Sperm chromatin integrity and DNA methylation in Norwegian Red bulls of contrasting fertility

In this study, the complexity of chromatin integrity was investigated in frozen‐thawed semen samples from 37 sires with contrasting fertility, expressed as 56‐day non‐return rates (NR56). Protamine deficiency, thiols, and disulfide bonds were assessed and compared with previously published data for DNA fragmentation index (DFI) and high DNA stainability (HDS). In addition, in vitro embryo development and sperm DNA methylation were assessed using semen samples from 16 of these bulls. The percentages of DFI and HDS were negatively associated with NR56 and cleavage rate and positively associated with sperm protamine deficiency (p < 0.05). Significant differences in cleavage and blastocyst rates were observed between bulls of high and low NR56. However, once fertilization occurred, further development into blastocysts was not associated with NR56. The differential methylation analysis showed that spermatozoa from bulls of low NR56 were hypermethylated compared to bulls of high NR56. Pathway analysis showed that genes annotated to differentially methylated cytosines could participate in different biological pathways and have important biological roles related to bull fertility. In conclusion, sperm cells from Norwegian Red bulls of inferior fertility have less compact chromatin structure, higher levels of DNA damage, and are hypermethylated compared with bulls of superior fertility.

including oxidative stress, abortive apoptosis, deficiencies in recombination, age, semen handling, thermal stress, vaccinations, and bacterial infections (González-Marín et al., 2012;Kumaresan et al., 2020). Studies have shown that the degree of sperm DNA fragmentation is negatively correlated with bovine field fertility (Gliozzi et al., 2017;Narud et al., 2020;Waterhouse et al., 2006) and the outcome of in vitro fertilization (IVF) (Fatehi et al., 2006;Simões et al., 2013). Further, the histone to protamine ratio in sperm cells affects male fertility, where infertile men possess sperm cells with higher proportions of retained histones compared to fertile men (X. Zhang et al., 2006). Protamine deficiency may be considered as a contributing factor to DNA instability and damage (Boe-Hansen et al., 2018). The highly compacted structure of sperm chromatin is dependent on the number of disulfide bonds within and between protamines (Oliva, 2006). Assessment of free thiols and disulfide bonds in combination with protamine deficiency analysis may provide useful information regarding sperm chromatin compaction (Martínez-Pastor et al., 2010). However, there are disagreements between studies whether or not the degree of protamination of bull spermatozoa are associated with sperm chromatin integrity (Castro et al., 2018;Fortes et al., 2014). Fertilization of an oocyte by a sperm cell with damaged DNA may affect embryo development negatively and contribute to diseases in future generations, as the oocyte only has the ability to correct a certain degree of DNA damage (Johnson et al., 2011;Ménézo et al., 2010). Sperm cells deliver epigenetic components to the oocyte, essential to successful fertilization and embryonic development (Kropp et al., 2017). The term epigenetics refers to the study of heritable changes in gene expression that occur without altering the DNA sequence (McSwiggin & O'Doherty, 2018).
The most thoroughly studied epigenetic modification is DNA methylation, which has emerged as a promising indicator of male infertility (Kumaresan et al., 2020). The mechanism behind DNA methylation is an addition of a methyl group to the 5th carbon of a cytosine immediately followed by a guanine (CpG dinucleotide) (Alkhaled et al., 2018;Urrego et al., 2014). Sperm DNA methylation signatures have been associated with the fertility status of bulls (Kropp et al., 2017), buffalo (Verma et al., 2014), and men (Alkhaled et al., 2018). Studies on DNA methylation in bull spermatozoa are, however, still limited. Increased knowledge within the field would benefit livestock breeding by providing information regarding possible heritable traits and predisposition of diseases (Triantaphyllopoulos et al., 2016). Reduced representation bisulphite sequencing (RRBS) is a high-throughput and cost-efficient DNA methylation analysis method, allowing the study of DNA methylation at a single-base resolution (Doherty & Couldrey, 2014;Meissner et al., 2005). Analysis of DNA methylation using RRBS has previously been conducted for bovine somatic tissues and spermatozoa Perrier et al., 2018;Zhou et al., 2016).
Recently, we showed that the chromatin integrity parameters DNA fragmentation index (DFI) and high DNA stainability (HDS) in sperm cells from Norwegian Red bulls were significantly negatively associated with field fertility, expressed as 56-day non-return rate (NR56) . The aim of the present study was to evaluate several parameters related to sperm chromatin integrity in Norwegian Red bulls of contrasting NR56. The analyses included evaluation of protamine deficiency, free thiols, and disulfide bonds. In addition, in vitro embryo development and sperm DNA methylation signatures were assessed.

