Microsatellites employed in the captive population of jaguars.
The construction of the pedigree of captive jaguars (Panthera onca) in zoological parks of Colombia was done using the analysis of the Regional Studbook for Jaguars and DNA analysis of 9 microsatellites of 20 Jaguars (n=20). The assignments for paternities and maternities were done with for the program CERVUS and the relationship between animals were established with the KINSHIP program. The analysis of the Studbook was done with SPARKS and PM2000 software generating the following values: genetic diversity for the population (GD=0.7832), potential genetic diversity (GD=0.9113), genic value (GV=0.7846), mean coefficient of inbreeding (F=0.0179), and the Mean KINSHIP (MK) for each individual. The averages of the observed and expected heterozygosity were 0.687 and 0.684 respectively. Nevertheless, a wild jaguar sample of 156 individuals obtained in Colombia substantially showed a higher degree of gene diversity (H = 0.87) than the Colombian captive jaguar population. Thus, the captive jaguar population retained 78 % of the gene diversity of the Colombian wild jaguar population. With this study the pedigree of the captive population of jaguars was built in order to develop an ex situ conservation plan for the species in the Colombian zoological parks.
- Panthera onca
During the last decades, genetic analyses have been conducted using microsatellites, on different species of wild felids, in order to solve questions regarding the origin and evolution of members of the
2. Materials and methods
2.1. Genetic analysis with microsatellites
Seeking to construct the pedigree of the population, blood samples were obtained from captive jaguars (
The samples of the 156 jaguars sampled in the wild in Colombia for comparative purposes were from the following localities as follows: representing the putative subspecies
Following a 24-h fast, an anesthesia protocol was conducted, using a combination of xylazine–ketamine. Xylazine, an agonist alfa 2 adrenergic sedative, was administered with an initial injection. After 5 min of its application, and once signs of sedation were observed, a second injection with a fixed dissociative anesthetic, derived from cyclohexylamines (ketamine), was administered. The administration of these medications was conducted using devices of drug injections at a distance (DIDD). The site of the impact of the dart was localized in the muscular mass of the upper and lower limbs; the former being preferred, seeking that the dart or the injection be applied in the most caudal muscular mass and avoiding a possible impact on the femoral bone or the sciatic nerve. The doses of the medications administered were calculated, bearing in mind the reports on weight recorded on the medical history and the doses reported for jaguars [17, 18].
2.1.3. Sample collection
Blood samples were obtained by puncturing the femoral vein or the saphenous vein, having previously disinfected the area with alcohol. The samples were collected in vials with ethylenediaminetetraacetic acid (EDTA) (10 ml) and were maintained at 4°C, until their arrival to the laboratory.
2.1.4. Microsatellites employed
The microsatellite primers selected were developed for domestic cats  (Table 1). The criteria for the selection for the microsatellites were based on the fact that all microsatellites should be in different chromosomes and that their employment be reported on jaguars [12, 13]. The microsatellites selected were marked by fluorescence with fluorochromes that do not overlap among themselves, depending on the microsatellite selected. Only the first of the forward sequence was fluoro-marked for the wild jaguars analyzed for comparative purposes, 12 microsatellites were employed, being them,
2.1.5. Extraction and quantification of DNA
The extraction of DNA was conducted from leucocytes isolated from samples of blood, through the salting-out technique. The concentration of DNA was quantified through spectrophotometry at 260 nm. As for the temperature for each microsatellite, it was calculated through the thermodynamic formula, obtaining that the average annealing temperature needed to conduct the coupling of bases was of 55.5°C. As for the hair samples, the extraction kit DNeasy Blood and Tissue of QIAGEN was employed, following the steps given.
2.1.6. Polymerase chain reaction
The reaction for all the markers were carried to a total volume of 20 μl; these consisted of 1.5 mM of MgCl2; 1.25 mM for each one of the dNTPs (dATP dGTP dCTP y dTTP) plus 0.4 units of DNA polymerase
After having conducted the PCR procedures, the products of amplification were evaluated in agarose gel at 1.5% stained with SYRB-GREEN (Invitrogen, USA). Once the amplification of the products was verified by direct visualization, they were taken to electrophoresis in capillary.
2.1.8. Electrophoresis in capillary
Electrophoresis in capillary was conducted in ABI PRISM® 310 Genetic Analyzer. The results of the electrophoresis in capillary and the naming of the alleles are determined by direct visualization of the chromatograms in the Softgenetics Gene Marker Version 1.97 program. All samples were amplified and Genotypified at least twice to minimize problems of no assignation.
2.2. Statistical analysis
2.2.1. Genetic analysis from records
Records from captive jaguars were analyzed with the information compiled in the Regional Studbook for Jaguars (
Statistics of individual jaguars, founders of the population, and of their descendants was calculated. The allelic
Records from captive jaguars were analyzed with the information compiled in the Regional Studbook for Jaguars (
The value of the
Individual statistics of captive jaguars with a known pedigree were calculated, and the average kinship
2.2.2. Genetic analysis from DNA
For the analysis of the information obtained, the
3.1. Genetic analysis from the regional Studbook of jaguars for Colombia
3.1.1. Demographic and individual statistics
The demographic statistics that determine the genetic characteristics of the population of captive jaguars in Colombia were calculated. By means of the genetic analysis from records, conducted with the PM2000 program, we found that the origin of 28.9% of the population starting in the year 1968 is known, and that there have been five founding animals (T1, T2, T5, T6, T27) for the population; likewise, three possible potential founders were the individuals T32 and T33 present at the Barranquilla zoo, and the T20 in the Piscilago zoological park. These founding animals, coming from wildlife, were either captured or seized and would represent the closest genetics to that that could be found among jaguars in the wild. In this way, individual statistics were obtained, of the jaguars founders of the population, where the number is presented on the Studbook, as well as the gender, age, representation (proportion of genes within the direct line living descendants of that founder of the population), contribution (number of copies of the founder’s genome that are present in the living descendants; each new generation of offspring contributes 0.5, each generation of grandchildren 0.25, etc.), allelic retention (the probability that a gene present in a founding individual exist in the living descendant animals), potential retention, and descendants. The jaguars T1 and T2 are the oldest for which there is any kind of information, they were part of the Santafé Zoo and their remains are kept in the museum of natural history of the Universidad de Antioquia; their genetic representation in the population is of 8.9% and have contributed with two descendants. Jaguars T5 and T6 are animals that remained in the Santacruz Zoological Foundation, until their death. Their genetic representation in the population is of 28 and 46% and has contributed with four and six descendants respectively. Jaguar T2, situated at the Jaime Duque Park, has a genetic representation of 7% in the population, and has contributed with a descendant.
It was also found that five founding animals have produced descendants and that jaguar T6 the one that presented the greatest number of descendants (
The current and potential allelic retention was calculated. Regarding the current allelic retention, it can be observed that this depends mainly on whether the founders have produced descendants and if there has been a genetic contribution from them. It can be observed, in the case of the potential allelic retention, the capacity of allelic retention of the population with the genes coming from those animals that still have not reproduced themselves within the population. (T33, T32, and T20). It can also be observed that individuals T1 and T2, although having contributed to the allelic retention, do not contribute to the potential allelic retention, due to the fact that they have died.
The average value of the current genetic diversity, of the population (GD = 0.7832), was obtained. Similarly, the value of the potential genetic diversity (GDp = 0.9113) was calculated, which is determined when conducting control on the production of cubs in the population, and if there is control over which genes are transmitted to the descendants by means of programs of the management of reproduction toward the future. In this way, it was possible to determine the variations of genetic diversity, during a span of time which permits appreciating that between 1995 and 1996, the genetic diversity of the population increased from 50 to 75%, due to a birth and other reproductive events that took place at that time. A slight increase from the years 1999–2002 can also be appreciated. A genetic value (GV) of 0.7846 was obtained, as was also obtained the value of the equivalent for the current founding genome (FGE = 2.31) and the potential (FGEp = 5.64). In the same way, the value of the equivalent of the current surviving founding genome (FGS = 3.14) and potential (FGSp = 5.64) was obtained. In the graph, an increment of FGE between the years 1995 and 2002 can be observed.
The average coefficient of
The size of the effective population or the effective demographic number was also calculated (Ne) based on the history of the population; the current demographic number (
3.2. Genetic analysis with microsatellites
The genotypes of the jaguars sampled were stabilized from the analysis of the chromatograms obtained after the capillary electrophoresis. It could be determined that the microsatellite, which amplified in fewer individuals, was FCA008 showing the highest index of presentation of null alleles (+0.16). For the 20 jaguars analyzed and the 9 microsatellites, an average of alleles of 5.67 per locus was found, finding the highest number of alleles for the locus FCA 224 (eight alleles) and the lowest for the FCA736 (two alleles). In the wild Colombian jaguar sample for 156 individuals and 12 microsatellites, the average allele number was 13
The average gene diversity (expected heterozygosity) found for the Colombian captive jaguar population was
In the captive population, FCA 391 and +FCA 220 presented the highest values of heterozygosity and information of polymorphic content (PIC) and the locus with least heterozygosity and PIC was the FCA736. In the wild sample,
3.2.2. The Hardy-Weinberg equilibrium
In this study, the test of probabilities for each locus was conducted; finding excess of homozygotes for the loci FCA 441, FCA 008, FCA 224, and FCA096 through the estimates
When assuming the Hardy-Weinberg equilibrium, the demographic
3.2.4. Disequilibrium by linkage
No significant differences for the de test of disequilibrium by linkage were found, which means that each locus, being independent from the others, can be employed as determiners of the genetic diversity of the population. The same was found for the 12 microsatellites applied in the wild jaguar sample.
The analysis of verisimilitude obtained in CERVUS resulted with assignments of maternity with no knowledge of the father by 34% for the strict confidence interval at 95% (LOD critical = 4) and with a 100% for the assignments for the flexible interval at 85%. The assignments of maternity with knowledge of the father were of 84% for the strict confidence interval at 95% (LOD = 2.05). Only the father (T5) of individuals T7, T8, and the father (T3) of individual T10 were known. Assignments are conducted according to the coupled LOD scores.
The analysis of verisimilitude obtained in CERVUS resulted with assignments of paternity with no knowledge of the mother of 32% for the strict confidence interval at 95% (LOD critical = 4) and with 100% of assignments for the flexible interval at 85%. Assignments of paternity with knowledge of the mother were of 81% for the strict confidence interval at 95% (LOD = 2.05). Only the mother (T6) of individuals T7, T8, T10 was known. The assignments were conducted according to the coupled LOD scores.
The probabilities of relationship and kinship assigned by KINSHIP were obtained to a prove
Taking into account the information of the records of the Studbook, the assignation of paternities and maternities for the CERVUS program and the relationships of kinship obtained by KINSHIP, the construction of the pedigree was conducted, for the population of captive jaguars in the zoological parks of Colombia (Figure 1). When conducting the construction of the pedigree, a great association was found, between the animals present at the Matecaña and the Santafe zoological parks. A high genetic relation among the jaguars present at the Santacruz zoological park could be determined, and additionally there were connections in the records of the Studbook between this zoo and the Piscilago, Barranquilla, and Jaime Duque. As for the
To sum it up, through the CERVUS program, four maternities (T15, T25, and T19 in two occasions) and five paternities (T17, T26, T3, and T16 in two occasions) were assigned. They were assigned by CERVUS as well as by KINSHIP four paternities (
In Figure 2, it is possible to observe as an example a paternity and maternity test in jaguars (
In this study, it could be proven that the population of captive jaguars in zoological parks in Colombia is a population that presents a high genetic variability and that is a population with the necessary conditions to be an important model as a genetic reservoir to revitalize natural populations given the case that they might be isolated and present low levels of genetic diversity. Nevertheless, this study also shows that the Colombian zoological captive jaguar population only has a fraction of the overall genetic diversity found in the wild jaguar population of this South-American country [7, 13, 14, 31].
