Effects of Dietary Supplementation of Zingiber officinale Root-Powder on Growth, Nutrient Utilization and Intestinal Microbes of African Mud Catfish (Clarias gariepinus) Fingerlings

Aquaculture, one of the most important options in animal protein production. It however requires high quality feeds with high protein content and some complementary additives or dietary supplements to keep fish healthy and to enhanced growth [1]. Medicinal plant products (phytobiotics) have been shown to act as anti-stress, growth promoters, appetite stimulators, tonic and immunostimulants and to have antimicrobial characteristics in finfish and shell fish culture [2]. Phytobiotics are plant-derived, natural compounds embedded into diets which enhanced animal productivity and they include: Aloe vera (Aloe barbadensis), garlic (Allium sativum) and ginger (Zingiber officinale) as growth promoters have been widely used in different species of fish [3]. All fish live in microbe-rich environment and are vulnerable to invasion by pathogenic and opportunistic micro-organisms. The environment of fish being aquatic is very challenging with fish in constant interaction with a wide range of pathogenic and non-pathogenic micro-organisms [4].

To develop alternative practices for growth promotion and disease management in aquaculture, attention has also been focused in identifying novel drugs, especially from plant sources. Rao et al. [5], reported that these drugs may be delivered to the cultivable organisms either through feed supplementation or oral delivery through diets mode.

(Z. officinale) belongs to the Zingiberaceae plant [6]. It is beneficial to growth and immune systems in aquatic animals [7]. The rhizome of Z. officinale has been reported to possess a broadspectrum of prophylactic and therapeutic activities [8]. It is important in fish diet in that it control infection [9]. The function the plant performed is linked to the active ingredients that are present in the plant. Themajor constituents in ginger rhizomes are carbohydrates (50-70 %), lipids (3-8 %), terpenes, and phenolic compounds [10]. Hence, this study aims at assessing the growth, nutrient utilization and anti-microbial potentials of cultured Clarias gariepinus [11] fed varying inclusion levels of Z. officinale root-powder.

The study was conducted at the fish farm (hatchery unit) of the Department of Aquaculture and Fisheries Management, College of Environmental Resources Management, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The experiment was conducted in twelve (12) rectangular plastic tanks each (60 litres) and each tank was two third filled (40 litres).

African mud catfish (C. gariepinus) fingerlings (2.33 g average weight) were used in this study. A total of one hundred and twenty (120) fingerlings were purchased at Motherhood Fish Farm, Abeokuta, Ogun State, Nigeria. The fish were randomly (completely randomized design) allotted into four (4) treatments in the plastic tanks at a stocking rate of ten fingerlings per tank in triplicates.

Four iso-nitrogenous diets containing 40 % Crude Protein (CP) were formulated to containing three diets containing different concentrations of Z. officinale root-powder and the control as listed below:

• Treatment 1 (Control) - 0 % 
• Treatment 2 (ZOP1) - 1 % Z. officinale root-powder
• Treatment 3 (ZOP2) - 2 % Z. officinale root-powder
• Treatment 4 (ZOP3) - 3 % Z. officinale root-powder

A ration of 40 % Crude Protein (CP) containing fishmeal (72 % CP), groundnut cake (45 % CP), soybean meal (42 % CP) and yellow maize (10 % CP) as the energy source and fixed ingredients including vitamin premix (1 %), lysine (0.5 %), methionine (0.5 %), di calcium phosphate (0.5 %); salt (0.5 %) and vegetable oil (4.0 %). The weight of each ingredient was calculated using Pearson Square method using the stipulated crude protein requirement of the fish. All the four diets formulated were carefully prepared which involves measuring the ingredients, thoroughly mixing the ingredients, and pelletizing them as described by Fagbenro and Adebayo [12]. The ginger rhizome was prepared separately before incorporation into the basal diets.

Fresh rhizomes of Z. officinale roots were bought from a local market in Abeokuta, Ogun state, Nigeria and were confirmed by a botanist. The plant was dried in the shade. The dried rhizomes were further grinded into powdered form using a household grinder and sieved using a household sieve as described by Haghighi and Rohani [13].

The powdered Z. officinale root produced was mixed directly with the basal diet and added into the diets at concentration of 1 %, 2 % and 3 % of feed. Compounded feeds were pelletized (2 mm) using the pelletizing machine from University fish farm, sun dried and allowed to cool then packed and stored in an opaque nylon bag according to the treatments. The percentages of all the feed ingredients used are shown in table 1.

