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.

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.

Figures

Morphology of control and thawed cPGCs. One day after thawing, male (a) and female control cPGCs (c) were compared with thawed male (b) and female cPGCs (d). After 7 days of thawing, male (e) and female control cPGCs (g) were compared again with thawed male (f) and female cPGCs (h). The eccentric refractive granules (white arrows) were observed in both control (i) and thawed cPGCs (j). Dead cell (black arrows) was observed and counted.Fig 1 dummy alt textFig. 2Characterization of established male and female cPGCs compared with chicken embryonic fibroblast (CEF). Pluripotency markers (*NANOG* a

Viability of control and thawed cPGCs during 10 days post-thawing. All data are presented as mean ± SEM. Data with different letters are significantly different (*P* < 0.05) on each day of cultivation.Fig 3 dummy alt text

Proliferation of male and female control cPGCs compared with thawed male and female cPGCs 10 days post-thawing. All data are presented as mean ± SEM.Fig 4 dummy alt text

The relative expression of germ cell markers (*CVH* and *DAZL*), pluripotency markers (*SOX2, POUV, NANOG*), *CXCR4*, and *TERT*. Data were evaluated in fresh, day 10 post-thawing, and day 20 post-thawing in male (a) and female cPGCs (b). All data are presented as mean ± SEM. Data with different letters are significantly different (*P* < 0.05) in each gene. Fresh cPGCs was used as a control group by adjusting the expression level to 1. Ribosomal Protein S17 (*RPS17*) was used as a reference gene.Fig 5 dummy alt text

Expression of DAZL (green), EMA-1 (magenta), and SSEA-1 (magenta) in fresh and thawed cPGCs from both male and female, visualized by immunofluorescence. Nuclei are shown in blue. Negative controls were processed with secondary antibody only. Scale bar, 25 µm.Fig 6 dummy alt text

Mean original FTIR spectra (a), mean second derivative spectra (b) of fresh, day 10 post-thawing, and day 20 post-thawing cPGCs in the spectral region 3100 – 900 cm^-1^.Fig 7 dummy alt textTable 1The band assignment of the FTIR spectrum of cPGCs in the mid-Infrared region (3100–900 cm^-1^).Table 1 dummy alt textWave number (cm^-1^)Band assignment3060*v*_as_ (=CH): unsaturated lipids2960*v*_as_ (CH_3_): lipids and proteins2920*v*_as_ (CH_2_): saturated lipid and proteins2870*v*_sym_ (CH_3_): lipids and proteins2850*v*_sym_ (CH_2_): saturated lipid and proteins1740*v* (C=O):) carbonyl ester of l

Principal component analysis score plot of fresh, day 10 post-thawing, and day 20 post-thawing cPGCs (a), loading plot PC-1 (b), loading plot PC-3 (c) from PCA model.Fig 8 dummy alt text

Full text

Introduction

Advances in maintaining chicken primordial germ cells (cPGCs) in vitro have led to this approach becoming popular in various laboratories for various purposes, including enhanced transgenic chicken development through gene editing approaches and mainly for poultry conservation and biodiversity management (Han and Park, 2018; Lázár et al., 2021; Lillico et al., 2007). Additionally, cPGCs represent the exclusive lineage capable of transferring genetic and epigenetic information from one generation to the next (Kress et al., 2016, 2024; Leitch et al., 2013). Consequently, these cells are a critical resource for avian germplasm preservation and the implementation of advanced reproductive biotechnologies.

The cryopreservation of cPGCs offers a significant advantage for the long-term preservation of avian genetic resources (Nandi et al., 2016; Tonus et al., 2016), thereby contributing to the security and sustainability of the poultry genetic management and avian industry. However, this technique presents challenges, primarily due to potential cell damage from cryoprotectant toxicity, osmotic stress, putative gene structure alteration and the formation of intra- and extracellular ice crystals (Chatterjee et al., 2017). Therefore, comprehensive quality evaluations—encompassing both physiological traits, such as viability and proliferation, and biochemical integrity, such as membrane and protein stability—are essential to confirm that cryopreserved cPGCs maintain the cellular homeostasis necessary for post-thaw recovery and subsequent biological use.

Various approaches have been used to characterize cell integrity and functionality after cryopreservation, including assessments of biological characteristics such as cell viability, growth, and colonization for functional gamete production, as well as molecular characteristics like gene and protein expression analysis (Lázár et al., 2021; Tonus et al., 2017). However, a comprehensive understanding of cryopreservation-induced cellular damage and recovery also necessitates that biomolecule alterations be taken into account as the fundamental building blocks and functional machinery of cells; the integrity and precise arrangement of biomolecules are paramount for maintaining cellular homeostasis, responding to stress, and ensuring long-term viability and functionality (Henderson, 2010; Hengartner, 2000). This highlights the importance of biomolecular alteration in cell biology.

As a particularly powerful method of molecular and biochemical analysis, Fourier Transform Infrared (FTIR) microspectroscopy offers a unique biochemical fingerprint of a sample by interrogating it with IR energy. Different biochemical bonds and functional groups within a molecule vibrate at characteristic energies. Their selective absorption of infrared radiation at specific wavenumbers (within the mid-infrared range of 4000 to 400 cm⁻^1^) contributes to this unique biochemical fingerprint, which includes information on lipids, proteins, nucleic acids, and carbohydrates. This technique also enables the simultaneous detection of changes in the quantity, conformation, and interaction of these key cellular components (Baker et al., 2014). FTIR has been widely utilized to characterize cell properties after various treatments, including drug-induced cytotoxicity, differentiation processes, and responses to physical stresses like temperature fluctuations or cryopreservation (Altharawi et al., 2019; Ami et al., 2008; Wang et al., 2018). Additionally, FTIR offers a non-invasive and label-free nature, which allows for direct analysis of cells without the need for extensive sample preparation or exogenous probes, thereby minimizing cellular perturbation capable of mimicking the physiological state of the cell without mechanical or environmental disturbances.

FTIR microspectroscopy is a proven tool for evaluating biomolecular changes in various cell types. However, it has not yet been applied to comprehensively assess the impact of cryopreservation on germinal cells, particularly cPGCs. Herein, we characterized cPGCs subjected to cryopreservation processes using FTIR to assess biomolecular alterations. Our findings enabled us to identify the most indicative spectral markers reflecting the post-thawing cellular status and establish a reference map for the variability of these cells after cryopreservation.

Material and methods

Chicken and egg incubation

Experiments were conducted at the Suranaree University of Technology (SUT) in Nakhon Ratchasima, Thailand. Fertilized eggs of Thai indigenous chicken, Lueng-Hang-Khao (LK) chicken, were obtained from a SUT farm and collected after 3 times of insemination and incubated in an incubator with automatic turning every 1 h. The temperatures and humidity were set at 37.7°C and 50–60 % respectively, for 48–54 h of incubation. All animal experiments were performed in accordance with the institutional guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee, SUT (Animal License No. U1-02631-2559).

