172 Hatch rate of Fowler’s toad (Anaxyrus fowleri) embryos as influenced by different culture media

Many endangered amphibian species rely on captive breeding to bolster their populations and could benefit from better developmental success through IVF. Although fertilised amphibian eggs may experience high cleavage rates, the number of eggs that reach neurulation and hatching is often much lower, resulting in markedly fewer tadpoles than expected. One possible contribution to this problem is the liquid in which eggs are raised. In the wild, amphibians breed in ponds containing a variety of algae, bacteria, and other organic and inorganic materials'all of which may contribute to hatching success. However, IVF performed in sterile laboratory settings may not sufficiently duplicate the environmental conditions present in these breeding ponds. This experiment sought to identify whether increasing the salt content and osmolality of solutions used to flood each dish would influence the hatching rates of Fowler’s toad (Anaxyrus fowleri) tadpoles by more closely mimicking environmental conditions. Two salt solutions were evaluated: 10% Holtfreter’s solution (0.67 ± 1.2 mmol kg⁻¹) and 10% Simplified Amphibian Ringer’s solution (SAR; 9.0 ± 5.6 mmol kg⁻¹). These mixtures contain differing amounts of sodium chloride, calcium chloride, potassium chloride, and sodium bicarbonate, with SAR having a higher salt content. Water treated with aquarium conditioner designed to remove chlorine and detoxify ammonia, nitrite, and nitrate was used as a control (0 mmol kg⁻¹). We hypothesised that 10% Holtfreter’s solution would result in the highest hatch rate due to its moderate salinity and osmolality compared with the other treatments. Females (N = 2) were administered 300 IU human chorionic gonadotrophin (hCG) and 15 μg of gonadotrophin-releasing hormone analogue intraperitoneally to induce oviposition, and males (N = 4) were similarly administered 300 IU hCG to promote spermiation. Eggs (60.3 ± 21.0) were collected from females and distributed to Petri dishes where sperm was immediately applied. Following a 5-min dry-fertilisation period, dishes were flooded with control water, 10% Holtfreter’s, or 10% SAR. Cleavage, neurulation, and hatching rates were recorded over a 5-day period. The effects of each solution were analysed using a generalised linear mixed model with solution type as a fixed effect and breeding pair as a random effect. Preliminary analysis indicated no significant differences (P > 0.05) in mean hatch rates between the three treatments: control water (85.5 ± 1.6%), Holtfreter’s (83.5 ± 1.1%), or SAR (75.1 ± 1.7%). However, it was found that SAR yielded lower hatch rates on average compared with control water (P = 0.07, t = −1.8) and Holtfreter’s solution (P = 0.11, t = −1.7). Overall, further research is needed to determine the optimal medium for sustaining A. fowleri embryos; however, these results can be applied to at-risk amphibian species in efforts to aid in their conservation.