RESULTS

Amino acid profile of fermented L. leucocephala

Fermentation of L. leucocephala significantly altered the amino acid profile. Most essential amino acids increased after fermentation, including lysine (+24.6%), leucine (+23.5%), phenylalanine (+28.7%), and valine (+16.3%). In contrast, the toxic non-protein amino acid mimosine was markedly reduced by 42.2%, indicating detoxification during fermentation (Table 1). Overall, the total essential amino acid content was higher in the fermented material than in the non-fermented material.

The inclusion of fermented L. leucocephala meal gradually altered the amino acid composition of the diets in a dose-dependent manner (Table 4). Most essential amino acids, including lysine, methionine, leucine, and threonine, showed a decreasing trend as the inclusion level increased from 0% to 20%. Lysine content decreased from 2711.91 mg/100 g in the control diet to 2132.84 mg/100 g in the 20% replacement group. A similar pattern was observed for methionine and threonine. Non-essential amino acids also showed a general decreasing trend with increasing inclusion levels, particularly glutamic acid and aspartic acid. Overall, dietary amino acid profiles were moderately affected by higher inclusion levels of fermented leaf meal.

**Antioxidant and phytochemical properties of experimental diets supplemented with fermented ** L. leucocephala

The antioxidant activity and phytochemical contents of diets supplemented with fermented L. leucocephala are shown in Table 7. Antioxidant capacity and phytochemical content increased dose-dependently with increasing levels of fermented L. leucocephala supplementation. The 20% fermented L. leucocephala diet showed the highest antioxidant activity in both DPPH and ABTS radical scavenging assays, as evidenced by the lowest IC50 values (1612.57 ± 19.87 and 305.38 ± 17.19 µg/mL, respectively). In contrast, the control diet showed the lowest activity. Likewise, FRAP values were significantly higher with increasing supplementation levels, ranging from 6.02 ± 0.50 mg FeSO₄/g extract in the control diet to 27.78 ± 2.23 mg FeSO₄/g extract in the 20% supplementation group. Phytochemical analysis also showed higher TPC and TFC after supplementation with fermented L. leucocephala. The highest TPC (11.62 ± 0.50 mg GAE/g extract) and TFC (1.364 ± 0.4 mg CE/g extract) values were observed in the 20% supplementation group compared with the control diet. Overall, the results showed that fermented L. leucocephala improved the antioxidant activity and phytochemical profiles of the experimental diets, particularly at the 20% supplementation level.

Immunological parameters

Hemocyte proliferation: The groups of _M. _rosenbergii fed diets supplemented with 10% and 20% fermented L. leucocephala had more circulating hemocytes than the control group and showed a statistically significant increase in circulating hemocytes during 6–72 h after infection with _A. _hydrophila (Figure 1A). At 96 h, only the 10% L. leucocephala group (3.22 × 10⁶ ± 1.94 × 10⁵ cells/mL) had significantly higher hemocyte circulation than the control (2.58 × 10⁶ ± 8.7 × 10⁴ cells/mL) and 20% L. leucocephala groups (2.54 × 10⁶ ± 1.77 × 10⁵ cells/mL) (p < 0.05, Figure 1A).

Hematopoietic cell division was significantly greater in the 20% L. leucocephala group than in the control group at 0 h (p < 0.001), 12–24 h (p < 0.05), and 72–120 h (p < 0.01) post-infection (Figure 1B). Although the 10% L. leucocephala group showed a trend toward a higher number of dividing cells, the difference was not statistically significant compared with the 20% L. leucocephala group and the control group (Figure 1B). Hematopoietic cells undergoing cell division are shown in Figure 1C and Supplementary Figure S1. Dividing hematopoietic cells were identified by nuclear membrane disappearance and chromosome condensation during prophase (Figure 1C, red arrow), metaphase (Figure 1C, white arrow), anaphase (Figure 1C, black arrow), and telophase (Figure 1C, blue arrow).

The expression of CHF in hemocytes at all time points was greater in the 10% and 20% L. leucocephala groups than in the control group (Figure 1D). However, a significant increase in CHF expression was observed in the 20% L. leucocephala group at 12 h (2.92 ± 1.00, p < 0.05), 24 h (7.90 ± 1.00, p < 0.001), 72 h (2.86 ± 0.36, p < 0.05), and 96 h (1.72 ± 0.37, p < 0.05) post-infection compared with the control group. In the 10% L. leucocephala group, CHF expression (5.83 ± 0.56) was significantly increased at 24 h (p < 0.01) compared with the control group (1.00 ± 0.15). However, no statistically significant difference in hemocyte CHF expression was observed between the 10% and 20% L. leucocephala groups.