Isolation and in Vitro culture of cPGCs

Chicken embryonic blood was collected from the vitelline arteries of Hamburger and Hamilton (HH) Stage 13–16 embryos (1951) using a capillary glass needle. Approximately 1–2 µL of collected blood was individually placed into 270 µL of cPGC medium in a 96-well plate. One-half of the medium was changed every two days with fresh medium. When the cells reached confluency, they were transferred to larger wells, and at least an equivalent volume of fresh medium was added to the cell suspension.

The FACs medium was prepared as previously described by Whyte et al. (2015) and adapted by Chen et al. (2019). Briefly, the medium consisted of Dulbecco’s Modified Eagle Medium (DMEM) diluted with ultrapure sterile water (a 1:2 ratio of ultrapure sterile water to calcium-free high-glucose DMEM) containing final concentrations of 1x penicillin-streptomycin, 1x L-glutamine, 1x sodium pyruvate, 0.1 mM β-mercaptoethanol, 1x B-27 supplement, 0.2 % chicken serum (all from Gibco®, Grand Island, NY, USA), 0.2 % ovalbumin, 0.1 mg/mL heparin sodium salt (both from Sigma-Aldrich, St. Louis, MO, USA), 30 ng/mL human Activin A, and 5 ng/mL human FGF-basic (both from PeproTech, Rocky Hill, NJ, USA). All cPGC lines were maintained at 37 °C in 5 % CO₂. After approximately 4 weeks of cultivation, each established cPGC line was maintained in a 6-well plate and placed on an orbital shaker at 80–100 RPM to prevent aggregation and subsequently used in downstream experiments.

Chicken embryonic fibroblast (CEF) derivation

CEFs were established from the same batch of fertilized embryos, but at a later developmental stage (8–9 days of incubation; E8–E9), to provide sufficient tissue for fibroblast derivation. After head/limb removal, remaining tissue was minced and dissociated in PBS with 0.5 % trypsin–EDTA for 5 min at RT, filtered (40 µm), pelleted (400 × g, 5 min), and plated in DMEM/F-12 supplemented with 5 % FBS, penicillin/streptomycin, and 10 % TPB (all Thermo Fisher; TPB from Sigma-Aldrich). Cells were cultured at 38°C, 10 % CO₂, 60–70 % humidity; medium was replaced every 2 d. CEFs at passages 1–3, mycoplasma-negative, were used as controls for cPGC marker assays.

cPGCs sexing

Genomic DNA was extracted from chicken embryonic tissue collected after blood sampling (DNeasy Blood & Tissue Kit, Qiagen) and quantified (NanoDrop 1000, Thermo Fisher Scientific). cPGC sex was determined by PCR using W-specific primers USP1/USP3 and an autosomal control CPE15F/CPE15R (Itoh et al., 2001; Ogawa et al., 1997), primer sequences shown in Table S1. Reactions (25 µL) contained 12.5 µL DreamTaq PCR Master Mix (2 ×), primers (10 µM each), 8 µL DNase-free water, and 2.5 µL genomic DNA. Cycling: 95°C 2 min; 35 cycles (95°C 30 s, 56°C 30 s, 72°C 30 s); and a final extension at 72°C 5 min. Products were run on 2 % agarose (1 × TBE) with ViSafe Red (Vivantis Kuala Lumpur, Malaysia) at 100 V for 30 min and visualized under UV transilluminator.

Cryopreservation and thawing

Seven cPGC lines (five males and two females) were cryopreserved at approximately 0.5 – 1 × 10⁶ cells per cryovial. Cells were initially resuspended in 250 μL of basal medium (DMEM diluted with ultrapure sterile water at a 2:1 ratio, v/v). A 2X freezing medium (10 % Dimethyl sulfoxide (DMSO) in fetal bovine serum (FBS); Gibco, Paisley, UK) was prepared. For cryopreservation, 250 µL of the 2X freezing medium was slowly added to the cell suspension, achieving final concentrations of 45 % FBS and 5 % DMSO (Ecker et al., 2023). Cryovials were placed in a Mr. Frosty™ Freezing Container (Thermo Fisher Scientific, Waltham, MA, USA) for a cooling rate of approximately −1°C/min and stored at −80°C overnight before moving to be kept in liquid nitrogen.

After at least 2 weeks of storage, frozen cPGCs were rapidly thawed by hand-warming for approximately 3–4 min until the ice disappeared. Samples were washed by adding 5 mL basal medium per cryovial, followed by centrifugation at 1,200 rpm for 5 min to discard the freezing medium. Cells were then resuspended in 3 mL culture medium, counted by trypan blue exclusion assay, and seeded into 6-well plates at approximately 0.5 × 10⁶ cells per well. Four cPGCs lines (two males and two females) were chosen and used for viability, proliferation, relative gene expression, and immunocytochemistry. Separately, five male cPGC lines were used to evaluate biochemical alterations via FTIR microspectroscopy, as there were unfortunately not enough female isolates to perform the analysis in parallel in the same experiment.

Proliferation assay

The proliferation rates of control and thawed cPGCs were evaluated for both male and female cPGC lines. A total of 1 × 10^5^ cells were seeded into each well of a 6-well plate, which contained 2 mL/well of completed medium. Two-thirds of the medium was changed every two days. Cells were counted every other day before medium change by taking 50 µL from each well, staining it with an equal volume of 0.4 % Trypan Blue Solution (Thermo Fisher Scientific, Waltham, MA, USA), and counting the cells using a hemocytometer.

RNA extraction and quantitative RT-PCR

Total RNA was extracted from ∼8 × 10^5^ cPGCs with TRIzol® (Invitrogen, Thermo Fisher Scientific) and quantified by NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); quality was verified by A260/280 (1.80–2.10). cDNA was synthesized from 2 µg RNA using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems). qPCR was run on a CFX Opus 96 (Bio-Rad) with SYBR™ Green PCR Master Mix (Applied Biosystems™, Foster City, CA, USA) to quantify germline markers (chicken vasa homologue; CVH and deleted in azoospermia-like; DAZL), pluripotency genes (SRY-box transcription factor 2; SOX2, POU class 5 homeobox 1; POUV, and Nanog homeobox; NANOG), migration (C-X-C chemokine receptor type 4; CXCR4), and immortality (telomerase reverse transcriptase; TERT) in the fresh, day 10 post-thawing, and day 20 post-thawing cPGCs (both sexes). Ribosomal Protein S17 (RPS17) served as the internal control. This set of primers was designed and validated by SBRI-U1208 INSERM, Lyon, France. Cycling: 95°C 20 s; 39 cycles of 95°C 3 s, 60°C 30 s. Relative expression was calculated by ΔΔCt, normalized to fresh cPGCs (set to 1).

