Reproductive status of endemic felid species in Latin American zoos and implications for ex situ...

Reproductive Status of Endemic FelidSpecies in Latin American Zoos andImplications for Ex Situ ConservationW.F. Swanson,1n W.E. Johnson,2 R.C. Cambre,3 S.B. Citino,4 K.B. Quigley,5

D.M. Brousset,6 R.N. Morais,7 N. Moreira,8 S.J. O’Brien,2 and D.E. Wildt3

1Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo andBotanical Garden, Cincinnati, Ohio2Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland3Conservation and Research Center, Smithsonian National Zoological Park,Washington, D.C.4White Oak Conservation Center, White Oak Plantation, Yulee, Florida5Hornocker Wildlife Research Institute, Moscow, Idaho6Facultad de Medicina Veterinaria y Zootechnia, Universidad Nacional Autonoma deMexico, Coyocan, Mexico7Setor de Ciencias Biologicas, Universidade Federal do Parana, Curitiba, Brazil8Curso de Medicina Veterinaria, Universidade Federal do Parana, Palotina, Brazil

Reproductive evaluations were conducted on 185 male cats representing eightendemic Latin American species that were maintained in 44 zoos and privatefacilities in 12 Latin American countries. Reproductive assessments (testicularmeasures, ejaculate quality, and blood testosterone/cortisol concentration) wereused to establish normative values for large- and small-sized cats in LatinAmerican collections. Data also were analyzed using multiple regression to studythe impact of proven breeder status, diet, and various animal housingcombinations. Most felids (495%) in the survey were of wild-born origin, ando20% had produced offspring in captivity. Larger felids had bigger testes andproduced more semen, but tended to produce low-sperm-density ejaculates. Theejaculates of small felids were more sperm-concentrated, but contained fewer totalspermatozoa. Sperm motility was unrelated to species size, and certain species(puma, margay, tigrina, and jaguarundi) consistently produced few (o40%)normal sperm forms. Across species, 450% of males had low sperm counts

nCorrespondence to: Dr. William F. Swanson, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine

St., Cincinnati, OH 45220. E-mail: [email protected].

Grant sponsor: Philip Reed Foundation; Grant sponsor: Friends of the National Zoo (FONZ); Grant

sponsor: Conservation Endowment Fund, American Zoo and Aquarium Association.

Received for publication July 30, 2002; Accepted September 20, 2002.

DOI: 10.1002/zoo.10093

Published online in Wiley InterScience (www.interscience.wiley.com).

Zoo Biology 22:421–441 (2003)

�c 2003 Wiley-Liss, Inc.

(o1 million total sperm per ejaculate). Among large cats (jaguars and pumas),proven breeders had larger (Po0.05) testes, greater semen volume, and morenormal sperm than nonbreeders. Males on adequate diets had higher (Po0.05)circulating cortisol. Among small-sized felids, proven breeders had higher(Po0.05) testosterone, and males housed alone or paired with a conspecificfemale had more (Po0.05) total sperm per ejaculate and greater (Po0.05)seminal and testicular volumes. Fifty-nine ejaculates (potentially representingB100 artificial insemination (AI) or 26,000 in vitro fertilization (IVF) procedures)were cryopreserved for a felid genome resource bank. In conclusion, breedingsuccess and reproductive traits for many endemic felids in Latin American zoosappear to be suboptimal, and likely would benefit from improvements in diet andexhibitry. Technology transfer and continued training of zoo staff and scientistsin Latin American countries are essential if these zoos are to achieve theirtremendous conservation potential for felids and other threatened endemicspecies. Zoo Biol 22:421–441, 2003. �c 2003 Wiley-Liss, Inc.

Key words: electroejaculation; semen; cats; breeding; testosterone; cortisol; nutrition;exhibitry; cryopreservation

INTRODUCTION

The Felidae display an extraordinary degree of phylogenetic diversity withinLatin America, a geographic region stretching more than 14,000 km from northernMexico to the southern tip of South America. Ten felid species are endemic to LatinAmerica, including two large-sized (435 kg body weight) species (the jaguar(Panthera onca) and puma (Puma concolor)), and eight small-sized (o15 kg bodyweight) felids (the ocelot (Leopardus pardalis), margay (Leopardus wiedi), tigrina(Leopardus tigrinus), Geoffroy’s cat (Oncifelis geoffroyi), pampas cat (Oncifeliscolocolo), kodkod (Oncifelis guigna), Andean mountain cat (Orealiurus jacobita), andjaguarundi (Herpailurus yaguaroundi)). Each of these species is consideredendangered or threatened with extinction in all or portions of their natural range[Nowell and Jackson, 1996; Oliveira, 1994]. Long-term conservation of LatinAmerican felids ultimately depends on preserving adequate habitat in rangecountries. However, captive management programs also play a significant role inspecies survival, serving as hedge populations and as educational and researchresources.

A prerequisite to the development of effective breeding programs is a basicknowledge of species biology, including fundamental reproductive traits and status[Wildt and Bush, 1984; Wildt et al., 1993]. Information on reproduction is basic andrelatively limited for virtually all Latin American felid species [Howard, 1993;Barone et al., 1994a; Morato et al., 2001; Moreira et al., 2001; Morais et al., 2002].Most individuals comprising these stocks are wild-born and are genetically valuablefounders, but to date, there has been little successful propagation within felidpopulations in Latin American zoos.

The overall aim of this study was to begin to understand the basic reproductivebiology of felids living in Latin American zoos, while gaining insights into husbandryor other management factors that may influence breeding success or failure in thesepopulations. In this context, our approach was similar to historic [Wildt et al., 1993]and more recent [Wildt et al., 2003] surveys of reproductive health in other species.Using a multidisciplinary, multi-institutional approach, vast amounts of species-

422 Swanson et al.

specific and region-specific data were accumulated, analyzed, and interpreted toestablish reproductive norms and examine management issues that most likelyinfluence reproductive fitness. Thus, our objectives were to: 1) evaluate past breedingsuccess of endemic male felids housed in Latin American zoos, 2) conduct detailedphysiological evaluations to determine current reproductive status, 3) identifymanagement factors that impact reproductive traits, and 4) begin conserving futurereproductive potential by cryopreserving high-quality sperm in a genome resourcebank.

MATERIALS AND METHODS

Survey Protocol and Anesthesia

A parallel survey of molecular genetic-based phylogeny of Latin Americanfelids [Johnson and O’Brien, 1997; Johnson et al., 1998, 1999; Eizirik et al., 1998,2001] provided an opportunity to simultaneously assess male reproductive traits. Forthe genetics survey, blood and skin samples were collected from male and female catsheld in Latin American zoos and private facilities, with the primary focus on wild-born individuals of known origin. Over a 2-year period, five research expeditions—each involving a field biologist, geneticist, veterinarian, and reproductive biologist—were completed.

