Dorota Hering

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.

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.

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.

Genome-wide association study for sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls

The aim of the study was to screen the entire bull genome to identify SNP markers and propose candidate genes potentially involved in the variation of sperm membrane integrity in Holstein-Friesian bulls. Two hundred eighty eight bulls kept in one AI center were included in the study. Each bull was genotyped for 54.001 Single Nucleotide Polymorpisms (SNP) by the Illumina BovineSNP50 BeadChip. Commercial straws of frozen-thawed semen were used for the evaluation of sperm plasma membrane integrity (SYBR-14/PI staining) and sperm mitochondrial function (JC1/PI staining). An additive model for Linear Regression Analysis was applied to estimate the effect of SNP marker for sperm membrane integrity (by the use of GoldenHelix SVS7 software). Five significant markers (encompassing 2,2 MB region located on chromosome 6) for SYBR-14/PI were found. Among them one marker-rs41570391 passed Bonferroni correction test. Within approximately 3 Mb genomic region including significant markers three candidate genes: SGMS2 (Sphingomyelin Synthase 2), TET2 (Methylcytosine dioxygenase 2) and GSTCD genes (Gluthatione S-transferase C terminal domain) were proposed as potentially involved in sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls.

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.

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.

Subset of SNPs for parental identification in European bison Lowland-Białowieża line (Bison bonasus bonasus)

Parentage testing and individual identification are essential for protection and efficient management of animal populations. Panels of highly polymorphic SNP markers have recently become available in microarrays addressed to domestic animals. Such SNPs may also be useful in closely-related species living in the wild. The European bison (Bison bonasus), as an extremely inbred extinct species, requires individual identification to sustain maximum genetic diversity. The aim of the work was to prepare a panel of highly polymorphic SNPs for parental and individual identification for the wisent. 163 bison were genotyped on Illumina BovineHD microarray. Data filtration resulted in the panel of 100 highly polymorphic, unlinked SNPs giving the probability of parentage exclusion equal to 3.90 × 10−6 for one and 3.55 × 10−14 for two known parents and a probability of identity equal to 1.14 × 10−40. The probabilities obtained in this study are sufficient for effective management of the genetic diversity of the Lowland-Białowieża line of the European bison.

Identification of genome regions determining semen quality in Holstein-Friesian bulls using information theory

Use of information theory can be an alternative statistical approach to detect genome regions and candidate genes that are associated with livestock traits. The aim of this study was to verify the validity of the SNPs effects on some semen quality variables of bulls using entropy analysis. Records from 288 Holstein-Friesian bulls from one AI station were included. The following semen quality variables were analyzed: CASA kinematic variables of sperm (total motility, average path velocity, straight line velocity, curvilinear velocity, amplitude of lateral head displacement, beat cross frequency, straightness, linearity), sperm membrane integrity (plazmolema, mitochondrial function), sperm ATP content. Molecular data included 48,192 SNPs. After filtering (call rate = 0.95 and MAF = 0.05), 34,794 SNPs were included in the entropy analysis. The entropy and conditional entropy were estimated for each SNP. Conditional entropy quantifies the remaining uncertainty about values of the variable with the knowledge of SNP. The most informative SNPs for each variable were determined. The computations were performed using the R statistical package. A majority of the loci had relatively small contributions. The most informative SNPs for all variables were mainly located on chromosomes: 3, 4, 5 and 16. The results from the study indicate that important genome regions and candidate genes that determine semen quality variables in bulls are located on a number of chromosomes. Some detected clusters of SNPs were located in RNA (U6 and 5S_rRNA) for all the variables for which analysis occurred. Associations between PARK2 as well GALNT13 genes and some semen characteristics were also detected.

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.