Effect of Roselle ( Hibiscus sabdariffa ) and ginger ( Zingiber officinale ) as feed additives , on growth and haematology of Clarias gariepinus Juvenile

1 Department of Fisheries and Aquaculture, Federal University, Ndufu Alike Ikwo, Ebonyi State, Nigeria. 2 Department of Agriculture, Federal University, Ndufu Alike Ikwo, Ebonyi State, Nigeria. 3 Department of Biological Science, Ibrahim Badamasi Babangida University, Lapai, Niger State, Nigeria. 4 Department of Zoology, Ahmadu Bello University, Zaria, Kaduna State, Nigeria. 5 Department of Zoology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. 6 Department of Fisheries, Modibbo Adama University of Technology, Yola, Adamawa State, Nigeria. 7 Department of Animal Science and Technology, Nnamdi Azikiwe University Awka, Anambra State, Nigeria.

use of land and water resources, lack of governance and regulation in production, poorly developed infrastructure, limited access to fish seed, feed, increased fish feed extraction from the world's ocean and diseases (World Bank, 2006).Disease which is one of the critical factors hampering aquaculture development ensued as a result of negative interactions between fish and pathogenic bacteria (Romero et al., 2012;Iheanacho et al., 2017a).
Antibiotics are chemical compounds used in treating and preventing diseases in aquaculture.However, the use of antibiotics in fish feeds has a good impact on growth (Rico et al., 2013).Antibiotics growth promoters (AGP) were supposed to increase growth rate as a result of improved gut health, resulting in better nutrients utilization and improved feed conversion (Visek, 1978).But now, antibiotics growth promoters are considered as human health risk factor for their possible role in the emergence of microbial resistance, breakage of the animal intestinal micro-ecological balance and the presence of antibiotics residues in resultant fish products (Okeke and Ososa, 2003).Feed supplements are costly to the extent that most fish farmers depend on it to supplement some essential minerals and vitamins found wanting in fish feeds but currently, can no longer afford to use them.
Natural materials such as medicinal plants could be widely accepted as feed additives to enhance feed utilization and aquaculture productive performance and sustainability (Levic et al., 2008).Phytogenic feed additives, also known as phytobiotics products are plant derived products, used in animal feeding to improve performance through amelioration of feed properties, promotion of production performance and improving the quality of animal origin food/feed (Alector and Osho, 2009).World Health Organization encourages using medicinal herbs and plants to substitute or minimize the use of chemicals through the global trend of go back to nature (Adewole, 2014).Roselle (Hibiscus sabdariffa) is an annual dicotyledonous, erect, herbaceous tropical plant.The plant is cultivated majorly in the northern part of Nigeria as edible vegetable and considered to be medicinal (Ijeomah et al., 2012).The chemical constituents of the flower include the flavonoids, gossypetine and sabdaretine (Pietta, 2000).Roselle is reported to be diuretic, digestive, antiseptic, sedative, purgative, emollient, demulcent and astringent (Adewole, 2014).The calyces have many medicinal applications in curing kidney stone, pyrexia, liver damage, hypertension and leukemia (Abu-Tarboush et al., 1997;Estrella et al., 2000).
Ginger (Zingiber officinale) is also a herbaceous perennial plant which grows annual stems about a meter tall, bearing narrow green leaves and yellow flowers (Kim et al., 2008).Ginger has been reported to be rich in mineral elements (magnesium, potassium, phosphorus, calcium and zinc), vitamins (retinol, cholecalciferol, ascorbic acid, thiamine, riboflavin, niacin, folic acid, pantothenic acid and pyridoxine) and phytochemicals (flavonoid and alkaloid) (USDA, 2014;Iheanacho et al., 2017a).Shubha (2015) reported that ginger rhizomes also contain gingerol, shogoal and a potent proteolytic enzyme called zingibain.The author also reported that ginger is anti-platelet, antibacterial, antifungal, antiviral, anti-worm, anti-inflammatory and has anti-oxidative activity.
The use of medicinal plants in aquaculture has not being widely practised.The current study seeks to evaluate the effects of roselle and ginger supplemented diets as feed additives, on growth and haematology of African catfish (Clarias gariepinus).

Study area
The study was carried out at the Animal House of the Department of Microbiology/Biotechnology, Federal University, Ndufu Alike Ikwo, Ebonyi State, Nigeria.

