Biological and Health impact of Astaxanthin powders in common carp Cyprinus carpio L

The effect of Astaxanthin powders on biological, health and growth parameter of common carp Cyprinus carpio L. Fish weight varied between (59.2 – 66.4 g) were studied for 10 weeks. Four treatments (triplicate) were employed as follows: T1: Diet without any addition, T2: adding 2.5 g Astaxanthin powders /kg diet, T3: adding 5 g Astaxanthin powders /kg diet, T4: adding 7.5 g Astaxanthin powders /kg diet. Twice daily feeding at 9:00 a.m. and 2:00 p.m. Blood samples were taken from the caudal vein of fish. The results showed no significant difference between treatments in terms of biological parameter (p>0.05). The results showed a significant difference between treatments in terms of Health indices, including gonadosomatic, but no significant difference (p>0.05) hepatosomatic, spleenosomatic index and Gill index. The results showed a significant difference between treatments in terms of growth performance (p<0.05), including weight gain, Relative growth rate and Specific growth rate. Group results of blood factors showed that between Granulocytes, Lymphocytes and Monocytes there was no significant difference between treatments (p>0.05). Lastly, we found that diets containing higher levels of astaxanthin powders improve the growth performance, biological and GSI in common carp Cyprinus carpio L.


Introduction
Numerous studies have shown that astaxanthin has potential health-promoting effects in the prevention and treatment of various diseases, such as cancers, chronic inflammatory diseases, metabolic syndrome, diabetes, diabetic nephropathy, cardiovascular diseases, gastrointestinal diseases, liver diseases, neurodegenerative diseases, eye diseases, skin diseases, exercise-induced fatigue, male infertility as reviewed by Yuan et al. (2010). It has been proven in over 65 clinical studies, featured in over 300 peer-reviewed publications. The main benefits for human health. "Eye fatigue relieve" (Yamashita, 2009), "skin aging defence" (anti-photoaging) (Tominaga et al., 2012) and "muscle resilience" (sports performance enhancement) (Yamashita, 2011;Earnest et al., 2011) have been most clinically substantiated.
The natural sources of astaxanthin are algae, yeast, salmon, trout, krill, shrimp and crayfish.
Astaxanthin from various microorganism sources. The commercial astaxanthin is mainly from Phaffia yeast, Haematococcus and through chemical synthesis. Haematococcus pluvialis is one of the best sources of natural astaxanthin (Ranga Rao et al., 2010).
Carotenoids are pigments found in plants and animals, but only plants, including algae, some bacteria and yeasts are able to produce them. Nonetheless, structural variants of carotenoids can still be found in animals. Perciformes and Salmonidae fish synthesize zeaxanthin from astaxanthin; however, this is reversed in Cyprinidae fish, with astaxanthin being synthesized from zeaxanthin (Maoka, 2011). Research has shown that carp fed high-carotenoid diets retained serum astaxanthin and red color better than did other groups and had a higher phagocyte concentration (Yuangsoi et al., 2010). It has been shown that astaxanthin and β-carotene improves the reproductive performance and egg quality of female goldfish (Tizkar et al., 2013). Some microorganisms are rich in astaxanthin -the Chlorophyte Haematococcus pluvialis is a green microalga, which accumulates high astaxanthin content under stress conditions such as high salinity, nitrogen deficiency, high temperature and light (Sarada et al., 2012). Astaxanthin is closely related to other well-known carotenoids, such as b-carotene, zeaxanthin and lutein, thus they share many of the metabolic and physiological functions attributed to carotenoids. Free astaxanthin is particularly sensitive to oxidation. In nature, it is found either conjugated to proteins, such as in salmon muscle or lobster exoskeleton, or esterified with one or two fatty acids, which stabilize the molecule. Astaxanthin in H. pluvialis offered the best protection from free radicals in rats followed by β-carotene and lutein (Ranga Rao et al., 2013;Ranga Rao et al., 2010).
Astaxanthin contains a unique molecular structure in the presence of hydroxyl and keto moieties on each ionone ring, which are responsible for the high antioxidant properties (Kidd, 2011). Astaxanthin cannot be synthesized by animals and must be acquired from the diet. Although mammals and most fish are unable to convert other dietary carotenoids into astaxanthin, crustaceans (such as shrimp and some fish species including koi carp) have a limited capacity to convert closely related dietary carotenoids into astaxanthin, although they benefit from being fed astaxanthin directly. The consumption of astaxanthin can prevent or reduce risk of various disorders in humans and animals (Kidd, 2011).
The use of astaxanthin as a nutritional supplement has been rapidly growing in foods, feeds, nutraceuticals and pharmaceuticals. This present review paper provides information on astaxanthin sources, extraction methods, storage stability, biological activities, and health benefits for the prevention of various diseases and use in commercial applications, based on the benefits of Astaxanthin the present experiment will dome to assess their effects Saraddin et al., Biological and Health impact of Astaxanthin powders in some biological and Health indices in common carp Cyprinus carpio L.

