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Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas)
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Theriogenology 73 (2010) 501–511 www.theriojournal.com
Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas) T.L. Roth *, D.C. Szymanski, E.D. Keyster Center for Conservation and Research of Endangered Wildlife (CREW), Cincinnati Zoo & Botanical Garden, Cincinnati, Ohio, USA Received 14 July 2009; received in revised form 28 August 2009; accepted 30 September 2009
Abstract The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (SD) weight of female toads producing eggs (58.9 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 7.0 g). Similarly, four of seven nonhibernated females (58.8 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment. # 2010 Elsevier Inc. All rights reserved. Keywords: Amphibian; Amplexus; Egg retention; Oviposition; Spermiation
1. Introduction Since 1997 when the Declining Amphibian Task Force of the International Union for the Conservation of Nature (IUCN) unanimously confirmed a significant worldwide decline in amphibians [1], zoos in conjunction with federal and state wildlife departments have
* Corresponding author. Tel.: +1 513 569 8220; fax: +1 513 569 8213. E-mail address: [email protected] (T.L. Roth). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.09.033
increased their commitment to conserving amphibians by establishing new breeding facilities and conducting research with these previously neglected species [2]. To date, there have been several successful programs in which captive breeding has provided adequate offspring for reintroductions. For example, the Puerto Rican crested toad (Peltophryne lemur), Wyoming toad (Bufo baxteri), and boreal toad (Bufo boreas boreas) breeding programs are all over a decade old and have provided continual infusion of captive-bred animals into the wild to boost population numbers and to establish new populations in previously inhabited locations [3–5].
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However, even these successful programs struggle with the inconsistency of annual breeding attempts and could benefit from the development of improved reproductive technologies. Amphibians propagate by a wide variety of methods, and cues triggering natural reproduction are typically related to seasonal provision of suitable habitat for larvae. However, knowledge of these cues is incomplete, preventing accurate duplication in captive systems. Therefore, the typical approach is to mimic natural reproductive cues in temperate species by subjecting animals to artificial hibernation, then rely primarily on exogenous hormones for inducing reproductive behavior, spermiation, and oviposition. The use of gonadotropins and gonadotropin releasing hormones for stimulating gamete production in amphibians dates back to an early pregnancy test developed in 1934 [6] and several subsequent studies that further characterized anuran responsiveness to gonadotrophs of various species [7–11]. In general, although mammalian or synthetic luteinizing hormone releasing hormone analogue (LHRHa) was most effective across a variety of anuran species for inducing the desired response, human chorionic gonadotropin (hCG) was highly effective in specific species. A few recent studies have involved testing a combination of hormones in succession or as cocktails, and some results have been encouraging, but overall, responses are mixed and appear species-specific [12–14]. Hibernation has traditionally been considered necessary in preparing temperate toads for successful reproduction, however its role in physiologic reproductive processes has not been well characterized. It has been reported that anurans prepare for reproduction fairly quickly after breeding and prior to hibernation [12,15–17]. Furthermore, animal care staff and conservationists working with highly endangered species are concerned hibernation stress could increase mortality. Therefore, ascertaining the importance of hibernation on breeding success in temperate anurans would be beneficial. The goal of this study was to further delineate the effects of age, hibernation, and exogenous hormone treatments on female and male temperate toads, using the boreal toad as a research model. This species was chosen because it is already a conservation concern with an active captive breeding, reintroduction, and recovery program that was established in 1994 [18]. Therefore, captive-bred toadlets were available for this study, and the results could have direct, immediate application to a species in need of conservation
breeding, regardless of any species-specific findings. However, the results could also be applicable to closely related endangered anurans, for example, the Wyoming toad (B. baxteri). 2. Materials and methods 2.1. Animals Seventy-seven captive-bred boreal toads (32 females and 45 males) were provided by the Colorado Division of Fish and Wildlife for this project. The toads were produced in spring 2001 and were sent to the Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo & Botanical Garden, in October of that year. Toads arrived in good condition and were allowed 1 mo to acclimate to their new surroundings before being moved into hibernacula for Experiment 1. During the acclimation period, photographs were taken of each toad’s back, and these were used throughout the study for identifying individuals, based on their unique wart patterns. When not in hibernacula, animals were maintained in small, samesex groups in a laboratory at 22 to 25 8C. Toads received 12 h of light daily, water ad libitum, and 4-wk-old crickets dusted with vitamin supplement 4 to 6 times per week. 2.2. Hibernation Hibernacula consisted of tanks with a base covered in charcoal, sand, and peat moss soaked with aged tap water. Holes in the bottom of the tanks allowed excess water to drain to minimize mold and bacterial growth. A piece of slotted pipe was inserted into the substrate to act as burrows for the toads. The tanks were soaked once or twice monthly with fresh water. The temperature and humidity of the hibernacula were monitored with the aid of battery-operated data loggers (readings were consistently 4 8C and 65% humidity). Toads were chosen at random, individually weighed, and then placed in the hibernation tanks (two or three toads/tank) and transferred to an environmental chamber set at 10 8C for 24 h before being moved into a walk-in cold room at 4 8C. In the walk-in cold room, all light bulbs were changed to red and a dark cloth was hung from the top shelf to further minimize light exposure. Toads were checked weekly and sprayed with simplified amphibian Ringers solution (113 mM NaCl, 1 mM CaCl2, 2 mM KCl, 3.6 mM NaHCO3, 220 mOsm/kg, pH 7.4; Years 1 and 2) or water (Years 3 to 5) to keep them hydrated.
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Table 1 Mean (SEM) boreal toad body weights prior to treatment (2002 through 2006). Year 2002 2003 2004 2005 2006 a–e
Toad age (yr) 1 2 3 4 5
Hibernated males (g) a
37.1 1.2 41.4 1.3 c 45.8 0.8 d 38.8 1.0a,c 47.7 2.2 d
Hibernated females (g) b
32.6 1.1 37.2 0.8 a 40.7 1.7a,c 42.1 2.0c,d NA
Nonhibernated females (g) NA NA 41.4 1.9 c 57.3 3.5 e 59.3 2.4 e
Values without a common superscript differed (P < 0.05). NA = not applicable based on study design.