Immunocytochemistry

cPGCs (2 × 10⁵ cells) were cytospun onto positively charged slides (Jiangsu Huida Medical Instruments Co., Ltd., Yancheng, China) at 500 rpm for 3–5 min using an MPW M-DIAGNOSTIC laboratory centrifuge (MPW Med. Instruments, Warsaw, Poland), then fixed in 4 % paraformaldehyde (RT, 10–15 min), and washed in DPBS. Samples were blocked (2 % BSA/DPBS, 30 min). For extracellular markers, slides were incubated overnight at +5°C with mouse anti-SSEA-1 (MC-480) at 1:65 dilution or anti-EMA-1 at 1:200 (both from The Developmental Studies Hybridoma Bank (DSHB)). For intracellular DAZL, cells were permeabilized (0.3 % Triton X-100/DPBS, 15 min) before blocking and incubating with rabbit anti-DAZL at 1:500 dilution (ab215718, Abcam). Secondary antibodies (goat anti-rabbit IgG H&L Alexa Fluor 488; goat anti-mouse IgG H&L Alexa Fluor 647; both 1:500 in blocking buffer; Abcam) were then applied. Confocal fluorescence images were acquired on a Nikon ECLIPSE Ni-E upright microscope (Nikon Instruments Inc., Tokyo, Japan) coupled to a Nikon AX Confocal Laser Scanning System. Images were collected using a 40X objective, specifically the CFI Plan Apochromat Lambda 40X (NA 0.95, dry), with all acquisitions and subsequent analysis performed using NIS-Elements software

Fourier transform infrared (FTIR) microspectroscopy

The cPGCs, approximately 8 × 10^4^ cells/µL, were washed and diluted in 0.9 % NaCl solution before mounting onto a BaF_2_ window (22 mm x 1 mm) (Crystran Ltd., Poole, Dorset, United Kingdom) by applying 3 µL of the cell suspension in triplicate. The samples were then dried in a desiccator overnight. To remove residual salts, each dried sample was gently washed with sterile water and re-dried and kept in a desiccator until spectra were acquired (Tanthanuch et al., 2010). Spectra were acquired with a Tensor 27 FTIR spectrometer (Bruker Optics, Ettlingen, Germany) coupled with a Hyperion 2000 IR microscope equipped with a 36x objective and a mercury cadmium telluride (MCT) detector. FTIR spectra were measured in transmission mode with 64 scans at 6 cm^−1^ over the spectral ranges 4000–800 cm^−1^. Spectral acquisition and instrument control were performed using OPUS 7.5 software (Bruker Optics Ltd, Ettlingen, Germany).

Raw spectra were processed for analysis, including vector normalization to correct variations in cell numbers and sample thickness across different spectra. A second derivative analysis was performed using the Savitzky–Golay algorithm (3rd polynomial, 13 smoothing points) to enhance spectral resolution and resolve overlapping bands in three key regions: 3200–2800 cm⁻^1^ (lipid area), 1800–1480 cm⁻^1^ (proteins and esters), and 1480–900 cm⁻^1^ (nucleic acids and carbohydrate region) (Bozkurt-Girit and Kılıç, 2025). The processed second derivative spectra were then used for Principal Component Analysis (PCA) using the Unscrambler X software (version 10.4, CAMO Software AS, Oslo, Norway). The integrated area of each of these regions and important individual peaks within them was calculated from the second derivative spectra using the software OPUS 7.5 (Bruker Optics Ltd, Ettlingen, Germany).

For protein secondary structure analysis, the amide I band was analyzed by combining multiple Gaussian and/or Lorentzian functions to identify and characterize each underlying component band combination. The program will adjust the parameters (position, width, area) of each peak under the amide I band from 1700–1600 cm^−1^, such as anti-parallel β-sheet (1695–1680 cm^−1^), β-turn (1675–1666 cm^−1^), α-helix (1660–1645 cm^−1^), and parallel β-sheet (1640–1610 cm^−1^) (Bozkurt-Girit and Kılıç, 2025; Ducic et al., 2021; Kreuzer et al., 2020).

Statistical analysis

To minimize genetic and batch variability, a paired experimental design was employed where each cPGC line served as its own internal control. Data are reported as mean ± SEM. Cell viability and proliferation were compared among the four treatment groups (control male, control female, thawed male, and thawed female) using one-way ANOVA followed by Tukey’s HSD post-hoc test. Gene expression and FTIR results were analyzed separately for each sex to evaluate the effect of culture time (fresh, day 10, and day 20 post-thawing) using repeated-measures ANOVA followed by Bonferroni post-hoc tests. Statistical significance was set at α = 0.05.

Results

cPGCs characterization

The established cPGCs displayed a spherical shape with diameters between 9 and 18 µm (Fig. 1a-h). On day 1 post-thawing, control samples (Fig. 1a and 1c) consisted primarily of spherical, refractive, and bright cells. Whereas both male (Fig. 1b) and female (Fig. 1d) thawed samples showed a noticeable presence of shrunken, dark, and fragmented cells. By day 7, however, dead cells were rarely observed for both male (Fig. 1f) and female (Fig. 1h) cPGCs. Additionally, lipid refractive granules were consistently present throughout (Figs. 1i and 1j). To characterize the established cPGCs, the expression of pluripotency markers, NANOG and POUV, and germ cell markers, CVH and DAZL, was assessed in comparison to CEF (Fig. 2). The cPGCs positively expressed CVH, DAZL, NANOG, and POUV, while CEF expressed only their gene marker, T-box transcription factor 3 (TBX3). Although the housekeeping gene actin was expressed in both cPGCs and CEF.Fig. 1. Morphology of control and thawed cPGCs. One day after thawing, male (a) and female control cPGCs (c) were compared with thawed male (b) and female cPGCs (d). After 7 days of thawing, male (e) and female control cPGCs (g) were compared again with thawed male (f) and female cPGCs (h). The eccentric refractive granules (white arrows) were observed in both control (i) and thawed cPGCs (j). Dead cell (black arrows) was observed and counted.Fig 1 dummy alt textFig. 2Characterization of established male and female cPGCs compared with chicken embryonic fibroblast (CEF). Pluripotency markers (NANOG and POUV), germ cell markers (CVH and DAZL), CEF marker (TBX3), and reference gene (Actin) were characterized.Fig 2 dummy alt text

Cell viability and proliferation

Cell viability was evaluated immediately after thawing (Day 0) and every other day until day 10 post-thawing (Fig. 3). At day 0 and day 2 post-thawing, the viability of thawed male and female cPGCs (84.38 ± 1.45 % and 87.24 ± 1.86 %, respectively) was significantly lower than that of control (fresh) male and female cPGCs (96.03 ± 0.40 % and 92.90 ± 0.86 %, respectively) (P < 0.05). Subsequently, the viability of thawed cPGCs increased until reaching the viability of control cPGCs by day 4 post-thawing.Fig. 3. Viability of control and thawed cPGCs during 10 days post-thawing. All data are presented as mean ± SEM. Data with different letters are significantly different (P < 0.05) on each day of cultivation.Fig 3 dummy alt text

