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Fecal androgens of bison bulls during the rut
Hormones and Behavior 46 (2004) 392 – 398 www.elsevier.com/locate/yhbeh
Fecal androgens of bison bulls during the rut M.S. Mooring, a,* M.L. Patton, b V.A. Lance, b B.M. Hall, a E.W. Schaad, a S.S. Fortin, a J.E. Jella, a and K.M. McPeak c b
a Department of Biology, Point Loma Nazarene University, San Diego, CA 92106, USA Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA 92101, USA c Fort Niobrara National Wildlife Refuge, U.S. Fish and Wildlife Service, Valentine, NE 69201, USA
Received 23 January 2004; revised 17 March 2004; accepted 19 March 2004 Available online 20 July 2004
Abstract The influence of sex hormones is a key proximate factor underlying male reproductive behavior in mammals. Effective conservation policies for the remaining purebred plains bison (Bison bison bison) herds require knowledge of the physiology underlying bison reproductive biology. We used fecal steroid analysis to characterize androgen levels in adult bison bulls before, during, and after the rut, and to examine androgen levels of bulls differing in reproductive status, age, and mating success. Fieldwork was carried out at the Fort Niobrara National Wildlife Refuge in north-central Nebraska. All adult bison in the herd were individually known by unique brands. Fecal samples were collected during 2003 from bulls during pre-rut (June), rut (July – August), and post-rut (September), and behavioral observations focused on reproductive status and mating success during the rut. Matched sample data indicated that androgen levels (ng/g feces) of bulls peaked during the rut, doubling from pre-rut to rut and then declining by 75% during post-rut. Dominant bulls that tended (guarded) cows maintained higher androgen levels than bulls that were not tending. There was a positive correlation between bull age (associated with mating success) and androgens, with higher androgen levels in prime-aged bulls compared with younger bulls. Nonetheless, there was no correlation between mating success (measured by number of copulations observed) and androgen level. This suggests that while androgens may provide the proximate motivation to compete for matings, other factors determine the mating success of bison bulls. D 2004 Elsevier Inc. All rights reserved. Keywords: Fecal steroid; Testosterone; Androgens; Bison; Pre-rut; Rut; Post-rut; Mating success; Age; Dominance
Introduction The influence of sex hormones is known to be a key proximate factor underlying male reproductive behavior in mammals. Almost nothing is currently known of the behavioral endocrinology of adult bull bison. Because most existing bison herds have been polluted with cattle genes through hybridization (Schnabel et al., 2000; Ward et al., 1999, 2001), the way that the few purebred herds are managed will be crucial for the preservation of bison genetic diversity in the future. The physiology underlying bison reproductive behavior must be established to understand the role of individual phenotype on reproductive success and genetic structure of bison populations. Because most aspects of reproduction are mediated through hormonal signals, * Corresponding author. Department of Biology, Point Loma Nazarene University, 3900 Lomaland Drive, San Diego, CA 92106. Fax: +1-619-8492598. E-mail address: [email protected] (M.S. Mooring). 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.03.008
reproductive status can be assessed by endocrine measurements (Lasley and Kirkpatrick, 1991). Fecal steroid techniques have recently emerged as a noninvasive and convenient means of determining the reproductive status of free-ranging wildlife (Kirkpatrick et al., 1991, 1992, 1993; Lasley and Kirkpatrick, 1991). Gonadal steroid hormones and their metabolites in feces accurately reflect endocrine activity, and fecal steroid profiles closely reflect plasma values (Desaulniers et al., 1989; Lasley and Kirkpatrick, 1991). Because hormones and metabolites in feces reflect hormone secretion over composite periods of time, they may better represent individual daily hormonal levels than do blood samples (Pelletier et al., 2003). In many species of artiodactyls, androgens are elevated during the breeding season (Brown et al., 1991; Bubenik et al., 1987; Hamasaki et al., 2001; Lund-Larsen, 1977; Mossing and Damber, 1981; Newman et al., 1991; Sanford et al., 1977; Schanbacher and Lunstra, 1976; Yamauchi et al., 1997), whereas in other species, androgens peak during pre-rut (Freudenberger et al., 1993; Pelletier et al., 2003). In
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either case, the seasonal elevation of androgens is associated with morphological, physiological, and behavioral changes in males (Fletcher, 1978; Imwalle et al., 2002; Li et al., 2000, 2001; Lund-Larsen, 1977; Sanford et al., 1977; Schanbacher and Lunstra, 1976). Androgen concentrations may be positively correlated with aggression, age, or social rank (Ahmad et al., 1992; Ditchkoff et al., 2001; Fletcher, 1978; Li et al., 2000, 2001; Oba et al., 1988; Patton et al., 2001; Pelletier et al., 2003). The goal of this study was to use fecal steroid analysis to characterize androgen levels in adult bull bison before, during, and after the rut, and to examine androgen levels of known bulls differing in reproductive status, age, and mating success during the rut. We hypothesized that fecal androgens would increase during the rut and influence rutting behavior in bison.
