Aquatic animals have benefited from Bacillus subtilis-based probiotics over the past few decades. This study evaluated the effects of B. subtilis DSM 32315 probiotics as a feed additive on growth, immune response and resistance to acute ammonia challenge in Nile tilapia. Specifically, four supplemental levels (0%, 0.1%, 0.2%, and 0.3%) of B. subtilis probiotics were tested under two dietary protein levels (32% and 28%). Five replicate tanks were randomly allotted to each dietary treatment, with each tank containing 30 Nile tilapia. After 8 weeks of feeding, Nile tilapia in each tank were exposed to 43.61 mg/L of total ammonia nitrogen for 48 h. The results revealed that reducing protein levels from 32% to 28% did not affect growth performance or antioxidant capacity. However, the low protein diet tended to induce an inflammatory effect shown by increased expressions of TGF-β and IFN-γ genes (P < 0.05) in the liver. The impact was alleviated by the probiotic supplementation. Compared with the non-supplemented group, 0.1% probiotic supplementation remarkably increased plasma lysozyme activity, total antioxidant capacity and complement C3 and interleukin-10 mRNA levels (P < 0.05) in the 28% protein diet, while higher supplementation of probiotics (0.3%) was shown to be beneficial for the high protein diet (32%). In both the dietary protein levels, 0.1% supplementation of probiotics promoted the antioxidant capacity of Nile tilapia before exposure to ammonia stress but higher probiotic supplementation (0.3%) proved to be necessary under ammonia stress as evidenced by higher fish survival rate. Results exhibited that supplementation with B. subtilis probiotics had a better effect on the intestinal morphology (villi height and width) regardless of protein levels. In conclusion, dietary supplementation of B. subtilis DSM 32315 probiotics at 0.1% in the low protein diet and up to 0.3% in the high protein diet showed beneficial effects on the growth, immunity, and antioxidant capacity of Nile tilapia. Under ammonia stress conditions, the higher supplementation of B. subtilis DSM 32315 probiotics at 0.3% improves stress tolerance of Nile tilapia despite the two dietary protein levels (32%; 28%).

An 8-week feeding trial was conducted in Pacific white shrimp (Litopenaeus vannamei) to evaluate the effects of dietary choline supplementation on choline transport and metabolism, hepatopancreas histological structure and fatty acid profile, and regulation of lipid metabolism. Six isonitrogenous and isolipidic diets were formulated to contain different choline levels of 2.91 (basal diet), 3.85, 4.67, 6.55, 10.70 and 18.90g/kg, respectively. A total of 960 shrimp (initial weight, 1.38 ± 0.01g) were distributed randomly into twenty-four 250-L cylindrical fiber-glass tanks, with each diet assigned randomly to 4 replicate tanks. The results indicated that dietary choline significantly promoted the deposition of choline, betaine and carnitine (P < 0.05). The diameters and areas of R cells, total lipid and triglyceride contents in hepatopancreas, and triglyceride and non-esterified fatty acid contents in hemolymph were negatively correlated with dietary choline level. The contents of functional fatty acids in hepatopancreas, the activity of acetyl-CoA carboxylase (Acc), and the mRNA expression of fas, srebp and acc were highest in shrimp fed the diet containing 4.67g/kg choline, and significantly higher than those fed the diet containing 2.91g/kg, the lowest level of choline (P < 0.05). The number of R cells, content of very lowdensity lipoprotein (VLDL), activities of carnitine palmitoyl-transferase (Cpt1), lipoprotein lipase and hepatic lipase, and the mRNA expression levels of cpt1, fabp, fatp, ldlr, and ampk in hepatopancreas increased significantly as dietary choline increased (P < 0.05). In addition, hepatopancreas mRNA expression levels of ctl1, ctl2, oct1, badh, bhmt, ck, cept, and cct were generally up-regulated as dietary choline level increased (P < 0.01). In conclusion, dietary choline promoted the deposition of choline and its metabolites by up-regulating genes related to choline transport and metabolism. Moreover, appropriate dietary choline level promoted the development of hepatopancreas R cells and maintained the normal accumulation of lipids required for development, while high dietary choline not only promoted hepatopancreas lipid export by enhancing VLDL synthesis, but also promoted fatty acid β-oxidation and inhibited de novo fatty acid synthesis by activating the Ampk/Srebp signaling pathway. These findings provided further insight and understanding of the mechanisms by which dietary choline regulated lipid metabolism in L. vannamei.

