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

Received 15 Dec, 2021

Accepted 01 Mar, 2022

Published 01 May, 2022

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 ^–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 ^–1 (R ² = 0.66) and 0.4 g 100 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 conditions and methods applied in digestibility trials². Digestibility of individual plant proteins (such as soyabean meal, groundnut cake and sesame cake) in the compound diets is considered as one of the important factors positively affecting the growth of fish³. But there is meagre information on the digestibility of plant-based diet most especially in soyabean meal-based diet in C. gariepinus.

Soyabean meal has an advantageous nutrient characteristic, which makes it an ideal ingredient in aquaculture feed. This is possible because of its well-balanced amino acid profile and low fiber content⁴. However, soyabean meal is constraint with lysine and methionine in fish diet⁵, despite it is readily available. These two limiting amino acids are required to improve feed quality, performance, digestibility and profitability of the aquaculture industry. Mai et al.⁶ reported that lysine is habitually the most required amino acids when replacing fish meal with plant protein sources in fish feed because it is required for normal growth and muscle turnover. Also, its limitation in fish feed affects performance and carcass quality C. gariepinus⁷. In many fish diets, the first limiting amino acid is usually methionine especially those having higher inclusion of plant protein sources^8,9.

Nwanna et al.⁸ posited that DL-methionine supplementation in Cyprinus carpio diets significantly improved protein digestibility, weight gain, feed conversion efficiency and carcass quality. Thu et al.¹⁰ reported similar observations when lysine utilization efficiency at marginal lysine intake in Oncorhynchus mykiss fry was assessed. Ajani et al.⁹ affirmed that complete substitution of fish meal with soyabean meal is achievable when methionine is supplemented in Oreochromis niloticus diet. Ochang et al.¹¹ reported that meeting the amino acid requirement of fish diet could improve digestibility of soyabean based diet when supplemented with dietary lysine and methionine. Therefore, this study investigated the feasibility of improving the utilization and digestibility of soyabean meal-based diet with lysine and DL-methionine supplementation in C. gariepinus.

MATERIALS AND METHODS

Study area: The feeding trial was conducted at Aquaculture Research Laboratory, Aquatech College of Aquaculture, Fodacis, Ibadan, Nigeria. The college is situated in the tropical rain forest zone of Nigeria with latitude 7°36 N and longitude 3°86 N with a mean altitude of 275 m above sea level. Temperature range and the average relative humidity of the location were between 20-37°C and 60%, respectively.

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. gariepinus¹². 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 g^–1) combinations in solvent extracted soyabean based diets (SESBD) were as follows S₁ (without supplemental lysine and DL-methionine), S₂ (0+1), S₃ (0.25+0.75), S₄ (0.5+0.5), S₅ (0.75+0.25) and S₆ (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 have been acclimatized for 14 days. The experiment lasted for 84 days and the fish were fed to satiation.

Table 1:

Gross composition of solvent extracted processed soyabean based diets

Ingredient (g 100 g–1)	Control	S2	S3	S4	S5	S6
Soyabean meal	70	70	70	70	70	70
Yellow maize	26	26	26	26	26	26
*Vit/min premix	0.25	0.25	0.25	0.25	0.25	0.25
Soyabean oil	1	1	1	1	1	1
CaCO3	0.5	0.5	0.5	0.5	0.5	0.5
Salt	0.5	0.5	0.5	0.5	0.5	0.5
Chromic Oxide	0.5	0.5	0.5	0.5	0.5	0.5
Lysine	0	0	0.25	0.5	0.75	1
Methionine	0	1	0.75	0.5	0.25	0
Total (%)	99	100	100	100	100	100
Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 g methionine, 0.75 g lysine, S6: 1 g lysine, *1 kg of premix contains Vitamin A: 22,000 I.U, Vitamin D3: 5,000 I.U, Vitamin E: 300 mg, Vitamin k3: 10 mg, Vitamin B1: 20 mg, Vitamin B2: 25 mg, Vitamin C: 300 mg, Niacin: 120 mg, Calcium pantothenate: 60 mg, Vitamin B6: 10 mg, Vitamin B12: 0.05 mg, Folic acid: 5 mg, Biotin: 1 mg, Choline chloride: 500 mg, Inositol: 50 mg, Manganese: 30 mg, Iron: 35 mg, Zinc: 45 mg, Copper: 3 mg, Iodine: 5 mg, Cobalt: 2 mg, Lysine: 85 mg, Selenium: 0.15 mg, Anti-oxidant: 80 mg and Methionine:100 mg

