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Seasonal levels of LH, FSH, testosterone and prolactin in adult male pudu (Pudu puda)
ISSN 0305-0491/96/$15.00 PI1 S0305-0491(96)00163-0
Comp. Biochem. Physiol. Vol. 115B, No. 4, pp. 417-420, 1996 Copyright © 1996 Elsevier Science Inc.
ELSEVIER
Seasonal Levels of LH, FSH, Testosterone and Prolactin in Adult Male Pudu (Pudupuda) George A. Bubenik,* Eugenia Reyes,l Dieter Schams,~- Alejandro Lobos,§ and Ludek Bartos** *DEPARTMENT OF ZOOLOGY,UNIVERSITYOF GUELPH, GUELPH, ONTARIO, CANADA, N1G 2W1; tDEPARTMENTOF MOLECULARBIOLOGY,AND §DEPARTMENTOF ANIMALPRODUCTIONAND REPRODUCTION,FACULTYOF VETERINARY MEDICINE, UNIVERSITYOF CONCEPCION, CONCEPCION, CHILE; ~DEPARTMENTOF PHYSIOLOGY,TECHNICALUNIVERSITYOF MUNICH, D 85350 FREISING--WEIHENSTEPHAN,GERMANY;AND **RESEARCHINSTITUTEOF ANIMALPRODUCTION, DEPARTMENTOF BIOLOGY,ETHOLOGYGROUP, PRAHA 10-UHRINEVES,CZECH REPUBLIC
ABSTRACT. Seasonal levels of LH, FSH, testosterone (T) and prolactin (PRL) were determined in plasma of six captive adult male pudu (Pudu puda) kept in Concepcion, Chile. Average PRL levels exhibited one peak (28 ng/ml) in December (summer); minimal levels (3 to 6 ng/ml) were detected between April and July. FSH concentrations remained at peak levels (54-63 ng/ml) from December until March; minimal values (25-33 ng/ml) were detected from April until October. T levels exhibited two, almost equal peaks; the first peak (2.8 ng/ml) was detected in March (rut) and the second one (2.7 ng/ml) in October (spring). Both T peaks were preceded by an earlier elevation of LH in February and July (both around 1.3 ng/ml). During the fall, only the alpha male exhibited a sharp peak of T (8.4 ng/ml), whereas in the spring five out of six bucks demonstrated an increase of T levels. Two peaks of LH and T and the 4 months of elevated FSH may be related to a long period of spermatogenesis observed in this species. Copyright© 1996 ElsevierScience Inc. COMemOCHEMPHYS1OL 115B;4:417-420, 1996. KEY WORDS. LH, FSH, testosterone, prolactin, testosterone, pudu, male, seasonality, deer
INTRODUCTION Pudu (Pudu puda, Molina) is the smallest member of the deer family (average weight 7-10 kg) and the smallest ungulate of South America (12). This deer lives mostly in Chile where its range is approximately between 35-49°S (9). Because of the destruction of the habitat, poaching and the introduction of non-native cervids, this species is threatened with extinction. Until recently this solitary, reclusive animal was one of the least studied deer (12); however a successful captive breeding program (20) initiated in several countries resulted in increasing numbers of pudu being available for studies. This provided an opportunity for basic anatomical, physiological and behavioral investigations (2,8,10,15,19,21). The following article summarizes the results of previous studies and expands our knowledge of the seasonal reproduction in pudu. We report the variations of seasonal levels of LH, FSH, testosterone (T) and prolactin (PRL) in a captive male population, relate them to the reproductive pattern and the antler cycle and compare them to other cervid species. Address reprint requests to: Dr G.A. Bubenik, Dept. Zoology, University of
Guelph, Guelph, Ontario, Canada, N1G 2W1. Fax (519) 767-1656; Email: [email protected] Received 6 February 1996; accepted 31 May 1996.
MATERIAL AND METHODS Anima/s and Sampling Procedures Six adult male pudu, ranging in age from 2 to 6 years were maintained at the University of Concepcion, Chile (latitude 36.6'S). The deer were fed freshly cut grass, native shrubs and leaves of deciduous trees ad libitum. During a 1year period the animals were anaesthetized once a month with a 1:1 mixture of Rompun (xylazine hydrochloride, Haver-Lockhard Labs. Bayvet Div., Mississauga, Ontario, Canada) and Rogarsetic (ketamine hydrochloride, Rogar, Montreal, Quebec, Canada) (5-10 mg/kg) injected manually. Then three consecutive blood samples were taken (510 minutes apart) from the jugular vein. The details of the immobilization and cannulation procedures were described in our previous publication (2). The samples were immediately centrifuged and the plasma was frozen for later determination of hormones by radioimmunoassays (RIA).
