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Effects of melatonin treatment on the onset of ovarian activity, reproductive parameters and PRL plasma levels of immature ewes
Animal Reproduction Science, 13 (1987) 283-290 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
283
Effects of Melatonin T r e a t m e n t on the Onset of Ovarian A c t i v i t y , R e p r o d u c t i v e P a r a m e t e r s and P R L P l a s m a L e v e l s of I m m a t u r e E w e s C. T A M A N I N I
1'3,A. P R A N D I 2,D. B I A C C H E S S I 2 and F. D E R E N S I S 2
lIstituto di Fisiologia Veterinaria, Universita ' di Bari, Via Caduti di tutte le guerre 1, 70126 Bari (Italy) 2Istituto di Fisiologia Vete. inaria, Universita' di Bologna, Via Belmeloro 8/2, 40126 Bologna (Italy) (Accepted 11 November 1986}
ABSTRACT Tamanini, C., Prandi, A., Biacchessi, D. and De Rensis, F., 1987. Effects of melatonin treatment on the onset of ovarian activity,reproductive parameters and P R L plasma levelsof immature ewes. Anita. Reprod. Sci.,13: 283-290. The aims of this study were to investigate the effect of chronic treatment with melatonin on prolactin plasma profiles, the onset of ovarian activity and the fertility of immature ewes. Beginning in late June, 30 maiden ewes were administered 2.5 mg melatonin (i.m.) daily until midSeptember. Progesterone and prolactin plasma concentrations were determined by validated radioimmunoassays on samples collected every 5 days from 17 June until 31 October. Prolactin (PRL) plasma concentrations in the control animals were highest at the beginning of the experiment and lowest towards its conclusion. In melatonin-treated ewes, PRL levels dropped just after treatment began and were similar to those observed in the control animals at the end of the experimental period. The ovarian activity was advanced by approximately one month by the administration of melatonin, while the mean date of lambings was advanced by about two weeks compared with the controls. Fertility in the treated animals was very similar to that of the controls; the prolificacy (lambs/pregnant ewe) was 2 in the treated and 1.62 in the control ewes.
INTRODUCTION E x p e r i m e n t a l d a t a s u p p o r t t h e view t h a t t h e p i n e a l h o r m o n e m e l a t o n i n is responsible for m e d i a t i n g t h e r e s p o n s e to c h a n g e s in t h e p h o t o p e r i o d in sheep ( K e n n a w a y et al., 1983 ). M a r k e d differences in p l a s m a m e l a t o n i n c o n c e n t r a t i o n s b e t w e e n b r e e d i n g a n d n o n - b r e e d i n g s e a s o n s in Suffolk cross ewes h a v e 3Correspondence and reprint requests.
0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
284 been described (Rollag et al., 1978; Arendt et al., 1981 ). On this basis, attempts have been made to shorten the anestrous season in this species by administering melatonin in order to induce changes in the reproductive activity similar to those occurring after the modification of the photoperiod. Encouraging results have been obtained by many authors (Nett and Niswender, 1982; Arendt et al., 1983; Lincoln and Ebling, 1985; Nowak and Rodway, 1985; Roche et al., 1985 ), who observed a lengthening of the breeding season by administering melatonin. It has also been proved that melatonin reduces PRL plasma levels which tend to be low during the breeding season and high in anestrus. This hormone does not seem to be directly involved in regulating reproductive activity (Worthy and Haresign, 1983; Worthy et al., 1985). Attempts to advance the breeding activity in ewes by reducing PRL plasma levels were ineffective (McNeilly and Land, 1979; Baird and McNeilly, 1981; Kennaway et al., 1982). Furthermore, it has been observed (Walton et al., 1977; Thimonier, 1981) that PRL plasma concentrations often increase towards the end of the breeding season without negatively affecting the estrous activity. Even if PRL does not seem to be decisive in regulating reproductive activity, the secretion of this hormone appears to be modulated by melatonin activity. The present experiment was designed to: (1) determine whether melatonin is effective in advancing the breeding season in immature ewes; (2) investigate the influence of melatonin on fertility and prolificacy; and (3) confirm melatonin's negative effect on the PRL plasma concentrations. MATERIALSAND METHODS
Animals and experimental design The experiment was performed in Northern Italy (45 ° N latitude) using 60 Friesian ewes recently imported from Northern Europe. The ewes were approximately 10-12 months old and their body weight averaged 70% of that of mature animals. The ewes were housed indoors under a natural photoperiod and were fed a normal ration of hay and concentrates. Two adult males, whose fertility had previously been proved, were penned under the same conditions. The ewes were randomly assigned either to be treated or to serve as a control. From 2 June to 15 September, the animals in the first group (A, n--30) were treated daily with an intramuscular injection of 2.5 mg melatonin dissolved in 0.2 ml of an ethanol/distilled water solution (30/70, v/v); injections of the vehicle only were given to the control animals (group B, n = 3 0 ) . Both the melatonin and the vehicle were always administered between 16:00 and 17:00. Reproductive parameters (fertility and prolificacy) were subsequently
285 recorded. The approximate date of fertile matings were established a posteriori by assuming a gestation length of 150 days.
