Stubborn Breeders

People have struggled to breed cheetahs for hundreds of years. The cats slinked through the ochre palaces of the Mughal Empire some 500 years ago as captive hunting pets. Yet there is only one record of their breeding in captivity before 1956 (7, 8). Zoos struggled to reliably breed cheetahs through the 1980s, even as wild populations saw steep declines from habitat loss and poaching (9).

Zoos overlooked natural history because of “a lot of historic baggage,” says Nadja Wielebnowski, conservation and research manager at the Oregon Zoo in Portland. Zoos began as menageries of exotic and often little-understood species. Although many facilities have made updates, most still have limited resources as they try to balance animal needs, construction costs, and the desires of the public, she says. The breeding setup of large, grassy cheetah yards at the Smithsonian in Virginia would probably look unkempt to tourists. But this kind of attention to natural history is the new guard in zoos.

Back in 1983, a landmark study began to explain why cheetahs wouldn’t breed in the typical zoo. It assessed genetic variation in 55 South African cheetahs at 47 genetic loci and in 155 proteins and showed that cheetahs have unusually low levels of polymorphisms at these sites, suggesting they have very low genetic variation—compared with not only other cats but with mammals in general. The finding was consistent with a genetic bottleneck, followed by inbreeding, at the end of the Pleistocene some 12,000 years ago (10).

In a concurrent analysis of the cheetahs’ sperm, the same study found that 70% was deformed, and sperm counts were 10 times lower than in other cats. It’s shocking cheetahs didn’t die out thousands of years ago, Saffoe says. After the 1983 study, “effectively the community said, well there’s your problem, that’s why they’re not breeding. They’ve got really jacked up sperm and they’re all identical twins.”

But then in 1992, researchers discovered the cats reproduced effectively in the wild despite their genetic bottleneck. Cheetahs could breed; they just weren’t doing it in captivity. An observational study of 20 wild female cheetahs on Tanzania’s Serengeti found that all but one had cubs (11). Zoos then began to scrutinize their own breeding programs, Saffoe says, asking how their efforts could mimic cheetahs’ natural history and reproductive biology in the wild.

To do so, researchers first needed to know how the cats breed in the wild. Research in the 1980s and 1990s found that wild females are solitary and roam large distances, whereas males live in coalitions of brothers in smaller territories. Females are induced-ovulators, meaning they don’t ovulate until after mating. Studies in the AZA-managed population have also found that females go into estrus every 12 to 14 days with occasional months-long periods of ovarian inactivity, possibly as constant preparation for the possibility of mating, to accommodate their solitary, induced-ovulator lifestyle. Females housed together can suppress ovulation unless the cats know and like each other. And despite their frequent cycles, females show few outward signs of estrus unless they’re interested in mating with a male (12).

This challenging reproductive biology inspired zoos to attempt assisted reproduction beginning in the 1990s via artificial insemination or embryo transfer. Artificial insemination—the introduction of sperm into the vagina or through the cervix into the uterus—is the more straightforward and more common method in zoo animals. But it’s difficult in cats because their sperm are propelled through the long horns of the wishbone-shaped uterus to the oviducts by muscle contractions. Anesthetized females don’t make these contractions, so sperm don’t meet eggs (13). Two breakthrough studies in the 1990s involved directly inseminating housecat uteruses and cheetah uteruses, successfully producing 15 cheetah pregnancies between 1992 and 2003 (14, 15).

But despite repeated attempts since 2003, it was the last successful cheetah artificial insemination ever conducted, says Bill Swanson, Director of Animal Research at the Cincinnati Zoo & Botanical Garden in Ohio. The reasons for the failures remain unclear. One possible explanation is a change in the manufacturer of the hormones that zoos use to induce ovulation before artificial insemination. But even after changing hormone sources, cheetahs still aren’t getting pregnant through artificial insemination. Even surgically putting sperm into the oviducts, onto the eggs—which has yielded pregnancies in ocelots, tigers, jaguars, and other wild cats—has not worked for cheetahs, Swanson says.

More recent research implies that a female’s age at first pregnancy strongly influences her reproductive success. For example, a study published this April followed 12 female cheetahs in European zoos throughout their lives and found that cats with a first pregnancy before the age of 5 1/2 had higher reproductive performance throughout their lives, even at advanced ages, compared with females with a first pregnancy at age 6 or older (16). The new findings agree with previous research led by the Smithsonian, suggesting that cheetahs tend to breed young and that captive programs should mimic the cats’ biological timing.

