Astaxanthin in the diet positively impacts the reproductive characteristics of rainbow trout
The effects of dietary astaxanthin supplementation on reproductive characteristics were studied in five groups of broodstock fed diets containing 0.07, 12.46, 33.33, 65.06 and 92.91 mg*, respectively. kg -1 astaxanthin and two groups of broodstock rainbow trout were fed this diet supplemented with 0.07 and 33.33 mg astaxanthin kg -1, respectively, for 6 months in an artificial lighting system until sexual maturity. . Eggs from each group of broodstock salmon were divided into two equal batches. One batch was inseminated with homogenized sperm from four males fed a diet containing 0.07 mg astaxanthin kg -1 and the remaining with sperm from four males fed a diet containing 33.3 mg*kg -1 astaxanthin. Females produced eggs with astaxanthin concentrations ranging from 2.03 to 29.79 mg kg -1 . Dietary astaxanthin supplementation has a positive effect on the reproductive characteristics studied. There were significant differences in the fertilization rate, the percentage of eggs with eyes, the percentage of eggs with eyes, and the percentage of eggs with eyes between treatments (P < 0.05), but there was no significant difference in the percentage of eggs with eyes. Eggs die before hatching (P > 0.05). A significant difference (P < 0.05) in fertilization rate was found for male groups fed 0.07 and 33.3 mg astaxanthin kg −1. The astaxanthin content in eggs and the fertilization rate, the rate of eggs with eyes and the hatching rate were significantly correlated (P < 0.05). It was concluded that dietary supplementation of astaxanthin is necessary for optimal reproduction in rainbow trout.
Carotenoids are fat-soluble pigments responsible for the coloration of many animals ranging from yellow to red. They are biosynthesized by plants and some bacteria and fungi, but can be absorbed and metabolized by animals such as fish (Schiedt, 1998). Astaxanthin (3,3¢-dihydroxy-b,b-carotene-4,4¢-dione) is one of the major carotenoids in aquatic animals (Matsuno and Hirao, 1989), including salmon (Khareet al. , 1973; Schiedt et al., 1981, 1986). However, fish and other animals cannot biosynthesize carotenoids de novo and must obtain them from their diet (see Davies, 1985). Astaxanthin and canthaxanthin (b,b-carotene-4,4¢-dione) are the carotenoids most commonly used to pigment farmed salmon; Ingested carotenoids are deposited in muscle, liver and skin along with their metabolites (Torrissen et al., 1989; Store bakken and No, 1992). Fish have the ability to convert some carotenoids into vitamin A (Morton and Creed, 1939). Therefore, in addition to being responsible for mating color characteristics during sexual maturation, 4-ketocarotenoid also serves as a precursor to retinol and dehydroretinol (vitamins A1 and A2) in salmonids (Schiedt et al. al., 1985; Al-Khalifa and Simpson, 1988; Whiteet al., 2003). Sexually mature salmonid fish redistribute the carotenoid pool in their bodies in a process that involves the transfer of flesh carotenoids primarily to the skin of males and to the gonads of females (Steven , 1949; Crozier, 1970; Ando and Hatano, 1996; Concomitant with this redistribution, a significant amount of carotenoids are lost in the body (Crozier, 1970; Bjerkeng et al., 1992), and evidence suggests that carotenoid mobilization and metabolism are affected. by the sex steroid hormones 11-ketotestosterone and 17b-estradiol (Bjerkeng et al., 1999). Thus, during oogenesis, carotenoids are mobilized from muscle, incorporated into the developing ovary, and found in unesterified form in mature eggs (Steven, 1949; Kitahara, 1983, 1984). . Because they accumulate in the reproductive organs of a variety of organisms, carotenoids have long been thought to play a role in reproduction (Goodwin, 1950).