At each collaborating institution, the local zoo veterinarian, curator, keepers,and/or director were asked to provide historical information on each male in thecollection. Information was obtained regarding the animal’s origin (i.e., wild- vs.captive-born, capture location in the wild, or origin of parents), length of time incaptivity, previous captive breeding success (i.e., proven vs. nonproven breeders) andmain components of the diet (i.e., beef, horse meat, chicken, bones, visceral organs,and vitamin and/or mineral supplementation). At most institutions, additional datawere acquired on the characteristics of the animal exhibit, specifically the presence,number, and species of cage mates.

In general, anesthesia was induced using a tiletamine-zolazepam combination(5–10 mg kg�1 body mass; Telazol, Fort Dodge Laboratories Inc., Fort Dodge, IA)administered i.m. by hand or pole syringe, blow-pipe, or CO2-powered pistol,depending on exhibit design and animal accessibility. This injectable anestheticprovided an adequate plane of anesthesia for electroejaculation of all species exceptthe puma and jaguarundi. Alternative anesthetic regimens that provided a greaterdepth of anesthesia and relaxation were required for the latter two species. Forpumas, males were anesthetized with an initial injection of ketamine hydrochloride(8–10 mg kg�1; Ketaset, Fort Dodge, IA) followed 10–15 min later by an injection oftiletamine-zolazepam (2–3 mg kg�1). For jaguarundis, males were injected with acombination of ketamine (8–10 mg kg�1) mixed with xylazine (0.2–2 mg kg�1;Rompun, Mobay Corp., Shawnee, KS). Each male was anesthetized on only oneoccasion for evaluation. When possible, males were fasted 12–24 hr beforeanesthesia.

Semen Collection and Evaluation

Reproductive evaluations followed a standardized protocol, consisting of pre-electroejaculation blood sampling, evaluation of penile morphology, measurement

Reproduction of Latin American Felids 423

of testicular volume, and regimented electroejaculation [Howard et al., 1986; Wildtet al., 1983]. A 2–3-ml blood sample was collected via jugular venipunctureimmediately after each anesthetized male became tractable (typically within 10–15min of initial anesthetic injection). The penis was extended from the prepuce andevaluated for general shape and any abnormalities (i.e., adhesions). The presence orabsence of penile spines was also noted, but spine dimensions were not determined.Laboratory calipers were used to measure the length and width of each testis, andthese values were used to calculate combined testicular volume [Howard et al., 1986].A lubricated rectal probe (1.0–2.3 cm in diameter, 13–29 cm in length, exact sizedepending on species) and an electrostimulator (PT Electronics, Boring, OR) wereused to deliver 80–120 electrical stimuli (voltage range¼2–6 V), divided into three orfour separate series. Semen was collected into a warmed (371C) sterile container andevaluated initially for volume, and presence or absence of sperm. Recovered spermwere immediately assessed microscopically (100� ) for percent sperm motility(0–100%) and rate of progressive motility (on scale of 0–5, with 0¼no motion,1¼stationary movement with no progression, and 5¼very rapid forward progres-sion). Both motility parameters were used to calculate a sperm motility index([SMI¼% motile+(20� rate of progressive motility)/2]) for each ejaculate [Howardet al., 1986].

For each spermic ejaculate, an aliquot of raw semen (5–10 ml) was fixed in 0.3%glutaraldehyde (100 ml) and 200 sperm were later evaluated by phase-contrastmicroscopy (640–1,000�magnification) for percentage of normal and pleiomorphicsperm forms [Howard et al., 1986]. The remaining raw semen was diluted with equalvolumes of warm (371C) Ham’s F10 culture medium (Irvine Scientific, Santa Ana,CA), that was supplemented with 5% fetal calf serum (Irvine Scientific). Dilutedspermic ejaculates from each electroejaculation series were combined, and spermconcentration for each male was determined by a white blood cell/hemacytometermethod [Howard et al., 1986].

Sperm Cryopreservation

Prior to study onset, a minimal sperm quality standard (i.e., total spermnumber/ejaculate of Z5� 106 motile sperm with an SMI of Z50) was identified forcryopreservation. Each ejaculate meeting this standard was centrifuged (200 g) toremove seminal fluid and culture medium, and the resulting sperm pellet wasresuspended in cryoprotectant medium (PDV, consisting of 11% lactose, 20% eggyolk, and 4% glycerol) [Platz et al., 1978] to a target concentration of 50–100� 106

motile sperm/ml. Diluted sperm were cooled in a refrigerator or ice chest for 30 minto 51C and, depending on the availability of dry ice and/or liquid nitrogen (LN2),frozen by one of two methods: 1) pelleting onto indentations in dry ice [Howard etal., 1986]; or 2) straw freezing at a controlled rate (�401C/min) over LN2 vapor[Wood et al., 1993]. Frozen pellets and straws were sealed in cryovials and stored inLN2 dry shippers (Taylor-Wharton, Theodore, AL) for transport.

Analysis of Serum Hormones

Collected blood samples were allowed to clot in a refrigerator, and serum washarvested after centrifugation and stored in labeled cryovials, initially in LN2 andlater at –801C. For analysis, each sample was thawed and assessed for testosteroneand cortisol concentrations using a double antibody testosterone 125I radio-

424 Swanson et al.

immunoassay kit (Diagnostic Products Corp., Los Angeles, CA) and a solid-phasecortisol 125I radioimmunoassay kit (Coat-A-Count, Diagnostic Products), respec-tively, as previously described [Carlstead et al., 1992; Brown et al., 1996; Morais etal., 2002].

Statistical Analyses

Differences between large (jaguar and puma) and small (all other species) catsin percentages that were wild-born, proven breeders, provided an adequate diet, andhoused alone or paired, were assessed by chi-square analysis (Statview, SASInstitute, Inc., Cary, NC). Mean values (7standard error of the mean (SEM)) werecalculated for reproductive and hormonal characteristics for each species. Valueswere compared using analysis of variance (ANOVA) and least significant difference(LSD) means comparison (Statview). The effect of independent factors (provenstatus, diet, and housing) on dependent reproductive/hormonal variables wasassessed by multiple regression analysis (Statview). Males that had sired offspring atany time during captivity were considered proven breeders. Diets were classified asnutritionally inadequate (all beef, horse meat, or chicken head/neck, withoutsupplementation with vitamin/mineral mixtures or whole carcasses more than onceper week) or adequate (the above diets with vitamin/mineral or whole carcasssupplementation more than once per week). For housing status, males were classifiedas being maintained alone or paired with a conspecific female, or held in some otherconfiguration (e.g., with a conspecific male, multiple conspecific females, and/orheterospecific males or females). For purposes of analysis, independent variableswere assigned a numerical value of 1 (for proven breeder, adequate diet, and alone/paired housing) or 2 (for nonproven male, inadequate diet, and other housingconfiguration). Multiple regression analysis was conducted initially for the completedata set (i.e., all species) and then for large- (jaguar and puma) and small- (all otherspecies) sized felids as separate groups.