Blood sampling From 17 June to the end of October, all of the ewes were bled by jugular venipuncture every 5 days. The blood was collected into heparinized tubes and centrifuged within 30 min of collection; the blood plasma was stored at - 20 ° C until required for the measurement of the concentrations of progesterone and PRL.
Assays The concentrations of progesterone and P R L in the blood plasma were measured using radioimmunoassays. Details of the methods are described elsewhere: progesterone in Bono et al, (1983) and PRL in Tamanini et al. (1985). The lowest limit of detection for the progesterone assays was 21 + 0.74 pg/tube (2+ SE) and the intra- and inter-assay coefficients ( % ) o f variation were 9.2 and 14.2 respectively. The ewes were assumed to be cycling when the progesterone plasma concentration was higher than 0.4 ng/ml for at least two consecutive samples. The lowest limit of detection for the P R L assay was 93 _+2.1 pg/tube ( 2 + SE) and the i n t r a - a n d inter-assay coefficients (%) of variation were 8.0 and 12.9 respectively; all of the samples from an individual animal were assayed together, using either 100 zl undiluted plasma or 100 ~l plasma diluted 1-10 depending on the month of collection.
Statistical analysis The data on the P R L plasma concentrations were subjected to an analysis of variance; the difference between the P R L levels observed in melatonintreated and control ewes were further analysed using the Student's t-test. RESULTS
Hormonal profiles The progesterone plasma concentrations were less than 0.2 ng/mt in all of the group A animals until the end of July; from then onwards, the number of ewes with high progesterone levels (1-4 ng/ml) progressively increased and almost all of the animals showed high levels by the end of August (Fig, 1). None of the ewes in group B showed progesterone plasma levels higher than 0.2 ng/ml before 14 August. These animals started to show progesterone levels ranging between 1 and 4 ng/.ml during the second half of August. All of the
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Fig. 1. Effectoftreatmentwith melatoninon the onsetofovarianactivity:continuousline,treated animals;brokenline,controls.Eweswereconsideredto be cyclingwhenprogesteroneplasmalevels exceeded0.4 ng/ml for at least two consecutivesamples. ewes had shown at least one progesterone plasma peak by the first week of October (Fig. 1 ). The difference between the dates when 50% of the treated or control ewes exhibited ovarian activity was statistically significant (P<0.01). PRL plasma variations in the melatonin-treated and control animals are reported in Fig. 2. In the control ewes, the PRL plasma levels ranged between 150 and 380 ng/ml at the beginning of the experiment and then progressively decreased; the lowest concentrations (10-100 ng/ml) were observed near the end of the experimental period. In the melatonin-treated ewes,the PRL plasma concentrations ranged between 150 and 400 ng/ml before the beginning of the melatonin treatment and showed a sudden and marked decrease after it had started. The PRL plasma levels remained markedly lower than those observed in the control animals until the end of the melatonin treatment; then the PRL plasma profiles reached values similar to those observed in the control ewes (Fig. 2).
