BIOLOGY OF RPRODUTIO 18, 279-285 (1978) Serum Melatonin oncentrations During Different Stages of the Annual Reproductive ycle in wes M. D. ROLLAG, P. L. O ALLAGHA and G. D. ISWDR2 Department of Physiology and Biophysics, olorado State University, Fort ollins, olorado 8523 ABSTRAT To determine the effect of reproductive status on photo-induced alterations in serum concentrations of melatonin, samples of blood were collected at 2 h intervals for 96 h from ewes during estrous cycles, anestrus, the transition from estrus to anestrus and the transition from anestrus to estrus. Two groups of ewes were sampled during the normal breeding season. One group was sampled during the luteal phase of the estrous cycle (Days 8-11 of a 16 day cycle) and the other group was sampled during the follicular and estrual phases of the cycle (Days 15, 16, 1, 2). At each of the 4 times of the year sampled, there was a distinct circadian rhythm in peripheral concentrations of melatonin. Average night-time concentrations (297 ± 46.5, S..M., pg/mi) were 2-3 times greater than average day-time concentrations (14 ± 16.8, S..M., pg/mi). oncentrations of melatonin remained constant throughout the dark phase. Due to alterations in duration of the photoperiod, the duration of elevated melatonin concentrations was longest when ewes were exhibiting estrous cycles and shortest when ewes were exhibiting anestrus. Mean concentrations of melatonin during the day or during the night did not differ significantly during different reproductive states. To determine precisely how serum melatonin concentrations reflect light-dark transitions, melatonin concentrations were quantified in samples of blood collected at 1 mm intervals from 3 ewes during the mid-luteal phase of the estrous cycle. Lights were left on 2 h into the normal night, turned off 1 h and then turned on. Sampling was initiated 3 mm before the initial light-dark transition and was terminated 3 mm after the return to light. When ewes were exposed to light, plasma concentrations of melatonin remained at baseline levels of 1-1 pg/mi. Upon initiation of darkness, there was a short lag (5-1 mm) after which melatonin concentrations elevated to levels of 15-3 pg/mi. This elevation occurred over a 2 mm interval in 2 ewes and over a 1 mm interval in the third. Upon return to light, melatonin concentrations declined to baseline levels within 5-1mm. ITRODUTIO In temperate latitudes sheep display behavioral estrus during those months surround ing the winter solstice and behavioral anestrus during those months surrounding the summer solstice (Hafez, 1952). This annual rhythm in reproductive competence persists when sheep are maintained under constant environmental conditions (Radford, 1961a, b; Wodzicka- Tomaszewska et al., 1967; Speedy and Owen, 1975) suggesting that the rhythm is driven by an endogenous mechanism. evertheless, the rhythm can be modified by alterations in Acceted August 29, 1977. Received April 11, 1977. Present address: Department of Anatomy, University of Texas Health Science enter, 773 Floyd url Drive, San Antonio, Texas 78284. 2 Send reprint requests to Dr. Gordon D. iswender. photoperiod. A shift in the phase of the annual photoperiodic rhythm results in a corresponding shift in the phase of the reproductive rhythm (Yeates, 1949; Yeates, 1956; Wodzicka-Tomaszewska et al., 1967). Small changes in the period of the annual photoperiodic rhythm result in corresponding changes in the period of the annual reproductive rhythm (esely, 1975). These effects indicate that although photoperiod does not drive the annual reproductive rhythm in sheep, it does act as an entraining agent. For photoperiod to be an effective entraining agent, sheep must be differentially responsive to the effects of photoperiod at different stages of the annual reproductive cycle. Such a differential responsiveness to photoperiod has been demonstrated (Hart, 195; Fraser and Laing, 1969). Short photoperiods are more effective in advancing the onset of estrous activity when ewes are near the 279
