Astaxanthin was compared with β-carotene on growth performance, antioxidant capacity and gene expression of black tiger shrimp under normal and hypoxic conditions.
Two trials were conducted to determine the effects of two different carotenoid sources on Penaeus monodon, first on growth performance, and second on the immune response of the studied shrimp to challenge. in the air. In trial 1, P. monodon (average initial wet weight approximately 2.07 g) was fed 5 diets in 3 divided doses; basal diet (D1) without carotenoids; The 2 diets were formulated to provide 0.1% astaxanthin (D2) alone, combined with 0.1% astaxanthin and 1% cholesterol (D3); diet containing only 0.25% β-carotene (D4), combined with 0.25% β-carotene and 1% cholesterol (D5). Growth performance (final weight, FBW; weight gain, WG; biomass gain, BG) and survival rate of shrimp fed D3 showed the highest values. The apparent digestibility coefficient (ADC) of carotenoids in carotenoid-supplemented diets (D2–D5) was high (>90%) and cholesterol supplementation did not further improve carotenoid ADC significantly. However, cholesterol supplementation significantly enhanced tissue carotenoid retention in the astaxanthin-supplemented dietary treatments (D3 vs. D2) but not in the patent treatments. β-carotene supplemented diet (D5 vs D4). Hepatopancreatic malondialdehyde (MDA) and blood clotting times of shrimp fed carotenoid-supplemented diet (D2-D5) were lower (P < 0.05) than shrimp fed basal diet (D1). . In contrast, the total blood cell count of shrimp fed the basal diet (D1) was lower (P < 0.05) than that of shrimp fed the carotenoid-supplemented diet (D2-D5). No significant differences (P > 0.05) were found in the expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia-inducible factor-1α (HIF-1α) mRNA in the hepatopancreas of shrimp among all diets. In trial 2, shrimp were exposed to air during simulated live transport for 36 h after rearing trial 1. There were no mortality cases in all diets after 36 h of simulated live transport. . The total blood cell count of shrimp fed the basal diet and supplemented with β-carotene (D1, D4 and D5) decreased significantly (P< 0.05) after trial 2 while it did not change in Shrimp were fed diets supplemented with astaxanthin (D2 and D3). The clotting time of shrimp fed the basal diet (D1) increased significantly (P < 0.05) after trial 2 and remained unchanged in shrimp fed the carotenoid-supplemented diet (D2-D5). ). The content of MDA and protein carbonyl in the hepatopancreas of shrimp in trial 2 was a record high (P < 0.05) compared with the content in trial 1. Expression profiles of Hsp 70 mRNA and HIF-1α mRNA of The hepatopancreas of shrimp fed the basal diet was significantly lower (P < 0.05) than that of shrimp fed the other diets. In summary, all the data suggest that astaxanthin is better than β-carotene either as a pigment or as an antioxidant in commercial diets of P. monodon, and that cholesterol supplementation can enhance shrimp performance. Extremely effective of astaxanthin but not β-carotene.
The maintenance of natural skin pigmentation is of great importance from a commercial point of view, directly related to consumer acceptance or rejection and the market price of the product (Shahidi et al., 1988). In crustaceans, color is provided by carotenoids, the main pigment usually being astaxanthin in free or esterified form (Niu et al., 2012). In the exoskeletons of living crustaceans, the red-orange color of astaxanthin (3,3′-dihydroxy-β,β-carotene4,4′-dione) can be transformed to brown, green or pine blue. Through the formation of carotenoprotein complexes, the red color is revealed when cooked. Carotenoids are synthesized from geranylgeranyl diphosphate by all photosynthetic organisms, and in their biosynthetic pathway, lycopene is reported to be converted to β-carotene, which is then further converted to astaxanthin (Giuliano et al. ., 2000). Photosynthetic plants can synthesize lycopene and β-carotene while astaxanthin is a non-plant carotenoid. Black tiger shrimp are not capable of de novo biosynthesis of carotenoids but can convert β-carotene and canthaxanthin synthesized in food into astaxanthin that accumulates in the body (Boonyarataplin et al., 2001). The desired color is achieved by including astaxanthin in the food, which is the carotenoid that creates the natural color. The cost of synthetic astaxanthin is high and significantly increases feed and production costs. Therefore, it is now commercially desirable to improve the efficacy of astaxanthin or identify alternative, more cost-effective means of achieving the desired color. Castillo (1980) showed that the hermit crab Clibanarius erythropus latreille (1818) has the ability to convert β-carotene in food into astaxanthin. Boonyarataplin et al. (2001) and Linan-Cabello et al. (2002) also reported that Penaeus monodon has the ability to convert β-carotene to astaxanthin. Liao et al. (1993) reported a food preparation of Spirulina, a cyanobacterium with β-carotene as the major carotenoid, resulted in effective apricot pigmentation in P. monodon.
Furthermore, our previously unpublished data suggest that astaxanthin is approximately 2.5 times more efficient in carotenoid resource utilization than β-carotene in the growth and survival of P. monodon. This raises the possibility that if the conversion process is sufficiently efficient, feeding β-carotene or products rich in β-carotene may provide an alternative and cheaper method of achieving the desired color in crustaceans. To date, no studies have been designed to compare the effects of dietary astaxanthin and β-carotene on pigmentation performance in P. monodon, and our previously unpublished study only compared the effect of astaxanthin versus β-carotene on the growth and survival of P. monodon.
