Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2.

Lysosomal storage diseases (LSDs) comprised ~50 monogenic diseases characterized by the accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic molecular phenotyping is lacking. Here, we develop a nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneously quantify HeLa cell proteomes and lipidomes from more than two dozen LSD mutants, revealing diverse molecular phenotypes. Defects in delivery of ferritin and its autophagic receptor NCOA4 to lysosomes (ferritinophagy) were pronounced in NPC2-/- cells, which correlated with increased lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography. Ferritinophagy defects correlated with loss of mitochondrial cristae, MICOS-complex components, and electron transport chain complexes rich in iron-sulfur cluster proteins. Strikingly, mitochondrial defects were alleviated when iron was provided through the transferrin system. This resource reveals how defects in lysosomal function can impact mitochondrial homeostasis in trans and highlights nMOST as a discovery tool for illuminating molecular phenotypes across LSDs.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

How Membrane Contact Sites Shape the Phagophore.

During macroautophagy, phagophores establish multiple membrane contact sites (MCSs) with other organelles that are pivotal for proper phagophore assembly and growth. In S. cerevisiae, phagophore contacts have been observed with the vacuole, the ER, and lipid droplets. In situ imaging studies have greatly advanced our understanding of the structure and function of these sites. Here, we discuss how in situ structural methods like cryo-CLEM can give unprecedented insights into MCSs, and how they help to elucidate the structural arrangements of MCSs within cells. We further summarize the current knowledge of the contact sites in autophagy, focusing on autophagosome biogenesis in the model organism S. cerevisiae.

In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Effects of an oral hydro-ethanolic purple coneflower extract on performance, clinical health and immune parameters in calves.

The objective of this randomized, placebo-controlled, double-blinded field trial was to investigate the effects of oral administration of purple coneflower (Echinacea purpurea L. (EP)) on performance, health and immune parameters in calves. Calves (n = 27) were enrolled to three groups (9 calves per group): 0.5 g EP/calf per day (ECL), 5 g EP/calf per day (ECH) or placebo. Calves were vaccinated with Bluetongue-Virus (BTV) serotype 4 vaccine to investigate EPs effects on seroconversion. Clinical and performance parameters, inter alia body weight, health and milk intake were recorded for 57 days. Blood samples were analyzed for BTV antibodies and IgG by ELISA, white and red blood cell counts by flow cytometry and mRNA abundance of various inflammatory markers in leukocytes (IL-1ß, IL-8, tumor necrosis factor α (TNFα), cyclooxygenase 2 (Cox-2) and prostaglandin E synthase) was studied. The findings demonstrated no differences between groups regarding performance parameters. In all groups, calves suffered from diarrhea for a minimum of 2 days, but EP reduced the number of diarrhea days by 44% in ECL and increased the body temperature. Interestingly, ECL resulted in an increased number of respiratory disease days during the follow-up period. EP did not change blood cell and IgG counts, whereas eosinophil granulocytes were reduced in ECL. Decreased levels of hemoglobin and hematocrit were found in ECH. Prostaglandin E synthase levels in leukocytes were higher in ECL and ECH, whereas no differences were obtained for IL-1ß, IL-8, TNFα and Cox-2. Due to the unexpected occurrence of BTV seropositive calves before the first vaccination, 13 calves were excluded from the evaluation on seroconversion and no statistical analyses could be performed regarding antibody production. BTV-4 antibodies were not produced in 4 placebo-calves, whereas 4 of 5 and 1 of 6 ECL- and ECH-calves produced antibodies. Further investigations are needed to draw final conclusions on mode of action and efficacy of EP in calves.

A multicomponent herbal feed additive improves somatic cell counts in dairy cows - a two stage, multicentre, placebo-controlled long-term on-farm trial.

In contrast to natural and historical diets of wild and domesticated ruminants, the diversity of plant species is limited in diets of modern dairy cows. Are "production diseases" linked to this? We conducted a trial to test the effects of a multicomponent herbal feed additive (HFA) on health, performance and fertility traits. A dose-finding study (DF) with 62 cows on 11 commercial farms compared a low (50 g) and a high (100 g) dose of HFA (HFA-50, HFA-100) with a placebo (PL). In a subsequent field trial (FT) with 280 cows on 30 commercial farms, HFA-100 was compared to PL. Cows were randomly assigned to HFA and PL groups and received HFA or PL individually daily from 14 days pre- to 300 days post-calving. Data were analysed with mixed effects models. No differences between HFA and PL were found regarding performance, body condition score and overall culling rates. A tendency towards lower milk urea for HFA-100 compared to PL (p = .06) was found in DF. HFA significantly reduced elevated milk acetone observations (≥10 mg/L) in the first 10 lactation weeks (HFA-100: 4%; HFA-50: 4%; PL: 12%) in DF. HFA-50 significantly reduced lameness incidence (HFA-100: 11%; HFA-50: 2%; PL: 14%) in DF. Calving intervals were 15 days shorter in HFA compared to PL in both trials, which could be confirmed by tendency (p = .07) in FT. In both trials, the proportion of test days with elevated somatic cell score (≥3.0) was significantly lower in HFA compared to PL (DF: HFA-100: 40%, HFA-50: 45% and PL: 55%; FT: HFA-100: 38% and PL: 55%) which is also reflected by tendency (p = .08) in lower culling rates due to udder diseases in FT. HFA showed no negative impact on any of the measured parameters. The effects of HFA indicate a potential of phytochemically rich and diverse feed additives for dairy cows' nutrition and physiology.

