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Background The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream ( Sparus aurata ) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite ( Enteromyxum leei ) challenge was tested. Results The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. Conclusions This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.

Background Accurate screening of new alternative antimicrobial compounds is essential for their use to control pathogens in swine production due to the replacement of antibiotics and zinc oxide. Most in vitro studies have separately reported the antimicrobial activity of organic acids and essential oils (EOs) using diverse methods for susceptibility testing. In addition, in vitro outcomes can help in the selection of the suitable antimicrobial compound and effective combinations of these compounds in the control of pathogens of interest in pork production. Therefore, the aim of this study is to determinate the antibacterial activity of six organic acids and six EOs against Escherichia coli , Salmonella spp. and Clostridium perfringens isolates, some of them multi-resistant to antibiotics, from swine origin. The synergistic effects between the products with higher activity for each bacteria were also calculated. Results All products tested showed activity against at least one bacterial species, except for black pepper EO. The results showed that formic acid with the shortest chain length was the most effective against E. coli and Salmonella spp. , while the sodium salt of coconut fatty acid distillates with long chain acids was the most effective against C. perfringens . The susceptibility of isolates tested to EOs was similar, a result that demonstrates a similar activity of these products against phylogenetically unrelated pathogens. In addition, an additive effect was shown for carvacrol-oregano EO for E. coli , formic acid-carvacrol and formic acid-thymol for Salmonella spp. and carvacrol-cinamaldehyde for C. perfringens . Conclusions The susceptibility of isolates to EOs was similar, a result that demonstrates a similar activity of these products against phylogenetically unrelated pathogens in contrast to organic acids. In addition, an additive effect was shown for several combinations of these compounds.

The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a -based probiotic, in gilthead sea bream (GSB, ), a marine farmed fish of high value in the Mediterranean aquaculture The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN ; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture. MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T. MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.

Nutritional strategies to improve gut health of broilers are under research. This study investigated the effect of dietary supplementation with sodium butyrate protected by sodium salts of medium-chain fatty acids as a feed additive on broiler gut health. The first experiment was conducted to evaluate the effect of supplementing at 0.5, 1, and 2 kg/t in broilers housed under optimal conditions. Supplementation at 0.5 and 1 kg/t maintained goblet cell counts at 10 days of age (p ≤ 0.05), and supplementation at 1 kg/t decreased intraepithelial lymphocyte counts compared to 2 kg/t at 39 days (p ≤ 0.10). Abdominal fat pad levels of lauric and myristic acids were gradually increased by supplement dose (p ≤ 0.05). In the second experiment, the feed additive at 1 kg/t was evaluated in coccidiosis-challenged broilers. Experimental treatments were as follows: non-challenged, control-challenged, and supplemented-challenged treatments. Coccidiosis negatively impact performance and modify histomorphometry and microbiota (p ≤ 0.05). The feed additive increased crypt depth at 7 days post-inoculation and goblet cell count at 14 days post-inoculation (p ≤ 0.05). Further, supplementation interacted with the microbiota modification led by the coccidiosis (p ≤ 0.05). These results suggest that this feed additive could be a useful strategy to reinforce the gut barrier, especially for birds under coccidiosis-challenge treatments.

Butyric acid has received great attention as a feed additive to maintain or increase the gut integrity and health of broiler chickens. Particularly, the protection of butyrate is under research to allow slow intestinal release of butyric acid and to promote its beneficial effects throughout the intestine. This study evaluated in vivo the intestinal release of butyric acid from sodium butyrate protected by salts of medium-chain fatty acid in broilers. Brilliant blue was used as an inert marker, so it was included in the feed additive that broilers ingested for two days. The gastrointestinal tract was then colored in blue from jejunum and backward. Considering the digesta color of the broilers non-supplemented as blank, it allowed quantification of the amount of brilliant blue, and consequently, butyric acid delivered in the intestine from the protected feed additive. Few traces of butyric acid were released in the duodenum and proximal jejunum, whereas the major amount (45.9%) was delivered in the distal ileum (p < 0.001). These results suggest that this in vivo approach allows for evaluation of the intestinal delivery of butyric acid supplemented as protected sodium butyrate by medium-chain fatty acids, showing a gradual intestinal release of butyric acid in broiler chickens.

