Antimicrobials in a rabbit semen extender: effects on reproduction

[EN] The use of fluoroquinolone antibiotics was legally restricted by the European Commission in March 2019. Since the extender for rabbit semen Galap® contained this antibiotic, it became necessary to modify it. The purpose of this study was to search for another molecule, based on its antimicrobial activity and also on the conservation, motility and fertility of semen diluted with the new extender. Several bacterial strains were isolated from 10 poor-quality ejaculates, including Enterococcus spp., Staphylococcus aureus and Proteus spp. They were then tested for antimicrobial susceptibility. Out of 15 antibiotics evaluated, gentamicin was the one targeting such bacteria. In vivo tests were then carried out to assess the effects of this antibiotic change on sperm parameters. Up to 26 pools of good quality semen (total motility >70%) were diluted in original Galap® or in this extender with gentamicin. Ejaculates were analysed on the day (D) of collection and up to 6 d of storage at +17°C. After 24 h storage, the motility in the new extender was reduced by 7.7% compared to the original; this decrease did not worsen by storing. After 6 d of storage, no difference between the two media was detected. A total of 360 females were then inseminated with pools of 4 to 5 good quality semen diluted 1:10 in both extenders, following the routine protocol of rabbit semen processing centres. There was no difference in female fertility or prolificacy between both formulations. In conclusion, these preliminary results suggest that the addition of gentamicin to this extender is useful and also has no adverse effect on fertility or prolificacy.This study was supported by IMV TechnologiesRouillon, C.; Camugli, S.; Carion, O.; Echegaray, A.; Delhomme, G.; Schmitt, E. (2022). Antimicrobials in a rabbit semen extender: effects on reproduction. World Rabbit Science. 30(4):295-308. https://doi.org/10.4995/wrs.2022.1713229530830

Mise au point de biopuces à peptides et à polysaccharides et étude d'interactions biomoléculaires

Le développement des méthodes de production et d'isolement des peptides et des polysaccharides a amené la création de nouveaux outils pour l'analyse d'interactions à haut débit. Les biopuces à peptides et à polysaccharides ont été conçues pour répondre à ce besoin avec des applications dans le domaine médical et dans le sérodiagnostic. Ces nouveaux outils nécessitent des développements et des améliorations pour exploiter pleinement leur potentiel. La première partie de ma thèse a consisté à caractériser des biopuces à peptides sur lames de verre obtenues par la réaction chimiosélective de peptides modifiés par une fonction a-oxo aldéhyde avec une surface présentant des fonctions semicarbazide. La caractérisation a été réalisée à l'échelle macroscopique (angle de goutte, test de chimisorption/physisorption) et microscopique (AFM). Les études ont permis de mettre en évidence les limites des biopuces sur lame de verre (nature et rugosité du support, surface spécifique, structure tridimensionnelle et nombre des biomolécules dans un dépôt). A partir de ces expériences, nous avons élaborer une biopuce sur polycarbonate grâce au dépôt de nanoparticules de silice modifiées qui assurent la microfonctionnalisation locale du support par des fonctions semicarbazides. Les études de chimisorption/physisorption et de la taille des nanoparticules ont permis d'optimiser l'efficacité et la spécificité de l'immobilisation. Des tests d'interactions biomoléculaires peptides/anticorps sont également décrits. La deuxième partie du travail a consisté à mettre au point de biopuces à polysaccharides pour étudier les interactions entre des polysaccharides modifiés, les DMCBSu (Dextran MéthylCarboxylate Benzylamide Sulfate) et le facteur de croissance PDGF-BB. Le PDGF-BB est impliqué dans la formation de tissus granuleux dans des processus de régénérescence de blessures curatives. Les DMCBSu testés sont des polymères semi-synthétiques, modifiés par des fonctions carboxyméthyle, benzylamide et sulfate, mimant les propriétés de glycoaminoglycannes comme les héparanes sulfates. L'ajout des DMCBSu permet de potentialiser l'action de PDGF-BB sur la prolifération de fibroblastes in vitro. Les résultats de l'interaction, entre PDGF-BB et la chimiothèque de DMCBSu, immobilisés par adsorption, démontrent que la préparation des biopuces à polysaccharides sur des lames de verre semicarbazide est un outil efficace pour l'étude des interactions polysaccharides/facteur de croissance. Cette étude a montré que l'utilisation des biopuces permet d'une part, d'établir rapidement la relation structure-activité et d'autre part, de sélectionner les meilleurs polysaccharides interagissant avec la protéine d'intérêt.The development of new methodologies for the production and isolation of peptides and polysaccharides has required the fabrication of new devices allowing a high-throughput screening. Peptides and polysaccharides microarrays have been developed to answer this need. Application of those techniques may be found both in the medical and serodiagnosis fields. Development and optimization of these new tools are needed to attain microarrays full analysis potential. The first part of my thesis consisted in the characterization of peptides microarrays on glass slides. To do so, we performed chemoselective reactions between modified a-oxo aldehyde peptides and semicarbazide coated surface. Characterisation as been done in a macroscopic scale (contact angle, chemisorption/physisorption) and microscopic scale (AFM). Studies showed the limits of glass slides microarrays (nature and rugosity of the medium, surface specificity, tridimentional structures and number of molecules in a spot). From those experiments, we design a new kind of microarrays based on the use of polycarbonates. The polymer was locally micro-functionalized using silica nanoparticules coated by semicarbazides functions. Chemisorption/physisorption and nanoparticules size studies allowed optimizations of the ligation process specificity and efficency. In this first step, we also described biomolecular interactions between peptides and antibodies. The second part of this work allowed the development of polysaccharides microarrays to study interactions between modified polysaccharides DMCBSu (dextranmethylcarboxylate benzylamide sulfate), and PDGF-BB growth factor. PDGF-BB is implied in the regeneration of tissues during chronic wound healing. Tested DMCBSu are semi-synthetic polymers, modified by carboxymethylation, benzylamidification, and sulfation, thus mimicking glycoaminoglycans, like heparan sulphate. DMCBSu allows in vitro potentializing of PDGF-BB effect on fibroblasts proliferation. The results of the interactions between PDGF-BB and the library of modified dextran, immobilized by adsorption, demonstrate the efficiency of polysaccharides microarrays on semicarbazydes slides as a tool for the study of interactions between them and growth factors. This study also showed that microarrays can establish quickly the relationship between the structure of DMCBSu and the modification of PDGF-BB activity and then to select the polysaccharides providing the stronger interaction level with the studied protein.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Chemical Micropatterning of Polycarbonate for Site-Specific Peptide Immobilization and Biomolecular Interactions

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CrAsH-quantum dot nanohybrids for smart targeting of proteins.

International audienceSmart nanohybrids were prepared by conjugation of CrAsH to hydrosoluble and biocompatible quantum dots (QDs). The resulting probes were shown to bind efficiently and selectively to Cys-tagged proteins. The interaction with the protein was detected by an increase of the fluorescence emission of CrAsH. While the latter faded rapidly under continuous excitation, emission of the QD remained unaffected. The persistent fluorescence of the QD should thus allow extended monitoring of the target protein

INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling

International audienceAmong biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions (“bad coolers”) are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C. The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer. Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P < .05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2. Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96. These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells

Oligomeric PEG-Phospholipids for Solubilization and Stabilization of Fluorescent Nanocrystals in Water

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Management of an outbreak of meningococcal meningitides in the Province of South Kivu, Democratic Republic of Congo

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