Strawberry Calyx and its Impact on Fish Production Performance

Background and Objective: The plant wastes (inedible parts) contain many functional and nutraceutical compounds and are a rich source of bioactive components and fiber. These by-products have a bad effect on the environment. This study was conducted to evaluate the nutritional value of Strawberry Calyces (SC) when added as an additive to the tilapia diet and to investigate its effect on growth performance, feed utilization and the physiological status of fish. Materials and Methods: A 70 days nutritional experiment was done on four groups of tilapia fry with an initial weight of 1.53±0.02 g, which were fed on test diets (40.03% protein, 399.61 kcal) as control (0%), SC1 (1%), SC2 (2%) and SC3 (3%) of strawberry calyces. Results: The results cleared those strawberry byproducts, rich in nutritional values with a high percentage of dry matter which consists of main components such as phenolics, vitamins, minerals and considerable amounts of fatty and amino acids, that are promising when used as feed additives. Growth performance (weight gain, specific growth rate and length gain) was improved by increasing the level of additive up to 3%, compared to control. Also, feed utilization and body composition were enhanced for the fish. At the same trend, the immune response (IgM and IgD) was improved for the three examined fish groups especially cleared in those fed SC2 and SC3. Conclusion: This article hopes to be applied to the other plant residues and extend such studies to the use of many different plant residues that pollute the environment if left and study the impact of their use in feeding aquatic animals at various levels.


INTRODUCTION
The processing of fruits and vegetables for discarding a huge amount of by-products, which consisted of inedible such parts as peel, seeds, stems and leaves. Almost, these unused tissues are engendering a bad impact on the environment 1 . These wastes contain many functional and nutraceutical components that can be reused in a lot of industries. They are sources of bioactive compounds such as vitamins, minerals, phenolic compounds, terpenes and fibers, mainly composed of lignocellulose 1,2 .
The strawberry, family Rosaceae and genus Fragaria are worldwide distributed 3 . Regarding to its nutrient value, it represents a good healthy food choice. Strawberries have fiber and fructose contents that may be considered a blood sugar level regulator and are a source of healthy essential fatty acids [4][5][6] . Strawberry is an excellent source of manganese, potassium and a good source of iodine, magnesium, copper, iron and phosphorus 5 .

Flesh condition indicators:
The normality of the fish body growth was measured using the formula 19 : 3 Fish wet weight (g) Condition Factor (CF) = × 100 Fish length (cm ) Hepato-Somatic (HSI) and Gastro-Somatic (GSI) were calculated as the following equations by 20 :  Liver weight (g) HIS = 100 Total body weight (g) Gut -weight (g) GSI = ×100 Total body weight (g) Digestibility: Apparent Protein Digestibility (APD) was measured by using 21 methods. Fish final weight was recorded, then the digestion trial was started where the uneaten diet and feces were collected once daily for 15 days by siphoning. Feces were collected separately, 2 hrs after feeding, then filtered and dried at 60°C and stored for determining the chemical composition. Crude protein contents of the diets and feces were determined. Feces were analyzed separately to determine their values of dry matter and CP.
Biochemical studies Plant materials analysis: Samples of the additive were analyzed for Crude Protein (CP), Ether Extract (EE), Crude Fiber (CF), ash and minerals using the 22 reference methods.

Assessment of the main vitamins in byproducts:
C Ascorbic acid determination: The sample (1.0 g) were extracted in 4% oxalic acid and the solution was brought to 100 mL (V1 mL) and centrifuged at 4032×g for 10 min. Then, 5 mL of the supernatant was mixed with 10 mL of 4% oxalic acid, then the solution was titrated against the dye (V2 mL) 23 : 0.5 mg/V1 mL ×V2/5 mL ×100 mL Ascorbic acid (mg / 100 g) = ×100 Sample weight (g) C Determination of vitamins: The essential vitamins A, E, K and B were measured in the Central Laboratory of Agricultural Ministry, Dokki, Giza Amino and fatty acids profiles: Fatty acid and amino acid compositions of the residuewere measured in the Central Laboratory of Agricultural Ministry, Dokki, Giza and were assessed by using the methods recommended in the studies of Bireche et al. 24 .
Liver health signal of fish: At the end of the trial, random samples of fish liver were taken from each aquarium, then liver tissues were homogenized by 5 mL distilled and liver glycogen was measured (g/100 g fresh tissue) using the following formula: Absorbance sample Volume of dilute factor Hepatic glycogen= ×Conc. of the standard × Absorbance standard Weight of tissues Total liver protein was extracted by homogenization in trichloroacetic acid, then centrifuged (1008×g) and the hepatic protein content was determined by 25 using the following formula: Absorbance sample Volume of dilute factor Hepatic protein = × Conc. of the standard × Absorbance standard Weight of tissues The samples of 0.5 g of liver were preserved in a 2:1 mix of chloroform/methanol for posterior lipid class analysis 26 , then the liver lipid content which detected by 25 : Absorbance sample Volume of dilute factor Hepatic lipid = × Conc. of the standard × Absorbance standard Weight of tissues Carcass analysis: At the end of the experiment, fish samples were taken randomly and analyzed for adjusting the moisture oven-dried (at 85°C till constant weight), crude protein using Kjeldahl, total lipids by the method of ether extraction and the total ash (550°C for 6 hrs) 22 .
Physiological status: Blood samples (0.5-1 mL blood), at the end of the investigation periods, were taken randomly, by puncture of the caudal vein, using the heparinized syringes for some plasma parameters and syringes without anticoagulant for obtained serum, which was separated by centrifuging at 3000 RPM for 10 min 27 . Plasma was submitted to measure plasma protein (PTP) according to Riche 28 . The immunoglobulin (IgM and IgD) were assessed according to Brydges 29 .

