Abstract: This study investigates the histological changes of kidney and liver of Clarias gariepinus fed diets containing Chrysophyllum albidum as maize replacer. Five isonitrogenous diets containing maize which was replaced by Chrysophyllum albidum at a rate of 0, 25, 50, 75 and 100% were made. The diets without Chrysophyllum albidum seedmeal served as the control. The diets were isonitrogenous and isolipidic. Experimental diets were assigned randomly to the tanks and each group of fish was fed 5% body weight in equal proportion per day. The results of this study showed that there was a marked vacuolation of hepatocytes among the treatments after the experiment which is not dietary treatment related. The experiment showed that it is technically feasible to replace maize with Chrysophyllum albidum seedmeal in the diet of Clarias gariepinus without adverse effect on the histology of kidney and liver.
INTRODUCTION
Carbohydrates are the cheapest source of dietary energy not only for fish but all domestic animals (Shiau and Lin, 2001). Fagbenro et al. (2003) reported that carbohydrates have the physical function of acting as a binder in the formulation of diets. It is essential to ensure adequate energy level is provided in fish diets so as to realize protein sparing effect so that higher percentage of amino acids in protein are made available for growth and other physiological function (Abu et al., 2009). The digestibility of carbohydrates has been shown to vary with their complexity, treatment and levels of inclusion (Adeparusi and Jimoh, 2002). Olurin et al. (2006a) reported that maize is one of the major sources of metabolizable energy in most compounded diets for catfish as it is readily digestible by fish. Moehl and Halwart (2005) reported that insufficient quantities of maize were provided in Nigeria because it is predominantly used for human consumption, that it was illegal to export maize from Nigeria. Thus the increasing prohibitive cost and scarcity of maize has necessitated the need to search for underutilized energy source feed ingredients.
The use of alternative energy source feed ingredients in fish feed is well documented; coffee pulp (Fagbenro and Arowosoge, 1991), sweet potato (Faturoti and Oyelese, 1989), cassava (Olurin et al., 2006b; Abu et al., 2009), biscuit waste (Aderolu et al., 2011).
Chrysophyllum albidum (G. Don) is a tropical edible fruit tree. It belongs to the family Sapotaceae (Ehiagbonare et al., 2008). It is a primary forest tree species and its natural occurrence has been reported in diverse ecological zones in Nigeria and other African countries (Oboh et al., 2009). The cotyledon from the seeds of Chrysophyllum albidum have been said to be an excellent source of carbohydrates, tannin, flavonoids, terpenoids and resin (Akaneme, 2008). However, paucity of information exists on the use of Chrysophyllum albidum seeds as dietary energy source of fish feed especially the effect it has on the histology of the kidney and the liver. This study, therefore, seeks to evaluate the histological changes in the liver and the kidney of Clarias gariepinus using diets containing Chrysophyllum albidum.
MATERIALS AND METHODS
Seed collection and processing: Dried matured Chrysophyllum albidum seeds were obtained from Bodija Market, Ibadan Oyo State and they were processed by boiling in water (100°C) for 30 min. They were prepared by grinding the samples in a laboratory mill, then mechanically defatted by the use of locally made screw press and sieved with a 200 mm mesh size sieve and put in polythene bags and stored at 4°C. The cakes therefore were analyzed for their proximate composition (AOAC., 1990). Fish meal, soybean meal and other feedstuffs obtained from commercial sources in Nigeria were separately milled screened to fine particles size and triplicate samples were analyzed for their proximate composition (AOAC., 1990). Based on the nutrient composition of the protein feed stuff (Table 1), a control diet and four test diets (40% crude protein, 12% crude lipid) were formulated. The control diets contained soybean meal, providing 50% of the total protein which was replaced by cooked Chrysophyllum albidum seed meal. The rate of substitution was 0, 25, 50, 75 and 100% (Table 2).
Culture condition: Clarias gariepinus fingerlings were acclimated to experimental condition for 7 days prior to the feeding trial. Groups of 15 catfish fingerlings were stocked into aquaria comprising 60 L capacity rectangular plastic tanks. Each diet was fed to the catfish in triplicate tanks twice daily (09.00, 16.00 h) at 5% body weight for 56 days. Fish mortality was monitored daily, total fish weight in each tank was determined at two weeks intervals and the amount of diet was adjusted according to the new weight.
| Table 1: | Proximate composition of the protein feed ingredients |
| CSM: Chrysophyllum albidum seedmeal | |
| Table 2: | Gross composition of experimental diets (g/100 g) containing Chrysophyllum albidum seedmeal fed to Clarias gariepinus |
CSM: Chrysophyllum albidum
seedmeal, *Specification: Each kg contains: Vitamin A: 4,000,000 IU, Vitamin
B: 800,000 IU, Vitamin E: 16,000 mg, Vitamin K3: 800 mg, Vitamin
B1: 600 mg, Vitamin B2: 2,000 mg, Vitamin B6:
1,600 mg, Vitamin B12: 8 mg, Niacin: 16,000 mg, Caplan: 4,000
mg, Folic acid: 400 mg, Biotin: 40 mg, Antioxidant: 40,000 mg, Chlorine
chloride: 120,000 mg, Manganese: 32,000 mg, Iron: 16,000 mg, Zinc: 24,000
mg, Copper: 32,000 mg, Iodine: 320 mg, Cobalt: 120 mg, Selenium: 800 mg
manufactured by DSM Nutritional Products Europe Limited, Basle, Switzerland |
|
Histological examination of test organ: At the end of the experiment, three fish per treatment was sampled for histological analysis; the test organism was killed with a blow on the head, using a mallet and was dissected to remove the kidney and liver. The organs was fixed in 10% formalin for three days after which the tissue was dehydrated in Periodic Acid Schiff’s Reagent (PAS) following the method of Huges and Perry (1976), in graded levels of 50, 70, 90 and 100% alcohol for 3 days, to allow paraffin wax to penetrate the tissue during embedding.
