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Endocrine changes in photostimulated willow ptarmigan (Lagopus lagopus lagopus) and Svalbard ptarmigan (Lagopus mutus hyperboreus)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
70, 169-177 (1988)
Endocrine Changes in Photostimulated Willow Ptarmigan (Lagopus lagopus lagopus) and Svalbard Ptarmigan (Lagopus mutus hyperboreus) KARL-ARNE STOKKAN, PETER J. SHARP,* IAN C. DUNN,* AND ROBERT W. LEA? Department of Arctic Biology and Institute of Medical Biology, University Tromso, Norway; *AFRC Institute of Animal Physiology and Genetics Station, Roslin, Midlothian EH25 9PS, Scotland; and fSchoo1 of Applied Preston PRl 2TQ, United Kingdom
of Tromso, P.O. Box 635, N-9001 Research, Edinburgh Research Biology, Lancashire Polytechnic,
Accepted December 1, 1987 Changes in plasma luteinizing hormone (LH), testosterone, thyroxine (T4), and triiodothyronine (T,) and the height of supraorbital combs were compared in captive willow ptarmigan (Lagopus lagopus lagopus) and Svalbard ptarmigan (Lagopus mutus hyperboreus) exposed to an artificial annual cycle of daylength simulating that at 70”N. Plasma LH and testosterone and comb height increased more slowly in Svalbard than in willow ptarmigan as daylength increased. In both species, plasma LH and testosterone fell abruptly, and the supraorbital combs regressed in June, marking the development of long-day refractoriness. Comparison with free-living Svalbard ptarmigan (K.-A. Stokkan, P. J. Sharp, and S. Unander (1986) Gen. Comp. Endocrinol. 61, 44M51) at 80”N, showed a similar slow increase in reproductive function before the onset of the breeding season. However, maximum plasma LH levels and comb size were higher in free-living than in captive Svalbard ptarmigan. Furthermore, long-day refractoriness developed earlier in captive than in freeliving Svalbard ptarmigan. In both species of ptarmigan, the development of long-day refractoriness was associated with increased plasma prolactin. This increase was larger and occurred earlier in the Svalbard than in the willow ptarmigan. Seasonal changes in thyroid hormones were not as marked as for the other hormones measured. In both species, plasma T, tended to increase and plasma T, to decrease as daylength increased. A small increase in plasma T, was seen after the development of long-day refractoriness in both species. It is concluded that captivity depresses the photoperiodic response of Svalbard ptarmigan more than that of willow ptarmigan. The association between increased plasma prolactin and the development of long-day refractoriness is similar to that reported for birds in other families. The changes in plasma thyroid hormones during the development of refractoriness cannot be readily related to changes seen in other species. 0 1988 Academic Press, Inc.
In most birds breeding at temperate zone latitudes, increasing daylengths in spring stimulate a rapid, final maturation of the gonads to initiate the breeding season. The same response to increasing daylength in long-day breeders at very high latitudes may not be appropriate. At these latitudes, daylengths become continuous long before environmental conditions such as temperature and snow cover allow successful breeding to occur. Thus, at 70”N, Norwegian willow ptarmigan (Lagopus Zagopus
Zagopus) breed in late May after exposure
to continuous light for nearly a month (Stokkan and Sharp, 1980a) while at 80”N, the Svalbard ptarmigan (Lagopus mutus hyperboreus) breeds in June after exposure to continuous light for about 2 months (Stokkan et al., 1986). In both the Svalbard ptarmigan (K. Lindgard and K.-A. Stokkan, unpublished observations) and the willow ptarmigan (Stokkan and Sharp, 1980a,b), the seasonal development of the testes depends on an increase in daylength.
169 0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The lag between increasing daylength and the onset of breeding in free-living Svalbard ptarmigan can be accounted for by an initial slow increase of plasma luteinizing hormone (LH) and gonadal growth (Stokkan et al., 1986). A similar lag in increase of plasma LH and comb growth after photostimulation is not so obvious in the willow ptarmigan (Stokkan and Sharp, 1980a,b; Sharp and Moss, 1981). However, a direct comparison between the two species exposed to identical lighting conditions has not been made. Seasonal breeding in both species of ptarmigan is terminated by the development of long-day refractoriness (Stokkan and Sharp, 1980a; Stokkan et al., 1986). Egg laying in free-living Svalbard ptarmigan may continue well into July (Stokkan et al., 1986) while in the willow ptarmigan, egg laying is rare after the beginning of July (Stokkan and Sharp, 1980a). It is therefore possible that the Svalbard ptarmigan becomes long-day refractory less quickly than the willow ptarmigan. In principle, the physiological mechanism responsible for the development of long-day refractoriness in the willow ptarmigan is probably the same as in passerine species (Nicholls et al., 1984). It involves a seasonal reduction in gonadotrophin secretion which is not dependent on changes in the inhibitory feedback effects of gonadal steroids (Stokkan and Sharp, 1984). However, the willow ptarmigan and the closely related red grouse (Lagopus lagopus scotiUS), differ from passerine species in that testicular hormones play a major role in maintaining low concentrations of plasma LH in long-day refractory birds (Sharp and Moss, 1977; Stokkan and Sharp, 1980b, 1984). The role of changes in the concentrations of hormones in the peripheral circulation in the development or maintenance of longday refractoriness is uncertain. In several species, the development of long-day refractoriness is associated with an increase in the concentration of plasma prolactin
ET AL.
