Periodic Reporting for period 1 - CRYOCHICK (Innovative cryopreservation methods for chicken germplasm)
Periodo di rendicontazione: 2023-04-10 al 2025-04-09
Sintesi del contesto e degli obiettivi generali del progetto
Poultry products are the most consumed source of animal protein globally. To enable current and future poultrygenetic improvement towards climate adaptation and sustainable farming practices to meet future nutritional demands it is essential to maintain and make available the existing genetic diversity within and between the breeds.
Gene banks can assist in conservingenetic diversity and reintroduction and implementation of important traits or genetic variants into poultry lines. The main goal of CRYOCHICK is to develop effective cryopreservation methods for ex-situ conservation of male and female chicken germplasm.
Existing strategies and methods for gene banking of chicken reproductive material are not adequate because 1) chicken sperm are very sensitive to cryopreservation protocols, which results in highly variable and mostly poor reproduction success rate with cryopreserved semen, especially in native breeds; 2) some native breeds (considered “bad freezers”), have very poor semen cryoresistance; 3) glycerol, the most effective sperm cryoprotectant, is contraceptive in the hen; 4) When only semen is stored in gene banks, six generations of back-crossing are needed to recover a lost original genotype, which is impractical and costly, hence we also need cryopreserved female germplasm 5) cryopreservation of chicken oocytes and embryos is currently impossible due to the very large egg yolk, high lipid content, and the polar organization of bird ova, while semen alone does not capture genes of mitochondria and the W sex-chromosome6; 6) cryopreservation of female primordial germ cells (PGCs) is possible, but is difficult, laborious, and costly. Therefore, new strategies for cost-effective gene banking, and new effective cryopreservation methods for both male and female chicken germplasms are required. The methods and strategies developed here will likely also benefit other poultry species and conservation of wild relatives
Gene banks can assist in conservingenetic diversity and reintroduction and implementation of important traits or genetic variants into poultry lines. The main goal of CRYOCHICK is to develop effective cryopreservation methods for ex-situ conservation of male and female chicken germplasm.
Existing strategies and methods for gene banking of chicken reproductive material are not adequate because 1) chicken sperm are very sensitive to cryopreservation protocols, which results in highly variable and mostly poor reproduction success rate with cryopreserved semen, especially in native breeds; 2) some native breeds (considered “bad freezers”), have very poor semen cryoresistance; 3) glycerol, the most effective sperm cryoprotectant, is contraceptive in the hen; 4) When only semen is stored in gene banks, six generations of back-crossing are needed to recover a lost original genotype, which is impractical and costly, hence we also need cryopreserved female germplasm 5) cryopreservation of chicken oocytes and embryos is currently impossible due to the very large egg yolk, high lipid content, and the polar organization of bird ova, while semen alone does not capture genes of mitochondria and the W sex-chromosome6; 6) cryopreservation of female primordial germ cells (PGCs) is possible, but is difficult, laborious, and costly. Therefore, new strategies for cost-effective gene banking, and new effective cryopreservation methods for both male and female chicken germplasms are required. The methods and strategies developed here will likely also benefit other poultry species and conservation of wild relatives
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
WP2 / SO1 – Development of novel cryopreservation protocols for rooster semen
• A comprehensive set of experiments was conducted to develop improved semen cryopreservation protocols for both commercial and native chicken breeds.
• The first stage of the project focused on understanding the effects of supercooling on rooster sperm. Initial studies showed that supercooling at −10 °C did not significantly affect sperm viability, motility, or DNA integrity. However, subsequent experiments revealed that acrosome integrity was significantly reduced after submitting the sperm to uncontrolled warming. This acrosome damage may result from structural alterations caused during supercooling that only become apparent upon warming, or from the effects of the warming process itself. This finding is highly relevant, as during artificial insemination sperm are exposed to an abrupt temperature shift—from low storage temperatures to approximately 41 °C inside the hen.
