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 |
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 |
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 |
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
for
the June and for the December files
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
|
|
|
|
|
|
where
A is the relationship matrix among animals, I is identity matrices, and
,
and
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
,
, and
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 |
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.
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|
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
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|
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).
 |
DISCUSSION |
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.
 |
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