How does deterioration of different sources of astaxanthin affect antioxidant activity in rainbow trout?

 In recent years, the culture development of rainbow trout (Oncorhynchus mykiss) in China has been rapid, and the culture output has reached 39.37 million tons by 2019, which is an important cold water economic fish in China. The muscle redness value of rainbow trout is an important criterion for the market to evaluate its quality, and in the production, astaxanthin is often added exogenously to improve the color of rainbow trout muscle. Currently, the main astaxanthin used in production is synthetic astaxanthin (Ast), which accounts for 10%-20% of the total cost of feed [1]. The safety of Ast has been debated due to the presence of different stereoisomers and possible residues of synthetic intermediates. Natural sources of astaxanthin include Haematococcus pluvialis [2], Phafia rhodozyma [3], Spirulina platen-sis [4], Chlorella zofingiensis [5], etc. Natural sources of astaxanthin are more stable, and the safety of astaxanthin has been discussed. Astaxanthin from natural sources is more stable and safe to use, but there are problems such as complicated extraction process and high price.

 

Summer side marigold (Adonis aestivalis L.), also known as Fuchsia, belongs to the buttercup family (Ranunculaceae), side marigold genus (Adon- is L.), its petals are rich in carotenoids, of which the astaxanthin content of the total amount of carotenoids accounted for more than 80%, about 1% of the dry weight of the flower petal, is a high-quality natural astaxanthin source [6]. It is a high quality natural source of astaxanthin [6]. However, studies on summer marigold have mainly focused on cardiac glycosides[7] , and little research has been done on its use as a colorant.Kamata et al.[8] fed rainbow trout with diets containing 5.05% of A. fumigatus petals (AF) and 0.01% of A. fumigatus extracts (AE) (converted to astaxanthin content of 100 mg/kg) for 3 months, and high mortality rates (30%) were observed in the AF group. As a result, the AF group had a high mortality rate (30%), and the AE group increased the muscle redness value, but the amount of astaxanthin deposited in the muscle was very low, only 1.17 mg/kg, which was not up to the market requirement (6 mg/kg).

 

Red algae, belonging to Chlorophyta, Volvocales, Haematococcus, are the organisms with the highest known accumulation of naturally occurring astaxanthin, with astaxanthin accumulation up to 5% of dry weight[9] . Astaxanthin in R. rainieri usually exists in the form of esters, with a pure levulinic structure (3S,3 'S), which is one of the most active configurations in antioxidant activity [10]. However, the thick cell wall of Rhodococcus pyrenoidus prevents fish from absorbing and utilizing the carotenoids [11] and reduces the utilization of the algal meal as bait directly. Therefore, the addition of Rhodococcus aurantium as astaxanthin source to feed, should use appropriate wall breaking method, if the wall breaking method is not appropriate, incomplete wall breaking, will affect the activity and utilization of astaxanthin. The application of Rhodococcus rainbowii algal powder in aquaculture has been reported in rainbow trout [12], Atlantic salmon (Salmo salar) [13], red sea bream (Pagrus pagrus) [14], croaker (Pseudosciaena cro- cea) [15], European catfish (Silurus glanis) [16], etc., and the studies showed that Rhodococcus rainbowii has a good effect on the utilization of astaxanthin. These studies have shown that red algae have good coloring and antioxidant effects, and can improve the growth performance and enhance the immunity of fish. Currently, most of the studies on R. rainbowii are based on algal powder, but there are relatively few studies on R. rainbowii extract (HE)[2,17] , especially in the coloration of salmon and trout.

 

How effective are AF, AE and HE as natural astaxanthin sources in improving meat color and antioxidant properties of rainbow trout? How do they compare with Ast? There is no clear report. Therefore, in this experiment, Ast, AF, AE and HE were added to the feed of rainbow trout to investigate their effects on the growth performance, pigment deposition and antioxidant capacity of rainbow trout, so as to provide a theoretical basis for the rational application of natural astaxanthin sources in aquatic feeds.

 

1 Materials and Methods

1.1 Test materials

Ast, AF, AE and HE (n-butane extraction) were provided by a Guangzhou biotechnology company, and the astaxanthin contents were 10.30%, 1.54%, 2.90% and 2.26%, respectively.