28 ROLLAG T AL end of the anestrous period than when ewes are in the middle of the anestrous period. We have postulated that in the sheep, as in the ferret (Herbert, 1972), an intact pineal gland is necessary for photoperiod to act as an entraining agent for the reproductive rhythm. To determine if the differential sensitivity of sheep to photoperiod could be attributed to an annual rhythm in the responsiveness of the pineal gland to light-dark transitions, peripheral concentrations of melatonin have been examined at different stages of the annual reproductive cycle. The dynamics of photo-induced alterations in plasma concentrations of melatonin have also been characterized to determine how accurately peripheral concentrations of melatonin reflect the duration of darkness. MATRIALS AD MTHODS Melatonin oncentrations at Different Times of Year xperimental animals were chosen from a population of orriedale crossbred western range ewes maintained in an outdoor pen. asectomized rams were used to detect sexual receptivity. strous activity began during the first and second week of September and ended during the last week of January and the first week of February. During the intervals of February 4-8, 1975, ovember 4-8, 1975; June 3-7, 1976 and August 19-23, 1976, groups of 4 ewes were moved into an indoor pen with overhead fluorescent lighting (4 watt cool white, Westinghouse F4OW; 12 feet from the floor). The intensity of illumination as determined by a Weston Illumination Meter, model 756 (Weston lectrical Instrument orp., ewark,.j.), ranged between 42 and 46 foot-candles. The light cycle in the indoor pen was synchronized with the natural photoperiod, the lights were turned on and off at the half-hour nearest to civil twilight. When the ewes were exhibiting normal estrous cycles (ovember 4-8, 1975) 2 groups of 4 ewes were sampled simultaneously. wes of 1 group were sampled during the follicular and estrual phases of the estrous cycle (samples collected from Day 15 until Day 2 of a 16 day cycle) and ewes of the other group were sampled during the luteal phase of the estrous cycle (samples collected from Day 8 until Day 11 of the cycle). Upon introduction to the indoor pen, a 1cm, 16 gauge teflon cannula was inserted into both the right and left jugular vein of each ewe. The ewes were allowed to adjust to their environment for 24 h, when 1 ml of blood were withdrawn at 2 h intervals for 96 h. The blood was stored overnight at 4#{176}. Serum was harvested following centrifugation at 2 X g for 3 mm and stored frozen. The melatonin content was quantitated by radioimmunoassay (Rollag and iswender, 1976). To monitor assay precision and accuracy, a set of 5 control samples (serum with a low, an intermediate and a high concentration of melatonin, assay buffer and buffer containing 1 ng/ml melatonin) was inserted after every 5 unknown samples prior to extraction. Hypotheses were tested utilizing analysis of variance or a t test. Dynamics of Photo-Induced Alterations in Melatonin oncentrations orriedale crossbred western range ewes in the mid-luteal phase of the estrous cycle (Day 9 of a 16 day cycle) were utilized to study the dynamics of photo-induced alterations in plasma concentrations of melatonin. On the day of the experiment, October 4, 1976, 3 ewes were transferred to a room with Westinghouse cool white fluorescent lamps (F4OGW, 4 watts) 3.6m above the ground. The intensity of illumination as measured by a Weston Illumination Meter, model 756 (Weston lectrical Instrument orp., ewark,.j.), was 25 foot-candles at the level of the sheep s eyes. When introduced to the indoor pen, a 1 cm, 16 gauge teflon cannula was inserted into both the right and left jugular vein of each ewe. Three hours prior to the initiation of the experiment, the ewes were placed into individual stalls (.45m X 1.2m) to limit mobility. At 212 h, 1, IU heparin (Sigma hemical o., St. Louis, MO) were injected i.v. and a 1.2m segment of #18 polyvinyl chloride tubing (Alpha Wire o., Torrance, A) having a luminal volume of 2 ml was connected to 1 cannula of each ewe. The 3 tubings were passed through a polystatic pump (Buchler Instruments, Fort Lee,.J.) and connected to fraction collectors (ISO, model 12 pup, Lincoln, ). Starting at 213 h, blood was withdrawn continuously over a 2 h interval at a rate of 2 mi/mm and pooled into 1 mm fractions. Due to problems with a cannula, samples were not collected from 1 ewe during the interval of 22-225 h. The lights were left on until 