Integrated transcriptomics and proteomics analysis reveals muscle metabolism effects of dietary Ulva lactuca and ulvan lyase supplementation in weaned piglets.

Seaweeds, including the green Ulva lactuca, can potentially reduce competition between feed, food, and fuel. They can also contribute to the improved development of weaned piglets. However, their indigestible polysaccharides of the cell wall pose a challenge. This can be addressed through carbohydrase supplementation, such as the recombinant ulvan lyase. The objective of our study was to assess the muscle metabolism of weaned piglets fed with 7% U. lactuca and 0.01% ulvan lyase supplementation, using an integrated transcriptomics (RNA-seq) and proteomics (LC-MS) approach. Feeding piglets with seaweed and enzyme supplementation resulted in reduced macronutrient availability, leading to protein degradation through the proteasome (PSMD2), with resulting amino acids being utilized as an energy source (GOT2, IDH3B). Moreover, mineral element accumulation may have contributed to increased oxidative stress, evident from elevated levels of antioxidant proteins like catalase, as a response to maintaining tissue homeostasis. The upregulation of the gene AQP7, associated with the osmotic stress response, further supports these findings. Consequently, an increase in chaperone activity, including HSP90, was required to repair damaged proteins. Our results suggest that enzymatic supplementation may exacerbate the effects observed from feeding U. lactuca alone, potentially due to side effects of cell wall degradation during digestion.

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.

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.

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.

Rapid diagnostics and ceftazidime/avibactam for KPC-producing Klebsiella pneumoniae bloodstream infections: impact on mortality and role of combination therapy.

This study was aimed at investigating risk factors for mortality in patients suffering from KPC-producing Klebsiella pneumoniae (KPC-Kp) bloodstream infections (BSIs), evaluating the impact of rapid diagnostics and ceftazidime/avibactam use. This observational retrospective study (January 2017-May 2021) included all patients with a KPC-Kp BSI. Uni-multivariable analyses were carried out to evaluate the effect of clinical variables on both in-hospital death (IHD) and 30-day all-cause mortality, and the role of the combination of ceftazidime/avibactam plus polymyxin. One hundred and ninety-six patients met the study's inclusion criteria. Older age, having undergone renal replacement therapy during the 30 days preceding the KPC-Kp BSI onset, having an INCREMENT-CPE score ≥ 8, and having suffered from a superimposed and/or following KPC-Kp BSI treatment candidemia were found to be the main factors associated with both mortality rates. Among protective factors, the centrality of ceftazidime/avibactam in monotherapy (IHD: OR: 0.34; CI 95%: 0.11-1.00-30-day all-cause mortality: OR: 0.18; CI 95%: 0.04-0.77) or combination (IHD: OR: 0.51; CI 95%: 0.22-1.19-30-day all-cause mortality: OR: 0.62; CI 95%: 0.21-1.84) emerged and became even more evident once the effect of ceftazidime/avibactam plus polymyxin was removed. Rapid diagnostics may be useful to adopt more effective strategies for the treatment of KPC-Kp BSI patients and implement infection control measures, even if not associated with higher patient survival. Ceftazidime/avibactam, even when used alone, represents an important option against KPC-Kp, while combined use with polymyxin might not have altered its efficacy. Patient comorbidities, severity of BSI, and complications such as candidemia were confirmed to have a significant burden on survival.

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.

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.

Membrane fractionation of Cynara cardunculus swine blood hydrolysate: Ingredients of high nutritional and nutraceutical value.

