The impact of dietary Laminaria digitata and alginate lyase supplementation on the weaned piglet liver: A comprehensive proteomics and metabolomics approach.

The brown seaweed Laminaria digitata, a novel feedstuff for weaned piglets, has potentially beneficial prebiotic properties. However, its recalcitrant cell wall challenges digestion in monogastrics. Alginate lyase is a promising supplement to mitigate this issue. This study's aim was to investigate the impact of incorporating 10% dietary Laminaria digitata, supplemented with alginate lyase, on the hepatic proteome and metabolome of weaned piglets. These diets introduced minor variations to the metabolome and caused significant shifts in the proteome. Dietary seaweed provided a rich source of n-3 PUFAs that could signal hepatic fatty acid oxidation (FABP, ACADSB and ALDH1B1). This may have affected the oxidative stability of the tissue, requiring an elevated abundance of GST for regulation. The presence of reactive oxygen species likely inflicted protein damage, triggering increased proteolytic activity (LAPTM4B and PSMD4). Alginate lyase supplementation augmented the number of differentially abundant proteins, which included GBE1 and LDHC, contributing to maintain circulating glucose levels by mobilizing glycogen stores and branched-chain amino acids. The enzymatic supplementation with alginate lyase amplified the effects of the seaweed-only diet. An additional filter was employed to test the effect of missing values on the proteomics analysis, which is discussed from a technical perspective. SIGNIFICANCE: Brown seaweeds such as Laminaria digitata have prebiotic and immune-modulatory components, such as laminarin, that can improve weaned piglet health. However, they have recalcitrant cell wall polysaccharides, such as alginate, that can elicit antinutritional effects on the monogastric digestive system. The aim of this study was to evaluate the effect of a high level of dietary L. digitata and alginate lyase supplementation on the hepatic metabolism of weaned piglets, using high throughput Omics approaches.

Enhancing meat quality of weaned piglets with the dietary incorporation of Ulva lactuca and carbohydrases supplementation.

The impact of the dietary incorporation of 7% Ulva lactuca, a green seaweed, on the quality and nutritional value of piglet's meat was assessed. U. lactuca is rich in nutrients and bioactive compounds but its cell wall is composed of complex polysaccharides that reduce their bioavailability. Therefore, the effect of supplementing piglet diets with exogenous carbohydrases was also assessed here. A total of 40 male weaned piglets were divided into four dietary groups, each with 10 piglets: control (wheat, maize and soybean meal-based diet), UL (7% U. lactuca replacing the control diet), UL + R (UL and 0.005% Rovabio®), and UL + E (UL and 0.01% ulvan lyase). The piglets were fed the diets for 2 weeks. The results showed that incorporating U. lactuca in piglet diets did not influence most of the meat quality traits (P > 0.05). However, the incorporation of U. lactuca with the commercial carbohydrase (UL + R) increased the amount of the docosahexaenoic acid (DHA; 22:6n-3) in their meat (P = 0.011) compared with the control, by 54%. In addition, meat from piglets fed seaweed diets showed a nearly two-fold increase in iodine contents (P < 0.001). Meat tenderness, juiciness and overall acceptability of piglets fed the control diet and the UL diet were lower than those fed the diets containing seaweed and carbohydrases (P < 0.001). Overall, the findings indicate that 7% U. lactuca in the diets of weaned piglets had no major detrimental effects on meat quality and their carbohydrase supplementation has the potential to improve meat sensory traits.

Dietary Ulva lactuca and CAZyme supplementation improve serum biochemical profile and hepatic composition of weaned piglets.

Ulva lactuca is a seaweed with antinutritional cell wall for monogastrics. Carbohydrate-Active enZymes (CAZymes) supplementation can potentially cause its disruption. This study evaluates four diets: Ctrl-control diet; UL-control + 7% U. lactuca (wild caught, powdered form); ULR-UL + 0.005% Rovabio® Excel AP; ULU-UL + 0.01% ulvan lyase on piglets' haematologic and serologic profiles, hepatic lipids and minerals. White blood cells and lymphocytes reached the highest values in piglets fed UL compared to control, and to control and ULR; respectively (P < 0.05). IgG levels were boosted by seaweed incorporation compared to control (P = 0.015). The glycaemic homeostasis was assured by the seaweed inclusion. Dietary seaweed decreased serum lipids (P < 0.001), with the exception of ULU, due to HDL-cholesterol increase (P < 0.001). Cortisol was decreased in ULR and ULU (P < 0.001). No systemic inflammation was observed (P > 0.05). While hepatic n-3 PUFA increased in piglets fed with seaweed diets due to increment of beneficial 22:5n-3 and 22:6n-3 fatty acids (P < 0.05), the opposite occurred for n-6 PUFA, PUFA/SFA and n-6/n-3 ratios (P < 0.05). Hepatic pigments were unchanged (P > 0.05). ULR reduced α-tocopherol levels (P = 0.036) and increased serum potassium levels (P < 0.001) compared to control. Seaweed contributed to overcome piglets' weaning stress, with some benefits of including CAZyme supplementation.