(e.g., Lincoln et al., 1980; Campbell et al., 1981; Dawson and Goldsmith, 1982, 1983; Ebling et al., 1982; Hissa et al., 1983; Haase et al., 1985, Sharp et al., 1986a,b) although no causal relationship has been established. Thyroid hormones have also been implicated in the development of longday refractoriness (Nicholls et al., 1984). However, changes in concentrations of plasma thyroid hormones during the development of long-day refractoriness in willow ptarmigan are not consistent in birds exposed to differing fixed daylengths (Klandorf et al., 1982) and differ between species (e.g., Sharp et al., 1986a,b). In the present study, changes in plasma hormone concentrations were compared in captive Svalbard and willow ptarmigan exposed to identical environmental conditions during a simulated annual lighting cycle to assess differences in the relative roles of photoperiodic, as opposed to nonphotoperiodic factors in the timing of seasonal breeding in the two species. Changes in plasma prolactin and thyroid hormones were also measured in relationship to the development of photorefractoriness to determine whether they differ from those reported for other species. MATERIALS
AND METHODS
Male willow ptarmigan (L. lagopus lagopus) and Svalbard ptarmigan (L. mutus hyperboreus) were hatched from eggs laid by captive females and were reared as described by Stokkan and Sharp (1980a). Beginning in January, six willow ptarmigan and five Svalbard ptarmigan were exposed to artificial changes in daylight similar to that experienced at 70”N for 1 year. During spring and autumn, the photoperiod was changed in a stepwise manner once each week. Between mid-May and mid-July, during the breeding season, each bird was given access to a breeding pen housing a female, for l-2 hr each day. The females were not allowed to incubate since their eggs were removed as they were laid. Between January and May, blood samples were taken each week from a wing vein. Thereafter, blood samples were taken approximately once each month. The height of a supraorbital comb was measured on each bird to the nearest 0.5 mm each time a blood sample was taken. All plasma samples, after separation, were stored at -20” prior to assay. The concentrations of plasma
ENDOCRINE
CHANGES
LH, testosterone, and prolactin were measured by radioimmunoassay as described by Gow et al. (1985), Sundby et al. (1975), and McNeilly et al. (1978), respectively. Thyroxine (TJ and triiodothyronine (TJ were measured using reagents and protocols provided by the Scottish Antibody Production Unit (Carluke, Glasgow) and standards were made up in ptarmigan plasma free of T, and T,. The Mann-Whitney U test was used to compare hormone levels between the species and between different sampling data in the same species, unless otherwise stated.
RESULTS
Fertile eggs were laid throughout June and between mid-May and mid-June by the partners of the Svalbard ptarmigan and willow ptarmigan, respectively. Plasma LH and Testosterone
Concentrations of plasma LH increased in response to increasing daylength in March and reached maximum values in April and May in both species of ptarmigan (Figs. 1 and 2). The increases in plasma LH were followed by increases in plasma tes-
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tosterone which reached maximum values in April (Figs. 1 and 2). In both species, plasma LH and testosterone fell steeply at the beginning of June, marking the development of long-day refractoriness, and remained depressed for the rest of the year (Figs. 1 and 2). The most striking difference between the two species was the significantly lower LH (P < 0.001) and testosterone (P < 0.01) concentrations in the Svalbard ptarmigan between January and June (Figs. 1 and 2). Another marked difference between the two species was seen in the way in which plasma LH and testosterone concentrations increased during photostimulation (Figs. 1 and 2). This photoperiodic response was slower in the Svalbard ptarmigan than in the willow ptarmigan (Figs. 1 and 2). Thus, in March, a steep increase in plasma LH and testosterone was seen in the willow ptarmigan whereas in the Svalbard ptarmigan, plasma LH levels were only just beginning to increase and plasma testosterone 24
18 $ 12
6 5 I”
6
‘J
‘F’M’A’M’J’J’A’S’O’N’D’ Months
1. Changes in concentrations of plasma testosterone (O), LH (0), and prolactin (A) in six captive, male Willow ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM. FIG.
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ET AL.
Months
FIG. 2. Changes in concentrations of plasma testosterone (O), LH (0), and prolactin (A) in five captive, male Svalbard ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
levels had not begun to change at this time (Figs. 1 and 2). Comb Height
The heights of the supraorbital combs in both species began to increase in February and continued to increase steadily until April. At the beginning of June, the combs began to regress rapidly (Fig. 3). As was noted for the changes in plasma LH and testosterone, the growth of the supraorbital combs of the Svalbard ptarmigan was slower than that of the willow ptarmigan (Fig. 3).
Svalbard than in the willow ptarmigan. In both species, maximum prolactin levels occurred between the end of May and the beginning of June, coinciding with steep decreases in the concentrations of plasma LH and testosterone. In addition to beginning to increase earlier in the annual cycle, (P < plasma prolactin was significantly 0.001) higher in May and June in the Svalbard ptarmigan than in the willow ptarmigan (Figs. 1 and 2). Plasma prolactin decreased steadily in both species during July and August to reach baseline values in September or October (Figs. 1 and 2).
Plasma Prolactin
Plasma Thyroid Hormones
Concentrations of plasma prolactin began to increase in March in Svalbard ptarmigan and in April in willow ptarmigan (Figs. 1 and 2). From the first week of March, concentrations of plasma prolactin were significantly (P < 0.05) higher in the
In both species, changes in plasma T4 and T, were not large during the simulated annual cycle (Figs. 4 and 5). However, certain changes were associated with increasing daylength and the development of photorefractoriness. During the period that day-
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24
6
1
‘J’F’M’A’M’J’J
A
I
S
I
0
1
Months
FIG. 3. Changes in comb height in captive male Svalbard (0, 12= 5) and Willow ptarmigan (a, n = 6) exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
lengths were increasing, plasma T4 tended to rise and plasma T, to decrease (Figs. 4 and 5). Thus, in the Svalbard ptarmigan, monthly mean plasma T, levels increased (P < 0.001) from 6.4 + 0.25 to 8.5 k 0.42 t&ml between February and April, while plasma T3 levels in the same samples decreased (P < 0.01) from 3.8 + 0.15 to 3.3 f. 0.13 rig/ml. In the willow ptarmigan, be-
tween March and April, plasma T, increased (P < 0.001) from 9.9 t 0.45 to 12.4 -C 0.55 rig/ml while T, decreased (P
1218
lo=‘ PE
9-
j
8A
12
E g % Y( 2
8 6
7-
f 650-I
1 Lo ‘J’F’M’A’M’J’J’A’S’O’M’DI Months
FIG. 4. Changes in concentrations of plasma thyroxine (T4, 0) and triiodothyronine (Ts, 0) in five captive, male Svalbard ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
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ET AL.
Months
FIG. 5. Changes in concentrations of plasma thyroxine (T4, 0) and triiodothyronine (T3, 0) in six captive, male Willow ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
Thus, between the beginning of May and the beginning of June, plasma T, increased (P < 0.05) from 3.12 + 0.09 to 4.85 k 0.51 rig/ml and from 3.29 k 0.14 to 4.79 -e 0.454 rig/ml in the Svalbard and willow ptarmigan, respectively. No significant change in plasma T4 was seen in either species between May and June when the birds became long-day refractory. The patterns of change in plasma T, between July and December were different between the two species. Whereas no significant change was seen during this period in the willow ptarmigan, in the Svalbard ptarmigan T, increased (P < 0.05) to a peak in October (Kruskall-Wallis test) and returned to baseline values in November and December. Plasma T, levels fell (P < 0.02; Kruskall-Wallis test) between September and December in the Svalbard but not in the willow ptarmigan (Figs. 4 and 5). DISCUSSION
This study shows that as daylength is artificially extended, gonadotrophic and gonadal function is stimulated earlier and more strongly in willow than in Svalbard
ptarmigan held under the same conditions. This difference may reflect the situation in free-living birds. Thus, the delayed increase in concentrations of plasma LH and comb height between March and May in captive Svalbard ptarmigan (Fig. 1) was similar to that seen in free-living birds (Stokkan et al., 1986). A similar difference in response to a progressive increase in daylength occurs in captive willow ptarmigan and red grouse. When kept under identical increasing artificial daylengths, red grouse come into breeding condition and develop long-day refractoriness some 3-4 weeks before willow ptarmigan (Sharp and Moss, 1981). This difference reflects an earlier onset and termination of the breeding season in freeliving red grouse than in free-living willow ptarmigan, Red grouse appear to come into breeding condition more quickly than willow ptarmigan after photostimulation because they have a more active hypothalamic-pituitary-testicular axis when exposed to nonstimulatory daylengths. As a result, plasma LH levels increase to their maximum more rapidly after photostimulation in red grouse than in willow ptarmigan (Sharp and Moss, 1981). A similar explanation
ENDOCRINE
CHANGES
might account for the difference in the photoperiodic responses of the Svalbard and willow ptarmigan. In support of this view, the hypothalamic-pituitary-testicular axis appeared to be more active in willow than in Svalbard ptarmigan when exposed to nonstimulatory daylengths as indicated by differences in plasma LH levels (Figs. 1 and 2) and comb size (Fig. 3). However, it is also possible that Svalbard ptarmigan are more sensitive to nonphotoperiodic inhibitory or stimulatory factors than the willow ptarmigan. Such a differential sensitivity could also account for the delayed photoperiodic response in the Svalbard ptarmigan. It seems that any such inhibitory or stimulatory factors operate to depress hypothalamic-pituitary-testicular function equally in both captive and free-living Svalbard ptarmigan. It has been previously assumed (Stokkan et al., 1986) that Svalbard ptarmigan rely on nonphotoperiodic environmental factors to modify photoinduced gonadotrophin release to ensure that the breeding season is timed appropriately. There was a