Amino Acid Digestibility of Catfish Clarias Gariepinus Fed Soyabean Meal Supplemented with Lysine and DL-Methionine

Background and Objective: Soyabean Meal (SBM) could be a suitable replacement for the expensive fishmeal but for its low digestibility in fish. Appropriate dietary supplements of amino acid can improve digestibility of SBM based diets. Therefore, the effect of supplementing lysine and DL-methionine in Solvent Extracted Soyabean Based Diets (SESBD) on performance and amino acid digestibility of Clarias gariepinus ( C. gariepinus ) was assessed. Materials and Methods: Juvenile fish (n = 360) with the weight of 17.74±0.29 g were fed experimental diet to satiation for 12 weeks. Six diets containing varied combinations of lysine+DL-methionine (g 100 g G 1 ) in SESBD were formulated, S 1 (without supplemental lysine and DL-methionine), S 2 (0+1 g), S 3 (0.25+0.75 g), S 4 (0.5+0.5 g), S 5 (0.75+0.25 g), S 6 (1+0 g). Each treatment was in triplicate. Results: Fish fed S 1 (43.35±4.03 g), S 3 (40.80±1.74 g) and S 5 (36.70±5.11 g) were not significantly different (p>0.05) but significantly higher (p<0.05) than those on S 2 (34.77±1.17). Lysine and DL-methionine supplementation had no significant effect (p>0.05) on feed conversion ratio. Supplemental lysine and methionine resulted in an increase (p<0.05) in both apparent (96.34±0.31) and true (96.51±0.31) protein digestibility in diet S 5 . The C. gariepinus on S 5 (95.75±0.26) had a significantly higher (p<0.05) apparent lysine digestibility than other treatments. The optimum estimates of supplemental lysine and methionine in SESBD for the weight gain of C. gariepinus occurred at 0.6 g 100 g G 1 (R 2 = 0.66) and 0.4 g 100 g G 1 (R 2 = 0.42), respectively. Conclusion: The findings suggest that lysine and DL-methionine supplementation could improve growth performance and amino acid digestibility in C. gariepinus fed SESBD.


INTRODUCTION
Evaluating the efficiency of feed ingredients could mostly be achieved through digestibility study. Ribeiro et al. 1 noted that the nutritional values to be applied in the formulation of nutritionally complex fish feeds could be determined through the evaluation of the digestibility coefficients of feed ingredients. However, the determination of digestibility coefficients is mostly established on fecal measurements, fish Feed ingredients and diet preparation: The experiment was conducted at the onset of the dry season between late September and early December, 2018. Six isonitrogenous chemical composition tested diets were formulated with varying levels of lysine and methionine in Table 1 and their chemical composition was showed in Table 2. The dietary protein level was fixed at 40% crude protein, as reported optimum for the growth of C. gariepinus 12 . The ingredients were thoroughly mixed together and each diet mixture was pelleted at 60°C, using 2 mm pellet die to form noodle-like strands, which were crumbled manually into appropriate size for C. gariepinus juveniles. The pellets were sundried, packed into labeled transparent bag and stored in a cool dry place to prevent fungal growth. Six dietary treatments of supplemental lysine+DL-methionine (g 100 gG 1 ) combinations in Solvent Extracted Soyabean Based Diets (SESBD) were as follows S 1 (without supplemental lysine and DL-methionine), S 2 (0+1), S 3 (0.25+0.75), S 4 (0.5+0.5), S 5 (0.75+0.25) and S 6 (1+0).
Fish and experimental procedure: The C. gariepinus juveniles (n = 360) pieces of aged 2 months weighing 17.74±0.29 g were purchased from a reliable fish farm in Ibadan, Nigeria. The feeding trial was conducted at research laboratory of Aquatech College of Aquaculture, Ibadan, Nigeria using 18 plastic tanks with the dimension measuring 60×45×30 cm. Up to 80% capacity of well water was supplied to each experimental tank and was replaced every 2 days to prevent fouling from feed residues and maintain relatively uniform physico-chemical parameters. There were six dietary treatments were replicated with 20 fish per replicate. The fish were weighed and randomly distributed into experimental tanks after they  Control: Without supplemental lysine and DL-methionine, S 2 : 1 g methionine, S 3 : 0.75 g methionine, 0.25 g lysine, S 4 : 0.5 g methionine, 0.5 g lysine, S 5 : 0.25 g methionine, 0.75 g lysine and S 6 : 1 g lysine have been acclimatized for 14 days. The experiment lasted for 84 days (12 weeks) and the fish were fed to satiation. After the 9 weeks feeding trial, faecal collection was carried out. This procedure was carried out until sufficient volume of faeces for a replication chemical analysis was obtained. The collection of faeces lasted for 3 weeks. During the faecal collection, the faeces were siphoned twice daily (07:00 and 16:00 hrs). Faeces were oven dried at 55°C and stored at -20°C until chemical analysis.
Proximate composition: Proximate composition of the diets and faecal were determined as described by Oyelese et al. 13 .
Growth studies: Calculations of the growth performance and feed utilization data were according to Gabriel et al. 14  Amino acid analysis: Samples of the diets and feaces were treated with performic acid at 0°C to oxidize methionine and cystine to methionine sulfone and cysteic acid prior to the hydrolysis 15 . The samples were prepared by 6 N HCL hydrolysis for 24 h at 110°C. After which the samples were vaporized in sodium citrate buffer (0.2 mol LG 1 Na + , pH 2.2) and the mixture was equalized to a 50 mL volume. The amino acids in the hydrolysate were determined by an AA Analyzer (Biochrom 30.30 Plus, Biochrom Ltd., Cambridge, UK).

