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Effect of injected astaxanthin on survival, antioxidant capacity, and immune response of the giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879) challenged with Lactococcus garvieae.

ABSTRACT This study evaluated the effects of astaxanthin (AX) injected at 1.34 nmol/g body weight on survival, antioxidant capacity, total hemocyte count, and hepatopancreas AX content of the giant freshwater prawn Macrobrachium rosenbergii (De Man 1879) challenged with Lactococcus garvieae. Injected AX significantly increased the survival of M. rosenbergii challenged with L. garvieae (P [less than or equal to] 0.05) and enhanced to some extent its antioxidant capacity (superoxide dismutase, glutathione peroxidase, and glutathione reductase) and total hemocyte count. AX-injected M. rosenbergii yielded significantly higher hepatopancreas AX content compared with non-AX-injected control. Overall results of this study indicate that AX plays an important role in enhancing M. rosenbergii resistance against L. garvieae.

KEY WORDS: glutathione peroxidase, glutathione reductase, superoxide dismutase, total hemocyte count, hepatopancreas, prawn, Macrobrachium, Lactocoecus

INTRODUCTION

The aquaculture production of the giant freshwater prawn Maerobrachium rosenbergii (De Man 1879) is a promising industry. Total world production for the farmed M. rosenbergii increased more than 3-fold during 1996-2006 (FAO 2008). Most M. rosenbergii is produced in South Asia (New 2005) because of its favorable tropical and subtropical climate, and vast area of inland waters. Moreover, it is more valuable than fin fish because demand is higher than the supply, and its development has attracted considerable attention because of its export potential and market price (Ahmed et al. 2008).

Diseases can be the main threat to sustaining growth of the industry. Several shrimp farming ventures collapsed in 1990s as a result of the degraded environment and diseases (Kutty 2005). One of the major pathogens of M. rosenbergii is Lactococcus garvieae (Chen et al. 2001), which causes disease outbreaks during the summer season, resulting in decreased production (Cheng & Chen 1998). In 1991-2003, a 37.98% decrease in M. rosenbergii production in Taiwan was reported, which was mainly caused by diseases (Cheng & Chen 1998).

One alternative solution to the disease problem is to enhance the animal's antioxidant capacity, which would consequently increase its resistance against stress (Pan et al. 2003, Chien & Shiau 2005) and microbial infection. The antioxidant system of M. rosenbergii helps in eliminating reactive oxygen species (ROS), which are produced under oxidative damage (Halliwell & Gutteridge 1989) when they are exposed to stressors. Astaxanthin (AX), a naturally occurring carotenoid pigment, is a powerful biological antioxidant. The enhancement of antioxidant capacity by dietary AX and, consequently, the improvement in recovery against stress demonstrated that AX is a semiessential nutrient for the black tiger prawn Penaeus monodon (Fabricius 1798) (Chien et al. 2003).

Antioxidant capacity and immune response can be assessed by measuring hemolymph antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR), and by determining the total hemocyte count (THC). SOD is involved in protective mechanisms within tissue injury following oxidative processes and phagocytosis (Bell & Smith 1993). GPx, on the other hand, protects cells from excessive levels of hydrogen peroxide ([H.sub.2][O.sub.2]) and intracellular lipid peroxides (Robertson & Harmon 2007), whereas GR catalyzes the reduction of glutathione to yield reduced glutathione, which is readily oxidized by ROS (Stohs & Bagchi 1995). THC is one of the major parameters used to evaluate immune response (Rodriguez & Le Moullac 2000). Hemocytes are associated with proteins like prophenol oxidase and phenol oxidase, which are involved in encapsulation and melanization, and which function as nonself-recognition systems (Johansson & Soderhall 1989, Rodriguez & Le Moullac 2000).

This study was conducted to determine whether body AX would affect antioxidant capacity, total hemocyte count, hepatopancreas AX content, and disease resistance of M. rosenbergii challenged with L. garvieae. SOD, GPx, and GR were used as indicators of antioxidant capacity, whereas THC was used as an indicator of immune response. Because variations in digestibility and efficiency of type and concentration of carotenoids are observed when they are introduced through the diet (Ytrestoyl & Bjerkeng 2007a), AX was administered by injection in this study.

