Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 78(2): 59-65, 2025 Firenze University Press https://riviste.fupress.net/index.php/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3420 Citation: de Oliveira Machado, L., Severo Salau, H., Rodrigues Pereira, L., Koslovski Sassi, A., Pimentel Tor- res, F., del Valle Garnero, A. & José Gunski, R. (2025). Avian DNA extraction: An economical and efficient alterna- tive for Farmer-fixed samples. Caryo- logia 78(2): 59-65. doi: 10.36253/caryo- logia-3420 Received: March 19, 2025 Accepted: October 10, 2025 Published: December 20, 2025 © 2025 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID: LDOM: 0000-0002-4172-2283 HSS: 0009-0009-0979-3661 LRP: 0000-0003-0031-0124 AKS: 0009-0000-6007-4659 FPT: 0009-0009-5514-0522 ADVG: 0000-0003-4252-8228 RJG: 0000-0002-7315-0590 Avian DNA extraction: An economical and efficient alternative for Farmer-fixed samples Lilian de Oliveira Machado1,2,*, Hybraim Severo Salau1,2, Larissa Rod- rigues Pereira1,2, Adriana Koslovski Sassi3, Fabiano Pimentel Torres1, Analía del Valle Garnero1,2, Ricardo José Gunski1,2,* 1 Laboratório de Diversidade Genética Animal, Universidade Federal do Pampa, São Gabriel, RS 97300-162, Brazil 2 Programa de Pós-graduação em Ciências Biológicas, Universidade Federal do Pampa, Campus São Gabriel, RS, Brazil 3 Universidade Federal do Pampa, Campus São Gabriel, RS, Brazil *Corresponding author. E-mail: lilianmachado.aluno@unipampa.edu.br, ricardogun- ski@unipampa.edu.br Abstract. Cytogenetics laboratories often accumulate vast collections of cells fixed in Farmer’s solution (3 parts methanol to 1-part glacial acetic acid), stored long-term in freezers. While many of these samples are unsuitable for conventional cytogenetic analyses, they hold potential for molecular applications, especially as ethical restric- tions around the collection of biological material through invasive procedures (e.g., biopsies, tissue excision, bone marrow aspiration) become increasingly stringent. How- ever, extracting DNA from these cells presents significant challenges, such as structural fragility induced by the fixative and potential genetic material degradation, which can compromise subsequent analyses, including PCR. This study developed and stand- ardized a protocol for extracting DNA from Farmer-fixed avian cells using accessible and low-cost reagents. The method proved economical and efficient, even for decades- old samples, recovering DNA suitable for cytogenomic and molecular studies. This approach significantly advances sustainable practices in science by utilizing long- stored samples that might otherwise be discarded, this approach provides a cost-effec- tive strategy that reduces the need for new collections and aligns with current ethical guidelines in molecular genetics research. Compared to commercial kits, the protocol demonstrated economic viability while expanding the use of biological collections in genetic research and evolutionary studies. Keywords: DNA extraction, cell preservation, cytogenomics, sustainable methods, 3Rs principles. INTRODUCTION Biological sample fixation is widely used to preserve cells and tissues, ensur- ing structural integrity essential for subsequent analyses (Tan and Yiap, 2009). In the field of cytogenomics, understood here as the integration of cytogenetic and genomic approaches, the primary fixation method employs Farmer’s solu- https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3420 https://doi.org/10.36253/caryologia-3420 https://doi.org/10.36253/caryologia-3420 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0002-4172-2283 https://orcid.org/0009-0009-0979-3661 https://orcid.org/0000-0003-0031-0124 https://orcid.org/0009-0000-6007-4659 https://orcid.org/0009-0009-5514-0522 https://orcid.org/0000-0003-4252-8228 https://orcid.org/0000-0002-7315-0590 mailto:lilianmachado.aluno@unipampa.edu.br mailto:ricardogunski@unipampa.edu.br mailto:ricardogunski@unipampa.edu.br 60 Lilian de Oliveira Machado et al. tion, a 3:1 mixture of methanol and acetic acid, which dehydrates and stabilizes cells (Coleman and