Cell proliferation was assessed by changes in cell number over 10 days post-thawing (Fig. 4). Proliferation results revealed that the cell number of control male and female cPGCs generally exhibited significantly higher cell numbers (P < 0.05) compared to their respective thawed counterparts from days 2 to 8 post-thawing. An exception was observed on day 4 post-thawing, where no significant difference in cell number was found between control male cPGCs and thawed female cPGCs. By day 10 post-thawing, no significant difference in cell number was observed among any of the groups. The average doubling time (DT) from day 2 to 10 post-thawing was determined for all groups, showing no significant difference (P > 0.05) in DT when compared between control cPGCs (male: 2.73 ± 0.17 days; female: 2.82 ± 0.14 days) compared to thawed cPGCs (male: 2.64 ± 0.14 days; female: 2.81 ± 0.17 days).Fig. 4. Proliferation of male and female control cPGCs compared with thawed male and female cPGCs 10 days post-thawing. All data are presented as mean ± SEM.Fig 4 dummy alt text

Alterations of specific transcript expression in cPGCs

Gene expression analysis of selected germline markers (CVH and DAZL), pluripotency-associated genes (SOX2, POUV, and NANOG), migration ability (CXCR4), and immortality (TERT) were quantified in fresh, day 10 post-thawing, and day 20 post-thawing from both male and female cPGCs. The results are summarized in Fig. 5a (male) and 5b (female).Fig. 5. The relative expression of germ cell markers (CVH and DAZL), pluripotency markers (SOX2, POUV, NANOG), CXCR4, and TERT. Data were evaluated in fresh, day 10 post-thawing, and day 20 post-thawing in male (a) and female cPGCs (b). All data are presented as mean ± SEM. Data with different letters are significantly different (P < 0.05) in each gene. Fresh cPGCs was used as a control group by adjusting the expression level to 1. Ribosomal Protein S17 (RPS17) was used as a reference gene.Fig 5 dummy alt text

In male cPGCs, the relative transcript levels of several genes remained similar after cryopreservation at all time points. Only CVH, SOX2, and TERT were initially significantly increased (P < 0.05) at day 10 post-thawing before returning to levels seen in fresh cPGCs at day 20 post-thawing. NANOG expression was the only marker that consistently decreased (P < 0.05) after cryopreservation at each time point. Conversely, female cPGCs showed an initial significant decrease (P < 0.05) in DAZL and POUV at day 10 post-thawing before recovering to the levels of fresh cPGCs. Meanwhile, SOX2 and NANOG remained underexpressed (P < 0.05) for both day 10 and day 20 post-thawing compared to fresh cPGCs.

Immunocytochemistry

We analyzed the expression of DAZL, EMA-1, and SSEA-1 in male and female cPGCs, both before and after cryopreservation, using immunocytochemistry (Fig. 6). Our results confirmed positive expression of all three markers in both sexes under all conditions. Specifically, DAZL expression was localized to the cytoplasm (green fluorescence), while EMA-1 and SSEA-1 were detected on the cell membrane (magenta fluorescence). All cPGC groups were counterstained with DAPI (blue fluorescence) to highlight cell nuclei. No nonspecific fluorescent signals were observed in the negative control group, confirming the specificity of the staining protocol.Fig. 6. Expression of DAZL (green), EMA-1 (magenta), and SSEA-1 (magenta) in fresh and thawed cPGCs from both male and female, visualized by immunofluorescence. Nuclei are shown in blue. Negative controls were processed with secondary antibody only. Scale bar, 25 µm.Fig 6 dummy alt text

FTIR spectroscopic profiles and principal component analysis (PCA)

The average original FTIR spectra of cPGCs at different post-thawing time points and their corresponding second derivative spectra within the fingerprint region (3100–2800 and 1800–800 cm⁻^1^) are presented in Figs. 7a and 7b, respectively. The major band assignments for each predominant wavenumber are summarized in Table 1.Fig. 7. Mean original FTIR spectra (a), mean second derivative spectra (b) of fresh, day 10 post-thawing, and day 20 post-thawing cPGCs in the spectral region 3100 – 900 cm^-1^.Fig 7 dummy alt textTable 1The band assignment of the FTIR spectrum of cPGCs in the mid-Infrared region (3100–900 cm^-1^).Table 1 dummy alt textWave number (cm^-1^)Band assignment3060vas (=CH): unsaturated lipids2960vas (CH_3_): lipids and proteins2920vas (CH_2_): saturated lipid and proteins2870vsym (CH_3_): lipids and proteins2850vsym (CH_2_): saturated lipid and proteins1740v (C=O):) carbonyl ester of lipids1700-1600Amide I: proteins1580-1480Amide II: proteins1480-1350δ(CH_2_): lipids and proteins1236νas (PO₂⁻): mainly nucleic acids, phospholipids1080νsym (PO₂⁻): mainly nucleic acids, phospholipids1200-950Glycogen and carbohydratesAbbreviation: as = asymmetric vibrations, sym = symmetric vibrations, δ = bending.

To objectively visualize the complex biochemical transitions within these spectral profiles, PCA was performed on the second derivative data. The PCA scores plot revealed clustering of fresh, day 10 or 20 post-thawing cPGCs (Fig. 8a). The combination of PC1, PC2, and PC3 explained 83 % of the overall data variability. The corresponding loading plots identified the key spectral features driving these distinctions. Among the corresponding loading plots, the most influential bands were amide I protein (1639 cm^−1^ and 1658 cm^−1^), lipid (2917 cm^−1^ and 2850 cm^−1^), and C=O ester (1741 cm^−1^) in PC1 (Fig. 8b), while in PC3 also showed that lipid (2927 cm^−1^ and 2911 cm^−1^), C=O ester (1741 cm^−1^ and 1712 cm^−1^), amide I protein (1641 cm^−1^), carbohydrate and glycogen (1068 cm^−1^), and nucleic acid (1083 cm^−1^) were the most prominent bands (Fig. 8c). Overall, these loading plot analyses indicated that the amide I and lipid bands were the most significant contributors to the variations observed in the PCA scores plot.Fig. 8. Principal component analysis score plot of fresh, day 10 post-thawing, and day 20 post-thawing cPGCs (a), loading plot PC-1 (b), loading plot PC-3 (c) from PCA model.Fig 8 dummy alt text

