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Plasma prolactin concentrations in barren, pregnant and lactating red deer (Cervus elaphus) given melatonin to advance the next breeding season
Animal Reproduction Science, 18 (1989) 77-86
77
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Plasma Prolactin Concentrations in Barren, Pregnant and Lactating Red Deer (Cervus elaphus) given Melatonin to Advance the Next Breeding Season C.L. ADAM, C.E. MOIR and P. SHIACH
Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB (Great Britain) (Accepted 18 July 1988)
ABSTRACT Adam, C.L., Moir, C.E. and Shiach, P., 1989. Plasma prolactin concentrations in barren, pregnant and lactating red deer (Cervus elaphus) given melatonin to advance the next breeding season. Anim. Reprod. Sci., 18: 77-86. The objectives of this trial were to investigate the effects of melatonin treatment initiated in May on plasma prolactin concentrations, growth rate of suckled calves and onset of seasonal oestrous cyclicity in red deer hinds which were either barren or gave birth on average 37 days after the start of treatment. Six barren and nine pregnant/lactating hinds were each given 5 mg melatonin daily in a feed at 15.30 h from 6 May to 22 September. Six barren and nine pregnant/ lactating deer served as controls by receiving the feed unsupplemented. Plasma prolactin concentrations in control deer increased during May (from about 10 to 40 ng/ml), remained high in June and July (barren: 30-40 ng/ml, lactating: 50-80 ng/ml), and decreased by the end of August to reach basal values (0-7 ng/ml) thereafter. Melatonin treatment significantly lowered plasma prolactin concentrations to basal levels after 21 days (i.e. end of May) in barren hinds, but not until 14 days post-partum in the pregnant/lactating animals (i.e. end of June) after an average of 51 days of treatment. Calves grew at the same rate (g/day, mean + SEM) whether suckled by melatonin-treated (321 _+5.9) or control (326_+8.1) hinds. Mean onset of seasonal ovarian activity was advanced by melatonin treatment in lactating hinds (38 days, P < 0.001) and non-lactating hinds (33 days, P<0.01). In a second concurrent experiment, barren hinds kept indoors in natural daylength were given melatonin as above (n = 2) or remained as untreated controls (n = 2). Prolactin showed two or three distinctive phases of increased plasma concentrations in blood samples taken hourly over intermittent 24-h periods, even when baseline values were undetectable for some of the time, and treatment with melatonin apparently induced a phase-shift in this diurnal pattern.
INTRODUCTION
Melatonin given daily from mid-summer advances the onset of seasonal breeding activity in short-day breeders such as sheep (e.g. Kennaway et al., 0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
78 1982; Nett and Niswender, 1982; Arendt et al., 1983) and red deer (Adam and Atkinson, 1984; Webster and Barrell, 1985; Adam et al., 1986). Results of treatments initiated earlier in the year, aimed at achieving a greater advance of the breeding season, are equivocal for the ewe (English et al., 1986; Nowak and Rodway, 1985; Wigzell et al., 1986) and unknown for the red deer hind. Exogenous melatonin treatments during long days rapidly reduce plasma prolactin concentrations in sheep (Kennaway et al., 1982; Symons et al., 1983) and in both barren and lactating red deer (Adam et al., 1987). Despite causing hypoprolactinaemia, melatonin did not affect milk yields and maintenance of lactation in the red deer. However, an earlier start to melatonin treatment, in late pregnancy, might be expected to compromise the initiation and establishment of lactation and subsequent milk yields. In the present trial, melatonin was administered daily from early May to both barren hinds and pregnant hinds which subsequently gave birth and nursed a calf during the course of treatment. The objectives were to determine the effects of such treatment on the time of onset of seasonal oestrous cyclicity (detected from plasma progesterone concentrations (Adam et al., 1985)), on plasma prolactin concentrations in blood samples taken twice-weekly throughout and taken hourly over intermittent 24-h periods, and on the growth rates of the suckled calves. MATERIALSAND METHODS
Experimental protocol (Experiment 1) Thirty-four tame, mature red deer hinds, aged 3-6 years and of average initial live weight ( _+SD ) 90 + 1.9 kg, were assigned to experimental groups on 5 May 1986 (Day 0). Eighteen of the hinds were pregnant, gave birth on average date 11 June (Day 37, range 12-60 days following the start of the experiment), and suckled their singleton calves during the trial. From Day 1 to Day 140, nine pregnant/lactating (Group ML) and six barren (Group MB) hinds were given 5 mg melatonin in 500 g pelleted concentrates daily per head at 15.30 h; thereafter no melatonin was added to the feed. Control hinds, comprising nine pregnant/lactating (Group CL) and six barren (Group CB) hinds, received the feed unsupplemented throughout. Hinds were initially assigned to Groups ML or CL by matching expected calving dates and to Groups MB or CB at random. Hinds in the four groups grazed adjacent ryegrass-clover paddocks on the Duthie Farm, Aberdeen, U.K. (57 ° 10'N). Calves were weighed at birth and weekly thereafter until weaning on 17 October (Day 165). Blood samples were collected from all hinds by jugular venipuncture into heparinized vacutainer tubes at 09.00-10.00 h on Mondays and Thursdays from
79 Day 0 to Day 190 (11 November). The plasma was then stored at - 2 0 °C until required for progesterone and prolactin assays.