| Animals and semen processing
Cryopreserved semen samples were provided by the breeding company Geno (Geno Breeding and Artificial Insemination (AI) Association). Bulls were raised and fed uniformly and cared for according to the Norwegian Animal Welfare Act (LOV 2009-06-19 no. 97). The semen production procedures were in compliance with European Union Directive 88/407.
Sires were selected based on their field fertility performance, measured as NR56, as previously described by Narud et al. (2020). In brief, data on AIs were collected from the Norwegian Dairy Herd Recording System (NDHRS). The General Linear Model (PROC GLM in SAS ® ) was used to calculate least square mean (LSmean) NR56 for 507 Norwegian Red bulls used for AI in the period 2013-2018. The model included effects of bull, month and year of AI, parity of the female and repeated AI within 1-4 days. Based on these results, a group of 19 bulls with high NR56 (hereafter referred to as HF) and a group of 18 bulls with low NR56 (hereafter referred to as LF) were selected. LSmean NR56 ranged from 0.76 to 0.78 for HF and from 0.46 to 0.65 for LF (Table 1). All bulls were in regular semen production, with a mean (±SD) age corresponding to the collection date of ejaculates analyzed in vitro being 517 (±162) days for HF and 459 (±35) days for LF bulls. The age of the bulls in the two groups was not different (p > 0.05). For the in vitro production (IVP) of embryos and the analysis of sperm DNA methylation by RRBS, eight bulls with high and low NR56 were selected (marked by asterisk in Table 1).
Pedigree information was considered to avoid including closely related individuals.
Post collection, ejaculates with motility above 70% and morphological abnormalities below 15% were diluted to a final concentration of 12 × 10 6 spermatozoa per AI dose in French mini straws (IMV). A two-step dilution procedure with Biladyl ® extender containing glycerol (Minitube, 13500/0004-0006) and fresh egg yolk was applied. Cryopreservation was performed according to standard procedures (Standerholen et al., 2014) and the semen doses were stored in liquid nitrogen (−196°C) until use.

| Preparation of samples for protamine deficiency and thiol assays
Two semen straws per bull were thawed at 37°C for 12 s and mixed. For four of the bulls, only one straw was available. The concentration of spermatozoa was determined with a Nucleocounter SP-100 (ChemoMetec). Further, each sample was split in two, one part of the sample was diluted in 400 µl TNE-buffer (0.01 mol/L of Tris-HCl, 0.15 mol/L of NaCl, and 1 mmol/L of EDTA, pH 7.4) and used for analysis of protamine deficiency, while the other was diluted in 1100 µL TNE-buffer and used for analysis of thiols and disulfide bonds. In both sample preparations, the final concentration was 2 × 10 6 cells/ml.
Quadruplicates of both sample types were then snap-frozen in liquid nitrogen.

| Analysis of protamine deficiency
The level of sperm protamine deficiency was assessed using chromomycin A 3 (CMA3; Sigma-Aldrich), as described by Zubkova et al. (2005) with minor modifications. Briefly, snap-frozen samples were thawed on ice and washed with phosphate-buffered saline (PBS) by centrifugation (300 x g for 10 min). The resulting sperm pellet was resuspended in 80 μl McIlvaine's buffer (17 ml 0.1 mol/L citric acid mixed with 83 ml 0.2 mol/L Na 2 HPO 4 and 10 mmol/L MgCl 2 , pH 7.0) containing 0.25 mg/ml CMA3. Further, the samples were incubated in the dark for 20 min at 37°C and washed in 400 μl PBS by centrifugation (300 x g for 10 min). Pellets were resuspended in 400 μl PBS containing 3.2 μl propidium iodide (PI, 2.4 mM solution; Molecular Probes). A flow cytometer (FACSVerse; BD Biosciences) equipped with a blue laser (488 nm) was utilized for analysis of the samples. Gating of the sperm cell population was performed using forward scatter (FSC) and side scatter (SSC) and sperm cells were further identified by PI positive signal collected via 586/42 bandpass filter. After excitation with a violet laser (405 nm), the CMA3 fluorescence from gated cells was collected through a 528/45 bandpass filter.

