Our research demonstrates that amphibian captive breeding programs that seek to utilize exogenous hormones for assisted reproduction need to carefully evaluate the optimal hormone concentration for their particular species. If the goal is assisted natural breeding, rather than in vitro fertilization, meticulous consideration must also be given to which hormones stimulate reproductive behaviors as well. In our study, the best hCG concentration tested, where 100% of the animals responded by producing sperm, was 300 IU. This same concentration of hCG has also been found to be extremely effective in stimulating spermiation in Anaxyrus baxteri [1, 2, 9], Anaxyrus boreas boreas , Anaxyrus fowleri , Anaxyrus houstonensis (personal communication, Paul Crump) and Peltophryne lemur (personal communication, Andrew Lentini). Although this concentration of hCG is extremely effective in stimulating male Bufonidae to produce spermic urine, species-specific differences can be found in the density of sperm produced. In our lab, we have found that seven Bufonidae species (in the 20–75 g range) produce spermic urine after hCG administration of 300 IU or higher; however, one Ranid species of similar weight (Rana pipiens) produced low quantities of sperm with often variable responses when treated with similar hCG concentrations . This further supports our concept that developing a protocol in one taxonomic species may not extrapolate exactly to another, but that fine-tuning is necessary on hormone efficacy to optimize a breeding strategy. However, the basic principles learned about one taxa may provide a good starting point for more developed studies.
By testing various concentrations of hCG from 50–300 IU we were able to show a clear dose-dependent and dramatic increase in the number of male Anaxyrus americanus producing sperm, along with an increase in concentration of sperm. In contrast, LHRH did not show a clear dose-dependent effect on the sperm parameters measured. None of the LHRH treatments tested produced as high a concentration of sperm as shown with the 100–300 IU hCG treatments. Moreover, the number of animals producing sperm in response to LHRH (~35%) was much less than the 100% achieved with 300 IU hCG. Interestingly, the individual sperm characteristics, motility, forward progression, and SMI from the optimal LHRH treatment group were not different from the 300 IU hCG group. This suggests that hormone type and concentration can be altered to induce more animals to produce greater quantities of sperm, but that these hormones did not directly affect the movement patterns or quality of mature ejaculated spermatozoa. While it is possible that increasing the concentration of hCG above 300 IU/animal might increase sperm concentration or possibly the number of animals in amplexus, we chose to stop at a point where 100% of the animals were producing sperm of good quality that would lead to high rates of natural or artificial fertilization without compromising the health of the animals by over-dosing them. Furthermore, we recognize that our behavioral responses were not optimized (75% of the pairs in amplexus with hCG compared to 91% with LHRH) and that higher doses may have increased numbers of males exhibiting amplexus. Future studies will examine the effect that combinations of the two hormones might have on reproductive behaviors at these concentrations without increasing hormone levels to possibly dangerous concentrations. This method of cautious experimentation and our stopping point within our treatments is necessary considering we are outlining a hormonal strategy for developing such protocols in critically endangered toad species where losses to the assurance colonies could be catastrophic.
Very few studies have been conducted in male amphibians directly testing these two different hormones at four or more concentrations. Michael et al.  evaluated different concentrations of hCG and LHRH in female Eluetherodactylus coqui and, in contrast to our results with male toads, found that LHRH stimulated more female coqui to ovulate than the hCG concentrations they tested. The difference between these results and ours are likely due to species or gender differences in the response to these hormones. Our lab has found that toads within the family Bufonidae tend to respond in a similar fashion to hCG across species; yet, Rana pipiens seem to respond better to LHRH [2, 12] and these results are often concentration dependent. Understanding the a priori hormone levels that stimulate optimal spermiation or ovulation is critical for long-term breeding programs where captive facilities may utilize these protocols for endangered or threatened species. Without this prior knowledge on how a species will respond to varying hormone concentrations (possibly spanning several orders of magnitude), investigators may be tempted to conclude that their species’ lack of ovulation or spermiation may limit hormone induction as a conservation tool. For example, Mann et al.  concluded that a suite of hormones tested, at a single concentration, did not work well in Litoria raniformis. However, a more robust level of hormone testing may have yielded a larger number of animals ovulating, and could be explored.