Effect of Laminaria digitata dietary inclusion and CAZyme supplementation on blood cells, serum metabolites and hepatic lipids and minerals of weaned piglets.

Seaweeds, such as Laminaria digitata, are a sustainable alternative to conventional feedstuffs for weaned piglet diets, improving their health and mitigating environmental impacts. L. digitata has a complex cell wall that can be difficult for monogastrics to digest. However, carbohydrate-active enzymes (CAZymes) such as Rovabio® Excel AP and alginate lyase can help break down these polysaccharides and render intracellular nutrients more accessible. This study aimed to evaluate the impact of 10% L. digitata feed inclusion and CAZyme supplementation on piglet blood cells, serum metabolites, liver lipid and mineral profiles. Forty weaned piglets were randomly assigned to one of four diets (n = 10 each): a control diet, 10% L. digitata (LA), 10% L. digitata + 0.005% Rovabio® Excel AP (LAR), and 10% L. digitata + 0.01% alginate lyase (LAL). After two weeks of trial, animals were slaughtered and liver and blood serum samples taken for analysis. The results showed that the LA and LAL diets increased blood lymphocytes, IgG and IgM, and decreased serum lipids, improving both cellular and humoral immune response and cardiovascular health. Dietary CAZymes reversed the anti-inflammatory and hematopoietic effects. Additionally, cortisol levels were reduced with seaweed inclusion compared to the control diet (P < 0.001). In the liver, total n-3 PUFA and n-6/n-3 ratio were increased and decreased, respectively, due to eicosapentaenoic acid and α-linolenic acid accumulation (P < 0.001). However, total liver mineral content was incorporated to a lesser extent with the combined seaweed and enzyme diets (P < 0.001), potentially indicating a negative effect on mineral bioavailability. Overall, results suggest that a 10% L. digitata inclusion can effectively improve piglet health by reducing stress during weaning, without the need for dietary CAZymes.

Impact of dietary Chlorella vulgaris and feed enzymes on health status, immune response and liver metabolites in weaned piglets.

In this study, we analysed the impact of dietary inclusion of Chlorella vulgaris and carbohydrases on general health, redox status, immune response, liver lipids and metabolites in weaned piglets. Forty-four male piglets were allocated into four diets: control (n = 11), CH (control diet with 5% CH, n = 10), CH+R (control diet with 5% CH plus 0.005% Rovabio Excel AP, n = 10), and CH+M (control diet with 5% CH plus 0.01% of a pre-selected four-CAZyme mixture, n = 11). After 15 days of trial, animals were slaughtered and samples of blood and liver collected. Spectrophotometry methods and commercial kits were used to determine blood parameters and gas and liquid chromatography for hepatic fatty acid and chlorophylls profiles, respectively. While total, LDL- and VLDL-cholesterol were increased by CH, the opposite was recorded for HDL-cholesterol (p < 0.001). Piglets fed CH-based diets presented an increase of IgG and a decrease of IgM (p < 0.001) which along with lymphocytes exacerbation contributed for piglets' survival after weaning. n-6 PUFA were reduced in piglets fed CH and the opposite occurred for n-3 PUFA (p < 0.001), thus benefiting n-6/n-3 ratio in the liver. Chlorophylls amount was not changed by the use of Rovabio or enzymatic mixture. The discriminant analysis applied to hepatic parameters revealed a clear separation between control and CH-based diets but failed to discriminate feed enzymes. Our findings indicate health promoting effects of CH as feed ingredient in piglets' nutrition at weaning, without negatively impacting on animals' performance.

Effect of dietary Spirulina (Arthrospira platensis) on the intestinal function of post-weaned piglet: An approach combining proteomics, metabolomics and histological studies.

