Growth evaluation of Oreochromis niloticus fed different concentrations of choline and methionine

11Animal Physiology, Development, and Molecular Laboratory, Department of Biology Faculty of Mathematics and Natural Sciences Mulawarman University, Jl. Barong Tongkok No 4 Gn Kelua, Samarinda, East Kalimantan, Indonesia 75123. 2Plant Physiology and Development Laboratory, Department of Biology Faculty of Mathematics and Natural Sciences Mulawarman University, Jl. Barong Tongkok No 4 Gn Kelua, Samarinda, East Kalimantan, Indonesia 75123.


Introduction
Choline is a pivotal nutrient and has an important responsible in cell structure, cell maintenance, and specific metabolic functions (Baldissera et al., 2019;Khosravi et al., 2015;NRC, 1993). It is also a part of acetylcholine, a neurotransmitter in the synapsis of neuron, which acts in the impuls transmission across synapses (Topal et al., 2016;Wauben and Wainwright, 1999). One of the essential functions of choline metabolism is a source of methyl groups for reaction transmethylation formation of methionine from homocysteine (Combs, 2008;Kim et al., 2019). Choline is a methyl group donor via its degradation products which is known as betaine (Day and Kempson, 2016;Workel, 2005). Some animals can produce choline if the methyl donor (Example: Methionine) is presented in the feed. Choline (example: phosphatidylcholine) can be synthesized through methylation of phosphatidylethanolamine assisted by S-adenosyl methionine (SAM) as a methyl donor (Abbasi et al., 2018;Combs, 2008).
Choline, also known as a vitamin-like nutrient, is identified as an important feed component especially in fish (NRC, 1993). Dietary choline supplementation enhanced growth indices, boosted protein and lipid deposition, increased hepatopancreatic and intestinal enzyme activities in Jian carp Cyprinus carpio var. Jian (Wu et al., 2011). Dietary choline requirement for several fish species have been predicted and reported to be at the range of 400 to 1,000 mg kg −1 diet (Fernandes et al., 2016;Luo et al., 2016;Qin et al., 2017). Previous study by Shiau and Lo (1999) stated that the optimum growth of tilapia can be achieved by addition 1,000 mg choline per kg diet. A previous study also confirmed that choline level about 230 mg kg −1 diet sufficient for juvenile parrot fish (Oplegnathus fasciatus) requirement (Khosravi et al., 2015).
Meanwhile, methionine is a pivotal amino acid metabolite that uses for protein synthesis and the formation of SAM (Ratriyanto et al., 2009). However, the rate of choline synthesis is not enough to achieve either the physiological or metabolic requirement in several animals (Wilson and Poe, 1988). This is due to the function of methionine for protein synthesis and the formation of mutual competence SAM to methionine available. Therefore, to meet the needs of choline and methyl donor for the methionine synthesis, choline should be supplemented in the diet. Niu et al. (2016) found that the substitution of fish meal using soybean meal and methionine supplementation resulted in significantly better growth in juvenile golden pompano (Trachinotus ovatus). In addition, Wang et al., (2013) stated that the methionine supplementation in for juvenile Pseudobagrus ussuriensis culture to obtain optimum growth and feed utilization is approximately 14 g kg -1 dry diet, respectively, in addition of 2.5 g kg -1 dry diets cysteine. However, the studies on nutrient requirement of the choline and methionine on the growth of Nile Tilapia (Oreochromis niloticus) are limited. Thus, the purpose of this research was to determine the effects of choline and methionine addition on the growth indices and carcass proximate composition of tilapia.
Tilapia is an omnivorous species and considered a valuable species because of high demand, easily maintained and cultivated ponds by utilizing natural feed and supplementary feeding in the form of artificial feed (Kordi, 2010). Tilapia is also considered to be the second most valuable fish culture in the globe, after carps (Moniruzzaman et al., 2015;Ridha, 2006), and its cultivation is practiced in most of the tropical, subtropical and temperate regions, including Indonesia (Phillips et al., 2015). In Indonesia, the biggest production of tilapia is still in West Java, reaching 8.524 ton in 2013 while East Kalimantan has 5.117 ton production of tilapia in the same year (Widiarti, 2015). The success and significantly improve in tilapia culture outcome have received attention among farmers, investors and especially researchers who attempts to boost productivity by optimizing their growth. Therefore, the objective of the present study was to determine the effects of choline and methionine supplementation on the growth performance of Nile tilapia Oreochromis niloticus.

