Nano xanthophyll improves the natural yellow color of catfish and Bagridae
This study investigated the effects of supplemental carotenoid pigments on growth and color performance in fathead catfish ( Clarias macrocephalus ). Two experiments were performed to determine the appropriate types, feeding times, and dosages of astaxanthin (As), canthaxanthin (Ca), and xanthophyll (Xa) pigments individually and in combination. In the first experiment, fish were fed one control diet (basal diet), six test diets including three diets of As, Ca and Xa with supplementation rate of 100 mg/kg respectively. and three diets combining As + Ca, As + Xa and Ca + Xa with a supplementation rate of 50 mg + 50 mg/kg. The results showed that there were no significant differences in weight gain (WG), specific growth rate (SGR), survival rate (SR) and food conversion coefficient of fish between treatments (p > 0 .05) after 6 weeks. L* (Brightness) and a* (redness) values in the Xa diet were significantly lower than in the other treatments, while b* (yellowness) was significantly higher than in the control and other treatments ( p < 0.05). These values peaked after 4 weeks and remained stable until the end of the trial. Consistently, the highest muscle carotenoid content (16.89 ± 0.60 mg/100 g) was found in fish fed the Xa diet. The Far diet was chosen for the second experiment. This experiment included four diets supplemented with Xa at rates of 25, 50, 75 and 100 mg/kg and one basal diet without Xa supplementation. The results showed no difference in SGR or SR of fish fed at different Xa levels ( p > 0.05). Fish fed the 75 mg/kg Xa diet are most popular with consumers because of the natural “yellowness” of the muscles. Therefore, the results showed that supplemented carotenoid pigments did not affect the growth performance of fish. However, the color development time of xanthophyll in 4 weeks is quite long and the concentration of 75 ppm is also quite good. Nano Xanthophyll can fix this problem with only 20 ppm and the time can be shortened by 10 days.
Bighead catfish Clarias macrocephalus is one of the most widespread and economically important native fish species in Southeast Asia [ 1 , 2 , 3 , 4 , 5 ]. The fish has become one of the most important freshwater fish species for the aquaculture industry in Vietnam [ 3 , 4 , 5 ]. The market value of this fish depends not only on meat quality and taste but also on skin and muscle pigmentation performance. Buyers and consumers alike prefer good quality bighead catfish because of its yellow skin and muscles. This is the most important characteristic of fish quality on the market. Other studies report that fish color is the first feature perceived and a decisive selection criterion, directly related to subsequent acceptance or rejection [ 6 , 7 ].
Farmed fathead catfish often have pale body color and do not have naturally attractive colors like wild fish. This problem negatively affects farmers’ profits due to falling market prices and reduced consumer demand. To overcome this problem, many applications have been implemented by farmers, such as supplementing diets with crude carotenoid ingredients such as pumpkin, sweet potato, and carrot [ 4 , 5 ]. However, these applications do not yield any successful color improvement in the fish due to the resulting fading of the color.
Similar to other species, bighead catfish are not capable of synthesizing new color pigments. Coloring pigments are related to carotenoid supplementation and should be included in the diet [ 8 , 9 ]. In nature, mackerel get their pigmentation from natural foods such as algae, crustaceans, etc. However, foods naturally change the amount of carotenoids. The carotenoid pigments, astaxanthin, canthaxanthin and xanthophyll, are widely used in aquafeeds [ 10 ]. Dietary supplementation of these pigments has been shown to enhance color and growth performance, improve survival, and reduce stress [ 11 , 12 , 13 ].
Studies have shown that incorporating synthetic carotenoid pigments or natural carotenoid-rich feed ingredients into fish diets enhances the growth and color performance of different fish species [ 12 , 14 , 15 , 16 , 17 , 18 ]. However, the optimal dietary intake levels for each carotenoid pigment (canthaxanthin, astaxanthin, xanthophyll) are species-specific. The application of canthaxanthin or astaxanthin has been shown to be effective on rainbow fillets Oncorhynchus mykiss at a dietary supplementation level of 80 mg/kg diet [ 19 ]. Other studies have found that both xanthophylls and astaxanthin (37.7–75 mg/kg diet) are effective carotenoid sources for improving skin color in large yellow croaker Larimichthys croceus [ 12 ]. However, there is currently a lack of scientific knowledge and literature on the effects of these carotenoids on the color performance of bighead catfish C. macrocephalus. Therefore, the purpose of this study was to examine the appropriate carotenoid pigmentation, supplementation dose and duration of use on the development and color expression of bighead catfish C. macrocephalus.
