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Pii: s0305-0491(97)00183-6

Comp. Biochem. Physiol. Vol. 116B, No. 2, pp. 269–277, 1997
ISSN 0305-0491/ 97/$17.00 Copyright  1997 Elsevier Science Inc.
Seasonal Levels of Reproductive Hormones and Their Relationship to the Antler Cycle of Male and Female Reindeer (Rangifer tarandus) George A. Bubenik,a Dieter Schams,b Robert J. White,c Janice Rowell,c John Blake,d and Ludek Bartosd Department of Zoology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1; Institut fuer Physiologie, Technische Universitaet Munchen, D-85350 Freising-Weihenstephan, Germany; Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99775, U.S.A.; Research Institute of Animal Production, Department of Biology, Ethology Group, CZ-104 00 Praha 10-Uhrineves, Czech Republic ABSTRACT. Seasonal levels of LH, FSH, testosterone (T), estradiol, progesterone (P), and prolactin (PRL)
were determined in the plasma of five adult bulls, and five barren and four pregnant cows of Alaskan reindeer
(Rangifer tarandus), which were sampled every 3 weeks for 54 weeks. The male reproductive axis was sequentially
activated; LH peaked in May–June (2 ng/ml), FSH in June (51 ng/ml), and T in September (11.8 ng/ml). LH
levels in females reached a maximum in both groups at the end of August (the beginning of the rut). Seasonal
variation in FSH was minimal in pregnant cows, but exhibited one elevation (41 ng/ml) in barren ones in
November. T levels in cows remained at barely detectable levels. The decrease of T values observed in both
groups in December and March was not significant. PRL peaked in May in cows (135 ng/ml pregnant, 140 ng/
ml non-pregnant) and in June in bulls (92 ng/ml). Estradiol was highest in bulls in the rut (August), in non-
pregnant cows in January and in pregnant cows in April, shortly before parturition. P levels in the pregnant
cows rose from September and peaked (9 ng/ml) shortly before parturition in April. In the non-pregnant females
P values increased and decreased several times before peaking (5 ng/ml) in March. In the males, the variation
of T and estradiol levels correlated relatively well with the antler cycle but in the females the variation of neither
estradiol, progesterone nor T appeared to be related to mineralization or casting of antlers. Copyright 1997
Elsevier Science Inc.
comp biochem physiol 116B;2:269–277, 1997.
KEY WORDS. LH, FSH, testosterone, prolactin, estradiol, progesterone, reindeer, pregnancy, seasonality, antler
dominant factor in determination of individual phases ofthe antler cycle, such as the growth period and the time of Seasonal variation of reproductive hormones [LH, FSH, tes- polishing of the velvet and casting of antlers (9,19,24,42).
tosterone (T), and prolactin (PRL)] has been investigated In the male reindeer this correlation between T levels and frequently in various cervids of temperate regions [white- the antler cycle is less pronounced. Although the growth tailed deer (9,10,28); roe deer (42); red deer (2,18); Pere Da- of male reindeer antlers occurs also during the period of low vid's deer (25); fallow deer (3)]. Conversely, studies of the concentrations of plasma T (58), out-of-phase polishing of reproductive hormones in the boreal cervids, e.g., moose, antlers can be induced by administration of large doses of reindeer, and caribou are relatively rare (4,23,26,40,57).
exogenous T (39) and antler casting will follow shortly after Reindeer/caribou (Rangifer tarandus) is the most northern castration (50), the timing of the individual phases of the cervid and the only one in which the females carry antlers.
antler cycle does not entirely relate to the variation of T Whereas the regulation of the antler cycle in male deer of in plasma (57). In addition, unlike in most other cervids, temperate zones has been studied extensively (7,21,49), and antlers are produced in both sexes, the pedicles and antlers is well understood, this is not the case for male and female developed well before puberty and gonadectomy does not prevent the growth and subsequent replacement of antlers In most cervids, the seasonal variation of T is the pre- (23,50,59). In addition, long-term administration of an an-drogen receptor blocker cyproterone acetate which causes Address reprint requests to: Dr. G. A. Bubenik, Dept. Zoology, University quick casting of antlers in other cervids (7,19), was not ef- of Guelph, 50 Stone Rd.-E., Guelph, Ontario, Canada, N1G 2W1. Tel.
fective in the reindeer male or female calves or yearlings (519) 824-4120; Fax (519) 767-1656; E-mail: [email protected].
