Fertile egg collection, processing, packaging, and shipping Christian K. Tiambo1, Christine Muhonja1,2, Marcel Blank3 and Mike McGrew3 1Centre for Tropical Livestock Genetics and Health, International Livestock Research Institute 2Kenya Agricultural and Livestock Research Organization 3Centre for Tropical Livestock Genetics and Health, Roslin Institute, University of Edinburgh c.tiambo@cgiar.org International Workshop on Primordial Germ Cell Biobanking and Surrogate Technology for Conservation and Development of Chicken Genetic Resources 27–31 October 2025, ICAR-CARI, Izatnagar, India Overall objective of egg sampling To identify and obtain appropriate embryonic material that allows to isolate PGCs as a cell population for conservation of indigenous chicken genetic resources of India, Study them, expand them under controlled conditions, or Biobank them for genetic conservation or downstream applications. 19 indigenous chicken breeds officially registered in India by the National Bureau of Animal Genetic Resources (NBAGR) Source: ICAR–NBAGR official breed registry (https://nbagr.icar.gov.in/en/chicken-breeds) ICAR Annual Reports & Breed Atlases (2023–2024) Having in mind: Sourcing / selection of fertile eggs from targeted flocks; Safely transfer them to the incubation center (after sorting, processing and packaging) Controlled embryo development (to reach the developmental window when PGCs are accessible); Isolation of PGC-enriched material (blood or gonadal tissue in different species/stages); Characterization (marker-based confirmation); and Downstream handling (culture/cryopreservation/genetic manipulation) Before start (3) Know your breeds/ecotypes/strains: Collect and critically evaluate available information on the breed/ecotype/strain you want to sample (1) Master the goals of the work: Preserving the diversity of indigenous poultry populations in India. (2) Involve local stakeholders: Central Avian Research Institute Central Poultry Development Organizations State agricultural universities and their poultry programs BAIF Development Research Foundation ICAR–Directorate of Poultry Research (Hyderabad) National Bureau of Animal Genetic Resources (NBAGR, Karnal) State Agricultural Universities (SAUs) and KVKs (Krishi Vigyan Kendras) (4) Seek regulatory, ethical, biosafety and animal-welfare requirements that must be met before any hands-on work is performed. SOP - LIVESTOCK SAMPLING IN INDIA Purpose: To ensure safe, ethical, and compliant collection, handling, transport, and documentation of livestock samples in India. Scope: Applicable to all field and laboratory personnel involved in livestock sampling for research, diagnostics, or surveillance. Regulatory references: CPCSEA (Animal Welfare Rules, 1998 & Compendium), DBT/ICMR Biosafety Guidelines, DAHD Veterinary Sampling & Quarantine Rules, Biomedical Waste Management Rules, 2016. FAILURE FOR NON-COMPLIANCE WITH ABS As governed by the Biological Diversity Act, 2002, and the Biological Diversity Rules, 2004 (amended 2014 and 2023): serious legal, financial, and criminal consequences: Penalties for non-compliance Non-compliance: up to ₹50 lakh (approx. USD 60,000). Repeated offence: an additional ₹1 lakh per day, up to a maximum of ₹1 crore (USD 120,000). Imprisonment up to 5 years Consequences beyond legal penalties Loss of research licenses or project suspension. Rejection or revocation of patents (if ABS clearance not provided to IPO). Reputational damage in academic and corporate circles. Blacklisting by NBA/SBBs for future access requests. International implications - non-compliance may affect eligibility under Nagoya Protocol cooperative frameworks. Remember: ONLY fresh fertilized eggs will be collected, considering Phenotypic traits, Production environment, Production system, Pedigree relationships, Agro-climatic zones, At least 10% percent of breed size, A minimum of 30 eggs per breed, Keep strict records, Etc. Collection of fertilized eggs Collect