Nocturnal variation of prolactin secretion in the Mouflon (Ovis gmelini musimon) and domestic sheep (Ovis aries): seasonal changes

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1 Animal Reproduction Science 64 (2000) Nocturnal variation of prolactin secretion in the Mouflon (Ovis gmelini musimon) and domestic sheep (Ovis aries): seasonal changes J. Santiago-Moreno a,, A. González-Bulnes a, A. Gómez Brunet a, A. del Campo a, R. Picazo b, A. López Sebastián a a Dpto. Reproducción Animal y Conservación de Recursos Zoogenéticos, SGIT-INIA, Avda., Puerta de Hierro km 5.9, Madrid, Spain b Dpto. Fisiología Animal, Facultad de Veterinaria, UCM, Ctra, Coruña km 5.2, Madrid, Spain Received 11 February 2000; received in revised form 30 June 2000; accepted 21 September 2000 Abstract Seasonal changes in nocturnal prolactin secretion and their relationship with melatonin secretion were monitored in wild (Mouflon, Ovis gmelini musimon) and domesticated sheep (breed Manchega, Ovis aries). Two groups of eleven adult females each, were maintained outdoors under natural photoperiod. Plasma concentrations of prolactin and melatonin were determined during the summer and winter solstices and the autumn and spring equinoxes. Blood samples were collected every 3 h during the night hours, and 1 h before and after the onset of darkness and sunrise. Maximum mean plasma concentrations of prolactin during the dark-phase in Mouflons were observed in the summer solstice, (P <0.001) and in the summer solstice and spring equinox in Manchega ewes (P <0.001). Mean plasma concentrations of prolactin were higher in the wild species (P <0.001) during the summer solstice. In contrast, during the spring equinox, mean levels of prolactin were higher in Manchega ewes than in Mouflons (P <0.05). Plasma prolactin concentrations showed a nocturnal rhythm in both breeds, with seasonal variations (P <0.001). The increase in plasma melatonin levels during the first hour after sunset was accompanied to increasing concentrations of PRL 1 h after the onset of darkness, only in the autumn and spring equinox for the Mouflon, and in the summer solstice and spring equinox for the Manchega ewes. In Mouflons, the fall of plasma PRL concentrations about the middle dark-phase in all the periods studied, coincided with high levels of melatonin. A similar relation was observed in Manchega ewes only in the winter solstice and spring equinox. The current study shows that the nocturnal rhythm of prolactin secretion exhibits seasonal variation; differences in the patterns of prolactin secretion between Mouflon and Manchega sheep are taken to represent the effects of genotype Elsevier Science B.V. All rights reserved. Keywords: Prolactin; Melatonin; Nocturnal; Mouflon; Sheep Corresponding author. Tel.: ; fax: address: moreno@inia.es (J. Santiago-Moreno) /00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (00)00213-X