Materials and methods
2.1.Experimental fish
Bighead pangasius fingerlings (46.11 ± 1.19 g) were obtained from a reliable hatchery (Tam Loc hatchery, Can Tho City, Vietnam). The fish were placed in a plastic tank with gentle aeration then transferred to the wet laboratory of the Department of Aquaculture, Can Tho University. Fish were acclimatized to the experimental conditions for 2 weeks before use for testing, and they were fed a basal diet during this period.
2.2. Experimental design
Two experiments were conducted to test the appropriate dietary pigmentation for optimal growth and coloration of catfish.
In the first experiment, fish were fed seven dietary levels or combinations of astaxanthin (As), canthaxanthin (Ca) and xanthophyll (Xa) (Manufacturer BASF, Germany), including a control diet without supplementation. pigment supplementation (Basic diet only, Table 1 ). Three diets were supplemented with As, Ca, Xa at 100 mg/kg feed and 3 combined diets were supplemented with 50 mg As + 50 mg Ca/kg feed, 50 mg As + 50 mg Xa/kg feed eat. feed and 50 mg Ca + 50 mg Xa/kg feed. Overall, the diets were labeled as control, representing the As, Ca, Xa, As + Ca, As + Xa and Ca + Xa diets, respectively. These reference levels are based on carotenoid pigment levels approved for use in aquaculture feeds in the United States and European Union and range from 80 to 135 mg/kg of feed (USA: Section 21CFR and EU Code (EC, 2003b), regulation no: CD70/524/EEC).
The feed formulation and proximate analysis of the basic test diets are presented in Table 1 . This diet meets the optimal nutritional needs of catfish [ 4 , 5 ]. Experimental diets were prepared and processed according to Hien et al. [ 4 , 5 ]. Briefly, the ingredients were ground in a hammer mill to pass through a 0.8 mm mesh. All ingredients are then thoroughly mixed using a mixer. The extruded feed (2.0 mm) was then dried in an oven at 45–50 °C for approximately 8–10 h and then stored at −20 °C until use. The chemical composition of the diet was analyzed according to the AOAC method [ 20 ].
Fish were raised in a circulating experimental tank system with a stocking density of 60 fish/200 L tank (about 13.8 kg/m3). A total of 28 round composite drums (250 L/drum) were used. In total, there are seven recycling systems, each consisting of four test tanks and a 350 L filter tank. The biofilter tank contains 87.5 liters of biofilter material (RKPlast Bioelement, Brorup, Denmark, area surface of 750 m 2 /m 3 ) and a tank settlement (solid tank) of 120 L. All tested recirculating systems were prepared at least 21 days before use, during which the rate The water flow was constant at 0.8–1 L/min. The experiment lasted for 6 weeks.
The second experiment, this experiment was designed to evaluate the effects of xanthophyll and Nano Xanthophyll pigments on growth and color in catfish. Results from Experiment 1 showed that dietary xanthophyll had the best color improvement after 4 weeks. This experiment was conducted to test the appropriate dosage of xanthophyll pigment supplementation for color development and performance to meet consumer preferences. Fish were fed with 5 diets including 0.0 mg (basal diet), 25, 50, 75 and 100 mg Xa/kg of feed, hereafter referred to as control, 25 mg Xa, 50 mg Xa , 75 mg Xa and 100 mg Xa. The baseline diet ( Table 1 ) and experimental design and setup were the same as Experiment 1. All experiments were conducted in accordance with national guidelines for the protection and welfare of laboratory animals in Vietnam, Law on Veterinary Medicine, 2015 (Report number: VM5068).
2.3. Experiment management
Experimental fish were fed ad libitum, twice a day at 8:00 a.m. and 4:00 p.m. Water parameters, including water temperature, pH and dissolved oxygen, were measured twice daily at 7 a.m. and 3 p.m. parameters measured (YSI 556, USA) and maintained within the appropriate range for normal growth, i.e. temperature (27.4–31.4 °C), pH (7.6–8 ,3) and DO (4.7–5.2 mg/L). Total ammonium nitrogen (TAN) and nitrite-nitrogen were measured weekly and maintained at 0.12–0.18 mg/L; 0.1–0.2 mg/L, respectively, throughout the experimental period.
2.4. Growth performance parameters
Fish weight was measured at the beginning of the experiment and periodically every two weeks until the end of the experiment. Growth performance parameters such as weight gain (WG), specific growth rate (SGR) and survival rate (SR) were calculated using the following equations:
Survival rate (SR, %) = (Final fish count)/(Initial number of fish)× 100 (3)
in there: Wf và Wi is the final and initial wet weight of catfish; T = experimental period; Ln = standard logarithm.
