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.

Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility.

In the dairy breeding industry, prediction of bull fertility in artificial insemination (AI) is important for efficient and economically sustainable production. However, it is challenging to identify bulls with superior fertility applying conventional in vitro sperm assays. In the present study, sperm functionality was investigated to identify a multivariate model that could predict fertility. Two groups of young Norwegian Red bulls were selected, one with inferior fertility (18 bulls) and one with superior fertility (19 bulls) based on non-return rate after 56 days (NR56). Frozen-thawed semen doses were analysed for sperm chromatin integrity, viability, acrosome integrity, motility, and ATP content. A targeted approach was used to study intracellular concentrations of amino acids and trace elements in viable sperm cells. Significant differences between the two groups of bulls were observed, both for sperm functional attributes and intracellular concentrations of metabolites. Pearson correlation analyses indicated a negative relationship between NR56 and chromatin integrity parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS). Several motility parameters correlated positively with NR56. The concentrations of cysteine and glutamic acid in sperm cells correlated negatively with NR56, while the concentrations of aspartic acid, leucine and serine showed a positive NR56-correlation. The sperm intracellular concentrations of the trace elements Fe, Al and Zn, correlated negatively with NR56. Correlations were observed between several sperm parameters and metabolites. Stepwise multiple regression analysis indicated that the best predictor of NR56 was a model containing %DFI, together with the intracellular sperm concentration of aspartic acid, Fe and Zn. This model explained 59% of the variability in NR56.

Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls.

Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation® RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.

Viability, motility, ATP content and fertilizing potential of sperm from Atlantic salmon (Salmo salar L.) in milt stored before cryopreservation.

Artificial fertilization is increasingly used in aquaculture, mostly applying short-term cold stored milt. Large scale cryopreservation of milt could be valuable for increased flexibility and acceleration of breeding progress. The aim of this study was to assess viability, motility and ATP content of sperm from Atlantic salmon as a function of storage time, before and after cryopreservation. The objective was also to investigate whether in vitro parameters were associated with sperm fertilizing ability after cryopreservation. Milt from six mature Atlantic salmon males were collected twice, one week apart. The milt was stored undiluted at 5 °C in cell culture flasks for six days. Samples were taken on days 1, 3 and 6 of storage for cryopreservation. In total, 36 batches were diluted to a standardized sperm concentration of 2 × 109 spermatozoa/mL, filled into 0.5 mL French medium straws and cryopreserved. In vitro analyses were assessed on the same sample for the 72 combinations of male, collection week, days of storage and cold stored or frozen-thawed. Fertilization trials with cryopreserved milt were carried out for all 36 batches in triplicate for each combination of male, collection week, storage time and sperm:egg ratios of either 2 or 4 × 106 sperm per egg, respectively, totally 218 experimental units, including two egg controls. There was a significant influence of storage and collection week on sperm quality parameters, both cold stored and cryopreserved, and cryopreservation had a significant effect on all tested sperm quality parameters. High correlations for cold stored vs cryopreserved samples was demonstrated for ATP content (p < 0.00001), motility and velocity parameters (p < 0.001), but not for viability, straightness and linearity. The overall percentage of fertilization achieved was 73.9 ± 1.7%. Sperm collected in week 2 showed significantly lower fertility when cryopreserved after six days of storage than after 1 or 3 days for sperm to egg ratios of 2 × 106 (p < 0.005), while there was no such effect for milt collected in week 1. Several post-thaw sperm parameters were correlated to fertilization rates, while curvilinear velocity best explained variations in fertilization by modelling. Our results suggest that cryopreservation of Atlantic salmon milt should be performed soon after milt collection to maximize the cryopreserved sperm quality. Fertilization results seems not to be compromised by storage for three days before cryopreservation.

DNA methylation patterns vary in boar sperm cells with different levels of DNA fragmentation.