 

1.2 Test feed

Five kinds of nitrogen and energy feeds were formulated, namely, the base feed and the test feed with 1.0 g/kg Ast, 6.5 g/kg AF, 3.4 g/kg AE, 4.4 g/kg HE added to the base feed, which were converted to 100 mg/kg of astaxanthin. Single-screw extruder pelleting into 2.0 mm diameter hard granular sinking feed [pelleting temperature (85 ± 5) ℃], with a blower drying oven at 40 ℃ drying to moisture content of less than 10%, sealed and stored for use. The contents of astaxanthin in these five feeds were 11.00, 95.23, 101.32, 104.25 and 93.52 mg/kg, respectively.

Table 1 Composition and nutrient levels of experimental diets (air-dry basis) g/kg

 

Project Items	control subjects Control group	Synthetic astaxanthin group Ast group	Fuchsia petal group AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Ingredients
Fish meal	250.0	250.0	250.0	250.0	250.0
Soybean meal	200.0	200.0	200.0	200.0	200.0
Soy protein concentrate	110.0	110.0	110.0	110.0	110.0
Flour	260.0	259.0	253.5	256.6	255.6
Pork meat powder	50.0	50.0	50.0	50.0	50.0
Brewers dried yeast	40.0	40.0	40.0	40.0	40.0
Fish oil	30.0	30.0	30.0	30.0	30.0
Soybean meal	30.0	30.0	30.0	30.0	30.0
Vitamin premix1)	5.0	5.0	5.0	5.0	5.0
Mineral premix2)	5.0	5.0	5.0	5.0	5.0
Calcium dihydrogen phosphate Ca(H2  PO )4 2	15.0	15.0	15.0	15.0	15.0

 

Choline chloride	5.0	5.0	5.0	5.0	5.0
Synthesis astaxanthin		1.0
Adonis aestivalis flower			6.5
Adonis aestivalis extract				3.4
Haematococcus pluvialis extract					4.4
Total	1 000.0	1 000.0	1 000.0	1 000.0	1 000.0
Nutrient levels3)
Moisture	39.3	39.0	38.0	41.5	41.7
Crude protein CP	451.3	446.2	445.7	442.9	441.1
Crude fat EE	134.8	134.7	134.5	124.4	125.1
Crude Ash Ash	85.5	84.8	86.8	85.4	85.5

 

 

 

1.3 Test fish and feeding management

The rainbow trout was purchased from Tiangui Aquaculture Farm, Dongpo District, Meishan City, Sichuan Province, China. The rainbow trout were temporarily reared for 2 weeks to acclimatize to the experimental environment. The feeding was stopped for 24 h before the formal experiment, and 375 rainbow trout of healthy and uniform size [average weight (6.28±0.07) g] were randomly assigned to 15 self-inflating recirculating glass tanks (0.60 m×0.60 m×0.50 m) with 25 trout in each tank, and there were 5 groups of 3 replicates in each group. Before the start of the culture experiment, 20 rainbow trout were stored at -20 ℃ for the initial analysis of the whole fish routine composition. During the culture period, the fish were fed twice a day (09:00 and 16:00), and the daily feeding rate was 2%~3% of the body weight, which was adjusted according to the feeding situation of the fish and the weather condition, and the feeding level of each group was basically the same, and no residual bait was preferred in each feeding. The water temperature was 13~18 ℃, the dissolved oxygen content was 6~7 mg/L, the pH was 7.24~7.78, the ammonia nitrogen content was ≤0.2 mg/L, and the nitrite content was ≤0.1 mg/L. The fish were fed for 1~2 h in the morning, and the feces at the bottom of the tank were removed by siphoning, and the water was changed twice a week, and the amount of water was 1/3 of the volume of water in the tank. The culture experiment was conducted in the Fish Nutrition Laboratory of Shanghai Ocean University for 6 weeks.

 

1.4 Sample collection

The samples were collected according to the method of Zhao et al.[18] At the end of the 2nd, 4th and 6th weeks of culture, three fish were randomly taken from each tank after 24 h of starvation and anesthetized with 100 mg/L MS-222. Blood was collected from the tail vein, centrifuged at 8000 r/min for 10 min, and the serum was stored at -80 ℃ for the determination of serum carotenoids. After blood collection, the skin of both dorsal parts was peeled off, the muscle between the lateral line and the dorsal fin was removed, and the color difference was measured, then stored with the skin and the caudal fin at -20 ℃ for determination of the astaxanthin content of the tissues. 6 weeks of the culture test was completed, and the fish were starved for 24 h. All the rainbow trout in the tanks were weighed, and the weights and numbers of tails were recorded, and 3 tails were taken from the tanks, and then anaesthetized and stored at -20 ℃ for determination of the whole fish conventional ingredients and astaxanthin content. The muscle, liver and serum of three fish were collected for the determination of antioxidant capacity, and 1.5 g of each muscle on both sides of the back of the other three fish were collected for the determination of drip loss and freezing loss, respectively.

