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Neuroendocrine-Associated Behavioral Patterns in the Male Asian Elephant (Elephas maximus)
Physiology & Behavior, Vol. 61, No. 5, pp. 771–773, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 / .00
PII S0031-9384(96)00563-X
BRIEF COMMUNICATION
Neuroendocrine-Associated Behavioral Patterns in the Male Asian Elephant (Elephas maximus) ROB D. DICKERMAN,* NANNEPEGA Y. ZACHARIAH,* MICHAEL FOURAKER† AND WALTER J. MCCONATHY*‡ 1 *Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 USA; †Fort Worth Zoo, Fort Worth, TX, USA; and ‡Department of Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 USA Received 3 July 1996; Accepted 18 October 1996 DICKERMAN, R. D., N. Y. ZACHARIAH, M. FOURAKER AND W. J. McCONATHY. Neuroendocrine-associated behavioral patterns in the male asian elephant (Elephas maximus). PHYSIOL BEHAV 61(5) 771–773, 1997.—Steroid-responsive behaviors have been reported in various species; however, the reports thus far on the male Asian elephant (bull) during musth are few in number and most have been conducted on single captive animals for short time periods. The purpose of this investigation was to perform a longitudinal study on steroid-responsive behavior in 3 male Asian elephants from a captive herd of 11 male Asian elephants in Nepal. Male Asian elephants were 18, 25, and 43 years old. The animals had serum collected for 11 months and were observed on a daily basis for aggressive behavior according to the Species Survival Plan (SSP) collection protocol on SSP data sheets. Testosterone (T) and dihydrotestosterone (DHT) were measured in each animal by radioimmunoassay. Testosterone levels rose during musth 26-fold compared to nonmusth, and DHT was elevated 12-fold in musth. Maximal aggressive behavior episodes occurred during peak elevations of T and DHT, with correlation coefficients of 0.82 and 0.89, respectively. Therefore, we suggest that the aggressive episodes are dependent on elevated circulating androgens acting on androgen-responsive neural tissues. q 1997 Elsevier Science Inc. Musth
Dihydrotestosterone
Testosterone
Aggression
MAMMALIAN male sexual behavior is directly controlled by the steroid hormone testosterone, which is secreted from the Leydig cells of the testis (5). Testosterone may be metabolized within target tissues by aromatization to estradiol or 5 alpha-reduction to dihydrotestosterone. Testosterone and its metabolites may then control various behaviors, by mechanisms not fully understood, possibly by regulation of various neuropeptides (10). Mature healthy male Asian elephants experience a temporary aggressive state that occurs sporadically, known as musth, the exact mechanisms for onset and/or triggers for musth remain a mystery to this day (6). Musth may last for 1 day or up to 10 months. The most obvious manifestations in musth are a sharp rise in aggressive behavior, enlargement and secretions from the temporal gland, a modified apocrine sweat gland, and frequent urine discharge (7). In the wild, musth is thought to be a period of heightened reproductive behavior; however, in captivity, male Asian elephants become aggressive towards other elephants, less responsive to handlers commands, and have been known to at-
Elephant
tack and kill their keepers (7,8). The purpose of our longitudinal study was to establish a relationship between aggressive behavior and altered steroid hormone levels. Behavioral data were collected according to the Species Survival Plan (SSP) procedures. The following data represents an attempt to gain a better understanding of steroid-responsive behaviors in male Asian elephants. METHODS
Three male Asian elephants (Elephas maximus) were utilized from a captive herd in Nepal. The elephants were 18, 25, and 43 years old. All animals had olfactory and visual contact with female Asian elephants and were fed similar rations of hay and grain. A protocol was followed for each sample collection. Time of collection was based on available personnel and animal temperaments. Blood was collected from a leg or ear vein in 10-ml vacutainer tubes and allowed to sit for 20 min, centrifuged, and serum decanted into 10-ml vials. Samples were immediately la-
1 To whom requests for reprints should be addressed. Walter J. McConathy, Ph.D., Department of Medicine, University of North Texas Health Science Center (UNTHSC), 3500 Camp Bowie Blvd., Fort Worth, Tx 76107 USA.