Hormonal Assays and Analysis LH, FSH, PRL and T levels were determined by heterologous RIA performed at the TU Munich-Weihenstephan. All assays showed no crossreactivity to other pituitary hormones. The assays were validated for the deer plasma by recovery experiments and dilution curves, which run par-
418
allel to the standard. The LH and prolactin RIA was a homologous bovine assay and the pituitary bovine reference preparation LH-DSA (biological activity 1.0 times NIHbLH-S1) and prolactin (USDA-P-B4) were used. The assay system for FSH was homologous for ovine FSH. Pituitary bovine FSH (USDA-bFSH-B1) was used as the reference. The details of the LH, FSH, PRL and T assays were published in the following publications (13,23,25). Because of the small amount of plasma obtained, the assays were performed in a pool made from all three samples. The data were subjected to the General Linear Models Procedure (GLM) for Unbalanced ANOVA (SAS). Classes were "Month" (January to December) and "Animal" (six males). Least-squares means (LSMEAN) were computed for each class and differences between classes were tested by t-test.
G.A. Bubenik et al.
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RESULTS
Luteinizing Hormone LSMEANS of LH levels exhibited two equal peaks, one in July and the second in February (both 1.3 ng/ml). These peaks preceded T peaks by 1 and 3 months, respectively (Fig. 1). Minimal levels (0.6 ng/ml) were recorded in May (Fall).
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Basal FSH levels of 25-33 ng/ml were detected from April to October. Then the concentrations elevated rapidly and stayed high (54-63 ng/ml) from December until the peak of the rut in March (Fig. 1). However, the average elevation was due to the exceptional increase of FSH in only two males. The minimal values (25 ng/ml) were observed in June.
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Testosterone LSMEANS of T levels exhibited two almost equally high peaks. First maximum (2.8 ng/ml) was detected in March (peak of the rut), the other maximum (2.7 ng/ml) was found in October (Fig. 1). Minimal concentrations (>0.1 ng/ml) were detected in June and July. Prolactin
LSMEANS of seasonal levels of PRL were maintained low in the fall and winter (3 to 6 ng/ml), then rose rapidly during the spring and finally reached peak concentrations (28 ng/ml) around the summer solstice in December (Fig. 1).
Statistic GLM showed significant variation for LH (F(16,64) = 2.27, P < 0.05; Month F/1~,64) = 2.18, P < 0.05, Animal F(5,64) = 2.53, P < 0.05). FSH (F(16,64) = 2.36, P < 0.01; Month FIll.64)
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= 2.58, P < 0.01, Animal F(5,641 = 1.81, NS). Testosterone 3.39, P < 0.001, Month F01,64) = 3,98, P < 0.001, Animal FI5.641-- 2.00, P = 0.096). Prolactin (F~16.64/= 8.32, P < 0.001, Month Fl1~.641 = 10.07, P < 0.001, Animal F(5,64) = 1.66, NS). LSMEANS (-+SE) of seasonal levels of LH, FSH, testosterone and prolactin are shown in Fig. 1. (F06,64) =
DISCUSSION
In many respects, the seasonal variations of hormones in pudu were similar to other cervids but in others they differed considerably. Unsurprisingly, the peak levels of PRL were reached around the summer solstice (Fig 1), the same period where maximum PRL levels also were found in most other deer species (4,14,16,17,28). However, the maximum sea-
LH, FSH, Testosterone and Prolactin in Pudu
sonal levels of pudu (28 ng/ml) were by far the lowest found in all cervids. Prolactin peak concentration in other subtropical deer were much higher: e.g. 110 ng/ml in Axis (6), 102 ng/ml in Eld's deer (17) and 70 ng/ml in Rusa deer (31). As the seasonal peak levels of PRL in the arctic dwelling reindeer reached only 91 ng/ml (7) and PRL concentration in the fallow deer, which originated in the Mediterranean area, was reported to be around 310 ng/ml (18), the low levels of PRL in pudu are probably not related to its habitat in the temperate climate. Conversely, the winter values, which appear to be uniformely low (mostly or below 1 ng/ml) in boreal cervids (5,26,28,29) are generally high in the temperate and tropical deer: around 3 ng/ml in pudu, and 20 ng/ml in the rusa and axis (6,31). The exception are PRL values reported for the tropical Eld's deer (17) when it was kept at the latitude of 38"N; its minimal concentration of PRL stayed around 1 ng/ml. More tropical cervid species must therefore be investigated before any definitive conclusions can be made about the relationship of seasonal PRL variations of the particular cervid and the latitude of their habitat. FSH levels were maintained relatively low for approximately 7 months and then, after a rapid elevation, stayed high for some 4 months, until the rut (Fig. 1). Lowest