Efficacy of a feed additive consisting of Saccharomyces cerevisiae DBVPG 48 SF (BioCell®) for ruminants (Mazzoleni S.p.A.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Saccharomyces cerevisiae DBVPG 48 SF (BioCell®) as a zootechnical feed additive for horses, pigs and ruminants. In a previous opinion, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive has the potential to be efficacious at the proposed conditions of use for horses, dairy ruminants and all pigs. However, the Panel was not in the position to conclude on the efficacy of BioCell® for calves, and, consequently, for other ruminants for fattening or rearing. The applicant provided three additional efficacy trials in veal calves to support the efficacy of BioCell® for ruminants for fattening or rearing. The three studies showed positive effects of the supplementation with the additive at 1.7 × 109 colony forming unit (CFU)/kg complete feed on the performance of veal calves. Considering the previously submitted studies in dairy cows and the new submitted trials, the FEEDAP Panel concluded that the additive has the potential to be efficacious for all ruminants at the proposed condition of use: 4.0 × 108 CFU/kg complete feed for dairy ruminants and 4.0 × 109 CFU/kg complete feed for ruminants for fattening and rearing.

Safety and efficacy of a feed additive consisting of 6-phytase produced by Aspergillus oryzae DSM 33737 (HiPhorius™) for all poultry, all Suidae and all fin fish (DSM Nutritional Products Ltd).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of 6-phytase produced by the genetically modified strain Aspergillus oryzae DSM 33737 (HiPhorius™ 10, 40, 20L and 50L) as a zootechnical feed additive for all poultry, all Suidae and all fin fish. The FEEDAP Panel concluded that the genetic modification of the production strain does not give rise to safety concerns. Based on the no observed adverse effect level identified in a subchronic oral toxicity study in rats, the additive was considered safe for all poultry, all Suidae and all fin fish at the proposed conditions of use. The Panel also concluded that the use of the product as a feed additive is of no concern for the consumers and the environment. The liquid formulations of the additive are not skin or eye irritants. The two solid ones are not skin irritants but are eye irritants. Owing to the lack of data, the Panel cannot conclude on the skin sensitisation of the final formulations of the additive. Due to the proteinaceous nature of the active substance (6-phytase), the additive is considered a respiratory sensitiser. The Panel concludes that the additive is efficacious when included in the diet of poultry for fattening or reared for laying/breeding, reproductive Suidae, and all fin fish. Due to the lack of sufficient data, the Panel could not conclude on the efficacy for laying and reproductive poultry and growing Suidae.

Safety and efficacy of a feed additive consisting of endo-1,4-ß-xylanase produced by Komagataella phaffii ATCC PTA-127053 (Xygest™ HT) for all Suidae (Kemin Europa N.V).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of endo-1,4-ß-xylanase produced by Komagataella phaffii ATCC PTA-127053 (Xygest™ HT) as a zootechnical feed additive (functional group: digestibility enhancers) for all Suidae at a recommended minimum level of 90,000 U/kg complete feed for pigs in the growing period and 45,000 U/kg complete feed for sows. The production strain is considered suitable for the qualified presumption of safety (QPS) approach to safety assessment. The FEEDAP Panel concluded that the additive is safe at the minimum recommended use level for all growing Suidae and all sows in the lactating phase. Xygest™ HT was considered safe for the consumers and for the environment when used in feed for all Suidae. The additive was considered not to be irritant to eyes and skin, but was considered as a dermal and respiratory sensitiser. In the absence of sufficient data, the FEEDAP Panel was not in the position to conclude on the efficacy of Xygest™ HT for all Suidae.

Efficacy of a feed additive consisting of Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866 (BIO-THREE®) for chickens for fattening and reared for laying, turkeys for fattening and reared for breeding, and all avian species for rearing/fattening or reared for laying/breeding (TOA BIOPHARMA Co., Ltd.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of Bacillus subtilis FERM BP-07462, Enterococcus lactis FERM BP-10867 and Clostridium butyricum FERM BP-10866 (BIO-THREE®) as a zootechnical feed additive to be used as a gut flora stabiliser for chickens for fattening and reared for laying, turkeys for fattening and reared for breeding, and all avian species for rearing/fattening or reared for laying/breeding. In a previous opinion, the FEEDAP Panel could not conclude on the efficacy of BIO-THREE® for the target species at the proposed conditions of use. The applicant has provided a new study in chickens for fattening as supplementary information to support the efficacy of BIO-THREE® for the target species. Considering the previously submitted studies and the new submitted trial, the Panel concluded that the additive is efficacious for chickens for fattening and reared for laying, turkeys for fattening and reared for breeding, and all avian species for rearing/fattening or reared for laying/breeding under the proposed conditions of use.

