Effects of rumen lipopolysaccharides on the growth and fermentation of the end products of pure cultured bacteria

All experimental procedures involving animals used as rumen fluid donors in the study were performed under protocols approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). Furthermore, all methods were performed in accordance with IACUC guidelines and regulations. The following study is reported according to ARRIVE guidelines.

statistical analysis

Each treatment was tested with at least three biological replicates (n ≥ 3), for a total sample size n = 12 for each strain tested. Significance has been declared in s ≤ 0.05, while the slope is at 0.05s ≤ 0.10.

The logistic function was used to predict the growth rate (μ) and lag time (delay). according to41the logistic function used was:

$$ Y = {y}_{0} + frac{C}{left(1+ {exp}^{left[4*mu max*frac{lag-t}{C}+2right]} right) } $$

Y is the real ODT, y0 is the initial OD0μmax is the maximum specific growth rate, and C is the OD increase from OD0 that ofR.

The expected maximum specific growth rate and lag time were used as inputs to the SAS analysis. Treatment effects on maximum specific growth rate, lag time, ammonia concentrations, and organic acids were analyzed using least squared analysis of variance (ANOVA) using the SAS mixed procedure.

The statistical model used is:

$${y}_{i}=mu +{T}_{i}+{E}_{j}+{varepsilon}_{ij}$$

where y is a dependent variable, μ is the general mean, ({T}_{i}) is a consistent effect of treatments, ({E}_{j}) is a trial run, and ({varepsilon}_{ij}) It is a random error. The trial run was considered as a random effect.

rumen liquid fractionation

Ruminal liquor was obtained from rumen-fed Holstein cows on a total mixed ratio (DM basis: 60% whole vegan corn forage, 12.5% ​​ground corn, 13% citrus pulp, 12% soybean powder, and 2.5% mineral and vitamin blended). ). About 3 h after morning feeding, rumen contents were collected manually (7 L) and filtered through four layers of cheesecloth in preheated thermos bottles and immediately transported to the laboratory. The contents were again strained through two-layer gauze, transferred to cups, and immersed in ice for 15 minutes. The strained rumen fluid (approximately 14 L) was centrifuged (Sorvall RC-5B ultra-refrigerated, DuPont Instruments® Wilmington, DE) three times in a row. first at 1000×g for 10 min, then the supernatant was collected and centrifuged again at 11250×g for 20 min, then a bacteria pellet was obtained, resuspended in Milli-Q water and centrifuged a third time at 16250×g for 20 min to obtain a bacteria pellet that was subsequently suspended in Milli-Q water. Finally, bacterial pellets were transferred to pyrogen-free tubes, homogenized, diluted to 15 ml with Milli-Q water and stored at −80 °C for subsequent rumen LPS extraction.

Extraction of rumen polysaccharides

A modified hot phenol extraction was used to extract LPS from rumen bacteria obtained from a cannulated rumen cow as previously described.42,43 But with some minor modifications and validation described below. Briefly to isolate total LPS from rumen fluid, bacterial pellets were boiled at 100–110 °C using a heat block for 30 min followed by addition of 50 ml Milli-Q water. The bacterial suspension was then treated with 50 mL of 90% phenol pre-warmed at 68 °C for 30 min. The preparation was then placed at -20 °C for 30 min to cool down and centrifuged at 5000×g for 10 minutes. The aqueous layer (upper) was then collected because it displays the largest concentration of LPS after being tested with silver dye (Thermo Scientific™ Pierce™ Silver Stain Kit). The aqueous layer was then transferred to a regenerated cellulose dialysis membrane (Fisherbrand™) for further dialysis against Milli-Q at 4 °C until no phenol was detected at 260 nm in Milli-Q. The precipitated samples were then treated with 5 mM MgCl .2 followed by 20 μg/ml Dnase I (M0303s, New England Biolabs) for 2 h at 37 °C to break down contaminating DNA. Then, 20 μg/mL Rnase H (T3018, New England Biolabs) was added for 2 h at 37 °C, to hydrolyze the contaminating RNA, and another 30 mg/mL Proteinase K (Fisher BioReagents™ Proteinase K, catalog number BP1700- 100) to remove protein contamination. The preparation was then lyophilized and the crude LPS mass was determined. After lyophilization, dry samples were suspended in 15 ml of Milli-Q water and centrifuged at 1110×gfor 10 minutes to remove any solids. The supernatant was treated with 0.15 mL 50 mM acetic acid, 95% ethanol and transferred using a glass Pasteur pipette to ultracentrifuge tubes (Quick-Seal® Round-Top polypropylene tube) and then spun for 8 h at 4 °C, 105,000× g in an ultracentrifuge (Optima XE, Beckman Coulter Life Sciences, Indianapolis, IN). The supernatant was removed and LPS gels were suspended in 2 ml of endotoxin-free water and lyophilized to determine the dry weight of pure LPS. To confirm the purity and normalization of rumen-derived LPS, the final products were visualized using a Pierce™ Silver Stain Kit (Thermo Scientific™) according to the manufacturer’s instructions. In all cases, the Pierce™ Silver Stain Kit indicated a purity similar to that of LPS purified from pure bacterial isolates.

LPS stock setting

concentrationscoli bacteria-LPS ( Escherichia coliO111: B4, L2630; Sigma-Aldrich Co. , Louis, MO), ruminal-LPS and MIX-LPS were 200,000 EU (1 ng/ml = 10 EU based on the Sigma-Aldrich protocol).

