# Biochemical components of chicken primordial germ cells are modified by cryopreservation: original analysis by Fourier Transform Infrared (FTIR) microspectroscopy

## TL;DR

This study shows that cryopreservation affects the biochemical makeup of chicken germ cells, with FTIR revealing changes in lipids and proteins that could help improve biobanking methods.

## Contribution

The study introduces FTIR microspectroscopy as a label-free method to detect cryopreservation-induced biochemical changes in chicken germ cells.

## Key findings

- Cryopreservation caused a temporary drop in cell viability and proliferation, but germ-cell markers remained stable.
- Female cPGCs showed more sensitivity in pluripotency markers compared to males.
- FTIR revealed lipid changes in male cPGCs, including increased saturation and lipid accumulation after thawing.

## Abstract

Chicken primordial germ cells (cPGCs) are a highly valuable resource for preserving chicken genetics and poultry biodiversity, as these diploid stem cells, derived from both male and female genotypes, can be maintained in long-term in vitro culture and are still capable of producing germline-transmitting progeny. However, the cryopreservation process, which guarantees that these cells can be used to maintain genetic resources, raises various questions about their quality after thawing. We assessed cryo-induced biological alterations in cPGCs derived from Lueng Hang Khao (LK), a Thai native chicken breed, using both Fourier transform infrared (FTIR) microspectroscopy along with traditional biological and molecular assays. Established male and female cPGC lines were cryopreserved for ≥ 2 weeks and assessed at day 10 and day 20 post-thawing against fresh control. The main findings of this study were that i) cryopreservation induced a transient decrease in viability and proliferation, while germ-cell gene markers remained stable in both sexes, ii) pluripotency markers in female cPGCs were more sensitive than in males, and more interestingly, iii) FTIR analysis of male cPGCs showed that cryopreservation induced biochemical changes. Those include membrane remodeling—increased lipid peroxidation, greater saturation and acyl-chain packing, and reduced unsaturation—with lipid accumulation that is most evident at day 20 post-thawing, suggesting a long-term effect. Additionally, a marked reduction of the ester C=O (∼1740 cm⁻1) band was also observed in cryopreserved cPGCs, suggesting constant modifications of key molecules. Concordantly, secondary-structure analysis indicated a shift toward native-like α-helical states, and nucleic-acid/carbohydrate signatures declined. These results highlight the overall post-thawing recovery of cPGCs and establish FTIR as a practical, label-free readout to complement standard assays, informing quality control and optimization of the quality of germ-cell biobanking.