Guided by the major sources of variance identified via PCA, we performed a targeted quantitative analysis of the specific biochemical regions driving these cellular distinctions. Quantitative analysis of the integrated areas obtained from the second derivative spectra revealed notable and widespread alterations in the biochemical composition of cPGCs across the various post-thawing periods, as detailed in Table 2.Table 2. Integrated FTIR band areas (mean ± SEM) for key biochemical regions in fresh, day 10 post-thawing, and day 20 post-thawing cPGCs. Values are reported × 10³ (i.e., raw areas = reported value × 10⁻³ a.u.). Different superscript letters within a row indicate significant differences among groups (P < 0.05).Table 2 dummy alt textBiomolecule (Wavenumber)Fresh cPGCsDay 10 post-thawingDay 20 post-thawing $[eqn]$ (∼3060 cm⁻^1^)3.070 ± 0.006^a^2.692 ± 0.024^c^2.880 ± 0.013^b^ $[eqn]$ (∼2960 cm⁻^1^)12.172 ± 0.040^a^11.144 ± 0.066^b^12.074 ± 0.015^a^ $[eqn]$ (∼2920 cm⁻^1^)28.427 ± 0.165^b^26.050 ± 0.133^c^30.884 ± 0.080^a^ $[eqn]$ (∼2870 cm⁻^1^)0.2423 ± 0.08550.0667 ± 0.0004ND $[eqn]$ (∼2850 cm⁻^1^)14.071 ± 0.037^c^14.759 ± 0.077^b^17.495 ± 0.028^a^ $[eqn]$ (3000 - 2800 cm⁻^1^)54.913 ± 0.160^b^52.020 ± 0.272^c^60.454 ± 0.087^a^ $[eqn]$ (∼1740 cm⁻^1^)0.935 ± 0.029NDND $[eqn]$ (1700-1600 cm⁻^1^)115.029 ± 0.126^a^111.551 ± 0.702^ab^112.800 ± 0.144^b^ $[eqn]$ (1580-1480 cm⁻^1^)54.905 ± 0.33253.470 ± 0.29753.884 ± 0.232 $[eqn]$ (∼1480-1350 cm⁻^1^)29.393 ± 0.263^a^26.880 ± 0.124^b^26.200 ± 0.076^b^ $[eqn]$ (∼1236, ∼1080 cm⁻^1^)29.518 ± 0.077^a^28.580 ± 0.062^b^27.739 ± 0.068^c^ $[eqn]$ (∼1200 - 950 cm⁻^1^)20.038 ± 0.292^a^19.191 ± 0.369^ab^16.720 ± 0.029^b^Abbreviation: A = integrated area, ND = not detected.

Unsaturated lipid (∼3060 cm⁻^1^), CH stretching bands (3000–2800 cm⁻^1^) and C=O ester (∼1740 cm⁻^1^). Table 2 showed that in the lipid region, $[eqn]$ , $[eqn]$ , and $[eqn]$ were the most predominant bands reflected as a high integrated area, while the integrated area of $[eqn]$ was very low and absent at day 20 post-thawing. In particular, $[eqn]$ and $[eqn]$ exhibited a similar trend, with significantly higher integrated areas observed at day 20 post-thawing. Whereas, $[eqn]$ , was significantly lower (P < 0.05) at day 10 and 20 post-thawing compared to fresh cPGCs. Moreover, the prolonged absence of $[eqn]$ was observed after thawing. The $[eqn]$ displayed an initial significant lower at day 10 post-thawing before surpassing fresh cPGCs at day 20 post-thawing (P < 0.05).

To quantitatively evaluate lipid alterations after cryopreservation, we used established ratios of FTIR lipid bands. Because the ester carbonyl area $[eqn]$ was undetectable in thawed cPGCs at either day 10 or day 20 post-thawing, it was excluded from analysis. Likewise, the symmetric methyl area ( $[eqn]$ ) showed very low or undetectable—particularly at day 20 post-thawing—and was omitted. The asymmetric methyl area $[eqn]$ was used as the methyl reference instead. The unsaturation index ( $[eqn]$ / $[eqn]$ ) reflected unsaturated phospholipid, ( $[eqn]$ / $[eqn]$ ) indicated saturation level, and ( $[eqn]$ / $[eqn]$ ) indicated hydrocarbon chain integrity (Magalhaes et al., 2023; Nešić et al., 2022; Saeed et al., 2015). The modified ratio of $[eqn]$ / $[eqn]$ indicated lipid chain packing. In addition, $[eqn]$ / $[eqn]$ was also used as an indicator of protein-to-lipid contents (Wilkins et al., 2023).

Table 3 showed that $[eqn]$ / $[eqn]$ and $[eqn]$ / $[eqn]$ were significantly decreased at day 20 post-thawing (P < 0.05). In particular, $[eqn]$ / $[eqn]$ revealed a prolonged decrease after thawing. In the other hand, $[eqn]$ / $[eqn]$ and $[eqn]$ / $[eqn]$ were significantly highest at day 20 post-thawing compared to fresh and day 10 post-thawing (P < 0.05). On addition, cryopreservation did not affect $[eqn]$ / $[eqn]$ , which indicates hydrocarbon chain integrity.Table 3. Characteristic ratios of integrated FTIR band areas in fresh, day 10 post-thawing, and day 20 post-thawing cPGCs (mean ± SEM). Different superscript letters within a row indicate significant differences among groups (P < 0.05).Table 3 dummy alt textRatioIndicationFresh cPGCsDay 10 post-thawingDay20 post-thawing $[eqn]$ / $[eqn]$ Unsaturated lipid content0.252 ± 0.001^a^0.242 ± 0.001^b^0.239 ± 0.001^b^ $[eqn]$ / $[eqn]$ Saturation level2.335 ± 0.006^b^2.338 ± 0.023^b^2.558 ± 0.003^a^ $[eqn]$ / $[eqn]$ Lipid chain packing1.156 ± 0.001^c^1.324 ± 0.004^b^1.449 ± 0.004^a^ $[eqn]$ / $[eqn]$ Hydrocarbon chain integrity0.518 ± 0.0020.501 ± 0.0040.511 ± 0.001 $[eqn]$ / $[eqn]$ Protein to lipid contents2.095 ± 0.008^a^2.144 ± 0.005^a^1.866 ± 0.005^b^

Amide I (1700–1600 cm⁻^1^), amide II (1580–1480 cm⁻^1^) and second derivative of amide I. The results revealed that at day 20 post-thawing, the integrated area of amide I ( $[eqn]$ ) was significantly decreased (P < 0.05) compared to fresh cPGCs, while no significant difference was observed in $[eqn]$ (P > 0.05) (Table 2). Amide I spectra were deconvoluted by curve fitting to resolve overlapping secondary-structure components—parallel β-sheet (1640–1610 cm⁻^1^), α-helix (1666–1645 cm⁻^1^), β-turn (1675–1666 cm⁻^1^), and antiparallel β-sheet (1695–1680 cm⁻^1^) (Bozkurt-Girit and Kılıç, 2025; Ducic et al., 2021; Kreuzer et al., 2020). Relative to fresh cPGCs, the α-helix contribution increased after cryopreservation and was significantly higher at day 20 post-thawing. In contrast, the parallel β-sheet decreased (P < 0.05) at both day 10 and day 20 post-thawing. Concurrently, no significant changes were detected in the β-turn and antiparallel β-sheet (P > 0.05) among each group (Table 4).Table 4. Integrated FTIR band areas of amid I secondary structure components in fresh, day 10 post-thawing, and day 20 post-thawing cPGCs (mean ± SEM). Different superscript letters within a row indicate significant differences among groups (P < 0.05).Table 4 dummy alt textBiomolecule (Wavenumber)Fresh cPGCsDay 10 post-thawingDay 20 post-thawingParallel β-sheet (1640-1610 cm^−1^)1.619 ± 0.008^a^1.445 ± 0.011^b^1.462 ± 0.011^b^α-helix (1660-1645 cm^−1^)1.441 ± 0.019^b^1.670 ± 0.018^ab^1.691 ± 0.013^a^β-turn (1675-1666 cm^−1^)1.229 ± 0.0131.263 ± 0.0141.208 ± 0.047Antiparallel β-sheet (1695–1680 cm^−1^)0.581 ± 0.0100.578 ± 0.0150.644 ± 0.023