The effect of dietary Spirulina (Arthrospira platensis) and CAZyme supplementation was assessed on the gut of weaned piglets, using an integrated NMR-metabolomics approach combined with Tandem Mass Tag labelled proteomics. Thirty weaned male piglets were assigned to one of the three following diets (n = 10): cereal and soybean meal basal diet (Control), basal diet with 10% Spirulina inclusion (SP) and SP diet supplemented with 0.01% lysozyme (SP + L). The experiment lasted 4 weeks and, upon slaughter, small intestine samples were collected for histological, metabolomic and proteomic analysis. No significant differences were found for the histology and metabolomics analysis between the three experimental groups. Lactate, glutamate, glycine and myo-inositol were the most abundant metabolites. Proteomics results showed 1502 proteins identified in the intestine tissue. A total of 23, 78, 27 differentially abundant proteins were detected respectively for the SP vs. Control, SP + L vs. Control and SP + L vs. SP comparisons. The incorporation of Spirulina and supplementation of lysozyme in the piglet's diets is associated to intestinal proteomic changes. These include increased protein synthesis and abundance of contractile apparatus proteins, related with increased nutrient availability, which has beneficial (increased glucose uptake) and detrimental (increased digesta viscosity) metabolic effects. SIGNIFICANCE: The use of conventional feedstuffs becomes increasingly prohibitive due to its environmental toll. To increase the sustainability of the livestock sector, novel feedstuffs such as microalgae need to be considered. However, its recalcitrant cell wall has antinutritional effects that can inhibit high dietary inclusion levels. The supplementation with CAZymes is a possible solution to this issue. The small intestine is a central piece in monogastric digestion and of particular importance for the weaned piglet. Studying the effect of dietary Spirulina and CAZyme supplementation on its histomorphology, metabolome and proteome allows studying relevant physiological adaptations to these diets.

Influence of Feeding Weaned Piglets with Laminaria digitata on the Quality and Nutritional Value of Meat.

Laminaria digitata is a novel feedstuff that can be used in pig diets to replace conventional feedstuffs. However, its resilient cell wall can prevent the monogastric digestive system from accessing intracellular nutrients. Carbohydrate-active enzyme (CAZyme) supplementation is a putative solution for this problem, degrading the cell wall during digestion. The objective of this work was to evaluate the effect of 10% L. digitata feed inclusion and CAZyme supplementation on the meat quality and nutritional value of weaned piglets. Forty weaned piglets were randomly allocated to four experimental groups (n = 10): control, LA (10% L. digitata, replacing the control diet), LAR (LA + CAZyme (0.005% Rovabio® Excel AP)) and LAL (LA + CAZyme (0.01% alginate lyase)) and the trial lasted for two weeks. The diets had no effect on any zootechnical parameters measured (p > 0.05) and meat quality traits, except for the pH measured 24 h post-mortem, which was higher in LAL compared to LA (p = 0.016). Piglets fed with seaweed had a significantly lower n-6/n-3 PUFA ratio compared to control, to which the higher accumulation of C20:5n-3 (p = 0.001) and C18:4n-3 (p < 0.0001) contributed. In addition, meat of seaweed-fed piglets was enriched with bromine (Br, p < 0.001) and iodine (I, p < 0.001) and depicted a higher oxidative stability. This study demonstrates that the nutritional value of piglets' meat could be improved by the dietary incorporation of L. digitata, regardless of CAZyme supplementation, without negatively affecting growth performance in the post-weaning stage.

Influence of Chlorella vulgaris on growth, digestibility and gut morphology and microbiota of weaned piglet.

The purpose of this study was to evaluate the impact of Chlorella vulgaris (5% in the diet), supplemented or not with two exogenous carbohydrase mixtures on piglets' performance, nutrient digestibility and gut morphology, fermentation products and microbiota. Forty-four male piglets weaned at 28 days of age, with 11.2 ± 0.46 kg of live weight, were used and assigned to 1 of 4 dietary treatments: cereal and soybean meal based-diet (control, n = 11), control diet with 5% of C. vulgaris (CH, n = 10), CH diet supplemented with 0.005% of Rovabio® Excel AP (CH + R, n = 10) and CH diet supplemented with 0.01% of a recombinant 4-carbohydrase mixture (CH + M, n = 11). Growth performance was not changed by the of C. vulgaris inclusion during 21 days of trial. However, total tract apparent digestibility of nutritional fractions was negatively impacted by the inclusion. In addition, the viscosity of duodenum plus jejunum contents slightly increased in all groups fed with the microalga. In contrast, dietary microalga increased duodenum villus height and promoted a healthier gut microbiota, with higher abundance of some specific bacterial taxa (Colidextribacter, Oscillospira and Lactobacillus). This study indicates that the dietary inclusion of 5% C. vulgaris improves piglets' gut health without impairing performance. Data also indicate that C. vulgaris reduces nutrient digestibility but promotes compensatory developments of gut mucosa and prebiotic effects. Dietary supplementation with exogenous carbohydrases does not seem to be necessary for this inclusion level. Therefore, the incorporation of CH as a sustainable feed ingredient in piglets' nutrition is a viable alternative approach.

