Newsletter March 2020

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. Data includes 14 qualitative interviews with farmers, and a representative questionnaire survey completed by 156 dairy farmers. The main findings from the questionnaire survey are that a clear majority of dairy farmers (78%) find it likely that they would use semen from bulls derived from the EliteOva technology. A smaller proportion (30%) finds it likely that they will use fertilized eggs from the technology. A very small proportion (5%) reports to be against the technology on a matter of principle. It is worth noting that a rather large minority (33%) report to be undecided about the technology. The reason for rejecting use or being against the technology on a matter of principle can partially be explained by objections to the ‘ethical soundness’ of the EliteOva technology. There is hardly any difference between organic and conventional farmers in reported likelihood to use, and accept, the technology – to the extent it is legal. Larger farms (measured as number of dairy cows) have a higher likelihood to report that they will use the technology.

Expected publications outlets regarding the collected farmer data: A peer-reviewed manuscript (likely journal: J DAIRY SCI), and a Danish report.

The second part of data collection on consumers’ readiness level, is expected to be initiated and carried out in the first half of 2020.

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 noted cleavage rates and day 6 and 7 blastocysts rates. Regarding blastocyst rates at day 7, no statistically significant difference between the two media has been recorded (ETB blastocyst rate day 7, n= 248: 40%; TCM-SOF blastocysts rate day 7, n= 243: 39%). Masterrind has now proceeded into the next phase where OPU/IVP will be implemented in the testing. The delivery time for these experiments are in May.

The EliteOva partners Trans Embryo Genetics (TEG), ETB and University of Copenhagen, Department of Veterinary and Animal Sciences (UCPH-DVAS) collaborate on the pipeline for OPU/IVP of embryos and establishment of pregnancies. The process includes OPU, which is performed at TEG in Tirsvad, IVP, which is performed either at ETB/UCPH-DVAS or at TEG, and embryo transfer and calving, which takes place at TEG. So far, 15 calves have been born from “Wave 1” to prove that this basic pipeline is functional. Subsequently, an additional IVP laboratory was established at TEG, and for “Wave 2” the IVP took place in this laboratory. During “Wave 2”, 31 pregnancies have been produced in this new laboratory setting, which has also proven functional, and calving is now ongoing.

During “Wave 3”, where OPU is conducted at TEG and IVP at UCPH-DVAS, acquisition of embryonic biopsies has now been implemented in the pipeline.

For acquisition of biopsies, the blastocysts are firmly mounted to a holding pipette after which the zona pellucida is penetrated using a biopsy pipette of 25 µm inner diameter. Approximately 5-10 trophectoderm cells are aspirated into the biopsy pipette. The blastocyst is then loosened from the holding pipette, which is then brought adjacent to the embryo and against the biopsy pipette. With several flicking movements of the blastocyst, the aspirated trophectoderm is detached from the embryo. Survival rate for OPU blastocysts of the biopsy procedure with this technique is 95%. A total of 50 embryos have now 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.  

DNA from the biopsies is amplified and shipped to Eurofins for SNP analyses and derivation of Nordic Total Merit (NTM) and other genomic information. So far 44 of the biopsied blastocysts have been analyzed for SNPs, out of which only 4 had call rates below 0.80 (threshold according to VikingGenetics). We have received NTM for 37 embryos (data from 3 embryos missing at present).

At present, it is negotiated how to complete “Wave 3” when activities will be re-initiated after COVID-19, as the resources of recipients at TEG have been exhausted for now.

The next technological process to be implemented is cryopreservation of the biopsied embryos. 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. Experimental trials are presently ongoing at ETB/UCPH-DVAS, where IVP based on slaughterhouse ovaries are conducted and two different methods for cryopreservation of biopsied embryos are tested: Conventional slow-freezing and vitrification using manufacturers cryopreservation protocols. In these experiments embryos are evaluated after cryopreservation with respect to: (1) re-expansion rate, (2) ICM, trophectoderm and apoptotic cell counts, (3) electron microscopy, and (4) pregnancy rates after transfer. The initial studies have shown that vitrification yielded better results than slow freezing with respect to re-expansion rates. In terms of ICM, trophectoderm and apoptotic cell numbers, however, there was no statistical difference between these two methods of cryopreservation. Abstract on these results has been submitted for the Symposium of the European College of Animal Reproduction (ECAR) August 2020 in Budapest, Hungary. Further analyses including electron microscopy and pregnancy results are still in progress but await the COVID-19 intermission.

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 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 B-M Secher. This project is ongoing and will support WP3 with valuable data as well as the introduction of batch codes on semen straws that will be initiated later in 2020. 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 index and is also a part of the NTM index for dairy cattle. Hence, the embryos from EliteOva now gets breeding values for all economical important traits in the selection programme 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 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 is 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, due to unforeseen reasons been delayed. So far, two in vivo Day 15 embryos and 3 IVP Day 15 embryos have been recovered, and the embryos have been dissected into pieces including the embryonic disc and pieces including TE. Further collections of in vivo and IVP embryos are in progress, but are hampered by the COVID-19 situation in Estonia.

Calves for the epigenetic studies are being produced at TEG using the same heifer-bull combination. Pregnancies were established in order to produce 3 calves from OPU/IVP and 3 from in vivo embryos. Unfortunately, one of the OPU/IVP pregnancies was aborted. Fortunately, two IVP replacement calves, produced with the same heifer but different bull, have been identified. One of them was born in January 22, the other one is expected to be born in early May. The remaining calves will be born during April-May. These calves will be euthanized at around two and a half months of age (initially the plan was 2 months, but this period has been extended due to the COVID-19 intermission), after determination of the breeding index. Samples will be collected from liver, brain and blood (and potentially other organs for later experiments). These samples will be processed for studies of gene expression, DNA methylation and histone modifications at DTU, Laval and Melior. The goal is to establish the blood as a marker for the DNA methylation of the inner organs, and the further objective is, with this tool in hand, to examine blood DNA methylation in a larger number of OPU/IVP calves from the project. These investigations will give a molecular epigenetic signature of the normality of the IVP calves.

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” (see WP2). One calf with malformations has been encountered, which is not alarming and considered within normal range. 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 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. This issue will be addressed at the upcoming Steering Group Meeting.  

Birth of calves in “Wave 3” is now in progress and blood samples are submitted to hematology and biochemistry to ensure normality. No deaths or diseased calves have been observed yet.

Euthanization of calves for epigenetic studies as well as autopsy was planned for week 12, 2020, but is on hold due to the COVID-19 situation as the veterinary university hospitals are closed for research and staff not allowed to enter the premises. The calves were planned to be euthanized at the age of 2 month, but initially this has been changed to 2.5 months. Further postponements due COVID-19 are carefully considered.

We have succeeded in implementing a set of advanced technologies into an embryo production pipeline that across geographical barriers efficiently results in calves on the ground. We have encountered certain delays in the project, but we are now moving into a very exciting phase of the project, where the cumulative set of technologies including OPU, IVP, biopsies, cryopreservation and embryo transfer will be applied, and where we will acquire molecular information on the embryo’s breeding indices as well as their epigenetic normality. However, due to the COVID-19 intermission we are facing challenging delays in processing the calves for epigenetic analyses and autopsies. This situation is critically evaluated on a running basis.

Finally, I would like to thank Project Manager Betina W Jensen and project leader trainee Jan B-M Secher for keeping meticulous track of the complex management and all EliteOva partners for investing enthusiasm and hard working hours into the project.