RESULTS

Figure 1 illustrates the geographic distribution of the collaborating zoos andprivate facilities across 12 countries. Institutions in Mexico and Central Americawere visited in January and February (winter), whereas South American facilitieswere visited January through September (summer, autumn, and winter). Reproduc-tive evaluations were conducted in 185 male cats representing eight felid species(Table 1). Neither the kodkod nor the Andean mountain cat was encountered at anyof the collaborating institutions. Of the 185 males, 172 (93%) were wild-born. Theremaining 13 individuals (four jaguars, six pumas, two ocelots, and one jaguarundi)were captive-born to wild-born parents. Virtually all of the small cats (126/129, 98%)were wild-born compared (Po0.05) to the larger cats (46/56, 82%). Only 37 males(20%) could be classified as proven breeders, the majority of which were jaguars,pumas, and ocelots (Table 1).

Diets at 29 of 44 (66%) facilities (representing 128 surveyed males) wereconsidered inadequate, and were comprised almost entirely of red meat (horse orbeef) or chicken heads and necks. Fifteen (34%) institutions provided fairly routine(more than once a week) diet supplements of whole prey carcasses, organ meat, orcommercial vitamin/mineral mixtures. Overall, only 57 of 185 (31%) cats received


nutritionally adequate diets, with no difference (P40.05) between large (29%, 16/56)and small (32%, 41/129) cats.

Males were housed either alone (26%), paired with a single conspecific female(43%) or single conspecific male (12%), or grouped with multiple conspecific malesand females (14%) or heterospecific cagemates (6%; three exhibits housing two malemargays with one female tigrina, one male margay with one male tigrina, and onemale and two female margays with two male jaguarundis). Similar (P40.05)proportions of large (79%) and small (65%) cats were housed alone or paired with aconspecific female vs. other combinations.

Table 2 presents a compilation of all male reproductive and endocrine data foreach species. Of the 185 males, 131 (71%) had sperm in their ejaculates. Except forthe pampas cat, aspermic individuals were observed in all species (ranging from 4%

Fig. 1. Geographic location of 44 zoological institutions and private facilities (identified bystars) in the 12 Latin American countries that participated in the reproductive survey. Thenumber of individual cats that were evaluated in each country are listed using three-lettercodes to designate species (Pon¼jaguar (Panthera onca), Pco¼puma (Puma concolor),Lpa¼ocelot (Leopardus pardalis), Lwi¼margay (Leopardus wiedi), Lti¼tigrina (Leopardustigrinus), Oge¼Geoffroy’s cat (Oncifelis geoffroyi), Oco¼pampas cat (Oncifelis colocolo), andHya¼jaguarundi (Herpailurus yaguaroundi)).

426 Swanson et al.

of Geoffroy’s cats to 43% of jaguarundis), with no difference (P40.05) betweenlarge (30%) and small (29%) cats. In general, sperm concentration was low acrossspecies, with only 87 (47%) having at least 1� 106 total sperm/ejaculate, and just 53(29%) with Z10� 106 total sperm. As expected, larger cats (jaguars and pumas) hadgreater (Po0.05) semen volume, total sperm/ejaculate, testicular volume, and/orserum testosterone than most of the smaller cats. Among the small cats, the ocelotwas notable for having the greatest semen volume, total sperm/ejaculate, percentageof normal sperm forms, and testicular volume. Although small-sized cats producedsmaller ejaculate volumes, they averaged higher (Po0.05) sperm concentrations thanthe large-sized felids.

Four of the five smallest cat species (o5 kg body weight) had higher (Po0.05)serum cortisol values (490 ng/ml) than most of the larger cats (Table 2). Theexception was the puma, which produced an overall cortisol average of 4250 ng/ml.Sperm motility indices generally were similar among species (Table 2), with thelowest values found in jaguarundis and pumas, and the highest values observed inpampas cats and ocelots.

The proportion of pleiomorphic sperm forms was more variable, with thepuma, margay, tigrina, and jaguarundi having fewer (Po0.05) structurally normalsperm (o40%) compared to the jaguar and ocelot (455% normal). Notable spermabnormalities (Table 3) included a high percentage (45%) of head anomalies(particularly abnormal acrosomes) in jaguars and jaguarundis. Midpiece anomaliesincluded high percentages of abnormal or absent midpieces (44% in the puma,tigrina, and jaguarundi), a bent midpiece with or without residual cytoplasmicdroplet (420% in the puma, tigrina, pampas cat, and jaguarundi), and a proximalor distal droplet (48% in the jaguar and jaguarundi). High percentages of flagellaranomalies (mainly bent or coiled tails (425%)) were observed in the margay, tigrina,and Geoffroy’s cat.

As regards penile morphology, spine formation in the jaguar and puma wasminimal, consisting of widely spaced ‘‘bumps’’ on the glans, with very small spines attheir center. While penile spines were prominent in the tigrina, Geoffroy’s cat,

TABLE 1. Origin, proven breeding success and diet/housing status of male felids in Latin

American zoos

SpeciesNumberof cats

Number wildborn (%)

Numberproven

breeders (%)a

Numberprovidedadequatediet (%)b

Number housedalone or

paired (%)c

Jaguar 21 17 (81) 7 (33) 3 (4) 14 (78)Puma 35 29 (83) 9 (26) 13 (37) 23 (79)Ocelot 38 36 (95) 9 (24) 14 (37) 26 (87)Margay 27 27 (100) 3 (11) 11 (41) 9 (50)Tigrina 17 17 (100) 2 (12) 4 (24) 9 (64)Geoffroy’s cat 24 24 (100) 4 (17) 3 (13) 11 (55)Pampas cat 2 2 (100) 0 (0) 0 (0) 2 (100)Jaguarundi 21 20 (95) 3 (14) 9 (43) 7 (47)

Total 185 172 (93) 37 (20) 57 (31) 101 (69)

aSired offspring at least once during captive history.bProvided with a whole prey item or vitamin-mineral supplement more than once per week.cFor males (n¼146) with known housing status only.


