Variations in Hematological, Biochemical and Immunological Parameters in Labeo rohita Fed with Different Levels of Aerva javanica Supplementation Diet

In the present study, Aerva javanica (in Powder form) was used to evaluate its effects on Hematological, Biochemical and Immunological Parameters of Labeo rohita fingerlings (fresh water fish). A total of 50 Labeo rohita were randomly distributed in five treatments including a control group and four experimental groups. Aerva javanica powder at the rate of 0.5 g, 1.0 g, 1.5 g, and 2.0 g were added to the feed. The fish were fed for 60 days in fiber water tanks. After 60 days, end of cultured the Hematological, Biochemical and Immunological studies were done in fish samples (control, Exp-1, 2, 3 and 4 groups). The results showed significant (p<0.05) differences in the amount of hemoglobin, Rbc, Wbc, neutrophil, lymphocyte, eosinophil, hct, mcv, mch, mchc, total protein, albumin, globulin, blood glucose, lysozyme, phagocyte, Nbt, Sod, and catalase, in the treatments compared to the control group at the end of the cultured. The results showed that Aerva javanica powder has the effect of an immune system booster and using the powder form in Particular at the level of 1.5g, 2.0g as an immune stimulant in the diet of Labeo rohita can be effective in preventing disease in this species.


Introduction
Aquaculture is playing a major role globally in tackling issues of food and nutritional security, livelihood and economy. Annual growth rate of aquaculture is more than 80% and producing about 4.4% of world fish production (FAO 2002). According to a recent report published by the FAO, world aquaculture has grown significantly in the past 50 years. It is mainly contributing to the increase in export earning, income, employment generation and food nutritional security for the developing countries. Emerging diseases constitutes an increasingly serious threat and health problem in aquaculture by limiting the survival, growth and production of farmed fish. In view of increased cost of production and reduced marginal profit in carp culture enterprising farmers is using fresh water fish, Labeo rohita as alternative species for culture. In commercial aquaculture, antibiotics were primarily used for deterrence and control of the diseases and hormones were utilized for improved growth, however, the main disadvantage of antibiotics and hormones are that they are not cost-effective. Several researchers across the world are trying to find novel, cost-effective and proficient compounds of plant origin for the disease prevention and to promote growth in aquaculture [1]. Eventually, all medicinally active herbs contain potential ingredients which possess several physiological activities [1]. Numerous medicinal palnts are being used in aquaculture which comprises of plants such as Cynodon dactylon, Azadirachta indica, Allium sativum, Tridax procumban and Phyllanthus emblica [2]. The aim of this paper is to review current research work being carried out this study was performed to evaluate the effects of four doses of Aerva javanica on hematological, biochemical, immunological parameters of this valuable species Labeo rohita.

Experimental Design
The study was carried out in the Post-Graduate and Research department of zoology, Sri Vasavi College, Erode, Tamilnadu. The experiment was conducted in laboratory condition for sixty days. Indian major carps Labeo rohita were obtained from government nursery fish farm in Bhavanisagar dam, Sathyamangalam, Erode district at Tamilnadu. The collected fish was transferred alive in polyethylene bags and brought to the laboratory and acclimated for three days feeding on mixed diet. Fifty acclimated Labeo rohita of similar size were randomly distributed in fiber tank filled with unchlorinated water. Constant aeration was provided to each container using air compressor.

Experimental Diets
The formulation of experimental diet was prepared using ingredients given in table 1.

Collection of Blood Samples
At the end of 60 days of fish cultures of Labeo rohita were fed with Aerva javanica in powder form to evaluate the hematological, bio chemical and immunological parameters and to compare with control and experimental groups. Blood samples from the caudal vein of 10 fishes from each of five groups were taken by using 2 ml tuberculin syringes with 24 gauge needles and the blood was collected in serological tubes. The blood (without anticoagulant) collected from fish was kept overnight at 4 0 C for serum separation. The serum was separated by spinning down at 3000 rpm for 15-20 min in centrifuge. The supernatant was collected in sterile vials and stored a -20 0 C for further hematological, biochemical, and immunological analysis.

