Influence of plane of winter nutrition on plasma concentrations of prolactin and testosterone and their association with voluntary food intake in red deer stags (Cervus elaphus)

Animal Reproduction Science, 8 (1985) 247--258

247

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

INFLUENCE OF PLANE OF WINTER NUTRITION ON PLASMA CONCENTRATIONS OF PROLACTIN AND TESTOSTERONE AND THEIR ASSOCIATION WITH VOLUNTARY FOOD INTAKE IN RED DEER STAGS (CER VUS ELAPHUS)

J.M. SUTTIE* and R.N.B. KAY

Physiology Department, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB (Great Britain) *Present address: Invermay Agricultural Research Centre, Private Bag, Mosgiel (New Zealand) (Accepted 21 May 1984)

ABSTRACT Suttie, J.M. and Kay, R.N.B., 1985. Influence of plane of winter nutrition on plasma concentrations of prolactin and testosterone and their association with voluntary food intake in red deer stags (Cervus elaphus). Anita. Reprod. Sci., 8: 247--258. Blood samples were taken at weekly or 3-weekly intervals from 12 red deer stags, from 3 weeks of age until 3 years of age. Six of the stags were fed to appetite throughout the study and six were maintained on 70% as much during each winter and to appetite each summer. All plasma samples were analysed for testosterone and prolactin. Food intake was measured daily. Annual rhythms of testosterone, prolactin and food intake were evident. Peaks of prolactin, on an increasing photoperiod tended to occur slightly before peaks of food intake and troughs of food intake on a decreasing photoperiod occurred at or slightly after peaks of testosterone. The reduced plane of winter nutrition tended to delay rather than alter the amplitude of the annual cycles of testosterone, prolactin or food intake. Several of the unrestricted stags showed biannual rhythms of testosterone, prolactin and food intake accompanied by additional cycles of coat development and antler growth. These additional cycles tended to be 6 months out of rhythm with normal cycles. It is considered that this is due to the stags being stimulated by a particular daylength irrespective of whether it is decreasing or increasing and that plane of nutrition may influence the control of phase blockers.

INTRODUCTION I n m a l e d e e r t h e a n n u a l c y c l e o f r e p r o d u c t i o n is c o n t r o l l e d b y a l t e r a t i o n s i n p h o t o p e r i o d m e d i a t e d b y c y c l e s o f v a r i o u s h o r m o n e s s u c h as t e s t o s t e r o n e ( S h o r t a n d M a n n , 1 9 6 6 ; L i n c o l n , 1 9 7 1 , M c E w a n , 1 9 7 3 , M c M i l l i n e t al., 1 9 7 4 ; M i r a r c h i e t al., 1 9 7 8 , L e a d e r - W i l l i a m s , 1 9 7 9 ) . T h e p a t t e r n i n all cases is t h a t levels o f t e s t o s t e r o n e are h i g h e s t d u r i n g t h e b r e e d i n g s e a s o n , a t w h i c h ever t i m e o f y e a r it o c c u r s , b u t d r o p t o l o w o r u n d e t e c t a b l e levels a t o t h e r

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248 times of the year. Annual cycles in plasma prolactin concentration have been shown for red deer (Cervus elaphus) (Brown et al., 1979). The nature of the seasonal cycle in the stag is similar to that of the ram except that in the ram amplitude is lower but mean testosterone levels during the breeding season may be higher (Simpson et al., 1984). Long term alterations or cycles of voluntary f o o d intake have recently received much attention in deer such as white tailed deer (Odocoileus virginianus) (Long et al., 1966), black tailed deer (O. hemionus) (Wood et al., 1962), reindeer (Rangifer tarandus) (McEwan, 1968), roedeer (Capreolus capreolus) (Drozdz and Osiecki, 1973), and red deer (Brown et al., 1979). In all cases intake of food in the summer m o n t h s was higher than in the winter. The only study to measure prolactin in conjunction with testosterone in red deer was by Brown et al. (1979). They showed that high levels of prolactin and high appetite occurred during long daylength and high levels of testosterone followed by low appetite during short days, but the results were partly c o n f o u n d e d due to the sequence of changes in daylength used. The present study formed part of a project designed to test the hypothesis that poo r winter nutrition was responsible for small sized Scots red deer stags (Suttie et al., 1983). The aim of this part of the study was to describe the effects of a restricted plane of nutrition during the winter on plasma levels of testosterone and prolactin. The extent to which annual changes in voluntary f o o d intake were associated with these hormones was also examined. MATERIALS AND METHODS

