اثرات ویتامین C کپسوله‌شده با نانوکیتوزان در جیره بر عملکرد رشد، کارایی خوراک و ترکیب لاشه قزل‌آلای رنگین کمان (Oncorhynchus mykiss)

Introduction: Rainbow trout (Oncorhynchus mykiss) is a premier cold-water aquaculture species and a valuable source of high-quality protein and long-chain n-3 polyunsaturated fatty acids. As trout farming intensifies, feeding strategies must sustain rapid growth and optimal feed efficiency while safeguarding carcass quality against physiological and environmental stressors. Vitamin C (ascorbic acid) is an indispensable micronutrient for teleosts because they lack L-gulonolactone oxidase, the terminal enzyme required for endogenous ascorbic acid biosynthesis. This vitamin plays a critical role in collagen synthesis, skeletal ontogeny, wound healing, and non-specific antioxidant defense; deficiency severely impairs growth and compromises immunocompetence. However, crystalline ascorbic acid exhibits poor stability during feed processing, prolonged storage, and transit through the digestive tract, resulting in diminished bioavailability. Chitosan is a biodegradable, biocompatible, and mucoadhesive biopolymer capable of encapsulating and stabilizing delicate bioactive compounds. Dietary administration of chitosan and its nanoscale derivatives have been shown to enhance growth performance, nutrient utilization, mucosal intestinal integrity, antioxidant status, and systemic immunity in aquatic species. Nanoencapsulation can effectively shield ascorbic acid against oxidative degradation while facilitating controlled gastrointestinal release. Although positive responses to nanochitosan-encapsulated vitamin C have been documented in warm-water species such as Nile tilapia and Pacific white shrimp, empirical evidence in salmonids remains scarce, as previous rainbow trout studies have primarily focused on conventional ascorbic acid or synergistic vitamin C-E regimens. Therefore, this study evaluated the effects of graded dietary inclusion levels of nanochitosan-encapsulated vitamin C (NC-VitC) on growth performance, feed utilization, and carcass proximate composition. It was hypothesized that NC-VitC supplementation would non-linearly improve growth and feed conversion efficiency, optimizing carcass quality by promoting protein deposition and attenuating lipid accumulation at intermediate inclusion levels.
Materials and Methods: NC-VitC nanoparticles were synthesized via ionic gelation using low-molecular-weight chitosan, sodium tripolyphosphate (TPP), and L-ascorbic acid. A chitosan solution (2 mg/mL; pH 4.8-5.0) was homogenized with vitamin C and cross-linked with TPP at a 5:1 chitosan: TPP mass ratio. The resulting colloidal suspension was isolated via centrifugation, washed, lyophilized, and comprehensively characterized using DLS, FE-SEM, FTIR, XRD, and EDS. A vitamin C-free basal diet meeting the nutritional requirements of rainbow trout was formulated to contain 0, 25, 50, 100, or 200 mg NC-VitC/kg diet. Juvenile rainbow trout (initial body weight: 30.0 ± 1.0 g) were randomly stocked into 15 fiberglass tanks (300 L; 20 fish/tank; three replicate tanks per dietary treatment). Following a 10-day acclimation period, fish were hand-fed to apparent satiation four times daily for 68 days under controlled rearing conditions (water temperature: 14 ± 1 °C; dissolved oxygen: 7.5 ± 0.3 mg/L; pH 7.2-7.6). Biometric indices, feed utilization parameters, survival, and Fulton’s condition factor were calculated using the tank as the experimental unit. Carcass moisture, crude protein, crude lipid, and ash were determined following standard AOAC protocols. Data were subjected to One-Way ANOVA, followed by Tukey’s post-hoc test (P<0.05) and orthogonal polynomial contrasts (quadratic regression).
Results and Discussion: Physicochemical profiling confirmed the successful synthesis and loading of NC-VitC. Characteristic alterations in FTIR spectra across the O-H/N-H stretching regions, the 1700-1500/cm band, and the polysaccharide fingerprint confirmed the chemical entrapment of ascorbic acid. XRD spectra exhibited broad, diffuse haloes lacking sharp crystalline reflections, denoting an amorphous dispersion within the polymer matrix. DLS analysis revealed an intensity-weighted hydrodynamic diameter of 6399.8 ± 671.0 nm, whereas FE-SEM depicted discrete primary spherical nanoparticles ranging from 25.79 to 47.09 nm residing within visible clusters. This variance indicates that DLS measured secondary agglomerates in liquid suspension, an expected artifact stemming from the distinct operational principles of dynamic light scattering versus electron microscopy. EDS spectra displayed dominant carbon, nitrogen, and oxygen peaks, confirming an organic, contaminant-free polysaccharide scaffold. Dietary inclusion of NC-VitC significantly influenced final body weight, total weight gain, daily weight gain, specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and condition factor, with no adverse effects on survival across treatments. Final body weight increased from 69.28 g in the unsupplemented control to 80.97 g in the 200 mg/kg group; overall weight gain escalated from 40.26 to 51.65 g, accompanied by an increase in SGR from 1.28 to 1.49%/day. The basal control diet yielded the highest FCR (1.50) and lowest PER (1.48), whereas the most favorable nutrient utilization indices were achieved at 50 and 200 mg/kg. Quadratic regression revealed non-linear plateau responses, predicting an optimum growth response at 150-170 mg NC-VitC/kg diet. This plateau phenomenon is consistent with micronutrient kinetics and tissue saturation profiles in teleosts, corroborating the biostimulatory effects of functional nano-carriers in aquatic feeds. However, because unencapsulated ascorbic acid and bare chitosan controls were not evaluated alongside, the specific carrier effects cannot be fully decoupled from the encapsulation synergy. Carcass moisture and ash contents were unaffected by dietary treatments. Conversely, whole-body crude protein peaked at 100 mg/kg (19.30%), coinciding with a minimum crude lipid level (8.80%), compared with 17.50% protein and 10.30% lipid in the control cohort. Quadratic models estimated optimal carcass composition within the 110-120 mg/kg inclusion range. Similar shifts in carcass composition have been documented following functional feed supplementation, reflecting enhanced nutrient deposition efficiency rather than verified alterations in hepatic lipogenesis. Overall, intermediate supplementation levels provided the optimal physiological balance between growth acceleration and carcass quality.
Conclusion: Dietary supplementation with nanochitosan-encapsulated vitamin C significantly improved somatic growth and feed utilization in juvenile rainbow trout under intensive culture conditions. Relative to the unsupplemented control, NC-VitC elevated final biomass, weight gain, and SGR, while concurrently lowering FCR and enhancing PER. Survival remained exceptionally high across all groups, demonstrating physiological safety. Second-order polynomial regression indicated an optimal dietary inclusion range of 150-170 mg/kg for maximal growth, whereas optimal carcass proximate composition was achieved at a lower threshold (110-120 mg/kg). Although the primary particles were nanoscale, their secondary aggregation in suspension highlights the necessity for advanced dispersion stabilization protocols in feed processing. While NC-VitC represents a potent functional aquafeed additive, parallel validation against free ascorbic acid and blank nanoparticles is recommended prior to commercial application.