An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats

Background: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. Results: Plasmid DNA with two resistance genes (nptII and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, where constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. Conclusions: The analyses showed that extensive ingestion of DNA (100 \ub5g plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit <1 transformant per 1.1 x 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed

Comparison of molecular cargo of follicular fluid exosomes from large dominant and small subordinate follicles in cows

Application: Taken in account a beneficial effect of follicular fluid exosomes (ffExo) in reproduction biotechnologies in bovine, out studywill enlighten potential factors relying their role on oocyte quality.Introduction: A beneficial effect of ffExo supplementation during IVM on oocyte quality and embryo development was reported in severalanimal species including cattle (Asaadi et al., 2021; Singina et al., 2022). However, this effect was not observed with ffExo extracted fromlarge dominant follicles (da Silveira et al., 2017). We aimed to compare protein and lipid cargo between ffExo preparations from small follicles(SF) and large dominant follicles (LF).Materials and Methods: Follicular fluid (FF) was aspirated from small antral follicles (3–6 mm) and dominant follicles (>8 mm) of 12slaughtered cows ovaries. Fractions enriched in ffExo were obtained by 4 serial centrifugations followed by ultracentrifugation at100 000g. Transmission electron microscopy (TEM) was performed on fixed ffExo. Presence of exosome markers was analyzed by Westernblot. Peptide/protein and lipid profiles of ffExo samples (n = 24) were acquired by MALDI-TOF mass spectrometer RapifleX Tissuetyper(Bruker Daltonics) in the 200–1 200 m/z range for lipids and 2 000–30 000 m/z range for proteins, in 9 technical replicates each. Spectralprocessing and statistical analyses were performed using home R software based on MALDIquant & MALDIquantForeign packages. Peaksannotation were obtained from Top-Down proteomics database and Lipidmaps.Results: Exosomes from SF and LF were similar in size (mean diameters 61.4 nm and 59.1 nm, respectively); however, abundance of exosomespecific marker CD81 was significantly higher in SF-ffExo preparations (p 1.5). 15 out of 17 differential peaks were annotated and contain protein proteolytic fragments or small proteinsknown involved in cumulus cells functions. Lipid profiles in negative and positive acquisition modes gathered in total 666 m/zfeatures correspondent to different lipid isoforms. 34 features were significantly up-regulated and 222 down-regulated in ffExo from LFcompared to SF (Wilcoxon test, p 2). According to annotation, lysophospholipids LPC and LPE were more abundantin LF exosomes, whereas different phosphatidylcholines and sphingomyelins were more abundant in small follicles.Conclusions: Preparations of follicular fluid exosomes from large dominant or small follicles differed more by their lipid composition thanby proteins. Significant difference in membrane lipids might explain different affinity of ffExo to target cells including oocyte