Methods Study site The Fort Niobrara National Wildlife Refuge (77 km2) is located along the Niobrara River near the town of Valentine in the Sandhills of north-central Nebraska (N 42j53.65V, W 100j28.47V). The topography of the refuge and surrounding region is flat or rolling hills of native grassland (mixed and sandhill prairie), providing excellent visibility for behavioral observations. Established in 1912 as a sanctuary for bison, elk, and native birds, the refuge supports a population of plains bison (Bison bison bison) that is currently maintained at 350 heads after the fall roundup, and up to f475 following calving. During the spring and summer, bison graze over about twothirds of the refuge, and are rotated among different pastures to avoid overgrazing. Unique among public bison herds, every adult in the Fort Niobrara herd is individually marked with unique brands. The composition of the herd in September 2003 was 126 calves, 68 yearlings, and 279 adults (z2 years). The sex ratio of adults was 0.8:1 (123 bulls, 156 cows). Age classes of adults ranged to 17 years for bulls, and 20 years for cows. Review of bison reproductive biology Bison exhibit male-dominance polygyny. Most breeding takes place from mid-July to mid-August, with the peak around 1 August (Lott, 1981; Meagher, 1986). During the rut (breeding season), bulls move through the mixed herds seeking cows that are approaching estrus by sniffing the anogenital region, licking urine and vaginal fluids, and performing flehmen. When a cow interests a bull, he tends (guards) her by staying close alongside her until she comes into estrus, then mates with her (Lott, 2002). During tending, bulls display frequently by bellowing, scent-urination, pawing, rubbing, and wallowing. Tending bulls are
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frequently challenged by rival bulls surrounding the tending pair (‘attending bulls’), and head-to-head fights are common. A tending bull may guard a cow for anywhere from a few minutes to a few days before being displaced by a more dominant bull or copulating. A cow may be tended by 10 or more different bulls that alternately displace the previous bull (Wolff, 1998; unpublished data). Copulations are quite brief, usually less than 10 s from mount to dismount, and most cows breed only once in a season (Lott, 1981). Immediately following a successful copulation, the cow arches her back, expels a small volume of clear or milky secretions from the vulva (presumably vaginal fluids and semen), and erects her tail (Berger, 1989; Berger and Cunningham, 1991; Komers et al., 1992b; Lott, 1981; Wolff, 1998). So reliable is this behavioral indicator of copulation that it can be used to infer copulations not directly observed (Berger, 1989; Berger and Cunningham, 1991; Wolff, 1998). After copulation with the cow, the bull may continue to guard her for anywhere from a few minutes up to 8 h, until he leaves to search for another cow (Wolff, 1998). Bison bulls and cows are sexually mature starting at 2 years (Meagher, 1986). However, most bulls capable of guarding a cow are >5 years old, with prime breeding age being 7 – 13 years old (Maher and Byers, 1987). Prior studies have established that the most dominant bulls have the greatest breeding success (Berger and Cunningham, 1994; Lott, 1979; Wolff, 1998). Behavioral observations We conducted observations during the rut from 15 July through 13 August 2003, which bracketed peak rut for this species (Meagher, 1986). All observations were conducted from 4WD vehicles from <100 m (often within 50 m) of focal animals. We used 10 binoculars or 15– 60 zoom telescopes to read brand numbers. Two to three observers took shifts to maintain continuous surveillance of the herd during daylight hours (0600 to 2000 h). Some breeding occurs at night, and therefore nighttime rutting behavior (which we did not observe) may have influenced hormone levels from feces collected during the day. During the rut, all of the herd was maintained in the same grazing unit, and herd members tended to aggregate in one or several large groups that could be monitored by 4WD vehicle. Observers drove around all parts of the herd so as to account for every tending pair every 1– 2 h. During these group observations, we monitored all tending bulls and cows, recorded tail-ups by cows, and documented copulations. A tending pair was recorded when a bull stood parallel to a cow and followed her movements closely, attempting to exclude competitors from the cow (Lott, 1974, 1981). Tending bulls were active in the rut for a mean (FSD) of 19F6 days. A copulation was recorded either when directly observed from mount to dismount (observed copulation), or when a cow displayed tail-up and was tended by the same bull before and after this behavior was noted (inferred copulation).
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Most cows displayed tail-up only once during the rut (i.e., they copulated once), although two cows that copulated early in the rut came into a second estrus 3 weeks later. Tail-ups persisted anywhere from 1 h to 1 week (mean F SD = 29 F 42 h). Whenever fecal samples were collected, the current reproductive status of the bull (tending, not tending) was recorded. The behavioral data reported here pertains only to bulls from which fecal samples were collected. Fecal androgen samples Fecal samples were collected from June to September 2003, from sexually mature bulls during all daylight hours. Samples were collected from bulls z3 years because younger bulls did not participate in the rut, although they may be sexually mature at 2 years (Meagher, 1986). Mean (FSEM) age of sampled bulls was 8.9 F 0.4 years (range = 3 – 17 years). We usually collected one sample per bull for each period (pre-rut, rut, and post-rut). Samples were opportunistically taken from bulls only when defecation was observed from a known individual. Fresh fecal material was transferred to a 70-ml polypropylene container with screw cap (Sarstedt, Inc., Newton, NC), with date and bull I.D. written directly on the container with an industrial super permanent Sharpee. Samples were placed immediately into an ice chest while in the field, and later transferred to a freezer at 20 jC for storage until shipped overnight on dry ice to San Diego. At the lab, the large samples were lyophilized for 120 h in a Flexi-Dry microprocessor manifold lyophilizer (FTS Systems, Inc., Stone Ridge, NY) to reduce variability in water content. Vegetation was removed from the lyophilized samples by sifting through a mesh screen (2 1.5 mm). A 0.2-g sample of the sifted feces was added to a 16 150 mm borosilicate culture tube, wetted with distilled water (2 ml) and vortexed (2 min). Five milliliters of diethyl ether anhydrous (Mallinckrodt, Paris, KY) were added to each tube, vortexed (2 min), and flash frozen in a methanol:dry ice bath. The supernatant was poured into 12 75 mm culture tubes and allowed to evaporate in a water bath (37jC). The ether extract was resolubilized in 1 ml absolute ethanol. Radioimmunoassays Androgen content was analyzed in the fecal extracts by radioimmunoassay (RIA) using an antibody produced against testosterone 19 carboxymethyl-ether:BSA at a working dilution of 1:12,000 and a final dilution of 1:84,000. This antibody was characterized to cross-react 100% with testosterone, 18.75% with 5a-dihydrotestosterone, 3.00% with 5a-androstane-3a,17h-diol, and 1.00% with 5-androstene-3h,17h-diol. Other hormones tested were found to cross-react <1.00%. These were androstenedione, 5aandrostane-3, 17-dione, androstenedione, progesterone, dihydroepiandrosterone, corticosterone, desoxy-corticosterone, estriol, and estrone (ICN, Costa Mesa, CA). Tritiated
testosterone (10,000 cpm/0.1 ml, Perkin Elmer, Boston, MA) was used to compete against standard testosterone (7 – 1000 pg, Sigma, St. Louis, MO). Ten microliters of ethanolic fecal extract was diluted 1:100 in 0.1 M phosphate-buffered saline pH 7.0 (PBS) and 100 Al of this diluent was assayed in duplicate. Following an overnight incubation at 4 jC, the competitive reaction was terminated by the addition of 0.25 ml of charcoal dextran solution (6.25 g charcoal: 0.625 g dextran in 1.0 l PBS) to separate bound from free hormone. The charcoal-treated samples were held for 30 min at 4 jC, then centrifuged at 1500 g at 4 jC for 15 min. The supernatant was decanted into scintillation vials and scintillation fluid (5 ml, Ultima Gold, Packard Instrument, Meriden, CT) was added and counted for 2 min in a Beckman liquid scintillation spectrometer (LS 6500). High-pressure liquid chromatography Reverse-phase high-performance liquid chromatography (HPLC) (Ultra Sphere C-18 Column; Beckman, San Ramon, CA) was used to characterize the immunoreactive fecal androgen metabolites. Tritiated testosterone (10,500 cpm) was added to a pooled sample and then analyzed in an HPLC run. Samples were first evaporated and then reconstituted 20:1 in 100% methanol (Fisher; Optima grade). Androgen metabolites were separated using isocratic methanol and distilled water (30:14) with 0.2 M potassium phosphate buffer, pH 5.35. Fractions were collected at a rate of 1 ml/min for 40 min, evaporated, and reconstituted in 500 Al PBS buffer. An aliquot (100 Al) of each was taken and counted in the LS 6500 to assess the elution profile of the reference 3H testosterone. Crossreactivity against the androgen antibody was tested in each fraction by RIA. Data analysis and animal welfare Data were analyzed using the SPSS 11.5 statistical package for Windows (Norusis, 2002). The level of significance was set at 0.05, and all tests were two-tailed. Statistical analysis made use of standard parametric tests: Student’s t tests for independent samples, one-way Analysis of Variance, and Pearson bivariate correlation. We have adhered to the guidelines for animal welfare specified in National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH, 1985).
