Ethiopian Journal of Science and Sustainable Development

e-ISSN 2663-3205 Volume 11 (1), 2024

ASTU Journal Home Page: www.ejssd.astu.edu.et

Research Paper

Bacterial-resistance, growth and immune competence of Oreochromis niloticus fed

Lactobacillus fermentum
Olugbenga Orisasona1, Kolawole Emmanuel Ajani2, Bamidele Oluwarotimi Omitoyin2, Omotola Jenyo-
Oni2, Elijah Friday Osho2, Kazeem Oladeji Kareem2, Traore Youssouf2, Abimbola Olumide Adekanmbi3
1
Department of Animal Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
2
Department of Aquaculture and Fisheries Management, University of Ibadan, Ibadan, Nigeria
3
Department of Microbiology, University of Ibadan, Ibadan, Nigeria

Article Info Abstract

Article History: Aquaculture production can be boosted with enhanced feed efficiency, growth and disease
Received 18 July 2023 resistance using probiotics. In this study, the effects of Lactobacillus fermentum (LF) probiotic
Received in revised on growth, intestine, immune competence of Oreochromis niloticus, and the resistance to
form 22 January 2024 virulent Aeromonas hydrophila were investigated. A basal (LF1) diet (35% crude protein) was
Accepted 04 February formulated and five treatments with L. fermentum included at 1 x 103 cfu/g (LF2), 1 x 105 cfu/g
2024 (LF3), 1 x 107 cfu/g (LF4), 1 x 109 cfu/g (LF5) and 1 x 1011 cfu/g (LF6). The six diets were
each allotted to three groups of Oreochromis niloticus (1.85±0.01g mean weight) for 90 days.
After which fish were injected with A. hydrophilia, fed and observed for 15 days. At the end
Keywords: of the 90 days, LF diets fed fish had higher (p<0.05) mean weight gain. Feed conversion ratio
gut, and specific growth rate were superior in fish fed LF3 and LF4. Villi height was significantly
innate immune, high in LF3, while microbial loads were more in the guts of fish fed probiotics. Blood packed
L. fermentum, cell volume and haemoglobin increased significantly in the groups fed LF. Hydrogen peroxide
and malondialdehyde reduced significantly (p<0.05) in the liver and kidney of fish fed L.
Nile Tilapia,
fermentum, while glutathione transferase and glutathione peroxidase activities were increased.
probiotics The challenge test with virulent Aeromonas hydrophila showed improved resistance in fish fed
probiotic. Thus, the study disclosed improved growth performance, enhanced health status and
better survival with the supplementation of Lactobacillus fermentum in the diets of
Oreochromis niloticus.

1. Introduction
Fish disease outbreaks in aquaculture is a major as to ensure increased production, more profit to
impediment to the continuous growth of fish food farmers, and quality fish and fish products.
producing sector, which has been described as the Bacterial infections are the major causes of mortality
world’s fastest growing (Subasinghe et al., 2003; FAO, in fish (Austin and Austin, 2007) and motile
2018). This is evident in the reduction in growth rate Aeromonas, especially Aeromonas hydrophila, has been
from the 10.8 and 9.5% recorded in the 80’s and 90’s, identified in most freshwater fish diseases (Pridgeon and
respectively to 5.8 % between 2001 and 2016 (FAO, Klesius, 2011). A. hydrophila comprises gram-negative
2018). It is therefore, important to establish ways to motile, straight rods belonging to the family
effectively control and treat diseases in cultured fish, so Aeromonadaceae. They are notorious for increased


