Missense mutation within cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with selected parameters of the frozen-thawed sperm in Holstein-Friesian bulls.

In our previous Genome-wise Association Study we found that Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) is a candidate gene for sperm motility in fresh semen of Holstein-Friesian bulls. Since in cows thawed semen is commonly used for the artificial insemination (AI) we have decided to find out whether functional polymorphism within CFTR gene coding sequence is associated with selected parameters of thawed sperm, including their motility evaluated by computer-assisted sperm analysis (CASA), the activity of three antioxidant enzymes: glutathione peroxidase (GPx) catalase (CAT), superoxide dismutase (SOD), ATP content and integrity of sperm membranes. One hundred twenty Holstein Friesian bulls kept in uniform environmental conditions (one AI company) were included in the study. Significant associations between genotypes of missense mutation within exon 11 of the CFTR gene (Met468Leu) and the activity of antioxidant enzymes and sperm mitochondrial function were revealed. No effect of CFTR genotypes on sperm motility was observed. Significant differences in CAT and SOD activity were found between AA and TT homozygous individuals. Bulls with TT genotype had the lowest activity of both antioxidant enzymes. The same bulls also showed the lowest number of sperm with active mitochondria. Our results demonstrate that missense mutation Met468Leu within CFTR gene is associated with antioxidant enzyme activity and mitochondrial function of bovine thawed sperm without affecting their motility.

Influence of splicing mutation within the lysophosphatidic acid receptor 1 gene (LPAR1) on semen quality in Holstein-Friesian bulls.

Effect of single nucleotide polymorphism (SNP) in splicing site of the LPAR1 (lysophosphatidic acid receptor 1) gene on selected quality traits was investigated in frozen-thawed semen of Holstein-Friesian bulls. Splicing mutation A/G in the LPAR1 gene (rs43581860) was identified in 120 Holstein-Friesian bulls using PCR-RFLP technique (Hph I). Heterozygotes AG were the most frequent (37.5%) compared with AA (30.8%) and GG (31.7%) homozygotes. Observed differences in total motility (TM), sperm membrane integrity (SYBR-14/PI) and ATP content were significant between homozygotes AA or GG and heterozygotes AG. For all three traits disadvantageous effect of heterozygotes AG was detected. This means that LPAR1 splicing mutation has significant effect on semen quality and should be considered as a new marker of semen quality in Holstein-Friesian bulls.

Assessment of genomic inbreeding in Polish Konik horses.

The aim of this study was to assess the inbreeding coefficient of Polish Konik horses based on runs of homozygosity (ROH). Ninety six horses kept in 6 herds located across Poland were genotyped with the use of EquineSNP60 BeadChip (Illumina). SNP markers with a Minor Allele Frequency lower than 0.01 and SNPs assigned to chromosome X or Y were excluded from the study. A total of 50 708 SNPs were included for statistical analysis (SVS software, Golden Helix). The analysis showed that the population is in genetic equilibrium, with He and Ho estimates both equal to 0.3086. Seven categories of Runs of Homozygozity (ROH) length were defined: >0.5, >1, >2, >4, >8, >16, >25 Mb. The genomic inbreeding coefficient derived from ROH (FROH) calculated for each ROH length ranged from 15.96% based on the shortest ROH (>0,5Mb) to 2.71% for the longest ROH (>25Mb). Among individual horses, the inbreeding coefficient ranged from 5.25% to 22.41% (for ROH >1Mb). Analysis of ROH in Polish Koniks allows for more effective management of their inbreeding in the future.

A polymorphism in the norepinephrine transporter gene is associated with affective and cardiovascular disease through a microRNA mechanism.

