Dietary inclusion effect of ginger (Zingiber officinale) and garlic (Allium sativum) blend on growth, feed nutrients utilization and retention in African Catfish (Clarias gariepinus) fry in intensive system

This study was conducted to evaluate the dietary inclusion effect of ginger-garlic mixture on growth, feed nutrient utilization and whole-body composition of Clarias gariepinus fry. Fry weighing 1.2±0.01g were divided into four triplicate treatments before being fed with diets containing 0mg (D0 or control), 50mg (D1), 100mg (D2) and 200mg (D3) of ginger-garlic mixture /kg diet (1:1 proportion) for 56 days. Fish were fed at the rate of 10% of their body weight and every 14 days, intermediate sampling was done during which fry per treatment were counted, measurements taken on a representative sample and the feeding rate adjusted. Main water parameters were recorded daily before feeding. The results obtained indicate that, adding ginger-garlic blend in the diet enhanced signifi cantly growth, feed nutrients utilization and retention as well as whole-body composition of C. gariepinus fry depending to the inclusion level. Fish fed with the highest supplemented level (200mg/kg diet) have recorded a fi nal weight of 25.5±0.16g, with a weight gain (WG) of 24.34±0.16g, feed conversion ratio (FCR) of 1.46±0.01 and protein effi ciency ratio (PER) of 1.53±0.02. These parameters were signifi cantly different to those recorded in fi sh fed with control diet (fi nal weight=17.02±0.27g, WG=15.23±0.28g, FCR=2.03±0.01, and PER=1.10±0.01). Similar trend to growth parameters was observed with macro-nutrient retention (protein=36.2±0.33%, lipid=29.1±0.33%, ash=23.13±0.68% and energy=24.99±0.25%) as compared to control (protein=24.94±0.01%, lipid=21.91±0.01%, ash=13.40±0.01% and energy=18.00±0.01%) Thus, the improvement in growth induced by the feed addition of gingergarlic mixture could be largely attributed to the synergistic actions of the bioactive molecules contained in each phyto-additive on the functioning of the digestive system as well as to their hypoglycaemic and antioxidant properties. Research Article Dietary inclusion effect of ginger (Zingiber offi cinale) and garlic (Allium sativum) blend on growth, feed nutrients utilization and retention in African Catfi sh (Clarias gariepinus) fry in intensive system Paulin Nyadjeu*, Noël Arlette Tamko Ndjuissi, Doriane Divine Mane Yemdjie, Nesrine Yolande Chamsy Dedou, Georges Fonkwa, Ghislain Nguimdop Nguenang and Minette Eyango Tabi-Tomedi Department of Aquaculture, Institute of Fisheries and Aquatic Sciences of Yabassi, University of Douala, PO Box: 7236-Douala, Cameroon Received: 12 May, 2021 Accepted: 26 May, 2021 Published: 27 May, 2021 *Corresponding authors: Paulin Nyadjeu, Department of Aquaculture, Institute of Fisheries and Aquatic Sciences of Yabassi, University of Douala, PO Box: 7236-Douala, Cameroon, Tel: (+237) 690872377; E-mail:


Introduction
African catfi sh, Clarias gariepinus (Burchell, 1822) is an air-breathing species when very active or under low dissolved oxygen conditions. It is indigenous to the inland waters of much of Africa, more precisely sub-Saharan Africa. Clarias gariepinus is cultured in several countries throughout Africa as well as in Europe, Asia and South America [1]; though its contribution to world aquaculture production (89.1%) is very low and represent only 0.33% [2,3]. In Africa on the other hand, out of the contribution of 16% to 18% to total fish production that represent about 2.7% of global production, most of that production (99%) is dominated by tilapia and African catfi sh [4,5]. In sub-Saharan Africa countries, particularly in Cameroon, although the statistics are currently unavailable, African catfi sh is the most cultured species mostly in small-scale fi sh farms [6,7]. Moreover, interest in growing C. gariepinus is increasing in all parts of the country and the success of its farming is linked to improvement of its artifi cial reproduction technic and its acceptability by consumers as well as by fi sh farmers due to benefi cial characteristics including fast growth and high market demand. Nevertheless, some challenges such as the unavailability of high quality feed at low cost is a major constraint which hinders its production.
The only way to overcome this challenge is to encourage local production of quality feeds with a high protein content, which should contain not only the necessary nutrients but also complementary additives to improve the quality of the feed, maintain the fi sh healthy and promote their growth.
Feed additives are edible substances that are supplemented to feeds in small amounts (alone or in combination) for a specifi c purpose, such as to improve fi sh performance and quality, to preserve the physical and chemical quality of the feed as well as that of the aquatic environment [8,9]. Several studies have proven the positive potentials of spices and medicinal plants as feed additives in aquaculture for better growth and survival [10,11]. Phyto-additives or Phytogenic feed additives are plantderived, natural compounds that are added to feed to improve it quality and animal performance. They may be both nutritive and non-nutritive ingredients and work by either direct or indirect methods on the animal's system [9,12]. Phytogenic feed additives can be classifi ed into several groups: sensory phyto-additives, which affect positively the sensory properties of animal products; technological phyto-additives, which have antioxidant and anti-mold properties; zootechnical phytoadditives, which are immunomodulators, digestive stimulants acting on the activity of digestive enzymes, growth promoters of non-microbial origin as well as substances increasing performance or quality of animal products; nutritional phytoadditives, which are exceptionally rich in vitamins, minerals, micro and macro molecules [13][14][15]. Garlic (Allium sativum) and ginger (Zingiber offi cinale) are among the spices most commonly used in aquaculture as phyto-additives for their nutritional, physiological and pharmacological properties.
Garlic (Allium sativum) is a perennial bulb-growing plant belonging to the family Liliaceae. It has been used for decades as a fl avouring agent, traditional medicine, and a functional food for improvement of physical and mental health. Research has been carried out on garlic in several forms: extracts (aqueous, ethanol) and dried powder [16,17)] Garlic contains a variety of organo-sulfur compounds like allicin, ajoene, S-allylcysteine, diallyl disulfi de, Smethylcysteine sulfoxide and S-allylcysteine [18]. It also contains minerals (phosphorus and calcium), vitamins (A, C and B complexes), linolenic acid, carbohydrates and many valuable compounds such as iodine and silicates with positive effects on circulatory, skeletal and cholesterolemia [19,20]. Earlier studies have reported that garlic, as a feed additive in fi sh feed, stimulate growth, improve antioxidant status, and enhance immunological, hematological and serum biochemical parameters [21].
Ginger (Zingiber offi cinale), an underground stem or rhizome belonging to the family Zingiberaceae, is widely used around the world in food as a spice. Ginger contains a wide variety of biologically active compounds like alkaloids, fl avonoids, polyphenols, saponin, steroids and tannin as well as nutritional molecules like fi ber, carbohydrate, vitamins, carotenoids and minerals [22]. The species is also rich in natural antioxidants components as gingerols, shogaols and Zingerone [23]. The diet enriched with ginger has been shown to promote growth, immunostimulation, digestion stimulation, improvement of protein and lipid metabolism as well as antioxidant, antihyperglycaemic, antiviruses, antimicrobial and parasites properties in rearing fi sh [24,25].
Garlic is generally paired with ginger to make stews and soups in most homes and restaurants all over the world. They are often used in a small quantity as food additives to fl avour or preserve food and to stimulate appetite by increasing the fl ow of gastric juice as well as enhancing food taste [26]. Owing to physiological and pharmacological properties of different bioactive ingredients contained in ginger and garlic, they are considered as safe herbal medicines with very little and insignifi cant adverse effects in human being, livestock, including fi sh [27,28]. Previous studies revealed that dietary supplementation of the combination of ginger and garlic in the diet of commercial broilers did not improved growth and feed utilization compared to control diet [29]. On the other hand [30] had previously indicated that feeding Rainbow Trout (Oncorhychus mykiss) with a combination of garlic and ginger have improved weight gain, specifi c growth rate and feed conversion ratio compared to control diet. Moreover [17] have shown that dietary inclusion of the mixture of garlic and ginger enhance growth, feed utilization parameters and survival in Clarias gariepinus post-larvae. Despite these few studies, in our knowledge, research regarding the combined effects of garlic and ginger as phytogenic feed additive in the diet of African catfi sh during the nursery phase are almost non-existent. Thus, the present study was aimed to evaluate the effect of different inclusion levels of the mixture of garlic and ginger powders as a feed additive on growth, feed nutrient utilization and whole body composition of African catfi sh fry in intensive system.