Corresponding author, e-mail: [email protected]
https://doi.org/10.20372/ejssdastu:v11.i1.2024.716
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© 2024 Adama Science & Technology University. All rights reserved
Orisasona et al. Ethiop.J.Sci.Sustain.Dev., Vol. 11 (1), 2024
capacity to uptake and exchange antimicrobial fish and used as probiotics (Nwanna et al., 2013).
resistance genes and they exhibit several antibiotics Gewaily et al (2021) reported improved performance in
resistance pattern (Ogbonne et al., 2020). According to Nile Tilapia fed on L. plantarum diets. L. fermentum
the authors, isolates of A. hydrophila were completely supplementation resulted in increased weight and
resistant to some antibiotics, while some resistance nutrient utilization, and improved immune system in
levels vary from 55 to 70%. Antimicrobial resistance is common carp, Cyprinus carpio (Govindaraj et al.,
a well acknowledged threat to public health globally, 2020). The variations in strains, species and the source
and Nigeria is not left out. According to FMAEH of isolation of bacteria, factors coupled with the fish a
(2017), the emergence of multidrug resistant microbes probiotic is administered to, will most likely influence
has resulted in exponential increase in mortality and the results from each trial. This study examines effects
economic losses to farmers in Nigeria. of Lactobacillus fermentum isolated from cheese-milk
The control and treatment of these pathogenic on the growth, gut micro flora and immune response of
bacteria is achieved in the past by routine application of an important freshwater fish in Nigeria and Sub-Saharan
antibiotics (Lewbart, 2001). However, inappropriate Africa, the Nile Tilapia.
and overuse of these compounds resulted in the
development of antibiotic resistance strains of bacteria 2. Materials and Methods
(Omitoyin et al., 2019). A survey on antibiotic resistant 2.1. Experimental site and procedure
disease revealed that A. hydrophila and A. sobria The study was carried out in the Department of
isolated from tilapia hybrid resisted oxytetracycline, Aquaculture and Fisheries Management, Wet
erythromycin and sulfadiazine, which were frequently Laboratory, University of Ibadan, Nigeria. Four
used in treatments and prevention of bacterial diseases hundred and fifty fingerlings of O. niloticus
in fish (Wang et al, 2003). This, coupled with the build- (1.85±0.01g) were procured and acclimatized for 14
up of antibiotic residues in cultured species and the days to laboratory condition during which they were fed
disruption of the micro flora and fauna in lakes receiving commercial food. Fish were distributed randomly into
effluents from ponds (Ringo et al., 2004), presented eighteen 50cm x 34cm x 27cm rectangular plastic
major challenges to the continued use of antibiotics and aquaria after acclimatization at 25 fish per tank.
its eventual ban in several parts of the world.
An approach to disease prevention is the reduction or 2.2. Isolation, identification of Lactobacillus
total elimination of pathogenic microbes from cultured fermentum, diet preparation and feeding
fish. This is mostly achieved by feeding live microbial Cheese produced from milk was procured in Ibadan
adjunct that are beneficial to the host through the and 500 µL of the liquid cheese sample was inoculated
modification of ambient microbial population, enhanced to MRS broth (10 µL) and incubated to obtain enriched
feed utilization and nutritional value, and improved host culture for 48 h at 37 °C. One ml of the culture was
response towards disease and modulating mucosal and thereafter inoculated into 10 ml PBS (pH 2.5) (Erkkila
systemic immunity (Vine et al., 2004). Bryan (2015) and Petaja, 2000) and allowed to incubate for 3 h. At
stated that not all probiotics produce the same effects for 2500 g, the culture was centrifuged for 5 min and the
all fish species; thus, more work is required to establish survived organisms inoculated in 10 ml MRS broth
the effect of specific probiotics (Nwanna et al., 2013). before incubating at 37oC for 24 h.
Lactobacillus fermentum is a probiotic candidate which The modified method of Gilliland et al (1984) was
is reported to produce bacteriocins that inhibits the used for screening against bile salt. MRS broth was used
growth of some pathogens in vitro (Ng et al., 2009). A to inoculate the overnight culture and incubated at 37 oC
number of works have been reported on the use of L. for 4 h. The enriched culture of the acid-/bile resistant
fermentum to improve fish resistance to diseases, cultivation was serially diluted and thereafter, 0.01ml of
causing better growth and boosting of immune response 10−5 dilution spread into MRS-agar plates and allowed
(Balcazar et al., 2006; Balcazar et al., 2007). Lactic acid to incubate for 24 h at 37 °C. A number of single
bacteria have been isolated from the skin, gill and gut of colonies were selected at random and then incubated in
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MRS broth (10 mL). Isolates were screened using Table 1: Gross composition of the diet for the control and
morphological evaluation of the single clones. Films the five treatments
were prepared using the pure isolates earlier obtained, Feed ingredient Amount (g/100g)
and Gram’s stain carried out for morphological Fish meal 17.13
characterization under the microscope. Suspected Soya bean meal 17.13
isolates were biochemically identified according to
Ground nut cake 8.56
Quinn et al. (1994). The Lactobacillus fermentum
isolates were sub-cultured using MRS broth. Yellow maize 26.50
A diet was formulated to have 30% crude protein Biscuits waste 26.50
using Pearson Square Method. Feed ingredients (Table Fish oil 2.00
1) were ground, weighed, thoroughly mixed and made Fish Premix* 0.50
into dough by making it moist using the proper amount Lysine 0.60
of oil and water. The dough was left for an hour and Methionine 0.50
autoclaved at 121 oC for 25 min, cooled and the sub-
Vitamin C 0.08
cultured L. fermentum suspended in oil was added to the
Salt 0.50
basal diet (control, LF1), at 1 x 103 cfu/g (LF2), 1 x 105
*Composition kg-1: Vit. A – 1,000,000 UI; Mg – 2,600 mg;
cfu/g (LF3), 1 x 107 cfu/g (LF4), 1 x 109 cfu/g (LF5) and Vit. B1 – 2,500 mg; Zn – 14,000 mg; Fe – 10,000 mg; Vit.
1 x 1011 cfu/g (LF6) and pelletized using a hand B2 – 2,500 mg; Cu – 1,400 mg; Co – 20 mg; I – 60 mg; Vit.
pelletizer with 2 mm die size. Prior to feeding, the B6 – 2,500 mg; Se – 60 mg; Vit. D3 – 400.00 UI;
Pantothenic acid – 5,000 mg; Vit. E – 10,000 mg; Vit. K3 –
viability of the putative probiotics was ascertained 500 mg; Biotin – 80,000 mcg; Vit. B12 – 3,000 mcg; Vit. C
(Folorunsho, 2010). A gram of each feed sample was – 35,000 mg; Folic acid – 500 mg; Niacin – 10,000 mg;
dissolved in 9 ml of distilled water. This was diluted Chorine – 200,000 mg; Methionine – 130 g; Instill – 5,000
serially from 101 – 109 cfu/ml. One millilitre was taken mg; Etoxiquin – 15,000 mg. LF = Lactobacillus fermentum
from 102, 104, 106, 108 and 1010 cfu/ml concentration and
2.3. Chemical analysis
poured on the MRS agar already prepared in a Petri dish
Water quality was monitored using Combined Probe
under a sterile environment. These were incubated in an
Model 57 (YSI) for dissolved oxygen and temperature,
anaerobic environment at 37 0C for 48 h and the total
digital pH meter for pH and commercial test kits for
count of colonies done.
ammonia and nitrite. The proximate analysis of the
For 90 days under a semi-static condition, triplicate
experimental diets and fish, after feeding experiment, on
groups of fish were fed the six diets to satiation daily
dry matter basis were carried out in accordance with
(8.00-8.20 a.m. and 3.00-3.20 p.m.). Water quality was
AOAC (2005) in duplicates (Table 2).
monitored throughout the trials and weight was
measured biweekly.