2.3. Exogenous hormones Two exogenous hormones were used in this study: LHRHa and hCG. The LHRHa (pGlu-His-Trp-Ser-Tyrp-Ala-Leu-Arg-Pro-NHEt; Sigma L-4513; Sigma Chemical Co., St. Louis, MO, USA) was dissolved with ddH20 to a concentration of 0.2 mg/mL. The hCG (Lot No. 020605; Sioux Biochemical Inc., Sioux Center, IA, USA) was diluted with ddH20 to a concentration of 2.5 IU/mL. Each year of the study, both hormones were diluted the day of use and any remaining aliquots were stored frozen at 80 8C. These aliquots were thawed and used for repeat treatments and/or sperm production tests conducted within that year. Hormones were administered intraperitoneally as previously described [19] using a 30-gauge needle. Toads treated with LHRHa received 6 mg in a total volume of 30 mL. Toads treated with hCG received 250 or 300 IU administered in a total volume of 100 to 120 mL. When eggs were produced, they were carefully removed from the breeding tanks within 24 h after oviposition, counted, and transferred into aquariums that had been acclimated for at least 1 wk prior to use. Tadpoles were counted once fully formed and swimming freely around the tank. 2.4. Experiment 1 Experiment 1 tested the hypothesis that both hibernation and hormone administrations are necessary to elicit breeding behavior in male boreal toads. Furthermore, the potential benefit of a second hormone administration was examined in males and females as well as the type of hormone (LHRHa or hCG) in females for inducing oviposition and tadpole production. For the first year of the study (2002), all female toads and half of the male toads were hibernated, for a total of 54 animals placed in hibernation. 2.4.1. Trial 1 Toads entered hibernation on November 29, 2001, and were removed April 26, 2002. After removal from
hibernation, toads were weighed (Table 1) and allowed a week of feeding before breeding was attempted. Toads were paired and assigned a treatment group (four or five pairs per treatment) at random. Treatment groups were as follows: (1) hibernated males and females receiving hormones; (2) hibernated males and females with only males receiving hormones; (3) hibernated males and females receiving no hormones; (4) nonhibernated males and hibernated females receiving hormones; (5) nonhibernated males and hibernated females with only males receiving hormones; and (6) nonhibernated males and hibernated females receiving no hormones. All hormone-treated females received 300 IU hCG for their first treatment, whereas males received 6 mg LHRHa. Two weeks later, a second treatment was applied; both male and female toads received LHRHa. 2.4.2. Trial 2 Toads entered hibernation on November 22, 2002, and were removed and weighed (Table 1) on April 27, 2003. Two days after removal from hibernation, toads were paired and assigned a treatment group (five or six pairs per treatment) at random. The six treatment groups were the same as those in Trial 1, however LHRHa was the hormone administered in all initial treatments requiring males and/or females to receive hormone. If no eggs were produced by 48 h after the start of the treatments, female toads in Treatments 1, 2, 4, and 5 received hCG (250 IU) and the males received a second dose of LHRHa. Female and male toads in Treatments 3 and 6 received LHRHa. After a 2-wk interval, toads that had not produced eggs received a second series of hormone treatments identical to the first. 2.4.3. Sperm production test Because the toads were young and the breeding age of boreal toads had not been documented, sperm production in response to LHRHa was used to assess the status of male sexual maturation after each trial in Experiment 1. Toads were rested 1 mo after breeding in Trial 1, and then seven hibernated and seven nonhibernated toads were chosen for the sperm
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production test. All male toads were weighed, and the four heaviest and three lightest hibernated males were treated with 6 mg LHRHa. Similarly, the three heaviest and four lightest nonhibernated males were treated with 6 mg LHRHa. At 8, 15, 22, and 28 h after treatment, each toad was picked up and held over a Petri dish until it urinated. The urine was examined under a microscope for the presence of sperm. To test for sperm production the following year, toads were rested 2 mo after the series of hormone treatments for Trial 2, and then all male toads (hibernated n = 18 and nonhibernated n = 18) received 6 mg LHRHa. Urine was collected approximately 12 h after treatment and examined for the presence of sperm cells. 2.5. Experiment 2 Based on 2003 results, it was determined that the most effective treatment for inducing mating behavior in male toads was to hibernate them and treat them with LHRHa. Therefore, all males in the 2004 study underwent that combined treatment, and the focus of the study design was on female toads. The objectives were to determine if female boreal toads needed to be hibernated to produce fertile eggs and to determine if hibernation increased the reproductive productivity of the females. Furthermore, the potential benefit of a second hormone treatment was further examined as well as the type of hormone (LHRHa or hCG) for inducing oviposition and tadpole production. The experimental design for Experiment 2 was complex (see flowchart in Fig. 1). Hibernation began November 21, 2003, and ended April 24, 2004. Two days after the hibernated subset of toads was removed from hibernation, toads were weighed (Table 1),
assigned a treatment group and paired (4 or 10 pairs per treatment) at random. Treatments applied to female toads in each group were as follows: (1) hibernated, no hormones (n = 4); (2) hibernated, LHRHa (6 mg) treatment (n = 10); (3) not hibernated, no hormones (n = 4); and (4) not hibernated, LHRHa (6 mg) treatment (n = 10). Forty-eight hour later, toads were treated a second time. Treatment for Groups 1 and 3 remained the same, with only the male receiving a second dose of LHRHa (6 mg). Females in Groups 2 and 4, that did not produce eggs were treated with hormones again, half with hCG (250 IU) and the other half with a second dose of LHRHa (6 mg). Males in Groups 2 and 4 also received a second dose of LHRHa (6 mg). Toads were checked every 12 h for up to 8 d after the last treatment of each trial, and breeding behavior (amplexus), egg production, and tadpole development were recorded. 2.6. Experiment 3 2.6.1. Trial 1 In response to both the poor success experienced in inducing oviposition in female toads during the previous experiments and the surprising production of eggs and tadpoles by a nonhibernated female, a simpler study design was followed for Experiment 3 that focused on the effect of hibernation on egg production. In 2004, half of the females (n = 10) were randomly chosen for hibernation, and the other half were maintained throughout the winter under laboratory conditions. All males were hibernated. Hibernation of all toads began on December 4, 2004. Female toads were brought out of hibernation on May 29, 2005, and males were brought out of hibernation on May 31, 2005.
Fig. 1. Flowchart depicting the experimental design for Experiment 2.
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All female toads were weighed prior to the start of hibernation and after hibernation on the day before hormones were administered. All hibernated and nonhibernated female toads and the hibernated male toads received LHRHa (6 mg) on June 1, 2005. Males and females were housed in pairs and observed for amplexus and egg production every 12 h for 6 d. 2.6.2. Trial 2 Trial 2 was designed to further test the hypothesis that hibernation is not a prerequisite to egg production by female boreal toads. Seven female toads were maintained under laboratory conditions throughout the winter. Male toads were hibernated from December 4, 2005, until May 7, 2006, and all toads were weighed just prior to treatment (Table 1). Twenty-four hours after their removal from hibernation, male toads were randomly paired with the seven nonhibernated female toads, and all toads were treated with LHRHa (6 mg). Pairs were housed separately and observed every 12 h for amplexus and egg production over a 4-d period. Sixteen days later, all females that had not produced eggs were treated with hCG (300 IU) and paired with males receiving LHRHa (6 mg). Pairs were observed every 12 h for amplexus and egg production over the following 4 d.
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treatments were analyzed using chi-square or Fisher’s exact test when appropriate. For Experiment 1, data were pooled across hormone-treated groups within first and second treatments to compare the percentage of male toads exhibiting amplexus after one or two treatments with LHRHa. In addition, data from Experiment 1, Trial 2, were further assessed by pooling data across female toad treatment groups and comparing male amplexus behavior in response to different treatments. Differences in mean values (i.e., weights and time in amplexus) were determined using unpaired Student’s t-tests. Mean body weights for each group across sexes, years, and hibernation treatment were compared in Table 1 using ANOVA. For all statistical analyses, P < 0.05 was considered significant. 3. Results Approximately 5 mo after toads were first moved into a hibernation environment, they were removed. Over the course of the 5-yr study, there were approximately 200 toad hibernations completed with only two mortalities (both female). In addition, a third female succumbed to infection upon removal from hibernation. All other toads appeared healthy at the end of the 5-mo period.
2.7. Statistical analysis
3.1. Experiment 1
Data were analyzed using the Macintosh software program Statview (1998, SAS Institute Inc., Cary, NC). Differences in proportion of toads responding to
3.1.1. Trial 1 Only 19% of toad pairs were observed in amplexus at any time during Trial 1 of Experiment 1 (Fig. 2), and
Fig. 2. Percentage of toad pairs in amplexus after their first and second treatments with exogenous hormones during Experiment 1, Trials 1 (2002) and 2 (2003), and Experiment 2 (2004). a–cColumns without a common superscript differ (P < 0.05).
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those observations were spread among treatment groups (Treatment 1, n = 3; Treatment 2, n = 4; Treatment 3, n = 1; Treatment 4, n = 1; Treatment 5, n = 1; Treatment 6, n = 1). No females in any treatment group produced eggs in response to hCG or LHRHa. 3.1.2. Trial 2 Although the percentage of toad pairs exhibiting amplexus in response to the first hormone treatment in Trial 2 (17.2%) was no better than that recorded in Trial 1, amplexus behavior by male toads increased in response to the second LHRHa treatment administered 48 h after the first (56.9%; P < 0.05; Fig. 2). As in Trial 1, amplectant pairs in response to the first and/or second hormone treatment were distributed among treatment groups (Treatment 1, n = 7; Treatment 2, n = 12; Treatment 3, n = 8; Treatment 4, n = 7; Treatment 5, n = 3; and Treatment 6, n = 6). However, by pooling the data across female toad treatments and comparing just the male treatments, differences in treatment effects were revealed (Fig. 3). Treatment with LHRHa appeared essential in inducing amplexus in male toads. In fact, no male toad exhibited amplexus without receiving LHRHa (Fig. 3B). Although hibernation alone did not appear to influence amplexus in male toads (P > 0.05; Fig. 3A), it enhanced the likelihood of amplexus in toads treated with LHRHa compared with those that were not hibernated and treated with LHRHa (P < 0.05; Fig. 3C). One female toad in Treatment 3 produced an egg mass (650 eggs) in response to the LHRHa she received 48 h after the initial treatment. Because the initial Treatment 3 consisted of hibernated males and females receiving no hormones, the 48-h follow-up administration of LHRHa was the first hormone received by this female that season. She was observed in amplexus with a male after the LHRHa treatment and during oviposition, which occurred approximately 48 h after LHRHa administration. One tadpole developed from the egg mass and survived to become an adult. 3.1.3. Sperm production test After Experiment 1, Trial 1, none of the seven hibernated or seven nonhibernated 1-yr-old male toads produced sperm in response to LHRHa despite repeated sampling during a 24-h period. In contrast, after Experiment 1, Trial 2, sperm production was confirmed in 26 of the 36 male 2-yr-old toads treated with LHRHa. Results from five males (three hibernated and two nonhibernated) were inconclusive because urine samples were not obtained, and no sperm were identified in urine from five males (one hibernated and four
Fig. 3. Percentage of male toads in amplexus when grouped across treatments by (A) those that were hibernated versus not hibernated, (B) those receiving LHRHa versus those not receiving hormones, and (C) those receiving LHRHa after hibernation (LHRH + H) versus those treated with LHRHa without undergoing hibernation (LHRH – H) in Experiment 1, Trial 2. a,bWithin each graph, columns without a common superscript differ (P < 0.05).
nonhibernated). Sperm production test results were similar (P > 0.05) in hibernated and nonhibernated toads with 14 of 15 samples collected from hibernated toads and 12 of 16 samples collected from nonhibernated toads containing sperm. 3.2. Experiment 2 Similar to results reported in Experiment 1, amplexus behavior in male toads increased (P < 0.05) in response to a second LHRHa treatment administered 48 h after the first. In this experiment, percentage of toad pairs in amplexus increased from 67.9% after the first treatment to 100% after the second treatment (Fig. 2).
