Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, OH, USA
Due to the continuous decline of amphibian populations worldwide, it has become increasingly important both to conserve extant wild populations and to facilitate reproductive success in captive animals. To overcome the challenges of stimulating reproduction in captive bufonids (true toads), several different hormonal treatments have been tested to induce oocyte maturation, ovulation and oviposition. In this study, hormonal treatments were tested using the common American toad (Bufo americanus), a useful research model for gaining a better understanding of the reproductive physiology of endangered toads. The overall goal of this study was to identify the most effective hormone regimen for inducing oviposition in non-hibernated American toads. Specific objectives included determining: 1) the type of hormone most effective for inducing ovulation and oviposition; 2) the dosage of hormones required; and 3) the number of injections necessary to elicit the desired response. A secondary goal of the project was to use ultrasonography to characterize the effect of treatment and oviposition on ovarian (egg) mass.
Twenty-seven female American toads were housed in plastic (Experiment 1) or terrarium (Experiment 2) containers in small groups (2–6) and photographed individually for identification. Water was provided ad libitum and shelter was available within each container. Crickets or wax worms were provided three times per week and animals were maintained in a laboratory at 23–25°C with 8–12 h light each day. Hormones tested included human chorionic gonadotropin (hCG), equine chorionic gonadotropin (eCG), luteinizing hormone (LH) and luteinizing hormone releasing hormone (LHRH). These four hormones were each diluted in ddH2O and delivered in 200 µl volumes by intracoelomic injection using a 30-ga needle. Oviposition was defined as the production of any eggs, and the number of eggs produced by each female was recorded. Ultrasound exams were conducted using an Aloka 500 and 900 with a 7.5 MHz linear array transducer. For ultrasound analysis, toads were placed in a tub of water while exams were performed. The widest view of the ovaries observed via ultrasonography was used for ovarian mass size measurements and was determined using the ellipse caliper measurement tool on the ultrasound machine. The average of the shortest and longest lengths was recorded due to the non-elliptical shape of the ovarian mass. Ovarian mass and body weights were averaged for both responders and non-responders across all treatments in Experiments 1 and 2. A paired t-test was used to compare mean body and ovarian mass pre- and post-treatment. An unpaired t-test was used to compare body and ovarian mass for responders and non-responders pre- and post-treatment.
In Experiment 1, the effect of hormonal priming was investigated. In July, 25 toads were randomly assigned (n=5/treatment) to one of five priming hormone treatments (LH 50 µg; LHRH 50 µg; eCG 50 IU; hCG 50 IU; and control [ddH2O]) delivered at time 0. All toads received 500 IU hCG at 48 h post-treatment in order to stimulate ovulation. If oviposition did not occur within 72 h, toads received a second injection of hCG (300 IU). All toads were weighed both prior to treatment, and after oviposition or at 120 h post-treatment. Ultrasound examinations were performed on all toads prior to the priming hormone injection and then approximately every 4 h following the injection of 500 IU hCG (48–72 h). Following the second hCG injection, ultrasound was performed twice a day from 72–120 h. Follow-up ultrasound examinations were conducted on all toads once monthly between July and December of the same year.
A total of 14 toads (14/25; 56%) responded to hormone stimulation by ovipositing egg masses. Oviposition occurred in only one toad following priming treatment (LHRH, n=1). Twelve toads (12/24; 50%) responded to the first ovulatory injection of hCG as determined by oviposition of eggs (LH n=1; LHRH n=2; eCG n=2; hCG n=3; and control n=4). Of those toads not responding to either a priming hormone or first injection of hCG (n=12), only one responded to a second injection of hCG. Hence, 11 toads (11/25; 44%) did not respond to any of the hormone treatments.
Experiment 2 was conducted in December, 5 mo after Experiment 1. This experiment focused on the effectiveness of three different dosages of hCG (100, 500, and 1000 IU) on eliciting ovulation and oviposition. Nineteen toads were randomly assigned to each of four treatments: Control (ddH2O n=5); 100 IU hCG (n=5); 500 IU hCG (n=4); and 1000 IU hCG (n=5). Toads were injected with their respective treatments and received a second injection of hCG (300 IU) 24 h later if oviposition had not occurred. Toads were weighed prior to hormone treatment, and after oviposition or at 72 h post-treatment. Ultrasound was performed on all toads prior to the initial hormone injection and then three times from 0–24 h. Following the second hCG injection at 24 h, ultrasound was performed daily until oviposition or until the end of the study (72 h).
Oviposition was induced in five toads from the initial hCG treatments (500 IU n=2 and 1000 IU n=3). None of the remaining four toads in these treatment groups responded to the second hCG injection. Six toads (6/10) from the other two treatments (100 IU hCG n=3 and control ddH2O n=3) responded to the second hCG injection. A total of 11 toads (11/19; 58%) responded to the combined hCG treatments. Of the 17 toads utilized in both Experiments 1 and 2, 8 underwent oviposition in both experiments.
There was no difference in the initial weight (p>0.05) and ending weight (p>0.05) of toads that responded to hormone treatment compared to those that did not respond. This may have been due, in part, to the wide range in number of eggs produced (11–7,489) by the females. Pre-treatment ovarian mass was not different (p>0.05) between toads that ultimately responded and those that did not respond to the hormonal treatments. Therefore, ovarian mass measurements are not likely to serve as useful predictors when trying to select toads most likely to respond to treatment. However, at the end of the experiments, toads that had undergone oviposition had a smaller average ovarian mass (p<0.001) than toads that had not produced eggs. By 1 mo after oviposition, toads that had no observable ovarian mass immediately following oviposition had recovered approximately 80% of their ovarian tissue.
In conclusion, a priming hormone is not necessary and does not appear to improve the success of hCG-induced oviposition in non-hibernated American toads. The optimal hCG dosage for inducing oviposition in this species appears to be 300–500 IU and only a single injection is necessary. The ovarian mass of toads is significantly reduced following oviposition, but the tissue mass recovers by 1 mo post-oviposition. Furthermore, oviposition can be hormonally induced in non-hibernated American toads multiple times during the year; however, the quality (fertilizability) of the eggs produced was not evaluated in these experiments.
The authors thank Helen Bateman, Diane Szymanski, Julia Laylor, Bernadette Plair and Monica Stoops for their assistance with the ultrasonography portion of this research and Jim Chrzan for the use of the Aloka 900. The authors also thank the many people that cared for the toads during this study, especially Dr. Ken Cameron, Robert Licht and Greg Ho. This research was supported by a grant from the Morris Animal Foundation.