EVALUATION OF THE POTENTIAL INCLUSION OF MALAYSIAN RUBBER SEED, Hevea brasiliensis Muell. Arg. (EUPHORBIACEAE) ON THE TAMBACU DIET

In intensive production systems, fish are exposed to stressful conditions, becoming more susceptible to diseases, which may reflect on their growth parameters. Alternative growth promotion methods without environmental impacts have become the target of researchers and fish farmers. For this reason, the objective of this work was to evaluate the potential of including Malaysian rubber seed in the feeding of tambacu raised in cages. The seeds were collected on a farm in the municipality of Imperatriz, Maranhão, a sample was sent for physical-chemical analysis of the fixed oil. For inoculation in the diet, the husks were removed, dried at room temperature, crushed and manually mixed with the commercial ration feed following the experimental design. The quality of the water was measured throughout the experiment. Biometric measurements were performed to infer growth gains with the tested diet. The results show an influence on the inclusion of 40% of the Malaysian rubber seed in the tambacu diet during rearing. In order to reduce costs resulting from the use of alternative ration feeds, we suggest the use of Malaysian rubber seed in an attempt to minimize barriers due to the high prices of commercial ration feeds within the fish farming production chain.


INTRODUCTION
Tambacu is a hybrid resulting from the breeding induced between the tambaqui female, Colossoma macropomum, and the pacu male, Piractus mesopotamicus (SOUZA, 1998). It has an omnivorous habit and its grayish color characteristics are similar to those of tambaqui. Its commercial creation has been growing due to the meat flavor, the low maintenance cost, and the high adaptability to cultivation in tanks and nurseries. However, in intensive production systems, fish are exposed to stressful conditions, decreasing their growth condition (ALVES et al., 2016), increasing the mortality rate, which can result in economic losses for producers (ADORIAN et al., 2018). This is one of the concerns of this sector, since the most recurrent strategy in aquaculture for prophylactic or therapeutic treatment is the use of antibiotics, but their negative impacts related to human health (bioaccumulation in animal tissues with consequent selection of resistant strains) and with the environment (discharge of untreated effluents and deposition of waste) have aroused great concerns (COSTA and SILVA-JÚNIOR, 2017;BORTOLOTTE et al., 2019).
One of the alternatives to its use is the inoculation of products of vegetal origin inoculated directly in the feed ration, where, despite the controversies, studies have demonstrated its efficiency to increase the mortality rate (SANTOS et al., 2015), in addition to acting as growth promoters, pathogen control (antimicrobial activity), antioxidant activity and stimulation of enzymatic activity (SANTOS, LUDKE and LIMA, 2009).
Among these nutritional prophylactic strategies, we highlight the use of garlic, rosemary and sage as digestive stimulants and antioxidants (RODRIGUES, MEURES and BOSCOLO, 2015;INOUE et al., 2016), in addition to green tea in improving health conditions of fish (CORRÊA et al., 2018).Despite the rubber tree, Hevea brasiliensis Muell. Arg., is included among the species of fruits and seeds consumed by Tambaqui, Colossoma macropomum, in the natural environment (SILVA, PEREIRA-FILHO and OLIVEIRA-PEREIRA, 2003), this is the first work that uses this food source inoculated in commercial ration feed during the cultivation of tambacu. Thus, the objective of this work is to evaluate the introduction of rubber tree seed in commercial ration feed as a growth promoter in tambacu.
Then, a sample was sent for analysis at the Institute of Scientific and Technological Research of Amapá, at the Food Science and Technology Center, to evaluate the chemical characteristics, such as acidity, iodine rate, refraction, saponification and content of lipids according to the techniques described by the Adolfo Lutz Institute (ZENEBON, PASCUET and TIGLEA, 2008).

Chemical analysis
The lipid content was carried out in a Soxhlet extractor. The determination of acidity was performed by titration with a neutral ether-alcohol solution and phenolphthalein indicator and the result was expressed in % oleic acid (m/m).The saponification index was determined by 4% titrating potassium hydroxide and hydrochloric acid. The iodine index was determined using titration with the Wijs solution, in which the amount, in mg, of iodine absorbed per 100g of oil was obtained by the difference between the volumes spent on the titration of the blank and the sample.The peroxide index was determined by the sample's ability to oxidize potassium iodide, and the results were expressed in milliequivalents. The refractive index was performed using an Abbe refractometer.

Ration Feed production with implementation of the rubber tree seed
The seeds were taken to the property to remove the husks, and according to Lee and Wendy (2017) research, they were put to dry at room temperature for two days and then crushed in a forage machine. After the end of this process, they were added manually to the commercial ration feed (Table 1), according to the experimental design. The ration was offered to the fish twice a day, in the morning (7am) and in the afternoon (5:30pm), paying attention to avoid the accumulation of ration in the bottom of the cages, as it is a pelleted diet.
Table1: Characteristics of the commercial ration feed used.