TABLE

2.Reproductivecharacteristics

ofmale

felidsmaintained

inLatinAmericanzoosn

Species

(no.cats)

Sem

envolume

(ml)

Sperm

concentration

(�106ml)

Total

sperm

(�106ml)

Sperm

motility

index

a

Norm

al

sperm

(%)

Testicular

volume

(cm

3)

Testosterone

concentration

(ng/m

l)

Cortisol

concentration

(ng/m

l)

Jaguar

6.2971.44b

4.371.7

b,f

39.3719.8

b65.872.1

b,c

57.375.0

b46.272.9

b3.7570.53b

57.175.5

c,d

(n¼21)

(0.33–23.50)

(0–34)

(0–406.0)

(50.0–70.0)

(21–90)

(28.7–72.6)

(0.57–10.60)

(10.9–110.3)

Puma

2.0570.22e

15.674.5

b,e,f

27.778.4

b,c

60.672.1

c33.573.4

c,e

18.270.7

a2.1370.34c

271.9718.7

b

(n¼35)

(0.26–6.08)

(0–139)

(0–246.0)

(52.5–80.0)

(3–65)

(12.2–26.6)

(0.06–5.85)

(100.0–592.8)

Ocelot

0.6270.08d

53.8717.9

c,d,f

34.2711.9

b,d

70.772.2

b58.475.8

b22.071.3

d2.0070.27c

35.274.8

c

(n¼38)

(0–2.32)

(0–483)

(0–307.2)

(45.0–80.0)

(19–91)

(4.1–34.0)

(0–7.47)

(0–97.4)

Margay

0.3170.05d

14.275.3

b,e,f

6.472.8

c62.875.3

b,c

39.577.7

c,d,e

4.870.4

e2.0770.33c

54.476.9

c,d

(n¼27)

(0.03–1.46)

(0–132)

(0–57.0)

(12.5–80.0)

(5–85)

(1.7–10.0)

(0.07–6.23)

(4.7–141.8)

Tigrina

0.1170.02d

83.0735.5

d10.073.8

b,c

62.175.7

b,c

35.676.0

c,d,e

2.870.3

e0.7670.14d,e

91.1711.3

d,e

(n¼17)

(0–0.29)

(0–526)

(0–57.4)

(25.0–80.0)

(9–70)

(0.9–4.9)

(0.08–2.03)

(46.5–214.3)

Geoffroy’s

0.2170.03d

66.5724.4

d8.571.7

c,d

64.074.7

b,c

46.975.0

d5.570.4

e0.4770.10d,e

109.0710.8

e

cat(n¼24)

(0.01–0.53)

(0–558)

(0–24.7)

(15.0–85.0)

(15–80)

(2.5–10.3)

(0.06–2.13)

(6.6–208.4)

Pampascat

0.0870.01c,d

364.07326.0

e22.6720.2

b,c,d

81.376.3

b,c

56.570.5

b–e

2.870.7

e0.1870.07c,d,e

125.3716.3

e

(n¼2)

(0.07–0.08)

(38–690)

(2.4–42.8)

(75.0–87.5)

(56–57)

(2.1–3.4)

(0.11–0.25)

(109.0–141.5)

Jaguarundi

0.0870.02d

7.274.0

f1.070.5

c57.872.5

c25.774.6

e3.070.3

e0.9670.19e

223.3718.2

f

(n¼21)

(0–0.37)

(0–77)

(0–9.3)

(45.0–65.0)

(9–50)

(1.1–5.4)

(0.07–3.26)

(78.5–389.3)

nMean7SEM;(rangein

values).

aSperm

motility

index¼[%

motility+

(20�sperm

progressivemotility)]/2.

b–f W

ithin

each

parameter,values

withdifferentsuperscripts

differ(Po0.05).

TABLE

3.Sperm

pleiomorphisms(m

ean%7SEM)observed

inejaculatesofmale

felidsmaintained

inLatinAmericanzoos

Felid

species

Sperm

pleiomorphism

Jaguar

(n¼12)

Puma

(n¼24)

Ocelot

(n¼18)

Margay

(n¼15)

Tigrina

(n¼12)

Geoffroy’scat

(n¼20)

Pampascat

(n¼2)

Jaguarundi

(n¼9)

Headanomaly

Macrocephalic

0.370.1

a0.270.1

a,e

0.170.1

a0.670.4

a,b

0.470.2

a0.170.0

a0.370.3

a0.770.4

b,c

Microcephalic

0.570.1

b0.870.2

a,b,e

0.470.2

a,b

1.570.6

b1.370.4

b,d

0.470.1

b0.370.3

b1.370.4

a

Bicephalic

0.170.0.1

c0.2701b,c,c

0.270.1

a,c

0.270.2

a0.370.1

a0.470.2

c0.370.3

b0.470.3

b

Abnorm

alacrosome

4.871.4

c2.470.6

a,b,e

0.870.3

b1.870.5

a,b

1.470.4

b,c,d

1.070.3

d2.071.5

b6.671.6

b,c

Midpiece

anomaly

Abnorm

almidpiece

2.971.2

c2.970.6

c2.370.4

a,b

3.270.6

a,b

4.371.1

b,c,d

1.470.6

d0.570.5

c,d

4.871.3

c,d

Absentmidpiece

0.370.2

c1.370.7

c0.070.0

b,c

0.270.1

a,b

0.570.3

c0.570.3

d0c,d

0.570.2

d

Bentmidpiece

with

cytoplasm

icdroplet

8.872.3

c22.772.1

e3.672.0

b9.072.4

b10.072.0

d12.572.3

c16.372.3

d17.873.5

e

Bentmidpiece

w/o

cytoplasm

icdroplet

2.870.8

c8.271.6

e2.670.6

b7.871.7

b11.372.9

d5.070.9

c5.075.0

d8.872.1

e

Proxim

aldroplet

4.970.9

b,c

4.571.0

d1.971.5

a,b,c

2.070.5

a,b

1.570.6

a–d

1.870.7

d2.872.3

b,c,d

5.871.5

d

Distaldroplet

5.971.2

c2.970.6

e3.771.7

b1.570.5

b1.570.4

d1.870.6

c0.870.8

d2.270.5

e

Flagellaranomaly

Bentflagellum

4.771.3

c3.870.6

e20.774.0

b22.676.0

b20.576.0

d16.873.9

c7.073.0

d6.370.9

e

Coiled

flagellum

4.871.3

c15.572.7

e3.971.4

b9.772.6

b11.374.6

d8.872.9

c4.874.8

d16.673.9

e

Totaloverall

42.775.0

a66.573.4

b,d

41.675.8

a60.577.7

b,c,d

64.476.0

b,c,d

53.175.0

c43.570.5

a–d

74.374.6

d

a–eWithin

each

parameter,values

withdifferentsuperscripts

differ(P

o0.05).

pampas cat, and jaguarundi, they were most developed in the ocelot (Fig. 2a) andliterally nonexistent in the margay (Fig. 2b).