Experimental protocol (Experiment 2) In a concurrent experiment, four barren hinds were maintained indoors on a hay-based ration and under conditions of natural daylight. Hinds 1 and 2 were controls (Group CBf) while hinds 3 and 4 received melatonin exactly as their outdoor counterparts (Group MBf). In addition to twice-weekly blood sampling as above for plasma progesterone determinations, on the mornings of Days 29, 57, 85, 113 and 148 they were each fitted with an indwelling jugular catheter for more frequent withdrawal of blood samples. The catheter was inserted under general anaesthesia (Rompun: Bayer, Bury St. Edmunds, Suffolk, U.K. ) which was reversed by an antidote (Yohimbine: Sigma, Poole, Dorset, U.K.). They were then penned individually and blood samples were withdrawn from the catheters with no further restraint necessary into heparinized tubes at 14.00 h and hourly for 24 h, after which the catheters were removed. During darkness, blood was collected under faint red illumination. All plasma samples were then stored at - 2 0 ° C until required for prolactin assay.
Prolactin assay Prolactin concentrations in plasma samples were measured in duplicate by a radioimmunoassay method based on that of Chesworth (1977) as described by Adam et al. (1987). Ovine prolactin (o-PRL-I-1) was used for iodination and standards, and rabbit ovine-prolactin antiserum (anti-o-PRL-1) was used at a final dilution of 1:200 000 (NIADDK, Bethesda, MA, U.S.A.). Donkey rabbit-antiserum (SAPU, Carluke, Lanarkshire, U.K.) was used as the immunoprecipitant. The antiserum was reported to have < 0.01% cross-reaction with ovine LH, GH, FSH and TSH. Increasing amounts of deer plasma gave a curve which paralleled the ovine prolactin standard curve, and recovery of ovine prolactin added to deer plasma was 100-112%. Inter- and intra-assay coefficients of variation were 13 and 8%, respectively, determined at a prolactin concentration of 23.8 ng/ml. The sensitivity of the assay, determined as 2 standard deviations of the zero standard, was better than 2 ng/ml.
Progesterone assay Progesterone concentrations in plasma were measured by a modification of the radioimmunoassay method ofHenricks et al. (1971), a s described by Adam and Atkinson (1984). The antiserum (HP/S/53-IIc from Guildhay Antisera,
80 University of Surrey, U.K. ) was raised in sheep to progesterone-11a-hemisuccinate-ovalbumen and cross-reactions were 0.3% with 17-hydroxyprogesterone, 0.8% with corticosterone, 0.9% with deoxycorticosterone, and negligible for other steroids. Extraction efficiency was measured by determining the recovery of tritiated progesterone added to deer plasma with each assay run. This averaged 95% and sample values were corrected accordingly. Inter- and intraassay coefficients of variation were 13 and 9%, respectively, determined at progesterone concentration 1.3 ng/ml. The sensitivity of the assay was 0.2 ng/ ml.
Statistical methods
Results are given as means and standard errors of the mean ( S E M ) , except where the S E M was less than the diameter of the points used in Fig. 1, and were analysed by Student's t test.
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Fig. 1. Mean ( + SEM) plasma prolactin concentrations in melatonin-fed (O) and control (©), barren (I) and pregnant/lactating (II) red deer hinds. Asterisks indicate significant differences between treatment groups within each graph (P < 0.05, Student's t test).
81 RESULTS
Experiment I Onset of seasonal ovarian activity was determined from a sustained increase in plasma progesterone concentrations above 1 ng/ml (Adam et al., 1985 ). The mean date of estimated first ovulation was significantly earlier for Group ML vs CL (38 days, P < 0 . 0 0 1 ) , for Group MB vs CB (33 days, P < 0 . 0 1 ) , and for Group CB vs CL (19 days, P < 0 . 0 0 1 ) (Table 1). Mean calf birth weight ( + SEM) was similar in Groups ML (n = 9 ) and CL (n = 9 ), at 8.7 + 0.4 and 8.8 + 0.4 kg, respectively, and the suckled calves grew at a similar rate to 105 days of age (i.e. age of youngest calf at weaning), averaging 321 + 5.9 and 326 _+8.1 g/day, respectively. Mean twice-weekly plasma prolactin concentrations for the four groups during the course of the trial are shown in Fig. 1. Data are related to calving date for individual hinds in Groups ML and CL and are arranged around the average calving date of 11 June (corresponding to Day 37 from the start of the experiment) for comparison with barren hinds in Groups MB and CB over the same period. Prolactin concentrations were lowered significantly by melatonin treatment ( P < 0.05 ) after 21 days in barren hinds, but not until 14 days after parturition in pregnant/lactating hinds, i.e. after an average of 51 days of melatonin. Prolactin concentrations thereafter remained lower in melatonin-fed hinds until control concentrations fell to similar values by Day 112 (25 August) for both barren and lactating animals.
Experiment 2 Seasonal ovarian activity commenced later for CBf hinds 1 and 2 (both on Day 161 ) than for MBf hinds 3 and 4 (Days 122 and 108, respectively). TABLE 1 Mean time of onset of breeding season for melatonin-fed (M) and control (C), barren (B) and lactating (L) red deer hinds Group
ML MB CL CB
n
9 6 9 6
Onset of breeding Day of experiment
SEM
Calendar date
134 120 172 153
4.8 10.0 3.0 1.9
16 Sept. a 2 Sept. b 24 Oct? 5 Oct. a
a vs ¢, c vs d, p < 0.001; b vs a, p < 0.01 (Student's t test).