Several functions of carotenoids in fish have been proposed beyond their role as precursors of vitamin A (Tacon, 1981; Craik, 1985; Choubert, 1986; Torrissen, 1990). Therefore, adding astaxanthin to the diet of shrimp broodstock helps improve the quality of yellowtail eggs (Seriola quinqueradiata) (Vera-kunpiriya et al., 1997), eel eggs (Pseudocaranx dentex) (Vassallo-Agius et al., 2001), sea urchin (Lytechinus) variegatus) eggs (George et al., 2001) and giant freshwater shrimp (Cherax quadrucarin-atus). In addition, carotenoids improve growth and survival in species such as tiger shrimp (Pangantihon-Ku¨hlmann et al., 1998) and Japanese abalone Haliotis discus (Tsushima and Matsuno, 1998). It is thought that egg carotenoids provide an indication of egg quality (Hartmann et al., 1947). Hartmann et al. (1947) suggested that astaxanthin acts as a fertilization hormone by stimulating sperm attraction, leading to higher fertilization rates. Higher fertilization rates, sexual maturation rates, and survival rates have been reported for fish fed diets containing carotenoids (Hubbs and Strawn, 1957; Hubbs and Stavenhagen, 1958; Deufel , 1965; Georgev, 1971; Mikulin and Soin, 1975; On the other hand, Quantz (1980), Torrissen (1984), Craik and Harvey (1986), Tveranger (1986), Christiansen and Torrissen (1997) found no relationship between dietary carotenoids and mortality. of eggs, hatching rate and survival rate of alevin fish. and Choubert et al. (1998). According to Harris (1984), canthaxanthin does not affect the reproductive ability of rainbow trout (Oncorhynchus mykiss). Considering the wide variation in reports on the biological functions of astaxanthin in the reproductive cycle of different fish species, the objective of this study was to investigate whether reproductive characteristics such as fertilization rate and egg mortality was correlated with dietary astaxanthin concentration (0.07–92.9 mg kg )1) in cultured male and female rainbow trout.
Materials and methods
Fish rearing conditions
Two-year-old rainbow trout broodstock from Ghezel mahi fish farm (Urmiah, Iran) with an average weight of 846 ± 112 g and length of 43 ± 8 cm were used as broodstock. Fish were randomly distributed into seven indoor concrete tanks (8·1·1m3) and photoperiod controlled. A total of 85 females were stocked in five tanks (17 fish in each pond) and two tanks were stocked with 20 males (10 fish in each tank). Fish were kept at 6:00 a.m. and 6:00 p.m. over a 3-month period using an automatic timer. The light intensity on the water surface was measured using a photometer and the average light intensity over the entire period was 76.5 lx. The tanks were supplied with fresh water from deep wells, oxygenated and filtered through sand filters to avoid zooplankton contamination that might have provided the source of astaxanthin. Average water flow, dissolved oxygen, pH, and water temperature in all tanks were measured three times a week and ranged from 0.8 to 1.1 L·s-1, 7.5 to 7.8 mg L-1, 7.5 to 7.7 and 11.6 to 14.8°C, respectively, for the entire period. Five steam pellet diets were produced by a commercial feed manufacturer (Behparvar Feed Manufacturing Company, Tehran, Iran; Table 1). Four of the diets were supplemented with varying levels of astaxanthin (Carophyll Pink, 8% astaxanthin; DSM, Basel, Switzerland); an unsupplemented diet with similar composition served as control. Five groups of female broodstock were fed diets containing 0.07, 12.5, 33.3, 65.1 or 92.9 mg astaxanthin kg -1, respectively; two groups of male broodstock were fed diets containing 0.07 or 33.3 mg astaxanthin kg -1, respectively. Fish were fed three times daily at a rate of 1% of body weight during light periods (09:00, 12:00, and 15:00) throughout the entire period.
