NEWS FEATURE
Getting the world’s fastest cat to breed with speed NEWS FEATURE Cheetahs 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 the Zoos once tried to breed cheetahs very differently, Smithsonian Conservation Biology Institute, in Front he says, keeping males and females together rather Royal, Virginia. Off exhibit from the public, the 1-year- than mimicking their lifestyle in the wild. Out on the old cats roam a large and grassy enclosure behind a savannah, females are solitary and roam vast distances. high, chain-link fence. They are the newest of 56 cubs Males defend smaller territories and mate when fe- born at this facility since 2007 (1). males pass through (2). Designing exhibits to reflect Breeding cheetahs to produce cubs such as these that natural history has been a major part of breeding was once famously difficult. The biggest problem was success over the last three decades, Saffoe says. that zoos were “not paying attention to natural his- Success has also come from a better understand- tory,” says Craig Saffoe, curator of lions, tigers, and ing of cheetah reproductive physiology (3). Reproduc- bears at the Smithsonian’s National Zoo in Washing- tive studies in the last 30 years have improved ton, DC, who in 2004 led the team that produced breeding by increasing understanding of males’ poor the zoo’s first litter. Tattoos of cheetahs now adorn sperm quality, females’ ovulation cycle, and mate his forearms. choice. 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 breeding facility suggests that a better understanding of natural history and reproductive physiology can dramatically improve captive reproduction. Image credit: Mehgan Murphy/Smithsonian Conservation Biology Institute.
Published under the PNAS license.
www.pnas.org/cgi/doi/10.1073/pnas.1918672116 PNAS | December 10, 2019 | vol. 116 | no. 50 | 24911–24915 Downloaded by guest on September 30, 2021 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 chee- tahs at 47 genetic loci and in 155 proteins and showed that cheetahs have unusually low levels of polymor- phisms 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). ’ Researchers bring males up this lover’s lane at the Smithsonian Conservation In a concurrent analysis of the cheetahs sperm, the Biology Institute to sniff around a potential mate’s enclosure. The approach same study found that 70% was deformed, and sperm mimics some of the cats’ wild behaviors. 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 majority of North American captive cheetahs are man- community said, well there’s your problem, that’s why aged by the Association of Zoos & Aquariums (AZA), they’re not breeding. They’ve got really jacked up whose member institutions include the Smithsonian’s sperm and they’re all identical twins.” National Zoo and other zoos and facilities around the But then in 1992, researchers discovered the cats country. The AZA-managed cheetah population aver- reproduced effectively in the wild despite their ge- ages 43 births per year. netic bottleneck. Cheetahs could breed; they just Despite those successes, breeding programs are weren’t doing it in captivity. An observational study of hitting a wall of sorts, and further progress will be key 20 wild female cheetahs on Tanzania’s Serengeti found to maintaining and expanding the genetic diversity that all but one had cubs (11). Zoos then began to of captive populations. More than one-third of AZA- scrutinize their own breeding programs, Saffoe says, managed captive cheetahs still don’treproduceduetoa asking how their efforts could mimic cheetahs’ natural mix of health, behavior, and reproductive problems (4, history and reproductive biology in the wild. 5). Between 1970 and 2011, more than 70% of females To do so, researchers first needed to know how the and more than 80% of males died cubless (6). cats breed in the wild. Research in the 1980s and Assisted reproduction would help maintain the 1990s found that wild females are solitary and roam diversity of captive populations, but success has been large distances, whereas males live in coalitions of ’ mixed. Artificial insemination hasn t worked well in re- brothers in smaller territories. Females are induced- cent years. Embryo transfer has shown signs of prog- ovulators, meaning they don’t ovulate until after ress, however. This past June, researchers completed mating. Studies in the AZA-managed population have the first-ever successful transfer in cheetahs. also found that females go into estrus every 12 to All these efforts underscore a common theme: When 14 days with occasional months-long periods of ovar- it comes to breeding cheetahs in captivity, natural history ian inactivity, possibly as constant preparation for the — matters a lesson that applies beyond the race to save possibility of mating, to accommodate their solitary, cheetahs to many other rare, captive cats. induced-ovulator lifestyle. Females housed together can suppress ovulation unless the cats know and like Stubborn Breeders each other. And despite their frequent cycles, females People have struggled to breed cheetahs for hun- show few outward signs of estrus unless they’re in- dreds of years. The cats slinked through the ochre terested in mating with a male (12). palaces of the Mughal Empire some 500 years ago as This challenging reproductive