Shrimp muscle color is due to the absorption, accumulation and metabolic transformation of dietary carotenoids (Nickell and Bromage, 1998). However, the effectiveness of carotenoids on muscle pigmentation is very low, with only about 5–15% of dietary carotenoids being used for muscle pigmentation (Nickell and Bromage, 1998). Low levels of utilization may be due in part to low rates of absorption in the gastrointestinal tract, deposition in other organs, and metabolic conversion to colorless compounds that may eventually be excreted (Niu et al., 2012). Furthermore, carotenoids are lipid-soluble compounds, so the amount and type of fat present in the diet can influence carotenoid bioavailability (Regost et al., 2004). Various studies have evaluated the importance of dietary fat compared to the complete absence of fat at the time of β-carotene digestion (Dimitrov et al., 1988; Prince et al., 1991 ). Borel et al. (1998) reported that the type of fat present in the diet also affects the bioavailability of carotenoids. As network lipids, neither medium-chain triglycerides nor long-chain triglycerides can improve the bioavailability of β-carotene (Borel et al., 1998). However, Niu et al. (2012) showed that astaxanthin is better than canthaxanthin as a source of carotenoids in commercial diets of P. monodon and that cholesterol supplementation can positively enhance the effectiveness of astaxanthin and canthaxanthin.
A common measure of gastrointestinal absorption is the apparent digestibility coefficient (ADC), typical values of the ADC of astaxanthin and canthaxanthin for salmon are between 30% and 60% (Kiessling et al. events, 2003), but the higher or lower value may be higher or lower. found depending on food dose and species differences. Niu et al. (2012) showed that the ADC of astaxanthin for P. monodon was quite high (N98%), adding dietary cholesterol could not improve the ADC of astaxanthin anymore; however, dietary cholesterol supplementation significantly improved canthaxanthin digestibility from 50% to 77% for P. monodon. Astaxanthin and canthaxanthin are more polar compounds, while β-carotene belongs to non-polar carotenoid hydrocarbons (Yeum and Russell, 2002), which are two different subclasses of carotenoids, there are no data on the ADCs of astaxanthin and β-carotene was compared in shrimp and whether dietary cholesterol supplementation could enhance the effectiveness of β-carotene. Besides pigmentation, growth enhancement or improvement of broodstock performance characteristics of carotenoids, increasing attention is being directed towards determining the biological function of carotenoids in aquatic animals. Among the biological functions of carotenoids in aquaculture indicated by Niu et al. (2012), antioxidant properties may be closely related to stress resistance. Recent studies suggest that increased resistance to dissolved oxygen depletion stress (Niu et al., 2009), salinity and heat stress (Chien et al., 2003), ammonia and pathological stress ( Pan et al., 2003a,b) in penaeid shrimp is associated with an increase in dietary and body carotenoids. Additionally, air exposure has been used as a more common stressor to assess the health of crabs (Cancer pagurus) (Lorenzon et al., 2008), lobsters (Panulirus cygnus) ( Fotedar et al., 2001), Western king shrimp (Penaeus latisulcatus) and black tiger shrimp (Penaeus esculentus) (Sang and Fotedar, 2005). However, the effect of this stressor on P. monodon has not yet been studied. This study was designed to evaluate the effects of astaxanthin or β-carotene on growth, survival, pigmentation, antioxidant capacity and gene expression of P. monodon and whether cholesterol supplementation in Diet can improve the efficacy of astaxanthin and β-carotene in terms of digestibility, retention efficiency, and tissue carotenoid content.
2.Materials and methods
2.1. Diet preparation and dietary treatment
In this study, two experiments (experiment 1 and experiment 2) were conducted. Approximate formula and composition of 5 test diets (D1: basal diet; D2: 0.1% astaxanthin; D3: 0.1% astaxanthin + 1% cholesterol; D4: 0.25% β- carotene; D5: 0.25% β-carotene +1% cholesterol) with different carotenoid sources (Carophyll® Pink, 10% astaxanthin and 10% β-carotene, DSM Nutritional Products France SAS) are shown in Table 1 and 2. The diet preparation method was the same as described by Niu et al. (2012). Briefly, all dry ingredients of the test diets were weighed, combined, and mixed thoroughly to homogeneity in a Hobart-style mixer. Then the oil is added and mixed thoroughly for 5 minutes. Deionized water (40% of the dry ingredient mixture) was added and mixed for another 5 minutes. The wet mixture was placed in a single screw extruder (Institute of Chemical Engineering, South China University of Technology, Guangzhou, PR China) and extruded through a 1.2 mm die. Y2O3 was used as an inert tracer at a concentration of 0.01% in the diet. The obtained pellets were dried at 25°C with the help of an air conditioner and an electric fan. All diets were stored at −20°C until use.
