Assessment of ultra-rapid freezing as a simplified, field-friendly technique for semen cryopreservation in wild ocelots (Leopardus pardalis) and bobcats (Lynx rufus) in southern Texas

Ocelot ( Leopardus pardalis ) populations in southern Texas have shown declines in genetic variability over time. Assisted reproductive technologies (ARTs 1 1 Assisted Reproductive Technologies ), such as semen cryopreservation and artificial insemination (AI 2 2 Artificial Insemination ), can help improve species sustainability by preserving genetic diversity and connecting populations by transport of frozen gametes. Traditionally, felid semen has been cryopreserved by slow freezing in plastic straws (STRAW) for liquid nitrogen storage. However, another technique, ultra-rapid freezing (URF 3 3 Ultra- rapid Freezing ), would decrease the time and effort needed to preserve samples and potentially facilitate cryopreservation in the field. The goal of this study was to compare the effectiveness of URF to traditional STRAW freezing with two wild felid species living in southern Texas. Semen was collected by urethral catheterization (UC 4 4 Urethral Catheterization ) and electroejaculation (EEJ 5 5 Electroejaculation ) and frozen using both cryopreservation methods from 12 free-ranging adult felids (n=6 ocelots; n=6 bobcats ( Lynx rufus )). Post-thaw, sperm samples were assessed for progressive motility, acrosomal integrity, and heterologous in-vitro fertilization (IVF 6 6 In-Vitro Fertilization ) of domestic cat oocytes. For both species, sperm acrosomal integrity, percent progressive motility, and rate of forward progression declined (p<0.001) over time, with no difference (p>0.05) between cryopreservation methods. Frozen-thawed spermatozoa from both species fertilized mature domestic cat oocytes (range, 8.3 – 100%), and oocyte cleavage percentage did not differ (p>0.05) between cryopreservation techniques. Our initial results suggest that URF, in combination with UC, may allow wildlife veterinarians to routinely collect and bank semen samples from free-ranging cats for conservation purposes.