marked difference between the endocrine status of captive and freeliving Svalbard ptarmigan in June when the free-living birds breed (Steen and Unander, 1985). At this time, plasma LH levels and comb height in free-living birds (Stokkan et al., 1986) exceed the maximum plasma LH levels and comb height observed in captive birds (Fig. 1). Even more striking was the observation that in captive but not in freeliving birds, plasma LH levels and comb height were decreasing in June. It therefore seems that captive birds became long-day refractory more rapidly than free-living birds. The accelerated development of long-day refractoriness in the Svalbard ptarmigan cannot be ascribed to the absence of a female breeding partner. It seems that other unidentified, nonphotoperiodic environmental factors suppress plasma LH levels and comb growth toward the end of the photoinduced breeding season in the captive birds. The increase in plasma prolactin during
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175
the development of long-day refractoriness in both species of ptarmigan is similar to that observed in other birds (see introduction). This observation is of interest because in another respect endocrine function in long-day refractory ptarmigan is dissimilar from that in passerine species. Thus, in long-day refractory ptarmigan but not passerine species, LH levels are kept low by the inhibitory action of gonadal hormones (see introduction). The close association between increasing concentrations of plasma prolactin and the development of long-day refractoriness in birds from different families encourages the view that this association is of functional. significance. The increase in plasma prolactin is unlikely to be a consequence of gonadal regression. Thus, when gonadal regression is induced by a decrease in daylength in the starling (Goldsmith and Nicholls, 1984) or ring dove (Lea et al., 1986), there is no associated increase in plasma prolactin. Although a role for prolactin in the development of long-day refractoriness remains to be established, it is unlikely that the hormone is solely responsible for the maintenance of low LH levels in long-day refractory birds. The fact that prolactin levels are increased throughout the summer (Figs. 1 and 2) suggests that prolactin could act in this way. However, plasma prolactin levels decrease in long-day refractory starlings (e.g., Dawson and Goldsmith, 1983) and drakes (Sharp et al., 1986a) kept for prolonged periods exposed to long days. In these circumstances, the decrease in plasma prolactin was not associated with an immediate recovery from long-day refractoriness. The changes in plasma T4 and T3 during the development of long-day refractoriness were similar in Svalbard and willow ptarmigan, with a progressive increase in plasma T, and decrease in T,. After the development of long-day refractoriness, plasma T4 remains high and plasma T, increases. There are few comparable data from other species for comparisons. Plasma T4 in-
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creases in response to lengthening photoperiods in the duck (Jallageas et al., 1978; Haase and Paulke, 1980), ruffed grouse (Garbutt et al., 1979)) and capercaille (Haase et al., 1985) but not in the rook (Lincoln et al., 1980) or partridge (Sharp et al., 1986b). Decreases in plasma T3 after photostimulation have been seen in ducks (Haase and Paulke, 1980), capercaille (Hissa et al., 1983), and partridge (Sharp et al., 1986b), but not in the rook (Lincoln et al., 1980). Difficulties in obtaining a consistent pattern of plasma thyroid hormone changes after photostimulation are not surprising in view of the variety of environmental factors, in addition to photoperiod, which influence thyroid function (Sharp and Klandorf, 1985). The possibility that an increase in plasma T, might be involved in the development of long-day refractoriness has been suggested by Dawson (1984). However, in the drake (Sharp et al., 1986a) and the willow ptarmigan (Klandorf et al., 1982), long-day refractoriness has been induced without any detectable change in plasma Tq. A similar observation was made in a more recent study in the starling by Dawson et al. (1985). It therefore seems probable that if thyroid hormones play a role in the development of long-day refractoriness, as has been shown in the starling (Nicholls et al., 1984), it may not be necessary for the concentrations of these hormones to increase in the peripheral circulation. A final, incidental observation made in this study concerns the relationship between the decrease in plasma LH and testosterone, marking the development of long-day refractoriness and breeding activity. Despite the fact that endocrine measurements indicated that the Svalbard ptarmigan were long-day refractory at the beginning of June, their partners continued to lay fertile eggs until mid-June and some to the end of the month. A similar observation was reported in male willow ptarmigan where successful mating continued after
ET AL.
plasma LH and testosterone levels had decreased (Stokkan and Sharp, 1980a). Parker (1981) found that female willow ptarmigan continued to lay fertile eggs for an average of 7.8 days after separation from males. These observations indicate that mating activity and viable spermatozoa do not disappear concomitantly with the seasonal fall in plasma LH and testosterone. ACKNOWLEDGMENTS We are grateful to Mr. J. Ness for rearing the birds, to Mrs. G. Main for carrying out the LH assays, and to Dr A. S. McNeilly for the reagents for the prolactin radioimmunoassay.