and highest FSH values in pudu (25 vs. 63 ng/ml) were similar to minimal and maximal concentrations found in the male reindeer (19.0 vs. 49.8 ng/ml) (7) or Axis deer (32 vs. 63 ng/ml) (6) but differed from white-tailed deer (20 vs. 105 ng/ml) (3) or roe deer (15 vs. 35 ng/ml) (24). Interestingly, only two males in the group of five exhibited a sharp elevation of FSH levels. Perhaps a quick elevation of a transitory nature might have been missed during the once-a-month sampling or the massive elevation occurred only in a few bucks, which later participated in the rut. Unlike in the boreal cervids, where peak FSH levels were achieved several months before the rut and then declined (3,7,16,24) in pudu, peak FSH levels stayed high until the rut (Fig. 1.). This time course of FSH in pudu was comparable to FSH in the tropical Axix and Eld's deer in which peak levels were detected at the time of the rut (6,17). Seasonal levels of T exhibited two, almost equally high, seasonal peaks (Fig. 1). The minimal and maximal levels (>0.1 vs. 2.6 ng/ml) were similar to values reported in Axis (0.1 vs. 1.8 ng/ml) (6) but slightly different from the roe deer (0.1 vs. 7.5 ng/ml) (24). The finding of peak T values during the rut (March) was not surprising. However, the second peak in the spring (October), albeit not entirely unexpected, is still interesting as it reaches an amplitude similar to the peak detected in the rut. A temporary spring increase of T levels was occasionally reported in various cervid species (3,22,27,32). Occasionally, a spring rutting behavior was observed in red deer stags (5) and a spring activation of testicular Leydig cells was reported by Frankenberger (11). The true significance of that phenomenon has not been yet elucidated. It has been speculated that this bian-
419
nual activation of the reproductive system, which occurs regularly only in the roe deer (24,28) and was also found in the castrates of white-tailed deer (3) and intact red deer stags (29), is a relic of the original reproductive pattern of earlier cervids that developed in the semitropical regions (1). The biannual T-pattern in pudu would support that hypothesis. LH levels exhibit only minimal variations throughout the year. However, two peaks (preceding the T peak by 1 and 3 months) were observed in February and July (Fig. 1). Whereas the fall peak occurred fairly uniformly in all bucks, the spring LH peak varied from individual to individual and the average elevation was the result of major LH increase in only three males. The average I peak levels of LH (1.86 ng/ml) found in this study were similar to peak levels ( 1.78 ng/ml) established in our previous publication (21). In that study a small elevation of LH was registered in September in addition to the major peak detected in February. The narrow range of minimal and maximal average values (1.1 vs. 1.9 ng/ml) is similar to values reported in roe deer, 0.92.6 ng/ml (24) but the minimal concentrations exceed substantially the values found in Axis (minimal 0.1, maximal 1.8 ng/ml) (6). Interestingly, the pre-rut LH peak in pudu preceded the peak of testosterone only by 1 month (Fig. 1). In most other cervids, LH maximum was found at least 3 months before the peak of T (3,16,17,27,30). The exception is the tropical Axis deer, where LH peaks only 1 month before T (5). The two seasonal elevations of T and the prolonged elevation of FSH reported in pudu may be related to the relatively long period of spermatogenesis, which was recently observed in this species (Reyes et al., in preparation). It could be speculated that the first peak of T, which coincides with the beginning of the FSH increase, is related to the activation of the reproductive system. Perhaps the rank of males is determined at this time and the spermatogenesis is then reactivated. Several infants (10) born out of season were reported in pudu (Reyes, unpublished observation), which indicates a relatively long window of opportunity for conception. However, as endocrine investigations of seasonal changes in the female pudu have not yet been reported, the mechanisms of these out-of-season conceptions remain obscure. We would like to thank Mr. Don Salvador de Silva for the exemplary care of animals, Peter Bubenik and Greg Humphreys for the preparation of data and Mrs. Yoko Imai for the preparation of the manuscript. The donation of reference preparation for FSH and PRL by Dr Bolt from USDA Animal Hormone Program, Beltsville, MD, U.S.A. is greatly appreciated. This study was supported by NSERC Canada, Granting Agency of the Czech Republic (grant # 505195/0291) and by a generous financial contribution of Dr W. Trense, Pullach, Germany.
fin order to compare studies, an average value was used instead of t.SMEANS.
