In Vitro Evaluation of Phytobiotic Mixture Antibacterial Potential against Enterococcus spp. Strains Isolated from Broiler Chicken.

Enterococcus spp. are normal intestinal tract microflorae found in poultry. However, the last decades have shown that several species, e.g., Enterococcus cecorum, have become emerging pathogens in broilers and may cause numerous losses in flocks. In this study, two combinations (H1 and H2) of menthol, 1,8-cineol, linalool, methyl salicylate, γ-terpinene, p-cymene, trans-anethole, terpinen-4-ol and thymol were used in an in vitro model, analyzing its effectiveness against the strains E. cecorum, E. faecalis, E. faecium, E. hirae and E. gallinarum isolated from broiler chickens from industrial farms. To identify the isolated strains classical microbiological methods and VITEK 2 GP cards were used. Moreover for E. cecorum a PCR test was used.. Antibiotic sensitivity (MIC) tests were performed for all the strains. For the composition H1, the effective dilution for E. cecorum and E. hirae strains was 1:512, and for E. faecalis, E. faecium and E. gallinarum, 1:1024. The second mixture (H2) showed very similar results with an effectiveness at 1:512 for E. cecorum and E. hirae and 1:1024 for E. faecalis, E. faecium and E. gallinarum. The presented results suggest that the proposed composition is effective against selected strains of Enterococcus in an in vitro model, and its effect is comparable to classical antibiotics used to treat this pathogen in poultry. This may suggest that this product may also be effective in vivo and provide effective support in the management of enterococcosis in broiler chickens.

MicroRNA expression and oxidative stress markers in pectoral muscle of broiler chickens fed diets supplemented with phytobiotics composition.

Phytobiotic compositions are commercially used in broiler production, mostly to improve general health and the production parameters. Moreover, some of their active substances may change the expression of miRNA in different tissues. Therefore, the purpose of this study was to evaluate the effect of the phytobiotic composition (PBC) containing white mustard, calamus, turmeric, and common ivy on production parameters, oxidative stress markers and expression of selected miRNAs in pectoral muscle of broiler chickens. The experiment was performed on broiler chickens fed the control diet (without PBC), and a diet supplemented with 60 or 100 mg/kg of PBC for 35 days. After the experiment, samples (blood and muscle) were collected for analyses. The analyzed production parameters included: feed conversion ratio, feed intake and body weight. There was no effect on growth performance of broiler chickens but feeding diet supplemented with 60 mg/kg phytobiotics significantly increased the expression of miR-30a-5p, miR-181a-5p, and miR-206, and decreased that of miR-99a-5p, miR-133a-5p, miR-142-5p, and miR-222 in pectoral muscle of chickens. The addition of 100 mg/kg phytobiotics significantly increased miR-99a-5p and miR-181a-5p expression, and caused down-regulation of the expression of miR-26a-5p and miR-30a-5p. Chickens fed diet supplemented with 100 mg/kg PBC had lower level of lipid peroxidation products in blood, while in the muscle tissue it was higher in birds fed a diet with the addition of 60 mg/kg as compared to the control group. The results suggest that this unique composition of phytobiotics does not affect productive traits but can change expression of miRNAs that are crucial for muscle physiology and pathology in broiler chickens. This additive may also protect against the oxidative stress but the effect is dose dependent.

The Impact of a Phytobiotic Mixture on Broiler Chicken Health and Meat Safety.

The purpose of the study was to assess the effects of different doses of a phytobiotic mixture on selected production parameters and meat quality and to assess the residue of the preparation in tissues and the possible toxic effects in broiler chickens. Broiler chicks aged 160 days, divided into four equal groups, were supplemented with the phytobiotic mixture at different doses, D1-0.5 mL/L, D2-1 mL/L, and D3-2 mL/L, four times during a 42-day trial. There were no statistically significant differences in weight gain per week of life and mortality in the birds. The study also demonstrated that the use of the mixture of phytobiotics had no significant effect on colour, pH, WHC, and natural leakage. However, a beneficial effect of the additive was found in the group treated with a dose of 1 mL/L, where less thermal leakage from the meat was demonstrated. Furthermore, significant differences in the change in thigh muscle tenderness were also observed. In the histopathological analysis of the liver no significant differences were observed. In addition, no residues of the mixture or its metabolites were found in the tissues analysed. In conclusion, the proposed scheme of administration of the phytobiotic additive, regardless of the dose, does not cause pathological changes in organs and does not carry the risk of residues of the product in tissues intended for human consumption.

In Vitro Assessment of Antimicrobial Activity of Phytobiotics Composition towards of Avian Pathogenic Escherichia coli (APEC) and Other E. coli Strains Isolated from Broiler Chickens.