As for the genetic analysis from the regional Jaguar Studbook, it could be determined that regarding the mean kinship (MK) on average (MK = 0.217), the values found for the captive population in Colombia are less than the MK reported by Drury  for captive jaguars in Europe (MK = 0.238), which offers a large indicative as future potential to reduce and/or events of inbreeding. It was found that there exists representation and genetic contribution of founders from wildlife in the current population due to breeding in captivity.
Regarding the genetic diversity according to the records found in the regional Studbook, a clear tendency was found, of an increase that went from 50% in the year 1994 to 72% in the year 1996 and reaching nearly 78% in the year 2002, due to the reproductive processes and the births that took place in this lapse of time.
As for the genetic diversity in the analysis of microsatellites, it was found that it is in the order of 68%. Such a result is found in the levels reported by Eizirik et al. , where he studied levels of genetic diversity between 62 and 73%. This last study analyzed a large fraction of jaguars living in the periphery of the geographical distribution of this species (Mexico and Central America and Southern Brazil and Argentina). However, these gene diversities were considerably minor than those found by Ruiz-García et al. [7, 13], who reported a gene diversity of 84%, or the gene diversity showed in this study and Ruiz-García et al.  for a larger Colombian wild jaguar sample around 87% . Henceforth, the Colombian captive jaguar sample contained around 78% of the gene diversity found in wild conditions. Due to this, it is possible to determine that the genetic diversity of captive jaguars in Colombia is at a high level; nevertheless, it is necessary to follow the alignments of reproductive population management for the species and thus reach a level close to 90% and maintain it for a 100-year period as it is proposed in the plans of survival of species .
As for the analysis conducted of the microsatellites, a smaller number of alleles were found, for locus FCA96 (7 alleles), FCA08 (7 alleles), and FCA391 (6 alleles), than the number reported by Ruiz-García et al.  where the number of alleles found was greater for FCA96 (15 alleles), FCA08 (15 alleles), and FCA391 (13 alleles) , and by Ruiz-García et al. , where it was found for Fca96 (13 alleles), Fca08 (13 alleles), and Fca391 (13 alleles) . Regarding locus Fca96, alleles of 185pb 187pb, and 189pb have been reported in Amazonian jaguars of the subspecies (
In this study, it was found that for the loci analyzed, there exists Hardy-Weinberg equilibrium, which contrasts with the findings of Ruiz-García et al. [13, 31] who reported the inexistence of the Hardy-Weinberg equilibrium for excess of homozygotes which was attributed to an effect of the demographic subdivision or Wahlund effect in the total wild jaguar population of Colombia and north-western South America. On the other hand, Eizirik  reported deviations from the Hardy-Weinberg equilibrium in 6 loci of the 29 analyzed from the population of jaguars studied. Thus, the estimated equilibrium Hardy-Weinberg is a point that needs greater approach in order to clarify and determine if the disequilibrium found by Ruiz-García is due to the effect of demographic subdivisions, or Wahlund effect, or if this finding is a dependent reflection of loci analyzed. However, notwithstanding that in this study we find the existence of equilibrium for the loci analyzed, the size of the sample is reduced; therefore, inference could not be extensively extrapolated to the populations of jaguars in the wild. In this way, it would be important to continue this kind of analysis in future investigations.
The results of the genetic analysis of the Regional Jaguar Studbook for Colombia in the PM2000 program clearly show that according to the records regarding inbreeding, the maximum value was found for
As for the value found for
Before conducting the study, the origin of the pedigree population of jaguars was known by 28.9% (
From this, it was possible to obtain a precise register of the origin of the live specimens within the population, which can be employed in the future programs of population management, involving controlled crossings in order to avoid the presence of inbreeding and thus maintain the jaguar population as a variable population, in genetic terms, as a model of investigation.
