Primordial Germ Cell-Mediated Chimera Technology Produces Viable Pure-Line Houbara Bustard Offspring: Potential for Repopulating an Endangered Species

BACKGROUND: The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. METHODOLOGY/PRINCIPAL FINDINGS: Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster. CONCLUSION: This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity

Progeny test of germline chimeric roosters by artificial insemination with female Houbara bustards.

<p>Progeny test of germline chimeric roosters by artificial insemination with female Houbara bustards.</p

Parents, offspring and progeny tests.

<p>(<b>a</b>) Germline chimeric rooster; (<b>b</b>) Female Houbara bustard (HB020154); (<b>c</b>) Houbara bustard chick generated from donor cells derived from Houbara bustard sperm produced by chimeric rooster (<b>d</b>) Dead Houbara bustard embryo from chimeric rooster; (<b>e</b>) species identification test by PCR; (<b>f</b>) molecular sexing by PCR of the dead Houbara embryo and the live Houbara chick.</p

Genotyping analysis of the Houbara bustard family using Houbara bustard microsatellites (STR) markers [26].

<p>*alleles from paternal genome.</p

Primer list for species identification test and molecular sexing.

<p>Primer list for species identification test and molecular sexing.</p

PCR gels with species-specific primers showing the detection of Houbara bustard DNA.

<p>(<b>a</b>) Detection of bustard DNA in the gonadal tissue of chimeric chicken embryos. Lanes 3, 7, 10, 13, and 16 show the bustard specific DNA; (<b>b</b>) Species identification sensitivity test: 6×10⁶ chicken sperm mixed with decreasing quantities of bustard sperm; (<b>c</b>) Detection of bustard DNA in the semen of chimeric roosters. Lanes 4, 5, 9, 18, 21, 26 and 28 show the bustard DNA.</p