| Analysis of thiols and disulfide bonds
Free thiols, total thiols, and disulfide bonds in bull spermatozoa were analyzed using monobromobimane (mBBr; Molecular Probes), according to the previously described method by Zubkova et al. (2005) and Seligman et al. (1994) with some modifications. Briefly, the samples were thawed on ice and divided into two tubes, each containing 1 × 10 6 sperm cells. The first tube was incubated with 1 mmol/L of 1,4dithiothreitol (DTT; Sigma-Aldrich) for 10 min at 37°C, while no DTT was added to the second tube. After centrifugation of both tubes (300 x g for 10 min), the pellets were resuspended in 100 μl PBS containing 0.5 mmol of mBBr solution. Both tubes were incubated in the dark for 10 min at 37°C, washed with 500 μl PBS twice by centrifugation (300 x g for 10 min). The pellets were resuspended in 500 μl PBS and analyzed with a FACSVerse flow cytometer (BD Biosciences) after excitation of mBBr by a 405 nm violet laser. The mBBr fluorescence was collected by a 528/45 bandpass filter, and gating of the sperm cell population was done using FSC and SSC. To calculate disulfide concentrations, the fluorescence signals of free thiols (mBBr fluorescence from non-DTT-treated sample) were subtracted from fluorescence signals of total thiols (mBBr fluorescence from DTT-treated sample), thereafter that value was divided by two. After the initial run, 4 µl PI (2.4 mM solution; Molecular Probes) was added to each sample, and the samples were analyzed again, this time also collecting the PI-fluorescence through a 586/42 bandpass filter. These data were used only as a further aid to discriminate debris from spermatozoa and were not used for quantitative purposes, since the PI was found to quench the mBBr-fluorescence.

| IVP of bovine embryos
IVP of embryos was conducted using media from IVF biosciences (Falmouth). Eight bulls with low NR56 and eight bulls with high NR56 were used for IVF. For each experiment, oocytes were randomly split into groups of 30 oocytes and two groups were fertilized with frozen-thawed semen from the same bull. Two LF and two HF bulls were used for fertilization in each test week. In addition, a reference bull with known IVF performance was used as control throughout the study. Each IVF experiment was repeated three times and a total of 180 oocytes per bull were included.
T A B L E 1 Field fertility (LSmean NR56), age (days) at semen collection for in vitro analyses and number of inseminations for the bulls used in the study

| IVF and culture
After maturation, the COCs were washed and transferred to new four-well plates containing 500 µl BO-IVF media (Falmouth), and kept in the incubator (38.8°C, 6% CO 2 , maximum humidity) while the semen samples were prepared. Two semen straws per bull were thawed in a water bath at 37°C for 1 min and the semen was transferred to the bottom of a 15 ml falcon tube containing preheated (36°C) BO-SemenPrep (Falmouth). The semen samples were centrifuged (330 x g for 5 min), the supernatant was removed, and 4 ml of BO-SemenPrep was added to the pellet. The centrifugation step was repeated, followed by removal of the supernatant, and computer-assisted sperm analysis (CASA) was used for assessing sperm motility and concentration. Sperm was added to each group of oocytes with a final concentration of 1 × 10 6 progressive motile spermatozoa/ml, followed by 18 h incubation (6% CO 2 , 38.8°C, maximum humidity). The presumptive fertilized oocytes were denuded by vortexing, before they were washed and transferred to culture plates containing 500 µl BO-IVC media (Falmouth) with oil overlay. Culture was performed in a humidified atmosphere of 7% O 2 , 6% CO 2 , and 87% N 2 at 38.8°C. At day 3 post-fertilization, the cleavage rate was recorded. Further, at day 8 the blastocyst rate was calculated based on the total number of oocytes. The number of cleaved cells that developed further into blastocysts was also recorded.

| RRBS library preparation and Illumina sequencing
Semen samples from eight bulls with low NR56 and eight bulls with high NR56 were utilized for the construction of RRBS libraries using a gel-free multiplexed technique (Boyle et al., 2012), previously optimized to study sperm DNA methylation in boar (Khezri et al., 2019) and bull . The protocol consisted of the following steps. The RRBS results were plotted using GraphPad Prism (v 6.01 for Windows, GraphPad Software). The Venny online platform (Oliveros, 2015) and ggplot2 package (v 3.1.0) in Rstudio (Wickham, 2016) were employed to construct Venn diagrams and plot pathway analysis results, respectively.