2.2. Shrimp and experimental model 1
In trial 1, P. monodon postlarvae (30,000 animals), obtained from a commercial hatchery near Hongsha Bay, Sanya, Hainan Province, were stocked in a 5-ton indoor concrete tank and fed a mixed diet. Commercial diets do not contain carotenoid supplements. 1–2 months or until reaching a weight of about 2 g. Furthermore, shrimp were acclimatized to the experimental conditions and fed the current basal diet for two weeks before the experiment began. A total of 600 healthy shrimp with an initial body weight of 2.08 g were randomly assigned to 15 fiberglass tanks (500 L, 3 tanks per diet, 40 shrimp per tank). Water changes in each aquarium were regulated at approximately 1.0 L/min with a running water filtration system. Each tank is covered with a plastic mesh lid to prevent shrimp from jumping out. Shrimp are raised outdoors under steel shelters and undergo a natural light cycle (12 h light/12 h dark). During the experiment, water temperature, salinity, dissolved oxygen, and total ammonia nitrogen ranged from 26.0 to 28.0 °C, 29.0 to 32.0 g L−1, 6.8 to 7 .5 mg L−1 and 0.22 to 0.51 mg L−1, respectively.
All shrimp in each tank were initially fed 6% of total body weight daily according to Shiau et al. (1991). Feeding frequency was three times daily at 07:00, 13:00 and 21:00 and lasted for 74 days. During the feeding trial, the amount of feed provided was gradually varied and adjusted to the shrimp’s taste by checking the bottom of the tank for excess feed remaining 2 hours after feeding. In this way, overfeeding is minimized and the shrimp are fed close to fullness. Every morning and afternoon, before each feeding time, all uneaten food, feces, molting and dead shrimp are sucked out of the tank. Every two weeks shrimp from each tank were weighed and counted to assess growth and survival. During the final 14 days, feces were vacuumed from each aquarium twice daily at 08:00 and 14:00 within 30 minutes of discharge. If present, red-stained food scraps are easily sorted from the fecal fibers and removed. The stool was then rinsed with double-distilled water to avoid salt contamination and frozen at −20 °C. Samples were freeze-dried pending analysis (Merican and Shim, 1995).
2.4. Sampling and preservation
Before farming, enough shrimp were randomly collected to analyze whole body, muscle and shell carotenoids. At the end of the feeding trial, six shrimp from each tank were randomly collected for whole body, muscle, skin and blood samples. See Barbosa et al. (1999), the time from the last meal to blood sampling was within 2 hours. Blood is withdrawn by inserting a needle into the pericardial cavity through the intervening membrane between the cephalothorax and abdomen. Hemolymph samples were prepared by mixing 400 μL of isotonic NaCl solution containing 0.94 m mol/L EDTA with 100 μL of hemolymph immediately after hemolymph withdrawal. Plasma was then separated by centrifugation (12,000 rpm, Eppendorf 5810R) and carotenoid concentrations were measured immediately. Skin, hepatopancreas, and muscle samples were dissected and frozen immediately in liquid nitrogen and stored at −70°C for later analysis.
2.5. Chemical analysis
2.5.1. Analyze the composition of food samples and shrimp samples
Samples from diets and feces were lyophilized (Advantage 2.0, VirTis, USA) and then ground. Moisture, crude protein, crude lipid and crude ash of the diets and manure were determined using standard methods (AOAC, 2001). Moisture was determined by drying at 105°C for 24 h and ash was determined by muffle furnace at 550°C for 24 h. Crude protein was analyzed using the Kjeldahl method after acid digestion (1030-Auto-analyzer, Tecator, Sweden). Crude lipids were determined using the Soxtec System HT ether extraction method (Soxtec System HT6, Tecator). Dietary and fecal energy was determined using an adiabatic small-sized bomb calorimeter (adiabatic calorimeter HR-15A, Changshan, China). Yttrium (Y) was analyzed by inductively coupled plasma mass spectrometry (ICP; model: IRIS Advantage (HR), Thermo Jarrel Ash Corporation, Boston, USA) as described by Refstie et al. (1997).
2.5.2. Test for malondialdehyde and protein carbonyl
As an index of lipid peroxidation, we used TBARS formation in the acid heating reaction, which is a widely applied method previously described by Draper and Hadley (1990). Results are expressed as malondialdehyde (MDA) equivalents (nmol/mg protein). For protein carbonylation, the supernatant was incubated at room temperature for 1 h with 10 mM 2,4-dinitrophenylhydrazine (DNTP) dissolved in 2 M HCl to allow DNTP to bind to carbonyl groups (Levine et al. , 1994).
The blank was run with HCl only. Proteins were then precipitated with 6% trichloroacetic acid (TCA) and centrifuged for 10 min at 11,000 g. Protein pellets were washed three times with ethanol/ethylacetate (1:1), resuspended in 6 M guanidine hydrochloride, 50% formic acid, and incubated at 37°C until complete resuspension. Carbonyl content was measured spectrophotometrically (Synergy HT, Bioteck) in the resulting suspension at 370 nm (molar extinction coefficient 22,000 M−1 cm−1). Results are expressed as nanomoles of DNPH incorporated mg protein−1. Total protein content was determined for each sample according to Lowry et al. (1951).