Background The search for alternatives to antibiotics in pig production has increased the interest in natural resources with antimicrobial properties, such as medium-chain fatty acids (MCFA) as in-feed additives. This study evaluated the potential of a novel blend of MCFA salts (DIC) from distilled coconut oil with a lauric acid content to reduce enteropathogens and control intestinal diseases around weaning. Two experimental disease models were implemented in early-weaned piglets, consisting of two oral challenges: Salmonella Typhimurium (1.2 × 10 8 CFU) or enterotoxigenic Escherichia coli (ETEC) F4 (1.5 × 10 9 CFU). The parameters assessed were: animal performance, clinical signs, pathogen excretion, intestinal fermentation, immune-inflammatory response, and intestinal morphology. Results The Salmonella challenge promoted an acute course of diarrhea, with most of the parameters responding to the challenge, whereas the ETEC F4 challenge promoted a mild clinical course. A consistent antipathogenic effect of DIC was observed in both trials in the hindgut, with reductions in Salmonella spp. plate counts in the cecum ( P = 0.03) on d 8 post-inoculation (PI) ( Salmonella trial), and of enterobacteria and total coliform counts in the ileum and colon ( P < 0.10) on d 8 PI (ETEC F4 trial). When analyzing the entire colonic microbiota (16S rRNA gene sequencing), this additive tended ( P = 0.13) to reduce the Firmicutes/Bacteroidetes ratio and enriched Fibrobacteres after the Salmonella challenge. In the ETEC F4 challenge, DIC prompted structural changes in the ecosystem with increases in Dialister , and a trend ( P = 0.14) to increase the Veillonellaceae family. Other parameters such as the intestinal fermentation products or serum pro-inflammatory mediators were not modified by DIC supplementation, nor were the histological parameters. Only the intraepithelial lymphocyte (IEL) counts were lowered by DIC in animals challenged with Salmonella ( P = 0.07). With ETEC F4, the IEL counts were higher with DIC on d 8 PI ( P = 0.08). Conclusions This study confirms the potential activity of this MCFA salts mixture to reduce intestinal colonization by opportunistic pathogens such as Salmonella or E. coli and its ability to modulate colonic microbiota. These changes could explain to some extent the local immune cell response at the ileal level.

The study was conducted to elucidate effects of feed supplementation with two sources of encapsulated sodium butyrate (ESB, ~30% sodium butyrate encapsulated with vegetable fat) respect to a control diet, on broilers performance. A total of 960 Ross308 one-day-old chicks were randomly distributed into 3 treatments with 16 replicates and 20 birds per replicate: CON (control diet), AD (CON + 2.19kg/tn of ESB from supplier 1) and GUSTOR (CON + 2.19kg/tn of ESB from supplier 2). Feed and water were offered ad libitum. Performance was recorded at 0, 21 and 42d at pen level, and the results presented were corrected by mortality; gut morphology from jejunum at 42 days was also recorded. Data were analyzed with one-way ANOVA using the GLM procedure of SAS. Regarding body weight, some numerical differences were observed between control and groups with ESB at 21d (777g, 786g, and 784g, for CON, AD and GUSTOR, respectively) and at 41d (2719g, 2727g and 2734g, for CON, AD and GUSTOR, respectively). From 0-21d, 22-42d and 0-42d average daily feed intake (ADFI, g/d) was significantly affected by treatment (0-42d: 101b, 100b, 98a, for CON, AD and GUSTOR, respectively, P = 0.045). Feed conversion ratio (FCR) followed the same trend and was significantly improved by GUSTOR at 21 (0-21d: 1.52b, 1.52b and 1.48a for CON, AD and GUSTOR, respectively, P = 0.002) and at 42 days (0-42d: 1.59b, 1.58b and 1.56a for CON, AD and GUSTOR, respectively, P < 0.001). No differences were found in villus height and density, crypt depth and density, number of goblet cells and mucus layer in jejunum at 42d. As expected, the results showed no differences on gut morphology due to a totally encapsulated active principle without activity in the small intestine. Even though both ESB were similar, birds supplemented with ESB-GUSTOR were more efficient than birds supplemented with ESB-AD.

