An organism-wide ATAC-seq peak catalog for the bovine and its use to identify regulatory variants.

We report the generation of an organism-wide catalog of 976,813 cis-acting regulatory elements for the bovine detected by the assay for transposase accessible chromatin using sequencing (ATAC-seq). We regroup these regulatory elements in 16 components by nonnegative matrix factorization. Correlation between the genome-wide density of peaks and transcription start sites, correlation between peak accessibility and expression of neighboring genes, and enrichment in transcription factor binding motifs support their regulatory potential. Using a previously established catalog of 12,736,643 variants, we show that the proportion of single-nucleotide polymorphisms mapping to ATAC-seq peaks is higher than expected and that this is owing to an approximately 1.3-fold higher mutation rate within peaks. Their site frequency spectrum indicates that variants in ATAC-seq peaks are subject to purifying selection. We generate eQTL data sets for liver and blood and show that variants that drive eQTL fall into liver- and blood-specific ATAC-seq peaks more often than expected by chance. We combine ATAC-seq and eQTL data to estimate that the proportion of regulatory variants mapping to ATAC-seq peaks is approximately one in three and that the proportion of variants mapping to ATAC-seq peaks that are regulatory is approximately one in 25. We discuss the implication of these findings on the utility of ATAC-seq information to improve the accuracy of genomic selection.

A multidisciplinary and structured investigation of three suspected clusters of transverse upper limb reduction defects in France.

INTRODUCTION: Between 2019-2021, facing public concern, a scientific expert committee (SEC) reanalysed suspected clusters of transverse upper limb reduction defects (TULRD) in three administrative areas in France, where initial investigations had not identified any risk exposure. We share here the national approach we developed for managing suspicious clusters of the same group of congenital anomalies occurring in several areas. METHODS: The SEC analysed the medical records of TURLD suspected cases and performed spatiotemporal analyses on confirmed cases. If the cluster was statistically significant and included at least three cases, the SEC reviewed exposures obtained from questionnaires, environmental databases, and a survey among farmers living near to cases' homes concerning their plant product use. RESULTS: After case re-ascertainment, no statistically significant cluster was observed in the first administrative areas. In the second area, a cluster of four children born in two nearby towns over two years was confirmed, but as with the initial investigations, no exposure to a known risk factor explaining the number of cases in excess was identified. In the third area, a cluster including just two cases born the same year in the same town was confirmed. DISCUSSION: Our experience highlights that in the event of suspicious clusters occurring in different areas of a country, a coordinated and standardised approach should be preferred.

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics.

A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Serial translocation by means of circular intermediates underlies colour sidedness in cattle.

Colour sidedness is a dominantly inherited phenotype of cattle characterized by the polarization of pigmented sectors on the flanks, snout and ear tips. It is also referred to as 'lineback' or 'witrik' (which means white back), as colour-sided animals typically display a white band along their spine. Colour sidedness is documented at least since the Middle Ages and is presently segregating in several cattle breeds around the globe, including in Belgian blue and brown Swiss. Here we report that colour sidedness is determined by a first allele on chromosome 29 (Cs(29)), which results from the translocation of a 492-kilobase chromosome 6 segment encompassing KIT to chromosome 29, and a second allele on chromosome 6 (Cs(6)), derived from the first by repatriation of fused 575-kilobase chromosome 6 and 29 sequences to the KIT locus. We provide evidence that both translocation events involved circular intermediates. This is the first example, to our knowledge, of a phenotype determined by homologous yet non-syntenic alleles that result from a novel copy-number-variant-generating mechanism.

Reverse genetic screen for loss-of-function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle.

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10-bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large-scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild-type allele at the co-dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named 'cool gray'. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.

Genome-wide next-generation DNA and RNA sequencing reveals a mutation that perturbs splicing of the phosphatidylinositol glycan anchor biosynthesis class H gene (PIGH) and causes arthrogryposis in Belgian Blue cattle.

BACKGROUND: Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants. RESULTS: We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis. CONCLUSIONS: This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.

A stop-gain in the laminin, alpha 3 gene causes recessive junctional epidermolysis bullosa in Belgian Blue cattle.