2.5.3. Tissue carotenoid analysis
Carotenoid analyzes were performed in triplicate and general precautions are recommended for carotenoid isolation and handling as Barbosa et al. (1999) was followed. Diet and fecal carotenoid extraction were performed according to the method of Schierle and Hardi (1994). Carotenoid extraction from skin samples was performed according to the method of Schiedt et al. (1995). Carotenoid extraction from muscle samples was performed according to the method of Boonyarataplin et al. (2001). Plasma carotenoids were extracted using a hand-held sonic probe according to the method of Kiessling et al. (2003). The total amount of carotenoids present per 100 g of tissue was calculated from the following equation (McBeth, 1972):
mg carotenoids/100 g tissue
= (OD × vol × 103)/(E× tissue weight(g))
where E, absorption coefficient, because the crude extract often contains many types of carotenoids, an average coefficient of 2500 is often used in calculations; OD, optical density at λmax (476 nm); vol, total solution volume (ml).
2.5.4. Total blood cell count (THC) and clotting time
Total blood cell counts were performed according to established protocols for rock lobsters (Fotedar et al., 2001). The pectoral part of each shrimp from each tank was cleaned with 70% alcohol. An aliquot of 0.2 mL of hemolytic solution was drawn into a 1 mL sterile syringe containing 0.2 mL of anticoagulant (450 mM NaCl, 100 mM KCl, 10 mM EDTA-2Na, 10 mM HEPES; pH7.45 ) and dispensed into the Eppendorf tube were kept intact. on the ice. The total blood cell count of each shrimp was estimated using a hemocytometer (Neubauer, Germany) under 100x magnification, from the anticoagulant/hemolytic mixture. Cells were counted in both grids and the average value was used as the blood cell count. Total blood cell count was calculated as THC = (number of cells counted × dilution factor × 1000)/volume of mesh (0.1 mm3).
Hemolytic clotting time was determined according to the procedure described by Fotedar et al. (2001) with some modifications. The hemolymph is drawn into a sterile syringe that is dispensed into an Eppendorf tube. A 30 mL aliquot was quickly transferred to another tube and drawn into the capillary tube. The tube was inverted several times until the hemolymph stopped moving and the time was recorded as the clotting time.
2.6. Exposure to air during live transport simulation-test 2
After 60 days of culture, shrimp were transported live and simulated. From each diet group, 18 shrimp (6 from each replicate) were marked with nail polish and placed in polystyrene boxes (60 × 40 × 30 cm) for 36 h. In the box there are two gel ice bags covered with a layer of foam. Shrimp are placed on this layer of foam and then covered with another layer of foam. Then the box is covered with a lid and sealed. After 36 hours in the box, the shrimp were placed back into the culture medium for 6 hours and then tested for total blood cell count and clotting time.
After the air exposure stress tolerance test, the surviving shrimp were collected and the digestive gland samples were also removed from the sampled shrimp and immediately frozen in liquid nitrogen and then preserved. Store at -70°C. The determination of malondialdehyde and protein carbonyl was the same as mentioned above. Determination of total blood cell count (THC) and clotting time are similar to those mentioned above.
2.7. Expression analysis of Hsp70 mRNA and HIF-1α mRNA
Heat shock protein 70 (Hsp 70) is an evolutionarily highly conserved molecular chaperone that is an important part of the cellular protein folding machinery and helps protect cells from stress (Joly et al ., 2010). To understand the antioxidant molecular mechanism of astaxanthin and β-carotene, in this study, the expression levels of Hsp70 mRNA and hypoxia-inducible factor-1α (HIF-1α) were measured by time-varying quantitative PCR in shrimp fed five different experimental diets, and processed by exposure to air during simulated live transport. Total RNA was extracted using RNeasy Mini Kit (QIAGEN Cat: no. 74104) according to the manufacturer’s instructions and treated with DNase I (QIAGEN Cat: no. 79254) to remove contaminated DNA. First-strand cDNA was then synthesized based on the manufacturer’s instructions for the PrimeScript™ RT (Perfect Real-Time) Reagent Kit (TaKaRa DRR037S) using total RNA as template. The cDNA mixture was diluted to a 1:5 ratio and stored at −80 °C for subsequent real-time quantitative RT-PCR. The primers for each gene were designed based on the published P. monodon cDNA using Primer 3 software (http://primer3.wi.mit.edu/) (Table 3). All primers were manufactured by Takara Biotechnology (Dalian) Co., Ltd. (Takara Dalian) and reaction conditions were also optimized. Quantitative real-time RT-PCR was performed in a total volume of 20 μL containing 10 μL of 2× SYBR Green Real-time PCR Master Mix (TaKaRa DRR041A), 1 μL of cDNA, 0.16 μM of each primer, and 8.2 μL double -distilled water. The real-time quantitative RT-PCR program consisted of a denaturation step at 95°C for 2 min, followed by 40 cycles of amplification with 15 s denaturation at 95°C, 15 s incubation at 58°C, and extension. 30 seconds long at 72°C. Fluorescence readings were performed at the end of each cycle. To analyze Hsp70 mRNA expression levels, the comparative CT method (2 – ΔΔCT method) was used. CT for targeted amplified Hsp70 and CT for internal control β-actin were determined for each sample. The difference in CT for the target and internal control, referred to as ΔCT, was calculated to normalize the difference in the total amount of cDNA added to each reaction and the efficiency of RT-PCR. The control group is used as a reference sample, called the calibrator. The ΔCT of each sample is subtracted from the ΔCT of the calibrator, the difference being called ΔΔCT. PoGal mRNA expression level can be calculated as 2 − ΔΔCT and the value represents the n-fold difference from the calibrator.