Experimental fish
The experiment will be done 10 weeks on 60 common carp C. carpio L. brought from local fish ponds in Daquq/ Kirkuk/ Iraq. Fish weight varied between (59.2 -66.4gm). Fish distributed among experimental plastic tanks with mean initial weight 61.45 gm. laboratory pre-acclimation and feeding with commercial pellets (their Percentage of ingredients and chemical composition are seen in Table 1 and 2) were for 21 days prior to the real feeding trials.

Experimental system
Twelve plastic tanks (70 L water) will be used in this trial for four treatments each with three replicates. Proper continuous aeration added to each tank by using Chinese's air compressors, Hailea ACO-318. Each replicate was stocked with six fish. The replicates will be randomly placed to reduced differences among treatments. A daily cleaning by siphoning method will be applied to remove remained feeds and feces from the system. the experimental trial represent four treatments with three replicates; each with five fish per replicate as bellow: T1: Diet without any adiition, T2: adding 2.5 gm Astaxanthin powder /kg diet, T3: adding 5 gm Astaxanthin powder /kg diet, T4: adding 7.5gm Astaxanthin powder /kg diet.

Diet formulation
Experimental diets contain standard ingredients found in Sulaimani city markets, enriched with Astaxanthin powder. The items will be mixed to obtain dough. Then, using electrical mincer for pelleting by Kenwood Multi-processors. Room temperature drying used for a four days and crushed to obtained fine particles. Twice daily feeding at 9:00 a.m. and 2:00 p.m. with 3% of body weight. Fish in every tank will be weighed together bimonthly. The feeding levels will be then recalculated according to new weights. The feeding trial continued for 10 weeks.

Health and biological parameters
All fish specimens will be dissected and the abdominal cavity will opened to weigh each organ alone.

Growth standards used in the study
Fish weighed (g) together for all replicate every two weeks. Feed consumption of each replicate will be read just by the obtained biomass at every two weeks.

Statistical analysis
The trial will be conducted by one way (ANOVA) with completely randomized design (CRD) and general linear models (GLM) procedure of XLSTAT 2016 Version.02.28451. Duncan's test will used to compare among treatments means.

Biological parameters
Biological performance: Fish fed T2 (2.5gm astaxanthin powder), T3 (5 gm Astaxanthin powder) and T4 (7.5gm Astaxanthin powder) achieved higher Intestine weight index % and Intestine length index% compared to the CON without significant differences (P>0.05) ( Table 2), There were no significant differences among treatments in the Condition factor. Also, condition factor did not show any significant differences among studied groups, and no specific signs of disease were observed (P>0.05). Meat weight index % and Fish weight index% (P>0.05) ( Table 2). All groups fed with astaxanthin powder diets had better feed performance compared to the CON.

Health parameter
Data on some Body indices are shown in (Table 4). No significant (p>0.05) differences were recorded in HIS, SSI and GI (%). The T3 (5gm astaxanthin powder/kg) had a significantly higher GSI than fish fed the CON (P<0.05).

Blood parameter
In this experiment, blood factors including Granulocytes %, Lymphocytes % and Monocytes % there was no significant difference between treatments (p>0.05) ( Table 5).

