2.1 Experimental location, feedstuff procurement and processing
The experiment was carried out in the hatchery and laboratories of the Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia, Terengganu, Malaysia. J. curcas seeds were obtained and the oil was mechanically extracted as described by Belewu, (2008) at the Jatropha farm of the University of Ilorin, Nigeria. The other ingredients were obtained from the local market in Kuala Nerus, Terengganu, Malaysia. The defatted J. curcas meal was mixed with distilled water to 66% moisture. The mixture was made into pastes, covered with aluminium foil and placed in an autoclave at 1210C for 30 minutes (Azzaz, et al., 2011). While the proximate composition of the raw and processed J. curcas cake meal was determined using the methods of the A.O.A.C, (2005) the phytochemical analysis was done following the steps of Saetae and Suntornsuk, (2010).
2.2 Diet formulation and nutritional analysis
Five diets (40% CP) were formulated for the feeding trial. The five diets include a control (SBM) and four other diets in which soybeans meal was replaced with J. curcas cake meal at 25%, (TTR25), 50% (TTR50), 75% (TTR75) and 100% (TTR100) (Fawole et al., 2022). All the feed ingredients were integrated into computing the required quantities to make up 100 units of the feed (Table 1). Feed preparation involved milling the grain ingredients separately, toasting the soybeans, sieving (using > 1 mm sieve), mixing all dry ingredients, the addition of fish oil before adding hot water (90oC – 95oC) and mixing to form doughs (Lall, 1991). The mixtures were pelleted using a 2mm diameter pelletizer. The strands were oven-dried at 60oC and packaged in waterproof cellophane until usage time. The diets were analyzed for proximate composition using standard proximate analysis methods (AOAC, 2005).
2.3 Experimental design and feeding of experimental fish
The design of the experiment was a simple random arrangement. The experiment comprised five treatments each of which had three replicates. A group of 150 juveniles of Clarias gariepinus were obtained from a commercial hatchery, acclimatized for 14 days and fed commercial pellets (40% CP) during the period (Ayanwale et al., 2017). After the period of acclimatization, 15 transparent static plastics (31x29x25cm dimension) were set and 10 fish were randomly assigned and labelled accordingly (Suleiman and Solomon, 2017). All selected experimental fish were fasted for 12 hours prior to the experiment, to empty their gut and increase their appetite for the reception of the new experiment diets. They were thereafter fed with the assigned test diets till satiation. Feeding was done twice daily (08.00h and 09.00h for the morning session and 17.00h and 19.00h for the evening session) (Falaye et al., 2014). This was done for 63 days. The faecal matter and uneaten food were siphoned out of the tanks and 30% fresh water was supplied every two days to maintain water quality.
2.4 Feeding and Growth Performances Assessment
The mean weight of the fish was taken fortnightly while the record of fish mortality for each treatment was recorded daily. The feeding and growth parameters were evaluated as below:
-
Weight gain (WG)
WG = Final weight of fish – Initial weight of fish
-
Average Daily Weight gain (ADWG)
$$\text{A}\text{D}\text{W}\text{G}=\frac{Final mean body weight-Initial mean body weight}{\text{C}\text{u}\text{l}\text{t}\text{u}\text{r}\text{e} \text{p}\text{e}\text{r}\text{i}\text{o}\text{d}}$$
-
Specific Growth Rate (SGR) %
$$\text{S}\text{G}\text{R}=\frac{In WF-In W1}{\text{T}\text{i}\text{m}\text{e} \left(\text{d}\text{a}\text{y}\text{s}\right)} \text{x} 100$$
-
Percentage Survival
$$\text{S}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l} \text{r}\text{a}\text{t}\text{e}=\frac{Total number of fish-Mortality}{Total number of fish} \text{x} 100$$
-
Feed conversion ratio (FCR)
$$\text{F}\text{C}\text{R}=\frac{ Dry feed consumed\left(g\right)}{Gain in weight \left(g\right)}$$
-
Protein efficiency ratio (PER)
$$\text{P}\text{E}\text{R}=\frac{Gain in weight \left(g\right)}{Protein fed \left(g\right)}$$
2.5 Haematological Analysis
The blood collected from the sampled fish was used for plasma analysis. The following parameters were determined by using blood analysis kits (AMES Blood Analyser and Randox Laboratory Limited, UK), following the manufacturer procedure including total protein, albumin, globulin, creatine, urea, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase. In addition, haemoglobin was estimated by cyanomethemoglobin method. Red blood cells (RBV) and white blood cells (WBC) were counted by Naubauer’s improved haemacytometer using Hyem’s and Turks solution as a diluting fluid respectively. Packed cell volume (PCV) was estimated by using haematocrit method. Mean corpuscular haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and mean cell volume (MCV) were calculated by using standard formula described by Joshi et al., (2002):