Tsongalis, 1997; Amorim et al., 2007). While effective for long-term preservation, fixation alters the chemical and physical properties of cells, making them more prone to fragmen- tation and chemical or cross-DNA contamination. These factors can compromise DNA quality and hinder molecu- lar analyses (Schrader et al., 2012; Floridia et al., 2023). Long-term storage exacerbates these challenges, as variations in temperature, exposure to contaminants, and infrequent fixative replacement can degrade genetic material (Pereira, 2015). Nonetheless, using fixed sam- ples offers significant bioethical advantages. These sam- ples enable genetic analysis without requiring new col- lections, contributing to species conservation, minimiz- ing impact on wild populations, and adhering to the 3Rs principles (replacement, reduction, and refinement) (Díaz et al., 2020; Hubrecht and Carter, 2019). While commercial DNA purification kits are available, most target blood samples are costly, have limited shelf lives, and are impractical for processing large sample volumes (Kulkarni et al., 2020). This study proposes a protocol adapted and opti- mized for Farmer-fixed avian cells, making use of acces- sible, low-cost reagents. Effective for cytogenomic and evolutionary research, the approach also emphasizes sus- tainable practices by preserving vital genetic data from existing biological collections. Furthermore, this work introduces an optimized protocol tailored for fixed avian samples, with potential applicability to other vertebrates. By utilizing affordable reagents, the protocol not only ensures efficiency but also promotes the expanded use of preserved biological collections in future studies. This method facilitates the preservation of essential genetic data while advancing sustainable and ethical practices in data collection (Srinivasan, 2002; Miyaki, 2001). MATERIALS AND METHODS Cell suspensions were obtained from avian sam- ples collected between 1998 and 2024 from Antarc- tic research stations (Carlini and Orcadas Bases) and Brazilian biomes (Pampa and Atlantic Forest) (Table 1). Lymphocyte cultures were prepared from blood col- lected with heparinized syringes following Moorhead et al. (1960). Cultures were incubated at 39°C for 72 hours in RPMI 1640 medium supplemented with 20% fetal bovine serum, 0.25 mL penicillin/streptomycin, and 0.2 mL phytohemagglutinin. Colchicine (0.05%) was added one hour before harvest. Hypotonic treatment (0.075 M KCl) and fixation with Farmer’s solution were performed as described in Garnero and Gunski (2000). Samples were stored at -20°C. The bone marrow was suspended in 10 mL of Hank’s balanced solution (HBSS), together with 0.1 mL of 0.05% colchicine, and incubated at 37°C for 1 hour. After this period, the suspension was centri- fuged at 120 g for 8 minutes and the supernatant was removed. Then, 10 mL of hypotonic solution (0.075 M KCl) was added and the sample was incubated again at 37°C for 30 minutes. After incubation, the sample was centrifuged again at 120 g for 8 minutes, discarding the supernatant. The cell pellet was fixed with a solution of methanol and acetic acid (3:1) and centrifuged at the same speed. This fixation procedure was repeated three times to ensure the integrity of the preparations (Table 1). Finally, the samples were stored in a freezer at -20°C. DNA Extraction Cell suspensions were centrifuged at 12,000 g for 5 minutes at 4°C, the supernatant was removed, and the cells were resuspended in 500 µL of 10% phosphate-buff- ered saline (PBS) at 4 °C (Amorim et al., 2007), repeat- ing washes up to three times to remove as much fixative residue as possible. Add 20 µL Proteinase K (20 mg/mL) and lyse cells with 400 µL lysis buffer (0.1 M Tris-HCl, 0.1 M EDTA, 1% SDS, 0.06 M NaCl) at 56°C for 30 min- utes. RNase A (20 µL at a concentration of 4 mg/mL) was added to all samples, except for the Colaptes mel- anochloros specimen from Santana da Boa Vista. Precipi- tated DNA with 2 volumes of ice-cold ethanol; incubated at -20°C overnight. Afterwards, centrifuged at 15,000 g for 10 min- utes at 4°C; washed pellet