Recent studies on Anaxyrus boreas boreas suggest that higher LHRH or hCG concentrations, or even priming regimens, may be more effective than what was historically employed in the captive breeding and reintroduction program for this threatened species (Natalie Calatayud and Kevin Thompson, unpublished). Knowing that the optimal hormone concentration could be orders of magnitude higher than historically employed for inducing spermiation or ovulation in this species, raises the question of how to interpret other studies that are dependent upon hormone therapy to answer other physiological processes, such as the importance of hibernation in captive breeding . Often times, how investigators arrived at optimal hormone levels is not clear and reports indicate that concentrations were: 1) chosen from previously published reports on other species; 2) determined in a pilot study; 3) established in a limited number of animals; 4) measured but the data was not shown; or 5) inconclusive leading to the need to go back and understand hormone efficacy due to less than hoped for results [15–17]. To be used as a conservation tool for the growing number of endangered amphibian species that will need reproductive intervention to prevent extinction, it is valuable that hormone efficacy trials be continued so that future studies on other species can be modeled likewise. Studies beginning with very low concentrations of hormone and working up to higher levels is imperative to strike a balance between using enough hormone to obtain gametes for reproduction while not administering too high a dose that will cause health problems or even possibly death of the animal.
Another important finding from our study was that the hormones’ effects on sperm concentration, motility and forward progression display a time-dependent trend within a treatment (more visibly obvious with hCG than LHRH). American toad sperm concentration peaks between 7–9 hrs post hCG administration at 12 x 106 sperm/ml and declines steadily to about 4.5 x 106 and 2 x 106 sperm/ml at 12 and 24 hrs, respectively. However, sperm concentrations for our LHRH treatment were low compared to hCG and typically were below 1 x 106 sperm/ml. This low sperm concentration following administration of 4.0 μg LHRH in our study is similar to levels reported by Obringer et al.  for Anaxyrus americanus administered 4.0 μg LHRH via subcutaneous injections. However, they report an average concentration of 4.9 x 106 sperm/ml when providing the same hormone dose via intra-peritoneal injections. The range in sperm concentration for their study was 0.1 to 24 x 106 sperm/ml, although 33% of their values were below 1 x 106 sperm/ml indicating that one third of their data readings were comparable to our values when using the same hormone concentration and route of administration. The average sperm concentration value reported by Obringer et al.  are nearly five times higher than what we observed, due to a couple animals with very high sperm concentration. The discrepancy in our results from theirs with LHRH may be due to seasonal timing issues. Obringer et al.  initiated their study on newly captured animals during the breeding season with all experiments conducted shortly thereafter (April-June). In contrast, our animals had been held in captivity for more than a year prior to the start of the study, were not hibernated, and all experiments were conducted outside of the breeding season. For endangered toads held long-term in captivity as part of a breeding program, our study scenario is likely more realistic of the challenges confronted by using non-hibernated individuals without the appropriate environmental cues to stimulate natural reproduction (hence the need for hormones in the first place).