Received 6 February 1996; accepted 14 June 1996.
(G. Bubenik et al., unpublished observation). Therefore, G. A. Bubenik et al.
based on the above data, Lincoln and Tyler (23) concluded individual animal temperament. Rutting bulls are exqui- that in males ‘‘the secretion of T by testes, although not sitely sensitive to xylazine and seem to be unable to properly essential for the growth and seasonal replacement of antlers, clear ketamine (J. Blake, unpublished data), therefore they has an overriding influence of the timing of the antler were immobilized using only 0.1–0.2 mg/kg of xylazine hy- drochloride. Drugs were administered via hand injection, In the female reindeer the regulation of the antler cycle pole syringe, or blow pipe. The method of administration has been investigated only rarely (22,23). The results of was determined by animal temperament and human safety these studies indicate that in intact females ovarian steroids, consideration with the primary intent to minimize excite- possibly estradiol, participate in antler mineralization. In ment. After blood collection and antler measurement the ovariectomized animals the hardening of antlers may have reindeer received an intravenous injection of yohimbine been induced by androstenedione, which is most likely of (0.125 mg/kg) to reverse the xylazine.
adrenal origin (23).
Once immobilized, three consecutive samples of blood In view of the many gaps which exist in the elucidation (35 ml each) were drawn from the jugular vein into heparin- of the endocrine regulation of the antler cycle in the male ized tubes at 10 min intervals. The blood was centrifuged and female reindeer we decided to perform a detailed inves- within 4 hr of collection and plasma was separated and fro- tigation of the seasonal changes of reproductive hormones zen at 220°C. Life history events were recorded for each in relationship to antler cycles of both sexes. As the timing animal throughout the study period. The antlers were usu- of casting of antlers differs in pregnant (PG) and non-preg- ally polished within a two-day period and mostly both at nant (NP) cows (26), our present study aimed to investigate the same time. The casting was also mostly synchronous; in each group separately.
cases of a desychronous casting an average date of bothdrops was recorded. As the date of initiation of antler re-growth we have chosen the period when a complete re-epi- MATERIAL AND METHODS
thelization of the pedicle wound occurred. This was fol- Animals and Sampling Procedure
lowed almost immediately by a vigorous antler growth. The Experimental protocols for this study were approved by the data of parturition were recorded daily for the PG group.
University of Alaska Fairbanks campus-wide animal welfarecommittee (IACUC #92-016).
Fourteen adult reindeer (Rangifer tarandus) were housed outside at the Large Animal Research Station and at the The concentrations of LH, FSH, testosterone, prolactin, es- Biological Reserve, Institute of Arctic Biology, Fairbanks, tradiol, and progesterone were determined in plasma in du- AK (latitude 66°N). The animals were divided into 3 plicate by radioimmunoassays, according the techniques de- groups: 2- to 6-year-old bulls (n 5 5), 2- to 5-year-old non- scribed in previous papers. For each hormone all samples pregnant cows (n 5 4), and 2- to 5-year-old pregnant cows were assayed at the same time.