CH bending (∼1480–1350 cm⁻^1^), nucleic acid (∼1236, ∼1080 cm⁻^1^), and carbohydrate (∼1200 - 950 cm⁻^1^). Table 2 showed that the integrated area of CH bending ( $[eqn]$ , which this band can be associated with both lipids and proteins (Saeed et al., 2015), was significantly decreased after day 10 post-thawing and persisted through day 20 post-thawing (P < 0.05). Similarly, the integrated area of nucleic acid ( $[eqn]$ ) was significantly progressively decreased after thawing (P < 0.05), while the integrated area of carbohydrate ( $[eqn]$ ) was significantly lowest at day 20 post-thawing (P < 0.05).

Discussion

The cryopreservation process enables long-term preservation of cPGCs, safeguarding valuable chicken genetics, and promoting poultry diversity maintenance. cPGCs quality is typically assessed by morphology, viability, proliferation, and gene/protein expression—and by colonization assays for functional gamete output (Tonus et al., 2016, 2017). Yet the biomolecular impact of cryopreservation remains underexplored. FTIR can sensitively profile cellular composition and structure—used for cell differentiation (Ami et al., 2008; Mata-Miranda et al., 2017), membrane integrity after cryostress (Giugliarelli et al., 2016), and infection-driven alterations (Elsheikha et al., 2019)—but, to our knowledge, cPGC biomolecular changes post-cryopreservation have not yet been investigated.

In this study, we established cPGCs from embryonic blood at 50–54 h of incubation from HH Stage 13–16 embryos (Fig. 1). We confirmed the identity of cPGCs after in vitro culturing by characterizing them with cPGCs gene markers in comparison with CEF (Fig. 2). Under a bright-field microscope, more visibly dead cells in thawed cPGCs at day 1 post-thawing were observed compared to day 7 post-thawing (Fig. 1b and d, Fig. 1f and 1h, respectively). This result is in line with lower viability during the first 2 days post-thawing (Fig. 3). This evidence may confirm that the stress during cryopreservation caused the cell membranes to become less intact, leading to cell apoptosis in the initial stage post-thawing. This phenomenon was similar to the findings of Tonus et al. (2017), which initially showed lower viability during the first 2 days post-thawing in cPGCs. However, the higher cell viability after the initial damage after thawing demonstrated the recovery ability of cPGCs. Additionally, the eccentric refractive granule was found in both control and thawed cPGCs, which is a characteristic morphological feature of cPGCs and serves as an energy reserve in cells (Macdonald et al., 2010). In this context, eccentric refractive granules may indicate stable cell quality after cryopreservation.

Proliferation also demonstrated the impact of cryopreservation, as shown in Fig. 4. In thawed cPGCs, the cell number remained nearly the same as immediately after thawing at day 2 post-thawing, which was significantly lower (P < 0.05) than control cPGCs in both sexes. Additionally, Fig. 4 illustrates the generally higher cell number of control cPGCs during the first 8 days post-thawing. It seems like the long-lasting effects of cryopreservation were reflected in the lower cell number of thawed cPGCs during 8 days of cultivation. However, the average DT from day 2 to day 10 post-thawing showed no significant difference (P > 0.05) among the groups, indicating the same growth rate after day 2 post-thawing. Similar results were reported by Tonus et al. (2017), indicating a slight decrease in cell number on day 1 post-thawing before recovery by day 5 post-thawing. Therefore, our evidence confirmed cPGCs' recovery ability after cryopreservation.

By examining key gene markers, our study was able to assess the molecular consequences of cryopreservation on cPGCs phenotypes. This study is also based on the expression of two key germline markers, CVH and DAZL, as a proxy for cPGC identity and developmental competence (Fig. 5a and 5b). As RNA-binding proteins, these genes are essential for germ cell specification and survival (Lavial et al., 2009; Lee et al., 2016). These germline marker expression results indicated that cryopreservation did not affect cPGCs from either sex and supported that immunocytochemistry was performed to illustrate the expression of DAZL protein after thawing, as shown in Fig. 6. Additionally, SSEA-1 (stage-specific embryonic antigen 1) and EMA-1, cell surface markers used to identify cPGCs, also maintained stable expression after thawing. These results indicate that the cryopreservation effects only appear in the initial period post-thawing on germline markers.

Regarding pluripotency genes, SOX2, POUV, and NANOG are well expressed to sustain proliferation, and as demonstrated, POUV and SOX2 form heterodimers to control downstream targets. In cPGCs, a study by Choi et al. (2021) demonstrated that POUV and SOX2 are crucial for the positive regulation of NANOG expression. Our results revealed that POUV expression seems to be the only gene that was stably maintained over SOX2 and NANOG in both sexes (Figs. 5a and 5b). This result may indicate that POUV plays an important role in maintaining proliferation at this stage. The conflict was found in male cPGCs that exhibited the initial upregulation of SOX2 expression before recovering at day 20 post-thawing. In contrast, the NANOG gene exhibited progressive downregulation over time in male cPGCs. These results may imply that NANOG-expressed regulation relies on more than one pathway, as Choi et al. (2021) also reported that the tumor protein p53 gene (TP53), primarily responsible for inducing cell cycle arrest or apoptosis in response to DNA damage, acts as a suppressor of NANOG expression in cPGCs. Additionally, female cPGCs exhibited SOX2 downregulation, which is in line with the study of Tonus et al. (2017), who discussed that such persistent downregulation of SOX2 may be due to fundamental metabolic changes caused by cryopreservation or, more likely, by cell selection events. Moreover, higher DNA lesion was found in the SOX2 gene rather than the POUV gene after cryopreservation in the zebrafish genital ridge, as reported by Riesco and Robles (2013). Interestingly, the expression levels of the core pluripotency genes (POUV, SOX2, and NANOG) did not directly correlate with the overall proliferation rate of the cPGC population. This apparent discrepancy highlights the complex relationship between gene expression and phenotypic outcome. This suggests that the survival cPGCs may not require the same high transcriptional levels of pluripotency genes to maintain their state, possibly relying on a unique, species-specific regulatory network that operates at a lower threshold. While male and female cPGCs exhibited distinct molecular responses to cryopreservation, their proliferation rates remained similar. This cellular buffering suggests that a high degree of redundancy in the stress response pathways allows both sexes to maintain a consistent functional outcome despite differing molecular challenges.

The stable expression of CXCR4 suggests that the thawed cPGCs retain their functional integrity and migratory potential. This is critical because CXCR4 is the receptor that guides the cells to the gonads (Lee et al., 2017). This reaffirms the suitability of these cryopreserved cPGCs for germline transplantation, as the essential machinery for gonadal homing remains intact. Similarly, the preserved expression of TERT is a strong indicator that the thawed cPGCs have maintained their telomerase activity and thus their self-renewal capacity (Coussens et al., 2006). Only male cPGCs exhibited initial upregulation of TERT before recovering to the fresh-cPGCs level at day 20 post-thawing. This pattern was similar to CVH and SOX2 expression, which may be due to a sex-specific stress-response pathway that is more acutely activated by the freeze-thawing process.

FTIR microspectroscopy was performed to evaluate the overall biochemical changes in cPGCs following cryopreservation. The infrared spectra provide a molecular fingerprint of the cells, with distinct absorption bands corresponding to major cellular components such as lipids, proteins, and nucleic acids (Ami et al., 2008; Zhong et al., 2022) (Table 1). Because these spectra were captured from intact cPGCs, they represent the biochemical signatures of integrated cellular membranes and the cytoplasmic environment, providing a direct readout of membrane integrity and fluidity (Abdelrazzak et al., 2021; Blat et al., 2019; Lewis and McElhaney, 2007).

Analysis of these spectral data via PCA identified lipids and proteins as the primary drivers of biochemical divergence, explained by PC-1 (58 % variance), PC-2 (18 % variance), and PC-3 (7 % variance) in the total variance of 83 % (Fig. 8a). The positive score plots from the fresh group were associated with negative loading plots on PC-1 from C=O ester (1741 cm^−1^), parallel β-sheet (1639 cm^−1^), CH bending (1394 cm^−1^), nucleic acid (1240 cm^−1^), and glycogen (1066 cm^−1^). Positive loading plot on PC-1 from α-helix (1656 cm^−1^), methylene vibrations (CH_2_; 2917 and 2851 cm^−1^) corresponded to the day 10 and day 20 post-thawing cPGCs (Fig. 8b). These results revealed that the post-thaw recovery is characterized by a significant transition in membrane lipid density and protein secondary structure."

Concurrently, the day 10 and day 20 post-thawing cPGCs were quite separated by PC-3 by which the day 20 post-thawing cPGCs associated methylene vibrations (CH_2_; 2927 and 2857 cm^−1^), C=O ester (1741 cm^−1^), α-helix (1656 cm^−1^), and glycogen (1068 cm^−1^), while the day 10 post-thawing cPGCs were corresponded to methylene vibrations (CH_2_; 2911 cm^−1^), C=O ester (1712 cm^−1^), parallel β-sheet (1641 cm^−1^), and nucleic acid (1214 cm^−1^ and 1083 cm^−1^) (Fig. 8c). These results demonstrate an overall increase in cellular lipid content on day 20 post-thawing cPGCs (2927 cm^−1^ and 2857 cm^−1^). The higher intensity of the phospholipid ester C=O ester (1741 cm^−1^), demonstrating the cellular commitment to membrane lipid assembly and repair. Conversely, the day 10 post-thawing cPGCs, defined by structural instability, evidenced by the 2911 cm^−1^ band (ordered/rigid lipid phase) (Lewis and McElhaney, 2013) and the C=O stretch at 1712 cm^−1^, may be associated with peroxidation (Aboualizadeh et al., 2017). Together, these results highlight a significant conformational transition in cellular lipids and proteins between day 10 and day 20 post-thawing, reflecting a gradual recovery process. However, the concurrent depression of nucleic acid (1214 and 1083 cm⁻¹) and glycogen (1068 cm⁻¹) signals underscores the persistent impact of the cryopreservation process on the cellular metabolic fingerprint.

To further substantiate the global PCA trends, targeted quantitative analysis of specific spectral bands was conducted. This revealed that cryo-induced stress significantly altered the chemical integrity of major biomolecular components, with the shifting integrated areas reflecting the cells' immediate response and subsequent adaptation to the freeze-thaw process (Table2).

In the lipid region revealed that $[eqn]$ decreased after thawing, indicating a reduction of unsaturated bonds, likely due to lipid peroxidation (Bozkurt-Girit and Kılıç, 2025; Magalhaes et al., 2023; Nešić et al., 2022). Consistently, the increase in $[eqn]$ and $[eqn]$ is consistent with oxidative stress, aligning with prior observations of higher vas (CH_2_) and vsym (CH_2_) intensities following RuCN exposure in A2780 cells (Nešić et al., 2022). Additionally, Saeed et al. (2015) also demonstrated the rise in the integrated area of $[eqn]$ and stable $[eqn]$ after irradiation treatment. Interestingly, $[eqn]$ declined dramatically post-thawing and was undetectable by day 20 post-thawing, whereas vsym (CH_3_) was already weak in the fresh state. In agreement with earlier studies, oxidative stress elevates the vsym (CH_2_)/vsym (CH_3_) ratio, indicating a reduction of vsym (CH_3_) and/or the increase of vsym (CH_2_) (Nešić et al., 2022; Saeed et al., 2015; Zhong et al., 2022). Furthermore, only fresh cPGCs exhibited a detectable ester C=O band at 1741 cm^−1^, as post-thawing cPGCs spectra did not meet the detection criteria. Prior work links higher ν (C=O) intensity (∼1740 cm⁻^1^) to lipid peroxidation (Magalhaes et al., 2023; Nešić et al., 2022), while reductions in ν (C=O) following irradiation have been reported as indicative of lipid degradation (Saeed et al., 2015). Collectively, the data are consistent with cryopreservation-induced oxidative stress and lipid peroxidation, which may, in turn, alter and weaken the membrane conformation and structure.

