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Title: Novel approaches of generating and selecting high genetic merit in vitro produced pig and cattle embryos
Author: Silvestri, Giuseppe
ISNI:       0000 0004 7227 9522
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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In vitro production (IVP) in agricultural species such as pigs and cattle is performed in a similar manner to human IVF, and involves ovum pick up or oocyte maturation, fertilization outside the body and subsequent transfer of viable embryos. Its application in agricultural breeding can significantly speed up genetic gain rates by reducing generational intervals, increasing selection intensity, and facilitating the dissemination of valuable genetics. IVP is an increasingly important tool for breeding companies given the ongoing emphasis on feeding a growing population with fewer resources. With the above in mind, the aim of this work was to improve the yield of competent, euploid, and high genetic merit IVP embryos available for transfer. Oocytes destined for IVP are commonly assessed for developmental competence by morphological screening. To determine whether it would be possible to improve the utilisation of oocytes per donor, the developmental competence of porcine oocytes with decreasing cumulus oocyte complex (COC) investment was examined. It was found that current IVP practice is wasteful, through the elimination of oocytes with slightly impaired morphology, which still have remarkable developmental potentials. Moreover, whilst it is accepted that embryo splitting (generating more than one embryo from a single fertilized zygote) could benefit the breeding industry by increasing the offspring of the most desirable parents, a comparative analysis of the different splitting methodologies available, including stage of the split and single versus serial splitting strategies, is currently missing in the literature. Here, the splitting of an 8-cell stage embryo into four identical twins was identified as the strategy producing the greatest output of good quality embryos. Additionally, time-lapse investigation of the embryo splits found evidence of the existence of a developmental clock that tightly regulates early cleavage events. Normally, only embryos that display satisfactory morphology are selected for transfer. However, in cattle and pigs, this assessment is complicated by the accumulation of lipid droplets within the embryo, which renders it opaque. Consequently, there is scope for the application of new imaging modalities, such as optical coherence tomography (OCT), which are able to image an embryo in full depth and non-invasively. In this work, swept source OCT was successfully tested for use in early stage bovine embryos to obtain both structural and functional imaging. Moreover, micron-scale movements were measured within blastocysts by OCT as a way to rapidly discriminate between living and deceased embryos, representing a novel application of this methodology. Embryo biopsies can be used to establish the genetic merit of an embryo through single nucleotide polymorphism (SNP) analysis, allowing the application of genomic selection soon after fertilisation rather than at birth. Moreover, SNP information can be analysed by karyomapping to select the most chromosomally normal embryos for transfer. Here, the birth of the first five karyomapped calves in the world is reported. Additionally, karyomapping was used to measure the incidence of aneuploidy in bovine blastocysts by parent of origin and to determine the recombination frequency for each chromosome, demonstrating the applicability of this methodology to basic research.
Supervisor: Griffin, Darren Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available