Abnormal penile morphology was noted in four males. One individual initiallyclassified on the basis of morphology as a tigrina had a glans penis distorted into a‘‘club shape ’’ distally (rather than tapering toward the tip). This male also had fewpenile spines and produced an aspermic ejaculate, despite having the largesttesticular volume (6.2 cm3) in the tigrina cohort (mean¼2.8 cm3). Molecular geneticanalysis later proved that this wild-born male was a tigrina-pampas cat hybrid[Johnson et al., 1999]. The data from this animal were therefore excluded from thereproductive data set.

Three ocelots demonstrated an abnormal penile condition consisting ofcircumferential preputial adhesions that prevented glans extension 41 cm fromthe prepuce (Fig. 2c). These individuals were all wild-born and housed at institutionsin Mexico, Panama, and Bolivia. One ocelot was aspermic, although the otherocelots produced low to moderate sperm numbers (1.5–29.6� 106 total sperm), butwith few (o25%) normal sperm forms. Two of the ocelots also had abnormallysmall testicular volumes (4.1 and 9.1 cm3) and low circulating testosterone (0.26 and0.61 ng/ml).

The results of multiple regression analysis based on proven breeder, diet, andhousing status are presented in Table 4. For all felid species combined, provenbreeders had greater (Po0.05) semen and testicular volumes and higher (Po0.05)circulating testosterone than nonproven counterparts. Animals maintained onnutritionally adequate diets produced fewer (Po0.05) normal sperm forms than didindividuals on poor diets. Further, males housed alone or paired with a conspecificfemale had more (Po0.05) total sperm per ejaculate than males housed in othercombinations.

Analysis of these data within cat size groups, however, provided a differentperspective (Table 4). Large felids that were proven breeders had bigger (Po0.05)testes and produced greater (Po0.05) ejaculate volume and more (Po0.05) normalsperm than non proven breeders. Large cats on a nutritionally adequate diet had alower (Po0.05) percentage of normal sperm, and higher (Po0.05) serum cortisolconcentrations than males on poor diets. Among small felids, proven breeders hadhigher (Po0.05) testosterone than nonproven males, and those individualsmaintained alone or with a single conspecific female had larger (Po0.05) testesand produced more (Po0.05) semen and sperm per ejaculate than males housed inother configurations. Multiple regression analysis indicated that dietary status hadno (P40.05) influence on male reproductive traits in small felids.

Fifty-nine of the ejaculates (32%) from eight species met the spermcryopreservation criteria (Table 5). Most (n¼48) were frozen by pelleting themonto dry ice, and the remainder (n¼11) were frozen in straws over LN2 vapor.Samples were stored in the National Zoological Park’s Felid Genome ResourceBank. Based on known pre-freeze motility and sperm concentration of each storedsample, and a minimal dose of 10� 106 motile sperm per artificial insemination (AI)

Fig. 2. Penile morphology: (a) ocelot penis with prominent penile spines, (b) margay peniswith a total absence of penile spines, and (c) ocelot penis with circumferential adhesionbetween the glans and the prepuce.

c

430 Swanson et al.

TABLE4.Probabilityvalues

formultiple

regressionanalysisoffactors

affectingmale

reproductiveparametersforlarge-

andsm

all-sized

felidspeciesin

LatinAmericanzoos

Allfelidspecies

Large-sizedfelidspecies

Small-sized

felidspecies

Dependentvariable

Proven

statusa

Diet

statusb

Housing

statusc

Proven

status

Diet

status

Housing

status

Proven

status

Diet

status

Housing

status

Sem

envolume

0.01n

0.73

0.53

0.05n

0.74

0.91

0.86

0.34

0.02n

Sperm

concentration

0.13

0.88

0.21

0.39

0.47

0.70

0.11

0.65

0.17

Sperm

per

ejaculate

0.46

0.85

0.03n

0.97

0.61

0.42

0.09

0.40

0.03n

SMId

0.50

0.65

0.46

0.47

0.36

0.73

0.48

0.33

0.45

%norm

alsperm

0.07

0.01n

0.83

0.05n

0.003n

0.32

0.16

0.43

0.41

Testicularvolume

0.03n

0.34

0.07

0.03n

0.13

0.48

0.80

0.92

0.01n

Testosteroneconcentration

0.001n

0.37

0.55

0.06

0.83

0.85

0.01n

0.16

0.45

Cortisolconcentration

0.45

0.33

0.77

0.56

0.02n

0.38

0.42

0.54

0.36

nP

valueo

0.05.

aSired

offspringduringcaptivehistory

ornon-proven.

bMaintained

onanutritionally-adequate

orinadequate

diet.

cHousedalone/paired

withfemale

orin

another

grouping.

dSperm

Motility

Index¼[%

motility

þ(20�rate

offorw

ard

progression]/2.

dose [Swanson et al., 1996], these cryopreserved samples represent B101 potentialAI procedures. Alternatively, if the samples were used for in vitro fertilization (IVF;each with 5� 104 motile sperm and 10–15 conspecific oocytes per insemination drop)[Donoghue et al., 1992], a total of B26,000 microdrops containing 4250,000conspecific oocytes could be inseminated.

DISCUSSION

This study represents the first survey of the reproductive status of endemicfelids maintained in Latin American zoos and private facilities. Because of thesignificant number of animals and institutions involved over a wide geographicrange, these data represent a fairly comprehensive assessment of basal reproductivetraits for eight Latin American felid species. We believe that the informationgenerated here helps to define the overall reproductive status of the captivepopulation, and has important implications for zoo-based activities in thesecountries. Notably, our findings highlight several management factors that shouldbe addressed before Latin American zoos can fulfill their enormous potential forparticipating in ex situ breeding and conservation of these rare felid species.

Previous surveys of wild felids focused primarily on a singular species. Thepresent study was unique in that, for the first time, comparisons could be madeacross average body size or species. For example, it was not surprising that largejaguars and pumas had larger testes and ejaculated more seminal volume, andtherefore had more sperm per ejaculate. However, smaller felids tended to producemore sperm per seminal volume, which suggests that these species have more efficientsperm output per testes—a characteristic that was previously noted in a comparisonof the smaller tigrina to the ocelot and margay [Morais et al., 2002].