(n 5 5). The females that were selected to become pregnant LH was determined by a homologous bovine assay with were kept with bulls during the entire rutting season and no crossreactivity to other pituitary hormones (43,44). The some time thereafter. The females selected to be non-preg- reference preparation was a bovine pituitary LH-DSA. The nant were kept apart from bulls in a separate enclosure ap- sensitivity was 0.05 ng/tube; intra-assay coefficient of varia- proximately 300 m away. All animals were kept in large tion (CV) averaged 8.6%, and interassay variation was paddocks with constant access to pasture grasses. They also 11.5–14%. The reference preparation was a pure bovine pi- received a diet composed of 85% textured pelleted reindeer tuitary extract prepared in our laboratory (LH-DSA with a feed (dry matter values ranged from 15–15.6% digestible biological activity of 1.0 times NIH-LH-S1). The assay was protein, 9.6–10.8% acid detergent fiber, 77.5–78.5% validated for reindeer plasma by recovery studies of 4 differ- TDN), and 15% chopped grass hay (dry matter values ent concentrations added to plasma and was on average 97.5 ranges from 5–9% digestible protein, 38.6–41% acid deter- 6 6.2%. Dilution curves of reindeer plasma with a high LH gent fiber, 56.8–57.9% TDN). Consumption in reindeer content run parallel to bovine standard.
varies seasonally (56) and ranged from approximately 1–4 FSH was determined by a system where pure ovine FSH kg/animal/day. Consumption of grass pasture was not mea- was used in labeling, and the antisera against ovine FSH sured. All animals were provided with ad libitum water in was produced in guinea pigs (43,46). As a reference prepara- the summer and snow in the winter.
tion we used USDA-bFSH-B1. The sensitivity was 5 ng/ Every 3 weeks, from June 5, 1992 to June 18, 1993, each tube and intra-assay CV was 7.5%, an interassay CV was animal was immobilized using 1–2 mg/kg xylazine hydro- 12–18%. Prolactin was determined by a homologous bovine chloride (Anased, Lloyd Labs. Shenandoah, IA, U.S.A.) assay. The antisera produced in rabbits exhibited no cross- and 1.5 mg/kg of ketamine hydrochloride (Ketaset, Aveco reactivity to other pituitary hormones. The reference prepa- Co., Fort Dodge, IA, U.S.A.). Doses varied depending on ration used was USDA-bPRL-B1 (biological activity 0.49 3 Reproductive Hormones in Reindeer 1). The sensitivity was 0.05 ng/tube, intra-assay Statistical values for analyzed hormones
CV was 8.4%, and the interassay CV was 14%. Mean recov- ery of added FSH to reindeer plasma (4 different concentra- tions) was 95.6 6 4.2% and the dilution curve of the rein- deer plasma run parallel to the standard.
PRL was determined by a homologous bovine assay (45).
The antisera produced in rabbits exhibited no crossreactiv-ity to other pituitary hormones. The reference preparation used was USDA-bPRL-B1 (biological activity 24.1 i.u./mg).
Validation experiment showed a mean recovery of added bovine prolactin to reindeer plasma of 98.1 6 4.1% andthe dilution curves of reindeer plasma run parallel to the Testosterone was determined by a highly sensitive en- zyme immunoassay (EIA) after extraction with tertiarybutylmethylether/petrolether 30/70 (V/V) (16). The sensi- tivity was 50 pg/ml. The interassay CV was 7.5–12% and the interassay CV was 3.5–5.9% respectively.
Estradiol-17 beta was determined after some modification of the original method (55) by EIA, 2 ml of plasma was extracted with 5 ml of tertiar-butyl-methylether, evaporated and redissolved in 0.5 ml of assay buffer and evaluated in 50 ul. The sensitivity was 0.1 pg/tube. The interassay CV was in range between 7.9–13.5% and the intra-assay CV was 4.1–7.2%, respectively.
Progesterone was evaluated directly in duplicate using 20 ul samples of blood plasma by an EIA in microtitre plates based on the double antibody technique (35). The sensitiv-ity was 200 pg/ml. The interassay CV was in the range of7.2–13.1% and the intra-assay CV was between 3.1–6.2%,respectively.
There were overall differences for all hormones (P , The determination of hormone concentrations was per- 0.001). All hormones differed by Category (P , 0.001) and formed individually in all three samples and the statistical Date (P , 0.001). Age differences were found in some cases calculations were then performed on all data.
but trends were unclear and inconsistent (Table 1).