Study location and experimental system
The study was carried out in the hatchery unit of a small-Citation: Nyadjeu  scaled private farm called "Massoma fi sh farm" at Bojongo-Bonaberi, Wouri Division, Littoral Region of Cameroon from April to May 2020. The experiment was conducted in twelve (12) circular plastic tanks each (10 litres) and each tank was two third fi lled (6.5 litres) with water. All the tanks were mounted in an open circuit connected by a pressurized piping system of diameter 25 mm and supplied with borehole water by means of a pump.

Experimental fi sh and housing
A total of 444 apparently healthy Clarias gariepinus fry weighing 1.2±0.01g were obtained from an artifi cial reproduction carried out in farm hatchery. They were randomly stocked into 12 circular plastic tanks of 37 fry each and allowed to acclimatize for 7 days before the beginning of the feeding trial. Commercial feed (Coppens, 0.2mm, Coppens International B.V, Netherlands) was used to feed the fi sh during the period of acclimatization. The breeding tanks were divided into four treatments as listed below. Each treatment was made up of three replicates. Fish faeces and residual feed in each of the rearing tanks were siphoned off daily in the morning by the use of a rubber tube and the water renewed by half. Prior to the beginning of the experiment, fi sh were starved for 24 hours after which, twenty four fi sh (2 per tank) were randomly pick up for initial determination of the carcass composition. The remaining fry (420) were hand-fed four times daily (08:00 a.m, 11:00 p.m, 14:00 p.m and 17:00 p.m respectively) with different diets at a rate of 10% of their body weight (BW) during the fi rst 28 days of feeding and 5% for the rest of the feeding period, with feeding ration being adjusted in accordance to weight gain of fi sh after random sampling at 14 days interval. Each day before feeding, the physico-chemical parameters such as temperature (T°C) was measured using a Maximum-minimum thermometer, dissolved oxygen (D.O), using JBL Test Kits, pH, nitrite (NO 2-) and nitrate (NO 3-), using Test strips (JBL Easy Test 6in1) ( Table 1). During intermediate samplings, fi fteen fi sh from each tank were randomly harvested to record growth in terms of total body length (TBL) and body weight (BW). At the end of the feeding trial, fi sh were harvested and then weighed as total fi sh weight of each replicate within dietary treatment and counted for the calculation of both the growth and feed effi ciency parameters. Fifteen fi sh (fi ve fi sh per tank) from each treatment were selected to analyse the fi nal proximal composition of the carcass according to the method of Association of Offi cial Analytical Chemistry [31].   (Table 2). To prepare the diets, the dried and grinded ingredients of each diet were weighed and mixed thoroughly in a bowl, palm oil and warm water were then added slowly along to the mixture and mixed manually for about 30 minutes to achieve a proper consistency. The resulting mixture was pelletized (2 mm) using an electric pelleting machine and allowed to dry for 24h by air circulation before being packed into airtight containers and stored at room temperature to be crumbled before use.

Formulation and preparation of experimental diets
Formulated diet samples (10g) were analysed following the procedures of [31]. Moisture was analysed by drying the sample in an air convection oven at 105 °C overnight. Crude protein was analysed by the Kjeldahl method after acid digestion (% crude protein = % nitrogen × 6.25) while crude lipid was determined by extraction with petroleum ether using the Soxhlet method.
The ash content in the diet was analysed by combustion of samples in a muffl e furnace at 550 °C for 12 h (Table 2) Growth, survival rate and feed utilization parameters Growth performances, survival rate, feed utilization and nutrients retention were assessed for each treatment by determination of weight gain (WG), length gain (LG), mortality

Statistical analysis
All results were expressed as mean ± SD (Standard Deviation). The data collected during every fi sh sampling were analysed by one-way analysis of variance (ANOVA-1) repeated measure followed by Tukey's multiple comparisons test with n=3 replications containing 35 fi sh each. Differences were regarded as signifi cant when p<0.05; Regression analysis was established between fi sh growth, feed intake as well as macronutrient retention and dietary supplementation level of the ginger-garlic mixture. All statistical analyses were conducted using GraphPad Prism version 6.0.