References 1. Bubenik, A.B. Epigenetical, morphological, physiological, and behavioral aspects of evolution of horns, pronghorns and antlers. In: Bubenik, G.A.; Bubenik, A.B. (eds). Horns, Pronghorns and Antlers. New York: Springer Verlag; 1990: pp. 3113. 2. Bubenik, G.A. Plasma levels of cortisol, testosterone and growth hormone in pudu (Pudu pud~ Moiina) after ACTH administration. Comp. Biochem. Physiol. 107A:523-527;1994. 3. Bubenik, G.A.; Morris, J.M.; Schams, D.; Claus, A. Photoperiodicity and circannual levels of LH, FSH, and testosterone in normal and castrated, male white-tailed deer. Can. J. Physiol. Pharmacol. 60:788-793;1982. 4. Bubenik, G.A.; Schams, D.; Leatherland, J.F. Seasonal levels of prolactin and its role in the antler cycle of white-tailed deer. In: Drew, K.; Fennessy, P. (eds). Biology of Deer Production. Bull. 22: The Royal Soc. of N.Z.; 1985: pp. 257-262. 5. Bubenik, G.A.; Bubenik, A.B.; Frank, A. Aussersaisonale Brunft beim Edelhirsch (Cervus ekLphmmaral). Z. Jagdwiss. 21: 122-133;1985b. 6. Bubenik, G.A.; Brown, R.D.; Schams, D. Antler cycle and endocrine parameters in male xis deer (Axis axis): Seasonal levels of LH, FSH, testosterone and prolactin and results of GnRH and ACTH challenge tests. Comp. Biochem. Physiol. 99A:645-650; 1991. 7. Bubenik, G.A.; Schams, D.; White, R.J.; RoweU, J.; Blake, J.; Bartos, L. Seasonal levels of reproductive hormones and their relationship to the antler cycle of male and female reindeer (Rangifer tarandus). Comp. Biochem. Physiol. 115B (in press), 1997. 8. Cortes, R.I.; Angulo, A.O.; Guzman, R.; Reyes, E. Compartamiento del pudu [Pudu puda (Molina)] en cautiverio (Mammalia, Cervidae). (Behavior of pudu in captivity). Gayana Zool. 52:3-14;1988. 9. Eldridge, W.; MacNamara, M.M.; Pacheco, N.V. Activity patterns and habitat utilization of pudus (Pudu puda) in south central Chile. In: Wemmer, C.M. (ed). Biology and Management of the Cervidae. Washington D.C.: Smithsonian Inst.; 1987: pp. 352-370. 10. Fradrich, H. The husbandry of tropical and temperate cervids in the West Berlin Zoo. In: Wemmer, C.W. (ed). Biology and Management of the Cervidae. Washington D.C.: Smithsonian Inst.; 1987: pp. 422-428. 11. Frankenberger, Z. Interstitialni bunky u jelena (Cervus elaphus L.). (Interstitial cells of the red deer). Ceskoslovenska Moffologie 2:36-41;1954. 12. Hershkovitz, P. Neotropical deer (Cervidae) Part 1. Pudus, genus Pudu Gray. Feldiana: Zoology, New Series, No. 11; 1982: pp. 43-86. 13. Karg, H.; Gimenez, T.; Hartl, M.; Hoffman, B.; Schallenberger, E.; Schams, D. Testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) in peripheral plasma of bulls: levels from birth through puberty and short term variations. Zentbl. Vet. Med. A 23:793-803;1976. 14. Loudon, A.S.I.; Milne, J.A.; Curlewis, J.D.; McNeilly, A.S. A comparison of the seasonal changes and patterns of growth, voluntary food intake and reproduction in juvenile and adult red deer (Cervus elaphus) and Pere David's deer (Elaphurus davidianus) hinds. J. Endocrinol. 122:733-745;1989. 15. MacNamara, M.W.; Eldridge, W. Behavior and reproduction in captive pudu (Pudu puda) and red brocket (Mazama americana), a descriptive and comparative analysis. In: Wemmer, C.W. (ed). Biology and Management of the Cervidae. Washington, D.C.: Smithsonian Inst.; 1987: pp. 371-387. 16. Mirarchi, B.E.; Howland, B.E.; Scanlon, R.E.; Kirkpatrick, R.L.; Sanford, L.M. Seasonal variations of LH, FSH, prolactin and testosterone concentrations in the adult white-tailed deer. Can. J. Zool. 56:121-127;1978.
