Introduction and Background
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. Of critical importance for the conservation of farm animal biodiversity will be the cryopreservation of reproductive tissues (germplasm), both for conservation purposes (gene banks) and for use in modern farm animal breeding programmes, underpinning sustainable and efficient animal production systems (food security). However, the cryopreservation and use of germplasm is still constrained by a number of technical problems. To tackle the loss of animal biodiversity there is a strong need for a new generation of collaboratively-minded internationally trained researchers who are capable of integrating the multidisciplinary fields of research encompassing cryobiology, reproduction/animal breeding and animal conservation, combining hands-on research and intersectoral placements.
Germplasm and conservation of farm animal biodiversity
As of 2018, 7745 out of 8803 reported livestock breeds are classed as local (occurring in one country only). Of these local breeds, 594 have become extinct, while 26% are classed as being at risk of extinction[1]. According to a recent Eurobarometer survey, the overwhelming majority of Europeans are now concerned about the loss of biodiversity and support stronger EU action to protect nature[2]. Critically, Europe owns an animal inheritance with huge genetic diversity, including hundreds of threatened or rare breeds of domestic farm animals, including fish. Animal production systems are constantly changing (intensification and industrialization), 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 and diverse 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 for naturally occurring traits will help overcome anticipated difficulties in livestock production due to climate change[3],[4], including global warming and more extreme weather patterns (droughts and floods). Examples of genetic advantages present within endangered local breeds include the slick-hair phenotype for heat tolerance in certain tropical bovine breeds[5], and natural resistance to gastrointestinal parasitism in Gulf Coast Native sheep[6]. As such, there is an urgent need to protect and conserve animal genetic resources, not just as an “insurance” against acute threats such as climate change and disease epidemics, or as a source of genes for contemporary breeding[7] (both in situ and ex situ conservation), but also as part of our national heritage[8]. The keystone of these conservation initiatives will be the cryopreservation of reproductive cells and/or tissues, collectively known as germplasm.
¨Germplasm¨ refers collectively to cells that, singly or in combination (sperm, oocytes, embryos, gonadal tissue, germ cells), lead to the development of offspring. The collection and cryopreservation of animal germplasm confers two critically important inter-connected functions, 1) the conservation of animal reproductive material in gene banks, and 2) its use in assisted reproductive technologies (ARTs) in farm animal breeding and conservation programs.
CryoStore builds on the results of the EU Horizon 2020 project IMAGE (Innovative Management of Animal Genetic Resources) that ended in 2019. The aim of IMAGE was to enhance the use of genetic collections and to upgrade animal gene bank management. IMAGE research focused on the latest developments in DNA technologies and reproductive physiology for collecting, storing and distributing biological resources, including germplasm, with the ultimate goal of demonstrating the benefits of gene banks to the development of more sustainable livestock farming systems. CryoStore will build on the IMAGE project focusing on those areas identified as being of high priority, including new developments for improved germplasm cryopreservation, and most critically the urgent need to train a new generation of young researchers needed to oversee future policies on the conservation of animal genetic resources.
Germplasm and animal production
The animal production sector, excluding aquaculture, contributes substantially to the European economy (€168 billion annually, 45% of the total agricultural activity), to trade balance and creates employment for almost 30 million people (ESCO INRA, 2016). Further, the EU aquaculture industry is worth almost €5 billion a year, while Norway alone exported €7.9 billion of farmed Atlantic salmon in 2021 (Norwegian Seafood Council). These industries also play an important role in European society, underpinning both food security and helping sustain human communities in Europe´s more marginal regions. Farm animal breeding and reproduction is a global, highly competitive and knowledge-intensive sector. Given the central role of reproduction in production efficiency and genetic selection, improvements in reproductive technologies are crucial to meeting the future needs of food security, as well as challenges to livestock production created by climate change. Europe has gained world leadership in the breeding of several commercially important species. However, reproductive technologies used in livestock production are evolving rapidly, and if Europe is to retain its competitive strength in the animal breeding sector, innovative research is needed into those reproductive technologies used to aid genetic selection and improve reproductive efficiency in the European livestock and aquaculture industries. In this respect, one crucial area of development within these industries is assisted reproductive technologies (ARTs), including artificial insemination, in vitro fertilization (IVF) and embryo transfer. Central to most ARTs is the development of effective procedures for the cryopreservation, storage and use of animal germplasm. Currently, artificial insemination is by far the most widely used method for breeding farm animals within the EU, accounting for 75% of all dairy cattle and 85% of all pigs. In most artificial insemination procedures, cryopreserved semen (-196°C) is preferred because it confers a number of important production benefits, including easier use of semen from genetically superior and valuable males, allowing assessment of semen sanitary status before use, and facilitating semen transport and trade which is an essential commercial component of many animal breeding industries. The use of cryopreserved sperm also has animal welfare benefits, because shipping sperm from one location to another is less stressful than transporting animals. The importance of embryo transfer techniques, increasingly using cryopreserved embryos, is growing, allowing farmers to obtain multiple progenies from genetically superior females.
In summary, the cryopreservation of animal germplasm is of critical importance for both the conservation of farm animal biodiversity (genetic resources) and the advancement of modern animal breeding practices. The application of frozen germplasm in ARTs has led to more efficient and profitable animal production, helping European breeders to maintain their competitive lead over global competitors. As such, the successful cryopreservation of farm animal germplasm conveys both major societal and economic benefits to the European Community.
Unfortunately, current procedures for the cryopreservation of animal germplasm, both for use in assisted reproductive technologies and for the conservation of animal genetic resources (biodiversity) are constrained by a number of technological limitations, which affects both efficiency and profitability. These notably include:
1. Cryopreservation procedures result in decreased viability and fertilizing capacity (cryodamage).
As cryodamage, induced by freeze-thaw procedures, results in reduced viability and fertilizing capacity of all reproductive cells/tissues[9],[10], 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 the safe and efficient long-term storage of these cells.
2. Major technological difficulties still exist in the successful cryopreservation of animal germplasm.
The ability to successfully cryopreserve animal germplasm not only varies between the different cell or tissue types, but also between different animal groups9. For more effective animal breeding procedures species-specific freezing protocols need to be developed for the different germplasm types. Due to their relatively large size, mosaic membrane structure and organelle content, both oocytes and embryos suffer considerable morphological and functional damage during cryopreservation, although the extent of cryodamage is highly variable between species, developmental stage and origin[11].
3. Epigenetic risks.
Epigenetic changes due to cryoprotective agents and freeze-thaw procedures have yet to be fully investigated. While freezing protocols are generally considered to be safe, emerging evidence now suggests that cryopreservation procedures can induce epigenetic modifications with the risk of long-term (transgenerational) effects on reproductive cells and their derivatives[12]. A better understanding of the underlying risk of inducing epigenetic modifications with different cryopreservation techniques would be beneficial for improving the safety and effectiveness of freezing protocols, leading to increased offspring quality via a reduction in putative generation effects.
Innovative technological advances in these key areas, resulting in more effective, efficient (profitable) and secure procedures for the cryopreservation of farm animal germplasm will help underpin the two important pillars of germplasm use, that is a) modern farm animal breeding practices involving ARTs, and b) the conservation of Europe’s unique farm animal biodiversity (genetic resource banking).
[1] FAO 2019. State of the World’s Biodiversity for Food and Agriculture. J. Belanger & D. Pilling (Eds). Rome. 572 pp.
[2] Attitudes of Europeans towards Biodiversity. Special Eurobarometer 481. December 2018.
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