2 212 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) Introduction Prolactin (PRL) shows an endogenous rhythm of secretion, which is modulated by phototoperiod among other influences, and clearly affected by time of year in mammals. Seasonal changes in plasma concentrations of PRL have been shown to be associated with changes in daylength (Thimonier et al., 1978), with highest and lowest concentrations of PRL occurring during summer (long days) and winter (short days), respectively (Pelletier, 1973; Ravault and Ortavant, 1977). The regulation of this seasonal rhythm of PRL is under pineal control by the secretion of melatonin (Reiter, 1991; Misztal et al., 1994; Lincoln and Tortonese, 1995) whose natural suppressive effect on PRL secretion is predominantly caused by a direct action on the pituitary gland (Lincoln and Clarke, 1994). There is also direct evidence of a genetic influence on the seasonal changes in plasma concentrations of PRL during the light-phase, between rams of wild (Mouflon), feral (Soay) and various domesticated breeds of sheep, which shows that selection for a more continuous pattern of reproduction is accompanied by variations on the annual PRL profile (Lincoln, 1990). In addition to a seasonal pattern of PRL secretion, earlier studies have shown a marked variation in plasma PRL levels between the light and dark-phases of the day (Walton et al., 1980). Seasonal changes in these circadian rhythms in plasma PRL of the ram have been confirmed by Ravault et al. (1987); however, whether melatonin participates in the circadian regulation of PRL secretion is still unclear. In the domestic sheep, the earliest studies showed that the major daily change in plasma PRL concentrations is a rise around dusk (Davis and Borger, 1974; Forbes et al., 1975), although this increase may vary depending on seasonal influences (Misztal et al., 1997; Walton et al., 1980). The Mouflon is the European wild sheep (Ovis gmelini musimon) originated in Sardinia and Corsica, and represents the wild ancestor of the current domesticated breeds of sheep (Ovis aries, Hiendleder et al., 1998). Although, marked seasonal changes in blood plasma concentrations of PRL have been established (Lincoln, 1990), the nyctohemeral variations in prolactinemia have not been estimated in this species. The aims of the current study were to assess the existence of a nocturnal rhythm of secretion of PRL in the Mouflon, and whether possible seasonal differences in the nocturnal rhythms of PRL could be affected by breed in sheep. Manchega breed was selected for this study because, despite it is originated in the Mediterranean area and at similar latitude than Mouflon (40 N), the domestication has modified its reproductive activity in comparison with the wild ancestor (Santiago-Moreno et al., 2000). In addition, this breed is a valuable model to study if the selection for a more continuous pattern of reproduction is accompanied by nyctohemeral variations of the annual PRL profile when compared with the wild ancestor. The temporal relationship between the secretion of PRL and melatonin was also studied. 2. Material and methods 2.1. Experimental design and animals Eleven adult (2 6 years of age), female Mouflons were allocated under natural daylength conditions at the facilities of the Departamento de Reproducción Animal, INIA, Madrid

3 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) (40 25 N) in a 250 m 2 enclosure. The Mouflons came originaly from El Hosquillo National Wildlife Reserve, in central Spain (40 6 N). An acclimatisation period of at least 3 months was allowed before the beginning of the experiment. A group of 11 adult (2 6 years of age) Manchega ewes (Spanish milking breed), were housed under similar conditions in an adjacent enclosure. Plasma PRL and melatonin concentrations were measured during the summer solstice (21 22 June, 15L:9D), the winter solstice (22 23 December, 9L:15D), the autumn equinox (22 23 September, 12L:12D) and the spring equinox (21 22 March, 12L:12D). Blood samples were collected by venepuncture from the jugular vein and plasma was separated within 1 h and stored at 20 C. Blood samples were collected every 3 h during the night hours, and 1 h before and after the onset of darkness and sunrise. Animals were physically restrained and confined in a small enclosure (6 m 2 ) to permit the collection of blood. To alleviate stress during experimental procedures, all animals had been previously restrained in the enclosure destined to blood sampling to get them accustomed to venepuncture. Dim red light (<3 lux) was used to facilitate blood collection at night. The National Observatory of Astronomy in Madrid kindly provided the precise time of dawn and dusk Hormone assays Plasma concentrations of PRL were determined by radioimmunoassay in duplicate 100 l aliquots by a previously described method (Gomez Brunet and Lopez Sebastian, 1991). The samples were analysed in a single assay. Assay sensitivity was 0.3 ng/ml and the intra-assay coefficient of variation was 9.3% (n = 8). Melatonin concentrations were estimated in duplicate aliquots of 100 l of blood plasma by radioimmunoassay using the technique of Fraser et al. (1983) with an antibody raised by Tillet et al. (1986). The sensitivity of the assay was 4 pg/ml of plasma. The inter- and intra-assay coefficients of variation, estimated from plasma pools every 50 unknown samples, were 15 and 9%, respectively Statistical analyses The results are presented as mean ±S.E.M. Plasma concentrations of PRL were analysed by ANOVA with repeated measurements for the effect of time. PRL concentrations were compared by two-way ANOVA for the effects breed and season. The nocturnal increase in PRL was determined by comparing plasma concentrations of PRL 1 h before and after the onset of darkness using a t-test. Timing of decrease in plasma PRL was considered as the time when three consecutive plasma samples had PRL concentrations below the preceding value as determined with a t-test. All statistical procedures were performed with the BMDP, Statistical Software, Inc. 3. Results The seasonal changes in plasma concentrations of PRL, during the dark-phase are illustrated for each breed in Fig. 1. In female Mouflons, the period of highest secretion of PRL

