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.

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.

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.

The aim of this study was to compare computer assisted sperm analysis (CASA) results of frozen thawed bull semen using three different chambers. Sixty bull frozen semen samples were thawed (37°C; 30 sec), extended in PBS (30×106 spermatozoa/mL; 37°C) and incubated (37°C; 2 min). Each semen sample was analyzed by CASA [total motility, progressive (pro)/ non-progressive/rapid/medium/slow movement spermatozoa, VCL, VSL, VAP, ALH, BCF, LIN, STR, WOB and hyperactive spermatozoa] using three different chambers: a Makler® chamber (MC; 10 μm); a Leja 4 chamber slide (LC; 20 μm); and a Glass slide covered with a coverslip (GSC; 10.3 μm). The Makler chamber gave higher values compared to both the LC and GSC for almost all examined parameters. No systematic effect was evident between LC and GSC for VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF. Method agreement between MC and LC was generally moderate, between MC and GSC poor and between LC and GSC moderate to good. In general, narrower limits of agreement were found in samples with lower values. In conclusion, the CASA outcomes could be influenced by the analysis chambers. This finding should be taken into consideration when comparing results from different laboratories.

In our previous Genome-wise Association Study we found that Cystic Fibrosis Transmem- brane 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 insem- ination (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 con- tent and integrity of sperm membranes. One hundred twenty Holstein Friesian bulls kept in uni- form environmental conditions (one AI company) were included in the study. Significant associ- ations 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 num- ber 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.

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.