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EFFECT OF EXOGENOUS MELATONIN AND PLANE OF NUTRITION AFTER WEANING
ON ESTROUS ACTIVITY, ENDOCRINE STATUS AND OVULATION RATE IN SALZ EWES LAMBING IN THE SEASONAL ANESTRUS
F. Forcada, L. Zarazaga and J.A. Abecia
Depat-tamento de Producci6n Animal y Ciencia de 10s Alimentos
Universidad de Zaragoza. Miguel Servet, 177. 50013 Zaragoza, Spain
Received for publication: ~prii! 26, 199g
Accepted: November 8, 1994
ABSTRACT
Forty-nine Spanish Salz ewes lambing in the second fortnight of March (20
March? 1.5 d) were used to determine the effects of exogenous melatonin and
postweaning nutrition on endocrine status, date of first estrus and ovulation rate.
Experimental design was a factorial defined by 2 postweaning planes of nutrition, 1.80
(high) and 1.35 (low) times the maintenance requirements, and treatment with a single 18-
mg subcutaneous implant of melatonin (M) 32 d after lambing or no treatment control (C).
Mean weaning to first estrus interval was shorter in treated than in control ewes (50.8 f 4.2
vs 87.626.3 d; P<O.Oi). Considering both the treated and control animals together, the
ratio between mean night and daytime plasma melatonin levels was significantly correlated
with the implant insertion-first estrus interval on Day 5 (0.67; P<O.Ol) and Day 35 (0.63;
P < 0.05) after implantation. Melatonin implants induced a significant increase of mean LH
concentrations at Days 14 and 33 after implantation (P<O.Ol) without any significant
influence of plane of nutrition. Ovulation rate was higher for treated than control ewes in
the second estrus (PcO.05). An interaction between plane of nutrition and exogenous
melatonin on ovulation rate at the second cycle after weaning was detected (P<O.Ol),
being close to the significance in the first, fourth and fifth cycles (PcO.1). These results
suggest that exogenous melatonin in April may be an effective way of advancing the
breeding season and enhancing ovulation rate associated with a low rather than a high
plane of nutrition.
Key words: sheep, melatonin, post pat-turn, anestrus, feeding level
Acknowledgments
The authors thank A. Daveau (INRA-Nouzilly) and A. Gomez (INIA-Madrid) for help with
melatonin and LH analysis, respectively. This study was supported by Grant Gang1 -0656
from C.I.C.Y.T.
Theriogenology 43:117%1193. 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691w95/$10.00 SSDI 0093-691X(95)00090-U
Theriogenology
INTRODUCTION
Reproductive seasonality greatly limits productivity in farm animal species which
are regulated mainly by photoperiod (48). However, Mediterranean sheep breeds show
reduced seasonal@, with a seasonal anestrus of nearly 100 d (16,44), especially when
the male animal is handled appropiately (36). The Salz breed is a genotype obtained (41)
by crossing a prolific breed (Romanov; 50%) with a local breed (Rasa Aragonesa; 50%)
that has a short anestrus period between May and June. Individual Salz ewes may exhibit
estrus nearly every month of the year (Forcada and Zarazaga, unpublished results). Thus
this breed is well adapted for intensive production and for an accelerated lambing rate.
Photoperiodic information is conveyed to the reproductive neuroendocrine system
by circadian secretion of melatonin from the pineal gland (7). Melatonin implants inserted
in June and July (or their equivalent in the Southern hemisphere) have been widely used
as a means of advancing the breeding season of adult ewes (22,25,29). However,
attemps to achieve and advance estrous activity by the insertion of melatonin implants
earlier in the season, in April, have failed in the United Kingdom (14) or have provided
contradictory results in Mediterranean genotypes (27).
Nutrition has an important impact on reproductive performance in sheep, but the
magnitude of nutritional or body fat reserve effects can vary with the season (1646) or
with the level of fecundity of the studied breed (20,42). The level of feed intake or protein
suplementation after weaning in reduced-seasonality ewes lambing in seasonal anestrus
did not reduce the remating interval, but did influence reactivation of the ovulation rate (l-
2). Since there are no references in the literature to the possible interactions between
melatonin treatment and nutrition on the resumption of reproductive activity, especially in
ewes mated shortly after weaning, this study was designed to investigate the role of these
2 factors on endocrine status, timing of estrous activity and ovulation rate in Salz ewes
lambing in the seasonal anestrus and having received melatonin implants at 32 d post
pat-turn.