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COMPARATIVE
ENDOCRINOLOGY
70, 169-177 (1988)
Endocrine Changes in Photostimulated Willow Ptarmigan (Lagopus lagopus lagopus) and Svalbard Ptarmigan (Lagopus mutus hyperboreus) KARL-ARNE STOKKAN, PETER J. SHARP,* IAN C. DUNN,* AND ROBERT W. LEA? Department of Arctic Biology and Institute of Medical Biology, University Tromso, Norway; *AFRC Institute of Animal Physiology and Genetics Station, Roslin, Midlothian EH25 9PS, Scotland; and fSchoo1 of Applied Preston PRl 2TQ, United Kingdom
of Tromso, P.O. Box 635, N-9001 Research, Edinburgh Research Biology, Lancashire Polytechnic,
Accepted December 1, 1987 Changes in plasma luteinizing hormone (LH), testosterone, thyroxine (T4), and triiodothyronine (T,) and the height of supraorbital combs were compared in captive willow ptarmigan (Lagopus lagopus lagopus) and Svalbard ptarmigan (Lagopus mutus hyperboreus) exposed to an artificial annual cycle of daylength simulating that at 70”N. Plasma LH and testosterone and comb height increased more slowly in Svalbard than in willow ptarmigan as daylength increased. In both species, plasma LH and testosterone fell abruptly, and the supraorbital combs regressed in June, marking the development of long-day refractoriness. Comparison with free-living Svalbard ptarmigan (K.-A. Stokkan, P. J. Sharp, and S. Unander (1986) Gen. Comp. Endocrinol. 61, 44M51) at 80”N, showed a similar slow increase in reproductive function before the onset of the breeding season. However, maximum plasma LH levels and comb size were higher in free-living than in captive Svalbard ptarmigan. Furthermore, long-day refractoriness developed earlier in captive than in freeliving Svalbard ptarmigan. In both species of ptarmigan, the development of long-day refractoriness was associated with increased plasma prolactin. This increase was larger and occurred earlier in the Svalbard than in the willow ptarmigan. Seasonal changes in thyroid hormones were not as marked as for the other hormones measured. In both species, plasma T, tended to increase and plasma T, to decrease as daylength increased. A small increase in plasma T, was seen after the development of long-day refractoriness in both species. It is concluded that captivity depresses the photoperiodic response of Svalbard ptarmigan more than that of willow ptarmigan. The association between increased plasma prolactin and the development of long-day refractoriness is similar to that reported for birds in other families. The changes in plasma thyroid hormones during the development of refractoriness cannot be readily related to changes seen in other species. 0 1988 Academic Press, Inc.
In most birds breeding at temperate zone latitudes, increasing daylengths in spring stimulate a rapid, final maturation of the gonads to initiate the breeding season. The same response to increasing daylength in long-day breeders at very high latitudes may not be appropriate. At these latitudes, daylengths become continuous long before environmental conditions such as temperature and snow cover allow successful breeding to occur. Thus, at 70”N, Norwegian willow ptarmigan (Lagopus Zagopus
Zagopus) breed in late May after exposure
to continuous light for nearly a month (Stokkan and Sharp, 1980a) while at 80”N, the Svalbard ptarmigan (Lagopus mutus hyperboreus) breeds in June after exposure to continuous light for about 2 months (Stokkan et al., 1986). In both the Svalbard ptarmigan (K. Lindgard and K.-A. Stokkan, unpublished observations) and the willow ptarmigan (Stokkan and Sharp, 1980a,b), the seasonal development of the testes depends on an increase in daylength.
169 0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The lag between increasing daylength and the onset of breeding in free-living Svalbard ptarmigan can be accounted for by an initial slow increase of plasma luteinizing hormone (LH) and gonadal growth (Stokkan et al., 1986). A similar lag in increase of plasma LH and comb growth after photostimulation is not so obvious in the willow ptarmigan (Stokkan and Sharp, 1980a,b; Sharp and Moss, 1981). However, a direct comparison between the two species exposed to identical lighting conditions has not been made. Seasonal breeding in both species of ptarmigan is terminated by the development of long-day refractoriness (Stokkan and Sharp, 1980a; Stokkan et al., 1986). Egg laying in free-living Svalbard ptarmigan may continue well into July (Stokkan et al., 1986) while in the willow ptarmigan, egg laying is rare after the beginning of July (Stokkan and Sharp, 1980a). It is therefore possible that the Svalbard ptarmigan becomes long-day refractory less quickly than the willow ptarmigan. In principle, the physiological mechanism responsible for the development of long-day refractoriness in the willow ptarmigan is probably the same as in passerine species (Nicholls et al., 1984). It involves a seasonal reduction in gonadotrophin secretion which is not dependent on changes in the inhibitory feedback effects of gonadal steroids (Stokkan and Sharp, 1984). However, the willow ptarmigan and the closely related red grouse (Lagopus lagopus scotiUS), differ from passerine species in that testicular hormones play a major role in maintaining low concentrations of plasma LH in long-day refractory birds (Sharp and Moss, 1977; Stokkan and Sharp, 1980b, 1984). The role of changes in the concentrations of hormones in the peripheral circulation in the development or maintenance of longday refractoriness is uncertain. In several species, the development of long-day refractoriness is associated with an increase in the concentration of plasma prolactin
ET AL.