BACKGROUND: Sperm DNA integrity is considered essential for successful transmission of the paternal genome, fertilization and normal embryo development. DNA fragmentation index (DFI, %) has become a key parameter in the swine artificial insemination industry to assess sperm DNA integrity. Recently, in some elite Norwegian Landrace boars (boars with excellent field fertility records), a higher level of sperm DFI has been observed. In order to obtain a better understanding of this, and to study the complexity of sperm DNA integrity, liquid preserved semen samples from elite boars with contrasting DFI levels were examined for protamine deficiency, thiol profile and disulphide bonds. Additionally, the DNA methylation profiles of the samples were determined by reduced representation bisulphite sequencing (RRBS). RESULTS: In this study, different traits related to sperm DNA integrity were investigated (n = 18 ejaculates). Upon liquid storage, the levels of total thiols and disulphide bonds decreased significantly, while the DFI and protamine deficiency level increased significantly. The RRBS results revealed similar global patterns of low methylation from semen samples with different levels of DFI (low, medium and high). Differential methylation analyses indicated that the number of differentially methylated cytosines (DMCs) increased in the low-high compared to the low-medium and the medium-high DFI groups. Annotating the DMCs with gene and CpG features revealed clear differences between DFI groups. In addition, the number of annotated transcription starting sites (TSS) and associated pathways in the low-high comparison was greater than the other two groups. Pathway analysis showed that genes (based on the closest TSS to DMCs) corresponding to low-high DFI comparison were associated with important processes such as membrane function, metabolic cascade and antioxidant defence system. CONCLUSION: To our knowledge, this is the first study evaluating DNA methylation in boar sperm cells with different levels of DFI. The present study shows that sperm cells with varying levels of DNA fragmentation exhibit similar global methylation, but different site-specific DNA methylation signatures. Moreover, with increasing DNA fragmentation in spermatozoa, there is an increase in the number of potentially affected downstream genes and their respective regulatory pathways.

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size.

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n = 693) and Norwegian Duroc (ND; n = 655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p < 0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p < 0.0001), parity (p < 0.05) and DFI (p < 0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p < 0.01). The DFI and the motility of the same semen samples were negatively correlated (p < 0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p = 0.0038 and 92.3% vs. 89.5%; p = 0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.

Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

RNA sequencing reveals candidate genes and polymorphisms related to sperm DNA integrity in testis tissue from boars.

BACKGROUND: Sperm DNA is protected against fragmentation by a high degree of chromatin packaging. It has been demonstrated that proper chromatin packaging is important for boar fertility outcome. However, little is known about the molecular mechanisms underlying differences in sperm DNA fragmentation. Knowledge of sequence variation influencing this sperm parameter could be beneficial in selecting the best artificial insemination (AI) boars for commercial production. The aim of this study was to identify genes differentially expressed in testis tissue of Norwegian Landrace and Duroc boars, with high and low sperm DNA fragmentation index (DFI), using transcriptome sequencing. RESULTS: Altogether, 308 and 374 genes were found to display significant differences in expression level between high and low DFI in Landrace and Duroc boars, respectively. Of these genes, 71 were differentially expressed in both breeds. Gene ontology analysis revealed that significant terms in common for the two breeds included extracellular matrix, extracellular region and calcium ion binding. Moreover, different metabolic processes were enriched in Landrace and Duroc, whereas immune response terms were common in Landrace only. Variant detection identified putative polymorphisms in some of the differentially expressed genes. Validation showed that predicted high impact variants in RAMP2, GIMAP6 and three uncharacterized genes are particularly interesting for sperm DNA fragmentation in boars. CONCLUSIONS: We identified differentially expressed genes between groups of boars with high and low sperm DFI, and functional annotation of these genes point towards important biochemical pathways. Moreover, variant detection identified putative polymorphisms in the differentially expressed genes. Our results provide valuable insights into the molecular network underlying DFI in pigs.

Gene expression of TLR homologues identified by genome-wide screening of the earthworm Dendrobaena veneta.

TLRs represent one of the most important components of innate immunity. Currently, these receptors have been extensively studied in vertebrates and insects, but our knowledge for annelids is very limited. Therefore, the aim of our study was to identify earthworm TLR homologs by genome-wide screening, and to determine the expression of candidate genes as a response to Gram-positive and Gram-negative bacteria. Using a combination of deep pyrosequencing and quantitative PCR we found six candidate genes, for which all were expressed in Dentrobaena veneta. Two of the candidates showed significant response to bacterial exposure. In conclusion, TLRs seem to have a role in earthworm immunology.