Four newborn purebred Belgian Blue calves presenting a severe form of epidermolysis bullosa were recently referred to our heredo-surveillance platform. SNP array genotyping followed by autozygosity mapping located the causative gene in a 8.3-Mb interval on bovine chromosome 24. Combining information from (i) whole-genome sequencing of an affected calf, (ii) transcriptomic data from a panel of tissues and (iii) a list of functionally ranked positional candidates pinpointed a private G to A nucleotide substitution in the LAMA3 gene that creates a premature stop codon (p.Arg2609*) in exon 60, truncating 22% of the corresponding protein. The LAMA3 gene encodes the alpha 3 subunit of the heterotrimeric laminin-332, a key constituent of the lamina lucida that is part of the skin basement membrane connecting epidermis and dermis layers. Homozygous loss-of-function mutations in this gene are known to cause severe junctional epidermolysis bullosa in human, mice, horse, sheep and dog. Overall, our data strongly support the causality of the identified gene and mutation.

A splice site variant in the bovine RNF11 gene compromises growth and regulation of the inflammatory response.

We report association mapping of a locus on bovine chromosome 3 that underlies a Mendelian form of stunted growth in Belgian Blue Cattle (BBC). By resequencing positional candidates, we identify the causative c124-2A>G splice variant in intron 1 of the RNF11 gene, for which all affected animals are homozygous. We make the remarkable observation that 26% of healthy Belgian Blue animals carry the corresponding variant. We demonstrate in a prospective study design that approximately one third of homozygous mutants die prematurely with major inflammatory lesions, hence explaining the rarity of growth-stunted animals despite the high frequency of carriers. We provide preliminary evidence that heterozygous advantage for an as of yet unidentified phenotype may have caused a selective sweep accounting for the high frequency of the RNF11 c124-2A>G mutation in Belgian Blue Cattle.

Natural intrauterine infection with Schmallenberg virus in malformed newborn calves.

We surveyed morphologic alterations in calves in Belgium that were naturally infected in utero by Schmallenberg virus (SBV) and born with deformities during January-March 2012. SBV-specific RNA was distributed unevenly in different tissues. Natural intrauterine SBV infection of calves might cause serious damage to the central nervous system and muscles.

Evaluation of Blood Lactate, Heart Rate, Blood Pressure, and Shock Index, and Their Association with Prognosis in Calves.

Diseases in young calves received as emergencies are often associated with hypovolemic and/or septic shocks. The objectives of our study were to (1) assess the correlation between clinical hemodynamic parameters and blood L-lactates (LAC), systolic blood pressure (SBP), and the shock index (SI) recorded upon arrival; and (2) to evaluate how these parameters were related to short-term outcomes in calves under 4 months of age presented as emergencies. We conducted a single-observer prospective observational study on calves aged from 1 day to 4 months, presented to the Veterinary Clinic for Ruminants of Liège from December 2020 to May 2022. Forty-five calves were included in the study. The statistical analysis revealed a significant correlation between LAC and heart rate (r = 0.570; p < 0.05) and LAC and SI (r = 0.373; p < 0.05). A high LAC value at admission was significantly associated with a negative outcome (death) (p < 0.05). In calves suffering from obstructive digestive diseases, the SI was related to the outcome and the analysis indicated a cut-off value of 1.13 (Se = 0.77, Spe = 1). In conclusion, in our study, the initial blood L-lactate value was correlated with heart rate, the shock index, and the clinical shock score, and admission hyperlactatemia was associated with a poor prognosis in calves under 4 months of age. In this cohort, the shock index in calves suffering from digestive diseases was linked with mortality.

GWAS reveals determinants of mobilization rate and dynamics of an active endogenous retrovirus of cattle.

Five to ten percent of mammalian genomes is occupied by multiple clades of endogenous retroviruses (ERVs), that may count thousands of members. New ERV clades arise by retroviral infection of the germline followed by expansion by reinfection and/or retrotransposition. ERV mobilization is a source of deleterious variation, driving the emergence of ERV silencing mechanisms, leaving "DNA fossils". Here we show that the ERVK[2-1-LTR] clade is still active in the bovine and a source of disease-causing alleles. We develop a method to measure the rate of ERVK[2-1-LTR] mobilization, finding an average of 1 per ~150 sperm cells, with >10-fold difference between animals. We perform a genome-wide association study and identify eight loci affecting ERVK[2-1-LTR] mobilization. We provide evidence that polymorphic ERVK[2-1-LTR] elements in four of these loci cause the association. We generate a catalogue of full length ERVK[2-1-LTR] elements, and show that it comprises 15% of C-type autonomous elements, and 85% of D-type non-autonomous elements lacking functional genes. We show that >25% of the variance of mobilization rate is determined by the number of C-type elements, yet that de novo insertions are dominated by D-type elements. We propose that D-type elements act as parasite-of-parasite gene drives that may contribute to the observed demise of ERV elements.