twice with 70% ethanol and once with 90% ethanol. The material was centrifuged at 15,000 g for 8 min at 4ºC, dry pellet at 45ºC for ~15 minutes and eluted in 50 µL of ultrapure water. Store at -20ºC. DNA quality and quantity were assessed using a Nanovue spectrophotometer and agarose gel electro- phoresis. PCR amplification targeted avian 18S rDNA (559 bp) with specific primers, evaluated under standard cycling conditions. RESULTS The DNA was isolated from samples collected and fixed up to 26 years ago on scientific bases in Antarctica, as well as more recent samples collected in 2015, 2022, and 2024 in the Pampa Biome and the Atlantic Forest. As is characteristic of materials fixed and stored for long periods, the extracted DNA showed a significant degree of degradation, as shown in Figure 1. 61Avian DNA extraction: An economical and efficient alternative for Farmer-fixed samples Cost analysis highlighted significant savings, with the protocol costing $0.05-$0.10 per sample compared to $2.50-$3.00 for commercial kits, such as the Reli- aPrep™ gDNA Tissue Miniprep System. This difference represents a saving of up to 98%, making the reagent- based protocol a viable option for large-scale studies or in laboratories with budget constraints. For com- parison purposes, DNA was also extracted using the ReliaPrep™ gDNA Tissue Miniprep System (Promega). Some degree of degradation was observed in both the samples isolated using the protocol developed in this study and those extracted using the commercial kit. However, the samples extracted with the commercial kit showed better integrity. In the samples extracted using the protocol developed, no significant differences were observed between the samples collected in 2024 and those collected between 1998-1999. Notably, the oldest sample, corresponding to Daption capense and stored for 26 years, showed integrity comparable to the most recent samples and to those extracted with the commercial kit (Figure 1). Despite the degree of degra- dation observed and the low amount of DNA obtained (Figure 1), these factors did not prevent efficient PCR amplification. The expected amplicon of around 559 bp was detected with good resolution in all the sam- ples analyzed (Figure 2). DISCUSSION DNA obtained from cell suspensions represents a practical and viable alternative for genomic studies, par- ticularly when derived from samples originally prepared for cytogenetic analyses, as demonstrated by Amorim et al. (2007). Contrary to the findings of Nogueira and Fre- itas (2013), this study shows that the cell lysis technique is an effective method for extracting avian DNA from fixed materials, offering significant advantages over com- mercial kits due to its affordability. This methodology is especially beneficial for challenging scenarios, such as accessing samples from remote regions like Antarctica or rare specimens, including endangered species. The use of fixed material samples provides both scientific and ethical benefits, allowing genetic stud- ies to proceed without the need for new sample collec- tions. This aligns with research ethics guidelines that emphasize reducing the number of animals used in sci- entific studies. Leveraging biological material stored for Table 1. Details of the analyzed samples: bird species, method of obtaining metaphases, origin, geographical location and year of collection. Species Method of obtaining metaphase Origin of the sample Coordinates Year of collection Daption capense Peripheral blood leukocyte culture Carlini Scientific Station, Antarctica 62° 14′ S, 58° 40′ O 1998 Pygoscelis papua Peripheral blood leukocyte culture Carlini Scientific Station, Antarctica 62° 14′ S, 58° 40′ O 1998 Catharacta lonnbergi Peripheral blood leukocyte culture Orkney Base, Antarctica 60° 44′ 17″ S, 44° 44′ 17″ O 1999 Chionis alba Peripheral blood leukocyte culture Orkney Base, Antarctica 60° 44′ 17″ S, 44° 44′ 17″ O 1999 Macronectes giganteus Peripheral blood leukocyte culture Orkney Base, Antarctica 60° 44′ 17″ S, 44° 44′ 17″ O 1999 Leucocarbo bransfieldensis Peripheral blood leukocyte culture Orkney