Periodic Reporting for period 1 - CryoStore (Innovation in germplasm cryopreservation for improved animal breeding and the conservation of Europe's livestock biodiversity)
Periodo di rendicontazione: 2023-09-01 al 2025-08-31
There is growing international concern about the continued loss of animal biodiversity. While most attention has focused on iconic species, the conservation of Europe’s numerous native breeds of farm animals and the need to maintain genetic diversity within breeds, has largely been overlooked. Animal production systems are constantly changing, and a decreasing number of breeds are now producing an increasing part of Europe’s total animal production. This has contributed to the disappearance of many native breeds, and within populations the genetic base is becoming increasingly narrow. The genetic resource base of Europe’s numerous native breeds is of great importance to the future prosperity of the European animal production industry as genetic biodiversity allows the expression of advantageous traits influencing adaptability to harsh environments, productivity, or disease resistance. It is likely that selecting naturally occurring traits will help overcome anticipated difficulties in livestock production due to climate change, including global warming and more extreme weather patterns. Of critical importance for the conservation of farm animal biodiversity will be the cryopreservation of reproductive cells and tissues (germplasm), both for conservation purposes (gene banks) and for use in modern farm animal breeding (assisted reproductive technologies), underpinning sustainable and efficient animal production systems (food security). The successful cryopreservation of farm animal germplasm conveys both major societal and economic benefits to the European Community. However, the cryopreservation and use of germplasm is still constrained by several technical problems, including reduced viability and fertilization rates of reproductive cells due to cryodamage following freeze-thaw procedures.
CryoStore addresses the technological limitations in the cryopreservation of farm animal germplasm through the following three inter-linked research tasks:
Task 1: Develop advanced techniques for the visualization and characterization of freeze-thaw damage at the cellular level in reproductive cells and tissues (WP2)
Task 2: Develop new procedures for the improved cryopreservation of problematic farm animal germplasm (WP3)
Task 3: Investigate potential epigenetic risks of cryopreservation procedures (WP4).
The Work Packages (WPs) and Doctoral Candidates (DCs) are currently making great progress towards fulfilling their respective research tasks. As cryodamage results in reduced viability and fertilizing capacity of all reproductive cells/tissues, a better understanding of subcellular alterations caused following cryopreservation is required. Further understanding the diffusion kinetics of cryoprotective agents (CPAs) and vitrifying agents (VAs) into reproductive cells is key to ensuring safe and efficient long-term storage. DC 3 has successfully developed a prototype temperature-controlled dynamic perfusion chamber compatible with RAMAN microscopy for monitoring CPA and VA diffusion into reproductive cells. DC 4 has developed a prototype for the motorized directional freezing cryostage to be used during cell imaging by multiphoton microscopy. The prototypes will be used to increase our understanding of the kinetics of cryodamage and help in designing less invasive cryopreservation procedures.
The ability to successfully cryopreserve animal germplasm not only varies between the different cell or tissue types, but also between different animal groups. Innovative advances in cryopreservation procedures are needed to overcome difficulties in particular problematic farm animal species and/or types of germplasm. DCs 7 and 10 are currently working to improve cryopreservation of porcine semen, an animal group with persistent cryopreservation limitations and difficulties, with the aim to improve sperm viability and fertilization rates. This includes investigating the use of ice binding proteins or antioxidants to modulate the effect of supercooling, ice formation and ice recrystallization. DC 1 has studied the effect of L-carnitine on teleost semen before and after cryopreservation and conducted initial studies on freeze-thaw damage in teleost ovarian tissue. DC 2 has been working to improve methods for cryopreservation and storage of bovine oocytes, as well as to characterize the molecular and functional changes of cryopreserved oocytes. The cryopreservation of primordial germ cells (PGCs) represents a reliable alternative to the impossibility of embryo and egg cryopreservation in avian species. DC 8 aims to use this technique to restore the complete genetic heritage of a poultry breed, and has been successful in determining a suitable host, injecting PGCs and developing chimeras.
There is a risk that cryopreservation can induce epigenetic modifications with the risk of long-term (transgenerational) effects on reproductive cells and their derivatives. DC 5 has developed a protocol and obtained results on the epigenetic effects of cryopreservation of teleost spermatozoa and embryos. DCs 2, 6 and 9 will investigate the epigenetic changes following cryopreservation in cattle, fish and sheep once all material has been acquired.
Task 1: Develop advanced techniques for the visualization and characterization of freeze-thaw damage at the cellular level in reproductive cells and tissues (WP2)
Task 2: Develop new procedures for the improved cryopreservation of problematic farm animal germplasm (WP3)
Task 3: Investigate potential epigenetic risks of cryopreservation procedures (WP4).
The Work Packages (WPs) and Doctoral Candidates (DCs) are currently making great progress towards fulfilling their respective research tasks. As cryodamage results in reduced viability and fertilizing capacity of all reproductive cells/tissues, a better understanding of subcellular alterations caused following cryopreservation is required. Further understanding the diffusion kinetics of cryoprotective agents (CPAs) and vitrifying agents (VAs) into reproductive cells is key to ensuring safe and efficient long-term storage. DC 3 has successfully developed a prototype temperature-controlled dynamic perfusion chamber compatible with RAMAN microscopy for monitoring CPA and VA diffusion into reproductive cells. DC 4 has developed a prototype for the motorized directional freezing cryostage to be used during cell imaging by multiphoton microscopy. The prototypes will be used to increase our understanding of the kinetics of cryodamage and help in designing less invasive cryopreservation procedures.
The ability to successfully cryopreserve animal germplasm not only varies between the different cell or tissue types, but also between different animal groups. Innovative advances in cryopreservation procedures are needed to overcome difficulties in particular problematic farm animal species and/or types of germplasm. DCs 7 and 10 are currently working to improve cryopreservation of porcine semen, an animal group with persistent cryopreservation limitations and difficulties, with the aim to improve sperm viability and fertilization rates. This includes investigating the use of ice binding proteins or antioxidants to modulate the effect of supercooling, ice formation and ice recrystallization. DC 1 has studied the effect of L-carnitine on teleost semen before and after cryopreservation and conducted initial studies on freeze-thaw damage in teleost ovarian tissue. DC 2 has been working to improve methods for cryopreservation and storage of bovine oocytes, as well as to characterize the molecular and functional changes of cryopreserved oocytes. The cryopreservation of primordial germ cells (PGCs) represents a reliable alternative to the impossibility of embryo and egg cryopreservation in avian species. DC 8 aims to use this technique to restore the complete genetic heritage of a poultry breed, and has been successful in determining a suitable host, injecting PGCs and developing chimeras.
There is a risk that cryopreservation can induce epigenetic modifications with the risk of long-term (transgenerational) effects on reproductive cells and their derivatives. DC 5 has developed a protocol and obtained results on the epigenetic effects of cryopreservation of teleost spermatozoa and embryos. DCs 2, 6 and 9 will investigate the epigenetic changes following cryopreservation in cattle, fish and sheep once all material has been acquired.
CryoStore is developing advanced techniques for the visualization and characterization of freeze-thaw damage, including RAMAN spectroscopy and multiphoton microscopy. Deep learning using artificial neural networks will be utilized to achieve reproducible analysis of images for better understanding of the effects of cryopreservation on specific reproductive cells and tissues. A further innovative aspect of CryoStore will be the application of multiple cryo-techniques in the development of improved cryopreservation protocols, which will be tested by breeding companies in the consortium. Lastly, three different cell types from three different species will be used to investigate DNA methylation damage and whether chromatin organization plays a role in epigenetic stability after cryopreservation.