Recovery of gene diversity using long-term cryopreserved spermatozoa and artificial insemination in the endangered black-footed ferret

One of the most significant challenges in the recovery of threatened species is the ability to maintain genetic diversity, avoid inbreeding and sustain population health and reproduction. Assisted reproductive techniques, including artificial insemination (AI), have been touted for decades as approaches that could contribute to the demographic and genetic management of rare species. Here, we report the first successful integration of AI with frozen semen into a formal recovery program and the positive impact on genetic diversity for the critically endangered black-footed ferret Mustela nigripes. Techniques developed in the taxonomically related domestic ferret Mustela putorius furo and Siberian polecat Mustela eversmannii were applied over time to selected black-footed ferrets, including semen banking from six of the last 18 survivors. After evaluation, processing and storage in liquid nitrogen (-196 degrees C/-321 degrees F), for as long as 20 years, sperm samples were thawed and transabdominally inseminated into the uterine horns of female conspecifics. Eight black-footed ferret offspring were produced using thawed sperm samples (including after two decades of cryopreservation) with inseminates containing as few as 3.4 x 10(6) motile spermatozoa. The incorporation of these offspring and/or their descendants into the ex situ breeding program prevented heterozygosity loss in the population and actually enhanced gene diversity (GD) significantly by 0.2% and lowered measures of inbreeding by 5.8%. This study demonstrates the utility and genetic diversity benefits of applying AI with cryopreserved spermatozoa 20 generations removed from the contemporary population for a wild animal revival program.