Table 2:

Proximate composition of the experimental diets DM (%)

Ingredient (%)	Control	S2	S3	S4	S5	S6
Dry matter	92.29	92.25	92.09	92.31	92.09	92.11
Crude protein	41.59	40.98	41.08	40.75	40.83	40.78
Ash	9.05	7.95	9.3	8.15	7.2	7.8
Ether extract	6.75	6.35	6.55	6.55	7.05	6.7
Crude fibre	5.85	5.15	6.05	5.25	6.7	5
Gross energy (kcal g–1)	3.99	4.06	3.98	4.11	4.02	3.98
Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 g methionine, 0.75 g lysine and S6: 1 g lysine

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:

Where:

W1	=	Initial weight of fish (g)
W2	=	Final weight of fish (g)
T2	=	Time
T1	=	Time

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 hydrolysis¹⁵. The samples were prepared by 6 N HCL hydrolysis for 24 hrs at 110°C. After which the samples were vaporized in sodium citrate buffer (0.2 mol L^–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 analyzed¹⁶. 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 of chromic oxide content in samples and digestibility coefficient was calculated as follows¹:

Apparent Nutrient Digestibility Coefficient (ANDC):

True Nutrient Digestibility Coefficient (TNDC):

Apparent Amino Acid Digestibility Coefficient (AAADC):

True Amino Acid Digestibility Coefficient (TAADC):

Where:

MFAA	=	Metabolic feaces amino acid (it was determined by feeding protein free diet)
Cr2O3	=	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, S₃ and S₅ were similar (p>0.05) and were significantly higher from C. gariepinus fed diet S₂ (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 (S₃)-3.12±0.31 (S₂) and 66.97±7.03 (S₅)-77.97±8.93 (S₃), respectively. Control diet (13.73±1.56) had a significantly higher value in protein intake (PI) and least in C. gariepinus on diet S₆ (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 S₂ (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 S₅ (0.79±0.02) and least in diet S₂ (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 S₃ (47.64±2.10). C. gariepinus fed diet S₆ (77.80±0.20) had the least survival rate during the experiment but significantly higher (p<0.05) in C. gariepinus on diet S₅ (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 g^–1, respectively are presented in Fig. 1.

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 S₅ (80.78±0.05). Supplemental amino acid significantly (p<0.05) improved crude protein digestibility in fish fed diet S₅ (96.34±0.31) compared to other diets. However, ash digestibility significantly reduced with amino acid supplementation except for fish fed diet S₅ that was significantly higher than other treatments. Ether extract digestibility of fish on S₅, control and S₆ were similar (p>0.05) but higher than fish on S₂, S₃ and S₄. Also, crude fibre digestibility value was significantly higher in C. gariepinus fed diet S₅ (90.34±2.27) while, fish on control, S₃ and S₆ were not significantly different from each other (Table 4). Energy digestibility values varied significantly (p>0.05) among the diets with the higher value observed in C. gariepinus on S₅ (89.04±0.00).