The study was conducted to elucidate effects of feed supplementation with two sources of encapsulated sodium butyrate (ESB, ~30% sodium butyrate encapsulated with vegetable fat) respect to a control diet, on broilers performance. A total of 960 Ross308 one-day-old chicks were randomly distributed into 3 treatments with 16 replicates and 20 birds per replicate: CON (control diet), AD (CON + 2.19kg/tn of ESB from supplier 1) and GUSTOR (CON + 2.19kg/tn of ESB from supplier 2). Feed and water were offered ad libitum. Performance was recorded at 0, 21 and 42d at pen level, and the results presented were corrected by mortality; gut morphology from jejunum at 42 days was also recorded. Data were analyzed with one-way ANOVA using the GLM procedure of SAS. Regarding body weight, some numerical differences were observed between control and groups with ESB at 21 d (777g, 786g, and 784g, for CON, AD and GUSTOR, respectively) and at 41d (2719g, 2727g and 2734g, for CON, AD and GUSTOR, respectively). From 0-21d, 22-42d and 0-42d average daily feed intake (ADFI, g/d) was significantly affected by treatment (0-42d: 101b, 100b, 98a, for CON, AD and GUSTOR, respectively, P = 0.045). Feed conversion ratio (FCR) followed the same trend and was significantly improved by GUSTOR at 21 (0-21d: 1.52b, 1.52b and 1.48a for CON, AD and GUSTOR, respectively, P = 0.002) and at 42 days (0-42d: 1.59b, 1.58b and 1.56a for CON, AD and GUSTOR, respectively, P 1< 0.001). No differences were found in villus height and density, crypt depth and density, number of goblet cells and mucus layer in jejunum at 42d. As expected, the results showed no differences on gut morphology due to a totally encapsulated active principle without activity in the small intestine. Even though both ESB were similar, birds supplemented with ESB-GUSTOR were more efficient than birds supplemented with ESB-AD.

Despite the limited use of antibiotics as growth promoters (AGPs) in a number of countries, many others find a useful tool in them. However, many producers in the later regions are transitioning to AGP-free production for commercial reasons. The aim of this trial was to determine whether a blend of essential oils (EO), like oregano or clove, could reach similar production/health parameters compared to an AGP treatment used in broiler production. A total of 1,200 one-day-old chicks were distributed in 2 treatments within 12 floor pens (n = 6). The treatments were: T1-basal feed + AGP (Enramycin 10ppm); T2-basal feed + EO (1kg/t). The trial lasted 35d and performance parameters were recorded weekly. On day 21 and 35, one bird per pen was euthanized for intestinal and carcass analysis. Data were analyzed by one-way ANOVA using the GLM procedure of SAS 9.0. There were no significant differences in the performance parameters at 35d. A trend was observed of higher BW (P = 0.062) at 28d for EO birds (1,708g) compared to AGP birds (1,671g), which relates to another trend (P = 0.071) of increased daily gain from 21-28d (685g and 653g for EO and AGP treatments respectively). A relevant finding was a significantly (P = 0.034) higher mortality in AGP birds from 0-28d (4.83%) compared to EO birds (3.00%). In the post-mortem analysis no differences were observed in intestinal segments in relative and total length. However, animals from T2 showed a trend (P = 0.077) to have heavier gizzards at 21d, which could indicate an effect of EO on their development, while T1 animals had a significantly heavier cecum at 35d (P = 0.026). No relevant findings were observed in carcass weights and yield. These results suggest that this EO blend could be a valid alternative to AGPs, although further trials under commercial conditions should be carried out to confirm its potential.

The aim of this study was to investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of organic acid salts against six field isolates of Streptococcus suis. The three products evaluated were sodium salt of coconut fatty acids distillate (DIC) alone and two combinations with sodium butyrate (NaBut): DIC70:30, being 70% of NaBut protected with 30% of DIC; and DIC50:50, being 50% of NaBut protected with 50% of DIC. Antimicrobial susceptibility testing was performed to estimate the MIC values for each product and strain by the broth microdilution method at pH 6.0. MBC values were also determined by sub-culturing supernatant from wells without evident bacterial growth. The values of MIC50/MBC50 were calculated as the concentration which inhibited/killed 50% of the isolates tested. The MIC50 showed DIC as the most effective (8 ppm) against S. suis followed by DIC50:50 (32 ppm) and DIC70:30 (64 ppm). The MBC50 demonstrated a similar trend, DIC being the most effective (16 ppm) followed by DIC50:50 (64 ppm) and DIC70:30 (64 ppm). It is well known that butyric acid is a short-chain fatty acid which has strong antimicrobial activity against Gram-negative bacteria. In contrast, coconut fatty acids distillate is a medium-chain fatty acid source (MCFA) rich in lauric acid which has strong antimicrobial activity against Gram-positive bacteria. Both products are generally available as salts to facilitate their application in feed. In this study, the results showed that DIC was the most effective against the Gram-positive bacteria tested, followed by DIC50:50 and DIC70:30, the sodium butyrate-based products. As expected, a higher concentration of MCFA in the tested product was associated with a higher inhibitory and bactericidal activity. Further studies would be required to better understand these interactions as well as in vivo studies to demonstrate the effects on microbial populations.