The ratio measurements were assessed to quantitatively examine cell membrane characteristics (Table 3). Our study revealed that $[eqn]$ / $[eqn]$ was declining after thawing, indicating reduced unsaturation and possible fluidity loss. In parallel, the lipid chain packing indicator $[eqn]$ / $[eqn]$ , increased, consistent with tighter acyl-chain packing after thawing. Additionally, at day 20 post-thawing, $[eqn]$ / $[eqn]$ ratio was highest compared to fresh and day 10 post-thawing, indicating a higher methylene-to-methyl content—consistent with increased saturation of the cell membrane. These results align with prior reports showing greater lipid chain packing (Magalhaes et al., 2023; Nešić et al., 2022; Saeed et al., 2015), increased saturation (Nešić et al., 2022), and reduced unsaturation (Magalhaes et al., 2023; Nešić et al., 2022) in treated samples. Meanwhile, the stable $[eqn]$ / $[eqn]$ ratio indicates that the average hydrocarbon chain integrity remains unchanged. In the CH-bending region, prominent bands at 1455 cm⁻^1^ and ∼1396 cm⁻^1^ were assigned to CH₃ asymmetric bending with CH₂ scissoring overlap (lipid-dominated) and the CH₃ symmetric ‘umbrella’ bend (mixed lipid + protein), respectively (Zhong et al., 2022). The reduced $[eqn]$ post-thawing further supports fewer terminal methyl groups, consistent with denser packing and greater saturation of lipid tails. Additionally, the decrease in $[eqn]$ / $[eqn]$ indicates a protein–lipid imbalance, reflecting lipid enrichment relative to protein. Previous studies in microalgae used lipids (3050–2800 cm^−1^)/amide I ratio as a neutral lipid accumulation indicator (Dean et al., 2010; Grace et al., 2020) and in yeast (Ami et al., 2014). In particular, Dean et al. (2010) found a strong linear relationship between FTIR (lipid/amide I ratio) and conventional Nile Red lipid estimates. While lipid accumulation can signify oxidative stress-induced metabolic disruption (Li et al., 2025), it likely serves as an adaptive reservoir for membrane repair during cPGC recovery. This dual role underscores the complex interplay between cryo-stress and the re-establishment of cellular homeostasis. Given that hallmark cPGCs naturally harbor abundant lipid droplets (Macdonald et al., 2010), post-thawing lipid accumulation likely reflects an adaptive response—providing a reservoir for membrane rebuilding and sequestering/neutralizing peroxidized fatty acids (Olzmann and Carvalho, 2019). Thus, our FTIR analysis may indicate changes in membrane composition and packing in cPGCs, accompanied by concurrent lipid accumulation. This observation could pave the way for supplementing the culture medium with lipids during thawing to help cells restore their membrane balance and reserves more quickly.

Cryopreservation shifted the amide I profile toward greater α-helix (∼1653 cm⁻^1^) and lower parallel β-sheet (1640–1610 cm⁻^1^). The integrated areas likewise showed increased α-helix in thawed cPGCs and a reduction in parallel β-sheet (Table 4). In contrast to studies in which oxidative stress induces an α-helix to β transition (Bischof et al., 2002; Jackson and Mantsch, 1991; Tunçer et al., 2018; Wolkers et al., 2007), this discrepancy may reflect timing: prior work examined immediate, oxidation-driven changes, whereas our analysis at day 10 post-thawing likely reflects repair-associated refolding. These changes suggest structural rebalancing toward native-like helical states, rather than oxidation-induced β-enrichment. The observed α-helical enrichment parallel to the peak total lipid area at day 20 post-thawing may indicate a coordinated restoration of lipid-transporter activity and membrane remodeling in recovering cPGCs. Consistent with this, Kampf and Kleinfeld (2007) reviewed several proteins that facilitate free-fatty-acid (FFA) transport in mammalian cells—FABPpm, CD36/FAT, FATP, and caveolin—all of which possess α-helix-rich transmembrane or membrane-anchoring domains that enable interaction with lipid bilayers and fatty-acid translocation (Lewis et al., 2001; Pepino et al., 2014; Pilch and Liu, 2011). Moreover, aquaporins, such as AQP3, also comprise multiple α-helical segments that contribute to membrane stability and solute flux during post-stress recovery, with emerging links to lipid metabolism and cryotolerance (Delgado-Bermúdez et al., 2021), supporting the spectral evidence for enhanced α-helical structures in post-thaw cPGCs. Collectively, these results may indicate that α-helix-dominated lipid transporters play a key role in re-establishing membrane order and lipid balance during late-stage recovery.

The nucleic-acid region comprises two bands located at 1236 and 1080 cm^−1^, which are assigned to the antisymmetric and symmetric phosphate diester stretches (νas (PO₂⁻) and νsym (PO₂⁻)), respectively. Both bands include contributions from nucleic acids and phospholipid headgroups (Magalhaes et al., 2023; Zhong et al., 2022). The progressive post-thawing decrease in these bands suggests a loss of total nucleic-acid content (Table 2), consistent with reports that low-dose DMSO reduces cellular nucleic acids (Tunçer et al., 2018) and more DNA damage was observed in cryopreserved sperm (Thomson et al., 2009; Xiao et al., 2012). These results highlight nucleic acids as additional cryo-stress targets, complementing the lipid and protein alterations detected by FTIR.

The 1200–900 cm⁻^1^ region is carbohydrate-dominated, with overlap from nucleic acids and phosphorylated proteins (≈984–948 cm⁻^1^) (Hackett et al., 2016; Zhong et al., 2022). Because cPGCs contain abundant cytoplasmic glycogen (Macdonald et al., 2010), this window likely has a strong glycogen contribution. Notably, Hackett et al. (2016) validated FTIR-derived glycogen by showing spatial concordance with PAS staining and loss of both signals after amylase digestion. The reduced $[eqn]$ at day 20 post-thawing (Table 2), therefore, likely reflects a decline in cellular glycogen, which may be due to post-thawing increases in membrane packing that transiently reduce permeability and nutrient influx, prompting cPGCs to mobilize intracellular glycogen to meet energy and redox demands during recovery.

While these findings establish a robust baseline for post-thaw recovery, the use of male cPGC lines highlights a valuable direction for future research. Because avian germ cells exhibit cell-autonomous sex determination and differences in Z-linked protein dosage as early as the pre-gonadal stage (Soler et al., 2021), future studies should validate these biochemical signatures in female lines. Such efforts will help confirm the universality of these recovery markers in both sexes.

Conclusion

In this study, we combined established biological and molecular assays with FTIR microspectroscopy to provide an integrated view of post-thawing changes in cPGCs after cryopreservation. Biological and molecular readouts revealed transient post-thawing perturbations, followed by recovery to a state comparable to fresh cPGCs. Although female cPGCs exhibited more persistent effects than males, the endpoint proliferation, viability, and protein expression profiles were comparable between the two sexes. Whether these molecular variations affect complex functional traits, such as in vivo gonadal colonization, requires further investigation. FTIR microspectroscopy revealed biochemical alterations consistent with membrane conformational remodeling, including post-thawing lipid accumulation and a marked reduction of the ester C=O signal. Concordantly, secondary-structure analysis indicated a shift toward native-like α-helical states, and nucleic-acid/carbohydrate signals declined. The outcomes of the current study deliver an integrated biological–molecular–biochemical reference for cPGCs post-thawing, supporting the robustness of the protocol and informing future improvements in germ-cell culture conditions and cryobanking. Additionally, this work has demonstrated the feasibility and utility of FTIR in cPGCs, delivering rapid, label-free biochemical readouts that complement standard assays and allow qualitative criteria to be applied to the conditions for growing and thawing cPGCs.

CRediT authorship contribution statement

Sirangkun Sornsan: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Kanjana Thumanu: Writing – review & editing, Visualization, Software, Methodology, Investigation. Kannika Siripattarapravat: Writing – review & editing, Methodology. Parinya Noisa: Writing – review & editing, Resources. Bertrand Pain: Writing – review & editing, Supervision, Methodology, Investigation. Amonrat Molee: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.