4 214 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) Fig. 1. Seasonal changes in plasma concentrations of PRL during the dark-phase in the Mouflon (n = 11) and Manchega ewes (n = 11). Each value represents the mean ± S.E.M. of plasma concentrations of PRL in each month. Asterisks denote significant differences ( P<0.001; P<0.05). was in June (P <0.001). The Manchega ewes showed the highest levels (P <0.001) in June and March. The highest plasma levels of PRL occurred in the summer solstice in both groups, and plasma concentrations of PRL were higher (P < 0.001) in the wild species than in Manchega ewes. In contrast, during the spring equinox, higher levels of PRL were found in Manchega ewes than in Mouflons (P <0.05). Plasma concentrations of PRL showed a considerable individual variation; however, the profiles of PRL exhibited a day/night rhythm of secretion in both breeds (Figs. 2 and 3), with seasonal variation (P <0.001). In Mouflons, the concentrations of PRL at the middle dark-phase were lower (P < 0.001) than the first sample collected after the onset of the dark-phase, in all the periods studied. In the autumn and spring equinoxes, an increase (P <0.05 and P<0.001, respectively) in PRL occurred 1 h after the onset of darkness, coincident with increasing concentrations of melatonin. Similarly, in the summer solstice and spring equinox, an increase (P <0.001 and P<0.05, respectively) in PRL plasma levels was found 1 h after sunset in Manchega ewes. In Manchega ewes, the concentrations of PRL at the middle dark-phase were lower (P <0.001) than the first sample collected after the onset of the dark-phase in the winter solstice and spring equinox. Day/night variations in prolactinemia were not detected in the autumn equinox. Plasma levels of melatonin in domestic and wild sheep increased (P < 0.01) above day-time levels during the first hour after sunset, and decreased around sunrise. During the light-phase, plasma concentrations of melatonin remained at basal levels (Figs. 2 and 3). 4. Discussion Mean plasma concentrations of PRL during the dark-phase, in Manchega ewes, decreased from June to December and increased again in March, in consistency with prior studies in

5 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) Fig. 2. Plasma concentrations of PRL (ng/ml) and melatonin (pg/ml) (mean±s.e.m.) in Mouflon (n = 11) during the winter solstice (22 23 December), the spring equinox (21 22 March), the summer solstice (21 22 June) and the autumn equinox (22 23 September). The asterisks represent the time of increase ( P<0.05; P<0.001) or decrease ( P<0.001) in plasma levels of PRL. Shaded areas depict darkness. ( ) Melatonin; ( ) PRL. the same breed (Gomez Brunet and Lopez Sebastian, 1991). However, in Mouflons, mean plasma concentrations of PRL in the dark-phase decreased from June to December, with no increase in March unlike in sheep. Unusual low mean levels during the dark-phase of the spring equinox are in contrast with previous studies that have reported an increase of PRL plasma levels in Mouflon rams in March (Lincoln, 1990). Differences in the time of blood sampling (light or dark-phase) might account for the discrepancies with the mentioned studies.