Although sperm motility was generally consistent among species, thepercentage of pleiomorphic sperm was highly variable, with the puma, margay,tigrina, and jaguarundi consistently producing o40% normal sperm forms. Types ofsperm abnormalities also varied significantly among species: serious head anomalieswere predominant in the jaguar and jaguarundi; midpiece malformations were morecommon in the puma, tigrina, and jaguarundi; and flagellar anomalies were more

TABLE 5. Genome resource banking of spermatozoa from Latin American felids

SpeciesNumbercollections

Numberejaculates frozena

Numberpotential AIsb

Number potentialIVFsc

Jaguar 21 6 20 4,573Puma 35 13 17 4,927Ocelot 38 12 44 10,028Margay 27 8 7 2,171Tigrina 17 6 4 1,244Geoffroy’s cat 24 11 7 2,488Pampas cat 2 1 2 425Jaguarundi 21 2 0 95

Total 185 59 101 25,951

aEjaculates with SMIZ50 and total sperm count Z5� 106.bBased on insemination dose of 10� 106 motile spermatozoa (pre-freeze motility) per AI.cBased on insemination dose of 5� 104 motile spermatozoa (pre-freeze motility) per IVF drop.


prevalent in the margay, tigrina, and Geoffroy’s cat. The causes of specificmorphological defects in cat sperm have not been identified, but a clear link has beenshown between limited genetic variation and high percentages of sperm pleiomorph-isms in certain species and populations (i.e., cheetah, Florida panther, and lion)[Wildt, 1994; Wildt et al., 1995]. More importantly, it is well known that thesestructurally deformed sperm do not participate in fertilization [for review seePukazhenthi et al., 2001].

Interestingly, testosterone concentrations tended to vary consistently withspecies size. The largest felid (the jaguar) had the highest circulating testosterone,followed by the next two largest cats (the puma and ocelot), and then by the smallestfelids (margay, tigrina, jaguarundi, pampas cat, and Geoffroy’s cat). In contrast (andwith the exception of the puma (see below)), cortisol concentrations were inverselyrelated to size, with the smallest cats typically having the highest circulating cortisol.Such a finding may indicate inherent species differences in basic adrenal function,and/or, more likely, that these wild-born small cats are stress-susceptible in captivity.For clarification, further studies are warranted. It would be especially useful tomonitor gonadal steroid patterns in various small felid females [Moreira et al., 2001],as normal profiles would be indicative of adaptation to zoo conditions.

Because blood samples were obtained from most individuals within 10–15 minof anesthetic injection, it was assumed that serum cortisol values reflected basal ornear-basal concentrations [Carlstead et al., 1993a]. The exception was the puma thatrequired pre medication with ketamine, and thus a longer time (an additional 10–15min) to obtain a blood sample. In a previous study [Wildt et al., 1988], we observedgradually rising cortisol concentrations over time in anesthetized pumas, so it wasnot surprisingly that this species had the highest cortisol levels of any speciesevaluated. A more recent study [Nogueira and Silva, 1997] also suggested thatelevated basal cortisol is a species-specific trait for the puma.

Historically, little attention has been devoted to penile morphology in felids,although the presence of penile spines in the domestic cat is known to be androgen-dependent and is presumed to be stimulatory for ovulation induction during mating[Aronson and Cooper, 1967]. Our survey revealed pronounced species differences inthe occurrence and size of these spines that were not connected to circulatingtestosterone. For example, although jaguars and pumas had some of the highestserum testosterone concentrations, their penile spines were relatively small andundeveloped. Among the smaller cats, ocelots had the most prominent spines, buttheir closest genetic relative, the margay [Johnson and O’Brien, 1997; Eizirik et al.,1998], had no spines. All other small felids had spines of various degrees. The femaleocelot is known to be an induced ovulator, in contrast to recent findings that themargay ovulates spontaneously [Moreira et al., 2001], which suggests that thepresence of penile spines is indeed species-specific and adaptive.

Three surveyed ocelots (all wild-born) had circumferential adhesions thatprevented penile extension from the prepuce, which likely prevented successfulcopulation. In the domestic cat, formation of the preputial cavity is testosterone-dependent [Bharadwaj and Calhoun, 1961], and male cats castrated before pubertyexperience penile abnormalities [Herron, 1971; Root et al., 1996] similar to thoseseen in the three ocelots. Because these animals originated in the wild in threedifferent countries, this defect is unlikely to have a familial genetic basis.Furthermore, the precise histories of these individuals, including age at capture,

434 Swanson et al.

were unknown. Male ocelots have a prolonged (42 years) prepubertal growth phase,and two of the three affected males had nadir (o1 ngml�1) testosteroneconcentrations. One possible explanation is that captive rearing of the youngocelots under substandard conditions retarded reproductive development, contribut-ing to defective preputial cavity formation, which essentially removed these malesfrom the breeding population. It is unlikely that hormonal therapy or surgicalintervention would be effective in correcting this developmental defect.

Compared to data reported for these same species housed in North Americanzoos and/or Brazilian zoos under more controlled conditions, reproductive valuesfrom the survey appeared more variable [Wildt et al., 1988; Barone et al., 1994a;Howard, 1993; Morato et al., 2001; Morais et al., 2002]. Such broad-scalecomparisons are difficult, largely due to variations in husbandry factors or studymethods, and, in this case, the wide geographic distribution of the collaboratinginstitutions. Nonetheless, some of these species housed in North American zoos(puma, margay, and Geoffroy’s cat) [Wildt et al., 1988; Barone et al., 1994a;Howard, 1993] appear to have greater sperm numbers per ejaculate than theirconspecifics in Latin American zoos. However, for others, the total sperm numbersper ejaculate (jaguar, ocelot, and jaguarundi) and proportion of pleiomorphicsperm forms (jaguar, puma, and Geoffroy’s cat) were nearly identical or eveninferior to those observed in the survey [Wildt et al., 1988; Barone et al., 1994a;Howard, 1993].

Nevertheless, a review of breeding records at Latin American zoos revealedthat few male felids (o20%) had ever reproduced during their time (months toyears) in captivity. Furthermore, the detailed reproductive assessment indicated thatmore than half of all ejaculates contained o1 million total sperm, which is anextraordinarily low number for felids [Wildt et al., 1998]. Clearly, these twoobservations (poor breeding success and low sperm count) indicate the existence ofother, undefined interference factors that must be understood and corrected beforethese populations can contribute to robust breeding programs.