Results Fecal extraction and radioimmunoassay Serial dilution of fecal extract yielded displacement curves parallel to those obtained with the testosterone standard (r = 0.988). Extraction efficiency of added tritiated testosterone was 68.3 F 2.7% (mean F SD, N = 8).
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Accuracy was determined as 95.4 F 6.8 (mean F SD, N = 6) by recovery of six known quantities of standard (7.8 –500 pg) that were equivalent to the standards used in the standard pool added to a pool of fecal extract. A diluted fecal sample from a study male was used for this pool, which contained an immunoreactive content just above the sensitivity of the assay. Assay sensitivity was 9.079 pg/tube (calculated as mean pg/tube at 90% B/BO, N = 10). Buffer blanks were below the assay sensitivity. Inter-assay coefficients of variation (%SD/mean, N = 6) were 19.5% based on duplicates of a bison fecal pool with an immunoreactive content that yielded a %B/BO >60% and 10.5% immunoreactive content that yielded a %B/BO > 25%. Intra-assay variation estimates (10 replicates of the same pools in a single assay) were 7.8% for the high pool and 7.7% for the low pool. Results are presented as ng/g (equal to ng/g dry fecal weight). High-pressure liquid chromatography (HPLC) separated fractions of fecal androgen metabolites and an immunoreactive peak at fraction 14 min coeluted with 3Htestosterone (Fig. 1). This peak (marked I on the figure) was followed by 5h-androstan-3-17-dione (the major immunoreactive peak at II), 5a-dihydroxytestosterone (III), an unidentified peak (IV), and 5h-androstan-3a-oI-17h-oI (V) at fractions 19, 23, 25, and 31, respectively (Fig. 1). Seasonal levels of androgens Androgen levels of bulls peaked during the rut (compared with pre-rut and post-rut) for matched samples of 41 bulls for which data were collected in all three periods (one-way ANOVA: F2,120 = 101.0, P = 0.0001; Scheffe multiple comparisons, P = 0.0001; Fig. 2). The mean (FSEM) ng androgens/g dry feces more than doubled from pre-rut (551 F 34) to rut (1466 F 93), and then sharply declined by more than 75% during post-rut (311 F 34). Androgen levels
Fig. 1. High-pressure liquid chromatography (HPLC) separated fractions of fecal androgen metabolites (- - - -, broken line, ‘pg/tube’) and an immunoreactive peak at fraction 14 min co-eluted with 3H-testosterone (——, solid line, ‘counts/min’). This peak (marked I on the figure) was followed by 5h-androstan-3-17-dione (the major immunoreactive peak at II), 5a-dihydroxytestosterone (III), an unidentified peak (IV), and 5handrostan-3a-oI-17h-oI (V) at fractions 19, 23, 25, and 31, respectively.
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Fig. 2. Mean (FSEM) androgen levels from fecal steroid analysis (ng/g feces) for bison bulls at Fort Niobrara NWR, Nebraska, during pre-rut (samples collected June 6 – 11), rut (July 14 – August 13), and post-rut (September 15 – 16). Matched samples involved 41 bulls for which fecal samples were collected from all three periods. Androgens peaked during the rut, being significantly higher during rut compared with pre-rut or post-rut.
during pre-rut were significantly greater than during postrut (matched t test: N = 42, t = 5.2, P = 0.0001). Matchedpair analyses of all bulls for which data were available in each period showed that androgen levels increased significantly from pre-rut to rut (matched t test: N = 84, t = 11.8, P = 0.0001) and then dramatically declined from rut to post-rut (N = 41, t = 11.3, P = 0.0001). Of the 84 bulls for which we had fecal samples for pre-rut and rut, only 5 (6%) did not show the predicted increase in androgens during rut. Of the 41 bulls for which both rut and post-rut data were available, only 2 (5%) failed to show the predicted decline in androgens following rut. Individual differences in androgens during the rut During the rut, bulls that were actively tending cows at the time of fecal collection had higher levels of androgen compared with bulls that were not tending at that time (t test: N = 95, t = 3.3, P = 0.001; Fig. 3). Bulls that were not tending were either attending a tending bull and cow, or not active in the rut at all. Tending bulls generally continued to sequentially tend cows throughout the time they were active in the rut (x¯ = 19 days), while nontending bulls generally continued to not tend during the rut period because they were too low-ranking to guard a cow from rival bulls. Two of the tending bulls in this data set had very high androgen levels (>4000 ng/g feces); however, when these outliers were omitted from the data, tending bulls still had higher androgen levels compared with nontending bulls (N = 93, t = 2.8, P = 0.005). During the postrut, when reproductive activity was almost over, fecal samples taken from three bulls that were still tending cows indicated that the tending bulls had higher levels of androgen on the day of collection compared with the nonrutting bulls (t test: N = 41, t = 2.9, P = 0.006; Fig. 3). Pearson correlation analysis indicated no significant correlation between mating success (number of copulations)
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Fig. 5. Androgen levels from fecal steroid analysis of bison bulls at Fort Niobrara NWR, Nebraska, according to age. Androgens were logtransformed for illustrative purposes to compress outlying values. Age of bull was positively correlated with level of androgen. Bulls z7 years (to right of dashed line) had significantly higher androgens compared with bulls <7 years.
Fig. 3. Mean (FSEM) androgen levels from fecal steroid analysis (ng/g feces) during the rut (July to August; upper frame) and post-rut (September; lower frame) for bison bulls at Fort Niobrara NWR, Nebraska, that were tending or not tending cows. Tending bulls had significantly higher levels of androgen compared with nontending bulls during both rut and post-rut. Sample sizes for tending and nontending bulls were 53 and 42 (rut), and 3 and 38 (post-rut), respectively.
and androgen level (N = 103, r = 0.03, P = 0.80); in fact, some bulls that were never observed to breed had higher levels of androgen compared with some of the top observed breeders (Fig. 4). However, there was a positive correlation between age of bull and androgen level (Fig. 5; N = 94,
Fig. 4. Androgen levels from fecal steroid analysis (ng/g feces) of bison bulls at Fort Niobrara NWR, Nebraska, according to their reproductive success (measured by copulations). There was no association between reproductive success and level of androgen.
r = 0.26, P = 0.01), and prime-aged bulls of 7 years or greater had significantly higher androgens compared with bulls <7 years (N = 95, t = 3.2, P = 0.002). Bull age was positively correlated with number of copulations observed (N = 94, r = 0.263, P = 0.02), indicating that older bulls had higher mating success. The age of tending bulls (x¯ = 10.5 years) was significantly greater than the age of nontending bulls (x¯ = 7.8 years; t test: N = 82, t = 3.4, P = 0.001).