| Sperm chromatin integrity analyses
Sperm quality parameters related to chromatin integrity for the two fertility groups are presented in Table 2. As previously reported in Narud et al. (2020), higher percentages of DFI (p < 0.001) and HDS (p < 0.01) were observed in sperm cells from LF bulls compared to HF bulls. The mean levels of free thiols, total thiols, disulfide bonds, and protamine deficiency were not different (p > 0.05) for LF bulls compared with HF bulls.
The chromatin integrity parameters were further subjected to Pearson correlation analysis to identify possible relationships with NR56 and among parameters. As presented in Narud et al. (2020), DFI and HDS correlated negatively with NR56. Furthermore, the results presented in Table 3 shows that DFI and HDS correlated positively with each other and with protamine deficiency. The number of disulfide bonds was strongly (positively) correlated with total thiols.

| Basic statistics of RRBS libraries
An overview of the basic statistics for the RRBS libraries is presented in Cluster analysis according to CpG 5x methylation value showed that the samples were not clustered based on fertility performance ( Figure 2).
Furthermore, Pearson's correlation coefficient based on the same criteria revealed a high positive correlation between samples regarding CpG 5x methylation profile (Pearson's correlation coefficient > 0.94) (Table S1).

| Differential methylation analysis
By applying a significance cut-off equal to q value < 0.05, a total number of 16,542 DMCs were detected with varying degree of methylation, ranging from 0% to 75%. Majority of DMCs (56%) were found to be hypermethylated in the LF group compared to the HF group ( Figure 3a). After applying a 10% methylation cut-off, 65% of DMCs had over 10% methylation difference in both hypo-and hypermethylated groups (Figure 3b). Applying a 10% methylation cutoff resulted in 10,772 DMCs (filtered DMCs), which were further used for downstream analyses (Figure 3c).

| Annotation of DMCs with gene and CpG features
Annotation of filtered DMCs in both hypomethylation and hypermethylation groups with gene features revealed similar trends. For instance, 70% of the filtered DMCs were present in the intergenic regions followed by introns, exons, and promoters ( Figure 4a).

Furthermore, over 85% of filtered DMCs in both hypomethylation
and hypermethylation groups were annotated within regions outside of CpG islands (CGI)/CGI shores. Only around 15% of filtered DMCs were annotated within CGI/CpG shores (Figure 4b).
We were particularly interested in genes associated with biological processes related to fertility. Table 6 shows that the number of such identified genes were higher in the hypermethylation group compared with the hypomethylation group. Genes related to the penetration of zona pellucida was identified exclusively in the hypermethylation group. In both hypo-and hypermethylation groups biological processes including in utero embryo development, followed by fertilization and embryo implantation, were represented by the highest numbers of genes (Table S2).
Pathway analysis showed that the majority of identified mutual pathways between hypomethylated and hypermethylated groups exhibited similar p values ( Figure S1). Moreover, the majority of exclusively identified pathways (32 pathways) were in association with genes close to hypermethylated cytosines. Only five pathways were linked to genes close to hypomethylated cytosines ( Figure 6). Several pathways were exclusively identified in the hypermethylation group of test samples (LF bulls). This included hormonal pathways such as oxytocin signaling and ion-related signaling pathways such as calcium signaling, ion channel/ transport, and voltage-gated channel, which all play a direct role in the fertilization process. Furthermore, pathways involved in embryonic development such as developmental proteins and vascular endothelial growth factor (VEGF) signaling pathway were exclusively identified in the hypermethylation group of LF bulls.