Digestibility indices:
Chromic oxide in diets and feaces were analyzed 16 . Samples were digested with concentrated nitric acid and oxidation of chromic oxide with 70% prechloric acid. The 50 mg of the samples was put in a Kjeldahl Flask. The 5 mL of concentrated nitric acid was added to the flask and the mixture was gently boiled for 20 min. The boiled sample was cooled at 3 mL of 70% perchloric acid was added to the flask. The resultant mixture was gently heated for another 10 min until the solution changed from green to orange to ensure complete oxidation. The oxidized solution was then put inside a 100 mL volumetric flask and diluted to 100 mL with distilled water. The absorbance of the solution was determined by means of a spectrophotometer at 350 nm. Percentage chromic oxide content in samples and digestibility coefficient was calculated as follows 1 :  Trends Agric. Sci., 2 (1): [1][2][3][4][5][6][7][8][9][10][11][12]2023 True Nutrient Digestibility Coefficient (TNDC): Where: MFAA = Metabolic feaces amino acid (it was determined by feeding protein free diet) Cr 2 O 3 = Chromic oxide AA = Amino acid Statistical analysis: The design of the experiment was a completely randomized design. Data were subjected to One-way ANOVA followed by Duncan's Multiple Range Tests were used to compare differences among individual means and polynomial regression. All statistics were performed using SPSS 20.0 (SPSS, Chicago, IL, USA).

RESULTS
Growth performance and nutrient utilization of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid shown in Table 3. The weight gain of C. gariepinus fed diet Control, S 3 and S 5 were similar (p>0.05) and were significantly higher from C. gariepinus fed diet S 2 (34.77±1.17 g). However, supplementation of lysine and methionine in solvent extracted soyabean based diet had no significant (p>0.05) influenced on FCR and GEFC and the values ranged from 2.27±0.32 (S 3 )-3.12±0.31 (S 2 ) and 66.97±7.03 (S 5 )-77.97±8.93 (S 3 ), respectively. Control diet (13.73±1.56) had a significantly higher value in Protein Intake (PI) and least in C. gariepinus on diet S 6 (11.24±0.72). Protein efficiency ratio and specific growth rate reduced significantly with amino acid supplementation in solvent extracted soyabean based diet as higher values were in control diet (14.45±1.34 and 0.59±0.08) and least in C. gariepinus fed diet S 2 (11.59±0.39 and 0.44±0.02), respectively. Also, gross protein retention increased as methionine decreased and lysine increased in the diet as there was a significant difference in the GPR with the higher value in S 5 (0.79±0.02) and least in diet S 2 (0.66±0.02) ( Table 3). Significantly (p<0.05) higher nitrogen retention efficiency was in control diet (49.49±6. 19) but similar (p>0.05) to fish on S 3 (47.64±2.10). Clarias gariepinus fed diet S 6 (77.80±0.20) had the least survival rate during the experiment but significantly higher (p<0.05) in C. gariepinus on diet S 5 (88.90±0.10). Regression of gross protein retention and supplemental lysine and methionine were both quadratic with the optimum dietary inclusion levels 0.6 and 0.4 g 100 gG 1 , respectively are presented in Fig. 1.    . Control: Without supplemental lysine and DL-methionine, S 2 : 1 g methionine, S 3 : 0.75 g methionine, 0.25 g lysine, S 4 : 0.5 g methionine, 0.5 g lysine, S 5 : 0.25 methionine, 0.75 lysine and S 6 : 1 g lysine Apparent nutrient digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid is shown in Table 4. Dry matter digestibility values varied significantly (p>0.05) among the diets with the higher value in C. gariepinus on S 5 (80.78±0.05). Supplemental amino acid significantly (p<0.05) improved crude protein digestibility in fish fed diet S 5 (96.34±0.31) compared to other diets. However, ash digestibility significantly reduced with amino acid supplementation except for fish fed diet S 5 that was significantly higher than other treatments. Ether extract digestibility of fish on S 5 , control and S 6 were similar (p>0.05) but higher than fish on S 2 , S 3 and S 4 . Also, crude fibre digestibility value was significantly higher in C. gariepinus fed diet S 5 (90.34±2.27) while, fish on control, S 3 and S 6 were not significantly different from each other (Table 4)  Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S 2 : 1 g methionine, S 3 : 0.75 g methionine, 0.25 g lysine, S 4 : 0.5 g methionine, 0.5 g lysine, S 5 : 0.25 methionine, 0.75 lysine and S 6 : 1 g lysine   Table 3 indicated that C. gariepinus fed soyabean based diet when supplemented with lysine and DL-methionine could improve fish growth performance and utilization of feed. Lu et al. 17 attributed it to the fact that, amino acid has a powerful feeding stimulant and it has been good in reducing feed intake while improving the body weights of fish. There have been similar reports of improved performance of fish fed diets supplemented with lysine and DL-methionine 18,19 . The authors noted that supplemental amino acid improved amino acids utilization for protein synthesis which shows that lysine and DL-methionine are essential for the growth of C. gariepinus.