Given the key role of methionine in biological processes, adequate methionine should be provided to meet the nutritional requirements. DL-2-hydroxy-4-(methylthio)-butanoic acid (DL-HMTBA) has been considered as an important source of methionine. However, the effects of different sources and levels of methionine on the intestinal health status have not been clarified yet. An experiment was carried out to investigate the effects of different dietary sources and levels of methionine on the intestinal epithelial barrier, inflammatory cytokines expression, ileal morphology, microbiota composition, and cecal short chain fatty acids (SCFA) profiles. For this purpose, 720 male Arbor Acre broiler chicks at 1d old were randomly assigned to a 2 × 3 factorial arrangement with 2 methionine sources (DL-methionine and DL-HMTBA) and 3 total sulfur amino acids (TSAA) levels (80%, 100%, and 120% of Arbor Acre recommendation). The results showed that DL-HMTBA supplementation promoted intestinal physical barrier at both gene expression level of claudin-1 and serum diamine oxidase level (P < 0.05), and the inflammatory cytokine IL-6 mRNA expression was down-regulated by dietary DL-HMTBA supplementation compared with the DL-methionine group (P < 0.05). Meanwhile, an upregulated gene expression of claudin-1 and zonula occluden-1 (ZO-1) were observed in the low-TSAA treatment on d 14 (P < 0.05), whereas this treatment increased the expression of IL-1β and IL-6 (P < 0.05). Villus height to crypt depth ratio was high (P < 0.05) in the middle-level TSAA group. Furthermore, DL-HMTBA supplementation optimized the microbiota of the ileum especially the relative abundance of Lactobacillus, where the digestion and absorption were completed, and elevated the concentrations of SCFA (acetate, propionate, and butyrate) in the cecal content on d 21 (P < 0.01). In conclusion, dietary DL-HMTBA supplementation improved the intestinal barrier function, immune homeostasis and optimized the microbiota to promote intestinal health status in broiler chickens.

To explore the effects of fermented rapeseed meal (FRSM) on growth performance and intestinal health, a total of 30 growing pigs were randomly allotted to three treatments consisting of corn-soybean meal diet (CSD), rapeseed meal diet (RSD), and fermented rapeseed meal diet (FRSD). Results showed that compared with RSD, FRSD feeding increased the average daily gain and final body weight in pigs (P < 0.01). Compared with RSD feeding, FRSD feeding elevated the apparent digestibility of crude protein, acid detergent fiber, and ether extract in pigs (P < 0.01). Moreover, the FRSD group exhibited greater apparent ileal digestibility of His, Thr, Lys, and Ser than the RSD group (P < 0.01). The digestible energy, metabolic energy, and nitrogen utilization were higher in the FRSD and CSD groups than in the RSD group (P < 0.01). As compared to the RSD, FRSD feeding decreased the serum concentration of leptin but significantly increased the concentrations of immunoglobulin (Ig) A, IgG, ghrelin, and enzyme activities of amylase, lipase, and trypsin in the pancreas (P < 0.05). Interestingly, the villus height, the ratio of villus height to crypt depth, and the activities of brush border enzymes (e.g., maltase and sucrase) in the small intestine were higher in the CSD and FRSD groups than in the RSD group (P < 0.05). As compared to the RSD, the FRSD feeding not only increased the expression level of the occludin in the small intestinal epithelium (P < 0.05) but also elevated the expression levels of claudin-1, MUC1, and PepT1 genes in the duodenum, and elevated the expression levels of SGLT1 and CAT1 genes in the jejunum (P < 0.05). Importantly, FRSD feeding significantly decreased the abundance of Escherichia coli, but increased the abundance of Lactobacillus and the content of butyrate in the cecum and colon (P < 0.05). These results indicated that compared with rapeseed meal, fermented rapeseed meal exhibited a positive effect on improving the growth performance and intestinal health in growing pigs, and the results may also help develop novel protein sources for animal nutrition and the feed industry.

