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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 ...

This research was co-funded by the European Fund of Regional Development of the European Union within the framework of the FEDER operating program of Catalunya 2014-2020 (project COMRDI16-1-0033) and managed by ACCIÓ. 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 × 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.

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.

Essential oils (EOs) have different mechanisms, most of them targeting the bacterial wall. This fact can explain differences in the effectivity of EOs between Gram-positive and Gram-negative bacteria. Therefore, combining certain EOs can broaden their individual spectrum of efficacy due to potential synergistic effects. This trial aimed to test the in vitro antibacterial activity of an EO combination (oregano and clove oils) against a collection of relevant bacterial pathogens in swine production. The Gram-negative bacterial species chosen were Salmonella enterica ssp. enterica, Escherichia coli and Brachyspira hyodysenteriae and the Gram-positive bacterial species were Clostridium perfringens and Streptococcus suis. In addition, Lactobacillus fermentum was included to compare the susceptibility between this beneficial intestinal bacteria and the pathogens tested. The broth microdilution method at pH 6 and the subculturing from wells without bacterial growth were used to determine the minimum concentration of active principle necessary to inhibit (MIC) or kill (MBC) the 50% and 90% (MIC50/90/MBC50/90) of the population of every bacteria. The results showed that the lowest MIC50/90 were obtained for B. hyodysenteriae (37.5/75 ppm) while for S. enterica ssp. enterica (150/300 ppm), C. perfringens (150/150 ppm), E. coli (300/300 ppm) and S. suis (150/300 ppm) results were similar. Regarding the MBC50/90; B. hyodysenteriae (18.8/75 ppm) was the most susceptible pathogen, again, compared to S. enterica ssp. enterica (300/300 ppm), C. perfringens (150/150 ppm), E. coli (300/300 ppm) and S. suis (150/300 ppm). In contrast, the highest bacteriostatic/bactericidal concentrations were obtained against L. fermentum (MIC50/90 600/1,200 ppm and MBC50/90 600/2,400 ppm). These results suggest that the bacterial category (Gram-positive or Gram-negative) did not have an influence on the MIC and MBC. It can also be concluded that B. hyodysenteriae is the most susceptible enteropathogen to this EO blend. However, the in vivo effect of this combination of EOs must be further studied.

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

The potential benefits of a commercial preparation of heptanoate (NOREL, HEPTON®) were evaluated in an 11‐week gilthead sea bream feeding trial (May–August), using a factorial design with four isoproteic and isoenergetic diets. Fish meal (FM) was added at 200 g/kg in D1–D2 diets and at 50 g/kg in D3–D4 diets, which also contained fish peptones and plant proteins as source of proteins. Heptanoate was added at 3 g/kg in D2 and D4 diets. All fish grew from 13–14 g to 81–84 g with an overall feed efficiency (FE) of 0.91–0.94. An early impairment of FE (weeks 1–4) was found with the standard FM‐based diet (D1), but this detrimental condition was reversed by heptanoate, increasing FE from 0.88 in D1 fish to 0.99 in D2 fish. Further improvements were progressively diluted over time, remaining D2 and D3–D4 fed fish almost undistinguishable through all the trial. Heptanoate supplementation produced higher hepatic glycogen depots, but no signs of histopathological damage were found in liver or intestine. Other lasting heptanoate effects included changes in plasma antioxidant capacity, plasma cortisol and growth hormone levels, and measures of respirometry in swimming performance tests. Altogether, it supports the potential use of heptanoate to speed up adaptive and healthy metabolic states of farmed fish to cope with challenging culture conditions., This work has been carried out with the financial support from NOREL S.A. and BioMar. Additional funding has been received by Spanish MINECO project no. AGL2013‐48560‐R and Generalitat Valenciana (PROMETEO‐FASE II/2014/085). MCP was supported by MINECO through grant FPDI‐2013‐15741 and by CSIC PIE project no. 201740E013. JAMS was supported by a postdoctoral research fellowship (Juan de la Cierva‐Formación, Reference FJCI‐2014‐20161) from MINECO