MATERIALS AND METHODS

Culture ofLactococcus garvieae

The bacterial strain L. garvieae isolated from diseased M. rosenbergii was used in this study (Chen et al. 2001). It was subcultured on tryptic soy agar (Difco, Detroit, MI) for 24 h at 25[degrees]C, and grown in 10 mL tryptic soy broth (Difco) for another 24 h. The broth culture was centrifuged at 6,000 g for 15 min at 4[degrees]C. The supernatant was discarded and the bacterial pellets were resuspended in phosphate buffered saline (PBS) to attain a concentration of 1 x [10.sup.9] cfu/mL and 1 x [10.sup.7] cfu/mL. These concentrations were used as stock bacterial suspensions for the succeeding experiments.

Preparation of AX Solution

AX (10% Carophyll Pink CWS, DSM Nutritional Products, Basel, Switzerland) solution was prepared by dissolving 8 mg in 10 mL PBS to obtain 80 ppm. This solution was then put in an ultrasonic bath at 45[degrees]C for 10 min. The concentration of AX used in this study was 1.34 nmol/g body weight (Chien 2005). This concentration was derived from the following computation:

0.01 mL/g body weight x 80 mg/1,000 mL / 596.8 mg/nmol = 1.34 nmol/g body weight,

where 596.8 mg/nmol is the molecular weight of CWS 10% AX.

Experimental Design

A factorial design with 7 sampling times (days postbacterial challenge), 1 dosage of AX (1.34 nmol/g body weight), and bacteria L. garvieae (2 x [10.sup.7] cfu/12.87 g body weight) was used for the susceptibility study. Likewise, a factorial design with 3 sampling times (1, 2, and 4 days postbacterial challenge), the same dosage of AX, and 1 dosage of bacteria (2 x [10.sup.5] cfu/26.05 g body weight) was used for the determination of antioxidant capacity and THC. A single-factor design with 1 sampling time (7 days postbacterial challenge) and the same dosage of AX and bacteria (2 x [10.sup.5] cfu/26.05 g body weight) was used for hepatopancreas AX content analysis.

Experimental Animals and Setup

Subadult prawns (12.87 [+ or -] 0.90 g) were used for susceptibility testing; adults (26.05 [+ or -] 1.95 g) were used to analyze antioxidant capacity, THC, and hepatopancreas AX content. The prawns were acclimatized indoors for 3 wk in 1 circular tank (2 m deep x 0.5 m high) for subadults and 10 concrete rectangular tanks (2 x 2 m x 0.5 m) for adults. Aeration was provided in each tank, and water quality such as dissolved oxygen (5.12-6.90 mg/ L), temperature (27.1 28.9[degrees]C), and pH (7.6-8.2) were monitored and kept within safe levels.

The prawns were fed a commercial diet at 5% body weight/ day, given at 0800, 1500 and 2200 h. Tank bottom debris was siphoned out, and one third of the water was replaced daily. Only prawns in the intermolt stage were used in this study. The molt stage was determined by examining the uropod in which partial retraction of the epidermis could be observed (Peebles 1977).

Susceptibility Study

Seven prawns were stocked in each 60-L glass aquaria containing 40 L freshwater. Aeration was provided in each aquarium and water was renewed daily. The procedures/timing on administering solution and bacteria to the prawn were based on the study of Hou and Chen (2005) with some modifications. Briefly, the prawns were injected with AX (0.01 mL/g body weight) individually into the ventral sinus of the cephalothorax on the 1st day. Control prawns were injected with 20 [micro]L sterile saline. On the 2hd day, both AX-injected and challenged control groups were injected with 20 ~L bacterial suspension (1 x [10.sup.9] cfu/mL). The control in both groups was injected with 20 [micro]L sterile saline. Each treatment and the control had 3 replicates. Mortality was recorded every day until 7 days postbacterial challenge.

Antioxidant Capacity and THC Analysis

Fifteen prawns in duplicate tanks for each of the experimental and the control groups were used in this study. The prawns were injected individually with AX and sterile saline as described earlier. At 1 day after AX injection, 20 [micro]L bacterial suspension (1 x [10.sup.7] cfu/mL) was injected in each prawn in both groups.

At 1, 2, and 4 days postbacterial challenge, hemolymph (100 [micro]L) was withdrawn from the ventral sinus (4 prawns/sampling) using a 1-mL sterile syringe (25 gauge) containing 0.9 mL anticoagulant solution (trisodium citrate, 30 mM; sodium chloride, 0.34 M; ethylenediamine tetraacetic acid, 10 mM; pH, 7.55; osmolality adjusted with glucose to 780 mOsm/kg). The hemolymph mixture was used for determinating antioxidant capacity and THC.