All LPS stocks were prepared under anaerobic conditions, for which 25 mg of coli bacteria-LPS was added to 62.5 mL of sterile, anaerobic, non-heat-generated water during CO2 flushing2to generate 0.4 mg/mL of coli bacteria-LPS. Ruminal stock-LPS (25 mg) was resuspended in 2 mL of sterile, unheated water and sonicated for 20 min. After sonication, rumen LPS stocks were prepared under anaerobic conditions and the final volume was raised to 62.5 mL in sterile, anaerobic, non-thermic water to generate 0.4 mg/mL stock. For stock MIX-LPS, 13.5 mL (equivalent to 5.4 mg of coli bacteria-LPS) from coli bacteria LPS stock was mixed with 13.5 mL (equal to 5.4 mg rumen-LPS) to generate 0.4 mg/mL MIX-LPS. All LPS stocks were then filtered through 0.45 μm followed by a 0.22 μm membrane syringe (PES) filter (Celltreat, Pepperell, MA) into serum bottles pre-washed with CO and sterilized. Volume 0.5 ml 0.4 mg/ml coli bacteria -, ruminal- and MIX- The LPS inventory contained 200,000 EU of LPS. We chose a concentration of 0.4 mg/mL assuming that rumen LPS would equal coli bacteria LPS by weight. This assumption was made to make all doses similar in weight, volume and endogenous toxicity. To verify the endotoxinity of the MIX stock solution, we performed an amebocyte lysate lysate assay, which showed an endotoxin close to 200,000 EU.

The media

The basal medium contains 240 mg of K .2HPO4240 mg of KH2after, after4480 mg of (NH4)2So4480 mg of NaCl, 100 mg of MgSO47 h2O, 64 mg of CaCl22H2O, 600 mg cysteine ​​hydrochloride, 1 g of trypticase peptone (Product 212750; BD), and 0.5 g of yeast extract (Product 212750; BD) per liter44; Ph 6.5; Autoclaved (121 °C, 15 min) to remove O2 and cooling under O2free company2. Sodium carbonate (4 g/L) was added as a buffer. Resazurin has been added as a redox indicator. Anaerobic growth substrates were prepared and introduced to the basal medium under sterile conditions. Glucose (final concentration 20 mM) was added as a growth substrate for S. Bovis JB1 and Himself. ruminants HD4 and 50 mM lactate (final concentration) were added as a growth substrate for M. Sydney T81. All media and media additions (glucose or lactate) were based on previous peer-reviewed studies of pure cultured rumen bacteria.44 It was set up at the same time to reduce variance between operations.

Organisms

lactate-producing bacteria The ruminant Selinomonas HD4 (Guardianship Series: Herbert J. Strobel and Michael de Vlathe) and Macular streptococcus JB1 (Chain of Guardianship: James B. Russell, Michael D. Flythe), and bacteria that use lactate Megasphaera elsdenii T81 (Chain of Guardianship: Paul J. Weimer, Michael D. Flythe) was obtained from a stock breeding collection held at the Animal Feed Production Research Unit, ARS, USDA on the University of Kentucky campus. The purity of all isolates was verified using Gram stain and microscopy45. Initial growth curve analyzes were performed to determine the lag, log, and persistence stages for each strain (data not shown).

Treatments and measurement of bacterial growth

Strains were inoculated in 10 ml growth medium (lactate producers: basal medium plus 20 mM glucose; lactate users: basal medium plus 50 mM lactate) and incubated at 39 °C overnight. The optical density (OD, absorbance 600 nm) was recorded for each night culture to determine inoculum for growth curve experiments. 100 microliters S. Bovis JB1, 100 μl Himself. ruminantsHD4, or 500 μl M. SydneyT81 was added to basal medium using 0.5 ml pretreatment or control LPS. Treatments were (1) CTRL, control group (LPS-free anaerobic water); (2) RUM, rumen – LPS (0.4 mg/mL rumen LPS); (3) E. coli, coli bacteria-LPS (0.4 mg/ml coli bacteria -LPS); and (4) mix, 1:1 coli bacteria: Ruminal-LPS (0.4 mg/ml MIX-LPS). The concentration of all three LPS (RUM, E. COLI, MIX) was chosen based on previous studies in which LPS concentration in cows was measured using SARA (200,000 EU)46. All experimental tubes were inverted for mixing, flame sterilized, and 2 mL of primary fermentation final products (NH .) removed3-N, organic acids) and the initial optical density (OD .)0). All anaerobic strains grown under O2free company2 in suspended tubes and incubated at 39°C without shaking. Optical densities (ODt) were recorded every hour except in the case ofS. BovisJB1, for which measurements were collected every 30 min, until bacterial growth reached a plateau. Once bacterial growth reached the intermediate exponential phase, 2 ml of culture medium was collected into Eppendorf tubes, and clarified by centrifugation (15,000×g2 min), and frozen at −20 °C to determine the final fermentation products for later (NH3-N and organic acids). Samples of the end products of fermentation were collected mid-exponential to represent the conditions of continuous fermentation as it occurs in the rumen (Figs 1, 2, 3).

Fermentation end product analyses

Media samples were thawed and clarified by centrifugation (15000× .).g2 min), and ammonia concentrations were determined by the phenolic acid/hypochlorite method47. Volatile fatty acids, lactate, and soluble sugar were quantified by HPLC (Dionex, Sunnyvale, CA, USA). The column (Aminex HP-87H, Bio-Rad, Hercules, CA) was run at 50 °C, at a flow rate of 0.4 ml/min and aqueous H.2So4 (0.17 N) mobile stage. A refractive index detector (Shodex/Showa Denko, Kanagawa, Japan) and a UV detector (Dionex, Sunnyvale, CA, USA) were used in tandem to detect the filter compounds.