Innovations in Reproductive and Cryopreservation Technologies in Pigs and Cattle

Innovations in Reproductive and Cryopreservation Technologies in Pigs and Cattle


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Landbouw, Water, Voedsel>Sleuteltechnologieën LWV>Biotechnologie en Veredeling






Innovative Reproductive Technologies (RT), including cryopreservation of reproductive cells, are essential for ex situ conservation of genetic diversity. For breeding companies, cryopreservation of embryos and related RT are needed for accelerated genetic progress in breeding towards climate neutral and circular livestock production and for biosecure dissemination of genetic improvement. This PPP will make a major contribution to increased efficiency and safety of RT in pigs and cattle.

Doel van het project

This project will address innovative Reproductive Technologies (RT) and will deliver efficacious, efficient, and practical methods for cryopreservation of embryos and ovarian tissue, and methods for better quality assessment of embryos. These methods will be applied by breeding industry partners and gene banks, to enable increased genetic progress in important traits, safe dissemination of genetic improvement, and long-term conservation of genetic diversity. This project thus contributes to Priority 44, Key Technologies ST2 Biotechnology and Breeding, and will directly or indirectly contribute to the Missions A-D.
The PPP is a unique collaboration between two leading Dutch animal breeding companies, Topigs-Norsvin (TN, pigs) and CRV (cattle), and Wageningen Livestock Research (WLR). Both breeding companies are at the forefront of novel developments in breeding. WLR is an internationally renowned centre of expertise in animal breeding, reproduction, and cryobiology, contributing to innovations in industry and to the national (government funded) cryopreservation program for farm animal species and breeds (CGN – Wageningen University & Research).

Relatie met missie (Motivatie)

Wider and more efficient use of RT in the pig and cattle breeding sector
Developing innovative vitrification protocols for cryopreservation of embryos is essential in both pig and cattle breeding to enable the wider use of embryo’s in breeding programs. Existing cryopreservation methods are suboptimal in terms of fertility results as well as in practical aspects, as effective direct transfer vitrification methods are lacking. Direct transfer (DT) methods allow a straw with an embryo to be thawed and used at the farm without the need of on-site equipment or expertise. Together with the development of direct transfer protocols for biopsied bovine embryos and improvements in direct transfer technology for porcine embryos, it is anticipated that this research will significantly improve the liveborn rates obtained with cryopreserved embryos. Advanced methods will be immediately implemented in the field.
Conservation of genetic diversity
Progress in RT is also important for the purpose of long-term conservation of genetic material in gene banks or biobanks. Further development of improved protocols for cryopreservation (vitrification) offers exceptional advantages for conserving genetic diversity of rare breeds, more widely used breeds, and commercial breeding lines, or biobanking of different species. The innovative knowledge and methodologies jointly developed by partners WLR, CRV and TN will contribute to the long-term Statutory Research Tasks (WOT) of the Centre for Genetic Resources, the Netherlands (CGN) to conserve genetic diversity of livestock species and breeds.
Better animal welfare and biosecurity
Global distribution and use of embryos, instead of movement of live breeding animals, strongly reduces biohazard risks and does not have the negative animal welfare and health effects associated with shipment of live animals and their local adaptation following transport.
Scientific advances
The project is expected to make a major contribution to better understanding and knowledge regarding RT, in particular the quality of gametes and embryos produced in vitro or in vivo and cryopreservation of embryos and ovarian tissue. Through this PPP the international profile of Wageningen University & Research (Wageningen Livestock Research) as an international centre of expertise and partner in further development of reproductive and cryoconservation technologies, will be strengthened.
Innovation and competitiveness of the animal breeding sector
The project addresses innovative RT which will have a positive impact on innovation and competitiveness in the animal breeding and livestock sector. The PPP will strengthen the leading role of the Dutch pig and cattle breeding sector at national and global level. Through the ability of improving cryopreservation outcomes and development of direct transfer protocols, the business competitiveness of CRV and Topigs Norsvin will be further enhanced by the ability to practically increase the number of offspring from genetically unique elite animals, and to distribute germplasm over time and distance.
Collaboration between cattle and pig breeding companies
Technology specifically for cryopreservation of porcine embryos is some years behind the current state of the art in the bovine industry. This PPP is an excellent opportunity for joint development of innovative protocols and exchange of knowledge and experiences between sectors.