Antioxidant capacity was analyzed spectrophotometrically using a U-2000 spectrophotometer (Hitachi Ltd., Tokyo, Japan) at 37[degrees]C at 505 nm absorbance (for SOD) and 340 nm absorbance (for GPx and GR). All assays were performed within 5 h after sampling using Randox Laboratories kits (Crumlin, Co., Antrim, UK) according to manufacturer instructions. Activities were expressed in units per milligram protein.

For hemolymph protein, soluble protein of the hemolymph sample was determined using a protein assay kit (no. 500-0006, Bio-Rad laboratories, Richmond, CA) with bovine serum albumin (66 kDa, Sigma, St. Louis, MO) as standard, a method derived from Bradford (1976).

Total hemocyte count was carried out with 100 [micro]L anticoagulant hemolymph mixture placed on a hemocytometer. Hemocytes were counted using an inverted phase-contrast microscope and were expressed in number of cells per milliliter.

Hepatopancreas AX Analysis

Seven days after AX injection, 3 prawns from each treatment were sampled for analysis of hepatopancreas AX content. The samples were kept in a freezer (-80[degrees]C) for 24 h to solidify the hepatopancreas prior to dissection. Dissected hepatopancreas was freeze-dried using a refrigerated air dryer (model FD-12-6P-D, Kingmech, Tuncheng, Taiwan, ROC) and then ground using a porcelain mortar and pestle. Samples were placed into a polypropylene centrifuge tube and 20 mL acetone (0.05% butylated hydroxytoluene) was added as antioxidant and solvent. The mixture was homogenized using the Polytron PT MR-3000 homogenizer (Kinematica AG, Littau, Switzerland) at 8,000 g for 1 min. The samples were then centrifuged in a high-speed refrigerated centrifuge (Himac CR-21, Hitachi, Japan) under 4[degrees]C at 10,000 g for 15 min. The liquid phase was transferred to a 250-ml separatory funnel, partitioned with 30 mL n-hexane, and washed 2 times with 25 ml 10% NaCl solution to remove residual acetone. The extract was put into a rotary evaporator (Rotavapor Model Rl14, BUCHI, Switzerland) at 30[degrees]C in a waterbath (model B480, BUCHI, Switzerland) to reduce the volume. The samples were further dried from excess water using nitrogen gas (N2) and filtered through a 0.22-[micro]m Millipore (Millipore: Nashua, NH) filter. Three-milliliter samples were stored in brown vials and allocated inside an autosampler (model L-7200, Hitachi, Japan) prior to high-performance liquid chromatography (HPLC) analysis.

The carotenoid analysis by HPLC was done using a Hitachi L-6200 pump, a silica column 250 x 4.6 mm (LUNA 5[mu] SILICA, Phenomenex, Torrance, CA), a Hitachi L-4250 UV/Vis detector, and a Hitachi D-2000 Chromato-integrator. This system was controlled by a chromatographic data system (Scientific Information Services Corporation). The program of solvents during the mobile phase included mixture A composed of 490 mL n-hexane, 5 mL methylene chloride, and 5 mL isopropyl alcohol; and mixture B composed of 460 mL n-hexane, 5 mL methylene chloride, and 35 mL isopropyl alcohol. The absorption maximum was set to 470 nm and the work volume at 1 mL/min.

Statistical Analysis

Assumptions of analysis of variance (ANOVA) were assessed using the Shapiro-Wilk test for normality on antioxidant capacity (SOD, GPx, and GR) and THC. One-way ANOVA was performed to determine the effects of AX on survival at 1-7 days postbacterial challenge, on antioxidant capacity and THC at 1,2, and 4 days postbacterial challenge, and on hepatopancreas AX content at 7 days postbacterial challenge. Duncan's multiple range test was used to compare differences among treatments. An arcsine square root transformation was used before processing percentage data (susceptibility test). The significant level applied to all analyses was set to 5% SAS version 9.0 software (SAS Institute, Inc., Cary, NC) was used for statistical analysis.

RESULTS

Survival

At 7 days postbacterial challenge, AX showed a significant positive effect on the survival of prawns. Ali the unchallenged control prawns survived. Mortality occurred at 1 day postbacterial challenge for the challenged group. Percentage survival of AX-injected prawns was significantly higher than the challenged control group (Table 1).

Antioxidant Capacity

SOD was found to be significantly different when prawns were injected with AX only compared with the control and challenged groups at 4 days postbacterial challenge. However, SOD of prawns injected with AX that then received the bacterial challenge demonstrated no significant differences compared with the control and challenged control group at each observation period (Table 2).