Feed utilization and mortality
Daily observations without measurement during feeding made it possible to note an acceptance of all the feeds by the fry which fed voraciously during the whole feeding period.
This is refl ected in Table 3   The mortalities recorded in different treatments during the study and presented in Figure 4 were not related to the inclusion level of garlic and ginger blend in the diet of Clarias gariepinus fry. The highest mortality rate registered at the end of the feed trial was observed with diet D2 (41±1.73%), followed by fi sh fed with diets D0 (35±4.58%) and D1 (35±1.73%), while the lowest mortality of 29±0.00% was observed in fi sh fed with diet D3.

Discussion
The performances of fi sh to feeds are due to more than just nutritional factors. Indeed, water quality parameters, which are the result of the rearing system have a great infl uence on fi sh survival as well as growth, especially at the fry stage. The physico-chemical parameters of the rearing medium such as temperature and dissolved oxygen recorded during the present study were observed to be within the range recommended for freshwater fi sh culture [32,33]. [33] recommended temperature values between 25 and 30°C for rearing of C. gariepinus. In addition, dissolved Oxygen level greater than 5mg/l is necessary to support good fi sh production. The values of the acidity degree registered during the study were oscillated between 6.6 and 6.7. [34] reported that the ideal pH level is between 7.5 and 8.5 and any value above or below this could be stressful to the fi shes, especially to fry. According to [35], when the pH value drops below 7, it has a negative infl uence on the nitrifi cation process. However, the nitrite concentration recorded in the present study shows a range between 0.0mg/L and 0.2mg/L. According to [36] concerning water quality for aquaculture pond and based on the research work carried out by [37] whose observations led to the development of guidelines on the useful water quality for the management of pond fi sh culture, the nitrite threshold for freshwater fi sh fry is 0.2mg/l. Excess nitrite compounds in water caused by faeces and unconsumed feeds are termed as an invisible killer of fi sh because it oxidizes haemoglobin to methemoglobin in the blood, turning the blood and gills brown and hindering respiration. Accordingly, the concentration of nitrite recorded during the present study still meets the proper range for optimum growth of catfi sh fry. Moreover, nitrite being a transitional form between ammonia and nitrate during nitrifi cation process, and between nitrate and nitrogen gas during denitrifi cation process is relatively less toxic [38]. The results of the present study have indicated that the concentration of nitrate ranged between 0.00mg / L and 10mg/L. This concentration still meets the proper range for optimum growth of catfi sh fry [39,40]. According to the above observations, the mortalities accounted in all the treatments would be attributed more to the poor conditions of the breeding environment than to the diet. Indeed, the absence of automatic siphoning of feed residues and faeces would be partly responsible for the acidity of the rearing medium. Moreover, water in the rearing tanks was half renewed each morning before feeding. Unfortunately, this daily action has being carried out sometimes manually due to power cuts to run the water pump. Thus, the low pH value mentioned above associated with the daily renewal of the water in the rearing tanks constitute potentially stressful conditions for the fry of C. gariepinus which would probably have caused a dysfunction of their physiological balance and would certainly have induces an increase in the production of free radicals whose effects on vital functions are known to cause death. On the other hand, it was observed a signifi cant decrease of the mortality rate after feeding fry with the diet containing the ginger- Ni: initial number of fi sh; Nf: fi nal number of fi sh; Wi: initial body weight of fi sh; Wf: fi nal body weight of fi sh; Li, initial length of fi sh; Lf: fi nal length of fi sh; FI: feed intake; PI: protein intake; K: condition factor; FCR: feed conversion ratio; FER: feed effi ciency ratio; PER: protein effi ciency ratio to eliminate free radicals, prevent lipid peroxidation and promote endogenous antioxidant defences, but also through their ability to improve palatability and feed consumption [42].  Direct observations during feeding permitted to notice that fry fed with the control diet were the smallest and less active in the hapas, compared to those fed on basal diet enriched with the ginger-garlic mixture. It was also observed that enriched diets with the mixture of ginger and garlic were more attractable to the fry than the control diet. This, because fry in the experimental hapas in addition to being the most vigorous and agitated during feeding, have also expressed the highest feed consumption values, compared to control. This indicate that inclusion of ginger and garlic blend in the diet of C. gariepinus fry would have improved the feed taste, leading to an increase in appetite stimulation. These observations corroborate the previous research works conducted by [17] during which it was observed that inclusion of the ginger-garlic mixture in the diet of C. gariepinus post-larvae in fertilized pond increased feed intake with respect to the inclusion level.
