Experiment On Fishes

Abstract
This study was performed to obtain the scientific evidence for biological effect of the Daemyung Bio-functional water (DB-functional water). Fifty two rockfish, Sebastes schlegeli, which had been proved to have fatty liver, was used in this study. After the exposure to the DB-functional water for one week, liver grossly showed uniformity in surface color, compared to control which was exposed to conventional seawater. Liver also showed significant improvement of severity of fatty change in individuals exposed the DB-functional water. Visceral fat in peritoneum was clearly reduced in this group. From these results it is suggested that DB-functional water is very effective in the relief or improvement of severity of fatty liver and in reducing the visceral fat. The DB-functional water is highly recommendable as drinking water for improving health and prevention of diseases of human.

Introduction
The social and economical development of the world gives rise to the ?ell-being? code, which means more upgraded life. So people prefer foods and drinks that keep healthy rather than simple food simply satisfying hunger and thirst. In addition, the style of enjoying food is going to change with time. Food materials have been regarded as substitute medical supplies rather than simple food source. Mushroom, milk and rice which have pharmacological effect are commercially available.
Recently people have had special interest in drinking water that they are consuming daily because water can keep their health. Various kinds of specified water were commercially available in the market of Korea. However there are no scientific or medical evidence that can verify their beneficial effect of their so-called specified water. So we need to find out real specified water that can be effective in health care.
Recently we became to know specified water produced by a company called Damyung Bio Co., Ltd.. This water is called functional water by producer. It has been proved to be effective in treating the various kinds of so called incurable diseases. The diseases include B and C hepatitis, diabetes mellitus, hypertension etc. which we can exemplify in human cases. In this study we were trying to obtain the effectiveness of the functional water on the liver by rearing a marine fish species suffering from fatty liver in the water artificially made by using the functional water.
These experimental results can be expected partly to support the effectiveness of the functional water in treating and relieving the incurable diseases, especially hepatitis type B and C.

Materials and Methods
Animals
Fifty two individuals of rockfish, Sebastes schlegeli, which had been suffering from fatty change of liver, were used as experimental animals in total three times of experiment (Table 1). They ranged 20-114g in body weight and maintained at a range of water temperature between 18~21 C with sufficient aeration. During experimentation fish were fed by pelleted food (Vision Ureok-Chimgang 7, Cheonhajeil, Co., Ltd.) at a rate of 3% of body weight.

Preparation of rearing water
Functional water was directly obtained from tap water using a functional water purifier made by Daemyung Bio Co., Ltd. Purified tap water was also directly obtained from tap water using a water purifier also made by the same company.
In advance two kinds of artificial seawater for rearing fish were made using the purified tap water, the functional water with the addition of 35% of Red Sea Salt(Red Sea, Israel) as sea salt.

Application of rearing water and histopathological examination of liver
In the 1st and 2nd trials of exposure of rearing water, animals were autopsied and examined liver 7 days after the exposure. But autopsy in the 3rd trial was carried out 2 weeks after the exposure. During the exposure, fish were clinically observed at least 2 times daily.
All fish were anesthetized with MS222 and weighed. After gross examination of external and internal findings, livers of each fish were photographed with a digital camera(COOLPIX 8400, Nikon, Japan) and then carefully removed from the body and weighed. A part of liver tissue was removed from fish body and pre-fixed in Bouin? fixative solution. Following post-fixation the liver tissue blocks were washed with tap water and then dehydrated in graded series of 70~100% alcohol. After cleaning and paraffination, about 4㎛-thickness tissue slices were obtained with use of microtome (Reichert-jung 820, Leica, Germany). All preparation of the slices was stained with Hematoxylin and Eosin (HE) for examining the histologic structure of liver with a light microscope (U-MDOB, Olympus optical Co. Ltd., Japan). As needed, photographs were taken using digital camera (Polaroid DMC-3, Polaroid, USA).

Results
1. Clinical findings
Clinically fish maintained in functional sea water showed uniformity in body color action among each other, compared to individuals of the control which maintained in conventional sea water. In functional sea water fish were more active and stable in behavior, less stressful to such environmental inevitable factors as noise and more rapid in food recognition.

2. Gross findings
There were no statistical difference in hepatosomatic index(HSI) between two experimental groups (Table 2). The color of liver surface was homogeneously light brown, compared to that of the control fish which showed uneven coloration due to mixed distribution of congested and white fatty parts (Fig. 1-1, 2, 3, 4, 2-1)
Elasticity of organs was very excellent, compared to that of the control. The hardness of tissue felt obviously excellent, especially when scissoring the peritoneal wall for autopsy. Fish maintained in functional sea water had little amount of mesenteric fat. But fish maintained in conventional artificial sea water had large amount of mesenteric fat so that differentiation between peritoneal organs was ambiguous.