Data statistical analysis:
The data were subjected to Analysis of Variance (ANOVA) using the General Linear Models (GLM) procedure, the software used was SPSS (Version 16.0). Duncan's multiple range tests were used to compare between means of the control and treated groups, the model of analysis was as follows: Where: μ = Overall mean T i = Effect of treatment E ij = Random error

Bioassay of strawberry residues
Proximate composition of the strawberry calyces: The present results ( Mineral and vitamin in the additive: Table 3 and Fig. 1 show that SC is a rich source of main minerals such as potassium, sodium, calcium, phosphorus and magnesium and contained vital vitamins like vit. C (20.2 mg/100 g) and minor levels from other vitamins (E, B6, A and K).
Fatty acids content of additive: Table 4 and Fig. 2 verified that calyces contain, saturated and monounsaturated fatty acids196.3 and 185.23% besides to considerable level of polyunsaturated group (523.83%) which consists of ω6 (307.03%) and ω3 (82.11%).    Essential amino acids of the strawberry by-product: The results in Table 5 and Fig. 3 cleared those strawberry calyces have a considerable content of essential amino acids, where glutamic acid was the dominant.
Fish growth performance: Water parameters were in within normal ranges through all the experimental days. Table 6 showed that fish growth parameters as the gain in weight, specific growth rate (Fig. 4)

Food utilization parameters:
The present data (Table 7 and Fig. 6, 7) cleared that PPV, PER, EER, FCE and FCR were the best values (p<0.05) for those fed the levels 2 and 3% SC, compared with the control one.      Carcass composition of test fish: Data of proximate analysis of fish clarified that the protein content was increased with the increase of the inclusion level of strawberry calyces, accompanied with decreasing of lipid content (Table 8 and Fig. 8). Present results recorded that fish fed (2 and 3%) SC had a higher content of Ash than the control (p<0.05).        Liver composition: Table 9 and Fig. 9 verified that adding the three levels of strawberry calyces for fish diet resulted in high hepatic protein and glycogen, on the other hand, hepatic lipid was decreased with SC inclusion level compared to the control (p<0.05).

Physiological measures in blood:
Blood parameters of the fish revealed that Immunoglobulins (IgM and IgD) and Plasma Total Protein (PTP) were enhanced (p<0.05) for the three test fish groups compared to control one (Table 10 and Fig. 10).