Efficacy of the feed additive consisting of 6-phytase (produced by Komagataella phaffiiCGMCC 7.19) (Nutrase P) for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds (Nutrex N.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of 6-phytase produced by Komagataella phaffii CGMCC 7.19 (Nutrase P) as a zootechnical feed additive for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds. In a previous opinion, the FEEDAP Panel concluded on the efficacy of Nutrase P for the target species at the level of 1,500 phytase units (FTU)/kg but could not conclude at the minimum recommended use level of 500 FTU/kg complete feed. The applicant has provided supplementary information, consisting in the statistical re-analysis of the long-term study assessed in the original opinion, in order to support the efficacy of the additive at the minimum recommended level of 500 FTU/kg complete feed. Considering the previously submitted studies and the re-analysis of the long-term study, the Panel concluded that the additive has a potential to be efficacious for chickens for fattening, other poultry for fattening or reared for laying and ornamental birds under the proposed conditions of use.

Safety of 41 flavouring compounds providing a herbal flavour and belonging to different chemical groups for use as feed additives in all animal species (FEFANA asbl).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety of 41 compounds to provide a Herbal flavour and belonging to different chemical groups, when used as sensory additives in feed for all animal species. Fourteen out of the 41 compounds were tested in tolerance studies in chickens for fattening, piglets, cattle for fattening and Atlantic salmon. No adverse effects were observed in the tolerance studies at 10-fold the intended level. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the 14 tested compounds were safe for these species at the proposed use level and conclusions were extrapolated to all animal species. For the remaining 27 compounds, read-across from structurally similar compounds tested in tolerance trials and belonging to the same chemical group was applied. The FEEDAP Panel concluded that these 27 compounds were safe for all animal species at the proposed use level. No safety concern would arise for the consumer and the environment from the use of the 41 compounds up to the maximum proposed use level in feed.

Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model.

The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species.

Assessment of the feed additive consisting of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) NCIMB 30083 for all animal species for the renewal of its authorisation (Chr. Hansen A/S).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the assessment of the application for renewal of authorisation of Lactiplantibacillus plantarum (previously Lactobacillus plantarum) NCIMB 30083 as a technological additive, silage additive for all animal species. The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. There is no evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concluded that the additive remains safe for all animal species, consumers and the environment under the authorised conditions of use. Regarding user safety, the additive should be considered as a respiratory sensitiser. No conclusions can be drawn on the skin sensitisation, and skin and eye irritancy potential of the additive. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Efficacy of the feed additive consisting of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) CECT 8350 and Limosilactobacillus reuteri (formerly Lactobacillus reuteri) CECT 8700 (AQ02) for suckling piglets (Aquilon Cyl S.L.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the feed additive consisting of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) CECT 8350 and Limosilactobacillus reuteri (formerly Lactobacillus reuteri) CECT 8700 (AQ02) as a zootechnical feed additive for suckling piglets. In a previous opinion the FEEDAP Panel concluded that the additive is considered safe for the target species, the consumer, and the environment. The Panel concluded that the additive should be considered a respiratory sensitiser but could not conclude on the skin/eye irritation potential or on its skin sensitisation potential. The Panel previously could not conclude on the efficacy of AQ02. The applicant has provided supplementary information to support the efficacy of the additive in suckling piglets. Based on the data provided, the FEEDAP Panel could not conclude on the efficacy of the additive.

Assessment of the safety of the feed additives acetic acid, calcium acetate and sodium diacetate for fish (FEFANA asbl).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety of acetic acid, calcium acetate and sodium diacetate as technological feed additives (preservatives) for salmonids/fish. The additives are already authorised for use for all animal species other than fish. In previous opinions, the FEEDAP Panel concluded that a maximum concentration of 2,500 mg acetic acid/kg complete feed (or 1,000 mg/L water for drinking) was safe for poultry, pigs and pet animals. Ruminants were considered to exhibit a higher tolerance. Due to lack of data for salmonids, the Panel could not conclude on the safety of acetic acid and its salts for fish. The applicant has provided supplementary information consisting in a tolerance study in Atlantic salmon (Salmo salar) and a literature search to support the safety of acetic acid in fish. Considering all the available information, the FEEDAP Panel concluded that acetic acid (and its salts by analogy) is considered safe for fish up to the maximum recommended supplementation level of 2,500 mg acetic acid/kg complete feed.