This work proposes an innovative approach to valorise swine blood based on enzymatic hydrolysis and membrane fractionations. Hydrolysis with Cynara cardunculus enzymes, followed by microfiltration and double nanofiltration generated three high protein fractions, retentate of microfiltration (RMF; >0.5 µm) and retentate of nanofiltration (RNF; >3 kDa) with approximately 90% of protein on a dry basis and filtrate of nanofiltrate (FNF; <3 kDa) with 65%. FNF, rich in low molecular weight peptides, showed excellent antioxidants (ABTS and ORAC of 911.81 and 532.82 µmol TE g-1 DB, respectively) and antihypertensive (IC50 of 28.51 µg mL-1) potential. By peptidomics and in silico analysis, 43 unique sequences of interest were found, among which LVV-Hemorphin-7 was identified. This hemorphin was demonstrated as the main responsible for the observed bioactivity. Complementary results showed a prebiotic effect mainly for the growth of Bifidobacterium animalis Bo, as well as interesting free amino acids (mainly glutamic acid, leucine, alanine, phenylalanine and aspartic acid) and mineral (e.g., Ca, Mg, P, K and Na) profiles. No antibacterial effect was verified for the seven pathogenic bacteria tested. This study allowed obtaining new ingredient of high nutritional and nutraceutical value for human consumption, with a perspective of sustainability and industrial viability.

Partial replacement of soybean meal with Chlorella vulgaris in broiler diets influences performance and improves breast meat quality and fatty acid composition.

Alternative feed ingredients, such as microalgae, may be more sustainable in comparison to conventional feedstuffs that need large amounts of arable land and are often imported. This study evaluates the effects of Chlorella vulgaris various inclusion levels in the diet of broiler chickens on performance, carcass yield, organ measurements, breast meat quality, fatty acids profile, and antioxidant capacity. A total of two hundred forty 5 d old male Ross 308 broilers were randomly allotted to 4 groups (6 replicates of 10 birds each). Each group received either a control diet or a diet where soybean meal was replaced with 10% (CV10%), 15% (CV15%), or 20% C. vulgaris for 40 d. Performance parameters, carcass and meat traits were evaluated. Compared to the control group, birds supplemented with C. vulgaris (15% and 20%) had lower body weight, weight gain, and feed intake (P < 0.0001), whereas no differences were observed between the control and CV10% groups (P > 0.05). Feed conversion ratio did not differ between control and CV groups. Diets containing C. vulgaris significantly increased ileal digesta viscosity, weight and size of several gastrointestinal compartments, as well as breast muscle yield (P < 0.0001). Incorporation of C. vulgaris resulted in yellower breast muscle (P < 0.0001), with significantly increased chlorophyll a (P < 0.05), chlorophyll b, and total carotenoids contents (P < 0.0001). Inclusion of C. vulgaris decreased bacterial count in meat samples in comparison to controls (P < 0.0001). A 20% C. vulgaris inclusion resulted in higher water holding capacity (P < 0.05) and lower cooking loss (P < 0.05). As dietary C. vulgaris increased, concentrations of DHA + EPA (P < 0.05) and n-3 PUFA (P < 0.0001) increased in breast meat, while the n-6/n-3 PUFA ratio decreased (P < 0.0001). Sensory analysis showed that breast meat from the CV10% group had the highest acceptance score. Overall, dietary concentrations of C. vulgaris of up to 20% improve breast meat quality, whereas 10% of C. vulgaris inclusion is recommended.

Digestibility of Meat Mineral and Proteins from Broilers Fed with Graded Levels of Chlorella vulgaris.

The incorporation of sustainable protein sources in animal feeding is a growing trend. So far, no study has investigated in vitro digestion of meat, from broilers fed microalgae, in a human model. This research aimed to evaluate the effect of incorporating Chlorella vulgaris in the broilers diet on human protein digestibility, and mineral bioaccessibility. The study used 240 male Ross 308 broilers randomly allocated to groups fed a control diet or a diet where soybean meal was replaced with 10% (CV10%), 15% (CV15%), or 20% (CV15%) of C. vulgaris for 40 days. The microalga supplementation increased the protein and lowered the fat content in the muscle. Results on the percentages of amino acids highlighted that arginine and threonine proportions increased and lysine and cysteine proportions decreased with microalga inclusion. CV15% and CV20% meat had higher amount of K, Ca, Mg, P, and Fe in raw breasts, improving the nutrient composition of the meat. Cooking caused a decrease in Na and K and an increase in other minerals. CV20% had higher bioaccessibility of K, Ca, Mg, P, and Mg, compared to the control. Replacing soybean meal in broiler feed with higher concentrations of C. vugaris could improve the digestibility of meat protein and minerals.