Plant derived products or by-products used as additives in fi sh feed are known to enhance feed taste or palatability as well as appetite stimulation, thereby inducing a positive effect on growth [43,44].
In the present study, growth performance and feed utilization were signifi cantly improved in fish fed on dietary supplementation of ginger-garlic mixture as compared to those fed with control diet; with the highest response being observed in fi sh fry fed with the highest level of phytoadditives combination. These results are in contradiction with those obtained by [29] who observed no improvement in growth and feed utilization in broilers fed a diet containing the mixture of ginger and garlic as compared to control. The confl icting results of the present study with those of [29] could therefore be attributed to the different between the species used, feeding program and even the culturing conditions.
On the other hand, these results agrees with the fi ndings of [45,30] as well as those of [17] where the mixture of ginger and garlic powders was included in the diet of Oncorhychus mykiss juveniles, Huso huso juveniles and Clarias gariepinus post-larvae respectively. The improvement in growth and utilization of feed nutrients in C. gariepinus fry induced by the gingergarlic blend inclusion over the performances of those fed with the control diet could have certainly emanated from the presence of bioactive compounds contained in both phytogenic additives. Indeed, ginger is known to stimulate the appetite, the secretion of pancreatic enzymes and the bile from the liver in addition to intervening in the balance of intestinal bacteria [46][47][48]. Garlic for its part is known to promote feed intake, the performance of the intestinal fl ora, enhance digestion as well as energy utilization [49]. Thus, it is easy to suggest that the presence of the mixture of ginger and garlic in the diets would have boosted nutrient utilization characterized by low feed conversion ratio and high protein effi ciency ratio; which was refl ected by signifi cantly higher weight gain as well as length gain. Moreover, because of the pharmacological properties of ginger and garlic on blood glucose reduction, it has been suggested that they would partially stimulate growth by increasing the infl ow of glucose into tissues [50]. According to the above mentioned physiological and pharmacological properties of ginger and garlic on the digestion and glucose metabolism, it can be agree that enhancement of growth performance as well as feed nutrient utilisation in C. gariepinus fry fed with experimental diets as compared to the control could be partially attributed to the combined effects of the bioactive compounds contained in each phytogenic additive which would have acted in synergy to increase the digestibility of macronutrients such as proteins, lipids and ash which are part of the major constituents of the feed. The probable improvement in digestibility induced by the synergistic effects of the bioactive molecules of the plant additives could be justifi ed by the low value of the feed conversion ratio obtained in C. gariepinus fry fed on the experimental diet, particularly that containing the highest level of the phyto-additives mixture. In addition to the possible involvement of ginger and garlic bioactive molecules in the digestion mechanism as well as glucose metabolism of experimental fry, other studies have indicated that the benefi cial effects of both phytogenic additives on the health status of fi sh when they are used as feed additive are also due to their antioxydant properties [51]. Biochemical analysis of the feeds showed no difference between different diets. Thus, signifi cant improvement in C gariepinus fry growth in the experimental treatments compared to control can only be explained by the physiological and pharmacological properties of the bioactive components contained in the mixture of ginger and garlic. These bioactive molecules would thus have induced their biological effects through their synergistic actions on the functioning of the digestive system, but also by their hypoglycaemic and antioxidant properties, which would have contributed to an improvement in general body homeostasis. This therefore, allow the fry to make the best use of the nutrients contained in the consumed feed, justifying thus the signifi cant improvement in the macronutrients retention as well as biochemical composition of the whole-body of the juveniles obtained at the end of the study as compared to control.

Conclusion
The results of the present study revealed that, independently of the parameter evaluated, the dietary inclusion of the gingergarlic mixture induced signifi cantly increased in the growth performance, feed nutrient utilization and retention as well as whole-body composition of the juveniles produced; the highest effect being obtained with the highest level of inclusion. These observed benefi cial effects have been attributed to the synergistic actions of the bioactive contents of ginger and garlic than to their nutritional properties.