Hélène Kiefer

Background: Bull fertility is a key parameter in the dairy cattle industry, with significant economic implications. DNA methylation is an epigenetic mark essential for fertility, and recent works have highlighted sperm DNA methylation as a promising source of biomarkers for male fertility, but no operational tool has been developed for predicting bull fertility based on DNA methylation. Here, we leveraged existing methylation data to evaluate the potential of DNA methylation for predicting bull fertility with machine learning models. Results: Our study was conducted on a cohort of 351 Holstein commercial bulls, classified as subfertile, neutral and fertile based on a field fertility index (non-return rate of the inseminated cows after 56 days post-insemination). This dataset includes the largest cohort assembled for bull fertility prediction from epigenetic markers. We developed classification models to discriminate subfertile and fertile individuals using a subset of predictors derived from reduced representation bisulfite sequencing data obtained on frozen semen. Four machine learning algorithms were compared: Least Absolute Shrinkage and Selection Operator (LASSO), Random Forest, Gradient Boosting, and Neural Networks. To optimize performances of each algorithm and conduct an unbiased assessment of classification accuracy, we used a three-fold cross-validation before final testing on an independent test set. To increase the robustness of the results, model performances were averaged over 20 partitions of the data into training and test sets, to account for variability in bull distribution across sets. All models performed better than random classification. The best model was LASSO according to classification accuracy (0.62) and Random Forest according to Area Under Curve (0.69) computed with the independent test set. Conclusions: Even with this moderate performance, our models would lead to a 12.4% point increase of the probability of identifying subfertile individuals. These results suggest that sperm DNA methylation holds potential as a relevant source of predictive markers for bull fertility.

Background: Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans- methylation QTLs (meQTLs). Results: Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis -meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans -meQTL hotspots. First, we identified CpG-SNP trans -associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans- CpG sites. Then, regions associated with at least 34 independent trans -CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans -CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes ( RUNX1 , NFIC and FOXA3 ) for which the associated trans -CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B , and their corresponding trans -CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions: By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

Background: Germline de novo mutations (DNMs) are rare events in mammals, typically occurring only a few dozen times per generation. These mutations are not entirely random; several factors are known to influence their rate, including DNA methylation. In this study, we leveraged the unique population structure of cattle with a few ancestors having a large contribution to the current gene pool, along with comprehensive genomic resources, to investigate mutational processes. We applied two complementary approaches: (1) identifying DNMs accumulated over generations in family trio segments (from identical-by-descent segments between descendants and direct ascendants spanning several generations) in Holstein and Montbéliarde breeds, and (2) detecting rare bi-allelic substitution variants (Minor Allele Frequency < 0.001) from a large panel of sequenced Holstein animals. Results: Overall, transitions were over-represented compared to transversions for both DNMs and rare substitutions. Considering the nucleotide context, a notable enrichment of C > T substitutions was observed within CpG sites (CpG > TpG). This enrichment was particularly pronounced in low CpG density regions and positively correlated with local methylation levels in both gametes and several somatic tissues. Additionally, several transposable elements exhibited higher mutation rates relative to the rest of the genome, particularly young SINE and LINE elements. Conclusions: Together, these results provide insights into the mutational landscape in cattle and reinforce observations made in other mammalian species.

Extensive DNA methylation and small non-coding RNA modifications occur during male germline differentiation, which are essential for normal spermatogenesis and sperm maturation. Recent evidence has underscored the role of these key regulators in male fertility. However, due to significant environmental and technical variations across the studies, the identification of epigenetic biomarkers relevant to fertility remains challenging. Nevertheless, recent studies using large cohorts and integrative approaches have highlighted sperm epigenetic signatures that improve fertility prediction. In addition, sperm-borne epigenetic marks have been proposed to influence early embryo development and offspring phenotype, supporting the concept of non-genetic inheritance across generations. In breeding sectors and particularly in cattle, while artificial insemination enables the dissemination of paternal genetics at the populational level, some of the broadly used reproductive biotechnologies may impact the sperm epigenetic profile. Therefore, deciphering sperm epigenetic mechanisms has become an important research topic for enhancing breeding efficiency. This review provides an overview of current knowledge on the bovine epigenetic dynamics in male germ cells, their association with fertility and their potential role in embryo development and offspring phenotype, with a focus on DNA methylation and small non-coding RNAs.

Background: DNA methylation (DNAm) plays an important functional role and is influenced by genetic variants known as methylation QTLs (meQTLs). The majority of meQTL studies have been conducted in human blood. Despite its unique landscape, the genetic regulation of sperm DNAm remains largely unexplored. In this study, we leveraged DNAm measured in sperm from 405 Holstein bulls using reduced representation bisulfite sequencing (RRBS) and performed sequence-level genome-wide association studies for 166,985 variable CpGs (s.d. >5%). We reported heritability estimates and have mapped both cis-meQTLs and trans-meQTLs. Results: Heritability estimates ranged from 0 to 1 and averaged 0.26 across all selected CpGs, with 76% of estimates above 0.1. The meQTL mapping revealed that 32.9% of the CpGs had a cis-meQTL, 3.6% had a trans-meQTL and 1.0% had both cis- and trans-meQTLs. The cis-CpGs were located on average 261 kb (absolute mean) from their cis-meQTL top SNPs (defined by the most significant association). MeQTLs were enriched in featured genomic annotations, including regions surrounding transcription start sites and ATAC-seq peaks. We also identified spurious trans-associations by analyzing data across multiple genome assemblies, including the construction of a partial pangenome. Additionally, eight trans-meQTL hotspots, defined as variants associated with at least 30 trans-CpGs, were identified and overlapped with genes involved in epigenetic regulation. Using peripheral blood mononuclear cell DNAm from 54 out of the 405 bulls, we did not observe a similar effect of the trans-meQTL hotspots to that one observed in sperm. Conclusions: For the first time, meQTLs have been detected and characterized in bovine sperm, contributing to a better understanding of the transmission of paternally inherited DNAm marks. These findings provide useful information for further research aimed at integrating epigenetic information into the prediction of performance traits.

