Oxidative stress (OS) is a significant cause of DNA fragmentation and is associated with poor embryo development and recurrent miscarriage. The aim of this study was to compare two different methods for assessing seminal OS and their ability to predict sperm DNA fragmentation and abnormal semen parameters. Semen samples were collected from 520 men attending for routine diagnostic testing following informed consent. Oxidative stress was assessed using either a chemiluminescence assay to measure reactive oxygen species (ROS) or an electrochemical assay to measure oxidation reduction potential (sORP). Sperm DNA fragmentation (DFI) and sperm with immature chromatin (HDS) were assessed using sperm chromatin structure assay (SCSA). Semen analysis was performed according to WHO 2010 guidelines. Reactive oxygen species sORP and DFI are negatively correlated with sperm motility (p = 0.0012, 0.0002, <0.0001 respectively) and vitality (p < 0.0001, 0.019, <0.0001 respectively). The correlation was stronger for sORP than ROS. Reactive oxygen species (p < 0.0001), sORP (p < 0.0001), DFI (p < 0.0089) and HDS (p < 0.0001) were significantly elevated in samples with abnormal semen parameters, compared to those with normal parameters. Samples with polymorphonuclear leukocytes (PMN) have excessive ROS levels compared to those without (p < 0.0001), but sORP and DFI in this group are not significantly increased. DNA fragmentation was significantly elevated in samples with OS measured by ROS (p = 0.0052) or sORP (p = 0.004). The results demonstrate the multi-dimensional nature of oxidative stress and that neither assay can be used alone in the diagnosis of OS, especially in cases of leukocytospermia.

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml–1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

BackgroundIn both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip.ResultsConception rates for each of the four rounds of AI were within a normal range: 70–73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6.ConclusionsThis study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-234) contains supplementary material, which is available to authorized users.

BackgroundThe central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility.ResultsIn total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total).ConclusionsThe results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

Embryonic mortality is a major constraint to improving reproductive efficiency and profitability in livestock enterprises. We previously reported differential expression of genes with identified roles in cellular growth and proliferation, lipid metabolism, endometrial remodeling, inflammation, angiogenesis, and metabolic exchange in endometrial tissue on day 7 of the estrous cycle (D7), between heifers ranked as either high (HF) or low (LF) for fertility. The aim of the current study was to further elucidate the underlying molecular mechanisms contributing to early embryo loss by examining differential endometrial gene expression in HF or LF heifers at a later stage of the estrous cycle;day 14(D14). A second objective was to compare these expression profiles with those from midluteal HF and LF endometrium. Using the same animal model as employed in the previous study, we slaughtered HF and LF animals on D14, harvested endometrial tissue, and carried out global gene expression analysis using the Affymetrix Bovine GeneChip. Microarray analysis detected 430 differentially expressed genes (DEG) between HF and LF animals. Ingenuity Pathway Analysis revealed enrichment for a host of biological pathways including lipid metabolism, molecular transport, immune response, cell morphology and development, and cell growth and proliferation. Important DEG includedALB, BMPR2, CCL28, COL4A3/4, FADS1, ITGA6, LDLR, PLCB3, PPARG, PTGS2, and SLC27A4 Furthermore, DEG expressed on both D7 and D14 included:PCCB,SLC25A24,DAP, and COL4A4 This study highlights some of the pathways and mechanisms underpinning late luteal bovine endometrial physiology and endometrial-related conception rate variance.

Seminal oxidative stress (OS) is a major contributing factor to male infertility. Semen analysis cannot identify reactive oxygen species (ROS), which can be measured using a chemiluminescence assay. Measurement of redox potential provides a more comprehensive assessment of OS, although the test has yet to be fully validated. This study aimed to validate the MiOX sys analyser for measuring static oxidation-reduction potential (sORP). Results demonstrated that duplicate measurements must be taken, sensors must be batch tested, and sockets should be regularly changed to avoid inconsistency in measurement. Measurement of sORP using MiOX sys exhibited good reproducibility across different operators (p = 0.469), analysers (p = 0.963) and days (p = 0.942). It is not affected by mechanical agitation (p = 0.522) or snap freezing and thawing (p = 0.823). The stability of sORP over time requires further verification, particularly in samples with high initial sORP. Measurement is temperature sensitive between 2 and 37°C, significantly increasing with increasing temperature (p = 0.0004). MiOX sys is a more stable assay for assessing OS than chemiluminescence methods and permits greater flexibility for sample handling. MiOX sys could be implemented to complement semen analysis as part of routine diagnostic testing for male infertility and may be useful in identifying contributing factors to idiopathic infertility.

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of D-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-g, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, D-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 mM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration ( P , 0.01). Expression of D-9 desaturase mRNA was decreased five-and seven-fold, respectively, following 50 and 100 mM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6-and 18-fold in cells supplemented with 50 and 100 mM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both D-9 desaturase ( P , 0.001) and SREBP-1c ( P , 0.001), while a significant positive relationship was observed between D-9 desaturase and SREBP-1c gene expression ( P , 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both D-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

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