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°C/−321°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 × 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.

The black-footed ferret (Mustela nigripes), which was extirpated from its native North American prairie habitat during the 1980s, is being reintroduced to the wild because of a successful captive-breeding program. To enhance propagation, the reproductive biology of this endangered species is being studied intensively. The typical life span of the black-footed ferret is approximately 7 yr. Female fecundity declines after 3 yr of age, but the influence of age on male reproduction is unknown. In this study, testis volume, seminal traits, sperm morphology, and serum testosterone were compared in 116 males from 1 to 7 yr of age living in captivity. Results demonstrated that testes volume during the peak breeding season was similar (P > 0.05) among males 1 to 5 yr of age, reduced (P < 0.05) among males 6 yr of age, and further reduced (P < 0.05) among males 7 yr of age. Motile sperm/ejaculate was similar in males 1 to 6 yr of age but diminished (P < 0.05) in those 7 yr of age. Males at 6 and 7 yr of age produced fewer (P < 0.05) structurally normal sperm than younger counterparts; however, serum testosterone concentrations were not reduced (P > 0.05) in older males. Histological comparison of testicular/epididymal tissue from 5- and 7-yr-old black-footed ferrets confirmed that the interval between these two ages may represent a transitional period to reproductive senescence. In summary, functional reproductive capacity of male black-footed ferrets exceeds that of females by at least 2 yr. Testes and seminal quality are indistinguishable among males 1 to 5 yr of age, with progressive reproductive aging occurring thereafter.

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In an attempt to save the species from extinction, the last remaining 18 black-footed ferrets (Mustela nigripes) were trapped up from the wild to initiate a captive breeding program. Nearly 30 years later more than 8,000 black-footed ferrets have been produced in captivity and approximately 4,100 animals have been reintroduced into 20 sites in eight US states (Arizona, New Mexico, Utah, Colorado, Kansas, Wyoming, South Dakota and Montana), Mexico and Canada. However, full recovery of the species has yet to be achieved, mainly due to limited viable habitat, disease and reduced fecundity. This chapter will highlight the advances in the black-footed ferret recovery program over the last 10 years including: (1) adaptive management techniques employed for the captive population; (2) development of new reintroduction sites and associated challenges facing wild black-footed ferrets; and (3) optimization of assisted reproductive techniques to secure the future of this rare species.

For selected species, conservation breeding has become integrated into recovery plans, most often through the production of offspring for reintroduction into nature. As these programs increase in size and scope, it is imperative that conservation managers retain the biological integrity of the species. This study investigated the causes of morphological changes that are known to occur in black-footed ferrets (Mustela nigripes) maintained ex situ. In a previous study, ferrets maintained in captivity were 5-10% smaller in body size than pre-captive, in situ animals. In the present study, the authors compared nine morphological characters among ex situ animals and their in situ descendants. Within the ex situ population, cage types were compared to determine whether housing influenced morphometry. Black-footed ferrets born to reintroduced individuals quickly returned to their pre-captive size suggesting that a diminutive morphology ex situ did not have a genetic basis. Furthermore, cage type affected overall body size and shape; ulnas and tibias were as much as 9% shorter for ex situ animals. The authors hypothesise that small cage size and environmental homogeneity inhibit the mechanical stimuli necessary for long bone development. These findings have ramifications for ex situ managers who need to create artificial captive settings that promote natural physical development. In the absence of such an environment, 'unnatural' morphologies can result that may contribute to poor fitness or perhaps even domestication.

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