Rosemary extracts have been widely used as feed additives in recent years. This study aimed to investigate the effects of rosmarinic acid (RA) and ursolic acid (UA), the main active components of rosemary, on growth performance, meat quality and lipid metabolism in finishing pigs. A total of 72 finishing pigs (Landrace; initial age of 150 d) were randomly divided into 3 treatments with 8 replicates of 3 pigs each, and fed a basal diet or diet containing 500mg/kg of RA or UA. The results showed that dietary supplementation of RA or UA had no significant effect on the growth performance and carcass traits of finishing pigs (P > 0.05). However, both RA and UA significantly increased the triglyceride (TG) level in soleus muscle (P < 0.001). Supplementation of RA increased the expression of genes related to lipogenesis and transport including fatty acid synthase (FAS) (P < 0.001), sterol regulatory element binding protein-1c (SREBP1c) (P < 0.001) and peroxisome proliferator-activated receptor γ (PPARγ) (P < 0.05), while UA increased the expression of fatty acid transport protein 1 (FATP1), a gene related to lipid uptake (P < 0.05). However, RA reduced the expression of adipogenesis-related gene acetyl-coenzyme A carboxylase α (ACCα) (P < 0.01). Characterization of cecal microbiota indicated that RA increased the microbial richness (chao 1, P < 0.001) and diversity (observed species, P < 0.01). Further analysis of the genera revealed that RA increased the relative abundance of Bacteroides and g-UCG-005 (P < 0.05), and UA enriched Prevotella (P < 0.001). Correlation analysis showed that g-UCG-005 was positively correlated with the expression of FAS, carnitine palmitoyl transferase 1B (CPT1B), SREBP1c and PPARγ (P < 0.01). In conclusion, dietary supplementation of RA or UA may increase fat deposition in muscle of finishing pigs by regulating lipid metabolism and gut microbiota.

This study was conducted to evaluate the effects of dietary crude protein (CP) and rumen-protected lysine (RPL) supplementation on lactation performance, amino acid (AA) balance, nitrogen (N) utilization and hindgut microbiota in dairy cows. Treatments were in a 2 × 2 factorial arrangement, and the main effects were CP concentration (16% vs. 18%) and RPL supplementation (with or without RPL at 40 g/cow per day). Forty cows were randomly allocated to 4 groups: low-CP diet (LP), low-CP diet plus RPL (LPL), high-CP diet (HP), high-CP diet plus RPL (HPL). The experiment was conducted for 8 weeks. Results showed that RPL increased the dry matter intake (P < 0.01), milk protein yield (P = 0.04) and energy corrected milk (P = 0.04), and tended to increase milk fat yield (P = 0.06) and fat corrected milk (P = 0.05). Cows in the HP group tended to have higher milk urea N (P = 0.07). Plasma concentrations of Arg, Ile, Lys, Met, Pro, total essential AA and total nonessential AA were increased by RPL (P < 0.05). The total essential AA, total nonessential AA and most AA (except Ile, Phe, Gly and Pro) were increased in the HP group (P < 0.05). N excretion was increased in the HP group through an increase in urea N excretion (P < 0.01) and an upward trend in plasma urea N (P = 0.07). In addition, RPL tended to increase milk protein N secretion (P = 0.08), milk N (P = 0.07) and microbial protein synthesis (P = 0.06), and decreased plasma urea N (P < 0.001). In the hindgut, the bacterial community were different between the LP and LPL groups (P < 0.01). The probiotic abundances of Christensenellaceae_R-7_group and Acinetobacter were increased by RPL (P = 0.03 and 0.03, respectively). The pathogenic abundances of Clostridium_sensu_stricto_1 (P < 0.001) and Turicibacter (P < 0.01) were decreased by RPL. In conclusion, supplementing RPL with low dietary CP could balance AA supply and increase milk protein yield, resulting in an improvement in N utilization efficiency, and altered the composition of the hindgut microbiota to favor the lactation performance of dairy cows.