Animals Twelve red deer stags were maintained on a nutrition trial described fully by Suttie et al. (1983). In brief six stags were fed to appetite for 3 years (unrestricted) and the remainder were fed 70% of appetite (restricted) from August to May of their first year of life and November to May therafter and to appetite during the summer. The trial was designed to test the hypothesis that poor winter nutrition and subsequent failure to compensate during the summer was responsible for the small size of Scots red deer stags. All stags were fed a pelleted diet at 08.00 h and uneaten residues were collected and weighed so that daffy food intake could be measured. At weekly or 3-weekly intervals from June 1977 when the stags were 2 weeks old until May 1980 when the stags were 150 weeks old antler status was recorded and blood samples were taken by jugular venopuncture into preheparinised evacuated tubes.

Rad ioimmunoassays The testosterone was analysed using the m e t h o d of Corker and Davidson (1978) as follows. The antiserum used was B3R3FT (Rowe et al., 1974),

249 which was raised in the rabbit against testosterone-3-0-carboxymethoxyloxime BSA. The labelled ligand was [1, 2, 6, 7-3H(N)] testosterone (New England Nuclear, Boston, MA) and the standard was testosterone (Sigma Chemicals). The apparent sensitivity of the assay was 0.2 ng/mi and inter assay variation was 13.6%. The efficiency of extraction which was calculated separately for each tube and was corrected for accordingly, was normally >90%; all values <80% were repeated. All estimations were carried out in duplicate. Prolactin was determined using the m e t h o d of Chesworth (1977), which was a radioimmunoassay using polymerised second antisera. The antiserum was raised in rabbits against ovine prolactin. The labelled ligand was prepared by reacting ~2sI (Radiochemical Centre, Amersham, Bucks.) with chloromine T for 12 sec. Iodinated fractions were separated on a column of Sephadex (G-50), the protein fraction showing highest activity being used w i t h o u t further purification. There was no cross reaction with ovine LH, ovine GH or ovine TSH but there was with bovine prolactin. The sensitivity of the assay was 0.1 ng per tube and the inter-assay coefficient of variation was 4.4% and intra-assay was 3.3%. The blank consisted of a tube containing only the radioactive immunogen and counts b o u n d in each assay tube were expressed as a percentage of the counts in this tube. This percentage was plotted aginst the log of concentration. A dilution curve for plasma samples from stags containing high levels of prolactin was parallel to the standard curve for the assay. RESULTS

Seasonal cycles The changes in food intake and plasma levels of prolactin and testosterone throughout the study are given in Fig. 1 (unrestricted stags) and Fig. 2 (restricted stags). Representative individual curves from one stag from each group, number 3 (unrestricted) and number 7 (restricted) are given in Figs. 3 and 4 respectively. It is apparent from Fig. 1 that during the study some stags exemplified by number 3 exhibited two cycles of testosterone and prolactin accompanied by two cycles of food intake during the first 15 months of life and thereafter two cycles of each during 1 calendar year, where only one cycle of each would have been expected. For analysis the cycles have been labelled A--E, where B and D were the expected cycles which occurred at the normal time and A, C and E were the additional or out of season rhythms. In all animals high levels of prolactin soon after birth fell to basal levels by October 1977. Subsequently prolactin levels rose and began to fall before testosterone reached a maximum. Prolactin peaks preceded testosterone peaks by 13--16 weeks for normal cycles and 6 weeks for extra cycles. The profile of the unrestricted stags shows that food intake increased when prolactin increased but prolactin levels reached a m a x i m u m

250

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Fig. 2. T e s t o s t e r o n e , prolactin and v o l u n t a r y f o o d intake levels, e x p r e s s e d as a mean ± standard error, for the restricted group t h r o u g h o u t the study. The letters B and D label each c y c l e o f h o r m o n e s and f o o d intake. Periods o f f o o d restriction are s h o w n by the cross h a t c h e d areas. Stages o f the antler c y c l e are s h o w n as for Fig. 1.