17. Monfort, S.L.; Brown, J.L.; Bush, M.; Wood, T.C.; Wemmer, C.; Varga, A.; Williamson, L.R.; Montali, R.J.; Wildt, D.E. Circannual inter-relationship among reproductive hormones, gross morphometry, behavior, ejaculate characteristics and testicular histology in Eld's deer (Cervus eldi thamin). J. Reprod. FertiL 98:471-480;1993. 18. Newman, R.E.; Foldes, A.; Maxwell, C.A.; Rigby, R.D.G.; Wynn, P.C. Identification of a seasonal elevation in the daytime melatonin levels associated with the rut in fallow bucks (Dama dama): The effect of the day length and exogenous melatonin. J. Pineal Res. 11:101-110; 1991. 19. Reyes, E.; Guzman, R.; Angulo, A.; Hermosilla, I.; Conejeros, Y.S. Ciclo de vida y madurez sexual de Pudu puda (Molina) (Mammalia, Cervidae). (Life cycle and sexual maturity of Pudu puda). Bol. Soc. Biol. Concepcion, Chile 59:143-150; 1988. 20. Reyes-Toledo, E.; Carrasco, C.; Torres, P.; Hermosilla, I. Program for the conservation of the species Pudu puda (Molina) (Mammalia, Cervidae). In: Ortiz, C. (ed). Cervidos Nativos i Introducidos en Chile. Osorno, Chile: Chilean Assoc. of Deer Breeders; 1992: pp. 115-118. 21. Reyes, E.; Munoz, P.; Recabarren, S.; Torres, P.; Bubenik, G.A. Seasonal variation of LH and testosterone in the smallest deer, the pudu (Pudu puda Molina) and its relationship to the antl cycle. Comp. Biochem. Physiol. 106A:683-685;1993. 22. Roll, H.J.; Fischer, K. Serum testosterone (T) and 5-alphadihydrotestosterone (DHT) in male fallow deer (Dama dama L.): seasonality and age dependence. Comp. Biochem. Physiol. 95A:445-452;1990. 23. Schams, D.; Karg, H. Radioimmunologische Bestimmung von Prolactin in Blutserum von Rind. Milchwissenschaft 24:262265;1969. 24. Schams, D.; Barth, D. Annual profiles of reproductive hormones in peripheral plasma of the male roe deer (Capreolus capreolus). J. Reprod. Fertil. 66:463-468;1982. 25. Schams, D.; Barth, D.; Karg, H. LH, FSH, and progesterone concentrations in peripheral plasma of the female roe deer during the rutting season. J. Reprod. Fert. 80:109-114;1980. 26. Schulte, B.A.; Seal, U.S.; Plotka, E.D.; Letellier, M.A.; Verme, L.J.; Ozoga, J.J.; Parsons, L.A. The effect of pinealectomy on seasonal changes in prolactin secretion in the whitetailed deer (Odocoileus vir~nianus borealis). Endocrinology 108:173-179;1981. 27. Sempere, A.J.; Lacroix, A. Temporal and seasonal relationship between LH, testosterone and antlers in fawn and adult male roe deer (Capreolus capreolus L.): a longitudinal study from birth to four years of age. Acta Endocrinol. 99:295-302; 1982. 28. Sempere, A.J. The annual antler cycle of the european roe deer (Capreolus capreolus) in relation to the reproductive cycle. In: Bubenik, G.A.; Bubenik, A.B. (eds). Horns, Pronghorns and Antlers. New York: Springer Verlag; 1990: pp. 396-415. 29. Suttie, J.M.; Kay, R.N.B. Influence of plane winter nutrition on plasma concentration of prolactin and testosterone and their association with voluntary food intake in red deer stags (Cervus elaphus). Anim. Repr. Sci. 8:247-258;1985. 30. Suttie, J.M.; Lincoln, G.A.; Kay, R.N.B. Endocrine control of antler growth in red deer stags. J. Reprod. Fert. 71:7-15;1984. 31. Van Mourik, S.; Stelmasiak, T. Seasonal variations of plasma prolactin concentrations in adult male rusa deer (Cervus rusa timorensis). Comp. Biochem. Physiol. 79A:581-584;1985. 32. West, N.O.; Nordan, H.C. Hormonal regulation of reproduction and the antler cycle in 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;1976.
Comp. Biochem. Physiol. Vol. 115B, No. 4, pp. 417-420, 1996 Copyright © 1996 Elsevier Science Inc.
