Circannual rhythm of plasma prolactin concentration in the goat

Animal Reproduction Science, 17 (1988) 85-94

85

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Circannual R h y t h m of P l a s m a P r o l a c t i n Concentration in the Goat A. PRANDI 1, M. MOTTA 1, F. CHIESA 1 and C. TAMANINI 2'3

lIstituto di Fisiologia Veterinaria, Universita' di Bologna, Via Belmeloro, 8/2, 40126 Bologna (Italy) ~Istituto di Fisiologia Veterinaria, Universita' di Bari, Via Caduti di tutte le Guerre, 1, 70126 Bari (Italy) 3To whom correspondence should be addressed. (Accepted 8 March 1988)

ABSTRACT Prandi, A., Motta, M., Chiesa, F. and Tamanini, C., 1988. Circannual rhythm of plasma prolactin concentration in the goat. Anim. Reprod. Sci., 17: 85-94. The aim of this experiment was to investigate the variations of prolactin plasma levels in goats throughout the year as related to photoperiod (45 °N latitude ), ambient temperature and reproductive status. Prolactin concentration was measured on samples collected from 50 goats every 35 days for 14 months; during the same period, the mean ambient temperature was recorded every day. PRL plasma levels were very low ( < 30 ng/ml) from November to March and increased to higher values (about 100 ng/ml) from March to May; the highest values (300-400 ng/ml) were observed during the summer period. In the next months, the PRL levels fell to the lowest values, closing the annual cycle. High PRL plasma concentrations were associated with the anestrous season, low PRL levels with the breeding season. Fluctuations in the mean temperature values were accompanied by correspondent fluctuations of PRL plasma levels: high temperature values were associated with high PRL plasma levels. No relationships were observed between PRL plasma concentrations and pregnancy or lactational anestrus. Doubts still exist about the effects of parturition on PRL levels.

INTRODUCTION

There is now compelling evidence that some photoperiodic seasonal breeders (like sheep) present circannual variations in prolactin plasma level with the highest concentrations during the summer and the lowest ones during the winter (Pelletier, 1973; Forbes et al., 1975; Ravault, 1976; Lincoln et al., 1978; Tamanini et al., 1987). In the ewe, high PRL plasma levels coincide with the anestrous season, low P R L plasma concentrations with the breeding season. It has been postulated (Walton et al., 1977) that the high levels of prolactin in the summer may negatively affect the ovarian function and the decrease in 0378-4320/88/$03.50

© 1988 Elsevier Science Publishers B.V.

86 PRL levels during a u t u m n is responsible for the beginning of the ovarian cyclicity. Nevertheless, attempts to manipulate breeding activity by the manipulation of PRL have been unsuccessful (Worthy et al., 1985). In a previous work (Prandi et al., 1987), we observed that, in the goats as well, P R L plasma levels are high during the summer and tend to decrease as the breeding season approaches. The present experiment was undertaken to investigate the profile of prolactin plasma levels throughout the year in the goat and to verify if other factors than photoperiod may influence the secretion of this hormone. In particular, the effects of the reproductive status (anestrus, pregnancy, parturition) and of an external factor (temperature) on PRL plasma variations have been taken into account. MATERIALSAND METHODS Animals and management The experiment was performed beginning in November, 1984 till December, 1985 in Northern Italy (45°N latitude). Fifty female goats of Alpine breed maintained under natural photoperiod conditions were used. The animals were housed outdoors; they were fed a normal ration of hay and concentrates and allowed free access to fresh water. The age ranged between three months and five years; more precisely, 42 animals had kidded at least once before the beginning of the experiment, while eight were immature; of these, four were three months old at the beginning of the experiment. No significant changes in body weight (except for the physiological ones related to different reproductive statuses) were observed throughout the experimental period. Throughout the whole experiment, any kind of interference with the normal management of the flock was avoided so that all the goats experienced their own physiological reproductive activity all through the period (i.e., attainment of puberty, breeding season, pregnancy and anestrus, depending on the age and the season). Neither hormonal treatments nor drugs were used. The dates of kiddings were recorded; furthermore, the mean ambient temperature of each day was recorded. The average temperatures were correctly obtained by integration of the continuous curve over the 24 h period. Thus, the daily values were not affected by statistical errors. Blood samples were collected by jugular venipuncture every 3-5 days from November, 1984 till December, 1985. 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 concentration of prolactin and progesterone.

