The large variability in melatonin blood levels in ewes is under strong genetic influence

Luis A. Zarazaga1, Benoît Malpaux1, Loys Bodin2, and Philippe Chemineau1

1 Institut National de la Recherche Agronomique, Neuroendocrinologie Sexuelle, Physiologie de la Reproduction, 37380 Nouzilly; and 2 Institut National de la Recherche Agronomique, Station d'Amélioration Génétique des Animaux, 31326 Castanet Tolosan, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. Three hundred twelve ewes born from 18 males and with known genealogy were sampled at the summer and the winter solstices. The nocturnal plasma melatonin concentration was defined as the mean of four plasma samples taken at hourly intervals in the middle of the night (2200-0200). Identity of the father (P < 0.001) and the solstice (P < 0.05) were significant. Melatonin concentrations varied considerably among individuals [338.4 ± 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml] and between rams regarding the melatonin concentrations of their daughters (range from 202.9 to 456.3 pg/ml). Inheritance was analyzed by a statistical model that allows discrimination of genetic effects from nongenetic effects and that estimates repeatability and heritability coefficients. Both the repeatability coefficient between solstices (0.60) and heritability coefficient [0.45 ± 0.07 (SE)] were high. These results demonstrate that the variability in plasma melatonin concentration in ewes is under strong genetic control.

pineal gland; photoperiod; ram; repeatability; heritability

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MELATONIN, THE MAIN secretory product from the mammalian pineal gland, transduces environmental light information received by the retina into a neuroendocrine signal. Studies of the importance of melatonin concentrations in the transduction of photoperiodic signals have focused mainly on its ability to regulate reproduction (1). There is, however, evidence that melatonin may regulate many other functions, such as body weight (15), pelage changes (12), immune system (13), thyroid activity (20), neoplastic growth (4), and circadian rhythms (2). However, these effects have been demonstrated generally by using either pinealectomized animals or melatonin injections/implantations, impairing the ability to distinguish, in many cases, the specific role of duration of melatonin presence in the blood from that of mean absolute melatonin concentration. To our knowledge, there is no available information on the specific role of mean absolute concentration, especially in sheep.

It is well established that melatonin blood levels are highly variable among individuals. The reason for this variation is unknown, but it is established that nocturnal plasma melatonin concentration is a very stable characteristic for each individual in humans (1) and in ewes (5) that may be indicative of a strong genetic basis.

The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. This species is particularly well suited because large variations in plasma melatonin concentrations have been reported, and repeated nighttime blood sampling can be performed easily without disturbance of the animals. In a very large set of animals for which the genealogy was known, it was decided to estimate two statistical coefficients describing the degree of genetic control of a trait. The first is the repeatability coefficient, which represents the proportion of the total variance attributable to the individual animal (equivalent to a correlation coefficient between successive measurements on the same animals). The second is the heritability coefficient, which is the proportion of the individual variation caused by the effects of genes.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. This experiment was carried out at the Institut National de la Recherche Agronomique, Research Center of Nouzilly, France (45° north), around the summer solstice (June 12-15, n = 399 ewes) and around the winter solstice (December 18-21, n = 351 ewes). The Ile-de-France breed was used for its already known important among-individual variability in melatonin levels (5). Ewes were born from 18 males (11-43 daughters/sire), and their genealogy was known over several generations. The physiological status (ewes suckling their lambs, n = 111, mean age 3.9 yr; ewes weaned from their lambs, n = 225, mean age 3.0 yr; pregnant ewes, n = 353, mean age 2.7 yr; and nonpregnant ewes, n = 61, mean age 3.2 yr), the age (n = 192, 1 yr old; n = 195, 2 yr old; n = 150, 3 yr old; n = 213, 4 yr old or more), and progesterone levels (determined in the samples obtained from melatonin determinations; <= 1 ng/ml, n = 327; >1 ng/ml, n = 423) were known and used in the statistical model to take into account their possible effects on melatonin concentrations, before calculation of repeatability and heritability coefficients. Ewes were maintained under a natural photoperiod, were fed daily with hay, straw, and corn, and had free access to water and mineral licks.

