Abstract:
It is an object of the invention to provide a method for processing a very large number of fish eggs for gene injection and so on, which employs a simple system with high performance and a reasonable construction expense. It is another object to provide the method having an water tank apparatus capable of producing a large number of fish eggs continuously. 
     A large number of water tank groups having an independent drainage passage each is lighted up in turn with a predetermined interval. An egg-collecting case with a net bottom is set in the drainage passage connecting to the water tank group to which the lighting is started. The water tank group consists of a plurality of tank unit accommodating fishes each. Each book-shaped tank unit has a rectangular-shaped upper portion and a cone-shaped lower portion. Rotating water stream is formed in each tank unit.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a method for processing a large number of fish eggs, in particular to an automated method for processing fertilized fish eggs. The invention can be employed for production method for producing genetically recombinant proteins and so on from fertilized fish eggs by gene injection. Furthermore, the invention can be employed to produce materials by means of using fertilized fish eggs. 
     2. Description of the Related Art 
     A method to produce specific protein is known by injecting gene material to the fertilized egg of the fish. This protein-producing method may have high productivity, because the specific fish such as the zebra fish lay eggs approximately every day. 
     The gene material must be injected into each fish eggs before dividing of the fertilized fish egg is started. However, a very large number of the fish eggs employed for the gene-injection is very small. For example, the eggs of the zebra fish have a diameter of about 1 mm. It is not easy to deal very large number of small fish eggs within a short time. Moreover, the injection apparatus for injecting the gene material into the small fish egg is expensive, because precision control is required for an injection needle to inject the gene material into the very small fish egg. 
     It is known that zebra fish lays eggs by means of lighting. Japan Unexamined Patent Publication No. 2001-120,110 proposes a rectangular-shaped water tank having square-shape in the horizontal direction. Water supplied to an upper portion of the water tank is drained out from a drainage pipe established in the bottom of the water tank. A square-shaped upper half portion of the water tank consists of four pieces of perpendicular wall plates. A reverse-pyramid-shaped lower half portion of the water tank consists of four pieces of slope wall plates. Each slope wall is slanted diagonally. 
     However, it is considered that the fish eggs is caught easily at corners between two slope wall plates of the reverse-pyramid-shaped lower half portion of the water tank. It is difficult to separate the caught fish eggs from the corners, because a fish-separating net is disposed between the upper portion and the lower portion of the water tank. The fish eggs staying for a long time at the corners deteriorate the quality of the water. 
     Japan Patent No. 3,769,680 illustrates a water tank having a cylinder-shaped upper portion and a cone-shaped lower portion. A pair of water-supplying pipes is disposed horizontally at the cylinder-shaped portion. A pair of drainage pipes is disposed at the cylinder-shaped upper portion. These pipes makes rotating water stream in the water tank. The rotating water stream rises upwardly. 
     U.S. Pat. No. 4,798,168 illustrates a water tank having a cylinder-shaped upper portion and a cone-shaped lower portion. A pair of water-supplying pipes is disposed horizontally at the cylinder-shaped upper portion of a water tank. A drainage pipe is connected to a bottom hole disposed at a top of the cone-shaped lower portion of the water tank. These pipes makes rotating water stream in the water tank. The rotating water stream descends downwardly. However, these water tanks described in the patent documents 2-3 needs a large horizontal space. 
     SUMMARY OF THE INVENTION 
     It is an object of the invention to provide a method with high performance, which processes a large number of fish eggs. It is another object of the invention to provide a method for producing genetically recombinant proteins and so on from a very large number of fertilized fish eggs with a simple system having a reasonable construction expense. 
     It is another object to provide the producing method employing a water tank apparatus capable of producing a large number of fish eggs continuously. It is another object to provide the producing method employing a water tank apparatus capable of removing the fish eggs from inner side walls of the tank unit easily. 
     According to an aspect of the present invention, the method comprises an egg-producing step, an egg-collecting step, an egg-arranging step and an injecting step, which are executed in turn. Especially, the drainage including the produced fish eggs is drained out from a water tank apparatus. 