Lack of genetic variation does not appear to be related to suboptimalreproductive status, because recent assessments of these same populations revealedample heterozygosity [Johnson and O’Brien, 1997; Eizirik et al., 1998, 2001; Johnsonet al., 1999; Culver et al., 2000], with the possible exception of the puma in northernLatin America [Culver et al., 2000]. Seasonality also does not appear to becontributing to poor breeding or sperm traits because 1) we observed similarmeasurements within species across three different seasons, and 2) most LatinAmerican felids do not exhibit pronounced circannual patterns in reproductivecapacity [Nowell and Jackson, 1996; Moreira et al., 2001; Morais et al., 2002]. Forthese reasons, our attention turned to examining findings on the basis of pastbreeding success, diet, and housing status.

Multiple regression analysis within large- and small-sized species groups wasmost revealing. This approach was justified because of differences between these twocohorts in reproductive performance, origin, and management in zoos. For example,compared to small cats, large cats reproduced more readily based on the percentageof proven breeders and overall number of captive-born individuals encountered.Excluding ocelots, 485% of all small cats were failing to breed in captivity. It wasalso possible that overall nutritional quality differed between the size groups, sincelarge felids are capable of ingesting large bones in daily diets (as a source of calcium).


Furthermore, unlike small cats, the large cats typically are not housed in groups ofconspecifics or heterospecifics.

For large felids, proven status and diet strongly influenced reproductive traits.Proven breeders had larger testes, produced more semen volume, and had ejaculateswith more structurally normal sperm than nonproven breeders. Proven breeders alsotended to have higher testosterone, although there was no significant difference(P¼0.063). Thus, at least for the jaguar and puma, we contend that these fourcharacteristics (testis size, proportion of normal sperm, semen volume, and bloodtestosterone concentration) may be useful predictors of fertility potential.

Oddly, large felid males maintained on nutritionally adequate diets ejaculatedfewer normal sperm forms and had higher circulating cortisol than their counter-parts on inadequate diets (large felids fed the poorer diet averaged B15%more normal sperm). We suspect that this finding is primarily attributable toregional genetic variation in pumas. Interestingly, and coincidentally, increases innormal sperm morphology within this species and the percentage of pumas giveninadequate diets tended to follow a north-to-south gradient from Mexicoand Central America (B20% normal sperm forms; B80% of pumas on adequatediets) to southern South America (B45% normal sperm forms; B17% on adequatediets). This apparent paradox may be related to levels of molecular genetic variation,which in the puma (and particularly the Florida panther) has been tightly linked tosperm quality [Miller et al., 1990; Barone et al., 1994a; Pukazhenthi et al., 2001].Genetic heterozygosity increases in pumas from north to south (Culver et al., 2000),and it probably is this significant geographic variation in genetic diversity thatconfounds attempts to interpret the impact of diet on sperm morphology in thisspecies.

In contrast, the higher cortisol concentration among large cats on nutritionallyadequate diets was found primarily in jaguars. The three males on adequate dietshad higher cortisol than the 18 males on inadequate diets. Although this may be aconsequence of the small sample size of the former subgroup, the effect may havereflected depressed steroid synthesis among males provided with inferior nutrition[Brown, 1994]. Regardless, a positive relationship between diet and adrenal functionon the basis of a one-time cortisol value was inconsistently observed in all other felidspecies.

For the small cats, proven breeder status and housing were the best overallpredictors of measured reproductive values. Proven breeders had higher testosteroneconcentrations, and males housed alone or with only one conspecific female hadlarger testes and produced more semen volume and more total sperm per ejaculate.More than one-third of the small cats surveyed were maintained in group exhibitswith multiple conspecific males and/or females in a single enclosure, or, occasionally,with heterospecific males and females. The housing of small cats in multiple maleand/or female groups in North American zoos has been associated with reducedreproductive success compared to that obtained with cats maintained as heterosexualpairs [Mellen, 1991]. Many small-cat exhibits in Latin American zoos also lackhiding places and den boxes, a deficiency that could contribute to stress and perhapspartially explain the elevated cortisol concentrations (4100 ng ml–1) in some species(e.g., Geoffroy’s cat, pampas cat, and jaguarundi). It is now well known that a lackof nest boxes or hiding places, and of visual or olfactory proximity to large felids(which may be their natural predators in the wild), can induce acute increases in

436 Swanson et al.

stress hormones in small cats [Carlstead et al., 1993b]. For the many small felids nowhoused in Latin American zoos, providing more exhibit ‘‘security’’ and evaluatingthe impact of housing regimens on adrenocortical activity (especially using fecalcortisol metabolite monitoring for longitudinal assessments) [Graham and Brown,1996; Morais et al., 1997] should be given high priority.

Surprisingly, diet was not a major variable influencing reproductive traits insmall felids. All meat and chicken-neck diets are known to be deficient in essentialvitamins and minerals, including vitamins A, D, and E, and have unbalancedcalcium: phophorus ratios [Ullrey and Bernard, 1989]. Vitamin A and taurinedeficiencies have reproductive consequences in female felids, primarily pregnancyloss and small litter size [Scott and Scott, 1964; Scott, 1968; Dieter et al., 1993].Although comparative data are lacking for male felids, vitamin A has a pronouncedeffect on spermatogenesis in other mammals. A 2-month deficiency was reported tocause complete aspermia in the rat [Huang and Hembree, 1979]. The effect of anunbalanced calcium: phosphorus ratio on felid reproduction is also unknown, butsecondary hyperparathyroidism and osteomalacia may produce skeletal defects thatinterfere with normal breeding and bearing of offspring [Pedersen, 1983]. Regardless,we have no definitive explanation for the disconnect between diet and reproductionin small felids.

Since almost all of our surveyed animals were housed in outdoor exhibits, it ispossible that some naturally supplemented their diets by capturing bird or rodentprey. More likely, the strong impact of housing with other small felids obscured aclearly defined dietary effect. Our recent findings from a Brazilian study [Morais etal., 2002] support the need to concurrently improve both exhibit quality and diet toinfluence reproductive success in small cats. In that study, ocelots, margays, andtigrinas maintained singly in off-exhibit enclosures, and provided vitamin-mineralsupplements, exhibited three- to sevenfold greater sperm numbers per ejaculate, and20–30% improvement in normal sperm morphology compared to species normsfrom the survey.