Materials and Methods Experimental diets preparation
Composition of the test diets formula is provided in Table 1. The test diets that were formulated and consisted of three diets (P2-P4) were prepared by supplementing 0.04; 0.08; 0.12 % choline, three diets (P6-P8) with same levels choline supplementation plus methionine (0.15 %), a diet with methionine (P5) and control diet without any supplementation (P1). The proximate analysis of test diet fish carcass was analysed using AOAC (1990).

Fish and feeding trial
Twenty-four cages (1 m x 1 m) were set up in a semi-intensive pond (17 m x 8 m) at a density of 50 fish per cage. The fish were fed a commercial diet (Hi Pro Vite FF-888, containing 36-38% protein, 2% lipid, 2% crude fibre, 10% ash and 12% moisture) for three days to become acclimated to the research condition and equipment. At final day of acclimation period, the fish (Initial body weight, 21.05±1.6 g) were randomly assigned to pond cage. The study was conducted in three replicates and fish were fed by hand to apparent satiation five times a day (08:00, 10:00, 11:30, 14:00 and 16:00 h) for 12 weeks. Two pumps ventury were used as aeration to maintain sufficient dissolved oxygen. Dissolved Oxygen (DO), temperature, and pH of pool water were measured twice a day (morning and afternoon). The levels of nitrite and TAN (total ammonia nitrogen) of pool water were checked once a week. Sampledfish growth was noted every four weeks, and feeding rate was calculated following the change of the weight of the fish. The fish were then fasted for 24 h before weight and the carcass of fish sampling.

Sample collection and analyses
At the final of week 12, the sampled-fish in each cage were weighed and counted to calculate the growth performance such as Final weight (FW x 100 Where, W0 is the initial fish weight (g) at time T1 (day), and Wt is the final fish weight (g) at time T2 (day).

Feed conversion ratio (FCR)
FCR is stated as the feed consumed in dry weight per unit live weight gain. Survival rate (SR) Where N0 is the number of fish at initial day and Nt is the number of fish dead during of experiment (Muchlisin et al., 2016).
Further, 1.2 kg to 1.5 kg of fish per cage was randomly selected samples for the measurement of the proximate fish carcass (crude protein and lipid).
The proximate fish carcass was analysed using AOAC (1990) standard methods in laboratories feed mill PT. Suri Tani Pemuka unit Gresik, East Java, Indonesia.

Statistical analysis
Resulted data were expressed as mean ± standard error (SE). Growth data and proximate analysis were analyzed by means of a one-way analysis of variance (ANOVA) using SPSS Statistics software, version 22 (SPSS, Inc., USA). Comparisons between treatment media were carried out using the Duncan test. Percentage data was arcsine transformed before ANOVA and then reversed. All significant tests were at levels P <0.05.

Growth, feed utilization and survival
Growth performance and feed utilization of tilapia fed different ratio combination of choline and methionine for 12 weeks are presented in Table 2  PER in Figure 1. After 12 weeks of the feeding trial, tilapia fed 0.04 % choline (P2) in the diet showed the highest final weight, ADG, SGR, and PER. In feed utilization, there was also significantly better (P<0.05) in the FI, FE, and FCR of tilapia fed 0.04 % choline (P2) in the diet. Meanwhile, the SR of tilapia was not significantly affected by dietary any combination ratio of choline and methionine ( Figure  2).

Carcass proximate composition
Besides the growth performance, dietary choline supplementation improved the percentage of crude protein in the carcass of tilapia. The proximate composition of the fish carcass from the experiment is shown in Table 3. Crude protein (52.50 ± 0.98 %) was significantly (P<0.05) higher in the tilapia fed choline 0.08 % (P3) in the diet compared to other groups. However, dietary 0.08% choline supplementation in the diet resulted the highest crude lipid (22.34 ± 2.41%) was found in the carcass of tilapia fed control diet, followed by fish fed 0.04 % choline in the diet. However, the administration choline and methionine in the diet may reduce lipid content in the carcass. This result is in agreement with past research stated that the addition of choline and methionine in the diet can reduce fat deposition in carp (Cyprinus carpio) (Wu et al., 2011).   (