This study aimed to investigate the feeding effect of wheat silage on growth performance, nutrient digestibility, rumen fermentation, and microbiota composition in feedlot lambs. Sixty-four male crossbred Chinese Han lambs (BW = 27.8 ± 0.67kg, 3 months of age) were randomly assigned to four ration groups with wheat silage replacing 0% (WS0), 36% (WS36), 64% (WS64), and 100% (WS100) of oaten hay on forage dry matter basis. The concentrate-to-forage ratio was 80:20 and the feeding trial lasted 52 d. Increasing wheat silage inclusion linearly decreased dry matter intake by 4% to 27% (P < 0.01). However, increasing the wheat silage replacement of oaten hay by no more than 64% improved the feed efficiency by 14% as noted by the feed-to-gain ratio (P = 0.04). Apparent digestibility of organic matter (P < 0.01), neutral detergent fibre (P = 0.04) and acid detergent fibre (P < 0.01) quadratically increased. Ammonia nitrogen (P = 0.01) decreased while microbial protein production (P < 0.01) increased with the increase of wheat silage inclusion. Total volatile fatty acids concentration increased quadratically with the increase of wheat silage inclusion (P < 0.01), and the highest occurred in WS64. The molar proportion of acetate (P < 0.01) and acetate-to-propionate ratio (P = 0.04) decreased while butyrate (P < 0.01) and isovalerate (P = 0.04) increased. Increasing wheat silage inclusion increased the Firmicutes-to-Bacteroidota ratio by 226% to 357%, resulting in Firmicutes instead of Bacteroidota being the most abundant phylum. The relative abundance of cellulolytic Ruminococcus numerically increased but that of amylolytic Prevotella (P < 0.01) decreased as increasing wheat silage inclusion. Taken together, increasing wheat silage replacement of oaten hay by no more than 64% exhibited greater feed efficiency and fibre digestion despite low feed intake by feedlot lambs due to the change of Firmicutes-to-Bacteroidota ratio in the rumen.

A 21-d experiment was conducted to study the effect of xylanase, protease, and xylo-oligosaccharides on growth performance, nutrient utilization, gene expression of nutrient transporters, cecal short-chain fatty acids (SCFA), and cecal microbiota profile of broilers challenged with mixed Eimeria spp. The study utilized 392 zero-d-old male broiler chicks allocated to 8 treatments in a 4 × 2 factorial arrangement, as follows: corn-soybean meal diet with no enzyme (Con); Con plus xylanase alone (XYL); Con plus xylanase combined with protease (XYL + PRO); or Con plus xylo-oligosaccharides (XOS); with or without Eimeria challenge. Diets were based on a high-fiber (100g/kg soluble fibers and 14g/kg insoluble fibers) basal diet. At d 15, birds in challenged treatment were gavaged with a solution containing Eimeria maxima, Eimeria acervulina, and Eimeria tenella oocysts. At d 21, birds were sampled. Eimeria depressed (P < 0.01) growth performance and nutrient utilization, whereas supplementation had no effect. There were significant Eimeria × supplementation interactions for the sugar transporters GLUT5 (P = 0.02), SGLT1 (P = 0.01), SGLT4 (P < 0.01), and peptide transporter PepT1 (P < 0.01) in jejunal mucosa. Eimeria challenge increased the expression of GM-CSF2 (P < 0.01) and IL-17 (P = 0.04) but decreased (P = 0.03) IL-1β expression in the cecal tonsil. Eimeria × supplementation interactions for cecal acetate, butyrate, and total SCFA showed that concentrations increased or tended to be greater in the supplemented treatments, but only in non-challenged birds. Birds challenged with Eimeria spp. had higher concentrations of isobutyrate (P < 0.01), isovalerate (P < 0.01), and valerate (P = 0.02) in cecal content. Eimeria challenge significantly (P < 0.01) decreased the microbial richness and diversity, and increased (P < 0.01) the proportion of Anaerostipes butyraticus, Bifidobacterium pseudolongum, and Lactobacillus pontis. In conclusion, Eimeria infection depressed growth performance, nutrient utilization with regulating nutrient transporters. Furthermore, Eimeria challenge shifted the microbial profile and reduced microbial richness and diversity. On the other hand, enzyme supplementation showed limited benefits, which included increased concentrations of SCFA.