Base, Antarctica 60° 44′ 17″ S, 44° 44′ 17″ O 1999 Turdus subalaris Bone marrow culture Porto Vera Cruz, Atlantic Forest 27° 44′ 09″ S, 54° 54′ 03″ O 2015 Colaptes campestris Bone marrow culture Porto Vera Cruz, Atlantic Forest 27° 44′ 09″ S, 54° 54′ 03″ O 2015 Colaptes melanochloros Bone marrow culture Santana da Boa Vista, Pampa Biome 30º 52’ S 53º 07’ O 2022 Colaptes campestris Bone marrow culture Porto Vera Cruz, Atlantic Forest 27° 44′ 09″ S, 54° 54′ 03″ O 2015 Turdus amaurochalinus Bone marrow culture Santana da Boa Vista, Pampa Biome 30º 52’ S 60° 41′ 47,88″ O 2022 Elaenia chilensis Bone marrow culture São Gabriel, Pampa Biome 30° 20′ 09″ S, 53º 07’ O 2024 62 Lilian de Oliveira Machado et al. extended periods reduces the impact on populations of endangered species and adheres to the 3Rs principles (replacement, reduction, and refinement) (Hubrecht and Carter, 2019; Díaz et al., 2020). These principles play a crucial role in minimizing animal suffering and decreasing the reliance on new animal collections for research purposes. However, extracting DNA from Farmer-fixed sam- ples presents inherent challenges. Cells treated with hypotonic solutions and fixatives such as methanol and acetic acid become structurally fragile and sus- ceptible to contamination, potentially compromising DNA quality and hindering analyses like PCR. Resi- dues from heparin and the culture medium can per- sist despite thorough washing, interfering with PCR efficiency depending on their concentration (Schrader et al., 2012). Nevertheless, in this study, no significant interference from heparin or residual culture medi- um was observed, as corroborated by Beránek et al. (2022). These authors reported that heparin, when present in appropriate concentrations, interferes less with PCR reactions compared to other anticoagulants like EDTA and citrate. Floridia et al. (2023) further emphasized that variations in anticoagulants, includ- ing heparin, minimally affect gene expression in quantitative PCR, highlighting their applicability in diverse experimental contexts. The low protein content observed in the extracted samples is attributed to the denaturing effects of the fixa- tive and the action of Proteinase K, which degrades pro- teins during the extraction process. Prolonged storage can exacerbate genetic material degradation; however, no significant differences were noted between recently collected samples and those stored since 1998. Proper maintenance of the fixative is essential, as inadequate replacement can compromise cell preservation and fur- ther degrade DNA. These findings highlight the impor- tance of strict storage and handling conditions to main- tain the integrity of genetic material and minimize deg- radation impacts on subsequent analyses. CONCLUSION This study validates the viability of extracting DNA from Farmer-fixed avian cells for molecular research, demonstrating significant cost and ethical advantages. The proposed method effectively extracts DNA from Farmer-fixed avian cells, offering a cost-effective alter- native to commercial kits. Its application aligns with Figure 1. 1% agarose gel showing DNA integrity. Lanes 1–3: Lambda DNA standards (25 ng, 50 ng, and 75 ng; 10 µL each). Remaining lanes: DNA extracted using the protocol developed in this study, all loaded with equal volumes (10 µL). Differences in band intensity reflect variation in extraction efficiency among species. Samples correspond to Turdus subalaris, Colaptes campestris, Colaptes melanochloros, Elae- nia chilensis, Catharacta lonnbergi, Chionis alba, Macronectes giganteus, Pygoscelis papua, Leucocarbo bransfieldensis, e Daption capense. DNA extracted with a commercial kit corresponds to T. subalaris, C. campestris, Turdus amaurochalinus, and E. chilensis. 