82
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Hind 2
Hind 3
Hind 4
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Fig. 2. Individual 24-h plasma prolactin profiles for two control (CBf) and two melatonin-fed (MBf) hinds taken on 29, 57, 85, 113 and 148 days from start of trial. The arrows indicate time of melatonin feeding and the solid horizontal bar indicates the duration of natural darkness. Individual profiles of plasma prolactin concentrations over 24 h showed distinctive bi- or tri-phasic patterns, even when baseline values were undetectable for some of the time (Fig. 2). Compared with C B f hinds, M B f hinds showed an advanced seasonal decline in overall prolactin values (as in the main groups), an earlier shift in the timing of the increases in circulating concentrations of prolactin, and increased prolactin concentrations on Day 148 when melatoninfeeding had ceased and ovarian cyclicity had commenced. DISCUSSION Red deer hinds given melatonin from early M a y in this trial showed a 5-week advance in onset of seasonal oestrous cycles. This was no greater advance than that shown by hinds given melatonin from J u n e (Adam and Atkinson, 1984; Adam et al., 1986) so that the inductive melatonin-feeding period was extended by about a m o n t h to 120-134 days. Furthermore, the May-initiated treatment induced a wider variation in response, as seen in English et al.'s (1986) ewes implanted with melatonin in May. It has been postulated that a minimum period of exposure to long daylength is apparently required before
83 sheep are sensitive to a subsequent short-day (or melatonin) signal (Foster, 1983; Lincoln and Ebling, 1985), and it is likely that the precise timing of seasonal development of this sensitivity varies between individuals (English et al., 1986 ). Thus in early May the hinds may have been, to a greater or lesser degree, in a transition phase from initial refractoriness to subsequent sensitivity to the melatonin signal, and this could explain the variability in the time taken to show the breeding response. An indication of this initial refractoriness comes from the relative delay in the inhibition by melatonin of prolactin secretion in barren hinds (21-28 days) compared with melatonin treatments which start in June (15 days: Adam et al., 1987). Furthermore, in the present experiment pregnancy caused complete refractoriness to the effects of melatonin on prolactin secretion. In addition, there was no indication from calf birth weights that melatonin had affected either calf growth rates in utero or the date of parturition. After parturition (average 11 June) plasma prolactin concentrations took just 2 weeks to decrease to basal levels, much the same as when melatonin-feeding is initiated in June (Adam et al., 1987). Although daylength (melatonin secretion pattern ) is of primary importance in regulating prolactin secretion, this signal may apparently be overridden by other factors as appropriate. High blood levels of prolactin are important for the normal initiation of copious lactation in the ruminant (Schams et al., 1972; Johke and Hodate, 1978), but are not required for maintaining an established lactation (Karg et al., 1972; Hart, 1973). Thus, at a time when their barren counterparts had basal values, the melatonin-fed hinds in the present trial had normal (high) plasma prolactin concentrations in late pregnancy; concentrations fell to basal values soon after parturition, and the onset of lactation and milk yields were apparently normal as judged by the suckled calf growth rates. Similarly, ewes lambing out-of-season have high prolactin before parturition at a time when barren ewes have low values in response to the prevailing winter photoperiod (J.J. Robinson, personal communication, 1987). Prolactin secretion from the anterior pituitary is under constant inhibition from the hypothalamus via dopamine production. Where melatonin acts on this system has yet to be identified but its effect is evidently blocked during late pregnancy. Circulating oestrogens in particular increase in concentration during pregnancy and are also known to increase prolactin secretion. For example, oestradiol administration causes a direct rise in circulating prolactin concentrations (Fell et al., 1972), and the normal pre-ovulatory rise in circulating prolactin is thought to be amplified by the coincident rise in circulating oestradiol concentrations (McNeilly, 1980). Although oestrogens may block the effects of dopamine at its pituitary receptor (Labrie et al., 1978), they also increase prolactin secretion by an action at the hypothalamus (see Lancranjan and Friesen, 1978). It is probable, therefore, that melatonin acts at the hypothalamic level to increase dopamine production, an action which is perhaps
84 prevented by the high concentrations of oestrogens present in the pregnant female, since direct administration of a dopamine agonist is indeed able to inhibit prolactin secretion during pregnancy (Schams et al., 1972; Johke and Hodate, 1978). Blood prolactin concentrations are known to fluctuate in sheep over 24-h periods, although a consistent pattern has not emerged. A rise in concentration generally occurs around dusk (e.g. Lincoln et al., 1978; Brown and Forbes, 1980) and bi- or tri-phasic diurnal patterns similar to that shown by the present deer have been reported (Wallace and McNeilly, 1986; Poulton et al., 1987). Interestingly, whatever the circadian pattern, it is not blocked by melatonin suppression through pinealectomy (sheep: Brown and Forbes, 1980) nor by melatonin administration (sheep: Symons et al., 1983; deer: this study). Although actual concentrations of prolactin were reduced in deer given melatonin, there remained distinct episodes of prolactin release, even when baseline values were undetectable. It is possible therefore that whilst melatonin (daylength) controls overall seasonal changes in prolactin secretion, the existence of underlying circadian rhythms of prolactin secretion may not be mediated by the pineal gland (Symons et al., 1983). However, in agreement with Lincoln et al. (1982), this trial indicates that the timing of these rhythms may indeed be sensitive to melatonin. The phase-shift induced by melatonin, which apparently did not occur as acutely as the reduction in prolactin concentrations, was evident on Days 57 and 85 and was similar to the seasonal phase-shift shown by control hinds on Days 113 and 148 (Fig. 2). That oestrous cyclicity had commenced for Hinds 3 and 4 on Days 122 and 108, respectively, might explain the increase in their plasma prolactin values on subsequent frequentsampling dates (Fig. 2), since prolactin levels increase in the pre-ovulatory period (Lamming et al., 1974) and to a lesser extent during the luteal phase in ewes (Wallace and McNeilly, 1986). In addition, the increase in prolactin values on Day 148 may also represent a 'rebound' effect following the end of the suppressive melatonin treatment (Poulton et al., 1987), although no such significant effect was detected in serial samples from the main groups of deer outdoors (Fig. 1). Whether or not these observed changes in prolactin secretion represent a mechanism whereby melatonin controls seasonal breeding is unknown. Circulating prolactin concentrations per se are not of primary importance since the prolonged administration of a dopamine agonist to suppress prolactin secretion at the pituitary level does not advance the breeding season (e.g. Schanbacher, 1980). However, alterations by melatonin at the hypothalamic level to prolactin secretion and its diurnal pattern of release may indeed be significant. ACKNOWLEDGEMENTS We thank NIADDK and SAPU for the provision of antisera, the staff of the Duthie Farm for assistance with feeding and handling the animals, and Mr. T. Atkinson for assistance with the hormone assays.