Blood sampling. Ewes were randomly distributed into groups of 25 ewes (bleeding group). Mean nocturnal melatonin plasma concentration of ewes was assessed on four plasma samples per ewe, taken at hourly intervals during the night (2300-0200 in June, 2200-0100 in December). Blood samples were obtained by venipuncture of jugular veins and were collected under dim red light (<1 lux at 20 cm), avoiding any direct illumination of the eyes. Plasma was immediately separated by centrifugation and stored at -20°C until assay.

Radioimmunoassays. Plasma melatonin concentrations were estimated in duplicate aliquots of 100 µl of blood plasma by radioimmunoassay using the technique of Fraser et al. (7) with an antibody raised by Tillet et al. (19). The sensitivity of the assay was 4 pg/ml. The inter- and intra-assay coefficients of variation, estimated from plasma pools every 100 unknown samples, were 2.2 and 13.6%, respectively. Plasma progesterone concentrations were estimated by radioimmunoassay using the technique of Saumande et al. (17). The sensitivity of the assay was 0.13 ng/ml. The inter- and intra-assay coefficients of variation were 16.6 and 3.4%, respectively.

Statistical analysis. The choice of performing four consecutive hourly samples taken in the middle of the dark phase was justified by further analyses done on a set of data generated in a previous experiment (5), in which we calculated a correlation coefficient of 0.84 between mean amplitude [calculated as Malpaux et al. (14) from hourly samples] during either short or long nights and mean nighttime concentration estimated from four samples in the middle of the night. Thus results from the four samples were averaged for each period to provide one measurement per night and per ewe. Statistical analysis was performed on these averaged data arranged in three different sets as follows: 1) data recorded in summer (n = 399); 2) data recorded in winter (n = 351); and 3) data of the ewes recorded in both summer and in winter (n = 312 ewes with 2 data/ewe). The effects of age, physiological status, bleeding group, and sire were tested independently on data collected in spring and summer using the General Linear Model (GLM) procedure (SAS). Data from animals collected in June (399 ewes) were used in the June analysis, data from animals collected in December (351 ewes) were used in the December analysis, and data from animals collected at both seasons (312 ewes) were used in the June plus December analysis.

As age effect was never significant, this effect was removed from the model for overall data, which included season, physiological status within season, bleeding group within season, and sire.

Genetic parameters (Y) for each data set were estimated using the following mixed linear models
Y<SUB><IT>ijkl</IT></SUB> = &mgr; + YB<SUB><IT>i</IT></SUB> + PB<SUB><IT>j</IT></SUB> + a<SUB><IT>k</IT></SUB> + e<SUB><IT>ijkl</IT></SUB>
for the June and for the December files
Y<SUB><IT>ijkl</IT></SUB> = &mgr; + S<SUB><IT>i</IT></SUB> + PB<SUB><IT>ji</IT></SUB> + a<SUB><IT>k</IT></SUB> + p<SUB><IT>k</IT></SUB> + e<SUB><IT>ijkl</IT></SUB>
for the June plus December analysis, with

  

µ The overall mean
YB The birth year effect of the ewe accounting for the ewe age (levels: i = 1, 7)
S A season effect (levels: i = 1, 2)
PB A combined effect accounting for the physiological status of the ewe at bleeding and the bleeding group, this effect was nested within the season effect for the overall analysis (levels: j = 1, 15)
ak The additive genetic value of animal k
pk The permanent environment for animal k (environment specific to each animal and common to all measurements on each animal)
eijkl The residual

a, p, and e were assumed as random effects normally distributed
a ∼ N(0, A&sfgr;<SUP>2</SUP><SUB>a</SUB> )
p ∼ N(0, I&sfgr;<SUP>2</SUP><SUB>p</SUB>)
e ∼ N(0, I&sfgr;<SUP>2</SUP><SUB>e</SUB>)
where A is the relationship matrix among animals, I is identity matrices, and &sfgr;<SUP>2</SUP><SUB>a</SUB>, &sfgr;<SUP>2</SUP><SUB>p</SUB> and &sfgr;<SUP>2</SUP><SUB>e</SUB> are the additive genetic, permanent, and error variances, respectively.