3. Histopathological findings
The finding of fatty change was prominently relieved in individuals of functional sea water group. Fat globules were distributed in much less number throughout the hepatic parenchyma and their size of the globules was small (Fig. 1-5, 6, 7, 8). Sinusoidal capillary areas were better defined, compared to those of the control (Fig. 1-5, 6). Most of hepatic cells in relieved area had centrally located nuclei which is normal (Fig. 1-6, 8)

Discussion
From this study, livers grossly and microscopically showed obvious changes by supply of the functional water, which meant to have the obvious effectiveness of the water on the fatty liver. Functional water is made by letting conventional tap water pass a series of filters. By passing a series of filters water is purified, energized by upbringing ray(4~14㎛ of wave length), and weakly alkalized. This energized functional water is known to have smaller molecular clusters so that water is well driven into cellular interior with Ca2+ to activate various cellular biochemical events.
The exact mechanism for improving fatty liver of fish can not be explained from this study but this histopathologic data using fish is sufficiently supportive to the clinical results that were proven to be effective in treating hepatic B and C hepatitis. This data is also expected to be fundamentals to obtain the experimental evidences of functional water effectiveness for other various curable and incurable diseases. Furthermore hepatic health is critically important because liver is well known to be responsible for above 90% of total body health.
Accordingly functional water made by the DB-functional water purifier is highly recommendable as daily drinking water for keeping people of worldwide healthy.

Table 1. Experimental groups of rockfish, Sebastes schlegeli

Experiment	Rearing water	Duration of rearing	Numbers of fish used
1st trial	CW	One week	8
	FW	One week	8
2nd trial	CW	One week	10
	FW	One week	10
3rd trial	CW	Two weeks	8
	FW	Two weeks	8

CW: conventional sea water, FW: functional sea water

Table 2. Hepatosomatic index(HIS) of rockfish, Sebastes schlegeli

	Average body weight(g)		Liver weight(g)		HSI(%)
Experiment	CW	FW	CW	FW	CW	FW
1st trial	71.40	86.38	0.67	0.53	0.99	0.69.85
2nd trial	41.65	49.42	0.63	0.67	1.48	1.35
3rd trial	71.63	63.50	1.33	1.98	1.73	1.98

HIS: hepatosomatic index, CW: conventional sea water, FW: functional sea water

Fig. 1. Gross and microscopic findings of rockfish liver one week after rearing in conventional sea water(1, 3, 5, 7) and functional sea water(2, 4, 6, 8)

Fig. 1-1. Gross finding of rockfish liver one week after rearing in conventional sea water in 1st trial. Liver shows severely uneven surface coloration due to congested and fatty parts.

Fig. 1-2. Gross findings of rockfish liver one week after rearing in functional sea water in 1st trial. Liver shows relatively homogeneous coloration.

Fig. 1-3. Gross finding of rockfish liver one week after rearing in conventional sea water in 2nd? trial. Liver shows uneven surface coloration due to severely fatty parts. Milky white area is part of fatty change and lightly brown area normal part.

Fig. 1-4. Gross finding of rockfish liver one week after rearing in functional sea water in 2nd trial. Liver shows nearly homogeneous coloration with minimal white milky area representing area of fatty change.

Fig. 1-5. Microscopic finding of rockfish liver one week after rearing in conventional sea water in 1st trial. Hepatic parenchyma is severely vacuolated with intracellular large fat globules. It is very difficult to differentiate the boundary among hepatic cells. Nuclei of the cells are dislocated eccentrically. H&E, X400.

Fig. 1-6. Microscopic finding of rockfish liver one week after rearing in functional sea water in 1st trial. Fatty state of parenchyma is greatly relieved. Intracellular fat globules are smaller in size and much less in number. Cellular boundary and sinusoidal capillaries are easily recognized. Most of the cells without fat globules have central nuclei which became normal. H&E, X400.

Fig. 1-7. Microscopic finding of rockfish liver one week after rearing in conventional sea water in 2nd trial. Liver shows severe fatty change. Thousands of large fat globules are distributed throughout the parenchyma and nuclei is eccentrically displaced. Sinusoidal capillaries cannot be easily differentiated. H&E, X400.

Fig. 1-8. Microscopic finding of rockfish liver one week after rearing in functional sea water in 2nd trial. The level of fatty change is prominently relieved. Numbers and size of fat globules decreased significantly. Many of cell have normally located nuclei and sinusoidal area is easily differentiated. H&E, X400

Fig. 2. Gross and microscopic findings of liver in rockfish, Sebastes schlegeli, just before the experiment

Fig. 2-1. Gross finding of liver in rockfish, Sebastes schlegeli, just before the experiment
Liver shows homogeneous milky white coloration. Large amount of visceral fat can be seen.

Fig. 2-2. Microscopic findings of liver in rockfish, Sebastes schlegeli, just before the experiment. Hepatic parenchyma is severely vacuolated with large fat globules. Sinusoidal area is hardly defined.