MATERIALS AND METHODS
This study was conducted in Spain at the experimental farm of the Veterinary
Faculty of the University of Zaragoza (latitude 41” 40’ N). Forty-nine adult (4 to 6 yr old)
Salz ewes lambing in the second fortnight of March (20 March+ 1.5 d) 1992 were used.
Ewes suckled single lambs and were weaned in 2 blocks, with an average duration of
lactation of 40.520.9 d. The animals were kept permanently in communal pens with an
uncovered area throughout the experiment, without supplementary light. Clean fresh water
Theriogenology 1181
was available ad libitum. During the lactation period, rams were placed in the same pen
but were separated from the ewes to prevent a ram effect.
The experiment was designed as a 2 x 2 factorial. During the suckling period, the
ewes were group-fed with a ration supporting 3.52 Meal metabolizable energy (ME) per
head per day. After weaning (April 30), the ewes were divided into 2 groups and were fed
0.6 kg (high, 24 ewes) or 0.3 kg (low, 25 ewes) of concentrate (barley 100%) and 1 kg
of ammonia treated straw per head per day. These amounts correspond with a daily
intake of 3.31 (high) or 2.46 Meal ME (low), for 1.80 and 1.35 times, respectively, the daily
maintenance requirements (3).
Half of the ewes in each of the 2 groups received a single, subcutaneous implant
at the base of the left ear containing 18 mg melatonin (Melovine,T%AMCO, Cambridge
Animal and Public Health, Hauxton, Cambridge, UK, and SANOFI, Sante Nutrition
Animale, Libourne, France) on Day 3221 after lambing (M group, 24 ewes). The
remaining animals served as untreated controls (C group, 25 ewes). All groups were
balanced for liveweight and body condition.
The experiment was divided into nine 17-d periods after weaning starting on April
20, and then estrus was detected daily using aproned rams. The ovulation rate was
measured by laparoscopy 6 d after positive identification of estrus. Liveweight and body
condition (40) were determined once a week. Blood samples were taken twice weekly
from Day 14 after lambing by jugular venipuncture and assayed for progesterone. In
addition, samples were collected hourly for 24 h on Days 5 and 35 after implant insertion
from 20 ewes (5 in each treatment group) via a jugular catheter, ipsilaterally to implant
insertion, and assayed for melatonin. During the hours of darkness, samples were
collected under dim red light (less than 3 lux). Further samples were taken on Days 0, 14
and 33 after melatonin treatment, at 20-min intervals for 8 h from 5 ewes in each
treatment group via a jugular catheter for LH determination. Plasma was separated by
centrifugation and stored at -20” C.
The criteria used for evaluating reproductive parameters after weaning were 1) date
of the first estrus detected by the rams and the length of interval from weaning to the first
behavioral estrus; 2) ovulation rate of each detected cycle and each 17-d period; 3)
percentage of ovulations unaccompained by estrus (silent ovulations: the minimum
plasma progesterone level for a luteal phase was 0.5 ng/ml, which is the minimum value
for monitoring cyclicity used in Salz ewes (17) and in other Mediterranean breeds (16,44);
4) percentage of ewes showing estrus (detected cyclicity) and silent ovulations during
each 17-d period.
1182 Theriogenology
Plasma progesterone concentrations were determined by radioinmunoassay (16).
Sensitivity of the assay was 0.05 ng/ml progesterone, and intra and inter-assay
coefficients of variation were 4.3 and 7.7%, respectively. Plasma LH and melatonin levels
were also determined by radioinmunoassay using the techniques of Mondain et al. (30)
and modified by Gomez (19) and by Fraser et al. (18), with the antibody raised by Tillet
et al. (43). The sensivities of the assays were 0.4 ng LH/ml (NIAMDD-oLH-22) and 5 pg
melatonin/ml (melatonin FLUKA-63610). The intra and inter-assay coefficients of variation
were 11.7 and 12.8% for LH and 16.0 and 17.0% for melatonin, respectively.
Blood samples taken at 20-min intervals for 8 h were used to test temporal
changes in LH mean concentrations and pulse frequency. Pulses of LH were defined
according to Baird et al. (6); a pulse was deemed to have occurred when 2 consecutive
values were higher than the 2 preceding values, and when the value of the highest (pulse
amplitude) exceeded the mean basal value by at least 4 times the coefficient of variation
of the assay.
For each 24-h series of samples, melatonin night levels were defined according to
Malpaux et al. (28). A melatonin elevation was defined as the interval between the first and
the last value that exceeded the baseline level preceding and following the dark period
by more than 3 standard deviations of those respective baselines (the baselines were
defined as the means of the daytime samples preceding and following the dark period).
