Eradication of carriers of Complex Vertebral Malformation (CVM) and Brachyspina in Polish Holstein-Friesian bulls.

CVM ( Complex Vertebral Malformations) and Brachyspina (BY) are the most common autosomal recessive genetic defects occurring in the last two decades in Holstein dairy cattle around the world. Beginning from 2004 and 2014, 3035 and 338 Polish Holstein-Friesian bulls were tested to find carriers of CVM and BY, respectively. Among analyzed bulls 191 CVM carriers (6.29%) and 20 BY carriers (5.92%) were identified. No CVM carriers were observed beginning from 2016, whereas only single BY carriers was identified annually for the last 5 years. One bull turned to be double CVM/ BY carrier as a son of also double CVM/BY top Dutch sire (JABOT 90676-4-9). It is shown that CVM and BY defects are practically eradicated from Polish dairy cattle although incidental testing should be continued if new bulls with CVM or BY carriers in sire or dam pedigree will unexpectedly appear.

SNP panel for evaluation of genetic variability and relationship in roe deer (Capreolus capreolus).

Blood samples from forty-six roe deer ( Capreolus capreolus) acquired during officially approved hunting in six hunting divisions throughout Poland were used to isolate the genomic DNA. All individuals were genotyped by MD_Bovine BeadChip (Illumina) for 46.750 Single Nucleotide Polymorphism (SNP) markers. SNPs of inappropriate clusters, with a marker call rate lower than 90% and with a minor allele frequency (MAF) lower than 0.01, located on sex chromosomes and mitochondrial DNA, were removed. Altogether, 21.033 SNP markers were included for further analysis. Observed and expected heterozygosity amounted to 0.098 and 0.119, respectively. Among 21.033 markers, a panel of 148 SNPs were selected for relationship analysis. They were unlinked and had a MAF higher than 0.2. This set of SNPs showed a probability of parentage exclusion of 1.29x10 -6 and 2.37x10 -19 for one, and two known parents, respectively. The probability of identity was estimated at 1.8x10 -40. The probabilities obtained in this study are sufficient for the monitoring and effective management of the genetic diversity of roe deer in Poland and is a cost-effective complementary tool for forensic applications.

Novel method for identification of the lethal mutation in bovine APAF1 gene and its preliminary prevalence in Polish Holstein-Friesian bulls.

The aim of the study was to develop a reliable and cost-effective method for detection of nonsense mutation in APAF1 gene causing lethal effect called HH1 (Holstein Haplotype1) and to evaluate its prevalence in a sample of Polish Holstein-Friesian bulls. One hundred seventy eight bulls born between 1996 and 2017 were included in the analysis. They were kept in four artificial insemination centers and have in the pedigree the known carrier of HH1. All bulls were diagnosed by novel PCR-SSCP technique. Specific amplicons of 261 bp APAF1 gene fragment were used to detect changes in single stranded conformation (SSCP) caused by nonsense mutation C/T responsible for HH1. Each new carrier was used to trace another potential carriers among their offspring available in Polish Holstein Bull Repository Database. Among 178 bulls, 85 HH1 carriers were found. Our results show that nonsense mutation in APAF1 gene is already transmitted and segregating in Polish Holstein-Friesian cattle and its frequency may increase if no action will be undertaken against actual carriers.

Follicle-stimulating hormone receptors expression in ovine corpora lutea during luteal phase: effect of nutritional plane and follicle-stimulating hormone treatment.

Corpus luteum (CL), a transient endocrine gland critical for reproductive cyclicity and pregnancy maintenance, is controlled by numerous regulatory factors. Although LH is widely recognized as the major regulator, other factors may also affect luteal functions. It has been demonstrated that FSH receptors (FSHR) are expressed not only in ovarian follicles but also in other tissues within the reproductive tract, including the CL. To evaluate FSHR expression in nontreated (nonsuperovulated; experiment 1) or FSH-treated (superovulated; experiment 2) sheep fed a control (C; maintenance), excess (O; 2 × C), or restricted (U; 0.6 × C) diet, CL were collected at the early, mid and/or late luteal phases (n = 5-7 per group). Protein and messenger RNA (mRNA) expression of FSHR were detected in the CL from all groups using immunohistochemistry followed by image analysis and quantitative RT-PCR, respectively. Follicle-stimulating hormone receptor was immunolocalized to steroidogenic small and large and nonsteroidogenic luteal cells. In both experiments, FSHR protein expression was not affected by stage of luteal development or diet. In experiment 1, expression of mRNA for all FSHR variants was greater (P <0.02 to 0.0003) at the late phase than mid or early luteal phase, and in experiment 2, it was greater (P < 0.001) at the mid than early luteal phase. Plane of nutrition did not affect FSHR mRNA expression. Comparison of FSH-treated with nontreated ewes demonstrated that FSH increased FSHR protein expression by 1.5- to 2-fold (P < 0.0001) in all groups, and mRNA expression by 7- to 30-fold (P < 0.001) for (1) FSHR-1 in all groups except U at the early luteal phase, (2) FSHR-2 in C, O, and U at the mid-phase, but not early luteal phase, and (3) FSHR-3 in U at the mid-luteal phase. Our data demonstrate that (1) FSHRs are expressed in ovine CL at several stages of luteal development, (2) FSHR protein expression does not change during the luteal phase and is not affected by diet, (3) FSHR mRNA expression not only depends on the stage of the estrous cycle but also not affected by diet in nonsuperovulated or superovulated ewes, and (4) in vivo FSH treatment enhanced FSHR protein and/or mRNA expression in the CL depending on diet and phase of the estrous cycle. Presence of FSHR in the CL indicates a regulatory role of FSH in luteal function in sheep. As very little is known about the possible role of FSH and FSHR in luteal functions, further studies should be undertaken to elucidate the endocrine, molecular, and cellular mechanisms of FSH effects on the CL.