Table 2: Gross composition of the diets (g/100g)

Composition (% Dry Mass)
Ingredient
LF1 LF2 LF3 LF4 LF5 LF6
Crude protein 35.70±1.0 34.25±2.0 34.05±1.0 35.10±2.0 35.10±1.0 34.30±2.0
Ash 8.70±1.0 9.30±0.5 8.50±1.0 8.10±1.0 8.50±2.0 8.40±1.0
Ether extract 7.70±1.0 8.10±1.5 7.90±1.0 7.70±2.0 8.20±2.0 7.80±1.0
Crude fiber 3.40±1.5 3.20±1.2 3.50±1.6 3.30±1.3 3.10±1.2 3.20±1.2
Moisture 7.80±0.5 7.71±1.1 7.59±0.7 8.24±1.4 7.82±0.4 8.25±1.2

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2.4. Growth evaluation using methods described by Reitman and Frankel
After 90 days of feeding, the biweekly weight and (1957), while the colorimetric method of Tietz et al
feed intake recorded were used to calculate the growth (1983) was used to measure alkaline phosphate (ALP).
parameters including treatment means for weight gain
(MWG), growth rate (SGR), percentage weight gain 2.7. Evaluation of gut-morphometric features in
(PWG), food conversion ratio (FCR),protein efficiency experimental fish
ratio (PER) according to Castell and Tiews (1980) and The gut-morphometric was carried out as described
percentage survival estimated. Feed intake (g) is sum of by Oladele et al (2012). Before measurements, villus
fed during experimental period. must have its base clearly embedded in the sub mucosa
(10x magnification) and there must not be discontinuity
𝐌𝐖𝐆 (g) = W2 − W1 or fold in the body (4x magnification). The villus should
SGR = {[Log e W2 − Log e W1] ÷ [T2 − T1]} × 100 also have simple columnar epithelium present at the tip
(40x magnification). Five villi were picked randomly
𝐅𝐂𝐑 = Feed Intake (g) ÷ Weight gain(g)
from each section and measured in each slide as a field.
PER = Mean weight gain ÷ Protein intake Five fields were used and the villi lengths, widths and
Survival rate = cryptal depths were measured (microns converted to
cm) with the aid of software in an Am scope camera
[No. of fish at Tz ÷ No. of fish at Ta] × 100
(MU900).
Protein intake =
Feed intake × %Protein in diets 2.8. Oxidative Stress
Excised kidney and liver tissues were washed in ice-
where: W2 = final weight, cold saline solution (normal) and then blotted with filter
W1 = initial weight, paper, weighed and homogenized (Teflon
Loge= Natural log, homogenizer). Homogenates resulting were cold-
Tz – Ta = experimental period in days, centrifuged for 10 min at -4 ˚C and 10,000 rpm to obtain
a post mitochondrial fraction (PMF). Biochemical
2.5. Analysis of gut microflora analyses were done using the supernatants collected.
At the start of the trial, 5 fish were picked randomly Protein concentrations of samples were determined
after acclimatisation for gut bacterial analysis. Fish were using the Biuret method described by Gornal et al.
stunned and aseptically gutted using sterile scissors and (1949). For evaluation of nitrogen oxide (NO)
forceps. Guts (intestine) was kept in sterile plates and production, the level of Nitrite (an indication of NO) in
taken for isolation, characterisation and identification of the liver and kidney tissues were measured using Griess
bacterial colonies (Ogunbanwo et al., 2003; Ogunshe et reagent (Olaleye et al., 2007). The activity of superoxide
al., 2007). This process was carried out for six randomly dismutase (SOD) in samples was determined according
picked fish samples from each treatment after 90 days to Misra and Fridovich (1972), while glutathione (GSH)
of feeding. and glutathione peroxidase (GPx) were estimated
following the method of Beutler et al., (1963). Reduced
2.6. Analysis of Blood indices in fish fed glutathione level was evaluated through the method of
experimental diets Ellman (1959). To determine lipid peroxidation, the
Nine fish randomly selected per treatment group formation of thiobarbituric acid reactive substances
were tranquilized in 150 mg/l of Tirana Methane (TBARS) were measured (Varshney and Kale, 1990).
Sulphonate for blood collection. Hematological The malondialdehyde (MDA) level was calculated
parameters were determined by the standard methods according to Adam-Vizi and Seregi (1982), while
(Kelly, 1979; Schalm, et al., 1975; Adeyemo et al., Gluthathione s-transferase activity was determined
2007). Alanine Aminotransferase (ALT) and Aspartate according to Habig et al. (1974).
Aminotransferase (AST) activities were determined

