Langhorne, C. J. | bioRxiv Science

bioRxiv2023

Hormonal induction, quality assessments and the influence of seasonality on male reproductive viability in a long-term managed ex situ breeding colony of Southern Rocky Mountain Boreal toads, Anaxyrus boreas boreas.

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. HIGHLIGHTSO_LISpermiation in the Southern Rocky Mountain Boreal toad, Anaxyrus boreas boreas, is elicited most effectively by hCG singly or in combination with GnRH-A. C_LIO_LISpermiation responses lasted up to 96 hours post injection (hpi) though quantity and quality parameters were highest in the first 12 hpi. C_LIO_LITotal motility, forward progressive motility and percentage live cells all indicated A. b. boreas sperm quality was in a good range. C_LIO_LIAcrosome integrity requires more research as it was comparatively lower than expected. C_LIO_LIResponses to hormone inductions are seasonally affected in this species but suggest semi-discontinuous cycling. C_LIO_LIEx situ breeding should perform better sperm assessments before designing breeding strategies. C_LI

zoology↗

bioRxiv2017

Induction of Reproductive Behaviors by Exogenous Hormones in Captive Southern Rocky Mountain Boreal Toads, Anaxyrus boreas boreas

Loss of reproductive viability, physiologically and/or behaviorally, can have profound effects on the fitness of a captive population and conservation efforts. The southern rocky mountain (SRM) population of the boreal toad has declined over the past 35 years, making captive breeding necessary to protect and augment the species in the wild. In recent years, a notable reduction in the incidence of amplexus and viable offspring from the captive breeding population has been observed. Hormone treatment protocols to stimulate gamete release in males and females are established in this species and in vitro fertilization has been performed successfully. However, successful hormone stimulation of reproductive behaviors and natural fertilization has not been well documented. During the breeding season of 2012, 24 males and 24 female toads were selected from a population of over 600 captive animals. Both sexes were treated with Human chorionic gonadotropin (hCG) and Gonadotropin Releasing Hormone (GnRH) or phosphate buffered saline (PBS). Females were primed twice with 3.7IU/g hCG and then injected with an ovulatory dose (OvD) of 13.5 IU/ g BW (Body weight) hCG and 0.4 g/ g BW GnRH. Males were injected a single time with 10 IU/g BW hCG and 0.4 g/ g BW GnRH, 12 h after females received their OvD. In 2013, knowing the approximate time when females oviposited after hormone treatments, we tested the best time to induce amplexus and spermiation. Males were divided into 4 groups and injected at 4 different times: (a) 12 h before females OvD; (b) at the same time as OvD; (c) 12 h after OvD; (d) control injected with PBS. Results from 2012 indicated that oviposition was solely dependent on females receiving hormone treatments not males. However, in 2013 we found that the duration of amplexus significantly influenced oviposition (P>0.05), and males injected 12 h prior to females spent more time in amplexus than males injected at the same time or 12 h after the females received hormones. Promoting reproductive behaviors and synchronizing gamete deposition continues to be imprecise and may require more than exogenous hormones. The complexity of promoting breeding behaviors may require a closer assessment of the captive environment.

zoology↗