85 REFERENCES Adam, C.L. and Atkinson, T., 1984. Effect of feeding melatonin to red deer (Cervus elaphus) on the onset of the breeding season. J. Reprod. Fertil., 72: 463-466. Adam, C.L., Moir, C.E. and Atkinson, T., 1985. Plasma concentrations of progesterone in female red deer (Cervus elaphus) during the breeding season, pregnancy and anoestrus. J. Reprod. Fertil., 74: 631-636. Adam, C.L., Moir, C.E. and Atkinson, T., 1986. Induction of early breeding in red deer (Cervus elaphus) by melatonin. J. Reprod. Fertil., 76: 569-573. Adam, C.L., Atkinson, T. and Moir, C.E., 1987. Melatonin lowers plasma prolactin levels in female red deer (Cervus elaphus). J. Pineal Res., 4: 13-20. 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. Brown, W.B. and Forbes, J.M., 1980. Diurnal variations of plasma prolactin in growing sheep under two lighting regimes and the effect of pinealectomy. J. Endocrinol., 84: 91-99. Chesworth, J.M., 1977. Radioimmunoassays of ovine LH and ovine prolactin using polymerized second antisera. Anal. Biochem., 80: 31-40. English, J., Poulton, A.L., Arendt, J. and Symons, A.M., 1986. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J. Reprod. Fertil., 77: 321-327. Fell, L.R., Beck, C., Brown, J.M., Cumming, I.A. and Goding, J.R., 1972. Radioimmunoassay for ovine prolactin. The secretion of prolactin as affected by milking, oestradiol administration and onset of parturition. J. Reprod. Fertil., 28."133-134. Foster, D.L., 1983. Photoperiod and sexual maturation in the female lamb: early exposure to short days perturbs oestradiol feedback inhibition of luteinizing hormone secretion and produces abnormal ovarian cycles. Endocrinology, 112:11-17. Hart, I.C., 1973. Effect of 2-bromo-~-ergocryptine on milk yield and the level of prolactin and growth hormone in the blood of the goat at milking. J. Endocrinol., 57: 179-180. Henricks, D.M., Dickey, J.F. and Hill, J.R., 1971. Plasma estrogen and progesterone levels in cows prior to and during estrus. Endocrinology, 89: 1350-1355. Johke, T. and Hodate, K., 1978. Effects of CB154 on serum hormone level and lactogenesis in dairy cows. Endocrinol. Jpn., 25: 67-74. Karg, H., Schas, D. and Reinhardt, V., 1972. Effects of 2-Br-~-ergocryptine on plasma prolactin level and milk yields in cows. Experientia, 28: 574-576. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 1982. Effect of melatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology, 110: 1766-1772. Labrie, F., Baulieu, M., Caron, M.G. and Raymond, V., 1978. The adenohypophyseal dopamine receptor. Specificity and modulation of its activity by estradiol. In: C. Robyn and M. Hatter (Editors), Progress in Prolactin Physiology and Pathology. Elsevier Biomedical Press, Amsterdam, pp. 121-136. Lamming, G.E., Moseley, S.R. and McNeilly, J.R., 1974. Prolactin release in the sheep. J. Reprod. Fertil., 40: 151-168. Lancranjan, I. and Friesen, H.G., 1978. The neural regulation ofprolactin secretion. In: W.L. Veale and K. Lederis (Editors), Current Studies of Hypothalamic Function. S. Karger, Basel, pp. 131-150. 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 effects of a sudden decrease or increase in daylength on prolactin secretion in the ram. J. Reprod. Fertil., 52: 305-311. Lincoln, G.A., Almeida, O.F.X., Klandorf, M. and Cunningham, R.A., 1982. Hourly fluctuations
86 in the blood levels of melatonin, prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone, tri-iodothyronine, thyroxine and cortisol in rams under artificial photoperiods, and the effects of cranial sympathectomy. J. Endocrinol., 92: 237-250. McNeilly, A.S., 1980. Prolactin and the control of gonadotrophin secretion in the female. J. Reprod. Fertil., 58: 537-549. Nett, T.M. and Niswender, G.D., 1982. Influence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology, 17: 645-653. Nowak, R. and Rodway, R.G., 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., 1987. Changes in plasma concentrations of LH, FSH and prolactin in ewes receiving melatonin and short-photoperiod treatments to induce early onset of breeding activity. J. Endocrinol., 112: 103-111. Schams, D., Reinhardt, V. and Karg, H., 1972. Effects of 2-Br-a-ergokryptine on plasma prolactin level during parturition and onset of lactation in cows. Experientia, 28: 697-699. Schanbacher, B., 1980. Relationship of daylength and prolactin to resumption of reproductive activity in anoestrous ewes. J. Anim. Sci., 50: 293-297. Symons, A.M., Arendt, J. and Laud, C.A., 1983. Melatonin feeding decreases prolactin levels in the ewe. J. Endocrinol., 99" 41-46. Wallace, J.M. and McNeiUy, A.S., 1986. Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle. J. Endocrinol., 111: 317-327. Webster, J.R. and BarreU, G.K., 1985. Advancement of reproductive activity, seasonal reduction in prolactin secretion and seasonal pelage changes in pubertal red deer hinds (Cervus elaphus) subjected to artificially shortened daily photoperiod or daily melatonin treatments. J. Reprod. Fertil., 73: 255-260. Wigzell, S., Robinson, J.J., Aitken, R.P. and McKelvey, W.A.C., 1986. The effect of the oral administration of melatonin at two times of the year on ovarian activity in ewes. Anim. Prod., 42:448 {abstract).
