In vitro embryo production in animal breeding: Enhancing oocyte quality from peri-pubertal donors to promote biosecure and sustainable food production

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences


The world population will increase from 7 to 9 billion in the next 30 years. Consumption of meat (over half of which comes from pigs and cattle) and milk (nearly all from cattle) will also rise by 20% per person. This will place an intolerable demand on food producers globally who are charged with generating more meat and milk from fewer animals, in less time. Livestock production also contributes 18% of global warming. Using genetic selection to improve livestock productivity offers the most sustainable means to match increased global demand for animal products and to mitigate harmful effects to the environment.

The UK leads the world in farm-animal genetics, but the pace at which genetic change must occur in future represents a challenge. The use of advanced reproductive technologies such as in vitro fertilised (IVF) embryos offers several benefits in this regard. Many IVF embryos can be generated from a small number of elite females allowing us to be more selective. Genetic traits can also be screened in IVF embryos just a few days after fertilisation. This speeds up the time to selection, prevents the birth of unwanted animals and has the added advantage of facilitating bio-banking of genetically precious stock, together with low-cost, high-welfare and biosecure international transportation. IVF embryos from young (peri-pubertal) donors can lead to a greater increase in the rate of genetic improvement by reducing the time to selection even further.

The ultimate aim of this project, therefore, is to make pig and cattle IVF work better from mothers that are as young as possible. Pig and cattle embryos are also good models for studying human IVF. By developing a better understanding of the biological processes that occur during IVF, particularly for the peri-pubertal donor, we aim to improve significantly the overall efficiency of this procedure, thereby making it the method of choice for genetic selection in future. We believe that we can improve the process in both species by focussing on reducing the level of gross genetic abnormalities (i.e. extra or missing chromosomes) in the embryo which is linked to energy metabolism by numerous small structures within the cell called mitochondria. Our central question therefore is can we improve the quality of the embryo produced by improving the oocyte (egg) maturation process? In cows, we will also investigate milder therapeutic regimes used to stimulate ovaries (similar to human IVF) in the hope that this will also improve egg and embryo quality, together with dietary regimes designed to do the same. This should reduce levels of gross genomic imbalance, metabolic problems and stress in our embryos, thereby increasing their developmental potential. Our second question is whether this actually leads to "on-farm" improvements in live-birth rates following embryo transfer. By combining our new regimes of IVF with screening and removal of embryos containing mitochondrial and chromosomal abnormalities we aim to establish this.

The academic and industry partners in this LINK proposal have enjoyed a successful track-record of collaboration in developing pig and cattle IVF in recent years. The current application will witness a step-change in improvements to this technology as we address a fundamental aspect of developmental biology associated with metabolism and chromosome behaviour during IVF. Our industry partners will work alongside us to collect eggs, undertake dietary studies and large-scale on farm embryo transfers to demonstrate improvements in live-birth rates. The unique "selling point" of this application, therefore, is the novel combination of approaches, academics and industrialists that will ensure success of the project all the way from research lab to farm.

Technical Summary

With increasing global population and per capita meat/milk consumption, in vitro produced (IVP), genomically evaluated (SNP chip) cattle and pig embryos can accelerate worldwide livestock genetic improvement and facilitate dissemination of genetics with minimal environmental, biosecurity and welfare problems. Further significant benefits will arise when oocytes are donated from younger, peri-pubertal donors, however, developmental competency of such oocytes is poor due to deficiencies in mitochondrial number/metabolism and related increased chromosomal abnormality. Through a fundamental study of biochemical and chromosomal events that occur during oocyte maturation this LINK proposal will address the hypothesis that peri-pubertal IVP can be improved with appropriate modifications to (i) in vitro maturation (IVM), (ii) ovarian stimulation, and (iii) diet that enhance mitochondrial number/function and minimise chromosome abnormalities. We further hypothesise that such modifications, in combination with judicious selection of embryos with normal chromosomal and mitochondrial number (preimplantation genetic diagnosis, PGD), will lead to improvements in live-birth rates in both species following embryo transfer. To test these hypotheses we will implement a series of experiments that will lead to a step-change in the nature of chemically-defined IVM media/protocols that promote mitochondrial biogenesis/metabolism during regulated meiotic progression. Improvements will be defined by measures of energy metabolism, oxidative stress, mitochondrial number, aneuploidy (by "Karyomapping") and embryo morphokinetics. Cattle studies will also integrate mild ('coasted') ovarian stimulation regimes and novel dietary strategies that advance puberty onset and enhance yields of developmentally competent oocytes. The proposal outcomes for each species will be the best strategies to emerge from these experiments combined with PGD of transferable embryos to improve live-birth rates.

