1. Introduction
Poultry meat is expected to represent 41% of all the protein meat sources by 2030 [
1]. Macroalgae, due to their high growth rates and richness in nutritive and bioactive compounds, have emerged as sustainable alternative resources to conventional feedstuffs (i.e., corn and soybean) that could hinder the existing food-feed competition for grains or legumes [
2]. Green macroalgae species, mostly
Ulva sp., are rich in carbohydrates (up to 65% of dry matter, DM) [
3], antioxidant chlorophylls and carotenoids [
4] They are also rich in minerals including calcium, magnesium and iron, and have a balanced sodium and potassium proportion [
5].
The protein content of
Ulva sp. is variable (4.8–41.8% DM) [
2], but with a good nutritional quality [
5]. However, lipids are present in low amounts in alga biomass (<6.6% DM), although they contain health-promoting polyunsaturated fatty acids (PUFA) [
6]. Moreover, ulvan is a sulphated soluble heteropolysaccharide, which is the major compound of the
Ulva sp. cell wall (up to 30% DM) [
7], followed by insoluble cellulose. This polymer has various bioactive functions as it can act as an anti-tumor, anti-coagulant, antihyperlipidemic, hepato-protective and immuno-stimulating agent [
7].
The inclusion of
Ulva sp. in poultry diets can influence animal growth performance and plasma biochemical parameters [
8,
9,
10,
11], with some studies showing a positive effect on the latter, such as the decrease of serum cholesterol [
8]. However, the level of macroalga used in these reports was only up to 3–3.5% [
8,
10,
11] and 6% [
9] in the feed. In fact, although there are sustainable seaweed production methods (i.e., Integrated Multitrophic Aquaculture, ITMA), macroalga cultivation technology is still in development in order to reduce its production costs and environmental impact [
2]. In addition, some studies suggested that the maximum level of
Ulva sp. incorporated in a chicken diet should not surpass 10% in the feed, due to the presence of cell wall polysaccharides that are indigestible for monogastric diets with consequent impairment of nutrient digestibility [
12,
13].
Feed enzymes appear as a solution, since Carbohydrate-Active enZymes (CAZymes) were previously supplemented in broiler chicken diets for their ability to degrade intricate matrices of carbohydrates present in microalgae [
14,
15], macroalgae such as
Laminaria digitata [
16,
17] and
Ulva sp. [
18,
19,
20] or grains [
21,
22]. Recently, an in vitro study showed the ability of a recombinant ulvan lyase from family 25 of polysaccharide lyases (PL25) to partially disrupt the
U. lactuca cell wall and, thus, release mono- and oligosaccharides and some monounsaturated fatty acids (MUFA) (e.g., 18:1
c9) [
23].
The objective of the present study was to test if
U. lactuca, when replacing 15% of corn and soybean meal, combined or not with a commercial carbohydrase mixture or recombinant ulvan lyase (PL25), would enhance a broiler’s metabolic state and relate that with results of growth performance previously discussed in a companion report [
18]. Thus, the effects of
U. lactuca, supplemented or not with CAZymes, on a broiler’s diet on plasma biochemical parameters and liver composition, including lipids, antioxidant pigments and mineral profile, were assessed.
3. Discussion
The majority of the studies carried out so far reported the dietary effect of low levels of
U. lactuca (up to 3.5% feed) on broilers’ growth and plasma biochemical parameters [
8,
19]. Previously, we reported that the dietary incorporation of high levels (15%) of
U. lactuca, especially when supplemented with ulvan lyase, slightly impaired broilers’ performance but improved meat composition through an accumulation of antioxidant carotenoids,
n-3 PUFA and total minerals [
18]. The negative effect of macroalga in animal performance was probably due to the presence of algal indigestible polysaccharides and high mineral content, which might have compromised the digestibility of feed compounds [
24]. Although not significant, the tendency for a decrease in feed intake in broilers fed with
U. lactuca-containing diets probably contributed to a reduction of average daily gain and final body weight, especially in those animals fed the diet supplemented with ulvan lyase. A numerical decrease in feed intake was also reported by Ventura et al. [
12] when feeding increasing levels of
Ulva sp. and, thus, it is possible that some algal compounds could have decreased feed palatability. Herein, we hypothesized that the addition of 15%
U. lactuca, alone or in combination with both exogenous enzymes, might improve the plasma biochemical profile and change lipid metabolism in broilers. Most of the plasma metabolites were largely affected by the experimental diets; however, the values are within the reference values reported for broilers [
25,
26]. Total lipids, TAG and VLDL-cholesterol were consistently increased in broilers that were fed
U. lactuca with the recombinant ulvan lyase, followed by reduced HDL-cholesterol levels, pointing towards a negative impact on the lipemia profile. In turn,
U. lactuca was responsible for a reduction in total and LDL-cholesterol, which agrees with the findings of some works using 1 to 3% [
27] and 2 to 6% [
9] of
U. lactuca, respectively, in broilers’ diet. It was reported higher total cholesterolemia levels (183.8 mg/dL) [
25] than the ones found in our study, probably, due to the use of chicken layers instead of broilers. Total cholesterol, TAG, HDL-cholesterol and LDL-cholesterol have been used as key indicators of lipid metabolism balance [
28,
29]. The reverse cholesterol transport is the mechanism by which the organism removes excess of cholesterol from peripheral tissues and delivers it to the liver, where it will be redistributed to other tissues or removed from the organism, being HDL-cholesterol the main lipoprotein responsible for this process, whereas LDL-cholesterol has the opposite function [
30]. Moreover, the majority of fatty acids is synthesized in the liver and transported via LDL for storage as triacylglycerols in the adipose tissue.