biology inspired captive hunting pets. Yet there is only one record of zoos to attempt assisted reproduction beginning in their breeding in captivity before 1956 (7, 8). Zoos the 1990s via artificial insemination or embryo transfer. struggled to reliably breed cheetahs through the Artificial insemination—the introduction of sperm into 1980s, even as wild populations saw steep declines the vagina or through the cervix into the uterus—is the from habitat loss and poaching (9). more straightforward and more common method in Zoos overlooked natural history because of “a lot zoo animals. But it ’s difficult in cats because their of historic baggage,” says Nadja Wielebnowski, con- sperm are propelled through the long horns of the servation and research manager at the Oregon Zoo in wishbone-shaped uterus to the oviducts by muscle Portland. Zoos began as menageries of exotic and contractions. Anesthetized females don’t make these often little-understood species. Although many facilities contractions, so sperm don’t meet eggs (13). Two have made updates, most still have limited resources as breakthrough studies in the 1990s involved directly
24912 | www.pnas.org/cgi/doi/10.1073/pnas.1918672116 McDermott Downloaded by guest on September 30, 2021 inseminating housecat uteruses and cheetah uteruses, transfer cheetahs among their facilities to match successfully producing 15 cheetah pregnancies be- potential mates. tween 1992 and 2003 (14, 15). To breed a female cat in Virginia, Crosier’s team But despite repeated attempts since 2003, it was first moves that target female out of her pen and into a the last successful cheetah artificial insemination ever new yard nearer to the males to stimulate estrus with a conducted, says Bill Swanson, Director of Animal Re- change of scenery. After a few days, the team moves search at the Cincinnati Zoo & Botanical Garden in the female indoors with a treat and brings a male or Ohio. The reasons for the failures remain unclear. One males down lover’s lane to sniff around the female’s possible explanation is a change in the manufacturer now-empty territory. In the wild, he’d smell her before of the hormones that zoos use to induce ovulation he saw her, Crosier explains. If the male stutter barks, a before artificial insemination. But even after changing mating vocalization like a pigeon’s coo, “then he’s hormone sources, cheetahs still aren’t getting preg- allowed to see her,” Crosier says. If he doesn’t bark, “ ” “ nant through artificial insemination. Even surgically then he just goes home. Today, she explains, is not ” putting sperm into the oviducts, onto the eggs— the day. which has yielded pregnancies in ocelots, tigers, jag- But even though the AZA suggests pairing certain uars, and other wild cats—has not worked for chee- males and females to make good genetic matches, tahs, Swanson says. cheetahs are picky, and about one-third of recom- ’ More recent research implies that a female’s age at mended pairs don t breed, Crosier says. One way that first pregnancy strongly influences her reproductive ongoing research attempts to improve reproduction success. For example, a study published this April fol- is through greater understanding of mate choice. A lowed 12 female cheetahs in European zoos through- study published last June, for example, tested scent as out their lives and found that cats with a first pregnancy a possible indicator of mate preference, available before the age of 5 1/2 had higher reproductive before cats meet (17). When researchers presented 12 female cheetahs with the scent of urine from performance throughout their lives, even at advanced 17 males, the females spent the most time investigating ages, compared with females with a first pregnancy at scents from the males least-related to them, regardless age 6 or older (16). The new findings agree with pre- of the males’ testosterone levels, the female’s age, or vious research led by the Smithsonian, suggesting that her point in the estrus cycle. Whether a female’surine cheetahs tend to breed young and that captive pro- preference actually predicts her mate preference has grams should mimic the cats’ biological timing. yet to be tested. But if it does, it could be a useful future Despite these many challenges, the state of cheetah indicator of mating success before cats are transferred breeding is much improved from 30 years ago. Zoos between zoos, which is expensive and stressful. now produce cubs every year, with nearly 400 cheetahs in North America’s captive breeding population today. A Helping Hand Emulating the wild has helped, but researchers will In the meantime, Crosier is pushing the envelope in need additional approaches to ensure the long-term assisted reproduction. In still-unpublished research, genetic diversity of captive populations. she led the first-ever embryo transfer in the cats this summer, taking eggs from a 7-year-old donor female, Wild Child fertilizing them, then implanting the embryos in a Beyond the high fence and heavy gate of the cheetah young female that’s a proven breeder, she explains. enclosure at the Smithsonian Conservation Biology Before surgery, both donor and recipient females re- Institute, a T-shaped dirt road is fringed by grassy ceived hormone injections to stimulate estrus. yards. Male cheetahs live in four separate yards at the “The challenging part,” Crosier says, “is that top of the T, while solitary females and mothers with they’re cheetahs, not domestic cats, not animals you cubs have their own yards at the bottom. This setup, can just walk up to and give an injection.” Behaviorally ’ called a lover s lane, was pioneered by facilities in conditioning wild animals to tolerate the process, South Africa, The Netherlands, and the United States which included carefully timed shots before surgery to to mimic life in the wild, where females are solitary and prepare the reproductive system and a trip in a car- mostly live out of sight and scent of the males, ex- rying crate from the cheetahs’ pens to the operating plains African and North American carnivore curator room, demanded cooperation by multiple cats, keepers, Adrienne Crosier. and veterinarians, making it the most logistically On the day of a recent visit, Crosier walked along challenging part of the procedure. Crosier says, “you the fence line of each cheetah yard, clucking the cats’ have to have all the stars align.” names as she passed. A few cheetahs lifted their Crosier had previously demonstrated the first two heads in seeming acknowledgment, and one young steps of the embryo transfer procedure. In 2011, she female, Zuri, jogged up and jumped against the fence, and collaborators laparoscopically extracted about which Crosier translated to mean “this is my space, 300 eggs from 29 donor females, ranging in age from stay out.” Zuri was born at this facility two years ago, 2 to 15, and successfully fertilized them with thawed, thanks to the AZA Species Survival Plan, a cooperative frozen semen, creating embryos (6). The final step, program that sets the annual pairing recommenda- transferring donor embryos into a recipient was a tions to maintain genetic diversity in the majority of the world-first this summer. Unfortunately, the pregnancy North American captive population. Member institutions didn’t take. But just completing this complex and
McDermott PNAS | December 10, 2019 | vol. 116 | no. 50 | 24913 Downloaded by guest on September 30, 2021 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.
carefully timed process is a success, Crosier says. of 74 cats and found that clouded leopards housed in “Now we know we can do it.” enclosures out of sight of the public and with higher In the future, embryo transfer could create new places to climb had lower concentrations of fecal stress opportunities to breed cheetahs, she says, especially hormones, suggesting lower-stress husbandry measures for older cats without offspring, whose genes are not for captive breeding, according to the study (18). well-represented in the population. The Smithsonian But even with husbandry improvements, natural zoo is already banking cheetah sperm but doesn’t breeding often fails for captive wildcat species. “That’s currently freeze eggs because egg cells are larger and why we need the possibility of artificial insemination,” more vulnerable to punctures by ice crystals during Comizzoli says. The technique is unreliable in not only the thawing process. Just as breeding studies may use cheetahs but many cat species—and for similarly per- urine as a proxy for mate choice before shipping cats plexing reasons. No cat species is consistently man- between zoos, frozen semen and eggs are also biological aged using artificial insemination today, he says, but materials that could make breeding more efficient and the technique has produced at least one pregnancy in cost-effective while minimizing transit for the cats. all eight big cats, the last of which were jaguars, born “We have so many cheetahs in our North American this March in Brazil. It’s only worked three times in population that behaviorally won’t participate in the clouded leopards since 1992. breeding program,” Crosier says. “Being able to re- To understand the failings of artificial insemination, produce those cats is what’s so important, because Comizzoli studies the basic physiology of cat preg- those genetics can’t be captured.” nancy, including embryo implantation in the uterus. “We don’t really know exactly how it works and when Larger Lessons it’s happening,” he says, even in domestic cats. Cheetahs are not the only captive cats that turn up Comizzoli coauthored a study this July using newly their whiskers at potential mates. “Most cats are difficult,” developed model uteruses to observe the effects of says reproductive biologist Pierre Comizzoli of the estrus hormones (19). First, he and colleagues col- Smithsonian’s National Zoo in Washington, DC, and lected the uteruses of spayed housecats, specifically conservation institute in Front Royal. The challenges the epithelial cells lining the inside surface of each are the same: replicating their natural history and un- one. Researchers kept the epithelial cells alive and derstanding their reproductive biology. in the uteruses’ three-dimensional shape in plastic Clouded leopards, for example, spend much of their dishes. These model uteruses survived for at least two lives in trees, an arboreal lifestyle that’s hard to mimic in weeks in incubators, the study found. zoos, explains Wielebnowski at the Oregon Zoo. The cats The researchers then treated the model uteruses are also choosy, and mismatched mates sometimes kill with synthetic hormones to replicate the natural estrus each other. Wielebnowski analyzed the stress responses cycle of domestic cats. They observed changes in