A similar pattern was observed for GPx, where values for prawns injected with AX only were higher than the control and challenged groups at 2 and 4 days postbacterial challenge. However, no significant differences existed between the challenged control group and AX-injected prawns at each observation period (Table 3).

GR was significantly higher in the AX-injected prawns than the challenged control group at 2 days postbacterial challenge. However, no significant differences were found at 1 and 4 days postbacterial challenge (Table 4).

Total Hemocyte Count

THC of AX-injected prawns subjected to bacterial challenge was significantly higher than the challenged control group at 4 days postbacterial challenge. However, no significant differences were observed at 1 and 2 days postbacterial challenge (Table 5).

Hepatopanereas AX Content

Seven days after AX injection, significantly higher diester AX (DA), monoester AX (MA), free AX (FA), and total AX concentrations in hepatopancreas were observed in the AX-injected prawns than in the control without AX. DA, MA, and FA contents of the hepatopancreas ranged from 35-45%, 49-61%, and 2-5%, respectively (Table 6).

DISCUSSION

Pathogens, such as L. garvieae, have a significant negative effect on the survival of M. rosenbergii, as shown in the decline of survival through time. However, carotenoids help to strengthen the immune system of M. rosenbergii under normal or stressed conditions. In this study, the greater survival of challenged AX-injected M. rosenbergii than the challenged control showed that AX was efficient in improving survival. In one study it was reported that dietary AX increased the resistance of P. monodon in its early stage against Vibrio damsela (Pan et al. 2003). It was also proved that P. monodon fed a diet of 300 mg/kg of the Dunaliella extract (a marine alga containing various carotenoids) for 8 wk exhibited higher resistance to white spot syndrome virus (WSSV) infection (Supamattaya et al. 2005). Moreover, a higher percentage survival was also observed in kuruma prawn, Marsupenaeus japonicus (Bate 1888), fed an AX-supplemented diet (Yamada et al. 1990). The current study demonstrated that AX could improve health in M. rosenbergii in terms of resistance against L. garvieae infection.

Response of antioxidant capacity such as SOD, GPx, and GR attributed to AX indicated stronger resistance against stress to some extent. The good antioxidant properties of AX may relieve oxidative stress and may have beneficial biological effects in relation to health as reviewed by Higuera-Ciapara et al. (2006). SOD is the primary cellular defense mechanism against superoxides. A decrease in SOD activity in response to bacterial infection is considered to enhance the generation of ROS, which may contribute to a higher mortality rate. It was reported that SOD levels of P. monodon with WSSV infection were below normal values (Lin 1998, Chang et al. 2003, Mathew et al. 2007). Moreover, SOD activity in a caridean shrimp, Palaemonetes argentinus (Nobili 1901), infected by a gill parasite was significantly reduced (Neves et al. 2000). On the other hand, GPx functions primarily to catalyze the decomposition of [H.sub.2][O.sub.2], thus it detoxifies [H.sub.2][O.sub.2] derived from SOD activity. It is commonly assumed that any significant increase in SOD must be accompanied by a comparable increase in GPx to prevent excessive buildup of [H.sub.2][O.sub.2] (Warner 1994). In the current study, M. rosenbergii injected with AX only showed higher SOD and GPx levels than the control and infected groups after bacterial challenge. However, considering only the challenged groups, AX elevated SOD and GPx, but not to a significant level.

In some studies, no significant effects were detected on some antioxidant capacity indicators when the organisms were exposed to stressful conditions or were supplemented with antioxidants (vitamin E) (Bainy et al. 1996, Dandapat et al. 2000). It has been suggested that Nile tilapia Oreochromis niloticus (Linnaeus 1758) from a polluted reservoir was under oxidative stress as shown in all the tissues investigated, such as erythrocytes, gills, liver, and kidney. No significant differences in SOD, GPx, and GR activities in the gill filaments were noted, but higher levels of other antioxidant indicators such as cytochrome B5, and diminished catalase and glucose-6-phosphate dehydrogenase were noted. These might be the causes of enhanced oxyradical production in stressed fish. Dandapat et al. (2000) concluded that dietary vitamin E could modulate the antioxidant defense system. However, SOD and GPx exhibited tissue-specific variable and dose-dependent responses to vitamin E. Similarly, no significance difference in SOD activity was observed for immunostimulant-fed white shrimp, Litopenaeus vannamei (Boone 1931), at the beginning, and after 3 and 6 days (Cheng et al. 2004, Fu et al. 2007). Nonetheless, SOD activity of immunostimulant-fed L. vannamei was significantly higher than the control after 9, 14, 21, and 28 days (Fu et al. 2007). In the current study, the nonsignificant effects found in SOD and GPx might also indicate that L. garvieae infection suppressed the activity of the hemolymph antioxidant system of infected M. rosenbergii. This study therefore suggests that additional antioxidant capacity indicators should be analyzed in the hemolymph and possibly from other tissues of M. rosenbergii. In addition, the period of observation should be extended to explain completely the oxidative stress mechanisms in this species.