Reproductive findings The reproductive parameters are reported in Table 1. The percentage of ewes lambing was 86.6 for the treated animals and 90.0 for the controls. The mean date of lambing was advanced by approximately 20 days (P<0.01) in the melatonin-treated animals compared with the controls. The prolificacy
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Fig. 2. Top panel: PRL plasma profiles (2+ S.D.) in melatonin-treated (continuous line) and control (broken line) ewes during the experimental period; bottom panel: levels of statistically significant differences (t-test) between PRL values observed in melatonin-treated and untreated ewes for each day of sampling. ( l a m b s / p r e g n a n t ewe) was 2 a n d 1.62 in groups A a n d B respectively; this difference was n o t significant. DISCUSSION A c i r c a n n u a l r h y t h m o f c i r c u l a t i n g p r o l a c t i n has b e e n p r e v i o u s l y d e s c r i b e d in sheep ( F o r b e s et al., 1975; R a v a u l t , 1976; L i n c o l n et al., 1978; T h i m o n i e r et TABLE 1 Reproductive parameters observed in ewes chronically treated with melatonin (group A) and in control animals (group B) Parameter
Group A
Group B
Ewes (n) Lambings (n) Date of lambing (2_+SE) Prolificacy (lambs/pregnant ewe)
30 26 (86.6%) 14 January + 2.6 2.00
30 27 (90.0%) 4 February _+5.6 1.62
288 al., 1978) with the highest levels observed in summer and the lowest in winter. The profile of PRL plasma levels we observed in the control ewes (with maximum values in June and m i n i m u m concentrations in October) was, therefore, due to the shortening days (Walton et al., 1977 ). The administration of melatonin was responsible for a sudden decrease in PRL plasma levels and the concentrations remained lower than those observed in the control animals until the end of the experiment. It is, therefore, likely that at least circannual variations in PRL secretion are mediated through melatonin concentrations and, consequently, through the pineal gland (Symons et al., 1983 ). Similar results were reported by Kennaway et al. (1982, 1983), Howland et al. (1984) and Poulton et al. (1986). The fall in P R L plasma concentrations was not coincident with the onset of ovarian activity; this situation is consistent with previous suggestions (Worthy et al., 1985) that the onset of the breeding season is independent of the decreasing PRL plasma concentrations and that it is not possible to manipulate breeding activity by the manipulation of P R L (Kennaway et al., 1982). T h a t the treatment of ewes with melatonin can be effective in advancing the breeding season has been demonstrated by other authors (Nett and Niswender, 1982; Kennaway et al., 1982; Arendt et al., 1983; Nowak and Rodway, 1985; Roche et al., 1985). In our experiment, the onset of ovarian activity was observed 5-6 weeks after the beginning of the treatment. A similar latent period has been reported by Nett and Niswender (1982). The latent period observed by Kennaway et al. (1982) and Nowak and Rodway (1985) was slightly longer. Most of these results were observed following treatment of mature animals with melatonin. To our knowledge, only Nowak and Rodway (1985) and Roche et al. (1985) have induced a significant advancement of ovarian activity, by giving melatonin intravaginally or orally, in immature ewes. Our experiment seems to confirm the data reported by the above-mentioned authors. Even though chronic treatment with melatonin has been investigated as far as ovarian activity is concerned, there are no data about the effect of the treatment on fertility. Our experiment seems to