ELSEVIER
Seasonal Levels of LH, FSH, Testosterone and Prolactin in Adult Male Pudu (Pudupuda) George A. Bubenik,* Eugenia Reyes,l Dieter Schams,~- Alejandro Lobos,§ and Ludek Bartos** *DEPARTMENT OF ZOOLOGY,UNIVERSITYOF GUELPH, GUELPH, ONTARIO, CANADA, N1G 2W1; tDEPARTMENTOF MOLECULARBIOLOGY,AND §DEPARTMENTOF ANIMALPRODUCTIONAND REPRODUCTION,FACULTYOF VETERINARY MEDICINE, UNIVERSITYOF CONCEPCION, CONCEPCION, CHILE; ~DEPARTMENTOF PHYSIOLOGY,TECHNICALUNIVERSITYOF MUNICH, D 85350 FREISING--WEIHENSTEPHAN,GERMANY;AND **RESEARCHINSTITUTEOF ANIMALPRODUCTION, DEPARTMENTOF BIOLOGY,ETHOLOGYGROUP, PRAHA 10-UHRINEVES,CZECH REPUBLIC
ABSTRACT. Seasonal levels of LH, FSH, testosterone (T) and prolactin (PRL) were determined in plasma of six captive adult male pudu (Pudu puda) kept in Concepcion, Chile. Average PRL levels exhibited one peak (28 ng/ml) in December (summer); minimal levels (3 to 6 ng/ml) were detected between April and July. FSH concentrations remained at peak levels (54-63 ng/ml) from December until March; minimal values (25-33 ng/ml) were detected from April until October. T levels exhibited two, almost equal peaks; the first peak (2.8 ng/ml) was detected in March (rut) and the second one (2.7 ng/ml) in October (spring). Both T peaks were preceded by an earlier elevation of LH in February and July (both around 1.3 ng/ml). During the fall, only the alpha male exhibited a sharp peak of T (8.4 ng/ml), whereas in the spring five out of six bucks demonstrated an increase of T levels. Two peaks of LH and T and the 4 months of elevated FSH may be related to a long period of spermatogenesis observed in this species. Copyright© 1996 ElsevierScience Inc. COMemOCHEMPHYS1OL 115B;4:417-420, 1996. KEY WORDS. LH, FSH, testosterone, prolactin, testosterone, pudu, male, seasonality, deer
INTRODUCTION Pudu (Pudu puda, Molina) is the smallest member of the deer family (average weight 7-10 kg) and the smallest ungulate of South America (12). This deer lives mostly in Chile where its range is approximately between 35-49°S (9). Because of the destruction of the habitat, poaching and the introduction of non-native cervids, this species is threatened with extinction. Until recently this solitary, reclusive animal was one of the least studied deer (12); however a successful captive breeding program (20) initiated in several countries resulted in increasing numbers of pudu being available for studies. This provided an opportunity for basic anatomical, physiological and behavioral investigations (2,8,10,15,19,21). The following article summarizes the results of previous studies and expands our knowledge of the seasonal reproduction in pudu. We report the variations of seasonal levels of LH, FSH, testosterone (T) and prolactin (PRL) in a captive male population, relate them to the reproductive pattern and the antler cycle and compare them to other cervid species. Address reprint requests to: Dr G.A. Bubenik, Dept. Zoology, University of
Guelph, Guelph, Ontario, Canada, N1G 2W1. Fax (519) 767-1656; Email: [email protected] Received 6 February 1996; accepted 31 May 1996.
MATERIAL AND METHODS Anima/s and Sampling Procedures Six adult male pudu, ranging in age from 2 to 6 years were maintained at the University of Concepcion, Chile (latitude 36.6'S). The deer were fed freshly cut grass, native shrubs and leaves of deciduous trees ad libitum. During a 1year period the animals were anaesthetized once a month with a 1:1 mixture of Rompun (xylazine hydrochloride, Haver-Lockhard Labs. Bayvet Div., Mississauga, Ontario, Canada) and Rogarsetic (ketamine hydrochloride, Rogar, Montreal, Quebec, Canada) (5-10 mg/kg) injected manually. Then three consecutive blood samples were taken (510 minutes apart) from the jugular vein. The details of the immobilization and cannulation procedures were described in our previous publication (2). The samples were immediately centrifuged and the plasma was frozen for later determination of hormones by radioimmunoassays (RIA).
