Cattle breeding populations are susceptible to the propagation of recessive diseases. Individual sires generate tens of thousands of progeny via artificial insemination. The frequency of deleterious alleles carried by such sires may increase considerably within few generations. Deleterious alleles manifest themselves often by missing homozygosity resulting from embryonic/fetal, perinatal or juvenile lethality of homozygotes. A scan for homozygous haplotype deficiency in 25,544 Fleckvieh cattle uncovered four haplotypes affecting reproductive and rearing success. Exploiting whole-genome resequencing data from 263 animals facilitated to pinpoint putatively causal mutations in two of these haplotypes. A mutation causing an evolutionarily unlikely substitution in SUGT1 was perfectly associated with a haplotype compromising insemination success. The mutation was not found in homozygous state in 10,363 animals (P = 1.79 × 10−5) and is thus likely to cause lethality of homozygous embryos. A frameshift mutation in SLC2A2 encoding glucose transporter 2 (GLUT2) compromises calf survival. The mutation leads to premature termination of translation and activates cryptic splice sites resulting in multiple exon variants also with premature translation termination. The affected calves exhibit stunted growth, resembling the phenotypic appearance of Fanconi-Bickel syndrome in humans (OMIM 227810), which is also caused by mutations in SLC2A2. Exploiting comprehensive genotype and sequence data enabled us to reveal two deleterious alleles in SLC2A2 and SUGT1 that compromise pre- and postnatal survival in homozygous state. Our results provide the basis for genome-assisted approaches to avoiding inadvertent carrier matings and to improving reproductive and rearing success in Fleckvieh cattle.

Background: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. Results: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. Conclusion: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Congenital malformations resulting in late abortions and stillbirths affect the economic wellbeing of producers and the welfare of cattle in breeding programs. An extremely high incidence of stillbirths of “half-sized” calves of normal karyotype and uninflated lungs was diagnosed in the progeny of the Finnish Ayrshire (Bos taurus) bull - YN51. No other visible anatomical abnormalities were apparent in the stillborn calves. We herein describe the positional identification of a 110 kb microdeletion in the maternally imprinted PEG3 domain that results in a loss of paternal MIMT1 expression and causes late term abortion and stillbirth in cattle. Using the BovineSNP50 BeadChip we performed a genome-wide half-sib linkage analysis that identified a 13.3 Mb associated region on BTA18 containing the maternally imprinted PEG3 domain. Within this cluster we found a 110 kb microdeletion that removes a part of the non-protein coding MER1 repeat containing imprinted transcript 1 gene (MIMT1). To confirm the elimination of gene expression in calves inheriting this deletion, we examined the mRNA levels of the three maternally imprinted genes within the PEG3 domain, in brain and cotyledon tissue collected from eight fetuses sired by the proband. None of the fetuses that inherited the microdeletion expressed MIMT1 in either tissue. The mutation, when inherited from the sire, is semi-lethal for his progeny with an observed mortality rate of 85%. The survival of 15% is presumably due to the incomplete silencing of maternally inherited MIMT1 alleles. We designed a PCR-based assay to confirm the existence of the microdeletion in the MIMT1 region that can be used to assist cattle breeders in preventing the stillbirths.

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10⁻²⁷), head (P = 2.0 x 10⁻⁴⁴) and tail anomalies (P = 7.2 x 10⁻⁴⁹) and insemination success (P = 9.9 x 10⁻¹³). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10⁻³²) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle. In cattle farming, artificial insemination is the most common method of breeding. To ensure high fertilization rates, ejaculate quality and insemination success are closely monitored in artificial insemination bulls. We analyse semen quality, insemination success and microarray-called genotypes at more than 600,000 genome-wide SNP markers of 794 bulls to identify a recessive allele that compromises semen quality. We take advantage of whole-genome sequencing to pinpoint a variant in the coding sequence of WDR19 encoding WD repeat-containing protein 19 that activates a novel exonic splice site. Our results indicate that cryptic splicing in WDR19 is associated with reduced male reproductive performance. This is the first report of a variant that contributes to quantitative variation in bovine semen quality.

Inherited developmental diseases can cause severe animal welfare and economic problems in dairy cattle. The use of a small number of bulls for artificial insemination (AI) carries a risk that recessive defects rapidly enrich in the population. In recent years, an increasing number of Finnish Ayrshire calves have been identified with signs of ptosis, intellectual disability, retarded growth and mortality, which constitute an inherited disorder classified as PIRM syndrome. We established a cohort of nine PIRM-affected calves and 38 unaffected half-siblings and performed a genome-wide association study (GWAS) to map the disease to a 700-kb region on bovine chromosome 17 (p = 1.55 × 10-9). Whole genome re-sequencing of an unaffected carrier, its affected progeny and 43 other unaffected animals from another breed identified a G > A substitution mutation at the last nucleotide of exon 23 in the ubiquitin protein ligase E3B encoding gene (UBE3B). UBE3B transcript analysis revealed in-frame exon skipping in the affected animals resulting in an altered protein lacking 40 amino acids, of which 20 are located in the conserved HECT-domain, the catalytic site of the UBE3B protein. Mutation screening in 129 Ayrshire AI bulls currently used in Finland indicated a high carrier frequency (17.1%). We also found that PIRM syndrome might be connected to the recently identified AH1 haplotype, which has a frequency of 26.1% in the United States Ayrshire population. We describe PIRM syndrome in cattle, which is associated with the mutated UBE3B gene. The bovine phenotype resembles human Kaufman oculocerebrofacial syndrome, which is also caused by mutations in UBE3B. PIRM syndrome might be connected with the recently identified AH1 haplotype, which is associated with reduced fertility in the US Ayrshire population. This study enables the development of a genetic test to efficiently reduce the high frequency of mutant UBE3B in Ayrshires, significantly improving animal health and reducing economic loss.

Hitchhiking mapping and association studies are two popular approaches to map genotypes to phenotypes. In this study we combine both approaches to complement their specific strengths and weaknesses, resulting in a method with higher statistical power and fewer false positive signals. We applied our approach to dairy cattle as they underwent extremely successful selection for milk production traits and since an excellent phenotypic record is available. We performed whole genome association tests with a new mixed model approach to account for stratification, which we validated via Monte Carlo simulations. Selection signatures were inferred with the integrated haplotype score and a locus specific permutation based integrated haplotype score that works with a folded frequency spectrum and provides a formal test of signifance to identify selection signatures.