24914 | www.pnas.org/cgi/doi/10.1073/pnas.1918672116 McDermott Downloaded by guest on September 30, 2021 cellular structure consistent with those already known even in closely related species. “We’ve done that in from pregnant cats, suggesting the model uteruses very few species, unfortunately,” he says. have promise as a new tool to study the earliest stages As for cheetahs, improved breeding efforts prom- of pregnancy, Comizzoli says. ise to preserve what little genetic diversity the species However, the hormones did not induce some has left. Factoring natural history into reproductive changes in gene expression known from live cats, programs continues to help as cheetah-breeding in- suggesting that other, still-unknown factors also pre- stitutions find new ways to mimic the wild. One on- pare the uterus for embryo implantation. Future studies going trend is a shift away from breeding in zoos, in these models may identify those factors, Comizzoli toward keeping reproductively important cheetahs in says, possibly revealing previously unknown prerequi- a handful of large breeding facilities such as the sites for pregnancy that could explain why artificial in- Smithsonian Conservation Biology Institute. The semination hasn’t worked since 2003 in cheetahs and is sprawling grounds can easily house many cats, of- inconsistent in other cats. fering a lineup of potential partners that better Efforts to improve assisted reproduction and nat- mimics mate choice in the wild. ural breeding programs in cheetahs and other wildcats Great mate choice encourages breeding but offer lessons beyond cats for many zoo animals, doesn’t guarantee it. If Crosier’s team succeeds with Comizzoli says. Captive birds, rodents, large carni- embryo transfer, it could allow even celibate cats to vores, marsupials, fish, and insects can all be choosy about their mates and breed more successfully in pass their genes into the next generation. Zoos ’ programs that offer mate choice rather than assigning wouldn t have to move animals to breed them and partners, according to a study published last fall (20). It could instead send sperm and embryos around the concluded that greater use of behavioral research in world to maximize the genetic diversity of global species survival plans could raise breeding success captive populations, and perhaps even bring sperm across many captive species. and eggs from wild cheetahs into captive cats, Crosier Assisted reproduction, says Comizzoli, must be says. Assisted reproduction is one vast, next frontier of tailored to individual species, which means researchers captive breeding. If it works, she says it would offer need to know how reproduction varies, down to the “many more options for genetic management of the fine detail of the molecular and physiological level, entire species around the world.”
1 Smithsonian’s National Zoo and Conservation Biology Institute, “Three cheetah cubs born at the Smithsonian Conservation Biology Institute” 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). 5 A. Crosier, E. Moloney, J. Andrews, Cheetah (Acinonyx jubatus) Species Survival Plan (Association of Zoos and Aquariums, 2017). 6 A. E. Crosier et al., Increasing age influences uterine integrity, but not ovarian function or oocyte quality, in the cheetah (Acinonyx jubatus). Biol. Reprod. 85, 243–253 (2011). 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). 11 M. K. Laurenson, T. Caro, M. Borner, Female cheetah reproduction. Natl. Geogr. Res. Explor. 8,64–75 (1992). 12 A. E. Crosier, P. Comizzoli, D. C. Koester, D. E. Wildt, Circumventing the natural, frequent oestrogen waves of the female cheetah (Acinonyx jubatus) using oral progestin (Altrenogest). Reprod. Fertil. Dev. 29, 1486–1498 (2017). 13 J. G. Howard, D. E. Wildt, Approaches and efficacy of artificial insemination in felids and mustelids. Theriogenology 71, 130–148 (2009). 14 J. G. Howard, M. A. Barone, A. M. Donoghue, D. E. Wildt, The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. J. Reprod. Fertil. 96, 175–186 (1992). 15 J. Howard, Successful induction of ovarian activity and laparoscopic intrauterine artificial insemination in the cheetah (Acinonyx Jubatus). J. Zoo Wildl. Med. 23, 288–300 (1992). 16 C. Ludwig et al., Asymmetric reproductive aging in cheetah (Acinonyx jubatus) females in European zoos. J. Zoo Aquar. Res. 7,87–93 (2019). 17 R. H. Mossotti et al., Reactions of female cheetahs (Acinonyx jubatus) to urine volatiles from males of varying genetic distance. Zoo Biol. 37, 229–235 (2018). 18 D. J. Shepherdson, K. C. Carlstead, N. Wielebnowski, Cross-institutional assessment of stress responses in zoo animals using longitudinal monitoring of fecal corticoids and behavior. Anim. Welf. 13,105–113 (2004). 19 K. Wilsterman, X. Bao, A. D. Estrada, P. Comizzoli, G. E. Bentley, Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system. Biol. Reprod. 10.1093/biolre/ioz145 (2019). 20 M. S. Martin-Wintle, N. J. P. Wintle, M. D´ıez-Le ´on,R. R. Swaisgood, C. S. Asa, Improving the sustainability of ex situ populations with mate choice. Zoo Biol. 38, 119–132 (2019).
McDermott PNAS | December 10, 2019 | vol. 116 | no. 50 | 24915 Downloaded by guest on September 30, 2021