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Fig. 4. (A) Percentage of hibernated versus nonhibernated female toads producing eggs and (B) time spent in amplexus after LHRHa treatment, (C) the body weights of hibernated versus nonhibernated toads and (D) the body weights of females that produced eggs versus those that did not in Experiment 3, Trial 1. a,bWithin each chart, columns without a common superscript differ (P < 0.05).
Only 2 of 28 female toads produced eggs in this experiment. Both females had been treated with a single LHRHa dose prior to oviposition. Interestingly, one of the females was not hibernated. Both hibernated and nonhibernated females were in amplexus during oviposition, which occurred 24 and 48 h after hormone treatment, respectively. Although the hibernated female produced more eggs than the nonhibernated female (2711 vs. 1567, respectively), none of the eggs produced by the hibernated female developed into tadpoles, whereas 36.5% of the eggs produced by the nonhibernated female developed to tadpoles. Many of these developed fully into toadlets. 3.3. Experiment 3 3.3.1. Trial 1 A total of 14 pairs of toads (70%) were observed in amplexus after the single LHRHa treatment. Of those males observed in amplexus, eight were with nonhibernated females, and six were with hibernated
females. A total of six females produced eggs 15 to 60 h after LHRHa treatment, and five of these were in amplexus with males during oviposition. Additionally, three females died within 6 wk of the study, and necrotic eggs were found in their coelomic cavities upon necropsy. Of the eight females that produced eggs during this experiment (retained or delivered), one was hibernated and seven were not (Fig. 4A). Five of the six females that exhibited oviposition were in amplexus at the time, and of these, four produced tadpoles, but fertility was low (2.6% to 16.1% of the eggs developed into tadpoles). In response to the surprising finding that more nonhibernated than hibernated female toads produced eggs (P < 0.05; Fig. 4A), an effort was made to identify contributing factors. Because the male toads could be inducing some response in females through amplexus behavior, the length of time toad pairs remained in amplexus was compared between hibernated and nonhibernated females, but no difference was found (P > 0.05; Fig. 4B). Additionally, because body
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condition is often linked to reproductive success, the body weight of hibernated and nonhibernated toads was compared. There was difference in body weight between groups: nonhibernated female toads weighed more than hibernated females (P < 0.05; Fig. 4C). Furthermore, female toads producing eggs were heavier on average than those not producing eggs (P < 0.05; Fig. 4D). 3.3.2. Trial 2 In Trial 2, all seven toad pairs (100%) were in amplexus within 12 h after receiving LHRHa. Of these seven nonhibernated females, four produced eggs while in amplexus 21 to 36 h after treatment, but none of the eggs developed into tadpoles. Similar to results from all previous experiments in this study, none of the females that failed to produce eggs in response to their first hormone treatment responded to the second hormone treatment. In contrast with Trial 1, none of the females in this trial died with retained eggs. 4. Discussion The most surprising study finding was the reproductive success experienced by nonhibernated female toads (Table 2). In a previous pilot study, we reported that American toads (B. americanus) were capable of oviposition in response to hormone treatments without first undergoing hibernation, but those toads were not with males, so the fertility of the eggs was unknown [12]. Herein, we have proved that nonhibernated female boreal toads can produce fertile eggs that develop into normal toadlets. These results contradicted the previously held belief that toads that hibernate in the wild must be hibernated in captivity to reproduce successfully. In fact, one female toad produced eggs two consecutive years without undergoing hibernation during either winter. It is well established that male temperate toads produce sperm year-round in response to exogenous Table 2 Mean (SD) outcomes from hibernated and nonhibernated female toads producing eggs after a single LHRHa administration (combined for all experiments and years, excluding three toads that died with retained eggs).
Body weight of females (g) Time in amplexus (h) Number producing eggs Interval postinjection (h) Number of eggs produced
Hibernated
Nonhibernated
44.7 7.2 40.0 13.9 n=3 24.0 10.8 1527 1064
56.5 7.2 33.9 45.3 n = 10 33.9 11.3 1530 1044
hormone treatments without undergoing hibernation [19–21], despite reports that cold exposure is an important modulator of male toad breeding activities [22]. However, hibernation has been considered a requirement for female temperate toads, in part, because temperature is considered an important environmental cue for triggering breeding [16,23,24]. It has even been suggested that keeping amphibians at lower temperatures for 4 to 6 wk can partially correct for abnormal vitellogenesis that otherwise occurs in captive animals [25]. However, given that oogenesis is well under way soon after breeding [17], a large portion of the ovarian mass is restored within 1 mo after oviposition [12], and vitellogenic oocyte production is complete prior to hibernation [15,16], the role of hibernation in preparing the female for reproduction may be insignificant. Herein, we demonstrated that appropriate reproductive processes occurred in female toads maintained in laboratory conditions, and eggs produced by these nonhibernated toads were developmentally competent. In fact, fewer hibernated females produced eggs and fewer of their eggs fertilized when compared with nonhibernated females. Therefore, although some ovarian and endocrine processes may naturally occur during hibernation or in response to temperature fluctuations in wild temperate toads, this relationship may be more of an association than a dependency in some species. Alternatively, perhaps environmental stimulatory cues involving hibernation are required in the wild, whereas dependency on those cues is eliminated under captive conditions or artificially induced with exogenous hormones. That sufficient body weight was critically important for female reproductive success was not surprising as positive relationships between body condition and reproduction can be demonstrated in most species including amphibians. One study examining the effects of population density, rainfall, temperature, and body condition on fecundity in common toads (B. bufo) found that body condition (particularly weight) was the most significant factor [26]. However, breeding protocols for endangered temperate toads detail conditions required for hibernation but offer no information regarding target or minimum body weights [5,25,27]. Throughout the study, only 3 of the 15 toads that produced eggs weighed less than 50 g. Therefore, we inferred that 50 g was a good target weight for female boreal toads in a breeding program. In contrast, for males, body weight did not appear to be a significant factor influencing spermiation once males reached sexual maturation. Based on the spermiation data, it was evident that male boreal toads reached sexual maturation at 2 yr of age. A
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few males might have matured at 1 yr, because not all males were tested for sperm production, and from those tested, urine samples were not consistently collected at the desired intervals. However, all samples collected from 1-yr-old toads were devoid of sperm, suggesting the majority of animals were not yet sexually mature. In contrast, most female boreal toads required 4 yr to reach sexual maturity, based on successful oviposition. However, there appeared to be a greater range during which females reached sexual maturation, as one female produced eggs at 2 yr and two others produced at 3 yr. Variation in exact age of sexual maturation was not surprising as the dependence of sexual maturation on a toad achieving a certain minimum body size has been recognized previously [28], and growth rates of individuals can differ. Toads in laboratory conditions with a consistent diet were less likely to experience extremes in body condition than were wild counterparts, but subjecting females to hibernation slowed their growth and could have been responsible for delaying sexual maturation. Although toads in our study did not appear to lose weight during hibernation, they stopped gaining weight. In contrast, nonhibernated toads continued to increase their body condition and weight throughout the year. Hibernation did not seem to affect age of sexual maturation in males, as both hibernated and nonhibernated males failed to produce sperm at 1 yr of age but succeeded in producing sperm at 2 yr. The sexual size dimorphism observed in the boreal toad (Table 1) was not unexpected, as females are larger than males in 90% of anuran species [29]. The reason for this difference between the sexes has been the topic of several large-scale studies and appears related to both the difference in energy investment required by each sex for successful reproduction and that males reach sexual maturation at a younger age when they are smaller [30]. Clearly, the minimum body mass required for male toads to reach sexual maturation was lower than that for females. Regardless of the confounding factor of female body condition and hibernation in this study, data supported the conclusion that male boreal toads reach sexual maturation at 2 yr and females at 4 yr. These results were consistent with those reported for the wild common toad, in which males achieve maturation at 2 to 6 yr of age and females usually a year after males, with the variation due largely to body size [28,31,32]. However, it is possible that by maintaining female boreal toads under laboratory conditions and feeding ad libitum throughout the year, more would be sexually mature in 3 yr. The most likely explanation for the low fertilization success with all egg masses produced was male-female