Residual correlation indicated significant relationship be- tween gonadotropins and sexual hormones mostly in males.
Fewer correlations were found for females (Table 2).
The data on seasonal changes in plasma concentrations ofhormones (except those of progesterone) were subjected Antler Cycles, Rut, and Parturition
to the General Linear Models Procedure for UnbalancedANOVA (SAS). Classes were ‘‘Order of sampling'' (one to The range of dates of antler polishing, the beginning of ant- three), ‘‘Category'' (males, NP and PG cows), ‘‘Date'' (19 ler growth, the casting, and the parturition are presented in dates of sampling), and ‘‘Age'' (Males 2, 3, or 5 and older, Females 2, 4 or 5 and older). Progesterone was analyzed forfemales only. The classes Date and Age were applied in the LH, T, and FSH in Bulls
model as Nested effects within the class Category. Least-square means (LSMEANS) were computed for each class In bulls, LH reached peak levels (1.98 ng/ml) in May (late and differences between classes were tested by t-test.
March vs April, P , 0.001, April vs May, P , 0.001) andthe minimum (,0.1 ng/ml) was observed in January (lateJune vs July, P , 0.001, July vs early August P , 0.001).
Mean T levels began to increase at the time when LH levels were already declining (Fig. 1). A steep increase of T fol- No differences (P . 0.05) were observed based upon Order lowed in August, achieving peak levels (11.8–11.5 ng/ml) of sampling. Table 1 shows results of the GLM analysis.
from the end of August until mid-September (late August G. A. Bubenik et al.
TABLE 2. Residual correlation coefficients between selected hormones by category
Estr 3 Test
centrations of LH, FSH, and T were observed in the twomost aggressive bulls.
LH, FSH, and T in Cows
In pregnant cows peak LH concentrations (0.84 ng/ml)were detected at the end of August, the beginning of therut, and then declined to levels below 0.1 ng/ml. In the NPcows LH reached also a peak in August but, due to a large(pre-ovulatory) peak (7.3 ng/ml) detected in one female,the LSMEANS reached 2.3 ng/ml. A second LH peak ob-served in that group in November matched a similar peakof FSH (both LH peaks being significantly higher than anyother value P , 0.001) (Fig. 1). The pregnant group exhib-ited a FSH peak (25 ng/ml) in mid-November (Fig. 1). Sim-ilarly, FSH concentrations in the NP group were almostidentical to FSH values in the PG group except for a sharppeak (40 ng/ml) detected in November (this FSH peak wassignificantly higher than any other value P , 0.001) (Fig.
1). Variations of T levels in females (,0.05–0.3 ng/ml) wasnot related to the antler cycle either in the PG or in theNP females. Interestingly, in both female groups T levels ex-hibited a very similar seasonal pattern with an unexplainedsharp decrease in December and May (Fig. 1, insert).
Peak estradiol (E2) levels in bulls (3.5 pg/ml) were reachedduring the rut in mid-August (P , 0.001) (Fig. 2).
In NP cows E2 reached a maximum (2 pg/ml) in January FIG. 1. Least-square means (6S.E.) of seasonal levels of LH,
and in the PG females the peak concentration (3 pg/ml) FSH and testosterone in the adult reindeer males and the
non-pregnant and pregnant females. Testosterone insert:

was detected in April, shortly before the parturition (P , values in females.
0.001). Unlike in males, E2 levels in females varied indepen-dently from the levels of testosterone (Table 2).
The elevation and decline of E2 in bulls correlated with vs early August (P , 0.001), the peak period of the rut. The the mineralization and casting period of antlers, but a simi- FSH levels began to rise significantly in April and achieved lar relationship was not seen in the females.
maximum (51 ng/ml) in late May (April vs early May, P, 0.001, early May vs late May, P , 0.001) and June (Fig.
