Enhanced ileum function in weaned piglets via Laminaria digitata and alginate lyase dietary inclusion: A combined proteomics and metabolomics analysis.

Laminaria digitata, a brown seaweed with prebiotic properties, can potentially enhance the resilience of weaned piglets to nutritional distress. However, their cell wall polysaccharides elude digestion by monogastric animals' endogenous enzymes. In vitro studies suggest alginate lyase's ability to degrade such polysaccharides. This study aimed to assess the impact of a 10% dietary inclusion of L. digitata and alginate lyase supplementation on the ileum proteome and metabolome, adopting a hypothesis-generating approach. Findings indicated that control piglets escalated glucose usage as an enteric energy source, as evidenced by the increased abundance of PKLR and PCK2 proteins and decreased tissue glucose concentration. Additionally, the inclusion of seaweed fostered a rise in proteins linked to enhanced enterocyte structural integrity (ACTBL2, CRMP1, FLII, EML2 and MYLK), elevated peptidase activity (NAALADL1 and CAPNS1), and heightened anti-inflammatory activity (C3), underscoring improved intestinal function. In addition, seaweed-fed piglets showed a reduced abundance of proteins related to apoptosis (ERN2) and proteolysis (DPP4). Alginate lyase supplementation appeared to amplify the initial effects of seaweed-only feeding, by boosting the number of differential proteins within the same pathways. This amplification is potentially due to increased intracellular nutrient availability, making a compelling case for further exploration of this dietary approach. SIGNIFICANCE: Pig production used to rely heavily on antibiotics and zinc oxide to deal with post-weaning stress in a cost-effective way. Their negative repercussions on public health and the environment have motivated heavy restrictions, and a consequent search for alternative feed ingredients/supplements. One of such alternatives is Laminaria digitata, a brown seaweed whose prebiotic components that can help weaned piglets deal with nutritional stress, by improving their gut health and immune status. However, their recalcitrant cell walls have antinutritional properties, for which alginate lyase supplementation is a possible solution. By evaluating ileal metabolism as influenced by dietary seaweed and enzyme supplementation, we aim at discovering how the weaned piglet adapts to them and what are their effects on this important segment of the digestive system.

Tissue-Specific Variation in Aquaporins and Cytokines Transcriptional Profiles in Piglets being LPS Challenged.

BACKGROUND: Lipopolysaccharide (LPS), an effective stimulator of the immune system, has been widely applied in an experimental pig model for human sepsis. Aquaporins (AQPs), a family of small integral membrane proteins responsible for facilitating water fluxes through the cell membrane, offer potential promising drug targets for sepsis treatment due to their role in water balance and inflammation. METHODS: In order to investigate the potential effect of a dietary amino acid mixture supplementation on LPS-challenged weaned piglets, a total of 30, 28-day-old, males were randomly allocated to 1 of 3 dietary treatments for a 5-week period, with 10 animals in each: diet 1 was a control (CTL) treatment; diet 2 was LPS treatment, where the piglets were intraperitoneally administered LPS (at 25 µg/kg body weight); diet 3 was LPS + cocktail treatment, where the piglets were intraperitoneally administered LPS and fed a diet supplemented with a mixture of arginine, branched-chain amino acids (BCAA, leucine, valine, and isoleucine), and cystine. Key organs that control sepsis were collected and processed by real time quantitative PCR (RT-qPCR) for the AQPs and cytokines transcriptional profiles. RESULTS: Minor variations were detected for AQPs and inflammatory markers mRNA levels, upon the dependence of LPS or the amino acid cocktail suggesting the piglets' immune recovery. Using a discriminant analysis tool, we report for the first time, a tissue-specific variation in AQPs and cytokines transcriptional profiles that clearly distinguish the small intestine and the kidney from the liver and the spleen. CONCLUSIONS: This study provides a novel insight into the gene expression signature of AQPs and cytokines in the functional physiology of each organ in piglets.

Integrated Omics analysis of pig muscle metabolism under the effects of dietary Chlorella vulgaris and exogenous enzymes.