The expression of CHF in the hematopoietic tissue of the 10% L. leucocephala group was significantly greater at 0 h (3.05 ± 0.60, p < 0.05), 24 h (42.24 ± 6.15, p < 0.01), and 72 h (3.46 ± 1.10, p < 0.05) post-infection compared with the control group (Figure 1E). The expression of CHF in the 20% L. leucocephala group tended to be higher than that in the control group at 0–120 h post-infection, with significant increases observed only at 12–24 h and 72 h (p < 0.05) (Figure 1E). However, CHF expression in the 20% L. leucocephala group was consistently lower than that in the 10% L. leucocephala group. PO activity: PO activity in the hemolymph of the three treatment groups of _M. _rosenbergii during _A. _hydrophila infection is shown in Figure 2A. Although _M. _rosenbergii receiving 10% and 20% L. leucocephala showed a trend toward increased PO activity during 0–96 h post-infection, significantly higher PO activity than that in the control group was observed only at 48 h (p < 0.01).

In addition, mRNA expression of proPO in hemocytes significantly increased during 0–72 h post-infection (Figure 2B). The expression of proPO in the 20% L. leucocephala group was significantly higher than that in the control group at 0–6 h (p < 0.01) and 24–72 h (p < 0.05) post-infection. In the 10% L. leucocephala group, proPO expression was elevated at 0 h but did not differ significantly from that in the control group; however, it became significantly higher at 12 h post-infection compared with the control group (p < 0.05) (Figure 2B).

Figure 1: Hemocyte counts and hematopoietic cell proliferation in Macrobrachiumrosenbergii fed fermented Leucaena leucocephala and infected with Aeromonashydrophila. (A) Total hemocyte count at different time points post-infection (n = 5). (B) Average number of mitotic hematopoietic cells at different time points post-infection (n = 5). (C) Representative hematopoietic tissue at 6 h post-infection undergoing cell division; red arrow = prophase, white arrow = metaphase, black arrow = anaphase, and blue arrow = telophase. (D) Relative mRNA expression of CHF in hemocytes. (E) Relative mRNA expression of CHF in hematopoietic tissue. Data are expressed as mean ± SD. For gene expression analyses, n = 3 (cDNA pooled from two prawns per sample). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2: Effects of dietary fermented Leucaena leucocephala on phenoloxidase activity and prophenoloxidase expression in Macrobrachiumrosenbergii infected with Aeromonashydrophila. (A) Phenoloxidase activity in hemolymph. (B) Relative mRNA expression of prophenoloxidase in hemocytes. Data are expressed as mean ± SD. For gene expression analyses, n = 3 (cDNA pooled from two prawns per sample). *p < 0.05; **p < 0.01; ***p < 0.001.

Immune-related gene expression in hemocytes and hematopoietic tissue

The mRNA expression levels of TRAF6 and Dorsal in hemocytes were significantly lower in the 10% and 20% L. leucocephala groups than in the control group during 6–48 h post-infection (Figure 3A and B). At 72 h, TRAF6 expression remained downregulated in both treatment groups; however, the differences were no longer statistically significant compared with the control group. TRAF6 expression subsequently returned to levels comparable to those of the control group at 96–120 h post-infection (Figure 3A). Similarly, Dorsal mRNA expression was downregulated in the 10% and 20% L. leucocephala groups during 6–72 h and then returned to near control levels at 96–120 h post-infection (Figure 3B).

In hematopoietic tissue, TRAF6 expression did not differ significantly among the three groups throughout the experimental period (Figure 3C). In contrast, Dorsal expression in hematopoietic tissue was significantly decreased at 6 h in the 10% L. leucocephala group (0.21 ± 0.04) compared with the 20% L. leucocephala group (0.91 ± 0.19) and the control group (1.04 ± 0.34) (p < 0.01, Figure 3D). Although a general trend toward reduced Dorsal expression was observed at other time points, significant reductions were detected only in the 20% L. leucocephala group at 12 h (0.60 ± 0.08) and 72 h (0.64 ± 0.12), and in the 10% L. leucocephala group at 96 h (0.46 ± 0.10), compared with the control group (1.09 ± 0.49) (Figure 3D).

Figure 3: Expression of Toll/Dorsal pathway genes in hemocytes and hematopoietic tissue of Macrobrachiumrosenbergii infected with Aeromonashydrophila. (A) Relative mRNA expression of TRAF6 in hemocytes. (B) Relative mRNA expression of Dorsal in hemocytes. (C) Relative mRNA expression of TRAF6 in hematopoietic tissue. (D) Relative mRNA expression of Dorsal in hematopoietic tissue. Data are expressed as mean ± SD. For gene expression analyses, n = 3 (cDNA pooled from two prawns per sample). *p < 0.05; **p< 0.01; ***p < 0.001.