(e.g., Lincoln et al., 1980; Campbell et al., 1981; Dawson and Goldsmith, 1982, 1983; Ebling et al., 1982; Hissa et al., 1983; Haase et al., 1985, Sharp et al., 1986a,b) although no causal relationship has been established. Thyroid hormones have also been implicated in the development of longday refractoriness (Nicholls et al., 1984). However, changes in concentrations of plasma thyroid hormones during the development of long-day refractoriness in willow ptarmigan are not consistent in birds exposed to differing fixed daylengths (Klandorf et al., 1982) and differ between species (e.g., Sharp et al., 1986a,b). In the present study, changes in plasma hormone concentrations were compared in captive Svalbard and willow ptarmigan exposed to identical environmental conditions during a simulated annual lighting cycle to assess differences in the relative roles of photoperiodic, as opposed to nonphotoperiodic factors in the timing of seasonal breeding in the two species. Changes in plasma prolactin and thyroid hormones were also measured in relationship to the development of photorefractoriness to determine whether they differ from those reported for other species. MATERIALS
AND METHODS
Male willow ptarmigan (L. lagopus lagopus) and Svalbard ptarmigan (L. mutus hyperboreus) were hatched from eggs laid by captive females and were reared as described by Stokkan and Sharp (1980a). Beginning in January, six willow ptarmigan and five Svalbard ptarmigan were exposed to artificial changes in daylight similar to that experienced at 70”N for 1 year. During spring and autumn, the photoperiod was changed in a stepwise manner once each week. Between mid-May and mid-July, during the breeding season, each bird was given access to a breeding pen housing a female, for l-2 hr each day. The females were not allowed to incubate since their eggs were removed as they were laid. Between January and May, blood samples were taken each week from a wing vein. Thereafter, blood samples were taken approximately once each month. The height of a supraorbital comb was measured on each bird to the nearest 0.5 mm each time a blood sample was taken. All plasma samples, after separation, were stored at -20” prior to assay. The concentrations of plasma
ENDOCRINE
CHANGES
LH, testosterone, and prolactin were measured by radioimmunoassay as described by Gow et al. (1985), Sundby et al. (1975), and McNeilly et al. (1978), respectively. Thyroxine (TJ and triiodothyronine (TJ were measured using reagents and protocols provided by the Scottish Antibody Production Unit (Carluke, Glasgow) and standards were made up in ptarmigan plasma free of T, and T,. The Mann-Whitney U test was used to compare hormone levels between the species and between different sampling data in the same species, unless otherwise stated.
RESULTS
Fertile eggs were laid throughout June and between mid-May and mid-June by the partners of the Svalbard ptarmigan and willow ptarmigan, respectively. Plasma LH and Testosterone
Concentrations of plasma LH increased in response to increasing daylength in March and reached maximum values in April and May in both species of ptarmigan (Figs. 1 and 2). The increases in plasma LH were followed by increases in plasma tes-
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tosterone which reached maximum values in April (Figs. 1 and 2). In both species, plasma LH and testosterone fell steeply at the beginning of June, marking the development of long-day refractoriness, and remained depressed for the rest of the year (Figs. 1 and 2). The most striking difference between the two species was the significantly lower LH (P < 0.001) and testosterone (P < 0.01) concentrations in the Svalbard ptarmigan between January and June (Figs. 1 and 2). Another marked difference between the two species was seen in the way in which plasma LH and testosterone concentrations increased during photostimulation (Figs. 1 and 2). This photoperiodic response was slower in the Svalbard ptarmigan than in the willow ptarmigan (Figs. 1 and 2). Thus, in March, a steep increase in plasma LH and testosterone was seen in the willow ptarmigan whereas in the Svalbard ptarmigan, plasma LH levels were only just beginning to increase and plasma testosterone 24
18 $ 12
6 5 I”
6
‘J
‘F’M’A’M’J’J’A’S’O’N’D’ Months
1. Changes in concentrations of plasma testosterone (O), LH (0), and prolactin (A) in six captive, male Willow ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM. FIG.
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STOKKAN
ET AL.
Months
FIG. 2. Changes in concentrations of plasma testosterone (O), LH (0), and prolactin (A) in five captive, male Svalbard ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
levels had not begun to change at this time (Figs. 1 and 2). Comb Height
The heights of the supraorbital combs in both species began to increase in February and continued to increase steadily until April. At the beginning of June, the combs began to regress rapidly (Fig. 3). As was noted for the changes in plasma LH and testosterone, the growth of the supraorbital combs of the Svalbard ptarmigan was slower than that of the willow ptarmigan (Fig. 3).