Planned Impact

Need for LINK: The successful development of in vitro produced embryo transfer (IVP-ET) using peri-pubertal livestock donors necessitates fundamental research into deficiencies in oocyte maturation. These deficiencies represent a barrier to the use of embryo-based reproductive technologies in pig and cattle breeding, thereby limiting the rate of genetic improvement that can be achieved. 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 represents 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 genomically evaluated embryos from peri-pubertal donors 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 implement emerging technical improvements, ultimately enhancing industrial processes by: (i) significantly reducing the time-interval to genetic selection; and (ii) facilitating international transport of genetically 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 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 industry-wide use of developmentally competent, genomically and cytogenetically evaluated embryos from peri-pubertal donors will increase the rate of slaughter stock value inflation. This is projected to be >50% increase in the net value of genetic improvement per slaughter pig (from £1.50 to £2.30/pig/annum). This benefit will arise because of the advances in genetic improvement, moving from male selection based on artificial insemination (AI) to female selection based on IVP-ET from peri-pubertal donors. The latter facilitates greater female 'selection intensity' (i.e. oocyte collections require fewer, more elite females for breeding) whilst reducing the age of donors when selection decisions are made (by around 12-15 months in cattle). 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): Aneuploidy is the leading cause of ART failure in humans (accounting for >50% embryo loss). Current systems for in vitro maturation (IVM) of human oocytes are suboptimal and the technique is not widely practiced. IVM can be of great benefit to women undergoing fertility preservation and is used when ovarian stimulation has been contraindicated. Thus, there is considerable interest in developing chemically-defined IVM systems and aneuploidy screening for use in human ART. New information from the current project will inform on improvements to both.


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Description Cattle embryo development is now well established and we are genetically screening
Pig blastulation rate is up to 35%

significant association between implantation rate and aneuploidy levels as per narrative below

Single Nucleotide Polymorphism (SNP) data obtained at the embryonic stage is used in cattle breeding to calculate the genetic value of an animal before a pregnancy is established, leading to improved selective breeding. However, the cost of the procedure can be high due to the number of recipients not getting pregnant after receiving a SNP typed embryo. Aneuploidies are the most common cause of embryo development arrest, so it might be beneficial to screen out aneuploid embryos by employing preimplantation genetic testing for aneuploidy (PGT-A).
Here, we applied a novel PGT-A algorithm, which employs the same SNP information used to calculate genetic merit, to obtain ploidy diagnoses. We performed a retrospective analysis in n=1129 bovine embryos of transferable quality produced by the company Boviteq (Saint-Hyacinthe, Canada).
Embryo morphology (good or excellent) affected pregnancy rates, which were 59.9% for excellent embryos and 44.9% for good embryos (general linear model, P=.035). Nevertheless, live birth rates were not improved by morphology-based selection (40.1% vs 39.1%, P=.88). In contrast, pregnancy rates were significantly higher in euploid rather than aneuploid embryos (60,9% vs 16,1%, P=1.3E-10) as were live birth rates (50.9% vs 12.5%, P=4.7E-8). The proportion of aneuploid embryos in this sample was 13.8% (156/1129); 25.3% (41/162) of chromosomal abnormalities had paternal origin and 74.7% (121/162) maternal origin. The incidence of monosomy appeared higher than that of trisomy, however the difference was not significant (58% vs 42%, chi-square, P=.1241).
Our results indicate that embryos classified as euploid by PGT-A have better developmental competence and their selection for transfer could reduce for breeders the economic losses associated with pregnancy loss. At present, a bigger database is being analysed to elucidate the effects of specific chromosomal abnormalities.

