Newsletter January 2023

The EliteOva partner IFRO (Department of Food and Resource Economics) investigates the societal readiness levels of EliteOva with a focus on the readiness to embrace the technologies among dairy farmers, and acceptance of them in the general Danish population (consumers). Based on data collection regarding the farmers’ readiness level including 14 qualitative interviews with farmers and a representative quantitative questionnaire survey completed by 156 dairy farmers a manuscript has been published in J Dairy Sci in July 2021.
A representative quantitative questionnaire survey on consumers’ readiness level was concluded by 2036 Danish citizens in the summer 2021. The manuscript on these results was published in J Dairy Sci in January 2023.
Conclusions: The conclusions from the two studies are that it is likely that semen from the OPU-IVP-GS technology (and similar technologies) will be widely adopted in both conventional and organic farming provided that costs can be kept low, and there are advantages in terms of achieving desired breeding goals. While Danish milk consumers are critical of advanced breeding methods in dairy production, only 1 in 5 is unwilling to drink milk from dairy cows bred with semen derived from such methods.

The EliteOva partner Masterrind had the mission of testing the ETB media series against an alternative production system. Masterrind did conclude 6 IVP experiments comparing TCM-SOF with ETB media. In these experiments, they did not find a statistically significant difference 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. In total, 15 healthy calves were 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 were born from Wave 2.
In Wave 3, OPU was performed at TEG, IVP and BIOPSY (this technology was 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 being submitted to this combined series of technologies, and the result was 21 live calves. This is a live born rate of 48%, which is highly encouraging. DNA from the biopsies 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 were subjected to pregnancy diagnosis by rectal palpation in early March 2021 revealing 3 pregnancies from vitrification and 2 from slow freezing. All calves were born, but one 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 resulting in a total of 63 live calves born over the complete the project.
NTM was calculated from 17 embryo-calf pair but showed discrepancies of up to 11 NTM points. To increase the NTM dataset for WP3 slaughterhouse experiments were conducted. From these, embryo biopsies and biopsied embryos were sent to Eurofins for SNP calculation to test the biopsy predictive efficiency. This resulted in additional 78 biopsy-embryo pairs with whole genome amplification (WGA) and 5 embryos without WGA for SNP-chip analysis.
Conclusions: The comparison of the ETB media series against the TCM-SOF media series showed no difference in blastocysts rates.
The full pipeline of OPU, IVP, BIOPSY, CRYO, NTM, ET and calving has been established. The SME TEG has established a fully functional IVP laboratory and can offer OPU, IVF and ET. It is, however, more expensive to and less stable to produce IVF calves than MOET calves, and, therefore, IVP will at present serve as an alternative to MOET, when this technology for different reasons is not applicable. The precision of the NTM from the embryo TE biopsies is not accurate enough and cannot be used as it is.

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 in the acquisition of the embryonic biopsies, WP3 suffered a delay. 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 of up to 11 NTM points between embryo and resulting calf biopsy. The embryo biopsy and embryo data also show discrepancies and interesting enough also call rates below 0,8 for the embryos. 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 Gebreyesus presented the results from the embryo biopsy SNP-data analysis at the annual meeting on March 24, 2021. The data on NTM on BIOPSY-calf and BIOPSY-embryo need to be analyzed in more detail.

The goal of finding potential genomic and functional markers for bull IVP performance was extended by the project EliteSemen, for which additional funding was secured from the Milk Levy Foundation. The EliteSemen project period terminated on Jan 1,2023. A total of 259 experimental IVP groups (with at least 50 oocytes in each) were analyzed in this project. This allowed testing of different ejaculates (sexed, conventional, different bull age at semen collection) from 123 different bulls. All embryos from the project have been snap frozen. A manuscript on the data on these experiments are being concluded and is expected to be submitted within the nearest future. Furthermore, a genome-wide association study was undertaken to identify genomic regions associated with the bull’s effect on the variability of a set of IVP performance traits including blastocyst rate, hatching rate and an index trait combining blastocyst rate, kinetic score, and morphology score (BL_M_K). While the study suffered from limited statistical power due to the small sample size (n ~ 259), some interesting regions were detected based on a less-stringent significant threshold criteria. Some of these regions included more than one IVP trait (example on BTA17, 18 and X-chromosome) and others were co-associated with non-return rate (example on BTA 6). These results were presented as poster presentation at the 32nd European Congress of AI VETS held at Chester, UK (October 26 – 26, 2022).

An additional article on field fertility of the involved bulls and its relationship with semen quality traits has been published J Dairy Sci in January 2021. The study showed moderate to high genetic and phenotypic correlations between some of the semen quality traits and service sire non-return rate. Moreover, while conventional semen quality traits have moderate to high heritability and repeatability, significant phenotypic variations were observed based on bull’s age. A further study was therefore undertaken to investigate the genetic variability and heritability trends across the age of the bulls. This study is now accepted for publication in J Dairy Sci. A small set of additional experiments on the repeatability of the IVP performance in a small subset of bulls needs to be performed at the Estonian University of Life Science and analyzed. This will be concluded in 2023.

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 has also been implemented in the NTM index for dairy cattle. Hence, the embryos from EliteOva were assigned 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 after EliteOva has been concluded.

