Maintaining intestinal health is crucial for the overall well-being and productivity of livestock, as it impacts nutrient absorption, immune function, and disease resistance. Oxidative stress and inflammation are key threats to intestinal integrity. This study explored the antioxidant, anti-inflammatory, and barrier-strengthening properties of a fermented plant macerate (FPM) derived from 45 local herbs, using a specifically developed fermentation process utilizing the plants’ inherent microbiota to enhance bioactivity and sustainability. In vivo, FPM significantly reduced ROS levels in Drosophila melanogaster and improved activity and LT50 values in Caenorhabditis elegans under oxidative stress, although it did not affect intestinal barrier integrity in these models.
Methods
Oxidative stress analysis in C. elegans. To evaluate protective effects of FPM under oxidative stress, an infrared micro-beam-based system was utilized to record locomotor activity over time. After 72 h treatment with FPM, adult N2 nematodes were washed off plates using S-Basal (+0.5% w/v PEG 3.350). The number of nematodes was adjusted with S-Basal to reach approximately 80 worms per well. Next, 92.5 μL worms in S-Basal were pipetted into individual wells of a flat bottom 96-well plate. The experiment was performed in eight well replicates per testing group. Basal nematode activity was measured for 60 min using WMicrotracker One to obtain reference values for individual well data normalization. Next, oxidative stress was induced by adding 7.5 μL of 1 mol/L paraquat (PQ) in dH2O stock solution resulting in 75 mmol/L final concentration per well. For non-stressed groups, dH2O was added instead of PQ. Nematode activity was captured for a total duration of 1,000 min using WMicrotracker One. Results are given as locomotive motility normalized to basal activity. For AUC calculation, data was normalized to the untreated control group. For lethal time 50% (LT50) value calculation, data was normalized to group and non-linear regression was performed.
Smurf Assay. To assess intestinal barrier integrity in C. elegans, we conducted a Smurf assay using Brilliant Blue FCF dye. Smurfs serve as models for leaky gut syndrome, as the non-absorbable dye penetrates surrounding tissues after ingestion in leaky guts, thereby staining the entire organism blue. We investigated whether FPM could modulate age-related disruption of gut integrity in C. elegans. The number of Smurfs was counted in each treatment group and expressed as a fraction of the total worm population per group.
Results
We analysed the protective effects of FPM on C. elegans under oxidative stress induced by PQ. Locomotive motility overtime served as the indicative readout parameter. Figure 6a presents the activity counts of untreated worms, stressed worms, and stressed worms treated with FPM over time. PQ-induced oxidative stress significantly decreased worm activity, which was counteracted by FPM. Worms pretreated with 0.5%, 1% and 2% FPM tolerated PQ-induced oxidative stress significantly longer than the PQ control group. Non-linear regression analysis revealed an increase in LT50-values, ranging from251 to 269 min, compared to the PQ control (LT50 =148 min), suggesting improved lifespan of C. elegans under oxidative stress. The respective area under the curve (AUC) values of the individual time courses are summarized in Fig. 6b. A significant and dose-dependent increase in worm activity under oxidative stress was documented in the worms treated with 0.5%, 1% and 2% FPM, relative to the stress control. At the highest tested concentration of 2% FPM, the activity of C. elegans was increased by 115% (P < 0.0001). As depicted in Fig. 6c, treatment with0.5%, 1%, or 2% FPM did not significantly enhance intestinal barrier integrity. These results suggest that while FPM effectively mitigates PQ-induced oxidative stress in C. elegans, it does not affect intestinal barrier integrity under the experimental conditions of our study.
The findings indicate that FPM shows promising application as a functional feed supplement for improving intestinal health in livestock by mitigating oxidative stress and inflammation.
J Anim Sci Biotechnol. 2025 Apr 6.
Heckmann M, Sadova N, Sandner G, Neuhauser C, Blank-Landeshammer B, Schwarzinger B, König A, Liang M, Spitzer M, Weghuber J, Stadlbauer V.