indicate that the onset of the breeding season is characterized by fertile estrus since the ewes given melatonin lambed, on average, 20 days before the control animals. This difference, however, does not exactly reflect the difference between the onset of ovarian activity (monitored by the progesterone determination) observed in the two groups. As already mentioned, the ewes given melatonin showed an ovarian activity advanced by more than one month. We may presume that this discrepancy may be due to the fact that some ewes ovulated without exibiting any behavioural estrus and, therefore, could not be mated. This kind of irregular cycle has often been described in ewes at the beginning of the breeding season; the results of this study seem to confirm this tendency only in melatonin-treated animals. In fact, in almost all of the control ewes, the first progesterone plasma increase was possibly strictly related to the putative date of mating. Recordings
289 of the dates of lambings allowed us to observe t h a t almost all of the animals had become pregnant without previously exhibiting any estrous cycles. The data on prolificacy are also interesting. The difference (2 and 1.62 lambs/pregnant ewe in the treated and control animals respectively) is high even if it is not statistically significant. The t r e a t m e n t with melatonin needs to be extended to a higher number of ewes in order to confirm this difference and assess its possible statistical incidence. The mechanism (s) by which melatonin is likely to improve the ovulation rate also have to be investigated. CONCLUSIONS The results of this experiment provide additional evidence t h a t P R L plasma concentrations, reduced by melatonin administration, do not seem to be directly involved in the resumption of ovarian activity at the end of the anestrous season. Secondly, the administration of melatonin to ewes is effective in advancing the breeding season not only in mature females but also in immature ones. F u r t h e r work is needed to confirm our preliminary results on the fertility and prolificacy of ewes given melatonin. ACKNOWLEDGEMENTS The authors are grateful to Prof. Leo E. Reichert for providing purified ovine P R L (LER-860-2) and Mr. S. Mongiorgi for his skillful technical help. This work was supported by ECC G r a n t No. 3311 and C.N.R., Italy, Special G r a n t I.P.R.A Sub-project 1, Paper No. 1035.
REFERENCES Arendt, J., Symons,A.M. and Laud, C.A., 1981. Pineal function in the sheep: evidencefor a possible mechanismmediating seasonal reproductiveactivity. Experientia, 37: 584-589. Arendt, J., Symons,A.M., Laud, C.A. and Pryde, S.J., 1983. Melatonin can induce early onset of the breeding season in ewes. J. Endocrinol., 97: 395-400. Baird, D.T. and McNeilly,A.S., 1981. Gonadotrophiccontrol of folliculardevelopmentand function during the oestrous cycleof the ewe. J. Reprod. Fertil. Suppl. 30: 119-133. Bono, G., Cairoli, F., Tamanini, C. and Abrate, L., 1983. Progesterone,estrogens, LH, FSH and PRL concentrations in plasma during the estrous cyclein goat. Reprod. Nutr. Dev., 23 (2A): 217-222. Forbes, J.M., Driver, P.M., El Shahat, A.A.,Boaz, T.G. and Scanes, C.G., 1975. The effect of day length and level of feedingon serum prolactin in growinglambs. J. Endocrinol.,64: 549-554. Howland,D.E., Palmer, W.A.and Vriend,J., 1984. Endocrinechangesin ewesfed melatonin. 10th Int. Congr. Anita. Reprod. Art. Insem. Urbana-Champaign, 2: 25. Kennaway,D.J., Gilmore, T.A. and Seamark, R.F., 1982. Effect of melatonin feedingon serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicityin sheep. Endocrinology, 110 (5): 1766-1772.