Monogastric feeding is dependent on costly conventional feedstuffs. Microalgae such as Chlorella vulgaris are a sustainable alternative; however, its recalcitrant cell wall hinders monogastric digestion. Carbohydrate Active Enzyme (CAZyme) supplementation is a possible solution. The objective of this work was to evaluate the effect of 5% dietary C. vulgaris (CV) and enzymatic supplementation (CV + R-Rovabio® Excel AP; CV + M-four CAZyme mix) on muscle transcriptome and proteome of finishing pigs, in an integrated approach. Control pigs increased the abundance of contractile apparatus (MYH1, MYH2, MYH4) and energy metabolism (CKMT1, NDUFS3) proteins, demonstrating increased nutrient availability. They had increased expression of SCD, characteristic of increased glucose availability, via the activation of SREBP-1c and ChREBP. CV and CV + R pigs upregulated proteolytic and apoptotic genes (BAX, DDA1), whilst increasing the abundance of glucose (UQCRFS1) and fatty acid catabolism (ACADS) proteins. CV + R pigs upregulated ACOT8 and SIRT3 genes as a response to reduced nutrient availability, maintaining energy homeostasis. The cell wall specific CAZyme mix, CV + M, was able to comparatively reduce Omics alterations in the muscle, thereby reducing endogenous nutrient catabolism compared to the CV + R and CV.

Combined effects of dietary Laminaria digitata with alginate lyase on plasma metabolites and hepatic lipid, pigment and mineral composition of broilers.

BACKGROUND: The Laminaria digitata is an abundant macroalga and a sustainable feedstock for poultry nutrition. L. digitata is a good source of essential amino acids, carbohydrates and vitamins, including A, D, E, and K, as well as triacylglycerols and minerals, in particular iron and calcium. However, the few studies available in the literature with broilers document the application of this macroalga as a dietary supplement rather than a feed ingredient. No study has addressed up until now the effects of a high-level incorporation (> 2% in the diet) of L. digitata on plasma biochemical markers and hepatic lipid composition, as well as minerals and pigments profile in the liver of broilers. Our experimental design included one hundred and twenty Ross 308 male birds contained in 40 wired-floor cages and distributed to the following diets at 22 days of age (n = 10) for 15 days: 1) a corn-soybean basal diet (Control); 2) the basal diet plus 15% of L. digitata (LA); 3) the basal diet plus 15% of L. digitata with 0.005% of Rovabio® Excel AP (LAR); and 4) the basal diet plus 15% of L. digitata with 0.01% of the recombinant CAZyme, alginate lyase (LAE). RESULTS: L. digitata compromised birds' growth performance by causing a reduction in final body weight. It was found an increase in hepatic n-3 and n-6 fatty acids, in particular C18:2n-6, C18:3n-6, C20:4n-6, C20:5n-3, C22:5n-3 and C22:6n-3 with the addition of the macroalga, with or without feed enzymes, to the broiler diets. Also, the beneficial C18:3n-3 fatty acid was increased by combining L. digitata and commercial Rovabio® Excel AP compared to the control diet. The sum of SFA, MUFA and the n-6/n-3 PUFA ratio were decreased by L. digitata, regardless the addition of exogenous enzymes. ß-carotene was enhanced by L. digitata, individually or combined with CAZymes, being also responsible for a positive increase in total pigments. Macrominerals, in particular phosphorous and sulphur, were increased in the liver of broilers fed L. digitata individually relative to the control. For microminerals, copper, iron and the correspondent sum were consistently elevated in the liver of broilers fed L. digitata, individually or combined with exogenous CAZymes. The powerful discriminant analysis tool based on the hepatic characterization revealed a good separation between the control group and L. digitata diets but failed to discriminate the addition of feed enzymes. CONCLUSIONS: Overall, this study highlights the value of L. digitata as a feed ingredient for the poultry industry. Moreover, we can conclude that the effect of L. digitata overpowers the effect of feed enzymes, both the Rovabio® Excel AP and the alginate lyase. Having in mind the negative effects observed on birds' performance, our main recommendation at this stage is to restraint L. digitata incorporation level in forthcoming nutritional studies.

