Haematological profile of Heterobranchus bidorsalis fingerlings fed processed Delonix regia seeds at different inclusion levels of diets

This study aimed at investigating the haematological profile of Heterobranchus bidorsalis fingerlings fed processed Delonix regia seeds at different inclusion levels of diets. Ten isonitrogenous diets (40% crude protein) were formulated with processed D. regia seed at 0% (Control), 10, 20 and 30% inclusion, respectively. The parameters analysed were pack cell volume (PCV), red blood cell (RBC), white blood cell (WBC), hemoglobin (HB), mean corpuscular volume (MCV), mean corpuscular heamoglobin (MCH) and mean corpuscular heamoglobin concentration (MCHC). Different among the groups were tested using analysis of variance. Raw Delonix regia seed meal had significant effect (P<0.05) on RBC, HB, MCV, MCH and MCHC respectively across the dietary treatments. RBC, MCV, MCH and MCHC differs significantly (P<0.05) across the dietary treatments for fish fed fermented D. regia seeds. All the haematological parameters differ significantly (P<0.05) across the dietary treatments with the exception of PCV, MCV and MCHC respectively for fish fed cooked D. regia seeds. It is therefore concluded that significant variations exist among the processing methods on the health status of the fish. It is recommended that inclusion of D. regia up to 20% will have no deleterious effect on their health status.


INTRODUCTION
Fish is an important source of high quality protein, providing approximately 16% of the animal protein consumed by the world's population (FAO, 1997).Fish evolved after several years of genetic improvement, and their relevance and success as a relatively cheaper and steady source of animal protein hinges on their higher carcass yield.Much progress in the productivity indices of fish are now achieved through improvement of several environmental factors regarding their growth, health and maintenance.
Among the Clariidae family, Heterobranchus bidorsalis is the second most important aquaculture species in Nigeria (Vanden Bossche and Bernacsek, 1990).H. bidorsalis is endemic to Africa and recent interest in culturing its species has been rising.H. *Corresponding author.E-mail: bigm4real@gmail.com.Tel: +2348023741725.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License bidorsalis, an active swimmer and predator (Fagbenro, 1992) which like all chariid catfishes is capable of aerial respiration.This species is found in turbulent or fast running streams, it feeds on fish, molluscs and alarming number of people, mostly in developing crustaceans.It attains a considerable size of over 120 cm.Physiological, including hematological, behavioral and biochemical parameters are useful diagnostic tools in the practice of veterinary medicine (Lemma and Moges, 2009).Haematological parameters are good indicators of the physiological status of animals under different conditions (Ambore et al., 2009).Heamatological studies are important when evaluating fish health diagnostically just as it is important in human health.Sampath et al. (1993) observed that studies on fish blood could reveal conditions within the body of fish long before an outward manifestation of disease or stress condition.Many hematological parameters can be used to assist in providing evidence and possible identification of any abnormality or disease condition.Hematological parameters have been acknowledge as valuable tools for monitoring fish health, confirming maturation and monitoring any changes in the quality of feed, water and related soil (Kumar et al., 2011).Low heamatological indices are indications of anemic conditions (Haruna and Adikwu, 2001).The quest for more economically viable, palatable and environmentally friendly feed among the fish farmers is highly desirable.This has redirected research interests toward the use of unconventional protein sources especially from plant products like leaves, seeds and other agricultural by products (Ali et al., 2003;Bake et al., 2009).In Nigeria, the high cost of formulated commercial fish feed is a major constraint to the growth and expansion of the aquaculture sector and this has prompted a concerted effort to seek for alternative feed ingredients.Hence, the objective of this study was to evaluate the haematological profile of H. bidorsalis fingerlings fed processed Delonix regia seeds at different inclusion levels of diets.

