Spain Records the World’s First In Vitro Births of Spanish Ibex

Researchers in Spain have achieved the world’s first births of Capra pyrenaica, the Spanish ibex, through in vitro fertilisation using reproductive cells collected from dead animals.

The team, from the National Institute for Agricultural and Food Research and Technology (INIA), part of Spain’s National Research Council (CSIC), produced viable embryos in the laboratory from eggs fertilised with sperm obtained post mortem. Those embryos were then transferred to recipient females.

The technique, applied successfully to an ibex species for the first time, represents a significant advance for wildlife conservation and for the preservation of valuable genetic lines.

How the Spanish ibex kids were produced

The gametes — eggs and sperm — were obtained from ovaries and testicles collected after death in two Spanish hunting reserves: the Sonsaz National Hunting Reserve in Madrid and the Andalusian Hunting Reserve of the Sierras de Tejeda and Almijara in Málaga.

Researchers from the Animal Genomic Engineering group and the Physiology and Reproductive Technologies in Wild Species group then applied assisted reproduction techniques to obtain the Spanish ibex births.

First, they used sperm cryopreservation, storing sperm from dead animals in liquid nitrogen at temperatures close to -200°C without deterioration. The method allows semen to be kept for later use in insemination.

“It is important for the sperm to be collected within the first eight hours after the animal’s death so that it maintains its fertilising capacity,” explained INIA-CSIC researcher Julián Santiago Moreno.

The researchers then obtained eggs from ovaries collected post mortem, matured them in vitro and fertilised them with thawed sperm in a culture medium known as TALP, originally developed for cattle reproduction.

After fertilisation, the zygotes were cultured in an environment designed to reproduce conditions in the reproductive tract, allowing them to develop to the blastocyst stage. At that point, they were cryopreserved until transfer to a recipient female.

“To achieve this, we optimised in vitro fertilisation techniques from a previous study in which sperm from male Spanish ibex (Capra pyrenaica) were used to fertilise eggs from domestic goats (Capra hircus),” explained Nuria Martínez de los Reyes.

A complete genetic reserve, not only males

The sperm cryopreservation procedure referred to by Santiago Moreno, developed in 2006, had one major limitation: it only allowed researchers to build a genetic reserve from males, not females.

The new technique makes it possible to develop a more complete genetic reserve, including females, because in vitro fertilisation of eggs obtained post mortem and cryopreservation of the resulting embryos could allow an entire population to be reconstituted.

Once embryos are obtained, they are frozen using vitrification, an ultra-rapid freezing process that protects cells by preventing ice crystal formation.

The achievement opens the door to creating banks of cryopreserved embryos obtained through in vitro fertilisation from gametes taken from animals that have been hunted or have died of natural causes.

“These banks could be used to recover ecotypes in the event of natural disasters or infectious disease outbreaks. In addition, we could apply the technique to conserve other endangered mountain ungulates,” said researcher Pablo Bermejo Álvarez.

The vitrified embryos are then thawed and transferred into the uterus of hormonally synchronised recipient females, so that their reproductive cycle is at the correct stage to accept the embryo. If implantation is successful, the recipient female carries the pregnancy as if it were her own offspring, although genetically the young belong to the donor animals.

After applying the technique to Spanish ibex eggs, the research team transferred cryopreserved embryos to five recipient females. The result was full-term pregnancies and the birth of three kids.

“Two of the three kids, both males, are in perfect health and continue to develop normally. The third died after birth due to a lack of colostrum — the first secretion of the mammary gland — from the recipient female,” explained Priscila Ramos-Ibeas.

The Spanish ibex in the Iberian Peninsula

The Spanish ibex is endemic to the Iberian Peninsula. Although it remains relatively common in some areas, two subspecies have already become extinct, and the isolation of some subpopulations leaves them vulnerable to disease outbreaks and other natural catastrophes.

In this context, generating embryos through in vitro fertilisation from gametes obtained post mortem could create a reserve of cryopreserved embryos for use in emergencies or for preserving animals with unique genetic value.

The techniques developed in this work could become a key tool in preventing the extinction of subspecies, as already happened in the Iberian Peninsula with the Portuguese ibex and, more recently, the bucardo, a Pyrenean subspecies of Spanish ibex whose last individual died in January 2000 in Ordesa y Monte Perdido National Park in Aragón.

“This technology, which until now had not been successfully applied to any ibex species, could be used in ex situ conservation programmes for other threatened mountain ungulates such as the Ethiopian ibex, the Nubian ibex or the Saharan and Egyptian aoudad,” the researchers concluded.

Cover photo: INIA.