77
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Plasma Prolactin Concentrations in Barren, Pregnant and Lactating Red Deer (Cervus elaphus) given Melatonin to Advance the Next Breeding Season C.L. ADAM, C.E. MOIR and P. SHIACH
Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB (Great Britain) (Accepted 18 July 1988)
ABSTRACT Adam, C.L., Moir, C.E. and Shiach, P., 1989. Plasma prolactin concentrations in barren, pregnant and lactating red deer (Cervus elaphus) given melatonin to advance the next breeding season. Anim. Reprod. Sci., 18: 77-86. The objectives of this trial were to investigate the effects of melatonin treatment initiated in May on plasma prolactin concentrations, growth rate of suckled calves and onset of seasonal oestrous cyclicity in red deer hinds which were either barren or gave birth on average 37 days after the start of treatment. Six barren and nine pregnant/lactating hinds were each given 5 mg melatonin daily in a feed at 15.30 h from 6 May to 22 September. Six barren and nine pregnant/ lactating deer served as controls by receiving the feed unsupplemented. Plasma prolactin concentrations in control deer increased during May (from about 10 to 40 ng/ml), remained high in June and July (barren: 30-40 ng/ml, lactating: 50-80 ng/ml), and decreased by the end of August to reach basal values (0-7 ng/ml) thereafter. Melatonin treatment significantly lowered plasma prolactin concentrations to basal levels after 21 days (i.e. end of May) in barren hinds, but not until 14 days post-partum in the pregnant/lactating animals (i.e. end of June) after an average of 51 days of treatment. Calves grew at the same rate (g/day, mean + SEM) whether suckled by melatonin-treated (321 _+5.9) or control (326_+8.1) hinds. Mean onset of seasonal ovarian activity was advanced by melatonin treatment in lactating hinds (38 days, P < 0.001) and non-lactating hinds (33 days, P<0.01). In a second concurrent experiment, barren hinds kept indoors in natural daylength were given melatonin as above (n = 2) or remained as untreated controls (n = 2). Prolactin showed two or three distinctive phases of increased plasma concentrations in blood samples taken hourly over intermittent 24-h periods, even when baseline values were undetectable for some of the time, and treatment with melatonin apparently induced a phase-shift in this diurnal pattern.
INTRODUCTION
Melatonin given daily from mid-summer advances the onset of seasonal breeding activity in short-day breeders such as sheep (e.g. Kennaway et al., 0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
78 1982; Nett and Niswender, 1982; Arendt et al., 1983) and red deer (Adam and Atkinson, 1984; Webster and Barrell, 1985; Adam et al., 1986). Results of treatments initiated earlier in the year, aimed at achieving a greater advance of the breeding season, are equivocal for the ewe (English et al., 1986; Nowak and Rodway, 1985; Wigzell et al., 1986) and unknown for the red deer hind. Exogenous melatonin treatments during long days rapidly reduce plasma prolactin concentrations in sheep (Kennaway et al., 1982; Symons et al., 1983) and in both barren and lactating red deer (Adam et al., 1987). Despite causing hypoprolactinaemia, melatonin did not affect milk yields and maintenance of lactation in the red deer. However, an earlier start to melatonin treatment, in late pregnancy, might be expected to compromise the initiation and establishment of lactation and subsequent milk yields. In the present trial, melatonin was administered daily from early May to both barren hinds and pregnant hinds which subsequently gave birth and nursed a calf during the course of treatment. The objectives were to determine the effects of such treatment on the time of onset of seasonal oestrous cyclicity (detected from plasma progesterone concentrations (Adam et al., 1985)), on plasma prolactin concentrations in blood samples taken twice-weekly throughout and taken hourly over intermittent 24-h periods, and on the growth rates of the suckled calves. MATERIALSAND METHODS
Experimental protocol (Experiment 1) Thirty-four tame, mature red deer hinds, aged 3-6 years and of average initial live weight ( _+SD ) 90 + 1.9 kg, were assigned to experimental groups on 5 May 1986 (Day 0). Eighteen of the hinds were pregnant, gave birth on average date 11 June (Day 37, range 12-60 days following the start of the experiment), and suckled their singleton calves during the trial. From Day 1 to Day 140, nine pregnant/lactating (Group ML) and six barren (Group MB) hinds were given 5 mg melatonin in 500 g pelleted concentrates daily per head at 15.30 h; thereafter no melatonin was added to the feed. Control hinds, comprising nine pregnant/lactating (Group CL) and six barren (Group CB) hinds, received the feed unsupplemented throughout. Hinds were initially assigned to Groups ML or CL by matching expected calving dates and to Groups MB or CB at random. Hinds in the four groups grazed adjacent ryegrass-clover paddocks on the Duthie Farm, Aberdeen, U.K. (57 ° 10'N). Calves were weighed at birth and weekly thereafter until weaning on 17 October (Day 165). Blood samples were collected from all hinds by jugular venipuncture into heparinized vacutainer tubes at 09.00-10.00 h on Mondays and Thursdays from
79 Day 0 to Day 190 (11 November). The plasma was then stored at - 2 0 °C until required for progesterone and prolactin assays.
Experimental protocol (Experiment 2) In a concurrent experiment, four barren hinds were maintained indoors on a hay-based ration and under conditions of natural daylight. Hinds 1 and 2 were controls (Group CBf) while hinds 3 and 4 received melatonin exactly as their outdoor counterparts (Group MBf). In addition to twice-weekly blood sampling as above for plasma progesterone determinations, on the mornings of Days 29, 57, 85, 113 and 148 they were each fitted with an indwelling jugular catheter for more frequent withdrawal of blood samples. The catheter was inserted under general anaesthesia (Rompun: Bayer, Bury St. Edmunds, Suffolk, U.K. ) which was reversed by an antidote (Yohimbine: Sigma, Poole, Dorset, U.K.). They were then penned individually and blood samples were withdrawn from the catheters with no further restraint necessary into heparinized tubes at 14.00 h and hourly for 24 h, after which the catheters were removed. During darkness, blood was collected under faint red illumination. All plasma samples were then stored at - 2 0 ° C until required for prolactin assay.