Production level, fertility, health traits, and longevity in local and commercial dairy breeds under organic production conditions in Austria, Switzerland, Poland, and Sweden.

Our aim was to map the performance of local (native) dairy cattle breeds in Austria, Switzerland, Poland, and Sweden with regard to production, fertility, longevity, and health-associated traits and to compare them with commercial (modern) breeds. For this purpose, we analyzed test-day records (July 1, 2011, to June 30, 2014) and treatment records (Austria, Sweden) of cows managed on organic farms. We performed country-wise comparisons of 123,415 lactations from Original Braunvieh (OB) and Grey Cattle (AL) with Braunvieh (BV; Brown Swiss blood >60%) in Switzerland; AL with BV (Brown Swiss blood >50%) in Austria; Polish Black and White (ZB), Polish Red and White (ZR), and Polish Red (RP) with Polish Holstein Friesian (PHF) in Poland; and Swedish Red (SRB) with Swedish Holstein (SH) in Sweden. Average milk yields were substantially lower for local compared with commercial breeds in all countries; differences ranged from 750 kg (Sweden) to 1,822 kg (Austria), albeit on very different average levels. Local breeds showed a longer productive lifetime by 0.64, 0.83, 1.42, and 0.20 lactations in Switzerland, Austria, Poland, and Sweden, respectively, again on very different levels in each country. Regarding fertility traits, calving interval was shorter in local than in commercial breeds by 13 (Sweden), 14 (Switzerland), and 20 d (Austria, Poland). Insemination index was lower in certain local breeds by 0.15 (Switzerland), 0.14 (Austria), 0.21 (Poland), and 0.13 (Sweden). Several local breeds showed a lower proportion of cows with >100,000 somatic cells/mL. This was the case in Switzerland (OB 24.2%; BV 35.8%), Austria (AL 25.3%; BV 36.9%), and Sweden (SRB 42.4%; SH 43.4%). In contrast, the respective proportion in Poland exceeded 82% in all breeds except the commercial PHF (76.1%). In Sweden, lactations with veterinary treatments were considerably less prevalent in SRB (15.6%) than in SH (21.7%). In Austria, breeds differed only in treatments for udder disorders, which favored AL. In conclusion, the markedly lower milk yields of local breeds are partly counterbalanced by (somewhat inconsistent) advantages in longevity, fertility, and health traits across 4 European countries. This indicates that the robustness of local breeds can contribute to improved sustainability of organic dairy systems.

Gd(III)-Gd(III) Relaxation-Induced Dipolar Modulation Enhancement for In-Cell Electron Paramagnetic Resonance Distance Determination.

In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron-electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms.

Placebo-controlled study on the effects of oral administration of Allium sativum L in postweaning piglets.

Postweaning diarrhoea (PWD) due to Escherichia coli is an economically important disease in pig production. In this placebo-controlled study performed in Switzerland, the effects of oral supplementation of Allium sativum L. (garlic, AS) on performance (bodyweight (BW) and daily weight gain (DWG)) and health (body condition and clinical score) were investigated in postweaning piglets. Piglets (n=600) were randomly assigned to the treatment groups (placebo, AS or colistin) and observed from birth until three weeks postweaning. The treatments were administered for the first two weeks postweaning. Faecal dry matter (FDM) and coliform bacteria on pen level were measured weekly. Data were analysed using generalised mixed-effect models in R. BW and DWG of the AS group were significantly higher compared with placebo in the third week postweaning. No differences in body condition and FDM were observed. The clinical score of AS-treated animals was significantly better compared with the colistin group. About 33 per cent of the piglets of the AS and the placebo group had to be treated with antibiotics due to the occurrence of severe PWD. The major finding of this study showed that AS supplementation increased growth performance and improved clinical health, but did not reduce the incidence and severity of PWD.

Light-induced dipolar spectroscopy - A quantitative comparison between LiDEER and LaserIMD.