Conclusions: The full pipeline of embryo TE biopsy, whole genome amplification, biopsy identification with IDNor, and analysis of the biopsy has been established. The discrepancies between the NTM from embryo TE biopsies and the NTM from biopsies from the resulting calves are up to 11 NTM points. Therefore, the embryo TE biopsy results cannot be directly implemented without refinements. This problem will be examined further after the conclusion of EliteOva. The work on finding potential genomic and functional markers for bull IVP performance has shown promising results, but the dataset needs to be extended.
The work on identifying traits related to reduced methane emission has gained immense public and political awareness after EliteOva was Initiated. AU and Viking Genetics will continue the work to identify such traits and incorporate them into the NTM after EliteOva has been concluded.

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 has been produced under strictly comparable conditions 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. A total of 16 embryos at the age of 15 days have been produced: 4 IVP-fresh embryos, 4 IVP-vitrification embryos, 4 morphological small in vivo embryos and 4 morphological large in vivo embryos. Each embryo has been divided into an embryonic disc sample and two TE samples and sent to the company BGI (Hong Kong) for whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). Unfortunately, the amount of DNA and RNA in the samples were low forcing us to use smart-seq2 library construction for some of the RNA-seq samples. The sequencing results will be sent to DTU in the nearest future, and former EliteOva postdoc, Maria Belen Rabaglino (now DTU guest researcher) has agreed to analyze them.

The normality of the born IVP calves was examined by evaluating male MOET calves 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 intermission). Samples were collected from the liver, pancreas, adrenals, brown and white fat, muscle, testes, brain regions (hypothalamus, hippocampus, cerebellum, brainstem, and cortex), pituitary, and whole blood. The following samples were submitted to BGI for RNA-seq and 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 have been completed, resulting in two publications. The first publication appeared in the FASEB journal in August 2021, describing the multi-omics analysis of data from the hypothalamus, pituitary, testis, and adrenal. Results suggested an early activation of the HPG axis in male IVP calves compared to their MOET counterparts. The second publication appeared in Biology of Reproduction and demonstrated epigenomic and transcriptomic upregulation of aerobic respiration of both liver and muscle, while protein synthesis was increased in the liver but down regulated in the muscles of IVP calves compared to MOET calves. Furthermore, we identified a set of biomarkers that allowed the prediction of the embryo origin based on the epigenomic profiles from blood or buffy coat samples, even when the latter were obtained at birth.
Samples for analyses of histone modifications were shipped to Melior. The initial analysis of the samples has been initiated with some challenges, however positive results have been obtained from pilot studies on liver samples. We are awaiting more results and hope to integrate them into the other studies on the calves mentioned above.

Conclusions: Transcriptomic and epigenomic analyses of 4 IVP and 4 MOET calves indicate that the HPG-axis is activated earlier in IVP calves compared to MOET calves. The analysis also demonstrated differences in activation of genes related to energy regulation in muscle and liver samples when IVP calves were compared to MOET calves. Finally, epigenomic analysis of buffy coat identified biomarkers that made it possible to identify IVP and MOET calves, respectively, at birth. Epigenomic and transcriptomic analysis of embryo samples will be concluded after EliteOva has ended.

The EliteOva partner UCPH, Department of Veterinary Clinical Sciences (UCPH-DVCS), has the mission of investigating the clinical normality of the IVP calves. All planed EliteOva calves have been born. Samples have been collected from calves born in Wave 1 as well as Wave 3 and 4. The samples taken include blood (D0 and ~D14) and placenta samples. Only one malformed calf has been found among the calves born in EliteOva, which is not alarming and considered within the normal range. Further, 3 calves have been born that appeared larger and heavier than usual for neonatal Holstein calves.
Blood samples and placenta samples have been analyzed and are being included in a manuscript on the health of IVP calves after ET, ET and BIOPSY, and ET, BIOPSY and CRYO. Interestingly, the study indicates larger variation in birth weight, weight at day 14 and weight gain correlated to the degree of embryo manipulation.
Conclusions: The calves born in the EliteOva project are healthy, however, it seems like there is larger variation in factors such as birth weight and biochemical profiles among the calves in the BIOPSY and BIOPSY/CRYO groups compared to the less manipulated IVP group.

EliteOva has implemented a series of technologies including OPU, IVP, BIOPSY, CRYO and ET resulting in born calves. Whereas the BIOPSY procedure had little or no impact on the calving rate (no biopsy 44%, biopsy 51%), the implementation of CRYO, in the form of vitrification, combined with BIOPSY resulted in lower calving rates (24%). However, this effect was not statistically significant. In general, the calves born in the project were healthy, but the birthweight of the calves, weight at day 14, and weight gain were more heterogeneous among the calves from BIOPSY and BIOPSY/CRYO groups compared to the less manipulated IVP group.

Discrepancies between the NTM from embryo TE biopsies and the NTM from biopsies from the resulting calves were too high for the technique to be immediately implemented in the industry without further refinement.
The work on finding potential genomic and functional markers for bull IVP performance has shown promising results, but the dataset needs to be extended.

Epigenenomic and transcriptomic analyses of calves born in the project indicate differences in the activation of the HPG-axis and energy regulation in muscle and liver when IVP calves are compared to MOET calves.

Overall, Danish dairy farmers are positive towards the EliteOva technologies, if relevant and costs are kept low. The milk consumers are more critical, but it will only to a limited degree affect their willingness to drink milk products based on the technologies in focus.