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Methionine supplementation

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Methionine supplementation
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  Effect of methionine supplementation to low protein diets on growth, protein utilization and amino acid profile of African Catfish ( Clarias gariepinus , Burchell 1822)  Nwanna, L.C and Falade, E.A Dept. of Fisheries & Aquaculture Tech., Federal University of Technology, PMB 704, Akure-Nigeria. Abstract A feeding trial was conducted to evaluate the effect of methionine supplementation to low protein diets on the growth, protein utilization and the amino acid profile of profile of African Catfish ( Clarias gariepinus ). Experimental diets; D1- D5 contained 25, 30, 35, 40 and 45% crude protein levels respectively. Diets 1-3 were supplemented with 1.12% DL- methionine while diets 4 and 5 were devoid of methionine. The fish were randomly distributed into glass tanks at 15 fish per tank and fed to apparent satiation twice daily for seventy days. Each group of fish was weighed bi-weekly for growth assessment. At the end of the experiment, carcass analysis of the fish was performed. Data analyses were carried out using one way analysis of variance. The result obtained shows that the weight gain, specific growth rate and the protein efficiency ratio of fish fed at 40% or 45% protein levels were the same but significantly higher (P<0.05) than those fed low (25%, 30% and 35%)  protein diets supplemented with methionine. FCR (1.10 or 1.01) obtained in fish fed 40 or 45% crude  protein diets were significantly better than in fish fed low protein diets supplemented with methionine. Carcass protein and lipids increased significantly with increasing protein levels while the moisture decreasing significantly with increasing protein levels in the diets. There were variations in the amino acid profile of the experimental diets and fish. Diets with 40 or 45% CP level had amino acid compositions which were higher than the diets with low protein contents supplemented with methionine. Amino acid levels in the fish also increased with increasing levels of protein in the diets. Results from the present study clearly demonstrated that supplementing low protein diets with DL- methionine will not be beneficial to juvenile African catfish ( Clarias gariepinus ) and also that feeding the African catfish with 45% crude protein diet would be uneconomical. INTRODUCTION Reducing the protein content of fish feeds is one strategy to increase the sustainability of catfish aquaculture via reducing feed costs as well as reducing the environmental impact if economic growth can be maintained with less nitrogen input (Gibson, 2008). The excess nitrogen excreted as ammonia  by fish may have a negative impact on the environment because it is a major contributor to water  pollution. Because every species of fish and the individual proteins within each species has its own unique amino acid composition, the ideal situation would be to formulate a low protein feed that would minimize nitrogen excretion and at the same time meet all requirements for essential amino acids. The most conventional protein sources used in fish feed such as soya bean, cotton seed, fish meal etc. are becoming expensive especially to small scale fish farmers in Nigeria. Methionine is an essential amino acid that cannot be synthesized in the fish body, but must be obtained from food sources or from dietary supplements. Methionine and cysteine are the principal source of sulphur in fish diets. Fish body can make cysteine from methionine, but not vice versa, so that methionine is a dietary essential. Methionine is important in the formation of blood proteins, globulins, and albumins, antibodies, neurotransmitters, immunoglobulin and hormones. It also assists in the breakdown of fats, preventing their build up in the liver and arteries, which can obstruct blood flow to the brain, heart, and kidneys.  The nutritional value of a soy protein isolate could be enhanced by supplementing it with the first limiting amino acid; i.e., methionine. The quantitative methionine requirement of Clarias garienpinus is 3.2g/100g protein (Fagbenro  , et al.,  1998). Since the primary objective of fish nutrition work is geared towards reducing protein cost in fish feed, it is of interest to investigate if low protein diets supplemented with methionine could be suitable for production of low-cost diets. Therefore, the  present study investigated the effects of methionine supplementation to low protein diets on the growth, protein utilization and amino acid profile of African Catfish ( Clarias garienpinus).   Materials and methods Sample collection and preparation Feed ingredients including fish meal, soybean meal, groundnut cake, yellow corn, cod liver oil, vitamin-mineral premix, binder and D ʟ -methionine were purchased from K  ₂  feed mill, Akure in Ondo State.  