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asynchrony in gamete release. Both male and female toads received their first hormone treatment simultaneously. The timing of sperm release by male toads in response to exogenous hormone treatments was nicely demonstrated first by Obringer et al. [19] in American toads and later by Browne et al. [13] in Wyoming toads. Results from both studies were similar, suggesting that although type of hormone and dosage can slightly modify the spermiation response, in general, spermiation occurred as quickly as 3 h posttreatment, and sperm production peaked 7 to 12 h posttreatment. Although sperm can still be recovered 24 h after treatment, both concentration and quality were reduced. One study reported peak sperm concentrations continuing 24 h after applying a very high dosage of LHRHa topically [20], but intraperitoneal injections were used in this study, which probably resulted in a spermiation pattern similar to that described in detail by Obringer et al. [19] and Browne et al. [13]. Because oviposition occurred 24 to 34 h after hormone treatment, sperm production and quality would have been suboptimal at that time, perhaps substantially reducing fertilization success. An attempt to synchronize gamete release and increase fertilization success by treating male boreal toads 12 h after females are treated is warranted, especially as premature egg deposition, a problem reportedly encountered with Puerto Rican crested toads after hormone injections [25], was never experienced in this species. Low fertility could not be attributed to the omission of hibernation, as the proportion of eggs fertilized after oviposition by hibernated females was no greater than that for nonhibernated females (Table 2). In fact, the highest fertilization recorded during this study (36.5%) was with an egg mass produced by a nonhibernated female. However, other factors associated with captive conditions could be responsible for overall poorer quality eggs and lower hatching rates. Others have reported a 45% decrease in hatching rates of boreal toad eggs produced by captive animals versus eggs produced by wild animals that are brought into captivity for hatching and development [27]. Interestingly, Toro and Michael [33] reported that approximately one third of coqui (Eleutherodactylus coqui) eggs produced in response to exogenous hormone treatment were artificially activated, which significantly reduced fertilization success. However, natural reproduction in the coqui involves internal fertilization, so the mechanical stress of oviposition is not typically experienced by eggs until after they are fertilized [34]. In contrast, eggs from external fertilizing species like the boreal toad naturally experience
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oviposition prior to fertilization and thus are unlikely to be sensitive to mechanical activation during the process. However, we did not assess egg autoactivation in this study and therefore cannot completely exclude it as a confounding factor in fertilization. The use of exogenous hormone treatments for inducing gamete release in anurans dates back to the 1930 s [6], but efforts to delineate and optimize speciesspecific responses have barely begun. Xenopus laevis, the most common laboratory amphibian, is somewhat unusual in responding reliably to hCG treatment, as many other anurans do not [7]. Interestingly, Browne et al. [13] reported that the female Wyoming toad did not respond to hCG unless first administered a priming hormone cocktail of LHRHa and hCG. Furthermore, Kouba et al. [14] suggested a cocktail and/or priming treatment may elicit similar results in B. fowleri and Rana sevosa. Regardless, LHRHa alone continues as the most common hormone for inducing reproductive activity in anurans, but there are also species specificities regarding the LHRH type employed. One detailed study by Michael et al. [11] tested the results of mammalian, avian, and fish LHRH, as well as a modified D-Ala6, desGly10, ethylamide LHRHa, and hCG for inducing egg deposition in the coqui. They found the species responds best to the synthetic D-Ala6, desGly10, ethylamide LHRHa, and that hCG was not only less effective but also led to serious side effects, including mortality. This study was not designed to specifically compare the effects of LHRHa and hCG, but both were incorporated in several of the trials. Although the early trials were confounded due to the small body size and immature status of the female toads, in the last few years of the experiment, the toads appeared to respond best to a single dose of LHRHa. If females did not respond to their first LHRHa treatment, they also failed to produce eggs in response to a second treatment of either LHRHa or hCG administered as soon as 48 h or as late as 16 d after the first treatment. These results were consistent with those reported for American toads by Johnson et al. [12], who found no benefit in multiple hormone injections. However, permeations of treatment timing and hormone dosages may lead to different outcomes. Unfortunately, such a multifaceted project was beyond the scope of this study. Regardless, based on our findings herein, any studies comparing hormone protocols in anurans will require close monitoring of the females to ensure that outcomes are not confounded by body weight and condition. Egg retention has long been identified as a serious concern in a variety of reptiles [35,36], however similar
cases are not well documented in amphibians, despite growing anecdotal evidence [14] associated with enhanced efforts to breed these species in captivity. During this project, two asymptomatic female toads were found dead, and necrotic egg masses were found in their coelomic cavities upon necropsy. The females had been treated with LHRHa 5 and 18 d prior to death. One female was obese (84.5 g), and her obesity may have contributed to the condition. However, the other female was 57.2 g, an ideal body mass for reproduction. One additional female died 40 d after producing 846 eggs, and a small mass of residual eggs was found in her during necropsy, consistent with partial egg retention. Interestingly, all cases of egg retention occurred in 2005 when females were 4 yr old. There were no subsequent cases the following year, suggesting young females that have recently become mature and are breeding for the first time may be more prone to the problem than older, experienced females. In conclusion, artificial hibernation alone was not sufficient to mimic the natural reproductive stimulatory cues required for successful reproduction in boreal toads. Both males and females required exogenous hormone treatment before appropriate breeding behaviors and gamete deposition were exhibited. Surprisingly, females that were not subjected to hibernation were more successful in producing eggs and tadpoles than females that had undergone hibernation. In female boreal toads, there were no indications that hibernation led to reproductive failure, but body mass was the most important factor associated with reproductive success. Therefore, it may be more productive to feed females throughout the winter instead of placing them in hibernation. In contrast, reproductive behavior was more readily displayed by males that had been hibernated and treated with LHRHa. As sperm production was not closely linked to body mass, it seems prudent to continue hibernating the males prior to the breeding season. Acknowledgments This study was funded, in part, by grants received from the Association of Zoos and Aquarium’s Conservation Endowment Fund and the Colorado Division of Wildlife. We are also very grateful for the toadlets provided for the project by the Colorado Division of Wildlife. The authors especially thank the Cincinnati Zoo & Botanical Garden reptile keepers and volunteers of the Center for Conservation and Research of Endangered Wildlife (CREW) who helped with the daily care of the toads, especially on weekends.
