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Integrated Omics analysis of pig muscle metabolism under the effects of dietary Chlorella vulgaris and exogenous enzymes
Publication . Coelho, Diogo; Ribeiro, David; Osório, Hugo; Almeida, André; Prates, José António Mestre
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
Infuence of Chlorella vulgaris on growth, digestibility and gut morphology and microbiota of weaned piglet
Publication . Martins, Cátia Falcão; Trevisi, Paolo; Coelho, Diogo F.; Correa, Federico; Ribeiro, David M.; Pinho, Mario; Pestana, Jose M.; Mourato, Miguel P.; Almeida, André; Fontes, Carlos M.G.A.; Freire, Joao P. B.; Prates, José A.M
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.
Impact of dietary Chlorella vulgaris and carbohydrate-active enzymes incorporation on plasma metabolites and liver lipid composition of broilers
Publication . Coelho, Diogo Francisco Maurício; Alfaia, Cristina Maria Riscado Pereira; Assunção, José Miguel Pestana; Costa, Mónica; Pinto, Rui Manuel Amaro; Fontes, Carlos Mendes Godinho de Andrade; Lordelo, Madalena; Prates, José António Mestre
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
Effect of dietary incorporation of Chlorella vulgaris and CAZyme supplementation on the hepatic proteome of finishing pigs
Publication . Ribeiro, David M.; Coelho, Diogo; Osório, Hugo; Martins, Cátia; Freire, João; Almeida, João; Moreira, Olga; Almeida, André; Prates, José A.M.
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
Effect of dietary Spirulina (Arthrospira platensis) on the intestinal function of post-weaned piglet: An approach combining proteomics, metabolomics and histological studies
Publication . Martins, Cátia Falcão; Ribeiro, D. M.; Matzapetakis, Manolis; Pinho, Mário A.; Kuleš, Josipa; Horvatić, Anita; Guillemin, Nicolas; Eckersall, Peter David; Freire, João; Almeida, André; Prates, José A. M.
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.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/CVT-NUT/5931/2014