The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea

Abstract

Reproductive technologies may assist amphibian conservation breeding programs (CBPs) to achieve propagation targets and genetic management goals. However, a trial-and-error approach to protocol refinement has led to few amphibian CBPs routinely employing reproductive technologies with predictable outcomes. Additionally, while injections can be safely administered to amphibians, perceived animal welfare risks, such as injury and disease transmission, warrant the development of alternative hormone administration protocols. The present study investigated the spermiation response of roseate frogs, Geocrinia rosea, administered various doses of human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone agonist (GnRH-a) via subcutaneous injection. This study also quantified the spermiation response of frogs administered both hormones via topical application. Total sperm, sperm concentration and sperm viability were assessed over a 12-h period post hormone administration. Males released sperm in response to the injection of hCG (88–100% response; 5, 10 or 20 IU), but all samples collected from males administered hCG topically (100, 100 + DMSO or 200 IU hCG) were aspermic. In contrast, males consistently released sperm in response to both the injection (100% response; 1, 5 or 10 μg), or topical application (80–100% response; 50, 50 + DMSO or 100 μg) of GnRH-a. Overall, the administration of GnRH-a was more effective at inducing spermiation than hCG. Mean total sperm and sperm concentration were highest in response to the optimal topically applied dose of 100 μg GnRH-a (mean total sperm = 2.44 × 103, sperm concentration = 1.48 × 105 sperm/ml). We provide novel evidence that topical application provides a viable alternative to injection for the administration of GnRH-a to induce spermiation in amphibians.

Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti

“…Immediately prior to hormone injection, frogs were weighed to the nearest 0.01 g, and the dose administered was adjusted according to an individual’s body mass. Hormones were diluted in 100 μL of simplified amphibian ringer (SAR; composition (in mM): NaCl 113; KCl 2; CaCl 2 1.35; NaHCO 3 1.2) and administered via subcutaneous injection into the dorsal lymph sac, using ultra-fine 31-guage needles, following hormone-injection protocols used previously [ 19 , 20 , 21 ]. All frogs were sexually mature and ranged in weight from 9.1 g to 20.7 g (mean± SEM male mass ( n = 20) = 11.56 ± 0.35 g; mean± SEM female mass ( n = 20) = 16.66 ± 0.52 g).…”

“…Urine volume at each collection time point ranged from 2 to 100 µL ( n = 140, mean ± SEM = 33 ± 2.2 µL). Spermic urine was collected and assessed according to methods adapted from previous studies [ 20 , 21 , 22 ]. Urine samples were collected by gently inserting the tip of a 50 μL glass microcapillary tube, which was fire polished and cooled, into the opening of the cloaca until urination occurred.…”

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

“…All sperm suspensions were stored in capped 1.5mL Eppendorf tubes and refrigerated at 5°C for a total of 12 days, with viability assessments occurring on days 0, 3, 6, 9 and 12 of storage and bacterial abundance assessments occurring on days 0, 6 and 12 of storage. Sperm suspensions were uncapped and gently agitated for 10 seconds every day during the 12-day storage period, to aerate the suspensions and reduce sperm sedimentation (Germano et al, 2013;Silla et al, 2020).…”

Topical application of hormone gonadotropin‐releasing hormone (GnRH‐A) stimulates reproduction in the endangered Texas blind salamander (Eurycea rathbuni)

“…While the primary role of GnRH-A is reproductive, other hormones and organ systems are affected by its release (Muñoz-Cueto et al, 2020) and it acts as a pheromone in some amphibian species (Moore et al, 2005;Vu & Trudeau, 2016). GnRH-A can be injected, inserted intranasally, or topically applied for use in amphibian breeding (Browne & Zippel, 2007;Julien et al, 2019;Kouba et al, 2009;Silla et al, 2020;Vu & Trudeau, 2016). While GnRH-A is naturally occurring in amphibians, the response to exogenous application is somewhat species specific and generally requires testing to understand its impacts and efficacy in inducing reproductive behavior, ovulation, and sperm release (Kouba et al, 2009;Vu & Trudeau, 2016).…”

“…Most commonly, GnRH-A has been provided as an injection in amphibian reproductive technologies. GnRH-A injection has proved helpful in inducing egg deposition of other salamander species (Guy et al, 2020;Marcec, 2016) and topical application has proven effective in two species of anurans (Silla et al, 2018(Silla et al, , 2020. The following pilot trial documents the first attempt at topical application of GnRH-A to a salamander species, utilizing the Endangered Plethodontid species, E. rathbuni, as an opportunistic and practical model.…”