Assessment of the feed additive consisting of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) NCIMB 30084 for all animal species for the renewal of its authorisation (Chr. Hansen A/S).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on assessment of the application for renewal of authorisation Lactiplantibacillus plantarum (previously Lactobacillus) NCIMB 30084 as a technological feed additive, silage additive for all animal species. The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. There is no new evidence that would lead the FEEDAP Panel to reconsider its previous conclusions. Thus, the Panel concludes that the additive remains safe for all animal species, consumers and the environment under the authorised conditions of use. Regarding user safety, the additive should be considered as a respiratory sensitiser. No conclusions can be drawn on the skin sensitisation, and skin and eye irritancy potential of the additive. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Safety and efficacy of a feed additive consisting of l-valine produced by Corynebacterium glutamicum CGMCC 18932 for all animal species (Xinjiang Fufeng Biotechnologies Co., Ltd.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of l-valine produced by fermentation using a non-genetically modified strain of Corynebacterium glutamicum (CGMCC 18932). l-Valine is intended to be used in feed and water for drinking as a nutritional additive, functional group amino acids, their salts and analogues, for all animal species and categories. The production strain is considered suitable for the qualified presumption of safety (QPS) approach to safety assessment. The FEEDAP Panel concluded that l-valine produced by C. glutamicum CGMCC 18932 is considered safe for the target species when supplemented to the diet in appropriate amounts according to the nutritional needs of the target species. The use of l-valine produced using C. glutamicum CGMCC 18932 in animal nutrition is considered safe for the consumer and for the environment. No conclusion could be drawn on the potential of l-valine produced using C. glutamicum CGMCC 18932 to be irritant to the skin or eyes, or a dermal sensitiser due to the lack of data. The FEEDAP Panel concluded that the l-valine produced by fermentation using C. glutamicum CGMCC 18932 is an efficacious source of the essential amino acid l-valine for non-ruminant nutrition. To be as efficacious in ruminants as in non-ruminants, supplemental l-valine requires protection against ruminal degradation. The FEEDAP Panel expressed concerns on the use of amino acids in water for drinking for hygienic reasons.

Debaryomyces hansenii supplementation in low fish meal diets promotes growth, modulates microbiota and enhances intestinal condition in juvenile marine fish.

BACKGROUND: The development of a sustainable business model with social acceptance, makes necessary to develop new strategies to guarantee the growth, health, and well-being of farmed animals. Debaryomyces hansenii is a yeast species that can be used as a probiotic in aquaculture due to its capacity to i) promote cell proliferation and differentiation, ii) have immunostimulatory effects, iii) modulate gut microbiota, and/or iv) enhance the digestive function. To provide inside into the effects of D. hansenii on juveniles of gilthead seabream (Sparus aurata) condition, we integrated the evaluation of the main key performance indicators coupled with the integrative analysis of the intestine condition, through histological and microbiota state, and its transcriptomic profiling. RESULTS: After 70 days of a nutritional trial in which a diet with low levels of fishmeal (7%) was supplemented with 1.1% of D. hansenii (17.2 × 105 CFU), an increase of ca. 12% in somatic growth was observed together with an improvement in feed conversion in fish fed a yeast-supplemented diet. In terms of intestinal condition, this probiotic modulated gut microbiota without affecting the intestine cell organization, whereas an increase in the staining intensity of mucins rich in carboxylated and weakly sulphated glycoconjugates coupled with changes in the affinity for certain lectins were noted in goblet cells. Changes in microbiota were characterized by the reduction in abundance of several groups of Proteobacteria, especially those characterized as opportunistic groups. The microarrays-based transcriptomic analysis found 232 differential expressed genes in the anterior-mid intestine of S. aurata, that were mostly related to metabolic, antioxidant, immune, and symbiotic processes. CONCLUSIONS: Dietary administration of D. hansenii enhanced somatic growth and improved feed efficiency parameters, results that were coupled to an improvement of intestinal condition as histochemical and transcriptomic tools indicated. This probiotic yeast stimulated host-microbiota interactions without altering the intestinal cell organization nor generating dysbiosis, which demonstrated its safety as a feed additive. At the transcriptomic level, D. hansenii promoted metabolic pathways, mainly protein-related, sphingolipid, and thymidylate pathways, in addition to enhance antioxidant-related intestinal mechanisms, and to regulate sentinel immune processes, potentiating the defensive capacity meanwhile maintaining the homeostatic status of the intestine.