Hormonal Assays and Analysis LH, FSH, PRL and T levels were determined by heterologous RIA performed at the TU Munich-Weihenstephan. All assays showed no crossreactivity to other pituitary hormones. The assays were validated for the deer plasma by recovery experiments and dilution curves, which run par-
418
allel to the standard. The LH and prolactin RIA was a homologous bovine assay and the pituitary bovine reference preparation LH-DSA (biological activity 1.0 times NIHbLH-S1) and prolactin (USDA-P-B4) were used. The assay system for FSH was homologous for ovine FSH. Pituitary bovine FSH (USDA-bFSH-B1) was used as the reference. The details of the LH, FSH, PRL and T assays were published in the following publications (13,23,25). Because of the small amount of plasma obtained, the assays were performed in a pool made from all three samples. The data were subjected to the General Linear Models Procedure (GLM) for Unbalanced ANOVA (SAS). Classes were "Month" (January to December) and "Animal" (six males). Least-squares means (LSMEAN) were computed for each class and differences between classes were tested by t-test.
G.A. Bubenik et al.
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RESULTS
Luteinizing Hormone LSMEANS of LH levels exhibited two equal peaks, one in July and the second in February (both 1.3 ng/ml). These peaks preceded T peaks by 1 and 3 months, respectively (Fig. 1). Minimal levels (0.6 ng/ml) were recorded in May (Fall).
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Basal FSH levels of 25-33 ng/ml were detected from April to October. Then the concentrations elevated rapidly and stayed high (54-63 ng/ml) from December until the peak of the rut in March (Fig. 1). However, the average elevation was due to the exceptional increase of FSH in only two males. The minimal values (25 ng/ml) were observed in June.
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Testosterone LSMEANS of T levels exhibited two almost equally high peaks. First maximum (2.8 ng/ml) was detected in March (peak of the rut), the other maximum (2.7 ng/ml) was found in October (Fig. 1). Minimal concentrations (>0.1 ng/ml) were detected in June and July. Prolactin
LSMEANS of seasonal levels of PRL were maintained low in the fall and winter (3 to 6 ng/ml), then rose rapidly during the spring and finally reached peak concentrations (28 ng/ml) around the summer solstice in December (Fig. 1).
Statistic GLM showed significant variation for LH (F(16,64) = 2.27, P < 0.05; Month F/1~,64) = 2.18, P < 0.05, Animal F(5,64) = 2.53, P < 0.05). FSH (F(16,64) = 2.36, P < 0.01; Month FIll.64)
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A
M
Month FIG. 1. Least.square means ( - SE) of seasonal levels of LH, FSH, testosterone and prolactin in male pudu.
= 2.58, P < 0.01, Animal F(5,641 = 1.81, NS). Testosterone 3.39, P < 0.001, Month F01,64) = 3,98, P < 0.001, Animal FI5.641-- 2.00, P = 0.096). Prolactin (F~16.64/= 8.32, P < 0.001, Month Fl1~.641 = 10.07, P < 0.001, Animal F(5,64) = 1.66, NS). LSMEANS (-+SE) of seasonal levels of LH, FSH, testosterone and prolactin are shown in Fig. 1. (F06,64) =
DISCUSSION
In many respects, the seasonal variations of hormones in pudu were similar to other cervids but in others they differed considerably. Unsurprisingly, the peak levels of PRL were reached around the summer solstice (Fig 1), the same period where maximum PRL levels also were found in most other deer species (4,14,16,17,28). However, the maximum sea-
LH, FSH, Testosterone and Prolactin in Pudu
sonal levels of pudu (28 ng/ml) were by far the lowest found in all cervids. Prolactin peak concentration in other subtropical deer were much higher: e.g. 110 ng/ml in Axis (6), 102 ng/ml in Eld's deer (17) and 70 ng/ml in Rusa deer (31). As the seasonal peak levels of PRL in the arctic dwelling reindeer reached only 91 ng/ml (7) and PRL concentration in the fallow deer, which originated in the Mediterranean area, was reported to be around 310 ng/ml (18), the low levels of PRL in pudu are probably not related to its habitat in the temperate climate. Conversely, the winter values, which appear to be uniformely low (mostly or below 1 ng/ml) in boreal cervids (5,26,28,29) are generally high in the temperate and tropical deer: around 3 ng/ml in pudu, and 20 ng/ml in the rusa and axis (6,31). The exception are PRL values reported for the tropical Eld's deer (17) when it was kept at the latitude of 38"N; its minimal concentration of PRL stayed around 1 ng/ml. More tropical cervid species must therefore be investigated before any definitive conclusions can be made about the relationship of seasonal PRL variations of the particular cervid and the latitude of their habitat. FSH levels were maintained relatively low for approximately 7 months and then, after a rapid elevation, stayed high for some 4 months, until