The incidence of ovarian activity and detected estrus between groups was
compared using a Chi-square test. Means (?SEM) were calculated for liveweight and
body condition, interval between weaning to first estrus, ovulation rate, LH concentrations
and pulse frequency, and plasma melatonin levels. Correlation coefficients for the ratio
between mean night and daytime plasma melatonin levels and the interval from implant
insertion to first estrus (10) on Days 5 and 35 after implantation were calculated. A 2 x 2
factorial analysis of variance was used to evaluate effects of postweaning nutrition level
and treatment or no treatment (control) with exogenous melatonin on the onset of estrous
activity, ovulation rate, LH plasma concentration and pulse frequency, and plasma
melatonin levels. Analysis of data was computed using the SPSS package (32).
RESULTS
No significant losses of liveweight and body condition from lambing (55.7? 1 .O kg
and 2.6OkO.04) to weaning (54.4k1.2 kg and 2.3920.04) were observed, indicating that
the level of nutrition was sufficient to allow for raising a single lamb in a high fecundity
genotype. Melatonin treatment did not influence mean liveweight and body condition after
Theriogenology 1183
_.--, (b)
6.00
2.71
2.60
2.2b
2.00
Figure 1. Changes in liveweight (a) and body condition (b) throughout the
experiment in Salz ewes lambing in March with a high (.) or low (t ) plane
of nutrition after weaning.
weaning, and the high plane of nutrition allowed for significant differences (P < 0.05) only
in the body condition from the seventh week to the end of the experiment (Figure 1).
Mean interval between weaning and first behavioral estrus was significantly
shortened by the melatonin treatment (50.8k4.2 vs 87.626.3 days for M and C group
ewes, respectively (P<O.OOl). No effect of level of nutrition on the resumption of sexual
activity was detected.
1184 Theriogenology
80
60
6 7 8 17-DAY PERIODS AFTER WEANING
Figure 2. Changes in the ovarian cyclic@ (%) after weaning in Salz ewes lambing in
March with a high (H) or low (L) plane of nutrition and treated (M) or not (C)
with exogenous melatonin. ** Effect melatonin P ~0.01.
Analysis of the plasma progesterone concentrations did not show any ovarian
activity during the suckling period, but did reveal the existence of at least 1 ovulation
unaccompained by estrus a few days before the onset of estrous activity in a 82% of the
ewes. Neither melatonin treatment nor the level of nutrition influenced the number of silent
ovulations.
Percentage of ewes exhibiting estrous cycles, as monitored by plasma
progesterone concentrations throughout the period of study is shown in Figure 2.
Exogenous melatonin caused a significant increase (PcO.01) in the percentage of cyclic
ewes in the second, third, fourth and fifth 17-d periods after weaning, without any
significant effect of level of nutrition, which could indicate that ewes given melatonin
implants had an early and more compact distribution of onset of ovarian activity than that
of the controls.
The 24-h plasma melatonin profiles in treated and control ewes in relation to the
plane of nutrition (5 animals from each experimental group) are given in Figure 3 (Days
5 and 35 after implant insertion). Mean day and night time plasma melatonin
concentrations in the implanted ewes were significantly higher than the correspondent
levels in the control animals, although the most remarkable effect of the implants seems
Theriogenology 1185
Figure 3. Twenty-four hour plasma melatonin profiles in Salz ewes lambing nn March
with a high (a) or low (b) plane of nutrition after weaning and treated (.) or
not (t ) with exogenous melatonin on Days 5 and 35 after implant insertion.
Means 2 SEM for n =5.
to be the higher mean daytime levels compared with those exhibited by the non treated
animals (PcO.01). There were no significant effects of plane of nutrition after weaning on
melatonin concentrations, except for the night time levels on Day 35 (P < 0.05) due to the
high concentrations exhibited by the implanted animals from the low plane of nutrition
group (more than 500 pg/ml).
Considering the data obtained from the animals for melatonin determination, the
ratio between mean night (endogenous t exogenous) and mean daytime plasma
concentrations (exogenous) were significantly correlated with the implant insertion to first
behavioral estrus interval on Day 5 (0.67; PcO.01) and Day 35 (0.63; PcO.05) after the
start of the melatonin treatment. This indicates that a reduced ratio is associated with an
early resumption of the sexual activity.
T T I A
0 1* 33
Days after implant insertion
Theriogenology
(b) /
0 14 33
Daya after implant insertion
Figure 4. Changes in LH pulse frequency (a) and plasma LH concentrations (b) in
Salz ewes lambing in March with a high (H) or low (L) plane of nutrition after
weaning and treated (M) or not (C) with exogenous melatonin on Days 0
(n=20), 14 and 33 (n=5) after implant insertion. Means + SEM.