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2.9. Challenge Test 3. Results and Discussion
After the feeding experiment, challenge test was 3.1. Results
conducted by exposing the survived fish to virulent 3.1.1 Water quality and Fish carcass composition
bacteria “Aeromonas hydrophila” following the Pooled mean of water quality parameters throughout the
procedures of Austin et al., (1995) with 20 fish selected 90-day experimental period for dissolved oxygen, pH,
per treatment. The fish per treatment were divided into temperature, ammonia and nitrite were 5.1 mg/l, 7.39,
two sets and the first sets injected inter-peritoneally with 28.09 oC, 2.00 mg/l and 0.20 mg/l, respectively. The
1 x 107 cell/ml (Omitoyin et al., 2019) virulent composition of carcass in fish fed diets shows
Aeromonas hydrophila carried in 0.5 ml phosphate significantly higher crude protein in the carcass of fish
buffer saline solution (PBS) while the second sets were fed L. fermentum (Table 3). The highest value of 74.8 %
injected with 0.5 ml PBS. Feeding with experimental crude protein was recorded in LF3 fish and the least
diets continued for 15 days. Mortality was monitored to value of 62.1 % was observed in LF1. Similarly, higher
ascertain clinical signs and survival rate of the fat deposits were observed in probiotic-fed fish when
experimental fish. compared with LF1. While fibre content was
statistically similar across treatments.
2.10. Statistical Analysis
Homogeneity of variance in data collected was 3.1.2 Nutrient utilization and fish growth indices
confirmed using Bartlett’s test. Data were analysed The indices of growth in tilapia fingerlings fed L.
using one‐ way analysis of variance (ANOVA) to fermentum supplements for 90 days are presented in
establish the effect of L. fermentum on fish. Differences Table 4. Significantly high final weights were observed
in mean were separated using Duncan’s test at the 5 % in LF3, LF4 and LF5, with LF3 having the highest value
probability level. The optimum inclusion levels were of 6.23 g. Mean weight gain (MWG) and percentage
determined using the Polynomial regression with the aid weight gain followed this same pattern. MWG was
of IBM Statistical Package for Social Science (SPSS) significantly high in LF3 and LF4, with values of 4.19
version 20. and 4.15g, respectively, and the least value of 3.09 g
recorded in LF1. Feed conversion ratio (FCR) ranged
from 2.24 (LF3) to 3.00 (LF1) and results shows better
FCR values in LF3 and LF4 compared to other
treatments. Specific growth rate (SGR) was highest in
LF4 followed by LF3. These two groups had
significantly higher protein efficiency ratios (PER) with
the least value of 0.95 observed in LF1.