Oak Acorn Poisoning in Cattle during Autumn 2022: A Case Series and Review of the Current Knowledge.

Oak poisoning is a known intoxication in grazing animals, but is slightly described in the literature. This case report describes 7 cattle from 3 different farms admitted to the clinic for ruminants of the University of Liège for suspected acorn poisoning in the autumn of 2022. The clinical signs were, anorexia, apathy with polyuria with low density. Further investigations led to the diagnosis of renal failure (blood urea 162 ± 88 mg/dL; blood creatinine 12 ± 4 mg/L). Supportive treatment, based on infusions (NaCl 0.9%) and electrolyte rebalancing, was administered and renal values were assessed every 24-48 h. Of these animals, 5/7 were euthanized. At necropsy, digestive erosions and ulcerations, oedema and renal hemorrhages, between the pyloric/caliceal cavity and the medulla were observed. Histopathological examination revealed necrosis of the renal tubules. The renal values of the two remaining animals were reduced, their general condition improved, and they were discharged. Acorn poisoning is a serious disease with no specific antidote or characteristic symptoms. Animals are identified as sick too late, when renal failure is already established. Farmers should be made more aware in order to prevent exposure, especially in years when acorns are abundant. Furthermore, there is no antidote for this intoxication.

Balancing selection of a frame-shift mutation in the MRC2 gene accounts for the outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle.

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed.

Blood Inflammatory, Hydro-Electrolytes and Acid-Base Changes in Belgian Blue Cows Developing Parietal Fibrinous Peritonitis or Generalised Peritonitis after Caesarean Section.

This study aimed to describe the inflammation, hydro-electrolyte and acid-base imbalances caused by generalised peritonitis (GP) and parietal fibrinous peritonitis (PFP) after caesarean section. After clinical examination, blood was sampled from 11 cows with PFP, 30 with GP and 14 healthy cows. Serum and plasma refractometry and glutaraldehyde tests were used to evaluate the inflammation level, while hydro-electrolytes and acid-base parameters were assessed using an EPOC® device. In addition to clinical signs of dehydration (>10%), blood analysis showed a high fibrinogen concentration (PFP: 8.64 ± 8.82 g/L; GP: 7.83 ± 2.45 g/L) and fast glutaraldehyde coagulation (<3 min) indicative of severe inflammation in both diseases compared to the control group (p < 0.05). Moreover, a severe decrease in electrolytes concentration (Na+: 126.93 ± 5.79 mmol/L; K+: 3.7 ± 1.3 mmol/L; Ca++: 0.89 ± 0.12 mmol/L; Cl−: 82.38 ± 6.45 mmol/L) and a significant increase in bicarbonate (30.87 ± 8.16 mmol/L), base excess (5.71 ± 7.42 mmol/l), L-lactate (8.1 ± 4.85 mmol/L) and creatinine (3.53 ± 2.30 mg/dL) were observed in cows with GP compared to the control group (p < 0.05). In contrast, few major perturbations were noticed in PFP, where only K+ (3.64 ± 0.25 mmol/L) and Ca++ (1.06 ± 0.09 mmol/L) were significantly modified (p < 0.05). In conclusion, a high dehydration and severe inflammation are induced by PFP and GP. Nevertheless, GP causes more electrolytes and acid-base disturbances than PFP.

Parietal fibrinous peritonitis in cattle: A literature review.

BACKGROUND: Parietal fibrinous peritonitis (PFP) is a complication of laparotomy in cattle, consisting of fluid and fibrin accumulation within a fibrous capsule between the parietal peritoneum and the abdominal muscles. Since scientific information on PFP is scarce, we aim to collect available information to help practitioners in its diagnosis and treatment, and to formulate research perspectives. METHODS: PubMed and GoogleScholar databases were scanned using "cattle" or "bovine", and one of the following keywords: "seroma", "parietal fibrinous peritonitis", "retroperitoneal abscess", or "wound infection". RESULTS: Although scientific information is often anecdotal, two recent larger studies shed more light on PFP symptoms, diagnosis and treatment. Symptoms vary according to the cavity's localisation and size, and include anorexia, weight loss and an inflammatory status. Rectal palpation is strongly indicative, but the definitive diagnosis is made by ultrasound. Trueperella pyogenes and Escherichia coli are frequently isolated germs, although it remains unclear whether they are primary or secondary agents. Good survival rates were reported after surgical drainage. CONCLUSION: Although the diagnosis and treatment seem clear, the exact pathogenesis of PFP should be the focus of ongoing research. This can be achieved by epidemiological data analysis focusing on risk factors like surgery technique, housing and ration.

