Newsletter March 2021
The overall goals of EliteOva are to implement ultrasound-guided ovum pick up (OPU) as well as in vitro production (IVP) and genomic selection of embryos in Danish cattle breeding and to secure that the resulting embryos and calves are healthy and the technologies are embraced by the industry and society.
The present Newsletter is a follow up on the latter from September 22, 2020, and serves as common information for the project participants as well as a project update for the Steering Committee.
The next EliteOva Steering Committee meeting will take place on March 23, 2021, the first day of the EliteOva Annual Meeting and due to the COVID-19 situation, it will be a virtual meeting via Zoom.
COVID-19 has had an impact on several of the activities included in EliteOva during 2020. Consequently, the EliteOva Steering Committee and Innovation Fund Denmark have granted a one-year non-cost project extension of EliteOva with the new project end date being November 30, 2022. The rebudget for this extension was completed and approved in December 2020 and a copy was sent to all partners.
Due to the retirement of Poul Hyttel, May 31, 2021, Jan Secher, who has served as Project Leader Trainee in EliteOva, took over the responsibilities as Project Leader on January 1, 2021, and Esben Østrup took over Poul Hyttel’s roles as representative for Department of Veterinary and Animal Sciences.
The project partner Embryo Trans Biotech (ETB) is closing and has therefore terminated the collaboration with EliteOva and will no longer participate in the Steering Committee meetings. CEO for ETB, Lotte Strøbech, has initiated three new companies. We would like to thank ETB for its contribution to EliteOva and to wish Lotte Strøbech the best of luck with her new companies.
In vitro production of embryos for EliteOva will continue in new laboratory facilities at Department for Veterinary Clinical Sciences, University of Copenhagen.
In the following, short updates on the status and further plans of each individual work package (WP) is presented.
The EliteOva partner IFRO (Department of Food and Resource Economics) investigates the societal readiness levels of the technologies included in EliteOva with a focus on the readiness to use the technology among dairy farmers, and acceptance of the technology in the general Danish population (consumers). Based on data collection regarding the farmers’ readiness level including 14 qualitative interviews with farmers and a representative questionnaire survey completed by 156 dairy farmers manuscript has been submitted to J Dairy SCI. The manuscript has been evaluated and is accepted for publication.
The second part of data collection on consumers’ readiness level has been delayed. It is, however, expected to be initiated and carried out in the first quarter of 2021. A questionnaire is under construction and data is expected to be collected in March 2021. Preliminary results may possibly be presented at the annual EliteOva meeting in late March 2021.
The EliteOva partner Masterrind has the mission of testing the ETB media series against an alternative production system. Masterrind has concluded 6 IVP experiments comparing TCM-SOF with ETB media. In these experiments, they have not found statistically significant differences between the production systems and, hence, it has been decided to suspend the further planned comparative OPU/IVP and embryo transfer (ET) experiments at Masterrind.
The full pipeline of OPU, IVP, acquisition of embryonic biopsies (BIOPSY), cryopreservation (CRYO), ET and calving pipeline has gradually been built up over 4 waves of activities.
In Wave 1, the EliteOva partners Trans Embryo Genetics (TEG), ETB and University of Copenhagen, Department of Veterinary and Animal Sciences (UCPH-DVAS) collaborated on the pipeline with OPU taking place at TEG in Tirsvad, IVP taking place at ETB/UCPH-DVAS, and ET and calving taking place at TEG. A total of 15 healthy calves have been born from Wave 1 to prove that this basic pipeline is functional.
In Wave 2, an additional IVP laboratory was established at TEG, and both OPU, IVP and ET took place at TEG. A total of 24 healthy calves have been born from Wave 2.