Broilers fed
U. lactuca with or without the recombinant ulvan lyase displayed an increased glycemic response. Even if the levels were higher than the ones reported elsewhere [
25], this small increment is thought to be devoid of clinical physiological relevance. The same rational applies for total protein. Unaffected renal function was proven by the non-variations of urea and creatinine across the experimental groups. The acute phase C-reactive protein (CRP) is produced by the liver and secreted into the blood in response to inflammation [
31]. Herein, we found that CRP was lower in broilers fed
U. lactuca, intermediate if supplemented with Rovabio
® Excel AP and recombinant ulvan lyase, and higher in the control group, pointing towards a beneficial anti-inflammatory effect of this macroalga. Notwithstanding, the highest value of CRP obtained for animals fed the control diet (0.026 mg/dL) was still below the normal averaged value found in the plasma of 35-d-old broilers (14 mg/dL) [
32]. Sodium, potassium and chloride are electrolytes positively affected by dietary
U. lactuca. While chloride and potassium presented a similar variation, with the highest levels in broilers fed
U. lactuca with recombinant ulvan lyase, sodium was increased by
U. lactuca, with or without enzymes. Potassium and sodium help the body maintaining fluid and blood volume so it can function normally [
33]. Sodium is responsible for the regulation of the membrane potential of cells and, along with potassium, is exchanged across cell membranes as part of active transport. Chloride is an anion occurring predominantly in the extracellular fluid whose serologic levels are mostly regulated by the kidney [
33]. AST, ALT and ALP were reduced by
U. lactuca, regardless of the CAZyme supplementation. These changes on aminotransferases activity are within the reference values reported for birds using both micro- and macroalga species,
Arthrospira platensis [
14] and
L. digitata [
17], respectively. GGT levels were higher in broilers fed
U. lactuca alone and supplemented with the recombinant ulvan lyase than the control diet, in resemblance to the variations observed previously by some authors [
14,
17], yet not implying any kind of liver injury or dysfunction.
Liver is the anatomical site for cholesterol synthesis and fatty acid oxidation in broilers. De novo lipogenesis, a highly regulated metabolic pathway, occurs in the liver but also in the adipose tissue [
34]. Hepatic total lipids did not change by dietary incorporation of 15%
U. lactuca, supplemented or not with exogenous feed enzymes, conversely to the plasma lipid profile. However, the addition of macroalga at this level in broilers’ diet promoted an increase of total cholesterol concentration in the liver, contrasting with our own results obtained with the dietary incorporation of 15%
L. digitata [
17]. Also, the dietary inclusion of the microalga
Chlorella vulgaris at 10% level, alone or supplemented with CAZymes, had no effect on total cholesterol content in the liver of finishing pigs [
35]. This discrepancy observed in different trials in response to total cholesterol in liver could be partly attributed to the dose and origin of the alga. Moreover, an effect of algal carotenoids, particularly xanthophylls, on increasing blood cholesterol levels might have occurred. This phenomenon was previously described in rodents [
36,
37], and then suggested when feeding the animals with extracted lipids from the brown seaweed,
Undaria pinnatifida [
38]. However, this mechanism of action is still unclear, and its occurrence in broilers lacks evidence.
