Bufonid Species Research Articles

Impacts of isolated or mixed Roundup® Original DI and Boral® 500 SC herbicides on the survival and metamorphosis of Melanophryniscus admirabilis tadpoles

The bufonid species Melanophryniscus admirabilis is restricted to a single location in the southern Atlantic Forest, Brazil. Although the site of occurrence of M. admirabilis is covered with native forest and it is not directly exposed to pesticides application, the area is surrounded by agricultural activity. Our objectives were to evaluate possible alterations in morphological parameters (body mass, snout-vent length, and body index), metamorphosis (time to reach Gosner stages 42, 46 and to complete metamorphosis), and survival of M. admirabilis exposed to isolated Roundup® Original DI (R1: 234 and R2: 2340 µg.L−1 of glyphosate) and Boral® 500 SC, (B1: 130 and B2: 980 µg.L−1 of sulfentrazone) or mixed (R1+B1, R2+B1, R1+B2, R2+B2). Spawns of M. admirabilis were collected in natural lakes in the municipality of Arvorezinha and taken to laboratory cultivation. After the tadpoles acquired free swimming, the animals were acclimated for five days and fed ad libitum. The aquariums were contaminated with herbicides on the sixth day of cultivation, and the animals stayed in these aquariums for four days. Afterwards, the tadpoles were transferred to aquariums with clean water and monitored until metamorphosis (Gosner stage 46), when they were weighed, measured (snout-cloacal length) and cryoeuthanized. We observed no alterations in morphological parameters; however, survival was reduced in exposed groups (mortality index: 71 % in R2 and 29–64 % in mixed groups), suggesting energy allocation for metamorphosis at the expense of survival. Boral did not alter metamorphosis time. Roundup isolated and mixed with Boral altered the timing of Gosner stages 42 and 46 and reduced metamorphosis time, suggesting endocrine disruption. Thus, monitoring the presence and limiting the use of these pesticides in the area where M. admirabilis occurs can be crucial for conservation strategies.

Mass Occurrence of Tadpole Deformities in Toad Species of the Genus <i>Duttaphrynus</i> (Bufonidae) in the Himalaya (Uttarakhand, India)

In April and May 2019, we examined seven tadpole samples of two bufonid species Duttaphrynus stomaticus (one site) and Duttaphrynus himalayanus (six sites) taken in the Yamuna River and its tributaries, and the Bhagirathi River at altitudes from 687 m up to 2448 m above sea level; the western part of the Garhwal Himalaya, Uttarakhand, India. Seven categories of larval deformities were recorded: scoliosis, kyphosis, shortened tail, rounded tip of the tail, bifurcated tip of the tail, opercular chamber defect, and edema. The occurrence of malformed tadpoles calculated from a sample size ranged from 3.3 to 13.6%. Water mineralization levels were fluctuated in the limits 37 – 330 mg/dm3, exceeding 100 mg/dm3 in three cases. There was no significant correlation between share of abnormal animals or abnormalities frequency and mineralization as well as with altitude. Mass occurrence of malformed tadpoles was revealed in two places: the Aglad Nadi (= Algar River, 10%, site 1, 687 m) and the town Purola (9.9 – 11.8%, sites 2 and 3, 1339 – 1349 m). Both places were associated with human activity: cattle grazing and residential agrolandscape, respectively. Enlarged rate of anomalies on the town territory may be explained by a synergy between complex of drivers (mineralization, fertilizers and pesticides) that can lead to amplification of general impact on larval morphogenesis. Thus, we reported the first record of mass larval abnormalities in amphibians for the Garhwal in particular and for the Himalaya in general. Formerly published data evidenced that another malformation hotspot of amphibians was situated at the Western Ghats, peninsular India.

Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management

The challenge of replicating environmental cues in amphibian captive assurance colonies has been shown to be one of the contributing factors leading to limited breeding success. To circumvent this challenge, assisted reproductive technologies (ART) have been developed in model organisms and can now be applied to at-risk species. This study tested the transferability of cryopreservation protocols developed in the Fowler's toad (Anaxyrus fowleri) to three threatened anurans, the Chiricahua leopard frog (Lithobates chiricahuensis), Puerto Rican crested toad (Peltophryne lemur), and Houston toad (Anaxyrus houstonensis). Males were administered human chorionic gonadotropin and/or gonadotropin releasing hormone-analog to elicit spermiation, and spermic urine was collected and analyzed for motility. Samples were cryopreserved with 10 % N,N-dimethylformamide + 10 % trehalose + 0.25 % bovine serum albumin and frozen at two rates, −32 to −45 °C/min and − 20 to −29 °C/min. Results show that sperm frozen at −32 to −45 °C/min recovered significantly higher post-thaw sperm motility than −20 to −29 °C/min for P. lemur (p = 0.04) and L. chiricahuensis (p = 0.03). When compared across species, L. chiricahuensis had significantly higher post-thaw motility at both freezing rates (p < 0.05) than the other species, yet there was no difference between sperm motility between the three bufonid species for either freezing rate (p > 0.06). These results indicate that transferability of a cryopreservation protocol between species is possible, and differences in sensitivity and response to cryopreservation may exist between genera.

Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri.

Briefly considered extinct in the wild, the future of the Wyoming toad (Anaxyrus baxteri) continues to rely on captive breeding to supplement the wild population. Given its small natural geographic range and history of rapid population decline at least partly due to fungal disease, investigation of the diversity of key receptor families involved in the host immune response represents an important conservation need. Population decline may have reduced immunogenetic diversity sufficiently to increase the vulnerability of the species to infectious diseases. Here we use comparative transcriptomics to examine the diversity of toll-like receptors and major histocompatibility complex (MHC) sequences across three individual Wyoming toads. We find reduced diversity at MHC genes compared to bufonid species with a similar history of bottleneck events. Our data provide a foundation for future studies that seek to evaluate the genetic diversity of Wyoming toads, identify biomarkers for infectious disease outcomes, and guide breeding strategies to increase genomic variability and wild release successes.

Larvicidal activity of the skin secretion ofRhinella marina and Rhaebo guttatus (Anura: Bufonidae) against the Brazilian malaria vector, Anopheles darlingi (Diptera: Culicidae).

Malaria, a mosquito-borne disease, is caused by protozoa of the genus Plasmodium and constitutes a serious public health problem. Because current insecticides used to control malaria face resistance due to continuous use, new alternatives are prompted. Considering this context, and the insecticidal potential of vertebrate venoms/secretions, crude and methanolic extracts from two frog species were tested as larvicides against Anopheles darlingi . Skin secretions of Rhinella marina and Rhaebo guttatus were obtained by manual stimulation. Then, methanol was added to obtain steroidal fractions from both venoms. Mosquitos were captured in suburban areas of Porto Velho and An. darlingi females were later fed with blood and stimulated to oviposit. The larvae were fed with fish food until the 3 rd and 4 th instars. For the larvicidal assays, crude secretions and methanolic fractions of both frog species were evaluated, and larvae mortality was recorded after 48 hours. Crude extracts and steroidal fractions from both species had larvicidal effects, with an LC 50 of 127.5 and 133 ppm for the crude extract and steroidal fraction of R. marina , and an LC 50 of 37.5 and 35.8 ppm for the crude extract and steroidal secretion of R. guttatus, respectively. The present work reports for the first time the larvicidal effects of the skin secretions from bufonid species occurring in the western Amazon region. Further studies should be carried out to investigate the purified components responsible for the observed activity.

Behavioural parental care repertoire in Frostius pernambucensis (Anura, Bufonidae)

ABSTRACT Parental care is a behavioural repertoire exhibited by parents to their offspring to ensure their survival. In anurans, parental care may be optional, maternal, paternal, alloparental or biparental, towards eggs, larvae and/or juveniles. Frostius pernambucensis is a bufonid species exclusively endemic to the Brazilian Atlantic Rainforests of northeastern Brazil, occurring in the states of Paraiba, Pernambuco, Alagoas and Bahia. This study describes the behavioural parental care repertoire displayed by F. pernambucensis at the Dois Irmãos State Park (Parque Estadual Dois Irmãos – PEDI), the species type locality, in the municipality of Recife, Pernambuco. Fieldwork was carried out between June 2015 and March 2017, in which parental care was observed for three F. pernambucensis males. In total, 45 behavioural acts were recorded (n = 526), distributed in six subcategories: agonistic behaviour (n = 228; 43.35%), vigilance (n = 92; 17.49%), offspring manipulation (n = 78; 14.82%), dislocation (n = 68; 12.93%), vocalisation (n = 51; 9.70%), and maintenance behaviours (n = 9; 1.71%). Descriptive parental care records for Brazilian amphibians are considered rare, emphasising the importance of the present study, which provides relevant information about this poorly known taxon.