| DISCUSSION
In the current study, various chromatin integrity parameters in semen samples from 37 Norwegian Red bulls of low and high NR56 were analyzed. Furthermore, 16 of these bulls were selected for IVP F I G U R E 1 Mean cleavage and blastocyst rates (SD given as whiskers) following in vitro fertilization of oocytes with sperm from bulls with high (HF) and low (LF) 56 days non-return rate (n = 8 in each group). Asterisks indicate a significant difference between the two fertility groups (p < 0.05) based on a linear mixed model. of embryos and investigation of sperm DNA methylation profiles by RRBS.
As we previously reported, significantly lower percentages of DFI and HDS were observed in sperm cells from bulls of high NR56 compared to bulls of low NR56 . In the present study, sperm DFI and HDS correlated positively with protamine deficiency. This corroborates the results of a previous study in bovine (Fortes et al., 2014). The protamine structure in the sperm nucleus is strongly stabilized through the formation of disulfide bonds between cysteine residues of adjacent protamine molecules (Oliva, 2006). Recently, our group discovered that DNA fragmentation had a significant but weak positive correlation with free thiols and disulfide bonds of boar spermatozoa (Khezri et al., 2019). In the present study, however, no associations were found between sperm thiols/disulfide bonds and DNA integrity or field fertility (NR56).
Spermatozoa from HF bulls displayed an increased ability to fertilize oocytes in vitro and resulted in significantly higher cleavage rates and total blastocyst rates than LF bulls. However, by measuring the number of cleaved cells that developed further into blastocysts, no differences were detected between the fertility groups. Our results corroborate the findings of Al Naib et al. (2011), which observed that once fertilization occurred the following embryo development into blastocysts was not influenced by the bulls' fertility status. Furthermore, Ward et al. (2001) observed that bull's field fertility measured as non-return rate after 150 days correlated with cleavage rate, while B. R. Zhang et al. (1997) found a positive correlation between NR56 and both cleavage and blastocyst rate. In contrast, Kropp et al. (2017) found no difference in cleavage or blastocyst rates between sires of contrasting fertility. The discrepancy between studies may be explained by factors such as the reliability of fertility data, number of ejaculates used per bull, how the experiments are conducted in lab and the range in fertility among the bulls included (Larsson & Rodrı́guez-Martı́nez, 2000).
The correlation analysis between NR56, chromatin integrity traits, and in vitro embryo development showed that both DFI and HDS were negatively correlated to the cleavage rate, but not to the blastocyst rate. This corroborated our findings that HF bulls had higher cleavage rates, but that the subsequent development of cleaved embryos into blastocysts was unaffected by bulls' fertility group. This is in agreement with the findings of a previous study, where sperm DNA damage, caused by increased oxidative stress, was found to affect cleavage rate (Simões et al., 2013). However, others have reported that DNA fragmentation in bull sperm does not impair IVF but rather the further embryonic development when the Note: Libraries were created from sperm DNA collected from bulls with high (HF) and low (LF) NR56 (n = 8 in each group). Clean reads were obtained after adapter and low-quality trimming of Illumina sequencing reads (total reads). Read coverage was calculated by dividing the number of bp in the clean reads by the number of bp at in silico MspI-digested bosTau9 genome. Mapping efficiency shows the percentage of clean reads that mapped with unique positions at bosTau9 genome. Global CpG methylation indicate the percentage of methylated CpGs regardless of depth in clean reads. Downstream analyses were performed based on CpGs with equal or more than 5x depth coverage. Bisulphite conversion rate shows the percentage of Cs that converted to uracil during the bisulphite conversion process.
blastocyst stage is reached (Fatehi et al., 2006). An important factor that may contribute to the observed results is the relatively low levels of DNA fragmentation in the Norwegian Red bulls of this study (ranging from 1.0% to 6.5%), which might be a result of selecting bulls with preferable semen quality for decades. It can be hypothesized that the level of fragmented DNA is within the range of damage that the oocytes manage to repair (García-Rodríguez et al., 2018).
However, such repair is usually followed by early embryonic death, implantation defect, chromosomal abnormalities, and higher abortion rate (Tesarik et al., 2004) In utero embryo development 30 33 levels of protamine deficiency in bovine spermatozoa (Castro et al., 2018;Kipper et al., 2017).
In this study, the average global CpG methylation level was 42.8%.
Recently, we reported similar results (global CpG methylation level of 40%) for bull sperm samples collected from young Norwegian Red bulls . These results are in agreement with previous studies, where average global CpG methylation levels of 35% and 45% in bull sperm cells have been reported, using RRBS and a luminometric methylation assay, respectively (Jiang et al., 2018;Perrier et al., 2018). In the present study, the differential methylation analysis showed higher level of CpG methylation (hypermethylation) in samples from bulls of low NR56 compared to bulls of high NR56. Similar results have previously been reported for humans, where 74% of DMCs were hypermethylated in infertile patients (Camprubí et al., 2016). In addition, the present findings are supported by a study in buffalo, where a higher number of genes were hypermethylated in sub-fertile compared with high-fertile bulls (Verma et al., 2014). In contrast, Kropp et al. (2017) detected a higher level of methylated regions in sperm cells from high fertility Holstein bulls. This may be explained by the different techniques used for the study of sperm DNA methylation.
