Dose rates of antimicrobial substances in boar semen preservation-time to establish new protocols.

To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37 mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5 mg/L, which is close to or below the epidemiological cut-off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512 mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic-free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready-to-use extenders. In conclusion, an end volume-based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.

Identification and characterization of intestinal lactobacilli strains capable of degrading immunotoxic peptides present in gluten.

AIM: Identify and characterize bacteria from the proximal gastrointestinal tract of pigs capable of degrading immunogenic gluten peptides. METHODS AND RESULTS: Bacteria were cultured from the small intestine of pigs fed a 20% gluten diet and from an enrichment media with the 18-mer peptide LQLQPFPQPQLPYPQPQL. Isolates were screened for the production of specialized proteolytic enzymes and the ability to degrade and remove metastable peptides from α-gliadin (16-mer and 33-mer) and ω-gliadin (17-mer), with established roles in the aetiology of coeliac disease. Degradation was determined by ELISA and mass spectrometry (UHPLC-MS/MS in MRM mode), and hydrolysis fragments were characterized by LC-MS/MS. Four strains from the species Lactobacillus ruminis, Lactobacillus johnsonii, Lactobacillus amylovorus and Lactobacillus salivarius showed the highest peptide-degrading activities. Strains displayed different degradation rates and cleavage patterns that resulted in reduction but not complete removal of immunotoxic epitopes. CONCLUSIONS: We employed a unique enrichment process to select for bacteria adapted to the conditions of the proximal gastrointestinal tract with the ability to partially detoxify well-characterized peptides involved in coeliac disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a basis for the selection of Lactobacillus strains for probiotic applications aimed to reduce epitope-containing gluten peptides before reaching the epithelium of the small intestine of patients with coeliac disease.

Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors In vivo by ultrasound-guided injection or transcoronary delivery.

BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R-specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus-treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade.