Capra pyrenaica pyrenaica

The bucardo (Capra pyrenaica pyrenaica) is an extinct species that was considered a subspecies of ibex. This subspecies of Iberian ibex was in danger of extinction since the beginning of the XX century, and was listed as a "critically endangered species". of extinction" (CR) by the IUCN Specialist Caprinae Group in 1997. On January 6, 2000, the last specimen, a female, living in Spain, was found dead. More recent molecular studies indicate that it could be a different species, but it is still the subject of debate and to this day it is still officially considered a subspecies.

Along with the mueyu, or Portuguese ibex (Capra pyrenaica lusitanica), which became extinct in 1892, it is one of only two subspecies of ibex to have gone extinct. It differed from the other subspecies mainly by its longer, denser fur in winter and the thicker base of the horns, both in males and females. The antlers of the bucardo were also the longest of the four subspecies of ibex that have inhabited the Iberian Peninsula.

The only record of the weight of a live buckeye (66 kg) indicates that it was larger than ibex (30-33 kg, data from captive animals at the Center for Agrifood Research and Technology of Aragon).

The karyotype of the bucardo shows the number of chromosomes characteristic of the species Capra pyrenaica (60 chromosomes). It is a monophyletic subspecies in origin. Phylogenetically, bucardo populations were closer to those of the ibex than to the Alpine Ibex or the domestic goat. It is worth remembering here the difference in the DNA of the bucardo with respect to other subspecies, which makes it a unique animal. The last specimens had a marked reduction of some major histocompatibility complex (MHC) alleles that were specific to this subspecies. Since low variation in MHC is associated with high susceptibility to infectious diseases, this may have influenced their survival and recent extinction.

History and extinction

This species was originally distributed on both slopes of the Pyrenees, extending south towards the mountainous areas of the Basque Country, Navarra, Huesca, Lleida and Gerona.

Vestiges that it was a common game item since Prehistoric times are already found in the caves of the area, inhabited during the Paleolithic period by Neanderthals first and then by our own species. Over the centuries it disappeared from the southernmost areas of its distribution until it was circumscribed to the Pyrenean area, where it was still especially abundant during the century XIX.

In the second half of that century, a multitude of European hunters, especially from France and Great Britain, came to the area in search of specimens to kill. The rarity of the ibex in the world (at that time distributed only in Spain, southern France and northwestern Portugal) and the impressive size of the antlers of the males, thicker and more separated than that of the Alpine ibex ( Capra ibex) multiplied its value and demand in hunting circles. Hunting was so intense that around the year 1900, the Pyrenean subspecies had already become extinct in France. In 1972 there was only a small group of less than 50 individuals left in Spain, too small to survive in the long term. Competition with other wild ungulates and domestic species for food resources may also have contributed to their extinction.

Celia, the last bucardo.

Despite this, the population remained stable at over thirty until 1981. It is not yet fully understood why, the animals declined rapidly. In 1999, the last male died. Misfortune struck the last female, "Celia". She died in January 2000, and her body was found under a fallen tree. So that its extraordinary size does not fall into oblivion, and so that the new generations can admire such a magnificent animal, Celia is kept in the Visitors Center of the municipality of Torla-Ordesa for the memory of some and the admiration of many more.

Work carried out to prevent the extinction of the bucardo

In 1913, the species was already going to be considered extinct, but it was learned that some animals had survived in the Ordesa y Monte Perdido Park Zone. One of the first measures aimed at the conservation of the Bucardo was the prohibition of its hunting in 1913 and the declaration of a national park in 1918 of a large part of the territories it inhabited. Starting in the 1960s, additional protection measures were taken, such as providing supplementary food in winter, which could not stop the regression of the population. In 1989, the Spanish Government financed two studies: the first was to "census the population of the bucardo", demonstrating that there were 11 ± 4 living individuals. The second study consisted of trying to multiply the number of individuals through assisted reproduction techniques. For this, in the facilities of the Center for Research and Agro-Food Technology (CITA) of the Government of Aragon, these techniques were perfected using goats of the subspecies of C. Hispanic Pirenaica, which is the closest genetically to the bucardo and is not in danger of extinction.

In 1990, the Decree regulating the National Catalog of threatened species, where the bucardo appears, was published. In 1993, the EU approved a project, the LIFE Project, in collaboration with the governments of Aragon and Central governments of Spain and France, to finance a recovery plan for the bucardo, with international advice. The main objectives of this plan were: 1) Capture and keep in captivity the entire live bucardo population, 2) Try to reproduce them in captivity, 3) Reintroduce the animals born, 4) Protect the bucardo habitat, 5) Information campaign and awareness. At the same time, other actions were carried out, such as epidemiological surveys of the wild and domestic ungulate species that share the habitat of the bucardo, and monitoring of individuals by specialized technical personnel.

Hybridization attempts

Between 1990 and 1995 males vasectomized and provided with a C radio transmitter were released. pyrenaica hispánica from the CITA to the area of the habitat of the Bucardo, in order to mix with the population of bucardas and facilitate their monitoring. In all cases, these animals failed to adapt and survived for a short time. Two intact males, equipped with a radio transmitter, were released in 1996 to attempt hybridization. They were given complementary feeding and survived for two years. It was possible to verify that some bucardas became pregnant by measuring the total estrogen levels in their feces collected in the field, but no parturition or calves were observed.

The cloning of the bucardo

In 1999 there was only one specimen left. Since the natural hybridization failed, it was decided to capture the last living buffalo and obtain skin cells, which were cultivated in two different laboratories (CITA de Aragón and CIEMAT, Madrid) and preserved in liquid nitrogen, with a view to trying to clone them. in the future. The animal was released after the biopsy. She was found dead in January 2000. The Government of Aragon held talks with the company ACT (Advanced Cells Technology) to attempt cloning, since this company had achieved the birth of a gaur (Bos gaurus) transferring a cloned gaur embryo into a domestic cow. No agreement was signed, but ACT supported the project.

In 2001, the National Institute for Agrarian and Agri-Food Research (INIA) financed a project to develop the necessary technology to carry out cloning in the domestic goat and apply said technology to the recovery of the Bucardo. The project was developed at CITA in Aragón with the collaboration of researchers from INIA and the Institut national de la recherche agronomique (INRA) of France. Genetic analyzes were performed at the Autonomous University of Barcelona (UAB). Pregnancy monitoring was carried out in collaboration with the University of Liège.

Experimental approach

The foundation of cloning resides in achieving the complete reprogramming of the genome of a diploid somatic cell, more or less differentiated (caryoplast), so that after its fusion with a mature, previously enucleated oocyte (cytoplast), a viable embryo. There are many factors that condition the success of this process, being its limiting phases: the coordination between the cycle of the caryoplast and that of the cytoplast, the fusion method between the two, the activation of the reconstructed embryo, the in vitro culture of the resulting embryos., its transfer, gestation and childbirth.

1. Generation of caryoplasts: Caryoplasts were obtained from bucardo skin biopsies (fibroblasts).

2. Cytoplast production: As oocytes from bucarda (Capra pyrenaica pyrenaica) were not available, those of superovulated adult domestic goats (Capra hircus) that were surgically recovered from the oviduct after ovulation (maturation in vivo). Therefore, “interspecific cloning” was performed, since the caryoplast and cytoplast belonged to different species.

3. Nuclear transfer: Each oocyte was enucleated and then a fibroblast was inserted into the perivitelline space (under the zona pellucida). The cytoplast-caryoplast assemblies were fused by two electrical pulses, resulting in reconstituted (clonal) bucarda embryos.

Transfer of cloned embryos

Normally, cloned embryos are transferred to recipient females of the same species for gestation. In the case of the bucardo, given the non-existence of individuals of the same subspecies, the cloned embryos were transferred to the closest ibex subspecies from the genetic point of view, that is, to C. pyrenaica hispanica, or crossbred goats (F1; ibex x domestic hybrids) as embryo recipients.

Results

Two sets of experiments were done. In the first series, 54 cloned embryos were transferred to 13 recipients, obtaining 2 pregnancies that were lost at an early stage (45 days of gestation). In the second series, 154 embryos were transferred to 44 recipients, obtaining five pregnancies, one of which reached term. The pregnant female was a hybrid goat. The birth occurred by caesarean section on July 30, 2003. The kid was born alive but died a few minutes later due to respiratory problems. The cloned kid was genetically identical to the bucarda.