 

1.5 Measurement indicators

1.5.1 Growth performance

The survival rate, weight gain rate and feed coefficient were calculated based on the first and last weights, number of tails and feeding rate of rainbow trout.

 

1.5.2 Feed and whole fish composition

After crushing the whole fish, the initial whole fish and the feed, the routine composition analysis was carried out with reference to AOAC (2000) as follows: moisture content was determined by drying at 105 ℃; crude protein content was determined by an automatic Kjeldahl nitrogen analyzer (2300-Auto-Analyzer, Fosstecator, Sweden); crude fat content was determined by chloroform-methanol extraction; crude ash content was determined by cauterization at 550 ℃ in a muffle furnace (SXL-1008 muffle furnace, Shanghai Jinghong Experimental Equipment Co., Ltd.). The crude fat content was determined by methanol extraction with chloroform, and the crude ash content was determined by cauterization in a muffle furnace (SXL-1008 muffle furnace, Shanghai Jinghong Experimental Equipment Co., Ltd.) at 550 ℃.

 

1.5.3 Muscle color difference values

After blood sampling and skinning, the muscle between the lateral line and the dorsal fin was taken, and the surface was dried with absorbent paper, then the probe of WSC-S colorimeter (WSC-S colorimeter, o/d light source, with luster, stability ΔY≤0.6, Shanghai Precision Scientific Instruments Co.

 

1.5.4 Astaxanthin content

The astaxanthin content of muscle and whole fish was determined by chloroform-ethanol (1:1) extraction according to the method of Zhang et al. [19], and the astaxanthin content of skin and fins was determined by dichloromethane-methanol (1:3) extraction according to the method of Song et al. [20]. The absorbance (OD) was measured and the astaxanthin content was calculated according to the standard curve of astaxanthin (Astaxanthin standard was purchased from Shanghai Jizhi Biochemical Technology Co.

 

1.5.5 Total serum carotenoids

After 0.2 mL of serum was mixed with 0.4 mL of 95% ethanol, 1 mL of n-hexane was added and centrifuged at 1,000 r/min for 5 min. The OD value of the supernatant was measured at 470 nm, and the total carotenoid content was calculated according to the standard curve of all-trans astaxanthin. The total carotenoid content was calculated from the standard curve of all-trans astaxanthin. The standard curve of all-trans astaxanthin was prepared according to Tolasa et al.

Serum carotenoid content (μg/mL) = OD value × OD value (μg/mL).

10,000/extinction coefficient E.

 

1.5.6 Muscle water holding capacity

The muscle of one side of the back of rainbow trout (1.5 g) was weighed (W1) and suspended by a thin wire in a refrigerator at 4 ℃. The muscle was removed from the refrigerator at 2, 4, and 6 h, and then weighed after gently wiping off the surface water with absorbent paper and the weight was recorded (W2). The other side of the back muscle (1.5 g) was weighed (W3), sealed in a bag and placed in the refrigerator at -20 ℃ for 24 h. After 24 h, the muscle was thawed at room temperature for 10 min, and then weighed after gently removing the surface water with absorbent paper, and the weight was recorded (W4). Drip loss and freezing loss were calculated as follows.

Drip loss (%) = 100 x [ ( W1 -W2 )/W1 ] ; Freezing loss (%) = 100 x [ ( W3 -W4 )/W3 ].

 

1.5.7 Antioxidant capacity of serum, muscle and liver

The liver and back muscle were thawed at 4 ℃, and the supernatant was made into 20% tissue homogenate with 0.9% saline, centrifuged at 2 500 r/min for 10 min. The antioxidant indices of serum, muscle and liver included total protein (TP), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and hydroxyl radical inhibition in serum, muscle and liver. The above indexes were determined according to the instructions of the kit (Nanjing Jianjian Institute of Biological Engineering), in which the total protein was determined by the Coomassie blue method, the MDA content was determined by the thiobarbituric acid (TAB) method, and the ability to inhibit hydroxyl radicals was determined by the Fenton reaction.

