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.

REFERENCES

1. Abecia JA, Forcada F, Zarazaga L A note on the effect of level of nutrition after weaning on the resumption of reproductive actMy by ewes of two Spanish breeds lambing in spring. Anim Prod 1993$X:273-276.

2. Abecia JA, Forcada F, Zarazaga I_, Lozano JM. Effect of plane of protein after weaning on resumption of reproductive activity in Rasa Aragonesa ewes lambing in late spring. Theriogenology 1993;39:463-473.

Theriogenology 1191

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

ARC. The Nutrient Requeriments of Ruminant Livestock. Slough, UK, Agricultural Research Council, 1980. Arendt J, Laud CA, Symons AM, Pryde SJ. Plasma melatonin in ewes after ovariectomy. J Reprod Fertil 1983;68:213-218. Arendt J, Symons AM, Laud CA. Pineal function in the sheep: Evidence for a possible mechanism mediating seasonal reproductive activity. Experientia 1981;37:584-586. Baird DT, Swanston IA, McNeilly AS. Relationship between LH, FSH and prolactin concentration and the secretion of androgens and estrogens by the preovulatory follicle in the ewe. Biol Reprod 1981;24:1013-1025. Biian EL, Karsch FJ, Hopkins JW. Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 1983;113:329-336. Chemineau P, Normant E, Ravault JP, Thimonier J. Induction and persistence of pituitary and ovarian activii in the out-of-season lactating dairy goats after a treatment combining a skeleton photoperiod, melatonin and the male effect. J Reprod Fertfl 1966;78:497-504. Chemineau P, Vandaele E, Brice G, Jardon C. Utilisation des implants de mefatonine pour I’amelioration des performances de reproduction chez la brebis. Ret Med Vet 1991; 167:227- 239. Chemineau P, Maurice F, Daveau A. Re-initiation of ovulatory activity by melatonin given as a constant-release implant in long-day treated Ile-de-France ewes depends on endogenous secretion of melatonin. In: Touitou Y, Arendt J, Pevet P (eds), Mefatonin and the Pineal Gland-From Basic Science to Clinical Application. Amsterdam, Elsevier Science Publishers BV, 1993; 247-250. Cowan RT, Robinson JJ, McDonald I, Smart R. Effects of body fatness at lambing and diet in lactation on body tissue loss, feed intake and milk yield of ewes in early lactation. J Agric sci 1980;95:497-514. Durotoye LA, Rajkumar R, Argo CM, Nowak R, Web@ GE, McNeil ME, Graham NB, Rodway RG. Effect of constant-release melatonin implants on the onset of oestrous activfty and on reproductive performance in the ewe. Anim Prod 1991;52:489-497. Eldon J. Effect of exogenous melatonin and exposure to a ram on the time of onset and duration of the breeding season in Iceland sheep. J Reprod Fertil 19X%99:1-6. English J, Poulton AL, Arendt J, Symons AM. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J Reprod Fettil 1986;77:321-327. Fabre-Nys CJ, Venier G. Quantitative analysis of oestrus behaviour through the breeding season in two breeds of sheep. Anim Reprod Sci 1989;21:37-51. Forcada F, Abecia JA, Sierra 1. Seasonal changes in oestrus activity and ovulation rate in Rasa Aragonesa ewes maintained at two diirent body condition levels. Small Rum Res 1992;8:313-324. Forcada F, Abecia JA, Zarazaga L A note on attainment of puberty of September-born early- maturing ewe lambs in relation to level of nutrition. Anim Prod 1991;53:407-409. Fraser S, Cowen P, Franklin M, Franey C, Arendt J. Direct radioinmunoassay for melatonin in plasma. Clin Chem 1983;29:396-397. Gbmez A. Estudio endocrine de las variaciones estacionales de la actividad reproductiva de la oveja y su efecto sobre el postparto y la lactation. Tesis Doctoral. Universidad Complutense Madrid, 1989.

1192 Theriogenology

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32. 33.

34.

35.

36.

37.

38.