• To further improve cryopreservation outcomes, the project investigated the use of Antifreeze Proteins (AFPs) and Antifreeze Glycoproteins (AFGPs). Testing of AFPI at concentrations of 0.3 and 3 µg/mL demonstrated significant enhancements in post-thaw motility, viability, and DNA integrity. In particular, for the Yellow Hungarian breed, AFPI supplementation increased DNA integrity from 49.1% to 70.2%. However, post-thaw fertility remained low (1.5% with AFPI-DMA), highlighting an unexpected loss of fertility that occurred during the chilling phase prior to freezing. These findings underscore the importance of carefully evaluating damage introduced during pre-freezing steps, as such damage might otherwise be incorrectly attributed to the freezing process itself. Furthermore, they highlight the need to specifically address fertility losses occurring during the chilling phase, as this stage may introduce critical damage that compromises the overall success of cryopreservation.
• Parallel studies evaluated Ice Nucleating Proteins (INPs) (Snowmax®) to reduce supercooling during the freezing protocol in chicken sperm. While INPs promoted ice nucleation at higher subzero temperatures, their use negatively impacted post-thaw sperm motility, likely due to presence of particles in suspension. These particles were observed as a result of the partial solubility of Snowmax® in the poultry extender, highlighting the need to identify fully soluble ice nucleators in order to properly evaluate the potential benefits of reducing supercooling on post-thaw sperm survival.
• Recognizing breed-specific challenges, the project developed tailored cryopreservation protocols for poor-freezing breeds. For Yellow Hungarian chickens, an optimized protocol using DMA (0.6 M), a 1:3 dilution ratio, and fast one-step freezing improved post-thaw viability to 56.7%, compared to 42.8% with standard protocols. This demonstrated the necessity of breed-specific adjustments to cryopreservation methods.
• Additionally, the influence of breed and insemination temperature on fertility was assessed. Fertility rates decreased when semen was cooled to 5 °C. Preliminary results suggested that Hungarian Yellow hens maintained higher fertility when inseminated with chilled semen compared to Leghorn hens. However, further experiments using standardized artificial insemination protocols with equal sperm doses across breeds are needed to determine whether this apparent difference reflects a genuine breed-related effect on fertility maintenance, or if it was influenced by variations in the number of spermatozoa used during insemination.
• Finally, studies on rewarming of chilled and frozen-thawed sperm showed that warming frozen-thawed sperm to 37 °C caused severe loss of acrosome integrity (~81% to ~11%), while chilled semen was unaffected
• These findings have been widely disseminated through presentations at major international conferences, including CRYO 2023, CRYO 2024, AERA 2024, ESDAR 2024, SLTB 2024, XXI AIDA 2025, and MCAA 2025, and will also be presented at CRYO 2025.
In addition, a draft manuscript entitled "Tailoring a glycerol-free cryopreservation protocol with anti-freeze (glycol)proteins for commercial and native breeds of chicken" prepared in collaboration with WUR, NBGK, TU/e, and INIA-CSIC, is ready for submission to the journal Cryobiology. A second manuscript, entitled "Changes in the quality of chilled and frozen-thawed chicken sperm when heated near the temperature of the female reproductive tract," is currently being prepared in collaboration with NBGK, WUR, and INIA-CSIC, and is expected to be submitted in the coming months. In addition, a third manuscript, entitled "Effect of supercooling on integrity of rooster sperm", is under preparation and will be submitted to the journal Cryobiology along this year.
WP3 / SO2 – Unravel innovative freezing methods for female chicken embryonic germplasm
• The researcher received hands-on training in the isolation, culture, and injection of primordial germ cells (PGCs) at NBGK. Afterwards, at the Roslin Institute, work focused on the collection of embryonic gonads and the development of a reliable vitrification and thawing protocol for gonadal germ cells (GGCs). The newly established vitrification protocol demonstrated improved post-thaw cell survival compared to the existing slow freezing method using StemCellBanker as cryoprotectant. The functional competence of thawed GGCs was further validated through migration assays in recipient embryos.
• These findings have been submitted for presentation at the 61st Meeting of the SLTB, 2025. The results of this work are also currently being prepared for joint publication in collaboration with NBGK and the Roslin Institute.
WP4 / SO3 – Analysis of alternative approaches to optimize the strategy and functionality of poultry gene banks
• WP4 aimed to rationalize the conservation strategy of the Centre for Genetic Resources, the Netherlands (CGN), providing recommendations for more effective poultry gene banking.