Prolactin assay Prolactin concentrations in plasma samples were measured in duplicate by a radioimmunoassay method based on that of Chesworth (1977) as described by Adam et al. (1987). Ovine prolactin (o-PRL-I-1) was used for iodination and standards, and rabbit ovine-prolactin antiserum (anti-o-PRL-1) was used at a final dilution of 1:200 000 (NIADDK, Bethesda, MA, U.S.A.). Donkey rabbit-antiserum (SAPU, Carluke, Lanarkshire, U.K.) was used as the immunoprecipitant. The antiserum was reported to have < 0.01% cross-reaction with ovine LH, GH, FSH and TSH. Increasing amounts of deer plasma gave a curve which paralleled the ovine prolactin standard curve, and recovery of ovine prolactin added to deer plasma was 100-112%. Inter- and intra-assay coefficients of variation were 13 and 8%, respectively, determined at a prolactin concentration of 23.8 ng/ml. The sensitivity of the assay, determined as 2 standard deviations of the zero standard, was better than 2 ng/ml.
Progesterone assay Progesterone concentrations in plasma were measured by a modification of the radioimmunoassay method ofHenricks et al. (1971), a s described by Adam and Atkinson (1984). The antiserum (HP/S/53-IIc from Guildhay Antisera,
80 University of Surrey, U.K. ) was raised in sheep to progesterone-11a-hemisuccinate-ovalbumen and cross-reactions were 0.3% with 17-hydroxyprogesterone, 0.8% with corticosterone, 0.9% with deoxycorticosterone, and negligible for other steroids. Extraction efficiency was measured by determining the recovery of tritiated progesterone added to deer plasma with each assay run. This averaged 95% and sample values were corrected accordingly. Inter- and intraassay coefficients of variation were 13 and 9%, respectively, determined at progesterone concentration 1.3 ng/ml. The sensitivity of the assay was 0.2 ng/ ml.
Statistical methods
Results are given as means and standard errors of the mean ( S E M ) , except where the S E M was less than the diameter of the points used in Fig. 1, and were analysed by Student's t test.
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00
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112
140
168
Days from start of trial
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Fig. 1. Mean ( + SEM) plasma prolactin concentrations in melatonin-fed (O) and control (©), barren (I) and pregnant/lactating (II) red deer hinds. Asterisks indicate significant differences between treatment groups within each graph (P < 0.05, Student's t test).
81 RESULTS
Experiment I Onset of seasonal ovarian activity was determined from a sustained increase in plasma progesterone concentrations above 1 ng/ml (Adam et al., 1985 ). The mean date of estimated first ovulation was significantly earlier for Group ML vs CL (38 days, P < 0 . 0 0 1 ) , for Group MB vs CB (33 days, P < 0 . 0 1 ) , and for Group CB vs CL (19 days, P < 0 . 0 0 1 ) (Table 1). Mean calf birth weight ( + SEM) was similar in Groups ML (n = 9 ) and CL (n = 9 ), at 8.7 + 0.4 and 8.8 + 0.4 kg, respectively, and the suckled calves grew at a similar rate to 105 days of age (i.e. age of youngest calf at weaning), averaging 321 + 5.9 and 326 _+8.1 g/day, respectively. Mean twice-weekly plasma prolactin concentrations for the four groups during the course of the trial are shown in Fig. 1. Data are related to calving date for individual hinds in Groups ML and CL and are arranged around the average calving date of 11 June (corresponding to Day 37 from the start of the experiment) for comparison with barren hinds in Groups MB and CB over the same period. Prolactin concentrations were lowered significantly by melatonin treatment ( P < 0.05 ) after 21 days in barren hinds, but not until 14 days after parturition in pregnant/lactating hinds, i.e. after an average of 51 days of melatonin. Prolactin concentrations thereafter remained lower in melatonin-fed hinds until control concentrations fell to similar values by Day 112 (25 August) for both barren and lactating animals.
Experiment 2 Seasonal ovarian activity commenced later for CBf hinds 1 and 2 (both on Day 161 ) than for MBf hinds 3 and 4 (Days 122 and 108, respectively). TABLE 1 Mean time of onset of breeding season for melatonin-fed (M) and control (C), barren (B) and lactating (L) red deer hinds Group
ML MB CL CB
n
9 6 9 6
Onset of breeding Day of experiment
SEM
Calendar date
134 120 172 153
4.8 10.0 3.0 1.9
16 Sept. a 2 Sept. b 24 Oct? 5 Oct. a
a vs ¢, c vs d, p < 0.001; b vs a, p < 0.01 (Student's t test).