Ingredients (%)	Control	ZOP1	ZOP2	ZOP3
Fishmeal	31.2	31.2	31.2	31.2
Soybean meal	15.6	15.6	15.6	15.6
Groundnut cake	15.6	15.6	15.6	15.6
Yellow Maize	30.5	30.5	28.75	27.75
Vitamin Premix	1.0	1.0	1.0	1.0
Lysine	0.5	0.5	0.5	0.5
Salt	0.5	0.5	0.5	0.5
Vegetable oil	4.0	4.0	4.0	4.0
Methionine	0.5	0.5	0.5	0.5
DCP	0.5	0.5	0.5	0.5
Z. officinale root-powder	0.0	1.0	2.0	3.0
Total	100	100	100	100

The Proximate analysis of the four diets and the fish were done following procedure as described by AOAC [14]. The proximate composition of Z. officinale root-powder [15] is shown in table 2.

Parameter	Amount (%)
Crude protein	34.13
Crude fat	4.02
Ether extract	4.07
Ash content	7.64
Moisture content	13.75
Vitamin C	1.036

The fish were acclimated to the experimental system for a period of 14 days before starting the experiment and were fed two times daily with a commercial diet (40 % CP). The fish were weighed according to the set up per treatments at the beginning of the experiment.

All fish were starved for 24 hours to increase the appetite of the fish before starting the experiment. Fish were fed with the diets two times daily, in the morning between 09:00 - 10:00 h and evening between 16:30 - 17:30 h, ad libitumfor 12 weeks.

Water temperature (°C) and dissolved oxygen (mg \ l) were measured every week using a combined digital YSI dissolved oxygen meter (YSI Model 57, Yellow Spring Ohio); pH was monitored weekly using pH meter (Mettler Toledo-320, Jenway UK).

Weighing of the fish were done in each tank weekly to monitor the fish growth and ensure adequate feed consumption using a sensitive electronic weighing scale (Mettler Toledo FB602, Jenway UK).

The ventral surface of the fish was dissected to removing the gut proximal section. The fish guts (intestines) were gotten for estimation the precision of serial dilution of viable bacteria count and fungi counts as described by Hedges [16].

The bacterial culture were isolated and were subjected to morphological and biochemical tests for their identification according to the methods of Buchanan and Gibbson [17]. The results were analyzed by cross reference to Bergey’s Manual of Systematic Bacteriology [17].

The fungal isolates were subjected to morphological characteristics according to Barnett and Hunter [18] and the isolated fungi were identified according to Campbell et al. [19].

Growth performance of fish was done as illustrated by Agbebi et al. [20], in term of mean weight gain, percentage weight gain, survival (%), specific growth rate (SGR % / day). The growth parameters calculated at the end of the experiment were:

The nutrient utilization parameters such as the Feed Conversion Ratio (FCR), Protein Efficiency Ratio (PER) and Net Protein Utilization (NPU) responses were calculated and data were obtained from them.

The data obtained were subjected to one way Analysis of Variance (ANOVA). Means were separated using Duncan Multiple Range Test [21] at a significant level of 0.05. Computations were subjected to SAS statistical package version 15.0.

Proximate compositions of the four diets formulated and prepared for the feeding trial are presented in table 3. Crude protein of the experimental diets is 40 % among the four treatments, moisture content ranged between 9.56 and 11.0 %, ether extract ranged between 4.70 and 5.42 %, fibre contents ranged between 1.23 and 1.67 %.

Proximate components (%)	Control (0 %)	ZOP1 (1 %)	ZOP2 (2 %)	ZOP3 (3 %)
Moisture	10.50	10.98	9.86	9.56
Crude protein	40.01	40.00	40.04	39.98
Fibre content	3.10	3.12	3.04	3.42
Ash	5.20	4.45	3.95	3.74
Ether extract	5.42	5.20	4.98	4.70
Nitrogen free extract	35.77	36.25	38.13	38.60

The initial and final carcass compositions of the fish fed with varying levels of ginger root-powder and the control is presented in table 4. The crude protein content of the fish carcass ranged between 43 and 52.5 %.