     The fish eggs in the drainage are collected on a porous plate of an egg-collecting case through which the drainage flows. The egg-collecting case is transferred from an egg-collecting apparatus to an egg-arranging apparatus after collecting the fish eggs from the drainage of the water tank apparatus. Accordingly, the water tank apparatus does not need a valve changing a drainage passage from a water-purifying apparatus to an egg-collecting apparatus. As the result, cleaning of the valve is abbreviated. 
     According to a preferred embodiment, each illumination period of water tank groups is started from each starting time points being different to each other. 
     According to a preferred embodiment, one half of the water tank groups are illuminated, and the other half of the water tank groups is not illuminated. Combination of the illuminated water tank groups is changed in turn. 
     Preferably, each illumination period of each water tank group is fourteen hours, and each non-illumination period of each water tank group is ten hours. The egg-collecting case is set in drainage passages of the water tank groups during a predetermined egg-collecting period stated from a time point when the water tank group is lighted up. As the result, the drainages from the other water tank groups except only one water tank group to which the lighting is started can be drained to the water-purifying apparatus. Moreover, a number of the fish eggs produced for a constant period approaches mostly constant. As the result, a gene-injection apparatus can inject gene material into a large number of the fish eggs a day. 
     In a preferable case, the injection must be done within less than twenty minutes, more preferably ten minutes. For example, a new water tank group is lighted up ten minutes later after starting of lighting of the previous water tank group. It means that the water tank apparatus preferably has one hundred and forty-four water tank groups. A construction expense and an operating cost of the expensive injection apparatus can be decreased. 
     According to one preferred embodiment, each of the water tank groups is surrounded by light-shielding members respectively. As the result, the water tank groups can be disposed nearly to each other. 
     According to another preferred embodiment, each drainage of the water tank groups flows out through each egg-collecting case collecting the fish eggs from each of drainages. As the result, the fish eggs can be collected easily. 
     According to another preferred embodiment, the drainage drained out from a bottom hole of each tank unit returns back to the tank units through a water purifying apparatus. In the other words, the tank unit does not have a water valve changing a water flow passage in order to collect the fish eggs. The fish eggs in the drainage are collected by the egg-collecting case disposed in a drainage passage. 
     According to another preferred embodiment, the egg-collecting case is set in a drainage passage of the lighted up water tank group during a predetermined egg-collecting period. The egg-collecting case is separated from the drainage passage after the egg-collecting period. As the result, Feces in the drainage are not collected by the egg-collecting case. 
     According to another preferred embodiment, a predetermined number of the tank units are arranged to one line in a horizontal direction on each shelf of a book-shelf-shaped frame. Each of the tank units has book-shape with a predetermined thickness. As the result, the water tank apparatus can have a high packing density of the tank units. 
     According to another preferred embodiment, the book-shaped tank unit has a rectangular-shaped upper portion and a cone-shaped lower portion. Moreover, the cone-shaped lower portion of the tank unit has a top portion having a bottom hole connected to a drainage conduit for draining the drainage. 
     The fish eggs circulate along the cone-shaped wall of the cone-shaped lower portion of the tank unit, because a specific gravity of the fish egg is larger than the water. As the result, the fish eggs are not caught by an inner surface of the cone-shaped lower portion of the tank unit, because cone-shaped lower portion does not have a corner. Moreover, a rotation angular velocity of the rotating water stream increases near the bottom hole, because a radius of the cone-shaped lower portion decreases near the bottom hole. 
     Furthermore, the tank unit has a large cavity for the fishes, because the upper portion of the tank, unit has rectangular-shape. Moreover, the book-shaped tank units can be arranged with a high value of the packing density. 
     According to another preferred embodiment, wherein the book-shaped tank unit has a water-supplying nozzle forming water stream rotating horizontally in the book-shaped tank unit. The water-supplying nozzle is disposed at a boundary portion between the rectangular-shaped upper portion and the cone-shaped lower portion. The water-supplying nozzle spouts out with water downwardly in order to force the rotating water stream downwardly. Accordingly, the water stream with a high speed rotates along an inner surface of the cone-shaped lower portion. The fish eggs remove the inner surface of the cone-shaped lower portion effectively. 