Fig. 1: Relationship between dietary supplement of lysine and methionine in a solvent extracted soyabean based diet and gross protein retention of Clarias gariepinus

Table 3:

Growth performance and feed utilization of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid

Parameters	Control	S2	S3	S4	S5	S6
IW (g)	17.85±0.49	18.03±0.38	17.50±0.10	17.77±0.32	17.50±0.00	17.80±0.17
WG (g)	43.35±4.03b	34.77±1.17a	40.80±1.74ab	36.13±1.27a	36.70±5.11ab	35.40±4.99a
FCR	2.35±0.15	3.12±0.31	2.27±0.32	2.88±0.26	2.95±0.60	2.88±0.62
GEFC	72.96±1.52	67.25±4.32	77.97±8.93	68.66±4.85	66.97±7.03	72.53±7.51
PI	13.73±1.56b	11.95±0.67ab	12.15±1.05ab	12.17±0.47ab	12.65±1.14ab	11.24±0.72a
PER	14.45±1.34b	11.59±0.39a	13.60±0.58ab	12.04±0.42a	12.23±1.70ab	11.80±1.66a
SGR	0.59±0.08c	0.44±0.02a	0.57±0.03bc	0.47±0.01abc	0.49±0.09abc	0.46±0.09ab
GPR	0.72±0.02b	0.66±0.02a	0.75±0.04bc	0.77±0.03cd	0.79±0.02d	0.78±0.03cd
NRE	49.49±6.19b	35.15±1.42a	47.64±2.10b	41.27±1.66ab	43.17±7.76ab	40.74±7.38ab
SR (%)	87.70±0.20d	87.70±0.20d	82.20±0.20b	85.60±0.20c	88.90±0.10e	77.80±0.20a
Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 methionine, 0.75 lysine, S6: 1 g Lysine, IW: Initial weight, WG: Final weight gain, TFI: Total feed intake, MWG: Mean weight gain, FCR: Feed conversion ratio, GEFC: Gross efficiency feed conversion, PI: Protein intake, FI: Feed intake, PER: Protein efficiency ratio, SGR: Specific growth rate, GPR: Gross protein retention, NRE: Nitrogen retention efficiency and SR: Survival rate

Table 4:

Apparent nutrient digestibility of C. gariepinus fed Solvent extracted soyabean based diets supplemented with amino acid

Parameters (%)	Control	S2	S3	S4	S5	S6
Dry matter	74.41±0.09e	49.71±0.05a	69.00±0.22c	51.41±0.02b	80.78±0.05f	71.56±0.03d
Crude protein	94.29±0.04c	91.49±0.07b	94.23±0.11c	90.27±0.14a	96.34±0.31e	95.95±0.02d
Ash	69.58±0.19e	51.62±0.70b	55.70±0.36c	44.51±2.03a	72.54±0.10f	65.89±1.39d
Ether extract	96.45±0.12b	93.70±0.01a	93.54±2.20a	93.56±0.23a	97.22±0.29b	96.12±0.05b
Crude fibre	84.94±1.06c	67.53±0.35a	82.61±1.28c	73.59±0.14b	90.34±2.27d	83.96±0.61c
Energy	85.56±0.03e	68.99±0.01a	82.12±0.30c	71.56±0.04b	89.04±0.00f	82.73±0.04d
Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 methionine, 0.75 lysine and S6: 1 g lysine

Table 5:

True nutrient digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid

Parameters (%)	Control	S2	S3	S4	S5	S6
Dry matter	97.39±0.01e	91.44±0.06a	97.26±0.01d	94.71±0.05b	98.60±0.01f	95.58±0.03c
Crude protein	94.39±0.05c	91.58±0.07b	94.33±0.11c	90.37±0.14a	96.51±0.31e	96.05±0.03d
Ash	69.93±0.19e	51.96±0.68b	56.09±0.35c	44.90±2.05a	72.94±0.13f	66.25±1.37d
Ether extract	96.56±0.12b	93.77±0.29a	93.67±2.17a	93.67±0.23a	97.34±0.01b	96.23±0.04b
Crude fibre	85.36±0.97c	67.98±2.19a	83.00±1.30c	73.96±0.18b	90.72±0.38d	84.36±0.64c
Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 methionine, 0.75 lysine and S6: 1 g lysine