There was no significant effect of melatonin treatment or plane of nutriiion after
weaning on the mean number of hourly LH pulses (Figure 4). Mean plasma LH
concentrations were not affected by the plane of nutrition. However, treatment with
exogenous melatonin induced a significant increase in this parameter in the last 2
sampling periods (PcO.01). Mean LH pulse frequency and concentration at the second
sampling period (14 d after implant insertion or 2 d after weaning) was significantly lower
than that obtained 19 d later (P<O.Ol).
Evolution of the ovulation rate after weaning in the successive cycles is shown in
Figure 5. There was a significant effect of exogenous melatonin on the ovulation rate of
the second sexual cycle after weaning (PcO.01). Moreover, the interaction between plane
of nutrition and melatonin treatment was significant (PcO.01) in the second cycle, being
close to the significance in the first, fourth and fifth cycles (PcO.1).
Theriogenology 1187
3.CC OVULATION RATE
2.75 i
1 2 3 4 5
NUMBER OF CYCLES
AHhI +LM + HC %- LC
Figure 5. Ovulation rate at each detected estrus after weaning in Salz ewes lambing
in March with a high (H) or low (L) plane of nutrition and treated (M) or not
(C) with exogenous melatonin.
DISCUSSION
The decrease of the liveweight after weaning shown in Figure 1 could be related
to the lower digestive content caused by a higher plane of nutrition during the suckling
period. On the other hand, the energy content of liveweight gain varies with physiological
stage and seems to be very high when the ewes recover their fat reserves after weaning
(11). This could explain the different evolution of the liveweight and body condition
throughout the experiment, and confirms the higher reliability of body condition as an
indicator of the level of fat reserves.
The results obtained in the present experiment indicate the ability of a reduced
seasonality breed to respond to melatonin implantation in April in advancing the breeding
season by 37 d. This positive effect of the implants on early resumption of breeding
activity has been repotted in the literature when subcutaneous implants were inserted
around the summer solstice (12,25,29). However, Nowak and Rodway (33) and English
et al. (14) found that the insertion of a continuous-release intravaginal implant in May or
a subcutaneous implant in April were ineffective in advancing the breeding season. These
authors concluded that the failure of melatonin implants inserted in April/May to induce
early sexual activity may arise from inadequate prior exposure to long hours of daylight.
1188 Theriogenology
On the other hand, a daily oral dose of 3 mg melatonin at 1500 to 1600 h in March/April
clearly advanced estrous activity (38,47).
The explanation for the differing results is two-fold. First, in previous studies on
daily melatonin administration, ewes were exposed to rams whereas in continuous
melatonin release, only plasma progesterone levels were used to detect ovarian activity;
Robinson et al. (37) showed that melatonin enhances the sensivity of the hypothalamic
GnRH pulse generator in the presence of the ram. Second, the efficacy of the melatonin
treatment in advancing the breeding season may be higher in reduced seasonality
breeds, as was the case in our present study. Thus, the melatonin implants placed in
March/April and the male effect 35 d after insertion increased fertility in French flocks (9);
in contrast, ewes exposed to large changes in seasonal photoperiods could be less
susceptible to the effects of exogenous melatonin than ewes exposed to small changes
(13).
The mean date of the first detected estrus was June 15 for ewes with melatonin
implants irrespective of their post-weaning plane of nutrition, while it was July 18 and 23
for the control high and low plane of nutrition groups, respectively. In a previous
experiment, the onset of the breeding season in Salz ewes lambing in May and raising
twin lambs was August 17 (1). The lack of influence of the plane of nutrition after weaning
on the onset of sexual activity is confirmed in earlier reports indicating that this parameter
is barely influenced by nutritional treatment (1,2,31).
Instances of ovulations unaccompained by estrus before the first detected cycle
of the breeding season were observed in 40 animals. Fabre-Nys and Venier (15) reported
that at the beginning of the breeding season the first estrus was preceded by at least 1
ovulation. Identical findings have been reported in other breeds with a reduced
seasonality (16). Progesterone is involved in the initiation of full-length cycles (23).
Analysis of plasma melatonin levels showed that the difference between night and
daytime concentrations were similar among the 4 groups studied, in spite of the higher
daytime levels exhibited by the ewes with melatonin implants. This shows that extra
melatonin in the circulation does not appear to suppress pineal secretion of the hormone
(26). Moreover, except for the daytime basal values in the nonimplanted animals, there
was a tendency toward a higher amplitude of melatonin concentrations at the second
sampling test (May 19), with a shorter night period. Arendt et al. (5) reported a lower
amplitude profile of melatonin in winter than in summer.