2.8. Calculation and statistical analysis
Biological parameters used to evaluate food quality are calculated using the following equations:
Weight gain (WG) % = 100 × (average final weight−average initial weight)/ average initial weight;
Biomass gain (BG) (g) = final biomass−initial biomass;
Survival rate (%) = 100 × final number of shrimp /initial number of shrimp;
mg carotenoids=100 g tissue =(OD× volume ×103) / E × weight of tissue (g)
where E, absorption coefficient, because the crude extract often contains many types of carotenoids, the average coefficient is 2500
often used in calculations;
OD, optical density at λmax (476 nm);
vol, total solution volume (ml);
ADC = [1-(yi / yf) × (nf / ni)]
Where yi is the yttrium trioxide content in the feed, yf is the yttrium trioxide content in the feces, ni is the nutrient content in the feed and nf is the nutrient content in the feces. All data from triplicate tanks of each diet were analyzed using one-way analysis of variance and Duncan’s multiple range test. A Generalized Linear Model (GLM) procedure was used to compare single dietary treatment in terms of tissue composition, oxidative stress parameters, total blood cell count (THC), and time. coagulation time, Hsp 70 mRNA and HIF-1α mRNA levels between the two trials. The software is SPSS (Version 16.0). The difference was considered significant at P < 0.05.
3.result
3.1. Test 1
3.1.1. Biological performance
The biological performance of shrimp is shown in Table 4. Growth performance (FBW; WG; BG) and survival rate of shrimp fed D3 and D1 showed the highest and lowest values, respectively, and there was a difference. significant difference between them (P < 0.05). The feed conversion ratio (FCR) of shrimp fed D2, D3 and D5 was significantly lower (P < 0.05) than shrimp fed D1 but there was no significant difference (P>0, 05) with shrimp fed D4.
3.1.2. Whole body and muscle composition
The whole body and muscle composition of shrimp is shown in Table 5. In trial 1, no significant differences were found in whole body and muscle moisture content as well as ash content between socks. both diets (P N 0.05). The whole body protein content of shrimp fed D3 was significantly higher (P < 0.05) than shrimp fed D1 and D4 but had no significant difference from shrimp fed D2 and D5 ( P > 0.05). The whole body lipid content of shrimp fed D4 and D5 was significantly higher than that of shrimp fed other diets (P b 0.05). The muscle protein content of shrimp fed D3 and D5 was significantly higher (P < 0.05) than that of shrimp fed D1 and D4 but had no significant difference compared to shrimp fed D2 (P > 0.05). The muscle lipid content of shrimp has the same trend as the whole body lipid content of shrimp.
3.1.3. Tissue carotenoid content and body color score
Carotenoid content of whole body, muscle, skin and plasma of shrimp
are presented in Table 6. Shrimp fed diets supplemented with astaxanthin (D2–D3) showed higher whole-body, muscle, skin, and plasma carotenoid concentrations (P b 0.05) than shrimp fed basal diet and β-carotene supplementation (D1 and D4–D5). ). Furthermore, cholesterol supplementation significantly increased carotenoid concentrations (Pb 0.05) in the whole body, muscle, shell, and plasma of shrimp in diets supplemented with astaxanthin (D3 vs. D2). However, cholesterol supplementation only significantly increased carotenoid concentrations (P b 0.05) in plasma but not in whole body, muscle, and shell shrimp in diets supplemented with β β-carotene (D5 vs. D4). At the end of the feeding trial, the red color of boiled shrimp in treatment D3 was higher than the other treatments and a light yellow color was observed in the basic diet treatment (D1) (Table 7).
3.1.4. Apparent digestibility of ingredients and dietary energy
The ADCs of dry matter, protein, lipids, carotenoids and energy of the experimental diets for young P. monodon are presented in Table 8. No differences were found in the ADCs of dry matter, protein, energy and carotenoids between the treatments. experimental diet for juvenile P. monodon (PN 0.05). Carotenoid ADC in carotenoid-supplemented diets (D2–D5) was high (N90%), and cholesterol supplementation did not further improve carotenoid ADC significantly. The ADC of lipids in the basal diet was significantly lower (P b 0.05) than the lipids in the other diets, cholesterol supplementation improved the ADC of lipids in the dietary treatments β carotene supplementation (D4 vs. D5) but no improvement in treatments with dietary astaxanthin supplementation (D3 vs. D2).
3.1.5. Effectively maintains tissue carotenoids
Carotenoid retention efficiency in tissues (whole body, muscle and shell) is shown in Table 9. Maximum carotenoid retention efficiency in tissues was observed in shrimp fed D3, unlike shrimp fed other diets (P < 0.05), followed by shrimp fed D2. Cholesterol supplementation improved tissue carotenoid retention in astaxanthin-supplemented dietary treatments (D3 vs. D2) but not in β-supplemented dietary treatments. β-carotene (D4 vs. D5).