Trophic Relationships among Five Species of Anura in the Colombian Caribbean Tropical Dry Forest: A Spatial and Temporal Approach

Species of Bufonidae and Leptodactylidae are common in the Colombian tropical dry forest. Although some of them are associated with active foraging and consumption of termites and ants, their trophic ecology is mostly unknown. The diet of five anuran species of Bufonidae (Rhinella horribilis, R. humboldti) and the leptodactylid subfamily Leiuperinae (Engystomops pustulosus, Pleurodema brachyops, and Pseudopaludicola pusilla) was examined at six sites of the Colombian Caribbean in fragments of dry forest and different land uses. A total of 310 food items were identified. The greatest contribution was represented by Coleoptera, Hymenoptera (Formicidae), Isoptera, and Diptera. The species differed in number and volume of prey. Except for Pseudopaludicola pusilla, which behaves as a generalist predator, species had high intake of termites and ants. Engystomops pustulosus preferred termites, Pleurodema brachyops had high consumption of ants and termites, and the two bufonid species were myrmecophagous. Except for Pseudopaludicola pusilla, predator and prey size was related. Most species have overlapping diets (spatially and temporally) when analyzing food items identified at the order level. However, the richness of different prey within these orders allows the coexistence of species. The diet of these species presents similar trends in different habitats throughout their distribution area.

Evaluation of different temporal periods between hormone-induced ovulation attempts in the female Fowler's toad Anaxyrus fowleri.

Many amphibian species reinitiate the processes of preparing for reproduction (e.g. oogenesis) soon after breeding indicating hormone-induced ovulation could potentially be achieved out-of-season, which would lead to higher annual fecundity compared to mono-seasonal breeding. Such strategies would be beneficial to captive breeding programs for threatened species that are short-lived, have aging populations or need large numbers of offspring to meet reintroduction goals for species recovery. Unfortunately, little is known regarding how female anurans respond to multiple ovulation events within a year, which could lead to higher annual fecundity compared to mono-seasonal breeding. Thus, we evaluated the effect of temporal period between exogenous hormone stimulation events on egg production using the Fowler's toad Anaxyrus fowleri as a model species. Female toads (n = 21) were administered hormone therapy twice in 1year with toads randomly assigned to a treatment of either a 4-, 8- or 12-month recovery period between hormone stimulations. Ovulation was induced using two priming doses of human chorionic gonadotropin (100IU; hCG) 72h apart, followed by a resolving dose of hCG (500IU) plus gonadotropin releasing hormone analogue (GnRHa; 15μg) given 24h after the second priming injection. Measured response variables include the number of females ovulating after treatment, total number of eggs produced and percent fertilization, neurula and tadpole development. No significant treatment effects were observed for any response variable (P > 0.05). Findings from this study suggest that hormone therapy can be administered in a bufonid species every 4 or 8months without significantly affecting the number of ovulating females, egg production, fertilization, neurulation or tadpole development. By collecting gametes out-of-season or multiple times throughout the year, captive breeding programs could potentially increase tadpole production for reintroductions as well as extend the breeding window in captivity.

A food spectrum analysis of three bufonid species (Anura: Bufonidae) from Uttarakhand region of the western Himalaya, India

The ecological diversity of insects and its predators like amphibians are important determinants in ecological balance. A total of 1,222 prey items in 84 specimens were examined to contribute the understanding of the diets of three Duttaphrynus species, viz., himalayanus, melanostictus, and stomaticus from Uttarakhand, the western Himalaya, India. Gut content analysis of three bufonids revealed acceptance of a wide range of terrestrial insects and other invertebrates as their food. The index of relative importance indicated that the most important preys were Formicidae, Coleoptera and Orthoptera. Duttaphrynus melanostictus had the broadest dietary niche breadth, followed by D. himalaynus and D. stomaticus. The wide prey spectrum well indicates that these species are the generalist and opportunist invertebrate feeder. Information pertaining to the food spectrum analysis contributes to understanding the ecological roles and used as a baseline data for future successful amphibian conservation and management programs in the Himalayan ecosystem.