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References [1] Heyer R. Declining amphibian populations task force. Species 1997;29:66. [2] Pavajeau L, Zippel KC, Givson R, Johnson K. Amphibian Ark and the 2008 Year of the Frog Campaign. Int Zoo Yrbk 2008;42:24–9. [3] Johnson B. Recovery of the Puerto Rican crested toad. In: U.S. Fish & Wildlife Service Endangered Species Bulletin, Bender M, Balis-Larsenj M (Eds.), USFWS, 1999; 24:8-9. [4] Spencer B. In: The Wyoming toad SSP. U.S. Fish & Wildlife Service Endangered Species Bulletin, Bender M, Balis-Larsenj M (Eds.), USFWS, 1999; 24:18-19. [5] Jackson T. Report on the status and conservation of the boreal toad (Bufo boreas boreas) in the southern Rocky Mountains 2006-2007, Colorado Division of Wildlife, 2008. [6] Shapiro HA, Zwarenstein H. A rapid test for pregnancy on Xenopus laevis. Nature 1934;133:762. [7] Creaser CW, Gorbman A. Species specificity of the gonadotropic factors in vertebrates. Quart Rev Biol 1939;14:311–31. [8] Galli-Mainini C. Pregnancy test using the male toad. J Clin Endocrinol Metab 1947;7:653–8. [9] Licht P, Papkoff H. Species specificity in the response of an in vitro amphibian (Xenopus laevis) ovulation assay to mammalian luteinizing hormones. Gen Comp Endocrinol 1976;70:83–90. [10] McCreery BR, Licht P. Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin releasing hormone during the ovarian cycle in the bullfrog Rana catesbeiana. Biol Reprod 1983;29:863–71. [11] Michael SF, Buckley C, Toro E, Estrada AR, Vincent S. Induced ovulation and egg deposition in the direct developing anuran Eleutherodactylus coqui. Reprod Biol Endocrinol 2004;2:6. doi: 10.1186/1477-7827-2-6. [12] Johnson CJ, Vance CK, Roth TL, Kouba AJ. Oviposition and ultrasound monitoring of American toads (Bufo americanus) treated with exogenous hormones. Proc Am Assoc Zoo Vet 2002;299–301. [13] Browne RK, Seratt J, Vance C, Kouba A. Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri). Reprod Biol Endocrinol 2006;4:34. doi: 10.1186/1477-7827-4-34. [14] Kouba AJ, Vance CK, Willis EL. Artificial fertilization for amphibian conservation: current knowledge and future considerations. Theriogenology 2009;71:214–27. [15] Jørgenson CB. Integrative functions of the brain. In: Lofts B, editor. Physiology of the Amphibia, II. Academic Press; 1974. p. 1–51. [16] Zug GR. Herpetology: An Introductory Biology of Amphibians and Reptiles. Academic Press; 1993. p. 197. [17] Harvey LA, Propper CR, Woodley SK, Moore MC. Reprdouctive endocrinology of the explosively breeding desert spadefoot toad, Scaphioppus couchii. Gen Comp Endocrinol 1997;105:102–13. [18] Colorado Division of Wildlife. Boreal Toad Recovery Plan, Colorado Division of Wildlife, 1994. [19] Obringer AR, O’Brien JK, Saunders RL, Yamamoto K, Kikuyama S, Roth TL. Characterization of the spermiation
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Theriogenology 73 (2010) 501–511 www.theriojournal.com
Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas) T.L. Roth *, D.C. Szymanski, E.D. Keyster Center for Conservation and Research of Endangered Wildlife (CREW), Cincinnati Zoo & Botanical Garden, Cincinnati, Ohio, USA Received 14 July 2009; received in revised form 28 August 2009; accepted 30 September 2009
Abstract The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (SD) weight of female toads producing eggs (58.9 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 7.0 g). Similarly, four of seven nonhibernated females (58.8 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment. # 2010 Elsevier Inc. All rights reserved. Keywords: Amphibian; Amplexus; Egg retention; Oviposition; Spermiation
1. Introduction Since 1997 when the Declining Amphibian Task Force of the International Union for the Conservation of Nature (IUCN) unanimously confirmed a significant worldwide decline in amphibians [1], zoos in conjunction with federal and state wildlife departments have
* Corresponding author. Tel.: +1 513 569 8220; fax: +1 513 569 8213. E-mail address: [email protected] (T.L. Roth). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.09.033
increased their commitment to conserving amphibians by establishing new breeding facilities and conducting research with these previously neglected species [2]. To date, there have been several successful programs in which captive breeding has provided adequate offspring for reintroductions. For example, the Puerto Rican crested toad (Peltophryne lemur), Wyoming toad (Bufo baxteri), and boreal toad (Bufo boreas boreas) breeding programs are all over a decade old and have provided continual infusion of captive-bred animals into the wild to boost population numbers and to establish new populations in previously inhabited locations [3–5].
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However, even these successful programs struggle with the inconsistency of annual breeding attempts and could benefit from the development of improved reproductive technologies. Amphibians propagate by a wide variety of methods, and cues triggering natural reproduction are typically related to seasonal provision of suitable habitat for larvae. However, knowledge of these cues is incomplete, preventing accurate duplication in captive systems. Therefore, the typical approach is to mimic natural reproductive cues in temperate species by subjecting animals to artificial hibernation, then rely primarily on exogenous hormones for inducing reproductive behavior, spermiation, and oviposition. The use of gonadotropins and gonadotropin releasing hormones for stimulating gamete production in amphibians dates back to an early pregnancy test developed in 1934 [6] and several subsequent studies that further characterized anuran responsiveness to gonadotrophs of various species [7–11]. In general, although mammalian or synthetic luteinizing hormone releasing hormone analogue (LHRHa) was most effective across a variety of anuran species for inducing the desired response, human chorionic gonadotropin (hCG) was highly effective in specific species. A few recent studies have involved testing a combination of hormones in succession or as cocktails, and some results have been encouraging, but overall, responses are mixed and appear species-specific [12–14]. Hibernation has traditionally been considered necessary in preparing temperate toads for successful reproduction, however its role in physiologic reproductive processes has not been well characterized. It has been reported that anurans prepare for reproduction fairly quickly after breeding and prior to hibernation [12,15–17]. Furthermore, animal care staff and conservationists working with highly endangered species are concerned hibernation stress could increase mortality. Therefore, ascertaining the importance of hibernation on breeding success in temperate anurans would be beneficial. The goal of this study was to further delineate the effects of age, hibernation, and exogenous hormone treatments on female and male temperate toads, using the boreal toad as a research model. This species was chosen because it is already a conservation concern with an active captive breeding, reintroduction, and recovery program that was established in 1994 [18]. Therefore, captive-bred toadlets were available for this study, and the results could have direct, immediate application to a species in need of conservation
breeding, regardless of any species-specific findings. However, the results could also be applicable to closely related endangered anurans, for example, the Wyoming toad (B. baxteri). 2. Materials and methods 2.1. Animals Seventy-seven captive-bred boreal toads (32 females and 45 males) were provided by the Colorado Division of Fish and Wildlife for this project. The toads were produced in spring 2001 and were sent to the Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo & Botanical Garden, in October of that year. Toads arrived in good condition and were allowed 1 mo to acclimate to their new surroundings before being moved into hibernacula for Experiment 1. During the acclimation period, photographs were taken of each toad’s back, and these were used throughout the study for identifying individuals, based on their unique wart patterns. When not in hibernacula, animals were maintained in small, samesex groups in a laboratory at 22 to 25 8C. Toads received 12 h of light daily, water ad libitum, and 4-wk-old crickets dusted with vitamin supplement 4 to 6 times per week. 2.2. Hibernation Hibernacula consisted of tanks with a base covered in charcoal, sand, and peat moss soaked with aged tap water. Holes in the bottom of the tanks allowed excess water to drain to minimize mold and bacterial growth. A piece of slotted pipe was inserted into the substrate to act as burrows for the toads. The tanks were soaked once or twice monthly with fresh water. The temperature and humidity of the hibernacula were monitored with the aid of battery-operated data loggers (readings were consistently 4 8C and 65% humidity). Toads were chosen at random, individually weighed, and then placed in the hibernation tanks (two or three toads/tank) and transferred to an environmental chamber set at 10 8C for 24 h before being moved into a walk-in cold room at 4 8C. In the walk-in cold room, all light bulbs were changed to red and a dark cloth was hung from the top shelf to further minimize light exposure. Toads were checked weekly and sprayed with simplified amphibian Ringers solution (113 mM NaCl, 1 mM CaCl2, 2 mM KCl, 3.6 mM NaHCO3, 220 mOsm/kg, pH 7.4; Years 1 and 2) or water (Years 3 to 5) to keep them hydrated.
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Table 1 Mean (SEM) boreal toad body weights prior to treatment (2002 through 2006). Year 2002 2003 2004 2005 2006 a–e
Toad age (yr) 1 2 3 4 5
Hibernated males (g) a
37.1 1.2 41.4 1.3 c 45.8 0.8 d 38.8 1.0a,c 47.7 2.2 d
Hibernated females (g) b
32.6 1.1 37.2 0.8 a 40.7 1.7a,c 42.1 2.0c,d NA
Nonhibernated females (g) NA NA 41.4 1.9 c 57.3 3.5 e 59.3 2.4 e
Values without a common superscript differed (P < 0.05). NA = not applicable based on study design.