This "animal" model (11) is the reference model for estimation of genetic parameters in the field of animal breeding. For single measurements on each animal, the relationship matrix A allows the prediction of additive genetic values by tying information on relatives. A multivariate multimodel restricted likelihood variance component estimation package (8) was used to estimate the variances &sfgr;<SUP>2</SUP><SUB>a</SUB>, &sfgr;<SUP>2</SUP><SUB>p</SUB>, and &sfgr;<SUP>2</SUP><SUB>e</SUB> and to predict the random effects of the models a and p. The estimated phenotypic variance was estimated by the sum of the estimated genetic, permanent environment and residual variances. Heritability was the ratio of estimated additive genetic variance on phenotypic variance; the higher the coefficient, the higher the variability of a trait is due to genetic cause. Repeatability coefficient between average melatonin concentrations in summer and winter was estimated by the ratio of the sum of estimated genetic and permanent environment variances on phenotypic variance.

Unless otherwise stated, data are presented as means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mean nocturnal melatonin plasma concentrations were highly variable among individuals [338.4 ± 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml]. The mean melatonin concentration in June during the long day photoperiod was significantly lower than in December (328.3 ± 15.6 vs. 400.4 ± 20.8 pg/ml, respectively, P < 0.05).

Figure 1 shows the distribution of melatonin concentrations for all ewes, before and after correction for fixed effects. This continuous distribution over a large range shows the great variability among individuals. This variability is maintained when averaging daughters of each sire, and highly significant differences among males regarding melatonin concentrations of their daughters were observed (range for raw data of values within each sire's offspring from 202.9 ± 18.6 to 456.3 ± 26.7 pg/ml; P < 0.001; Fig. 2).


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Fig. 1.   Number of Ile-de-France ewes per class of mean nighttime plasma concentration of melatonin, after blood collection in June (399 ewes) and December (351 ewes). Raw data are presented in A, and data after correction for fixed effects (312 ewes) are shown in B.


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Fig. 2.   Distribution of sires regarding the nighttime nocturnal plasma melatonin concentration (mean ± SE) of their daughters, ranked in order, in the Ile-de-France breed. Each sire has from 11 to 43 daughters, which were collected in June and in December. Raw data (312 ewes with 2 data/ewe, unadjusted) from both seasons were pooled.

The variance components estimated for different samples are shown in Table 1. In the three samples, the model used in the statistical analysis allowed us to explain a large part (>82%) of the global variance. The component of permanent environment was low in relation to the animal component and leads to a high repeatability coefficient (r = 0.60) of mean concentration between the two seasons. In the three sets of data, heritability coefficients were very high, and their estimates were not different between June and December (from 0.40 to 0.48).

                              
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Table 1.   Variance components, percentage of global variance explained by the model, heritability, repeatability, and SE of h2 for mean nighttime plasma concentration of melatonin in Ile-de-France ewes at 2 periods in the year

Breeding values of ewes were distributed from 137.2 to 611.0 pg/ml. Four sires had >60% of their daughters in the first quartile of the general distribution of ewe breeding values, whereas four other sires had >50% of their daughters in the upper quartile, and the remaining 10 sires had their daughters spread over three or four quartiles (Table 2).

