Frequency of exogenous hormone therapy impacts spermiation in male Fowler's toad (Bufo fowleri).

Amphibians are experiencing a global extinction crisis and captive assurance colonies, along with reintroduction programs, are necessary to prevent further losses. Assisted reproductive technologies (ART), such as hormone-stimulated gamete collection and in vitro fertlisation (IVF), are conservation methods that can be used to increase reproductive output for breeding and reintroduction programs when animals fail to breed naturally. In order to maximise the production of offspring using ART, it is important to establish the physiological limitations on the frequency that hormone therapy can be used to collect gametes for IVF or assisted breeding. The present study examined the effects of the frequency of hormone-induced spermiation on sperm quantity and quality in Fowler's toad (Bufo fowleri) by comparing four levels of hormone injection frequencies: twice a week, once a week, every other week, and every 3 weeks. Sperm release was induced with an intraperitoneal injection of 300IU human chorionic gonadotropin (hCG). Spermatozoa were collected at three time points after injection (5, 7 and 9h) and sperm concentration, motility and quality of forward progressive movement were measured. A significant decrease in sperm concentration (P<0.01) was observed with the most frequent treatment (twice a week hormone injections). However, there was no negative effect of the treatments on sperm motility (P=0.06) or forward movement (P=0.06). We also observed a significant decrease in the concentration (P<0.01), motility (P=0.02) and quality of forward progressive movement (P=0.01) of spermatozoa at the 9h collection compared with earlier collection times. These results have clear implications for amphibian captive breeding programs, where more frequent hormone-induced spermiation could have a negative effect on male performance. We recommend that hormone injections be spaced a minimum of 2 weeks apart to optimise the health of the animals, assisted breeding, IVF or collection of gametes for genome resource banking.