the rut (Fig. 1). Lowest and highest FSH values in pudu (25 vs. 63 ng/ml) were similar to minimal and maximal concentrations found in the male reindeer (19.0 vs. 49.8 ng/ml) (7) or Axis deer (32 vs. 63 ng/ml) (6) but differed from white-tailed deer (20 vs. 105 ng/ml) (3) or roe deer (15 vs. 35 ng/ml) (24). Interestingly, only two males in the group of five exhibited a sharp elevation of FSH levels. Perhaps a quick elevation of a transitory nature might have been missed during the once-a-month sampling or the massive elevation occurred only in a few bucks, which later participated in the rut. Unlike in the boreal cervids, where peak FSH levels were achieved several months before the rut and then declined (3,7,16,24) in pudu, peak FSH levels stayed high until the rut (Fig. 1.). This time course of FSH in pudu was comparable to FSH in the tropical Axix and Eld's deer in which peak levels were detected at the time of the rut (6,17). Seasonal levels of T exhibited two, almost equally high, seasonal peaks (Fig. 1). The minimal and maximal levels (>0.1 vs. 2.6 ng/ml) were similar to values reported in Axis (0.1 vs. 1.8 ng/ml) (6) but slightly different from the roe deer (0.1 vs. 7.5 ng/ml) (24). The finding of peak T values during the rut (March) was not surprising. However, the second peak in the spring (October), albeit not entirely unexpected, is still interesting as it reaches an amplitude similar to the peak detected in the rut. A temporary spring increase of T levels was occasionally reported in various cervid species (3,22,27,32). Occasionally, a spring rutting behavior was observed in red deer stags (5) and a spring activation of testicular Leydig cells was reported by Frankenberger (11). The true significance of that phenomenon has not been yet elucidated. It has been speculated that this bian-
419
nual activation of the reproductive system, which occurs regularly only in the roe deer (24,28) and was also found in the castrates of white-tailed deer (3) and intact red deer stags (29), is a relic of the original reproductive pattern of earlier cervids that developed in the semitropical regions (1). The biannual T-pattern in pudu would support that hypothesis. LH levels exhibit only minimal variations throughout the year. However, two peaks (preceding the T peak by 1 and 3 months) were observed in February and July (Fig. 1). Whereas the fall peak occurred fairly uniformly in all bucks, the spring LH peak varied from individual to individual and the average elevation was the result of major LH increase in only three males. The average I peak levels of LH (1.86 ng/ml) found in this study were similar to peak levels ( 1.78 ng/ml) established in our previous publication (21). In that study a small elevation of LH was registered in September in addition to the major peak detected in February. The narrow range of minimal and maximal average values (1.1 vs. 1.9 ng/ml) is similar to values reported in roe deer, 0.92.6 ng/ml (24) but the minimal concentrations exceed substantially the values found in Axis (minimal 0.1, maximal 1.8 ng/ml) (6). Interestingly, the pre-rut LH peak in pudu preceded the peak of testosterone only by 1 month (Fig. 1). In most other cervids, LH maximum was found at least 3 months before the peak of T (3,16,17,27,30). The exception is the tropical Axis deer, where LH peaks only 1 month before T (5). The two seasonal elevations of T and the prolonged elevation of FSH reported in pudu may be related to the relatively long period of spermatogenesis, which was recently observed in this species (Reyes et al., in preparation). It could be speculated that the first peak of T, which coincides with the beginning of the FSH increase, is related to the activation of the reproductive system. Perhaps the rank of males is determined at this time and the spermatogenesis is then reactivated. Several infants (10) born out of season were reported in pudu (Reyes, unpublished observation), which indicates a relatively long window of opportunity for conception. However, as endocrine investigations of seasonal changes in the female pudu have not yet been reported, the mechanisms of these out-of-season conceptions remain obscure. We would like to thank Mr. Don Salvador de Silva for the exemplary care of animals, Peter Bubenik and Greg Humphreys for the preparation of data and Mrs. Yoko Imai for the preparation of the manuscript. The donation of reference preparation for FSH and PRL by Dr Bolt from USDA Animal Hormone Program, Beltsville, MD, U.S.A. is greatly appreciated. This study was supported by NSERC Canada, Granting Agency of the Czech Republic (grant # 505195/0291) and by a generous financial contribution of Dr W. Trense, Pullach, Germany.
fin order to compare studies, an average value was used instead of t.SMEANS.
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