Overall, IMD expression was elevated in the 10% and 20% L. leucocephala groups, although expression patterns varied across time points (Figure 4A). At 0 h, IMD expression in hemocytes did not differ significantly between the 10% L. leucocephala group and the control group. However, the 20% L. leucocephala group (7.25 ± 1.74) showed significantly higher IMD expression than both the 10% L. leucocephala group (1.46 ± 0.29) (p < 0.05) and the control group (0.99 ± 0.6) (p < 0.01) (Figure 4A). During _A. _hydrophila infection, IMD expression remained upregulated in the 10% L. leucocephala group, although the increase was not statistically significant compared with the control group. In contrast, the 20% L. leucocephala group (2.20 ± 0.01) exhibited significantly higher IMD expression at 6 h post-infection than the control group (1.14 ± 0.63) (p < 0.05) (Figure 4A). IMD expression further increased in both treatment groups during 12–24 h post-infection, with the highest expression observed at 12 h (Figure 4A). Thereafter, although IMD expression remained higher than that in the control group, statistically significant differences were observed only at 72 h in both the 10% and 20% L. leucocephala groups (Figure 4A).

Relish expression was significantly increased in the 20% L. leucocephala group at 0 h (1.40 ± 0.08) compared with the control group (1.01 ± 0.17) (p < 0.05) (Figure 4B). At 6 h after bacterial challenge, Relish expression was significantly elevated in both the 10% (11.64 ± 5.49) and 20% L. leucocephala groups (12.69 ± 4.93) compared with the control group (1.13 ± 0.67) (p < 0.05) (Figure 4B). At 12 h, significantly higher Relish expression was observed only in the 20% L. leucocephala group (1.77 ± 0.10) compared with the 10% L. leucocephala group (1.23 ± 0.11) and the control group (1.04 ± 0.35) (p < 0.05) (Figure 4B). At 24 h, Relish expression was significantly upregulated in the 10% L. leucocephala group compared with the control group (p < 0.05) (Figure 4B). Although slight increases in Relish expression were observed in both treatment groups during 48–96 h post-infection, the differences were not statistically significant. At 120 h, Relish expression was significantly higher in the 20% L. leucocephala group (3.19 ± 1.04) than in the control group (1.00 ± 0.17) (p < 0.05) (Figure 4B).

HSP70 expression was significantly higher in the 20% L. leucocephala group than in the 10% L. leucocephala and control groups throughout the 0–120 h post-infection period (p < 0.001) (Figure 4C). HSP70 expression was highest at 0 h and gradually decreased until 48 h post-infection. Thereafter, expression gradually increased during 72–96 h, and by the end of the experimental period, no significant differences in HSP70 expression were observed among the groups.

Cu/Zn-SOD mRNA expression was significantly upregulated in the 20% L. leucocephala group compared with the 10% L. leucocephala and control groups during 0–12 h and at 120 h post-infection (p < 0.001) (Figure 4D). At 24 h post-infection, Cu/Zn-SOD expression was markedly elevated in both the 10% (50.44 ± 29.87) and 20% L. leucocephala groups (40.48 ± 26.46) compared with the control group (0.92 ± 0.17), although no significant difference was observed between the two treatment groups. During 72–120 h post-infection, both L. leucocephala-supplemented groups showed a trend toward increased Cu/Zn-SOD expression relative to the control group.

In addition, the marked upregulation of Cu/Zn-SOD expression in the 20% L. leucocephala group was associated with significantly lower ABTS and DPPH IC50 values than in the control group, indicating enhanced radical-scavenging activity. This group also exhibited higher FRAP values and increased TPC and TFC compared with the control group (Table 7). These findings suggest that elevated antioxidant gene expression corresponded with improved antioxidant capacity and phytochemical content.