Proximate components (%)	Initial	Control (0 %)	ZOP1 (1 %)	ZOP2 (2 %)	ZOP3 (3 %)
Moisture	11.54	11.84 ± 0.23b	11.92 ± 0.02a	11.85 ± 0.03a	11.97 ± 0.04a
Crude protein	43.50	47.37 ± 0.55c	52.48 ± 0.65a	50.83 ± 0.17b	50.19 ± 0.38b
Fibre content	0.90	1.23 ± 0.02c	1.34 ± 0.05b	1.60 ± 0.12a	1.39 ± 0.04b
Ash	0.98	4.18 ± 0.31a	3.83 ± 0.13b	3.49 ± 0.01b	3.57 ± 0.04b
Ether extract	8.50	12.44 ± 0.08a	9.85 ± 0.20b	10.01 ± 0.28b	9.58 ± 0.06b
Nitrogen free extract	34.58	22.95 ± 0.09a	19.58 ± 0.38c	21.22 ± 0.60b	23.30 ± 0.36a

The physicochemical parameters of the water recorded during the experimental period are shown in table 5.

Week	pH	Dissolved Oxygen (Mg / L)	Temperature (°C)
0	6.00	6.10	25.00
1	6.90	6.20	25.41
2	7.15	6.18	25.60
3	7.03	6.24	25.78
4	7.34	6.35	25.83
5	7.41	6.50	25.61
6	7.48	7.21	26.13
7	7.31	7.25	25.53
8	7.22	7.40	26.20
9	7.28	7.56	26.13
10	7.50	7.45	26.30
11	7.55	7.61	26.05
12	7.56	7.70	26.02

The growth performance and nutrient utilization of C. gariepinus fed Z. officinale root-powder at three varying levels of dietary supplementation and the control is shown in table 6. There were differences in the mean Weight Gains (WG) among fish fed Z. officinale root-powder. There were significant differences in the mean weight gain among fish fed with the Z. officinale root-powder when compared to the fish fed with the control diets. The highest WG (18.0 g) and Percentage With Gain, PWG (838 %) was recorded in fish fed with 1 % Z. officinale root-powder while the lowest WG (11.9 g) and PWG (518 %) was recorded in fish fed with control diet. The highest Feed Conversion Ratio, FCR (1.57) was observed in fish fed with 1 % Z. officinale root-powder while the lowest FCR was recorded in fish fed with control diet. There were significant no differences in the protein efficiency ratio among fish fed with the Z. officinale root-powder.

Parameters	Control (0 %)	ZOP1 (1 %)	ZOP2 (2 %)	ZOP3 (3 %)
Initial weight (g)	2.30 ± 0.06a	2.33 ± 0.09a	2.33 ± 0.03a	2.33 ± 0.09a
Final weight (g)	14.22 ± 1.12c	20.27 ± 0.92a	18.49 ± 1.09b	20.14 ± 1.15a
Weight gain (g)	11.92 ± 1.16c	17.95 ± 0.90a	16.15 ± 1.06b	17.82 ± 1.07a
Percentage weight gain (%)	518.3 ± 10.21c	840.8 ± 7.70a	691.4 ± 10.63cb	762.3 ± 17.69b
Feed intake (g / fish)	19.17 ± 2.29c	27.11 ± 1.49a	25.2 ± 1.65b	25.64 ± 1.96b
Feed conversion ratio	1.40 ± 0.07b	1.57 ± 0.04a	1.56 ± 0.02a	1.55 ± 0.02a
Specific growth rate (% / day)	2.31 ± 0.19b	2.57 ± 0.10a	2.46 ± 0.06a	2.56 ± 0.02a
Protein efficiency ratio	1.80 ± 0.09a	1.65 ± 0.04b	1.60 ± 0.02b	1.61 ± 0.03b
Apparent net protein utilization (%)	50.29 ± 1.38c	83.04 ± 3.92a	68.270 ± 5.12b	65.60 ± 5.05b
Survival rate (%)	86.67 ± 8.82a	80.00 ± 5.77b	63.33 ± 3.33c	63.33 ± 3.33c

The total bacteria count and fungal counts in the intestine of fish fed Z. officinale root-powder diets and the control are presented in table 7. The microbial population of fish fed diet containing varying levels of ginger roots powder was greatly reduced compared to the control (p < 0.05). A significant difference (p < 0.05) occurred in the total bacterial and a fungal count was recorded among the treatment groups with the control having the highest count. Fish fed 2 % Z. officinale root-powder diet recorded the least bacterial and fungal counts followed by 3 % Z. officinale root-powder fed diet.

Parameters	Control 0 %	ZOP1 1 %	ZOP2 2 %	ZOP3 3 %
Total Bacteria Count (CFU / ml) × 105	23.67 ± 0.88a	17.33 ± 4.76b	7.4 ± 5.31c	8.84 ± 7.58c
Total Fungal Count (CFU / ml) × 105	7.67 ± 0.44a	2.83 ± 1.36d	2.00 ± 0.00e	2.50 ± 0.00e

The cultural and colonial morphology of bacteria found in the intestine of fish fed varying level of Z. officinale root-powder are shown in tables 8 and 9.