DISCUSSION
The present result showed that Strawberry Calyces (SC) contain ambient amounts of macro-nutrients indicating that, SC acts as a good ingredient for fish diets 5,6,30 . At the same trend, results cleared that the SC contains a lot of some micro-nutrients (minerals and vitamins), which play a vital role in fish performance 30,31 . Stated that the micro-nutrients play an important key in fish growth and immunity.
Strawberry by-products had a similar nutrient composition as strawberry fruit 5,6 .
Giampieri et al. 5 mentioned that strawberry fruit is considered a good source of manganese and potassium and also contains iodine, magnesium, copper, iron and phosphorus. The strawberry contains considerable amounts of vitamin C, even higher than citrus fruits, also some of many other vitamins in minor amounts as a vitamin E, A, B6 and K, thiamine, riboflavin, folate acid and niacin 5,8,32 .
It should be noted from the present data, the residue (SC) is considered the best source of antioxidant agents, these results are consistent with the interpretation by Nowicka et al. 33 , who mentioned that antioxidant agents in strawberry wastes can neutralize different free radicals and other reactive oxygen species.
The nutritional composition of fish (as lipids and fatty acids) substantially varies due to the differences in their habitats. In general, freshwater fish require either linolic acid (18:2 n-6), linolenic acid (18:3 n-3) or both, which are stored in muscle tissue to meet physiological needs and are also related to fish growth and survival rates 34 .
Results with respect to fatty acids recorded that SC contains a high level of ω-6, so the SC acts as a good ingredient for fish feeding, which it agrees with 30 , when they studied other plant calyces.
The present data showed that SC consists of a balanced profile of amino acids, so, our results are similar to that recorded by Attalla et al. 30 , who mentioned that some plant calyces (eggplant, pepper and tomatoes) contain an ambient profile of amino acids and glutamic acid was the dominant non-essential amino acid.
A promise growth performance in our results cleared that, the test fish which fed on three levels of SC, especially, in the second and third levels, these results are similar to the results by Attalla et al. 30 , who fed Nile tilapia a diet containing different plant calyces.
These findings may be due to the plant by-products (SC) containing a nutritional value as mentioned previously ( Table 2-5) and also being rich in a bioactive compound 5,6 . Many studies by other scientific researchers confirmed that bioactive compounds improved fish growth performance, body condition and survival numbers fish 30,31 .
Optimum results of feed efficiency were accompanied by the lowing of the amount of feed consumed by the test fish for the three examined fish groups, when compared with control, these promising data may lead to saving a considerable part of the cost of feed which represents about 70 % of total fish culture cost.
The digestibility depends mainly on the feed intake and fiber content which increase the movement of the fish intestine causing an increased digestibility. These results may owe to the high fiber content of SC that enhanced the health of the digestive canal 30,31 .
Although, fiber does not provide the body with nutrients, it is essential in the diet of fish, which lead to promotes some health benefits when using the plant residues and is considered a good food ingredient. Insoluble fiber acts as a regulator and for maintaining digestion efficiency 30,31,[35][36][37] .
The chemical composition of the examined fish was positively improved by SC inclusion, which may be due to the presence of bioactive components and essential amino acid content of the additive that enhanced the flesh quality of fish 5,6,30 . Also, the high percentage of minerals in the strawberry calyces may increase the mineral content in the fish flesh 30,31 .
The fish fed on SC showed enhancement of liver composition, which could be due to the considerable fiber content in SC that may act as liver health protectors, these results agree with those recorded by 5,6,30,31 .
The blood parameters were improved by the additive inclusion level in the fish diet (p<0.05), these findings are due to the great contents of phenolics, minerals, vitamins (C, K, E, and B) and amino acids in the additive, these results at the same trend with data wrote by 5,6,30,38 when they added that, vitamin C has an antioxidant activity that improved immunity with lowing IgM. At the same trend 39,40 observed that IgM start to increase when an animal was infected harmfully and then returned to the optimal ranges when infection was reduced, this fact verified our findings, where the additive (SC) may reduce infection or enhance the immune response system of examined fish, with minimizing IgM.
The strawberry contains considerable amounts of vitamin C, even higher than citrus fruits, contributing to 24% of the antioxidant capacity of the strawberry, also it is a good source of many other vitamins such as vitamin E, A, B6 and K, thiamine, riboflavin, folate acid and niacin 5,8,32 . Also [8][9][10] added that vitamin C and folate are considered important factors in health promotion and disease prevention, where strawberry is the richest natural source of this essential micronutrient.
The presence of the mucous layers on the skin surfaces of the teleost fish causes permanent exposure to a large number of pathogens and high densities of commensal microorganisms in the aquatic environment. The B cells and intra-mucosal immunoglobulins associated with lymphoid tissue play key roles in local mucosal adaptive immune responses. To date, three Ig isotypes (that is, IgM, IgD and IgT/Z) have been identified from the genetic sequences of different species of teleost fish. Crucially, teleost IgT represents the oldest class of AB specialized in mucosal immunity and plays important roles in removing mucosal pathogens and maintaining microbial homeostasis 41 .

CONCLUSION
The use of the strawberry residue in the tilapia diet can obtain improving fish growth and immunity with optimum quality of flesh and may also reduce environmental pollution.

SIGNIFICANCE STATEMENT
This study discovered the strawberry calyces that can be beneficial for fish nutrition as feed additive that may enhance fish growth performance and immunity. This study will help the researchers to uncover the critical areas of the shortage of fish feed ingredients and may also contribute to reduce environmental pollution and enhance economical profits that many researchers were not able to explore. Thus a new theory on processing of strawberry calyces may be arrived and using as important additive in fish nutrition.