Table 3: Proximate Analysis of Fish Fed L. fermentum Supplemented Diet

Parameter (%) LF1 LF2 LF3 LF4 LF5 LF6
Crude protein 62.14±0.17a 65.15±0.32b 74.79±0.36d 73.37±0.59d 66.77±0.44c 74.16±0.35d
Ash 6.50±0.15d 6.20±0.17c 4.90±0.15b 4.50±0.11a 6.30±0.15c 5.10±0.2b
Ether extract 6.43±0.11a 6.63±0.15ab 7.80±0.11d 7.4±0.11cd 6.8±0.15b 7.5±0.15cd
Crude fibre 0.02±0.01a 0.02±0.01a 0.02±0.01a 0.03±0.01a 0.02±0.01a 0.03±0.01a
Moisture 54.53±1.9a 59.41±2.7ab 60.58±1.1b 56.64±5.5ab 61.32±0.5b 57.63±3.4ab
Dry matter 45.47±1.7b 40.59±2.7ab 39.41±1.1a 43.36±5.5ab 38.68±0.5a 42.37±3.4ab
Means with the same superscripts across row are not significantly different (p>0.0)

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Table 4: Growth response and nutrient utilization of Oreochromis niloticus fed experimental diet
Growth parameters LF1 LF2 LF3 LF4 LF5 LF6
Initial Weight (g) 1.85±0.01 1.97±0.01 1.98±0.05 1.70±0.2 1.98±0.01 1.94±0.01
Final Weight (g) 4.94±0.02a 5.20±0.03a 6.23±0.02c 5.85±0.03b 5.62±0.02b 5.16±0.03a
Weight gain (g) 3.09±0.01a 3.23±0.02a 4.19±0.03c 4.15±0.02c 3.64±0.01b 3.22±0.01a
% Weight gain 167.0±2.0a 163.9±1.0a 199.5±2.0c 244.1±2.0d 183.8±3.0b 165.9±3.0a
Feed Intake (g) 9.28±0.00a 9.39±0.00bc 9.51±0.00d 9.38±0.00b 9.39±0.00bc 9.43±0.03c
Feed conversion ratio 3.00±0.00d 2.90±0.05c 2.24±0.00a 2.26±0.01a 2.57±0.00b 2.92±0.03c
Specific growth rate 1.09±0.00b 1.07±0.00a 1.27±0.00d 1.37±0.00e 1.16±0.00c 1.08±0.00ab
Protein efficiency ratio 0.95±0.00a 0.98±0.00b 1.27±0.00d 1.26±0.00d 1.09±0.01c 0.98±0.00b
Means with the same superscripts across the rows are not significantly different at p<0.05

3.1.3 Fish gut morphometry experimental fish varied significantly (p<0.05) and
As shown in Figure 1, there was no significant suggests a dose dependent response with the least load
variation in the villi height in the intestine of fish across of 3.84 x 105 cfu/g recorded in LF1 group and the
treatments. And for the exception of the LF3 group with highest load of 8.27 x 108 cfu/g in LF6.
a significantly higher villi width, others were While Table 6 shows the identified bacteria in fish
statistically similar. guts. L. fermentum was present in experimental fish
irrespective of the assigned treatment. The LF6 group
3.1.4 Microbial Assay of the gastrointestinal tract of O.
recorded occurrence of 9 bacteria while LF1 to LF3 had
niloticus fed diets
7 each, LF4 had 8 and LF5 had 5. All the isolates were
The pH for sensitivity test ranged from 6.53 to 6.84
gram positive and short rod.
as shown in Table 5. Microbial load in the gut of

0.25
Villi height and width (cm)

0.19 0.19 0.2 0.19 0.19

0.2 0.18

0.15
Villi Height
0.1 0.07 0.08 0.07 0.07
0.06 0.06 Villi Width
0.05

0
LF1 LF2 LF3 LF4 LF5 LF6
Treatments

Figure 1. Histomorphometry of O. niloticus intestine fed L. fermentum supplemented diets

Table 5: Microbial Assay of Gastro-Intestinal Tract of O. niloticus fed experimental diets

Treatments LF1 LF2 LF3 LF4 LF5 LF6
Bacterial Count 0.0038±0.03 4.77±0.05 5.22±0.04 6.16±0.04 7.61±0.02 8.27±0.05
(CFU/g) x108a x108b x108c x108d x108e x108f
pH sensitivity test 6.84±0.04 6.73±0.03 6.64±0.02 6.53±0.02 6.61±0.01 6.58±0.01
Means with the same superscripts down the column are not significantly different at p<0.05

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Table 6: Bacteria identified in the gut of fish fed L. fermentum diets for 90 days

S. thermophiles

L. coryniformis
L. leichmannii

L. cellobiosus
L. delbrueckii

L. coagulans
L. fermentum

L. plantarum

L. salivarius
L. cremoris
L. hilgardii
S. faecalis
Sample

S. lactis
L. casei
LF1 - + + - - - + - - - + + + +
LF2 - + + - - - + - - - + + + +
LF3 + + - + + + - + - + - - - -
LF4 + + + + + - - + + + - - - -
LF5 - + - - - - + - - - - + + +
LF6 - + - + + + + + + + + - - -
+ means present; – means absent

