30 Development of hormone therapy and cryopreservation techniques for the genetic management of spotted salamanders (Ambystoma maculatum)

Several assisted reproductive technologies (ARTs) such as hormone therapy, ultrasound, IVF, and the cryopreservation of spermatozoa have all been established for the common tiger salamander (Ambystoma tigrinum). Unfortunately, the transferability of these technologies to other species within the same genera is unknown. It is critical to understand how these technologies can be transferred to other threatened ambystomid species in captive breeding and conservation programs. Here, we applied proven technologies in the tiger salamander to the closely related spotted salamander (Ambystoma maculatum). The objectives of this study were to (1) test two exogenous hormone treatments and a control via intramuscular injection on spotted salamander sperm production, and (2) evaluate post-thaw sperm parameters after treatment with one of two sperm cryoprotectants. To address the first objective, salamanders were injected with (1) 0.1 μg g⁻¹ bodyweight (BW) gonadotrophin-releasing hormone (GnRH), (2) 0.1 μg g⁻¹ BW (GnRH) + 0.4 μg g⁻¹ BW human chorionic gonadotrophin (hCG), or (3) saline control. To address the second objective, salamander sperm samples were treated with (1) 10% dimethyl sulfoxide (DMSO) + 0.5% bovine serum albumin (BSA), or (2) 10% dimethylformamide (DMFA) + 0.5% BSA. Following sperm collection with the two hormone treatments, GnRH (n = 7), GnRH + hCG (n = 7), or control (n = 5), prefreeze sperm samples were evaluated for concentration, velocity of movement, progressive motility, stationary motility, and total (progressive + stationary) motility. Samples were then mixed 1:1 with one of the two cryoprotectant treatments and frozen in liquid nitrogen vapor at a height of 10 cm for 10 min. Post-thaw sperm parameters were evaluated similar to the prefreeze parameters listed above, with the inclusion of morphology. A generalized additive model for location, scale, and shape was used for regression modeling, with hormone or cryoprotectant treatment as fixed effects and individual as a random effect. Results showed that 43% of the males treated with GnRH produced sperm compared with 71% for GnRH + hCG and 0% for saline treatment. There were no differences (P > 0.05) between the GnRH and GnRH + hCG treatment groups for prefreeze sperm concentration, velocity of movement, progressive motility, and total motility. In comparison, there was no difference (P > 0.05) between DMSO and DMFA post-thaw total motility (30% vs. 30%, respectively) or normal morphology (62% vs. 60%, respectively), although there was a difference (P < 0.05) in the percentage of abnormal heads between DMSO and DMFA (2.8 ± 0.8% vs. 1.0 ± 0.4%, respectively). Here, we show that ARTs developed in the tiger salamander were successfully transferred to the spotted salamander, resulting in sperm production and release followed by cryopreservation with post-thaw motility. Furthermore, this study suggests that standardized protocols for ambystomids can be used for the genetic management of other closely related salamanders of conservation concern.