212 J. Santiago-Moreno et al. Animal Reproduction Science 64 2000 211–219
1. Introduction
Prolactin PRL shows an endogenous rhythm of secretion, which is modulated by photo- toperiod 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 di-
rect 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
J. Santiago-Moreno et al. Animal Reproduction Science 64 2000 211–219 213
40
◦
25
′
N in a 250 m
2
enclosure. The Mouflons came originaly from “El Hosquillo” Na- tional 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 condi- tions 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.
2.2. Hormone assays Plasma concentrations of PRL were determined by radioimmunoassay in duplicate 100 ml
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 ngml and the intra-assay
coefficient of variation was 9.3 n = 8. Melatonin concentrations were estimated in duplicate aliquots of 100 ml 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 pgml of plasma. The inter- and
intra-assay coefficients of variation, estimated from plasma pools every 50 unknown sam- ples, were 15 and 9, respectively.
2.3. 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
