20 Paths less travelled: novel oral and nasal hormone administration routes for eliciting spermiation in male eastern tiger salamanders (Ambystoma tigrinum)

Hormone therapies using intramuscular (IM) injections are typically performed in salamanders to elicit spermiation. However, injections may cause stress responses in animals and are challenging to perform with smaller-bodied species. We investigated the spermiation response to a gonadotrophin-releasing hormone analogue (GnRHa) when administered orally, intranasally (IN), and IM in eastern tiger salamanders (Ambystoma tigrinum). We hypothesised that oral and IN administration of GnRHa would cause a spermic response with motility and concentration comparable to that of IM hormone administration. Males (N = 12) were orally administered a priming dose of 0.25 μg g⁻¹ bodyweight (BW) GnRHa and randomly assigned to an oral spermiation dose 24 h later of either 0.2 (n = 4), 1.0 (n = 5), or 2.5 (n = 3) μg g⁻¹ BW GnRHa. The hormone dosage was pipetted into the abdomen of a 5/8–1′ banded cricket (Gryllodes sigillatus), which was readily consumed by A. tigrinum. A separate group of males (N = 10) was randomly assigned to either 0.5 μg g⁻¹ BW GnRHa via IM injection (n = 5) or 20 μL (1 μg/μL) of GnRHa administered IN (10 μL/nare) (n = 5). Sperm was collected and total motility and concentration (spermatozoa mL⁻¹) were determined at 0, 1, 3, 6, 9, 12, 24, and 48 h (oral) or 0, 1, 3, 5, and 7 h (IN/IM) time points after hormone administration. Time points differed due to a delay in spermiation response to the oral treatment. Sperm motility and concentration did not differ between oral hormone concentrations (P > 0.21) according to a linear mixed model (LMM), so their results were averaged for comparison with IN and IM administration. Treatments caused spermiation in 83.3% (oral), 60% (IN), and 100% (IM) of males. Average total sperm motility was compared across treatment groups using an LMM, and average concentration was compared with a generalised LMM with salamander cohort set as a random effect. Total sperm motility was similar (P > 0.82) in oral (52.2 ± 24.9%), IN (45.1 ± 32.2%), and IM (49.3 ± 20.0%) treatment groups. There were differences in sperm concentration between the oral (3.26 × 10⁵ ± 4.59 × 10⁵), IN (3.16 × 10⁶ ± 2.12 × 10⁶), and IM (1.61 × 10⁷ ± 3.59 × 10⁷) treatments, where IN and IM administration were similar to each other (P = 0.24), but both resulted in higher concentrations than oral administration of hormone (P < 0.01). A time-course analysis indicated that peak sperm concentration occurred at 3 (IM), 5 (IN), and 12 (oral) h after the final hormone dose, indicating a delay in the mechanism of GnRHa entering the digestive tract, whereas IN and IM administration result in a quicker reaction. These preliminary results suggest that GnRHa can be administered via alternative routes and undergo successful uptake and diffusion to the pituitary to induce spermiation. Overall, this innovative approach to hormone administration shows that both oral and IN delivery of GnRHa are effective at stimulating a spermic response in salamanders, and IN delivery is comparable to IM injection of GnRHa while posing a lower risk to animal health.