Discussion Based on fecal steroid analysis, androgen levels of bison bulls at Fort Niobrara peaked during the rut in late July and early August. Androgens more than doubled from pre-rut levels in June, and then declined over 75% during post-rut in September. Although we did not attempt to measure androgens at other times of the year, we assume that androgen level of bulls remain low throughout the nonbreeding season. The elevation of androgens during the breeding season agrees with the pattern seen in a wide range of artiodactyls (Brown et al., 1991; Bubenik et al., 1987; Hamasaki et al., 2001; Lund-Larsen, 1977; Mossing and Damber, 1981; Newman et al., 1991; Sanford et al., 1977; Schanbacher and Lunstra, 1976; Yamauchi et al., 1997). A seasonal rise in circulating androgens of male artiodactyls is associated with morphological and physiological changes related to male reproduction, such as increased body mass, hypertrophy of neck muscles, enlarged scrotal size, and elevated ejaculate volume (e.g., Lund-Larsen, 1977; Sanford et al., 1977). We would predict that similar changes occur in bison bulls as they approach the time of peak rut. In contrast to a seasonal androgen peak during rut, some species exhibit a peak in androgens during the pre-rut (Freudenberger et al., 1993; Pelletier et al., 2003). In these
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species, androgen levels may be associated with aggressive behavior and the attainment of dominance rank, which in these species is established during pre-rut (Pelletier et al., 2003). Although bison bulls engage in dominance interactions during pre-rut, the frequency and intensity of male – male competition reaches a climax during peak rut (Wolff, 1998). During rut and post-rut, bison bulls that were actively tending cows on the day of fecal collection had higher androgen levels compared with bulls that were not tending. Nontending bulls were either attending bulls (surrounding a tending pair), or had retired from rutting behavior altogether. Because the level of steroid hormones in feces may represent circulating levels in blood 10 h to 4 days before collection (Morrow et al., 2002; Mo¨stl and Palme, 2002; Mo¨stl et al., 1999; Palme et al., 1996; Shaw et al., 1995), interpretation of these results is not completely straightforward. Because we had to keep track of up to 40 tending pairs on a given day, it was impossible to continually monitor the reproductive status (tending or not tending) of all sexually mature bulls before opportunistic fecal collections. However, because the average tending bull was in the rut for 19 days, we are confident that in the majority of cases the bulls we sampled had been active in the rut for at least 4 days prior. Most tending bulls, following copulation, moved directly to tend another cow. Thus, we believe that the rutting status observed at the time of collection was generally representative of that bull’s rutting activity at the time hormones were circulating in the blood. Assuming bull reproductive status at the time of fecal collection was the same as that a few days earlier, these results suggest that bulls actively guarding cows from other bulls are operating under the influence of higher androgen levels. Such an effect would be consistent with behavioral changes that accompany the seasonal elevation of androgens in other mammals, such as increased frequency of sniffing, flehmen, urine spraying, wallowing, herding, vocalizing, mounting, and copulating (Fletcher, 1978; Imwalle et al., 2002; Li et al., 2000, 2001; Sanford et al., 1977; Schanbacher and Lunstra, 1976). To successfully tend bison cows, bulls must outcompete any rivals that approach, either by exerting previously established dominance rank, or by escalating aggressive behavior (threat displays, charging, fighting). Fecal androgen concentrations are positively correlated with aggressive and agonistic behavior (e.g., horning, chasing, biting, threats) in artiodactyls (Fletcher, 1978; Li et al., 2000, 2001; Patton et al., 2001). Bison bulls that succeed in tending are older, more dominant individuals (Komers et al., 1992a; Wolff, 1998). In some species, fecal testosterone is positively correlated with social rank, whereas in other species, it is not (Pelletier et al., 2003). Because social rank and fighting ability often increase with age (up to a point), fecal and serum androgens have been found to be positively correlated with age in bovids and cervids (Ahmad et al., 1992; Ditchkoff et al., 2001; Oba et al., 1988; Pelletier et al.,
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2003). Thus, the positive correlation between fecal androgens and age in bison bulls found in this study is consistent with previous reports. In our study, older bulls with higher androgen levels were more likely to tend cows and enjoyed higher mating success. Surprisingly, however, androgen level was not directly related to mating success as measured by total observed copulations per season. Similarly, in cattle bulls, mounting activity was not positively correlated with androgen concentration (Imwalle et al., 2002). Indeed, some bison bulls at Fort Niobrara that were completely unsuccessful in breeding (according to our observations) had higher levels of androgen than the top-breeding bull in the herd. This suggests that, while androgens may provide the motivation to compete for matings, other factors (e.g., size, strength, skill, experience, perseverance, motivation) determine the mating success of particular bulls.
Acknowledgments We thank the Fort Niobrara National Wildlife Refuge and the United States Fish and Wildlife Service for permission to study the Fort Niobrara bison herd, and for making available housing and 4WD vehicles. Special gratitude goes to Royce Huber and Bernie Petersen for their support, to Dana Harty for assisting with fecal collection during postrut, and to all the refuge staff for their assistance. Helpful comments from two anonymous reviewers improved the manuscript. This research was supported with funds from Research Associates, a PLNU Research and Special Projects grant, and a PLNU Provost’s grant.