Description of Plasma Penicillin G Concentrations after Intramuscular Injection in Double-Muscled Cows to Optimize the Timing of Antibiotherapy for Caesarean Section.

In order to improve the efficacy of penicillin injection during caesarean section, we aimed to identify the optimal timing of its preoperative administration. A study was conducted in 12 adult, non-pregnant Belgian Blue cows. To evaluate the plasma penicillin concentrations, blood samples were taken from the jugular vein at -5, 15, 30, 45, 60, 120, 240, 480 min relative to the intramuscular (IM) injection of 21,000 IU/kg of body weight of penicillin G. Results showed that plasma concentrations at 15 min after IM injection (668.3 ± 73.7 ng/mL) largely exceeded the minimal inhibitory concentration (MIC) of penicillin-sensitive bacteria (MIC < 125 ng/mL). With increasing time, plasma concentrations continued to rise, attaining an increasing proportion of moderately sensitive bacteria (250 ng/mL > MIC < 2000 ng/mL). The maximal concentration was reached between 1 and 4 h (average: 1.495.1 ± 181.7 ng/mL) after IM injection in the majority of cows, and decreased non-significantly to 1002.1 ± 93.2 ng/mL at 8 h. In conclusion, plasma penicillin concentrations at 15 min after an IM injection inhibit penicillin-sensitive bacteria. However, in order to obtain the maximal protective effect of the antibiotherapy, surgery should be started at 1 to 2 h after IM penicillin injection.

Antimicrobial Susceptibility Profile of Several Bacteria Species Identified in the Peritoneal Exudate of Cows Affected by Parietal Fibrinous Peritonitis after Caesarean Section.

The aim of this study was to identify the species and antimicrobial susceptibility of bacteria involved in parietal fibrinous peritonitis (PFP). We studied 156 peritoneal fluid samples from cows presenting PFP after caesarean section. Bacteria were cultured in selective media and their antimicrobial susceptibility was tested by disk diffusion assay. Bacteria were isolated in the majority (129/156; 83%) of samples. The majority (82/129; 63%) of positive samples contained one dominant species, while two or more species were cultured in 47/129 (36%) samples. Trueperella pyogenes (T. Pyogenes) (107 strains) was the most identified species, followed by Escherichia coli (E. coli) (38 strains), Proteus mirabilis (P. mirabilis) (6 strains), and Clostridium perfringens (C. perfringens) (6 strains). Several other species were sporadically identified. Antimicrobial susceptibility was tested in 59/185 strains, predominantly E. coli (38 strains) and P. mirabilis (6 strains). Antibiotic resistance, including resistance to molecules of critical importance, was commonly observed; strains were classified as weakly drug resistant (22/59; 37%), multidrug resistant (24/59; 41%), extensively drug resistant (12/59; 20%), or pan-drug resistant (1/59; 2%). In conclusion, extensive antibiotic resistance in the isolated germs might contribute to treatment failure. Ideally, antimicrobial therapy of PFP should be based upon bacterial culture and susceptibility testing.

Infectious Agents Identified by Real-Time PCR, Serology and Bacteriology in Blood and Peritoneal Exudate Samples of Cows Affected by Parietal Fibrinous Peritonitis after Caesarean Section.

The aim of this study was to identify the pathogens potentially involved in parietal fibrinous peritonitis (PFP). PFP is a complication of laparotomy in cattle, characterized by an accumulation of exudate inside a fibrinous capsule. We have studied 72 cases of PFP in Belgian blue cows, confirmed by a standard diagnostic protocol. Blood was collected to evaluate the presence of antibodies for Mycoplasma bovis(M. bovis), Coxiella burnetii(C. burnetii) and Bovine Herpesvirus 4(BoHV4) by enzyme-linked immunosorbent assays. Peritoneal exudate was obtained from the PFP cavity to perform bacteriological culture, and to identify the DNA of M. bovis, C. burnetii and BoHV4 using real time polymerase chain reaction (qPCR). Bacteriological culture was positive in most peritoneal samples (59/72); Trueperella pyogenes (T. pyogenes) (51/72) and Escherichia coli (E. coli) (20/72) were the most frequently identified. For BoHV4, the majority of cows showed positive serology and qPCR (56/72 and 49/72, respectively). Contrariwise, M. bovis (17/72 and 6/72, respectively) and C. burnetii (15/72 and 6/72, respectively) were less frequently detected (p < 0.0001). Our study proves that PFP can no longer be qualified as a sterile inflammation. Moreover, we herein describe the first identification of BoHV4 and C. burnetii in cows affected by PFP.