Study fish
One hundred and fifty (150) C. gariepinus juveniles were procured from a private fish farm in Abakaliki and transported in a 50 L gallon filled with water to the Animal House of the Department of Microbiology/Biotechnology, Federal University, Ndufu Aliko Ikwo within 30 and 40 min.The fish were acclimated in a tarpaulin tank (4 m x 2 m x 1 m) for two weeks and were fed commercial fish feed (coppens) throughout the 2 weeks acclimation period.

Collection, preparation and processing of Roselle flower (Hibiscus sabdariffa) and ginger (Zingiber officinale)
The dried flower (calyx) of roselle and rhizomes of ginger were purchased from Abakpa Market located at Abakalik town, Ebonyi State, Nigeria.Both materials were ground into powder, using electric blender (Philips, model HRI701/BC, made in China), thus sieved, and stored separately in an air tight container until use.

Experimental diets
Five iso-nitrogenous diets were formulated to yield 37% crude protein (Table 1).Roselle and ginger were included in the diets as feed additives at varying inclusion levels and coded as HS1 and HS2 for Roselle, while G1 and G2 were for ginger.The different inclusions were HS1 (2.00 g), HS2 (4.00 g), G1 (2.00 g) and G2 (4.00 g).The control diet had 0% inclusion of both roselle and ginger.Other feed ingredients used in the diet formulation includes fish meal (FM) soy bean meal (SBM), wheat offal, yellow maize (YM), vitamin/mineral premix, cassava starch, palm oil, bone meal and salt (Table 1).Pearson square method was used in feed formulation.Samples of the experimental diets were sent to the laboratory for proximate analysis.Samples were analysed following the procedure of A.O. A.C (2000).

Experimental design
A total of one hundred and fifty fish (150) (initial weight 35.41±1.53g) were randomly distributed to 15 aquarium plastic tanks (1 m x 1 37.00 37.00 37.00 37.00 37.00 m x 1 m) at ten fish per treatment.Each tank contained 30 L of water.Experimental diets were randomly assigned using completely randomized design to triplicate tanks.The fish were fed 5% body weight per day in two portions (by 9.00 am and 5.00 pm) for 8 weeks.Quantity of feed was adjusted forth nightly after batchweighing of experimental fish.Water in the tanks was partly removed by siphoning and replaced with fresh water every three days to avoid fouling resulting from faeces and uneaten food.Water quality parameters such as temperature, dissolved oxygen and pH of the experimental water tanks were monitored and measured daily using water testing kits (PRO-LAB TM Flourida).Temperature, pH and dissolved oxygen were maintained at 27.12±0.04°C,6.98±0.01 and 6.05±0.21mg L -1 , respectively.

Growth parameters
Weight measurement of the fish was obtained at the end of the experiment and the following growth parameters were determined according to the formula of Iheanacho et al. (2018) Condition factor = K = 100W/Lb 3 (Gomiero and Braga, 2005) Where, K = condition factor; W = the weight of the fish in gram (g); L = the total length of the fish in centimetre (cm); b = the value obtained from the length-weight equation.

Haematological analysis
Three fish per tank were sampled for blood collection at the end of the experiment.Blood was collected from the caudal vein into an EDTA lithium tubes.Blood samples were immediately transported to the haematology laboratory unit of the Federal University, Ndufu Alike Ikwo Medical Centre for Haematological Analysis.The blood was analyzed to determine the packed cell value (PCV) with micro haematocrit using heparinised capillary tube (25 mm), while red blood cell (RBC), white blood cell (WBC) counts, haemoglobin (Hb) concentration and red cell indices (mean corpular volume (MCV), mean corpular hemoglobin (MCH) and mean corpular haemoglobin concentration MCHC) were determined as described by Dacie and Lewis (2011).

Statistical analysis
Data obtained from the experiment were subjected to one-way analysis of variance (ANOVA), using Statistical Package for Social Science (SPSS 2006, version 22).Duncan multiple range test (DMRT) was used to compare the differences between means at p<0.05.Data were presented as mean ± SE.