Growth performances
Growth data of the Growth rate (GR) in fish fed the T2, T3, and T4 diets was comparable with the CON (P>0.05) (Table 6). However, fish fed the T4 (7.5gm Astaxanthin  Values are listed as mean± SD (n=3 in each group)  Comparisons of means (control and treated fish) Significant at 5% level (p <0.05). Con, control diet T, treatment and CF, Condition factor  Intestine weight index % = Intestine weight (gm) / Fish weight (gm) x 100  Intestine length index % = Intestine length (gm) / Fish length (gm) x 100  Condition factor = Fish weight (gm) / Fish length ( cm) 3  Meat weight index % = Fish weight without Viscera & Head (gm) / Fish weight (gm) x 100  Fish weight index % = Fish weight without Viscera (gm) / Fish weight (gm) x 100   powder/kg) and T3 (5 gm Astaxanthin powder/kg) achieved higher weight gain (WG), Relative growth rate (RGR%) and specific growth rate (SGR%) were significantly higher compared to the T2 (2.5gm of astaxanthin powder/kg) and CON (P<0.05).
The biological and physiological condition of the fish is among the key factors underlying the attainment of the required performance levels. This is why monitoring the biological state of the common carp (Cyprinus carpio L.) has become an integral part of the routine examination of the health of the fish, and won a permanent position within the complex of methods serving to interpret the results of feeding trials, involved in the testing of the biological. For the carp (Cyprinus carpio L.). Li et al., (2014) reported that diets containing astaxanthin and Haematococcus pluvialis efficiently improved growth factors in large yellow croaker. Shapoori et al., (2012) believe that the difference in the color intensity was caused by natural and artificial pigments are due to the quality, concentration, and the absorption period of these materials. Specifically, weight gain was significantly higher in fish fed with H. pluvialis than those fed with the diet containing astaxanthin. The present results show that astaxanthin powder can intervene, to a certain extent, with the biological and growth performance.
Carotenoids present in the diet, when consumed by fish, accumulate in the liver before being transferred to the ovaries in the late stages of maturity (Tizkar et al., 2013). Previous studies that examined the effects of carotenoid supplementation in rainbow trout, gilthead seabream and yellow tail cichlid broodstock (Lakeh et al., 2010;Scabini et al., 2010;Güroy et al., 2012) and carp (Tizkar et al., 2013) diets have been undertaken. Our results show that dietary supplementation of astaxanthin powder in common carp (Cyprinus carpio L.) Increased the GSI but did not affect the HSI. The pattern of carotenoid utilization between the gonads and the liver is in agreement with the results of similar studies with other fish (Sadekarpawar & Parikh, 2013). The carotenoid enriched diets produced a positive effect on goldfish GSI and could lead to an increase in gonad weight brood stock (Chainapong & Traichaiyaporn ,2013).The GSI and HSI indices are useful indicators of the effects of exposure to different substances, growth promoters and carotenoid in fish.
Considering the results of the leukocyte examination, we believe it is important to point out the significantly high levels of Granulocytes, Lymphocytes and Monocytes in the plasma of the fish fed the astanxanthin powder diet, however there was no significant difference between the groups fed astanxanthin powder and control in leukocyte count. Faghani et al., (2009) reported the level of Hct 21.1±0.65, HB 5.23±0.64 and Lym 82±5.1. Generally, increased lysozyme activity in the serum and mucus of fish can be indicative of the immune system stimulation and improvement of the immune response. By enhancing the complement system and lysozyme, carotenoid pigments increase the total number of leukocytes and phagocytes and thereby cause the stimulation of the immune system, increased immunity, and resistance to pathogens. Wang et al., (2015) reported that dietary astaxanthin in Pacific white shrimp (Litopenaeus vannamei) significantly affects the hemolymph immunological index, including total haemocyte counts, phagocytic activity of haemocyte, serum anti-superoxide radical activity, serum phenoloxidase activity, serum antibacterial activity and serum bacteriolytic activity.
Astaxanthin are reported to improve growth performance of fish with the reason that carotenoids may exert a positive influence on intermediary metabolism in aquatic animals. There are different views about the effect of carotenoid pigments on growth factors of different fish species. While some results indicate that these pigments cannot improve growth factors in rainbow trout, Atlantic salmon, cod, Arctic char, and red porgy (Sheikhzadeh et al., 2012), the results of some other researchers suggest the positive effect of these pigments on growth improvement. This difference may be attributed to fish species, development stages, and type of carotenoid.
(2011) believed that astaxanthin powder could enhance lipid utilization in whole fish and liver, providing more energy and consequently enhancing growth performance. The relative growth rate was higher in the fish feed astaxanthin powder throughout the experiment. In addition, they might lead to negative effects on the taste of food, physical quality of the pellets and nutrition balance of diets (Lim, 1989). However, the degree of this effect naturally depends on the feeding habit of the fish and the preparation of the diet. Carp fish is herbivorous fish and, therefore, adding plants to their diets will be naturally restricted.
Based on the results, the highest growth was related to treatment T4 and T3 and respectively lowest treatments T2 and T1 that similar conditions were attributed together. Our results relating to the study of growth are similar to the conclusions published by (Foss et al.,1984). Who in trials with rainbow trout weighting 0.35 kg, found no differences in growth rate when testing astaxanthin and canthaxanthin In the present experiment, the growth performance (WG, RGR and SGR) and feed utilization of fish fed diet with supplemental astaxanthin powder were significantly higher that of fish fed the control diet. However, effect of carotenoids on fish growth is controversial. Many earlier studies have reported that dietary astaxanthin has no significant influence on growth and flesh composition of fish (Zhang et al. 2012;Pham et al., 2014;Yi et al., 2014). Tukmechi et al., (2011) stated that some factors such as environmental factors, especially due to the coldness of the fish, seasons, salinity, photoperiod, temperature, density, physiological parameters, species, reproductive cycle, puberty status, age, gender, nutritional conditions, sampling time and method, and the accuracy and sensitivity of measurement methods can affect growth factors and survival and make a difference in the interpretation of researchers. Our results are also in keeping with the data of the growth trial conducted by (Rehulka and Zak, 1986).

Conclusion
In conclusion, treatments that fed with astaxanthin powder in terms of biological, health and growth parameter were in better condition than the control. According to the study, effective dose for the astaxanthin powder can be 7.5 gm/kg. Due to the conditions of rearing, doses may be changed. astaxanthin in the diet for the common carp Cyprinus carpio L. Our results have again the justification of using the methods of clinical hematology and biochemistry within the complex of methods employed in assessing the biological and productive effectiveness of feeds.