Quality Traits and Nutritional Value of Pork and Poultry Meat from Animals Fed with Seaweeds.

Seaweeds have caught the attention of the scientific community in recent years. Their production can mitigate the negative impact of anthropogenic activity and their use in animal nutrition reduces the dependency on conventional crops such as maize and soybean meal. In the context of monogastric animals, novel approaches have made it possible to optimise their use in feed, namely polysaccharide extraction, biomass fermentation, enzymatic processing, and feed supplementation with carbohydrate-active enzymes (CAZymes). Their bioactive properties make them putative candidates as feed ingredients that enhance meat quality traits, such as lipid oxidation, shelf-life, and meat colour. Indeed, they are excellent sources of essential amino acids, polyunsaturated fatty acids, minerals, and pigments that can be transferred to the meat of monogastric animals. However, their nutritional composition is highly variable, depending on species, harvesting region, local pollution, and harvesting season, among other factors. In this review, we assess the current use and challenges of using seaweeds in pig and poultry diets, envisaging to improve meat quality and its nutritional value.

Impact of dietary Chlorella vulgaris and carbohydrate-active enzymes incorporation on plasma metabolites and liver lipid composition of broilers.

BACKGROUND: Chlorella vulgaris has been proposed as a sustainable green feedstock in poultry nutrition due to its ease of cultivation, minimal environmental impact and balanced nutritional composition. However, the majority of studies documents the use of C. vulgaris as a dietary supplement in broilers instead of a feed ingredient. To the best of our knowledge, no report has shown the effect of a high-level incorporation (>2 % in the diet) of C. vulgaris on plasma metabolites and hepatic lipid composition of broilers. One hundred and twenty Ross 308 male birds were housed in 40 wired-floor cages and randomly distributed by the following experimental diets at 22 days of age (n = 10) during 15 days: (1) a corn-soybean meal based diet (control); (2) based diet with 10% of C. vulgaris; (3) diet 2 supplemented with 0.005% Rovabio® Excel AP; and (4) diet 2 supplemented with 0.01% of a pre-selected four-CAZyme mixture. RESULTS: The inclusion of C. vulgaris at 10% in the diet, regardless of the presence of exogenous CAZymes, changed plasma metabolites but did not compromise broilers growth. Plasma total lipids increased in broilers fed C. vulgaris combined with the two feed CAZymes (p < 0.001) compared with the control diet. Moreover, the supplementation with Rovabio® increased total cholesterol and LDL-cholesterol, while the addition of the four-CAZyme mixture increased triacylglycerols, VLDL-cholesterol and ALP activity. In opposition, HDL-cholesterol levels decreased in broilers fed microalga alone (p = 0.002). Regarding hepatic composition, the inclusion of C. vulgaris in broiler diets, individually or combined with exogenous CAZymes, had a minor effect on fatty acids but improved the n-6/n-3 ratio and total carotenoids. CONCLUSIONS: In summary, the inclusion of a high level (10%) of C. vulgaris in broiler´s diet, regardless of the presence of exogenous CAZymes, improved hepatic antioxidant composition and did not impair broiler's performance. In addition, the feed supplementation with CAZymes increased broilers lipemia. Therefore, dietary C. vulgaris at this incorporation level seems to be safe for animal health and do not compromise performance traits, with no need of CAZymes supplementation.

Dietary Arthrospira platensis improves systemic antioxidant potential and changes plasma lipids without affecting related hepatic metabolic pathways in post-weaned piglets.