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.

Effect of dietary incorporation of Chlorella vulgaris and CAZyme supplementation on the hepatic proteome of finishing pigs.

The combination of several factors, including an increase in world population and living standards in developing countries and world dependency on conventional crop imports drive a search for alternative feedstuffs for poultry and pig diets. This would reduce the environmental impact associated with the foreseeable increase in the demand for animal products. One of such alternatives are microalgae, a diverse group of aquatic organisms with interesting nutritional properties. Chlorella vulgaris is a green microalga with a crude protein content comparable to that of soybean meal. However, its recalcitrant cell wall prevents it from being used as a nutrient source in monogastric diets. CAZyme supplementation is a putative strategy to increase its nutritional value, aiming at disrupting the cell wall and make intracellular nutrients available for digestion. The impact of these dietary strategies on the hepatic metabolism is currently unknown. The objective of this study was to evaluate the hepatic proteome of pigs fed with 5% C. vulgaris with or without CAZyme supplementation. Microalga inclusion has affected lipid metabolism and oxidative stress. CAZyme supplementation has caused higher oxidative stress in the liver, possibly caused by the higher digestive availability and consequent hepatic oxidation of fatty acids. SIGNIFICANCE: C. vulgaris, a microalga, is a novel feedstuff that is an alternative to conventional crops such as maize and soybean meal. Its recalcitrant cell wall may cause antinutritional effects when included in monogastric diets. This can be prevented by using exogenous enzyme supplementation, namely CAZymes, aimed at degrading this cell wall during digestion. Liver proteomics was used to identify the impact of these diets in finishing pig metabolism.

Upcycling Fish By-Products into Bioactive Fish Oil: The Suitability of Microwave-Assisted Extraction.

The seafood industry is often left out of the food waste discussion, but this sector is no exception, as it generates large amounts of various by-products. This study aimed to explore the potential of the microwave-assisted extraction (MAE) technique to obtain high-quality oil from fish by-products. The independent variables, which were time (1-30 min), microwave power (50-1000 W), and solid/liquid ratio (70-120 g/L) were combined in a 20-run experimental design coupled with the response surface methodology (RSM) for process optimization. The obtained oil yield values were fitted to a quadratic equation to build the theoretical models, which were statistically validated based on statistical criteria and used to predict the optimal MAE condition. The oil yields were significantly affected by the three independent variables through linear, quadratic, and/or interactive effects. Compared to a conventional Soxhlet extraction (SE), the optimal MAE conditions allowed between 60 and 100% of oil to be recovered in less than 19 min and with less solvent consumption. The fatty acid profiles of the oils obtained through SE and optimized MAE were characterized by gas chromatography with flame ionizing detection (GC-FID) after a derivatization process. These oils were constituted mainly of health, beneficial unsaturated fatty acids, such as oleic, docosahexaenoic (DHA), linoleic, and eicosapentaenoic (EPA) acids, which were not affected (p > 0.05) by the extraction methods. Interestingly, the oils obtained through MAE showed the best microbial growth inhibition results may have been due to thermolabile compounds, preserved via this unconventional non-thermal method. The oils also exhibited anti-inflammatory effects via nitric oxide production inhibition and cytotoxic potential especially, against breast and gastric adenocarcinoma cells. However, the threshold of toxicity should be further investigated. Overall, this work emerges as a future-oriented approach to upcycling fish by-products into high-quality oils that can be used in the formulation of pet food and other products.

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.

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.