Despite these many challenges, the state of cheetah breeding is much improved from 30 years ago. Zoos now produce cubs every year, with nearly 400 cheetahs in North America’s captive breeding population today. Emulating the wild has helped, but researchers will need additional approaches to ensure the long-term genetic diversity of captive populations.

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Researchers bring males up this lover’s lane at the Smithsonian Conservation Biology Institute to sniff around a potential mate’s enclosure. The approach mimics some of the cats’ wild behaviors.

Wild Child

Beyond the high fence and heavy gate of the cheetah enclosure at the Smithsonian Conservation Biology Institute, a T-shaped dirt road is fringed by grassy yards. Male cheetahs live in four separate yards at the top of the T, while solitary females and mothers with cubs have their own yards at the bottom. This setup, called a lover’s lane, was pioneered by facilities in South Africa, The Netherlands, and the United States to mimic life in the wild, where females are solitary and mostly live out of sight and scent of the males, explains African and North American carnivore curator Adrienne Crosier.

On the day of a recent visit, Crosier walked along the fence line of each cheetah yard, clucking the cats’ names as she passed. A few cheetahs lifted their heads in seeming acknowledgment, and one young female, Zuri, jogged up and jumped against the fence, which Crosier translated to mean “this is my space, stay out.” Zuri was born at this facility two years ago, thanks to the AZA Species Survival Plan, a cooperative program that sets the annual pairing recommendations to maintain genetic diversity in the majority of the North American captive population. Member institutions transfer cheetahs among their facilities to match potential mates.

To breed a female cat in Virginia, Crosier’s team first moves that target female out of her pen and into a new yard nearer to the males to stimulate estrus with a change of scenery. After a few days, the team moves the female indoors with a treat and brings a male or males down lover’s lane to sniff around the female’s now-empty territory. In the wild, he’d smell her before he saw her, Crosier explains. If the male stutter barks, a mating vocalization like a pigeon’s coo, “then he’s allowed to see her,” Crosier says. If he doesn’t bark, then he just goes home. “Today,” she explains, “is not the day.”

But even though the AZA suggests pairing certain males and females to make good genetic matches, cheetahs are picky, and about one-third of recommended pairs don’t breed, Crosier says. One way that ongoing research attempts to improve reproduction is through greater understanding of mate choice. A study published last June, for example, tested scent as a possible indicator of mate preference, available before cats meet (17). When researchers presented 12 female cheetahs with the scent of urine from 17 males, the females spent the most time investigating scents from the males least-related to them, regardless of the males’ testosterone levels, the female’s age, or her point in the estrus cycle. Whether a female’s urine preference actually predicts her mate preference has yet to be tested. But if it does, it could be a useful future indicator of mating success before cats are transferred between zoos, which is expensive and stressful.

A Helping Hand

In the meantime, Crosier is pushing the envelope in assisted reproduction. In still-unpublished research, she led the first-ever embryo transfer in the cats this summer, taking eggs from a 7-year-old donor female, fertilizing them, then implanting the embryos in a young female that’s a proven breeder, she explains. Before surgery, both donor and recipient females received hormone injections to stimulate estrus.

“The challenging part,” Crosier says, “is that they’re cheetahs, not domestic cats, not animals you can just walk up to and give an injection.” Behaviorally conditioning wild animals to tolerate the process, which included carefully timed shots before surgery to prepare the reproductive system and a trip in a carrying crate from the cheetahs’ pens to the operating room, demanded cooperation by multiple cats, keepers, and veterinarians, making it the most logistically challenging part of the procedure. Crosier says, “you have to have all the stars align.”

Crosier had previously demonstrated the first two steps of the embryo transfer procedure. In 2011, she and collaborators laparoscopically extracted about 300 eggs from 29 donor females, ranging in age from 2 to 15, and successfully fertilized them with thawed, frozen semen, creating embryos (6). The final step, transferring donor embryos into a recipient was a world-first this summer. Unfortunately, the pregnancy didn’t take. But just completing this complex and carefully timed process is a success, Crosier says. “Now we know we can do it.”

In the future, embryo transfer could create new opportunities to breed cheetahs, she says, especially for older cats without offspring, whose genes are not well-represented in the population. The Smithsonian zoo is already banking cheetah sperm but doesn’t currently freeze eggs because egg cells are larger and more vulnerable to punctures by ice crystals during the thawing process. Just as breeding studies may use urine as a proxy for mate choice before shipping cats between zoos, frozen semen and eggs are also biological materials that could make breeding more efficient and cost-effective while minimizing transit for the cats.