For some species where populations in the wild are highly diminished, the survival of the species depends on the propagation of captive animals. In these cases, the entire genetic pool of a species represented by the genetic contribution of the captive founders to the following generations of individuals bred and raised in captivity is presented as a closed system. In these cases, the new mutations that occur within the population provide the only new source of genetic variation. This process takes place at an extremely low rate and provides significant changes only at long intervals of time or when populations are extremely large. In the cases where the wild populations are stable in number and distribution so that potentially they can be sampled and could provide immigrants to the captive population, this access to new sources of variation provides the opportunity to preserve larger proportions of wild genetic pool, employing fewer captive individuals in a greater way than in closed populations. It is at this point where starting from the historical knowledge of animal populations is possible to pose concrete solutions with the purpose of conserving all the species that mankind has placed under threat, as is the jaguar. The implementation of programs of genetic analysis in the collections present in captivity contributes to the conservation of the species threatened, through the maintenance of viable populations as genetic reservoirs that can be used periodically to reinforce, revitalize, or reestablish captive populations and when necessary in cases of wild populations with low genetic variability. It also permits the identification of subspecies that even though phenotypically might seem similar, they have different geographical origins and have suffered a particular process of speciation. Although in the case of the jaguar, both morphological and molecular studies put in doubt the existence of subspecies [12–14, 34, 38, 39].
The scope of the instauration of programs of genetic investigation in the zoological parks and centers of rehabilitation of fauna, contributor to the ordering of captive collections, provides clear tools in the face of the movement of specimens from one place to another, in order to guarantee efficient reproductive and viable processes.
In addition, it contributes to the liberation of animals after the processes of rehabilitation in zones corresponding to the natural genetic origin of their congeners. The implementation of a genetic analysis of a population in captivity requires systematic steps in order to obtain the greatest quantity of possible information, such as the construction of a “pedigree” in the population, the identification of founding parental, the genetic contribution of each parental to the population, the estimate of the loss of alleles due to endogamy or “bottleneck” effects, and estimates of coefficients of inbreeding and making of tests employing molecular markers. In this way, adequate genetic techniques can be implemented to clarify the blanks of information about unknown specimens.
Therefore, this was the first study of records of captive jaguars in Colombia where it was possible to obtain relevant information regarding the identification of individual’s founders of the population and their descendants. This kind of studies could be carried out in other Latin American countries, where the captive populations of jaguars are large (for instance, Peru, Bolivia, and Brazil), and thus these captive populations could be decisive to the conservation of the largest wild cat of the Americas.
The authors would like to express their acknowledgments to the Colombian Association of Zoological Parks and Aquariums (ACOPAZOA): the Zoológico Santacruz Foundation: Dr. Angelica Moreno, John Vidal. Zoológico Santafé: Dr. Marta Ocampo. Zoológico Matecaña: Dr. Delio Orjuela, Sandra Correa.Zoológico Piscilago: Dr. Ana Carolina Falla. Fundación zoológica de Cali: Dr. Jorge Gardeazabal, Dr. Juliana Peña. International Species Information System ISIS: Laurie Bingaman. Venezuelan Institute of Scientific Investigations (IVIC): Dr. Kate Rodríguez Clark. Institute of Genetics of the National University: Dr. Victor Vera, Dr. Luis Fernando Cadavid, Miguel Novoa, Dr. Fernando Ariza. Dr. Paul Bloor. Dr. Carolina Corrales. National Zoo, Smithsonian Institution: Dr. Christine Bozarth, Dr. Joseph Kolowski, Dr. Francisco Dallmeier. National Cancer Institute: Dr. Stephen J. O’Brien, Dr. Warren Johnson, Victor David, Bailey Kessing. Maria Clara Espinel, Consuelo Espinel, Julia E González.