The study animals were fish of barbus (Labeo barbus) that was collected from Lake Tana, Amahara region, Ethiopia. Sampled fishes were selected by simple random sampling technique. Large, medium and small sized fishes were included in my study. Large, medium and small sized fishes were selected by simple random sampling techniques associations that are found in western part of Lake Tana. The study population, small (1 to 10 cm), medium (10.5 to 20cm) and large size fishes (≥20 cm) were categorized according to .
Sample collection techniques, gross and histopathological examination
A total of 384 randomly selected fish, Labeo barbus, were sampled and examined. The length, date and site of collection of host specimens were recorded. Gross lesion and health conditions of each specimen were recorded and monogenean parasite (Gryodactylus and Dactylogyrus ssp.) of gills were collected with forceps. Postmortem examination of gills of the investigated fishes was examined according to . Damaged fish tissues (gills) were taken from the parasite attachment area of infested fishes and were cut out in fresh condition fixed in 10% buffered neutral formalin. In addition, monogeneans parasites of gills were collected and preserved10% buffered neutral formalin. Then, the necessary information were leveled on the sampling bottles. Finally, the samples were transported to Faculty of Veterinary Medicine Pathology and parasitology laboratory, University of Gondar, for identification of parasites and histopathological examination of tissue specimens. After used the fixative, the tissue were washed in running water for 24hr to remove the fixative entirely .The tissues were then processed using a 18 hr automatic tissue processor . The tissue processor contains 12 beakers (8 glass beakers contains alcohol, 2 glass beakers contains xylene and 2 glass beakers containing paraffin wax).
The tissues after being processed are embedded using an automatic embedding centre. Embedding is a process of submerging a tissue in a metal plastic disposable embedding mould containing molten paraffin wax, which became solidified when it was cold. This formed a support medium for the tissue during sectioning. The blocked tissues were sectioned at 4-5 microns a rotary microtome (LEITZ 2535, Germany), and floated into pre coated slides and were placed in a clean grease free slide which was then placed on a hot plate for 30 min in order for section to adhere to the slides. The staining method used was the H&E staining method. Standard histological procedure was followed as described by . This method was used in order to demonstrate the general structure of the tissues. These were then dewaxed in xylene. The processed section were later taken to water by using descending grades of alcohol, that is, from absolute alcohol, 95% alcohol, 70% alcohol,50 %,for 5 min each and rinse tap water for 10 min. It was stained in haematoxylin for 6 min, then put in running water for 20 min.
This was counter stained in 1% Eosin for 15 min and, then dehydrated using ascending grades of alcohol (95% alcohol and absolute alcohol). These were cleared in xylen, mounted using D.P.X (a mountant) and viewed under light microscope . Lesions were described and scored as none for no lesion, mild, or severe depends on the type of lesion . Preserved in 10% formaline and labeled with all necessary information for further identification. The collected parasites placed on glass micro-scope slides with a drop of 10% formalin and slightly compressed between a slide and a cover slip prior to being examined under stereomicroscope (4x,10x.40x) and observe morphological feature of the parasite . The monogeneas parasites were identified microscopically using the identification guideline of .
The data were entered and managed in Microsoft Excel. All the data analysis was done by SPSS software version 12. Descriptive statistics was applied for the analysis of the data obtained. Descriptive statistics such as percentages were used to describe the nature and the characteristics of the data. The prevalence of monogenea parasite (Gryodactylus and Dactylogyrus spp.) was analyzed using Chi-square test and <0.05 was considered as significant.