Objective 1 - IVM
Trophectoderm biopsies accurately represent the ploidy status of the overall embryo
Incidence of aneuploidy declines in developmentally more advanced embryos
High degree of variability in the incidence of aneuploidy between donors
Metabolic regulation of mitochondrial function linked to lipid metabolism during IVM and oocyte quality
Objective 2 - Ovarian Stimulation
In vitro oocyte maturation rather than ovarian stimulation contributes to aneuploidy
Considerable between donor variability in ovarian response to stimulation - particularly in peri-pubertal donors
Progesterone support during ovarian stimulation key to enhancing oocyte quality and embryo development
Objective 3 - Nutrition/metabolism
Removal of complex proteins during IVM emphasises the importance of donor nutrition in determining oocyte quality
Donor lipid metabolism and/or lipid metabolism during IVM key to future success with defined IVM media
Objective 4 - Screening for pregnancy outcomes
Aneuploidy screening by SNP typing and Karyomapping established to cattle embryos
SNP-array analyses facilitate simultaneous genomic evaluation and aneuploidy screening
Maternal meiotic errors are the prevalent cause of aneuploidy
PGT-A significantly improves ET outcomes by improving pregnancies per embryo transferred

Objective 1
Developed a serum-free IVM system to mature porcine oocytes from both peri and pre pubertal gilts
Working with pre-pubertal animals resulted in higher experimental variability
Donor selection might be a tool to overcome this
Mitochondrial activity enhancers did not provide a significant advantage
While additional growth factors appear unnecessary, oxidative stress control might bring further improvements

Objective 4
Aneuploidy screening by SNP typing and Karyomapping now possible in pig embryos
Aneuploidy incidence appears very high in IVP generated embryos
OCT overcomes traditional barriers to animal embryo imaging and allows for multidimensional reconstruction of the samples in study
Exploitation Route We hope these will eventually be commercialised and we are in advanced discussions to this effect
Sectors Agriculture, Food and Drink

Description Some public talks, some live births. In cattle, IVF and PGT (using single nucleotide polymorphism information) is usually performed in high volumes from genetically elite parents. In this context, transfer of embryos detected to have a lower genetic merit (e.g. low milk yield, poor disease resistance) reduces the financial return achieved by the breeding programme. Underlying research in the Griffin lab, initiated in 2015, sought to apply Karyomapping for PGT-A for the first time to improve cattle IVF success rates. Application of this technology published in 2019 led to the first live born calves [4] and, to date, a highly significant (6%) increase in pregnancy rates (unpublished results). Karyomapping is now being applied, experimentally, in the pig IVF lab set up by Griffin in 2012 (unpublished results). Commercial Impact of Karyomapping on IVF service providers in clinical and agricultural settings Karyomapping was first patented by BlueGnome in 2010 (Alan Handyside named as inventor) and BlueGnome acquired by Illumina in 2013. In 2019, Vitrolife acquired the rights to market and distribute Karyomapping services as one of their portfolio of products [a] to diagnostic laboratories such as Cooper [b] and Igenomix [c]. Karyomapping has had significant positive impact on a number of companies offering IVF services. In a medical context this includes Cooper Genomics who calculate that the >10,000 clinical cases they have processed to date account for 65-75% of all Karyomapping tests thus far performed world-wide [b]. A conservative estimate therefore is that the total number of Karyomapping cases performed has exceeded 15,000 at the time of writing. Igenomix, have, more recently, adopted Karyomapping as a Flagship Product in their UK and Italian operations and have applied the technology to approximately 2000 embryos (300+ clinical cases) to date [c]. The companies confirm that this has increased profitability [a,b,c], enhanced he company profile, become an important marketing tool [a,b,c], and has contributed to job creation [a]. In preliminary studies, Karyomapping has been applied to 2000 cattle embryos, demonstrating conclusively a tenfold improvement in pregnancy rates in normal vs. aneuploid embryos. Commercial impact [d,e] includes company investment in the university, increased profitability, establishment of a new company, Activf-ET [d] and increased company profile.
First Year Of Impact 2019
Sector Agriculture, Food and Drink
Impact Types Economic

Description Making of a TV documentary entitled "Generation Genome" 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Production of Generation Genome - a six episode BFI supported science series started filming and saw us travel to many parts of the country filming exciting content with young people and lead presenters Prof Darren Griffin and Ogo Anokwuru.
Due for release April 2022
Year(s) Of Engagement Activity 2021