The aim of this PhD thesis was to characterize the variation of developmental competency in high-yielding cows when exposed to metabolically adverse conditions induced by negative energy balance during post-partum lactation in oocytes and embryos at the epigenetic level.
To do so, a whole genome epigenetic profile was performed on oocytes collected from lactating cows experiencing negative energy balance at early and late post-partum time points as well as control heifers using next-generation sequencing. In order to investigate the role of oviductal metabolic conditions on embryo epigenetic plasticity, in vivo culture of embryo in the oviduct of metabolically profiled lactating cows and heifers was performed followed by whole genome bisulfite sequencing. It was found that, during early lactation, cows exhibit significant different metabolic status at different stages postpartum compared to heifers. From these metabolically divergent animals, a general hypomethylation was observed in genome features of oocytes during early postpartum when compared to mid postpartum cows and heifers. Inversely, overall genomic features were found to be hypermethylated in embryos from lactating cows compared to heifers. Further characterization of differentially methylated regions (DMRs) in the genome revealed highest variation DMRs in genes involved in DNA binding, embryo development and metabolic pathways in both oocytes and embryos. This chapter discusses the differences and similarities of both experiments.
4.1 Energy balance profiling
In the first experiment, result analysis demonstrated that oocytes are sensitive to metabolic conditions in a time specific manner, which results in different methylation profiles. Cows during week 5-6 postpartum had a significantly increased metabolic profile of NEFA and BHB compared to week 9-10, consistent with mid-postpartum recuperation of negative energy balance in lactating cows. Even though NEFA levels of mid-postpartum cows were
significantly different from heifers, such difference was not found in BHB. Since ketone bodies like BHB are a byproduct of incomplete fatty oxidation, it appears that NEFA might be a better indicator of overall lipomobilization and by extension negative energy balance in the cow, and overall metabolic variability between animals. A previous study found that a higher ratio of cows were experiencing lipomobilization through NEFA screening than cows experiencing ketosis with BHB screening, further supporting the idea that NEFA provide a broader indication of the lipomobilization physiological status than BHB reading (González et al., 2011).
We found a similar profile when investigating embryos cultured in cattle, where lactating cows exhibited averages of BHB over a lower threshold than observed in cows used for oocyte collection, while maintaining NEFA level above the original threshold. Additionally, one should note that while negative energy balance is a transient metabolic stress in the lactating cows, positive energy balance is not experienced in the same magnitude during lactation. As cows are differentially fed with concentrates based on their milk production, feed efficiency in the cow aims to reach a net balance of zero MJ/day, and so recuperation from negative energy balance is more accurate term than cows reaching positive energy balance. Accordingly, both set of cows selected at mid-postpartum points or time of transfer did not reach an average positive energy balance, but remained rather closer to net balance of zero MJ/day. Interestingly, some cows experienced immediate weight gain upon post-partum, which one could speculate they could be associated with an unperturbed metabolic profile. Although following these cows in the breeding program to assess their reproductive ability is crucial, investigating the oocyte methylation profile of such animals would expose the epigenome of gametes in animals resilient to this transient metabolic stress and could be used to improve animal selection. Nevertheless, coupled with body weight assessment and
energy balance profiling, all animals selected exhibited weight loss and negative energy balance prior to week 5 and had their metabolite significantly different from heifers at the time of collection and transfer, indicating different metabolic profiles in both cows used for oocyte and embryo collection.
4.2 Global methylation characterization
Upon selection of animals exhibiting metabolic stress, global methylation characterization was performed from sequencing data in oocytes and embryos. To assess variability of samples, PCA clustering of quantitation was performed. This PCA clustering showed heterogeneity between conditions and extreme variation within conditions of oocytes, where oocytes from mid postpartum and heifers were clustered together and early postpartum oocytes clustered far apart. In the case of embryos, PCA clustering of samples reveal a close relation between samples, even if samples from separate conditions did not overlap each other, indicative of smaller differences to be observed between conditions.
Still, principal components accounted for 80% and 48% of the variation in oocytes and embryos, which suggest that single-cell sequencing may be a great avenue to investigate further variations within the conditions not accounted for in embryos. The characterization of methylation features looked similar across features in the genome and indicated an overall hypomethylation in early oocytes when comparing with late and heifer oocytes.
Global methylation levels were found to be higher than some previously reported studies, ranging from ~50% and 53.8% in WGBS human oocytes of different developmental stages (Okae et al., 2014; Yu et al., 2017), ~30 to 40% in RRBS bovine oocytes (Jiang et al., 2018) and
~30% in WGBS bovine oocytes (Duan et al., 2019). While differences in RRBS CpG methylation levels of features have been shown to be lower than WGBS derived methylation, with as much as an 11% difference (Doherty and Couldrey, 2014), the
methylation levels observed in our oocytes do not account for such a high variation. As most of these studies filter out CpG sites with less than five reads, this could possibly remove some methylation information data still present in our analysis. Further sequencing of our samples to permit such filtering of coverage depth could further characterize the methylation status of these oocytes with respect to other findings in the literature. In embryos, methylation of features revealed a slight hypermethylation found in morulae grown in lactating cows when compared to heifers, which was further reflected in the number of hypermethylated DMRs compared to hypomethylated DMRs in lactating cows.
The global average CpG methylation was constant with levels observed in literature, where morulae from lactating cows and heifers (33.1 and 31.3%, respectively) were hypermethylated compared to 8 cell embryos and similar to 16 cell embryos, early and compact morulae, indicative of post-embryonic genome activation methylation changes observable in our morulae (Duan et al., 2019; Jiang et al., 2018). Similarly, Jiang et al. report demethylation occurring from morula to blastocyst, suggesting that methylation levels are the highest at the morula stage. The results presented in this thesis seem to show that morulae grown in heifers can demethylate properly towards blastocyst stage methylation profile, with morulae grown in cows “lagging” behind to demethylate genes involved in proper development. Two mechanisms are thought to be involved in demethylation of genes upon fertilization, a post cellular division “passive” demethylation based on non-maintenance of epigenetic marks by DNMT1, and an active demethylation occurring through the T5-methylcytosine hydroxylases (TET) family of enzymes (Wu and Zhang, 2017). The investigation of the downregulation or inefficiency of such machinery in morulae grown under metabolic stress could be of great interest to shed some light into the pressure of lactation on basic molecular machinery involved in embryo development. Although the