Table 6:

Apparent amino acid digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid

Parameters	Control	S2	S3	S4	S5	S6
Essential amino acids
Methionine	85.09±0.36a	96.96±0.13c	92.33±2.10b	92.18±0.15b	96.86±3.68c	96.55±1.65c
Lysine	94.01±0.21c	86.21±0.17a	94.06±0.25c	86.08±1.05a	95.75±0.26d	92.80±0.14b
Threonine	97.04±0.21c	97.67±0.19cd	92.48±0.85a	96.09±0.71b	92.69±0.41a	98.18±0.31d
Tryptophan	94.88±0.25d	92.74±0.38c	97.83±0.67e	90.12±1.07b	85.14±0.69a	92.45±1.38c
Isoleucine	83.67±0.11e	86.55±0.53f	78.42±0.51d	64.41±0.27a	67.85±0.79b	74.93±2.62c
Leucine	90.38±0.02e	92.97±0.84f	78.11±0.39c	69.66±2.39a	72.96±0.24b	83.44±0.26d
Valine	96.87±0.51de	97.88±1.08e	89.66±0.88a	92.55±0.57b	96.20±0.75d	94.70±0.78c
Histidine	84.37±0.83c	88.40±0.53e	86.21±0.30d	66.83±0.58a	75.87±0.83b	83.85±0.02c
Phenylalanine	92.46±0.30c	96.96±0.18d	89.80±1.11b	89.44±0.61b	86.16±0.67a	92.99±0.23c
Arginine	88.74±0.26e	91.96±0.27f	87.40±0.29d	55.38±0.66a	74.55±65b	86.18±0.37c
Non-essential amino acids
Glycine	93.01±0.06f	90.71±0.13e	73.36±0.93a	76.08±0.11b	78.00±0.45c	83.03±0.06d
Serine	95.77±0.23c	98.24±0.12d	94.74±0.31bc	93.06±1.54a	94.06±0.51ab	97.44±0.10d
Proline	95.35±0.61d	93.66±0.06c	94.03±0.21cd	87.74±0.39a	94.91±0.33cd	91.36±1.89b
Alanine	90.43±0.19e	93.88±0.26f	79.73±0.04c	75.62±1.03b	67.25±0.12a	83.00±0.87d
Aspartic	84.55±0.20d	90.33±0.82e	82.37±0.62c	75.18±1.19b	71.33±0.35a	83.43±0.30cd
Glutamic	85.09±0.22e	89.72±0.58f	77.34±0.06c	64.37±0.04a	67.20±0.64b	82.98±0.34d
Cysteine	91.56±0.34c	96.95±0.49d	84.90±0.36b	70.92±0.90a	84.17±0.89b	90.49±0.36c
Pyrrolysine	94.55±26c	97.65±0.25d	93.96±0.23bc	91.78±1.26b	85.27±3.34a	96.13±0.45cd
Tyrosine	96.82±0.21d	95.25±0.16d	82.75±0.80b	72.22±1.89a	81.08±1.96b	86.94±0.90c
Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 methionine, 0.75 lysine and S6: 1 g lysine

Table 7:

True amino acid digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with amino acid

