1. Introduction
Soybean meal is one of the major protein sources for herbivorous fishes [
1] due to its high crude protein content and comparatively balanced amino acid composition [
2]. However, some limitations to its utilization have been encountered, drawing attention to antinutritional factors, such as the urease, soybean lectin, trypsin inhibitor and so on, which negatively affect the intestinal health and reduce the utilization nutrients [
3]. The huge gap between domestic soybean production and consumption had been filled by soybean import trade, which accounted for about 60% of the world’s annual imports [
4]. Therefore, it is of great significance to develop an alternative protein source to reduce soybean meal use.
Single-cell microalgae are an emerging non-grain protein source with great development prospects. Some studies showed that microalgae promoted growth [
5], improved immunity and antioxidant activity [
6], restrained the growth of the pathogenic bacterium and ameliorated intestine health [
7]. Among unicellular microalgae,
Chlorella vulgaris (
C. vulgaris) is well known for its high reproductive rate, short growth cycle, strong environmental tolerance, easy artificial cultivation and independence from geographical and climatic restrictions [
8]. Additionally,
C. vulgaris powder’s protein content reaches up to 50~60% and is rich in 18 kinds of amino acids. Especially, the threonine, glycine and proline contents of
C. vulgaris are higher than those of soybean meal. Moreover,
C. vulgaris powder contains a variety of beneficial substances, involving polysaccharides, pigments, vitamins, minerals and antioxidants [
9]. Some related studies have verified that
C. vulgaris promoted growth and relieved intestinal inflammation in African catfish (
Clarias gariepinus) and Atlantic salmon (
Salmo salar L.) [
10,
11]. It is worth mentioning that the polysaccharide, ferrum and aluminum accumulation in
C. vulgaris powder inhibited the growth of fish [
12] when they were fed diets with high levels of
C. vulgaris powder inclusion. However, research about
C. vulgaris powder inclusion in aquatic animals is still limited.
Grass carp (
Ctenopharyngodon idella) is widely bred for its rapid growth, tender meat, rich nutrition and moderate price [
13]. In practical production, the content of soybean meal as feed material in grass carp is 20~50%, accounting for about 21~53% of the cost. Therefore, this research aimed to explore the optimal proportion of
C. vulgaris powder inclusion and its effect on the growth performance, intestinal structure, intestinal microbiota and immunity in grass carp. The results of this research will provide a theoretical foundation for the development and utilization of
C. vulgaris powder as a novel non-grain protein source in aquatic animals.
4. Discussion
In the present study, the growth performance was observably improved in the X50 group of grass carp, which was similar to the results conducted on Nile tilapia (
Oreochromis niloticus) [
16]. This was likely because the higher content of threonine, glycine and proline in
C. vulgaris powder relative to soybean meal promoted grass carp growth, which was consistent with the results in shrimp (
Penaeus vannamei) and large yellow croaker (
Larimichthys crocea) [
17,
18]. Moreover,
C. vulgaris was rich in unsaturated fatty acids and the
Chlorella growth factor (CGF), which was beneficial to fish growth and protein efficiency [
19]. Furthermore, the antinutritional factors decreased with the decrease in the soybean meal content, which may be one of the reasons for the best growth of grass carp in the X50 group [
20]. However, the total substitution of
C. vulgaris powder with soybean meal reduced the growth performance of grass carp. This was in agreement with the result of juvenile yellow perch (
Perca flavescens) [
21] and blunt snout bream (
Megalobrama amblycephala) [
22]. This might be caused by a negative feedback system for a high concentration of active polysaccharides from high levels of
C. vulgaris powder in the body, which made the growth of fish back to a normal level or even below a normal level [
23]. In addition, ferrum and aluminum accumulation with high levels
C. vulgaris powder affected the feed utilization in the intestine by preventing the absorption of potassium, calcium, magnesium and other elements of grass carp, which was unfavorable for growing [
24]. An increased proportion of
C. vulgaris led to an amino acid imbalance, which was also one of the reasons [
25].
Additionally, the intestine plays a crucial role in the digestion and absorption of nutrients directly defining the growth of grass carp [
26]. The intestinal villi are formed by the surface epithelium and the lamina propria below it, protruding into the lumen. Higher villi indicated faster tissue turnover for permitting the renewal of the intestinal epithelium, which increased the contact area to improve nutrient absorption [
27]. In this experiment, the villus height increased when the 50% soybean meal was replaced by
C. vulgaris powder. It was the same with the study of
Spirulina and
Chlorella by-products in broiler chickens [
28,
29]. However, high levels of
C. vulgaris powder replacement of soybean meal showed intestinal villus adhesion, goblet cells hyperplasia and capillary congestion. It may be related to the content of metal elements in
C. vulgaris [
30]. Digestive enzymes facilitate the absorption of nutrients by fish [
31]. In this study, digestive enzyme activities increased when
C. vulgaris powder replaced the 50% soybean meal. This was consistent with the results of giant freshwater prawn (
Macrobrachium rosenbergii) and grey mullet (
Mugil cephalus) [
32,
33]. Supposedly, the improved growth by the
C. vulgaris powder replacement of soybean meal might be related to the increase in intestinal villi and digestive enzyme activities.