Norepinephrine released from sympathetic nerves is removed from the neuroeffector junction via the action of the norepinephrine transporter (NET). NET impairment is evident in several clinically important conditions including major depressive disorder (MDD), panic disorder (PD), essential hypertension and the postural orthostatic tachycardia syndrome (POTS). We aimed to determine whether a single nucleotide polymorphism (SNP) in the 3' untranslated region (UTR) of the NET gene is associated with NET impairment and to elucidate the mechanisms involved. The analyses were carried out in two cohorts of European ancestry, which included healthy controls and MDD, PD, hypertensive and POTS patients. Compared with controls, cases had significantly higher prevalence of the T allele of rs7194256 (C/T), arterial norepinephrine, depression and anxiety scores, larger left ventricular mass index, higher systolic and diastolic blood pressures, and heart rate. Bioinformatic analysis identified that the microRNA miR-19a-3p could bind preferentially to the sequence created by the presence of the T allele. This was supported by results of luciferase assays. Compared with controls, cases had significantly lower circulating miR-19a-3p, which was associated with pathways related to blood pressure and regulation of neurotransmission. In vitro norepinephrine downregulated miR-19a-3p. In conclusion, the T allele of the rs7194256 SNP in the 3'UTR of the NET gene is more prevalent in diseases where NET impairment is evident. This might be explained by the creation of a binding site for the microRNA miR-19a-3p. A defect in NET function may potentiate the sympathetic neurochemical signal, predisposing individuals with affective diseases to increased risk of cardiovascular disease development.

Soluble vascular endothelial growth factor receptor-1 is reduced in patients with resistant hypertension after renal denervation.

Renal denervation (RDN) has been shown in several studies to reduce blood pressure (BP) in patients with resistant hypertension (RH). Data on potential biomarkers associated with BP changes remain scarce. We evaluated whether soluble vascular endothelial growth factor receptor (sVEGFR-1) is affected by the procedure. A total of 57 patients with RH participated in this study. BP and heart rate were recorded at baseline and at 3 months follow-up, at which time blood samples were collected to determine the levels of sVEGFR-1, VEGF-A, VEGF-C, nitric oxide (NO), soluble vascular adhesion molecule 1 and soluble intracellular adhesion molecule 1. None of the biomarkers had a predictive value that could identify responders vs non-responders to RDN. However, sVEGFR-1 concentration was dramatically reduced after RDN (5913±385 vs 280±57 pg ml-1, P<0.001). At the same time VEGF-A levels were significantly increased (10.0±3.0 vs 55.5±7.9 pg ml-1, P<0.001), without significant changes in VEGF-C. NO levels were significantly increased after RDN in the whole group (82.6±6.2 vs 106.9±7.8 µM, P=0.021). Interestingly, the elevation in NO levels at 3 months was only seen in patients who demonstrated a reduction in systolic BP of ⩾10 mm Hg (78.9±8.3 vs 111.6±11.7 µM, P=0.018). We report a significant reduction in sVEGFR-1 levels after RDN procedure, which was accompanied by a significant increase in VEGF-A concentration as well as NO. Changes in plasma cytokines were not quantitatively linked to magnitude of BP reduction. An RDN-induced reduction in sVEGFR-1 plasma levels and increase in VEGF-A would raise the VEGF-A/sVEGFR-1 ratio, thereby increasing VEGF-A bioavailability to act on its full-length receptor and may contribute to the BP-lowering effect potentially via NO-mediated pathways.

Association between SOX5 Genotypes and Semen Quality in Polish Holstein-Friesian Bulls.

The aim of this study was to evaluate the effect of C/T missense mutation (SOX5) gene on selected parameters of non-frozen semen in Polish Holstein - Friesian bulls. Three hundred and sixty eight bulls were genotyped by the PCR-Rsa I technique. Semen was collected and evaluated by standardized procedure used in an AI station. Significant associations were found between SOX5 genotypes and sperm concentrations (p=0.020) and sperm motility (p=0.014). Bulls with TT genotype had the lowest values of both traits. Our results demonstrate that C/T missense mutation within the SOX5 gene is involved in bull sperm quality.

Genome-wide association study for host response to bovine leukemia virus in Holstein cows.