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Mossing, T., Damber, J.-E., 1981. Rutting behavior and androgen variation in reindeer Rangifer tarandus. J. Chem. Ecol. 7, 377 – 390. Mo¨stl, E., Palme, R., 2002. Hormones as indicators of stress. Domest. Anim. Endocrinol. 23, 67 – 74. Mo¨stl, E., Messmann, S., Bagu, E., Robia, C., Palme, R., 1999. Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. J. Vet. Med., Ser. A 46, 621 – 631. Newman, R.E., Foldes, A., Maxwell, C.A., Rigby, R.D.G., Wynn, P.C., 1991. Identification of a seasonal elevation in daytime melatonin levels associated with the rut in fallow bucks Dama dama: the effect of day length and exogenous melatonin. J. Pineal Res. 11, 101 – 110. NIH, 1985. National Institutes of Health Guide for the Care and Use of Laboratory Animals. DHEW Publication, vol. 80-23. Office of Science and Health Reports, DRR/NIH, Bethesda, MD. Norusis, M., 2002. SPSS 11.0 Guide to Data Analysis. Prentice-Hall, Upper Saddle River, NJ. Oba, E., Define, R.M., Muniz, L.M.R., Ramos, A.D.A., 1988. Determination of the serum levels of FSH luteinizing hormone and testosterone in Nellore cattle at different ages using radioimmunoanalysis. Arq. Bras. Med. Vet. Zootec. 40, 25 – 34. Palme, R., Fischer, P., Schildorfer, H., Ismail, M.N., 1996. Excretion of infused 14C-steroid hormones via faeces and urine in domestic livestock. Anim. Reprod. Sci. 43, 43 – 63. Patton, M.L., White, A.M., Swaisgood, R.R., Sproul, R.L., Fetter, G.A., Kennedy, J., Edwards, M.S., Rieches, R.G., Lance, V.A., 2001. Aggression control in a bachelor herd of fringe-eared oryx (Oryx gazella callotis), with melengestrol acetate: behavioral and endocrine observations. Zoo Biol. 20, 375 – 388. Pelletier, F., Bauman, J., Festa-Bianchet, M., 2003. Fecal testosterone in bighorn sheep (Ovis canadensis): behavioural and endocrine correlates. Can. J. Zool. 81, 1678 – 1684. Sanford, L.M., Palmer, W.M., Howland, B.E., 1977. Changes in the profiles of serum luteinizing hormone, follicle stimulating hormone and testosterone in mating performance and ejaculate volume in the ram during the ovine breeding season. J. Anim. Sci. 45, 1382 – 1391. Schanbacher, B.C., Lunstra, D.D., 1976. Seasonal changes in sexual activity and serum levels of luteinizing hormone and testosterone in Finnish Landrace and Suffolk rams. J. Anim. Sci. 43, 644 – 650. Schnabel, R.D., Ward, T.J., Derr, J.N., 2000. Validation of 15 microsatellites for parentage testing of North American bison Bison bison L. and domestic cattle. Anim. Genet. 31, 360 – 366. Shaw, H.J., Green, D.I., Sainsbury, A.W., Holt, W.V., 1995. Monitoring ovarian function in scimitar-horned oryx (Oryx dammah) by measurement of fecal 20a-progestagen metabolites. Zoo Biol. 14, 239 – 250. Ward, T.J., Bielawski, J.P., Davis, S.K., Templeton, J.W., Derr, J.N., 1999. Identification of domestic cattle hybrids in wild cattle and bison: A general approach using mtDNA markers and the parametric bootstrap. Anim. Conserv. 2, 51 – 57. Ward, T.J., Skow, L.C., Gallagher, D.S., Schnabel, R.D., Nall, C.A., Kolenda, C.E., Davis, S.K., Taylor, J.F., Derr, J.N., 2001. Differential introgression of uniparentally inherited markers in bison populations with hybrid ancestries. Anim. Genet. 32, 89 – 91. Wolff, J.O., 1998. Breeding strategies, mate choice, and reproductive success in American bison. Oikos 83, 529 – 544. Yamauchi, K., Hamasaki, S., Takeuchi, Y., Mori, Y., 1997. Assessment of reproductive status of sika deer by fecal steroid analysis. J. Reprod. Dev. 43, 221 – 226.
Fecal androgens of bison bulls during the rut M.S. Mooring, a,* M.L. Patton, b V.A. Lance, b B.M. Hall, a E.W. Schaad, a S.S. Fortin, a J.E. Jella, a and K.M. McPeak c b
a Department of Biology, Point Loma Nazarene University, San Diego, CA 92106, USA Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA 92101, USA c Fort Niobrara National Wildlife Refuge, U.S. Fish and Wildlife Service, Valentine, NE 69201, USA
Received 23 January 2004; revised 17 March 2004; accepted 19 March 2004 Available online 20 July 2004
Abstract The influence of sex hormones is a key proximate factor underlying male reproductive behavior in mammals. Effective conservation policies for the remaining purebred plains bison (Bison bison bison) herds require knowledge of the physiology underlying bison reproductive biology. We used fecal steroid analysis to characterize androgen levels in adult bison bulls before, during, and after the rut, and to examine androgen levels of bulls differing in reproductive status, age, and mating success. Fieldwork was carried out at the Fort Niobrara National Wildlife Refuge in north-central Nebraska. All adult bison in the herd were individually known by unique brands. Fecal samples were collected during 2003 from bulls during pre-rut (June), rut (July – August), and post-rut (September), and behavioral observations focused on reproductive status and mating success during the rut. Matched sample data indicated that androgen levels (ng/g feces) of bulls peaked during the rut, doubling from pre-rut to rut and then declining by 75% during post-rut. Dominant bulls that tended (guarded) cows maintained higher androgen levels than bulls that were not tending. There was a positive correlation between bull age (associated with mating success) and androgens, with higher androgen levels in prime-aged bulls compared with younger bulls. Nonetheless, there was no correlation between mating success (measured by number of copulations observed) and androgen level. This suggests that while androgens may provide the proximate motivation to compete for matings, other factors determine the mating success of bison bulls. D 2004 Elsevier Inc. All rights reserved. Keywords: Fecal steroid; Testosterone; Androgens; Bison; Pre-rut; Rut; Post-rut; Mating success; Age; Dominance
Introduction The influence of sex hormones is known to be a key proximate factor underlying male reproductive behavior in mammals. Almost nothing is currently known of the behavioral endocrinology of adult bull bison. Because most existing bison herds have been polluted with cattle genes through hybridization (Schnabel et al., 2000; Ward et al., 1999, 2001), the way that the few purebred herds are managed will be crucial for the preservation of bison genetic diversity in the future. The physiology underlying bison reproductive behavior must be established to understand the role of individual phenotype on reproductive success and genetic structure of bison populations. Because most aspects of reproduction are mediated through hormonal signals, * Corresponding author. Department of Biology, Point Loma Nazarene University, 3900 Lomaland Drive, San Diego, CA 92106. Fax: +1-619-8492598. E-mail address: [email protected] (M.S. Mooring). 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.03.008
reproductive status can be assessed by endocrine measurements (Lasley and Kirkpatrick, 1991). Fecal steroid techniques have recently emerged as a noninvasive and convenient means of determining the reproductive status of free-ranging wildlife (Kirkpatrick et al., 1991, 1992, 1993; Lasley and Kirkpatrick, 1991). Gonadal steroid hormones and their metabolites in feces accurately reflect endocrine activity, and fecal steroid profiles closely reflect plasma values (Desaulniers et al., 1989; Lasley and Kirkpatrick, 1991). Because hormones and metabolites in feces reflect hormone secretion over composite periods of time, they may better represent individual daily hormonal levels than do blood samples (Pelletier et al., 2003). In many species of artiodactyls, androgens are elevated during the breeding season (Brown et al., 1991; Bubenik et al., 1987; Hamasaki et al., 2001; Lund-Larsen, 1977; Mossing and Damber, 1981; Newman et al., 1991; Sanford et al., 1977; Schanbacher and Lunstra, 1976; Yamauchi et al., 1997), whereas in other species, androgens peak during pre-rut (Freudenberger et al., 1993; Pelletier et al., 2003). In
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either case, the seasonal elevation of androgens is associated with morphological, physiological, and behavioral changes in males (Fletcher, 1978; Imwalle et al., 2002; Li et al., 2000, 2001; Lund-Larsen, 1977; Sanford et al., 1977; Schanbacher and Lunstra, 1976). Androgen concentrations may be positively correlated with aggression, age, or social rank (Ahmad et al., 1992; Ditchkoff et al., 2001; Fletcher, 1978; Li et al., 2000, 2001; Oba et al., 1988; Patton et al., 2001; Pelletier et al., 2003). The goal of this study was to use fecal steroid analysis to characterize androgen levels in adult bull bison before, during, and after the rut, and to examine androgen levels of known bulls differing in reproductive status, age, and mating success during the rut. We hypothesized that fecal androgens would increase during the rut and influence rutting behavior in bison.
