Newsletter September 2020
Introduction
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 March 18, 2020, and serves as common information for the project participants as well as project update for the Steering Committee. The next EliteOva Steering Committee meeting is appointed to September 30, 2020. Due to the COVID-19 situation, we will set up for a virtual meeting, via Zoom.
COVID-19 has had an impact on several of the activities included in EliteOva during the spring 2020, and at the EliteOva Steering Committee meeting, March 25, 2020, a potential one-year extension of EliteOva was discussed. It was decided that the project leader, Poul Hyttel, together with Jan Secher and Betina W Jensen, would formulate an overview of the scientific and budgetary consequences in relation to COVID-19. This document has been circulated within the Steering Committee and the one year project extension has subsequently been granted by Innovation Fund Denmark with the new project end date being November 30, 2022. The rebudgetting is now also in its final phase with all partner having received their budget for approval by October 1, 2020. We are expecting to have the rebudget completed by mid-October.
Due to the retirement of Poul Hyttel May 31, 2021, it is recommended to the Steering Committee that Jan Secher, who has served as Project Leader Trainee in EliteOva from the initiation of the activities, steps in as project leader. Furthermore, it is recommended that the EliteOva activities, allocated to Department of Veterinary and Animal Sciences, are transferred to the leadership of Associate Professor Esben Østrup.
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). The team has finalized data collection regarding the farmers’ readiness level including 14 qualitative interviews with farmers and a representative questionnaire survey completed by 156 dairy farmers. A manuscript presenting the results has been submitted to J Dairy Sci and is under revision.
The data collection on consumers’ readiness level is the next challenge.
The EliteOva partner Masterrind has the mission of testing the Embryo Trans Biotech (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.
Wave 1
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 17 calves have been born in Wave 1. One calf was stillborn and one displayed malformations and died 3 days after birth, leaving 15 healthy calves, which proves that this basic pipeline is functional.
Wave 2
In Wave 2, an additional IVP laboratory was established at TEG, and both OPU, IVP and ET took place at TEG. A total of 27 calves have been born in Wave 2. Two calves were stillborn and one died within the first day leaving 24 healthy calves.
Wave 3
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 50 biopsied embryos have been transferred to recipients after this combined series of technologies, which has resulted in 23 pregnancies. This is a pregnancy rate of 46% which is highly encouraging. Calving in Wave 3 is still ongoing. 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. Normally, call rates are set to minimum of 0.95, but to ensure that retrieval of genomic enhanced breeding values (GEBVs), in this case NTM, for as many embryos as possible the threshold is decreased to 0.80 for the embryonic biopsies. Accordingly, we have received NTMs for all 66 embryos.
Wave 4
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 late 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 very early pregnancy diagnosis by rectal palpation on day 34, resulting in currently 4 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 Vahid, are presently ongoing 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 are still ongoing and will be concluded during the progress of the MSc projects during the fall 2020.
The EliteOva partners VG and Aarhus University (AU) have the missions of deriving breeding indices according to the Nordic Total Merit (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 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 bull’s 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.
In WP2 IVP production waves 3 and 4, NTM is derived from first the blastocyst biopsy and later from the born calf. Such NMT combinations are now emerging, and it has, in a couple of cases, been noted that the biopsy-derived NTM is significantly lower than its calf-derived counterpart. This is a remarkable finding and EliteOva is very focused on dissecting the methodological background for such discrepancies.
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 with respect to their transcriptome 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). The experiments have, unfortunately, been delayed due to technical problems and the COVID-19 situation in Estonia, and at the moment we have decided to concentrate our efforts on analyzing the unique calf material presented in the next paragraph. The embryonic material from Estonia will later be used for candidate gene studies of potentially aberrant patterns of DNA methylation identified in the calves described below.
Calves for the epigenetic studies have been produced at TEG using the same heifer-bull combination (as far as possible) resulting in 4 IVP and 4 MOET calves. The calves were euthanized when they around 103 days of age (initially the plan was 2 months, but this period was extended due to the COVID-19 intermission). Samples have been collected from liver, pancreas, adrenals, fat, muscle, testes, brain and blood. Selected samples will be processed for studies of gene expression, DNA methylation and histone modifications at DTU, Laval and Melior. The goals are (1) to establish the blood as a proxy for the DNA methylation of the inner organs, and (2) to compare the patterns of gene expression, DNA methylation and histone modifications of IVP and MOET calves in order to detect potentially underlying epigenomic aberrancies imposed by IVP and its associated procedures.
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). One calf with malformations has been encountered in Wave 1, which is not alarming and considered within the normal range.
The birth of calves in Wave 3 is now almost concluded and blood samples are submitted to hematology and biochemistry to ensure normality. No deaths or diseased calves have been observed yet, although one large calf with a birth weight of 59 kg has been recorded. Interestingly, this calf had a full sibling (same bull and cow) born from artificial insemination on the same day at a birth weight of 37 kg.
Autopsy of 4 IVP and 4 MOET calves, euthanized for epigenetic studies, has been concluded. Except for minor spontaneous lesions such as mild bronchopneumonia, the calves were normally developed.
EliteOva has successfully implemented a set of advanced technologies into an embryo production pipeline that, across geographical barriers, has resulted in calves on the ground. 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 will be submitted for RNA sequencing and analyses of DNA methylation and histone modifications as soon as possible. Calves produced in the project in general appear normal, although recently a healthy calf with a birth weight on 59 kg was born.
Finally, I would like to thank Project Manager Betina W Jensen and Project Leader Trainee Jan Secher for keeping meticulous track of the complex management and all EliteOva partners for investing enthusiasm and hard-working hours into the project.
Jan Secher and Poul Hyttel