771 / eh0c 2609 Mp 771 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609
772
DICKERMAN ET AL.
beled with the animal’s name, date of collection, and subsequently frozen at 0807C. Samples were shipped to the University of North Texas Health Science Center packed in dry ice. The male elephants were observed according to Species Survival Plan protocol for aggressive behavior on a daily basis for 11 months. Aggressive behavior was ranked 1 to 4 with 1 being least aggressive. Method of collection is similar to that previously described (9). The aggression ranking provides a practical, comparative method of assessing the severity of aggression and simplifies statistical procedures for correlation with steroid levels. Aggression, judged in elephants, was focused on elephant personnel, observers, other elephants, or inanimate objects, and recorded by intensity. Aggressive behavior included throwing saliva, rocks, or other objects, hitting trees or gates with their trunks, head butting, grabbing objects forcefully with their trunks, and spinning/charging quickly while intensively staring at a potential victim. Serum testosterone and dihydrotestosterone were measured on each collected sample in duplicate by basic radioimmunoassay, [125 I] kits provided by Diagnostic Systems, Webster, TX. Steroid assays were conducted without knowledge of age or behavioral data on each sample. We report absolute hormone levels for the male Asian elephant. The cross reactivity for the testosterone antibody is 6.6% with dihydrotestosterone and õ 0.9% for androstenedione and estradiol. Mean recovery values for testosterone and dihydrotestosterone were 85% { 7.6% based on extraction/recovery procedures with radioactive-labeled steroid in elephant serum. The sensitivities of the assays were 100 pg/ ml (n Å 10) for testosterone and 25 pg/ml (n Å 10) for dihydrotestosterone. We utilized multiple range/comparison tests to compare the mean testosterone, mean dihydrotestosterone, and aggression scores. In addition, mean steroid levels and aggressive episodes were analyzed by Pearson and Spearman correlation analysis through the use of statistical analysis system (SAS) program. RESULTS
The relationship between aggressive episodes and serum androgen concentrations is demonstrated in Table 1. The younger bulls, 18 and 25 years old, maintained higher aggression scores out of musth, 1.4 and 1.6 respectively, with corresponding higher nonmusth testosterone levels, although the older bull had the highest levels of aggression during musth. A significant relationship between plasma testosterone and aggression score was present in each musth period (r Å 0.82, p õ 0.05). Of most interest was the more significant relationship between dihydrotestosterone and aggression score (r Å 0.89, p õ 0.025). One must note that, although a statistically significant relationship between testosterone and aggression exists, peak aggression scores in the bulls were most often accompanied by a peak in dihydrotestosterone concentration.
TABLE 1 ANDROGEN LEVELS AND AGGRESSION SCORE DURING MUSTH Age (years)
Testosterone (ng/ml) u–m
Dihydrotestosterone (ng/ml) u–m
Aggression (1–4) u–m
18 25 43
3.8–7.7 (14)b 2.0–17.5 (17) 0.7–16.4 (14)
0.12–1.1 0.23–2.5 0.14–2.3
1.4–2.8 1.6–3.0 1.0–3.3
n Å 3 male Asian elephants; u, nonmusth; m, musth. * Number of samples analyzed for steroids, mean testosterone, dihydrotestosterone, and aggression scores (1–4). Statistical comparison of nonmusth to musth testosterone p õ 0.05, and nonmusth to musth dihydrotestosterone p õ 0.05. Correlation for testosterone, aggression, r Å 0.82, p õ 0.05; correlation for DHT, aggression, r Å 0.89, p õ 0.025.