Another significant observation is that injected AX modulated a substantial elevation of GR. GR is another scavenging enzyme involved in the removal of toxic metabolites by glutathione conjugation reactions. Considering only the challenged groups, GR of AX-injected M. rosenbergii increased by 33% at 2 days postbacterial challenge compared with the control. The decrease in the activity of GR may lead to the formation of [O.sub.2] and [H.sub.2][O.sub.2] (Yu 1994) and later on formation of hydroxyl radical (OH.) that bring harmful reactions to cell membranes.

AX can influence the immune response of M. rosenbergii, as evidenced by the increase in THC of AX-injected prawns that also underwent the bacterial challenge compared with challenged control prawns at 4 days postbacterial challenge. It has been observed in other studies that THC was higher in non-stressed crustaceans compared with stressed groups (Le Moullac & Hafner 2000, Flores et al. 2007). In crustaceans, an increase in THC is considered to enhance the immune response during periods of stress (Truscott & White 1990), leading to disease resistance (Le Moullac et al. 1998). It was reported that a reduction in THC might be an indication of suppressed resistance in M. rosenbergii (Yeh et al. 2006). The immune-enhancing effect of AX can be attributed to their antioxidant properties, such as protection of cells from oxidative damage. The immune system generates reactive oxygen metabolites as part of its regular defense functions. These reactive immune species are necessary for immune cells to kill pathogens and clear dead tissues, but chronic overproduction of them, as seen in a variety of pathological conditions, can cause damage to the immune cells and compromise their functions (Wu & Meydani 1999). In the current study, the THC of M. rosenbergii injected with AX did not change compared with control group. This result suggests further studies to point out the specific response of THC on the immune system of M. rosenbergii.

The injection of AX in this study resulted in increased hepatopancreas AX content of M. rosenbergii. Up to 180 mg/ kg AX was detected 7 days after AX injection with or without bacterial challenge. Moreover, a 400% increase in total hepatopancreas AX content was observed among the challenged and unchallenged groups. Despite the fact that FA was injected, DA and MA increased more than 4-fold, whereas FA increased by less than 2-fold, indicating the conversion of FA to MA and DA. This result corroborates the study of Ho (2007) in L. vannamei supplemented with different dosages of AX and [beta]-carotene for 6 wk. Regardless of dietary carotenoid type, ester AX dominated the total body AX by 98% and free AX only by 2%. In crustaceans, including M. rosenbergii, AX is present as DA, MA, and FA or is bound to protein, caroteno-protein, or lipoproteins (carotenolipoproteins) (Higuera-Ciapara et al. 2006). When total carotenoid content in P. monodon increases, DA increases proportionally, MA increases exponentially, but FA increases in a decaying exponential trend until an upper limit is reached (Okada et al. 1994, Chien 1996). An accumulation of dietary AX in the integument (carapace and epidermis) and hepatopancreas of Marsupenaeus japonicus by supplementing synthetic AX and canthaxanthin has also been observed (Negre-Sadargues et al. 1993). In Atlantic salmon, Salmo salar (Linnaeus 1758), Atlantic cod, Gadus morhua (Linnaeus 1758), and rainbow trout, Onchorhyncus mykiss (Walbaum 1792), a higher and more rapid uptake of AX was observed in plasma, muscle, skin, kidney, and liver after intraperitoneal injection of AX (Ytrestoyl & Bjerkeng 2007a, Ytrestoyl & Bjerkeng 2007b). The highest AX concentrations were found in the liver of S. salar, with up to 200 mg/kg detected after injection of 50 mg AX.