Nanometric distance measurements with EPR spectroscopy yield crucial information on the structure and interactions of macromolecules in complex systems. The range of suitable spin labels for such measurements was recently expanded with a new class of light-inducible labels: the triplet state of porphyrins. Importantly, accurate distance measurements between a triplet label and a nitroxide have been reported with two distinct light-induced spectroscopy techniques, (light-induced) triplet-nitroxide DEER (LiDEER) and laser-induced magnetic dipole spectroscopy (LaserIMD). In this work, we set out to quantitatively compare the two techniques under equivalent conditions at Q band. Since we find that LiDEER using a rectangular pump pulse does not reach the high modulation depth that can be achieved with LaserIMD, we further explore the possibility of improving the LiDEER experiment with chirp inversion pulses. LiDEER employing a broadband pump pulse results in a drastic improvement of the modulation depth. The relative performance of chirp LiDEER and Laser-IMD in terms of modulation-to-noise ratio is found to depend on the dipolar evolution time: While LaserIMD yields higher modulation-to-noise ratios than LiDEER at short dipolar evolution times (τ=2µs), the high phase memory time of the triplet spins causes the situation to revert at τ=6µs.

Impact of US Brown Swiss genetics on milk quality from low-input herds in Switzerland: Interactions with season.

This study investigated the effect of, and interactions between, US Brown Swiss (BS) genetics and season on milk yield, basic composition and fatty acid profiles, from cows on low-input farms in Switzerland. Milk samples (n = 1,976) were collected from 1,220 crossbreed cows with differing proportions of BS, Braunvieh and Original Braunvieh genetics on 40 farms during winter-housing and summer-grazing. Cows with more BS genetics produced more milk in winter but not in summer, possibly because of underfeeding potentially high-yielding cows on low-input pasture-based diets. Cows with more Original Braunvieh genetics produced milk with more (i) nutritionally desirable eicosapentaenoic and docosapentaenoic acids, throughout the year, and (ii) vaccenic and α-linolenic acids, total omega-3 fatty acid concentrations and a higher omega-3/omega-6 ratio only during summer-grazing. This suggests that overall milk quality could be improved by re-focussing breeding strategies on cows' ability to respond to local dietary environments and seasonal dietary changes.

Meta-analysis of genome-wide association studies for cattle stature identifies common genes that regulate body size in mammals.

Stature is affected by many polymorphisms of small effect in humans 1 . In contrast, variation in dogs, even within breeds, has been suggested to be largely due to variants in a small number of genes2,3. Here we use data from cattle to compare the genetic architecture of stature to those in humans and dogs. We conducted a meta-analysis for stature using 58,265 cattle from 17 populations with 25.4 million imputed whole-genome sequence variants. Results showed that the genetic architecture of stature in cattle is similar to that in humans, as the lead variants in 163 significantly associated genomic regions (P < 5 × 10-8) explained at most 13.8% of the phenotypic variance. Most of these variants were noncoding, including variants that were also expression quantitative trait loci (eQTLs) and in ChIP-seq peaks. There was significant overlap in loci for stature with humans and dogs, suggesting that a set of common genes regulates body size in mammals.

Short communication: Genomic prediction using imputed whole-genome sequence variants in Brown Swiss Cattle.

The accuracy of genomic prediction determines response to selection. It has been hypothesized that accuracy of genomic breeding values can be increased by a higher density of variants. We used imputed whole-genome sequence data and various single nucleotide polymorphism (SNP) selection criteria to estimate genomic breeding values in Brown Swiss cattle. The extreme scenarios were 50K SNP chip data and whole-genome sequence data with intermediate scenarios using linkage disequilibrium-pruned whole-genome sequence variants, only variants predicted to be missense, or the top 50K variants from genome-wide association studies. We estimated genomic breeding values for 3 traits (somatic cell score, nonreturn rate in heifers, and stature) and found differences in accuracy levels between traits. However, among different SNP sets, accuracy was very similar. In our analyses, sequence data led to a marginal increase in accuracy for 1 trait and was lower than 50K for the other traits. We concluded that the inclusion of imputed whole-genome sequence data does not lead to increased accuracy of genomic prediction with the methods.

Genome-wide association studies of fertility and calving traits in Brown Swiss cattle using imputed whole-genome sequences.