The time an amphibian takes to respond to a hormone treatment has direct bearing on how amphibian captive breeding programs time their series of hormone injections. Information from our studies highlights not only the importance of testing various hormone concentrations, but also the importance of carrying out a timed experimental sampling protocol such that the peak behavioral or physiological responses being measured are known. For example, studies in our lab with Rana pipiens discovered that sperm production following hormone administration occurred within 30 minutes and had stopped by 2 hours (unpublished data). If the same sampling period had been followed in the Rana pipiens experiments as with this study on Anaxyrus americanus, one might incorrectly assume that the hormone was ineffective, when the reality would be that the optimal time for sperm collection had been missed. Studies like these allow for protocol development on gamete synchronicity or the “timed release” of both sperm and eggs to maximize artificial fertilization or assisted natural breeding. After a review of several Bufonid captive breeding programs it became apparent that both male and female amphibians were receiving hormone stimulation simultaneously during the morning. In the case of the Anaxyrus americanus, this would result in an asynchronous release of gametes. Typically, female Anaxyrus americanus release eggs 12–24 hours post-hormone administration, but optimal sperm production in the male occurs 7–9 hours post-hormone administration and would be declining rapidly after 12 hours. These results indicate that the effect of hormone on spermiation has a limited duration. Several U.S. zoos working with the authors report that often females lay eggs following hormone stimulation but that poor fertilization was noted. This poor fertilization may be a result of the males already being in the refractory period and no longer producing sperm. Thus, an understanding of optimal or peak gamete release in relation to hormone administration assists in the development of a timed AI program for amphibians and how best to stagger the hormone injections for optimal fertilization and inception of reproductive behaviors.
While we were fairly confident that hCG was the optimal hormone for collecting sperm from Anaxyrus americanus to use for artificial fertilization experiments we needed to test whether replacing LHRH with hCG in several captive breeding programs for other Bufonids would affect their induced mating strategies. Several pilot studies were conducted at Central Park Zoo, Fort Worth Zoo and Sybille Wildlife Recovery Center in Wyoming to see whether amplexus or the percent of fertilized eggs increased in Anaxyrus baxteri or Peltophryne lemur. Every institution reported seeing fewer animals in amplexus; thus, fewer fertilized eggs, when administering hCG instead of LHRH. Hence, we went back and created a series of experiments to understand the impact these two hormones had on reproductive behavior. We found that over 91% of the pairs given LHRH were in amplexus 9 hrs post-administration, while 75% of the animals given hCG were in amplexus at the same time period. These two protein hormones have very different binding receptors and modes of action, yet both have non-gonadal targeted tissues with potential overlapping results on reproductive behaviors with varying degrees of responsiveness.
In the rat brain hippocampus, LHRH acts as a neurotransmitter linking actions inside the central nervous system where it facilitates reproductive behaviors to peripheral endocrine effects . In addition, LH/hCG receptors have also been found in the rat hippocampus, impacting specific reproductive behaviors . One of the hippocampus’ roles is chemical and hormonal sensing, whereby it exerts cognitive control over specific aspects of the hypothalamic-pituitary-adrenal axis impacting reproduction as well as memory cognition and arousal . In some amphibian species, it is likely that similar actions are occurring and that both LHRH and LH/hCG homologues modify neuronal excitability within the hippocampus, thereby modulating hippocampal function and downstream behaviors, although at varying levels of stimulation. Androgen receptors are also located within the hippocampus  and steroid biosynthesis following exogenous hormone administration may be another indirect route for stimulating amphibian spermiation or onset of reproductive behaviors. Binding of hCG to receptors in the testis has been shown to initiate steroid production and spermiation in several amphibians including Rana nigromaculata, Xenopus laevis and Bufo marinus. Similarly, administration of LHRH has been shown in several amphibian species to increase androgen production, resulting in increased reproductive behaviors and spermiation [24, 25]. Hence, both LHRH and hCG stimulate similar steroidogenic pathways although the effectiveness of each exogenous hormone on reproductive behaviors or spermiation may be species-, dosage-, or hormone-specific.
Our results suggest that although hCG may be optimal for collecting gametes for artificial fertilization, the use of this hormone alone is not sufficient for inducing breeding and overcoming reproductive behavioral challenges. Previous studies by our lab have used both hormones in a cocktail mixture to induce Anaxyrus baxteri to ovulate large numbers of eggs when given as a priming hormone . Future studies will test whether combining the two hormones in a cocktail formula may provide higher fertilization rates by increasing the sperm concentration while not sacrificing the number of animals that can be induced to amplex. Although we were able to achieve a 91% amplexus rate with LHRH up to 9 hrs in this series of experiments with Anaxyrus americanus, many programs using these same hormones, sometimes at higher concentrations than we tested, still report low numbers of animals in amplexus.