Haematological and Histological Examination of Oreochromis niloticus Fed Oxalic Acid Supplemented Diets Challenge with Escherichia coli

Introduction

Heamatological and pathohistological evidences have been used as important biomarkers in environmental monitoring that allows examining specific target organs in livestock production. supplementation of diets with organic acids have been reported to boost growth through improved digestion, absorption, and retention of a varieties of nutrients and minerals [1, 2]. Pathohistology analyses of internal organs stand out as a tool to indicate the health status of aquatic organism in their environment [3, 4]. Many toxicants have been shown affecting the growth parameters and reproduction with evidence of tissue damage [5, 6, 7], E. coli is bacterial infection of freshwater parasite that infecting physiological structure aquatic organism [8], thus post a tract on health status of the fish. It is only by examining appropriate tissues under high magnification that these problems can be identified. The tissues are essential before a competent histopathologist can identify genuine pathological change.

Fish sampled from different ages and species of fish will have a different appearance and these must be recognised in order to interpret sections correctly [9]. Recognition of these differences and a degree of common sense is paramount when it comes to interpretation and drawing any conclusions for any specific case examination [10].

If global aquaculture industry to continue its growth, there must be a response to the challenges that limiting the industry e.g environmental burdens, fish feed value, fish growth and health status. Research seems to be the feasible solutions to these challenges as innovations are needed in several areas to realize aquaculture potentials and to assess the haematology indices of O. niloticus fed varying levels of oxalic acid supplemented diets and investigate the immune response of O. niloticus fed oxalic acid supplemented diets and challenged with entero-toxigenic bacteria (Escherichia coli).

Materials and Methods

The research work was carried out at the Department of Fisheries and Aquaculture Technology Teaching and Research Farm, The Federal University of Technology, Akure, (FUTA), School of Agriculture and Agricultural Technology, FUTA. Haematology indices of O. niloticus fed with various supplement diets of oxalic acid was carried out on experimental fish (O. niloticus) at the Animal Production and Health Laboratory, The Federal University of Technology, Akure, Nigeria for the residue effects of oxalic diets on O. niloticus for 90 days indoor fed trial. The blood from the fish was collected from the cardiac puncture/cutting the cardiac peduncle using different 5 ml heparinized syringes, with ethylene diamine tetra acetic acid (10 ml EDTA) as anticoagulant. Fifteen blood specimens (each per tank) were taken from experimental fish for blood analysis. The heamatology parameters that were carried out is as follows.

White Blood Cells Count: The total white blood cell count was performed by taking one drop of the blood smeared on slide and air dried at room temperature. This was later fixed in 95% methanol and stained with Giemsa stain (Analar grade) for 20 minutes and mounted. White blood cells were identified using Olympus BX 50 microscope (Olympus UK).

Red Blood Cells Count: The blood cells (erythrocytes) was counted in Neubauer hemocytometer counting chamber using Olympus BX 50 microscope (Olympic UK). Number of cells counted was expressed as (106 mm-3).

Haemoglobin Concentration: This was performed with aid of Haemoglobinometer (Sigm, England). Standard Shilonometer N/10 HCL and 0.02ml pipette were used for the estimation. The graduated tube was filled to 20ml mark; 0.02ml of blood was added and mixed thoroughly until the colour matches the standard. Haemoglobin concentration was determined by the amount of solution in the graduated tube expressed in percentage.

Pack Cell Volume: Non-clotted blood was drawn by capillary action into micro-haemocrit tubes to determine the Pack Cell Volume (PCV). One end of the tube was sealed with a synthetic sealant. The sealed tube was centrifuged in a micro-haemocrit centrifuge at 10500 revolution per time (rpm). The micro-haematocrit reader was used to measure the PCV and expressed in percentage.

Mean Corpuscular Haemoglobin Concentration (MCHC): This is the concentration of haemoglobin in a unit of erythrocytes. It was calculated from the haemoglobin value (HB) in gL-1 and from the haematocrit value (PCV). MCHC=Hb/PVC x 100 Mean Corpuscular Haemoglobin (MCH): The corpuscular haemoglobin concentration expresses as the concentration of haemoglobin in unit volume of erythrocyte. It was calculated from the haemoglobin value (Hb) and from the Red Blood Cell (RBC) according to the following formula. MCH= Hb/ RBC x 100 Mean Cell Volume (MCV): This was calculated from pack cell volume (PVC) and from Red Blood Cell (RBC) according to the following formula. MCV= PVC/RBC x 100 Pathohistological Examination: Histopathological examinations were carried out to assess gills, livers, kidneys, heart and intestines of test fish for each treatment. These organs and tissues were preserved in sampling bottles containing 10% formalin before examination. These organs and tissues were cut out of the fish. The organs were dehydrated in periodic acid Schiff”s reagent (PAS) 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 cleaned and embedded in melted wax and carefully sliced into thin sections with a rotatory microtome (5μm thick). The cut sections were again cleaned by placing them in warm water (38 °C) from where they were transferred into clean slides and oven- dried at 58°C for 30 minutes to melt the wax and stained with Harris’ haematoxylin–eosin (H and E) stain. The slides containing sectioned tissues was cleaned using xylene and graded levels of 50%, 70%, 90%, 95% and 100% alcohol for two minutes each and stained in haematoxylin-eosin for ten minutes and mounted in dipterex on glass slides, to obtain their photomicrography, the stained sections were examined and photographed at different magnifications (x40, x100 and x400) by means of a binocular light microscope (Olympus Japan 312545) fitted with a digital camera (Olympus CH XSZ- 107BN), a photographic attachment (Olympus C35 AD4) and an automatic light exposure unit (Olympus PM CS5P).