The mechanisms of leukemogenesis induced by bovine leukemia virus (BLV) and the processes underlying the phenomenon of differential host response to BLV infection still remain poorly understood. The aim of the study was to screen the entire cattle genome to identify markers and candidate genes that might be involved in host response to bovine leukemia virus infection. A genome-wide association study was performed using Holstein cows naturally infected by BLV. A data set included 43 cows (BLV positive) and 30 cows (BLV negative) genotyped for 54,609 SNP markers (Illumina Bovine SNP50 BeadChip). The BLV status of cows was determined by serum ELISA, nested-PCR and hematological counts. Linear Regression Analysis with a False Discovery Rate and kinship matrix (computed on the autosomal SNPs) was calculated to find out which SNP markers significantly differentiate BLV-positive and BLV-negative cows. Nine markers reached genome-wide significance. The most significant SNPs were located on chromosomes 23 (rs41583098), 3 (rs109405425, rs110785500) and 8 (rs43564499) in close vicinity of a patatin-like phospholipase domain containing 1 (PNPLA1); adaptor-related protein complex 4, beta 1 subunit (AP4B1); tripartite motif-containing 45 (TRIM45) and cell division cycle associated 2 (CDCA2) genes, respectively. Furthermore, a list of 41 candidate genes was composed based on their proximity to significant markers (within a distance of ca. 1 Mb) and functional involvement in processes potentially underlying BLV-induced pathogenesis. In conclusion, it was demonstrated that host response to BLV infection involves nine sub-regions of the cattle genome (represented by 9 SNP markers), containing many genes which, based on the literature, could be involved to enzootic bovine leukemia progression. New group of promising candidate genes associated with the host response to BLV infection were identified and could therefore be a target for future studies. The functions of candidate genes surrounding significant SNP markers imply that there is no single regulatory process that is solely targeted by BLV infection, but rather the network of interrelated pathways is deregulated, leading to the disruption of the control of B-cell proliferation and programmed cell death.

Missense mutation in glutathione-S-transferase M1 gene is associated with sperm motility and ATP content in frozen-thawed semen of Holstein-Friesian bulls.

Glutathione-S-transferase genes (GSTs) encode enzymes that are involved in detoxification and neutralization of reactive oxygen species (ROS) in male reproductive system and play protective role during spermatogenesis. The aim of the study was to evaluate whether C/G missense mutation (rs135955605) within glutathione-S-transferase M1 (GSTM1) gene is associated with selected parameters of frozen-thawed semen in 309 Holstein-Friesian bulls. Single nucleotide substitution C/G was identified by amplification of GSTM1 gene fragment followed be digestion with restriction enzyme DdeI. Bulls with GG genotype were the most frequent (67.96%), in comparison to CC (2.59%) and GC (29.45%). Significant associations were found between GSTM1 genotypes and ATP content and total sperm motility. Bulls with GG genotype had the highest values for both traits. Rare variant C of GSTM1 was associated with significant decrease of sperm motility and ATP content. Our results demonstrate that C/G missense mutation within GSTM1 gene is involved in bull sperm quality.

Selected qualitative and biochemical parameters of cryopreserved semen of Holstein-Friesian (HF) AI bulls.

Selected qualitative and biochemical parameters were determined in cryopreserved semen used for artificial insemination, sampled from 120 bulls reared at the Animal Breeding and Insemination Center in Bydgoszcz. The total average motility of the analyzed sperm samples was determined at 62.51%. The percentage of motile spermatozoa displaying progressive forward motility was 21.65%. Analyzed samples were characterized by a high percentage of sperm cells with a intact plasma membrane (71.21%) and active mitochondria (71.32%). High efficiency of the enzymatic antioxidant system of the evaluated sperm cells was demonstrated by high activity of CAT, GPx and SOD (494.37, 2847.83 and 5.31U/1x10(9) spermatozoa, respectively) values and low values of the DNA Fragmentation Index (9.32). The results of the study, obtained with the involvement of advanced analytical methods, indicate a high fertilizing capability of the analyzed sperm samples.

Association between ETFA genotype and activity of superoxide dismutase, catalase and glutathione peroxidase in cryopreserved sperm of Holstein-Friesian bulls.