Findings from
Also, Nwanna et al. 8 Table 4. The results observed corroborated with Nwanna et al. 8 findings who detected that supplemental methionine and lysine significantly enhanced protein digestibility in Cyprinus carpio and Oreochromis niloticus, respectively. Also, Ribeiro et al. 1 reported similar trend when determining the digestibility of feedstuffs used in tilapia feed. The improvement in protein digestibility suggested that supplemental methionine and lysine in soyabean based diet increased availability of amino acid for maintenance and protein retention as revealed in the regression model of gross protein retention of this study (Fig. 1). Though, the value of protein digestibility observed in this experiment was greater than what Nwanna et al. 8  Trends Agric. Sci., 2 (1): [1][2][3][4][5][6][7][8][9][10][11][12]2023 The true digestibility of C. gariepinus fed solvent extracted soyabean based diet with supplemental amino acid had few literatures reported, apart from studies of Yamamoto et al. 27 , with Cyprinus carpio, Ribeiro et al. 1,28 and Wilson et al. 29 with Ictalurus punctatus ( Table 5). The values observed in true digestibility were higher than what was observed in apparent digestibility of this experiment because the nutrients in the feacal are intact and has not leached away as observed in apparent digestibility. Also, it revealed that study of true digestibility has the potential to correct endogenous losses that do occur in apparent digestibility monitoring. The study showed that true crude protein digestibility value was higher than those revealed by Yamamoto et al. 27 and Ribeiro et al. 1 . This increase observed could be as a result of supplemental amino acid in soyabean based diets were effectively utilized by the experimental fishes.
Also, the dissection method of feacal collection used in the study could have contributed to the increase observed in the study and it was corroborated by Ribeiro et al. 1 .
The apparent amino acid digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid as observed in reported relatively lower values when common feedstuffs were fed to channel catfish and tilapia, respectively. The high values noted in this study might be ascribed to the fact that thermal treating of soyabean meal enhanced release the encapsulated protein structure and inactivates the anti-nutritional factors which could have enhanced soyabean based diet with supplemental lysine and methionine to be more digestible by proteolytic enzymes. Higher methionine content presented in the study could be as a result of the higher sulfur amino acids (pancreatic secretions and mucin layer) equated with additional amino acids 30 . Isoleucine and arginine presented the least apparent amino acid digestibility values among essential amino acid for all the studies. Similar trend was observed by Ribeiro et al. 28 for soyabean meal.
The lease values obtained could be due to the fact that isoleucine is a hydrophobic amino acid situated in protein, deterring the hydrolysis of its peptide bonds, which may explain its low digestibility. Table 7 showed the true digestibility of C. gariepinus fed solvent extracted soyabean based diets with supplemental amino acid. True amino acid digestibility helped to consider the role of endogenous amino acids, quantity of amino acids and values of true digestibility used by fish in more precise, resulting in precise formulation of diets for C. gariepinus. Results observed showed a relatively small difference from the apparent digestibility. This could be due to higher levels of digestive enzyme secretions and its inclusion in the feaces from the protein free diets for C. gariepinus in this study. Similar trend was observed by Pozza et al. 30 , Ribeiro et al. 28 . The values acquired were within the ranged stated by Yamamoto et al. 27 and Pozza et al. 30 . Amino acid digestibility values observed in this study revealed that supplemental methionine and lysine in soyabean based diet served as a stimulant which enhance the digestibility of the diet and nutrient utilization as observed in the growth parameter.