3.1.6. Immune parameters
Antioxidant capacity (total blood cell count and lymph clotting time), oxidative stress parameters (malondialdehyde and protein carbonyl content of the digestive gland) and gene expression (Hsp 70 mRNA and HIF-1α mRNA of shrimp is shown in Table 10. In trial 1, the total blood cell counts of shrimp fed the basal diet were significantly lower (P b 0.05). compared to shrimp fed other diets. In contrast, the clotting time of shrimp fed the basal diet was significantly higher (P b 0.05) than that of shrimp fed the other diets. In trial 1, malondialdehyde content in the digestive glands of shrimp fed the basal diet was significantly higher (P b 0.05) than shrimp fed the other diets; moreover, no significant difference (PN 0.05) in malondialdehyde content in the digestive gland. shrimp in treatments D2–D5. There was no significant difference (PN 0.05) in protein carbonyl content in the shrimp digestive glands among all diets. In trial 1, no significant differences (P N 0.05) were found in the expression of Hsp 70 mRNA and HIF-1α mRNA in the shrimp digestive gland among all diets.
3.2. Test 2
3.2.1. Survival rate of shrimp in the air exposure stress tolerance test
After 36 hours of live transport simulation, no shrimp mortality was observed in any diet group.
3.2.2. Whole body and muscle composition
The whole body and muscle composition of shrimp is shown in Table 5. In trial 2, no significant differences were found in whole body and muscle moisture content as well as ash content between socks. both diets (P N 0.05). The whole body protein content of shrimp fed the basal diet (D1) was significantly lower (P b 0.05) than shrimp fed D3 but did not differ significantly from shrimp fed D3. eat D2, D4 and D5 (P N 0.05). The whole body lipid content of shrimp fed D4 and D5 was significantly higher than that of shrimp fed other diets (P b 0.05). The muscle protein content of shrimp fed D2 and D3 was significantly higher (P b 0.05) than shrimp fed other diets (P N 0.05). The muscle lipid content of shrimp has the same trend as the whole body lipid content of shrimp.
3.2.3. Immune parameters
Antioxidant capacity (total blood cell count and hemolytic coagulation), hemolysis time, oxidative stress parameters (malondialdehyde and protein carbonyl content of the digestive gland) and gene expression (Hsp 70 level digestive gland mRNA and HIF-1α mRNA) of shrimp are presented in Table 10. In trial 2, total hemocytes of fed shrimp
basal diet was significantly lower (P b 0.05) than shrimp fed the other diets, moreover, the total blood cell counts of shrimp fed the astaxanthin-supplemented diet (D2 and D3) were significantly higher (P b 0.05) than shrimp fed the basal diet. Dietary supplementation with β-carotene (D4 and D5). In contrast, the clotting time of shrimp fed the basal diet was significantly higher (P b 0.05) than that of shrimp fed the other diets.
In trial 2, malondialdehyde and protein carbonyl contents in the digestive glands of shrimp fed the basal diet were significantly higher (P b 0.05) than shrimp fed the other diets, moreover , no significant difference (P N 0.05) was found in malondialdehyde and carbonyl. Protein content in shrimp digestive glands in treatments D2–D5.
In trial 2, digestive gland Hsp 70 mRNA expression of shrimp fed the basal diet was significantly higher (P b 0.05) than shrimp fed the other diets, moreover, No significant difference (P N 0.05) was found in shrimp digestive gland Hsp 70 mRNA expression among treatments D2–D5. The expression profile of HIF-1α mRNA of the digestive gland of shrimp fed the basal diet was significantly (P b 0.05) lower than that of shrimp fed other diets, moreover, the Cholesterol supplementation significantly improved gastrointestinal HIF-1α mRNA levels in astaxanthin and β-carotene-supplemented dietary treatments (D3 vs. D2; D5 vs. D4)
4.DISCUSS
4.1. Shrimp growth performance
In this study, growth performance of P. monodon was affected by different experimental diets (Table 4). Segner et al. (1989) suggested that carotenoids have a positive role in intermediary metabolism and have beneficial effects on the development of aquatic animals. Similarly, Amar et al. (2001) and Niu et al. (2009) reported that dietary carotenoids, which serve as pigment sources, can also enhance nutrient utilization and may ultimately help improve shrimp growth. Different dietary sources of carotenoids have different results on shrimp growth and survival. In the work of Yamada et al. (1990), astaxanthin was reported to be more effective against pigmentation in Penaeus japonicus than β-carotene or canthaxanthin. Also for P japonicus, Chien and Jeng (1992) reported higher survival rates in shrimp fed diets supplemented with astaxanthin than in shrimp fed diets supplemented with β carotene or algae. This is consistent with the present results.