Methods Study site The Fort Niobrara National Wildlife Refuge (77 km2) is located along the Niobrara River near the town of Valentine in the Sandhills of north-central Nebraska (N 42j53.65V, W 100j28.47V). The topography of the refuge and surrounding region is flat or rolling hills of native grassland (mixed and sandhill prairie), providing excellent visibility for behavioral observations. Established in 1912 as a sanctuary for bison, elk, and native birds, the refuge supports a population of plains bison (Bison bison bison) that is currently maintained at 350 heads after the fall roundup, and up to f475 following calving. During the spring and summer, bison graze over about twothirds of the refuge, and are rotated among different pastures to avoid overgrazing. Unique among public bison herds, every adult in the Fort Niobrara herd is individually marked with unique brands. The composition of the herd in September 2003 was 126 calves, 68 yearlings, and 279 adults (z2 years). The sex ratio of adults was 0.8:1 (123 bulls, 156 cows). Age classes of adults ranged to 17 years for bulls, and 20 years for cows. Review of bison reproductive biology Bison exhibit male-dominance polygyny. Most breeding takes place from mid-July to mid-August, with the peak around 1 August (Lott, 1981; Meagher, 1986). During the rut (breeding season), bulls move through the mixed herds seeking cows that are approaching estrus by sniffing the anogenital region, licking urine and vaginal fluids, and performing flehmen. When a cow interests a bull, he tends (guards) her by staying close alongside her until she comes into estrus, then mates with her (Lott, 2002). During tending, bulls display frequently by bellowing, scent-urination, pawing, rubbing, and wallowing. Tending bulls are
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frequently challenged by rival bulls surrounding the tending pair (‘attending bulls’), and head-to-head fights are common. A tending bull may guard a cow for anywhere from a few minutes to a few days before being displaced by a more dominant bull or copulating. A cow may be tended by 10 or more different bulls that alternately displace the previous bull (Wolff, 1998; unpublished data). Copulations are quite brief, usually less than 10 s from mount to dismount, and most cows breed only once in a season (Lott, 1981). Immediately following a successful copulation, the cow arches her back, expels a small volume of clear or milky secretions from the vulva (presumably vaginal fluids and semen), and erects her tail (Berger, 1989; Berger and Cunningham, 1991; Komers et al., 1992b; Lott, 1981; Wolff, 1998). So reliable is this behavioral indicator of copulation that it can be used to infer copulations not directly observed (Berger, 1989; Berger and Cunningham, 1991; Wolff, 1998). After copulation with the cow, the bull may continue to guard her for anywhere from a few minutes up to 8 h, until he leaves to search for another cow (Wolff, 1998). Bison bulls and cows are sexually mature starting at 2 years (Meagher, 1986). However, most bulls capable of guarding a cow are >5 years old, with prime breeding age being 7 – 13 years old (Maher and Byers, 1987). Prior studies have established that the most dominant bulls have the greatest breeding success (Berger and Cunningham, 1994; Lott, 1979; Wolff, 1998). Behavioral observations We conducted observations during the rut from 15 July through 13 August 2003, which bracketed peak rut for this species (Meagher, 1986). All observations were conducted from 4WD vehicles from <100 m (often within 50 m) of focal animals. We used 10 binoculars or 15– 60 zoom telescopes to read brand numbers. Two to three observers took shifts to maintain continuous surveillance of the herd during daylight hours (0600 to 2000 h). Some breeding occurs at night, and therefore nighttime rutting behavior (which we did not observe) may have influenced hormone levels from feces collected during the day. During the rut, all of the herd was maintained in the same grazing unit, and herd members tended to aggregate in one or several large groups that could be monitored by 4WD vehicle. Observers drove around all parts of the herd so as to account for every tending pair every 1– 2 h. During these group observations, we monitored all tending bulls and cows, recorded tail-ups by cows, and documented copulations. A tending pair was recorded when a bull stood parallel to a cow and followed her movements closely, attempting to exclude competitors from the cow (Lott, 1974, 1981). Tending bulls were active in the rut for a mean (FSD) of 19F6 days. A copulation was recorded either when directly observed from mount to dismount (observed copulation), or when a cow displayed tail-up and was tended by the same bull before and after this behavior was noted (inferred copulation).
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Most cows displayed tail-up only once during the rut (i.e., they copulated once), although two cows that copulated early in the rut came into a second estrus 3 weeks later. Tail-ups persisted anywhere from 1 h to 1 week (mean F SD = 29 F 42 h). Whenever fecal samples were collected, the current reproductive status of the bull (tending, not tending) was recorded. The behavioral data reported here pertains only to bulls from which fecal samples were collected. Fecal androgen samples Fecal samples were collected from June to September 2003, from sexually mature bulls during all daylight hours. Samples were collected from bulls z3 years because younger bulls did not participate in the rut, although they may be sexually mature at 2 years (Meagher, 1986). Mean (FSEM) age of sampled bulls was 8.9 F 0.4 years (range = 3 – 17 years). We usually collected one sample per bull for each period (pre-rut, rut, and post-rut). Samples were opportunistically taken from bulls only when defecation was observed from a known individual. Fresh fecal material was transferred to a 70-ml polypropylene container with screw cap (Sarstedt, Inc., Newton, NC), with date and bull I.D. written directly on the container with an industrial super permanent Sharpee. Samples were placed immediately into an ice chest while in the field, and later transferred to a freezer at 20 jC for storage until shipped overnight on dry ice to San Diego. At the lab, the large samples were lyophilized for 120 h in a Flexi-Dry microprocessor manifold lyophilizer (FTS Systems, Inc., Stone Ridge, NY) to reduce variability in water content. Vegetation was removed from the lyophilized samples by sifting through a mesh screen (2 1.5 mm). A 0.2-g sample of the sifted feces was added to a 16 150 mm borosilicate culture tube, wetted with distilled water (2 ml) and vortexed (2 min). Five milliliters of diethyl ether anhydrous (Mallinckrodt, Paris, KY) were added to each tube, vortexed (2 min), and flash frozen in a methanol:dry ice bath. The supernatant was poured into 12 75 mm culture tubes and allowed to evaporate in a water bath (37jC). The ether extract was resolubilized in 1 ml absolute ethanol. Radioimmunoassays Androgen content was analyzed in the fecal extracts by radioimmunoassay (RIA) using an antibody produced against testosterone 19 carboxymethyl-ether:BSA at a working dilution of 1:12,000 and a final dilution of 1:84,000. This antibody was characterized to cross-react 100% with testosterone, 18.75% with 5a-dihydrotestosterone, 3.00% with 5a-androstane-3a,17h-diol, and 1.00% with 5-androstene-3h,17h-diol. Other hormones tested were found to cross-react <1.00%. These were androstenedione, 5aandrostane-3, 17-dione, androstenedione, progesterone, dihydroepiandrosterone, corticosterone, desoxy-corticosterone, estriol, and estrone (ICN, Costa Mesa, CA). Tritiated
