Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen

Summary Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P &lt; 0.05), while protamine deficiency and lipid peroxidation were higher (P &lt; 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P &lt; 0.05 and fold change &gt;1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.

Global Methylation and Protamine Deficiency in Ram Spermatozoa Correlate with Sperm Production and Quality but Are Not Influenced by Melatonin or Season

This study assessed whether the seasonal effects of melatonin that upregulate ram reproductive function alter sperm global methylation or protamine deficiency and whether these parameters corresponded to ram endocrinology, semen production and quality. Ejaculates were assessed from rams that received melatonin implants (n = 9) or no implants (n = 9) during the non-breeding season. Ejaculates (n = 2/ram/week) were collected prior to implantation (week 0), 1, 6 and 12 weeks post implantation and during the following breeding season (week 30). Flow cytometry was used to assess the sperm global methylation and protamine deficiency in each ejaculate, which had known values for sperm concentration, motility, morphology, DNA fragmentation, seminal plasma levels of melatonin, anti-Mullerian hormone and inhibin A. Serum levels of testosterone and melatonin were also evaluated. Though there was no effect of melatonin or season, sperm protamine deficiency was negatively correlated with sperm production and seminal plasma levels of anti-Mullerian hormone and positively correlated with sperm DNA fragmentation and morphology. Global methylation of spermatozoa was positively correlated with sperm DNA fragmentation, morphology and serum testosterone and negatively correlated with sperm motility. These moderate associations with sperm production and quality suggest that sperm protamine deficiency and global methylation are indicative of ram testicular function.

272 Proposing a combination of heritable fertility traits for bull selection

Traits such as sperm morphology and motility are routine in veterinarian evaluations of bull fertility. However, they rarely are included in livestock breeding programs, which typically use only scrotal circumference (SC) and some female traits for fertility selection. We studied 25 male fertility traits measured in two research populations of bulls (1,099 Brahman, and 1,719 Tropical Composite) and one commercial population (2,490 Santa Gertrude bulls). Measurements included standard semen evaluation (e.g. sperm motility and morphology) and SC. In the research data, we also measured sperm DNA fragmentation and sperm protamine deficiency for about 50% of the bulls. Using a mixture of genomic and pedigree analyses, we estimated heritabilities and genetic correlations for all traits, in each population. Our analyses suggest that bull fertility traits have a heritable component, which makes selective breeding possible. The phenotype variation in sperm DNA fragmentation and sperm protamine deficiency traits also have a heritable component (h2 ~ 0.05–0.22). These first estimates for heritability of sperm chromatin phenotypes require further studies, with larger datasets, to corroborate present results. In all three populations, we observed genetic correlations across traits that were favorable, but not high. For example, the percentage of normal sperm (PNS) from the sperm morphology evaluation was positively correlated with SC. In the research data, sperm DNA fragmentation was negatively correlated with PNS (r2 ~ 0.23–0.33), meaning that bulls with a higher PNS had less DNA fragmentation, being therefore more fertile according to both indicators. Given the favorable and yet not high genetic correlations between traits, it is possible to envision that sperm chromatin phenotypes might form a panel, together with PNS and SC, for a comprehensive bull fertility index. Selection indices that include fertility traits are being implemented in the dairy industry and could be recommended for beef cattle, too. An index that benefits from the favorable genetic correlations between traits that describe different aspects of bull fertility is a sensible approach to selective breeding. The clinical use of complementary indicators for male fertility is largely accepted, when deciding on bull fitness for the mating season. We propose extending this rationale to create a multi-trait index that captures genetic merit for bull fertility. In addition, we performed genome-wide association analyses in the research data and identified eight QTLs in the X chromosome. Correlations and shared SNP associations support the hypothesis that these phenotypes have the same underlying cause: abnormal spermatogenesis. In conclusion, it is possible to improve bull fertility through selective breeding, by measuring complementary fertility traits. Genomic selection for bull fertility might be more accurate if the X chromosome mutations that underlie the discovered QTL are included in the analyses. Polymorphisms associated with fertility in the bull accumulate in the X chromosome, as they do in humans and mice, thus suggesting specialization of this chromosome.

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.

Relationship of DNA integrity to HRG C633T SNP and ART outcome in infertile couples

The status of sperm DNA fragmentation, protamine deficiency, free thiols and disulphide bonds in colloid-selected samples and its relationship to ART outcome orHRGC633T SNP is not known. The objective of this study was to determine these relationships in spermatozoa from men with male factor or unknown factor infertility (n = 118) undergoingin vitrofertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Sperm DNA integrity was analysed by flow cytometry using three fluorescent probes (acridine orange, monobromobimane and chromomycin A3). Principal component analysis (PCA) was used to identify the parameters that most influenced fertility. The relationships of sperm DNA integrity with seminal parameters,HRGC633T SNP and ART outcome were established using ANOVA andt-test. Sperm concentration and yield after preparation accounted for 27% of the total variance; sperm DNA integrity (%DFI and disulphide bonds) accounted for 16% of the variance in men from infertile couples. Sperm %DFI was significantly higher (P < 0.05) in older men than in younger men. A significant difference (P < 0.01) was observed in %DFI between smokers and non-smokers. Sperm %DFI was significantly higher (P < 0.01) in male factor infertility compared to either female factor or unknown factor infertility while free thiols were significantly higher (P < 0.01) in unknown infertility factor. No significant difference was observed between IVF success/failure in any of the seminal parameters studied. There was a tendency for protamine deficiency to be higher and disulphide concentration to be lower in men withHRG633T. Such assessments may provide additional useful information about the prognosis for ART outcome, although more research is needed before clinical guidelines can be provided.