In Wave 3, OPU was performed at TEG, IVP and BIOPSY (see former Newsletter; technology established by postdoc Vahid Najafzadeh) at UCPH-DVAS, and ET and calving at TEG. A total of 44 biopsied embryos were transferred to recipients after this combined series of technologies, and resulted in 21 live calves. This is a live born rate of 48%, which is highly encouraging. DNA from the biopsies has been amplified and shipped to Eurofins for SNP analyses and derivation of Nordic Total Merit (NTM) and other genomic information. So far, 66 of the biopsied blastocysts have been analyzed for SNPs, out of which only 7 had call rates below 0.80 (threshold according to VikingGenetics). We have received Nordic Total Merit (NTM) for all embryos.
In Wave 4 OPU was performed at TEG, IVP, BIOPSY and CRYO at UCPH-DVAS, and ET at TEG. Two different cryopreservation methods have been tested: Conventional slow freezing and vitrification. The experiments have been conducted over the spring and summer 2020, where 22 embryos submitted to BIOPSY and CRYO (11 vitrified and 11 slow frozen) have been processed to ET. The recipients have been subjected to pregnancy diagnosis by rectal palpation resulting in currently 3 pregnancies from vitrification and 2 from slow freezing. Cryopreservation is needed in order to “park” the embryos for 1-2 weeks before transfer to recipients, while the SNP analyses are performed allowing for selection of the elite embryos for transfer. Additional experimental trials, including 4 MSc projects under daily supervision by postdoc Vahid Najafzadeh, has been concluded at ETB/UCPH-DVAS, where IVP, based on slaughterhouse ovaries, are conducted and the two different methods for cryopreservation of biopsied embryos are tested with respect to: (1) re-expansion rate, (2) inner cell mass, trophectoderm and apoptotic cell counts, and (3) electron microscopy. These investigations have been concluded and resulted in a poster presented at IETS 2021 and a manuscript submitted to Theriogenology.
The EliteOva partners VG and Aarhus University (AU) have the missions of deriving breeding indices according to the NTM from the biopsy SNP data, finding potential genomic markers for bull IVP performance, and implementing novel traits as e.g. methane-emission in the breeding indices. Due to the delay of the acquisition of the embryonic biopsies, WP3 has also suffered a delay but has now been initiated. The NTM from 17 embryonic biopsies and the NTM from biopsies from the resulting calves have been calculated and compared. The comparison shows a difference up to 11 NTM points between embryo and resulting calf biopsy. The reason for these discrepancies is being explored, and Søren Borchersen has initiated a collaboration with Finnish LUKE about this challenge. Gert Pedersen Aamand and Grum Gebreysus will present results from the embryo biopsy SNP-data analysis at the annual meeting on March 24, 2021.
The goal of finding potential genomic markers for bull IVP performance has been extended by the project EliteSemen, for which additional funding has successfully been secured from the Milk Levy Foundation by Jan Secher. The first project year of EliteSemen has been concluded and analyses of the first batch of IVP experimental data (89 records from 51 bulls) have been performed to estimate the repeatability of the bulls’ IVP traits and thereby the chance of identifying SNPs related to bull IVP performance.
The implementation of novel traits, as e.g. feed-efficiency and reduced methane-emission, in the selection of IVP embryos for transfer has been an important societal and environmental aspect of EliteOva. The saved feed index has been implemented as a selection criterion and is also a part of the NTM index for dairy cattle. Hence, the embryos from EliteOva now gets breeding values for all economically important traits in the selection program and can, therefore, be selected for improved resource efficiency and indirectly for lower methane emission. These methods will be further developed and improved throughout the project.
The EliteOva partners Danish Technical University (DTU), Université Laval (Laval), Canada, and Melior Life Sciences (Melior), US, have the mission of evaluating the normality of the IVP embryos and calves concerning their transcriptomic and epigenetic signatures.