Diversity and evolution of the parotoid macrogland in true toads (Anura: Bufonidae)

AbstractSkin glands in amphibians are either distributed throughout the skin or aggregated in multiglandular structures such as the parotoids typical of most species of Bufonidae. Although many early divergent and derived bufonids lack a discrete parotoid in the postorbital–supratympanic (PoSt) region, they have a great macroscopic diversity in the skin morphology of this region. To understand the origin and evolution of this diversity, in particular of the parotoids, we describe the histomorphology of the skin of the PoSt and dorsal regions in 17 species of bufonids, with or without external evident parotoid, and compare it with previously published descriptions. The survey results in 27 characters that were optimized on a phylogenetic hypothesis of Bufonidae. Our results reveal that the PoSt region has a noteworthy morphological diversity of types of glands, spatial organization and differences in the secretion products. Some morphological characters represent putative synapomorphies of internal clades of Bufonidae and are related to the progressive differentiation towards defined structures (macroglands, parotoids). These morphological results, along with published information on the toxicity of the skin secretions and defensive behaviours in some representative species, allow us to infer possible relationships between these features.

Helminth Fauna of Incilius marmoreus (Anura: Bufonidae) in a Neotropical Locality of Mexico

In June 2014, 30 marbled toads (Incilius marmoreus) from Oaxaca, Mexico were examined for helminths. We collected a total of 3,783 helminths belonging to 13 taxa: 4 digenean, 1 cestode, 1 acanthocephalan, and 7 nematoda. The nematode Physocephalus sp. was the most prevalent (63.3%) followed by Aplectana sp., which had the highest mean intensity (114.3) and mean abundance (63.8) that we recorded. The taxonomic composition of I. marmoreus mainly consists of generalist helminths, with nematodes being the predominant parasites found, confirming the terrestrial habits of this toad. The main route of helminth acquisition was ingestion of larvae and/or intermediate hosts, a process that also likely contributes to the high abundance of individuals in the toads. The knowledge of the helminth fauna associated to the 39 toad species distributed in Mexico is scarce and fragmentary, and our study presents results for only the second bufonid species studied in this region.

Description of the advertisement calls of four species of Amazophrynella (Anura:Bufonidae).

Amazophrynella comprises 11 small bufonid species with a pan-Amazonian distribution (Fouquet et al. 2012a, b; Rojas et al. 2016, Rojas et al. 2018). All species inhabit the forest leaf litter, breed in seasonal puddles and are diurnally and nocturnally active (Fouquet et al. 2012b; Rojas et al. 2014; 2015; 2016). Until now only one nominal species, A. javierbustamantei, and two putative lineages-A. moisesii (Rio Yuyapichis, Peru) and A. siona (Santa Cecilia, Ecuador)-had their advertisement calls formally described (Duellman 1978; Schlüter 1981; Rojas et al. 2016). Herein, we described for the first time the advertisement calls from additional four species of Amazophrynella.

Processes structuring amphibian assemblages along a subtropical arid gradient

Biotic assemblages are organized according to multiple factors, including environmental filtering and species interactions. However, the roles of abiotic and biotic factors in the organization of amphibian assemblages in arid regions are still not completely understood. Here, I examined the distribution patterns of seven species of amphibians that occur in Tunisia (north-western Sahara Desert) and the composition of 328 amphibian assemblages, including their meta-structural organization. The species showed different responses to environmental gradients, predominantly the aridity and vegetation gradients. The assemblages in xeric regions were composed of nested subsets of the species present in mesic regions. Most of the significant species associations were explained by shared environmental responses, indicating that they can be largely attributed to environmental filtering. The only exception was between two species of generalist bufonids, Bufotes boulengeri and Sclerophrys mauritanica, which showed a residual correlation greater than their shared association with the gradient. In this case, phylogenetic and morphological similarities could favour patterns of mutual exclusion in habitats such as desert oases, where the resources are limited.

Nematodes of Five Species of Bufonids (Anura: Bufonidae) from Peninsular Malaysia

Abstract Five species of toads (Bufonidae) (n = 22) from Peninsular Malaysia were examined for helminths: Duttaphrynus melanostictus, Ingerophrynus parvus, Leptophryne borbonica, Phrynoidis asper, and Rentapia hosii. The helminth community contained 10 species of Nematoda, consisting of mature individuals of Abbreviata bufonis, Aplectana macintoshii, Cosmocerca ornata, Falcaustra pahangi, Meteterakis singaporensis, Oswaldocruzia filiformis, Oswaldocruzia hoepplii, Seuratascaris numidica, and larvae assigned to Ascarididae and Physalopteridae. Nematoda reported in this study are mainly Old World in distribution. Bufonid species from Peninsular Malaysia are infected by generalist nematodes that also infect other anurans. Seven new host records are reported.