Effects of Chlorella vulgaris as a Feed Ingredient on the Quality and Nutritional Value of Weaned Piglets' Meat.

Chlorella vulgaris (CH) is usually considered a feed supplement in pig nutrition, and its use as an ingredient is poorly studied. Among many interesting characteristics, this microalga has high protein levels and can be a putative alternative for soybean meal. Our aim was to study the effect of a 5% CH incorporation in the diet, individually or combined with two carbohydrases, on meat quality traits and nutritional value. Forty-four post-weaned male piglets individually housed, with an initial live weight of 11.2 ± 0.46 kg, were randomly distributed into four experimental groups: control (n = 11, without CH) and three groups fed with 5% CH incorporation, plain (n = 10), with 0.005% Rovabio® Excel AP (n = 10), and with 0.01% of a pre-selected four-CAZyme mixture (n = 11). After two weeks of trial, piglets were slaughtered and longissimus lumborum collected. CH had no effect on piglets' growth performance. In turn, incorporation of CH improved the nutritional value of meat by increasing total carotenoids and n-3 PUFA content, thus contributing to a more positive n-6/n-3 fatty acid ratio. The supplementation with Rovabio® benefited tenderness and increased overall acceptability of pork. Our results show beyond doubt the viability of the utilization of this microalga as a feed ingredient for swine production.

Influence of dietary Spirulina inclusion and lysozyme supplementation on the longissimus lumborum muscle proteome of newly weaned piglets.

Arthrospira platensis (Spirulina) is a microalga with a high content of crude protein. It has a recalcitrant cell wall that limits the accessibility of the animal endogenous enzymes to its intracellular nutrients. Enzymatic supplementation aiming to degrade cell walls could benefit microalgae digestibility. The objective of this study was to evaluate the impact of dietary Spirulina and lysozyme supplementation over the muscle proteome of piglets during the post-weaning stage. Thirty piglets were randomly distributed among three diets: control (no microalga), SP (10% Spirulina) and SP + L (10% Spirulina +0.01% lysozyme). After 4 weeks, they were sacrificed and samples of the longissimus lumborum muscle were taken. The muscle proteome was analysed using a Tandem Mass Tag (TMT)-based quantitative approach. A total of 832 proteins were identified. Three comparisons were computed: SP vs Ctrl, SP + L vs Ctrl and SP + L vs SP. They had ten, four and twelve differentially abundant proteins. Glycogen metabolism and nutrient reserves utilization are increased in the SP piglets. Structural muscle protein synthesis increased, causing higher energy requirements in SP + L piglets. Our results demonstrate the usefulness of proteomics to disclose the effect of dietary microalgae, whilst unveiling putative mechanisms derived from lysozyme supplementation. Data available via ProteomeXchange with identifier PXD024083. SIGNIFICANCE: Spirulina, a microalga, is an alternative to conventional crops which could enhance the environmental sustainability of animal production. Due to its recalcitrant cell wall, its use requires additional measures to prevent anti-nutritional effects on the feeding of piglets in the post-weaning period, during which they endure post-weaning stress. One of such measures could be CAZyme supplementation to help degrade the cell wall during digestion. Muscle proteomics provides insightful data on the effect of dietary microalgae and enzyme activity on piglet metabolism.

Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets.

The regulation of glycerol permeability in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. Knowing that the amino acid glutamine is a physiological regulator of gluconeogenesis, whereas cystine promotes adiposity, herein we investigated the effects of dietary supplementation with glutamine and cystine on the serum biochemical parameters of piglets fed on amino acid-enriched diets, as well as on the transcriptional profile of membrane water and glycerol channels aquaporins (AQPs) in the ileum portion of the small intestine and its impact on intestinal permeability. Twenty male piglets with an initial body weight of 8.8 ± 0.89 kg were allocated to four dietary treatments (n = 5) and received, during a four week-period, a basal diet without supplementation (control) or supplemented with 8 kg/ton of glutamine (Gln), cystine (Cys) or the combination of the two amino acids in equal proportions (Gln + Cys). Most biochemical parameters were found improved in piglets fed Gln and Cys diet. mRNA levels of AQP3 were found predominant over the others. Both amino acids, individually or combined, were responsible for a consistent downregulation of AQP1, AQP7 and AQP10, without impacting on water permeability. Conversely, Cys enriched diet upregulated AQP3 enhancing basolateral membranes glycerol permeability and downregulating glycerol kinase (GK) of intestinal cells. Altogether, our data reveal that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target for adipogenesis regulation.