• The project began with a detailed analysis of CGN’s current semen-only strategy, identifying its limitations for complete breed restoration. A comprehensive comparison of alternative strategies was conducted, including:
Semen-only
PGC
Combined Semen + PGCs approaches
The analysis assessed total costs, restoration time, number of generations required, and technical complexity. Results indicated that combined Semen + female PGCs approaches offer the best balance, enabling full genome preservation and breed recovery within 2–3 generations at acceptable costs. GGC-based strategies emerged as a particularly promising and practical solution.
A stakeholder acceptance survey was conducted in 9 countries (Netherlands, Hungary, Spain, Portugal, Italy, France, Ecuador, Mexico, Colombia). The survey showed broad acceptance of PGC/GGC technologies for conservation, with ethical considerations and costs being the main concerns.
To enhance public awareness about PGC/GGC techology, multilingual infographics (English, Dutch, Spanish, Hungarian, French) were disseminated via MEDNIGHT, WUR, LinkedIn, and Facebook.
The outcomes have been compiled into a comprehensive technical report, which includes concrete recommendations for CGN — such as adopting combined strategies, promoting PGC/GGC protocols, and fostering ethical and regulatory frameworks. The report has been submitted to the Head of CGN (supervisor of WP4) and the Project Leader of CGN, and has also been uploaded to Zenodo (with restricted access pending their approval to make it public). https://zenodo.org/records/15622574(si apre in una nuova finestra)
• A comprehensive set of experiments was conducted to develop improved semen cryopreservation protocols for both commercial and native chicken breeds.
• The first stage of the project focused on understanding the effects of supercooling on rooster sperm. Initial studies showed that supercooling at −10 °C did not significantly affect sperm viability, motility, or DNA integrity. However, subsequent experiments revealed that acrosome integrity was significantly reduced after submitting the sperm to uncontrolled warming. This acrosome damage may result from structural alterations caused during supercooling that only become apparent upon warming, or from the effects of the warming process itself. This finding is highly relevant, as during artificial insemination sperm are exposed to an abrupt temperature shift—from low storage temperatures to approximately 41 °C inside the hen.
• To further improve cryopreservation outcomes, the project investigated the use of Antifreeze Proteins (AFPs) and Antifreeze Glycoproteins (AFGPs). Testing of AFPI at concentrations of 0.3 and 3 µg/mL demonstrated significant enhancements in post-thaw motility, viability, and DNA integrity. In particular, for the Yellow Hungarian breed, AFPI supplementation increased DNA integrity from 49.1% to 70.2%. However, post-thaw fertility remained low (1.5% with AFPI-DMA), highlighting an unexpected loss of fertility that occurred during the chilling phase prior to freezing. These findings underscore the importance of carefully evaluating damage introduced during pre-freezing steps, as such damage might otherwise be incorrectly attributed to the freezing process itself. Furthermore, they highlight the need to specifically address fertility losses occurring during the chilling phase, as this stage may introduce critical damage that compromises the overall success of cryopreservation.
• Parallel studies evaluated Ice Nucleating Proteins (INPs) (Snowmax®) to reduce supercooling during the freezing protocol in chicken sperm. While INPs promoted ice nucleation at higher subzero temperatures, their use negatively impacted post-thaw sperm motility, likely due to presence of particles in suspension. These particles were observed as a result of the partial solubility of Snowmax® in the poultry extender, highlighting the need to identify fully soluble ice nucleators in order to properly evaluate the potential benefits of reducing supercooling on post-thaw sperm survival.
• Recognizing breed-specific challenges, the project developed tailored cryopreservation protocols for poor-freezing breeds. For Yellow Hungarian chickens, an optimized protocol using DMA (0.6 M), a 1:3 dilution ratio, and fast one-step freezing improved post-thaw viability to 56.7%, compared to 42.8% with standard protocols. This demonstrated the necessity of breed-specific adjustments to cryopreservation methods.
• Additionally, the influence of breed and insemination temperature on fertility was assessed. Fertility rates decreased when semen was cooled to 5 °C. Preliminary results suggested that Hungarian Yellow hens maintained higher fertility when inseminated with chilled semen compared to Leghorn hens. However, further experiments using standardized artificial insemination protocols with equal sperm doses across breeds are needed to determine whether this apparent difference reflects a genuine breed-related effect on fertility maintenance, or if it was influenced by variations in the number of spermatozoa used during insemination.