testosterone (10,000 cpm/0.1 ml, Perkin Elmer, Boston, MA) was used to compete against standard testosterone (7 – 1000 pg, Sigma, St. Louis, MO). Ten microliters of ethanolic fecal extract was diluted 1:100 in 0.1 M phosphate-buffered saline pH 7.0 (PBS) and 100 Al of this diluent was assayed in duplicate. Following an overnight incubation at 4 jC, the competitive reaction was terminated by the addition of 0.25 ml of charcoal dextran solution (6.25 g charcoal: 0.625 g dextran in 1.0 l PBS) to separate bound from free hormone. The charcoal-treated samples were held for 30 min at 4 jC, then centrifuged at 1500 g at 4 jC for 15 min. The supernatant was decanted into scintillation vials and scintillation fluid (5 ml, Ultima Gold, Packard Instrument, Meriden, CT) was added and counted for 2 min in a Beckman liquid scintillation spectrometer (LS 6500). High-pressure liquid chromatography Reverse-phase high-performance liquid chromatography (HPLC) (Ultra Sphere C-18 Column; Beckman, San Ramon, CA) was used to characterize the immunoreactive fecal androgen metabolites. Tritiated testosterone (10,500 cpm) was added to a pooled sample and then analyzed in an HPLC run. Samples were first evaporated and then reconstituted 20:1 in 100% methanol (Fisher; Optima grade). Androgen metabolites were separated using isocratic methanol and distilled water (30:14) with 0.2 M potassium phosphate buffer, pH 5.35. Fractions were collected at a rate of 1 ml/min for 40 min, evaporated, and reconstituted in 500 Al PBS buffer. An aliquot (100 Al) of each was taken and counted in the LS 6500 to assess the elution profile of the reference 3H testosterone. Crossreactivity against the androgen antibody was tested in each fraction by RIA. Data analysis and animal welfare Data were analyzed using the SPSS 11.5 statistical package for Windows (Norusis, 2002). The level of significance was set at 0.05, and all tests were two-tailed. Statistical analysis made use of standard parametric tests: Student’s t tests for independent samples, one-way Analysis of Variance, and Pearson bivariate correlation. We have adhered to the guidelines for animal welfare specified in National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH, 1985).
Results Fecal extraction and radioimmunoassay Serial dilution of fecal extract yielded displacement curves parallel to those obtained with the testosterone standard (r = 0.988). Extraction efficiency of added tritiated testosterone was 68.3 F 2.7% (mean F SD, N = 8).
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Accuracy was determined as 95.4 F 6.8 (mean F SD, N = 6) by recovery of six known quantities of standard (7.8 –500 pg) that were equivalent to the standards used in the standard pool added to a pool of fecal extract. A diluted fecal sample from a study male was used for this pool, which contained an immunoreactive content just above the sensitivity of the assay. Assay sensitivity was 9.079 pg/tube (calculated as mean pg/tube at 90% B/BO, N = 10). Buffer blanks were below the assay sensitivity. Inter-assay coefficients of variation (%SD/mean, N = 6) were 19.5% based on duplicates of a bison fecal pool with an immunoreactive content that yielded a %B/BO >60% and 10.5% immunoreactive content that yielded a %B/BO > 25%. Intra-assay variation estimates (10 replicates of the same pools in a single assay) were 7.8% for the high pool and 7.7% for the low pool. Results are presented as ng/g (equal to ng/g dry fecal weight). High-pressure liquid chromatography (HPLC) separated fractions of fecal androgen metabolites and an immunoreactive peak at fraction 14 min coeluted with 3Htestosterone (Fig. 1). This peak (marked I on the figure) was followed by 5h-androstan-3-17-dione (the major immunoreactive peak at II), 5a-dihydroxytestosterone (III), an unidentified peak (IV), and 5h-androstan-3a-oI-17h-oI (V) at fractions 19, 23, 25, and 31, respectively (Fig. 1). Seasonal levels of androgens Androgen levels of bulls peaked during the rut (compared with pre-rut and post-rut) for matched samples of 41 bulls for which data were collected in all three periods (one-way ANOVA: F2,120 = 101.0, P = 0.0001; Scheffe multiple comparisons, P = 0.0001; Fig. 2). The mean (FSEM) ng androgens/g dry feces more than doubled from pre-rut (551 F 34) to rut (1466 F 93), and then sharply declined by more than 75% during post-rut (311 F 34). Androgen levels
Fig. 1. High-pressure liquid chromatography (HPLC) separated fractions of fecal androgen metabolites (- - - -, broken line, ‘pg/tube’) and an immunoreactive peak at fraction 14 min co-eluted with 3H-testosterone (——, solid line, ‘counts/min’). This peak (marked I on the figure) was followed by 5h-androstan-3-17-dione (the major immunoreactive peak at II), 5a-dihydroxytestosterone (III), an unidentified peak (IV), and 5handrostan-3a-oI-17h-oI (V) at fractions 19, 23, 25, and 31, respectively.
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Fig. 2. Mean (FSEM) androgen levels from fecal steroid analysis (ng/g feces) for bison bulls at Fort Niobrara NWR, Nebraska, during pre-rut (samples collected June 6 – 11), rut (July 14 – August 13), and post-rut (September 15 – 16). Matched samples involved 41 bulls for which fecal samples were collected from all three periods. Androgens peaked during the rut, being significantly higher during rut compared with pre-rut or post-rut.
during pre-rut were significantly greater than during postrut (matched t test: N = 42, t = 5.2, P = 0.0001). Matchedpair analyses of all bulls for which data were available in each period showed that androgen levels increased significantly from pre-rut to rut (matched t test: N = 84, t = 11.8, P = 0.0001) and then dramatically declined from rut to post-rut (N = 41, t = 11.3, P = 0.0001). Of the 84 bulls for which we had fecal samples for pre-rut and rut, only 5 (6%) did not show the predicted increase in androgens during rut. Of the 41 bulls for which both rut and post-rut data were available, only 2 (5%) failed to show the predicted decline in androgens following rut. Individual differences in androgens during the rut During the rut, bulls that were actively tending cows at the time of fecal collection had higher levels of androgen compared with bulls that were not tending at that time (t test: N = 95, t = 3.3, P = 0.001; Fig. 3). Bulls that were not tending were either attending a tending bull and cow, or not active in the rut at all. Tending bulls generally continued to sequentially tend cows throughout the time they were active in the rut (x¯ = 19 days), while nontending bulls generally continued to not tend during the rut period because they were too low-ranking to guard a cow from rival bulls. Two of the tending bulls in this data set had very high androgen levels (>4000 ng/g feces); however, when these outliers were omitted from the data, tending bulls still had higher androgen levels compared with nontending bulls (N = 93, t = 2.8, P = 0.005). During the postrut, when reproductive activity was almost over, fecal samples taken from three bulls that were still tending cows indicated that the tending bulls had higher levels of androgen on the day of collection compared with the nonrutting bulls (t test: N = 41, t = 2.9, P = 0.006; Fig. 3). Pearson correlation analysis indicated no significant correlation between mating success (number of copulations)
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Fig. 5. Androgen levels from fecal steroid analysis of bison bulls at Fort Niobrara NWR, Nebraska, according to age. Androgens were logtransformed for illustrative purposes to compress outlying values. Age of bull was positively correlated with level of androgen. Bulls z7 years (to right of dashed line) had significantly higher androgens compared with bulls <7 years.