 

Definition of Serum and Tissue T-SOD Activity Units (U/mL): The amount of SOD per milliliter of reaction solution and per milligram of tissue protein that results in 50% inhibition of SOD in 1 mL of reaction solution is defined as one SOD activity unit (U).

 

1.6 Statistics and analysis of data

The experimental data were analyzed by one-way ANOVA using SPSS 22.0 and Tukey's method for multiple comparisons.

 

2 Results

2.1 Effects of different sources of astaxanthin on growth performance of rainbow trout (Oncorhynchus mykiss)

As shown in Table 2, after 6 weeks of culture, there were no significant differences in weight gain rate, feed coefficient and survival rate among the Con, Ast, AE and HE groups (P>0.05), while the AF group had a significantly lower weight gain rate (P<0.05) and higher feed coefficient (P<0.05) than the other groups, and the weight gain rate of the AF group was reduced by 13.5% and increased by 0.10 (P<0.05) when compared with the control group. Compared with the control group, the weight gain rate of AF group decreased by 13.5% and the feed coefficient increased by 0.10 (P<0.05).

 

2.2 Effect of different sources of astaxanthin on the conventional composition of whole rainbow trout (Oncorhynchus mykiss)

As shown in Table 3, there was no significant difference (P>0.05) in the composition of whole rainbow trout, including moisture, crude protein, crude ash and crude fat content, among the groups.

 

2.3 Effects of different sources of astaxanthin on the color difference of rainbow trout muscles

As shown in Figure 1, with the increase of culture time, the muscle brightness and redness of rainbow trout decreased and increased in all groups; the muscle brightness of Ast, AF, AE and HE groups was significantly lower than that of the control group (P<0.05), and the redness and yellowness values of Ast, AF, AE and HE groups were significantly higher than those of the control group (P<0.05) at all time points; in the 6th week, there was no significant difference (P>0.05) in the values of muscle brightness and redness of astaxanthin-added groups; the muscle yellowness values of AE and HE groups were significantly higher than those of the Ast and AF groups (P<0.05). At week 6, there was no significant difference between the muscle brightness and redness values of the astaxanthin-added groups (P>0.05), and the muscle yellowness values of the AE and HE groups were significantly higher than those of the Ast and AF groups (P<0.05).

 

Table 2 Effects of different astaxanthin sources on growth performance of rainbow trout

 

sports event	control subjects	Synthetic astaxanthin group	Fuchsia Flower Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Items	Control group	Ast group	AF group	AE group	HE group
Initial weight IBW/g	6.24±0.04	6.34±0.07	6.25±0.05	6.25±0.03	6.24±0.04
Final weight FBW/g	27.62±0.31b	27.16±0.94b	24.10±1.05a	26.19±0.92b	26.27±0.78b
Weight gain rate WGR/%	339.45±1.29b	328.54±12.54b	293.74±16.98a	322.64±10.88b	325.16±6.57b
Survival ratio/%	100	100	100	100	100
Feed coefficient FCR	0.99±0.04a	1.01±0.02a	1.09±0.02b	1.03±0.04a	1.03±0.03a

 

 

Table 3 Effects of different astaxanthin sources on whole body routine composition of rainbow trout g/kg

 

sports event	control subjects	Synthetic astaxanthin group	Fuchsia Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Items	Control group	Ast group	AF group	AE group	HE group
Moisture	715.18±2.45	708.04±9.05	716.56±6.04	710.08±5.82	713.54±7.60
Crude protein CP	161.98±4.46	162.73±6.84	160.03±3.58	162.96±4.12	164.43±6.67
Crude fat EE	75.85±3.05	81.27±9.10	82.28±5.28	76.54±3.13	79.30±4.94
Crude Ash Ash	24.23±0.97	24.81±1.13	24.76±0.51	24.75±0.85	23.95±0.63

 

 

2.4 Effects of different sources of astaxanthin on astaxanthin content and deposition rate in rainbow trout tissues

As shown in Table 4, the astaxanthin content in different tissues of all astaxanthin-added groups increased with the increase of culture time. At weeks 2, 4 and 6, the astaxanthin content in muscle, skin and caudal fin of Ast, AF, AE and HE groups was significantly higher than that of the control group (P<0.05); at week 6, the muscle astaxanthin content was the highest in the HE group, and the skin and caudal fin of the AF group were the highest; there was no significant difference in whole fish astaxanthin content and astaxanthin deposition rate between Ast, AF, AE and HE groups (P>0.05); whole fish astaxanthin content was significantly higher than that of the control group (P>0.05); and whole fish astaxanthin content was significantly higher than that of the control group (P>0.05). In terms of whole fish astaxanthin content and astaxanthin deposition rate, there was no significant difference between the Ast, AF, AE and HE groups (P>0.05), while the whole fish astaxanthin content was significantly higher than that of the control group (P<0.05), and the astaxanthin deposition rate was significantly lower than that of the control group (P<0.05).

