NEWS FEATURE

Getting the world’s fastest cat to breed with speedCheetahs once rarely reproduced in captivity. Today, cubs are born every year in zoos.

Breeding programs have turned their luck around—but they aren’t done yet.

Amy McDermott, Science Writer

Three fluffy cheetah cubs lounge under a tree at theSmithsonian Conservation Biology Institute, in FrontRoyal, Virginia. Off exhibit from the public, the 1-year-old cats roam a large and grassy enclosure behind ahigh, chain-link fence. They are the newest of 56 cubsborn at this facility since 2007 (1).

Breeding cheetahs to produce cubs such as thesewas once famously difficult. The biggest problem wasthat zoos were “not paying attention to natural his-tory,” says Craig Saffoe, curator of lions, tigers, andbears at the Smithsonian’s National Zoo in Washing-ton, DC, who in 2004 led the team that producedthe zoo’s first litter. Tattoos of cheetahs now adornhis forearms.

Zoos once tried to breed cheetahs very differently,he says, keeping males and females together ratherthan mimicking their lifestyle in the wild. Out on thesavannah, females are solitary and roam vast distances.Males defend smaller territories and mate when fe-males pass through (2). Designing exhibits to reflectthat natural history has been a major part of breedingsuccess over the last three decades, Saffoe says.

Success has also come from a better understand-ing of cheetah reproductive physiology (3). Reproduc-tive studies in the last 30 years have improvedbreeding by increasing understanding of males’ poorsperm quality, females’ ovulation cycle, and matechoice. Zoos now consistently produce cubs. The

Cheetahs have a reputation as stubborn breeders. But the study of cats such as Fatir at the Smithsonian’s breedingfacility suggests that a better understanding of natural history and reproductive physiology can dramatically improvecaptive reproduction. Image credit: Mehgan Murphy/Smithsonian Conservation Biology Institute.

Published under the PNAS license.

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majority of North American captive cheetahs are man-aged by the Association of Zoos & Aquariums (AZA),whosemember institutions include the Smithsonian’sNational Zoo and other zoos and facilities around thecountry. The AZA-managed cheetah population aver-ages 43 births per year.

Despite those successes, breeding programs arehitting a wall of sorts, and further progress will be keyto maintaining and expanding the genetic diversityof captive populations. More than one-third of AZA-managed captive cheetahs still don’t reproduce due to amix of health, behavior, and reproductive problems (4,5). Between 1970 and 2011, more than 70% of femalesand more than 80% of males died cubless (6).

Assisted reproduction would help maintain thediversity of captive populations, but success has beenmixed. Artificial insemination hasn’t worked well in re-cent years. Embryo transfer has shown signs of prog-ress, however. This past June, researchers completedthe first-ever successful transfer in cheetahs.

All these efforts underscore a common theme: Whenit comes to breeding cheetahs in captivity, natural historymatters—a lesson that applies beyond the race to savecheetahs to many other rare, captive cats.

Stubborn BreedersPeople have struggled to breed cheetahs for hun-dreds of years. The cats slinked through the ochrepalaces of the Mughal Empire some 500 years ago ascaptive hunting pets. Yet there is only one record oftheir breeding in captivity before 1956 (7, 8). Zoosstruggled to reliably breed cheetahs through the1980s, even as wild populations saw steep declinesfrom habitat loss and poaching (9).

Zoos overlooked natural history because of “a lotof historic baggage,” says Nadja Wielebnowski, con-servation and research manager at the Oregon Zoo inPortland. Zoos began as menageries of exotic andoften little-understood species. Although many facilitieshavemade updates, most still have limited resources as

they try to balance animal needs, construction costs,and the desires of the public, she says. The breedingsetup of large, grassy cheetah yards at the Smithsonianin Virginia would probably look unkempt to tourists. Butthis kind of attention to natural history is the new guardin zoos.

Back in 1983, a landmark study began to explainwhy cheetahs wouldn’t breed in the typical zoo. Itassessed genetic variation in 55 South African chee-tahs at 47 genetic loci and in 155 proteins and showedthat cheetahs have unusually low levels of polymor-phisms at these sites, suggesting they have very lowgenetic variation—compared with not only othercats but with mammals in general. The finding wasconsistent with a genetic bottleneck, followed byinbreeding, at the end of the Pleistocene some12,000 years ago (10).