87 Hormone assays The concentration of P R L and progesterone in the blood plasma was measured by RIA; details of the methods are described elsewhere (progesterone, Bono et al., 1983; PRL, Tamanini et al., 1985). For PRL measurement 100/A of plasma or 100 pl of plasma diluted 1 : 10 were used, depending on the period of the year in which the samples had been collected. The lowest limit of detection was 93 _+2.1 pg/tube; the inter- and intra-assay C.V. (%) were 13.1 and 8.5, respectively. Progesterone plasma levels were measured only in samples collected in two different periods of the year: February-March and AugustSeptember in order to determine exactly the transition periods from breeding to anestrous season and from anestrous to breeding season. These months were chosen on the basis of the results of previous experiments (Tamanini et al., 1985; Prandi et al., 1987). Goats were considered to be cycling when the progesterone plasma levels were higher than 0.4 ng/ml for at least four consecutive samples. The lowest limit of detection for progesterone assay was 21 + 0.74 pg/ tube; both inter- and intra-assay C.V. ( % ) were < 12.0. Mathematical analysis The original values of PRL levels observed throughout the experiment were not evenly spaced and therefore were forced to become evenly spaced through the well-known mathematical procedure of the least square cubic splines approximation so that it was possible to compare them with the daily recorded temperature mean values. Splines are piecewise cubic polynomials fitted together so that the first and second derivatives are continuous. In a few words, for every set of four points on the plane, a cubic polynomial is constructed with the least square method and the interval of the two central points is taken as valid for the interpolation. Starting from the initial four points, the procedure is repeated, shifting one step in the sequence of experimental points with the condition of equal derivatives in the junction points. For the first and the last points of the sequence, a zero value of the second derivative is generally imposed. This piecing together allows the drawing of smooth curves even when there is a high number of points. The statistical errors are taken into account with the least square procedure applied in the determination of each cubic polynomial (Reinsch, 1967; Garbow, 1968; Knuth, 1973). The correlation between PRL and temperature shapes, mainly regarding the coincidence of peaks and valleys (turning points), was checked out by calculation of the derivatives of both the curves, for which the zero points give the exact positions of the turning points in the primitives. Moreover, the level of correspondence, one to one, of the turning points in both temperature and prolactin curves was checked through the calculation of the Spearman rank correlation coefficient among the above mentioned zeros of

88 the derivatives. The test is particularly useful when we want to examine whether two variables (zero positions in our case) are independent or vary in the same or in the opposite directions. For a test of null hypothesis, that there is no correlation, the usual correlation coefficient r may be used, provided that one of the variables is normal. When, as in our case, neither variable seems normal, the best-known procedure is that in which the two variables, say X, Y, are both rankings. The substitution of the exact zero positions X, Y for prolactin and temperature derivatives with the correspondent ranks evidently must reflect the order relationship between the observed values of X and Y since we are interested in the interdependence of the variables (Snedecor and Cochran, 1971; Kendall and Stuart, 1979). RESULTS Progesterone plasma levels were less than 0.4 n g / m l in all the non-pregnant goats by the end of February (beginning of the anestrous season); not one goat exhibited progesterone plasma levels exceding 0.4 n g / m l until the end of August (onset of the breeding season). PROLACTIN AND DAYLIGHT PERIOD TIME RANSE OF THE EXPERIMENT: 414 DAYS

(unsmoothed original data) PROLACTIN

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Fig. 1. Average values of prolactin plasma concentration with the quoted mean standard errors, observed during the time experiment (continuous line) and variations of day length as related to the natural photoperiod at 45°N latitude (broken line).

89

PROLACTIN AND EXTERNAL TEMPERATURE MEAN VALUES TIME WwNGE OF THE EXI~INB~T: 414 DAYS (cubic spllne smoothed data) PROLACTIN

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Fig. 2. Smoothed curves of prolactin plasma concentration (continuous line) and mean temperature values (broken line) as obtained by a least square cubic spline procedure from the original data with errors.