     According to another preferred embodiment, the water-supplying nozzle is disposed at a corner between adjacent two side walls of the rectangular-shaped upper portion. Preferably, the rectangular-box-shaped upper portion consists of a pair of wide plates and a pair of narrow plates. The water-supplying nozzle is disposed at a corner between one wide plate and one narrow plate being adjacent to each other. Moreover, the water-supplying nozzle spouts out with the water stream toward a center portion of the wide plate in the horizontal cross-section of the rectangular-box-shaped upper portion. As the result, the water stream is rotated effectively. 
     According to another preferred embodiment, the tank unit has a separating net disposed at a boundary portion between the rectangular-shaped upper portion and the cone-shaped lower portion in order to separate the fish eggs from fishes. As the result, the separating net for keeping the fishes in the upper portion of the tank unit is hold easily. 
     According to another aspect of the present invention, the method comprises an egg-producing step, an egg-collecting step, an egg-arranging step and an injecting step, which are executed in turn. Especially, the drainage including the produced fish eggs is drained out from a water tank apparatus. 
     Water in a tank unit of the water tank apparatus is rotated horizontally. The tank unit has a rectangular-box-shaped upper portion and a cone-shaped lower portion. Furthermore, the water in the tank unit is drained from a bottom hole disposed at a top of the cone-shaped lower portion of the tank unit. 
     The inventor found that fish egg in the rotating water stream in the tank unit never adhere on an inner surface of the cone-shaped lower portion of the tank unit. The reason is explained later. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a schematic diagram of a protein-producing system. 
         FIG. 2  is a flow diagram of a protein-producing process. 
         FIG. 3  is a schematic plan view of the protein-producing system shown in  FIG. 1 . 
         FIG. 4  is a front view of one bookshelf-shaped frame. 
         FIG. 5  is a schematic plan view of two water tank groups accommodated in two bookshelf-shaped frames. 
         FIG. 6  shows a schematic front view of a part of one bookshelf-shaped frame. 
         FIG. 7  is a schematic side view of a part of one bookshelf-shaped frame. 
         FIG. 8  is a schematic cross-section showing one egg-collecting portion. 
         FIG. 9  is a schematic front view of the water tank group. 
         FIG. 10  is a schematic plan view of the water tank group. 
         FIG. 11  is a block diagram showing a light control circuit topology. 
         FIG. 12  is a timing chart of the light control circuit shown in  FIG. 11 . 
         FIG. 13  is a schematic perspective view showing an outer surface of tank unit. 
         FIG. 14  is a schematic side view of tank unit. 
         FIG. 15  is a schematic front view of tank unit. 
         FIG. 16  is a perspective view of a designed tank unit. 
         FIG. 17  is a perspective view of a designed tank unit with a separating net. 
         FIG. 18  is a side view of a designed tank unit. 
         FIG. 19  is a plan view of designed tank unit. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     (Explanation of the Production Method) 
     A production method for producing genetically recombinant proteins from fertilized fish eggs is explained referring to  FIGS. 1 and 2 .  FIG. 1  shows a schematic diagram of a protein-producing system.  FIG. 2  shows a flow diagram of a protein-producing process. 
     The protein-producing apparatus has an egg-producing apparatus  1 , an egg-collecting apparatus  2 , an egg-arranging apparatus  3  and an injection apparatus  4 . The egg-producing apparatus  1  executing an egg-producing stage S 100  has a water tank apparatus for breeding a predetermined number of pairs of zebra fishes. 
     The egg-collecting apparatus  2  executing an egg-collecting stage S 102  collects fish eggs from drainage of the water tank apparatus. The drainage returns to the water tank apparatus. The egg-arranging apparatus  3  executing an egg-arranging stage S 104  arranges the collected fish eggs. The injection apparatus  4  executing a gene-injecting stage S 106  injects gene material into the fish eggs each. 
