154 RESEARCH IN ZOOS: REPRODUCTIVE AND GENETIC TECHNOLOGIES

Int. Zoo Yb. (2011) 45: 154–159 DOI:10.1111/j.1748-1090.2010.00126.x

An overview of the assisted reproduction and genome banking activities of Center, Yulee, FL, in the service of species conservation L. M. PENFOLD White Oak Conservation Center, Yulee, Florida 32097, USA E-mail: [email protected]

Assisted reproduction includes simple strategies, such as tion and genetics, and although oral progestin to maintain pregnancy and hormone mon- remain one of the primary taxa studied, itoring to predict oestrus for breeding introductions, as well as complex procedures, such as oestrous synchroni- species as diverse as White rhinoceros zation and artificial insemination (AI). The primary focus Ceratotherium simum simum (Metrione of research at White Oak Conservation Center, Yulee, FL, et al., 2007; Christensen et al., 2009), Black has been to work towards techniques allowing movement rhinoceros Diceros bicornis (Christensen of frozen semen to manage metapopulations rather than et al., 2009), Okapia johnstoni (Stan- translocate . Using the Gerenuk Litocranius wal- leri walleri as a model for threatened antelope, oestrous ton et al., 2010), Flying fox Pteropus hypo- synchronization and AI were refined to produce four live melanus (Metrione, Verstegen et al., 2008) offspring (from six attempts) using hand restraint rather and lemurs (Chatfield & Penfold, 2007), and than anaesthesia for inseminations. Conversely, similar Acinonyx jubatus, Jaguar Panthera progress with Okapi Okapia johnstoni has been slower. Okapi sperm are highly susceptible to osmotic changes onca and Kori bustards Ardeotis kori also and the physical pressures of the freezing process, which have been investigated. Assisted reproduc- has limited the ability to develop suitable cryopreserva- tion can be a useful conservation manage- tion protocols. Storage of frozen semen from highly ment tool and includes an array of techniques, threatened animals provides insurance against loss of the such as artificial insemination (AI), oestrous individual’s genes to the population and, if used for future insemination, can potentially provide new ‘foun- synchronization and embryo transfer. Al- ders’. Biomaterials from both species, including blood though more often associated with the more and blood products, are preserved in genome resource complex strategies, assisted reproduction also banks together with samples from other threatened encompasses simple strategies, such as the species, including the Florida panther Felis concolor coryi. It is important to note that assisted reproduction in use of daily oral progestin to sustain pregnan- novel species requires significant commitment and con- cies (Okapi: Schwarzenberger et al., 1999) or tinuity from zoological institutions for optimal results. faecal hormone analysis to monitor oestrous cycles to time introduction to the < for Key-words: artificial insemination; cryopreservation; breeding (Okapi: L. M. Penfold, unpubl. data). genome resource banking; sperm. There is no doubt that captive management INTRODUCTION of non-domestic animals in a zoological set- ting can be enhanced by assisted reproduc- Over the last 12 years a research programme tion, as it is critical for small-population at White Oak Conservation Center (WOCC), management that genetic representation is ob- Yulee, FL, USA, has been developed, initially tained from as many individuals as possible to address a paucity of reproductive research (Lacy, 1994, 1997). Inbreeding in small, on antelope species. Gradually the scope of fragmented populations can be minimized in the research has increased to include the two ways. One is by intensive management disciplines of nutrition, behaviour, contracep- and the frequent movement of animals among

Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London WHITE OAK CONSERVATION CENTER: ASSISTED REPRODUCTION & GENOME RESOURCE BANKING 155 populations for natural breeding. The other is and the Okapi as a flagship species for both to assist the natural breeding process by the Democratic Republic of Congo and WOCC. using, when appropriate and needed, as- Although GRBs are often associated with sisted-breeding techniques. In particular, AI cryopreserved semen, it is important to re- with cryopreserved sperm can augment ge- member that they may also store other biolo- netic management. Genome resource banks gical samples, such as whole blood, blood (GRBs), containing frozen samples from ge- products, tissue and urine. These biomaterials netically valuable << would provide protec- can be an important resource for future stu- tion for existing captive populations (see also dies, including epidemiological studies of Clarke, 2009; Jewgenow et al., 2011; Sara- disease incidence, nutrition analyses and ge- gusty et al., 2011). In the event of a disease, netics studies. As such, a repository of biolo- epidemic or natural catastrophe, sperm would gical samples can be an important resource be safeguarded in liquid nitrogen storage for a threatened species. There are several dewars. These samples then could be easily excellent reviews on the full scope of genome distributed from one holding institution to resource banking (Wildt, 1992; Holt et al., another to be artificially inseminated into 1996; Wildt et al., 1997), and this paper genetically valuable ,,. This would eliminate focuses briefly on two areas: biological sam- the need to ship animals between institutions pling (Okapi) and long-term semen preserva- and, because cryopreserved sperm can be tion (Florida panther Felis concolor coryi). stored for decades, the genes of valuable << could be used for years to come. AI combined DEVELOPING A PROTOCOL FOR with GRB can also be used with a goal of MANAGING METAPOPULATIONS BY moving frozen semen between as well as MOVING FROZEN SEMEN within countries. Imported species have the po- A prominent goal of WOCC has been to work tential to carry any number of foreign towards ruminant semen importation to man- diseases that are of concern to a country’s age hoofstock populations. If frozen sperm agricultural industry and semen can also together with AI can be used to move new carry many of them (Kirkwood & Colen- genetic material between and within popula- brander, 2001). However, live animal im- tions, theoretically this should not only have ports have the added risk of arthropod reduced risk but also should be less expensive transmitted diseases, such as trypanosomo- than moving live animals. The Gerenuk was sis, theileriosis, African swine fever as well selected as an understudied species that could as foreign arthropod species, such as the serve as a model species for other antelope. Screwworm fly Cochliomyia hominivorax. The effective use of assisted reproduction in Eradicated from the United States in the late antelope species is not simple. Techniques 1950s (Wyss, 2000), the severe outbreak of that work well in some species, such as Screwworm in 1972 demonstrated the vul- or humans, are not necessarily easily adapted nerability of US livestock to external para- to other species because of unique physiolo- sites. Thus, there is inherent reduced disease gies. Successful assisted reproduction is fa- risk importing frozen semen versus a live cilitated by first understanding the basic animal because semen cannot inadvertently reproductive biology of an unstudied species; contain arthropod species. One of the main this was accomplished by longitudinal faecal programme areas at WOCC has been to hormone analysis of ,, to identify that they develop a protocol for the importation of were a polyoestrous, aseasonal species ruminant semen for AI to manage ruminant (Penfold et al., 2005). Knowing the basic metapopulations. Efforts have focused on reproductive biology of a species is important the Gerenuk Litocranius walleri walleri,a because seasonal species may not cycle all small antelope species with ,, weighing year. For example, the , Jackson’s c. 27–31 kg, as a model species for antelope, Alcelaphus buselaphus jacksoni are a

Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London 156 RESEARCH IN ZOOS: REPRODUCTIVE AND GENETIC TECHNOLOGIES polyoestrous, seasonal species, undergoing a nated with fresh sperm gave birth to live period of acyclicity in the spring (Metrione, offspring. Norton et al., 2008). It is important to note Parallel studies continued to work towards that although the Hartebeest Alcelaphus bu- the importation of Gerenuk semen from Ken- selaphus and Gerenuk share the same geo- ya. The political aspects of this project, graphic region and habitat, and are exposed to namely working with governments to secure the same periods of light and rainfall, their protocols and permits has proven extraordi- reproductive strategies differ (see also Berger, narily challenging. Because no precedent 2011). Thus, although Gerenuk can poten- existed for importation of ruminant semen tially be inseminated year round, assisted from a country where foot-and-mouth disease reproduction in the Hartebeest must be (FMD) is endemic, a protocol defining how avoided in the spring. the animals should be handled in quarantine, Further studies have revealed Gerenuk together with disease testing requirements ejaculates contained high numbers of pleio- and other relevant procedures, was required morphic sperm (Penfold et al., 2005) and to be generated by the US Department of genetic diversity of Gerenuk in captivity was Agriculture (USDA). Although a protocol reduced compared to free-ranging Kenyan was released for the project in 2004 (5 years Gerenuk; therefore supporting the theory that after the process started), subsequent revi- Gerenuk would benefit from the addition of sions to protocol by USDA still have not new genetic material to the population. Stu- been completed to date, a total of 12 years dies to develop an AI protocol for Gerenuk, since the initial request. Ironically, protocols based on that of Domestic cattle taurus, for importation of live animals from the same have used faecal hormone analysis to investi- region are readily available. So importing a gate oestrous synchronization protocols. Two live Gerenuk antelope is theoretically much prostaglandin injections administered 11 days easier than importing its semen alone. To apart were shown to be effective in inducing provide balance to this issue it should be oestrus in the majority of ,,, and this was mentioned that a semen importation protocol confirmed behaviourally using a vasecto- would contain various requirements regard- mized < and noting mounting behaviour that ing the incidence of disease outbreaks in the occurred c. 48 hours after the second injec- country from which the semen was being tion. Owing to their small stature, insemina- exported. For example, one requirement is tion was accomplished transcervically under ‘no case of FMD or Rinderpest has occurred anaesthesia, stabilizing the cervix with Allis in the last 5 years in the quarantine facility or forceps before inserting a catheter to deliver within a radius of 5 km of the facility’. In the semen (Morrow et al., 2009). Insemina- turn, USDA is reliant on in-country expertise tion of four ,, after 72 hours with frozen– to provide assurances that there are no unac- thawed sperm resulted in one pregnancy that ceptable disease risks with importing the se- went full-term but the calf died during the men, which may inherently introduce more birth process after the , went into dystocia. ‘risk’ to the project. The reproductive scien- Subsequent inseminations (n 5 24) over the tist is thus also required to have an under- next 9 years did not yield live offspring even standing of the disease landscape of the when fresh rather than frozen semen was country of importation and disease-risk ana- used; although early pregnancy, confirmed lysis, as well as some epidemiologic back- by faecal hormone analysis, was noted on at ground in ruminant diseases. In the case of least three occasions. In 2009, the insemina- ruminant species, accomplishing a project tion protocol was changed to eliminate the involving the seemingly straightforward anaesthesia component and, following syn- concept of semen importation, can be an chronization, Gerenuk were hand restrained enormous undertaking, requiring a broad and inseminated in the field. Using this understanding of not only the reproductive approach four out of six (67%) ,, insemi- biology of the species but also multiple other

Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London WHITE OAK CONSERVATION CENTER: ASSISTED REPRODUCTION & GENOME RESOURCE BANKING 157 disciplines, including politics (of both the these were minimized by adding cryoprotec- importing and exporting country), disease tants incrementally at 4 1C, further mechan- epidemiology and risk assessment and animal ical pressures induced during ice-crystal management during quarantine, all of which formation and the cryopreservation process have high demands in terms of time, effort damaged the majority of the Okapi sperm and funding. plasma membrane, resulting in an extremely Concerns about airport irradiation on the poor yield of post-thaw motile and plasma- DNA of frozen semen prompted an investiga- membrane intact cells. Current studies are tion into the effects of X-radiation on Domes- investigating sperm-chilling studies rather tic bull sperm fertilizing ability and embryo than cryopreservation, with a goal to preserve development (Hendricks et al., 2010). No sperm motility for 72 hours for international apparent effects were observed on Domestic transport. bull sperm, which is encouraging for , Similarly, oestrous synchronization proto- although previous studies have indicated that cols were not as easily adapted from Domes- X-radiation may be detrimental for felid tic cattle protocols, which are based on an sperm (Gloor et al., 2006). To date, Gerenuk average cycle of 21 days (Morrow et al., sperm has not been imported for use in AI 2009). The 14 day cycle of Okapi eventually trials but this project highlighted difficulties yielded a synchronization protocol of two with ruminant semen importation into the prostaglandin injections 6 days apart (L. M. United States and WOCC has taken the lead Penfold, unpubl. data). Although some Okapi with working with the USDA and other cycle regularly, it has not been possible to institutions interested in semen importation predict oestrus accurately for natural insemi- by developing a task force to liaise with the nation. AI trials conducted both at WOCC USDA on ruminant and ruminant biological (n 5 5) and at the Okapi Research Station, sample importation. Epulu, Democratic Republic of Congo (n 5 3), have not resulted in any pregnancies to date. CHALLENGES WITH ASSISTED REPRODUCTIVE TECHNIQUES INSURANCE FOR THE FUTURE: GRB FOR OKAPI AND FLORIDA PANTHER In contrast to the methodical progress with Gerenuk, culminating this year with offspring While assisted reproductive techniques were production, development of a similar pro- unsuccessful in the Okapi maintained in gramme with the Okapi has been far more captivity in the Democratic Republic of Con- challenging. In contrast to all other go, these animals were utilized to collect studied to date, Okapi semen has proven valuable biological samples for GRB. An extremely difficult to collect, with average array of samples was collected from the ejaculate volumes of only 1 Á 3 ml, mean Okapi, including serum, plasma, skin biop- sperm motilities of 29% and normal morphol- sies, urine and whole blood. Blood was also ogies of 48% (Penfold, 2008). Investigation processed to bank serum for use in mineral of the effects of cryopreservation on Okapi analysis for nutrition studies. Faecal samples spermatozoa revealed that they are highly that were collected from the Okapi, stored in susceptible to detrimental influences of the alcohol and sun-dried, and transported to the cryopreservation process, and possess an United Kingdom were useful in developing a acute sensitivity to changes in osmotic condi- microsatellite library for Okapi genetics stu- tions when treated with addition of anisosmo- dies (Stanton et al., 2010), demonstrating the tic phosphate-buffered saline (Penfold, advantages of multinational collaborations. 2008). This directly translated to poor post- Unlike the relatively unknown Okapi, the thaw sperm survivability as large cell-volume plight of the Florida panther has been well excursions usually are expected upon addi- documented (O’Brien et al., 1990; Culver tion of permeating cryoprotectants. Although et al., 2000; McBride et al., 2008). In brief,

Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London 158 RESEARCH IN ZOOS: REPRODUCTIVE AND GENETIC TECHNOLOGIES low numbers of Florida panther in south- CONCLUSION central Florida faced likely extinction before Continuity and commitment are requisites for the introduction of eight Texas panthers Felis institutions if successful research in threatened concolor stanleyana, five of which bred and species is to be accomplished. Species knowl- produced 20 kittens whereon they were re- edge is gained in incremental steps over moved from the population. Various para- extended periods of time to build a database meters (health, offspring production, that, with persistence, can translate into suc- population size) were used to study the cessful assisted reproductive techniques. It is effects of this genetic introgression, including hoped that in the not too distant future the reproductive fitness of the <, assessed by concept of genome resource banking can be examining numbers of morphologically nor- put to practical use by transportation of frozen mal spermatozoa (Barone et al., 1994; John- gametes to manage metapopulations through son et al., 2010). When a < cat died, often assisted reproductive techniques, such as AI. through vehicular trauma or interspecific ag- In the meantime, they serve as important gression, the testes were removed and repositories of valuable genetic material, con- shipped overnight on cold packs to the serving genes for the future. WOCC. Spermatozoa were recovered from All studies described in this paper received the epididymides and a small fraction placed Institutional Animal Care and Use Commit- in 0 Á 3% glutaraldehyde fixative for morphol- tee approval before commencement. ogy analysis (Howard et al., 1986). If the samples were fresh enough, motile sperm could often to be recovered (a process known ACKNOWLEGEMENTS as gamete rescue), extended with an egg- Zoo research relies heavily on the collaboration of other yolk-based cryodiluent and frozen in liquid institutions for access to animals and/or biological samples nitrogen using a protocol previously shown to and our institution has been fortunate enough to work with be effective for felid species (Crosier et al., many tremendous institutions and outstanding researchers who have helped us accomplish our research goals, in 2006). Analysis of sperm morphology re- particular: Busch Gardens Tampa Bay, Cincinnati Zoo & vealed the spermatozoa of intergrade cats Botanical Gardens, Disney’s Animal Kingdom, Jackson- (Florida panthers containing Texas panther ville Zoo & Gardens, Lubee Bat Conservancy, Smithso- genes) was improved compared with that of nian’s National Zoo and Conservation & Research Center, St Catherine’s Wildlife Foundation (formerly Wildlife pure Florida panthers. As sperm quality in Conservation Society) and the University of Florida. felids is associated with genetic fitness (Wildt Special thanks to Marcie Oliva and all the White Oak staff et al., 1983; Barone et al., 1994), this was one who have played a critical role in developing the research of several parameters that confirmed the programme. Last but not least, thanks to each and every success of the genetic introgression. Also, one of the wonderful students with whom I have worked, especially Martine de Wit, Lara Metrione, Andre´sRojas because Florida panther numbers are still and Serena Barnes, all of whom deserve credit for helping low and there are no options for new genetic develop the research programme. material to be introduced into the population, banked sperm samples may effectively serve REFERENCES as new founders through reintroduction into BARONE, M. A., ROELKE, M. E., HOWARD, J. G., BROWN, the future population via AI. Cryopreserved J. L., ANDERSON,A.E.& WILDT, D. E. (1994): Reproduc- sperm from << that subsequently died with- tive characteristics of male Florida panthers: comparative out producing offspring ensures that their studies from Florida, Texas, Colorado, Latin America, and North American Zoos. Journal of Mammalogy 5: genes are not lost to the population. If the 150–162. samples were received within 24–36 hours, BERGER, A. (2011): Activity patterns, chronobiology and testicular tissue was also cryopreserved, the assessment of stress and welfare in zoo and wild further preserving genetic material for a future animals. International Zoo Yearbook 45: 80–90. CHATFIELD,J.& PENFOLD, L. M. (2007): Prevention of where more advanced techniques may poten- urethral blockage following semen collection in two tially be able to reintroduce the genes back into species of lemur. Journal of Zoo and Wildlife Medicine the population. 38: 280–284.

Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London WHITE OAK CONSERVATION CENTER: ASSISTED REPRODUCTION & GENOME RESOURCE BANKING 159

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Int. Zoo Yb. (2011) 45: 154–159 c 2010 The Authors. International Zoo Yearbook c 2010 The Zoological Society of London