Hematological Examination
Blood samples were analyzed following routine cmethods cadopted cinc hematology [3,4] . The total red blood cell counts (RBCx10 6 /ml) were determined in a 1:200 dilution of the blood sample in Hayem's solution and total white blood cell counts (WBC x10 3 /ml) in a 1:20 dilution of the blood sample with a Neubauer hemocytometer. The hematocrit and leucocrit Percentages were determined in duplicate using micro hematocrit heparinized capillary tubes of 75µl volume and a micro hematocrit centrifuge at 15000 rpm for 5 min [5] . The percentages of erythrocyte and leucocyte volumes were calculated by overlaying the tubes on a sliding scale hematocrit reader. The hemoglobin (Hb, gdL -1 ) concentrations determined by the cyanomethaemoglobin method [6] using a haemoglobin reagent set (pars Azmun Diagnostics). All the values of red blood cell indices, the mean values of cell hemoglobin (MCH pg), cell hemoglobin concentration (MCHC%) and cell hemoglobin volume (MCV fl) were calculated according to wintrobe formulae [7]. The differential leukocytes count was carried out using blood smears stained with wright-giemsa. The percentage composition of leukocytes was determined based on their identification characters listed [8].

Biochemical Examination
For Biochemical analysis, the blood collected was centrifuged at 4000 rpm for 10 min and blood serum was separated, then the serum extracted was analyzed on the spectrophotometer (T80+ UV/VIS) using an analyzer (Bioanalytic Diagnostic industry,co.) [6,9].The Biochemical parameters that were detected during the test included glucose, total protein, albumin and globulin.

Immunological Examination
Advances in Zoology and Botany 8 (3): 75-82, 2020 77 Phagocytic activity was estimated by using the method modified by Siwicki and Anderson [10]. Approximately 100 cells were counted in random fields of view, recording the number of phagocytic cells, and phagocytic activity (PA) was determined. PA = (number of phagocytic cells with engulfed bacteria/ number of total cells) x 100. The respiratory burst of the neutrophils and monocytes was quantified by the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the production of oxygen radicals (Siwicki and Anderson 1993) [11]. Serum lysozyme was assessed using the turbidometric assay (Ellis 1990) [3] . Superoxide dismutase (SOD) activity was determined according to the method of MC cord and Firdovich (1969) [12]. Catalase (CAT) activity was determined according to the method of Aebi (1974) [13].

Statistical Analysis
Statistical analysis was performed by using spss (version 23). Data are presented as mean ± SD. Data were analyzed by one way of variance analysis ANOVA. The significant means were compared by Duncan test and a p<0.05 was considered statistically significant.

Discussion
Haematological, Biochemical, Immunological parameters are important factors that could be considered for the fish diet quality assessment. Sahu et al., reported higher Rbc counts in Labeo rohita fingerlings fed with Mangifera indica [14]. Hajibeglu and Sudagar (2010) reported an increase in the number of Wbc in common carp fish with the use of dietary supplements containing herbal immune stimulants [15]. Cyprinus carpio fed with formulated feed with plant extract of Andrographis paniculata showed gradually increased the haemoglobin content in different day of treatment [16]. Abalaka [19]. A variety of medicinal herbs like Curcuma longa, zingiber officinale, Allium sativum are known to stimulate phagocyte cells [20]. Pavaraj et al., reported that Ocimum sanctum leaf extract enhance the phagocytic activity in the C.carpio [21]. Treatment of albino rats with aqueous extract of freshly plucked Ageratum conyzoides leaves displayed escalation catalase and Sod activity [22]. Lysozyme activity significantly (p<0.05) enhanced in fish fed with 1% of Origanum vulgare extract in Oncorhynchus mykiss [18]. Nbt increased significantly in the group fed 1% powdered zingiber officinale compared to control group in the fish Oncorhynchus mykiss [23].

Conclusions
The results and discussion of present study Prove a role of Aerva javanica (in powder form) supplemented diet has enhanced Hematological, Bio chemical and Immunological parameters in the fish of Labeo rohita fingerlings. Conclusively the Aerva javanica herbal feed supplements promote growth, minimizes stress improves immunity which could help prevent various infections in fishes.