changes (2–4). The present study enhanced previous investigations by monitoring testosterone and dihydrotestosterone throughout an 11-month period in 3 bulls, while collecting daily aggression scores on the same bulls. These findings strengthen prior reports (2–4) that testosterone and aggressive episodes are significantly related. However, we believe that the statistical correlation of dihydrotestosterone with aggression may be more important. A study reporting on the manipulation of hormone levels in castrated male sheep demonstrated that dihydrotestosterone alone has little effect on the central nervous system, estradiol alone mediated 75% of the total sexual behavior of a testosterone-replaced castrate, and estradiol plus dihydrotestosterone potentiated the most sexual behavior (1). Thus, it appears that dihydrotestosterone may enhance the effects of estradiol on sexual behavior by peripheral stimulation of the penis or by centrally promoting the more aggressive sexual behaviors, such as mounting. There is a definite need to differentiate the relative importance of estradiol and dihydrotestosterone in sexual behavior in the captive bull elephant. Due to the numerous control mechanisms involved in central nervous system steroid transport (11,12), one must caution against the use of direct relationships between peripheral steroid levels and central nervous system levels (13). Whether or not testosterone and/or dihydrotestosterone produce the aggression or whether it is a coexistent event has not been absolutely delineated; however, this report and previous investigations have displayed inferential evidence that increased androgen levels do promote aggression (14). Researchers may use 5 alpha-reductase or aromatase inhibitors on captive elephants to determine the role/need of dihydrotestosterone in sexual behavior. Through continued research efforts and the use of modern medicine, we may be able to further preserve and foster reproduction of this amazing animal in captivity. ACKNOWLEDGEMENTS
DISCUSSION
Previous studies have documented altered testosterone levels during musth and accompanying behavioral and physiological
The authors thank Dr. C. J. Turczynski, Dr. Sunder Shresta, Tarran Wagoner, and the elephant keepers of the Fort Worth Zoo for assistance in the study.
REFERENCES 1. Crichton, J. S.; Lishman, A. W.; Hundley, M.; Aimes, A. Role of dihydrotestosterone in the control of sexual behavior on castrated male sheep. J. Reprod. Fert. 93:9–17;1991. 2. Hall-Martin, A. J. Role of musth in the reproductive strategy of the African elephant (Loxodonta africana ). S. Afr. J. Sci. 83:616– 620;1987.
3. Hess, D. L.; Schmidt, A. M.; Schmidt, M. J. Reproductive cycle of the Asian elephant (Elephas maximus) in captivity. Biol. Reprod. 28:767–773;1983. 4. Jainudeen, M. R.; McKay, G. M.; Eisenberg, J. F. Observations of musth in the domesticated Asiatic elephant (Elephas maximus). Mammalia 36:247–261;1972.
/ eh0c 2609 Mp 772 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609
STEROIDS AND BEHAVIOR IN THE MALE ELEPHANT
773
5. Luttge, W. G. Endocrine control of mammalian male sexual behaviour: An analysis of the potential role of testosterone metabolites. In: Beyer, G., ed. Endocrine control of sexual behaviour. New York: Raven Press; 1979:341–363. 6. Niemuller, C. A.; Liptrap, R. M. Altered androstenedione to testosterone ratios and LH concentrations during musth in the captive male Asian elephant (elephas maximus ). J. Reprod. Fertil. 91:139–146; 1991. 7. Poole, J. H.; Kasman, L. H.; Ramsay, E. C.; Lasley, B. L. Musth and urinary concentrations in the African elephant (Loxodonta africana). J. Reprod. Fertil. 70:255–260; 1984. 8. Poole, J. H. Announcing intent: the aggressive state of musth in African elephants. Anim. Behav. 37:140–152; 1989. 9. Poole, J. H. Rutting behavior in African elephants: The phenomenon of musth. Behaviour 102:283–316; 1987.
10. Wang, Z.; De Vries, G. J. Testosterone effects on paternal behavior and vasopressin immunoreactive projections in prairie voles (Microtus ochrogaster). Brain Res. 631:156–160; 1993. 11. Marynick, S. P.; Haven, W. W.; Ebert, M. H.; Loriaux, D. L. Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the rhesus monkey. Endocrinology 99:400–405; 1976. 12. Sheridan, P. J. Androgen receptors in the brain: What are we measuring? Endocrine Rev. 4:171–178; 1983. 13. Martini, L. The 5-alpha reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocrine Rev. 3:1–25; 1982. 14. Guhl, A. M. Gonadal hormones and social behavior in infrahuman vertebrates. In: Sex and Internal Secretions, Vol. 2; Young, N. L; Comer, J. K., eds. Baltimore: Wilkins & Wilkins; 1961:1240–1267.