2.5. Skin and muscle pigmentation
Color performance in catfish was evaluated by a combination of three different methods, colorimeter, sensory evaluation method and by testing carotenoids accumulated in fish muscle as follows:
2.5.1. Color measurement method
The color change was examined using a CR200 Colorimeter (Minolta Camera Ltd., Osaka, Japan) [ 21 , 22 ]. Fish were measured at the beginning of the experiment and every two weeks until the end of the experiment. Here, 12 fish per treatment (3 per tank) were evaluated. All measurements are expressed in the colorimetric space L*, a*, b* according to the guidelines of the Commission Internationale de l’Éclairage [ 23 ]. Each measurement is determined and recorded as standard values L*, a*, b*. L*, a*, b* values were measured at different locations on each fish (body skin, belly skin, and muscle), as shown in Figure 1 . Each measurement was repeated three times for body skin, abdominal skin, and muscle. Finally, the average color values L*, a*, b* of the experimental fish were calculated and recorded. The L* value represents brightness from black to white on a scale of 0 to 100, while the a* value represents hue from red (+) to green (-), and the b* value represents hue from yellow (+) to blue (-) in fish color measurement.
To evaluate the effectiveness of different treatments on the color of catfish, the average color value (L*, a*, b*) of bighead catfish in each treatment was Compare with the value of the control group. Comparative metrics are color differences determined by the International Commission on Illumination (CIE) in 1976, widely applied in various studies related to food color measurement and comparison. [ 24 , 25 , 26 ]. Accordingly, the average color difference between control catfish with average color c(L*, a*, b*) and treated catfish with average color c(L t *, a t *, b t *) in their color space L*, a*, b* are calculated as in Equation (4).
2.5.2. Sensory evaluation method
The appearance of catfish color was also evaluated through the sensory evaluation method of Meilgaard et al. [ 27 ]. The apparent color of the fish is scored on a scale of 1 to 9. The color of the control is given a score of 6, while the other test samples are coded and scored for comparison. The score increases more than 6 if the yellow color is darker than the control and decreases below 6 if the yellow color is lighter than the control. This evaluation method was performed by 10 independent evaluators who had normal color vision and were able to consistently detect abnormalities in the fish’s appearance.
2.5.3. Carotenoid analysis
At the end of the experiment, the accumulated carotenoid levels in the meat (muscle tissue and skin) of the fish were also analyzed according to the method described by previous studies [ 28 , 29 ]. Samples were randomly taken from three fish in a tank. After anesthesia with clove oil, each fish was carefully dissected and muscle was immediately sampled for carotenoid analysis. Briefly, meat samples were randomly collected from 12 fish per treatment (three fish per tank). Meat samples were frozen at −20 °C and then homogenized under cryogenic conditions using a grinder. Carotenoids were extracted from 5.0 g representative samples using 3 × 25 mL acetone. Extraction with acetone was performed three times until the solvent was colorless. After adding the final acetone extraction and stretching for 24 h, the samples were centrifuged at 4000 rpm for 5 min. The absorbance of the extract was recorded at 470 nm using a Hitachi U5100 spectrophotometer (Tokyo, Japan), and the carotenoid concentration (mg/mL) was determined by referring to a standard curve. The carotenoid concentration in the muscle sample (mg/100 g) was calculated based on the dilution and weight of the muscle sample.
2.6. Statistical analysis
All data were calculated as mean and standard deviation (Mean ± SD) using Microsoft Excel 2013. Two factorial analyzes of different pigments and times were used by two-way ANOVA ( IBM SPSS Statistic 21, SPSS Inc., Chicago, IL, USA ). Mean comparisons between treatments were performed using one-way ANOVA. Differences between means were assessed for significant differences using Duncan’s test at p < 0.05.
3. Result
3.1. Effects of different nutritional pigments on growth and color performance of bighead catfish
3.1.1. Growth performance, feed utilization and survival rate
The growth performance, feed utilization, and survival rate of fathead catfish fed with different pigments are shown in Table 2 . Results showed that the highest final weight (W f ) was found in fish fed the xanthophyll diet. However, statistical analysis using one-way ANOVA showed no significant difference in specific growth rate (SGR) between dietary pigment treatments ( p > 0.05). Average final weight (W f ) ranged from 56.8 to 59.5 g/fish and SGR ranged from 0.35 to 0.43%/day. High survival rates from 80.8 to 89.2% were recorded in all treatments.