Sample collection and sample preparation for carotenoid analysis Egg samples were transported to AKVAFORSK (Sunn-dalsøra, Norway) in a tank of liquid nitrogen to reduce carotenoid degradation. The determination of astaxanthin in feed was based on the procedure for analyzing astaxanthin in fish feed described by Weber (1990). A homogeneous sample (approximately 5 g) was accurately weighed. The gelatin of Carophyll Pink microgranules consisting of a starch-coated matrix of gelatine, carbohydrates, and antioxidants was degraded by enzymatic treatment with encapsulated bacterial protease (30 mg; Maxatase P, Genencor International BV, Delft, the Netherlands) in distilled water (10 ml) in an ultrasonic water bath (30 minutes, 50oC). Ethanol(100 ml) was added and the sample was diluted to a volume of 250 ml with dichloromethane. The extract was purified by open column chromatography on silica gel 60 (Merck, Darmstadt, Germany; no. 7733), and the eluate was evaporated. After dissolving the sample in n-hexane:acetone (86:14), astaxanthin content was determined by high-performance liquid chromatography (HPLC), as described below. Egg samples (1.8–4.2 g) were added to methanol [2.0 ml, containing 500 ppm butylated hydroxytoluene (BHT) as antioxidant] and deionized water (1.0 ml), and the Samples were mixed (Whirlmixer, Fisons, England) for 20 seconds. Chlorine-form (6 ml) was added and the sample was mixed again for 20 s (cf. Bjerkeng et al., 1997; Wathne et al., 1998). After settling for 10 min, the sample was mixed again and centrifuged (approximately 1700 g, 10 min). A small amount (2 ml) of hypophase containing astaxanthin dissolved in chloroform was pipetted into an atest tube and the solvent was evaporated on a water bath (approximately 40oC) using a gentle stream of nitrogen gas. After evaporation, the sample was dissolved in 20% acetone in n-hexane (3 ml). The solution was filtered (0.45 lm; Minisart SRP15, Sartorius, Germany) directly into the sample vial and immediately sealed. Samples were analyzed by HPLC on the same day, as described below. by enzymatic treatment with encapsulated bacterial protease (30 mg; Maxatase P, Genencor International BV, Delft, Netherlands) in distilled water (10 ml) in an ultrasonic water bath (30 min, 50oC). Ethanol(100 ml) was added and the sample was diluted to a volume of 250 ml with dichloromethane. The extract was purified by open column chromatography on silica gel 60 (Merck, Darmstadt, Germany; no. 7733), and the eluate was evaporated. After dissolving the sample in n-hexane:acetone (86:14), astaxanthin content was determined by high-performance liquid chromatography (HPLC), as described below. Egg samples (1.8–4.2 g) were added to methanol [2.0 ml, containing 500 ppm butylated hydroxytoluene (BHT) as antioxidant] and deionized water (1.0 ml) and the Samples were mixed (Whirlmixer, Fisons, UK) for 20 seconds. Chlorine-form (6 ml) was added and the sample was mixed again for 20 s (cf. Bjerkeng et al., 1997; Wathne et al., 1998). After settling for 10 min, the sample was mixed again and centrifuged (approximately 1700 g, 10 min). A small amount (2 ml) of hypophase containing astaxanthin dissolved in chloroform was pipetted into an atest tube and the solvent was evaporated on a water bath (approximately 40oC) using a gentle stream of nitrogen gas. After evaporation, the sample was dissolved in 20% acetone in n-hexane (3 ml). The solution was filtered (0.45 lm; Minisart SRP15, Sartorius, Germany) directly into the sample vial and immediately sealed. Samples were analyzed by HPLC on the same day, as described below.
Feed analysis
The approximate composition of the feed is analyzed at the Behparvar Food Manufacturing Company laboratory. Crude lipid content was determined gravimetrically using soxhlet extraction with diethyl ether as solvent. Crude protein content (N·6.25) was determined using the Kjeldahl method. Dry matter content was determined after heating the sample to 105°C for 24 hours; Ash content was determined after burning at 550oC for 12 hours. The approximate composition of the diet was determined three times.