Results

The haematology indices of blood of O. niloticus fed oxalic acid supplemented diets in Table 1 shown that, there were significant variations (p<0.05) in the haemoglobin (Hb) of the fish on diets, however there is different in treatment OAC2, OAC3 and OAC1. The PCV increase across from control to other treatments, but their slight relationship in the PCV treatments OAC2 and OAC4 also OAC3, OAC5 and OAC1. While highest WBC was recorded in OAC1 and lowest RBC was recorded in OAC1. The highest MCHC was recorded in OAC4 followed by OAC3, OAC2 and OAC5 which was significant different from CTR likewise highest MCV was record in the CTR. However, there were no significant variations (p>0.05) in MCH.

Discussion

Histological changes are generally associated with the response of hepatocytes to toxicants. The liver, kidney and intestine of fish exposed to challenge test exhibited diffused, necrotized, hepatic tissues with fatty changes in the hepatic parachyma and vacuolation of hepatic parenchyma cell wall. The liver and pancreatic tissues showed necrosis and fatty changes in the hepatic parenchyma which corroborate to the earlier observation in the used of buyric acid in O. niloticus challenged with Aeromonas sobria [11]. According to Abdel- Aziz MFA, et al. [9] and Roy, et al. [12] the hepatic tissues was damage by vacuolation and generation of inner epithical layer ranging from mild to moderate with deviation from the inner epithelial layer. Fish tissues have limited range of reactions in any particular disease process. It is imperative that as full as clinical history is provided with samples the heamatology and histology indices indicate the varying alteration in the dietary provision. This clinical information is essential in allowing the histopathologist to arrive at a diagnosis of the health status for fish.

The use of a histopathology service by fish farmers and others can allow an investigation to proceed without the requirement for an initial visit. This can be especially important for farms situated in remote areas. The only requirement is that the person taking the samples is properly trained in this critical area.

In liver, the cellular structures of hepatocytes, sinusoids, and cental vein were similar to those in control group with bath culture of E. coli, while the kidney the cellular structure of bronchial, alveoli, alveolar duct and blood vessel were normal in treatment with oxalic acid. Furthermore, signs of injury, necrosis, congestion, or haemorrhagic regions around the section or sinusoids of the intestine were not observed in bath culture. The hepatocytes arranged in cords were clearly visible. The cross-section of the liver showed no lyses in the cells, or infiltration of neutrophil, lymphocyte, or macrophage in the liver, this is in confirmed the report of [9].

As for the kidneys, histologically there was no morphological damage for the control treated group until challenge test with E. coli, the appearance of the glomerular architecture was normal similar to the control groups. The glomeruli, distal, and proximal tubules in the kidney appeared normal in treatments with dietary oxalic 1.0,1.5 and 2.0 supplementation. There was no interstitial and intraglomerular congestion or tubular atrophies. All the nephron cells were normal and showed clearly visible nucleoli with no degeneration, bleeding, necrosis, or infiltration with lymphocytes this findings is similar to Koh CB, et al. [13] whom put forward that fish fed the OAB diets had significantly lower colony forming units of adherent gut bacteria compared to the control or OTC treatments while those fed the 1.0% OAB diet had the lowest total faecal bacterial counts. Tilapia fed the 0.5% OTC or OAB diet had significantly higher resistance to S. agalactiae than those fed the control diet.

The liver and kidney are important organs, which are responsible for the metabolism, detoxification, storage, and excretion of xenobiotics and their metabolites and are susceptible to damage by external substances [12]. However, the liver as a complex organ which is comprised from several cell types performing various functions, and those cells can be damaged by different pathways. Once the hepatic cell membrane is damaged, the cytosol enzymes are released into the blood, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), however, both culture and bath culture with E.coli are intracellular enzymes of which appearance in the blood is an indicative of a cellular damage, this finding is corresponding to Yacoub AM, et al. [6] and Biswadeep D, et al. [14]. Therefore, the determination in serum could be used to assess any incident organic damage in haematology examination in aquaculture particularly that there are established normal ranges of universal markers for the detection of organic damage El-Murr A, et al. [7].

Otherwise, there is no single biochemical marker that can be relied on as a universal test of liver damage (Olurin et al 2006), although AST and ALT are the serum enzymes that have been shown to be the most effective and sensitive indicators of hepatocellular injury. Unfortunately, AST also can exist in many organs including the heart and muscles; therefore, its release is not specific for acute liver diseases Limbu SM, et al. [15]. Unlike AST, ALT is primarily found in the liver Biswadeep D, et al. [14]. The serum level of histology is ubiquitous in several organs including liver, bone, kidney, intestine, and placenta and its exact role differs from one tissue to another [16, 17, 18].

Conclusion

Histopathology and heamatology evidence in liver, kidney and intestine of the sample fish were within acceptable limits and the normal functioning of respective organs base on the DTC protocol. However, the level of pollution of extogenic bacteria of this study is early warning calls for routine montoring of the pond water especially. The used of organic acid should be monitor but as an adjunct along with any number of other considerations including gross observations of fish behaviour, pattern of mortality, identification of potential factors such as haematology, histology examination of fish. In nutshell an histopathological evaluation was carried out to confirm the biochemical findings as shown to identify any structural changes. Light microscopic examination of the vital organs liver, kidney, intestine and control groups for plates did not reveal any gross pathological lesions in feed fish with supplemented oxalic acid. The photomicrographs of the liver, kidney and intestine of the control and varying dietary 4 with inclusion of 1.5g is recommended as best inclusion level-treated groups showed significant variation or morphological architecture.