 

Table 4 Effects of different astaxanthin sources on astaxanthin content and retention in tissue of rainbow trout

 

Project Items	Farming time Breeding time	control subjects Control group	Synthetic astaxanthin group Ast group	Fuchsia petal group AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Tissue astaxanthin content Astaxanthin		content in tissue/(mg/	kg)
Initial		0.79±0.07A	0.79±0.07A	0.79±0.07A	0.79±0.07A	0.79±0.07A
Muscle Week 2 Week		2 0.81±0.06a,A	1.85±0.30b,B	1.39±0.32b,B	2.97±0.79c,B	3.36±0.17c,B
Flesh Week 4 Week		4 0.87±0.06a,A	4.57±0.33b,C	4.56±0.25b,C	4.55±0.52b,C	4.12±0.44b,B
Week 6 Week		6 1.35±0.50a,B	4.96±0.79b,C	4.81±0.54b,C	5.20±0.91b,C	5.26±0.91b,C
Initial		2.49±0.18A	2.49±0.18A	2.49±0.18A	2.49±0.18A	2.49±0.18A
Skin Week 2 Week		2 1.73±0.48a,A	3.19±0.26b,B	3.04±0.89b,A	2.70±0.84b,A	2.95±0.68b,AB
Skin Week 4 Week		4 2.26±0.37a,A	3.90±0.15b,C	3.78±0.48b,AB	3.87±0.33b,AB	3.23±0.57b,AB
Week 6 Week		6 2.63±0.59a,A	4.02±0.25b,C	4.83±0.52c,B	4.13±0.17bc,B	3.74±0.36b,B
Initial		2.63±0.46A	2.63±0.46A	2.63±0.46A	2.63±0.46A	2.63±0.46A
Caudal Fins Week 2 Week		2 1.77±0.07a,A	10.08±1.13c,B	12.08±1.15c,B	7.11±0.95b,B	9.85±1.94c,B
Caudal fin Week 4 Week		4 1.93±0.37a,A	11.74±0.41c,B	15.35±1.80d,C	10.24±0.96b,C	12.85±0.71c,BC
Week 6 Week		6 2.73±0.52a,A	15.25±0.90b,C	17.84±0.61c,C	15.78±0.41b,D	15.82±0.56b,C
Whole body		5.59±0.27a	7.73±0.39b	7.69±0.35b	7.40±0.16b	7.21±0.06b
Astaxanthin deposition rate Astaxanthin retention/%		63.16±3.49b	10.37±0.57a	8.89±0.36a	9.71±1.67a	9.28±0.08a

 

 

2.5 Effects of different sources of astaxanthin on serum carotenoids in rainbow trout (Oncorhynchus mykiss)

As shown in Table 5, the serum carotenoid content of each group increased with the increase of incubation time, and the serum carotenoid content of Ast, AF (except week 2), AE and HE groups was significantly higher than that of the control group at weeks 2, 4 and 6 (P<0.05), and there was no significant difference between them at weeks 4 and 6 (P>0.05).

 

2.6 Effects of different sources of astaxanthin on the antioxidant capacity of muscle, liver and serum of rainbow trout (Oncorhynchus mykiss)

As shown in Table 6, the T-SOD activity and MDA content of muscle and serum of all astaxanthin-added groups were significantly lower than those of the control group (P<0.05), and the hydroxyl radical inhibition capacity of all tissues of Ast, AF, AE and HE groups was significantly higher than that of the control group (P<0.05). In the AF group, muscle antioxidant indices were not significantly different from those of the AE group (P>0.05), but the ability of liver to inhibit hydroxyl radicals was significantly lower than that of the AE group (P<0.05), and the activity of serum T-SOD was significantly higher than that of the AE group (P<0.05); there were no significant differences in the antioxidant indices of the AE and HE groups in terms of muscle, liver and serum antioxidant indices between these two groups and those of the Ast group (P>0.05).