                              
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Table 2.   Distribution of per sire average of ewe breeding values in four quartiles and sire breeding value

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study confirms the existence of large and repeatable differences among individuals in sheep nocturnal plasma melatonin concentrations and indicates that plasma melatonin concentrations are a strong individual characteristic (5). Large but repeatable differences among individuals regarding a trait may originate either from a permanent environmental effect such as during development (in utero or during early stages of life), which modifies the individual animal for all of its lifetime, or from a permanent genomic effect coming from the alleles of specific genes.

The assessment of nocturnal plasma melatonin concentration in a large number of animals with known genetic links, and particularly among groups of daughters with common fathers, showed that a major factor of variation was the sire effect. Furthermore, the experimental design allowed us to estimate the heritability coefficient with a low standard error. This study extends previous observations (5, 14) by demonstrating that the high repeatability of melatonin secretion in ewes is the result of a strong additive genetic control rather than a large permanent environmental effect. With such a high heritability coefficient, this trait could be considered as ranking among the most heritable traits within the same species (between 0.03 and 0.32 for the date of the onset of the breeding season; see Ref. 10).

This estimate of heritability of plasma melatonin concentration is consistent with the value observed in humans for the melatonin excretion in urine (21). Although it is unclear in that study whether melatonin itself or one of its metabolites was measured in urine, a heritability estimate of 0.53 was found. Wetterberg et al. (21) hypothetized that a major gene may control the expression of this trait in humans. Our results might suggest, in sheep, a similar type of inheritance, which would be easily tested by observing the segregation of the putative alleles of the gene in the progeny of some selected sires.

The physiological origin of the very high among-individual variability in nocturnal plasma melatonin concentration is not known, but the differences may originate either from the degradation side (catabolism in the liver) and/or from the production side (synthesis/liberation by the pineal). The first possibility is supported by studies in cows (3) and pony mares (9) in which a wide variability in half-life among animals was reported. The second possibility also has some support in the literature. In ewes, melatonin production rate in the plasma may be highly variable among individuals (16). This variability may originate from differences in pineal gland development, since there is a wide range in the pineal weight among ewes of the same breed (18). Alternately, the genetic differences may originate in the pinealocyte enzymatic equipment responsible for the transformation of tryptophan into melatonin. Arylalkylamine N-acetyltransferase recently cloned in various species including sheep (6) and hydroxyindole-O-methyltransferase may be good candidates for such interindividual variability.

This question of the difference due to melatonin production or catabolism/elimination could be addressed by injecting a bolus dose of melatonin during the day in several ewes from the high- and low-level ends of the spectrum to assess whether there are differences in melatonin clearance (supporting a difference in catabolism). However, preliminary data from our laboratory (L.A. Zaragaga, B. Malpaux, D. Guillaume, and P. Chemineau, unpublished observation) suggest that the variability observed in the present study is not due to differences in catabolism.

In summary, the high heritability of melatonin blood levels indicates a very important genetic control of this trait. It remains to be determined if this genetic control is exerted at the level of pineal or liver (or both) and if it is linked to a productive or reproductive trait. A divergent selection on nocturnal melatonin levels may also be useful for generating an animal model that could be used to elucidate the physiological mechanisms underlying regulation of melatonin blood levels in mammals.

    ACKNOWLEDGEMENTS

We thank Agnès Daveau and Françoise Maurice-Mandon for advice in the technical procedures, the breeders of the Institut National de la Recherche Agronomique (INRA) Research Center of Nouzilly for the supply and care of experimental animals, all people of the Neuroendocrinology unit who helped with collection of the nightime blood samples, and Dr. D. Skinner for help in the preparation of this manuscript.

    FOOTNOTES

L. A. Zarazaga was supported by a postdoctoral grant from Diputación General de Aragón and INRA-Direction Scientifique des Productions Animales.

Address for reprint requests: P. Chemineau, Institut National de la Recherche Agronomique, Neuroendocrinologie Sexuelle, Physiologie de la Reproduction, 37380 Nouzilly, France.

Received 18 August 1997; accepted in final form 4 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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