The Smithsonian Conservation Biology Institute was the first organization outside of Wyoming to receive offspring from the 18 surviving black-footed ferrets in 1987. Since then, our scientists have made significant breakthroughs in understanding the unique reproductive biology and breeding of the species. Because of the small number of original survivors and the need to maintain high genetic diversity in the population, SCBI filled a critical need by developing successful artificial insemination techniques, including using frozen-thawed sperm. SCBI achieved a major milestone in successfully producing kits via artificial insemination using sperm that had been frozen in liquid nitrogen for more than 20 years. SCBI scientists have developed methods for collecting, freezing and artificially inseminating the sperm of these small mammals. They have also gained extensive knowledge about the ferrets’ reproductive endocrinology by measuring hormone profiles in the animals’ feces. Learning and integrating this information eventually allowed increasing reproductive success both by natural and artificial breeding.
As a result of SCBI’s diligence, partnerships and innovative science, more than 670 black-footed ferrets have been born at the Zoo’s facility in Front Royal, Va. More than 200 have been reintroduced to their natural habitat in one of the National Zoo’s most successful conservation endeavors to date. About 1,000 black-footed ferrets live in the wild today.
Since 1995, the Smithsonian Conservation Biology Institute has focused on breeding black-footed ferrets that have been unable to reproduce naturally. These individuals are extremely valuable genetically, but are often unable to mate with their computer-designated mate due to sexual incompatibilities. According to the Association of Zoos and Aquariums’ Species Survival Plan for black-footed ferrets, nearly half of the population of males fails to sire offspring through natural breeding, thus limiting genetic variation in the population. Many male ferrets display aggression toward females or can’t figure out the proper positioning for mating. Artificial insemination (AI) offers the perfect alternative for ensuring that valuable, but underrepresented, ferrets reproduce. The development of the AI procedure was led by an SCBI scientist, the late Dr. JoGayle Howard, who was a pioneer in wildlife infertility. Howard developed a laparoscopic technique (using a fiber optic endoscope) to deposit the sperm through the female’s abdominal wall and directly into the uterus. To date, 139 kits have been produced by this laparoscopic artificial insemination method. The 70 to 80 percent rate of pregnancy from artificial insemination with fresh sperm has kept pace with that of natural breeding.
Ferrets are induced ovulators, which means that mating itself causes the ovary to release its eggs. So SCBI researchers first had to develop a hormone treatment that would artificially cause ovulation to occur. During the actual AI, the female is anesthetized and a small incision made at the umbilicus through which a small diameter laparoscope is inserted. The fiberoptics in the endoscope allows the entire abdominal cavity to be scanned and for an insemination catheter to be inserted into the uterine horns to inject the 100 microliters of sperm solution. The process is repeated in each of the two uterine horns.
Because there were so few surviving wild animals to work with, researchers have to carefully match animals to retain as much gene diversity as possible. As some ferrets failed to mate naturally, one of the major accomplishments of SCBI’s scientists was developing AI for such a small animal. The eventual successful procedure involved using a fiber optic endoscope to pass the sperm through the abdominal wall and directly into the uterus. More than 145 kits have been produced by AI, including using sperm that have been frozen and stored in liquid nitrogen (at minus 196 C) for many years. One of the Institute’s major milestones was producing kits from sperm collected and frozen from one of the original wild-caught males (named Scarface) 20 years later and long after his death.
SCBI researchers first developed assisted-reproduction techniques for black-footed ferrets using domestic ferrets (Mustela putorius) and later Siberian polecats (M. eversmanni), their closest relatives, as models. Like Siberian polecats, black-footed ferrets display few visible signs of estrus, veiling the proper timing for artificial insemination procedures. Instead, SCBI researchers test the readiness of female ferrets for ovulation by monitoring changes in their vaginal epithelial cells. When these cells demonstrate the readiness of the ovary’s follicles to release an egg, veterinarians administer the hormone to the female that stimulates ovulation. Howard discovered that the anesthesia necessary during artificial insemination procedures prevents sperm from being naturally transported through a ferret’s reproductive tract.
Reproductive technologies are also used routinely for management, including checking male fertility before mating, overcoming infertility and ensuring that every genetically valuable individual reproduces to maintain as much genetic diversity as possible.
Poor semen quality, perhaps due to inbreeding, only partly explains the failure of many natural breeders to conceive. Research at SCBI has shown that one-year-old ferret males that actively mate with females normally don’t produce sperm until near the end of their first breeding season. Yet the act of breeding can induce ovulation, triggering a biological response known as “pseudo-pregnancy” in which female ferrets ovulate without conceiving and refuse to mate with other males then actually begin building a nest. Ferrets breed during one short period each spring, so if a young male mates with a female, the female may lose her opportunity to bear offspring for an entire year. Fertility in female ferrets drops off dramatically after their third year of life, so the window of opportunity is narrow.
SCBI maintains the only genome resource bank for black-footed ferrets that preserves frozen semen from genetically valuable males. This semen is used for artificial insemination by Zoo scientists to maintain and even enhance genetic diversity by infusing valuable genes into the population. Each year, sperm are collected from male ferrets and frozen. Depending on its genetic value, this sperm may be used five to 20 years in the future.
In species that have short life spans like the black-footed ferret, the use of cryopreserved, or frozen, sperm extends an individual’s reproductive life. The bank’s contents help maintain and even enhance genetic diversity by infusing new genes into the population. A genetically healthy and diverse population has a greater chance of survival in the wild. The bank also serves as insurance against catastrophes in the wild populations, such as disease outbreak.
As the total number of black-footed ferrets has blossomed through the breeding program to more than 670, scientists including SCBI’s Jon Ballou, a researcher specializing in small population biology, have had to monitor carefully the ferret family tree in order to preserve as much genetic variety as possible, and stave off the ill effects of inbreeding.
In 1986, the U.S. Fish & Wildlife Service asked Ballou to provide consulting for developing the black-footed ferret captive-breeding program. Ballou had engineered software that uses algorithms to pair mates as distantly related as possible. He and other researchers originally set out to maintain 95 percent of the genetic diversity among black-footed ferrets for 50 years. The 18 left represented the genetic diversity of 9 to 10 unrelated individuals, which, under ideal circumstances, would allow scientists to maintain more than 90 percent of the species’ genetic diversity. However, as time passed, some lineages of the surviving wild ferrets did not breed as well as others. So about 85 to 87 percent of the diversity remains today. Ballou remained the genetic advisor to the black-footed ferret breeding program until about 1998.
Posted September 2011