2.7 Effect of different sources of astaxanthin on water holding capacity of rainbow trout muscle

As shown in Table 7, the drip loss of each group increased with time, and the drip loss (except for the drip loss of Ast group at 2 h) and freezing loss of Ast, AE and HE groups were significantly lower than that of the control group (P<0.05); in addition, the drip loss of AF group at 4 and 6 h was also significantly lower than that of the control group (P<0.05), while the freezing loss was not significantly different from that of the control group (P>0.05). gt;0.05).

 

 

Table 5 Effects of different astaxanthin sources on serum carotenoid contents of rainbow trout μg/mL

 

Farming time	control subjects	Synthetic astaxanthin group	Fuchsia Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Breeding time	Control group	Ast group	AF group	AE group	HE group
Initial	0.14±0.05A	0.14±0.05A	0.14±0.05A	0.14±0.05A	0.14±0.05A
Week 2	0.26±0.04a,AB	0.50±0.07bc,B	0.35±0.06ab,B	0.42±0.07bc,A	0.53±0.09c,B
Week 4	0.26±0.16a,A	1.09±0.18b,C	0.95±0.08b,C	0.90±0.08b,B	1.01±0.14b,C
Week 6	0.32±0.07a,A	1.24±0.07b,C	1.13±0.05b,D	1.18±0.17b,B	1.30±0.12b,D

 

 

Table 6 Effects of different astaxanthin sources on antioxidant ability of flesh , liver and serum of rainbow trout

 

Tissue	Project Items	control subjects Control group	Synthetic astaxanthin group Ast group	AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Muscle Flesh	Total superoxide dismutase T-SOD/(U/mg prot)	8.66±0.41b	6.45±0.66a	6.69±0.74a	6.24±0.53a	6.14±0.51a
	malondialdehyde MDA/(nmol/mg prot)	11.39±1.45b	7.04±0.91a	7.79±0.73a	6.44±0.65a	6.44±0.76a
	Ability to inhibit hydroxyl radicals
	Inhibition ability of -OH/(U/mg prot)	7.25±0.96a	12.84±1.06b	11.26±1.26b	12.23±0.78b	12.99±1.24b
Liver	Total superoxide dismutase T-SOD/(U/mg prot)	5.32±0.16b	4.44±0.17a	4.87±0.19ab	4.57±0.59a	4.58±0.03a
	malondialdehyde MDA/(nmol/mg prot) Ability to inhibit hydroxyl radicals	2.68±0.42c	1.89±0.32ab	2.41±0.50bc	1.63±0.57a	1.85±0.49ab
	Inhibition ability of -OH/(U/mg prot)	12.75±2.34a	19.02±0.98bc	16.84±1.81b	21.74±3.12c	19.70±2.63bc
	Total superoxide dismutase T-SOD/(U/mL)	249.17±8.73c	211.52±5.86ab	216.79±2.43b	204.16±7.00a	208.72±8.27ab
Serum	malondialdehyde MDA/(nmol/mL) Ability to inhibit hydroxyl radicals Inhibition ability of -OH/(U/mL)	13.11±1.05c   89.48±9.88a	4.39±0.71a   193.35±10.78b	7.48±0.91b   190.56±4.19b	5.47±1.55ab   205.60±1.05b	5.77±0.97ab   200.64±9.90b

 

 

3 Discussion

3.1 Effects of different sources of astaxanthin on growth performance of rainbow trout (Oncorhynchus mykiss)

There are different reports on the effects of Ast and HP on the growth of fish, Christiansen et al [21] and Wang Lei et al [22] showed significant improvement in the growth performance of Atlantic salmon (1.75 g) and rainbow trout by adding 50, 70 and 100 mg/kg of Ast to their diets, respectively. However, the addition of 50, 70 and 100 mg/kg Ast by Page et al. [23], Yanar et al. [24] and Amar et al. [25] had no significant effect on the growth performance of rainbow trout (Oncorhynchus mykiss), while the addition of 2.0 g/kg HE (100 mg/kg astaxanthin) by Pham et al. [2] also had a significant effect on the weight gain, specific growth rate and survival of juvenile flounder. There was no significant effect on the weight gain rate, specific growth rate and survival rate of juvenile toothfish. In the present experiment, the additions of Ast and HE also had no significant effect on the growth performance of rainbow trout, although the feed coefficients of the two additions were slightly higher than those of the control group, but there was no significant difference, which might be caused by experimental error. The effects of astaxanthin on the growth performance of fish may be related to fish species, sex, growth stage, feed composition and culture conditions.