290 Kennaway, D.J., Dunstan, E.A., Gilmore, T.A. and Sea_mark, R.F., 1983. Effect of shortened daylength and melatonin treatment on plasma prolactin and melatonin levelsin pinealectomised and sham-operated ewes. Anita. Reprod.Sci., 5: 287-294. Lincoln, G.A. and Ebling, F.J.P., 1985. Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams. J. Reprod. Fertil.,73: 241-253. Lincoln, G.A., McNeilly, A.S. and Cameron, C.L., 1978. The effectsof a sudden decrease or increase in daylength on prolactin secretion in the ram. J. Reprod. Fertil.,52: 305-311. McNeilly, A.S. and Land, R.B., 1979. Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in L H - R H treated anoestrous ewes. J. Reprod. Fertil., 57: 601-609. Nett, T.M. and Niswender, G.D., 1982. Influence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology, 17: 645-652. Nowak, R. and Rodway, G.R., 1985. Effect of intravaginal implants of melatonin on the onset of ovarian activity in adult and prepubertal ewes. J. Reprod. Fertil., 74: 287-293. Poulton, A.L., English, J., Symons, A.M. and Arendt, J., 1986. Effects of various melatonin treatments on plasma prolactin concentrations in the ewe. J. Endocrinol., 108: 287-292. Ravault, J.P., 1976. Prolactin in the ram: seasonal variations in the concentrations of blood plasma from birth until three years old. Acta Endocrinol., 83" 720-725. Roche, J.F., Hanrahan, J.P., Quirke, J.F. and Ronayne, E., 1985. Effects of melatonin on the time of onset of the breeding season in different breeds of sheep. In: F. Ellendorff and F. Elsaesser (Editors), Endocrine Causes of Seasonal and Lactational Anestrus in Farm Animals. Martinus Nijhoff, The Hague, pp. 55-65. Rollag, M.D., O'Collaghan, P.L. and Niswender, G.D., 1978. Serum melatonin concentrations during different stages of the annual reproductive cycle in ewes. Biol. Reprod., 18: 279-285. Symons, A.M., Arendt, J. and Laud, C.A., 1983. Melatonin feeding decreases prolactin levels in the ewe. J. Endocrinol., 99: 41-46. Tamanini, C., Bono, G., Cairoli, F. and Chiesa, F., 1985. Endocrine responses induced in anestrous goats by the administration of different hormones after a fluorogestone acetate treatment. Anita. Reprod. Sci., 9: 357-364. Thimonier, J., 1981. Control of seasonal reproduction in sheep and goats by light and hormones. J. Reprod. Fertil. Suppl., 30: 33-45. Thimonier, J., Ravault, J.P. and Ortavant, R., 1978. Plasma prolactin variations and cyclic ovarian activity in ewes submitted to different light regimens. Ann. Biol. Anita. Biochim. Biophys., 17: 459-473. Walton, J.S, McNeiUy, J.R., McNeilly, A.S. and Cunnigham, F.J., 1977. Changes in concentrations of follicle stimulating hormone, luteinizing hormone, prolactin and progesterone in the plasma of ewes during the transition from anestrus to breeding activity. J. Endocrinol., 75: 127-136. Worthy, K. and Haresign, W., 1983. Evidence that the onset of seasonal anoestrus in the ewe may be independent of increasing prolactin concentrations and day length. J. Reprod. Fertil., 69: 41-48. Worthy, K., Haresign, W., Dodson, S., McLeod, B.J., Foxcroft, G.R. and Haynes, N.B., 1985. Evidence that the onset of the breeding season in the ewe may be independent of decreasing plasma prolactin concentrations. J. Reprod. Fertil., 75: 237-246.
283
Effects of Melatonin T r e a t m e n t on the Onset of Ovarian A c t i v i t y , R e p r o d u c t i v e P a r a m e t e r s and P R L P l a s m a L e v e l s of I m m a t u r e E w e s C. T A M A N I N I
1'3,A. P R A N D I 2,D. B I A C C H E S S I 2 and F. D E R E N S I S 2
lIstituto di Fisiologia Veterinaria, Universita ' di Bari, Via Caduti di tutte le guerre 1, 70126 Bari (Italy) 2Istituto di Fisiologia Vete. inaria, Universita' di Bologna, Via Belmeloro 8/2, 40126 Bologna (Italy) (Accepted 11 November 1986}
ABSTRACT Tamanini, C., Prandi, A., Biacchessi, D. and De Rensis, F., 1987. Effects of melatonin treatment on the onset of ovarian activity,reproductive parameters and P R L plasma levelsof immature ewes. Anita. Reprod. Sci.,13: 283-290. The aims of this study were to investigate the effect of chronic treatment with melatonin on prolactin plasma profiles, the onset of ovarian activity and the fertility of immature ewes. Beginning in late June, 30 maiden ewes were administered 2.5 mg melatonin (i.m.) daily until midSeptember. Progesterone and prolactin plasma concentrations were determined by validated radioimmunoassays on samples collected every 5 days from 17 June until 31 October. Prolactin (PRL) plasma concentrations in the control animals were highest at the beginning of the experiment and lowest towards its conclusion. In melatonin-treated ewes, PRL levels dropped just after treatment began and were similar to those observed in the control animals at the end of the experimental period. The ovarian activity was advanced by approximately one month by the administration of melatonin, while the mean date of lambings was advanced by about two weeks compared with the controls. Fertility in the treated animals was very similar to that of the controls; the prolificacy (lambs/pregnant ewe) was 2 in the treated and 1.62 in the control ewes.