Svalbard than in the willow ptarmigan. In both species, maximum prolactin levels occurred between the end of May and the beginning of June, coinciding with steep decreases in the concentrations of plasma LH and testosterone. In addition to beginning to increase earlier in the annual cycle, (P < plasma prolactin was significantly 0.001) higher in May and June in the Svalbard ptarmigan than in the willow ptarmigan (Figs. 1 and 2). Plasma prolactin decreased steadily in both species during July and August to reach baseline values in September or October (Figs. 1 and 2).
Plasma Prolactin
Plasma Thyroid Hormones
Concentrations of plasma prolactin began to increase in March in Svalbard ptarmigan and in April in willow ptarmigan (Figs. 1 and 2). From the first week of March, concentrations of plasma prolactin were significantly (P < 0.05) higher in the
In both species, changes in plasma T4 and T, were not large during the simulated annual cycle (Figs. 4 and 5). However, certain changes were associated with increasing daylength and the development of photorefractoriness. During the period that day-
ENDOCRINE
CHANGES
IN
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PTARMIGAN
24
6
1
‘J’F’M’A’M’J’J
A
I
S
I
0
1
Months
FIG. 3. Changes in comb height in captive male Svalbard (0, 12= 5) and Willow ptarmigan (a, n = 6) exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
lengths were increasing, plasma T4 tended to rise and plasma T, to decrease (Figs. 4 and 5). Thus, in the Svalbard ptarmigan, monthly mean plasma T, levels increased (P < 0.001) from 6.4 + 0.25 to 8.5 k 0.42 t&ml between February and April, while plasma T3 levels in the same samples decreased (P < 0.01) from 3.8 + 0.15 to 3.3 f. 0.13 rig/ml. In the willow ptarmigan, be-
tween March and April, plasma T, increased (P < 0.001) from 9.9 t 0.45 to 12.4 -C 0.55 rig/ml while T, decreased (P
1218
lo=‘ PE
9-
j
8A
12
E g % Y( 2
8 6
7-
f 650-I
1 Lo ‘J’F’M’A’M’J’J’A’S’O’M’DI Months
FIG. 4. Changes in concentrations of plasma thyroxine (T4, 0) and triiodothyronine (Ts, 0) in five captive, male Svalbard ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
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STOKKAN
ET AL.
Months
FIG. 5. Changes in concentrations of plasma thyroxine (T4, 0) and triiodothyronine (T3, 0) in six captive, male Willow ptarmigan exposed to seasonal changes in daylength (upper panel). The values are means and the vertical bars are SEM.
Thus, between the beginning of May and the beginning of June, plasma T, increased (P < 0.05) from 3.12 + 0.09 to 4.85 k 0.51 rig/ml and from 3.29 k 0.14 to 4.79 -e 0.454 rig/ml in the Svalbard and willow ptarmigan, respectively. No significant change in plasma T4 was seen in either species between May and June when the birds became long-day refractory. The patterns of change in plasma T, between July and December were different between the two species. Whereas no significant change was seen during this period in the willow ptarmigan, in the Svalbard ptarmigan T, increased (P < 0.05) to a peak in October (Kruskall-Wallis test) and returned to baseline values in November and December. Plasma T, levels fell (P < 0.02; Kruskall-Wallis test) between September and December in the Svalbard but not in the willow ptarmigan (Figs. 4 and 5). DISCUSSION
This study shows that as daylength is artificially extended, gonadotrophic and gonadal function is stimulated earlier and more strongly in willow than in Svalbard
ptarmigan held under the same conditions. This difference may reflect the situation in free-living birds. Thus, the delayed increase in concentrations of plasma LH and comb height between March and May in captive Svalbard ptarmigan (Fig. 1) was similar to that seen in free-living birds (Stokkan et al., 1986). A similar difference in response to a progressive increase in daylength occurs in captive willow ptarmigan and red grouse. When kept under identical increasing artificial daylengths, red grouse come into breeding condition and develop long-day refractoriness some 3-4 weeks before willow ptarmigan (Sharp and Moss, 1981). This difference reflects an earlier onset and termination of the breeding season in freeliving red grouse than in free-living willow ptarmigan, Red grouse appear to come into breeding condition more quickly than willow ptarmigan after photostimulation because they have a more active hypothalamic-pituitary-testicular axis when exposed to nonstimulatory daylengths. As a result, plasma LH levels increase to their maximum more rapidly after photostimulation in red grouse than in willow ptarmigan (Sharp and Moss, 1981). A similar explanation
ENDOCRINE
CHANGES
might account for the difference in the photoperiodic responses of the Svalbard and willow ptarmigan. In support of this view, the hypothalamic-pituitary-testicular axis appeared to be more active in willow than in Svalbard ptarmigan when exposed to nonstimulatory daylengths as indicated by differences in plasma LH levels (Figs. 1 and 2) and comb size (Fig. 3). However, it is also possible that Svalbard ptarmigan are more sensitive to nonphotoperiodic inhibitory or stimulatory factors than the willow ptarmigan. Such a differential sensitivity could also account for the delayed photoperiodic response in the Svalbard ptarmigan. It seems that any such inhibitory or stimulatory factors operate to depress hypothalamic-pituitary-testicular function equally in both captive and free-living Svalbard ptarmigan. It has been previously assumed (Stokkan et al., 1986) that Svalbard ptarmigan rely on nonphotoperiodic environmental factors to modify photoinduced gonadotrophin release to ensure that the breeding season is timed