Diet preparation Five experimental diets were formulated and labelled D1 (25%), D2 (30%), D3 (35%), D4 (40%) and D5 (45%) crude protein levels using locally available feed ingredients. Diets with 25%, 30% and 35% CP were supplemented with 1.12% crystalline methionine while diets 4 and 5 serve as the reference. (Table1). The various ingredients were ground with hammer mill, weighed, mixed to homogeneity and pelleted using a pelleting machine (Hobart Mixer Machine, Model a 200, Hobart, CA, USA with a 2mm die holes. The pellets were sundried to a constant moisture level (< 10 percent), packed in airtight polyethylene bags and stored at - 20˚C before use. Diets were analysed for their proximate composition (Table 2) using the method of AOAC (1990).  Experimental tanks and feeding trial Fifteen glass tanks (75x45x30cm) filled with 60L of tap water were used for the study. The experiment carried out at FUTA Research Farm unit was made up five dietary treatments replicated thrice. A dissolved oxygen level at 7.5-8.35 was maintained throughout the experimental period while the pH ranged between 6.5 and 8.5. Apparently healthy fingerlings of African catfish (4.5  0.68g)  purchased from Silas Fish Farm, Oba-Ile estate Akure were acclimatized for seven days and were stocked at fifteen fish per tank. The fish were fed to satiation twice daily between 7 am  –   8 am and 4  pm  –   5 pm for seventy days. Weight measurements were taken bi-weekly for assessment of fish growth performance. Each day before feeding, the faecal matter in the tanks were siphoned while complete draining and cleaning of the tanks was carried out once in three days to ensure a healthy environment for the fish and to maintain good water quality. The growth performance of the fish was calculated according to Castell and Tiews (1980) as follows: mean weight gain = final mean weight  –   initial mean weight; specific growth rate =100 x (log e  final weight-Log e  initial weight/culture period (days)); feed conversion ratio = weight of feed fed (g)/fish weight gain. Analytical methods  Proximate and mineral analysis of fish samples At the end of the experiment, fish were not fed for 24h. The fish were weighed and an equal number of fish from each tank was collected. A total of twelve fish from each treatment were sacrificed. Three replicates of the fish carcass ( whole body) were analysed for proximate and minerals composition according to the methods of AOAC (1990). About 2.0g of the samples were ashed for 48h at 480 o C. After the ash had cooled to room temperature, 6ml of HCl was added and the mixture was brought to  boiling point. After cooling to room temperature, another 2.5ml of 6N HCl was added and the  mixture was warmed to dissolve all the solutes. The solution was then cooled and diluted to 25ml with distilled deionized water. Then the minerals ( Mg, Ca, K and Fe) were measured in Atomic Absorption Spectrophotometer (AAS). Phosphorus content of the fish was analysed using the Vanadomolybophosphoric acid colorimetric method 4500-p with slight modifications. To 3ml of the diluted solution of the sample, 3ml of vanadatemolydate reagent was added and phosphorus concentration was measured spectrophotometrically at 430nm, after the reaction mixture was thoroughly mixed and allowed to stand at room temperature for 10min.   Statistical analysis Growth, carcass, minerals data were analysed using one-way analysis of variance (ANOVA), followed by Duncan’s new multiple range test (Duncan 1955) at the 5% level of significance to detect differences among treatment means.  Amino acid profile determination The amino acid profile in the known samples of feed and fish was determined using the methods described by Benitez (1984). The known samples were dried to a constant weight, defatted, hydrolysed, evaporated in a rotary evaporator and loaded into the Technicon sequential Multi-Sample Amino Acid Analyzer (TSM). Results The proximate composition of the experimental diets as shown in table 4.1 reveals that the values are marginally different (P>0.05). However, the crude protein levels of the experimental diets varied according to the protein percentage as the protein content increased from 25.50 (Diet 1) to 45.0 (Diet 5). The overall results on variation in the amino acid composition of the experimental diets as presented in table 4 inferred that it was much influenced by the variation in the dietary protein content. This result confirmed that experimental diets 4 and 5 with 40% and 45% crude protein levels respectively exhibited higher amino acids composition than in low protein diets supplemented with methionine. However there were slight differences in the amino acid composition of diet 4 (40% CP) and diet 5 (45%CP). Growth response of fish fed diets containing 40-45% crude protein (Table 5) were statistically the same, but significantly better than those of fish fed low protein diets (25%-35%) supplemented with methionine. The mean final weight, mean weight gain, specific growth rate and feed conversion ratio were sensitive to the levels of protein in the diets and improved significantly (P<0.05) as dietary  protein levels increased from 25 to 45%. Highest weight gain (54.9) and specific growth rate (4.50) and best FCR (1.01) were obtained for the fish fed diet with 45% protein. However there was no significant difference (P>0.05) in the weight gain, specific