DISCUSSION
Proximate examination of roselle and ginger based diets revealed significant difference (p˂0.05) in proximate composition (Table 2).Higher values for crude protein, ash, dry matter and fat were found in ginger based diets followed by roselle based diets and the control.Kumar et al. (2014) reported 41.14 to 42.32 4% crude protein for ginger based diets.
Medicinal plants have been reported to be growth promoters and immune boosters in livestock and fish nutrition (Levic et al., 2008;Kumar et al., 2014;Reverter et al., 2014;Iheanacho et al., 2017a).The findings of the present study on growth performance of C. gariepinus juvenile fed roselle and ginger supplemented diets revealed that there were increases in treated groups as compared to the control, although, insignificant (p˃0.05)(Table 3).Highest values in terms of final weight (67.51±0.44)and weight gain (31.82±0.63)were observed in fish group fed 4.0 g ginger supplemented diet followed by 4.0 g roselle treated groups.Significant increase in terms of SGR was seen in 4.0 g ginger treated group as compared to the control.Food conversion ratio (FCR) was observed to be highest in fish group fed 2.0 g roselle supplemented diet and lowest in 4.0 g ginger treated groups.Kumar et al. (2014) reported enhanced growth in Indian catfish fingerling (Mystus montanus) fed ginger supplemented diets when compared with the control diet.Iheanacho et al. (2017a) reported significant increases in weight gain, specific growth rate and final weight when C. gariepinus juvenile were exposed to varying concentrations (0.25, 0.50, 0.75 and 1.0 g/35 mgL) of ginger as compared to the control.Adewole (2014) reported significant increases (p˂0.05) in growth parameters (final weight, weight gain, specific rate and relative growth rate) in C. gariepinus fed roselle supplemented diets when compared with the control.The positive response to growth in treated fish especially at 4.0 g inclusion level of ginger could be attributed to the high proximate content of ginger (Table 2).Ginger is a good source of protein, mineral elements, vitamins and contains good number of phytochemical constituent that enhance growth and health of animals.
Hematological assays might give a useful guide on the physiological condition of fish (Haghighi and Rohani, 2013).The present study revealed significant changes in hematological parameters in the study fish.Highest values for PCV, Hb and RBC were seen in fish fed 4.0 g ginger supplemented diet as compared to other treatments and the control.There were no significant changes (p˃0.05) in MCV, MCH and MCHC among treated groups as compared to the control.The present study revealed that ginger and roselle enhanced non specific immune response in C. gariepinus.The result of the present study collaborates the findings of Haghighi and Rohani (2013) who reported significant increases in RBC, PCV, Hb and WBC values in rainbow trout (Oncorhynchus mykiss) fed ginger supplemented diets when compared to the control.Kumar et al. (2014) also reported significant increases (p˂0.05) in RBC, Hb, PCV and WBC values in Indian catfish (Mystus montanus) fed ginger based diets as compared to the control.Iheanacho et al. (2017a) reported similar findings when C. gariepinus were exposed to varying concentrations of ginger bath for 12 weeks.Yahaya et al. (2012) opined that medicinal plants (roselle, ginger, ugwu and moringa) significantly increased haematological parameters in albino rat as compared to the control.

Conclusion
The present study revealed that both roselle and ginger supported the growth and hematology of C. gariepinus juvenile especially, 4.0 g ginger supplemented diets.The use of costly feed supplements should be discouraged.Since ginger and roselle are readily available all year round, there is need to encourage the local fish farmers to imbibe this feed biotechnology in order to minimize cost of fish production.
: Mean weight gain (SWG); Specific growth rate (SGR); Food conversion ratio (FCR); Mean weight gain (SWG) = final weight -initial weight Specific growth rate (SGR) (Ln mean final weight -Ln mean initial weight) fed (Dry gram weight) FCR = Weight gain of fish (Wet gram weight)

Table 4 .
Significant differences (p˂0.05) were seen among treated groups as compared to the control.

Table 2 .
Proximate composition (%) of experimental diets.Nitrogen free extract (NFE) = (100 -(crude protein + ash+ crude fibre + moisture + fat)).Data presented represents mean values of parameters and values in the same rows with the same alphabet superscript are not significantly different (p˃0.05).
e *

Table 3 .
Growth response of C. gariepinus juvenile fed Roselle and ginger supplemented diets.
a Data represents mean ± (SE) values of parameters estimated.Values in the same rows with the same alphabet superscript are not significantly different (p˃0.05).

Table 4 .
Hematological data of C. gariepinus fed Roselle and ginger supplemented diets.Packed cell value; Hb, haemoglobin; RBC, red blood cell; WBC, while blood cell; MCV, mean corpular volume; MCH, mean corpular hemoglobin; MCHC, mean corpular haemoglobin concentration.*Datarepresents mean ± (SE) values of parameters estimated.Values in the same rows with the same alphabet superscript are not significantly different (p˃0.05).
a PCV,