22 h, turned off for 1 h and then turned on until 233 h. Prior to this experiment the ewes had been maintained in an open air pen and were adapted to a light cycle in which the dark phase began at approximately 19 h. Plasma was harvested, stored and assayed for melatonin as described previously. RSULTS In those radioimmunoassays utilized to determine the concentrations of melatonin in samples obtained at different times of year, serum samples with melatonin concentrations of 181 pg/ml, 488 pg/mi and 1139 pg/ml had intra-assay coefficients of variation of 19.3%, 15.8% and 11.7%, respectively. The respective inter-assay coefficients of variation were 2 1.2%, 16.4% and 22.2%. The melatonin content of the 1 ng/ml solution of melatonin in buffer was radioimmunoassayed as 957 ± 218 (SD) pg/ml. The least detectable concentration of melatonin ranged between 7 and 17 pg/ml. In those radioimmunoassays utilized to determine the dynamics of photo-induced alterations in peripheral melatonin concentrations, serum samples with melatonin concentrations of 116 pg/ml, 396 pg/ml and 114 pg/ml had intra-as-
MLATOI OTRATIOS I WS 281 say coefficients of variation of 21.1%, 12.4% and 11.2%, respectively. The melatonin content of the 1 ng/mi standard solution was radioimmunoassayed as 845 ± 97 (SD) pg/ml. The least detectable concentration of melatonin ranged from 6 to 36 pg/ml. The levels of melatonin in serum of ewes during the different times of year are depicted in Figs. 1-5. The values represent the mean ± one standard error for the 4 animals sampled. During each of the 4 times of year there was a distinct circadian rhythm in peripheral concentrations of melatonin. Within a particular group there was no difference (P>.5) between the concentrations of melatonin in serum collected at the different times during darkness. Similarly, there was no difference (P>O.5) between the concentrations of melatonin in serum collected at the different times during light. The average concentrations of melatonin from each animal in those samples collected in darkness and in those samples collected in light are shown in Table 1. There was always a significant difference (P>.5) when the average daytime concentration of melatonin was compared to the average night-time concentration of melatonin within each animal. o difference (P<.5) was found between the mean night-time concentrations of melatonin during the different times of year. Similarly, there was no difference (P<.5) in the mean day-time concentrations of melatonin during the different times of year. The high variance in the estimates associated with the concentrations found in the follicular and estrual phases of the estrous cycle can be attributed to 27 24 21. 18 15. 12 a 9 6 3 12 24 36 48 6 72 84 96 18 Hour of xperiment FIG. 2. oncentrations of melatonin in ovine serum obtained during the interval of ovember 4-8, 1975. wes were in the follicular (Days 15 and 16) and estrual (Days 1 and 2) phases of the 16 day estrous cycle. Darkness is indicated by the solid bar on the time axis. alues represent the mean ± SM for 4 ewes. The high variance is due to exceptional values of a single ewe (animal 4, Table 1). exceptional values for a single ewe (Table 1, animal 4). The concentrations of melatonin in plasma as a function of time in the 3 ewes utilized to characterize the dynamics of photo-induced alterations in peripheral concentrations of melatonin are represented in Fig. 6. During periods of light, concentrations of melatonin remained at baseline levels which ranged between 1 and 1 pg/ml. Upon initiation of darkness there was a short lag of approximately 5-1 mm 5) 12 24 36 48 6 72 84 96 18 Hour of xperiment FIG. 1. oncentrations of melatonin in ovine serum obtained during the interval of ovember 4-8, 1975. wes were in the luteal phase of the estrous cycle (Day 8-11 of a 16 day cycle). Darkness is indicated by the solid bars on the time axis. alues represent the mean ± SM for 4 ewes. = 6 5-4 c 3 2 a 1 #{149} #{149} 12 24 36 48 6 72 84 96 18 Hour of xperiment FIG. 3. oncentrations of melatonin in ovine serum obtained during the interval of June 3-7, 1976. wes were exhibiting behavioral anestrus. Darkness is indicated by the solid bars on the time axis. alues represent the mean ± SM for 4 ewes.