INTRODUCTION E x p e r i m e n t a l d a t a s u p p o r t t h e view t h a t t h e p i n e a l h o r m o n e m e l a t o n i n is responsible for m e d i a t i n g t h e r e s p o n s e to c h a n g e s in t h e p h o t o p e r i o d in sheep ( K e n n a w a y et al., 1983 ). M a r k e d differences in p l a s m a m e l a t o n i n c o n c e n t r a t i o n s b e t w e e n b r e e d i n g a n d n o n - b r e e d i n g s e a s o n s in Suffolk cross ewes h a v e 3Correspondence and reprint requests.
0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
284 been described (Rollag et al., 1978; Arendt et al., 1981 ). On this basis, attempts have been made to shorten the anestrous season in this species by administering melatonin in order to induce changes in the reproductive activity similar to those occurring after the modification of the photoperiod. Encouraging results have been obtained by many authors (Nett and Niswender, 1982; Arendt et al., 1983; Lincoln and Ebling, 1985; Nowak and Rodway, 1985; Roche et al., 1985 ), who observed a lengthening of the breeding season by administering melatonin. It has also been proved that melatonin reduces PRL plasma levels which tend to be low during the breeding season and high in anestrus. This hormone does not seem to be directly involved in regulating reproductive activity (Worthy and Haresign, 1983; Worthy et al., 1985). Attempts to advance the breeding activity in ewes by reducing PRL plasma levels were ineffective (McNeilly and Land, 1979; Baird and McNeilly, 1981; Kennaway et al., 1982). Furthermore, it has been observed (Walton et al., 1977; Thimonier, 1981) that PRL plasma concentrations often increase towards the end of the breeding season without negatively affecting the estrous activity. Even if PRL does not seem to be decisive in regulating reproductive activity, the secretion of this hormone appears to be modulated by melatonin activity. The present experiment was designed to: (1) determine whether melatonin is effective in advancing the breeding season in immature ewes; (2) investigate the influence of melatonin on fertility and prolificacy; and (3) confirm melatonin's negative effect on the PRL plasma concentrations. MATERIALSAND METHODS
Animals and experimental design The experiment was performed in Northern Italy (45 ° N latitude) using 60 Friesian ewes recently imported from Northern Europe. The ewes were approximately 10-12 months old and their body weight averaged 70% of that of mature animals. The ewes were housed indoors under a natural photoperiod and were fed a normal ration of hay and concentrates. Two adult males, whose fertility had previously been proved, were penned under the same conditions. The ewes were randomly assigned either to be treated or to serve as a control. From 2 June to 15 September, the animals in the first group (A, n--30) were treated daily with an intramuscular injection of 2.5 mg melatonin dissolved in 0.2 ml of an ethanol/distilled water solution (30/70, v/v); injections of the vehicle only were given to the control animals (group B, n = 3 0 ) . Both the melatonin and the vehicle were always administered between 16:00 and 17:00. Reproductive parameters (fertility and prolificacy) were subsequently
285 recorded. The approximate date of fertile matings were established a posteriori by assuming a gestation length of 150 days.
Blood sampling From 17 June to the end of October, all of the ewes were bled by jugular venipuncture every 5 days. The blood was collected into heparinized tubes and centrifuged within 30 min of collection; the blood plasma was stored at - 20 ° C until required for the measurement of the concentrations of progesterone and PRL.