This was the world's first live birth of an extinct subspecies. These works have shown two important things: 1) that frozen bucardo cells are alive and can be used for cloning; 2) That the technique used consisting of using domestic goat oocytes as cytoplasts and hybrid goats (Ibex x Domestic goat) as recipients is adequate to clone the bucardo when appropriate.

Voices for and against cloning

Within the scientific sector, bucardo cloning work is generally considered to be of great interest, as well as the experience derived from them on interspecific cloning. The journal Theriogenology recognized the work published by Folch et al. (2009), as one of the most consulted works this year. Cloning is an ineffective way to preserve species and should only be used when other reproductive methods have failed. However, in cases such as the bucardo, cloning is the only possibility to avoid its complete disappearance. In recent years, much progress has been made in understanding both the cellular mechanisms involved in the cloning technique and the physiology of animals. The work carried out with the bucardo encourages the conservation of tissues and cells of endangered species as they may be necessary in the future to apply recovery plans based on cloning.

On the contrary, a sector close to naturalism, ecology, scientists and biologists, considers it unfeasible at the present time to create a bucardo population that can survive in the wild, arguing that, even if bucarda females were born, there would be no males to cross with them. In 2005, the Catalog of Threatened Species of Aragon was modified and, among other novelties, the category of “extinct” species was created to include the bucardo. However, this cataloging entailed the need to draw up a reintroduction plan for the species "whenever technical, scientific and social conditions allow it". One solution for its recovery would be to successively cross the bucarda clones with C. pyrenaica hispánica males (introgression by backcrossing). This technique has been successful in breeds of several domestic species but has not been applied to wild animals. In addition, research is advancing very rapidly in the technique of replacing the X chromosome with a Y chromosome from another subspecies, these procedures having been patented.

In the future, other bucardos (both male and female) could also be cloned from DNA reconstruction obtained from fossil remains or from samples obtained from bucardos that are kept in museums.

Study to reintroduce the ibex in the Aigüestortes National Park

There are economic and hunting interests for the introduction of the subspecies C. pyrenaica hispanica in Pyrenean areas where the bucardo lived. The suitability is based on the fact that the ibex is linked to rock and water, needs undergrowth and is in a protected area. However, as we have indicated previously, in previous studies animals of the subspecies C. pyrenaica hispanica to high mountain areas of the Pyrenees and survived for a short time, showing a lack of adaptation to these conditions.

The study is cross-border and those most interested in the reintroduction are France (which has been pressing since it lost the bucardo) and in Spain, Navarra, while Aragon is the most reluctant. Catalonia, where the ideal habitat for Aigüestortes is found, is not opposed to reintroduction but considers that a problem must first be solved: the overpopulation of mouflon and fallow deer in the area. Other enclaves that have been considered as a possible place of reintroduction are Montsec and Valle de Arán. The study began in October and was discussed at a recent cross-border meeting in Toulouse, France, where it was also proposed to reintroduce more bears to the Pyrenees. Already at the IUCN French Nature Congress of April 2012, twelve motions were accepted, among which was the reintroduction of the Iberian ibex in the Pyrenees.

Some people hope that with the introduction of the Iberian ibex of the "hispánica" That gap left by the bucardo after its extinction will be filled, both in the history and culture of the people of the Pyrenees, and for its biological work lost within the ecosystem. In the towns on both sides of the border, they believe that this initiative could be another engine for the promotion of ecological tourism in some areas of the Pyrenees. However, others consider that this decision should not be taken yet, since technological advances predict the possible recovery of the bucardo.

This argument must be discarded, since the subspecific regime of the bucardo is not accepted and the biological material (DNA of the recovered dead bucardos) is inappropriate due to the crossing and inbreeding that, at least, since the century XVII had been occurring in Ordesa, Gabarnie and Monte Perdido as attested by the sources, pathologies and morphology of the goats killed and hunted since that century.

It must be remembered that there are and have always been ibex in the ports of Tortosa-Beceite, barely 100 km from the Pyrenees. Likewise, the ibex is versatile, inhabiting both rocky areas and abandoned pine forests or terraces, so there is no biophysical impediment to its reestablishment in the Pyrenees.

Meanwhile, since 2014 it has been reintroduced in the Pyrenees, specifically in the Ariège and in the French Pyrenees National Park, from individuals of the western ibex breed, from the Sierra de Guadarrama.