     Water of the water tank apparatus is drained to the egg-collecting apparatus  2 . The fish eggs are collected by an egg-collecting case  5  in the egg-collecting apparatus  2 . The egg-collecting case  5  is transferred from the egg-collecting apparatus  2  to the egg-arranging apparatus  3 . The fish eggs are arranged on an egg-arranging case  6  in the egg-arranging apparatus  3 . The egg-arranging case  6  is transferred from the egg-arranging apparatus  3  to a gene-injecting apparatus  4  after arranging the fish eggs. 
     (Explanation of Egg-Producing Apparatus  1 ) 
     The egg-producing apparatus  1  is explained referring to  FIGS. 3 and 4 .  FIG. 3  is a schematic plan view of the protein-producing system shown in  FIG. 1 . The egg-producing apparatus  1  has a water tank apparatus  1 A, a water-circulating apparatus  1 B and a lighting apparatus  1 C. 
     However, the water-circulating apparatus  1 B and the lighting apparatus  1 C are not illustrated in  FIG. 3 . Water tank apparatus  1 A has seventy-two water tank groups  10  arranged horizontally and vertically. The egg-collecting apparatus  2  collects the fish eggs accommodated in the drainage drained from the water tank apparatus  1 A. 
     The drainage in which the fish eggs are removed is returned to the water tank groups  10  after purifying the drainage. The collected fish eggs in the egg-collecting apparatus  2  are transferred to the egg-arranging apparatus  3 . The arranged fish eggs in the egg-arranging apparatus  3  are transferred to the injection apparatus  4 . 
     Water tank apparatus  1 A has six bookshelf-shaped frames  7 . Each of the bookshelf-shaped frames  7  accommodates twelve water tank groups  10  arranged horizontally and vertically.  FIG. 4  is a front view of one bookshelf-shaped frame  7 . 
     (Explanation of the Water Tank Apparatus  1 A) 
     The water tank apparatus  1 A shown in  FIG. 3  is explained referring to  FIGS. 5 and 6 .  FIG. 5  is a schematic plan view of two water tank groups  10  accommodated in two bookshelf-shaped frames  7 .  FIG. 6  shows a schematic front view of a part of one bookshelf-shaped frame  7 . 
     Seventy two of water tank groups  10  consist of five tank units  100  each. Three water tank groups  10  are arranged horizontally to one line on one shelf board  70  of the frame  7 . Each of the frames  7  has four steps of the shelf boards  70 . Accordingly, each of the frames  7  accommodates twelve water tank groups  10  consisting of five tank units  100  each. As the result, water tank apparatus  1 A has three hundred sixty tank units  100 . Each frame  7  has vertical wall plates  71 - 74  supporting shelf boards  70 . The vertical wall plates  71 - 74  are extending vertically between adjacent two water tank groups  10 . 
     (Explanation of the Water-Circulating Apparatus  1 B) 
     Water-circulating apparatus  1 B is explained referring to  FIGS. 5-7 .  FIG. 7  is a schematic side view of a part of one bookshelf-shaped frame  7 . Water-circulating apparatus  1 B has a drainage passage  81 , a water-purifying apparatus  82 , a water pump  83 , a water-supplying pipe  84  and nozzles  85 . However,  FIG. 5  does not illustrate the nozzles  85  for supplying purified water to each tank unit  100 . 
     Each drainage of tank units  100  flows to the water purifying apparatus  82  via the drainage passage  81 . The water pump  83  sends the purified water from the water purifying apparatus  82  to nozzles  85 . Each tank unit  100  has a pair of nozzles  85 . 
     (Explanation of the Drainage Passage  81 ) 
     Drainage passage  81  has drainage pipes  811 , upper gutters  812 , egg-collecting portions  813  and lower gutters  814 . Each tank unit  100  has a bottom hole  11  connected to each drainage pipe  811  each. An outlet of the drainage pipe  811  reaches an upper position of the upper gutters  812 . An outlet of upper gutters  812  reaches the egg-collecting portion  813 . 