HPLC analysis
HPLC analyzes were performed on a Shimadzu LC-10ASLid liquid chromatograph connected to a Shimadzu SPD-M6A photodiode array UV-VIS detector and a Shimadzu CBM-10A Communications bus module and detection wavelength was set to 470 nm. Sample application performed by aShimadzu SIL-10 autoinjector. All chromatograms were re-integrated (CLASS LC10 software CLASS LC10; Shimadzu, Japan) for baseline correction. Two isocratic HPLC systems were used to separate and quantify carotenoids (see Bjerkeng et al., 2000). System I consisted of a nitrilecolum Spherisorb S5-CN column (PhaseSep, Queensferry, Clywd, UK; length 250 mm,Ø 4.6 mm, particle size 5 lm), using 20% acetone in n-hexane mobile phase ( flow rate 1.5 ml-1 min, pressure approximately 52 bar). System II consisted of an H3PO4-modified silica gel column (Hibar, LiChrosorb Si 60, 5 lm particles, Ø 4.6 mm, 125 mm length; Merck, Darmstadt, Germany) as described by Vecchi et al. (1987). The mobile phase was 14% acetone in hexane, and the flow rate was 1.2 ml min-1 (pressure about 36 bar). The mobile period is renewed daily. The HPLC I system is used for the quantification of polar carotenoids that may include the 3¢,4¢-cis to 3¢,4¢-trans glycolic isomers of idoxanthin (3,3¢,4¢-trihydroxy- b,b-carotene-4¢- one). Standards of idoxanthin (3¢,4¢)-cis and -trans glycolic isomers were prepared according to Aas et al. (1997). An HPLC II system was used to quantify all-E/Z isomers of astaxanthin. Individual carotenoids were quantified from HPLC chromatogram areas and corrected for differences in molecular absorbance (E1%,1 cm) at the detection wavelength (470 nm). The E1%,1 cm values used were 2100 for all-E-astaxanthin (Britton, 1995), and 1350 and 1750 for 13Z- and 9Z-astaxanthin, respectively. The E1 cm,1% values for 13Z- and 9Z-astaxanthin were estimated based on the HPLC response factors associated with all-E-astaxanthin reported by Schu¨ep and Schierle (1995). Standards of known concentrations were prepared from all-E-astaxanthin crystals (Hoffmann-LaRoche Ltd, Basel, Switzerland) and these standards were run each time the samples were analyzed. The concentration of the standard solution was measured spectrophotometrically (UV-260;Shimadzu) using E1%,1 cm ¼ 2100 at maximum absorbance (kmax ¼ 472 nm)
Statistical methods
The statistical design used in this study was a factorial experiment in a completely randomized design. The first factor was the treatments that fed the broodstock a diet containing five levels of astaxanthin (0.07, 12.46, 33.33, 65.06, and 92.97 mg kg−1; ). The number of replicates was not the same for all treatments, as there were only three adult females in the group fed a diet containing 0.07 mg.kg-1 astaxanthin during the maturation test period. genital. Five fish per treatment were used from the other groups. Fertilization rate is calculated = 100 *fertilized eggs (total number of eggs-1 ), and percentage of eggs with eyes = 100 *eggs with eyes. (total number of eggs-1 ), mortality rate to eye stage = 100 *number of dead eggs (total number of eggs-1 ), hatching percentage = 100 *number of hatched eggs (total number of eggs-1 ) and number Dead eggs from the eye stage to hatching = 100 *number of dead eggs from the eye stage to hatching (total number of eggs-1). Data were analyzed completely at random using the GLM method and assessed for normal distribution using the Kolmogorov–Smirnov method (Minitab Statistical Package, Version 13.1, Minitab Inc., PA, USA). The data were normally distributed and no transformations were performed. Data were subjected to ANOVA processing using SAS software (Version 6.03). The Duncan test was used to rank the mean values. The effect of astaxanthin concentration in eggs on the fertilization rate, the rate of eggs with eyes, the hatching rate and the effect of astaxanthin concentration in food on astaxanthin concentration in eggs were investigated by linear regression analysis ( SPSS version 11, SPSS Inc., Chicago, IL, USA). The difference was considered significant when P < 0.05. Results are presented as mean ± SE
Result