growth rate and the feed conversion ratio of fish fed at 40% and 45% crude protein levels. Protein efficiency ratio (PER) was higher in fish fed 40% CP diet followed by fish fed 45% but there was no significant difference (P> 0.05) between the two values. Fish fed with low protein diets supplemented with methionine had a significantly lower (P<0.05) PER values when compared with the reference group. Fish fed low protein diets supplemented with methionine exhibited significantly less (P<0.05) growth and reduced feed utilization efficiency. Table 4 presents the biochemical composition of African catfish fed the experimental diets. At the end of the experiment, significant differences (P<0.05) in body compositions were noted among the  groups. The overall results on the variation in carcass composition inferred that it was much influenced by the difference in the dietary protein content and less influenced by methionine supplementation. The carcass protein content of fish fed at high protein levels (40%-45%) were significantly (P<0.05) higher than those fish fed with protein diets (25%-35%). Carcass moisture content showed a continuous decrease with increasing body protein. Fish fed diets containing 40-45% crude protein did not show any significant difference (P>0.05) in body protein and lipids. However fish fed with 35% crude protein had ash content marginally higher than the values from other treatments. The carcass lipid also increased significantly with increasing levels of protein in the diets. Carcass mineral contents were significantly (P<0.05) affected by the dietary treatments. Body mineral composition in response to dietary treatments revealed an increase in the carcass calcium, phosphorus and sodium contents with an increasing level of protein in the diets. Fish fed 35, 40 and 45% protein diets had phosphorus deposition of 32.0, 31.6, and 32.0 respectively which were significantly different (P<0.05) from the phosphorus content of fish fed 25 and 30% crude protein diets. Also fish fed (35 and 40% CP) diets had calcium values of 36.01 and 32.0, followed by the fish fed 40% CP diet. However, manganese deposition increased in the methionine supplemented group in comparison with the deposition in fish fed diets without methionine supplementation. The amino acid composition of the experimental fish is presented in table 4.6. The overall results on variation in the amino acid composition of the experimental fish inferred that it was much influenced  by the variation in the dietary protein content. This result confirmed that fish fed high protein diets (40% and 45%) exhibited higher amino acid deposition in the tissue than fish fed with low protein diets supplemented with methionine.   Discussion The present study evaluated the possibility of feeding African catfish ( Clarias gariepinus ) with low  protein diets supplemented with methionine as protein is the most expensive component in fish feeds. In this study, proximate composition of the experimental diets were marginally (P>0.05) different. Moisture and ash content varied slightly but within the range recommended by Faturoti and Ajiboye (2011) for good growth. The lipid content of the diets was within the range considered to be good enough for Clarias gariepinus  as fat is an important macronutrient for fish. However, the crude  protein levels of the diets varied significantly in accordance with the inclusion levels in the diets. Aside lysine, methionine is generally the first limiting amino acid in aqua feeds (NRC. 2011), and its supply from plant protein sources is low making its supplementation increasingly important for modern aqua feed formulation (Nwanna et al.,2012).Nwanna (2012) further explained that aquaculture nutritionist often doubt the effectiveness of supplemental amino acids compared to  protein bound amino acids. This is because free amino acids are absorbed faster which may result in an unbalanced amino acid profile in the blood and at the site of protein synthesis. In the present study, despite methionine supplementation to diets having low protein content, methionine and other amino acids concentration in the reference diets (diet 4 and 5) were higher. This result means that dietary amino acid concentration was much influenced by the variations in the protein content of the diet. The effects of essential amino acids supplementation for animal growth performance are markedly affected by the nutritional status of the diet, especially the protein and EAA levels (Yamamoto et al  ., 2005). In pig and poultry diets, (Verstegen and Jongbloed, 2003) reported that supplementation of EAA achieved significantly better growth and feed utilization compared with the reference diets without supplemented EAA when the dietary protein levels were much lower than their requirements. However, in fish diets, the supplementation of EAA to a low protein diet is seldom reported by investigators. In channel catfish (Li and Robinson, 1998) and rainbow trout (Yamamoto et al  ., 2005), these researchers showed that the supplementation of EAA in an insufficient protein diet improved the feed efficiency but did not enhance fish growth. However in Asian