Theriogenology 1189
One possible explanation for the higher plasma melatonin levels in treated ewes
of the low vs high plane of nutrition in the second sampling period could be related to the
hormonal metabolism. Melatonin is metabolized in the liver and kidneys and excreted in
the urine as hydroxymelatonin. Another hormone that is metabolized in the liver,
progesterone, shows up at high levels in undernourished ewes (35). Thus, the higher
melatonin concentrations observed in our implanted low feeding intake group could
indicate that the pattern of clearance of this hormone differs according to the plane of
nutrition.
The high, positive correlation coefficient between the relative concentration of
melatonin (ratio exogenous+endogenous/exogenous levels) and the early resumption of
estrous activity observed in our present study was first reported by Chemineau et al. (lo),
and could be useful in explaining the mode of action of a continuous-release melatonin
implant. Melatonin implantation results in a hormone pattern that would not normally be
found in the ewe (39). It has generally been assumed that melatonin implantation conveys
a short-day photoperiod signal, the effect of exogenous melatonin is comparable to the
exposure to a short photoperiod on the time of reproductive transitions (14,26). However,
O’Callaghan et al. (34) demonstrated that this assertion was only true in prolonging the
breeding season, indicating the interest in evaluating more accurately whether melatonin
implants also act to advance sexual activity as a short day or whether they render ewes
nonphotoperiodic, this last effect being comparable to that of pinealectomy (45). Our
results seem to indicate that Salz ewes accelerated their response in sexual activity
according to the relative concentration rather than to the absolute plasma levels of
melatonin, showing that a relative perception of melatonin concentrations could be
involved in the physiological mechanisms acting under natural photoperiod in the studied
animals around the summer solstice.
No effect of exogenous melatonin on number of LH pulses was detected in our
study, similar to the findings reported by Robinson et al. (38) after daily oral melatonin
treatment starting in March, indicating that in the ewe melatonin initiates estrous activity
by an abrupt rather than a progressive increase in the activity of the GnRH pulse
generator. However, according to the data reported by Chemineau et al. (8), melatonin
implantation induced a significant increase of mean plasma LH concentrations in dairy
goats treated daily with melatonin injections at Day 40 after the start of treatment. This
indicates that melatonin could induce a shift in response to factors that regulate LH
secretion (e.g., gonadal steroids; 24).
No significant effect of the plane of nutrition after weaning on ovulation rate was
observed, which is in agreement with previous data on Salz ewes lambing in the seasonal
1190 Theriogenology
anestrus and in contrast with the higher ovulation rate reported in another Spanish breed
(Rasa Aragonesa) associated with a high plane of nutrition after weaning (1). Other
authors have also observed a reduced effect of nutrition on the reproductive parameters
of high fecundity breeds (42). Gunn (20) reported that, within limits, these genotypes are
more consistent producers of lambs in a nutritionally variable premating environment than
low fecundity breeds. The effect of exogenous melatonin on the ovulation rate was
significant at the second estrus after weaning (P ~0.05). Mher authors have reported an
increase in the ovulation rate after treatment with melatonin (21,37), probably due to an
effect at ovarian level. Thus, raised plasma melatonin levels were measured in
ovariectomized ewes, suggesting that the gonads exert an inhibitory influence on
melatonin secretion (4). However, the mechanisms involved in this process remain
unknown.
A significant interaction was found in this study in the plane of nutrition by
melatonin treatment for the ovulation rate. To our knowledge, our findings are the first of
this type observed in melatonin-implanted ewes. Thus, the effects of melatonin implants
in enhancing the ovulation rate are more pronounced in ewes on a low vs high plane of
feed intake, in agreement with those reported by Robinson et al. (37) involving daily oral
melatonin administration. The mechanisms involved in this response are not known. In
post-pubertal beef heifers, however, lipogenesis seems to be stimulated by melatonin
(49). The practical implications of this interaction could be important in the reactivation of
estrous activity and ovulation rate in ewes adapted for intensive production and
accelerated lambing systems.
In conclusion, the results of the present study indicate that melatonin implantation
at the end of the suckling period in reduced seasonality ewes lambing in spring could be
useful in advancing estrous activity. The ovulation rate can be also increased, especially
in ewes receiving a low vs high (1.35 vs 1.8 times the maintenance requirements) plane
of nutrition after weaning. These findings are important to commercial sheep producers
using an intensive breeding system (3 lambings in 2 years) to increase the reproductive
performances in each mating period.
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