In a concurrent analysis of the cheetahs’ sperm, thesame study found that 70% was deformed, and spermcounts were 10 times lower than in other cats. It’sshocking cheetahs didn’t die out thousands of yearsago, Saffoe says. After the 1983 study, “effectively thecommunity said, well there’s your problem, that’s whythey’re not breeding. They’ve got really jacked upsperm and they’re all identical twins.”

But then in 1992, researchers discovered the catsreproduced effectively in the wild despite their ge-netic bottleneck. Cheetahs could breed; they justweren’t doing it in captivity. An observational study of20 wild female cheetahs on Tanzania’s Serengeti foundthat all but one had cubs (11). Zoos then began toscrutinize their own breeding programs, Saffoe says,asking how their efforts could mimic cheetahs’ naturalhistory and reproductive biology in the wild.

To do so, researchers first needed to know how thecats breed in the wild. Research in the 1980s and1990s found that wild females are solitary and roamlarge distances, whereas males live in coalitions ofbrothers in smaller territories. Females are induced-ovulators, meaning they don’t ovulate until aftermating. Studies in the AZA-managed population havealso found that females go into estrus every 12 to14 days with occasional months-long periods of ovar-ian inactivity, possibly as constant preparation for thepossibility of mating, to accommodate their solitary,induced-ovulator lifestyle. Females housed togethercan suppress ovulation unless the cats know and likeeach other. And despite their frequent cycles, femalesshow few outward signs of estrus unless they’re in-terested in mating with a male (12).

This challenging reproductive biology inspiredzoos to attempt assisted reproduction beginning inthe 1990s via artificial insemination or embryo transfer.Artificial insemination—the introduction of sperm intothe vagina or through the cervix into the uterus—is themore straightforward and more common method inzoo animals. But it’s difficult in cats because theirsperm are propelled through the long horns of thewishbone-shaped uterus to the oviducts by musclecontractions. Anesthetized females don’t make thesecontractions, so sperm don’t meet eggs (13). Twobreakthrough studies in the 1990s involved directly

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

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inseminating housecat uteruses and cheetah uteruses,successfully producing 15 cheetah pregnancies be-tween 1992 and 2003 (14, 15).

But despite repeated attempts since 2003, it wasthe last successful cheetah artificial insemination everconducted, says Bill Swanson, Director of Animal Re-search at the Cincinnati Zoo & Botanical Garden inOhio. The reasons for the failures remain unclear. Onepossible explanation is a change in the manufacturerof the hormones that zoos use to induce ovulationbefore artificial insemination. But even after changinghormone sources, cheetahs still aren’t getting preg-nant through artificial insemination. Even surgicallyputting sperm into the oviducts, onto the eggs—which has yielded pregnancies in ocelots, tigers, jag-uars, and other wild cats—has not worked for chee-tahs, Swanson says.

More recent research implies that a female’s age atfirst pregnancy strongly influences her reproductivesuccess. For example, a study published this April fol-lowed 12 female cheetahs in European zoos through-out their lives and found that cats with a first pregnancybefore the age of 5 1/2 had higher reproductiveperformance throughout their lives, even at advancedages, compared with females with a first pregnancy atage 6 or older (16). The new findings agree with pre-vious research led by the Smithsonian, suggesting thatcheetahs tend to breed young and that captive pro-grams should mimic the cats’ biological timing.

Despite thesemany challenges, the state of cheetahbreeding is much improved from 30 years ago. Zoosnow produce cubs every year, with nearly 400 cheetahsin North America’s captive breeding population today.Emulating the wild has helped, but researchers willneed additional approaches to ensure the long-termgenetic diversity of captive populations.

Wild ChildBeyond the high fence and heavy gate of the cheetahenclosure at the Smithsonian Conservation BiologyInstitute, a T-shaped dirt road is fringed by grassyyards. Male cheetahs live in four separate yards at thetop of the T, while solitary females and mothers withcubs have their own yards at the bottom. This setup,called a lover’s lane, was pioneered by facilities inSouth Africa, The Netherlands, and the United Statesto mimic life in the wild, where females are solitary andmostly live out of sight and scent of the males, ex-plains African and North American carnivore curatorAdrienne Crosier.

On the day of a recent visit, Crosier walked alongthe fence line of each cheetah yard, clucking the cats’names as she passed. A few cheetahs lifted theirheads in seeming acknowledgment, and one youngfemale, 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 cooperativeprogram that sets the annual pairing recommenda-tions to maintain genetic diversity in the majority of theNorth American captive population. Member institutions

transfer cheetahs among their facilities to matchpotential mates.