MATERIALS AND METHODS
The study was conducted at the aquaculture production technology unit of the skill acquisition and development centre, National Agricultural Extension and research Liason Services, Ahmadu Bello University, Zaria, located at latitude 11° 09 45.2 N and longitude 7° 38 17.9 E.
Matured and dry pods of D. regia containing the seeds were collected from the annex campus of Nuhu Bamalli Polytechnic Zaria.Seeds were collected by opening the pods manually.

Fermentation of D. regia seeds
The seeds were soaked in water for 12 h.The drained soaked seeds were allowed to ferment naturally by tying in polythene bag and kept in a dark cupboard for 72 h without the addition of yeast (Udensi et al., 2006).The fermented seeds were allowed to air dry for two days before grinding into homogenous powder using a hammer mill.

Cooking of D. regia seeds
The seeds were boiled at 100°C for 80 min and were allowed to cool by sun drying and later grounded to homogenous powder using a hammer mill (Bake et al., 2013).

Raw D. regia seeds
The raw seeds were sundried for two days and were milled into a homogenous powder using a hammer mill.

Determination of haematological parameters of experimental fish
At the end of 26 weeks of feeding trials, a total of ninety fishes were randomly selected from the ten treatments used in this study.Nine fish was selected per treatment.The blood was collected from live fish by putting it on a tray.It was handled carefully to minimize stress.A damp cloth was used to cover the head of the fish.Blood was collected in the morning hours to avoid diurnal variation.The blood was drawn from the caudal vein using syringe.The collected blood was transferred from the syringe into an anti-coagulant, ethylene diaminetetraacetic acid (EDTA) bottles for heamatological analysis.

Heamatological procedures
All the heamatological parameters were determined using standard techniques.The heamatological parameters determined include red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV) and hemoglobin (HB).

Determination of red blood cell rbc and white blood cell (WBC) counts
Red blood cell counts (RBC) and white blood cell count (WBC) were determined by use of the Neubeur improved counting chamber (Kelly, 1979).The blood was diluted 1:200 with Dacies fluid (99 ml of 3% aqueous solution of sodium citrate and 1 ml of 40% formal dehydrate).This keeps and preserves the shape of red blood cell which was then estimated using the counting chamber for RBC.For the total white blood cell count the dilution was 1:20 using 2 to 3% aqueous solution of acetic acid to which a tinge of Gentian violet was added.The blood smear was stained using Wright-Giemsa stain, a total of 100 white blood cells were enumerated and differentiated (Schalm et al., 1975).

Determination of packed cell volume (PCV)
The packed cell volume was determined using a micro heamatocrit centrifuge.The blood was placed into capillary tubes and filled to ¾ of the tubes; one end was sealed with plasticine.They were centrifuged for 5 min at 12,000 rpm.The PCV was read by the use of heamatocrit reader.
The volume was made up to 1 L with distilled water.The mixture was allowed to stand for 3 min and the HB concentration was read photonutrically by comparing with a cyanomethaemoglobin standard with a yellow-green filter at 625 mm.

Determination of physicochemical parameters of experimental water
Water temperature was recorded daily in the morning using thermometer.Hydrogen ion concentration (pH) was taken with a pH meter model pH -009(111).Dissolved oxygen (DO) was recorded using a dissolved oxygen model meter -DO 510.Turbidity of the water was determined by the method of AOAC (2003).

Statistical model
Model equation for analysis of variance used in this study includes: Where, µ is the effect population mean; Ti is the effect of treatments (Processing methods = cooking and fermentation), and Wij is the random error associated with the record heamatology of the l th fish.

Data analysis
Data obtained were subjected to one way analysis of variance (ANOVA) using general linear model (GLM of SAS 9.2, 2008).Duncan multiple range test (DMRT) was used to test difference between levels of means and mean separation was considered significant at P<0.05.

