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

Abstract

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.

From petri dishes to politics – a multi-pronged approach is essential for saving endangered species

“…Additionally, sperm and embryos preserved from individuals that fail to reproduce in their lifetimes could be vehicles for infusing their unrepresented genes into populations long after death. The feasibility of this approach has been demonstrated recently in the Indian rhino 9 , Pallas’ cat 10 and black-footed ferret 11 . Furthermore, despite best efforts, physical and behavioral obstacles occasionally interfere with natural mating between optimal genetic matches, and ART may be needed as a remedy.…”

The black-footed ferret recovery program: a strong advocate for establishing semen banking programs as support tools for small population welfare

“…By having the foresight in the early days to cryopreserve semen samples from a few of the original males, it has been possible to minimize the loss of genetic diversity within the extant population. This is clear in Howard et al (2016), as animals bred from stored semen showed lower inbreeding coefficients and mean kinships than other individuals in the modern ex situ population. The establishment of a species-specific genetic resource bank and its use for genetic supplementation provides tangible and useful support to species conservation programs.…”

“…These colleagues shared the same vision and sense of optimism; back in the early to mid-1980s, it would have been all too easy for sceptics to have pointed out, citing a series of valid reasons, that their mission was hopelessly doomed to failure. Thus, Howard et al (2016) vindicates the enthusiasm shown by the originators of the black-footed ferret program.The success of this comprehensive program can be traced back to the initial establishment of a suitable semen freezing and artificial insemination (AI) protocol for the black-footed ferret (for reviews, see Howard, Marinari & Wildt, 2003;Santymire et al, 2014). Without these technologies, it is much less likely that the recovery program would have succeeded using conventional captive breeding techniques alone.…”

“…Without these technologies, it is much less likely that the recovery program would have succeeded using conventional captive breeding techniques alone. Howard et al (2016) highlights the genetic benefit to the black-footed ferret population that has been gained through the use of AI. By having the foresight in the early days to cryopreserve semen samples from a few of the original males, it has been possible to minimize the loss of genetic diversity within the extant population.…”

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The black-footed ferret rescue and reintroduction program described in this issue by Howard et al (2016) represents an outstanding, and possibly unique, conservation project. It has involved genuinely valuable collaboration between veterinarians, field biologists, ecologists, epidemiologists, regulatory authorities and reproductive technologists, all of whom focused their expertise on solving a single problem, the rescue of the black-footed ferret from extinction.

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