Effects of dietary crude protein level and sodium butyrate protected by medium-chain fatty acid salts on performance and gut health in weaned piglets.

To reduce the use of antibiotics, research into nutritional strategies designed to improve the gut health of weaned pigs is underway. This study sought to examine the effects of reducing dietary crude protein (CP) and/or supplementing the feed with sodium butyrate protected by the sodium salts of medium-chain fatty acids on the growth performance and gut health of weaned piglets. Ninety-six weaned piglets (Landrace × large white, 21 days of age) were allotted to four experimental treatments for 14 d. The experimental design was factorial with 2 CP levels and 2 feed-additive doses (0 vs. 1 kg/t). Results showed that reducing CP from 22.2% to 18.8% diet had no effect on piglet growth performance parameters during the first post-weaning week (P > 0.05), but did compromise growth in the second week (P = 0.011), impacting overall growth performance results (P = 0.019). Nonetheless, dietary CP level reduction led reducing crypt depth (P = 0.03657). In addition, Lactobacillus counts that were increased in the ileum (P = 0.032) and reduced in the colon (P = 0.032). Furthermore, apparent ileal digestibility of organic matter (P = 0.026) and fecal consistency (P < 0.05) were improved throughout the experiment. Moreover, in piglets fed diets containing 22.2% CP, the use of the feed-additive tended to improve the gain-to-feed ratio (P = 0.091) compared to those fed supplemented diets containing 18.8% CP. In addition, feed supplementation increased ileal numbers of goblet cells (P = 0.036), as well as apparent ileal digestibility of dry matter (P = 0.057) and organic matter (P = 0.003). Supplementation also had beneficial effects on the microbiota of the colon, increasing Lactobacillus counts (P = 0.006) and diminishing Enterobacteriaceae counts (P = 0.003), as well as affecting microbial metabolite profiles in that acetic acid concentrations tended to be increased (P = 0.088) and valeric acid concentrations were reduced (P = 0.002). These findings support the use of both strategies can improve the gut health of weaned piglets and prompt further research into the possible benefits of combining these two nutritional strategies on gut health and growth performance.

Reducing dietary levels of crude protein (CP) and the use of feed-additives such as sodium butyrate protected by medium-chain fatty acid salts are currently under investigation as nutritional strategies with beneficial effects on the intestinal barrier, and consequently on the health of weaned piglets. The intestinal barrier is a dynamic complex ecosystem that includes morphological structure and microbial composition. Reducing CP intake from 22.2% to 18.8% in piglets was found here to compromise their growth 2 wk after weaning. However, considering the effect of reducing CP on gut health, crypt depth was reduced and the Lactobacillus population was expanded in the ileum and diminished in the colon. In addition, organic matter digestibility and fecal consistency were improved. Supplementation with sodium butyrate protected by the sodium salts of medium-chain fatty acids at 1 kg/t increased the number of mucin-secreting cells, thereby reinforcing the intestinal barrier, and improving ileal digestibility. In addition, it modified the microbiota in the colon. These findings on different parameters of intestinal barrier prompt further investigation into the effects of both strategies on gut health and growth performance of piglets.

Short Communication: Evaluation of Intestinal Release of Butyric Acid from Sodium Butyrate Protected by Salts of Medium-Chain Fatty Acids in Broiler Chickens.

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.

Impact of Dietary Supplementation with Sodium Butyrate Protected by Medium-Chain Fatty Acid Salts on Gut Health of Broiler Chickens.

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.

Impact of in-feed sodium butyrate or sodium heptanoate protected with medium-chain fatty acids on gut health in weaned piglets challenged with Escherichia coli F4.