Overall, AX content was analyzed from the hepatopancreas because this lipid-rich organ, with its high metabolic rate, may undergo spontaneous auto-oxidation; thus, the generation of [O.sub.2.sup.-] and [H.sub.2][O.sub.2] may be comparatively higher than in other organs (Dandapat et al. 2000). Moreover, the hepatopancreas is the main digestive gland responsible for the regulation of overall body metabolism (Muriana et al. 1993). Although this study is not practical for commercial conditions, it justifies the need to increase hepatopancreas AX content to resist L. garvieae infection. These findings could be used as reference for future studies with regard to the potential effectiveness of AX on the enhancement of an organism's antioxidant capacity and immune response through dietary supplementation. Moreover, quantification of oral dosage on a commercial scale would be of great value.

ACKNOWLEDGMENTS

This work was supported by National Science Council project no. 96-2313-B-019-009-MY3 and Center of Marine Bioscience and Biotechnology. We thank the Bureau of Fisheries and Aquatic Resources, Science City of Munoz, Philippines, for assistance in rearing M. rosenbergii. The technical assistance of Dr. Eduardo Leano is highly appreciated.

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Ytrestoyl, T. & B. Bjerkeng. 2007a. Dose response in uptake and deposition of intraperitoneally administered astaxanthin in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.). Aquaculture 263:179-191.

Ytrestoyl, T. & B. Bjerkeng. 2007b. Intraperitoneal and dietary administration of astaxanthin in rainbow trout (Oncorhynchus mykiss): plasma uptake and tissue distribution of geometrical E/Z isomers. Comp. Biochem. Physiol. B 147:250-259.

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ISAGANI P. ANGELES, JR., (1) YEW-HU CHIEN (1) * AND APOLINARIO V. YAMBOT (2)

(1) Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China; (2) College of Fisheries-Freshwater Aquaculture Center, Central Luzon State University, Science City of Munoz 3119, Philippines
TABLE 1.
Percent survival of the giant freshwater prawn Macrobrachium
Rosenbergii injected with astaxanthin at 1.34 nmol/g body weight
at 7 days postbacterial challenge with Lactococcus garvieae.

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    1
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]          76.2 [+ or -] 12.6 (a)
    1.34            2 x [10.sup.7]          90.5 [+ or -] 9.5 (a)

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    2
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]         57.1 [+ or -] 8.3 (b)
    1.34            2 x [10.sup.7]         81.0 [+ or -] 12.6 (ab)

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    3
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]          38.1 [+ or -] 4.8 (c)
    1.34            2 x [10.sup.7]          76.2 [+ or -] 9.5 (b)

Treatment                               Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    4
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]          19.0 [+ or -] 4.8 (c)
    1.34            2 x [10.sup.7]          47.6 [+ or -] 4.8 (b)

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    5
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]                14.3 (c)
    1.34            2 x [10.sup.7]          38.1 [+ or -] 9.5 (b)

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    6
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]           4.8 [+ or -] 4.8 (c)
    1.34            2 x [10.sup.7]          38.1 [+ or -] 9.5 (b)

      Treatment                    Days After Bacterial Challenge

Astaxanthin           L. garvieae
(nmol/g body        (cfu/mL/prawn)                    7
  weight)

   Saline               Saline                    100.0 (a)
    1.34                Saline                    100.0 (a)
   Saline           2 x [10.sup.7]           4.8 [+ or -] 4.8 (c)
    1.34            2 x [10.sup.7]          38.1 [+ or -] 9.5 (b)

Means [+ or -] SEM in the same column with different letters are
significantly different (P [less than or equal to] 0.05). n = 21.

TABLE 2.
Mean ([+ or -] SEM) superoxide dismutase (measured in units per
milligram protein) of the giant freshwater prawn Macrobrachium
rosenbergii injected with astaxanthin at 1.34 nmol/g body weight at
1, 2, and 4 days postbacterial challenge with Lactococcus garvieae.

      Treatment                    Days After Bacterial Challenge

 Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                   1
   weight)

   Saline            Saline           0.272 [+ or -] 0.01 (ab)
   Saline        2 x [10.sup.5]       0.254 [+ or -] 0.01 (b)
    1.34             Saline           0.301 [+ or -] 0.01 (a)
    1.34         2 x [10.sup.5]       0.282 [+ or -] 0.01 (ab)

      Treatment                    Days After Bacterial Challenge

 Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                 2
   weight)

   Saline            Saline           0.268 [+ or -] 0.02 (ab)
   Saline        2 x [10.sup.5]       0.249 [+ or -] 0.05 (b)
    1.34             Saline           0.359 [+ or -] 0.02 (a)
    1.34         2 x [10.sup.5]       0.279 [+ or -] 0.O1 (ab)

      Treatment                    Days After Bacterial Challenge

 Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                  4
   weight)

   Saline            Saline           0.277 [+ or -] 0.02 (b)
   Saline        2 x [10.sup.5]       0.266 [+ or -] 0.02 (b)
    1.34             Saline           0.381 [+ or -] 0.03 (a)
    1.34         2 x [10.sup.5]       0.274 [+ or -] 0.02 (b)

Data in each observation time with different letters are significantly
different (P [less than or equal to] 0.05). n = 4.