1). In males, T levels correlated relatively well with the development of antlers. The polishing occurred at the timeof rising T levels and the antlers were cast at the time of In the PG females, progesterone (P) concentrations reached minimal T concentrations. Interestingly, the highest con- peak levels (8.8 ng/ml) at the end of March, about one Reproductive Hormones in Reindeer TABLE 3. The range of dates of antler polishing, antler casting, new antler growth, and parturition
Antlers cast
New antler growth
Aug. 8–Sep. 1 (Aug. 19) Dec. 7–Jan. 17 (Dec. 27) Feb. 5–Mar. 20 (Mar. 3) Aug. 30–Sep. 15 (Sep. 9) Mar. 26–Apr. 9 (Apr. 2) Apr. 22–Apr. 28 (Apr. 25) Apr. 4–May 1 (Apr. 24) Apr. 25–May 4 (Apr. 29) Apr. 4–May 9 (Apr. 23) NP 5 non-pregnant; PG 5 pregnant.
and bulls in May and June (Fig. 2). Both female groupsreached almost identical peak concentrations (135 NP vs140 ng/ml PG) as compared to the significantly lower peakof bulls (92 ng/ml). One female that gave birth 3 weeksbefore the rest of the group exhibited an early elevationof prolactin. However, during the late lactation period theconcentrations in both female groups were very similar.
In previous studies, the seasonal levels of reproductive hor-mones investigated in reindeer of both sexes followed a pat-tern established earlier in other cervids. However, there wasoften little information available about the relationship be-tween the peripheral levels of sexual steroids and the indi-vidual phases of the reindeer antler cycle.
LH, FSH, T. In bulls, the sequential activation of the re-
productive system starts with an almost simultaneous eleva-tion of LH and FSH in April. LH achieved peak levels inMay, about one month before FSH (Fig. 1). Similar to val-ues reported by Stokkan and co-workers (51), LH valuesdecline from May on and were already below 1 ng/ml dur-ing the rut. As in other cervids (9,12,49,53), T exhibitedpeak levels in August and September (i.e., the rutting sea-son), more than 3 months after the LH maximum was de-tected. LSMEANS peak levels of T in our study (11.8 ng/ml) were in the range similar to 10 ng/ml observed by Stok- FIG. 2. Least-square means (6S.E.) of seasonal levels of es-
kan and co-workers (51). Highest T levels (60 ng/ml) re- tradiol, progesterone and prolactin in the adult reindeer
ported by Whitehead and McEwan (57) were also compara- males and the non-pregnant and pregnant females.
ble with the highest concentration observed in one of ourbulls (41 ng/ml).
Generally, the highest individual concentrations of LH, month before parturition. After parturition P levels dropped FSH, and, subsequently, T were detected in the most aggres- to non-detectable levels (,0.2 ng/ml) (P , 0.001).
sive bulls, exhibiting also the largest antlers. These observa- In NP females, P concentrations increased and decreased tions are in accordance with our study in white-tailed deer, several times before reaching peak levels (4.8 ng/ml) in where highest T and LH levels were detected in the most March (Fig. 2). However, statistically the early March peak aggressive bucks of prime breeding age (10). Similar correla- did not differ from the February values.
tion between T concentrations and the fighting rank wasreported in reindeer by Stokkan and co-workers (51).
The increase of T levels observed during the rapidly de- Prolactin concentration exhibited a typical seasonal varia- clining concentrations of LH is mostly due to an altered tion with significant peak levels detected in all cows in May sensitivity of the reproductive system to the stimulation by G. A. Bubenik et al.