The regional analyses showed that on average 70% of the filtered DMCs were present in intergenic regions and over 85% of filtered DMCs were annotated within regions outside of CGI/CGI shores. These results are in agreement with previous studies on sperm DNA in bull (Jiang et al., 2018;Khezri et al., 2020;Perrier et al., 2018) and boar (Khezri et al., 2019). However, the results for intergenic regions are in contrast with data published in infertile human patients where only 33% of identified DMCs were annotated with intergenic regions (Camprubí et al., 2016).
In this study, functional analysis was used to identify some of the gene's biological processes related to fertility. The results indicated that a high number of genes represent biological processes such as in utero embryo development, fertilization, and embryo implantation, in both hypo-and hypermethylation groups, but with most genes represented in the hypermethylation group. For instance, genes such as transition protein 2 (TNP2) and T-box transcription factor T (TBXT), involved in penetration of zona pellucida, were exclusively identified in the hypermethylation group. Previous studies in mice have reported a relationship between premature translation of TNP2 mRNA and the number of immobile and deformed sperm cells F I G U R E 6 Exclusively identified pathways for annotated genes to differentially methylated cytosines (DMCs) ≥ 10%. Pathways are plotted in function of their Benjamini corrected p value (x-axis) and fold enrichment (y-axis). Gene count size key shows the number of genes involved in each pathway. Hypo, hypomethylated cytosines (referring to transcriptional start sites [TSSs] annotated with hypomethylated cytosines in test group); Hyper, hypermethylated cytosines (referring to TSSs annotated to hypermethylated cytosines in test group) NARUD ET AL.
| 197 (Tseden et al., 2007), and that deletion of the TNP2 gene may result in less condensed sperm chromatin (Zhao et al., 2001). Data from Chinese Holstein bulls showed that the relative mRNA expression of TNP1 gene was significantly associated with the degree of sperm cell deformities (S. Zhang et al., 2015). Currently, there are no data available on the role of TBXT gene in male fertility; therefore, this would be a fruitful area for further research.
One of the challenges in interpreting functional analysis results is that genes could be involved in several biological functions, simultaneously. Although several genes involved in fertilization were identified in this study, only three genes were identified with a single and specific biological process related to fertilization. These genes were zona pellucida binding protein (ZPBP) and regulated endocrine specific protein 18 (RESP18) in hypermethylated group and glioma pathogenesis-related 1-like protein 1 (GLIPR1L1) in hypomethylated group. Previous studies clearly demonstrated the significant role of GLIPR1L1 in sperm-oocyte binding in mice and bovine (Caballero et al., 2012;Gaikwad et al., 2019;Gibbs et al., 2010). Furthermore, mice lacking ZPBP genes produced abnormal sperm cells with decreased ability to penetrate zona pellucida (Lin et al., 2007). In this study, the RESP18 gene was connected to in utero embryo development. Previous studies reported that RESP18 may have an important role in the development of nervous, cardiovascular, endocrine, renal, and reproduction systems (Atari et al., 2019). Furthermore, in agreement with our result, RESP18 was previously identified as hypermethylated in low fertile buffalo bulls (Verma et al., 2014).
We have identified pathways related to fertilization and embryonic development that might explain the lower fertility performance of bulls with low NR56. For instance, oxytocin signaling, calcium signaling, ion channel/transport, voltage-gated channel, developmental proteins, and VEGF signaling pathways were discovered exclusively in the hypermethylation group of the test group (bulls with low NR56). In addition, we have recently shown that several intracellular sperm amino acids and trace elements are associated with field fertility and suggested that metabolomics may be a useful tool in the identification of biomarkers for male fertility . In accordance with this hypothesis, metal binding and metabolic pathways were found exclusively in the hypermethylation group, which might also explain the lower fertility in LF bulls. However, due to the high degree of transcriptome inactivity of sperm cells and unknown reproductive competence of females in this study, conclusion of involved pathways/genes in fertility output of LF bulls must be done with caution.
In conclusion, we show here that sperm DNA integrity is significantly associated with IVF capacity of Norwegian Red bulls.
Spermatozoa of low-fertility bulls are hypermethylated compared with those of high-fertility bulls. Genes annotated to differentially methylated Cs were identified to participate in different biological pathways important for bull fertility.