the blood of cocks and hens using EDTA tube，FTA card or feather. Phenotype data collection Digital photo GPS Questionnaire, production environment descriptors identify the indigenous chicken Collect the fresh egg (Candle Test） sample-specific information (e.g. ID numbers). Package eggs Shipment The import permit, export permit and health certificate !!!! On the field Date and geographic location. Select villages with less introgression from exotic populations. Description of genetic resource utilized. Associated traditional knowledge and any subsequent information. Label eggs and report the code to the sample sheet Metadata associated with egg collections Visual and Physical Indicators fresh eggs Check What to Look For What It Means Shell appearance Clean, intact, slightly rough/matte surface Fresh egg Shell strength Not thin, cracked, or overly shiny Shiny = older egg Float test Place in a bowl of water - Sinks and lays flat: very fresh ✅ good - Tilts upward or stands: 5–10 days old ⚠ still usable - Floats: stale or bad ❌ discard Sampling fertile eggs before incubation Fertile eggs cannot be reliably identified just by external appearance. Choose eggs from flocks with active roosters (1 male: 8–10 hens), well-fed, healthy, and not stressed. Avoid eggs more than 7 days old before incubation Taking the Perfect Poultry Picture No filter Take photos at the same level as the bird Confine birds to a small run or show pen to take good photos Don’t stress the chicken and allow it to showcase itself. Take photos at wacky angles, below the fowl, close-ups, etc. Get your bird in nice and close for the best photos Avoid light reflection and shade Head shot – side on showing size of comb and wattles Hackle feather shape (feathers around the neck) Saddle feather shape (feathers covering the lower back and tail base) Complete photo – to show chickens’ overall shape and build Key areas to take photos of your chicken. Full head Hackle or cape Saddle Egg grading Egg shape and colours Small, medium and large eggs Egg should be between 53g - 63g Should not be incubated. Discard Strange eggs Adulteration and other abnormalities Labelling Use a pencil The code should be reported on your recording sheet X/EIN 002 X/NIN 001 X/WIN 004 X/CIN 003 Country State/Division/District… Egg ID number Breed or type of chicken A= Normal B= Feathered legs C= Naked neck D= Crested E= Frizzle F= Dwarf G=… Other important information on the eggs: Date when the egg was laid, Date of collection Fertilized eggs The fertilized eggs anatomy The Infertile egg and fertile egg Eggs should be processed at the collection site. They should be dry cleaned prior to packaging. Eggs should be grouped according to similar characteristics. Processing of eggs Size Shape Soundness of the shell. Cleanliness Texture Condition of the egg white and yolk Air cell size The eggs must be clean; they must not be washed or be wet. Packaging material should be clean Loss of water due to evaporation should be minimized. The storage room must be free from tainting products or materials and should be cleaned regularly with odourless detergent sanitizers. The storage room must be kept at a constant temperature and appropriate humidity. There should be air circulation in the storage room. Eggs should be able to ‘breathe’ while in storage. Storage of eggs Eggs need to be kept below 24°C (‘Physiological Zero’ – the temperature at which embryo development does not advance) after they have been laid. Incubation period and recommended temperature and humidity ranges for various poultry species   Chicken Turkey Duck Muscovy Duck Goose Pheasant Guinea Fowl Quail Incubation period (days) 21 28 28 35 28 23-28 28 23-24 Incubation Temp. (°C) 37.6 37.4 37.5 37.5 37.4 37.6 37.6 37.6 Wet bulb temp. (°C) 29.4–30.5 28.3–29.4 28.8–30 28.8–30 30–31.1 30–31.1 28.3–29.4 28.8–30 Relative humidity (%) 56–62 51–56 53–60 53–60 60–65 60–65 51–56 53–60 Number of turns per day 18 25 25 31 25 21 25 21 Incubation