RESULTS
Table 1 shows the effect of raw D. regia at different inclusion levels of diets on the haematological parameters of H. bidorsalis fish.Raw D. regia seed meal had significant effect (P < 0.05) on RBC, HB, MCV, MCH and MCHC respectively across the dietary treatments.PCV and WBC were statistically similar (P > 0.05) across the dietary treatments.The fish fed control diet had higher numerical values (51.00 ± 1.00%) as compared to other dietary treatments for packed cell volume.The control group had significantly (P < 0.05) the highest volume of red blood cell as compared to R 10 , R 20 and R 30 respectively.Fish fed raw D. regia seed meal at 30% inclusion level had highest concentration of WBC (6.87± 0.35) while the least concentration was recorded in R 10 (6.00 ± 1.00 10 9 /L).Haemoglobin levels were higher in fish fed the control and R 10 diets (16.00 ± 1.00 and 14.80 ± 0.20 g/dl) which were statiscally different (P < 0.05) from fish fed raw D. regia at 20 and 30% inclusion levels 14.40 ± 0.40 and 14.20 ± 0.80 g/dl).Fish fed raw D. regia at 30% inclusion level had the highest quantity of MCV (188.46±0.04fl) while the control group recorded the least value (121.43 ± 0.77 fl).R 30 had the highest concentration of MCH (54.62 ± 0.02) which was statistically significant (P < 0.05) from the control, R 10 and R 20 respectively.Fish fed the control diet (31.37 ± 0.03 g/dl) had the highest concentration of MCHC which was statistically different from control, R 10 , R 20 and R 30 , respectively.
The effect of fermented D. regia seed meal at different inclusion levels of diet on the haematological parameters of H. bidorsalis are shown in Table 2. RBC, MCV, MCH and MCHC differs significantly (P < 0.05) across the dietary treatments.PCV, WBC and HB were statistically similar across the dietary treatments.
Fish fed the control diet was significantly (P < 0.05) higher (4.20 ± 0.20 10 6 mm -3 ) than fish fed fermented D. regia seed meal at 10, 20 and 30% inclusion levels respectively.Fish fed 30% inclusion level of fermented D. regia seed meal had the highest concentration of MCV (138.39) and MCH (45.28 ± 0.04 Pg) which differs significantly from other dietary treatments.Fish fed 10% inclusion level of fermented D. regia seed meal had significantly (P < 0.05) higher concentration (33.59 ± 0.50 g/dL) of MCHC from fish fed control diet, F 20 and F 30 respectively.
The effect of cooked D. regia seed meal at different inclusion levels of diets on the haematological parameters of H. bidorsalis are presented in Table 3.All the haematological parameters differs significantly (P < 0.05) across the dietary treatments with the exception of PCV, MCV and MCHC respectively.The highest concentration of PCV was recorded in the fish fed cooked D. regia seed meal at 10, 20 and 30% inclusion levels respectively which were statistically different (P < 0.05) from the control (51.0± 1.00%).C 20 and the control group had the highest levels of MCV (121.86 ± 0.04 and 121.43 ± 0.77 fl) while C 10 recorded the least concentration (119.32±0.03 fL).Fish fed cooked D. regia seed meal at 20% inclusion levels had the highest quantity (33.59 ± 0.02 g/dL) of MCHC which was statistically different (P < 0.05) from fish fed diets containing cooked D. regia seed meal at 0, 10 and 30% inclusion levels.

Table 1 .
Means ±standard error of heamatological profile in Heterobranchus bidorsalis fed raw Delonix regia seeds meal at different inclusion levels of diet.

Table 2 .
Means ±standard error of heamatological profile in H. bidorsalis fed fermented D. regia seeds meal at different inclusion levels of diet.Means with different superscripts across the treatments differs significantly (P<0.05).ns, Not significant; PCV, packed cell volume; WBC, white blood cell; RBC, red blood cell; HB, haemoglobin; MCV, mean cell volume; MCH, mean cell haemoglobin; MCHC, mean corpuscular haemoglobin concentration. abc

Table 3 .
Means ±standard error of heamatological profile in H. bidorsalis fed cooked D. regia seeds meal at different inclusion levels of diet.