282 ROLLAG T AL. = 6 5 r-1cc -,.,scc t-.s cc,-.cc 4 3. <2 2 5) 1. be - t-_ X O 12 24 36 48 6 72 84 96 18 Hour of xperiment FIG. 4. oncentrations of melatonin in ovine serum obtained during the interval of August 19-23, 1976. Darkness is indicated by the solid bars on the time axis. alues represent the mean ± SM for 4 ewes. wes were experiencing the transition between anestrus and estrus... be cc SO cc t -.-, cc cc rc after which concentrations of melatonin elevated to peak levels ranging from 15 to 3 pg/ml. This elevation occurred over a 2 mm interval in 2 of the ewes and over a 1 mm interval in the other. When the lights were turned on 1 h later, melatonin concentration rapidly declined to baseline level. This decline was completed within 5-1 mm., o.l. oa,c -*** orc cc t- cc)rc * DISUSSIO The magnitude of photo-induced alterations in peripheral concentrations of melatonin was shown not to differ during the different stages of the annual reproductive cycle in sheep. However, it is shown that peripheral concentrations of melatonin do accurately reflect the.. O as cc. cc cc - * (ji. be - OO * o *.* - - - - * - = 6 5. - 4 3 2 a) 1.)t *Q*b. -*,-.,-,- 12 24 36 48 6 72 84 96 18 Hour of xperiment FIG. 5. oncentrations of melatonin in ovine serum obtained during the interval of February 5-8, 1975. Darkness is indicated by the solid bars on the time axis. alues represent the mean ± SM for 4 ewes. wes were experiencing a transition from behavioral estrus to behavioral anestrus. U U -4 I- <. be X O-. - cc*
MLATOI OTRATIOS I WS 283 a. 4 3 2 1 4 3 2 1 4 3 2 1 during the follicular phase (ardinali et al., 1974) apparently does not cause a significant elevation in peripheral melatonin concentrations. Data described in this report differ from those reported for humans (Wetterberg et al., 1976). In women, daytime melatonin concentrations are highest at mensus and lowest at ovulation. Based on the photo-induced response of the pineal gland, a hypothetical mechanism for photoperiodic control of ovine reproductive competence has been developed and is presented in Fig. 7. According to this mechanism, Photoperiodic ontrol of Reproduction 213 223 233 Time (hi light Retina FIG. 6. Melatonin concentrations in samples collected at 1 mm intervals from 3 ewes. Darkness is indicated by the solid bar. alues represent the mean concentration ± 95% confidence intervals for the radioimmunoassay of duplicate samples. Samples were not obtained from the ewe represented in the bottom frame during the interval of 22 to 225 h. duration of darkness with the pineal gland responding rapidly to light-dark transitions. Perhaps sheep monitor the duration of the dark phase by monitoring the duration of elevated melatonin concentrations. The precipitous decline in serum concentrations of melatonin following the transition from dark to light could not occur if melatonin was not rapidly removed from the blood. This removal does not necessarily imply metabolism since melatonin may be partitioning into a large pool such as body fat. However, if such a pool exists, it is not saturated under physiological conditions. The response of the pineal gland to photostimulation may reflect changes in the activity of -acetyserotonin transferase (Klein and Weller, 1972; Deguchi and Axelrod, 1972), alterations in blood flow to the pineal gland (Quay, 1972; Rollag, 1977) or control of an underlying secretory mechanism. In ewes, no significant difference was found between the concentration profiles for melatonin during the luteal phase and the follicular and estrual phases of the estrous cycle. The elevation of hydroxyindole-o-methyltransferase activity reported in the pineal gland of sheep Dec. Sept. 8 h,,_-..24 h L#{128}) 12h- 6h March June Pineat Suprochiasmotic ucleus Phase urve Hypothalamohypophyseai axis Gland P Response FIG. 7. Hypothetical mechanism for photo-induced alterations in ovine reproductive competence. The solid bar in the pineal compartment indicates darkness. The shaded area of the suprachiasmatic nucleus represents that circadian time at which the hypothalamus is sensitive to melatonin. The phase response curve refers to the sensitivity of the circannual rhythm in reproductive competence to melatonin input as a function of the phase of the rhythm. The striped bar indicates estrus. A positive value indicates that melatonin increases the rate of the circannual oscillation (shortens the period) and a negative value indicates that melatonin decreases the rate of oscillation. The striped area in the circle representing the hypothalamo-hypophyseal axis indicates estrus and the clear area indicates anestrus.