Fig. 3. Mean (FSEM) androgen levels from fecal steroid analysis (ng/g feces) during the rut (July to August; upper frame) and post-rut (September; lower frame) for bison bulls at Fort Niobrara NWR, Nebraska, that were tending or not tending cows. Tending bulls had significantly higher levels of androgen compared with nontending bulls during both rut and post-rut. Sample sizes for tending and nontending bulls were 53 and 42 (rut), and 3 and 38 (post-rut), respectively.
and androgen level (N = 103, r = 0.03, P = 0.80); in fact, some bulls that were never observed to breed had higher levels of androgen compared with some of the top observed breeders (Fig. 4). However, there was a positive correlation between age of bull and androgen level (Fig. 5; N = 94,
Fig. 4. Androgen levels from fecal steroid analysis (ng/g feces) of bison bulls at Fort Niobrara NWR, Nebraska, according to their reproductive success (measured by copulations). There was no association between reproductive success and level of androgen.
r = 0.26, P = 0.01), and prime-aged bulls of 7 years or greater had significantly higher androgens compared with bulls <7 years (N = 95, t = 3.2, P = 0.002). Bull age was positively correlated with number of copulations observed (N = 94, r = 0.263, P = 0.02), indicating that older bulls had higher mating success. The age of tending bulls (x¯ = 10.5 years) was significantly greater than the age of nontending bulls (x¯ = 7.8 years; t test: N = 82, t = 3.4, P = 0.001).
Discussion Based on fecal steroid analysis, androgen levels of bison bulls at Fort Niobrara peaked during the rut in late July and early August. Androgens more than doubled from pre-rut levels in June, and then declined over 75% during post-rut in September. Although we did not attempt to measure androgens at other times of the year, we assume that androgen level of bulls remain low throughout the nonbreeding season. The elevation of androgens during the breeding season agrees with the pattern seen in a wide range of artiodactyls (Brown et al., 1991; Bubenik et al., 1987; Hamasaki et al., 2001; Lund-Larsen, 1977; Mossing and Damber, 1981; Newman et al., 1991; Sanford et al., 1977; Schanbacher and Lunstra, 1976; Yamauchi et al., 1997). A seasonal rise in circulating androgens of male artiodactyls is associated with morphological and physiological changes related to male reproduction, such as increased body mass, hypertrophy of neck muscles, enlarged scrotal size, and elevated ejaculate volume (e.g., Lund-Larsen, 1977; Sanford et al., 1977). We would predict that similar changes occur in bison bulls as they approach the time of peak rut. In contrast to a seasonal androgen peak during rut, some species exhibit a peak in androgens during the pre-rut (Freudenberger et al., 1993; Pelletier et al., 2003). In these
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species, androgen levels may be associated with aggressive behavior and the attainment of dominance rank, which in these species is established during pre-rut (Pelletier et al., 2003). Although bison bulls engage in dominance interactions during pre-rut, the frequency and intensity of male – male competition reaches a climax during peak rut (Wolff, 1998). During rut and post-rut, bison bulls that were actively tending cows on the day of fecal collection had higher androgen levels compared with bulls that were not tending. Nontending bulls were either attending bulls (surrounding a tending pair), or had retired from rutting behavior altogether. Because the level of steroid hormones in feces may represent circulating levels in blood 10 h to 4 days before collection (Morrow et al., 2002; Mo¨stl and Palme, 2002; Mo¨stl et al., 1999; Palme et al., 1996; Shaw et al., 1995), interpretation of these results is not completely straightforward. Because we had to keep track of up to 40 tending pairs on a given day, it was impossible to continually monitor the reproductive status (tending or not tending) of all sexually mature bulls before opportunistic fecal collections. However, because the average tending bull was in the rut for 19 days, we are confident that in the majority of cases the bulls we sampled had been active in the rut for at least 4 days prior. Most tending bulls, following copulation, moved directly to tend another cow. Thus, we believe that the rutting status observed at the time of collection was generally representative of that bull’s rutting activity at the time hormones were circulating in the blood. Assuming bull reproductive status at the time of fecal collection was the same as that a few days earlier, these results suggest that bulls actively guarding cows from other bulls are operating under the influence of higher androgen levels. Such an effect would be consistent with behavioral changes that accompany the seasonal elevation of androgens in other mammals, such as increased frequency of sniffing, flehmen, urine spraying, wallowing, herding, vocalizing, mounting, and copulating (Fletcher, 1978; Imwalle et al., 2002; Li et al., 2000, 2001; Sanford et al., 1977; Schanbacher and Lunstra, 1976). To successfully tend bison cows, bulls must outcompete any rivals that approach, either by exerting previously established dominance rank, or by escalating aggressive behavior (threat displays, charging, fighting). Fecal androgen concentrations are positively correlated with aggressive and agonistic behavior (e.g., horning, chasing, biting, threats) in artiodactyls (Fletcher, 1978; Li et al., 2000, 2001; Patton et al., 2001). Bison bulls that succeed in tending are older, more dominant individuals (Komers et al., 1992a; Wolff, 1998). In some species, fecal testosterone is positively correlated with social rank, whereas in other species, it is not (Pelletier et al., 2003). Because social rank and fighting ability often increase with age (up to a point), fecal and serum androgens have been found to be positively correlated with age in bovids and cervids (Ahmad et al., 1992; Ditchkoff et al., 2001; Oba et al., 1988; Pelletier et al.,
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2003). Thus, the positive correlation between fecal androgens and age in bison bulls found in this study is consistent with previous reports. In our study, older bulls with higher androgen levels were more likely to tend cows and enjoyed higher mating success. Surprisingly, however, androgen level was not directly related to mating success as measured by total observed copulations per season. Similarly, in cattle bulls, mounting activity was not positively correlated with androgen concentration (Imwalle et al., 2002). Indeed, some bison bulls at Fort Niobrara that were completely unsuccessful in breeding (according to our observations) had higher levels of androgen than the top-breeding bull in the herd. This suggests that, while androgens may provide the motivation to compete for matings, other factors (e.g., size, strength, skill, experience, perseverance, motivation) determine the mating success of particular bulls.
Acknowledgments We thank the Fort Niobrara National Wildlife Refuge and the United States Fish and Wildlife Service for permission to study the Fort Niobrara bison herd, and for making available housing and 4WD vehicles. Special gratitude goes to Royce Huber and Bernie Petersen for their support, to Dana Harty for assisting with fecal collection during postrut, and to all the refuge staff for their assistance. Helpful comments from two anonymous reviewers improved the manuscript. This research was supported with funds from Research Associates, a PLNU Research and Special Projects grant, and a PLNU Provost’s grant.
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