The diets contained an average of 81.3, 3.6, and 15.0 percent of total astaxanthin of the all-E-, 9Z-, and 13Z geometric isomers, respectively. A similar isomeric composition was found in eggs of all treatment groups (mean all-E: 84.3 ± 1.4% total astaxanthin and mean total Z isomers: 15, 7 ± 1.4% of total astaxanthin, respectively). b-Adonixanthin (3,3¢-dihydroxy-b,b-carotene-4-one) was detected in eggs of all treatment groups and on average comprised 18.2%, 5.7%, 3, 5%, 2.5% and 1.1% of total carotenoids in the groups by increasing dietary astaxanthin levels. Idoxanthin comprises <0.2% of total carotenoids in eggs. Only three individuals fed a diet with 0.07 mg astaxanthin kg-1 were found to be sexually mature; All fish in the other treatment groups reached sexual maturity within the 30-day control period following routine light treatment. The average egg astaxanthin concentration ranged between 2.0 ± 0.4 and 29.8 ± 4.8 mg kg−1 astaxanthin (Table 2). Astaxanthin concentrations in food and eggs were significantly correlated (P < 0.05, R2=0.78; Figure 1a). Fertilization rate and astaxanthin concentration in the feed were significantly related curvilinearly (P < 0.05, R2¼0.35; Fig. 1b), and fertilization rate showed a maximum for eggs of treated females. fed the diet at 65.1 mg*kg-1 astaxanthin . Fertilization rates were significantly lower in eggs from females fed a diet containing 0.07 mg kg−1 astaxanthin than in the other treatments (P < 0.05). Similarly, the percentage of eggs with eyes increased with increasing dietary astaxanthin concentration (P < 0.05). ; Table 3). The relationship between astaxanthin content of eggs and the percentage of eggs with eyes was significant (P < 0.05; Figure 1c). Egg hatching rate also increased with increasing astaxanthin content in the diet (P < 0.05). Regression analysis showed a significant relationship between egg astax-anthin concentration and hatching rate (P < 0.05; Fig. 1d). Eggs from females fed the 0.07 mg*kg-1 astaxanthin diet had significantly higher mortality than eggs from females fed other diets (P < 0.05; Table 3). Fertilization rates were 2.5% higher for eggs fertilized with sperm from males fed a diet containing 33.3 mg*kg-1 astaxanthin compared to eggs fertilized with sperm from males fed fed a diet containing 0.07 mg astaxanthin kg)1 (P < 0.05; Table 4). However, the eyed eggs, fertilization mortality at the eyed stage, and hatching rates of eggs fertilized by sperm from males fed diets supplemented with different levels of astaxanthin was significantly affected by dietary astaxanthin concentration (P > 0.05; Table 4).
DISCUSSIONS
Carotenoid content and composition
Variation in carotenoid content in both muscle and eggs is large between individuals and between species of salmon (Craik, 1985). Most astaxanthin deposited in wild salmon eggs is not esterified (Glover et al., 1951; Craik, 1985; Craik and Harvey, 1986). Only unesterified carotenoids were detected in this study, and total concentrations ranged from 2.0 to 29.8 mg kg-1 wet weight in a dose-dependent manner. Wild Atlantic salmon (Salmo salar) eggs contain approximately 6.4 ± 1.75 mg kg−1 astaxanthin (Craik, 1985), but concentrations up to 21.2 mg kg−1 canthaxanthin have been reported for with eggs of farmed Atlantic salmon fed diets containing canthaxanthin (Craik and Harvey, 1986). Astaxanthin concentrations ranging from 0 to 14.7 mg kg) 1 wet weight have been reported in eggs of Atlantic salmon fed diets containing 0–100 mg.kg-1 astaxanthin (Christiansen and Torrissen , 1997). The poorer utilization of astaxanthin in Atlantic salmon compared with rainbow trout (see Storebakken et al., 1986) is also reflected in high carotenoid concentrations in eggs of the latter species. E/Z geometric isomers of astaxanthin are used differently in rainbow trout. Therefore, the apparent digestibility coefficient (ADC) of all-E-astaxanthin is higher than that of 9Z- and 13Z-astaxanthin, and the ADC of 13Z-astaxanthin is significantly higher than that of 9Z-astaxanthin (Bjerkenget al., 1997 ). Furthermore, all-E-astaxanthin accumulates in plasma and muscle (>92% of total astaxanthin, regardless of dietary composition), whereas 13Z-astaxanthin accumulates in the liver (Østerlie et al., 1999). Thus, the eggs in the present experiment appear to have a slightly higher affinity for astaxanthin Z isomers than plasma and muscle tissues, but the reason for this remains unclear. Therefore, astaxanthin concentration rather than isomeric composition appears to account for the observed therapeutic effects. Astaxanthin is extensively transformed in rainbow trout, and some metabolites are present in the skin (Schiedt et al., 1985). The presence of b-adonixanthin and small amounts of idoxanthin in eggs indicates that this tissue also has a certain affinity for astaxanthin metabolites. Significant differences in the distribution patterns of astaxanthin metabolites can be found in eggs of different salmon species (Miki et al., 1982). While idoxanthin and cruaxanthin(3,4,3¢4¢-tetrahydroxy-b,b-carotene) comprised more than 70% of the total carotenoids in the eggs of Arctic char Salvelinusalpinus (Bjerkeng et al., 2000), idoxanthin comprised <0.2% of total carotenoids and rustaxanthin were not detected in the present study.