6 216 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) Fig. 3. Plasma concentrations of PRL (ng/ml) and melatonin (pg/ml) (mean±s.e.m.) in Manchega ewes (n = 11) during the winter solstice (22 23 December), the spring equinox (21 22 March), the summer solstice (21 22 June) and the autumn equinox (22 23 September). The asterisks represent the time of increase ( P < 0.05; P<0.001) or decrease ( P<0.001) in plasma levels of PRL. Shaded areas depict darkness. ( ) Melatonin; ( ) PRL. Several studies in domestic sheep have indicated that there is a peak in plasma concentrations of PRL at the time of lights off or dusk (Davis and Borger, 1974; Forbes et al., 1975; McMillen et al., 1987, 1995). Our results show a rise around dusk in the autumn and spring equinoxes for the Mouflon and in the spring equinox and summer solstice for Manchega ewes. In Mouflons, plasma concentrations of PRL remained high by 1 h after sunset in all animals, and fell to reach basal levels at the middle of the dark-phase (Barrell

7 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) and Lapwood, 1978). This is in agreement with findings in other small ruminants, such us Pygmy goats, in which plasma levels of PRL also declined in the dark-phase of the L/D cycle throughout the year, except in August, when a noticeable rise in serum PRL occurred during the dark-phase (Muduuli et al., 1979). Like in Mouflon, Suffolk Greyfaces ewes show a consistent decrease of plasma PRL concentrations around dawn (Brinklow and Forbes, 1984). In Suffolk Greyfaces ewes, the fall of plasma PRL concentrations around dawn is followed by an increase in the middle of the dark-phase, that we have not observed in our experiment. It has been established that the circadian pattern of PRL secretion varies with season in the ewe (Misztal et al., 1997). In the current study, variations in the pattern of PRL secretion in both wild and domestic sheep throughout solstices/equinoxes suggest genetic and seasonal influences on PRL secretion. Seasonal changes in the daily profile of PRL secretion have been reported in Préalpes du Sud rams (Ravault et al., 1987). These authors found an increase in PRL secretion coincident with the transition from the light to dark-phase, which exhibited a circadian rhythmicity in January and April, whereas no such pattern was found in June and September. Therefore, animals seemed to adjust their rhythmic patterns of PRL secretion to increasing (January, April) rather than to decreasing (June, September) daylength. Similarly, in our group of Manchega ewes, the rise of PRL resulting from the change from light to dark, was clearly found in the spring equinox and summer solstice, coinciding with maximum daylength, but not in autumn equinox, when daylength was decreasing. In contrast, in German Merino breed, a neat circadian rhythm of PRL secretion was described in autumn, and not in winter (Serón-Ferré et al., 1989). The influence of daylength on changes in plasma concentrations of PRL has also been evidenced in Cross-breed ewes in which exposure to short-days abolished the nocturnal rise in PRL (Walton et al., 1980). Variations among experiments are considerable and these disparities in the daily rhythms of PRL secretion might be explained by genetic influences. Significant differences in mean plasma concentrations of PRL, between Mouflon and Manchega ewes during the summer solstice and spring equinox, reinforce this suggestion. Our experiment does not allow us to establish a clear relation between the pattern of melatonin secretion and the nocturnal variations of PRL secretion. First, the increase in PRL plasma level found 1 h after the onset of darkness in the autumn and spring equinoxes in the Mouflon, and in the winter solstice and spring equinox in Manchega ewes, were accompanied with increasing concentrations of melatonin. On the other hand, the fall of plasma PRL concentrations to basal levels at the middle of the dark-phase coincided with high levels of melatonin. In this regard, it has been suggested that melatonin might stimulate the evening rise of PRL secretion in Polish Lowland ewes, and that this effect might turn to be inhibitory depending on time of year (Misztal et al., 1997). Although, different studies described an influence of melatonin on the circadian rhythm of PRL (Barrell and Lapwood, 1978; Parraguez et al., 1991), controversy surrounds to the possible mechanisms whereby changes in photoperiod regulate the daily profile of PRL secretion (Poulton et al., 1989; McMillen and Walker, 1991; Houghton et al., 1995). In conclusion, the present study has described for the first time, the day/night variations of PRL secretion and the seasonal differences in the nocturnal rhythm of secretion of this hormone, in a wild and domesticated breed of sheep. The results clearly demonstrate that the time of year determine the changes in the nocturnal variations of PRL secretion; the

