Bovine Digestive Contents

Digestive contents (DCs) from rumen, small intestine and rectum compartments were collected on eight healthy “Salers” bulls from the “Herbipole” experimental Unit at the INRAE (Saint-Genès-Champanelle, France) as previously described [21]. Briefly, bulls, approximatively 2 years of age and 562 (±26) kg mean weight, were raised according to current INRAE ethical guidelines for animal welfare. These animals were fed a mixed diet composed of hay (8.6 kg /head/day) and concentrate (2.5 kg/head/day) which composition is given in Additional file 1, Table S9. The bulls had not received any antibiotic treatment in the year prior to slaughter. The bulls were slaughtered in the experimental slaughterhouse of the “Herbipole” (Permit number: 63345001). The animals had received their last meal the day before slaughter. The experiments were approved by the local ethics committee (Comité d’Ethique pour l’Expérimentation Animale en Auverggne, C2E2A, Permit Number: C6334517).

DCs from rumen and small intestine (jejunum and ileum) were collected at slaughter whereas rectum contents were collected two days before slaughter by rectal palpation. All DCs were rapidly collected and immediately brought to the laboratory. Small intestine contents were directly distributed in sterile tubes without any particular attention paid regarding anaerobiosis while rumen and rectum contents were processed under strictly anaerobic conditions as previously described [21]. Briefly, rumen contents were filtered through four layers of cheesecloth to remove large feed particles, and rectum contents were diluted 1:1 in reduced potassium phosphate buffer (50 mM potassium phosphate, resazurin 0.1%, 40 mM Na₂CO₃, 3 mM cysteine, pH 7.6) in order to maintain a low redox potential. Rumen and rectum samples were distributed in sterile O₂-free CO₂-saturated Hungate tubes (Bellco, USA). The endogenous microbiota was then removed as previously described [21]. Briefly, DCs were centrifuged twice for 15 min at 10,000 x g, and supernatants were filtered successively through membranes pore size 0.45 and 0.22 μm (Millipore). Filtrates from rumen and rectum contents were then placed in an anaerobic chamber (JACOMEX, Lyon, France) under CO₂ atmosphere (< 80 ppm of O₂) during three days at room temperature. After getting out of the anaerobic chamber, the samples were filtered again (0.22 μm pore-size filters, Millipore) and placed into new O₂-free CO₂-saturated sterile Hungate tubes. Sterility was verified on Luria Bertani (LB) agar plates after overnight incubation at 37°C. Filtered DCs were stored at 4°C until use.