GnRH. In the early stages of the reproductive activation a Tyler (23) concluded that E2 is the main regulator of the small dose of GnRH induces a rapid elevation of LH but a antler cycle in the female reindeer and adrenal androgen small increase of T; in the later stages, the same dose GnRH androstenedione may be the secondary steroid involved in will cause a massive increase of T but a small one of LH antlerogenesis. As the relationship between the testoster- one and antler cycle in the male reindeer is less pronounced Unlike in other cervids, some males did not finish the than in other cervids, it could be speculated that in addition polishing of antlers before T levels reached a seasonal maxi- to T, estradiol could play a role of a secondary steroid in- mum of 11.8 ng/ml (see Fig. 1). Conversely, the casting of volved in the male reindeer antlerogenesis.
antlers occurred in some bulls more than 6 weeks after adecline of T levels to a baseline (,0.5 ng/ml). Similar delay in antler casting after a decline of T to basal levels (lastingfor 2–3 months) was observed also in caribou by Whitehead LH, FSH, T. In females, the evaluation of hormonal data
and McEwan (57). This differs from the observations in in relationship to their reproductive activity is difficult, as white-tailed deer, where T levels detected during the time the 3 week period between the samples is too long to detect of antler cleaning (around 1.5 ng/ml) are similar to levels rapid changes in the fluctuation of reproductive hormones.
detected during the time of antler casting (7). In addition, In all cows, peak levels of LH were detected during the rut antler casting was not achieved with an intramuscular ad- (28 August); however, the presence of 2 peaks of LH in the ministration of 100 or 200 mg/animal/week of androgen re- NP cows may indicate that subsequent estrouses occurred ceptor blocker cyproterone acetate given to yearling male in that group. This assumption is further supported by the and female reindeer (G. Bubenik et al., unpublished obser- data on FSH where in the NP cows several oscillations were vation). These data indicate that androgen receptors in detected before peak levels were observed on November 6 reindeer antlers are relatively slow to respond to rapid changes of circulating androgen levels.
The much higher elevation of average LH levels observed The onset of new antler growth varied considerably in the non-pregnant group was largely due to a massive in- within our bull group (February 5–March 20) but it usually crease of LH (7.2 ng/ml) observed in one cow. Such a rapid started about 8 weeks after casting (December 7–January (preovulatory) increase of LH which lasts only several hours 20). As expected, the two more aggressive bulls cast the (3,33) is difficult to detect and was probably missed in most antlers earlier than the three tamer ones. Our casting dates of our other animals.
were in variance with the data of Lincoln and Tyler (23) Seasonal variation of testosterone levels in both groups who reported the casting of antlers in their bulls already in of females was remarkably similar, with both groups exhib- November–December but the new antler growth began iting a non-significant trend with a decrease in December only 3–4 months later, in April. This difference could be and May. The time course of T was not in any case related either due to the more northern latitude of norvegian rein- to the milestones of antlerogenesis, such as the polishing or deer (69.2°N), different nutritional conditions or due to the casting of antlers (Fig. 1, insert).
genetic differences between groups. Persisting individualdifferences in the casting dates (up to 2 months apart) have ESTRADIOL. Seasonal levels of estradiol stayed below 1
been reported in white-tailed deer (6).
pg/ml for most of the year. Only in the pregnant cows,LSMEANS of E2 rose to 3 pg/ml at the beginning of parturi- ESTRADIOL. Maximum male estradiol levels (3.5 ng/ml),
tion, three weeks after peak concentration of progesterone reached during the peak of the rut, were surprisingly higher were detected (Fig. 2). This slow elevation of E2 was in than peak levels observed in pregnant females (3.0 ng/ml) agreement with Baksi and Newbrey (4) who reported that detected shortly before parturition in April (Fig. 2). Simi- E2 concentrations in pregnant reindeer cows in January larly, concentrations of estradiol in the feces of caribou bulls were only 50% higher than values detected in June. Similar were reported to be comparable to levels of non-pregnant slow rise in E2 levels in Rangifers was also reported by Mess- cows (27). A significant correlation found between T and ier and co-workers (27) who noted that until day 150 of E2 may indicate that, similar to stallions (36), reindeer tes- pregnancy no difference was detected between E2 of preg- tes are aromatizing considerable amount of T into estradiol.
nant and non-pregnant cows. Conversely, the rapid eleva- Whether estrogens are involved in the mineralization of tion of E2 shortly before parturition is in agreement with reindeer antlers has not yet been investigated, however ex- data reported by Blom and co-workers (5) in reindeer cows, perimental evidence indicates that E2 is 10–503 more po- Plotka and co-workers (32) in white-tailed does, and by tent in the mineralization of antlers than T (14,30). In addi- Kelly and co-workers (18) in red deer hinds. The variation tion, growing antlers exhibit estradiol receptors (20) and an in LSMEANS of E2 concentration in our study (1–3 pg/ experimental blockade of E2 receptors interrupts the miner- ml) was very similar to data of Lincoln and Tyler (23) who alization process in the compact antler bone mantle, re- reported average E2 values varying seasonally between 2– sulting in severe reduction of its thickness (8). Lincoln and 6 pg/ml. However, probably because of species differences, Reproductive Hormones in Reindeer variations in laboratory techniques and because of an infre- PG and NP caribou during the first 50 days of pregnancy.
quent sampling intervals, our peak values of E2 (3 pg/ml) Finally, P levels were also found elevated in NP cows in the were much lower than the ones reported by Plotka and co- study of McEwen and Whitehead (26).
workers (31) in the white-tailed deer (180 pg/ml) or by It could be argued that a sharp drop in progesterone levels Kelly and co-workers (18) in the red deer (35 pg/ml).
at the end of pregnancy may be related to antler casting.
The rapid decline of E2 levels, which correlated with the However, whereas there is plenty of evidence about the role time of parturition was usually followed by the casting of of estradiol in the antler cycle (as mentioned above), there antlers (Fig. 2). Similar to reports given by McEwen and is little evidence that progesterone levels can decisively in- Whitehead (26) and Lincoln and Tyler (23), the NP cows fluence antler mineralization and retention. Whereas Jac- cast antlers earlier than the pregnant ones (Table 3). Based zewski (15) reported partial or complete velvet shedding on the results in ovariectomized cows, Lincoln and Tyler and retention of antlers in some castrated red deer stags (23) concluded that ovarian E2 is the major steroid regulat- treated with large doses of P, neither Waldo and Wislocki ing the antler cycle in the female reindeer. This appears (54) in white-tailed deer nor Goss (13) in sika deer were to be true in some females, which cast their antler after able to induce velvet shedding in castrated bucks treated parturition. However, in our non-pregnant females no de- with P. Furthermore, McEwen and Whitehead (26) ob- cline of E2 was observed before their casting (Fig. 1) and in served antler casting in captive pregnant reindeer from Jan- the pregnant group two cows cast their antlers several days uary until March (the period of high P) whereas in the wild before parturition. As on few occasions, we have observed females the casting occurred mostly after calving in June or antler casting in the pregnant cows more than 2 weeks be- July (the period of very low P). Therefore, it appears that P fore parturition and McEwan and Whitehead (26) reported does not play a crucial role in the antler cycle of the female a casting period 2 months before the calving, it was con- cluded that a sudden drop in E2 levels is probably not theonly factor initiating the casting of antlers in the female PROLACTIN IN MALES AND FEMALES. Prolactin levels ex-
hibited a typical seasonal curve with peak levels observedin May and June and trough concentrations detected from PROGESTERONE. The seasonal variations of progesterone
September until March (12). In both female groups, maxi- concentrations detected in our pregnant cows (Fig. 2) con- mum concentration exceeded that of the bulls (Fig. 2). This firmed the expected time course with highest levels (9 ng/ is in agreement with PRL data on white-tailed deer and ml), observed shortly before parturition (Table 3) [for re- red deer, where peak female levels were higher than ones view, see (38)]. Similarly to some bovids, ovaries are the observed in males (18,47). Time courses of PRL levels in essential source of P in the pregnant cervid female (34).