Embryos for these investigations are being produced in the experimental herd of a subcontractor, Estonian University of Life Sciences, Tartu, Estonia, where an extensive series of OPU/IVP and in vivo embryo collection experiments have been conducted. The goal was to collect 15 days old IVP and in vivo embryos for the transcriptomic and epigenomic analyses (DNA methylation and histone modifications). Unfortunately, the experiments have been delayed due to technical problems and the COVID-19 situation in Estonia. Therefore, we have currently decided to concentrate our efforts on analyzing the unique calf material presented in the next paragraph.
Male calves were produced at TEG using the same heifer-bull combination (as far as possible), resulting in 4 IVP and 4 MOET calves. These animals were euthanized at 102 ± 2 days of age (one more month than initially planned due to the COVID-19 interruption). Samples were collected from the liver, pancreas, adrenals, brown and white fat, muscle, testes, brain regions (hypothalamus/hippocampus/cerebellum/brainstem/cortex), pituitary, and whole blood. The following samples were submitted to the BGI company (Hong Kong) for RNA-sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS): Hypothalamus, pituitary, adrenals, testes, liver, pancreas, buffy coat (white blood cells), blood, and muscle. Determination of the samples’ molecular integrity showed that the RNA-seq could not be performed on pancreas, blood, and buffy coat samples. This was not surprising, as the blood and BC samples were not snap-frozen immediately, and mRNA is degraded extremely fast in pancreas samples. Nevertheless, WGBS was successfully applied to all the samples. Up to now, the transcriptomic analyses are almost completed, while the epigenomic analyses are undergoing. Once this is finished, both transcriptomic and epigenomic results will be compared and integrated.
Samples for analysis of histone modifications are ready to be shipped to Melior, and we are awaiting an import permit from FDA.
The goals of WP4 are (1) to establish the blood as a proxy for the DNA methylation of the inner organs and (2) to compare the transcriptomic and epigenomic patterns in key organs of IVP and MOET calves, to detect potentially underlying epigenomic aberrancies imposed by IVP.
The EliteOva partner UCPH, Department of Veterinary Clinical Sciences (UCPH-DVCS), has the mission of investigating the clinical normality of the IVP calves. Blood and placenta samples have been obtained from the calves born in Wave 1, set on hold in Wave 2, but re-initiated in Wave 3 and Wave 4 (see WP2). Only one malformed calf has been found, which is not alarming and considered within the normal range, further 3 calves have been born that appeared longer and heavier than usual for neonatal Holstein calves. Due to a delay in the implementation of the embryonic biopsies, the production of calves for WP5 has also been delayed. Combined with the need in WP5 to follow the calves for 6 months after birth, this delay has sparked the need for a non-cost extension to secure a time frame, within which all calves reach the age of 6 months and the collected data can be analyzed.
The birth of calves in Wave 4 is now almost completed as only five calvings remain. Blood samples are submitted to hematology and biochemistry to ensure calf normality.
Conclusion
EliteOva has successfully implemented a set of advanced technologies into an embryo production pipeline that across geographical barriers results in calves on the ground. Generally, calves born during the project appear normal, but three calves from the IVP biopsy group appeared longer and heavier than usual at birth. We have encountered inevitable delays in the project, but have reached the phase, where the cumulative set of technologies including OPU, IVP, BIOPSY, CRYO and ET have resulted in pregnancies. Whereas the BIOPSY procedure had little or no impact on the pregnancy rate, the implementation of CRYO may result in a reduced rate.
Due to the COVID-19 intermission, we were challenged with respect to processing the calves for epigenetic analyses and autopsies, but the last planed autopsy has just been completed and the material has been submitted for RNA sequencing and analyses of DNA methylation and will be shipped for analyses of histone modifications as soon as possible.
I would like to use this opportunity to thank Poul Hyttel for his enormous contribution to EliteOva and wish him all the best with his retirement. I would also like to thank Project Manager Betina W Jensen and Poul Hyttel for keeping meticulous track of the complex management and all EliteOva partners for investing enthusiasm and hard-working hours into the project.
By Poul Hyttel and Jan Secher.