Hormonal induction, quality assessments and the influence of seasonality on male reproductive viability in a long-term managedex situbreeding colony of Southern Rocky Mountain Boreal toads,Anaxyrus boreas boreas

ABSTRACT The Southern Rocky Mountain Boreal Toad ( Anaxyrus boreas boreas) is an ex situ managed species which relies heavily on the use of assisted reproductive technologies to augment existing in situ populations. Despite the use of ARTs to manipulate reproduction of this species, the ex situ program continues to face challenges with annual reproduction. Human chorionic gonadotropin (hCG) at 10 IU/gbw singly or in combination with 0.4 ug/gbw GnRH-A have been successfully reported for this species, however, with a number of other available hormones, it is not clear if these are the most effective protocols for this species. Moreover, there is no information on how exogenous hormone administration is affected by other factors such as seasonality. Therefore, in the first part of this study, we compare the efficacy of the 10 IU/g hCG and 0.4 Lg/g Gonadotropin Releasing Hormone agonist (GnRH-A) administered singly or in combination, as well as GnRH-Apd + Amphiplex (0.4 Lg/g GnRH-A + 10 Lg/g Metoclopramide [MET] - a dopamine antagonist), or 10 Lg/g MET alone. Spermiation responses and sperm viability were compared across treatments with hormonal curves then correlated to seasonality. The results of this study suggest that the optimal hormonal stimulation protocol, across all treatments, in terms of sperm quality was 10 IU/g hCG + 0.4 Lg/g GnRH-A. Further optimization is required, in particular the exploration of higher doses of GnRH-A. Lastly, we observed that the effect of seasonality influenced the hormonal efficacy and magnitude of the spermiation response. As was expected, quality and concentration parameters were affected by the month in which hormone administration occurred. HIGHLIGHTS Spermiation in the Southern Rocky Mountain Boreal toad, Anaxyrus boreas boreas, is elicited most effectively by hCG singly or in combination with GnRH-A. Spermiation responses lasted up to 96 hours post injection (hpi) though quantity and quality parameters were highest in the first 12 hpi. Total motility, forward progressive motility and percentage live cells all indicated A. b. boreas sperm quality was in a good range. Acrosome integrity requires more research as it was comparatively lower than expected. Responses to hormone inductions are seasonally affected in this species but suggest semi-discontinuous cycling. Ex situ breeding should perform better sperm assessments before designing breeding strategies.