BACKGROUND: The detection of quantitative trait loci has accelerated with recent developments in genomics. The introduction of genomic selection in combination with sequencing efforts has made a large amount of genotypic data available. Functional traits such as fertility and calving traits have been included in routine genomic estimation of breeding values making large quantities of phenotypic data available for these traits. This data was used to investigate the genetics underlying fertility and calving traits and to identify potentially causative genomic regions and variants. We performed genome-wide association studies for 13 functional traits related to female fertility as well as for direct and maternal calving ease based on imputed whole-genome sequences. Deregressed breeding values from ~1000-5000 bulls per trait were used to test for associations with approximately 10 million imputed sequence SNPs. RESULTS: We identified a QTL on BTA17 associated with non-return rate at 56 days and with interval from first to last insemination. We found two significantly associated non-synonymous SNPs within this QTL region. Two more QTL for fertility traits were identified on BTA25 and 29. A single QTL was identified for maternal calving traits on BTA13 whereas three QTL on BTA19, 21 and 25 were identified for direct calving traits. The QTL on BTA19 co-localizes with the reported BH2 haplotype. The QTL on BTA25 is concordant for fertility and calving traits and co-localizes with a QTL previously reported to influence stature and related traits in Brown Swiss dairy cattle. CONCLUSION: The detection of QTL and their causative variants remains challenging. Combining comprehensive phenotypic data with imputed whole genome sequences seems promising. We present a QTL on BTA17 for female fertility in dairy cattle with two significantly associated non-synonymous SNPs, along with five additional QTL for fertility traits and calving traits. For all of these we fine mapped the regions and suggest candidate genes and candidate variants.

The genetic basis for the selection of dairy goats with enhanced resistance to gastrointestinal nematodes.

Gastrointestinal nematodes (GIN) severely affect small ruminant production worldwide. Increasing problems of anthelmintic resistance have given strong impetus to the search for alternative strategies to control GIN. Selection of animals with an enhanced resistance to GIN has been shown to be successful in sheep. In goats, the corresponding information is comparatively poor. Therefore, the present study was designed to provide reliable data on heritabilities of and genetic correlations between phenotypic traits linked to GIN and milk yield in two major dairy goat breeds (Alpine and Saanen). In all, 20 herds totalling 1303 goats were enrolled in the study. All herds had (i) a history of gastrointestinal nematode infection, (ii) uniform GIN exposure on pasture and (iii) regular milk recordings. For all goats, individual recordings of faecal egg counts (FEC), FAMACHA© eye score, packed cell volume (PCV) and milk yield were performed twice a year with an anthelmintic treatment in between. The collected phenotypic data were multivariately modelled using animal as a random effect with its covariance structure inferred from the pedigree, enabling estimation of the heritabilities of the respective traits and the genetic correlation between them. The heritabilities of FEC, FAMACHA© and PCV were 0.07, 0.22 and 0.22, respectively. The genetic correlation between FEC and FAMACHA© was close to zero and -0.41 between FEC and PCV. The phenotypic correlation between FEC and milk yield was close to zero, whereas the genetic correlation was 0.49. Our data suggest low heritability of FEC in Saanen and Alpine goats and an unfavourable genetic correlation of FEC with milk yield.

The 3'-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria.

Besides its primary informational role, the sequence of the mRNA (mRNA) including its 5'- and 3'- untranslated regions (UTRs), contains important features that are relevant for post-transcriptional and translational regulation of gene expression. In this work a number of bacterial twister motifs are characterized both in vitro and in vivo. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3'-UTR has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show dependence on motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3'-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their activity via ligand-dependent cleavage of the 3'-UTR and the removal of the protective intrinsic terminator. Our results provide insights into possible biological functions of these recently discovered and widespread catalytic RNA motifs and offer new tools for applications in biotechnology, synthetic biology and metabolic engineering.

Genetic and genomic analysis of hyperthelia in Brown Swiss cattle.

Supernumerary teats (SNT) are any abnormal teats found on a calf in addition to the usual and functional 4 teats. The presence of SNT has also been termed "hyperthelia" since the end of the 19th century. Supernumerary teats can act as an incubator for bacteria, infecting the whole udder, and can interfere with the positioning of the milking machine, and consequently, have economic relevance. Different types of SNT are observed at different positions on the udder. Caudal teats are in the rear, ramal teats are attached to another teat, and intercalary teats are found between 2 regular teats. Not all teats are equally developed; some are completely functional but most are rudimentary and not attached to any mammary gland tissue. Recently, different studies showed the poly/oligogenic character of these malformations in cattle as well as in other mammalian species. The objective of this study was to analyze the genetic architecture and incidence of hyperthelia in Swiss Brown Swiss cattle using both traditional genetic evaluation as well as imputed whole genome sequence variant information. First, phenotypes collected over the last 20 yr were used together with pedigree information for estimation of genetic variance. Second, breeding values of Brown Swiss bulls were estimated applying the BLUP algorithm. The BLUP-EBV were deregressed and used as phenotypes in genome-wide association studies. The gene LGR5 on chromosome 5 was identified as a candidate for the presence of SNT. Using alternative trait coding, genomic regions on chromosome 17 and 20 were also identified as being involved in the development of SNT with their own supernumerary mammary gland tissue. Implementing knowledge gained in this study as a routine application allows a more accurate evaluation of the trait and reduction of SNT prevalence in the Swiss Brown Swiss cattle population.