82
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Hind 2
Hind 3
Hind 4
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Fig. 2. Individual 24-h plasma prolactin profiles for two control (CBf) and two melatonin-fed (MBf) hinds taken on 29, 57, 85, 113 and 148 days from start of trial. The arrows indicate time of melatonin feeding and the solid horizontal bar indicates the duration of natural darkness. Individual profiles of plasma prolactin concentrations over 24 h showed distinctive bi- or tri-phasic patterns, even when baseline values were undetectable for some of the time (Fig. 2). Compared with C B f hinds, M B f hinds showed an advanced seasonal decline in overall prolactin values (as in the main groups), an earlier shift in the timing of the increases in circulating concentrations of prolactin, and increased prolactin concentrations on Day 148 when melatoninfeeding had ceased and ovarian cyclicity had commenced. DISCUSSION Red deer hinds given melatonin from early M a y in this trial showed a 5-week advance in onset of seasonal oestrous cycles. This was no greater advance than that shown by hinds given melatonin from J u n e (Adam and Atkinson, 1984; Adam et al., 1986) so that the inductive melatonin-feeding period was extended by about a m o n t h to 120-134 days. Furthermore, the May-initiated treatment induced a wider variation in response, as seen in English et al.'s (1986) ewes implanted with melatonin in May. It has been postulated that a minimum period of exposure to long daylength is apparently required before
83 sheep are sensitive to a subsequent short-day (or melatonin) signal (Foster, 1983; Lincoln and Ebling, 1985), and it is likely that the precise timing of seasonal development of this sensitivity varies between individuals (English et al., 1986 ). Thus in early May the hinds may have been, to a greater or lesser degree, in a transition phase from initial refractoriness to subsequent sensitivity to the melatonin signal, and this could explain the variability in the time taken to show the breeding response. An indication of this initial refractoriness comes from the relative delay in the inhibition by melatonin of prolactin secretion in barren hinds (21-28 days) compared with melatonin treatments which start in June (15 days: Adam et al., 1987). Furthermore, in the present experiment pregnancy caused complete refractoriness to the effects of melatonin on prolactin secretion. In addition, there was no indication from calf birth weights that melatonin had affected either calf growth rates in utero or the date of parturition. After parturition (average 11 June) plasma prolactin concentrations took just 2 weeks to decrease to basal levels, much the same as when melatonin-feeding is initiated in June (Adam et al., 1987). Although daylength (melatonin secretion pattern ) is of primary importance in regulating prolactin secretion, this signal may apparently be overridden by other factors as appropriate. High blood levels of prolactin are important for the normal initiation of copious lactation in the ruminant (Schams et al., 1972; Johke and Hodate, 1978), but are not required for maintaining an established lactation (Karg et al., 1972; Hart, 1973). Thus, at a time when their barren counterparts had basal values, the melatonin-fed hinds in the present trial had normal (high) plasma prolactin concentrations in late pregnancy; concentrations fell to basal values soon after parturition, and the onset of lactation and milk yields were apparently normal as judged by the suckled calf growth rates. Similarly, ewes lambing out-of-season have high prolactin before parturition at a time when barren ewes have low values in response to the prevailing winter photoperiod (J.J. Robinson, personal communication, 1987). Prolactin secretion from the anterior pituitary is under constant inhibition from the hypothalamus via dopamine production. Where melatonin acts on this system has yet to be identified but its effect is evidently blocked during late pregnancy. Circulating oestrogens in particular increase in concentration during pregnancy and are also known to increase prolactin secretion. For example, oestradiol administration causes a direct rise in circulating prolactin concentrations (Fell et al., 1972), and the normal pre-ovulatory rise in circulating prolactin is thought to be amplified by the coincident rise in circulating oestradiol concentrations (McNeilly, 1980). Although oestrogens may block the effects of dopamine at its pituitary receptor (Labrie et al., 1978), they also increase prolactin secretion by an action at the hypothalamus (see Lancranjan and Friesen, 1978). It is probable, therefore, that melatonin acts at the hypothalamic level to increase dopamine production, an action which is perhaps
84 prevented by the high concentrations of oestrogens present in the pregnant female, since direct administration of a dopamine agonist is indeed able to inhibit prolactin secretion during pregnancy (Schams et al., 1972; Johke and Hodate, 1978). Blood prolactin concentrations are known to fluctuate in sheep over 24-h periods, although a consistent pattern has not emerged. A rise in concentration generally occurs around dusk (e.g. Lincoln et al., 1978; Brown and Forbes, 1980) and bi- or tri-phasic diurnal patterns similar to that shown by the present deer have been reported (Wallace and McNeilly, 1986; Poulton et al., 1987). Interestingly, whatever the circadian pattern, it is not blocked by melatonin suppression through pinealectomy (sheep: Brown and Forbes, 1980) nor by melatonin administration (sheep: Symons et al., 1983; deer: this study). Although actual concentrations of prolactin were reduced in deer given melatonin, there remained distinct episodes of prolactin release, even when baseline values were undetectable. It is possible therefore that whilst melatonin (daylength) controls overall seasonal changes in prolactin secretion, the existence of underlying circadian rhythms of prolactin secretion may not be mediated by the pineal gland (Symons et al., 1983). However, in agreement with Lincoln et al. (1982), this trial indicates that the timing of these rhythms may indeed be sensitive to melatonin. The phase-shift induced by melatonin, which apparently did not occur as acutely as the reduction in prolactin concentrations, was evident on Days 57 and 85 and was similar to the seasonal phase-shift shown by control hinds on Days 113 and 148 (Fig. 2). That oestrous cyclicity had commenced for Hinds 3 and 4 on Days 122 and 108, respectively, might explain the increase in their plasma prolactin values on subsequent frequentsampling dates (Fig. 2), since prolactin levels increase in the pre-ovulatory period (Lamming et al., 1974) and to a lesser extent during the luteal phase in ewes (Wallace and McNeilly, 1986). In addition, the increase in prolactin values on Day 148 may also represent a 'rebound' effect following the end of the suppressive melatonin treatment (Poulton et al., 1987), although no such significant effect was detected in serial samples from the main groups of deer outdoors (Fig. 1). Whether or not these observed changes in prolactin secretion represent a mechanism whereby melatonin controls seasonal breeding is unknown. Circulating prolactin concentrations per se are not of primary importance since the prolonged administration of a dopamine agonist to suppress prolactin secretion at the pituitary level does not advance the breeding season (e.g. Schanbacher, 1980). However, alterations by melatonin at the hypothalamic level to prolactin secretion and its diurnal pattern of release may indeed be significant. ACKNOWLEDGEMENTS We thank NIADDK and SAPU for the provision of antisera, the staff of the Duthie Farm for assistance with feeding and handling the animals, and Mr. T. Atkinson for assistance with the hormone assays.