To breed a female cat in Virginia, Crosier’s teamfirst moves that target female out of her pen and into anew yard nearer to the males to stimulate estrus with achange of scenery. After a few days, the team movesthe female indoors with a treat and brings a male ormales down lover’s lane to sniff around the female’snow-empty territory. In the wild, he’d smell her beforehe saw her, Crosier explains. If the male stutter barks, amating vocalization like a pigeon’s coo, “then he’sallowed to see her,” Crosier says. If he doesn’t bark,then he just goes home. “Today,” she explains, “is notthe day.”

But even though the AZA suggests pairing certainmales and females to make good genetic matches,cheetahs are picky, and about one-third of recom-mended pairs don’t breed, Crosier says. One way thatongoing research attempts to improve reproductionis through greater understanding of mate choice. Astudy published last June, for example, tested scent asa possible indicator of mate preference, availablebefore cats meet (17). When researchers presented12 female cheetahs with the scent of urine from17males, the females spent the most time investigatingscents from the males least-related to them, regardlessof the males’ testosterone levels, the female’s age, orher point in the estrus cycle. Whether a female’s urinepreference actually predicts her mate preference hasyet to be tested. But if it does, it could be a useful futureindicator of mating success before cats are transferredbetween zoos, which is expensive and stressful.

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

“The challenging part,” Crosier says, “is thatthey’re cheetahs, not domestic cats, not animals youcan just walk up to and give an injection.” Behaviorallyconditioning wild animals to tolerate the process,which included carefully timed shots before surgery toprepare the reproductive system and a trip in a car-rying crate from the cheetahs’ pens to the operatingroom, demanded cooperation by multiple cats, keepers,and veterinarians, making it the most logisticallychallenging part of the procedure. Crosier says, “youhave to have all the stars align.”

Crosier had previously demonstrated the first twosteps of the embryo transfer procedure. In 2011, sheand collaborators laparoscopically extracted about300 eggs from 29 donor females, ranging in age from2 to 15, and successfully fertilized them with thawed,frozen semen, creating embryos (6). The final step,transferring donor embryos into a recipient was aworld-first this summer. Unfortunately, the pregnancydidn’t take. But just completing this complex and

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carefully timed process is a success, Crosier says.“Now we know we can do it.”

In the future, embryo transfer could create newopportunities to breed cheetahs, she says, especiallyfor older cats without offspring, whose genes are notwell-represented in the population. The Smithsonianzoo is already banking cheetah sperm but doesn’tcurrently freeze eggs because egg cells are larger andmore vulnerable to punctures by ice crystals duringthe thawing process. Just as breeding studies may useurine as a proxy for mate choice before shipping catsbetween zoos, frozen semen and eggs are also biologicalmaterials that could make breeding more efficient andcost-effective while minimizing transit for the cats.

“We have so many cheetahs in our North Americanpopulation that behaviorally won’t participate in thebreeding program,” Crosier says. “Being able to re-produce those cats is what’s so important, becausethose genetics can’t be captured.”

Larger LessonsCheetahs are not the only captive cats that turn uptheir whiskers at potential mates. “Most cats are difficult,”says reproductive biologist Pierre Comizzoli of theSmithsonian’s National Zoo in Washington, DC, andconservation institute in Front Royal. The challengesare the same: replicating their natural history and un-derstanding their reproductive biology.

Clouded leopards, for example, spend much of theirlives in trees, an arboreal lifestyle that’s hard to mimic inzoos, explainsWielebnowski at theOregon Zoo. The catsare also choosy, and mismatched mates sometimes killeach other. Wielebnowski analyzed the stress responses

of 74 cats and found that clouded leopards housed inenclosures out of sight of the public and with higherplaces to climb had lower concentrations of fecal stresshormones, suggesting lower-stress husbandry measuresfor captive breeding, according to the study (18).

But even with husbandry improvements, naturalbreeding often fails for captive wildcat species. “That’swhy we need the possibility of artificial insemination,”Comizzoli says. The technique is unreliable in not onlycheetahs but many cat species—and for similarly per-plexing reasons. No cat species is consistently man-aged using artificial insemination today, he says, butthe technique has produced at least one pregnancy inall eight big cats, the last of which were jaguars, bornthis March in Brazil. It’s only worked three times inclouded leopards since 1992.