4.2. Tissue composition of shrimp
In the present experiment, chemical analyzes revealed smaller increases in whole-body and muscle protein with dietary astaxanthin or β-carotene, and in the whole-body and muscle lipid content of shrimp from test 2 (air exposure stress tolerance test) showed lower (P b 0.05). ) compared to shrimp in trial 1 (normal culture time) (Table 5). Furthermore, fat content was significantly higher in shrimp fed diets containing β-carotene (D4 and D5) compared to basal and astaxanthin-containing diets (D1, D2 and D3). Vitamin A is widely involved in various biochemical processes in the body, and β-carotene can be converted into vitamin A as a major source of vitamin A (Ong and Chytil, 1975). In the study by Yang et al. (2007) also showed that the whole body lipid and protein content of white shrimp Litopenaeus vannamei was positively affected by dietary vitamin A supplementation. Singh et al. (1969) found elevated plasma free fatty acids and increased liver lipids in rats fed vitamin A and suggested that these changes were due to mobilization of fatty acids from adipose tissue. In the study by Shiau and Chen (2000), high body fat content and reduced blood triglyceride concentrations were also found in shrimp fed a diet high in vitamin A. However, a phenomenon The opposite effect has been reported in fish and body fat percentage decreased in salmon fed diets with excess vitamin A (Poston, 1970). The mechanism of dietary carotenoids on lipid nutrition in crustaceans needs further investigation.
4.3. Bioavailability of astaxanthin and β-carotene
In the present experiment, the high ADC and low retention efficiency of carotenoids meant that dietary astaxanthin could be efficiently digested in the gastrointestinal tract of P. monodon, but could not accumulate in the tissue as pigment. This may be partly due to low incorporation of carotenoids into chylomicrons, low excretion of carotenoids and their metabolites associated with chylomicrons into lymph, or metabolic conversion to colorless compounds. which can eventually be excreted (Van Het Hof et al., 2000). Regarding the mechanism by which dietary cholesterol supplementation may have the effect of increasing the bioavailability of astaxanthin in shrimp, it can be concluded that, first of all, the presence of dietary cholesterol in the intestine stimulates favors bile acid release (Horton et al., 1995). Inclusion of bile acids in the diets of ferrets (Lakshman et al., 1996) and rats (Schweigert et al., 2002) significantly increases the absorption and tissue accumulation of carotenoids. The amphiphilic properties of bile acids are important in the intestinal lumen for the formation of mixed micelles and liposomes during the emulsification and digestion of lipids and lipid-soluble compounds such as astaxanthin (Ódoherty et al., 1973). Furthermore, a recently reported study by Tyssandier et al. (2001) pointed out that bile lipids are thought to be the only lipids that solubilize carotenoids in the aqueous phase of the intestine, which may influence carotenoid transport. Second, dietary cholesterol may promote carotenoid transport by increasing lipoprotein formation. In intestinal cells, carotenoids are incorporated into chylomicrons, the chylomicrons are eventually released into the blood, and carotenoids are mainly transported by lipoproteins, especially very low density lipoproteins (VLDL) in the blood (Deming and Erdman, 1999; Herbeth et al., 2007). Furthermore, apolipoprotein B is the major structural protein of VLDL and chylomicrons, and cholesterol is an important determinant of apolipoprotein B synthesis (Kumar et al., 1992). Furthermore, an increase in dietary cholesterol may also affect hepatic lipoprotein recycling through down-regulation of hepatic LDL receptor activity (Karnaukhov, 1979).
However, this study found no positive effect of dietary cholesterol on β-carotene. Regarding the difference between astaxanthin and β-carotene, it may first be due to the different subclasses of astaxanthin and β-carotene. Astaxanthin is a more polar compound and β-carotene belongs to the group of nonpolar hydrocarbon carotenoids (Yeum and Russell, 2002). It is possible that less polar carotenoids, located mainly at the lipoprotein surface of lipid droplets, are more easily transported than less polar carotenoids, located mainly at the lipoprotein core of droplets (Tyssandier et al. events, 2001). Van Het Hof et al. (2000) stated that the breakdown of food structure and release of carotenoids is the first step in carotenoid absorption; and a second step in carotenoid absorption that may influence their bioavailability involves incorporation of released carotenoids into mixed micelles. As lipid-soluble compounds, consuming fats along with carotenoids is thought to be important. A large amount of carotenoid-rich fat added to a meal (3 g fat/meal) was as effective as a single dose of carotenoid-rich fat (35 g fat/meal) in enhancing α- carotene and β-carotene in plasma; however, for the polar compound lutein, added as lutein ester, the plasma response was greater than 100% after total fat consumption (Van Het Hof et al., 2000). Therefore, the amount of dietary cholesterol required to ensure carotenoid absorption appears to depend on the physicochemical properties of the ingested carotenoids, especially for β-carotene which currently does not provide bioavailability. similar to that observed for the present astaxanthin. Furthermore, the absorption of different carotenoid subclasses is triggered by different pathways such as different transporters, such that in humans, β-carotene absorption is limited by lyso- phosphatidylcholine but not by cholesterol (During et al., 2005). Second, as we know that β-carotene is the nutritional precursor of vitamin A, β-carotene can be cleaved to produce two molecules of vitamin A (retinol) in the intestine (Wolf, 1984). A large proportion of consumed β-carotene is absorbed intact, converted to vitamin A, or excreted (Parker, 1996).