The Taxonomic Status ofBufo intermediusGünther, 1858: Forensic Entomology Confirms What Was Long Suspected from Morphology

Bufo intermedius Günther, 1858, has a complex taxonomic history, with the type series being reported from Guayaquil and the Andes of Ecuador. However, these specimens are not referable to any known South American bufonid species. We examined the type series and found, based on morphology, that the specimens likely represent a Mesoamerican species, as earlier authors had suggested. We further explored this possibility by analyzing the stomach contents of one of the syntypes. The unique combination of arthropods, particularly the presence of the beetle species Megalostomis dimidiata and Zygogramma signatipennis, confirmed that the Ecuadorian locality data are erroneous and that the type series was collected in Mexico. We here present that the types of Bufo intermedius are conspecific with the Mexican species Incilius occidentalis (Camerano, 1879) and propose a new synonymy between Bufo intermedius Günther, 1858 and Incilius occidentalis (Camerano, 1879). An application has been made to the International Commission on Zoological Nomenclature to preserve prevailing usage of the junior name Incilius occidentalis for this widespread Mexican species.

Morphological and histochemical studies of Bidder’s organ in Rhinella schneideri (Amphibia: Anura) males

Bufonids have an organ that produces female germ cells in both sexes, known as the Bidder’s organ (BO). In males, BO is located in the anterior pole of the testis and it has been compared to a rudimentary ovary. It has been demonstrated that in some species the bidderian follicles can accumulate vitellogenin in males, while in other species, the development of follicles is inhibited by the differentiation of the corresponding gonad. This study describes the anatomical, histochemical and ultrastructural aspects of the BO in males of the neotropical frog Rhinella schneideri during the breeding season. A topographic model has also been built using three-dimensional (3D) reconstruction. BO has an irregular shape with lobes varying in size and number. There is no physical barrier between the BO and male gonads and, for this reason, female cells are in intimate association with seminiferous locules. Histologically, two distinct regions are observed in the BO: the cortex, and medulla. In the cortex, bidderian oocytes are surrounded by follicle cells forming bidderian follicles, which are in pre-vitellogenic stage. The ooplasm of bidderian oocytes is rich in cytoplasmic organelles. Microvilosities are formed in the oolemma, where the follicular cells are juxtaposed with oocytes, and amorphous extracellular material is deposited. Lipofuscin and myelin bodies occur in the medulla as a result of the cellular degradation. Pigmented cells were also detected in the medullar region. The oogonias observed in the BO periphery, and the significant amount of degenerating oocytes in the cortex, showed the renewal capacity of BO cells in R. schneideri males. The BO anatomical pattern in R. schneideri is similar to that observed for most species of bufonids. This work describes for the first time specific aspects related to the morphological description with emphasis on architecture, morphometry and histochemistry.

Intraspecific morphological variation and its implications in the taxonomic status of ‘Bufo pisanoi,’ a Pliocene anuran from eastern Argentina

ABSTRACTThe true toad family Bufonidae is one of the most speciose and widespread clades of neobatrachian anurans. Despite being well represented in South America at present, bufonids have a poor fossil record on this continent. ‘Bufo pisanoi’ from Pliocene outcrops of coastal Buenos Aires Province, Argentina, is the only extinct South American bufonid taxon recognized to date. In this study, we investigated the validity of the putative diagnostic characters of this extinct species to confirm its taxonomic status based on revision of the holotype and referred material. Comparisons with several extant bufonid species led us to include the taxon represented by this fossil material within the Rhinella marina clade, close to R. arenarum. Further comparisons and a geometric morphometric analysis based on a relatively large sample of the latter species demonstrated that the morphology of ‘B. pisanoi’ falls within the variation range of R. arenarum. Therefore, we consider that the names ‘B. pisanoi’ and R. arenarum represent the same taxon, with the latter having priority. This work emphasizes the importance of considering intraspecific variation whenever possible, because this leads to a more rigorous approach to the identification of fossil specimens and tests the taxonomic value of different osteological characters.SUPPLEMENTAL DATA—Supplemental materials are available for this article for free at www.tandfonline.com/UJVP