TABLE 3.
Mean ([+ or -] SEM) glutathione peroxidase (measured in units per
milligram protein) of the giant freshwater prawn Macrobrachium
rosenbergii injected with astaxanthin at 1.34 nmol/g body weight
at 1, 2, and 4 days postbacterial challenge with Lactococcus garvieae.

          Treatment                Days After Bacterial Challenge

Astaxanthin       L. garvieae
(mnol/g body     (cfu/mL/prawn)                 1
  weight)

   Saline            Saline          0.414 [+ or -] 0.02 (ab)
   Saline        2 x [10.sup.5]      0.271 [+ or -] 0.01 (b)
    1.34             Saline          0.532 [+ or -] 0.09 (a)
    1.34         2 x [10.sup.5]      0.325 [+ or -] 0.06 (b)

          Treatment                Days After Bacterial Challenge

Astaxanthin       L. garvieae
(mnol/g body     (cfu/mL/prawn)                 2
  weight)

   Saline            Saline          0.407 [+ or -] 0.02 (b)
   Saline        2 x [10.sup.5]      0.264 [+ or -] 0.01 (c)
    1.34             Saline          0.601 [+ or -] 0.02 (a)
    1.34         2 x [10.sup.5]      0.287 [+ or -] 0.01 (c)

          Treatment                Days After Bacterial Challenge

Astaxanthin       L. garvieae
(mnol/g body     (cfu/mL/prawn)                 4
  weight)

   Saline            Saline          0.401 [+ or -] 0.01 (b)
   Saline        2 x [10.sup.5]      0.278 [+ or -] 0.02 (c)
    1.34             Saline          0.611 [+ or -] 0.04 (a)
    1.34         2 x [10.sup.5]      0.298 [+ or -] 0.03 (c)

Data in each observation time with different letters are significantly
different (P [less than or equal to] 0.05). n = 4.

TABLE 4.
Mean ([+ or -] SEM) glutathione reductase (measured in units per
milligram protein) of the giant freshwater prawn Macrobrachium
rosenbergii injected with astaxanthin at 1.34 nmol/g body weight at
1, 2, and 4 days postbacterial challenge with Lactococcus garvieae.

          Treatment                 Days After Bacterial Challenge

Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                  1
  weight)

   Saline            Saline          0.010 [+ or -] 0.0001 (ab)
   Saline        2 x [10.sup.5]      0.009 [+ or -] 0.0006 (b)
    1.34             Saline          0.011 [+ or -] 0.0002 (a)
    1.34         2 x [10.sup.5]      0.010 [+ or -] 0.0003 (b)

          Treatment                 Days After Bacterial Challenge

Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                  2
  weight)

   Saline            Saline          0.010 [+ or -] 0.0003 (ab)
   Saline        2 x [10.sup.5]      0.006 [+ or -] 0.0004 (c)
    1.34             Saline          0.011 [+ or -] 0.0002 (a)
    1.34         2 x [10.sup.5]      0.008 [+ or -] 0.0011 (b)

          Treatment                 Days After Bacterial Challenge

Astaxanthin       L. garvieae
(nmol/g body     (cfu/mL/prawn)                  4
  weight)

   Saline            Saline          0.011 [+ or -] 0.0012 (ab)
   Saline        2 x [10.sup.5]      0.008 [+ or -] 0.0006 (b)
    1.34             Saline          0.013 [+ or -] 0.0011 (a)
    1.34         2 x [10.sup.5]      0.008 [+ or -] 0.0005 (b)

Data in each observation time with different letters are
significantly different (P [less than or equal to] 0.05). n = 4.

TABLE 5.
Mean ([+ or -] SEM) total hemocyte count (x105/mL) of the giant
freshwater prawn Macrobrachium rosenbergii injected with astaxanthin
at 1.34 nmol/g body weight at 1, 2, and 4 days postbacterial
challenge with Lactococcus garvieae.

