5-Dimensional High-Resolution non-invasive assessment of mammalian Embryos (5DHiResE)

The global population will rise from 7 to 9 billion by 2050 with an increasing pressure on natural resources. Diets are also changing towards more meat and dairy products meaning that there is increasing global demand for food, land, energy, water and other resources, e.g. phosphate fertilisers. The livestock sector is challenged to find new ways of accommodating this need while agriculture is competing for resources as urbanisation and industrialisation put more pressure on land use. It is routine practice for pig and cattle breeding companies to transport animals to countries with underdeveloped breeding programs to enhance the quality of their local breeds. Such procedures however have a high cost financially, environmentally and logistically, and raise ethical concerns, despite evidence-based assurances of the companies involved. These factors have resulted in the in vitro production (IVP) of pig and cattle embryos being of increased interest to producers, given that great financial and environmental benefits could be made. Pigs and cattle account for ~60% of meat and ~85% of milk consumption and speed and efficiency at which genetic improvement can be introduced can be much improved through the use of these embryos. It also allows us to facilitate the national and international transport of genetically superior stock in a high animal-welfare, low-cost and bio-secure manner. This is an issue of great global significance, particularly to the industrial partners involved in this application. Significant improvements are however essential to realise the potential of pig and cattle embryos for this use. While cattle (and to some extent pig) IVP continues along a parallel path to human IVF, success rates are relatively low compared to human. Novel and innovative approaches developed with animal embryos in mind from the outset are thus urgently needed and these may, in turn, ultimately inform future developments in human IVF. Most modern human IVF units now own devices that are both incubator and camera for monitoring embryo development and some studies show that their use improves birth rates. While we have made significant strides in this area for pigs and cattle, the imaging is limited, in part because pig and cattle embryos are harder to see because of their high fat content. To improve this, we have recently made use of a new technique called Optical Coherence Tomography (OCT). This provides a 3D image of the embryo and we can extend the analysis into 4 and 5 dimensions to look at how "active" the embryos are (this is called tiny movement analysis - TMA) and how "elastic" the embryos are (using OCT for elastography measurements). In essence, we will ask the question whether embryos that are "better looking"(in 3D) more "lively" (TMA) and more "elastic" are more likely to survive. This is a cross-disciplinary collaboration between Biosciences and Physical Sciences and complements the work we have ongoing to improve pig and cattle IVP generally. The proposal builds on the widespread acceptance of time lapse imaging systems in IVF clinics but augments it from 2 to 5 dimensional analysis. It builds on the world leading expertise of two very different disciplines at Kent. Recent research has shown that 2D monitoring can significantly improve human IVF. We intend to demonstrate whether 5D monitoring can improve the equivalent process in pigs and cattle.

World population will rise from 7-9 billion by 2050 and diets are changing towards more meat and dairy products, challenging the livestock sector to find new ways of accommodating this need. It is routine for animal breeding companies to ship breeding stock to countries with underdeveloped breeding programs to enhance local breeds, however this involves financial, environmental and logistical costs, and ethical concerns. These resulted in increased interest in in-vitro production (IVP) of cattle and pig embryos to mitigate these concerns, increase selection intensity and promote biosecure stock movement. Significant improvements in IVP are however essential to realise its potential given that success rates are relatively low compared to human IVF. Most IVF units possess devices that are both incubator and time lapse camera and morphokinetic criteria are associated with IVF success, however the nature of the imaging is limited. Optical Coherence Tomography (OCT) is an 'optical biopsy' method for obtaining sub-surface cross-sectional 3D live images of translucent or opaque materials at high resolution. A 4th dimension is added by tiny movement assessment (TMA), giving indications on minute changes over time and a 5th dimension by measurement of elasticity properties using optical coherence elastography (OCE), thereby identifying embryos with the highest implantation potential. This involves altering the shape of the embryo and measuring its subsequent 3D deformation. OCT has a proven track record in evaluating inside tissue volume and is widely used medically as it requires no prior sample preparation, fluorescence nor radiation. To date however, there is no systematic study of the added value of OCT in monitoring IVP/IVF embryos. The purpose of this project will thus be to establish whether OCT, with adapted signal processing protocols can evaluate and monitor early mammalian development, and subsequently improve IVP/IVF success rates.

The successful development of in vitro produced embryo transfer (IVP-ET) necessitates fundamental research into embryo screening and the development of enhanced, ideally non-invasive and label-free, 3D screening/imaging modalities that aid selection of the 'best' embryos and enhance IVP success rates. Herein is presented a screening tool, 5DHiResE, which focuses OCT technologies on the screening of embryos for IVP. These developments will augment the adoption and use of such screening methodologies and use of embryo-based reproductive technologies in pig and cattle breeding. Our industry partners are global leaders in IVP of livestock embryos and have been instrumental in driving technological innovations within the sector. Their technical expertise and access to animal resources represent an essential contribution to this project that facilitates the required scale of activity and industry-relevant endpoints for successful delivery of the scientific objectives. Uniquely, they provide technical expertise in live-animal egg recovery (cattle) and large-scale ETs (cattle and pigs) necessary to measure the ultimate endpoint of our scientific hypotheses - that the transfer of OCT-screened embryos (ie. using 5DHiResE) improves outcome and leads to live-birth rates comparable to natural conception in sexually mature animals.

Breeding companies: Our industry partners are among the largest global livestock breeding companies operating within the UK with a 28% (pigs) and 35% (cattle) share of the domestic market. They will evaluate the quality of the imaging outputs with a view to downstream implementation of 5DHiResE modality, ultimately enhancing industrial processes by: (i) augmenting the selection of better embryos for IVP-ET; and (ii) facilitating international transport of screened superior stock in a high animal-welfare, low-cost and bio-secure manner. The assurance of enhanced live-birth rates following embryo transfer is central to the success of this project and the companies' business models. IVP-ET will generate a new global market for livestock embryos and place the UK livestock breeding industry at the forefront of technical innovation in this field. In the pig sector we anticipate that the UK market share of our partners will increase from 28 to 40% over 5 years post-project. For cattle the current global semen market exceeds US$1.5 bn annually, with nearly 200 m doses/year sold at an average price of US$10. The potential for IVP-ET to infiltrate this market is significant - if 1% of the semen market was replaced by IVP-ET that would equate to 2 m embryo transfers per annum (5-fold greater than the current 400 K global IVP-ET estimate).

Livestock breeders and producers: We expect that use of screened embryos from OCT will increase the rate of slaughter stock value inflation. This is projected to be >10% increase in the net value of genetic improvement per slaughter pig (from £1.50 to £1.65/pig/annum).This benefit will arise because of the advances in embryo selection. We estimate that for 1.9 million UK dairy cows with a replacement rate of 30%, the Profitable Life Index (PLI) could be increased by £20/heifer/annum or £11.3 million.

Human assisted reproduction (ART): Current systems exist for time-lapse monitoring of embryos [e.g. Embryoscope(R), Vitrolife and Miri(R)] and, despite being widely practised, this has yet to reach its potential. OCT could in future be of great benefit to couples undergoing IVF. Thus, there is considerable interest in developing novel methods for non-invasive embryo screening for use in human ART. New information from the current project will inform on improvements to this.