Evaluating the Functional Properties of Spray-Dried Porcine Plasma in Gilthead Seabream (Sparus aurata) Fed Low Fish Meal Diets.

Blood by-products are an untapped source of high-quality ingredients for aquafeeds, containing a broad variety of cytokines, hormones, growth factors, proteins, bioactive peptides, and amino acids. The effects of the spray-dried porcine plasma (SDPP), a type of processed animal protein on several immune parameters, were evaluated in sea bream using ex vivo and in vitro assays. In this study, fish were fed with two isoproteic, isolipidic, and isoenergetic diets: control diet (7% fish meal, FM) and SDPP diet (2% FM and 5% SDPP). At the end of the 92-days trial, those fed the SDPP diet were larger in body weight (p < 0.05) without differences in feed conversion ratio (p > 0.05). The ex vivo immune stimulation of splenocytes indicated that SDPP had a beneficial effect in promoting systemic immunity, since the surface cell marker (cd4), pro- (il-1ß), and anti-inflammatory (tgf-ß1) cytokines, and genes involved in humoral immunity (IgM) were up-regulated. The co-culture assays of skin mucus corroborated that SDPP enhanced the antibacterial capacity of mucus against V. anguillarum. In addition, main mucus biomarkers did not show significant differences, except for cortisol levels which were lower in the SDPP diet. The present study indicated that SDPP may be considered a functional ingredient in aquafeeds formulated with low FM levels.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

Impact of dietary porcine blood by-products in meagre (Argyrosomus regius) physiology, evaluated by welfare biomarkers and the antibacterial properties of the skin mucus.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

Porcine Protein Hydrolysates (PEPTEIVA®) Promote Growth and Enhance Systemic Immunity in Gilthead Sea Bream (Sparus aurata).

The effects of porcine plasma protein hydrolysate (PPH) on growth, feed efficiency, and immune responses was evaluated in Sparus aurata. Fish were fed two isoproteic (48% protein), isolipidic (17% fat), and isoenergetic diets (21.7 MJ/kg) diets, one of them containing 5% PPH at the expense of fishmeal. Both diets were tested for 92 days. A significant increase in growth was observed in fish fed the PPH diet in comparison to the control group (182.2 ± 4.4 vs. 173.8 ± 4.1 g), as well as an increase in feed intake without worsening FCR values. An ex vivo assay, with splenocytes incubated with lipopolysaccharide, was conducted to evaluate the cellular immune competence of fish. Genes involved in humoral immunity (lys, IgM), pro- (tnf-α, il-1ß), and anti-inflammatory (tgf-ß1, il10) cytokines were upregulated in the PPH group in comparison to the control group. The inclusion of PPH in diets enhanced the antibacterial capacity of skin mucus, as the co-culture of selected bacteria (E. coli, V. anguillarum, and P. anguilliseptica) with skin mucus indicated. The present results showed that the PPH in low fishmeal diets (2%) promoted growth and feed efficiency, as well as enhancing the immune response, which indicates that this is a safe and functional ingredient for aquafeeds.

Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity.

Aquaculture growth will unavoidably involve the implementation of innovative and sustainable production strategies, being functional feeds among the most promising ones. A wide spectrum of phytogenics, particularly those containing terpenes and organosulfur compounds, are increasingly studied in aquafeeds, due to their growth promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative properties. This trend relies on the importance of the mucosal barrier in the fish defense. Establishing the phytogenics' mode of action in mucosal tissues is of importance for further use and safe administration. Although the impact of phytogenics upon fish mucosal immunity has been extensively approached, most of the studies fail in addressing the mechanisms underlying their pharmacological effects. Unstandardized testing as an extended practice also questions the reproducibility and safety of such studies, limiting the use of phytogenics at commercial scale. The information presented herein provides insight on the fish mucosal immune responses to phytogenics, suggesting their mode of action, and ultimately encouraging the practice of reliable and reproducible research for novel feed additives for aquafeeds. For proper screening, characterization and optimization of their mode of action, we encourage the evaluation of purified compounds using in vitro systems before moving forward to in vivo trials. The formulation of additives with combinations of compounds previously characterized is recommended to avoid bacterial resistance. To improve the delivery of phytogenics and overcome limitations associated to compounds volatility and susceptibility to degradation, the use of encapsulation is advisable. Besides, newer approaches and dedicated methodologies are needed to elucidate the phytogenics pharmacokinetics and mode of action in depth.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.