Experimental design
The experiment was conducted in a fish farm, located on a farm in the In the first phase, 30 tambacu fish fry whose size varied between 25 to 30 cm in length were selected to compose random sampling and biometrics were monitored for a period of 60 days, alternating the measurement record every twenty days, this period was standardized in order not to cause stress in animals. The abiotic characteristics of the water were monitored such as dissolved oxygen, nitrite, hardness, toxic ammonia and turbidity of the water weekly and pH and temperature daily according to the kit (Labcon Test).
After acclimatizing for seven days, the fish started to be fed with food supplemented with rubber tree seed, and the experimental design consisted of a group (40% seed + 60% commercial ration feed) with three replicates and the control (Commercial ration feed) according to the studies by Lee and Wendy (2017) for eight weeks

Statistical analysis
The results of the experimental growth study were presented as mean ± standard error through the Statistical Analysis Program (SPSS).

Chemical characterization of the ration feed
The values of the chemical analyzes performed are shown in table 2. In this study, a value of 0.68 mg KHO.g-1 of acidity was found in the rubber tree seed.It is worth mentioning that the grain maturation and the storage time of the raw material are factors directly proportional to the acidity index, being a factor related to the quality of the raw material (BORDIGNON, 2009;SANTOS et al., 2017).The high values found are indicative of high hydrolytic breakdown in the seeds, suggesting that the sample was not well preserved, and the seeds did not preserve their chemical quality (MARTIN et al., 2006;MORAIS et al., 2017). The iodine index found was 18.9 ± 1.2 g 100 g -1 . In a study carried out by Eka, Tajul and Wan (2010), they found values of iodine in the rubber tree seeds of 28.07 g.100 g -1 .The low iodine value of the rubber tree seed oil recorded in our study may be related to exposure to ambient light during storage, causing its oxidation, resulting, therefore, in a decrease in iodine levels.
The refractive index was 1.37 ± 0.1. This fat index rises with the increase in the length of the glyceride chain and also with unsaturation and correlates with the iodine index that allows to know the degree of unsaturation of the molecules (FERRARI et al., 2005). The refractive index is characteristic for each type of oil (BARROSO et al., 2014).
As for the saponification index, the values recorded in this study were lower than those observed by Silva et al. (2018) (182.57 mg KHO g-1) for Jatropha Curcas.
Lower saponification rates indicate the presence of esters of high molecular weight fatty acids in the sample. The higher the saponification rate, the more useful it is for food (SARMENTO et al., 2015).
It is important to highlight that although the seed used in this study is considered a protein source, there is the presence of a toxic factor, the cyanogenetic glycoside. And that can be an impediment to making use of it as a food source. As an alternative to reduce the amount of cyanogenetic glycoside in these seeds, it is necessary to put them to dry at room temperature for a few days (EKA, TAJUL and WAN (2010), as was done in this work.

Evaluation of the inclusion of the seed in the tambacu feed ration
The physical-chemical variables did not change during the experimental time, being adequate to the well-being of the fish (ARIDE, ROUBACH and VAL, 2007).The average water temperature (29.9 ± 0.78 °C), dissolved oxygen (OD) (10.6 ± 0 ppm) and pH (7.4 ± 0.1) were within the range recommended by Faria et al . (2013) for the species, where the ideal temperature for a good growth of tropical fish species is 28 to 32 °C. Therefore, the data demonstrate that this parameter was presented in adequate conditions for the creation of tambacu. and ammonia values were obtained of 1.6 ± 1.1mg L -1 .
After eight weeks of experiment, weight and growth gains corresponded to 0.509 ± 0.44g and 10 ± 6.92 cm, respectively, as shown in Figure 1. It is worth mentioning that the price of the ration feed, represents 60% to 70% of the aquaculture production cost (ROTTA, 2002), and the supply of nutritionally balanced food has a great impact on the fish growth rate (CHAGAS et al., 2005) .Hence the importance of alternative foods to supply the nutritional needs of each species, based on the idea that efficient and low-cost food can become the difference in productive success (RIBEIRO et al., 2016).

CONCLUSION
The results show an influence on the inclusion of 40% of the rubber tree seed in the tambacu diet during rearing. In order to reduce costs resulting from the use of alternative ration feeds, we suggest the use of rubber tree seed in an attempt to minimize barriers due to the high prices of commercial ration feeds within the fish farming production chain. It is worth mentioning that it is necessary to continue the work with the evaluation of food digestibility in fish and attention to the anti-nutritional factors present in vegetables to meet the nutritional requirements of the species.