Parameters	Control	S2	S3	S4	S5	S6
Essential amino acids
Methionine	85.51±0.34a	97.32±0.13c	92.70±2.09b	92.62±0.19b	97.25±3.69c	96.90±1.66c
Lysine	94.20±0.21c	86.37±0.17a	94.22±0.25c	86.24±1.04a	95.91±0.26d	92.97±0.14b
Threonine	97.33±0.21c	98.02±0.20cd	92.85±0.84a	96.41±0.69b	92.99±0.42a	98.53±0.32d
Tryptophan	95.23±0.24d	93.17±0.38c	98.07±0.67e	90.43±1.07b	85.51±0.68a	92.87±1.38c
Isoleucine	83.75±0.11e	86.63±0.91f	78.49±0.51d	64.48±0.27a	67.92±0.79b	75.02±2.62c
Leucine	90.57±0.02e	93.21±0.84f	78.31±0.39c	69.90±2.39a	73.19±0.24b	83.64±0.26d
Valine	97.07±0.51de	98.10±1.07e	89.93±0.87a	92.79±0.58b	96.48±0.75d	94.88±0.78c
Histidine	85.22±0.79c	89.01±0.54e	86.56±0.29d	67.53±0.58a	76.67±0.82b	84.72±0.02c
Phenylalanine	92.66±0.30c	97.13±0.17d	90.00±1.11b	89.61±0.61b	86.33±0.66a	93.18±0.22c
Arginine	89.04±0.26e	92.19±0.26f	87.65±0.29d	55.67±0.66a	74.80±0.64b	86.45±0.37c
Non-essential amino acids
Glycine	93.39±0.06f	91.16±0.13e	73.69±0.93a	76.57±0.12b	78.50±0.45c	83.51±0.06d
Serine	96.06±0.23c	98.54±0.12d	95.01±0.30bc	93.37±1.48a	94.32±0.51ab	97.66±0.11d
Proline	95.57±0.60d	93.84±0.06c	94.21±0.21cd	87.95±0.39a	95.09±0.34cd	91.52±1.89b
Alanine	90.50±0.18e	93.96±0.26f	79.80±0.04c	75.68±1.03b	67.31±0.12a	83.05±0.87d
Aspartic	84.84±0.19d	90.58±0.82e	82.65±0.62c	75.44±1.17b	71.61±0.35a	83.68±0.30cd
Glutamic	85.93±0.22e	90.30±0.56f	77.83±0.05c	65.18±0.04a	68.27±0.60b	83.72±0.34d
Cysteine	91.74±0.34c	97.20±0.49d	85.15±0.35b	71.18±0.89a	84.42±0.89b	90.73±0.36c
Pyrrolysine	94.73±0.26c	97.84±0.25d	94.15±0.23bc	91.98±1.25b	85.49±3.33a	96.32±0.45cd
Tyrosine	97.16±0.20d	95.68±0.17d	83.27±0.78b	72.89±1.87a	81.58±1.96b	87.37±0.90c
Means with different superscripts on the same row are significantly different (p<0.05), Control: Without supplemental lysine and DL-methionine, S2: 1 g methionine, S3: 0.75 g methionine, 0.25 g lysine, S4: 0.5 g methionine, 0.5 g lysine, S5: 0.25 methionine, 0.75 lysine and S6: 1 g lysine

True nutrient digestibility of C. gariepinus fed solvent extracted soyabean based diets supplemented with varying inclusion of dietary amino acid is shown in Table 5. Crude protein digestibility values varied significantly within the diets with the higher value in C. gariepinus on S₅ (96.51±0.31). Amino acid supplementation significantly decreased ash digestibility value except for fish on diet S₅ (72.94±0.13) that was higher significantly (p>0.05) than others. Ether extract digestibility values were significantly lower in fish fed diet S₂ (93.77±0.29), S₃ (93.67±2.17) and S₄ (93.67±0.23) than those on S₅, S₆ and control (Table 5). Crude fibre and dry matter digestibility values differs among diets with the higher value in fish on S₅ (90.72±0.38 and 98.60±0.01), respectively.