temp. (°C) (last 3 days) 37.4 37.2 37.3 37.3 37.2 37.4 37.4 37.4 Wet bulb temp. (°C) (last 3 days) 32.2–34.4 32.2–34.4 32.2–34.4 32.2–34.4 32.2–34.4 33.3–35 32.2–34.4 32.2–34.4 Relative humidity (%) (last 3 days) 70–83 70–83 70–83 70–83 70–83 76–90 70–83 70–83 Hatchery operation and sanitation The incubation and hatchery room (dirty lab) should be separate from the cell harvesting and injection room (clean lab). The timing of eggs setting in the incubator should carefully consider the time & type of experiment, as well as the capacity of the operator or the team to work with that number of eggs within the period of egg development stage required. Avoid using substandard eggs (weighing less than 32 grams, cracked or dirty). Egg incubation process Factors affecting incubation outcomes The four main requirements for incubation of good quality fertile eggs are: Correct and even temperature monitored Correct humidity controlled Correct O2 and CO2 concentrations controlled Turning (approximately 90°) of fertile eggs several times per day Cleaning and culling Do not incubate eggs that are cracked, misshapen, soiled, or unusually small or large Do not wash or wipe eggs with a damp cloth. The incubator The incubator room should have no drafts or direct sunlight; the room’s temperature and humidity should be controlled and stable. The incubator and hatcher should also be isolated from other poultry facilities. Sanitizing and setting the incubator Two to three days before incubation Sanitize and stabilize the incubators before setting the eggs. automatic turner. temperature humidity. Resting the egg before the work starts The day eggs are set Let stored eggs warm to room temperature for 4 hours to 8 hours before setting them in the incubator. Once the eggs are in the incubator, do not adjust the temperature or humidity for a few hours. Incubation temperature and relative humidity ISOLATION OF PGCS FROM CHICKEN BLASTODISC Incubate fertile eggs for 6 hours at 38.4°C. DERIVATION OF PGCs FROM BLOOD Eggs supposed to be at stage 16–17 (56 hours). DERIVATION OF PGCs FROM EMBRYONIC GONADS e.g: incubate Monday at 9:00 AM Remove on Wednesday at 1700 hours (56 h) and leave them overnight at room temperature On Thursday morning, put the eggs into the incubator for 2 hours to warm up before PGC derivation. Eggs are best incubated for 9 days. Egg turning /rotation Egg turning in vertical (A) and horizontal (B) incubators. Ideal, minimum and maximum percentage weight loss of eggs during incubation. Incubator chart Day by Day Chicken Embryonic Development https://www.youtube.com/watch?v=PedajVADLGw https://www.youtube.com/watch?v=5mDNWJRxg-I Candling of incubated eggs A set of fixed and stained embryos staged according to Hamburger and Hamilton (1951). Isolation PGC cells form different stage of Embryo CTLGH Funders THANK YOU image3.png image1.png image2.png image10.png image11.png image12.png image5.png image6.png image13.png image14.png image15.png image16.png image17.jpeg image18.png image19.jpeg image20.jpeg image21.jpeg image22.png image23.png image24.png image25.png image26.jpeg image27.png image28.jpeg image29.png image30.png image31.png image32.png image33.png image34.png image35.jpeg image36.png image37.jpeg image38.emf image39.png image40.jpeg image41.png image42.png image43.png image44.emf Study no: 2017-21 INCUBATOR DATA CHART Time = 24 hour clock Date format dd/mmm/yyyy Day# Date Turner Works 1 (Y/N) Morning Afternoon Temp ( o C) RH H 2 O 2 Temp ( o C) RH H 2 O Candling 3 Observation Initial/Data 1 2 3 Room Incub. Room Incub. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 xx xx xx 19 xxx xx xx 20 xx xx xx 21 xx xx xx 1 Check the turner three times each day except days 19 through 21. Eggs are not turned on these days. 2 Check water in bottom of incubator 3 Confirm candling performed image45.png image46.png image47.png image48.jpeg image49.png image50.jpeg image51.jpeg image52.png image53.png image9.png image54.jpeg