R. (1973) Puberty and early reproduc tive performance in sheep. II. Ram lambs. Anim. Breed Abstr. 41, 419^130. Fisher, M.W. & Lapwood, K.R. (1981) Effects of cranial cervical ganglionectomy and castration of male lambs. I. Influences on longitudinal endocrine pro files and growth. Theriogenology 16, 607-60. Foster, D.L., Roach, J.F., Karsch. F.J., Norton, H.W., Cook, B. & Nalbandov, A.V. (197) Regulation of LH in the fetal and neonatal lamb. I. LH concentrations in blood and pituitary. Endocrinology 90, 10-111. Kennaway, D.J. & Gilmore, T.A. (1984) Effects of melatonin implants in ewe lambs. J. Reprod. Fert. 70, 39-45. Kennaway, D.J., Obst, J.M., Dunstan, E.A. & Friesen, H.G. (1981) Ultradian and seasonal rhythms in plasma gonadotropins, prolactin, cortisol and testos terone in pinealectomized rams. Endocrinology 108, 639-646. Kennaway, D.J., Gilmore, T.A. & Seamark, R.F. (198a) Effects of melatonin implants on the circadian rhythm of plasma melatonin and prolactin in sheep. Endocrinology 110, 186-188. Kennaway, D.J., Gilmore, T.A. & Seamark, R.F. (198b) Effects of melatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology 110, 1766-177. Lee, V.W.K., Cumming, I.A., de Kretser, D.M., Findlay, J.K., Hudson, B. & Keogh, E.J. (1976) Regulation of gonadotrophin secretion in rams from birth to sexual maturity. I. Plasma LH, FSH and testosterone levels. J. Reprod. Fert. 46, 1-6. Lincoln, G.A. & Short, R.V. (1980) Seasonal breeding: Nature's contraceptive. Recent Prog. Horm. Res. 36, 1-5. Oldham, CM., Martin, G.B. & Knight, T.W. (1978/1979) Stimulation of seasonally anovular merino ewes by rams. I. Time from the introduction of the rams to the preovulatory LH surge and ovulation. Anim. Reprod. Sci. 1, 83-90. Ravault, J.P. (1976) Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old. Acta, endocr., Copenh. 83, 70-75. Ravault, J.P., Courot, M., Garnier, D., Pelletier, J. & Terqui, M. (1977) Effect of -bromo-ergocryptine (CB-154) on plasma prolactin, LH and testosterone levels, accessory reproductive glands and spermatogenesis in lambs during puberty. Biol. Reprod. 17, 19-197. Rollag, M.D. & Niswender, G.D. (1976) Radioimmuno assay of serum concentrations of melatonin in sheep exposed to different lighting regimes. Endocrinology 98, 48-489. Received 15 March 1984