Results Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol64.Issue3-4.Dec2000:

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

The seasonal changes in plasma concentrations of PRL, during the dark-phase are illus- trated for each breed in Fig. 1. In female Mouflons, the period of highest secretion of PRL 214 J. Santiago-Moreno et al. Animal Reproduction Science 64 2000 211–219 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 daynight 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. Daynight 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