• Finally, studies on rewarming of chilled and frozen-thawed sperm showed that warming frozen-thawed sperm to 37 °C caused severe loss of acrosome integrity (~81% to ~11%), while chilled semen was unaffected
• These findings have been widely disseminated through presentations at major international conferences, including CRYO 2023, CRYO 2024, AERA 2024, ESDAR 2024, SLTB 2024, XXI AIDA 2025, and MCAA 2025, and will also be presented at CRYO 2025.
In addition, a draft manuscript entitled "Tailoring a glycerol-free cryopreservation protocol with anti-freeze (glycol)proteins for commercial and native breeds of chicken" prepared in collaboration with WUR, NBGK, TU/e, and INIA-CSIC, is ready for submission to the journal Cryobiology. A second manuscript, entitled "Changes in the quality of chilled and frozen-thawed chicken sperm when heated near the temperature of the female reproductive tract," is currently being prepared in collaboration with NBGK, WUR, and INIA-CSIC, and is expected to be submitted in the coming months. In addition, a third manuscript, entitled "Effect of supercooling on integrity of rooster sperm", is under preparation and will be submitted to the journal Cryobiology along this year.
WP3 / SO2 – Unravel innovative freezing methods for female chicken embryonic germplasm
• The researcher received hands-on training in the isolation, culture, and injection of primordial germ cells (PGCs) at NBGK. Afterwards, at the Roslin Institute, work focused on the collection of embryonic gonads and the development of a reliable vitrification and thawing protocol for gonadal germ cells (GGCs). The newly established vitrification protocol demonstrated improved post-thaw cell survival compared to the existing slow freezing method using StemCellBanker as cryoprotectant. The functional competence of thawed GGCs was further validated through migration assays in recipient embryos.
• These findings have been submitted for presentation at the 61st Meeting of the SLTB, 2025. The results of this work are also currently being prepared for joint publication in collaboration with NBGK and the Roslin Institute.
WP4 / SO3 – Analysis of alternative approaches to optimize the strategy and functionality of poultry gene banks
• WP4 aimed to rationalize the conservation strategy of the Centre for Genetic Resources, the Netherlands (CGN), providing recommendations for more effective poultry gene banking.
• The project began with a detailed analysis of CGN’s current semen-only strategy, identifying its limitations for complete breed restoration. A comprehensive comparison of alternative strategies was conducted, including:
Semen-only
PGC
Combined Semen + PGCs approaches
The analysis assessed total costs, restoration time, number of generations required, and technical complexity. Results indicated that combined Semen + female PGCs approaches offer the best balance, enabling full genome preservation and breed recovery within 2–3 generations at acceptable costs. GGC-based strategies emerged as a particularly promising and practical solution.
A stakeholder acceptance survey was conducted in 9 countries (Netherlands, Hungary, Spain, Portugal, Italy, France, Ecuador, Mexico, Colombia). The survey showed broad acceptance of PGC/GGC technologies for conservation, with ethical considerations and costs being the main concerns.
To enhance public awareness about PGC/GGC techology, multilingual infographics (English, Dutch, Spanish, Hungarian, French) were disseminated via MEDNIGHT, WUR, LinkedIn, and Facebook.
The outcomes have been compiled into a comprehensive technical report, which includes concrete recommendations for CGN — such as adopting combined strategies, promoting PGC/GGC protocols, and fostering ethical and regulatory frameworks. The report has been submitted to the Head of CGN (supervisor of WP4) and the Project Leader of CGN, and has also been uploaded to Zenodo (with restricted access pending their approval to make it public). https://zenodo.org/records/15622574(si apre in una nuova finestra)
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
• Demonstrated importance of rewarming of sperm, AF efficacy; possible breed-dependent cryopreservation resilience.
• Provides a viable vitrification protocol for integrating female germplasm into poultry gene bank strategies, complementing semen-based conservation methods.
• First comprehensive analysis of combined poultry gene bank strategies.
• Provides a viable vitrification protocol for integrating female germplasm into poultry gene bank strategies, complementing semen-based conservation methods.
• First comprehensive analysis of combined poultry gene bank strategies.