2.3. Exogenous hormones Two exogenous hormones were used in this study: LHRHa and hCG. The LHRHa (pGlu-His-Trp-Ser-Tyrp-Ala-Leu-Arg-Pro-NHEt; Sigma L-4513; Sigma Chemical Co., St. Louis, MO, USA) was dissolved with ddH20 to a concentration of 0.2 mg/mL. The hCG (Lot No. 020605; Sioux Biochemical Inc., Sioux Center, IA, USA) was diluted with ddH20 to a concentration of 2.5 IU/mL. Each year of the study, both hormones were diluted the day of use and any remaining aliquots were stored frozen at 80 8C. These aliquots were thawed and used for repeat treatments and/or sperm production tests conducted within that year. Hormones were administered intraperitoneally as previously described [19] using a 30-gauge needle. Toads treated with LHRHa received 6 mg in a total volume of 30 mL. Toads treated with hCG received 250 or 300 IU administered in a total volume of 100 to 120 mL. When eggs were produced, they were carefully removed from the breeding tanks within 24 h after oviposition, counted, and transferred into aquariums that had been acclimated for at least 1 wk prior to use. Tadpoles were counted once fully formed and swimming freely around the tank. 2.4. Experiment 1 Experiment 1 tested the hypothesis that both hibernation and hormone administrations are necessary to elicit breeding behavior in male boreal toads. Furthermore, the potential benefit of a second hormone administration was examined in males and females as well as the type of hormone (LHRHa or hCG) in females for inducing oviposition and tadpole production. For the first year of the study (2002), all female toads and half of the male toads were hibernated, for a total of 54 animals placed in hibernation. 2.4.1. Trial 1 Toads entered hibernation on November 29, 2001, and were removed April 26, 2002. After removal from
hibernation, toads were weighed (Table 1) and allowed a week of feeding before breeding was attempted. Toads were paired and assigned a treatment group (four or five pairs per treatment) at random. Treatment groups were as follows: (1) hibernated males and females receiving hormones; (2) hibernated males and females with only males receiving hormones; (3) hibernated males and females receiving no hormones; (4) nonhibernated males and hibernated females receiving hormones; (5) nonhibernated males and hibernated females with only males receiving hormones; and (6) nonhibernated males and hibernated females receiving no hormones. All hormone-treated females received 300 IU hCG for their first treatment, whereas males received 6 mg LHRHa. Two weeks later, a second treatment was applied; both male and female toads received LHRHa. 2.4.2. Trial 2 Toads entered hibernation on November 22, 2002, and were removed and weighed (Table 1) on April 27, 2003. Two days after removal from hibernation, toads were paired and assigned a treatment group (five or six pairs per treatment) at random. The six treatment groups were the same as those in Trial 1, however LHRHa was the hormone administered in all initial treatments requiring males and/or females to receive hormone. If no eggs were produced by 48 h after the start of the treatments, female toads in Treatments 1, 2, 4, and 5 received hCG (250 IU) and the males received a second dose of LHRHa. Female and male toads in Treatments 3 and 6 received LHRHa. After a 2-wk interval, toads that had not produced eggs received a second series of hormone treatments identical to the first. 2.4.3. Sperm production test Because the toads were young and the breeding age of boreal toads had not been documented, sperm production in response to LHRHa was used to assess the status of male sexual maturation after each trial in Experiment 1. Toads were rested 1 mo after breeding in Trial 1, and then seven hibernated and seven nonhibernated toads were chosen for the sperm
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production test. All male toads were weighed, and the four heaviest and three lightest hibernated males were treated with 6 mg LHRHa. Similarly, the three heaviest and four lightest nonhibernated males were treated with 6 mg LHRHa. At 8, 15, 22, and 28 h after treatment, each toad was picked up and held over a Petri dish until it urinated. The urine was examined under a microscope for the presence of sperm. To test for sperm production the following year, toads were rested 2 mo after the series of hormone treatments for Trial 2, and then all male toads (hibernated n = 18 and nonhibernated n = 18) received 6 mg LHRHa. Urine was collected approximately 12 h after treatment and examined for the presence of sperm cells. 2.5. Experiment 2 Based on 2003 results, it was determined that the most effective treatment for inducing mating behavior in male toads was to hibernate them and treat them with LHRHa. Therefore, all males in the 2004 study underwent that combined treatment, and the focus of the study design was on female toads. The objectives were to determine if female boreal toads needed to be hibernated to produce fertile eggs and to determine if hibernation increased the reproductive productivity of the females. Furthermore, the potential benefit of a second hormone treatment was further examined as well as the type of hormone (LHRHa or hCG) for inducing oviposition and tadpole production. The experimental design for Experiment 2 was complex (see flowchart in Fig. 1). Hibernation began November 21, 2003, and ended April 24, 2004. Two days after the hibernated subset of toads was removed from hibernation, toads were weighed (Table 1),
assigned a treatment group and paired (4 or 10 pairs per treatment) at random. Treatments applied to female toads in each group were as follows: (1) hibernated, no hormones (n = 4); (2) hibernated, LHRHa (6 mg) treatment (n = 10); (3) not hibernated, no hormones (n = 4); and (4) not hibernated, LHRHa (6 mg) treatment (n = 10). Forty-eight hour later, toads were treated a second time. Treatment for Groups 1 and 3 remained the same, with only the male receiving a second dose of LHRHa (6 mg). Females in Groups 2 and 4, that did not produce eggs were treated with hormones again, half with hCG (250 IU) and the other half with a second dose of LHRHa (6 mg). Males in Groups 2 and 4 also received a second dose of LHRHa (6 mg). Toads were checked every 12 h for up to 8 d after the last treatment of each trial, and breeding behavior (amplexus), egg production, and tadpole development were recorded. 2.6. Experiment 3 2.6.1. Trial 1 In response to both the poor success experienced in inducing oviposition in female toads during the previous experiments and the surprising production of eggs and tadpoles by a nonhibernated female, a simpler study design was followed for Experiment 3 that focused on the effect of hibernation on egg production. In 2004, half of the females (n = 10) were randomly chosen for hibernation, and the other half were maintained throughout the winter under laboratory conditions. All males were hibernated. Hibernation of all toads began on December 4, 2004. Female toads were brought out of hibernation on May 29, 2005, and males were brought out of hibernation on May 31, 2005.
Fig. 1. Flowchart depicting the experimental design for Experiment 2.
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All female toads were weighed prior to the start of hibernation and after hibernation on the day before hormones were administered. All hibernated and nonhibernated female toads and the hibernated male toads received LHRHa (6 mg) on June 1, 2005. Males and females were housed in pairs and observed for amplexus and egg production every 12 h for 6 d. 2.6.2. Trial 2 Trial 2 was designed to further test the hypothesis that hibernation is not a prerequisite to egg production by female boreal toads. Seven female toads were maintained under laboratory conditions throughout the winter. Male toads were hibernated from December 4, 2005, until May 7, 2006, and all toads were weighed just prior to treatment (Table 1). Twenty-four hours after their removal from hibernation, male toads were randomly paired with the seven nonhibernated female toads, and all toads were treated with LHRHa (6 mg). Pairs were housed separately and observed every 12 h for amplexus and egg production over a 4-d period. Sixteen days later, all females that had not produced eggs were treated with hCG (300 IU) and paired with males receiving LHRHa (6 mg). Pairs were observed every 12 h for amplexus and egg production over the following 4 d.
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treatments were analyzed using chi-square or Fisher’s exact test when appropriate. For Experiment 1, data were pooled across hormone-treated groups within first and second treatments to compare the percentage of male toads exhibiting amplexus after one or two treatments with LHRHa. In addition, data from Experiment 1, Trial 2, were further assessed by pooling data across female toad treatment groups and comparing male amplexus behavior in response to different treatments. Differences in mean values (i.e., weights and time in amplexus) were determined using unpaired Student’s t-tests. Mean body weights for each group across sexes, years, and hibernation treatment were compared in Table 1 using ANOVA. For all statistical analyses, P < 0.05 was considered significant. 3. Results Approximately 5 mo after toads were first moved into a hibernation environment, they were removed. Over the course of the 5-yr study, there were approximately 200 toad hibernations completed with only two mortalities (both female). In addition, a third female succumbed to infection upon removal from hibernation. All other toads appeared healthy at the end of the 5-mo period.
2.7. Statistical analysis
3.1. Experiment 1
Data were analyzed using the Macintosh software program Statview (1998, SAS Institute Inc., Cary, NC). Differences in proportion of toads responding to
3.1.1. Trial 1 Only 19% of toad pairs were observed in amplexus at any time during Trial 1 of Experiment 1 (Fig. 2), and
Fig. 2. Percentage of toad pairs in amplexus after their first and second treatments with exogenous hormones during Experiment 1, Trials 1 (2002) and 2 (2003), and Experiment 2 (2004). a–cColumns without a common superscript differ (P < 0.05).