251

some 10--11 weeks before f o o d intake in normal cycles and 6 weeks before in extra cycles. At or before prolactin reached basal levels of around 10 ng/ ml plasma levels of testosterone began to increase and appetite decreased. Minimum f o o d intake occurred after testosterone had reached a peak in the plasma, but while the secretory function of the testis was still active. The stags showed two peaks of testosterone, before rutting time in August and during the rut in October.

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252

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Fig. 4. T e s t o s t e r o n e , p r o l a c t i n a n d v o l u n t a r y f o o d i n t a k e levels for o n e r e p r e s e n t a t i v e res t r i c t e d stag, n u m b e r 7. T h e l e t t e r s B a n d D label e a c h cycle o f h o r m o n e s a n d f o o d intake. " a " refers t o a p e a k of p r o l a c t i n b e f o r e access to feeding t o a p p e t i t e , " b " refers t o a p e a k of p r o l a c t i n w h i c h o c c u r r e d a f t e r this time. Stages of t h e a n t l e r cycle are as s h o w n for Fig. 1.

Peak levels o f prolactin were highest during the first year o f life and fell to half these values during subsequent cycles. Peak testosterone levels t ended to either stay th e same or increase as t he stags became older. Maximum f o o d intake was observed when plasma levels of b o t h prolactin and testosterone were low, immediately after a prolactin peak b u t before a testosterone peak. Minimum f o o d intake occurred also when levels of b o t h h o r m o n e s were low b u t immediately after a t es t os t er one peak and before a prolactin peak.

253 The h o r m o n e profiles for the restricted stags with m o n t h l y fluctuations in food intake are shown in Fig. 2. Prolactin levels rose before access to unrestricted food intake. In Fig. 4, prolactin cycles B and D show bifurcated peaks, labelled a and b. Prolactin rose before and after access to feeding to appetite; this relationship was seen in all restricted but not in the unrestricted stags. Testosterone, prolactin and food intake relationships followed the same basic pattern as seen in the unrestricted stags. From Figs. 3 and 4 it is apparent that cycles of antler development occur in synchrony with hormonal and f o o d intake cycles. The hormonal control of antler development has been considered elsewhere (Suttie et al., 1984a), suffice to say that all are associated and form part of a complex cyclic chain of events such that once initiated will tend to go to completion.

Additional cycles The additional cycles identified in Fig. 1 require further comment. Cycle A was shown by three unrestricted stags but by no restricted stags. Cycle C was shown by five unrestricted and two restricted stags and Cycle E by one of the two remaining unrestricted stags. These cycles resembled those that occurred at the normal season in all ways except t h a t they were 6 months out of season. Prolactin was slightly at variance with this in that both peaks occurred when daylength was increasing and were not therefore exactly 6 m o n t h s apart. During Cycle B, the prolactin levels of the restricted group were significantly higher than the unrestricted group (P~0.05 -- unpaired two tailed t-test) and during Cycle D, the food intake of the previously restricted group was significantly higher (P<0.05) than that of the unrestricted group. Otherwise there was no significant difference in either peak height of the hormones or food intake or trough depth of food intake or the timing of the B and D cycles. Despite the winter restriction on food intake, the peaks and troughs of the restricted group occurred at the same time as those of the unrestricted group. As there was no significant difference in age at each event for each group, data from both groups were combined (Table I). Cycles A and E were omitted from further analysis due to insufficient data. The daylength at which each peak or trough occurred was then read off the daylength curve for 56°N. In order to consider all cycles on the same basis it was decided to compare each daylength with the immediately preceding solstice. The values were subtracted from the m a x i m u m daylength at 56°N 17.67 h if the light was decreasing at the time of the event and 6.50 h, the minimum number of hours of daylight at 56°N, was subtracted from the value if light was increasing at the time of the event. These data are given in Table II. The peaks of testosterone and food intake and the trough of food intake occurred at significantly the same time after each solstice irrespective of whether the prevailing photoperiod was increasing or decreasing. Although prolactin Cycles B and D occurred at the same time after the solstice, this m e t h o d of analysis