Juveniles of the protandrous hermaphrodite gilthead sea bream (Sparus aurata) were fed from early life stages to completion of sex maturation with plant-based diets formulated by BioMar. Total fish meal (FM) was included at 25% in the control diet (D1) and at 5% in the other three diets (D2, D3, D4). Added oil was either fish oil (FO) (D1) or a blend of vegetable oils replacing 58% (D2) and 84% (D3, D4) of FO. A commercial butyrate preparation (BP-70® NOREL) was added to the D4 diet at 0.4%. All fish grew fast through a three-year ongrowing cycle with overall specific growth rates of 1.4%, 0.7% and 0.5% for fish harvest at 300 g, 1 kg and 1.5–1.7 kg, respectively. Overall feed efficiency decreased progressively as fish size increased from 0.99 in 300 g fish to 0.8–0.7 in 1–1.7 kg fish. At the last stage, a clear sexual dimorphism was found for body weight and hepatosomatic index when all sampled fish were considered as a whole. A sexual dimorphism was also found for sex steroids with a peak of estradiol in the females sampled in October–December, whereas the peak of 11-ketotestosterone was delayed in males to December. Plasma levels of testosterone were similar in both sexes. The two first components of principal component analysis (PCA) explained >90% of total variance of plasma levels of sex steroids. The displacement along X-axis clearly separated males and females, whereas the movement along Y-axis was related to sampling time. An androgenic effect in the steroid plasma profile of fish fed plant-based diets was also shown, which was especially evident for the low FM/FO diet formulation (D3). This effect was reversed by butyrate and the female/male ratio of D4 fish (age class +3) did not differ from that of control fish, whereas the proportion of females in D2 + D3 fish was higher (P, This work has been carried out with financial support from the European Union (ARRAINA, FP7-KBBE-2011-5-288925, Advanced Research Initiatives for Nutrition and Aquaculture). Additional funding was obtained from the Spanish MINECO (MI2-Fish, AGL2013-48560; Pubertrait, AGL2016-75400) and from Generalitat Valenciana (PROMETEO FASE II-2014/085 and 051).

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.

This study evaluated the effect of two feed lauric acid (LA) based additives on faecal microbiota of piglets. A total of 256 piglets were distributed (from 25 to 66d of age) in 4 treatments: CON-, diet without additives nor antibiotics; CON+, diet with Doxycycline; DIC, CON- diet plus sodium salt of coconut fatty acids distillate at 3kg/t and 1kg/t in starter and grower diets; MON, CON- diet plus monolaurine at 2.1kg/t and 0.7kg/t in starter and grower diets. The dosage of both products was made at the same level of LA (0.96kg LA/t starter and 0.32kg LA/t grower). At the end of the study, the 16S rDNA sequencing was performed. The result showed a drastic decrease in the abundance of the dominating genus Prevotella within the MON group compensated by other genera increase, mostly Selenomonadales. Streptococcus, Treponema, and unclassified Erysipelotrichaceae, Dialister or Bacillus increased their respective abundances. Treponema was more pronounced in the DIC, however, all other mentioned genera did not show a similar increase in abundance. Dialister (a marker for disease in humans) showed significantly lower abundance in DIC. In the bacterial fingerprints, a trend for clustering according to treatment was visible. Six of eight CON- samples formed a separate cluster. Six of eight samples of the DIC formed a cluster that shared very low similarity to the CON-. Four of eight CON+ samples formed a separate cluster. MON samples were interspersed among all samples and showed no distinct clustering. Even though quantitative microbial comparisons were only anecdotal, observations may indicate a more “healthier” microbiota in the DIC than was present in the MON. DIC also showed a much closer similarity of bacterial fingerprints for the pooled samples in the qualitative analysis indicating that DIC had a higher potential to modify the faecal (hindgut) microbiota than MON.

Effect of feed additives based on lauric acid (LA) on performance and faecal score in piglets. A total of 192 piglets were distributed in 24 pens from 25 to 66d of age (42d feeding period). The study was divided into two feeding phases; starter (25 to 38d) and grower period (39 to 66d). Treatments were: CON-, diet without additives nor antibiotics; CON+, diet with Doxycycline (0.25g/kg and 0.13g/kg in starter and grower diets); DIC, CON- diet plus sodium salt of coconut fatty acids distillate (DICOSAN) at 3kg/t and 1kg/tn in starter and grower diets; MON, CON- diet plus monolaurine at 2.1kg/t and 0.7kg/tn in starter and grower diets. The dosage of both products was made at same level of LA (0.96kg LA/t starter and 0.32kg LA/t grower). All parameters were analyzed using a one-way ANOVA via SPSS, with pen as the experimental unit. At 38d, CON+ and DIC piglets had significantly lower FCR than CON- (1.534a, 1.353b, 1.380b and 1.526ab, for CON-, CON+, DIC and MON, P = 0.021). At 66d, CON+ tended to have lower FCR than CON- (1.613x, 1.541y, 1.560xy and 1.605xy, for CON-, CON+, DIC and MON, P = 0.054). For the overall period, DIC and CON+ piglets had significantly lower FCR than CON- (1.603a, 1.514b, 1.537b and 1.589ab, for CON-, CON+, DIC and MON, P = 0.003), there was also a tendency to lower daily weight gain in MON treatment when compared with CON+ (443xy, 468y, 449xy and 440x g/d, for CON-, CON+, DIC and MON, P = 0.079). Finally, the faecal score showed higher values for DIC and CON+, meaning more consistent faeces (3.71a, 3.93b, 3.84b and 3.82ab, for CON-, CON+, DIC and MON, P = 0.001). The results demonstrated that DICOSAN, administered in feed at the same dosage of lauric acid, achieved better results than monolaurine and CON- and without significant differences with CON+.

The aim of this study was to evaluate the By-Pass fraction (BPF) of the dry matter (DM) and nitrogen (N) of coated methionine with different fat matrix (FM) and particle size (PS). Two FM (Hydrogenated Stearin, HS, and Hydrogenated Palm Fatty Acid Distillate, HPFAD) and four PS (< 500, 500–750, 750–1400, and >1400 µm) were evaluated in an in situ trial with three rumen-cannulated sheep. Intact samples (1 g) were weighed into nylon bags and incubated in the rumen for 2, 4, 8, 12 and 24 h; process repeated twice in each sheep. After extraction from the rumen, the bags were washed with cold water, frozen and lyophilized, before determining N content in the incubation residue. Effective degradability of DM and N was calculated for a rumen passage rate of 0.08/h-1 according to Ørskov and McDonald (1979), and the BPF was calculated as 100 minus the effective degradability. Data were analyzed using a mixed-effect model, with FM, PS and FMxPS interaction as fixed effects and sheep as random; means were compared with LSD test (P ≤ 0.05). HS-coated methionine had higher values of BPF of DM and N (78.2 and 49.6%, respectively; values averaged for all PS; P < 0.001) than HPFAD-coated methionine (64.4 and 17.7%, respectively). There was an interaction between FM and PS (P < 0.001) for BPF of DM and N. Increasing PS of HS-methionine augmented the BPF from 68.9 to 90.3% for DM and from 22.9 to 79.8% for N, whereas the increases for PFAD-methionine were lower (from 66.3 to 66.3% for DM, and from 7.5 to 28.6% for N). It is concluded that the use of HS as FM and the increase of PS are valid strategies to improve methionine protection from ruminal degradation, reaching BPF values of 90.3 and 79.8 % for DM and N, respectively.

