Response of Clarias gariepinus to Allium sativum-based diet on growth performance and Staphylococcus aureus challenge infection

The response of Clarias gariepinus to Allium sativum on growth performance and as anti-bacterial agent in Staphylococcus aureus challenge infection was evaluated. A. sativum was included at 0% (control), 1.5%, 3.0%, and 4.5% in fish diet. Twenty fish samples each were randomly distributed into four tanks (T1, T2, T3 and T4). Fish were fed twice daily at 5% body weight for twelve weeks. All fish were challenged with 0.5ml of pure culture of S. aureus. A significant (P<0.05) increase in body weight, total length and standard length was observed. A. sativum at 3.0% inclusion promoted highest growth with feed conversion ratio (1.25-1.41), protein efficiency ratio (1.72-0.56), specific growth rate (1.92-2.09), condition factor (0.83-1.99) and survival rate (60-95). Fish-fed A. sativum diet showed that 4.5% inclusion had the least S. aureus activity. Bacteria load significantly (P<0.05) decrease at week 12. Culture water had dissolved oxygen of 4.0 4.2 mg/L, pH 6.0 7.0 and temperature of 26.028.0C. This study has shown that A. sativum supplemented diets enhanced growth of C. gariepinus and functioned as antibacterial agent against S. aureus. A. sativum at 3.0% inclusion in diet is recommended for better fish growth and 4.5% A. sativum for antibacterial action. DOI: https://dx.doi.org/10.4314/jasem.v24i5.4 Copyright: Copyright © 2020 Nwabueze et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 10 March 2020; Revised: 17April 2020; Accepted: 20April 2020

Clarias gariepinus is a dominant freshwater fish and popular in commercial aquaculture due to its ability to grow rapidly and high tolerance to environmental conditions (Mohamed et al., 2017). However, C. gariepinus has been found to be susceptible to both microbial and parasitic infections particularly in intensive culture systems (Sudheesh et al., 2012;Opiyo et al., 2018). For decades antibiotics have been used for the treatment of bacterial infection in fish. The adverse effects associated with the use of antibiotics include drug residue, bioaccumulation and resistance of pathogens, which threaten human consumers (Lee and Gao, 2012). Hence, herbs are now being used as probiotics in preventing bacterial infections and are gaining success because they are cost effective, ecofriendly and have minimal side effects (Carusol et al., 2013). Herbs exhibit anti-microbial, anti-stress, appetite stimulation, immunostimulation, and aphrodisiac and antipathogenic effects which facilitate growth and maturation of cultured species (Harikrishnan et al., 2011). Alluim sativum is a pungent herb and has been reported to inhibits bacterial growth, promote fish growth and enhancement of blood parameters (Nya and Austin, 2009;Dikel, 2015). In aquaculture, A. sativum has been observed to promote growth, enhance immunity, stimulate appetite and strengthens the control of bacteria and fungi pathogens (Harris et al., 2001;Lee and Gao, 2012). S. aureus is one of the major bacterial agents causing food-borne diseases in humans worldwide and has been found on skin of healthy people and animals including fish (Fetsch and Johler, 2018).The response of Clarias gariepinus to A. sativum based diet on growth performance and Staphylococcus aureus challenge infection was investigated.  Acclimation and Experimentation: Fish specimens were acclimated for 14 days in a stock tank (45 cm x 45 cm x 90 cm) containing 120 L of bole hole water. Fish were fed commercially available fish feed at 5% body weight twice daily. Stock tank was well aerated and water temperature maintained at a ranged of 27.0 0 C -28.0 0 C. Twenty experimental fish each were randomly distributed into four tanks, T1 as control (0%) and T2 (1.5%), T3 (3.0%) and T4 (4.5%) as treatments dietary inclusion of A. sativum. The experiments were in duplicates and maintained in a weekly half renewal static bioassay. Experimental diets (Table 1) were formulated according to Ndong and Fall (2011). Prior to administration of experimental diet, fish were starved for 24 hours. Experimental diets were analyzed (AOAC, 1984) and proximate composition as presented in Table 2.
Growth Parameters and Nutrients Utilization: Growth parameters were calculated following the method described by Bagenal (1978). Growth indices considered were percentage weight gain and total length gain, specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), condition factor, K and survival rate, SR.  Gram Staining and biochemical characterization were done according to Olutiola et al. (1991).The isolates were identified by comparing their characteristics with those of known taxa, as described by Oyeleke and Manga (2008). Experimental fish were sampled biweekly for a period of six months for bacterial examination for possible infection. The average count on plates was multiplied by dilution factor and expressed as colony forming unit per milliliter (Cfug -3 ) of the original factor. Water quality parameters such as temperature, dissolved oxygen, hydrogen ion concentrations, pH and ammonia were analyzed according to (ALPHA, 2010).
Data Analysis: Data collected were subjected to one way analysis of variance with significant means (p<0.05) separated using Duncan's Multiple Range Test.

RESULTS AND DISCUSSION
Mean growth in body weight, standard length and total length were progressive during the experimental period ( Figure 1).   This study shows that fish fed (3.0%) of A. sativum inclusion in diet had the highest specific growth rate.This finding is in agreement with the study of Javadzadeh et al., (2012) who observed high specific growth rate in L. vannami when fed Artemia nauplii enriched with 200 mg garlic extract/ L. Jasour et al., (2018) reported that biogenic diet increased feed intake, feed conversion ratio (FCR) and PER in fish which could have attributed to the better growth performance observed in fish fed 3.0% of garlic. PER and FCR are utilized as quality indicators for fish diet and its amino acid balance. Therefore, these factors are used to evaluate protein utilization and turnover (Shalaby et al., 2006). Table 3 shows the nutrient utilization of C. gariepinus-fed dietary inclusions of A. sativum. The biweekly mean bacteria load of fingerlings of C. gariepinus-fed dietary inclusion of A. sativum is presented in Table 4.This study revealed a significant decrease (P<0.05) of bacteria load of C. gariepinus-fed experimental diet. This finding is in line with the report of Rahman et al. (2008) who found that young Thai silver barb, Barbonymus gonionotus fed a diet supplemented with 8 mg mL -1 garlic showed the best recovery rate (90%) during the 10-days experimental period. Deresse (2010) reported that dilute solutions of garlic completely inhibited the growth of S. aureus at concentrations greater than 7.50 mg mL -1 . C. gariepinus in treatment tanks, were positively infected with S. aureus. However, 4.5% garlic inclusion was observed to be more effective in reducing S. aureus counts/loads. Aly and Mohamed (2010) found that O. niloticus-fed 3% garlic supplemented feed showed a significantly increased survival rate (85%) even after infection with A. hydrophila. The water quality parameters were within the levels recommended for fish culture (Boyd and Lichtkoppler, 1990).

Conclusion:
This study has shown that garlic supplemented diets in fish enhanced growth performance and reduced bacterial load in Clarias gariepinus. Three percent (3.0%) inclusion of garlic in diet is recommended for increase in fish growth performance while 4.5% garlic inclusion can be used to reduce Staphylococcus aureus counts/loads in Clarias gariepinus fish culture.