254

TABLE I Mean age (weeks) for both unrestricted and restricted stags of peak of testosterone, prolactin and food intake and trough of food intake in the stags. The cycles are described in Fig. 1. Age (weeks)

At At At At

testosterone peak prolactin peak food intake peak food intake trough

Cycle B

Cycle C

Cycle D

X-+S.D.

n

X-+S.D.

n

X-+S.D.

n

66.0 -+ 3.52 49.0 -+ 2.71 58.0-+ 2.94 70.0 -+ 1.44

12 12 12 12

88.0 82.0 85.0 94.0

7 7 5 5

114.0 101.0 109.0 119.0

10 9 9 9

-+ 4.19 -+ 5.15 -+ 3.10 -+ 3.80

± 3.95 -+ 5.51 -+ 3.91 -+ 2.47

TABLE II Timing of peaks from preceding solstice Time (h)

Cycle B

Cycle C

Cycle D

Levels of significance of inter-cycle differences

At testosterone peak At prolactin peak

4.34 -+ 1.58 9.20 -+ 1.25

2.75 -+ 2.08 0.75_+ 2.00

2.95 -+ 2.09 9.55-+ 1.62

At food intake peak At food intake trough

0.59 -+ 1.16 6.50 -+ 0.50

1.75 + 1.00 5.55 _+ 2.62

0.59 -+ 1.16 5.40 -+ 1.78

n.s. Bv . C P < 0 . 0 0 1 ; C v. D P<0.001 t = 10.09 t = 9.47 n.s. n.s.

In order to analyse timing of all peaks and troughs in relation to the photoperiod, each peak was compared to the preceding solstice. If light was decreasing, the day length at the time the peak occurred was subtracted from 17.67 h; if light was increasing, 6.5 h was subtracted from the day-length value. These figures represent length o f light at the longest and shortest days respectively at 56 ° North, the closest tabulated values to Aberdeen at 57°N. An unpaired two tailed t-test was used.

showed that prolactin Cycle C does not conform to the pattern shown by testosterone and food intake. DISCUSSION

Seasonal cycles T h e a n n u a l f l u c t u a t i o n s in t e s t o s t e r o n e r e s e m b l e t h o s e d e s c r i b e d f o r r e d d e e r b y L i n c o l n (1971). Cyclic f l u c t u a t i o n s o f circulating levels o f p r o l a c t i n w e r e s h o w n b y M i r a r c h i e t al. ( 1 9 7 8 ) f o r w h i t e t a i l e d d e e r , w h o f o u n d p e a k p r o l a c t i n l e v e l s o f 1 4 0 n g / m l in M a y , a n d r e d d e e r b y B r o w n e t al. ( 1 9 7 9 )

255 who found peak values of 100 ng/ml. The present study shows that prolactin levels were highest before the restricted stags were given free access to food, further peaks of prolactin occurred at the same time in both groups. This indicates that prolactin release in stags is a consequence of the photoperiod and n o t of f o o d intake. Bartke et al. (1978) have shown that changes in prolactin release may play an important role in mediating the effects of photoperiod on testicular activity in hamsters by increasing testis weight, spermatogenesis and the n u m b e r of LH receptors in the testis. It is suggested that prolactin shares this function in short day breeding deer as it does in long day breeding hamsters, that is to prepare the testis for androgenesis and spermatogenesis. Forbes et al. (1979) have shown significant positive effects of long daylength on prolactin secretion, f o o d intake and weight gain in sheep. In the present study, peaks of prolactin preceded peaks of food intake. Prolactin has been suggested as a metabolic hormone in cattle by McAtee and Trenkle (1971) and it seems likely that it is involved in mediating the effect of daylength on food intake in ruminants. Peters and Tucker (1978) showed that 16L : 8D increased prolactin levels in the plasma of cattle compared to cattle on a natural p h o t o p e r i o d b u t low temperature (d0°C) suppressed the response. As maximum f o o d intake occurs while prolactin levels are high in the present study but falling and testosterone levels are rising it is suggested that although prolactin may be involved in initiating the increase in food intake in response to increasing daylength, moderate levels of testosterone are needed for maximum food intake. Conversely, only high levels of testosterone are associated with the abrupt decline in f o o d intake during the rut. This would explain w h y food intake cycles occur in the presence of prolactin, but in the absence of testosterone in castrated animals, but to a reduced extent and without showing the precipitous fall seen in entire stags in October. The function of this precipitous fall in intake is of some interest. In the wild state, while guarding a harem of hinds, it could be argued a stag has no time to feed. Although no measurements of intake have been made on active rutting stags, the rumens of shot stags contain no food, only peat and mud (B. Mitchell, personal communication, 1979) which supports the theory that stags outdoors eat little at this time. But, as captive stags eat little or nothing in the absence of hinds, it would appear that this self-imposed fast need have nothing to do with actual mating activity. Instead it would appear the fast is driven by some testicular factor, probably testosterone. Were testosterone, the levels of which may require to be elevated to maintain the intense breeding activity if there is access to hinds, to act to inhibit appetite ceptres, then this may explain the fast. In this case the fast may have no function, but merely be an unavoidable consequence of high levels of testosterone. Rams in the breeding season also decrease food intake (Suttie et al., 1984b).