Reproduction
Carotenoids may play several important roles in the growth and reproduction of crustaceans (reviewed by Lin˜a´n-Cabello et al., 2002), and improve hatching and prevent malformations. disability and increased survival in the sea urchin Pseudocentrotus depressus (Tsushima et al., 1997; Kawakami et al., 1998), and improved growth and reproductive outcomes in the guppy Poecilia reticulata (Karino and Haijima, 2004). . In line with this, we found positive effects of egg concentration and diet on fertilization rate, eyed egg hatching rate, and eyed egg mortality of fish. return. In addition, in ferrets, Mustela vison, astaxanthin reduces the rate of stillbirth (Hansen et al., 2001). Astaxanthin can act as a fertilization hormone, increasing the rate of fertilization by stimulating and attracting sperm (Hartmann et al., 1947). Several studies have been conducted to test this hypothesis but the results from these studies are conflicting (Christiansen and Torrissen, 1997). High concentrations of astax-anthin and canthaxanthin in eggs increase fertilization rates in rainbow trout (Deufel, 1975; Craik, 1985). According to Mikulin and Soin (1975), astaxanthin can improve egg quality through its role in metabolism during embryonic development. However, there was no difference in the fertilization rate of eggs from rainbow trout fed different levels of canthaxanthin and astaxanthin before spawning, as discovered by Quantz (1980) and Christiansen and Torrissen (1997) . Likewise, no significant differences were found in the fertilization rates of eggs from rainbow trout fed 10% krill meal as a source of astaxanthin and a zero control group (Tveranger, 1986). . However, dietary levels of astaxanthin are low. Differences in fertilization rates between eggs fertilized by sperm from males fed astaxanthin and eggs from the same females fertilized by sperm from males fed an unsupplemented control diet , suggesting that astaxanthin may have a positive impact on the sperm quality of male broodstock, b-Carotene has been shown to improve the semen quality of male rats by reducing lipid peroxidation (El-Demerdash et al., 2004). Whether the positive effects of astaxanthin may be due to reduced lipid oxidation in male rainbow trout remains to be elucidated.
Large eggs of different species require higher amounts of carotenoids than small eggs for metabolic processes during the embryonic stage, and there is a correlation between the length of the embryonic development period and the carotenoid content of eggs in each species. (Mikulin, 2003). If astaxanthin is used during embryonic development, there will be a decrease in astaxanthin concentration from the incubation period to the active feeding period. A positive effect of egg carotenoid content and survival rate during embryonic development was found. Hubbs and Strawn (1957) found increased survival of pigmented eggs in Etheostoma lepidum compared to pale eggs of this species. However, the large variation in survival rates of eggs with low astaxanthin content may be due to farm-specific factors (Craik, 1985). Therefore, Craik (1985) suggested a critical level of carotenoids ranging from 1 to 3 mg kg−1), below which hatchability may decrease. We found that astaxanthin content in eggs and survival rate from incubation to hatching were correlated, eggs with eyes and hatching rate were higher, and mortality rate up to the eye stage was higher. lower in offspring from parents supplemented with astaxanthin in the diet. The average astaxanthin concentration in the eggs of the control group (2.0 ± 0.4 mg kg−1) was within the range indicated by Craik (1985), and the fertilization rate, the proportion of eggs with eyes and Survival to hatching was lower in this group than in the treatment groups with the highest astaxanthin levels. In conclusion, dietary astaxanthin supplementation increases egg astaxanthin concentrations and appears to be an effective factor in improving egg quality, and thus survival and mortality. during embryonic development. Although astaxanthin concentrations in the male reproductive organs are low, the present results indicate that dietary astaxanthin supplementation has a positive effect on fertility and corroborate the recommendation of Torrissen et al. Christiansen (1995) on supplementing dietary astaxanthin levels above 10 mg kg-1) to ensure fish health.
By M. R. Ahmadi1, A. A. Bazyar2,S.Safi3, T. Ytrestøyl4and B. Bjerkeng41Department of Health and Aquatic Diseases, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran;2Department ofFisheries and Environmental Sciences, Faculty of Natural Resources, Tehran University, Karaj, Iran;3Department of ClinicalPathology, Faculty of Veterinary Specialized Sciences, Science and Research Campus, Islamic Azad University, Tehran, Iran;4AKVAFORSK, Institute of Aquaculture Research AS, Sunndalsøra, Norway