Most of the kiddings were observed between February and April; only a few kiddings were recorded in the following three months. The PRL plasma levels observed around parturition (about ten days ) were quite variable among these animals; in some cases, but not constantly, a peak of PRL was observed. In order to avoid a spurious source of variations due to these irregular data, all the PRL values observed around parturition were omitted. The statistical analysis of the data showed that PRL concentrations in the samples collected during the five months preceding parturition are not significantly different from those observed in non-pregnant and immature goats in the same period of the year. The PRL plasma levels observed during the lactational anestrus were also quite similar to those observed in non-lactating females in the same period of the year. Therefore, according to the above-mentioned observations, Fig. 1 shows the PRL plasma profile, on a circannual basis, obtained by pooling the values of the PRL plasma levels of all the goats for each day of bleeding (with the exclusion of samples collected from goats around parturition) and the variations of day length as related to the natural photoperiod. The PRL plasma levels are low ( < 30 ng/ml) from November to February, then start to increase and reach values around 300 ng/ml in June. During the summer

90

months, the PRL plasma concentrations range between 200 and 400 ng/ml and a sudden decrease is observed in September. In the last three months, the PRL plasma levels are again very low ( < 20 ng/ml). Fig. 2 shows the profiles of PRL plasma levels and of the mean daily temperature observed throughout the experimental period, which resulted from the mathematical elaboration (see above); obviously, low and high temperatures were recorded during the winter and summer months, respectively. The variations of the mean temperature values are accompanied by variations of PRL plasma levels. The correspondence between plasma PRL fluctuations and temperature variations is presented in Fig. 3, where the zeros of the derivatives of the PRL and temperature functions give the peak and valley positions; the fluctuations of PRL plasma levels are clearly related to fluctua-

DERIVATIVES OF PROLACTIN AND TEMPERATURE MEAN VALUES PEAK POSITIONS AT THE CROSSING POINTS OF X-AXIS (ZEROS) (derivatives PFIOLACTZN

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Fig. 3. The derivatives functions of prolactin plasma concentration (continuous line) and of ambient temperature (broken line), obtained from the smoothed curves of the original data. The peak and valley positions of the original smoothed data are given by the zero points of the plotted derivatives.

91 RANK CORRELATION OF PROLACTIN AND TEMPERATURE PEAKS AND VALLEYS LINE Y - i . 3 2 X - 2 . 3 7 . SPEARMAN CORRELATION COEFFICIENT - 0.992 (ranked points: the zeros of the d e r i v a t i v e curves) ~T

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Fig. 4. The rank correlation of the zero points of the derivatives functions giving the level of correspondencebetween the peaks (and valleys) of temperature and prolactin plasma concentration. The Spearman correlation coefficientwas 0.992, showinga high one to one correspondence of temperature and prolactin fluctuations. tions of the temperature values, with a one-to-one correspondence, at least in conditions of a strong variation of the parameters (high derivatives). Fig. 4 shows the regression line for the rank correlation among peaks and valleys of ambient temperature and prolactin plasma concentration. The rank value of the zeros of the derivatives (see Fig. 3), from which the straight line is deduced, are also plotted for more clarity. The Spearman coefficient is very high (0.992) and significant ( >> 1% level ) (see Table 7.11.2, p. 195, and Table A.II p. 557; Snedecor and Cochran, 1971 ). DISCUSSION Data from this study strongly confirm that goats, like sheep, exhibit a circannual r h y t h m in P R L secretion, with low prolactin levels in winter (short days) and high values in summer (long days). Photoperiod, therefore, greatly affects prolactin secretion in female goats as it does in ewes {Worthy and Haresign, 1983). Similar but incomplete and inconclusive data were obtained in previous studies done in this (Bono et al., 1983; Tamanini et al., 1985; Prandi et al., 1987) and other laboratories (Mori et al., 1985). The months during