63Avian DNA extraction: An economical and efficient alternative for Farmer-fixed samples ethical research principles, reducing the need for new sample collections and minimizing ecological impact. Despite degradation challenges, the protocol consist- ently yielded DNA suitable for PCR, emphasizing its potential for cytogenomic studies. Additionally, adher- ence to strict storage conditions can further enhance DNA integrity in future studies. By utilizing existing biological collections, this approach provides a sustain- able framework for advancing genetic and evolutionary studies in avian species. ACKNOWLEDGMENTS The authors would like to thank the Coorde- nação de Aperfeiçoamento de Pessoal de Nível Supe- rior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - grant 407285/2021-0), (A.D.V.G.), and FAPERGS for funding support. STATEMENT OF ETHICS The study protocol was reviewed and approved by the Ethics Committee on the Use of Animals, under approval numbers CEUA 019/2020 and CEUA 024/2023. Additional approvals were obtained from the Biodiversi- ty Authorization and Information System (SISBIO), with authorization numbers 61047-3, 33860-2, and 81564-1. Some samples were collected with authorization from the Argentine Antarctic Institute. FUNDING SOURCES This research was supported by FAPERGS through the Program for Supporting the Retention of Young PhDs in Brazil (EDITAL FAPERGS/CNPq 07/2022, awarded to L.O.M.) and by the National Council for Sci- entific and Technological Development (CNPq), Grant Number 407285/2021-0 (A.D.V.G.). Figure 2. A 1.2% agarose gel with PCR products obtained from amplified DNA using specific primers for the avian 18S rDNA gene. The first and last wells contain a 100 bp ladder. The first 10 samples correspond to extracted DNA using the protocol developed in this study, while the next 4 samples refer to extracted DNA using a commercial kit. The penultimate well corresponds to the negative control of the reaction. 64 Lilian de Oliveira Machado et al. AUTHOR CONTRIBUTIONS L.O.M., A.V.G., and R.J.G. contributed to the concep- tion and design of the study. Methodology was carried out by L.O.M. and L.R.P. Formal analysis, data curation, and investigation were conducted by L.O.M. and F.P.T. Visualization was prepared by L.O.M. and A.S.K. The original draft was written by L.O.M. and H.S.S. Review and editing were performed by H.S.S., L.R.P., F.P.T., A.S.K., A.V.G., and R.J.G. Funding acquisition was han- dled by R.J.G. and A.V.G. REFERENCES 1. Tan SC, Yiap BC (2009). DNA, RNA, and protein extraction: The past and the present. J Biomed Bio- technol 2009:1–10. 2. Lott JA, Coleman WB, Tsongalis GJ, eds. (1997). 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Information on the quality of the extracted DNAs Species Extraction Protocol A260/A280 A260/A230 Concentration (ng/ µl) Daption capense lysis 1.6 1.8 236.5 Pygoscelis papua lysis 1.5 2.1 466 Catharacta lonnbergi lysis 1.6 1.8 2424 Chionis alba lysis 1.6 1.9 713 Macronectes giganteus lysis 1.7 2.0 1163 Leucocarbo bransfieldensis lysis 1.7 2.0 1558 Turdus subalaris lysis 1.7 1.6 1890 Colaptes campestris lysis 1.5 1.5 2009 Colaptes melanochloros lysis 1.6 1.5 368.5 Elaenia chilensis lysis 1.7 1.8 1126 Turdus subalaris Commercial Kit 1.6 1.7 417 Colaptes campestris Commercial Kit 1.8 1.8 1138 Turdus amaurochalinus Commercial Kit 2.1 3.0 42 Elaenia chilensis Commercial Kit 1.8 1.9 161.5 Chromosome, ploidy analysis, and flow cytometric genome size of caper (Capparis spinosa) medicinal plant Parviz Radmanesh, Ghasem Karimzadeh* Karyotype analysis and chromosome evolution in Menyanthaceae using FISH Hye-rin Kim, Kweon Heo* Karyological data of five autumn-flowering Crocus L. species from Iran Alireza Dolatyari Divergence in the chromosomal distribution of repetitive sequences in Neotropical cichlid species of the genus Lugubria Luan Felipe da Silva Frade1, Carlos Eduardo Vasconcelos dos Santos1, Bruno Rafael Ribeiro de Almeida2, Cleusa Yoshiko Nagamachi3, Julio Cesar Pieczarka3, Luís Adriano Santos do Nascimento5, Cesar Martins4, Adauto Lima Cardoso4, Renata Coelho Rodrigues Nor Cytotoxic effects of %70 Thiophanate methyl fungicide Yasin Eren Avian DNA extraction: An economical and efficient alternative for Farmer-fixed samples Lilian de Oliveira Machado1,2,*, Hybraim Severo Salau1,2, Larissa Rodrigues Pereira1,2, Adriana Koslovski Sassi3, Fabiano Pimentel Torres1, Analía del Valle Garnero1,2, Ricardo José Gunski1,2,*