Protocol establishment for cryopreservation of somatic cells from poultry for the future use in conservation biotechnologies

• Grant number: 24/06219-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: January 01, 2025
• End date: July 31, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine
• Principal Investigator: Daniele dos Santos Martins
• Grantee: Bruna Dias Mota
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

Brazil ranks third in the world in terms of bird diversity, with 1971 species of the total found on the planet. However, it is the country that has most of its avifauna under threat. Brazil has three routes for migratory waders with around forty species already recorded. Two hundred and ninety-three of these species are endemic, ranking second in terms of endemism on the planet. Therefore, actions that promote the conservation and genetic variability of wild species are extremely important. Given this broad demand, ex situ conservation methods such as cryopreservation of genetic resources through biobanks are an alternative for preserving endangered and/or critically endangered species. However, there are many problems with cryopreservation, the main one being the products and concentrations used for freezing and thawing. Therefore, our research aims to establish the correct components and concentrations that should be present in the cryopreservation medium for the maintenance of somatic cells from Brazilian avifauna. To do this, cells present in the feather follicle of domestic birds will be cultured following the protocol adapted from Ferzeneh (2017), recommended by the Laboratory of Immunohistochemistry and Experimental Physiology (LIFE/FZEA/USP). And in the P0 to P2 passages, the samples will be cryopreserved using the modified method of Lucia Suárez López (2022). To obtain the ideal cryopreservation medium, we will evaluate different protocols composed of concentrations ranging from 10-80% chicken serum (Chicken Serum Gibco¿, New Zealand) and 2.5-10% dimethyl sulfoxide (DMSO), as well as basal medium (DMEM) (Dulbecco's Modified Eagle Medium Gibco¿) inversely proportional to the above. Post-freezing cell viability will be tested at 5, 7, 15, 30 and 60 days, where 3x104 cells will be plated in wells in triplicate. The aim is to obtain the growth curve of the different protocols at intervals of 24, 48 and 72 hours after plating. In addition, mitochondrial activity will be assessed using the Thiazolyl Blue Tetrazolium Bromide® (MTT) assay. The results will be analyzed by age variance considering the day as a fixed effect and comparison of means by Tukey's test to evaluate the differences in cell numbers between the P0 and P2 passages. Thus, our research aims to obtain the best post-thaw cell viability for use in conservation and biotechnology, aiding future research involving cellular products from national and international avifauna.