3.1.2. Color performance
Statistical analysis by two-way ANOVA showed that there were significant differences in L*, a* and b* values for body skin, abdominal skin and muscle of catfish in all treatments in the week of sampling ( p < 0.001) ( Table 3 ). Fish fed the xanthophyll-supplemented diet had the yellowest coloration of the body skin, abdominal skin, and muscles compared to the other diets ( Figure 2 and Figure 3 ). The highest values for fish fed the Xa diet were achieved at week 4 and remained stable or gradually decreased at week 6. These values were relatively improved and consistent for the body skin. , abdominal skin and muscle in all diets after two weeks of feeding. testing. Among the diets, L* (Brightness) and a* (redness) values in the xanthophyll-supplemented diet (Xa diet) were significantly lower than in the other treatments, but not significantly compared to with the control treatment, while b* (yellowness) was significantly higher than the control treatment and other treatments ( p < 0.05).
The brightness values of the body skin, belly skin, and fish muscles are shown in Figure 4 . Statistical analysis by one-way ANOVA showed no significant difference in L* values of body skin, abdominal skin and muscle with respect to the control treatment in the feeding trial ( p > 0.05). The brightness of the body skin of fish fed with carotenoid-supplemented diets showed a slight increase as feeding time increased, while the brightness of the abdominal skin and muscles slightly decreased.
Abdominal skin redness values in all treatments tended to increase during the experiment, while this phenomenon was not present on the body or in muscle tissue ( Figure 5 ). Both the Ca and Ca combination groups showed lower abdominal skin a* values than the As and Xa groups at the end of the experiment. Regarding the red color of the body and belly skin, fish fed with carotenoid supplemented diets were redder than the control group.
The yellowness value (b*) of fish fed diets supplemented with carotenoid pigments tended to increase in experiments on body skin, abdominal skin and muscle tissue ( Figure 6 ). The peak body jaundice of the Xa group was significantly higher than that of the control group ( p < 0.05) after 2, 4, and 6 weeks ( Figure 6 A). However, b* values in the ventral skin of fish fed with the control treatment showed no significant difference ( p > 0.05) compared to the values of As, Ca and/or the combined groups ( Figure 6 B). In contrast to the As and As combination groups, the Xa and Ca + Xa groups exhibited significantly higher b* values in muscle tissue. Fish fed the Xa group reached the highest b* value in muscle at week 4, which was significantly higher ( p < 0.05) than the other groups ( Figure 6 C).
The sensory evaluation and accumulation of carotenoids in the muscle of fish fed with different pigmented diets are presented in Table 4 . Sensory evaluation showed that the skin and muscle color of catfish fed the Xa diet were rated highest, followed by the combination of As + Xa pigments. Statistical analysis showed a significant change in color ( p < 0.05) of fish fed the Xa diet compared to other pigmented diets. Similarly, the accumulated carotenoid level in muscle (16.89 mg/100 g) of fish fed the Xa diet was significantly higher ( p < 0.05) than the other treatments.
3.2. Effects of dietary Xanthophyll on growth and color performance of bighead catfish
3.2.1. Growth performance and survival
Growth performance and survival of fathead catfish fed different Far diets are summarized in Table 5 . Statistical analysis showed no difference in SGR or SR of fish fed different levels of Xa ( p > 0.05). However, the feed utilization of the Xa diet at 75 mg/kg of feed was more efficient than other diets.
3.2.2. Color performance
The colors of catfish fed at different levels of Xa supplementation are shown in Table 6 and Figure 7 . The L* value of the body was statistically significant (p < 0.05) compared to the control treatment after 4 weeks. In contrast, the a* value of the pigmentation treatments decreased. Furthermore, b* values for fish fed diets supplemented with 75 mg (b*: 10.6) and 100 mg (b*: 11.3) xanthophyll were significantly higher than ( p < 0 .05) other treatments.
Sensory evaluation and accumulation of carotenoids in muscle tissue of fish fed different levels of dietary xanthophylls are presented in Table 7 and Figure 8 . The highest scores were also recorded for skin and muscle color in fish fed a diet supplemented with 75 mg xanthophyll followed by a 100 mg/kg diet. Carotenoid accumulation in the muscle of fathead catfish fed diets containing 75 mg/kg feed was significantly higher ( p < 0.05) than other treatments.