4.4. Antioxidant capacity of astaxanthin and β-carotene
Enhance the resistance of penaeid shrimp to stress due to lack of oxygen (Chien et al., 1999; Niu et al., 2009), stress due to salinity (Chien et al., 2003), stress due to ammonia (Pan et al., 2003a) and again, air exposure during simulated live transport in our present study was found to be associated with dietary carotenoids. Whether in trial 1 or trial 2, MDA as an index of lipid peroxidation was significantly lower in shrimp fed diets containing carotenoids (D2–D5) than in shrimp fed basic diet (D1); Furthermore, the levels of MDA and protein carbonyl in the digestive gland of shrimp from trial 2 were higher (P b 0.05) (Table 10). Carotenoids have good singlet oxygen quenching properties and can act as antioxidants in systems containing unsaturated fatty acids by quenching free radicals (Martin et al., 1993). This suggests that carotenoids may serve to protect the polyunsaturated fatty acids of tissues from the adverse effects of oxidation, especially in shrimp exposed to hypoxia. The consideration of protein carbonyls as biomarkers of oxidative damage to proteins is quite recent for fish (Parvez and Raisuddin, 2005) and is not commonly used for shrimp. However, oxidative modification of proteins is one of many consequences of oxidative stress (Stadtman, 1986), and assaying carbonyl groups in proteins provides a convenient technique for detecting and quantifying oxidative stress. chemistry (Levine et al., 1994). Overall, all of these supported lower levels of oxidative stress in shrimp fed carotenoid-containing diets (D2-D5) compared to basal diets (D1). Total blood cell count and clotting time are also used as indicators of crustacean health (Fotedar et al., 2001). Coagulation time and circulating blood cell counts have been reported to vary in crustaceans due to time spent in storage tanks and live transport (Fotedar et al., 2006; Le Moullac and Haffner, 2000). . Total hemocytes counts of shrimp fed basal and β-carotene supplemented diets (D1, D4, and D5) decreased significantly (P b 0.05) after 36 h of simulated live transport while it did not change in shrimp fed diets supplemented with astaxanthin (D2 and D3). The clotting time of shrimp fed the basal diet (D1) increased significantly (P b 0.05) after simulated live transport and remained unchanged in shrimp fed the carotenoid-supplemented diet ( D2-D5) (Table 10). The decline in total blood cell count after exposure to air during simulated live transport may be related to hemolysis due to defense activities (van de Braak et al., 2002 ). The total blood cell count of shrimp fed diets containing astaxanthin (D2 and D3) did not decrease after simulated live transport. Sequeira et al. (1996) showed that P. japonicus hemocytes have the ability to proliferate and the proliferation rate can be tripled when shrimp are fed immune stimulants. In the present study, the incorporation of astaxanthin in the diet could stimulate and increase the proliferation rate of shrimp blood cells to compensate for the loss of blood cells due to exposure to air during transportation. simulated life, resulting in a constant total blood cell count ratio. Exposure to air has been shown to reduce the health status of rock lobsters (Panucilus lygnus) (Fotedar et al., 2001). Trial 2 showed that shrimp fed diets containing astaxanthin (D2 and D3) were healthier than shrimp fed other diets as indicated by higher total blood cell counts and leukocyte clotting times. Blood pressure was lower after 36 hours of simulated live transport.
4.5. Gene expression of Hsp 70 and HIF-1α
Hsp 70 is an evolutionarily highly conserved molecular chaperone that is an important part of the cell’s protein folding machinery, which can aid in repair and protect cellular proteins from stress-induced damage. stress induces and reduces protein synthesis (Franzellitti and Fabbri, 2005). Woo et al. (2011) and Xu et al. (2011) showed that hypoxia represents a high level of stress that can lead to the induction of genes involved in cellular responses such as heat shock proteins. The expression level of Hsp 70 mRNA was significantly up-regulated under hypoxia compared with normoxia in shrimp fed all tested diets; However, the Hsp 70 mRNA expression level of shrimp fed a basal diet was significantly higher than that of shrimp fed a carotenoid-containing diet (D2–D5) under hypoxic conditions, indicating showed that dietary astaxanthin or β-carotene can partially reduce the hypoxic stress response. Little is known about the presence and role of HIF-1 under hypoxic conditions in crustaceans. Heidbreder et al. (2003) proposed that HIF-1α may contribute to protection during early and/or moderate hypoxia or against severe and/or prolonged hypoxia. Treinin et al. (2003) demonstrated that HIF-1α is a transcription factor that regulates dozens of genes involved in responses to hypoxia, which molecular responses then constitute a series of biochemical and physiology, helping animals survive better in hypoxic conditions. In the present experiment, the expression level of shrimp HIF-1α mRNA was significantly reduced under hypoxia compared with normoxia in shrimp fed all the tested diets, however, expression levels of HIF-1α mRNA of shrimp fed a diet containing dietary astaxanthin or β-carotene was higher than that of shrimp fed a basal diet under hypoxic conditions, which also indicates that astaxanthin or β-carotene in the diet may partially reduce the hypoxic stress response in P. monodon by enhancing the efficiency or utility of oxygen transport.
5.conclusion
In summary, growth assay and immune response data suggest that astaxanthin is superior to β-carotene in improving growth performance, health status, and defense against stress exposure. air. Replacing astaxanthin treatments with antibiotics could provide valuable new, safe and effective culture techniques for on-farm aquaculture in the near future. Furthermore, cholesterol supplementation can positively enhance the effect of astaxanthin but not β-carotene.