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those observations were spread among treatment groups (Treatment 1, n = 3; Treatment 2, n = 4; Treatment 3, n = 1; Treatment 4, n = 1; Treatment 5, n = 1; Treatment 6, n = 1). No females in any treatment group produced eggs in response to hCG or LHRHa. 3.1.2. Trial 2 Although the percentage of toad pairs exhibiting amplexus in response to the first hormone treatment in Trial 2 (17.2%) was no better than that recorded in Trial 1, amplexus behavior by male toads increased in response to the second LHRHa treatment administered 48 h after the first (56.9%; P < 0.05; Fig. 2). As in Trial 1, amplectant pairs in response to the first and/or second hormone treatment were distributed among treatment groups (Treatment 1, n = 7; Treatment 2, n = 12; Treatment 3, n = 8; Treatment 4, n = 7; Treatment 5, n = 3; and Treatment 6, n = 6). However, by pooling the data across female toad treatments and comparing just the male treatments, differences in treatment effects were revealed (Fig. 3). Treatment with LHRHa appeared essential in inducing amplexus in male toads. In fact, no male toad exhibited amplexus without receiving LHRHa (Fig. 3B). Although hibernation alone did not appear to influence amplexus in male toads (P > 0.05; Fig. 3A), it enhanced the likelihood of amplexus in toads treated with LHRHa compared with those that were not hibernated and treated with LHRHa (P < 0.05; Fig. 3C). One female toad in Treatment 3 produced an egg mass (650 eggs) in response to the LHRHa she received 48 h after the initial treatment. Because the initial Treatment 3 consisted of hibernated males and females receiving no hormones, the 48-h follow-up administration of LHRHa was the first hormone received by this female that season. She was observed in amplexus with a male after the LHRHa treatment and during oviposition, which occurred approximately 48 h after LHRHa administration. One tadpole developed from the egg mass and survived to become an adult. 3.1.3. Sperm production test After Experiment 1, Trial 1, none of the seven hibernated or seven nonhibernated 1-yr-old male toads produced sperm in response to LHRHa despite repeated sampling during a 24-h period. In contrast, after Experiment 1, Trial 2, sperm production was confirmed in 26 of the 36 male 2-yr-old toads treated with LHRHa. Results from five males (three hibernated and two nonhibernated) were inconclusive because urine samples were not obtained, and no sperm were identified in urine from five males (one hibernated and four
Fig. 3. Percentage of male toads in amplexus when grouped across treatments by (A) those that were hibernated versus not hibernated, (B) those receiving LHRHa versus those not receiving hormones, and (C) those receiving LHRHa after hibernation (LHRH + H) versus those treated with LHRHa without undergoing hibernation (LHRH – H) in Experiment 1, Trial 2. a,bWithin each graph, columns without a common superscript differ (P < 0.05).
nonhibernated). Sperm production test results were similar (P > 0.05) in hibernated and nonhibernated toads with 14 of 15 samples collected from hibernated toads and 12 of 16 samples collected from nonhibernated toads containing sperm. 3.2. Experiment 2 Similar to results reported in Experiment 1, amplexus behavior in male toads increased (P < 0.05) in response to a second LHRHa treatment administered 48 h after the first. In this experiment, percentage of toad pairs in amplexus increased from 67.9% after the first treatment to 100% after the second treatment (Fig. 2).
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Fig. 4. (A) Percentage of hibernated versus nonhibernated female toads producing eggs and (B) time spent in amplexus after LHRHa treatment, (C) the body weights of hibernated versus nonhibernated toads and (D) the body weights of females that produced eggs versus those that did not in Experiment 3, Trial 1. a,bWithin each chart, columns without a common superscript differ (P < 0.05).
Only 2 of 28 female toads produced eggs in this experiment. Both females had been treated with a single LHRHa dose prior to oviposition. Interestingly, one of the females was not hibernated. Both hibernated and nonhibernated females were in amplexus during oviposition, which occurred 24 and 48 h after hormone treatment, respectively. Although the hibernated female produced more eggs than the nonhibernated female (2711 vs. 1567, respectively), none of the eggs produced by the hibernated female developed into tadpoles, whereas 36.5% of the eggs produced by the nonhibernated female developed to tadpoles. Many of these developed fully into toadlets. 3.3. Experiment 3 3.3.1. Trial 1 A total of 14 pairs of toads (70%) were observed in amplexus after the single LHRHa treatment. Of those males observed in amplexus, eight were with nonhibernated females, and six were with hibernated
females. A total of six females produced eggs 15 to 60 h after LHRHa treatment, and five of these were in amplexus with males during oviposition. Additionally, three females died within 6 wk of the study, and necrotic eggs were found in their coelomic cavities upon necropsy. Of the eight females that produced eggs during this experiment (retained or delivered), one was hibernated and seven were not (Fig. 4A). Five of the six females that exhibited oviposition were in amplexus at the time, and of these, four produced tadpoles, but fertility was low (2.6% to 16.1% of the eggs developed into tadpoles). In response to the surprising finding that more nonhibernated than hibernated female toads produced eggs (P < 0.05; Fig. 4A), an effort was made to identify contributing factors. Because the male toads could be inducing some response in females through amplexus behavior, the length of time toad pairs remained in amplexus was compared between hibernated and nonhibernated females, but no difference was found (P > 0.05; Fig. 4B). Additionally, because body
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condition is often linked to reproductive success, the body weight of hibernated and nonhibernated toads was compared. There was difference in body weight between groups: nonhibernated female toads weighed more than hibernated females (P < 0.05; Fig. 4C). Furthermore, female toads producing eggs were heavier on average than those not producing eggs (P < 0.05; Fig. 4D). 3.3.2. Trial 2 In Trial 2, all seven toad pairs (100%) were in amplexus within 12 h after receiving LHRHa. Of these seven nonhibernated females, four produced eggs while in amplexus 21 to 36 h after treatment, but none of the eggs developed into tadpoles. Similar to results from all previous experiments in this study, none of the females that failed to produce eggs in response to their first hormone treatment responded to the second hormone treatment. In contrast with Trial 1, none of the females in this trial died with retained eggs. 4. Discussion The most surprising study finding was the reproductive success experienced by nonhibernated female toads (Table 2). In a previous pilot study, we reported that American toads (B. americanus) were capable of oviposition in response to hormone treatments without first undergoing hibernation, but those toads were not with males, so the fertility of the eggs was unknown [12]. Herein, we have proved that nonhibernated female boreal toads can produce fertile eggs that develop into normal toadlets. These results contradicted the previously held belief that toads that hibernate in the wild must be hibernated in captivity to reproduce successfully. In fact, one female toad produced eggs two consecutive years without undergoing hibernation during either winter. It is well established that male temperate toads produce sperm year-round in response to exogenous Table 2 Mean (SD) outcomes from hibernated and nonhibernated female toads producing eggs after a single LHRHa administration (combined for all experiments and years, excluding three toads that died with retained eggs).