         Treatment                   Days After Bacterial Challenge

Astaxanthin        L. garvieae
(nmol/g body     (cfu/mL/prawn)                    1
  weight)

   Saline            Saline             172.3 [+ or -] 2.5 (ab)
   Saline        2 x [10.sup.5]         149.5 [+ or -] 1.4 (c)
    1.34             Saline             182.3 [+ or -] 7.8 (a)
    1.34         2 x [10.sup.5]         159.4 [+ or -] 2.76 (bc)

         Treatment                   Days After Bacterial Challenge

Astaxanthin        L. garvieae
(nmol/g body     (cfu/mL/prawn)                    2
  weight)

   Saline            Saline             173.6 [+ or -] 2.2 (a)
   Saline        2 x [10.sup.5]         143.8 [+ or -] 1.8 (b)
    1.34             Saline             180.9 [+ or -] 8.3 (a)
    1.34         2 x [10.sup.5]         149.6 [+ or -] 4.0 (b)

         Treatment                   Days After Bacterial Challenge

Astaxanthin        L. garvieae
(nmol/g body     (cfu/mL/prawn)                    4
  weight)

   Saline            Saline             167.5 [+ or -] 3.4 (a)
   Saline        2 x [10.sup.5]          92.4 [+ or -] 3.7 (c)
    1.34             Saline             162.0 [+ or -] 1.9 (a)
    1.34         2 x [10.sup.5]         112.9 [+ or -] 7.0 (b)

Data in each observation time with different letters are significantly
different (P [less than or equal to] 0.05). n = 4.

TABLE 6.
Concentration and percentage (in parenthesis) of diester astaxanthin
(DA), moncester astaxanthin (MA), free astaxanthin (FA), and total
astaxanthin (TA) in hepatopancreas of the giant freshwater prawn
Macrobrachium rosenbergii 7 days after astaxanthin injection at 1.34
nmol/g body weight challenged with Lactococcus garvieae.

Treatment                         Concentration (%) of astaxanthin
                                         in hepatopancreas
Astaxanthin      L. garvieae
  (nmol/g      (cfu/mL/prawn)                 DA (mg/kg)
body weight)

  Saline           Saline           13.73 [+ or -] 3.7 (b) (45.23)
  Saline       2 x [10.sup.5]       10.86 [+ or -] 2.2 (b) (37.69)
   1.34            Saline           72.07 [+ or -] 11.9 (a) (45.18)
   1.34        2 x [10.sup.5]       54.72 [+ or -] 4.5 (a) (35.84)

Treatment                          Concentration (%) of astaxanthin
                                        in hepatopancreas
Astaxanthin      L. garvieae
  (nmol/g      (cfu/mL/prawn)                 MA (mg/kg)
body weight)

  Saline           Saline           15.11 [+ or -] 1.3 (b) (49.78)
  Saline       2 x [10.sup.5]       16.80 [+ or -] 1.9 (b) (58.32)
   1.34            Saline           83.39 [+ or -] 8.1 (a) (52.28)
   1.34        2 x [10.sup.5]       94.26 [+ or -] 16.0 (a) (61.73)

Treatment                          Concentration (%) of astaxanthin
                                        in hepatopancreas
Astaxanthin      L. garvieae
  (nmol/g      (cfu/mL/prawn)                 FA (mg/kg)
body weight)

  Saline           Saline            1.51 [+ or -] 0.8 (a) (4.99)
  Saline       2 x [10.sup.5]        1.15 [+ or -] 0.2 (a) (3.99)
   1.34            Saline            4.05 [+ or -] 1.4 (a) (2.54)
   1.34        2 x [10.sup.5]        3.72 [+ or -] 1.1 (a) (2.44)

Treatment                         Concentration (%) of astaxanthin
                                        in hepatopancreas
Astaxanthin      L. garvieae
  (nmol/g      (cfu/mL/prawn)                 TA (mg/kg)
body weight)

  Saline           Saline            30.36 [+ or -] 5.3 (b) (100)
  Saline       2 x [10.sup.5]        28.81 [+ or -] 3.3 (b) (100)
   1.34            Saline           159.50 [+ or -] 6.5 (a) (100)
   1.34        2 x [10.sup.5]       152.71 [+ or -] 20.7 (a) (100)

Means [+ or -] SEM in the same column with different letters are
significantly different (P [less than or equal to] 0.05). n = 3.
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Author:Angeles, Isagani, P., Jr.; Chien, Yew-Hu; Yambot, Apolinario V.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2009
Words:6811
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