3.1.5 Haematology of O. niloticus fingerling fed NO values increased significantly with probiotic
different level of L. fermentum addition. In the liver, TP was significantly increased in
Values for packed cell volume (PCV), haemoglobin the LF3 to LF6 groups, however, hydrogen peroxide and
(Hb), mean corpuscular volume (MCV and, mean MDA reduced significantly with inclusion of probiotic
corpuscular haemoglobin (MCH) were significantly (Table 9). There were significant increases in the values
higher (p<0.05) in groups fed L. fermentum compared to of GSH, GPx, GST, SOD and NO with the inclusion of
the control (Table 7). The reverse is observed in the L. fermentum.
results of red blood cells, where the control group had a
significantly higher RBC. White blood cells including 3.1.7 Challenge Test
the granulocytes constituents of neutrophils, eosinophils The control group recorded 95 % (19 out of 20)
and basophils and the non-granulocytes lymphocytes mortality or 5% survival after 15 days challenge test
and monocytes were not affected by the probiotic with Aeromonas hydrophila. No mortality was recorded
inclusions and levels in diets. Values for aspartate in the groups fed L. fermentum diets.
aminotransferase and alanine aminotransferase were
significantly lower in fish fed probiotics. 3.2. Discussion
The experimental diets fed to fish for 90 days met the
3.1.6 Oxidative Assay of the Liver and Kidney dietary requirement for Oreochromis niloticus for
Results of the assay of oxidative stress indicators optimal growth and performance (Britz, 2008).
analysed in the kidney presented in Table 8 reveals that Similarly, water parameters in all experimental tanks
total protein showed no significant difference (p>0.05) fall within the recommended range for tropical fish
across groups. There was significant reduction in culture (Ajani et al, 2011). The use of probiotics as
hydrogen peroxide levels with inclusion of L. dietary supplements has been reported to cause a
fermentum. The highest value of 48.01µmol/mg was colonization of host’s gut and thus increase feed
recorded in LF1, while LF4 had the least value of 41.29 utilization through the synthesis of growth factors, co-
µmol/mg. MDA was reduced significantly in fish fed factors, amino and fatty acids and augmentation of the
diets LF2, LF3 and LF4 with the least value observed in immune response of the cultured species (Farzanfar
LF3. Glutathione peroxidase (GPx), glutathione 2006; Talpur et al., 2013).
s-transferase (GST), superoxide dismutase (SOD) and

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Table 7: Haematological parameter of Oreochromis niloticus fingerling fed with diet of different level of probiotics

Indicators LF1 LF2 LF3 LF4 LF5 LF6

PCV 24.77±0.10a 26.33±0.00c 27.39±0.06d 26.09±0.00b 26.33±0.00c 26.38±0.04c
Hb 7.87±0.00a 8.50±0.05b 9.26±0.03e 8.71±0.04c 8.95±0.05d 9.143±0.01e
RBC 3.53±0.08e 2.74±0.00bc 2.66±0.02ab 2.57±0.00a 2.95±0.00d 2.80±0.00c
WBC 16.60±0.15c 7.14±0.05a 16.81±0.04c 16.66±0.49c 14.88±0.72b 14.13±0.15b
Platelet 176.00±1.0b 169.00±1.0ab 193.00±4.0c 194.00±4.0c 163.00±1.0a 189.50±0.5c
Lymphocyte 61.91±0.24c 61.45±0.45bc 61.38±0.2a 67.83±0.16d 60.38±0.29b 57.50±0.50a
MCV 79.61±0.39a 103.17±0.05e 97.95±0.37c 106.39±0.4f 95.96±0.13b 99.35±0.27d
MCHC 33.85±0.05d 32.09±0.01a 32.19±0.01b 32.32±0.00c 34.04±0.03e 34.66±0.00f
MCH 26.74±0.00a 33.59±0.01d 31.51±0.22b 35.53±0.01f 32.43±0.02c 34.44±0.01e
Neutrophils 33.00±0.00c 34.01±0.01d 32.00±0.00b 29.67±0.34a 32.99±0.00c 38.00±0.01e
Monocytes 2.68±0.01c 2.33±0.00b 3.27±0.06e 3.06±0.06d 2.72±0.05c 2.01±0.01a
Eosinophil 2.32±0.01d 1.33±0.00a 2.00±0.00c 1.68±0.01b 2.99±0.01f 2.68±0.01e
Basophil 0.33±0.01a 0.33±0.01a 0.33±0.02a 0.00 0.67±0.01a 0.33±0.01a
AST 215.15±0.17e 201.66±0.46b 193.50±0.50a 218.33±0.34f 213.11±0.42d 206.99±0.01c
ALT 32.83±0.16e 24.32±0.00b 25.37±0.04c 25.99±0.01d 20.58±0.25a 23.94±0.05b
ALP 206.50±0.5c 214.67±0.22e 198.50±0.50a 213.43±0.23d 202.70±0.30b 205.71±0.06c
Means with the same superscripts across the rows are not significantly different at p<0.05
PCV, packed cell volume; Hb, haemoglobin; RBC, red blood cell; WBC. White blood cell; MCV, mean corpuscular volume;
MCHC, mean corpuscular haemoglobin concentration; MCH mean corpuscular haemoglobin; AST, aspartate aminotransferase;
ALT, alanine aminotransferase; ALP, alkaline phosphate.