Body weight of females (g) Time in amplexus (h) Number producing eggs Interval postinjection (h) Number of eggs produced
Hibernated
Nonhibernated
44.7 7.2 40.0 13.9 n=3 24.0 10.8 1527 1064
56.5 7.2 33.9 45.3 n = 10 33.9 11.3 1530 1044
hormone treatments without undergoing hibernation [19–21], despite reports that cold exposure is an important modulator of male toad breeding activities [22]. However, hibernation has been considered a requirement for female temperate toads, in part, because temperature is considered an important environmental cue for triggering breeding [16,23,24]. It has even been suggested that keeping amphibians at lower temperatures for 4 to 6 wk can partially correct for abnormal vitellogenesis that otherwise occurs in captive animals [25]. However, given that oogenesis is well under way soon after breeding [17], a large portion of the ovarian mass is restored within 1 mo after oviposition [12], and vitellogenic oocyte production is complete prior to hibernation [15,16], the role of hibernation in preparing the female for reproduction may be insignificant. Herein, we demonstrated that appropriate reproductive processes occurred in female toads maintained in laboratory conditions, and eggs produced by these nonhibernated toads were developmentally competent. In fact, fewer hibernated females produced eggs and fewer of their eggs fertilized when compared with nonhibernated females. Therefore, although some ovarian and endocrine processes may naturally occur during hibernation or in response to temperature fluctuations in wild temperate toads, this relationship may be more of an association than a dependency in some species. Alternatively, perhaps environmental stimulatory cues involving hibernation are required in the wild, whereas dependency on those cues is eliminated under captive conditions or artificially induced with exogenous hormones. That sufficient body weight was critically important for female reproductive success was not surprising as positive relationships between body condition and reproduction can be demonstrated in most species including amphibians. One study examining the effects of population density, rainfall, temperature, and body condition on fecundity in common toads (B. bufo) found that body condition (particularly weight) was the most significant factor [26]. However, breeding protocols for endangered temperate toads detail conditions required for hibernation but offer no information regarding target or minimum body weights [5,25,27]. Throughout the study, only 3 of the 15 toads that produced eggs weighed less than 50 g. Therefore, we inferred that 50 g was a good target weight for female boreal toads in a breeding program. In contrast, for males, body weight did not appear to be a significant factor influencing spermiation once males reached sexual maturation. Based on the spermiation data, it was evident that male boreal toads reached sexual maturation at 2 yr of age. A
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few males might have matured at 1 yr, because not all males were tested for sperm production, and from those tested, urine samples were not consistently collected at the desired intervals. However, all samples collected from 1-yr-old toads were devoid of sperm, suggesting the majority of animals were not yet sexually mature. In contrast, most female boreal toads required 4 yr to reach sexual maturity, based on successful oviposition. However, there appeared to be a greater range during which females reached sexual maturation, as one female produced eggs at 2 yr and two others produced at 3 yr. Variation in exact age of sexual maturation was not surprising as the dependence of sexual maturation on a toad achieving a certain minimum body size has been recognized previously [28], and growth rates of individuals can differ. Toads in laboratory conditions with a consistent diet were less likely to experience extremes in body condition than were wild counterparts, but subjecting females to hibernation slowed their growth and could have been responsible for delaying sexual maturation. Although toads in our study did not appear to lose weight during hibernation, they stopped gaining weight. In contrast, nonhibernated toads continued to increase their body condition and weight throughout the year. Hibernation did not seem to affect age of sexual maturation in males, as both hibernated and nonhibernated males failed to produce sperm at 1 yr of age but succeeded in producing sperm at 2 yr. The sexual size dimorphism observed in the boreal toad (Table 1) was not unexpected, as females are larger than males in 90% of anuran species [29]. The reason for this difference between the sexes has been the topic of several large-scale studies and appears related to both the difference in energy investment required by each sex for successful reproduction and that males reach sexual maturation at a younger age when they are smaller [30]. Clearly, the minimum body mass required for male toads to reach sexual maturation was lower than that for females. Regardless of the confounding factor of female body condition and hibernation in this study, data supported the conclusion that male boreal toads reach sexual maturation at 2 yr and females at 4 yr. These results were consistent with those reported for the wild common toad, in which males achieve maturation at 2 to 6 yr of age and females usually a year after males, with the variation due largely to body size [28,31,32]. However, it is possible that by maintaining female boreal toads under laboratory conditions and feeding ad libitum throughout the year, more would be sexually mature in 3 yr. The most likely explanation for the low fertilization success with all egg masses produced was male-female
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asynchrony in gamete release. Both male and female toads received their first hormone treatment simultaneously. The timing of sperm release by male toads in response to exogenous hormone treatments was nicely demonstrated first by Obringer et al. [19] in American toads and later by Browne et al. [13] in Wyoming toads. Results from both studies were similar, suggesting that although type of hormone and dosage can slightly modify the spermiation response, in general, spermiation occurred as quickly as 3 h posttreatment, and sperm production peaked 7 to 12 h posttreatment. Although sperm can still be recovered 24 h after treatment, both concentration and quality were reduced. One study reported peak sperm concentrations continuing 24 h after applying a very high dosage of LHRHa topically [20], but intraperitoneal injections were used in this study, which probably resulted in a spermiation pattern similar to that described in detail by Obringer et al. [19] and Browne et al. [13]. Because oviposition occurred 24 to 34 h after hormone treatment, sperm production and quality would have been suboptimal at that time, perhaps substantially reducing fertilization success. An attempt to synchronize gamete release and increase fertilization success by treating male boreal toads 12 h after females are treated is warranted, especially as premature egg deposition, a problem reportedly encountered with Puerto Rican crested toads after hormone injections [25], was never experienced in this species. Low fertility could not be attributed to the omission of hibernation, as the proportion of eggs fertilized after oviposition by hibernated females was no greater than that for nonhibernated females (Table 2). In fact, the highest fertilization recorded during this study (36.5%) was with an egg mass produced by a nonhibernated female. However, other factors associated with captive conditions could be responsible for overall poorer quality eggs and lower hatching rates. Others have reported a 45% decrease in hatching rates of boreal toad eggs produced by captive animals versus eggs produced by wild animals that are brought into captivity for hatching and development [27]. Interestingly, Toro and Michael [33] reported that approximately one third of coqui (Eleutherodactylus coqui) eggs produced in response to exogenous hormone treatment were artificially activated, which significantly reduced fertilization success. However, natural reproduction in the coqui involves internal fertilization, so the mechanical stress of oviposition is not typically experienced by eggs until after they are fertilized [34]. In contrast, eggs from external fertilizing species like the boreal toad naturally experience
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oviposition prior to fertilization and thus are unlikely to be sensitive to mechanical activation during the process. However, we did not assess egg autoactivation in this study and therefore cannot completely exclude it as a confounding factor in fertilization. The use of exogenous hormone treatments for inducing gamete release in anurans dates back to the 1930 s [6], but efforts to delineate and optimize speciesspecific responses have barely begun. Xenopus laevis, the most common laboratory amphibian, is somewhat unusual in responding reliably to hCG treatment, as many other anurans do not [7]. Interestingly, Browne et al. [13] reported that the female Wyoming toad did not respond to hCG unless first administered a priming hormone cocktail of LHRHa and hCG. Furthermore, Kouba et al. [14] suggested a cocktail and/or priming treatment may elicit similar results in B. fowleri and Rana sevosa. Regardless, LHRHa alone continues as the most common hormone for inducing reproductive activity in anurans, but there are also species specificities regarding the LHRH type employed. One detailed study by Michael et al. [11] tested the results of mammalian, avian, and fish LHRH, as well as a modified D-Ala6, desGly10, ethylamide LHRHa, and hCG for inducing egg deposition in the coqui. They found the species responds best to the synthetic D-Ala6, desGly10, ethylamide LHRHa, and that hCG was not only less effective but also led to serious side effects, including mortality. This study was not designed to specifically compare the effects of LHRHa and hCG, but both were incorporated in several of the trials. Although the early trials were confounded due to the small body size and immature status of the female toads, in the last few years of the experiment, the toads appeared to respond best to a single dose of LHRHa. If females did not respond to their first LHRHa treatment, they also failed to produce eggs in response to a second treatment of either LHRHa or hCG administered as soon as 48 h or as late as 16 d after the first treatment. These results were consistent with those reported for American toads by Johnson et al. [12], who found no benefit in multiple hormone injections. However, permeations of treatment timing and hormone dosages may lead to different outcomes. Unfortunately, such a multifaceted project was beyond the scope of this study. Regardless, based on our findings herein, any studies comparing hormone protocols in anurans will require close monitoring of the females to ensure that outcomes are not confounded by body weight and condition. Egg retention has long been identified as a serious concern in a variety of reptiles [35,36], however similar
cases are not well documented in amphibians, despite growing anecdotal evidence [14] associated with enhanced efforts to breed these species in captivity. During this project, two asymptomatic female toads were found dead, and necrotic egg masses were found in their coelomic cavities upon necropsy. The females had been treated with LHRHa 5 and 18 d prior to death. One female was obese (84.5 g), and her obesity may have contributed to the condition. However, the other female was 57.2 g, an ideal body mass for reproduction. One additional female died 40 d after producing 846 eggs, and a small mass of residual eggs was found in her during necropsy, consistent with partial egg retention. Interestingly, all cases of egg retention occurred in 2005 when females were 4 yr old. There were no subsequent cases the following year, suggesting young females that have recently become mature and are breeding for the first time may be more prone to the problem than older, experienced females. In conclusion, artificial hibernation alone was not sufficient to mimic the natural reproductive stimulatory cues required for successful reproduction in boreal toads. Both males and females required exogenous hormone treatment before appropriate breeding behaviors and gamete deposition were exhibited. Surprisingly, females that were not subjected to hibernation were more successful in producing eggs and tadpoles than females that had undergone hibernation. In female boreal toads, there were no indications that hibernation led to reproductive failure, but body mass was the most important factor associated with reproductive success. Therefore, it may be more productive to feed females throughout the winter instead of placing them in hibernation. In contrast, reproductive behavior was more readily displayed by males that had been hibernated and treated with LHRHa. As sperm production was not closely linked to body mass, it seems prudent to continue hibernating the males prior to the breeding season. Acknowledgments This study was funded, in part, by grants received from the Association of Zoos and Aquarium’s Conservation Endowment Fund and the Colorado Division of Wildlife. We are also very grateful for the toadlets provided for the project by the Colorado Division of Wildlife. The authors especially thank the Cincinnati Zoo & Botanical Garden reptile keepers and volunteers of the Center for Conservation and Research of Endangered Wildlife (CREW) who helped with the daily care of the toads, especially on weekends.
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