256

Additional cycles The fact that there are two endocrine cycles in one year accompanied by two cycles of food intake, and in some cases by two cycles of antler growth is most interesting and by no means w i t h o u t precedent. Whitehead and McEwan (1973) presented data from an adult reindeer bull who exhibited two antler cycles and two cycles of testosterone in one year, the peaks of testosterone being 6 months apart. Bubenik et al. (1979) showed that white tailed deer bucks showed two peaks of oestradiol in 1 calendar year, in May and November although they were unable to demonstrate elevated levels of testosterone in May and no additional antler cycles were evident. Captive red deer stags in zoological gardens have been known to grow and cast two separate sets of antlers in one year (Gillet, 1904; Petzsch, 1959). In the present study, red deer calves, juveniles and adult stags exhibited reproductive, appetite and antler cycles 6 m o n t h s out of season. With the exception of prolactin which in any case reaches a peak at or near each solstice, these events occurred at the same daylength, irrespective of whether it is increasing or decreasing. Goss (1976) has shown that sika deer (Cervus nippon) show normal antler cycles whether daylength is continually decreasing or increasing. His result suggests that antler cycles, and by inference other cycles as well, need not be tied to either the decreasing or increasing phase of the light cycle but may occur at a fixed daylength irrespective of prevailing daylength. So having given possible reasons for the occurrence of additional out of season cycles in the present study, why have they not been shown more frequently in previous studies and why do they not occur in free living red deer? Many detailed studies have been carried out on the reproduction and nutrition of domestic animals and had such an effect existed it would surely have been discovered. However, domestic animals have been subjected, by man, over the millennia, to varying degrees of selection either for seasonal {hill sheep) or aseasonal (dairy cattle and pigs) breeding. Any tendency to show a bimodal reproductive pattern would either be selected out or selected for respectively. In non-domestic mammals bimodal reproduction or a type of it has been shown to have adaptive significance. A u t u m n sexuality, which the effect is called in birds, has been recorded in 67 species of British birds and has been studied in such diverse species as eider ducks (Somateria mollissima) (Gorman, 1977) and rooks (Corvus frugilegus) (Lincoln et al., 1980). Several reasons may be advanced to explain why the effect has not been noticed more frequently before in non-domestic animals, which are not mutually exclusive: (a) It is possible that insufficient detailed study has been carried out on some species to detect bimodal reproduction; (b) The physiological possibility to exhibit bimodal reproduction may not exist; (c) Studies of bimodal birth peaks may be due to a heterogeneous population where some males and females breed at different times of the year. Thus although each individual exhibits unimodal reproduction, the population may be bi-

