Blood Characteristics and Tissue Histology of Nile Tilapia (Oreochromis Niloticus Niloticus) Fed A Diet Containing Cheese Skipper (Piophila Casei) Larvae

A three-month laboratory feeding trial was conducted to evaluate the suitability of cheese skipper larvae [maggots] as an alternative protein source for Nile tilapia (Oreochromis niloticus niloticus) instead of fishmeal. The diets tested were a commercial diet (Diet 1, 0% maggot inclusion) and a maggot diet (Diet 2, 100% maggot inclusion). The values obtained for both treatment groups indicated nutritional adequacy of the diets. Non-significant differences were observed between both treatment groups for nearly all the hematological parameters. A marked increase in the total protein, ALT, AST and triglyceride levels was observed in the blood of fish fed the maggot diet compared to the levels in the blood of fish fed the commercial diet, suggesting health improvement in fish fed the maggot diet. The replacement of fishmeal with maggot meal is acceptable from a growth perspective and in terms of the observed histological architecture. The results show that the maggot diet can be conveniently used as a total replacement for fishmeal in the diet of Nile tilapia.

farm animals. Alteration in fish histology has been examined to identify changes, if any, in the tissue of fishes fed alternative feeds.
The digestive system of teleostean fishes has been widely studied and described morphologically to determine the functions of many specialized anatomical structures in relation to different feeding adaptations [22]. Histological analysis of the digestive system is considered to be a good and immediate indicator of the nutritional status of fish [23,24]. Histological methods used for assessment of different feed effects on the livers and intestines of fishes were reviewed and explained by Raskovic et al. [25]. The intestine and the liver are the most important organs involved in the digestion and absorption of nutrients from ingested food; therefore, monitoring of these organs is considered to be necessary [26] for assessing the effects of ingredients used as raw materials of animal/plant origin. In this study, blood characteristics and tissue histology were used to evaluate the suitability of cheese skipper larvae [maggots] as an alternative protein source for tilapia (Oreochromis niloticus niloticus).

Experimental design
A total of 120 healthy Nile tilapia (O. niloticus niloticus) were used in the present work. Nile tilapias [average initial weight 28-41 g and length 11-13.4 cm] were collected from the Nile River at Assiut, Egypt. The fish were acclimated in the laboratory for at least 21 days. During acclimatization, the fish were fed a commercial pellet diet twice per day and kept in a recirculation system, ensuring high water quality (dissolved oxygen: 5.6 mg/L, pH: 7.9, total NH3-N: 0.097 mg/L, and temperature: 25.5 °C). The composition of the commercial pellet diet is provided in Table 1.
After acclimatization, the fish were divided into two groups. The first group was fed a commercial diet (Diet 1), and the second group was fed a maggot diet (Diet 2) ( Table 1). Each group was assessed in triplicate. Experimental tanks were regularly cleaned, and faecal matter was siphoned out daily.

Sources of ingredients and diet preparation
Soybean meal, wheat bran, rice bran, mix oil, premix, dicalcium phosphate, and fishmeal were obtained locally from the market. The maggot meal used for this study was prepared in the laboratory during the experiment using the larval stage of Piophila casei skippers (fly larvae). The larvae were collected from conventionally prepared cheese by the floating method. The homemade cheese was mixed with running water, and the larvae that floated were collected with a sieve. Maggots were harvested, washed, killed in tepid water and dried for 36 hours at 60 °C in an oven. Dried samples were milled using mortar and pestle. The maggot powder was added to the components of the experimental fish food according to the recommended amounts listed in Table   1. Two test diets were formulated. Diet 1 (commercial diet) was formulated with the highest inclusion level of fishmeal and without maggot meal. Diet 2 (maggot diet) was formulated with the highest inclusion level of maggot meal and without fishmeal ( Table 1). All dry diet components, including vitamins and mineral mixtures, were thoroughly mixed with oil. Water was added, and the feed was pressed into pellets that were 1 mm in diameter. The wet pellets were dried for 3 days at room temperature and stored at -2 °C until use.

Biochemical analysis
Colorimetric determination of the selected biochemical parameters was performed using a spectrophotometer [Jasco-V530]. The absorbance of the sample was examined at an appropriate wavelength within a range of 340 to 546 nm according to the parameter tested. Commercial diagnostic kits from Biomatrix chemicals were used for assays of total protein content (g/dl) as described by Henry [29], cholesterol (mg/dl) as described by Thomas [30], triglycerides (mg/dl)as described by Friedewald et al. [31], calcium(Ca, mg/dl) as described by Fiereck [32], creatinine and urea (mg/dl) as described by Henry [29], glucose (mg/dl) as described by Trinder [33], and aspartate aminotransferase (AST, U/I)and alanine aminotransferase (ALT, U/I) as described by Reitman [34].