 

Table 7 Effects of different astaxanthin sources on flesh water-holding capacity of rainbow trout %

 

Project Items	Drip loss			Thawing loss
	2 h	4 h	6 h
Control group	9.74±1.06b	18.69±1.78c	23.91±1.71c	6.19±0.47b
Synthetic astaxanthin group Ast group	8.73±0.85ab	13.71±1.04b	17.62±2.02b	4.66±0.69a
AF group	8.23±0.63ab	14.18±0.39b	16.96±1.33b	5.57±0.84ab
AE group	7.50±1.11a	10.78±1.51a	14.52±1.50a	4.80±0.55a
Erythrocystis japonicus extract group HE group	7.55±1.27a	12.33±1.61ab	17.47±1.46b	4.86±0.41a

 

 

In this experiment, although the addition of AF did not affect the survival rate of rainbow trout (100%), it reduced the growth performance of the fish, which to a certain extent reflected the negative effects of alkaloids, cardiac glycosides and other toxic and harmful substances contained in the petals on feeding and feed utilization, and indicated that AF should not be added to rainbow trout feed directly. In order to minimize or eliminate the effects of toxic substances in AF, the extraction of astaxanthin is an effective way. Cardiac glycosides are water-soluble substances, and the extraction of astaxanthin is an organic solvent extraction process, so AE is basically free of cardiac glycosides. A study showed that the addition of 0.01% of AE (converted to 100 mg/kg of astaxanthin) to feed had no significant effect on the growth performance of rainbow trout[8] , and the addition of 3.4 g/kg of AE to feed in the present experiment did not have any adverse effect on the growth performance of rainbow trout. In the future, the development and utilization of fuchsia resources should follow the path of active substance extraction.

 

3.2 Effects of different sources of astaxanthin on meat color and astaxanthin deposition in rainbow trout (Oncorhynchus mykiss)

The muscle redness value of rainbow trout is an important quality criterion, which depends on astaxanthin deposition in the body.Rahman et al. [26] significantly increased the muscle redness value of rainbow trout (Oncorhynchus mykiss) weighing 18.5 g fed 100 mg/kg Ast for 10 weeks with muscle astaxanthin content of 6.1 mg/kg.Zhang et al. [19] significantly increased the muscle redness value of rainbow trout (Oncorhynchus mykiss) weighing 101 g fed 100 mg/kg Ast for 60 d, and the astaxanthin content of 8.03 mg/kg was found in the muscle of rainbow trout. Zhang et al. [19] fed 101 g of rainbow trout with 100 mg/kg Ast feed for 60 d, which resulted in a significant increase in muscle redness value and astaxanthin content of 8.03 mg/kg, and De La Mora et al. [27] fed 80 mg/kg Ast feed for 6 weeks to rainbow trout weighing 161 g, which resulted in muscle astaxanthin content of 8.8 mg/kg. After 6 weeks of culture, the muscle redness value and astaxanthin content of both the HE and AE groups increased significantly and reached the same level as that of Ast, but the muscle astaxanthin content was low, ranging from 4.96 to 5.26 mg/kg, which might be related to the short culture period (6 weeks) and the small size of the experimental fish (initial weight of 6.28 g). In aquaculture production, the muscle of rainbow trout is usually colored at a period of time before marketing, and the small size of rainbow trout used in this experiment was mainly because it was easy to culture small-size fish under laboratory conditions, and it could be used to screen astaxanthin sources on a larger scale, thus laying a foundation for the coloration test of rainbow trout adults.

 

In addition, the addition of Ast, AF and AE to the diets significantly increased the muscle redness and yellowness values of rainbow trout in the present study. Among them, there was no significant difference in the muscle redness value and astaxanthin content between the Ast and AE groups at the end of 6 weeks of culture, which was in agreement with the results of the study conducted on rainbow trout by Kamata et al[28] . However, Kamata et al[8] found that the addition of 5.05% AF (100 mg/kg astaxanthin) to the diet did not have a significant effect on the meat color of rainbow trout, which might be due to the slight deterioration in taste of the diets after 5-6 d, which had a greater impact on the intake of rainbow trout, and resulted in poorer deposition of the pigmentation.