Assays The concentrations of progesterone and P R L in the blood plasma were measured using radioimmunoassays. Details of the methods are described elsewhere: progesterone in Bono et al, (1983) and PRL in Tamanini et al. (1985). The lowest limit of detection for the progesterone assays was 21 + 0.74 pg/tube (2+ SE) and the intra- and inter-assay coefficients ( % ) o f variation were 9.2 and 14.2 respectively. The ewes were assumed to be cycling when the progesterone plasma concentration was higher than 0.4 ng/ml for at least two consecutive samples. The lowest limit of detection for the P R L assay was 93 _+2.1 pg/tube ( 2 + SE) and the i n t r a - a n d inter-assay coefficients (%) of variation were 8.0 and 12.9 respectively; all of the samples from an individual animal were assayed together, using either 100 zl undiluted plasma or 100 ~l plasma diluted 1-10 depending on the month of collection.
Statistical analysis The data on the P R L plasma concentrations were subjected to an analysis of variance; the difference between the P R L levels observed in melatonintreated and control ewes were further analysed using the Student's t-test. RESULTS
Hormonal profiles The progesterone plasma concentrations were less than 0.2 ng/mt in all of the group A animals until the end of July; from then onwards, the number of ewes with high progesterone levels (1-4 ng/ml) progressively increased and almost all of the animals showed high levels by the end of August (Fig, 1). None of the ewes in group B showed progesterone plasma levels higher than 0.2 ng/ml before 14 August. These animals started to show progesterone levels ranging between 1 and 4 ng/.ml during the second half of August. All of the
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Fig. 1. Effectoftreatmentwith melatoninon the onsetofovarianactivity:continuousline,treated animals;brokenline,controls.Eweswereconsideredto be cyclingwhenprogesteroneplasmalevels exceeded0.4 ng/ml for at least two consecutivesamples. ewes had shown at least one progesterone plasma peak by the first week of October (Fig. 1 ). The difference between the dates when 50% of the treated or control ewes exhibited ovarian activity was statistically significant (P<0.01). PRL plasma variations in the melatonin-treated and control animals are reported in Fig. 2. In the control ewes, the PRL plasma levels ranged between 150 and 380 ng/ml at the beginning of the experiment and then progressively decreased; the lowest concentrations (10-100 ng/ml) were observed near the end of the experimental period. In the melatonin-treated ewes,the PRL plasma concentrations ranged between 150 and 400 ng/ml before the beginning of the melatonin treatment and showed a sudden and marked decrease after it had started. The PRL plasma levels remained markedly lower than those observed in the control animals until the end of the melatonin treatment; then the PRL plasma profiles reached values similar to those observed in the control ewes (Fig. 2).
Reproductive findings The reproductive parameters are reported in Table 1. The percentage of ewes lambing was 86.6 for the treated animals and 90.0 for the controls. The mean date of lambing was advanced by approximately 20 days (P<0.01) in the melatonin-treated animals compared with the controls. The prolificacy
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iN I I III1
P
Fig. 2. Top panel: PRL plasma profiles (2+ S.D.) in melatonin-treated (continuous line) and control (broken line) ewes during the experimental period; bottom panel: levels of statistically significant differences (t-test) between PRL values observed in melatonin-treated and untreated ewes for each day of sampling. ( l a m b s / p r e g n a n t ewe) was 2 a n d 1.62 in groups A a n d B respectively; this difference was n o t significant. DISCUSSION A c i r c a n n u a l r h y t h m o f c i r c u l a t i n g p r o l a c t i n has b e e n p r e v i o u s l y d e s c r i b e d in sheep ( F o r b e s et al., 1975; R a v a u l t , 1976; L i n c o l n et al., 1978; T h i m o n i e r et TABLE 1 Reproductive parameters observed in ewes chronically treated with melatonin (group A) and in control animals (group B) Parameter
Group A
Group B
Ewes (n) Lambings (n) Date of lambing (2_+SE) Prolificacy (lambs/pregnant ewe)
30 26 (86.6%) 14 January + 2.6 2.00
30 27 (90.0%) 4 February _+5.6 1.62