257 m o d a l . A f u r t h e r a l t e r n a t i v e is t h a t t h e e f f e c t is a s s o c i a t e d w i t h o n l y males or females. A c c o r d i n g t o B u n n i n g {1958}, a n i m a l s w h i c h are p h y s i o l o g i c a l l y c a p a b l e o f r e a c t i n g t o b o t h decreasing a n d increasing light at a given d a y length do n o t do so as o n e o r t h e o t h e r p o t e n t i a l l y s t i m u l a t i n g p h o t o p e r i o d is p h a s e b l o c k e d . B u n n i n g ( 1 9 5 8 ) u s e d t h e e x a m p l e o f low t e m p e r a t u r e ( < 5 ° C ) releasing a b l o c k t o d e v e l o p m e n t in plants. I t is suggested t h a t high p l a n e o f n u t r i t i o n m a y also act as a b l o c k releasing agent. I n t h e p r e s e n t s t u d y a higher p r o p o r t i o n o f t h e u n r e s t r i c t e d stags s h o w e d b i m o d a l r e p r o d u c t i o n c o m p a r e d w i t h t h e r e s t r i c t e d stags. This result w o u l d be e x p e c t e d if p o o r n u t r i t i o n d u r i n g a critical p h a s e o f t h e a n n u a l cycle o f p h o t o p e r i o d w e r e c a p a b l e o f b l o c k i n g b i m o d a l r e p r o d u c t i o n a n d a s s o c i a t e d p h e n o m e n a . In t h e p r e s e n t s t u d y t h e r e s t r i c t e d stags w e r e r e t u r n e d t o r e d u c e d i n t a k e in N o v e m b e r f o l l o w i n g a s u m m e r o f high f o o d i n t a k e while t h e u n r e s t r i c t e d stags s h o w e d little w i n t e r i n a p p e t a n c e a n d increased t h e i r f o o d i n t a k e . T h e r e was a v e r y r a p i d regression o f r u t t i n g b e h a v i o u r o f t h e u n r e s t r i c t e d stags culm i n a t i n g in antlers b e i n g cast as early as t h e e n d o f D e c e m b e r . I t is post u l a t e d t h a t t h e critical p h a s e is i m m e d i a t e l y a f t e r the rut: if p l a n e o f nutrit i o n increases t h e n t h e s t i m u l a t o r y d a y l e n g t h is n o t b l o c k e d ; if p l a n e o f nut r i t i o n decreases, as w o u l d be f o u n d in t h e wild s i t u a t i o n , t h e n it is b l o c k e d .

REFERENCES Bartke, A., Goldman, B.D., Bex, F.J. and Dalterio, S., 1978. Mechanism of reversible loss of reproductive capacity in a seasonally breeding mammal. Int. J. Androl., Suppl., 2: 345--353. Brown, W.B., Forbes, J.M., Goodall, E.D., Kay, R.N.B. and Simpson, A.M., 1979. Effects of photoperiod on food intake, sexual condition and hormone concentrations in stags and rams. J. Physiol., 296:58--59 P. Bubenik, G.A., Bubenik, A.B. and Zamecnik, J., 1979. The development of a circannual rhythm of estradiol in plasma of white tailed deer (Odocoileus virginianus). Comp. Biochem. Physiol., 62A: 869--872. Bunning, E., 1958. The Physiological Clock. Longmans/Springer Verlag, Berlin. Chesworth, J.M., 1977. Radioimmunoassays of ovine LH and ovine prolactin using polymerised second antisera. Anal. Biochem., 80: 31--40. Corker, C.S. and Davidson, P.W., 1978. A radioimmunoassay for testosterone in various biological fluids without chromatography. J. Steroid. Biochem., 9: 373--374. Drozdz, A. and Osiecki, A., 1973. Intake and digestibility of natural feeds by roe deer. Acta. Theriol., 18: 81--91. Forbes, J.M., Driver, P.M., Brown, W.B., Scanes, C.G. and Hart, I., 1979. The effect of day length on the growth of lambs 2. Blood concentrations of growth hormone, prolactin, insulin and thyroxine and the effect of feeding. Anita. Prod., 29: 43- 51. Gillet, F., 1904. Exhibited antlers of Altai stag from London Zoo. Proc. Zool. Soc. London, 1904: 179. Gorman, M.L., 1977. Sexual behaviour and plasma androgen concentrations in the male eider duck (Sornateria mollissima). J. Reprod. Fertil., 49: 225--230. Goss, R.J., 1976. Photoperiodic control of antler cycles in deer. III. Decreasing versus increasing day lengths. J. Exp. Zool., 197: 307--312.