Missense mutation in SDE2 gene - new lethal defect transmitted into Polish Holstein-Friesian cattle.

The aim of the study was to find out whether carriers of new lethal mutation in SDE2 gene occur in the population of Polish Holstein-Friesian bulls. Eighty seven bulls were included in the analysis. Bulls were selected as having in the pedigree known carrier of SDE2 mutation (bull Mountain USAM000002070579). All bulls were diagnosed by PCR amplification of 524 bp fragment of SDE2 gene followed by digestion of Bcc I restriction enzyme. Heterozygotes (carriers) were confirmed by sequencing. Each new carrier was used to trace another potential carriers among its offspring available in Polish Holstein Bull Repository Database. Among 87 bulls, 50 new SDE2 carriers were found. The study has shown that mutation in SDE2 gene causing early embryo mortality is already transmitted to Polish Holstein-Friesian cattle. The results are sufficient to initiate the screening program to reveal new carriers and to avoid further spreading of SDE2 lethal mutation.

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.

[Precision pulse capsulotomy : The new capsulorhexis?] / Präzisionspulskapsulotomie : Die neue Kapsulorhexis?

The manual capsulorhexis created by Neuhann is still the standard procedure for opening the anterior capsule for cataract surgery. A limitation is the inaccuracy in the size and placement of the opening due to manual execution. In addition to the femtosecond laser a possible improvement in the standardization of capsulorhexis is provided by the Zepto procedure (precision pulse capsulotomy, PPC). In this case a 5.25 mm rhexis is created in a standardized fashion with a flexible suction adapter in which a nitinol ring is located. Whether the strength of PPC is comparable or better than that of the manual technique and how it behaves in terms of capsule shrinkage has not yet been finally clarified.

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.

Differences between horse selection based on two forms of osteochondrosis in fetlock.

Horses lose potential opportunities because of health problems. Available breeding strategies are not effective enough, probably also because of the different definition used and its genetic usefulness. The aim of the study was to compare the genetic background estimated by the genome-wide association study (GWAS) for osteochondrosis using two different scaling osteochondrosis (OC)/healthy and osteochondrosis dissecans (OCD)/healthy systems for evaluating the disease status of investigated fetlock joints. Two hundred one Warmblood horses trained for performance tests (87 stallions and 114 mares) were phenotyped and genotyped. Four fetlock x-ray images per horse were collected using the RTG Girth HF 80 and Vet Scan ray 3600. The DNA of each horse was genotyped using the BeadChip 70K. To identify SNPs that significantly affect the probability of osteochondrosis, two different methods were applied: the Cochran-Armitage test based on an additive mode of inheritance and logistic regression. The genetic background for osteochondrosis, expressed in the number of SNPs found with significant associations with osteochondrosis, was higher by evaluation in the scale of OCD/healthy horses (16 SNPs on several chromosomes mainly on the ECA1 and ECA10) than OC/healthy (2 SNPs on the ECA15 and one SNP on the ECA10). Detailed definition of osteochondrosis is needed in breeding and in veterinary practice. The genetic background for osteochondrosis and osteochondrosis dissecans seems not the same. Suggestive SNPs could be the candidate markers for osteochondrosis but should be checked on a larger population before usage.

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.

Effects of the platelet-activating factor (PAF) supplementation on ATP content of cryopreserved bull spermatozoa (AI).