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

Comizzoli coauthored a study this July using newlydeveloped model uteruses to observe the effects ofestrus hormones (19). First, he and colleagues col-lected the uteruses of spayed housecats, specificallythe epithelial cells lining the inside surface of eachone. Researchers kept the epithelial cells alive andin the uteruses’ three-dimensional shape in plasticdishes. These model uteruses survived for at least twoweeks in incubators, the study found.

The researchers then treated the model uteruseswith synthetic hormones to replicate the natural estruscycle of domestic cats. They observed changes in

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

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cellular structure consistent with those already knownfrom pregnant cats, suggesting the model uteruseshave promise as a new tool to study the earliest stagesof pregnancy, Comizzoli says.

However, the hormones did not induce somechanges in gene expression known from live cats,suggesting that other, still-unknown factors also pre-pare the uterus for embryo implantation. Future studiesin these models may identify those factors, Comizzolisays, possibly revealing previously unknown prerequi-sites for pregnancy that could explain why artificial in-semination hasn’t worked since 2003 in cheetahs and isinconsistent in other cats.

Efforts to improve assisted reproduction and nat-ural breeding programs in cheetahs and other wildcatsoffer lessons beyond cats for many zoo animals,Comizzoli says. Captive birds, rodents, large carni-vores, marsupials, fish, and insects can all be choosyabout their mates and breed more successfully inprograms that offer mate choice rather than assigningpartners, according to a study published last fall (20). Itconcluded that greater use of behavioral research inspecies survival plans could raise breeding successacross many captive species.

Assisted reproduction, says Comizzoli, must betailored to individual species, which means researchersneed to know how reproduction varies, down to thefine detail of the molecular and physiological level,

even in closely related species. “We’ve done that invery few species, unfortunately,” he says.

As for cheetahs, improved breeding efforts prom-ise to preserve what little genetic diversity the specieshas left. Factoring natural history into reproductiveprograms continues to help as cheetah-breeding in-stitutions find new ways to mimic the wild. One on-going trend is a shift away from breeding in zoos,toward keeping reproductively important cheetahs ina handful of large breeding facilities such as theSmithsonian Conservation Biology Institute. Thesprawling grounds can easily house many cats, of-fering a lineup of potential partners that bettermimics mate choice in the wild.

Great mate choice encourages breeding butdoesn’t guarantee it. If Crosier’s team succeeds withembryo transfer, it could allow even celibate cats topass their genes into the next generation. Zooswouldn’t have to move animals to breed them andcould instead send sperm and embryos around theworld to maximize the genetic diversity of globalcaptive populations, and perhaps even bring spermand eggs from wild cheetahs into captive cats, Crosiersays. Assisted reproduction is one vast, next frontier ofcaptive breeding. If it works, she says it would offer“many more options for genetic management of theentire species around the world.”

1 Smithsonian’s National Zoo and Conservation Biology Institute, “Three cheetah cubs born at the Smithsonian Conservation BiologyInstitute” Newsroom, 1 October 2018. https://nationalzoo.si.edu/news/three-cheetah-cubs-born-smithsonian-conservation-biology-institute-0. Accessed 25 October 2019.

2 T. M. Caro, Behavioral solutions to breeding cheetahs in captivity: Insights from the wild. Zoo Biol. 12, 19–30 (1993).3 D. E. Wildt, C. Wemmer, Sex and wildlife: The role of reproductive science in conservation. Biodivers. Conserv. 8, 965–976 (1999).4 S. E. Alves et al., Full-term pregnancy with vaginal birth following dystocia and caesarean section in two cheetahs (Acinonyx jubatus).Vet. Rec. Case Rep. 6, 1–4 (2018).

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7 N. Wielebnowski, J. L. Brown, Behavioral correlates of physiological estrus in cheetahs. Zoo Biol. 17, 193–209 (1998).8 M. E. Akhtar, The cheetah and the Mughals, Proc. Indian Hist. Congr. 57, 381-386 (1996).9 M.Menotti-Raymond, S. J. O’Brien, Dating the genetic bottleneck of the African cheetah. Proc. Natl. Acad. Sci. U.S.A. 90, 3172–3176(1993).

10 S. J. O’Brien, D. E. Wildt, D. Goldman, C. R. Merril, M. Bush, The cheetah is depauperate in genetic variation. Science 221, 459–462(1983).

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