Advertisement call of species of the genus Frostius Cannatella 1986 (Anura: Bufonidae)

Frostius pernambucensis and F. erythrophthalmus are cryptic bufonid species recognized mainly by the iris color: yellow in F. permambucensis and red in F. erythrophthalmus . However, field studies showed that the iris color of F. erythrophthalmus could vary between yellow and red. To improve the recognition of these species we described the advertisement call of Frostius pernambucensis and Frostius erythrophthalmus and we tested if call characteristics are influenced by temperature, male size and perch height. We also report on a physical interaction between two males of F. pernambucensis and the associated vocalization, suggesting that F. pernambucensis has not a territorial call or encounter call. Comparing the advertisement calls, the call of F. pernambucensis was lower, shorter and with a smaller number of notes than the call of F . erythrophthalmus . Dominant frequency and fundamental frequency variation of the F. pernambucensis advertisement call were related to the male’s size, while the call emission rate was related to air temperature. However, we could not find relationship among the acoustic characteristic of F. erythrophthalmus and male size or temperature.

Interspecific Variation in Innate Immune Defenses and Stress Response of Toads from Botucatu (São Paulo, Brazil)

Glucocorticoid hormone levels increase in the blood stream in response to a large range of social and physical stressors, affecting variables associated to fitness, such as reproductive output and immunocompetence. Although quantification of plasma levels of glucocorticoids has been used as an indicator of environmental stress in different clades of vertebrates, the existence of interspecific differences in magnitude of stress response, their functional bases and ecological correlates remain poorly investigated. In the present study, we tested the following hypotheses: (1) phyllogenetically close species of Bufonids differ in innate immunocompetence, baseline activity of the hypothalamic-pituitary-interrenal (HPI) axis, and response of the HPI axis to stressors; and (2) species showing higher baseline activity of the HPI axis are characterized by lower innate immunocompetence. To test these hypotheses, we compared plasma bactericidal ability against Escherichia coli, baseline field levels of corticosterone (CORT) during reproductive season, and levels of CORT under stressful conditions (captive maintenance plus physical restraint) of three species of toads from the genus Rhinella from Botucatu (SP/Brazil): R. icterica, R. schneiden, and R. ornata. We found interspecific variation in bactericidal ability and CORT levels, with R. ornata showing higher baseline and post-stress CORT levels, and R. schneiden showing higher plasma bactericidal ability. Bactericidal ability and baseline CORT levels are inversely related among species, suggesting that lower innate immunocompetence might be associated to immunosupressive effects of elevated CORT levels. Possible associations between these results and interspecific differences in reproductive behavior and sensitivity to environmental changes are discussed.

Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the Barred Frog Mixophyes fasciolatus (Myobatrachidae)

BackgroundProtocols for the hormonal induction of ovulation and oviposition are essential tools for managing threatened amphibians with assisted reproduction, but responses vary greatly between species and even broad taxon groups. Consequently, it is necessary to assess effectiveness of such protocols in representative species when new taxa become targets for induction. The threatened genus Mixophyes (family Myobatrachidae) has amongst the highest proportion of endangered species of all the Australian amphibians. This study developed and optimised the induction of oviposition in a non-threatened member of this taxon, the great barred frog (Mixophyes fasciolatus).MethodsGravid female M. fasciolatus were induced to oviposit on one or more occasions by administration of human chorionic gonadotropin (hCG) with or without priming with pregnant mare serum gonadotropin (PMSG). Treatments involved variations in hormone doses and combinations (administered via injection into the dorsal lymph sacs), and timing of administration. Pituitary homogenates from an unrelated bufonid species (Rhinella marina) were also examined with hCG.ResultsWhen injected alone, hCG (900 to 1400 IU) induced oviposition. However, priming with two time dependent doses of PMSG (50 IU, 25 IU) increased responses, with lower doses of hCG (200 IU). Priming increased response rates in females from around 30% (hCG alone) to more than 50% (p = 0.035), and up to 67%. Increasing the interval between the first PMSG dose and first hCG dose from 3 to 6 days also produced significant improvement (p<0.001). Heterologous pituitary extracts administered with hCG were no more effective than hCG alone (p = 0.628).ConclusionsThis study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.