Escherichia coli infections (including APEC) in broiler chickens are not only a health and economic problem of the flock, but also a significant health threat to poultry meat consumers. The prophylactic and therapeutic effects of the phytobiotic composition on E. coli in broiler chickens were previously described. However, most of the data were related to the reference strains (for both in vitro and in vivo models). Based on the previous studies in human and animals, E. coli strains seem to be multidrug resistance. This, in turn, makes it necessary to develop effective alternative methods of treating this type of infection already at the stage of poultry production. In the present study, the antibacterial activity against various strains of E. coli (including APEC) was assessed for two innovative phytobiotics mixtures: H1, containing thymol, menthol, linalool, trans-anethole, methyl salicylate, 1,8-cineol, and p-cymene; H2, in addition to compounds from H1, containing terpinen-4-ol and γ-terpinene. The unique mixtures of phytobiotics used in the experiment were effective against various strains of E. coli, also against APEC, isolated from broiler chickens from traditional industrial breeding, as well as against those showing colistin resistance. The minimum inhibitory concentration (MIC) values for these unique mixtures were: For H1 1:512 for APEC and non-APEC E. coli strains isolated from day old chicks (DOCs), 1:512 for non-APEC, and 1:1024 for non-APEC isolated from broilers sample. For mixture H2, MIC for APEC from both type of samples (DOCs and broilers) was 1:1024 and for non-APEC (DOCs and broilers) was 1:512. The results suggest that phytobiotic compositions used in this study can be successfully used as a natural alternative to antibiotics in the treatment of E. coli infections in broiler chickens. The promising results may be a crucial point for further analyses in broiler flocks exposed to E. coli infections and where it is necessary to reduce the level of antibiotics or completely eliminate them, thus reducing the risk of foodborne infections.

Sustainable Future Protein Foods: The Challenges and the Future of Cultivated Meat.

Global pressure from consumers to improve animal welfare, and reduce microbiological risks or the use of antibiotics pose new challenges for the meat industry. Today's livestock production, despite many undertaken measures, is still far from being sustainable. This forced the need to work on alternative protein types that come from plants, insects, fungi, or cell culture processes. Due to some technical and legal barriers, cultivated meat is not present on the European market, however, in 2020 it was approved in Singapore and in 2022 in the USA. While the technology of obtaining cell cultures from animal muscles has been known and successfully practiced for years, the production of a stable piece of meat with appropriate texture, taste, and smell, is still a problem for several scientific groups related to subsequent companies trying to obtain the highest quality product, in line with the expectations of customers. Although the work on optimal cell meat production has been going on for years, it is still in an early stage, mainly due to several limitations that represent milestones for industrial production. The most important are: the culture media (without animal serum), which will provide an environment for optimal muscle development, natural or close to natural (but still safe for the consumer) stable scaffolds for growing cells. Here, we review the actual knowledge about the above-mentioned challenges which make the production of cellular meat not yet developed on an industrial scale.

Effect of Phytobiotic Composition on Production Parameters, Oxidative Stress Markers and Myokine Levels in Blood and Pectoral Muscle of Broiler Chickens.

The aim of this study was to evaluate the effect of dietary level of a phytobiotic composition (PBC) on production parameters, oxidative stress markers and cytokine levels in the blood and breast muscle of broiler chickens. The experiment was performed on 48 one-day-old female Ross 308 broiler chickens divided into three groups (n = 16) fed the control diet (without PBC), and a diet supplemented with 60 or 100 mg/kg of PBC. After 35 days of feeding, blood and breast muscle samples were collected for analyses. There was no effect on final body weight and feed intake but PBC addition (100 mg/kg) improved feed efficiency as compared to the control. Also, this dietary level of PBC contributed to an increase in interlukin-6 content in blood and a reduction in tumor necrosis factor-α concentrations in pectoral muscle in comparison with the control group. In conclusion, the addition of 100 mg/kg PBC improved the production parameters of broiler chickens and beneficially influenced the regeneration and protection of pectoral muscle against pathophysiological processes that may occur during intensive rearing.

Simultaneous miRNA and mRNA Transcriptome Profiling of Differentiating Equine Satellite Cells Treated with Gamma-Oryzanol and Exposed to Hydrogen Peroxide.

Gamma-oryzanol (GO) is a popular supplement for performance horses, dogs, and humans. Previous studies indicated that GO supplementation decreases creatine kinase activity and lactate level after exercise and may affect oxidative stress in Thoroughbred horses. GO may change genes expression in equine satellite cells (ESC). The purpose of this study was to evaluate the effect of GO on miRNA, gene expression, oxidative stress, and cell damage and viability in differentiating ESC pretreated with hydrogen peroxide (H2O2). ESCs were obtained from a young horse's skeletal muscle. ESCs were pre-incubated with GO (24 h) and then exposed to H2O2 for one hour. For the microRNA and gene expression assessment, the microarray technique was used. Identified miRNAs and genes were validated using real time-quantitative polymerase chain reaction. Several tests related to cell viability, cell damage, and oxidative stress were performed. The microarray analysis revealed differences in 17 miRNAs and 202 genes between GO-treated and control ESC. The tests related to apoptosis, cell viability, and oxidative stress showed that GO affects these processes to varying degrees. Our results suggest that GO can change miRNA and gene expression and may impact the processes involved in tissue repairing after an injury.

Effect of ß-hydroxy-ß-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide.

BACKGROUND: Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. ß-Hydroxy-ß-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H2O2. We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H2O2-related injury by changing the expression of miRNAs. METHODS: Equine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H2O2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry. RESULTS: Analysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H2O2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition. CONCLUSIONS: Our results suggest that ESC pre-incubated with HMB and exposed to H2O2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after injury. Enrichment analyses for targeted genes revealed that a large group of genes was associated with the regulation of signaling pathways crucial for muscle tissue development, protein metabolism, muscle injury, and regeneration, as well as with oxidative stress response.