Chimeras of birds (Gallus gallus) obtained using the rocks Pantsirevskaya black and Kokhinhin karlikovy

 Progress in creating new forms of poultry is possible due to the rapid development of methods of cell and molecular biology. The creation of chimeric organisms is necessary to solve many problems of fundamental biology, to increase the population of endangered birds, or to restore extinct species and obtain transgenic organisms. The purpose of the work is the development and optimization of the technique for producing chimeras obtained from the Pantsirevskaya black, Kokhinhin karlikovy breeds and the study of their phenotype. 
The research material was, obtained from the «Genetic Collection of Rare and Endangered Breeds of Chickens» (VNIIRGZH Saint Petersburg).  Blastodisks were isolated from donors at the stage of freshly eggs (stage X according to Hamilton), which were crushed and cultured for two days at a temperature of 38°C in DMEM nutrient medium supplemented with 10% fetal bovine serum and gentamicin antibiotic. At the same time, the embryos of the recipients were cultured in the incubator, in which a small window was  cut out after two days. Using a micromanipulator, 5-7 μl of a suspension of donor cells was injected, they were glued with adhesive tape and placed in an incubator until the hatching stage.
To obtain chimeric organisms, 20 eggs were used as donors and 20 recipients for each breed. All were analyzed by the following indicators:  fertility, hatchability of recipients, and experiments on transplantation of blastoderm cells in order to obtain chimeras (Table 1). On average, 90% of  the eggs in the breeds studied were fertilized. The hatchability of experimental birds varied from 25% to 40%. The average number of chimeric organisms obtained by birds of two breeds was 8.3%.
With the selection options Kokhinhin - donor, Pantsirevskaya - the recipient  received chimera (Fig. 1) with unusual color. The ends of the feathers and   part of the head are bluish in color, obtained after transplantation of  cochinquin donor cells.
Two chimeras obtained after transplantation of blastoderm cells of an armored breed to cochinhin karlikovy recipients are shown in Fig. 2-3. In
Сochinchins with the gene for the weakening of the black color Bl (Blue), the black pigment turns into blue, so black spots on the beak and skin of
two experimental chickens are a sign of chimerism. 
Thus, by the method of transplantation of blastodermal cells isolated at the  stage of development X from one breed and transplantation into an embryo
of another breed, interbreed chimeras with a unique phenotype were obtained. 

1. Козикова Л.В. 2017. Трансгенные животные./Л.В. Козикова //Учебное пособие для студ. высш. учеб. завед. под ред. К.В. Племяшова. СПб. Проспект науки.-2017.-224 С.

2. Zinovieva N.A., Volkova N.A., Bagirov V.A. Genome Editing: State of the Art and Application to Animal Husbandry. / Zinovieva N.A., Volkova N.A., Bagirov V.A.// Biotekhnologiya.- 2018.-P. 9-22

3. Han J.Y. Germ cells and transgenesis in chickens. /Han J.Y. //Sci. Reprod. -2018.- V. 8(1).- PP.15126. doi: 10.1038/s41598-018-33244-x.

4. Nandi S., Whyte J., Taylor L. Cryopreservation of specialized chicken lines using cultured primordial germ cells. / Nandi S., Whyte J., Taylor L // Poult. Sci. -2016. -V.95.- PP.1905–1911.

5. Nakamura Y. Poultry genetic resource conservation using primordial germ cells. / Nakamura Y. // J. Reprod. Dev. -2016.- V.62(5). -PP.431– 437.

6. Macdonald J., Glover J.D., Taylor L, Sang H.M., McGrew M.J. Characterisation and germline transmission of cultured avian primordial germ cells. / Macdonald J., Glover J.D., Taylor L, Sang H.M., McGrew M.J.// PLoS One. -2010. - V.5(11).- e15518. https://doi.org/10.1371/journal.pone.0015518 cells. V.5(11).

7. Bednarczyk, M., Kozłowska, I., Łakota, P. et al. Generation of transgenic chickens by the non-viral, cell-based method: effectiveness of some elements of this strategy. / Bednarczyk, M., Kozłowska, I., Łakota, P. // J. Appl. Genetics.- 2018.- V.59.- PP. 81. https://doi.org/10.1007/s13353-018-0429-6

8. Pleshanov N., O. Stanishevskaya, K. Plemyashov. A method of evaluation and selection of cocks for cryotolerance of their sperm with aim of gene pool preservation. / Pleshanov N., O. Stanishevskaya, K. Plemyashov. // Reproduction in Domestic Animals. - 2016. - Vol. 51. Supplement 2. – P.131.

9. Park K.J., Kang S.J., Kim T.M., Lee Y.M., Lee H.C., Song G., Han J.Y. Gammairradiation depletes endogenous germ cells and increases donor cell distribution in chimeric chickens in vitro cell. / Park K.J., Kang S.J., Kim T.M., Lee Y.M., Lee H.C., Song G., Han J.Y. // Dev. Biol. Animal. -2010. -V.46.- PP. 828-833 (doi: 10.1007/s11626-010-9361-8

10. Ono, T., Yokoi, R. and Aoyama, H. Transfer of male or female primordial germ cells of quail into chick embryonic gonads. / Ono, T., Yokoi, R. and Aoyama, H. // Exp. Anim.,-1996.-V.45.-PP. 347-352.