$$MCV \left(fl\right) = PCV x 10/RBC$$
(i)
$$MCH \left(pg\right) = Hb x 10/RBC$$
(ii)
$$MCHC \left(g/dl\right) = Hb x 100/PCV$$
(iii)
2.6 Liver and Intestine Enzymes Activity Analyses
The fish were decapitated and dissected at the end of the experiment after being immobilized in ice. The intestines were gotten and washed in distilled water to remove blood stains. The liver and intestine samples were macerated and homogenized using ice-cold, distilled water with a Poltler-Elvehjem homogenizer (Kamran et al., 2023). The homogenates were centrifuged at 16000rpm for 10min at 4°C and the supernatant was transferred into a clean micro-plate and stored at -80°C until enzymatic assays were carried out (Leong et al., 2019). Amylase activity was evaluated using 1% starch solution in 20 mM sodium phosphate buffer pH 6.9 for the liver sample and pH 8 for the intestine, containing 6.0 mM NaCl as substrate (Worthington, 1993). A total of 0.5 ml of substrate solution was added to 0.5 ml of enzyme preparation followed by 3 min of incubation. This was followed by the addition of 0.5 ml of dinitrosalicylic acid and incubation in a boiling water bath for 5 min. The absorbance value at 540 nm was recorded. The amount of maltose released from this assay was determined from the standard curve. Specific enzyme
activity (U) was calculated as follows:
$$\text{A}\text{m}\text{y}\text{l}\text{a}\text{s}\text{e} \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} (\text{U}/\text{m}\text{l})=\frac{ \left(\text{m}\text{g} \text{o}\text{f} \text{m}\text{a}\text{l}\text{t}\text{o}\text{s}\text{e} \text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\text{d}\right)\times \text{D}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{F}\text{a}\text{c}\text{t}\text{o}\text{r}}{\left(\text{R}\text{e}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} \text{t}\text{i}\text{m}\text{e} \right(\text{m}\text{i}\text{n}\left)\right) \times (\text{E}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\left(\text{m}\text{l}\right)}$$
(i)
Lipase activity was assayed based on the measurement of fatty acids release due to
enzymatic hydrolysis of triglycerides in stabilized emulsion of olive oil (Borlongan, 1990). The assay was carried out by the addition of 1 ml of crude enzyme extract to 1 ml of
stabilized lipase substrate in 1.5 ml of 0.1 M Tris–HCl buffer at pH 8.0. Mixture was
incubated for 6 h at 370C, after which hydrolysis was stopped by the addition of 3 ml of
95% ethyl alcohol. The mixture was then titrated with 0.01 N NaOH using 0.9% (w/v)
thymolphthalein in ethanol as an indicator. Blank determinations were conducted in a
similar manner except that the crude enzyme extracts were introduced into the assay
system after the 6-h incubation and immediately before titration. A unit of lipase activity
(U) was defined as the volume of 0.01 N NaOH required to neutralize the fatty acids
released during the 6-h incubation with the substrate and after correction by the
appropriate blank (Natalia et al., 2004):
$$\text{L}\text{i}\text{p}\text{a}\text{s}\text{e} \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} (\text{U}/\text{m}\text{l})=\frac{ \left(\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{e}\text{n}\text{c}\text{e}\right)\times \text{N}\text{a}\text{O}\text{H} \text{v}\text{o}\text{l}\text{u}\text{m}\text{e} \times \text{A}\text{c}\text{i}\text{d} \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}}{\left(\text{R}\text{e}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} \text{t}\text{i}\text{m}\text{e} \right(\text{m}\text{i}\text{n}\left)\right) \times (\text{E}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\left(\text{m}\text{l}\right)}$$
(ii)
Protease Activity was assessed using buffers of pH 9 and 6.9 for intestine and liver samples respectively (Natalia et al., 2004). The reaction was initiated by the addition of 0.5 ml of 1% azocasein to 50 µl of enzyme extract in 0.5 ml buffer and stopped 60 min later by adding 0.5 ml of 20% trichloroacetic acid (TCA). After 10 min of precipitation, the reaction mixture was
centrifuged at 12 000 g for 5 min. The absorbance was recorded at 366 nm. Amount of
tyrosine released from this assay was determined from the standard curve.