Geplande acties

Aim of the project
This project is aimed at development of efficacious, efficient, and practical methods for cryopreservation of embryos and ovarian tissue, and of methods for better non-invasive quality assessment of embryos. The developed methods will be applied by breeding industry partners and gene banks to improve in vitro production and use of embryos, as well as cryopreservation of germplasm for safe, cost-efficient, and animal friendly dissemination of genetic improvement and conservation of genetic diversity.

The corresponding tasks
1. Vitrification methods porcine and bovine embryos: Mathematical simulations.
We will a our mathematical model to simulate vitrification of porcine and bovine embryos and predict the consequences of chosen cryoprotectants, conditions, and treatments. The mathematical modelling can indicate which empirical experiments seem most meaningful so that the number of (invasive, laborious, time consuming, expensive) experiments can be reduced.
- For swine, we will address both non-direct transfer and direct transfer vitrification methods for in vivo and in vitro derived swine embryos
- For bovine embryos we will focus on direct transfer vitrification methods for bovine in vitro produced biopsied and non-biopsied embryos.
2. Selected vitrification protocols for porcine and bovine embryos developed in Task 1 will subsequently be tested empirically. We will use existing methods for in vitro assessment of embryo quality after vitrification, complemented with non-invasive EFA and TLI (see Task 5), to evaluate conditions of and protocols for embryo vitrification.
3. The direct transfer vitrification protocol for bovine embryos that appears successful in the lab (in vitro assessment), will then be tested in a small field trial in which 50-100 direct transfer vitrified biopsied embryos are transferred to cows and pregnancy rates will be recorded.
4. Investigation of vitrification of porcine ovary cortex. The entry of cryoprotectants into, and efflux of water from, porcine ovarian cortex fragments will be measured, using isotope labelled cryoprotectants and water. This knowledge is critically needed for improvement of vitrification protocols for tissue fragments and also has relevance for improving vitrification of oocytes and embryos.
5. Develop and validate methods for non-invasive quality assessment of embryos
- Measurement of aerobic (respiration) and anaerobic (glycolysis) energy metabolism of embryos by Extracellular Flux Analysis (EFA).
- Time-lapse imaging (TLI) to study the physical ‘behaviour’ (movements) of embryos, (rate of) embryo development, and (rate of) changes of organisation of cytoplasm components.
Above variables will be studied in relation to culture conditions, stress, cryopreservation treatment, parental donors, and, most importantly, to the success of embryo development.
6. Publication, communication, dissemination and implementation
- We aim to publish scientific results in scientific journals and congresses. We will use other media, such as trade magazines, newsletters, social media, workshops and other means for direct interaction, to communicate with stakeholders and the wider audience. Stakeholders include organisations active in conservation of genetic diversity of farm animals and wild (zoo) animals. Implementation of the developed methods in the field is an integral part of this project.
- In the last year (M42), we will organise a special workshop to explore and discuss the emerging field of in vitro gamete production from induced pluripotent stem cells. The workshop will discuss recent scientific developments in human and animal model studies, the potential for future application in farm animals, and steps that may be taken to develop this field further.

D1. Protocol for direct transfer vitrification of bovine embryos
D2. Improved protocols for vitrification of porcine embryos
D3. Validation of possibilities for non-invasive quality assessment of embryos.
D4. Kinetic parameters of CPA and water fluxes in ovarian cortex fragments
D5. Workshop on future possibilities of RT, including ‘in vitro breeding’

Milestones: (month in brackets
M1. (M3) Experimental set up for Extracellular Flux Analysis (EFA) and time-lapse imaging (TLI).
M2. (M12) First recommended protocol(s) for vitrification of embryos from mathematical modelling.

Go/no-go. There will be a go for Task 3 (field trial) if in vitro assessment (Task 2) shows an advantage of experimental vitrification protocol(s) over current protocols. If not, new experimental protocols will be designed and will be tested by simulations and by in vitro assessment of embryo vitrification.

- Monthly: project team – researcher from WLR, TN and CRV
- Quarter: steering committee meeting

Task Year 1 Year 2 Year 3 Year 4
M1 M4 M7 M10 M13 M16 M19 M22 M25 M8 M31 M34 M37 M40 M43 M46
1. Vitrif. Math. modelling
2. Vitrif. In vitro testing
3. Vitrif. Field trial
4. Vitrif. Ovary cortex
5. Quality assessment
6. Comm. Dissem. Implem.

Naam projectleider

Henri Woelders