Diet | DPPH IC₅₀ ( μg /mL) | ABTS⁺ IC₅₀ ( μg /mL) | FRAP (mg FeSO ₄/g extract) | TPC (mg GAE/g extract) | TFC (mg CE/g extract)
0% fermented L. leucocephala | 6,436.66 ± 88.19ᵃ | 1,604.48 ± 51.51ᵃ | 6.02 ± 0.50ᵃ | 2.40 ± 0.15ᵃ | −0.134 ± 0.1ᵃ
10% fermented L. leucocephala | 2,189.23 ± 52.06ᵇ | 496.55 ± 12.64ᵇ | 15.13 ± 2.06ᵇ | 7.88 ± 0.42ᵇ | 0.882 ± 1.2ᵃ
20% fermented L. leucocephala | 1,612.57 ± 19.87ᶜ | 305.38 ± 17.19ᶜ | 27.78 ± 2.23ᶜ | 11.62 ± 0.50ᶜ | 1.364 ± 0.4ᵃ

ALF mRNA expression was generally upregulated in the 10% and 20% L. leucocephala groups compared with the control group throughout the 0–120 h post-infection period (Figure 4E). The 20% L. leucocephala group exhibited significantly higher ALF expression than both the 10% L. leucocephala and control groups at 0 h (p < 0.01), 6 h (p < 0.01), 24 h (p < 0.05 and p < 0.01, respectively), and 120 h post-infection (p < 0.01) (Figure 4E). In contrast, ALF expression in the 10% L. leucocephala group (2.55 ± 0.32) was significantly higher than that in the control group (1.00 ± 0.12) only at 24 h post-infection (p < 0.05) (Figure 4E). In contrast to ALF, C-lectin expression was generally lower in the 10% and 20% L. leucocephala groups than in the control group at all time points. However, significant reductions were observed only during 6–12 h and 72–96 h post-infection (Figure 4F).

Intestinal morphology and intestinal immune-related gene expression

The histology of _M. _rosenbergii intestines showed normal structures in all groups (Figure 5A and Supplementary Figure S2). Alterations in epithelial cell height (Figure 5B), microvilli height (Figure 5C), and muscular wall thickness (Figure 5D) were not observed.

Overall, IMD expression was higher in the 20% L. leucocephala group than in the control and 10% L. leucocephala groups throughout the experimental period (Figure 6A). Compared with the control group, IMD expression in the 20% L. leucocephala group was significantly increased at 6 h (p < 0.05), 24 h (p < 0.001), and 48 and 96 h post-infection (p < 0.01), and at 120 h post-infection (p < 0.01). However, IMD expression in the 20% L. leucocephala group did not differ significantly from that in the 10% L. leucocephala group. In the 10% L. leucocephala group, IMD expression was significantly higher than that in the control group at 48 h (p < 0.05) and 120 h post-infection (p < 0.01) (Figure 6A).

Relish expression (Figure 6B) was generally higher in the 20% L. leucocephala group than in the control and 10% L. leucocephala groups throughout the experimental period. Specifically, Relish expression in the 20% L. leucocephala group was significantly elevated (p < 0.01) at 0 h (2.34 ± 0.34) compared with the control (1.04 ± 0.32) and 10% L. leucocephala groups (1.02 ± 0.35). Significant increases were also observed at 6 h (2.37 ± 0.77) compared with the control group (1.01 ± 0.17) and 10% L. leucocephala group (1.28 ± 0.21), at 48 h (2.29 ± 0.64) compared with both the control (1.01 ± 0.19) and 10% L. leucocephala groups (1.28 ± 0.21) (p < 0.01 and p < 0.05, respectively), and at 120 h (2.74 ± 0.50) compared with the control group (1.06 ± 0.43) and 10% L. leucocephala group (1.28 ± 0.62) (p < 0.01) post-infection. Although Relish expression in the 10% L. leucocephala group tended to be higher than that in the control group, significant differences were observed only at 24 h (2.25 ± 0.66) and 72 h (1.42 ± 0.25) post-infection (p < 0.05) (Figure 6B).

Figure 4: Effects of dietary fermented Leucaena leucocephala on IMD/Relish pathway gene expression in hemocytes of Macrobrachiumrosenbergii infected with Aeromonashydrophila. (A) Relative mRNA expression of IMD. (B) Relative mRNA expression of Relish. (C) Relative mRNA expression of HSP70. (D) Relative mRNA expression of Cu/Zn-SOD. (E) Relative mRNA expression of ALF. (F) Relative mRNA expression of C-lectin. Data are expressed as mean ± SD. For gene expression analyses, n = 4 (cDNA pooled from two prawns per sample). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5: Effects of dietary fermented Leucaena leucocephala on intestinal morphology of Macrobrachiumrosenbergii infected with Aeromonashydrophila. (A) Representative hematoxylin and eosin-stained intestinal sections. (B) Epithelial cell height. (C) Microvillus height. (D) Muscular wall thickness. Data are expressed as mean ± SD (n = 5).

