29 Application of assisted reproductive technologies for eastern hellbender (Cryptobranchus alleganiensis alleganiensis) conservation

Conservation breeding programs are an essential tool for augmenting wild populations of threatened amphibians. Unfortunately, captive breeding of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) is challenging owing to poor reproduction and could benefit from assisted reproductive technology (ART) and biobanking efforts to increase reproductive output and maintain genetic diversity. Here, we evaluated the transferability of ART protocols validated in tiger salamanders (Ambystoma tigrinum), such as hormone therapy for sperm collection and cryopreservation, to eastern hellbenders. A total of 24 juvenile male eastern hellbenders (age = 5–6 years old) were treated with a combination of human chorionic gonadotrophin (hCG) and gonadotrophin-releasing hormone (GnRH) at either a Low dose (300 IU kg⁻¹ bodyweight [BW] + 3 μg kg⁻¹ BW), Medium dose (900 IU kg⁻¹ BW + 9 μg kg⁻¹ BW), or High dose (1500 IU kg⁻¹ BW + 15 μg kg⁻¹ BW). Semen was collected on Days 3–7 postadministration and sperm were analyzed for progressive motility, nonprogressive motility, total motility, and sperm cell concentration. We then evaluated the effectiveness of two cryoprotectant treatments on preserving motility and morphology: (1) 2.5% dimethyl sulfoxide (DMSO) + 0.5% bovine serum albumin (BSA), and (2) 2.5% dimethylformamide (DMFA) + 0.5% BSA. Differences in post-thaw motility and morphology were analyzed using a generalized additive model for location, scale and shape (GAMLSS). Overall, 5 out of 24 (21%) hellbenders produced sperm in response to hormone treatment (Low, n = 1; Medium, n = 2; High, n = 2). The Medium treatment yielded sperm with the highest prefreeze total motility (23.7 ± 7.9%) compared with the Low (13.8 ± 4.9%) and High (17.7 ± 4.8%) treatments, as well as sperm with the highest prefreeze progressive motility (2.8 ± 1.8%), compared with the Low and High treatments (both <1%). The Low, Medium, and High treatments produced samples with an average concentration of 4.4 ± 2.4 × 10⁵, 0.6 ± 0.3 × 10⁵, and 0.9 ± 0.5 × 10⁵ sperm mL⁻¹, respectively, with Day 7 posthormone administration yielding the highest concentrations. A total of 101 straws, representing four males and both cryoprotectant treatments, were cryopreserved and 60 straws (32 DMSO and 28 DMFA) were thawed for analyzing motility and morphology metrics. We found that the DMFA (19.6 ± 7.1%) treatment resulted in sperm with higher (P < 0.05) post-thaw relative total motility compared with DMSO (7.9 ± 2.3%), though actual post-thaw progressive motility (<2% for both) was not different (P > 0.05) between the two treatments. There was also no difference (P > 0.05) in normal morphology between the DMSO-treated (60.1 ± 2.5%) and DMFA-treated (59.3 ± 2.7%) sperm samples after thawing, indicating that hellbender sperm morphology might not be as sensitive to cryoprotectant type as motility is. Ultimately, we were able to adapt hormone protocols from another caudate species to induce spermiation in eastern hellbenders, as well as cryopreserve the sperm. These results are encouraging for enhancing captive breeding and establishing a biobanking effort for this threatened species.