Discuss
Besides color enhancement, carotenoids have been found to have many other beneficial functions in aquatic species, including improving broodstock performance [30, 31], improving disease resistance [32, 33 ] and improve growth performance [ 34 , 35 ]. In this study, growth performance and survival of catfish were not significantly affected by dietary supplementation of carotenoid pigments. Similarly, previous studies also found that dietary carotenoid supplementation did not affect the growth and survival of Atlantic salmon [36, 37, and rainbow trout [33]. , gilthead seabream [ 38 ] and red dwarf gourami Colisa lalia [ 20 ]. Similar research was conducted on the yellow cichlid Labidochromis caeruleus . Fish growth performance and survival were not affected by xanthophyll diets, and the addition of xanthophylls to feed could improve feed ratio [ 39 ]. Furthermore, the results of this study showed that feed efficiency in fish fed pigments was improved.
The color performance of fish fed dietary carotenoid supplements varies depending on the type and duration of feeding of the pigment in the diet [ 7 ]. In the present study, statistical analysis by two-way ANOVA showed clear significant differences in color performance of fathead catfish fed various species and feeding time according to dietary pigmentation. servings ( Table 3 ). Among the diets, catfish had a golden yellow color after 4 weeks of feeding on the xanthophyll diet and was significantly different from fish fed diets containing other carotenoid pigments. Color lasted for 6 weeks ( Figure 2 and Figure 3 ). Another study showed that adding astaxanthin pigment to sea bream feed took 8 weeks [38 ], while adding astaxanthin pigment to flounder feed or adding carotenoids to catfish feed hybridization requires 12 weeks [ 40 ]. The difference may be due to food intake and its utilization by the fish in this study compared to previous studies. Consistent with previous research, the ability to metabolize, absorb, and accumulate pigment in skin and muscle tissue varies by species [ 7 ].
High a* values in the presence of astaxanthin were found in this study. Fathead catfish is a species characterized by its yellow color, and when astaxanthin was added to this experiment, it lost its natural color due to increased redness. Similar results were found in yellow croaker when xanthophyll and astaxanthin were combined [ 12 ]. When astaxanthin is added, a red color appears and xanthophyll supplements appear more yellow. The b* value is yellow; Therefore, it has high value in treatments supplemented with Xa. Therefore, to improve the appearance and marketability of catfish, it is recommended to supplement xanthophyll, nano xanthophyll because it gives the skin and muscle a yellow color compared to the addition of other carotenoids.
The level of pigmentation in muscle tissue of aquatic animals when pigment is added to food depends on the species [ 7, 12 ]. In the present study, the amount of carotenoids accumulated in muscle varied according to the type of carotenoid administered ( Table 4 ). Carotenoid content was lowest in the control group, while higher carotenoid and muscle accumulation were found in treatments with dietary xanthophyll supplementation. Similar results were found in other fish species [8, 34, 35, 41], and additional synthetic colorants and/or natural sources of carotenoids also increased carotenoid levels in the muscle of Asian seabass. European [ 17 ] and Spinefoot rabbitfish [ 18 ]. Other studies reported that fish are better able to accumulate yellow pigments (lutein and zeaxanthin) than red pigments (canthaxanthin and astaxanthin) [ 14 , 42 , 43 ]. Adding astaxanthin pigment (120 mg/kg feed) to plaice after 30 days showed the clearest color and highest accumulation in fish muscle tissue.
Additionally, sensory evaluation also provided clear evidence of the yellowness of fathead catfish fed the xanthophyll diet ( Table 4 and Table 7 ). In other treatments, where only astaxanthin or canthaxanthin (orange) was added to the diet, the fish had a dark color but the yellow color was not evident. The combination of two pigments As + Xa also gives a golden yellow color with a good sensory score but not as high as Xa alone. Similarly, the combination of astaxanthin and xanthophyll in a 1:1 ratio also showed improvement in large yellow croaker [ 44 ].
The amount of pigment added to food affects the color and level of carotenoid accumulation in fish. At 25 and 50 mg Xa/kg of feed, fish color was unsatisfactory, as shown by low sensory scores and carotenoid accumulation in the meat. Application of 75 mg Xa/kg gave the best results in catfish. A similar conclusion regarding the use and ratio of Xa (75 mg) to achieve the best color was also made for yellow croaker [ 12 ].
CONCLUSION
This study examined suitable carotenoid pigments that enhance the color performance of catfish. Dietary xanthophyll showed the best results in terms of color performance after 4 weeks. Xanthophyll pigment supplementation at 75 mg/kg of feed is recommended over a 4-week feeding period to achieve golden yellow skin and muscle tissue of C. macrocephalus catfish, consistent with market demand and high consumer appreciation.
Nano xanthophyll helps increase absorption, which will help reduce the concentration used as well as speed up the gold formation process for catfish.