Histological analysis
At the end of the experimental period, three fish from each tank were sacrificed by decapitation. The livers and intestines were immediately dissected, fixed in 10% neutral buffered formalin, processed by conventional methods, sectioned at 3-5-µm thickness using a rotary microtome, and stained with hematoxylin-eosin

Statistical analysis
Data are presented as the means ± standard deviations. The data were analysed by one-way analysis of variance [ANOVA] using a data analysis software system [37]. Means were tested using Fisher's least significant difference [LSD] test. Two levels of significance were reported; *p<0.05; **p<0.01.

Results
Haematological parameters: Significantly (P<0.05) increased RBC, Hb, and MCH values were recorded in the blood of fish fed the commercial diet and those fed the maggot diet in the final samples compared to the initial blood samples ( in the blood samples from fish fed the commercial diet than in the blood samples from those fed the maggot diet (Table 2). In contrast, the final Hct concentration was higher in the blood samples from fish fed the maggot diet than in the blood samples from those fed the commercial diet ( Table 2). The final lymphocyte concentration was lower than the initial lymphocyte concentration in the blood of fish fed the commercial diet. The lymphocyte concentration exhibited a non-significant (P>0.05) increase in the blood of fish fed the maggot diet compared to those fed the commercial diet ( Table 2). The neutrophil concentration exhibited a non-significant decrease in the blood of fish fed the maggot diet. The final neutrophil concentration was higher in the blood of fish fed the commercial diet than in the blood of fish fed the maggot diet ( Table 2). The monocyte concentration exhibited a marked decrease in the blood of fish fed the commercial diet and those fed the maggot diet. The final monocyte concentration was the same for both diets ( Table   2). A significant (P<0.05) increase in the final concentration of eosinophils was observed compared to the initial concentration in the blood of fish fed the commercial diet and those fed the maggot diet. No significant difference was observed in the concentration of eosinophils between the fish fed the commercial diet and those fed the maggot diet (Table 2).    (Table 3).   (Table 3). In contrast, glucose, cholesterol, and calcium concentrations were significantly higher in the blood of fish fed the commercial diet than in the blood of those fed the maggot diet (Table 3). Non-significant changes were observed in the levels of urea and ALT in the blood of fish fed the commercial diet compared to the levels in the blood of those fed the maggot diet (Table 3).     The anterior and posterior intestines of fish fed the commercial diet showed detachment at the base of the villi between the circular muscles and villi (Figures 1 & 2). Lymphocyte infiltration in the

