Newsletter December 2021

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, a paper has been published in J Dairy SCI.

The second part of data collection on consumers’ readiness level was concluded in the summer 2021. Initial data analysis has been concluded and the results will be presented at the annual EliteOva meeting in March 2022. A manuscript on the attitudes to, and willingness to drink milk from cows, produced by advanced dairy breeding technologies, by Danish consumers is being prepared for submission to J Dairy SCI.

The EliteOva partner Masterrind had the mission of testing the ETB media series against an alternative production system. Masterrind concluded 6 IVP experiments comparing TCM-SOF with ETB media. In these experiments, they did not find statistically significant differences between the production systems and, hence, it was 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 (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.

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 was subjected to pregnancy diagnosis by rectal palpation in early march 2021: 3 pregnancies from vitrification and 2 from slow freezing was diagnosed. All calves have now been born. 1 of the calves from the vitrified group was delivered to early with assistance and died during birth. Therefore, 4 living calves were born in wave 4.

In total 63 live calves born in the project.

NTM was calculated from 17+4 embryo-calf pair. To increase the dataset for WP3 4 slaughterhouse experiments was conducted. From these, embryo biopsies and biopsied embryos were sent to Eurofins for SNP analysis. This resulted in additional 78 biopsy-embryo pairs and 5 embryos for SNP-chip analysis.

The EliteOva partners VG and Aarhus University (AU) have the missions of assessing genomic predictions in early embryo stage 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, we are awaiting data from the last five calves from EliteOva. The comparison shows a difference of up to 11 NTM points between embryo and resulting calf biopsy.

We have received NTM calculations from the 45 biopsy embryo pairs mentioned under WP2 and are awaiting results from 33 pairs. We have received call rates from all samples and done initial comparisons of NTM from the 45 biopsy embryo pairs. The comparison indicates a correlation coefficient of 0.66 between NTMs of embryo and that of resulting calf biopsy, with a difference up to 33 NTM points between the embryo-biopsy pairs. The reason for discrepancies between NTM from biopsies, calves and embryos is being explored, and Søren Borchersen has initiated a collaboration with Finnish LUKE about this challenge. AU have not received NTM data from LUKE yet.

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 screening of bulls in EliteSemen has been concluded and analyses of IVP experimental data (258 records from 123 bulls) have been performed to quantify the effects of bull’s age and semen type on the bulls’ IVP traits as well as estimate the heritability and repeatability of these traits. The results indicate significant effects of bulls age and semen type (Sexed or not) on most of the IVP traits for the bulls. More importantly, the results also suggest low to moderate heritability and moderate to high repeatability for the bull’s contribution to IVP traits presenting the possibility of genetic and environmental improvement to enhance IVP traits . Most importantly, the estimated heritabilities could also indicate the possibility of identifying single nucleotide polymorphisms (SNPs) related to bull IVP performance. We have decided to apply for an extension of EliteSemen until the end of 2022 to be able to analyze the data, and present the results in conferences and submit three manuscripts to scientific journals.

The implementation of novel traits, as e.g. improved feed-efficiency and reduced methane- emission by 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 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 reduced 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, but the last embryo flush have been scheduled for the 23 of December. We are therefore currently concentrating 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. The transcriptomic and epigenomic analyses are completed. A manuscript showing the results of the data analysis from hypothalamus, pituitary, gonads, adrenals and the use of blood as a proxy was already published in the FASEB Journal:

Epigenomic and transcriptomic analyses reveal early activation of the HPG axis in in vitro-produced male dairy calves

Also, another manuscript about the results of the data analysis from muscle and liver tissues is being concluded and will be sent for revision soon. Furthermore, these data have been presented in two international conferences: as an oral presentation at the Domestic Animal DOHaD and Epigenetics meeting, which was held online last October, and as a poster at the 48th Annual Conference of the International Embryo Transfer Society, which will be held in Savannah, USA, next January. Once this is finished, both transcriptomic and epigenomic results will be compared and integrated.

Samples for analysis of histone modifications has arrived at Melior, and we are awaiting results from them. We aim to make an integrated analysis of WGBS, RNA-seq and Chips-seq data from the calves. Former EliteOva PostDoc, Maria Belen, will perform the analysis.

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. All calves have now been born. Blood and placenta samples have been obtained from the calves born in Waves 1 and 3-5. A total of 69 calves have been born (all waves). 6 were stillborn or euthanized shortly after delivery (8.7%). Only one malformed calf has been found from waves 1 and 3-5 and one calf has been reported to lack epididymis from wave 2, which is within normal background level. Three calves appeared longer and heavier than usual for neonatal Holstein calves. Analysis of the obtained materials will be finalized in Q1-2, 2022.