92 which P R L levels are low coincide with the breeding season in this species, while high P R L concentrations are characteristic of the anestrus. These data could support the view that prolactin negatively affectsthe reproductive activity in goats, as postulated by Walton et al. (1977) for the ewe. Nevertheless, previous data (Tamanini et al.,1985) suggest that ovulation can be induced during the anestrous season even if P R L levels are stillhigh and, at the same time, that the reduction of P R L plasma levels induced by melatonin treatment (Prandi et al.,1987 ) is not followed by the initiationof the breeding activity. Therefore the role of P R L does not seem to be decisive in inhibitingthe ovarian activity, as postulated in a previous work (Prandi et al.,1987). W e may hypothesize that P R L plasma levels are a 'sign' (and therefore an effect) of the breeding (low levels) or anestrous (high levels) season rather than a cause of the reproductive seasonality. This hypothesis may be strengthened by considering that an inverse relationship exists between P R L and melatonin plasma levels (Kennaway et al.,1982a, b) and, therefore, P R L concentrations somewhat depend on the photoperiod; in addition, ithas been shown that melatonin administration significantlyreduces P R L plasma levels (Prandi et al.,1987). Our results, surprisingly, do not show any relationship between P R L plasma levels and reproductive status (i.e.,pregnancy, estrous cycle,etc.) or lactation. As we have already said, goats in this study experienced their normal physiological reproductive activity,so that they kidded between February and April and, consequently, the lactation started in animals with either low or high P R L plasma concentrations, without any apparent difference as far as milk production was concerned. More specific experiments are at present in progress in our laboratory on the relationship between lactation and PRL; preliminary data confirm our surprising results since prolactin does not seem to increase during lactational anestrus, unlike observations in other species. Some data allow us to suppose that parturition can induce a transient increase in P R L plasma concentration, but these data are neither constant nor conclusive and the P R L plasma pattern around kidding needs a more detailed study with more frequent collectionof blood samples. The relationships between P R L plasma variations and temperature are very interesting. Irrespective of the levels of P R L in plasma (high in summer and low during the breeding season), variations of the mean temperature are always accompanied by variations of P R L concentrations. Obviously, the fluctuations of P R L levelsare much wider and more evident when the concentration is high, even though the percent variation we observed in winter was quite similar to that in summer. A direct effectof the temperature on the P R L concentrations has been already observed in cattle (Wetteman and Allen Tucker, 1974 ) and also in goats (Mori et al.,1985; Maeda et al.,1986). In female Saanen goats subjected to artificialshort or long days, Mori et al. (1985) observed low and high prolactin levels,respectively (these data clearly reflect the different photoperiod). However, these authors observed marked increases of

93 plasma P R L in all goats (irrespective of light t r e a t m e n t ) when ambient temperature exceeded 27 ° C. These and our data let us hypothesize t h a t the thermal stress induces an increase of P R L plasma levels; this parameter is not decisive, but is responsible for inducing some modifications of the P R L plasma profile, which is mainly dependent on the photoperiod. We may conclude from this study that, in the goat: (1) P R L plasma levels show a circannual rhythm, with high levels during anestrus and low levels during the breeding season; (2) the events related to the reproductive statuses (pregnancy, estrous cycle, lactation, etc.) seem not to modify the circannual profile, even if some doubts exist about the P R L pattern around parturition, and (3) variations of the mean ambient temperature induce a modification in the general profile of P R L plasma concentrations. ACKNOWLEDGEMENTS The authors are grateful to prof. Leo E. Reichert for providing purified ovine P R L (LER-860-2). This work was supported by C.N.R., Italy, special grant I.P.R.A.-Sub-project 1. Paper N. 1649, and ECC G r a n t No. 3311.

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94 Pelletier, J., 1973. Evidence for photoperiodic control of prolactin release in rams. J. Reprod. Fertil., 35: 143-147. Prandi, A., Romagnoli, G., Chiesa, F. and Tamanini, C., 1987. Plasma prolactin variations and onset of ovarian activity in lactating anestrous goats given melatonin. Anim. Reprod. Sci., 13: 291-297. Ravault, J.P., 1976. Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old. Acta Endocrinoi., 83: 720-725. Reinsch, C.H., 1967. Smoothing by Spline Functions, Numerische Mathematik, Vol. 10, Berlin, p. 177. Snedecor, G.W. and Cochran, W.G., 1971. Statistical Methods, Sixth edition. Iowa University Press, Ames, IA, pp. 194. 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. Anim. Reprod. Sci., 9: 357-364. Tamanini, C., Prandi, A., Biacchessi, D. and De Rensis, F., 1987. Effects of melatonin treatment on the onset of ovarian activity, reproductive parameters and PRL plasma levels of immature ewes. Anim. Reprod. Sci., 13: 283-290. Walton, J.S., McNeilly, J.R., McNeilly, A.S. and Cunningham, 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. Wetteman, R.P. and Allen Tucker, H., 1974. Relationship of ambient temperature to serum prolactin in heifers. Proc. Soc. Exp. Biol. Med., 146: 908-911. 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.