Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated g-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line. Reproduction (2007) 134 445-453
Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa.Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.
The aim is to optimize the dimethylacetamide ( DMA ) straw freezing technology of Black silkies rooster semen through the handy patent equipment, screening the formula of freezing basic extender and optimizing the DMA addition method, and then by comparing the fertility of DMA straw frozen semen with the pellet frozen semen. After the DMA straw freezing technology is optimized, it is extended to the Youxian Partridge drake semen. The result showed that the frozen sperm motility of Lake and Ravie ( LR ) group is 64%, the fertility 49.57% and the hatchability 91.52%, all of which are superior to those of FEB, Beltsville Poultry Semen Extender ( BPSE ) and Lake ( P < 0.05). The sperm motility of adding DMA stock solution is 59%, which is superior to adding DMA directly into diluted semen ( P > 0.05). The fertility and hatchability of DMA straw group are 77.61% and 92.30%, respectively, and it is significantly higher than those in the pellet group ( P < 0.01; P < 0.05). The fresh drake sperm motility of induction collection method is 71%, the massage collection method 61% and the frozen drake sperm motility of induction 33% while the massage 19%. The fertility of frozen drake semen group is 85.93%, while that of the fresh semen group is 88.17%. The frozen drake semen fertility of the highest batch is 93.8%. In conclusion, the world's advanced fertility of frozen semen can be obtained both in the chicken and drake through the optimized DMA straw freezing technology and the method of screening freeze-resistant individuals.
Mucksová, J.; Brillard, J. P.; Hejnar, J.; Poplštein, M.; Kalina, J.; Bakst, M.; Yan, H.; and Trefil, P., "Identification of various testicular cell populations in pubertal and adult cockerels" (2009 a b s t r a c tPrecise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in singlecell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular * Corresponding author. Tel.: +420 261 395 234; fax: +420 241 950 503. This article is a U.S. government work, and is not subject to copyright in the United States. 416J. Mucksová et al. / Animal Reproduction Science 114 (2009) [415][416][417][418][419][420][421][422] cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.
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