The aim of this study was to determine whether C/T missense mutation within the ETFA gene is associated with sperm antioxidant enzymatic activity. One hundred and twenty Holstein-Friesian bulls were genotyped by the PCR-RFLP technique (MwoI). Commercial straws of frozen-thawed semen were used to evaluate the activity of three antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase. Among all bulls investigated, genotype CT was the most frequent (44.2%), in comparison with CC (42.5%) and TT (13.3%). Significant differences in glutathione peroxidase activity were observed between homozygous individuals (CC vs TT) with heterozygous CT having intermediate values. Dismutase activity was significantly associated with ETFA genotype, although only bulls with the CT genotype were significantly different from bulls carrying the CC genotype. The activity of catalase showed a similar trend (but was not statistically significant). In conclusion, we found that bulls with the ETFA TT genotype produce sperm with the highest glutathione peroxidase activity and can therefore be more efficiently protected from reactive oxygen. The mechanism of this interaction needs to be elucidated in future research.

Genome-wide association study for semen volume and total number of sperm in Holstein-Friesian bulls.

In artificial insemination industry bulls producing high volume of semen with relatively high concentration of sperm are very desirable since they ensure stable production of commercial straws especially in case of top bulls. The aim of the study was to screen the entire bull genome to identify markers and candidate genes underlying semen volume (SV) and total number of sperm (TNS) in ejaculate produced by Holstein-Friesian bulls. Data on semen production were retrieved from records of AI center and included a population of 877 Holstein-Friesian bulls. Each bull was genotyped using the Illumina BovineSNP50 BeadChip. Genome-wide association analysis was performed with the use of GoldenHelix SVS7 software. An additive model for Linear Regression Analysis was used to estimate the effect of SNP marker for SV and TNS. After Bonferroni correction, 3 markers located on chromosome 22 reached the highest significance (rs41625599, rs41584616, rs42012507) for both traits. In the vicinity of these significant markers 3 genes are located (DCP1A, SFMBT1, TMEM110). Moreover, marker rs110109069 located on chromosome 25 was significantly associated with TNS and marker rs42438348 located on chromosome 10 has been found to be associated with SV. Some additional candidate genes were suggested to be potentially involved in analyzed traits (GALC, PRKCD, PHF7, TLR9, SPATA7). Identifying SNPs associated with the lower total number of sperm may be very useful for early recognition of a young sire as less suitable for effective semen production in artificial insemination centers.

Single nucleotide polymorphism within arylsulfatase D gene (ARSD) is associated with selected kinematic parameters of sperm motility in Holstein-Friesian bulls.

The aim of the study was to find out whether the single nucleotide polymorphism (SNP) within arylsulfatase D (ARSD) gene is associated with kinematic parameters of sperm motility in Holstein-Friesian bulls. 367 Holstein-Friesian bulls kept in one AI center were included in the study. Point mutation C/T at position 139037255 on chromosome X (rs42207167) was identified by PCR-RFLP method (Pflm I). Significant associations were found between ARSD genotypes and CASA-derived sperm motility parameters: average TM (Total Motility), average VSL (Straight Velocity), average VCL (Curvilinear Velocity) and for fraction of sperms showing progressive motility (a) of sperms (VSLa, VCLa and BCFa -Beat Cross Frequency). Most significant differences were observed between alternative homozygotes (CC vs TT). Our results suggest new role of arylsulfatase D gene as being involved in sperm motility.

Genome-wide association study for sperm concentration in Holstein-Friesian bulls.

The aim of the study was to screen the entire bull genome to identify markers and candidate genes underlying sperm concentration. The analysed data set originates from a population of 877 Polish Holstein-Friesian bulls. Based on sperm concentration value, two extreme groups of bulls were created: Low (L, n = 126) and High (H, n = 140). Each bull was genotyped using the Illumina BovineSNP50 BeadChip. Genome-wide association analysis was performed with the use of GoldenHelix SVS7 software. An additive model with a Cohran-Armitage test, Correlation/Trend adjusted by a Bonferroni test, was used to estimate the effect of SNP marker for sperm concentration. Thirteen markers reached genome-wide significance. The most significant SNPs were located on chromosome 3 (rs109154964 and rs108965556), 14 (rs41621145) and 18 (rs41615539), in the close vicinity of protein arginine methyltransferase 6 (PRMT6), Sel1 repeat containing 1 (SELRC1), triple QxxK/R motif containing (TRIQK) and zinc finger homeobox 3 (ZFHX3) genes, respectively. For three other candidate genes located close to significant markers (within a distance of ca 1 Mb), namely histone deacetylase 9 (HDAC9), an inhibitor of DNA binding 2 (ID2) and glutathione S-transferase theta 1 (GSTT1), their potential role in the production of male germ cells was confirmed in earlier studies. Six additional candidate genes (Vav3, GSTM1, CDK5, NOS3, PDP1 and GAL3ST1) were suspected of being significantly associated with sperm concentration or semen biochemistry. Our results indicate the genetic complexity of sperm concentration but also open the possibility for finding causal polymorphism useful in marker-assisted selection.