/ eh0c 2609 Mp 773 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609
PII S0031-9384(96)00563-X
BRIEF COMMUNICATION
Neuroendocrine-Associated Behavioral Patterns in the Male Asian Elephant (Elephas maximus) ROB D. DICKERMAN,* NANNEPEGA Y. ZACHARIAH,* MICHAEL FOURAKER† AND WALTER J. MCCONATHY*‡ 1 *Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 USA; †Fort Worth Zoo, Fort Worth, TX, USA; and ‡Department of Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 USA Received 3 July 1996; Accepted 18 October 1996 DICKERMAN, R. D., N. Y. ZACHARIAH, M. FOURAKER AND W. J. McCONATHY. Neuroendocrine-associated behavioral patterns in the male asian elephant (Elephas maximus). PHYSIOL BEHAV 61(5) 771–773, 1997.—Steroid-responsive behaviors have been reported in various species; however, the reports thus far on the male Asian elephant (bull) during musth are few in number and most have been conducted on single captive animals for short time periods. The purpose of this investigation was to perform a longitudinal study on steroid-responsive behavior in 3 male Asian elephants from a captive herd of 11 male Asian elephants in Nepal. Male Asian elephants were 18, 25, and 43 years old. The animals had serum collected for 11 months and were observed on a daily basis for aggressive behavior according to the Species Survival Plan (SSP) collection protocol on SSP data sheets. Testosterone (T) and dihydrotestosterone (DHT) were measured in each animal by radioimmunoassay. Testosterone levels rose during musth 26-fold compared to nonmusth, and DHT was elevated 12-fold in musth. Maximal aggressive behavior episodes occurred during peak elevations of T and DHT, with correlation coefficients of 0.82 and 0.89, respectively. Therefore, we suggest that the aggressive episodes are dependent on elevated circulating androgens acting on androgen-responsive neural tissues. q 1997 Elsevier Science Inc. Musth
Dihydrotestosterone
Testosterone
Aggression
MAMMALIAN male sexual behavior is directly controlled by the steroid hormone testosterone, which is secreted from the Leydig cells of the testis (5). Testosterone may be metabolized within target tissues by aromatization to estradiol or 5 alpha-reduction to dihydrotestosterone. Testosterone and its metabolites may then control various behaviors, by mechanisms not fully understood, possibly by regulation of various neuropeptides (10). Mature healthy male Asian elephants experience a temporary aggressive state that occurs sporadically, known as musth, the exact mechanisms for onset and/or triggers for musth remain a mystery to this day (6). Musth may last for 1 day or up to 10 months. The most obvious manifestations in musth are a sharp rise in aggressive behavior, enlargement and secretions from the temporal gland, a modified apocrine sweat gland, and frequent urine discharge (7). In the wild, musth is thought to be a period of heightened reproductive behavior; however, in captivity, male Asian elephants become aggressive towards other elephants, less responsive to handlers commands, and have been known to at-
Elephant
tack and kill their keepers (7,8). The purpose of our longitudinal study was to establish a relationship between aggressive behavior and altered steroid hormone levels. Behavioral data were collected according to the Species Survival Plan (SSP) procedures. The following data represents an attempt to gain a better understanding of steroid-responsive behaviors in male Asian elephants. METHODS
Three male Asian elephants (Elephas maximus) were utilized from a captive herd in Nepal. The elephants were 18, 25, and 43 years old. All animals had olfactory and visual contact with female Asian elephants and were fed similar rations of hay and grain. A protocol was followed for each sample collection. Time of collection was based on available personnel and animal temperaments. Blood was collected from a leg or ear vein in 10-ml vacutainer tubes and allowed to sit for 20 min, centrifuged, and serum decanted into 10-ml vials. Samples were immediately la-
1 To whom requests for reprints should be addressed. Walter J. McConathy, Ph.D., Department of Medicine, University of North Texas Health Science Center (UNTHSC), 3500 Camp Bowie Blvd., Fort Worth, Tx 76107 USA.