Comparison between generalised peritonitis and parietal fibrinous peritonitis in cows after caesarean section.

BACKGROUND: Parietal fibrinous peritonitis (PFP) and generalised peritonitis (GP) are two postoperative complications in cows, characterised by fluid and fibrin accumulation throughout the peritoneum (GP) or in an encapsulated cavity (PFP). Unlike GP, PFP is scarcely documented. METHODS: Twenty-one GP cases and 12 PFP cases were confirmed by ultrasound in cows referred to the Veterinary Clinic (Liège University) for complications after caesarean section. All cows underwent a standardised examination protocol. Blood samples were analysed for metabolic and inflammatory markers. Bacteriology was performed on peritoneal fluid samples. Treatment consisted of surgical drainage of the abdominal cavity (GP) or the encapsulated cavity (PFP). Variables concerning anamnesis, clinical findings and treatment outcomes were compared. RESULTS: Perioperative complications had occurred in 9/21 GP cows but 0/12 PFP cows (P<0.05). Biochemical analysis indicated pronounced inflammation and did not differ between groups. Peritoneal fluid samples of both groups were contaminated and contained similar bacteria (Trueperella pyogenes and Escherichia coli). While 11/12 PFP cows were discharged, all patients with GP died or were euthanased (P<0.05). CONCLUSIONS: We hypothesise that PFP and GP are two different manifestations of perioperative peritoneal contamination. The severity and spread of the contamination determine the clinical presentation and the prognosis.

Prophylactic antibiotic usage by Belgian veterinarians during elective caesarean section in Belgian blue cattle.

The use of prophylactic antibiotics in veterinary surgery reduces the occurrence of postoperative complications. In order to limit the development of antimicrobial resistance in bacteria and to maximize therapy efficacy, antibiotics should be used prudently and efficiently. A survey was sent to Wallonian rural veterinarians in order to assess the use of antibiotics before, during and after bovine elective caesarean section, the most common surgical procedure in Belgian rural practice. Results were analysed in the light of the current guidelines formulated by the Belgian Centre of Expertise on Antimicrobial Consumption and Resistance in Animals. Among 380 contacted veterinarians, 113 answered the survey. All veterinarians use antibiotics during caesarean section. Veterinarians predominantly use penicillin as the first drug of choice, whereas a minority use drugs considered as second or third choice, such as amoxicillin, oxytetracyclin or lincomycin-spectinomycin. Also, 31/113 veterinarians simultaneously use molecules from different antibiotic classes. The majority (100/113) of veterinarians inject antibiotics during or after surgery, while a minority (13/113) administer antibiotics preoperatively. Most veterinarians (67/113) limit the duration of their antibiotic treatment to 1 day. Considering the administration route, most veterinarians (88/113) systematically use the intraperitoneal injection route, which is not registered. Intramuscular injection and injection between muscle layers during surgery are carried out by 82/113 and 43/113 respondents, respectively. Twenty-two respondents apply intra-uterine antibiotics. Most veterinarians (87/113) combine several administration routes. The dosage of antibiotics varies enormously and excessive injection volumes are common, especially when multiple injection routes are combined. Our results show a striking lack of consistency in the antibiotic therapy during elective CS by rural veterinarians. Whereas the drug of choice and the treatment duration were largely in line with the current guidelines, this is certainly not the case for the dosage and the administration route. The intraperitoneal injection of antibiotics cannot be justified. Incorrect dosage of antibiotics should be avoided at all cost. The use of second and third line molecules and the simultaneous use of different antibiotic classes should be discouraged. A major challenge lies in the education of veterinary students and the sensitization of practitioners to avoid or unlearn unnecessary habits concerning antibiotic use. Finally, more clinical trials are needed in order to refine the current guidelines for antibiotic use and to determine the optimal drug of choice, treatment moment and application route in elective caesarean section in cattle.