Table 8: Oxidative Stress Assay of O. niloticus Kidney Fed L. Fermentum Supplemented Diet
Parameter LF1 LF2 LF3 LF4 LF5 LF6
Total protein (U/mg) 0.33±0.01a 0.36±0.01a 0.37±0.01a 0.35±0.02a 0.31±0.01a 0.30±0.02a
H2O2 (µmol/mg) 48.01±1.5a 47.14±2a 44.42±1c 41.29±1.2b 41.52±2.1b 45.98±1c
MDA (U/mg) 2.79±0.2a 1.65±0.01cd 1.59±0.01d 1.86±0.02c 2.44±0.01b 2.61±0.01ab
GSH (U/mg wet tissue) 67.58±0.2b 67.56±3.2a 68.27±2.2a 68.08±2.1b 71.52±2.3a 69.05±2a
GPX (U/mg wet tissue) 561.20±5.4a 579.87±6.3b 638.09±5.3c 624.62±6.1c 668.19±4d 716.36±5e
GST (U/mg wet tissue) 0.15±0.01b 0.05±0.01d 0.24±0.03b 0.48±0.03a 0.63±0.02c 0.45±0.04a
SOD (U/mg tissue) 19.75±5b 23.58±2ab 21.29±4a 24.57±4a 25.79±3a 21.79±3b
NO (µmol/mg) 0.48±0.04c 0.50±0.01c 0.63±0.02a 0.68±0.011a 0.83±0.01b 0.67±0.02a
Means with the same superscripts across row are not significantly different at p<0.05
H2O2, hydrogen peroxide; MDA, malonaldehyde; GSH, glutathione; GPx, glutathione peroxidase; GST, glutathione
s-transferase; SOD, superoxide dismutase; NO, nitrogen oxide

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Orisasona et al. Ethiop.J.Sci.Sustain.Dev., Vol. 11 (1), 2024
Table 9: Oxidative Stress Assay of O. niloticus Liver Fed L. Fermentum Supplemented Diet
Parameter LF1 LF2 LF3 LF4 LF5 LF6
Total protein (U/mg) 0.17±0.03b 0.17±0.02b 0.23±0.01a c
0.19±0.02 0.19±0.02 c
0.21±0.01d
H2O2 (µmol/mg) 48.81±3.2a 35.33±2b 39.5±1.2c 39.06±1.4c 34.29±1b 44.33±1d
MDA (U/mg) 5.88±0.03b 4.13±0.01a 4.62±0.03a 5.12±0.03b 4.79±0.01a 4.46±0.02a
GSH (U/mg wet tissue) 88.17±3.2a 95.61±5.1b 95.56±4.3b 93.84±4.6c 98.77±3.1d 93.68±2.3c
GPX (U/mg wet tissue) 1002.19±125a 1263.57±102b 1174.68±78.5c 1187.14±98c 1377.13±86.3d 1112.99±117c
GST (U/mg wet tissue) 0.04±0.01d 0.43±0.04a 0.92±0.12c 0.72±0.14b 0.77±0.2b 0.49±0.31a
SOD (U/mg tissue) 35.05±2.1a 43.98±2.2b 40.19±2b 41.23±1.2b 47.21±3.2c 38.85±4a
NO (µmol/mg) 1.69±0.14a 1.61±0.02a 1.87±0.04b 1.33±0.02c 0.88±0.01d 1.54±0.02e
Means with the same superscripts across row are not significantly different at p<0.05
H2O2, hydrogen peroxide; MDA, malonaldehyde; GSH, glutathione; GPx, glutathione peroxidase; GST, glutathione s-
transferase; SOD, superoxide dismutase; NO, nitrogen oxide