Discussion
Choline plays a pivotal role in many metabolic functions such as transmission nerve impulse across synapsis (Wauben and Wainwright, 1999) and methyl donor in the anabolism of various methylated metabolites (Combs Jr and McClung, 2016). Similar to choline, methionine is an essential amino acid which benefit to fish (Niu et al., 2016) and acts as methyl donor (Bender, 2003;Furuya, 2017 (Luo et al., 2016); blunt snout bream (Megalobrama amblycephal) (Li et al., 2016) and grouper (Epinephelus coioides) (Qin et al., 2017). In addition, the choline supplementation without methyl methionine in the diet might not optimal to increase crude protein level in the carcass as shown in the P2 group in present findings. This result is supported by the statement that methionine is the first important amino acid in the catabolic pathway and has several potential interactions with choline and other dietary constituents (Kasper et al., 2000). According to NRC (1993) choline supplementation should be added to the diet of fish, although most of the animals could synthesize choline in the liver. That is because the choline production in the body does not meet the metabolic and physiological needs of the animal. In addition, choline has been identified as an important intermediate in the catabolism of methionine (Vemury et al., 1980). As reported by Luo et al. (2016), choline involves in lipid homeostatic in muscle and liver by influencing metabolism in mRNA levels. Thus, the addition of adequate choline in the diet may benefit to improve growth performance of fish by increasing the transport of lipids from the liver to muscle for storage and enhancing muscle lipid content. In contrast, deficiency choline in the diet may result in poor growth performance and poor feed intake in hybrid tilapia (Shiau and Lo, 2000).
Furthermore, PER value of the fish shows a protein digestibility of the ratio between the number of acidic amino acids that can be absorbed by the small intestine in the body and the amount of protein consumed by the formation of body tissues. In the process of digestion and absorption of food, intestinal enzymes have a pivotal role in the digestive activity and growth of the fish. Yang et al. (2006) stated that choline might increase the activity of the hepatopancreas in producing trypsin that plays a pivotal role in changing the trypsin peptides of proteins into amino acids. Thus, the increase in this enzyme activity can increase the uptake of amino acids by the small intestine.
In the current research, feed intake and FCR of tilapia fed 0.04 % in the diet was significantly better than other groups. It is stated by And and David (2005) that there is an interrelationship between choline and methionine in the portion of choline and methionine requirement. Choline can be a spare of the portion of methionine requirement if no sufficient methionine. However, methionine cannot be a spare of choline requirement in the presence of adequate choline. Therefore, the addition of choline in the diet of fish is compulsory to increase growth performance, including feed intake and FCR. Choline can also replace the role of methionine as a donor of methyl groups via betaine homocysteine methyltransferase (BHMT) (Zeisel, 2017). It is further explained that dietary adequate choline 0.04% can be added in the diet of fish without methionine supplementation.
On the other hand, a study in Pangasius catfish (Pangasius bocourti) fed diet 0.71 g 100 g -1 DLmethionine revealed a significantly increased weight gain and FCR in test group after 10 weeks of the feeding study (Yuangsoi et al., 2016). Bakke et al. (2010) revealed that fish growth is affected by the activity and capacity of the fish digestive tract. The digestive tract enzymes activity in the brush border such as amylase, trypsin, Chymotrypsin, lipase, Na+/K+-ATPase, and γ-GT improved in trial fish which fed the methionine supplementation in the diet and enhanced in fish fed optimal value of methionine (Wu et al., 2017;Xiao et al., 2011).
Current study has also shown that dietary choline and methionine in any ratio combination had no effects on the survival rate ( Figure 2). This was in agreement with the previous study on Pangasius catfish that dietary either methionine (Yuangsoi et al., 2016) or choline (Yeh et al., 2015) had no effect on the survival rate. The reason for this may be attributable to the occurrence of choline and/or methionine that plays an important role in lipid metabolism and stress tolerance (Yeh et al., 2015).
The deposition of liver fat in low dietary choline animals shows not only impaired liver lipoprotein secretion but also subsequent accumulation of triglycerides. According to Machlin (1991), and Twibell and Brown (2000) fat deposition in the liver of fish can be a tool of choline status in a variety of animals, though is not consistent in several of fish. For example: inclusion choline in the diet at or above the requirement resulted in significantly lower liver fat concentrations in channel catfish (Ictalurus punctatus) (Wilson and Poe, 1988), lake trout (Salvelinus namaycush) (Ketola, 1976), and hybrid striped bass (Morone chrysops x Morone saxatilis) (Griffin et al., 1994), while higher liver fat deposition has been found in red drum (Sciaenops ocellatus) (Craig and Gatlin III, 1996) and liver fat deposition of rainbow trout (Oncorhynchus mykiss) (Rumsey, 1991) and yellow perch (Perca flavescens) (Twibell and Brown, 2000) were not affected by dietary choline in the diet.

Conclusions
Based on the data results, it seems clear that juvenile tilapia requires choline supplementation in certain dietary formulations. Furthermore, it is suggested that dietary supplementation of 0.04-0.08% choline in the diet is useful to improve growth indices and protein carcass proximate composition of tilapia. Meanwhile, dietary supplementation methionine in tilapia feed is optional.