Enhanced Reproductive Efficiency and Pregnancies After Artificial Insemination in Black-Footed Ferrets
The black-footed ferret (Mustela nigripes) is an endangered species, once considered extinct until a remnant population was discovered in Wyoming in 1981. Between 1985 and 1987, the last remaining 18 black-footed ferrets were captured to begin a multi-institutional propagation program. Since 1987, ∼2,200 ferrets have been produced by natural breeding, and ∼300 animals currently reside in 7 breeding facilities (National Black-Footed Ferret Conservation Center, Laramie, WY; National Zoological Park’s Conservation & Research Center, Front Royal, VA; Omaha’s Henry Doorly Zoo, Omaha, NE; Cheyenne Mountain Zoological Park, Colorado Springs, CO; Louisville Zoological Garden, Louisville, KY; The Phoenix Zoo, Phoenix, AZ; Metropolitan Toronto Zoo, Ontario, Canada). The ability to produce ferrets in captivity has allowed for the reintroduction of black-footed ferrets in four states (Wyoming, Montana, South Dakota, Arizona). To date, ∼80 black-footed ferrets survive in the wild and young have been produced from reintroduced animals. Although captive breeding is successful, the goal of the recovery program (1,500 breeding ferrets in >10 free-ranging locations by year 2010) will not be achieved at the current rate of propagation. Recent assessment of breeding records revealed that a remarkably high proportion of males (∼50%) fail to reproduce in captive breeding situations due to behavioral incompatibility. Additionally, certain original ferret “founders” are poorly represented in the current population, and some of their descendants have never sired young due to aggression towards females. These issues prompted the use of assisted reproduction to improve black-footed ferret propagation to meet reintroduction demands and maintain maximum genetic diversity. A Black-Footed Ferret Genome Resource Bank (repository of cryopreserved sperm) was established to preserve valuable germ plasm. The objectives of the program are to use an intrauterine artificial insemination (AI) technique with fresh or cryopreserved semen to: 1) breed behaviorally incompatible animals; 2) produce offspring from genetically-valuable ‘non-proven’ males that have failed to reproduce; and 3) enhance founder representation in under-represented lineages. As a research tool, AI also is being applied to Siberian polecat (Mustela eversmanni) females using black-footed ferret semen to produce hybrids for testing the efficacy of a new “killed” canine distemper vaccine. For these procedures, electroejaculates are diluted in an egg-yolk cryodiluent and used either immediately for AI or pellet frozen on dry ice. Females in natural estrus with maximum vulvar swelling and >90% cornified vaginal epithelial cells are given 90 IU human chorionic gonadotropin to induce ovulation. For AI, females are laparoscopically inseminated in utero with fresh or frozen-thawed semen. In 1996 and 1997, a total of 20 females (17 black-footed ferrets and 3 Siberian polecats) maintained at the Conservation & Research Center (14 black-footed ferret females) or National Black-footed Ferret Conservation Center (3 black-footed ferret and 3 Siberian polecat females) were inseminated with fresh (n=16) or frozen-thawed (n=4) semen from black-footed ferret males meeting at least one criterion listed in the objectives above. In spring of 1996, 5 of 6 (83.3%) black-footed ferrets inseminated with fresh semen became pregnant and produced 16 kits (mean litter size, 3.2). In spring of 1997, 6 of 8 (75.0%) black-footed ferrets inseminated with fresh semen and 2 of 3 (66.7%) inseminated with frozen-thawed semen became pregnant, resulting in a total of 19 kits (mean litter size, 2.4). Pregnancies also were achieved in 2 of 3 (66.7%) Siberian polecat females inseminated with fresh or frozen-thawed black-footed ferret semen; a total of 10 hybrid kits (5 kits/litter) were produced for the vaccine research. These results demonstrate that: 1) laparoscopic intrauterine AI using fresh or cryopreserved black-footed ferret semen is successful (overall pregnancy rate, 75%) for enhancing reproductive efficiency; and 2) the integration of assisted reproduction and a Genome Resource Bank can play a beneficial role in the management and recovery of the endangered black-footed ferret.