Short and medium-chain fatty acids (SCFA and MCFA, respectively) are commonly used as feed additives in piglets to promote health and prevent post-weaning diarrhoea. Considering that the mechanism and site of action of these fatty acids can differ, a combined supplementation could result in a synergistic action. Considering this, it was aimed to assess the potential of two new in-feed additives based on butyrate or heptanoate, protected with sodium salts of MCFA from coconut distillates, against enterotoxigenic Escherichia coli (ETEC) F4+ using an experimental disease model. Two independent trials were performed in 48 early-weaned piglets fed a control diet (CTR) or a diet supplemented with MCFA-protected sodium butyrate (BUT+; Trial 1) or sodium heptanoate (HPT+; Trial 2). After 1 week of adaptation, piglets were challenged with a single oral inoculum of ETEC F4+ (minimum 1.4 · 109 cfu). One animal per pen was euthanised on days 4 and 8 post-inoculation (PI) and the following variables assessed: growth performance, clinical signs, gut fermentation, intestinal morphology, inflammatory mediators, pathogen excretion and colon microbiota. None of the additives recovered growth performance or reduced diarrhoea when compared to the respective negative controls. However, both elicited different responses against ETEC F4+. The BUT+ additive did not lead to reduce E. coli F4 colonisation but enterobacterial counts and goblet cell numbers in the ileum were increased on day 8 PI and this followed higher serum TNF-α concentrations on day 4 PI. The Firmicutes:Bacteroidetes ratio was nevertheless increased. Findings in the HPT+ treatment trial included fewer animals featuring E. coli F4 in the colon and reduced Enterobacteriaceae (determined by 16S RNA sequencing) on day 4 PI. In addition, while goblet cell numbers were lower on day 8 PI, total SCFA levels were reduced in the colon. Results indicate the efficacy of MCFA-protected heptanoate against ETEC F4+ and emphasise the potential trophic effect of MCFA-protected butyrate on the intestinal epithelium likely reinforcing the gut barrier.

Efficacy of medium-chain fatty acid salts distilled from coconut oil against two enteric pathogen challenges in weanling piglets.

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 × 108 CFU) or enterotoxigenic Escherichia coli (ETEC) F4 (1.5 × 109 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.

Response of gastrointestinal fermentative activity and colonic microbiota to protected sodium butyrate and protected sodium heptanoate in weaned piglets challenged with ETEC F4.

This study aimed to evaluate the potential of two new fat-protected butyrate or heptanoate salts to improve gut health and control post-weaning colibacillosis in weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4+, particularly focusing on their impact on intestinal microbiota and fermentative activity along the gastrointestinal tract (GIT). Seventy-two 21-d-old pigs were fed a plain diet (CTR) or supplemented with sodium butyrate (BUT) or sodium heptanoate (HPT), both at 0.3%. After a week of adaptation, animals were orally challenged at days 8 and 9 with 5.8 · 109 and 6.6 · 1010 cfu, respectively, and were euthanised on d 4 and d 8 post-inoculation (PI) (n = 8) to collect blood, digesta and tissue samples and characterise microbial groups, pathogen loads (qPCR), fermentation, ileal histomorphometry and immune markers. Colonic microbiota was analysed by 16S rRNA gene MiSeq sequencing. Supplementing both acid salts did not compensate clinical challenge effects nor performance impairments and neither histomorphometry nor serum biomarkers. Changes in the gastric fermentative activity were registered, BUT reducing lactic acid concentrations (day 8 PI), and with HPT fewer animals presenting detectable concentrations of propionic, butyric and valeric acids. At ileum BUT increased acetic acid concentration (day 8 PI), and both additives reduced short-chain fatty acids (SCFA) in the colon. Increases in enterobacteria and coliforms counts in ileal digesta (day 4 PI, p < 0.10) and mucosa scrapes (p < 0.05) were registered although E. coli F4 gene copies were unaffected. Regarding changes in the colonic microbiota (day 4 PI), Prevotellaceae and Prevotella were promoted with BUT supplementation whereas only minor groups were modified in HPT-treated animals. Summarising, although the pathogen loads or inflammatory mediators remained unresponsive, butyrate and heptanoate showed a significant impact on microbial fermentation along the whole GIT, being able to modify different bacterial groups at the colon. It could be hypothesised that these effects might be mediated by a carry-over effect of the changes observed in gastric fermentation, but possibly also to a better nutrient digestion in the foregut as a result of the reduced colonic SCFA concentrations.

Antimicrobial activity of a selection of organic acids, their salts and essential oils against swine enteropathogenic bacteria.

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.

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

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.

Sodium salt medium-chain fatty acids and Bacillus-based probiotic strategies to improve growth and intestinal health of gilthead sea bream (Sparus aurata).

BACKGROUND: 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 Bacillus-based probiotic, in gilthead sea bream (GSB, Sparus aurata), a marine farmed fish of high value in the Mediterranean aquaculture. METHODS: 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 Bacillus amyloliquefaciens 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. RESULTS: 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. DISCUSSION: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic B. amyloliquefaciens 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.