771 / eh0c 2609 Mp 771 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609
772
DICKERMAN ET AL.
beled with the animal’s name, date of collection, and subsequently frozen at 0807C. Samples were shipped to the University of North Texas Health Science Center packed in dry ice. The male elephants were observed according to Species Survival Plan protocol for aggressive behavior on a daily basis for 11 months. Aggressive behavior was ranked 1 to 4 with 1 being least aggressive. Method of collection is similar to that previously described (9). The aggression ranking provides a practical, comparative method of assessing the severity of aggression and simplifies statistical procedures for correlation with steroid levels. Aggression, judged in elephants, was focused on elephant personnel, observers, other elephants, or inanimate objects, and recorded by intensity. Aggressive behavior included throwing saliva, rocks, or other objects, hitting trees or gates with their trunks, head butting, grabbing objects forcefully with their trunks, and spinning/charging quickly while intensively staring at a potential victim. Serum testosterone and dihydrotestosterone were measured on each collected sample in duplicate by basic radioimmunoassay, [125 I] kits provided by Diagnostic Systems, Webster, TX. Steroid assays were conducted without knowledge of age or behavioral data on each sample. We report absolute hormone levels for the male Asian elephant. The cross reactivity for the testosterone antibody is 6.6% with dihydrotestosterone and õ 0.9% for androstenedione and estradiol. Mean recovery values for testosterone and dihydrotestosterone were 85% { 7.6% based on extraction/recovery procedures with radioactive-labeled steroid in elephant serum. The sensitivities of the assays were 100 pg/ ml (n Å 10) for testosterone and 25 pg/ml (n Å 10) for dihydrotestosterone. We utilized multiple range/comparison tests to compare the mean testosterone, mean dihydrotestosterone, and aggression scores. In addition, mean steroid levels and aggressive episodes were analyzed by Pearson and Spearman correlation analysis through the use of statistical analysis system (SAS) program. RESULTS
The relationship between aggressive episodes and serum androgen concentrations is demonstrated in Table 1. The younger bulls, 18 and 25 years old, maintained higher aggression scores out of musth, 1.4 and 1.6 respectively, with corresponding higher nonmusth testosterone levels, although the older bull had the highest levels of aggression during musth. A significant relationship between plasma testosterone and aggression score was present in each musth period (r Å 0.82, p õ 0.05). Of most interest was the more significant relationship between dihydrotestosterone and aggression score (r Å 0.89, p õ 0.025). One must note that, although a statistically significant relationship between testosterone and aggression exists, peak aggression scores in the bulls were most often accompanied by a peak in dihydrotestosterone concentration.
TABLE 1 ANDROGEN LEVELS AND AGGRESSION SCORE DURING MUSTH Age (years)
Testosterone (ng/ml) u–m
Dihydrotestosterone (ng/ml) u–m
Aggression (1–4) u–m
18 25 43
3.8–7.7 (14)b 2.0–17.5 (17) 0.7–16.4 (14)
0.12–1.1 0.23–2.5 0.14–2.3
1.4–2.8 1.6–3.0 1.0–3.3
n Å 3 male Asian elephants; u, nonmusth; m, musth. * Number of samples analyzed for steroids, mean testosterone, dihydrotestosterone, and aggression scores (1–4). Statistical comparison of nonmusth to musth testosterone p õ 0.05, and nonmusth to musth dihydrotestosterone p õ 0.05. Correlation for testosterone, aggression, r Å 0.82, p õ 0.05; correlation for DHT, aggression, r Å 0.89, p õ 0.025.