Isolates Details	A1	B1	C1	D1
Colour	Creamy	Dull Cream	Dull Cream	White
Shape / Margin	Irregular	Entire	Entire	Entire
Arrangement	Cocci in long chain	Baccilli	Bacilli	Cocci in pair
Surface Appearance	Smooth and Glistening	Smooth	Smooth	Smooth and Glistening
Elevation	Raised	Raised	Raised	Raised
Texture	Dry	Moist	Moist	Dry
Opacity	Translucent	Translucent	Translucent	Translucent

Table 10 shows the biochemical test of the bacteria isolated from the intestine of the fish fed Z. officinaleroot-powder and the control showing the occurrence of the bacteria in the fish intestine.

The morphological characters of fungal isolated from the intestine of C. gariepinus are presented in tables 11 and 12.

Treatment	Colour of spore	Appearance of mycelia	Type of spores	Arrangement of spores	Shape of spore	Type of hyphae	Identified organism
A11	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	Aspergillus niger
A12	Yellow black	Fluffy	Sporangiospore	In masses	Globuse	Aseptate	Mucor mucedo
B1	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger
C1	Yellow black	Fluffy	Sporangiospore	In masses	Globuse	aseptate	Mucor mucedo
D1	Black	Grainy	Conidiosphore	In masses	Cylindrical	septate	A. niger

Treatment	Colour of spore	Appearance of mycelia	Type of Spores	Arrangement of spores	Shape of spore	Type of hyphae	Identified organism
A21	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger
A22	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger
B2	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger
C2	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger
D2	Black	Grainy	Conidiosphore	In masses	Cylindrical	Septate	A. niger

The values of the physico-chemical parameters observed in the experimental tanks during this study were within the range recommended for C. gariepinus [22,23]. The achievement of this was as a result of optimum water management practices.

There was a general increase in weight gain with the highest growth performance observed in fish fed 1 % and 3 % Z. officinale root-powder. Fish fed 1 % Z. officinale root-powder diet recorded the highest and showing best growth responses in terms of Percentage Weight Gained (PWG), Specific Growth Rate (SGR) and Apparent Net Protein Utilization (ANPU). The fish fed the control diet recorded the lowest values of PWG and ANPU. This is in agreement with the work of Bello et al. [24], who recorded similar increase in weight gain of fish when fed diets supplemented with Walnut leaf and Onion bulb residues. There was a rapid growth rate of C. gariepinus in the first few weeks of culture in the study; this could probably due to initial starvation of the fish which made them more metabolically active. This is similar to report of Obasa and Faturoti [25] who recorded similar observation in the growth of juvenile Heterotis niloticus. They recorded an increase in growth of the fish as they were subjected to delay in feed distribution.

The remarkable performances of fish fed the supplemented diets in PWG, SGR and ANPU over control diet could be due to the presence of growth promoters: zingiberene, glycosides and terpenoids in 1 % Z. officinale root-powder. This is in agreement with the result of Onibi et al. [26], suggested that ginger and garlic supplements collectively or individually improved growth performance of broilers. This was corroborated by Haghighi and Rohani [13].

There was a reduction in the total feed intake at higher levels of (2 % and 3 %) of Z. officinale root-powder; this could probably due to lower palatability of the two diets as a result of the presence of tannin in the Z. officinale root-powder. The work of Fasasi et al. [12], stated that tannins interfere with digestion by displaying anti-trypsin and anti- amylase activity, forming complexes with vitamin B12 and interfering with the bioavailability of proteins. Also, authors such as Azaza et al. [27], are of opinion that the presence of 2.4 % tannin in faba beans (Vicia faba L. var. minuta) might be responsible for low palatability and consequently low feed intake in Nile tilapia. The increased FCR as revealed in fish fed Z. officinaleroot-powder and higher than the fish fed control diet is similar to the report of Bello et al. [24], who revealed that inclusion of 1.5 % walnut leaf increased FCR in the supplemented groups than the control. This was also corroborated by the work of Abd El - Rahman [28] which indicated that the inclusion of Propolis - ethanolic extract and crude propolis increased the FCR, FER and PER in the supplemented groups when compared with the control. Also the findings of Zomrawi et al. [29], who showed that were no significant differences (p > 0.05) in FCR among all dietary Z. officinale root-powder treatments which corroborated with the result obtained in this present study.