the individual cows indicate that the period of parturition/ Peak P levels (9 ng/ml) detected in pregnant reindeer, were lactation coincides with the increase of PRL levels, however higher than 5 ng/ml reported in pregnant red deer (2), 4.6 a continuation of lactation in ruminants does not appar- ng/ml found in white-tailed deer (31) or 3.2 ng/ml observed ently require peak PRL concentrations (17). This was con- in roe deer by Schams et al. (43). However, they were very firmed in our study, as the sharp decline in PRL levels was similar to 10.9 pg/ml reported in reindeer by Blom and co- observed in both the lactating as well as the non-lactating workers (5) or 8.4 ng/ml observed in roe deer by Sempere cows. However, this finding was in contrast to data of Adam (48). Whereas in red deer P concentrations were main- and co-workers (1) who, later in the lactation period, re- tained high for most of the second and third trimester and ported higher PRL concentrations in the plasma of lactating calving occurred only after P dropped precipitously (2,18), hinds, as compared to the non-lactating ones.
in our reindeer P levels rose steadily until peak levels in Because in some cervids new antler growth coincides March, some 3–4 weeks before parturition and then de- with PRL increase in the spring, Lincoln and Tyler (23) clined sharply. Similar steady rise in P values during the suggested that PRL may be involved in the reinitiation of pregnancy, followed by parturition and a subsequent rapid antler growth. This hypothesis is not supported by our data decline, was also reported by Blom and co-workers (5).
as a new antler growth was observed in some reindeer bulls In contrast, the peak levels in the NP cows were not only already in February, the time of minimal levels of PRL de- smaller (6.0 ng/ml) but also exhibited an irregular variation tected in our study (see Fig. 2). In addition, new antler (Fig. 2). The time course of P levels in our NP females may growth in tropical rusa deer is initiated during the decline be the result of seasonal polyestrous as demonstrated by of PRL levels (53) and in the subtropical Eld's deer, PRL Plotka and co-workers (33) in white-tailed deer and re- levels rise when the antlers are hard (29).
cently by Ropstadt and co-workers (37) in reindeer. Simi- In conclusion, our studies indicate that in reindeer, the larly, during the first trimester, peak P levels in NP white- individual milestones of their antler cycle are less related tailed does were not different from concentrations recorded to the seasonal variation of circulating levels of sexual hor- in the pregnant ones (31). Similar data were reported by mones than in most other cervids. In that case, either other Messier and co-workers (27) who investigated P in feces of hormones than those investigated in this study are involved G. A. Bubenik et al.
of progesterone on the antler cycle in red deer (Cervus ela- or the final regulation of the antler cycle occurs at the tissue phus). Z. Jagdwiss. 25:150 –159;1979.
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Animal Hormone Program, Beltsville, MD, U.S.A.) for the generous Plasma concentrations of LH, prolactin, oestradiol and pro- gift of pituitary hormones. The project was supported by NSERC Can- gesterone in female red deer (Cervus elaphus) during preg- ada, Institute of Arctic Biology and the Granting Agency of the Czech nancy. J. Reprod. Fertil. 64:475–483;1982.
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say for bovine follicle-stimulating hormone and its application



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Advanced Software Protection Now Diego Bendersky1,2, Ariel Futoransky1, Luciano Notarfrancesco1, Carlos Sarraute1,3, and Ariel Waissbein1,3 1 Corelabs, Core Security Technologies; 2 Departamento de Computaci´on, FCEyN, Universidad de Buenos Aires (UBA), 3 Departamento de Matem´atica, FCEyN, UBA Argentina. Abstract. We introduce a novel software-protection method, which canbe fully implemented with today's technologies, that provides traitortracing and license-enforcement functionalities, and requires no addi-tional hardware nor inter-connectivity other than those needed to ex-ecute the respective protected software.In [1] authors introduce the secure triggers and show that it is secure,modulo the existence of an ind-cpa secure block cipher. In this work,we present a framework for license enforcement and fingerprinting ofcomputer programs in which these capabilities are coupled with securetriggers to produce a secure software protection system.