Genome-wide association study for poor sperm motility in Holstein-Friesian bulls.

The aim of the study was to screen the whole bull genome to identify markers and candidate genes underlying poor sperm motility. The analyzed data set originates from the Polish Holstein-Friesian bull population and consists of 41 Case and 279 Control bulls (selected from 1581 bulls). The most distinguishing trait of case group was very poor sperm motility (average 25.61%) when compared to control samples (average 72.95%). Each bull was genotyped using the Illumina BovineSNP50 BeadChip. Genome-wide association analysis was performed with the use of GoldenHelix SVS7 software. An additive model with a Cohran-Armitage test, Correlation/Trend adjusted by Bonferroni test were used to estimate the effect of Single Nucleotide Polymorphism (SNP) marker for poor sperm motility. Markers (n=34) reached genome-wide significance. The most significant SNP were located on chromosome 24 (rs110876480), 5 (rs110827324 and rs29011704), and 1 (rs110596818), in the close vicinity of melanocortin 4 receptor (MC4R), PDZ domain containing ring finger 4 (PDZRN4) and ethanolamine kinase 1 (ETNK1), olfactory receptor 5K3-like (LOC785875) genes, respectively. For five other candidate genes located close to significant markers (in distance of ca. 1 Mb), namely alkaline phosphatase, liver/bone/kidney (ALPL), tripartite motif containing 36 (TRIM36), 3-hydroxyisobutyrate dehygrogenase (HIBADH), kelch-like 1 (KLHL1), protein kinase C, beta (PRKCB), their potential role in sperm motility was confirmed in the earlier studies. Five additional candidate genes, cystic fibrosis transmembrane conductance regulator (CFTR), insulin-like growth factor 1 receptor (IGF1R), steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2), cation channel, sperm associated 1 (CATSPER1) calpain 1 (mu/I) large subunit (CAPN1) were suggested to be significantly associated with sperm motility or semen biochemistry. Results of the present study indicate there is a genetic complexity of poor sperm motility but also indicate there might be a causal polymorphism useful in marker-assisted selection. Identifying genomic regions associated with poor sperm motility may be very important for early recognition of a young sire as unsuitable for effective semen production in artificial insemination centers.

Screening of Polish Holstein-Friesian bulls towards eradication of complex vertebral malformation (CVM) carriers.

The effectiveness of a program aimed at eradicating carriers of the recessive disorder Complex Vertebral Malformation (CVM) from the population of Holstein-Friesian bulls is reported. Among 1823 bulls, 1268 young and 555 proven bulls were examined. Three hundred and three bulls appeared to be CVM carriers (16.62%). The highest number of carriers occurred in the sons of a CVM sire, 55.51% and 61.90%, for proven and young bulls, respectively. This very high incidence of CVM carriers forced us to implement a strategy of screening young bulls offered by individual breeders to insemination centers. In effect, the number of CVM carriers dramatically dropped among proven bulls born in 2004 and disappeared in bulls born in 2006.

River restoration success: a question of perception.