The better SGR recorded in the supplemented diets is in correlation with the result of Abou-Zeid [30] which showed that Allium sativum supplementation positively affected O. niloticus biomass and Specific Growth Rate (SGR).

There is a reduction in survival rate in fish fed the Z. officinale root-powder diets as seen in this experiment could be as a result of some phytochemicals present in the phytobiotic. Fish fed 2 % and 3 % Z. officinale root-powder recorded the highest mortality rate. The findings of Ashade et al. [31], also revealed that the mortality rate of fish fed untreated ginger peel increased with respect to the different concentrations and the highest concentration having more mortality rate.

The bacteria flora of the intestine of C. gariepinus consisted of Bacillus substilis, Streptococcus agalactiaeand Enterococcus faecialis. This could indicate that Z. officinale root-powder favours or promote the growth of gram positive bacteria in the intestine of C. gariepinus. This is in the agreement with the work of Olojo et al. [32] who observed similar bacteria flora in the intestine of C. gariepinus. This is corroborated by Nwabueze [11] who observed similar bacteria flora from the epidermal mucus of C. gariepinus fed ginger powder. Pandy et al. [33], also reported that all medicinal plants are able to stimulate only non-specific immune responses and suggested that the medicinal plants could be used to treat diseases. This gastrointestinal microbes could also be advantageous in that they help in digestive processes of fish such as microbial breakdown of chitin, collagen, cellulose and they may also supply fatty acids and other vitamins to the host and hence promoting growth of the fish [34]. These might have taken place in the fish fed the dietary supplement. The gastrointestinal microbes also prevent colonization of the fish by other microbes that might otherwise be pathogenic. B. substilis has been reported to promote performance and immune responses which might have taken place in fish fed ZOP1 and ZOP3. Tannins and alkaloid are present in ginger roots powder. Tannins and saponins are responsible for antibacterial activity, and able to permeate cells without destroying cell morphology. Tannins inhibit microbial proliferation by denaturation of enzymes of involved in microbial metabolism [35]. This might have occurred in the intestine of fish fed the dietary supplement because majorly the bacteria flora in the intestine of C. gariepinus are mostly gram positive bacteria and thus these phytochemicals probably inhibited the growth of gram negative bacteria and promoted the growth of gram positive bacteria. 

There were differences (p < 0.05) in the reductions in the total bacteria count of fish fed varying levels of Z. officinale root-powder than the control. This is in agreement with the report of Bello et al. [24], who observed that there was a decrease in values of the bacterial load of the supplemented groups (onion bulb and walnut leaves) as the level of inclusion (0.5 %, 1.0 % and 2.0 %) increased and as the months increased. The enterobacteriaceae load in the intestine of C. gariepinus fed Z. officinale root-powder were lower than the control with significant decrease (p < 0.05) as recorded in this study. This decrease in bacteria load in fish as observed in this study has been linked to the presence of antimicrobial properties in Z. officinale root-powder. This study suggests that Z. officinale root-powder is more effective as an antibacterial. Tannins also have shown potential antiviral, antibacterial properties [36]. 

The fungi flora of the intestine of C. gariepinus fed diets containing varying levels of Z. officinale root-powder revealed the presence of Aspergillus niger and Mucor mucedo. A. niger and M. mucedo are associated to food spoilage. Their presence in the fish intestine could indicate that some of the experimental feeds might have been rancid and support the growth of these microbes.

Also, there were differences (p < 0.05) in the reductions in the total fungal count of fish fed diets supplemented with varying levels of Z. officinale root-powder than the control diet. Also, this study indicated that Z. officinale root-powder could act effectively as an antifungal. This is in line with the work of Idris et al. [37], which worked on the effect of different concentration of ginger on smoke-dried C. gariepinus, and found that ginger reduced the free fatty acid values, tri-methylamine values and reduced the fungi load of the processed fish.

The results of this present study revealed that dietary supplementation of Z. officinale root-powder for C. gariepinus fingerlings are encouraged to improve the growth performance; nutrient utilization and gastrointestinal microbes of C. gariepinus fingerlings. This could be attributed to the growth promoting activity and immunostimulation properties of the dietary supplement. Inclusion level of 1 % Z. officinaleroot-powder in the diet of fish is best considered because of its good FCR, high ANPU, SGR and weight gain. Based on this present study, inclusion of 2 % Z. officinale root-powder is relatively considered to act as an antibacterial and antifungal in the fish and improving the gastrointestinal microbes of C. gariepinusfingerlings.