The tight junction is a complex structure formed by the interaction of multiple proteins for closing the spaces between intestinal epithelial cells [
34]. Among tight junctions between cells,
zo-1,
zo-2 and
occludin were important for restoring the intestinal mechanical barrier [
35]. In this study, the mRNA expression levels of the
zo-1,
zo-2 and
occludin genes were upregulated when the 50% soybean meal was replaced by a
C. vulgaris powder diet, demonstrating the improved integrity of the mucosal barrier structure. This result was similar to the study in mice in which that microalgae improved the intestinal structure through improving the tight junction proteins expression [
36]. Goblet cells secrete mucin and trefoil peptides to protect the mucosal structure, which covers on the surface of the intestinal epithelium to strengthen intestinal mechanical barrier function [
37]. In the study, the variation in the number of goblet cells was consistent with the expression of the tight junction protein. It was speculated that the increase in goblet cells may be related to the improvement of the mucosal structural integrity induced by
C. vulgaris.
The antioxidative defense system can scavenge oxygen free radicals constantly produced by the body in the process of metabolism in order to keep the homeostatic regulation [
38]. The X50 group’s antioxidant capacity was improved. Likewise,
C. vulgaris and
Chlorella pyrenoidosa polysaccharides contributed to the antioxidative capacity [
39,
40]. The polyphenols [
41], flavonoids [
42] and phytopigments [
43] in
C. vulgaris were able to chelate redox-active metals and accept electrons from reactive oxygen species. This may be a reason for the enhanced antioxidant activity in the
C. vulgaris, whereas the
C. vulgaris powder completely replacing the soybean meal showed an opposite trend. Similar to this experiment, antioxidant enzyme activities reduced with higher dietary levels of
Nannochloropsis [
44,
45]. The
Nrf2-keap1 signaling pathway maintains the oxidation-reduction reaction balance and metabolism of cells [
46]. In the present study,
keap1 was downregulated and
nrf2 was upregulated in the X50 group, which was in line with the changes in studies in rats [
47]. The results revealed that the
C. vulgaris powder of proper substitution levels could promote the intestinal antioxidant capacity through the activation of the
nrf2-keap1 signaling pathway.
AKP and ACP are two important hydrolytic enzymes which are involved in the metastasis and metabolism of the phosphate group. LZM dissolves cell walls to engulf bacteria and mediates protection against microbial invasion. The three enzyme activities are common indexes for assessing the nonspecific immune function of the body [
48]. A variety of evidence has demonstrated dietary
C. vulgaris to be a potent immunostimulant on juvenile rainbow trout (
Oncorhynchus mykiss) [
49], Nile tilapia [
50] and gibel carp (
Carassius auratus gibelio) [
51]. In this experiment, the ACP and LZM activities in the X50 group increased. This may be ascribed to the involvement of polysaccharides and the
Chlorella growth factor (CGF) in regulating the non-specific immune response of grass carp [
52].
The inflammatory response regulates the immune system by enhancing or inhibiting the expression levels of cytokines [
53]. The regulation of microalgae regarding the inflammatory response has been reported.
Chlorella pyrenoidosa decreased the levels of the inflammatory factor
il-6 in the serum of rats with a high-fat diet [
54]. The microalgae aqueous extracts inhibited inflammatory effects in
il-1β, stimulating Caco-2 cells [
55]. In this study, the mRNA expression levels of
il-6 and
il-1β were downregulated after the 50% soybean meal was replaced by a
C. vulgaris powder diet. This was similar to the results in the glycolytic enzyme extract of microalgae residues [
56]. The findings showed that moderate
C. vulgaris powder replacing soybean meal could relieve the intestinal inflammation of grass carp. Toll-like receptors (
tlrs) played a pivotal role in regulating the secretion of inflammatory cytokines and the activation of the inflammatory response [
57]. In the present study, the mRNA expression levels of
tlr-4,
tlr-7 and
tlr-8 were downregulated after the 50% soybean meal was replaced by a
C. vulgaris powder diet. This was consistent with the gibel carp fed with dietary
Scenedesmus ovalternus [
58]. The proinflammatory cytokines expression was closely correlated with the
tlrs, revealing that the anti-inflammation of
C. vulgaris may be partially via
tlrs signaling.
Intestinal microbial populations colonize the intestinal tract, forming a biological barrier which influences various physiological processes [
59]. The
Firmicutes are mostly beneficial bacteria, and the
Bacteroidetes can produce enterotoxins. Compared with the group X50, the abundance of the
Firmicutes decreased and the
Bacteroidetes increased in group X100. This was a reason why the growth performance of the X50 group was better than that of the X100 group [
60]. It was shown that the intestinal microbial intricacy and abundance were affected by the
C. vulgaris powder replacement of the 50% soybean meal. The same observation could be seen in the study of
Chlorella pyrenoidosa regarding the human colon [
61]. LEfse showed that the
Desulfobaccerota was remarkedly reduced in the X100 group. The
Desulfobacterium played an essential role in the butyrate metabolism [
62]. The butyrate is a key compound for both microbial and body epithelial cell growth, which improves the growth performance of Chinese striped-neck turtles (
Mauremys sinensis) with dietary supplementation [
63]. Therefore, it was inferred that the
Desulfobacterium producing butyrate metabolites may affect the growth performance of grass carp with an excessive
C. vulgaris powder replacement of soybean meal. Moreover, the
Fusobacteriota was enriched in the X100 group of intestinal microbials.
Fusobacteriota was connected with diarrhea and enteritis in newborn piglets [
64,
65]. These results indicate that the
C. vulgaris complete replacement of soybean meal could also influence the intestinal homeostasis caused by enriching conditioned pathogens. Meanwhile, at the genus level, the results displayed that
Rhodobacter increased remarkably in the X50 group of intestinal microbials.
Rhodobacter is a kind of probiotic bacteria which contributes to promoting the growth of seawater red tilapia [
66]. The
Rhodobacter sphaeroides protein maintained the intestinal health [
67]. Therefore, it was inferred that appropriate
C. vulgaris powder strengthened the intestinal biologic barrier to limit the access of pathogenic bacteria and maintain intestinal health, contributing to the growth performance of grass carp.