Experimental data on the integrity and survival of primordial germ cells after cryopreservation in various chicken breeds.

PC-100 Narishige capillary puller tool. To prevent the pointed end from sticking together, a filament needle is used.</p><p dir="ltr">4. Narishige EG-45 Microgrinder for needle sharpening.</p><p dir="ltr">5.

Thermostat TC-1/20 SPU (Russia) for maintaining the medium used for seeding blood collected by microinjector.</p><p dir="ltr">6. Binocular Micromed MS-20 ZOOM (Russia) to control blood sampling from the dorsal aorta of the embryo.</p><p><br></p><p dir="ltr"><i>2.1.3 Cell culture cabinet</i></p><p dir="ltr">1. The Lamsystems SAVVY microbiological safety laminar flow box for sterile cell culture operations.</p><p dir="ltr">2.

Heal Force HF-90 CO₂ incubator (China).</p><p dir="ltr">3. DMi8 Leica inverted microscope (Germany).</p><p dir="ltr">4. TC20 cell counter (BioRad).</p><p dir="ltr">5.

A refrigerator-freezer in which PGC is incrementally frozen in cryonic containers at -80°C.</p><p dir="ltr">6. MiniSpin centrifuge</p><p><br></p><p dir="ltr"><i>2.2</i><i> </i><i>Research material</i></p><p dir="ltr">1. Embryos derived from eggs of ten breeds of meat-egg and egg production (Orloff, Pushkin, Ukrainian Muffed, Pantsirev, New-Hampshire, Campin, Black Austrolorp, Pervomay), as well as ornamental - New-Pavlovo and Moscow Game breeds.</p><p dir="ltr">2.

Primordial germ cells from various chicken breeds which were incubated in medium for 21 days.</p><p><br></p><p dir="ltr"><i>2.2.1 Reagents and kits for cell culture</i></p><p dir="ltr">1. A multicomponent medium was prepared for PGC cultivation. Base medium preparation: Invitrogen's Gibco KnockOut™ DMEM/F-12 (1x).</p><p dir="ltr">2.

Reagents added to the medium (final concentration):</p><p dir="ltr">- 1 M sodium pyruvate (Applichem, USA) - 1µl/ml,</p><p dir="ltr">- Mercaptoethanol-2 (NF, VWR) - 0.01%,</p><p dir="ltr">- Chicken serum (Gibco, USA) - 2%,</p><p dir="ltr">- Fibroblast growth factors (FGF (Thermo Fisher, USA) - 10 ng/μl,</p><p dir="ltr">- Activin A (Thermo Fisher, USA) - 25 ng/μl,</p><p dir="ltr">- Nucleosides (Millipore, USA) - up to x1,</p><p dir="ltr">- Antibiotic-antimycotic 100x (Thermo Fisher, USA) - up to x1.</p><p dir="ltr">The prepared medium was sterilised by filtering through 0.22µm diameter nylon filters.</p><p dir="ltr">3.

Thermo Scientific Nunclon Delta Surface (Denmark) 48-cell culture plates with adhesive layer.</p><p dir="ltr">4. CoolCell SV2 cell culture cryobox, Corning (BioCision, USA).</p><p dir="ltr">5. Cryogenic storage dewar for long-term preservation of PGCs.</p><p><br></p><p dir="ltr"><b>3.

Methods</b></p><p dir="ltr">1. Eggs for incubation are collected within two days, treated with 70% ethanol, weighed, signed, and placed in incubation trays with the blunt end facing upwards.</p><p dir="ltr">2. Eggs are incubated within laboratory incubators at a temperature ranging from 37.5 to 38.0°C, with a humidity level of 70%.

The tray angle is adjusted by 90° every hour. The fertilization rate of eggs varies from 60% to 89% depending on the breed.</p><p dir="ltr">3. Egg dissection on the 4th-5th day of egg incubation, using an engraving machine to drill a 1.0-1.5 cm borehole.</p><p dir="ltr">4.

Collection of blood sample (5-7 µl) under binocular vision using a micromanipulator and microinjector with a 30 nm diameter needle from the dorsal aorta of the embryo at stage 14-15 HH (Hamburger-Hamilton).</p><p dir="ltr">5. Cultivation in 300 µl tubes with sterile medium in a thermostat at +37.0°C. Then transfer to culture plates.</p><p dir="ltr">6. Transfer cell culture to culture plates in laminar.</p><p dir="ltr">7.

Cultivation in CO2 incubator (mode: +37.0°C, CO2 concentration - 4%, humidity - 90-95%).</p><p dir="ltr">8. The culture medium is replaced every three days at the rate of ½ the volume of medium in the well.</p><p dir="ltr">9. The initial counting of cells on day 10 - 12 of incubation (blood formations in the sample are degraded by day 10 - 12, PGCs become available for visualisation).</p><p dir="ltr">10.

Cryopreservation on day 20 - 21 of cultivation, in 2 ml cryo-tubes, with the addition of cryoprotectant DMSO at a concentration of 10%. The tubes were placed in a CoolCell SV2 container for freezing cell cultures, transferred to a -80°C freezer (temperature reduction rate of 1°C/min). After 24 hours cryo-tubes were transferred to liquid nitrogen for long-term storage.</p><p dir="ltr">11.

Thawing: Cryo-tubes with cell suspension are taken out of liquid nitrogen, kept at room temperature until the nitrogen vapour dissipated. Then they placed in a water bath at +4°C. The medium containing cryoprotectant is removed through centrifugation (1000 rpm, 4 min).</p><p dir="ltr">12. Cell Culturing: The cells are placed in a culture medium and maintained at a temperature of +37°C, then resuspended and transferred to culture plates before being placed in a CO₂ incubator.</p><p dir="ltr">13.

Cell viability is assessed by counting trypan blue-stained cells after 3-5 days of cultivation.</p><p dir="ltr"><br></p>