2026-10-11T18:38:45Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33923425 2026-09-18T17:51:14Z category_23848 item_type_3 month_year_09_2026

Experimental data on the integrity and survival of primordial germ cells after cryopreservation in various chicken breeds. Anna Krutikova (25070827) Agricultural biotechnology not elsewhere classified chicken, breeds, primordial germ cells, cryopreservation, conservation of biological resources Experimental data on the integrity and survival of primordial germ cells after cryopreservation in various chicken breeds.Birds are kept in collections by the floor method in nests with a capacity of 15-20 birds. The sex ratio is maintained at 1:4.The investigation regarding this subject has been carried out in three distinct laboratory cabinets, which were equipped with the necessary facilities and inventory. 2.1.1 Egg incubation cabinetThe cabinet is supplied with a single chamber laboratory incubator IFH-250NS (Russia) for incubation of eggs of the studied chicken breeds. 2.1.2 Embryo sampling cabinet.1. The Dremel engraving machine (Budget Germany), mounted on the table in an upside-down vertical position for easy cutting of eggshells. The attachment is a diamond-coated disc.2. The Narishige IM-11-2 micromanipulator and pneumatic microinjector were utilized, in conjunction with prepared puncture needles having a thickness of 0.9 mm, a length of 70 mm, and a pointed part diameter of 30 µm.3. PC-100 Narishige capillary puller tool. To prevent the pointed end from sticking together, a filament needle is used.4. Narishige EG-45 Microgrinder for needle sharpening.5. Thermostat TC-1/20 SPU (Russia) for maintaining the medium used for seeding blood collected by microinjector.6. Binocular Micromed MS-20 ZOOM (Russia) to control blood sampling from the dorsal aorta of the embryo. 2.1.3 Cell culture cabinet1. The Lamsystems SAVVY microbiological safety laminar flow box for sterile cell culture operations.2. Heal Force HF-90 CO2 incubator (China).3. DMi8 Leica inverted microscope (Germany).4. TC20 cell counter (BioRad).5. A refrigerator-freezer in which PGC is incrementally frozen in cryonic containers at -80°C.6. MiniSpin centrifuge 2.2 Research material1. 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.2. Primordial germ cells from various chicken breeds which were incubated in medium for 21 days. 2.2.1 Reagents and kits for cell culture1. A multicomponent medium was prepared for PGC cultivation. Base medium preparation: Invitrogen's Gibco KnockOut™ DMEM/F-12 (1x).2. Reagents added to the medium (final concentration):- 1 M sodium pyruvate (Applichem, USA) - 1µl/ml,- Mercaptoethanol-2 (NF, VWR) - 0.01%,- Chicken serum (Gibco, USA) - 2%,- Fibroblast growth factors (FGF (Thermo Fisher, USA) - 10 ng/μl,- Activin A (Thermo Fisher, USA) - 25 ng/μl,- Nucleosides (Millipore, USA) - up to x1,- Antibiotic-antimycotic 100x (Thermo Fisher, USA) - up to x1.The prepared medium was sterilised by filtering through 0.22µm diameter nylon filters.3. Thermo Scientific Nunclon Delta Surface (Denmark) 48-cell culture plates with adhesive layer.4. CoolCell SV2 cell culture cryobox, Corning (BioCision, USA).5. Cryogenic storage dewar for long-term preservation of PGCs. 3. Methods1. 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.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.3. Egg dissection on the 4th-5th day of egg incubation, using an engraving machine to drill a 1.0-1.5 cm borehole.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).5. Cultivation in 300 µl tubes with sterile medium in a thermostat at +37.0°C. Then transfer to culture plates.6. Transfer cell culture to culture plates in laminar.7. Cultivation in CO2 incubator (mode: +37.0°C, CO2 concentration - 4%, humidity - 90-95%).8. The culture medium is replaced every three days at the rate of ½ the volume of medium in the well.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).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.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).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.13. Cell viability is assessed by counting trypan blue-stained cells after 3-5 days of cultivation. 2026-09-18T17:51:14Z Dataset Dataset 10.6084/m9.figshare.33923425.v1 https://figshare.com/articles/dataset/Experimental_data_on_the_integrity_and_survival_of_primordial_germ_cells_after_cryopreservation_in_various_chicken_breeds_/33923425 CC BY 4.0