Piglets are particularly susceptible to oxidative stress, which causes inferior growth performance and intestinal damage. Squalene (SQ), a natural bioactive substance enriched in shark liver oil, shows excellent antioxidant properties and can currently be obtained at a low cost from deodorizer distillate during the production of plant oil. This study aimed to evaluate the effects of plant-derived SQ supplementation on the growth performance of piglets and explore the beneficial roles of SQ against oxidative stress and intestinal injury in diquat-challenged piglets. Forty piglets were randomly divided into five groups and fed a basal diet supplemented with SQ at 0, 500, 1000, or 2000mg/kg for 5 weeks. Acute oxidative stress was induced in the piglets with diquat (10mg/kg BW) at the fourth week of the experiment, followed by a 7-d recovery period. Results showed that before the diquat challenge, SQ supplementation significantly improved growth performance (average daily gain and feed conversion ratio) and serum antioxidant status, and after the diquat challenge, SQ supplementation significantly mitigated diquat-induced growth arrest, intestinal villous atrophy, intestinal epithelial cell apoptosis, intestinal hyperpermeability, and deficiency of intestinal epithelial tight junction proteins (zonula occludens-1, occludin, and claudin-3). Under oxidative stress induced by diquat, SQ supplementation consistently improved the antioxidant status of the small intestine, liver, and muscle. In vitro, SQ was shown to alleviate hydrogen peroxide (H₂O₂)-induced increase of the levels of intracellular reactive oxygen species and apoptosis of porcine intestinal epithelial cells. Taken together, SQ supplementation improves growth performance and effectively alleviates acute oxidative stress-induced growth retardation and intestinal injury via improving antioxidant capacity in piglets. Our findings may provide an efficient strategy for alleviating oxidative stress-induced inferior growth performance and intestinal damage in piglets.

This study aimed to investigate the potential mitigating effects of N-acyl homoserine lactonase (AHLase) on the virulence of Salmonella typhimurium and its induction of intestinal damages in broilers. In vitro study was firstly conducted to examine if AHLase treatment could attenuate the virulence of S. typhimurium. Then, an in vivo experiment was performed by allocating 240 broiler chicks at 1 d old into 3 groups (8 replicates per group): negative control (NC), positive control (PC), and PC supplemented with 10,000 U/kg AHLase. All chicks except those in NC were orally challenged by S. typhimurium from 8 to 10 d of age. Parameters were measured on d 11 and 21. The results showed that treatment with 1 U/mL AHLase suppressed the biofilm-forming ability (including biofilm biomass, extracellular DNA secretion and biofilm formation-related gene expression), together with swarming motility and adhesive capacity of S. typhimurium. Supplemental 10,000 U/kg AHLase counteracted S. typhimurium-induced impairments (P < 0.05) in broiler growth performance (including final body weight, average daily gain and average daily feed intake) during either 1-11 d or 12-21 d, and increases (P < 0.05) in the indexes of liver, spleen and bursa of Fabricius on d 11, together with reductions (P < 0.05) in ileal villus height and its ratio to crypt depth on both d 11 and 21. AHLase addition also normalized the increased (P < 0.05) mRNA expression of ileal occludin on both d 11 and 21 in S. typhimurium-challenged broilers. However, neither S. typhimurium challenge nor AHLase addition altered (P > 0.05) serum diamine oxidase activity of broilers. Noticeably, S. typhimurium challenge caused little change in the mRNA expression of ileal inflammatory cytokines except for an increase (P < 0.05) in interleukin-8 expression on d 11, whereas AHLase addition normalized (P < 0.05) this change. In conclusion, AHLase treatment could attenuate the virulence and pathogenicity of S. typhimurium, thus contributing to alleviate S. typhimurium-induced growth retardation and intestinal damages in broilers.