Major variations were observed in hepatic
n-3 fatty acids of broilers fed
U. lactuca, supplemented or not with CAZymes. These functional fatty acids are of particular interest in animal feeds due to their anti-microbial and antioxidant properties [
39]. The enhancement of
n-3 PUFA and their antioxidant properties in the liver have been associated with the downregulation of PUFA oxidation-related genes expression and mitigation of lipid peroxidation [
40]. The percentage of α-linolenic acid (18:3
n-3), EPA and DHA increased in the liver about 2-fold, 4.5-fold and 3-fold, respectively, in broilers fed
U. lactuca, with or without CAZyme supplementation, relative to the control. Also,
U. lactuca alone or in combination with the commercial Rovabio
® increased the amounts of 18:4
n-3. The raise in 18:3
n-3 and 18:4
n-3 is in line with the composition of the diets, but EPA was only 1.3-fold higher in macroalgae-containing diets than in the control and DHA was not even present in the diets (
Table S1. Therefore, the increase of the sum
n-3 PUFA, and particularly of
n-3 long-chain PUFA, in the liver of broilers fed
U. lactuca, alone or supplemented with exogenous CAZymes, could be explained by de novo lipogenesis through the intake of the precursor 18:3
n-3 in the biosynthetic pathway of
n-3 long-chain PUFA [
41]. These findings corroborate those of Costa et al. [
17], who also documented an increase in hepatic
n-3 fatty acids, as well as a decrease of
n-6/
n-3 ratio, with the incorporation of 15%
L. digitata, with or without feed enzymes, to broiler diets. Similarly, Costa et al. [
18] reported an increase in meat
n-3 PUFA, including EPA, DPA and DHA, with the addition of
U. lactuca combined with ulvan lyase. In contrast, the combination of
U. lactuca and the recombinant CAZyme decreased
cis-MUFA, particularly 18:1
c9, which is not in agreement with the in vitro release of MUFA from
U. lactuca biomass [
23].
The impact of 15%
U. lactuca incorporation in broilers’ diet, with or without feed CAZymes, on hepatic levels of tocopherols and pigments was also explored. α-Tocopherol is the major vitamin E compound with the highest antioxidant activity [
42]. The combination of
U. lactuca and ulvan lyase reduced both α- and γ-tocopherol contents compared to the control. Recently, a decrease of hepatic α-tocopherol was reported with 15% of dietary
L. digitata and the commercial carbohydrase Rovabio
® [
17]. Similarly, a reduction of α-tocopherol was observed in the meat of broilers fed 15% of
U. lactuca [
18]. Explaining the present results remains a challenge, since literature documenting the effect of macroalgae incorporation in broiler diets on hepatic vitamin E is scarce [
17]. There is a high variability of vitamin E levels between and within macroalgae species [
2], which, therefore, compromises the amount of vitamin deposited in the liver. The chemical composition of macroalgae biomass varies not only between species, but also with location and season of cultivation and maturity of the algae [
43]. However, the concentration of α-tocopherol found in
U. lactuca biomass was 0.793 mg/kg DM, which is below the value previously reported for
Ulva intestinalis (8.8 mg/kg DM) [
44]. In contrast, a consistent increase of the sum of carotenoids, including β-carotene, and chlorophylls-
a and -
b was observed in the liver with macroalgae treatments relative to the control, which is supported by the 72 and 23-fold higher total chlorophylls and carotenoids, respectively, in
U. lactuca-containing diets. In general,
Ulva sp. are rich in carotenoids, such as β- and α-carotenes and xanthophylls, and chlorophylls [
4]. Carotenoids are natural lipophilic compounds that have been used in animal feed due to their potential to modify meat and yolk color and health benefits including antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic activities [
45,
46]. The antioxidant potential of chlorophylls is less studied, although there is evidence of their ability to scavenge peroxyl radicals with a synergistic effect with vitamin E [
46].
Regarding trace elements, the incorporation of 15%
U. lactuca in broiler diets was not enough to promote major modifications on hepatic mineral concentrations, despite their high amounts in this microalga. Only iron and manganese, as well as total microminerals, were influenced by dietary inclusion of macroalga. These trace metals play an important role in metabolic processes by acting as cofactors of antioxidant enzymes [
47]. Unfortunately, iodine and bromine were not determined in the present study, in spite of a high bioaccumulation of these elements by
U. lactuca reaching up to 45.1 mg/kg DM of iodine and 694 mg/kg DM of bromine. However, other studies reported an increase of these minerals in the meat of broilers fed 15% of
L. digitata {16] or
U. lactuca [
18], although within safe levels for human health. A similar result occurred in piglets fed 10%
L. digitata [
48]. In further studies, these minerals should be analyzed due to the well-known benefits of iodine in the general metabolism [
49] and the toxicity of bromine [
50].