appropriately. There was a marked difference between the endocrine status of captive and freeliving Svalbard ptarmigan in June when the free-living birds breed (Steen and Unander, 1985). At this time, plasma LH levels and comb height in free-living birds (Stokkan et al., 1986) exceed the maximum plasma LH levels and comb height observed in captive birds (Fig. 1). Even more striking was the observation that in captive but not in freeliving birds, plasma LH levels and comb height were decreasing in June. It therefore seems that captive birds became long-day refractory more rapidly than free-living birds. The accelerated development of long-day refractoriness in the Svalbard ptarmigan cannot be ascribed to the absence of a female breeding partner. It seems that other unidentified, nonphotoperiodic environmental factors suppress plasma LH levels and comb growth toward the end of the photoinduced breeding season in the captive birds. The increase in plasma prolactin during
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175
the development of long-day refractoriness in both species of ptarmigan is similar to that observed in other birds (see introduction). This observation is of interest because in another respect endocrine function in long-day refractory ptarmigan is dissimilar from that in passerine species. Thus, in long-day refractory ptarmigan but not passerine species, LH levels are kept low by the inhibitory action of gonadal hormones (see introduction). The close association between increasing concentrations of plasma prolactin and the development of long-day refractoriness in birds from different families encourages the view that this association is of functional. significance. The increase in plasma prolactin is unlikely to be a consequence of gonadal regression. Thus, when gonadal regression is induced by a decrease in daylength in the starling (Goldsmith and Nicholls, 1984) or ring dove (Lea et al., 1986), there is no associated increase in plasma prolactin. Although a role for prolactin in the development of long-day refractoriness remains to be established, it is unlikely that the hormone is solely responsible for the maintenance of low LH levels in long-day refractory birds. The fact that prolactin levels are increased throughout the summer (Figs. 1 and 2) suggests that prolactin could act in this way. However, plasma prolactin levels decrease in long-day refractory starlings (e.g., Dawson and Goldsmith, 1983) and drakes (Sharp et al., 1986a) kept for prolonged periods exposed to long days. In these circumstances, the decrease in plasma prolactin was not associated with an immediate recovery from long-day refractoriness. The changes in plasma T4 and T3 during the development of long-day refractoriness were similar in Svalbard and willow ptarmigan, with a progressive increase in plasma T, and decrease in T,. After the development of long-day refractoriness, plasma T4 remains high and plasma T, increases. There are few comparable data from other species for comparisons. Plasma T4 in-
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STOKKAN
creases in response to lengthening photoperiods in the duck (Jallageas et al., 1978; Haase and Paulke, 1980), ruffed grouse (Garbutt et al., 1979)) and capercaille (Haase et al., 1985) but not in the rook (Lincoln et al., 1980) or partridge (Sharp et al., 1986b). Decreases in plasma T3 after photostimulation have been seen in ducks (Haase and Paulke, 1980), capercaille (Hissa et al., 1983), and partridge (Sharp et al., 1986b), but not in the rook (Lincoln et al., 1980). Difficulties in obtaining a consistent pattern of plasma thyroid hormone changes after photostimulation are not surprising in view of the variety of environmental factors, in addition to photoperiod, which influence thyroid function (Sharp and Klandorf, 1985). The possibility that an increase in plasma T, might be involved in the development of long-day refractoriness has been suggested by Dawson (1984). However, in the drake (Sharp et al., 1986a) and the willow ptarmigan (Klandorf et al., 1982), long-day refractoriness has been induced without any detectable change in plasma Tq. A similar observation was made in a more recent study in the starling by Dawson et al. (1985). It therefore seems probable that if thyroid hormones play a role in the development of long-day refractoriness, as has been shown in the starling (Nicholls et al., 1984), it may not be necessary for the concentrations of these hormones to increase in the peripheral circulation. A final, incidental observation made in this study concerns the relationship between the decrease in plasma LH and testosterone, marking the development of long-day refractoriness and breeding activity. Despite the fact that endocrine measurements indicated that the Svalbard ptarmigan were long-day refractory at the beginning of June, their partners continued to lay fertile eggs until mid-June and some to the end of the month. A similar observation was reported in male willow ptarmigan where successful mating continued after
ET AL.
plasma LH and testosterone levels had decreased (Stokkan and Sharp, 1980a). Parker (1981) found that female willow ptarmigan continued to lay fertile eggs for an average of 7.8 days after separation from males. These observations indicate that mating activity and viable spermatozoa do not disappear concomitantly with the seasonal fall in plasma LH and testosterone. ACKNOWLEDGMENTS We are grateful to Mr. J. Ness for rearing the birds, to Mrs. G. Main for carrying out the LH assays, and to Dr A. S. McNeilly for the reagents for the prolactin radioimmunoassay.
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