Among epigenetic modifications, DNA methylation is extensively studied in sperm, and variations in sperm DNA methylation patterns have been associated with fertility, sperm quality, and in vitro fertilization outcomes in several species. Reduced representation bisulfite sequencing (RRBS) is a cost-effective method that has been successfully applied to sperm. However, RRBS library preparation remains sensitive and labor-intensive and can be subjected to diverse sources of technical variation. In order to improve RRBS library preparation performance, as well as its reproducibility, we adapted the reference protocol and implemented it on a Hamilton pipetting automaton.

The performance of an adult dairy cow may be influenced by events that occur before her birth. The present study investigated potential effects of 2 prenatal groups of factors, Assisted Reproductive Techniques (ART) and maternal characteristics (e.g., dam parity), on offspring performance during their first lactation, in populations of 2 dairy cow breeds: French Holstein and Montbéliarde. The different ART studied included the type of semen (conventional or X-sorted) used for Artificial Insemination (AI) and the technology of conception used (AI, embryo transfer, or in vitro fertilization). Three maternal characteristics were considered: (1) the dam age at first calving, (2) dam parity number, and (3) indicators of dam udder health during gestation (somatic cell score and events of clinical mastitis). First, we investigated whether heifer survival from 3d to 18 mo old was associated with any of the prenatal factors considered. We then estimated the associations of these prenatal factors with 8 traits of commercial interest: (1) stature, (2-4) milk, fat, and protein yields, (5) somatic cell score, (6) clinical mastitis, and (7-8) heifer and cow conception rate, all measured on genotyped cows. Linear models were used for this study with the prenatal factors as covariates in the model, and for the 8 traits, phenotypes were adjusted for their corresponding genomic estimated breeding value. The results indicated that the survival rate of heifers born from embryo transfer was significantly higher than that of heifers born from AI (probably due to preferential management practices), while the other prenatal factors did not explain differences in heifer survival. Among the Montbéliarde cows born from AI, those born from X-sorted semen showed a lightly but significantly lower milk yield than those born without X-sorting of the semen (-52 kg of milk in the first lactation). Among the Holstein cows, those born from embryo transfer presented significantly lower milk performance than cows born from AI. Regarding the maternal characteristics, none or very weak associations were found between the dam age at first calving and the offspring performance in both breeds. Dam parity, on the other hand, was associated with offspring performance for milk, fat, and protein yield in both breeds, however not in the same direction. In the Holstein breed, an increase in dam parity was favorable for offspring performance for milk, fat, and protein yield, whereas in the Montbéliarde breed, an increase in dam parity was associated with lower milk and protein yield and no association was found for fat yield. The udder health of the dam during gestation was not or only weakly associated with the traits studied in the offspring. Although some significant associations were identified due to the large sample size, the effects were modest, typically less than 1% of the phenotypic mean, and were not consistently observed across the 2 breeds.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55–59 and 69–71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, “regulation of Rho protein signal transduction”, implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Background: Conflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility. Results: A unique collection of 100 sperm samples was constituted by pooling 2–5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls. Conclusion: This study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.

DNA methylation plays an important role in the structure and stability of the genome when associated with heterochromatin and repetitive elements such as transposable elements (TEs; mobile elements in the genome of viral origin that have accumulated during evolution) and satellites (sequences located near or at the centromeric regions of chromosomes). DNA methylation also regulates transcription and is dynamic as a function of cell type, developmental stage, the animal’s physiology, or the environment. Finally, DNA methylation is involved in the genomic imprinting of genes expressed in a mono-allelic manner depending on the parental origin of the allele, a phenomenon that is essential for harmonious growth of the fetus. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) that use S-adenosylmethionine as a methyl donor (produced from dietary folic acid). DNMT3A and DNMT3B enzymes are involved in de novo DNA methylation, while DNMT1, which recognizes the hemi-methylated cytosine-phosphate-guanine (CpG) sites resulting from DNA replication, ensures the maintenance and propagation of methylation patterns through cell division. The erasure of DNA methylation can result not only from a lack of DNMT1 activity but also from the conversion of 5-methylcytosines (5meCs) by ten-eleven translocation (TET) enzymes into oxidized derivatives such as 5-hydroxymethylcytosine (5hmC), which are then diluted during replication or replaced by unmethylated cytosines by the DNA repair machinery. Genomic DNA is wrapped around octamers of histones to form the nucleosome. Histones contain N-terminal tails targeted by different types of modifications, such as acetylation and methylation. These modifications affect different amino acids, producing dozens of posttranslational variants with different functional roles. The addition or removal of these modifications is highly flexible processes that directly affect the accessibility of genomic DNA to the transcription machinery and hence the activation or repression of gene expression. The combinatorial nature of the different histone marks, together with DNA methylation and the presence of certain transcription factors or RNA polymerase, define specific chromatin states associated with specific transcriptional states. These chromatin states are transmitted to daughter cells, thus ensuring the continuity of cell identity through mitosis. Finally, small noncoding RNAs (sncRNAs) play an important role in posttranscriptional regulations, and their expression is tightly regulated in a cell type-specific manner.