changes (2–4). The present study enhanced previous investigations by monitoring testosterone and dihydrotestosterone throughout an 11-month period in 3 bulls, while collecting daily aggression scores on the same bulls. These findings strengthen prior reports (2–4) that testosterone and aggressive episodes are significantly related. However, we believe that the statistical correlation of dihydrotestosterone with aggression may be more important. A study reporting on the manipulation of hormone levels in castrated male sheep demonstrated that dihydrotestosterone alone has little effect on the central nervous system, estradiol alone mediated 75% of the total sexual behavior of a testosterone-replaced castrate, and estradiol plus dihydrotestosterone potentiated the most sexual behavior (1). Thus, it appears that dihydrotestosterone may enhance the effects of estradiol on sexual behavior by peripheral stimulation of the penis or by centrally promoting the more aggressive sexual behaviors, such as mounting. There is a definite need to differentiate the relative importance of estradiol and dihydrotestosterone in sexual behavior in the captive bull elephant. Due to the numerous control mechanisms involved in central nervous system steroid transport (11,12), one must caution against the use of direct relationships between peripheral steroid levels and central nervous system levels (13). Whether or not testosterone and/or dihydrotestosterone produce the aggression or whether it is a coexistent event has not been absolutely delineated; however, this report and previous investigations have displayed inferential evidence that increased androgen levels do promote aggression (14). Researchers may use 5 alpha-reductase or aromatase inhibitors on captive elephants to determine the role/need of dihydrotestosterone in sexual behavior. Through continued research efforts and the use of modern medicine, we may be able to further preserve and foster reproduction of this amazing animal in captivity. ACKNOWLEDGEMENTS
DISCUSSION
Previous studies have documented altered testosterone levels during musth and accompanying behavioral and physiological
The authors thank Dr. C. J. Turczynski, Dr. Sunder Shresta, Tarran Wagoner, and the elephant keepers of the Fort Worth Zoo for assistance in the study.
REFERENCES 1. Crichton, J. S.; Lishman, A. W.; Hundley, M.; Aimes, A. Role of dihydrotestosterone in the control of sexual behavior on castrated male sheep. J. Reprod. Fert. 93:9–17;1991. 2. Hall-Martin, A. J. Role of musth in the reproductive strategy of the African elephant (Loxodonta africana ). S. Afr. J. Sci. 83:616– 620;1987.
3. Hess, D. L.; Schmidt, A. M.; Schmidt, M. J. Reproductive cycle of the Asian elephant (Elephas maximus) in captivity. Biol. Reprod. 28:767–773;1983. 4. Jainudeen, M. R.; McKay, G. M.; Eisenberg, J. F. Observations of musth in the domesticated Asiatic elephant (Elephas maximus). Mammalia 36:247–261;1972.
/ eh0c 2609 Mp 772 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609
STEROIDS AND BEHAVIOR IN THE MALE ELEPHANT
773
5. Luttge, W. G. Endocrine control of mammalian male sexual behaviour: An analysis of the potential role of testosterone metabolites. In: Beyer, G., ed. Endocrine control of sexual behaviour. New York: Raven Press; 1979:341–363. 6. Niemuller, C. A.; Liptrap, R. M. Altered androstenedione to testosterone ratios and LH concentrations during musth in the captive male Asian elephant (elephas maximus ). J. Reprod. Fertil. 91:139–146; 1991. 7. Poole, J. H.; Kasman, L. H.; Ramsay, E. C.; Lasley, B. L. Musth and urinary concentrations in the African elephant (Loxodonta africana). J. Reprod. Fertil. 70:255–260; 1984. 8. Poole, J. H. Announcing intent: the aggressive state of musth in African elephants. Anim. Behav. 37:140–152; 1989. 9. Poole, J. H. Rutting behavior in African elephants: The phenomenon of musth. Behaviour 102:283–316; 1987.
10. Wang, Z.; De Vries, G. J. Testosterone effects on paternal behavior and vasopressin immunoreactive projections in prairie voles (Microtus ochrogaster). Brain Res. 631:156–160; 1993. 11. Marynick, S. P.; Haven, W. W.; Ebert, M. H.; Loriaux, D. L. Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the rhesus monkey. Endocrinology 99:400–405; 1976. 12. Sheridan, P. J. Androgen receptors in the brain: What are we measuring? Endocrine Rev. 4:171–178; 1983. 13. Martini, L. The 5-alpha reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocrine Rev. 3:1–25; 1982. 14. Guhl, A. M. Gonadal hormones and social behavior in infrahuman vertebrates. In: Sex and Internal Secretions, Vol. 2; Young, N. L; Comer, J. K., eds. Baltimore: Wilkins & Wilkins; 1961:1240–1267.
/ eh0c 2609 Mp 773 Thursday Apr 03 11:14 PM EL–PB (v. 61, no. 5) 2609