The organs were then embedded in malted wax. The tissue was sectioned into thin sections (5-7 μm), by means of a rotatory microtome and was dehydrated and stained with Harris haematoxylin-eosin (H&E) stain (Bancroft and Cook, 1994), using a microtome and each section was cleared by placing it in warm water (38°C), where it was picked with clean slide and oven-dried at 58°C for 30 min to melt the wax. The slide containing sectioned materials/tissue was cleared using xylene and graded levels of 50, 70, 90, 95 and 100% alcohol for 2 min each.
The section was stained in haematoxylin eosin for 10 min. The stained slide was observed under a light microscope at varying X100 magnification, sections was examined and photographed using an Olympus BH2 microscope fitted with photographic attachment (Olympus C35 AD4), a camera (Olympus C40 AB-4) and an automatic light exposure unit (Olympus PM CS5P).
RESULTS
Proximate composition of the experimental diets: Table 3 shows the proximate composition of the experimental diets containing Chrysophyllum albidum seed meal fed to Clarias gariepinus. It reveals the diets to be isonitrogenous and isolipidic as there was no significant difference (p>0.05) in the crude protein and crude lipid content of the diet. The protein and lipid requirement of Clarias gariepinus was met by the 40 and 12% provided in the experimental diets. All the fish responded well to the dietary treatment given to them.
Histological changes in the liver and kidney of Clarias gariepinus fed diets containing Chrysophyllum albidum seed meal: Figure 1 shows the histological changes in the liver and kidney of Clarias gariepinus fed diet D1. There are few foci of fatty infiltration in the parenchyma with central vein severely congested while its kidney had severe interstitial congestion. Same observation was recorded in the liver and kidney of Clarias gariepinus fed diet D2 as shown in Fig. 2. Figure 3 showed there was mild diffuse hepatic vacuolation in the liver of Clarias gariepinus fed diet D3 with no visible lesion in its kidney.
| Fig. 1(a-b): | Photomicrograph of (a) Liver and (b) Kidney of C. gariepinus fed diet D1. Magnification: X400. There are few focl of fatty infiltration in the parenchyma. The central veins are severely congested (liver). There is severe interstitial congestion (kidney) |
| Fig. 2(a-b): | Photomicrograph of (a) Liver and (b) Kidney of C. gariepinus fed diet D2. Magnification: X400. There are large focal of hepatic vacuolation in the parenchyma (liver). There is severe interstitial congestion (kidney) |
| Table 3: | Proximate composition of experimental diets containing Chrysophyllum albidum seed meal fed to Clarias gariepinus |
| Row means without superscript are not significantly different (p>0.05) from one another | |
| Fig. 3(a-b): | Photomicrographs of (a) Liver and (b) Kidney of Clarias gariepinus fed diet D3. Magnification: X400. There is mild diffuse hepatic vacuolation (liver). No visible lesions seen (kidney) |
| Fig. 4(a-b): | Photomicrographs of (a) Liver and (b) Kidney of C. gariepinus fed diet D4. Magnification: X400. There are areas of severe congestion and haemorrhage in the hepatic parenchyma. There is also a diffuse vacuolation of hepatocytes (liver). There are few areas of mild interstitial congestion (kidney) |
Figure 4 showed areas of severe congestion and haemorrhage were seen in the hepatic parenchyma of the liver of Clarias gariepinus fed diet D4 with diffuse vacuolation of hepatocyte. Its kidney has few areas of mild interstitial congestion. There are mild diffuse vacuolation of hepatocytes in the liver of fish fed diet D5 as shown in Fig. 5 with no visible lesions seen in the kidney. The changes observed in the liver and kidney of Clarias gariepinus under study was not dietary treatment related.
| Fig. 5(a-b): | Photomicrographs of (a) Liver and (b) Kidney of C. gariepinus fed DS. Magnification: X400. There is a very mild diffuse vacuolation of hepatocytes (liver), no visible lesions seen (kidney) |
DISCUSSION
The histology of the kidney and the liver of fish fed the diets containing Chrysophyllum albidum replacing maize seemed not to be altered by Chrysophyllum albidum substitution similar to results reported by Merida et al. (2010) and that reported by Pereira et al. (2002) in rainbow trout fed diets with a partial substitution of Brassica by-products likewise, Hansen et al. (2006). Although, the results of this study showed that there was a marked vacuolation of hepatocytes among the treatments after the experiment which was not dietary treatment related, same observation was made by Olukunle (2011) and Jimoh (2012). The high vacuolation of the liver may be attributed to high lipid content of the organ traceable to high lipid in the diets. Same observation was made by Martins et al. (2007), Valente et al. (2011) and Gatta et al. (2011) that the presence of numerous and voluminous lipid droplets in hepatocytes is physiological response to dietary lipid excess or increased lipogenesis. Olurin et al. (2006b) reported that the shrinkage of the hepatic cells can result in the contracting of the blood vessels thereby impeding the portal flow through the liver which negatively affect the normal physiological functioning of the liver. Marchand et al. (2009) stated that large numbers of toxicants often induce histological alteration in the liver. Liver is a target organ of different xenobiotic substances (Cabot, 2000). Nero et al. (2006) reported that liver tissue damage occur when it is constantly exposed to toxicants. Hence it can be concluded that replacement of maize by cooked Chrysophyllum albidum seedmeal left some vacuolation symptoms in the hepatocytes and not much stress is placed on the health of Clarias gariepinus fed cooked Chrysophyllum albidum seedmeal based diets.