$$\text{P}\text{r}\text{o}\text{t}\text{e}\text{a}\text{s}\text{e} \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} (\text{U}/\text{m}\text{l})=\frac{ \left({\mu }\text{m}\text{o}\text{l} \text{t}\text{y}\text{r}\text{o}\text{s}\text{i}\text{n}\text{e} \text{e}\text{q}\text{u}\text{i}\text{v}\text{a}\text{l}\text{e}\text{n}\text{t}\text{s} \text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\text{d}\right) \times \text{T}\text{o}\text{t}\text{a}\text{l} \text{R}\text{e}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} \text{V}\text{o}\text{l}\text{u}\text{m}\text{e}}{\left(\text{R}\text{e}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} \text{t}\text{i}\text{m}\text{e} \right(\text{m}\text{i}\text{n}\left)\right) \times (\text{E}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\left(\text{m}\text{l}\right) \times \text{V}\text{o}\text{l}\text{u}\text{m}\text{e} (\text{m}\text{l}\left) \text{u}\text{s}\text{e}\text{d} \text{i}\text{n} \text{C}\text{o}\text{l}\text{o}\text{r}\text{i}\text{m}\text{e}\text{t}\text{r}\text{i}\text{c} \text{D}\text{e}\text{t}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \right)}$$
(iii)
All studies on the used fish were carried out in strict compliance with the recommendations of the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes (MYCODE). The study received the approval of the animal ethics committee of UMT under the code UMT/JKEPHMK/2022/70.
2.7 Water quality analysis
Water samples were taken and evaluated for selected parameters (dissolved oxygen, pH, temperature, nitrate, nitrite, and ammonia) once in a week in line with standard methods (APHA, 2005). While nitrate, nitrite, ammonia and pH were determined using an API freshwater master test kit, DO and temperature were determined water quality measurement meter (Eutech DO 6+. Thermo Scientific)
2.8 Statistical analysis
An independent two-sample t-test to compare the means of the proximate composition and anti-nutrients of the thermally processed Jatropha curcas kernel meal against the raw sample was used. Other data were subjected to one-way analysis of variance using the Statistical Analysis System. Means of parameters were separated using the New Duncan’s Multiple Range Test at 5% level of probability. The optimum inclusion level of J. curcas for growth was determined using polynomial regression.
Table 1
Percentage of ingredients composition of the thermally (TJCM) treated J. curcas based diets.
Ingredients | SBM | TTR 25 | TTR 50 | TTR 75 | TTR 100 |
Maize meal | 20.96 | 20.96 | 20.96 | 20.96 | 20.96 |
Soybean meal | 40.70 | 30.03 | 17.35 | 9.67 | 0.00 |
Fishmeal | 24.34 | 24.34 | 24.34 | 24.34 | 24.34 |
J. curcas meal | 0.00 | 16.67 | 27.35 | 38.03 | 48.70 |
Cellulose | 8.00 | 2.00 | 4.00 | 1.00 | 0.00 |
Starch | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 |
Fish oil | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
Salt | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
Pre-mix | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
Methionine | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
Lysine | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
Total | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 |
SBM – 100% soybean based diet, TTR 25 – Thermally processed J. curcas meal at 25% replacement level of soybean meal, TTR 50 - Thermally processed J. curcas meal at 25% replacement level of soybean meal, TTR 75 - Thermally processed J. curcas meal at 75% replacement level of soybean meal, TTR 100 - Thermally processed J. curcas meal at 100% replacement level of soybean meal |