Fish carcass recorded increased crude protein and fat effect of probiotics on fish growth were observed in
with Lactobacillus fermentum inclusion in this study. Fagundes et al (2016) and Tachibana et al (2012) when
This shows improved protein synthesis and enhanced Nile tilapia was fed diets containing L. plantarum at
lipid production caused by the presence of probiotic in concentrations of 104, 106 and 108 CFU/g and B. subtilis,
fish diets. According to Fountoulaki et al (2003), higher respectively.
nutrient deposit is usually associated with increase The villi morphometric presented in Figure 3
efficiency of metabolism. Growth as indicated by final revealed statistical similarities in all treatments except
weight and the mean weight gain were significantly for the LF3 group with significantly higher dimension.
highest in LF3 and LF4 groups, with a decrease Higher villi height and width will translate to greater
observed at higher concentration of L. fermentum as areas of absorption and improved nutrient utilization
seen in LF5 and LF6 groups. Feed intakes were also (Omitoyin et al., 2019). It has also been hypothesised
higher in the groups fed probiotics and were equally that Lactobacilli produces fatty acids that are short-
well transformed into flesh showing by the superior feed chained as products of carbohydrate metabolism and
conversion ratio in the L. fermentum groups. The results serves as main energy source in intestinal epithelial
of the study may be attributed to improved appetite cells. Pirarat et al (2011) suggested that this production
shown in increased feed intake and better digestibility in the cells causes an increase in villi height in the
and absorption of nutrients evident in the food to flesh digestive tract and subsequently improves nutrient
conversion and thus better growth (Eshaghzadeh et al., absorption by providing a greater absorptive surface
2015). Stimulation of growth in many aquatic animals area.
fed L. fermentum based diets, have been reported; this There were variations in the gut intestinal bacterial
includes white shrimp (Litopenaeus vannamei), Giant load, revealing a dose dependent pattern with the highest
freshwater prawn (Macrobrachium rosenbergii), load observed in the LF6 group. Some of the identified
Orange-spotted grouper (Epinephelus coioides) and bacteria include L. fermentum, L. brevis and L casei.
Red Sea bream (Pagrus major) (Kongnum and According to Olanrewaju et al (2018), blood is
Hongpattarakere 2012; Dash et al, 2015; Son et al., described as a pathos-physiological reflector and the
2009; Dawood et al., 2016). Similar result was reported count of its indices give an indication of the health status
when Nile tilapia was fed probiotic diets containing of fish. In the current study, the effects of probiotic, L.
Steptococcus faecium and Lactobacillus acidophilus fermentum on haematological parameters of
(Lara-Flores et al., 2003). In the contrary, insignificant Oreochromis niloticus was examined. The PCV, Hb,

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Orisasona et al. Ethiop.J.Sci.Sustain.Dev., Vol. 11 (1), 2024
MCV and MCH were higher in fish fed probiotic and Gutteridge, 1990). The reduction in MDA in this
supplemented diets, indicating better health status. study is an indication of lower occurrence of free
Increase in Hb levels of Nile tilapia fed Saccharomyces radicals in the groups fed probiotics, and therefore
cerevisiae and Bacillus spp. treated diets were earlier reduced peroxidation of lipids, suggesting improved
reported (Selim and Reda, 2015; Abu-Elala et al., 2013). innate immune system in fish fed probiotic. In contrast,
Red blood cells which are essential component of the the values recorded for GPx, GST, SOD and NO
innate and also the adaptive immune response systems increased significantly with probiotic addition, which
were significantly lower in fish fed L. fermentum in the shows the activation of antioxidant protection needed in
study. This is contrary to the findings of Opiyo et al all fish including the healthy ones during their life cycle
(2019) where increased RBC and WBC were recorded (Omitoyin et al. (2019). The effect of all these was
in fish fed S. cerevisiae or B. subtilis. The differences in tested via a challenge test with Aeromonas hydrophila
the findings may be due to variations in the probiotic for fifteen days. Fish fed diets supplemented with L.
species and the applied dosages. There were also fermentum recorded no mortality indicating that the
reductions in the aspartate aminotransferase and alanine immune system was boosted. The use of probiotics was
aminotransferase in the serum of fish fed L. fermentum reported to improve the non-specific immune of tilapia
in this study. According to Omitoyin et al (2019), the thus, enhancing resistance to Edwardsville trade
release of these blood enzymes suggests cell damage infection (Abd El-Rhman et al., 2009).
and lose of cellular integrity. Therefore, the fed
probiotic confers better health status and cell integrity to 4. Conclusion
fish. Lactobacillus fermentum resulted in increased
The innate immune system of the fish is considered growth performance of O. niloticus fingerling, and there
very important, as it normally present the first line of was also an increase in the gut microbial load. This
defence against stressors. The immune competence of a study also revealed better immune system in fish fed
fish can be ascertained using the oxidative biomarkers diets supplemented with L. fermentum resulting in the
which act as first line of antioxidant defence (Carvalho resistance of virulent Aeromonas hydrophila. An
et al., 2012). Hydrogen peroxide and malondialdehyde inclusion level of 1 x 105 cfu/g is recommended when
levels in the kidney and liver were reduced with L. all parameters measured are considered.
fermentum inclusion in fish diets. Lipid peroxidation of
Acknowledgement: The authors would like to thank all
cell membranes is reported to result in the production of
participants in this study for their collaborative support
metabolites like malondialdehyde (Borkan and
during the study.
Schwartz, 1989), which are measured and used as
indirect markers of oxidant-induced injury (Halliwell

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