The Effect of Suplementation Chlorella Vulgaris on Feed to Albumin Levels in Serum Nilem Fish (Osteochilus vittatus)

Rekam Jejak Artikel: Diterima : 21/11/2019 Disetujui : 24/10/2020 Abstract Immunostimulant is a substance that is capable of increasing the mechanism of non-specific immune response of fish one of them is by adding Chlorella vulgaris extract to the feed. This research objective is to determine the influence of Chlorella vulgaris supplementation on total blood albumin in nilem fish. This research was conducted by sampling method consisting of 5 treatments with further 4 replications. The treatments were feed supplementation of 0 g.kg, 2 g.kg, 3 g.kg, 4 g.kg, and 6 g.kg. Independent and dependent variables were used in this research; the independent variable was the dose of Chlorella vulgaris in the feed and the dependent variable was blood albumin concentration. The measuring of plasma albumin concentration was done using the Dyasis kit method with a spectrophotometer. The obtained data were then analyzed by ANOVA, followed by the Least Significant Difference (LSD) at the 95% confidence level if there is a real difference then followed by Tukey's test at the same confidence level. The results showed that the average value of serum albumin concentration of nilem fish from all treatments was between 4.2761 – 7.1826 g/dL. The normal albumin concentration was found in the treatment with a dose Chlorella vulgaris supplementation at 4 g.kg-1 of feed which was 4.77 g/dL. Meanwhile, the highest concentration was found from the dose Chlorella vulgaris supplementation at 3 g.kg-1 of feed which was 6.677 g/dL. Thus, it can be concluded that, Chlorella vulgaris supplementation in feed with different doses can increase the serum albumin concentration of nilem fish in normal range.


INTRODUCTION
Nilem is a freshwater fish commodity that has been widely cultivation. It has a very specific and savory taste compared to other freshwater fish because it contains sodium glutamate which may be caused by its eating habits of natural plankton feed, especially algae which grows due to pond fertilization (Cholik, 2005). Quality feed works as a primary energy source and improves the digestive system of fish, so that the growth and health are optimal. Information concerning the health status of cultivation nilem to prevent diseases caused by bacterial infections has been done by giving antibiotics. However, giving antibiotics can lead to problems. Antibiotics can trigger various pathogenic microbes to become resistant if their use is not in accordance with the dose due to lack of knowledge. Therefore, alternative feeds are needed to improve the growth and health of cultivation nilem fish by utilizing immunostimulant ingredients (Pratiwi et al., 2011).
One of the immunostimulants which is capable to increase the mechanism of non-specific immune response of fish is by adding Chlorella vulgaris to feed. The mechanism can improve growth performance and control disease by increasing fish immunity. In addition, it can stimulate the proliferation, differentiation and activation of lymphocytes to increase the ability of fish to fight foreign objects entering its body, so antibodies are produced (Simanjuntak et al., 2006).

Research Pond Preparation
A pond with the size of 1 m x 1 m x 30 cm was cleaned and poured with 30 Liter of water.

Supplementation Feed Preparation
Chlorella vulgaris supplementation to commercial feed was carried out by the following procedures: 500 g.kg -1 commercial feed was placed Then, 75 ml of water was added and dissolved until it became homogeneous. The solution was mixed to commercial feed in evenly spread manner. Then, all supplemented feed was dried in the sun. After the feed dried, it was left at room temperature and put in a jar which was tightly closed.
Supplementation feed was given as much as 3% for 56 days per total body weight and was given 2 times a day and was ready to be tested on nilem fish.

Nilem Fish Preparation
160 nilem fish were placed in 20 ponds with a density of 8 fish per pond. The fish were acclimatized for a week to adapt to the environment before the treatments. During acclimatization, the fish were fed commercially twice a day.

Sampling
Nilem fish samples were taken from rearing tanks using a net. Blood sampling was done once after the nilem has been given treatments. The blood was taken with a 3ml syringe and collected using an eppendorf tube. It was ensured to be ± 30 minutes minutes at a room temperature. The sample was centrifuged at 4000 rpm for 20 minutes. Then, the supernatant in the form of blood serum was taken using a dropper pipette and moved to a new eppendorf tube.

Data Collecting
Data collection on serum albumin was carried out after 56 days treatments of Chlorella vulgaris feed supplementation with different dosages -0 g.kg -1 , 2 g.kg -1 , 3 g.kg -1 , 4 g.kg -1 , 6 g.kg -1 . The measuring was done at the Laboratory of Animal Physiology, Faculty of Biology of Jenderal Soedirman University

Serum Albumin
The blood serum albumin of nilem fish was examined using a photometer with the Diasys Kit method with the following specification:

Serum Preparation
Blood samples were put into a centrifuge tube and then centrifuged for about 20 minutes at 4000 rpm. The blood serum was taken and put in a test tube.

Blank Absorbent Measurement
Prepared equipments and materials were piped 10 μL aquadest, and then put into cuvette, then subsequently added 1000 µL of Albumin reagent. It was then incubated at 25 o C for 10 minutes. The absorbent was then measured on a spectrophotometer with a wavelength of 546 nm.

Standard Absorbent Measurement
Prepared equipments and materials were piped 10 μL aquadest, and then put into cuvette, then subsequently added 1000 µL of Albumin reagent. It was then incubated at 25 o C for 10 minutes. The absorbent was then measured on a spectrophotometer with a wavelength of 546 nm.

Sample Absorbent Measurement
Prepared equipments and materials were piped 10 μL aquadest, and then put into cuvette, then subsequently added 1000 µL of Albumin reagent. It was then incubated at 25 o C for 10 minutes. The absorbent was then measured on a spectrophotometer with a wavelength of 546 nm.
The albumin concentration can be calculated using the following equation: Albumin Concentration (g/dl): Alb. Conc. = Conc. of STD (g/dl)

Data Analysis
The obtained data were then analyzed by ANOVA, followed by the Least Significant Difference (LSD) at the 95% confidence level.  Rehulka et al. (1993) ie the normal range of total protein values is 6-8 g.dLsch-1, albumin values 3.5 -5 g.dL-1, globulin is 1.5 -2.5 g.dL-1 and the ratio the A / G ratio is 0.7 -1.18 g.dL-1. The A / G ratio is best in the range of 0.7-1.18 g.dL-1, so the results of the albumin ratio calculation on day 56 determine the dose of Chlorella vulgaris supplementation 4 g.kg-1 in the feed which reaches 4.77 g. dL -1 is the best albumin ratio because it collects an albumin value of 3.5 -5 g.dL-1 in the normal albumin ratio rating. Increased value and number of blood cells will have an effect on increasing the concentration of total protein, albumin and globulin.

RESULT AND DISCUSSION
The total amounts of protein, albumin, and globulin have an important role in the immune system of animals (Yang et al., 2014). Increased concentrations of protein, albumin and globulin will associate well with the fish's innate immune system (Akrami et al., 2015). Albumin is a protein that can easily be found and can act as a connecting protein. It is a protein that works as a regulator of blood osmotic pressure and serves as a transport medium of metabolic products (fatty acids, hormones, bilirubin). In addition, albumin in fish can play a role in the immune system (Guardiola, et al., 2018, Kumar et al., 2017.
Albumin is a large part of the ionic protein found in vertebrate animals. The amount of albumin includes 52% of the total plasma protein.
Albumin has a role in the transportation of endogenous and xenobiotic ligands. In addition, it has an important role in the osmotic system as it preserves fish health (Silva et al., 2015). Research

Suggestion
The suggestion for further research is that in fish cultivation, it is necessary to use feed supplemented with Chlorella vulgaris in order to increase fish immunity.