HSP70 expression (Figure 6C) was generally higher in the 20% L. leucocephala group than in the control and 10% L. leucocephala groups throughout the experimental period. At 0 h, HSP70 expression in the 20% L. leucocephala group (3.62 ± 0.84) was significantly higher than that in the control (1.03 ± 0.31) and 10% L. leucocephala groups (1.46 ± 0.67) (p < 0.001 and p < 0.01, respectively). At 6 h post-infection, HSP70 expression in the 20% L. leucocephala group (2.59 ± 0.98) was significantly higher than that in the control group (1.00 ± 0.14) (p < 0.05). Markedly elevated expression was also observed at 12 h (53.11 ± 20.19) and 24 h (16.77 ± 6.19) (p < 0.001), as well as at 96 h (7.33 ± 5.00) and 120 h (6.97 ± 1.03) post-infection compared with the control group (p < 0.001).

In the 10% L. leucocephala group, HSP70 expression tended to be higher than that in the control group, although significant differences were observed only at 6 h (4.36 ± 0.85) and 120 h (5.83 ± 1.73) post-infection (p < 0.001). Notably, at 6 h post-infection, HSP70 expression was higher in the 10% L. leucocephala group than in the 20% L. leucocephala group.

Cu/Zn-SOD expression (Figure 6D) was generally higher in the 20% L. leucocephala group than in the control and 10% L. leucocephala groups throughout the experimental period. Significant upregulation in the 20% L. leucocephala group was observed at 0 h (7.02 ± 2.93) compared with the control group (1.07 ± 0.49) (p < 0.001) and the 10% L. leucocephala group (3.04 ± 0.30) (p < 0.01). At 6 h post-infection, Cu/Zn-SOD expression increased further to 15.32 ± 2.38 and remained significantly higher than in the control group (p < 0.001). Significant increases were also observed at 24 h (3.25 ± 1.33) compared with both the control and 10% L. leucocephala groups (p < 0.01 and p < 0.05, respectively), and at 48 h (3.79 ± 2.28) compared with the control group (1.06 ± 0.40) (p < 0.05).

In the 10% L. leucocephala group, Cu/Zn-SOD expression also tended to be higher than that in the control group, although the increase was less pronounced than that observed in the 20% L. leucocephala group. Significant differences between the 10% L. leucocephala and control groups were detected only at 6, 24, and 96 h post-infection (p < 0.01).

ALF expression (Figure 6E) was generally higher in the 10% and 20% L. leucocephala groups than in the control group. Significant upregulation was observed at 0, 12, 48, 72, and 120 h post-infection in both treatment groups compared with the control group, although no significant differences were detected between the 10% and 20% L. leucocephala groups at these time points. At 6 h post-infection, ALF expression in the 20% L. leucocephala group (18.44 ± 16.15) also showed an increasing trend; however, the difference was not statistically significant compared with the control group. In contrast, the 10% L. leucocephala group (23.01 ± 4.31) exhibited significantly higher ALF expression than the control group (1.89 ± 1.86) at this time point.

C-lectin expression (Figure 6F) at 0 h was significantly lower in the 10% L. leucocephala (0.16 ± 0.07) and 20% L. leucocephala groups (0.24 ± 0.17) than in the control group (1.04 ± 0.33). However, at 12 h and 48 h post-infection, C-lectin expression was markedly elevated in the 20% L. leucocephala group, reaching 52.56 ± 10.57 and 11.24 ± 3.54, respectively, which were significantly higher than those in the control group (1.03 ± 0.26 and 1.04 ± 0.37, respectively) and the 10% L. leucocephala group (7.60 ± 2.58 and 1.81 ± 1.58, respectively) (p < 0.001).

C-lectin expression in the 10% L. leucocephala group also showed an increasing trend during the 12–96 h post-infection period, similar to that observed in the 20% L. leucocephala group. At 72 h, expression in the 10% L. leucocephala group (30.26 ± 6.47) was significantly higher than that in the control group (1.01 ± 0.20) and the 20% L. leucocephala group (6.27 ± 2.26) (p < 0.001). After 72 h post-infection, C-lectin expression in the 20% L. leucocephala group gradually decreased, resulting in a significant difference compared with the 10% L. leucocephala group (p < 0.05).

Mortality rates

The effect of 10% and 20% fermented L. leucocephala in fishmeal replacement on the survival of adult _M. _rosenbergii during _A. _hydrophila infection is shown in Figure 7. Survival of _M. _rosenbergii tended to be higher in the 10% and 20% L. leucocephala groups than in the control group; however, the differences were not statistically significant (p = 0.167 and p = 0.204, respectively). No dose-dependent effect was observed.