Discussion
The primary objective in fish nutrition is to provide a nutritionally balanced mixture of ingredients to support the vital functions of fishes at an acceptable cost [38]. Blood parameters are an important tool for monitoring both the nutritional status and health status of fishes [39]. In recent years, increasing attention and acceptance of this biomaterial [43]. The result also corroborates previous observations that maggot meal, similar to other animal protein sources, is well accepted and utilized by fish [44][45][46]. Some haematological values observed here appeared to be slightly lower than those reported for tilapia by some authors but were within the acceptable range. Notably, the variations in haematological values within species can be influenced by environmental conditions, sex, age, origin, breeding system, and feeding, among other factors [47].
There was no significant difference in WBC in fish fed the maggot diet ranged from 1.3% to 2.3%. This finding was consistent with the results of Kelly [50], who reported that monocytes constitute less than 10% of the total WBC in animals of all species.
Basophils were not observed in the blood of fish in this study. This finding is similar to the results obtained in most livestock. Kelly [50] reported that basophils rarely occur in the blood of all species of livestock. Biochemical parameters vary among species and can be influenced by many biotic and abiotic factors, such as water temperature, seasonal pattern, food, age and sex [18]. A marked increase in the total protein level was observed in the blood of fish fed the maggot diet compared to that in the blood of fish fed the commercial diet. The low plasma protein level observed in Nile tilapia fed the commercial diet may be a consequence of decreased protein absorption by the relatively short villi observed in these fish. These results suggested that fish health was improved when the fish were fed the maggot diet. Although there was an increase in serum protein levels, the increased levels of ALT and AST suggest protein catabolism at the high dietary protein levels present in the maggot diet [51,52]. The increase in ALT and AST activities observed in Nile tilapia fed the maggot diet may reflect the use of excess hydrocarbons from amino acids to meet energy demands.
Similar responses were observed in Oncorhynchus mykiss for ALT [53] and in Rhamdia quelen for AST and ALT [54]. Blood glucose levels may vary according to season and water temperature and may decrease with increasing ages and sizes of fishes [55]. Plasma glucose levels in fishes increase during stress, probably due to the action of catecholamine on stored glycogen in liver and other tissues [56]. Here, elevation of plasma glucose was not observed in either feeding group. The level of glucose was lower in the blood of fish were within the normal range, possibilities of anorexia, diabetes, liver dysfunction and malabsorption of fat, which are symptoms of abnormal glucose and glucose levels in the blood [57], were ruled out. The triglyceride levels in the blood of fish fed the maggot diet were significantly higher and nearly twice those found in fish fed the commercial diet. Similar results were obtained in Liza klunzingeri by Mohammadizadeh et al. [58]. The triglyceride levels increased significantly due to the increase in protein levels in the maggot diet, which may be because the muscle is a pivotal compartment that is directly linked to amino acid turnover. The level of calcium was significantly low in the blood of fish fed the maggot diet. The creatinine level was high in the blood of fish fed the maggot diet.
The urea level exhibited non-significant changes in the blood of fish fed the commercial diet and those fed the maggot diet during the experimental period. The histological structure of the fish digestive system has been well documented [59,60]. Examination of the intestinal histology of aquaculture species is important for understanding pathological alterations related to infectious diseases or promoted by nutritional sources [61]. Although fish histological studies provide much information regarding the gastro-intestinal tract [62], further information is needed regarding morphological adaptations to variations in diet nutrients, which could affect diet formulation. Histological analysis of the digestive system is considered to be a good method to determine the nutritional status of fishes [23,24], and identification of the structural variations is useful for studies on nutritional development [63,64].
Histopathological changes in the intestine may vary depending on the species and feed used in the experiments [65]. The flexibility of the piscine gastro-intestinal tract for adaptation to food availability has been well studied [65,66]. In the present study, the intestines of fish fed the commercial diet exhibited normal architecture, with short villi, a wide lamina propria, a large number of small goblet cells, lymphocyte infiltration, and blood congestion. In the case of Nile tilapia fed the maggot diet, the anterior intestine exhibited normal architecture, with long villi, a narrow lamina propria, few small goblet cells, and slight lymphocyte infiltration and blood cell congestion. The histological alteration observed in the intestines of fish fed the commercial diet, that is, shortening of the villi, widening of the lamina propria, lymphocyte infiltration, blood cell congestion, and increasing number of goblet cells, was previously identified as enteritis [67,37]. The same changes were also observed in the intestines of salmonids fed full-fat diets [68,67]. Generally, widening of the lamina propria was accompanied by profound infiltration of a mixed population of inflammatory cells such as lymphocytes, neutrophilic granulocytes, macrophages, and eosinophilic granular cells [67,68]. Similar results were observed when fishmeal was replaced with sunflower meal in the feed of sharpsnout sea bream (Dyploduspuntazzo Cetti) [69]. Villus length is a useful histological parameter that can be monitored not only in experiments regarding the replacement of fishmeal but also in the evaluation of different types of commercial feed.
Based on the present results, we can conclude that the selected commercial diet is rich in fat and proteins of plant origin compared to the maggot diet, which is a 100% animal protein product. An increase in villus length is associated with an increase in the surface area for absorption of nutrients [70]. The long villi found in fish fed the maggot diet indicate high efficiency in the absorption process [24,71]. This high efficiency was evidenced by the improved growth performance of fish fed this diet [12]. This finding shows that the maggot diet promoted an increase in villus length in these fish. A decrease in villus length leads to reduced surface area for nutrient absorption [71], which may also explain the poor condition of the liver observed in fish fed the commercial diet. The number of goblet cells could vary with feeding habits or starvation [72]. A higher number of goblet cells was observed in the intestines of fish fed the commercial diet than in those of fish fed the maggot diet. Goblet 226 cells are associated with the immune system and act through the mucus as a lubricant. The increase in the number of goblet cells may be an indication of increased irritation [71], as these cells produce the mucus lining the brush border. This mucus serves as a lubricant, providing protection against chemical and mechanical damage. The increase in goblet cell number may also be an immune response against anti-nutrients [73]. The liver is of significant importance for nutrition and homeostasis in fishes. The liver of O. niloticus niloticus fed the commercial diet showed normal architecture, with normal hepatocytes and blood sinusoids. Large intra-cytoplasmic vacuoles and blood cell congestion in the sinusoidal blood vessels were observed. Low glycogen levels were recorded. Additionally, a normal architecture was observed in the liver of Nile tilapia fed the maggot diet, with normal hepatocytes and blood sinusoids, small intracytoplasmatic vacuoles, and high glycogen content. In conclusion, the haematological and blood biochemical analyses suggest improvement of fish health upon dietary administration of maggotsupplemented feed. The obtained results showed no negative effect on the histology of the visceral organs of Nile tilapia fed the maggot diet, suggesting that this diet is essentially good in terms of growth and utilization [72][73][74]. The replacement of fishmeal with maggot meal is acceptable from a growth perspective and in terms of the observed histological architecture. Thus, maggot meal could be an alternative animal protein source in fish diets to lower the production cost of fish diets. In future experiments in the area of fish nutrition, the histological status of the intestine should always be considered. This analysis should provide additional information regarding the state of this organ if any of the mentioned methods are used [75,76]. These methods are valuable in field experiments as well as in the laboratory. Maggots are a good alternative protein source for O. niloticus niloticus. However, further studies with tilapia and other fish species are needed to validate this conclusion.