284 ROLLAG T AL. the primary effect of light is to inhibit the pineal gland s secretion of melatonin. Due to this inhibitory effect, elevated melatonin concentrations accurately reflect the duration of darkness. onsequently, during the fall and winter months elevated rates of melatonin secretion persist for a greater length of time than occurs during the spring and summer months. The target tissue for melatonin is thought to be the hypothalamo-hypophyseal axis (Berndtson and Desjardins, 1974; Turek et al., 1975). According to the proposed mechanism for photoperiodic control of reproductive competence, the response of the hypothalamohypophyseal axis to melatonin stimulation is dependent upon two endogenous rhythms, a circadian rhythm, perhaps driven by the suprachiasmatic nucleus (Moore and icher, 1972; Stephan and Zucker, 1972), and a circannual rhythm. These rhythms in the sensitivity of the hypothalamo-hypophyseal axis to melatonin stimulation may be due to variations in the number of melatonin receptors. As a consequence of the endogenous circadian rhythm, melatonin concentrations must be elevated in the late afternoon of the subjective day to be effective. Presuming that melatonin concentrations are elevated at the proper time of day, the resultant response of the reproductive system is dependent upon the phase of the annual reproductive cycle which sheep are experiencing. If sheep are exhibiting behavioral estrus, melatonin stimulation will lengthen the period of the annual reproductive cycle, whereas, if sheep are exhibiting behavioral anestrus, the period of the cycle will be shortened. In this scheme it is not the duration of elevated melatonin concentrations which cues photo-induced reproductive changes, but instead the time of day during which melatonin concentrations are elevated. It is postulated that during the long nights of winter months, elevated melatonin concentrations overlap the sensitive portion of the circadian oscillation, whereas, during summer months they do not. This inclusion of a daily critical period in the hypothetical scheme for photoperiodic control of ovine reproductive competence explains the observation of Hart (195) that a 4L:2D:4L: 14D photoperiod is more effective than a 4L:8D:4L:8D photoperiod in advancing the onset of estrous cyclicity in Suffolk ewes. Although the ratio of light to dark is the same in both of these photoperiodic regimens, the time of day during which the light phases occur differ. A similar difference in the photoperiodic regimen significantly affects the reproductive response of both hamsters (Stetson et al., 1975) and voles (Grocock and larke, 1974). The role of melatonin in the interpretation of photoperiodic cues is supported by the observation that melatonin injections in the morning do not affect reproductive competence in hamsters, whereas, injections in the afternoon cause gonadal regression (Tamarkin et al., 1976). If this hypothetical mechanism is correct, pinealectomy of sheep would not prevent the transition between estrus and anestrus nor would it affect the 16 day estrous cycle. Instead, pinealectomy would prevent photoperiodic entrainment of the annual reproductive rhythm. Indeed, the length of the estrous cycle in pinealectomized ewes did not differ from controls, nor are the transitions from estrus to anestrus and from anestrus to estrus prevented by pinealectomy (Roche et al., 197a). The hypothesis would predict that melatonin administration would not affect the 16 day estrous cycle, but would, if administered every day in the late afternoon, result in a significant alteration in the timing of the onset of the transitions between estrus and anestrus. Administration of milligram quantities of melatonin during the latter half of the anestrous phase would advance the onset of reproductive competence and administration in the latter half of the estrous phase would delay the onset of anestrus. When melatonin has been administered to ewes during estrus (Roche et al., 197b), there has been no effect upon ovulation or the timing of LH release. The effects of melatonin administration upon the transitions between estrus and anestrus in sheep have not been examined, however, daily administration of melatonin to ferrets during the anestrus phase has advanced the onset of estrous activity (Thorpe and Herbert, 1974). AKOWLDGMTS This research was supported by funds from the olorado State University xperiment Station. RFRS Berndtson, W.. and Desjardins,. (1974). irculating LH and FSH levels and testicular function in hamsters during light deprivation and subsequent photoperiodic stimulation. ndocrinology 95, 19 5-25. ardinali, D. P., agle,. A. and Rosner, J. M. (1974). hanges in the pineal indole metabolism and
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