What defines success and failure of river restoration measures is a strongly debated topic in restoration science, but standardized approaches to evaluate either are still not available. The debate is usually centered on measurable parameters, which adhere to scientific objectivity. More subjective aspects, such as landscape aesthetics or recreational value, are usually left out, although they play an important role in the perception and communication of restoration success. In this paper, we show that different perceptions of restoration success exist by analyzing data from 26 river restoration measures in Germany. We addressed both objective parameters, such as hydromorphological changes and changes in fish and benthic invertebrate assemblages, from field investigations, and subjective parameters, such as opinions and perceptions, from water managers via an online survey. With regard to the objective hydromorphological and biotic parameters, our results agree with many studies that have reported improvements in the hydromorphology following restoration; however, there is no similar agreement between results concerning changes in the benthic invertebrate and fish assemblages. The objective results do not correspond to the subjective parameters because self-evaluation of the restoration projects by water managers was overly positive. Indeed, 40% of the respondents admitted that their evaluation was based on gut feeling, and only 45% of the restoration measures were monitored or occasionally checked. This lack of objectively recorded data meant that the water managers were not able to reasonably evaluate restoration success. In contrast, some self-evaluation responses reflected a different perception of the restoration success that was based on landscape aesthetic values or on benefit for the public; others adopted a general "condemned to success" attitude. Based on our data, we argue (1) that goals should be thoughtfully formulated prior to restoration implementation and (2) that it is necessary to monitor river restoration success from different perspectives.

Adaptive servoventilation in diastolic heart failure and Cheyne-Stokes respiration.

A high prevalence of nocturnal Cheyne-Stokes respiration (CSR) has been documented in patients with heart failure with normal left ventricular ejection fraction (HFNEF). The aim of the present study was to investigate the effects of adaptive servoventilation (ASV) for treatment of CSR in these patients. In 60 patients with HFNEF, defined according to current European Society of Cardiology guidelines, CSR was documented by polysomnography (apnoea/hypopnoea index (AHI) of >15 events x h(-1)). ASV treatment was offered to all patients; 21 initially rejected treatment, withdrew from treatment or presented noncompliant during follow-up (controls), whereas ongoing ASV therapy was initiated in 39 patients (ASV group). Echocardiography, cardiopulmonary exercise testing and measurement of N-terminal-pro-brain natriuretic peptide were performed at baseline and follow-up (11.6+/-3 months). ASV therapy led to a significant reduction in AHI, longest apnoea and hypopnoea length, maximum and mean oxygen desaturation by pulse oximetry, percentage of study time with an oxygen saturation of <90% and arousal index. In addition, significant positive effects could be confirmed on absolute and predicted peak oxygen consumption, oxygen consumption at the individual aerobic-anaerobic threshold, oxygen pulse, as well as left atrial size, and transmitral flow patterns (mean early diastolic lengthening velocity and the ratio of peak early Doppler mitral inflow velocity to this lengthening velocity). ASV effectively attenuates CSR in patients with HFNEF and improves heart failure symptoms and cardiac function. Whether or not this is accompanied by an improved prognosis remains to be determined.

New aspects on the assessment of left ventricular dyssynchrony by tissue Doppler echocardiography: comparison of myocardial velocity vs. displacement curves.

The purpose of this study is to assess the incremental value of tissue Doppler (TDI) derived displacement curves (TDint) compared to TDI velocity curves (TDvel) for the evaluation of left ventricular (LV) dyssynchrony (LVD). About 25 patients (pts.) with systolic heart failure were studied by TDvel and TDint. Four TDI sample volumes were placed at the basal and four at the mid ventricular myocardium, utilising two imaging planes. LV dyssynchrony (LVD) was defined as an interregional delay of >40 ms corrected for heart rate. 10 pts. had synchronous contraction, 15 pts. LVD as defined by two experts (EC). To determine diagnostic accuracy and intra-observer variability two identical sets of 100 documents (25 pts. x two imaging planes x two modalities) were produced and presented in random order to one trained (TR) and two untrained (UR) readers. The TR more frequently classified documents as unreadable (7.5 vs. 3.5%, P < 0.05) but more often as correct, i.e., consistent with EC (72.0 vs. 57.8%, P < 0.001). 8.7% of the documents were classified as unreadable using TDvel, 1.0% when applying TDint (P < 0.001). The mean value of correct classification of all 3 readers was 54.3% (TDvel only), 70.7% (TDint only), and 77.7% (combining both modalities), (P < 0.001). The kappa value for TR and TDint was 0.68, for TDvel 0.29. For UR, kappa did not differ (TDint: 0.58; TDvel 0.51). TDint is superior to TDvel in accuracy, reproducibility, and applicability for skilled and unskilled investigators when evaluating LVD by TDI. The combined application of TDint and TDvel is optimal.