258 Leader-Williams, N., 1979. Age related changes in the testicular and antler cycles o f reindeer, Rangifer tarandus. J. Reprod. Fertil., 57: 117--126. Lincoln, G.A., 1971. The seasonal reproductive changes in the adult red deer stag (Cervus elaphus L.) J. Zool., 163: 105--123. Lincoln, G.A., Racey, P.A., Sharp, P.J. and Klandorf, H., 1980. Endocrine changes associated with spring and autumn sexuality of the rook Corvus frugilegus. J. Zool., 190: 137--153. Long, T.A., Cowan, R.L., Strawn, G.D., Wetzel, R.S. and Miller, R.C., 1966. Seasonal fluctuations in feed consumption of the white tailed deer. Penn. State Univ, University Park, PA, Progress Report 262. McAtee, J.W. and Trenkle, A., 1971. Effects of feeding, fasting, glucose and arginine on plasma prolactin levels in the bovine. Endocrinology, 89: 730--734. McEwan, E.H., 1968. Growth and development of the barren ground caribou. II Postnatal growth rates. Can. J. Zool., 46: 1023--1029. McMillin, J.M., Seal, U.S., Keenlyne, K.D., Erickson, A.W. and Jones, J.E., 1974. Annual testosterone rhythm in the adult white tailed deer (Odocoileus virginianus borealis). Endocrinology, 94: 1034--1040. Mirarchi, R.E., Howland, B.E., Scanlon, P.F., Kirkpatrick, R.L. and Sanford, L.M., 1978. Seasonal variation in plasma LH, FSH, prolactin and testosterone concentrations in adult male white tailed deer. Can. J. Zool., 56: 121--127. Peters, R.R. and Tucker, J.A., 1978. Prolactin and growth hormone responses to photoperiod in heifers. Endocrinology, 103: 229--234. Petzsch, H., 1959. Zwei Geweihe bei einem Isubrahirsch-6 innerhalb eines Jahres (Cervus xanthopygus, Milne-Edwards). Zool. Gart., 24: 517--518. Rowe, P.H., Lincoln, G.A., Racey, P.A., Lehane, J., Stephenson, M.J., Shenton, J.C. and Glover, T.D., 1974. Temporal variations of testosterone levels in peripheral blood plasma of men. J. Endocrinol., 61: 63--73. Sempere, A., 1978. The annual cycle of plasma testosterone and teritorial behaviour in the roe deer. In: I. Assenmacher and D.S. Farner (Editors), Environmental Endocrinology. Springer-Verlag, Berlin, pp. 73--74. Short, R.V. and Mann, T., 1966. The sexual cycle of a seasonally breeding mammal, the roebuck (Capreolus capreolus). J. Reprod. Fertil., 12: 337--351. Simpson, A.M., Suttie, J.M. and Kay, R.N.B., 1984. The influence of artificial photoperiod on the growth, appetite and reproductive status of male red deer and sheep. Anim. Reprod. Sci., 6: 291--299. Suttie, J.M., Goodall, E.D., Pennie, K. and Kay, R.N.B., 1983. Winter feed restriction and summer compensation in red deer stags (Cervus elaphus). Br. J. Nutr., 50: 737--747. Suttie, J.M., Lincoln, G.A. and Kay, R.N.B., 1984a. The endocrine control of antler growth in red deer stags. J. Reprod. Fertil., 71: 7--15. Suttie, J.M., Kay, R.N.B. and Goodall, E.D., 1984b. The influence o f superior cervical sympathetic ganglionectomy on cycles of appetite and growth in Soay rams on a sixmonth photoperiod. Livest. Prod. Sci., 11: 529--534. West, N.O. and Nordan, J.C., 1976. Hormonal regulation of reproduction and the antler cycle in the male Columbian black tailed deer (Odocoileus hemionus columbianus) Part 1. Seasonal changes in the histology of the reproductive organs, serum testosterone, sperm production and the antler cycle. Can. J. Zool., 54: 1617--1636. Whitehead, P.F. and McEwan, E.H., 1973. Seasonal variation in the plasma testosterone concentration of reindeer and caribou. Can. J. Zool., 51: 651--658. Wood, A.J., Cowan, I. McT. and Nordan, H.C., 1962. Periodicity of growth in ungulates as shown by deer of the genus Odocoileus. Can. J. Zool., 40: 593--603.