The aim of this trial was to evaluate the effect of prenatal and postnatal inclusion of a combination of essential oils (EO) on performance parameters and diarrheas after 2 weeks post-weaning. The trial followed a 2x3 factorial design; 2 sow diets: basal or with EO at 375 g/t x 3 piglet diets: basal diet, medicated with 200 ppm of colistin or treated with 375 g/t of EO. A total of 168 weaned piglets were randomly distributed in the 6 groups: 3 from basal sows (CC, CA and CT) and 3 from EO sows (TC, TA and TT), being the animals from the TT treatment the ones imprinted. The incidence of diarrhea was recorded twice a day and calculated using the percentage of animals with diarrhea over the total period of observations carried out. The animals were weighed at 14 days post-weaning. Average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated. Data were analyzed by two-way ANOVA using GLM procedure of SAS. Animals from the TT group had significantly lower incidence of diarrheas from 0 to 14 days post weaning. Diarrhea was recorded on 4.7b% of the observations (TT group), being significantly lower (P ≤ 0.001) than the animals from CC (10.8a%), CA (13.7a%), CT (10.7a%), TC (14.4a%) and TA (12.9a%). Performance parameters indicated that there were non-significant differences in BW, ADFI, ADG with numerically lower FCR (kg/kg) 1.14 in TT group (CC 1.23, CA 1.33, CT 1.27, TC 1.22, TA 1.23). Despite the non-significant differences in performance parameters, in this study the effect of imprinting with essential oils reduced diarrhea incidence.

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.

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.

The objective of this study was to determine the effects of protected sodium butyrate (SB), and protected sodium butyrate plus essential oils (carvacrol and ginger; SBEO) on the cecal microbiota of broilers challenged with Eimeria maxima and Clostridium perfringens. Birds were assigned to 4 treatments (8 replicates pens of 58 birds/pen): unchallenged control; challenged control; challenged and supplemented with SB; challenged and supplemented with SBEO. On d 13, challenged birds were orally inoculated with ~5,000 Eimeria maxima oocysts. On d 18-19, the same birds were exposed to Clostridium perfringens via drinking water (~ 8 log CFU/ml). Cecal excreta was collected at d 12, 18, 21, and 28 for microbiome analysis through 16s rRNA sequencing using Illumina MiSeq platform and analyzed using QIIME v. 1.9.1 The cecal microbiota was analyzed over time within each experimental group. The inclusion of SB alone or in combination with EO contributed to larger variations in the cecal microbiota over time than the unsupplemented treatments, as shown by the diversity indices. The community structure and abundance of the cecal microbiota were significantly different across ages, especially in the groups supplemented with SB and SBEO. As shown in the PCoA analysis, the supplementation of SB led to a more stable microbial community and lower between-sample variability over time. In the unchallenged control birds, Ruminococcus decreased (p = 0.006), whereas Bacteroides and Clostridiales increased (p ≤ 0.02) as the birds aged. In the challenged control group, however, the frequency of Coprococcus and Blautia decreased as birds aged (p ≤ 0.01), and, Clostridiales did not increase. Supplementation of SB, but not SBEO, increased the frequency of Lactobacillus (p = 0.01) on d 12 compared to d 18 and d 28, and prevented the reduction in the frequency of Blautia as the birds aged. Nevertheless, supplementation of SB and SBEO contributed to unique changes in the predicted functions of the cecal microbiota over time, which was not observed in the unsupplemented birds. SB and SBEO modulated the diversity, composition, and predictive function of the cecal microbiota which may have lowered the negative impact of necrotic enteritis (NE).

[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., This work has been carried out with financial support from the European Union under grant projects ARRAINA (FP7-KBBE-2011-288,925) to JPS, MP and VK and ParaFishControl (H2020-634429) to ASB. Additional funding has been received from Spanish Ministry of Economy and Competitiveness (MINECO) project no. AGL2013- 48560-R to JPS and ASB, and Generalitat Valenciana (PROMETEOII/2014/085) to ASB. MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the “Generalitat Valenciana”., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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 R ; 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., Spanish MINECO: AGL2013-48560-R. ., Generalitat Valenciana: PROMETEO-FASE II/2014/085., MINECO: FPDI-2013-15741., CSIC PIE: 201740E013