sea bass, (Williams et al  ., 2001)  demonstrated that crystalline-EAA supplementation to an EAA deficient diet increased fish growth and the response was more pronounced for the low protein diets. In the present study, growth  performance of experimental fish was markedly affected by the nutritional status of the diet, especially the protein level. Growth of the fish increased significantly (P<0.05) with increasing dietary protein. Although growth rate of the fish was highest at 45% CP, this increase in growth was not statistically different (P>0.05) from the growth of fish fed diet containing 40% crude protein and hence, feeding catfish with 45% protein in the diet would be uneconomical. Therefore inclusion of 40% protein diet for African catfish juvenile is appropriate. Faturoti et al., (1986) and Fagbenro et al., (1998) reported that 40% crude protein diet was optimum for growth and nutrient utilization in Clarias gariepinus , Olufeagba (1999) recommended 45% for triploid  H. longifilis , Adebayo (2005) reported the maximum growth performance of hybrid catfish ( Clarias gariepinus ×  Heterobranchus   bidorsalis ) fingerlings in the fish fed 40% protein diet. Jamabo and Ockiya (2008) reported 40% crude protein for  H.   bidorsalis . The trend in growth rate in this feeding trial agreed with Dahlgren (1979) findings that suggested that an increase in dietary protein levels may lead to increase in growth rate in channel catfish (  Ictalurus punctatus ). The food conversion ratio (FCR) recorded in this study showed that fish fed on 40-45% crude protein diets most efficiently converted feed into flesh. The FCR became better with increasing dietary protein levels. This result agrees with the work of Jaucey (1982) on Sarotherondon mossambicus , Arowosoge (1987) on Clarias lazera,  De-Gani (1987) on Clarias gariepinus  and Diyaware (2009) on hybrid catfish (  Heterobranchus bidorsalis x Clarias anguillaris ). Lovell (1979) stated that fish are able to assimilate diets with higher percentage of  protein due to lower energy requirements. Also in this study, protein efficiency ratio (PER) increased with the increase in dietary protein content. This is also evident in other studies (Lee and Putnam 1973; Bromley 1980; Pongmaneerat and Watanabe 1991; Lee et al  ., 2002, Tabassum et al.,  2008, Diyaware et al.,  2009). Carcass protein and lipids increased significantly with increasing protein levels while the moisture decreasing significantly with increasing protein levels in the diets. Similar findings have been reported for young  Heteropneutes fossilis  (Tabassum et al  ., 2008),  Etroplus suratensis  (Palavesam et al.,  2008), juvenile grass carp (Li et al., 2009), Synodontis nigrita (Ajiboye et al.,  20011). The mineral composition of the experimental fish explains the high ash content in fish fed 35-45% crude protein diets. The phosphorus, calcium and sodium contents of the fishes varied marginally, increasing with increase in dietary protein content. Indeed calcium and phosphorus are the dominant inorganic components in the whole fish and about 90% and 80% of calcium and phosphorus are found in the bones (Hertrampf et al., 2000). However, in tilapia and salmonids, Skonberg et al., (1997) documented that whole body calcium and phosphorus concentrations are responsive to increased levels of dietary protein content. Methionine requirement of different fish species exhibits wide variation, being 9.4g/kg for Channel catfish, Cai and Burtle (1996), 3.55g/100g protein for Catla catla , (Ravi and Devaraj, 1991), 2.56g/100g protein for Seriola quinqueradiata  (Ruchinat et al.,  1997), 3.34g/100g protein for  Pseudosciaena crocea,  (Mai et al., 2005), 3g/100g protein for Cirrhinus mrigala , (Ahmed et al., 2006), 73g/100g protein for  Epinephelus coioides  ( Luo et al., 2005), 2.64g/100g protein for  Rachycentron canadum  (Zhou et al  ., 2005)  ,  2.0g/100g protein for  Dicentrachus labrax  ( Thebault et al.,  1985), 2.2g/100g protein for Oncorhynchus mykiss , (Walton et al., 1982), 2.3g/100g protein for Channel catfish (Nose 1989), 2.7g/100g protein for Oreochromis niloticus  (Santiago and Lovell 1988), 2.9g/100g protein for  Anguilla japonicas (Harding et al.,  1977), 3.1g/100g protein for Cyprinus carpio (Harding et a l., 1977), 3.2g/100g protein for Clarias gariepinus  (Fagbenro et al., 1998), 2.4g/100g protein for Clarias gariepinus (Un-prasert 1994), 2.3g/100g protein for Clarias  gariepinus  (Uys 1989)  , 4.0g/100g protein for Sparus aurata  (Walton et al.,  1982), 3.1-3.4g/100g  protein for Seriola quinqueradiata , (Twibell et al.,1999), 2.97g/100g protein for Clarias gariepinus  (Ovie et al.,  2009) and 8.6g/kg for common carp (Nwanna et al.,  2012). Such variations are attributed to differences in metabolic requirements of the species and in the daily protein intake by fish, caused  by the variation, dietary formulation (types and amount of protein used), and feeding regimes used in classical dose response experiments (Cowey, 1993). In the present study, fish fed low protein diets

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Jun 13, 2018
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