Methionine composition revealed that C. gariepinus on S₂, S₅ and S₆ were similar (p>0.05) but were higher (p<0.05) than other treatments in Table 6. Lysine digestibility was higher (p<0.05) in C. gariepinus on S₅ (95.75±0.26) and least value of 96.08±1.05 was in diet S₄. Threonine and valine were least (p<0.05) digested when fed diet S₃ (92.48±0.85 and 89.66±0.58) with the higher values in diet S₅ (98.18±0.31 and 96.20±0.75), respectively. Also, significantly (p<0.05) least digested values for tryptophan and isoleucine were in C. gariepinus fed S₅ (85.14±0.69 and S₄ (64.41±0.27) and higher in fish on S₃ (97.83±0.67) and S₂ (86.55±0.53), respectively as shown in Table 6. Leucine had the higher digestible value of 92.97±0.84 in diet S₂. Histidine and phenylalanine had higher (p<0.05) values in C. gariepinus on diet S₂ (88.40±0.53 and 96.96±0.18) and least in C. gariepinus on diet S₄ (66.83±0.58) and S₅ (86.16±0.67), respectively.

Apparent digestibility values for glycine revealed that fish on S₁ (93.01±0.06) were higher (p<0.05) than other treatments (Table 6). Significantly (p<0.05) least digested values for serine, proline, glutamic, cysteine, arginine and tyrosine were observed in C. gariepinus on S₄ (93.06±1.54, 87.74±0.39, 64.37±0.04, 70.92±0.90, 55.38±0.66 and 72.22±1.89) than other treatments. Furthermore, alanine and aspartic acid had the least (p<0.05) value of 67.25±0.12 and 71.33±0.35 in C. gariepinus fed S₅ and higher values were observed in diet S₂. Finally, higher (p<0.05) value of pyrrolysine was in diet S₂ (95.25±0.16) and least in diet S₅ (85.27±3.34).

Clarias gariepinus fed supplemental amino acid improved (p<0.05) methionine digestibility with higher value in diet S₅ (97.25±3.69) as shown in Table 7. Similar trend was also observed in lysine with the higher values in fish fed diet S₅ (95.91±0.26) and least value of 86.24±1.04 was in diet S₄. Higher value was recorded for threonine from C. gariepinus on S₆ (98.53±0.32) but similar (p>0.05) to C. gariepinus on diet S₂ (98.02±0.20). Tryptophan had a significantly (p<0.05) higher value in C. gariepinus on S₁ (95.23±0.24) and least in diet S₅ (85.51±0.68). Isoleucine, leucine and histidine had least (p<0.05) values in fish on diet S₄ (64.48±0.27, 69.90±2.39 and 67.53±0.58) and higher values of 86.63±0.91, 93.21±0.84 and 89.01±0.54 were in diet S₂, respectively (Table 7). Additionally, supplementation of amino acid altered (p<0.05) valine and phenylalanine in C. gariepinus on diet S₂ (98.10±1.07 and 97.13±0.17) and least in S₃ (89.93±0.87) and S₂ (97.13±0.17), respectively.

Also, Glycine was higher (p<0.05) in C. gariepinus fed S₁ (93.39±0.06) and the least value in diet S₃ (73.69±0.93). Significantly least (p<0.05) values for serine, proline, glutamic, cysteine, arginine and tyrosine were observed in C. gariepinus fed S₄ (87.95±0.39, 65.18±0.04, 71.18±0.89, 55.67±0.66 and 72.89±1.87). Serine value was similar in C. gariepinus on diet S₄ (93.37±1.48) and S₅ (94.32±0.51) but differed significantly from other treatments. However, alanine, aspartic acid and Pyrrolysine had least values in diet S₅ with the higher values of 93.96±0.26, 90.58±0.82 and 97.84±0.25 in diet S₂ (Table 7).

DISCUSSION

Findings from 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.