 

Most of the astaxanthin in AF and HP is in the form of esters [29], while that in artificial Ast is in the free state [9]. Some studies have shown that esterified astaxanthin is better absorbed by animals, which may be related to the low polarity of astaxanthin esters and their good solubility in the digestive tract[30-31] ; while Henmi et al.[32] suggested that free astaxanthin has better coloring effect than esterified astaxanthin at the same astaxanthin dosage, which may be attributed to the fact that free astaxanthin binds tightly to actin, while mono esterified astaxanthin is weakly bound to actin, while di ester is not bound at all. This may be due to the fact that free astaxanthin binds tightly to actin, monoesterified astaxanthin binds weakly to actin, and diester does not bind at all [32], resulting in poor deposition of esterified astaxanthin. However, in the present study, astaxanthin deposition in the AF, AE and HE groups was not significantly different from that in the Ast group, which is consistent with the findings of Bowen et al.[33] who fed mono-, di- and Ast-enriched feeds to rainbow trout. Schiedt[34] and Zhou[35] concluded that astaxanthin esters need to be hydrolyzed after entering the animal body in order to be absorbed and utilized. In a study by Su Fang[36] , it was found that astaxanthin from red algae Rainbow trout was de-esterified during its delivery in the rainbow trout. These studies showed that the esterification of astaxanthin did not affect its absorption and utilization by rainbow trout.

 

3.3 Effect of different sources of astaxanthin on the antioxidant capacity of rainbow trout (Oncorhynchus mykiss)

Rahman et al.[26] and Zhang et al.[19] significantly reduced serum SOD activity in rainbow trout fed diets supplemented with 50 and 100 mg/kg of astaxanthin, respectively, and muscle MDA levels were significantly reduced in rainbow trout fed diets supplemented with red fife yeast[37] and Ast[18-19] . In addition, the addition of Ast to the diets of horse fat carp (Hyphessobrycon eques Stein- dachner), blue carp (Hyphessobrycon callistus), and spotted prawn (Penaeus monodon) also significantly enhanced the antioxidant capacity of the organisms[38-40] . Meanwhile, the addition of astaxanthin to the diets of rainbow trout (Oncorhynchus mykiss) enhanced the hydroxyl radical inhibition capacity of serum, muscle and liver[18,26] . In the present experiment, the hydroxyl radical inhibition ability of rainbow trout muscle, liver and serum was significantly increased, and the T-SOD activity and MDA content were significantly reduced to the same level as that of Ast when AE and HE were added to the feeds respectively. The antioxidant property of astaxanthin is related to the unsaturated ketone and hydroxyl groups on the violet ketone ring at both ends of the astaxanthin, which have active electronic effects and can attract free radicals or provide electrons to free radicals to ultimately scavenge free radicals and improve antioxidant effects[41] .

 

Hydraulic force refers to the ability to maintain the original water when the muscle is subjected to external forces such as pressure, freezing, etc., is an important indicator of muscle quality, when the muscle is exposed to air, it will be oxidized to a certain extent, resulting in the evaporation of water from the surface of the muscle, so that the drip loss increases; when there are antioxidant substances in the muscle, such as astaxanthin, vitamin E and other antioxidant substances can reduce the degree of oxidation of the cell membrane, enhance the muscle hydraulic force [ 19 ,42]. When antioxidants such as astaxanthin and vitamin E are present in the muscle, it can reduce the oxidation of cell membrane and enhance the muscle system. In this experiment, the drip loss and freezing loss of Ast, AE and HE groups were significantly reduced compared with the control group (except for the 2-h drip loss of Ast group). It can be seen that the addition of Ast, AE and HE to the feed can prolong the shelf life of rainbow trout muscle. It was also found that the muscle drip loss at 6 h in the AE group was lower than that in the Ast and HE groups, which implies that the ability of AE to improve the shelf-life of muscle may be stronger than that of Ast and HE, and whether it is related to other antioxidant substances in AE needs to be further investigated.

 

4 CONCLUSIONS

Addition of AE and HE to the feed can effectively improve the muscle color of rainbow trout and enhance the antioxidant ability of the organism, which can achieve the same effect as that of adding Ast, but AF is not suitable to be used directly as a colorant for rainbow trout.

 

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