     The lower gutters  84  are extended under the egg-collecting portions  83 . An outlet of the lower gutters  84  reaches at an upper portion of an inlet aperture of the water-purifying apparatus  82 . Three upper gutters  812  are extending to one direction in parallel along each bookshelf-shaped frame  7 . The drainage in five drainage pipes  811  of one water tank group  10  reaches to water-purifying apparatus  82  via one of three upper gutters  812 , one of three egg-collecting portions  813  and one of three lower gutters  814 . Each gutter  812  and  813  are slanted. 
     (Explanation of the Egg-Collecting Portions  813 ) 
       FIG. 8  is a schematic cross-section showing one egg-collecting portion  813 . The egg-collecting portion  813  has a rectangular box  8131  having a shallow bottom and an upper opening. An intermediate plate  8132  extending horizontally is posted in an upper portion of the shallow bottom. The intermediate plate  8132  has holes  8133  and  8134 . An intermediate  8135  extending vertically is posted from the shallow bottom. 
     The intermediate plate  8135  comes into contact with an edge of the intermediate plate  8132 . Three chambers  8136 ,  8137  and  8138  are formed in the rectangular box  8131  by the intermediate plates  8132  and  8135 . The inlet chamber  8136  is formed in the upper space of the intermediate plate  8132 . 
     The middle chamber  8137  is formed in the lower space of the plate  8132 . The intermediate plate  8135  stands between the outlet chamber  8138  and the chambers  8136  and  8137 . The hole  8133  connects chambers  8136  and  8137 . The hole  8134  connects chambers  8137  and  8138 . The drainage in chamber  8138  is drained downwardly through a hole of the bottom plate of rectangular box  8131 . 
     The egg-collecting case  5  having a bottom net  51  and an upper opening are set on the intermediate plate  8132 . Accordingly, the drainage in the chamber  8136  flows down into the chamber  8137 . The drainage flows down from the outlet of the upper gutter  812  into the egg-collecting case  5 . The fish egg  100  in the drainage is collected on the bottom net  51 . The drainage overflows into the chamber  8138  through the chamber  8137 . The drainage is always drained into water-purifying apparatus  82  via the lower gutter  814 . Egg-collecting case  5  is transferred to egg-arranging apparatus  3  manually or automatically. 
     (Explanation of the Lighting Apparatus  1 C) 
     The lighting apparatus  1 C is explained referring to  FIGS. 9-12 .  FIG. 9  is a schematic front view of the water tank group  10 .  FIG. 10  is a schematic plan view of the water tank group  10 .  FIG. 11  is a block diagram showing a light control circuit topology.  FIG. 12  is a timing chart of the light control circuit  2000  shown in  FIG. 11 .  FIG. 12  is a timing chart showing case-setting periods for setting the egg-collecting case in each drainage passage in turn, too. 
     A LED lamp  91  is fixed to a down surface of shelf boards  70 . Water tank group  10  is surrounded with shelf boards  70  and vertical wall plates  72  and  73 . Furthermore, a front surface of the water tank group  10  is shielded with a black curtain  92 . A back surface of the water tank group  10  is shielded with a black plate  93 . 
     The frame  7  is painted with black color paint. As the result, each water tank group  10  is accommodated in each independent dark room by means of closing the curtain  92 . Each water tank group  10  is illuminated by each LED  91 . The lighting apparatus  1 C has LEDs  91 , transistors  95 , a DC power supply  94  and a controller  96 . The DC power source  94  applies a predetermined DC voltage to LEDs  91 . 
     Each LED  91  is connected to each transistor  95  in series. The controller  96  controls the transistors  95 . By controlling the LEDs  91 , each of water tank groups  10  is lighted in turn. The first water tank group  10  is lighted up during fourteen hours from a time point t 1  and in the darkness during ten hours after the lighting. The second water tank group  10  is lighted up during fourteen hours from a time point t 2  and in the darkness during ten hours after the lighting. The third water tank group  10  is lighted up during fourteen hours from a time point t 3  and in the darkness during ten hours after the lighting. Similarly, the 71st water tank group  10  is lighted up during fourteen hours from a time point t 71  and in the darkness during ten hours after the lighting. The 72nd water tank group  10  is lighted up during fourteen hours from a time point t 72  and in the darkness during ten hours after the lighting. 