The aim of this study was to investigate the effect of PAF supplementation in semen extender on ATP content in cryopreserved bull spermatozoa used for artificial insemination at different time intervals. Cryopreserved semen was treated with different concentrations of PAF: 1×10-5M, 1×10-6M, 1×10-7M, 1×10-8M and 1×10-9M at 37°C. In the present work we showed that content of ATP in cryopreserved semen supplemented with 1×10-9M PAF was statistically significantly higher at 90 and 120 minutes of incubation in comparison to the control group (p≤0.05). Present study indicates the potential influence of PAF on ATP content in male spermatozoa via it's protective role towards mitochondria metabolic activity.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-µmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

Microarray analysis of differential gene expression profiles in blood cells of naturally BLV-infected and uninfected Holstein-Friesian cows.

The aim of the present study was to examine gene expression changes in response to bovine leukemia virus (BLV) infection, in an effort to determine genes that take a part in molecular events leading to persistent lymphocytosis (PL), and to better define genes involved in host response to BLV infection. Using bovine 70-mer oligonucleotide spotted microarrays (BLOPlus) and qRT-PCR validation, we studied global gene expression profiles in blood cells in vivo of 12 naturally BLV-infected Polish Holstein cows, and 12 BLV non-infected controls of the same breed and reared in herds with high BLV seroprevalence. With an arbitrary cut-off value of 1.5-fold change in gene expression, we identified the down-regulation of 212 genes (M value ≤-0.585) and the up-regulation of 158 genes (M value of ≥0.585) at 1% false discovery rate in BLV-positive animals in comparison to the BLV-negative group. The gene set enrichment analysis demonstrated that the differentially expressed (DE) genes could be classified to diverse biological processes, including immune response of host blood cells. Interestingly, our data indicated the potential involvement of the innate immunity, including complement system activation, NK-cell cytotoxicity and TREM-1 signaling, during the BLV-induced pathogenesis. We showed the occurrence of numerous regulatory processes that are targeted by BLV-infection. We also suggest that a complex network of interrelated pathways is disturbed, causing the interruption of the control of B-cell proliferation and programmed cell death.

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.

Effects of the platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen (AI) with reduced sperm motility.

The aim of the study was to determine the effects of platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen with reduced sperm motility used for artificial insemination. The aim of experiment 1 was to identify the optimal concentration of the phospholipid able to preserve sperm viability. Cryopreserved semen was treated with different PAF concentrations: 1×10(-5) M, 1×10(-6) M, 1×10(-7) M, 1×10(-8) M and 1×10(-9) M. The experiment demonstrated that PAF at concentration 1×10(-9) M increased most the sperm viability parameters (motility parameters, plasma membrane integrity and mitochondrial function) after 120 min of incubation of thawed semen at 37°C. Cryopreserved bull semen with reduced sperm motility (below 70%) was supplemented with PAF in a concentration of 1×10(-9) M. A statistically significant increase in sperm motility, percentage of linear motile spermatozoa and VSL value was observed after 120 min incubation of sperm with 1×10(-9) M PAF. Sperm supplementation with PAF also had positive effects on plasma membrane integrity and percentage of spermatozoa with preserved mitochondrial transmembrane potential, but the differences were not statistically significant. The results indicated positive effects of PAF supplementation at a concentration of 1×10(-9) M on the selected sperm quality parameters in cryopreserved bull semen with reduced motility.

Cholesterol Deficiency - new genetic defect transmitted to Polish Holstein-Friesian cattle.

The aim of the study was to find out whether carriers of new genetic defect Cholesterol Deficiency (CD) occur in the population of Polish Holstein-Friesian bulls. Twenty seven bulls were included in the analysis. Bulls were selected as having in the pedigree known carrier of CD (Maughlin Storm CANM000005457798). All bulls were diagnosed by the test described by Menzi et al. (2016) by using allele-specific PCR. Among 27 bulls, 9 new CD carriers were found. Our results show that causal mutation for CD is already transmitted to Polish Holstein-Friesian cattle. The results are sufficient ground to take practical action in order to avoid further spreading of mutation causing CD.

Detection of Brachyspina carriers within Polish Holstein-Friesian bulls.

The aim of this paper was to verify the hypothesis whether carriers of genetic defect Brachyspina occur in the Polish Holstein-Friesian Cattle. PCR method was used to screen 78 Polish Holstein-Friesian bulls. Eight bulls were identified as heterozygotes for 3,3 kb deletion in the FANCI gene - the mutation causing Brachyspina defect. All carriers were sons of 3 sires: Cleitus Jabot, Sandy-Valley Bolton ET and Coyne-Farms Dorcy ET which were descendants of the US sire Sweet Haven Tradition (HOUSAM 1682485). Systematic screening of young bulls having in the pedigree Barchyspina carrier is necessary to prevent spreading of the recessive mutation in the dairy cattle population in Poland.

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.