85 REFERENCES Adam, C.L. and Atkinson, T., 1984. Effect of feeding melatonin to red deer (Cervus elaphus) on the onset of the breeding season. J. Reprod. Fertil., 72: 463-466. Adam, C.L., Moir, C.E. and Atkinson, T., 1985. Plasma concentrations of progesterone in female red deer (Cervus elaphus) during the breeding season, pregnancy and anoestrus. J. Reprod. Fertil., 74: 631-636. Adam, C.L., Moir, C.E. and Atkinson, T., 1986. Induction of early breeding in red deer (Cervus elaphus) by melatonin. J. Reprod. Fertil., 76: 569-573. Adam, C.L., Atkinson, T. and Moir, C.E., 1987. Melatonin lowers plasma prolactin levels in female red deer (Cervus elaphus). J. Pineal Res., 4: 13-20. 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. Brown, W.B. and Forbes, J.M., 1980. Diurnal variations of plasma prolactin in growing sheep under two lighting regimes and the effect of pinealectomy. J. Endocrinol., 84: 91-99. Chesworth, J.M., 1977. Radioimmunoassays of ovine LH and ovine prolactin using polymerized second antisera. Anal. Biochem., 80: 31-40. English, J., Poulton, A.L., Arendt, J. and Symons, A.M., 1986. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J. Reprod. Fertil., 77: 321-327. Fell, L.R., Beck, C., Brown, J.M., Cumming, I.A. and Goding, J.R., 1972. Radioimmunoassay for ovine prolactin. The secretion of prolactin as affected by milking, oestradiol administration and onset of parturition. J. Reprod. Fertil., 28."133-134. Foster, D.L., 1983. Photoperiod and sexual maturation in the female lamb: early exposure to short days perturbs oestradiol feedback inhibition of luteinizing hormone secretion and produces abnormal ovarian cycles. Endocrinology, 112:11-17. Hart, I.C., 1973. Effect of 2-bromo-~-ergocryptine on milk yield and the level of prolactin and growth hormone in the blood of the goat at milking. J. Endocrinol., 57: 179-180. Henricks, D.M., Dickey, J.F. and Hill, J.R., 1971. Plasma estrogen and progesterone levels in cows prior to and during estrus. Endocrinology, 89: 1350-1355. Johke, T. and Hodate, K., 1978. Effects of CB154 on serum hormone level and lactogenesis in dairy cows. Endocrinol. Jpn., 25: 67-74. Karg, H., Schas, D. and Reinhardt, V., 1972. Effects of 2-Br-~-ergocryptine on plasma prolactin level and milk yields in cows. Experientia, 28: 574-576. Kennaway, D.J., Gilmore, T.A. and Seamark, R.F., 1982. Effect of melatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology, 110: 1766-1772. Labrie, F., Baulieu, M., Caron, M.G. and Raymond, V., 1978. The adenohypophyseal dopamine receptor. Specificity and modulation of its activity by estradiol. In: C. Robyn and M. Hatter (Editors), Progress in Prolactin Physiology and Pathology. Elsevier Biomedical Press, Amsterdam, pp. 121-136. Lamming, G.E., Moseley, S.R. and McNeilly, J.R., 1974. Prolactin release in the sheep. J. Reprod. Fertil., 40: 151-168. Lancranjan, I. and Friesen, H.G., 1978. The neural regulation ofprolactin secretion. In: W.L. Veale and K. Lederis (Editors), Current Studies of Hypothalamic Function. S. Karger, Basel, pp. 131-150. 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 effects of a sudden decrease or increase in daylength on prolactin secretion in the ram. J. Reprod. Fertil., 52: 305-311. Lincoln, G.A., Almeida, O.F.X., Klandorf, M. and Cunningham, R.A., 1982. Hourly fluctuations
86 in the blood levels of melatonin, prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone, tri-iodothyronine, thyroxine and cortisol in rams under artificial photoperiods, and the effects of cranial sympathectomy. J. Endocrinol., 92: 237-250. McNeilly, A.S., 1980. Prolactin and the control of gonadotrophin secretion in the female. J. Reprod. Fertil., 58: 537-549. Nett, T.M. and Niswender, G.D., 1982. Influence of exogenous melatonin on seasonality of reproduction in sheep. Theriogenology, 17: 645-653. Nowak, R. and Rodway, R.G., 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., 1987. Changes in plasma concentrations of LH, FSH and prolactin in ewes receiving melatonin and short-photoperiod treatments to induce early onset of breeding activity. J. Endocrinol., 112: 103-111. Schams, D., Reinhardt, V. and Karg, H., 1972. Effects of 2-Br-a-ergokryptine on plasma prolactin level during parturition and onset of lactation in cows. Experientia, 28: 697-699. Schanbacher, B., 1980. Relationship of daylength and prolactin to resumption of reproductive activity in anoestrous ewes. J. Anim. Sci., 50: 293-297. Symons, A.M., Arendt, J. and Laud, C.A., 1983. Melatonin feeding decreases prolactin levels in the ewe. J. Endocrinol., 99" 41-46. Wallace, J.M. and McNeiUy, A.S., 1986. Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle. J. Endocrinol., 111: 317-327. Webster, J.R. and BarreU, G.K., 1985. Advancement of reproductive activity, seasonal reduction in prolactin secretion and seasonal pelage changes in pubertal red deer hinds (Cervus elaphus) subjected to artificially shortened daily photoperiod or daily melatonin treatments. J. Reprod. Fertil., 73: 255-260. Wigzell, S., Robinson, J.J., Aitken, R.P. and McKelvey, W.A.C., 1986. The effect of the oral administration of melatonin at two times of the year on ovarian activity in ewes. Anim. Prod., 42:448 {abstract).