Gunn RG. A note on the comparative reproductive performance of Friesland x North Country Cheviot and North Country Cheviot ewes on two levels of pasture prior to mating. Anim Prod 1986;42:287-289. Haresign W. Responses of ewes to melatonin implants: importance of the interval between treatment and ram introduction on the synchrony of mating, and effects on ovulation rate. Anim Prod 1992;54:41-45. Haresign W, Peters AR, Staples LD. The effect of melatonin implants on breeding activity and liier size in commercial sheep flocks in the UK. Anim Prod 1990;50:111-121. I’Anson H, Legan SJ. Changes in LH pulse frequency and serum progesterone concentrations during the transition to breeding season in ewes. J Reprod Fertll1988;82:341- 351. Karsch FJ, Biiman El_ Foster DL, Goodman RL, Legan SJ, Robinson JE. Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 1984;40:185-225. Koumitzis SA, Belibasaki S, Doney JM. Melatonin advances and condenses the onset of seasonal breeding in Greek dairy ewes. Anim Prod 1989;48:399-405. Lincoln GA, Ebling FJP. Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and mounting in rams. J Reprod Fertil 1985;73:241-253. Lopez A, lnskeep EK. Response of ewes of Mediterranean sheep breeds to subcutaneous implants of melatonin. Livest Prod Sci 1991;27:177-184. Malpaux B, Robinson JE, Brown MB, Karsch FJ. Reproductive refractoriness of the ewe to inductive photoperiod is not caused by inappropiate secretion of melatonin. Biol Reprod 1987;36:1333-1341. McMillan WH, Sealey RC. Do melatonin implants influence the breeding season in Coopworth ewes?. Proc NZ Sot Anim Prod 1989;49:43-46. Mondain M, Bonim M, Canivenc R, Scholler R. Heterologous radioinmunoassay of fox LH: Levels during the reproductive season and the anoestrus of the wild fox (Vu/pas vu/pea). Gen Comp Endocrinol 1984;55:125-132. Montgomery GW, Scott IC, Johnstone PD. Seasonal changes in ovulation rate in Coopworth ewes maintained at different llleweights. Anim Reprod Sci 1988;17:197-205. Norusis MJ. SPSS/PC t . Chicago, SPSS Inc, 1986. Nowak R, Rodway RG. Effect of intravaginal implants of melatonin on the onset of ovarian activity in adult and prepubertal ewes. J Reprod Fertll 1985;74:287-293. O’Callaghan D, Karsch FJ, Boland MP, Roche JF. What photoperlodic signal is provided by a continuous-release melatonin implant?. Biol Reprod 1991;45:927-933. Parr RA, Davis IF, Fairclough RJ, Miles MA. Overfeeding during early pregnancy reduces peripheral progesterone concentration and pregnancy rate in sheep. J Reprod Fertil 1987;80:317-320. Quirke JF, Hanrahan JP. Breed differences in the breeding season in sheep. In: Ellendorff F, Elsaesser F (eds), Endocrine Causes of Seasonal and Lactational Anoestrus in Farm Animals. Dorchecht, Martinus Nijhoff Publishers, 1985; 29-44. Robinson JJ, Wigzell S, Aiiken RP, Wallace JM, Ireland S, Robertson IS. The modifying effects of melatonin, ram exposure and plane of nutrition on the onset of ovarian activity, ovulation rate and the endocrine status of ewes. Anim Reprod Sci 1991;26:73-91. Robinson JJ, Wigzell S, Aitken RP, Wallace JM, Ireland S, Robertson IS. Daily oral administration of melatonin from March onwards advances by 4 months the breeding season of ewes maintained under the ambient photoperiod at 57” N. Anim Reprod Sci 1992;27: 141- 160.

Theriogenology 1193

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

Ronayne E, Jordan B, Quirke JF, Roche JF. The effect of frequency of administration of melatonin on the time of onset of the breeding season in anoestrous ewes. Anim Reprod Sci 1989;18:13-24. Russel AFJ, Doney JM, Gunn RG. Subjective assessment of body fat in live sheep. J Agric Sci 1969;72:451-454. Sierra I. Formation de una nueva raza sintetica a partir de las razas Romanov y Rasa Aragonesa. Zootechnia 1982;XXXI:14-20. Smith JF. Protein, energy and ovulation rate. In: Land RB, Robinson JJ (eds), Genetics of Reproduction in Sheep. London, Butterwonhs, 1985; 349-359. Tillet Y, Ravault JP, Selve C, Eving G, Castro 8, Dubois, MP. Conditions d’utilisation d’anticorps specifiques pour la visualisation immunohistochimique de la serotonine et de la melatonine dans la glande pineale du mouton. C R Acad Sci Paris 1986:303:77-82. Vosniakou AG, Doney JM, Tsakalof P. A note on the seasonal oestrus period in three breeds of Greek dairy sheep. Anim Prod 1989;49:147-150. Wayne NL, Malpaux B, Karsch FJ. Photoperiodic requirements for timing onset and duration of the breeding season of the ewe: Synchronization of an endogenous rhythm of reproduction. J Comp Physiol 199O;A166:835-842. White DH, Bowman PJ, Morley FHW, McManus WR, Filan SJ. A simulation model of the breading ewe flock. Agric Syst 1983;10:149-189. Wrlliams H, Ward S. Melatonin and light treatment of ewes for autumn lambing. Reprod Nutr Dev 1988;28:423-429. Yeates NTM. The breeding season of the sheep with particular reference to its modification by artificial means using light. J Agric Sci 1949;39: l-43. Zinn SA, Chapin LT, Enright WJ, Schroeder AL, Stanisiewski EP, Tucker HA. Growth, carcass composition and plasma melatonin in postpubertal beef heifers fed melatonin. J Anim Sci 4988;66:21-27.