Even though most males failed to provide high-quality ejaculates, othersproduced sufficient numbers of good- to excellent-quality sperm suitable forcryopreservation. The latter samples were consistent with those already used toproduce offspring successfully by assisted breeding with fresh or thawed aliquots[Barone et al., 1994b; Swanson et al., 1996; Moraes et al., 1997]. When the totalnumbers of viable sperm are considered in the context of the numbers that werepreviously required to produce offspring by AI or IVF, the value of systematic spermbanking becomes even clearer. The technology is approaching feasibility. AI withfresh or thawed sperm has been used to produce offspring in the puma [Moore et al.,1981; Barone et al., 1994b], ocelot [Swanson et al., 1996; Moraes et al., 1997], andtigrina [Moraes et al., 1997]. IVF embryos have been produced in the jaguar, puma,ocelot, tigrina, Geoffroy’s cat, and jaguarundi using fresh or thawed sperm [Milleret al., 1990; Pope, 2000; Morato et al., 2000; Swanson, 2001; Swanson et al., 2002](Swanson et al., unpublished data), and transfer of frozen-thawed embryos has beensuccessful in the ocelot [Swanson, 2001]. The potential value of such strategies is evengreater considering that most donors in the present survey were wild-born (i.e.,founders). Overall, this represents a powerful conservation resource, especially ifnational, regional, or even global management plans could be developed andimplemented for these species [Ballou, 1992; Wildt, 1992].


Finally, there were two other crucial components to this study. One was thegenerous collaboration of a wide range of hospitable and dedicated people andinstitutions. The establishment of partnerships to conduct such intensive, multi-disciplinary studies is essential. The other component involved technology transferand capacity building in the range countries. This survey fostered interactionsbetween American scientists and their counterparts in Latin America, initiallyinvolving hands-on, short-term training that, in some cases, evolved into moreextensive training and collaborations (especially in Mexico and Brazil) [Swanson etal., 1999]. The rewards attained by training zoo staff in husbandry and captivebreeding have long been recognized [Waugh, 1988; Wemmer et al., 1990]. However,the findings of the current study, and the significance of certain husbandry factors onreproductive success in felids support the notion that even more zoo-biology trainingprograms are warranted throughout Latin America. An important, parallelapproach is the mentoring of highly promising trainees, another avenue thatemerged from this survey. Several Latin American scientists who participated in thisoriginal study have since applied their new skills to related, advanced-degreeresearch. In doing so, their investigations have greatly broadened our understandingof the reproductive biology of Latin American felids, while helping to developassisted reproductive technologies for species management [Moraes et al., 1997;Brousset et al., 2000; Morato et al., 2001; Moreira et al., 2001; Morais et al., 2002].Most importantly, there now exists a cadre of highly skilled reproductive specialistsin Latin America who are connected to a North American network of colleagues,and are focused on the study and conservation of rare felid species in their nativecountries.

CONCLUSIONS

1. A broad-based reproductive survey of 185 male cats representing eightendemic species located at 44 facilities in 12 countries has allowed the characteriza-tion of reproductive norms and, most importantly, assessment of reproductivestatus.

2. Less than 20% of male felids in Latin American zoos are proven breeders,450% of males have a low sperm count (o1� 106 total sperm), and several species(puma, margay, tigrina, and jaguarundi) produce a high proportion of pleiomorphicsperm.

3. Multiple regression analysis revealed that among large felids (jaguars andpumas), proven breeders had larger testes, produced greater ejaculate volume, andhad more normal sperm forms than their non-proven counterparts. Malesmaintained on nutritionally adequate diets had a lower percentage of normal spermforms (which likely was related to a geographic–genetic interaction confounding thereproductive data), and had higher circulating cortisol.

4. Among small felids, proven breeders had higher testosterone, and maleshoused alone or paired with a conspecific female had larger testes and producedmore seminal volume and total sperm per ejaculate than males housed in othercombinations.

5. Although the majority of semen samples failed to meet sperm cryopreserva-tion critiera, 59 ejaculates (potentially representing about 100 AI or 26,000 IVFprocedures) were frozen for a felid genome resource bank.

438 Swanson et al.

6. Improvements in diet and exhibitry in Latin American zoos, coupled withcapacity building among resident zoo staff and scientists, are essential if LatinAmerica is to contribute significantly to the conservation of biologically imperiledfelid species.

ACKNOWLEDGMENTS

The authors thank Drs. Joe Lopez, Marilyn Menotti-Raymond, MelanieCulver, and Jill Pecon-Slattery (Laboratory of Genomic Diversity, National CancerInstitute) for their assistance with the reproductive evaluations, and Drs. JanineBrown and Laura Graham (Conservation and Research Center) for their help withthe endocrine assays. The authors also thank the following institutions and/orindividuals in Latin America for their cooperation: Universidad NacionalAutonoma de Mexico, Africam Safari, Centro Zoologico de Sonora, ProyectoBalam, Zoologico de Leon, Zoologico el Centenario, Zoologico San Juan deAragon, Zoologico Tamatan, Zoologico Zoochilpan, and Zoomat (Mexico); ParqueZoologico La Aurora, and Autosafari Chapin (Guatemala); Parque ZoologicoEdgar Lang, and Zoologico de Juigalpa (Nicaragua); Werner and Lilly Hagnauer,and Zoologico Simon Bolivar (Costa Rica); CEPEPE o Zoologico del Summit(Panama); Emperor Valley Zoo and Guptee Lutchmedial (Trinidad and Tobago);Parque Aquarium J.V. Seijas, Fundacion Nacional de Parques Zoologicos yAcuarios, Jardin Acuario de Merida, Jardin Zoologico Las Delicias, ParqueZoologico Miguel Romero Antoni, and Zoologico Leslie Pantin (Venezuela);Sociedade de Zoologicos Brasil, Bosque dos Jequitibas, Fundacao Zoobotanica doRio Grande do Sul, Itaipu Binacional, Jardim Zoologico de Brasılia, ParqueZoologico de Goiania, and Parque Zoologico Municipal Quinzinho de Barros(Brazil); Itaipu Binacional (Paraguay); Zoologico Municipal Vesty Pakos andZoologico Municipal Santa Cruz (Bolivia); Parque Pereira de Rossell, Estacion deCria de Fauna Autoctona, Zoologico de Durazno, and Zoologico de Mercedes(Uruguay); and Acuario Municipal de Mendoza, Jardin Zoologico de la Ciudad deBuenos Aires, Zoo Cordoba, Zoologico la Plata, and Zoologico de Rawson(Argentina). This study was funded in part by a grant to S.J. O’Brien from theAmerican Zoo and Aquarium Association’s Conservation Endowment Fund.

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Reproductive status of endemic felid species in Latin American zoos and implications for ex situ...

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