BACKGROUND: The ability of a high level of dietary Arthrospira platensis, individually or in combination with two exogenous carbohydrate-degrading enzymes (lysozyme and Rovabio®), to improve systemic antioxidant potential and hepatic lipid metabolism was tested in piglets. Forty male post-weaned piglets, sons of Large White × Landrace sows crossed with Pietrain boars, were allocated into 4 groups (n = 10) and fed during 28 days one of the following diets: 1) a control basal diet (cereal and soybean meal); 2) a basal diet with 10% of A. platensis (AP); 3) the AP diet supplemented with 0.005% of Rovabio® (AP + R); 4) the AP diet supplemented with 0.01% of lysozyme (AP + L). RESULTS: Arthrospira platensis decreased BW gain of piglets, regardless the addition of feed enzymes. The majority of plasma metabolites were affected by diets. A. platensis increased total lipids, total cholesterol and LDL-cholesterol, without changing hepatic fatty acid content or modulating, in an expressive manner, the transcriptional profile of lipid sensitive mediators. The antioxidant potential in general, and total carotenoids in particular, were improved by the microalga, regardless lysozyme or Rovabio®. CONCLUSIONS: Summing up, A. platensis, individually and combined with feed enzymes, impacts negatively on piglets' growth but improves the systemic antioxidant potential and changes plasma lipids with a minor modulation on related hepatic metabolic pathways.

Arginine supplementation modulates pig plasma lipids, but not hepatic fatty acids, depending on dietary protein level with or without leucine.

BACKGROUND: In the present study, the effect of arginine and leucine supplementation, and dietary protein level, were investigated in commercial crossbred pigs to clarify their individual or combined impact on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid sensitive factors. The experiment was conducted on fifty-four entire male pigs (Duroc × Pietrain × Large White × Landrace crossbred) from 59 to 92 kg of live weight. Each pig was randomly assigned to one of six experimental treatments (n = 9). The treatments followed a 2 × 3 factorial arrangement, providing two levels of arginine supplementation (0 vs. 1%) and three levels of basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet with 2% of leucine, RPDL). RESULTS: Significant interactions between arginine supplementation and protein level were observed across plasma lipids. While dietary arginine increased total lipids, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and triacylglycerols in NPD, the inverse effect was observed in RPD. Overall, dietary treatments had a minor impact on hepatic fatty acid composition. RPD increased 18:1c9 fatty acid while the combination of leucine and RPD reduced 18:0 fatty acid. Arginine supplementation increased the gene expression of FABP1, which contributes for triacylglycerols synthesis without affecting hepatic fatty acids content. RPD, with or without leucine addition, upregulated the lipogenic gene CEBPA but downregulated the fat oxidation gene LPIN1. CONCLUSIONS: Arginine supplementation was responsible for a modulated effect on plasma lipids, which is dependent on dietary protein level. It consistently increased lipaemia in NPD, while reducing the correspondent metabolites in RPD. In contrast, arginine had no major impact, neither on hepatic fatty acids content nor on fatty acid composition. Likewise, leucine supplementation of RPD, regardless the presence of arginine, promoted no changes on total fatty acids in the liver. Ultimately, arginine, leucine and dietary protein reduction seem to be unrelated with fatty liver development.

Dietary conjugated linoleic acid isomers change the unsaturation degree of hepatic fatty acids in neutral lipids but not in polar lipids.

The fatty acid composition of phospholipids plays a key role in the structural and functional properties of cellular membrane. In this study, it was hypothesized that conjugated linoleic acid (CLA) isomer supplementation changes the unsaturation degree of the fatty acids of neutral lipids (NLs) but not those of polar lipids (PLs). Thus, the main goal was to determine the pattern of fatty acid incorporation into hepatic PL and NL fractions. Wistar male rats were fed cis(c)9,trans(t)11 and t10,c12 CLA isomers, separately or as a mixture. Whereas the t10,c12 isomer incorporation in the PL fraction was similar when supplemented either individually or as a mixture, the c9,t11 isomer reached the highest values of incorporation when combined with t10,c12. In the PL fraction, the linoleic acid did not change; but the arachidonic acid decreased, especially in the rats given the mixture. Also in this fraction, the t10,c12 isomer, either separately or as a mixture, decreased the amounts of n-6 long-chain (LC) polyunsaturated fatty acids (PUFA) and increased those of the n-3 LC PUFA relative to the control. In the NL fraction, linoleic acid incorporation followed the diet composition, whereas the arachidonic acid was similar among treatments. Facing CLA isomer supplementation, the present study suggests that fatty acid incorporation into phospholipids, through the balance between n-6 and n-3 LC PUFA, is dependent upon maintaining the unsaturation degree of cellular membrane.