8 218 J. Santiago-Moreno et al. / Animal Reproduction Science 64 (2000) wide difference in the patterns of PRL secretion between Mouflon and Manchega are taken to represent the effects of genotype. Acknowledgements This work was supported by DGICYT Grant PB References Barrell, G.K., Lapwood, K.R., Effects of pinealectomy of rams on secretory profiles of luteinizing hormone, testosterone, prolactin and cortisol. Neuroendocrinology 27, Brinklow, B.R., Forbes, J.M., The effect of short and skeleton long photoperiods on the plasma concentrations of prolactin and cortisol in sheep. Reprod. Nutr. Dev. 24, Davis, S.L., Borger, M.L., Dynamic changes in plasma prolactin, luteinizing hormone and growth hormone in ovariectomized ewes. J. Anim. Sci. 38, Forbes, J.M., Driver, P.M., El Shahat, A.A., Boaz, T.G., Scanes, C.G., The effect of daylength and level of feeding serum prolactin in growing lambs. J. Endocrinol. 64, Fraser, S., Cowen, P., Franclin, M., Franey, C., Arendt, J., Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29, Gomez Brunet, A., Lopez Sebastian, A., Effect of season on plasma concentrations of prolactin and cortisol in pregnannt, non-pregnant and lactating ewes. Anim. Reprod. Sci. 26, Hiendleder, S., Mainz, K., Plante, Y., Lewalski, H., Analysis of mitochondrial DNA indicates that domestic sheep are derived from two different ancestral maternal sources: no evidence for contribution from Urial and Argali sheep. J. Hered. 89, Houghton, D.C., Young, I.R., McMillen, I.C., Evidence for hypothalamic control of the diurnal rhythms in prolactin and melatonin in the fetal sheep during late gestation. Endocrinology 136, Lincoln, G.A., Correlation with changes in horns and pelage, but not reproduction, of seasonal cycles in the secretion of prolactin in rams of wild, feral and domesticated breeds of sheep. J. Reprod. Fertil. 90, Lincoln, G.A., Clarke, I.J., Photoperiodically-induced cycles in the secretion of prolactin in hypothalamo-pituitary disconnected rams: evidence for translation of the melatonin signal in the pituitary gland. J. Neuroendocrinol. 6, Lincoln, G.A., Tortonese, D.J., Does melatonin act on dopaminergic pathways in the mediobasal hypothalamus to mediate effects of photoperiod on prolactin seretion in the ram? Neuroendocrinology 62, McMillen, I.C., Thorburn, G.D., Walker, D.W., Diurnal variation in plasma concentrations of cortisol, prolactin, growth hormone and glucose in the fetal sheep pregnant ewe during late gestation. J. Endocrinol. 114, McMillen, I.C., Walker, D.W., Effects of different lighting regimes on daily hormonal and behavioural rhythms in the pregnant ewe and sheep fetus. J. Physiol. 442, McMillen, I.C., Houghton, D.C., Young, I.R., Melatonin and the development of circadian and seasonal rhythmicity. J. Reprod. Fertil. 49 (Suppl. 49), Misztal, T., Romanowicz, K., Barcikowski, B., Seasonal changes of rhythms of melatonin and prolactin secretion in sheep have an opposite character. Eur. J. Endocrinol. 130 (Suppl. 2), Misztal, T., Romanowicz, K., Barcikowski, B., Natural and melatonin-stimulated changes in the circadian rhythm of prolactin secretion in the ewe during seasonal anestrus. Neuroendocrinology 66, Muduuli, D.S., Sanford, L.M., Palmer, W.M., Howland, B.E., Secretory patterns and circadian and seasonal changes in luteinizing hormone, follicle stimulating hormone, prolactin and testosterone in the male pygmy goat. J. Anim. Sci. 49, Parraguez, V.H., Vergara, M., Holmgren, N., Cardinali, D.P., Riquelme, R., Llanos, A.J., Serón-Ferré, M., Effect of a melatonin implant on the circadian variation of plasma prolactin and rectal temperature in newborn sheep. J. Dev. Physiol. 16,

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