Also, Nwanna et al.⁸ reported that supplementation of DL-methionine in DL-methionine deficient diet significantly increased feed utilization with better feed conversion ratio which resulted in significant improvements in weight gain and feed efficiency of common carp. Similar trends were noted by Gao et al.²⁰, Ren et al.²¹ and Sardar et al.²² in juvenile grass carp (Cyprinus carpio), juvenile blunt snout bream (Megalobrama amblycephala) and rohu (Labeo rohita) fingerlings when fed increasing levels of dietary histidine, isoleucine and met+cystine, respectively. Also, the supplementation of lysine and DL-methionine improved feed efficiency and decreased the amount of feed consumption necessary for fish growth which could result in production cost reduction. Yuan et al.²³, Nwanna et al.⁸ and Wang et al.²⁴ reported lower FCR which was due to lysine and DL-methionine supplementation in the diet of Myxocyprinus asiaticus, Cyprinus carpio and Pseudo bagr usussuriensis, respectively. These conformed to the result observed in this present study.

Zhou et al.²⁵ observed reduced growth, feed utilization and PER when lysine was supplemented to diets of Sparus macrocephalus. It may be due to the negative effects of excessive or insufficient amount of free lysine and DL-methionine and poor palatability. Wang et al.²⁴ suggested that DL-methionine levels used in the diet were not high enough to induce toxic effect. However, the relationship between solvent extracted soyabean based diet and gross protein retention of C. gariepinus fed dietary amino acid was shown by the regression model in Fig. 1. From the growth response curve, the estimated optimum level of lysine 0.6 and 0.4 g 100 g^–1 DL-methionine for gross protein retention.

Supplemental methionine and lysine in solvent extracted soyabean based diet improved crude protein digestion as shown in 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 and Ribeiro et al.¹ stated. This suggested that combining supplemental methionine and lysine in soyabean based diet is imperative in C. gariepinus diets.

Apparent digestibility of the ether extract, crude fibre and dry matter of C. gariepinus fed solvent extracted soyabean based diets supplemented with methionine and lysine suggested an increased digestibility though no particular trend were observed. This indicated that supplemental methionine and lysine in soyabean based diet had little influence on the experimental fish. Supplemental methionine and lysine in soyabean based diet fed to C. gariepinus further revealed that it supplementation could improve the energy digestibility. This result was in agreement with Sotolu and Faturoti²⁶ and Nwanna et al.⁸, who observed improved energy digestibility in C. gariepinus and common carp, respectively.

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.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 Table 6. The values observed from the soyabean based diets increased from 85.09±0.36 (S₁)-97.49±0.47 (S₅) for methionine, lysine values increased from 86.08±1.05 (S₄)-95.75±0.26 (S₅). The increased observed in fish fed diet S₅ are coherent with Yamamoto et al.²⁷ result, who perceived comparative values when several protein sources were fed to fingerlings, red sea beam, rainbow trout and common crap. However, Wilson et al.²⁹ and Ribeiro et al.¹ 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 acids³⁰. 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.

Results observed in 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 and 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.

CONCLUSION

It can be concluded that C. gariepinus are able to utilize solvent extracted soyabean based diet with lysine and DL-methionine supplementation. Supplementation of lysine at 0.6 and 0.4 g 100 g^–1 of DL-methionine in solvent extracted soyabean based diet when fed to C. gariepinus improved growth and feed utilization. However, lysine 0.75 and 0.25 g 100 g^–1 of methionine in soyabean based diet could improve digestibility of experimental fish. Further studies should, therefore, be conducted on the effect of true digestibility in C. gariepinus when fed other plant protein-based diet with or without amino acid supplementation.

SIGNIFICANCE STATEMENT

This study discovered that supplementation of lysine and DL-methionine in soyabean based diet could be utilize by C. gariepinus for its optimum growth and improve digestibility. The inclusion levels of 0.6 (lysine) and 0.4 g 100 g^–1 (DL-methionine) for growth and lysine 0.75 and 0.25 g 100 g^–1 of methionine in soyabean based diet for digestibility can be beneficial to researchers and fish feed industries for further investigations on how to improve utilization of plant protein-based diets. This study will help the researchers to uncover the critical areas of total replacement of fishmeal that many researchers were not possible able to explore.

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