“We have so many cheetahs in our North American population that behaviorally won’t participate in the breeding program,” Crosier says. “Being able to reproduce those cats is what’s so important, because those genetics can’t be captured.”

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Adrienne Crosier led the first complete embryo transfer in cheetahs this summer. Her team moved embryos from a donor female into a recipient, though no pregnancy resulted. Image credit: Roshan Patel/Smithsonian Conservation Biology Institute.

Larger Lessons

Cheetahs are not the only captive cats that turn up their whiskers at potential mates. “Most cats are difficult,” says reproductive biologist Pierre Comizzoli of the Smithsonian’s National Zoo in Washington, DC, and conservation institute in Front Royal. The challenges are the same: replicating their natural history and understanding their reproductive biology.

Clouded leopards, for example, spend much of their lives in trees, an arboreal lifestyle that’s hard to mimic in zoos, explains Wielebnowski at the Oregon Zoo. The cats are also choosy, and mismatched mates sometimes kill each other. Wielebnowski analyzed the stress responses of 74 cats and found that clouded leopards housed in enclosures out of sight of the public and with higher places to climb had lower concentrations of fecal stress hormones, suggesting lower-stress husbandry measures for captive breeding, according to the study (18).

But even with husbandry improvements, natural breeding often fails for captive wildcat species. “That’s why we need the possibility of artificial insemination,” Comizzoli says. The technique is unreliable in not only cheetahs but many cat species—and for similarly perplexing reasons. No cat species is consistently managed using artificial insemination today, he says, but the technique has produced at least one pregnancy in all eight big cats, the last of which were jaguars, born this March in Brazil. It’s only worked three times in clouded leopards since 1992.

To understand the failings of artificial insemination, Comizzoli studies the basic physiology of cat pregnancy, including embryo implantation in the uterus. “We don’t really know exactly how it works and when it’s happening,” he says, even in domestic cats.

Comizzoli coauthored a study this July using newly developed model uteruses to observe the effects of estrus hormones (19). First, he and colleagues collected the uteruses of spayed housecats, specifically the epithelial cells lining the inside surface of each one. Researchers kept the epithelial cells alive and in the uteruses’ three-dimensional shape in plastic dishes. These model uteruses survived for at least two weeks in incubators, the study found.

The researchers then treated the model uteruses with synthetic hormones to replicate the natural estrus cycle of domestic cats. They observed changes in cellular structure consistent with those already known from pregnant cats, suggesting the model uteruses have promise as a new tool to study the earliest stages of pregnancy, Comizzoli says.

However, the hormones did not induce some changes in gene expression known from live cats, suggesting that other, still-unknown factors also prepare the uterus for embryo implantation. Future studies in these models may identify those factors, Comizzoli says, possibly revealing previously unknown prerequisites for pregnancy that could explain why artificial insemination hasn’t worked since 2003 in cheetahs and is inconsistent in other cats.

Efforts to improve assisted reproduction and natural breeding programs in cheetahs and other wildcats offer lessons beyond cats for many zoo animals, Comizzoli says. Captive birds, rodents, large carnivores, marsupials, fish, and insects can all be choosy about their mates and breed more successfully in programs that offer mate choice rather than assigning partners, according to a study published last fall (20). It concluded that greater use of behavioral research in species survival plans could raise breeding success across many captive species.

Assisted reproduction, says Comizzoli, must be tailored to individual species, which means researchers need to know how reproduction varies, down to the fine detail of the molecular and physiological level, even in closely related species. “We’ve done that in very few species, unfortunately,” he says.

As for cheetahs, improved breeding efforts promise to preserve what little genetic diversity the species has left. Factoring natural history into reproductive programs continues to help as cheetah-breeding institutions find new ways to mimic the wild. One ongoing trend is a shift away from breeding in zoos, toward keeping reproductively important cheetahs in a handful of large breeding facilities such as the Smithsonian Conservation Biology Institute. The sprawling grounds can easily house many cats, offering a lineup of potential partners that better mimics mate choice in the wild.

Great mate choice encourages breeding but doesn’t guarantee it. If Crosier’s team succeeds with embryo transfer, it could allow even celibate cats to pass their genes into the next generation. Zoos wouldn’t have to move animals to breed them and could instead send sperm and embryos around the world to maximize the genetic diversity of global captive populations, and perhaps even bring sperm and eggs from wild cheetahs into captive cats, Crosier says. Assisted reproduction is one vast, next frontier of captive breeding. If it works, she says it would offer “many more options for genetic management of the entire species around the world.”