In ovo — isolation of primordial germ cells from embryonic blood of gene pool breeds of chickens

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Abstract

The article presents a new method (in ovo — inside the egg) for isolating primordial germ cells (PGC) from embryonic blood of gene pool chicken breeds, which is of great importance for preserving the genetic resources of birds and creating new innovative tools in the field of biotechnology. Despite the key role of PGC in the targeted modification of the genome, there are significant difficulties with the efficiency of their isolation. To analyze the efficiency of the in ovo method, experiments were conducted on collecting blood samples from 192 chicken embryos of various breeds, as well as subsequent cultivation of PGC and cell counting. It was found that the efficiency of sampling depends on the egg weight and breed. A high efficiency rate was observed in the Chinese Silkie breed (89.5 %), with an influence strength of η2 = 98 %, p-value <0.004. Analysis of PGC cultivation data showed an increase in the concentration of living cells and their survival by the 20th day. The highest survival rates were observed in the Chinese Silkie breed (69.1±2.56 %), and the Tsarskoye Selo (68.78±3.39 %) and Rhode Island (67.9±2.52 %) chicken breeds also had high rates. The results confirmed the importance of the in ovo method for optimizing the process of isolating PGCs from 4-day chicken embryos to increase the number of cells during further cultivation.

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About the authors

G. K. Peglivanyan

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Author for correspondence.
Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

T. A. Larkina

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

N. R. Reinbach

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

A. P. Dysin

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

A. V. Gabova

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

Е. A. Polteva

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

А. Е. Ryabova

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

A. I. Azovtseva

Branch of the Ernst Federal Science Center for Animal Husbandry (RRIFAGB)

Email: Peglivanian_grig@mail.ru

Russian Research Institute of Farm Animal Genetics and Breeding

Russian Federation, St. Petersburg, Pushkin

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2. Fig. 1. Demonstration of in ovo blood collection. a — Four-day embryo. b — Positioning under a binocular and inserting a microneedle; microinjection control under a video camera.

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3. Fig. 2. Appearance of cell cultures on the first day of blood collection (a) and on the 15th day of cultivation (b, c). a — With formed elements of the blood and PGC; b, c — only PGC. LEICA DMI8 microscope (Nicon, Germany), 40× magnification (a, b) and AXIOVERT 200M fluorescence microscope with a high-resolution color digital camera DFC420 (Germany), 100× magnification (c).

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