288 al., 1978) with the highest levels observed in summer and the lowest in winter. The profile of PRL plasma levels we observed in the control ewes (with maximum values in June and m i n i m u m concentrations in October) was, therefore, due to the shortening days (Walton et al., 1977 ). The administration of melatonin was responsible for a sudden decrease in PRL plasma levels and the concentrations remained lower than those observed in the control animals until the end of the experiment. It is, therefore, likely that at least circannual variations in PRL secretion are mediated through melatonin concentrations and, consequently, through the pineal gland (Symons et al., 1983 ). Similar results were reported by Kennaway et al. (1982, 1983), Howland et al. (1984) and Poulton et al. (1986). The fall in P R L plasma concentrations was not coincident with the onset of ovarian activity; this situation is consistent with previous suggestions (Worthy et al., 1985) that the onset of the breeding season is independent of the decreasing PRL plasma concentrations and that it is not possible to manipulate breeding activity by the manipulation of P R L (Kennaway et al., 1982). T h a t the treatment of ewes with melatonin can be effective in advancing the breeding season has been demonstrated by other authors (Nett and Niswender, 1982; Kennaway et al., 1982; Arendt et al., 1983; Nowak and Rodway, 1985; Roche et al., 1985). In our experiment, the onset of ovarian activity was observed 5-6 weeks after the beginning of the treatment. A similar latent period has been reported by Nett and Niswender (1982). The latent period observed by Kennaway et al. (1982) and Nowak and Rodway (1985) was slightly longer. Most of these results were observed following treatment of mature animals with melatonin. To our knowledge, only Nowak and Rodway (1985) and Roche et al. (1985) have induced a significant advancement of ovarian activity, by giving melatonin intravaginally or orally, in immature ewes. Our experiment seems to confirm the data reported by the above-mentioned authors. Even though chronic treatment with melatonin has been investigated as far as ovarian activity is concerned, there are no data about the effect of the treatment on fertility. Our experiment seems to indicate that the onset of the breeding season is characterized by fertile estrus since the ewes given melatonin lambed, on average, 20 days before the control animals. This difference, however, does not exactly reflect the difference between the onset of ovarian activity (monitored by the progesterone determination) observed in the two groups. As already mentioned, the ewes given melatonin showed an ovarian activity advanced by more than one month. We may presume that this discrepancy may be due to the fact that some ewes ovulated without exibiting any behavioural estrus and, therefore, could not be mated. This kind of irregular cycle has often been described in ewes at the beginning of the breeding season; the results of this study seem to confirm this tendency only in melatonin-treated animals. In fact, in almost all of the control ewes, the first progesterone plasma increase was possibly strictly related to the putative date of mating. Recordings
289 of the dates of lambings allowed us to observe t h a t almost all of the animals had become pregnant without previously exhibiting any estrous cycles. The data on prolificacy are also interesting. The difference (2 and 1.62 lambs/pregnant ewe in the treated and control animals respectively) is high even if it is not statistically significant. The t r e a t m e n t with melatonin needs to be extended to a higher number of ewes in order to confirm this difference and assess its possible statistical incidence. The mechanism (s) by which melatonin is likely to improve the ovulation rate also have to be investigated. CONCLUSIONS The results of this experiment provide additional evidence t h a t P R L plasma concentrations, reduced by melatonin administration, do not seem to be directly involved in the resumption of ovarian activity at the end of the anestrous season. Secondly, the administration of melatonin to ewes is effective in advancing the breeding season not only in mature females but also in immature ones. F u r t h e r work is needed to confirm our preliminary results on the fertility and prolificacy of ewes given melatonin. ACKNOWLEDGEMENTS The authors are grateful to Prof. Leo E. Reichert for providing purified ovine P R L (LER-860-2) and Mr. S. Mongiorgi for his skillful technical help. This work was supported by ECC G r a n t No. 3311 and C.N.R., Italy, Special G r a n t I.P.R.A Sub-project 1, Paper No. 1035.
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