     A time difference between adjacent two lighting-up timing points is twenty minutes. The egg-collecting case  5  shown in  FIG. 8  is set in a predetermined drainage passage connected each tank unit  100  during twenty minutes from the starting of the lighting-up. Four egg-collecting periods C 1 , C 2 , C 71  and C 72  are shown in  FIG. 12 . As the result, egg-collecting apparatus  2 , egg-arranging apparatus  3  and injection apparatus  4  are always operated. 
     (Explanation of Tank Unit  100 ) 
     The tank unit  100  is explained referring to  FIG. 13-19 .  FIG. 13  is a schematic perspective view showing an outer surface of tank unit  100 .  FIG. 14  is a schematic front view of tank unit  100 .  FIG. 15  is a schematic side view of tank unit  100 . 
     Tank unit  100  has four flat walls  101 - 104  and one cone-shaped bottom plate  105 . The four flat walls  101 - 104  forms a rectangular-shaped upper portion  106 . The cone-shaped bottom plate  105  forms a cone-shaped lower portion  107 . The cone-shaped lower portion  107  has a top portion having a bottom hole  11  connected to the drainage pipe  811 . 
     However, two of flat walls  101  and  102  are wider than two of flat walls  103  and  104 . Accordingly, the cone-shaped lower portion  107  includes a pair of lower portions of two wide flat walls  101  and  102 . In the other words, the cone-shaped lower portion  107  includes two of flat wall portions. 
       FIG. 16  is a perspective view of a designed tank unit  100 .  FIG. 17  is a perspective view of a designed tank unit  100  with a separating net  89 . Fish eggs pass through the separating net  89 , but zebra fishes can not pass through the separating net  89 . 
     Moreover, the wall  104  has an overflow window  108  for over-flowing water. In  FIG. 16 , two nozzles  85  are set at two of a boundary portion between the rectangular-shaped upper portion  106  and the cone-shaped lower portion  107 . Each of two nozzles  85  is set at each of two corners between one of two wide flat walls  101  and  102  and one of two narrow flat walls  103  and  104 . 
       FIG. 18  is a side view of designed tank unit  100 . Two of nozzles  85  spout water stream  87  each. The water stream  87  is mostly flows along an inner surface of the cone-shaped bottom plate  105 . As the result, the fish eggs remove the inner surface of the cone-shaped bottom plate  105 . An angle between the water stream  87  and the inner surface of the cone-shaped bottom plate  105  is in a range of 10-30 degree. 
       FIG. 19  is a plan view of designed tank unit  100 . Two of nozzles  85  spout water stream  87  each. An angle between the water stream  87  and one of flat walls  101 - 102  is in a range of 30-60 degree. As the result, rotating water stream is produced in the cone-shaped lower portion  107 . 
     cone-shaped lower portion In the above embodiment, the injection of gene-material is explained. However, any materials can be injected in fish eggs instead of the gene materials. 
     It is explained one reason that an fish egg in rotating water is not adhere on the inner surface of the tank unit. The fish eggs have a little larger specific gravity than water. Accordingly, the rotating fish eggs moves near an inner surface of the cone-shaped lower portion. However, a rotating speed of a boundary layer of water rotating very near the inner surface of the cone-shaped lower portion has a low speed by friction. Consequently, the fish egg near the inner surface of the cone-shaped lower portion is forced toward the center portion in the radial direction with the Magnus effect. In the other words, a lifting force toward the center portion in the radial direction forces the fish eggs. 
     Another reason is explained. The fish egg has a diameter of about 1 mm. Accordingly, two parts of the rotating water stream coming into contact with two portions of the outer surface of fish egg have different speeds to each other. The one part of the water stream has a lower speed than the other part of the water stream. In the other words, the inner part of the water stream has a lower speed than the outer part of the water stream, when the water stream rotating horizontally. Accordingly, the fish egg rotates strongly in accordance with a difference of the speed difference between the two parts of the water stream, which come into contact with two portions of the fish egg respectively. Consequently, the rotating fish egg rotates along the inner surface of the cone-shaped lower portion can not stick on the inner surface.