Approximately one million in vitro produced (IVP) cattle embryos are transferred worldwide each year as a way to improve the rates of genetic gain. The most advanced programmes also apply genomic selection at the embryonic stage by SNP genotyping and the calculation of genomic estimated breeding values (GEBVs). However, a high proportion of cattle embryos fail to establish a pregnancy. Here, we demonstrate that further interrogation of the SNP data collected for GEBVs can effectively remove aneuploid embryos from the pool, improving live births per embryo transfer (ET). Using three preimplantation genetic testing for aneuploidy (PGT-A) approaches, we assessed 1713 cattle blastocysts in a blind, retrospective analysis. Our findings indicate aneuploid embryos have a 5.8% chance of establishing a pregnancy and a 5.0% chance of given rise to a live birth. This compares to 59.6% and 46.7% for euploid embryos (p < 0.0001). PGT-A improved overall pregnancy and live birth rates by 7.5% and 5.8%, respectively (p < 0.0001). More detailed analyses revealed donor, chromosome, stage, grade, and sex-specific rates of error. Notably, we discovered a significantly higher incidence of aneuploidy in XY embryos and, as in humans, detected a preponderance of maternal meiosis I errors. Our data strongly support the use of PGT-A in cattle IVP programmes.

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology 125 : 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were “perfectly concordant” (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation.

Summary In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed ‘FLI’. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Study question Can we visualise model oocytes and embryos during embryo development using OCT non-invasively? Summary answer This approach, new to embryology, OCT, allows visualising cells in a 3D perspective, providing more detailed information than standard microscopy from the oocyte or embryo. What is known already Time-lapse is a well-established technique in human IVF, but it is limited in its depth of view due to the limitations of classical microscopy. To overcome this limitation, the approach of Optical Coherence Tomography (OCT), allows non-invasive visualisation through optical cross-sections of the embryo or oocyte to produce 3D images. The obtained information can be extended to 4 or 5 dimensions to track movement and elasticity, using a low-power light source and no staining to ensure the minimum effect on the cell. Here, a pilot study was performed in model (porcine) samples using a newly developed “in incubator” system. Study design, size, duration A pilot basic study was performed where 16 oocytes and 16 cleavage embryos were obtained and prepared to be visualised using OCT. Participants/materials, setting, methods Pig ovaries were obtained from a slaughterhouse, from which follicles were aspirated in order to retrieve oocytes. The best-quality oocytes were cultured for maturation for 44 hours and 16 oocytes were selected for OCT imaging. before fertilisation for 2 hours. Sperm was previously prepared by Percoll gradient. Zygotes were cultured for 6 days until visualisation through OCT. For imaging, samples were prepared in a 16-well Primo Vision dish. Main results and the role of chance Oocytes and embryos were successfully imaged, allowing the identification of distinct cellular features. In oocytes, it was possible to identify the germinal vesicle (nucleus) and polar bodies, individual blastomeres in cleavage stage embryos and trophectoderm, ICM and blastocoel in blastocysts. As images were taken throughout different optical sections, it was possible to correlate the position of the areas within the oocytes/embryos and create a 3D image from a blastocyst, understanding the size of the inner cell mass compared with the embryo's overall size. Interestingly, images were obtained non-invasively and under optimal conditions (in an incubator), demonstrating the future utility of OCT for embryo imaging. Limitations, reasons for caution An experimental OCT system was developed and placed in the incubator, and all images obtained were in a format of a pilot study. Future imaging sessions are planned to obtain more data points and assess the viability of the embryo. Wider implications of the findings Although in the present study, OCT was applied in pig oocytes and embryos, this new approach can be employed in future for human IVF to overcome the current imaging limitations, proving more information on the oocytes and embryos’ quality and assisting Artificial Intelligence analysis. Trial registration number not applicable

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