JP2006230328A - Method for sterilizing rotifera monogenital egg - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for sterilizing rotifera monogenital eggs, which can easily be applied to fish or shellfish seedling production sites needing large amounts of rotiferas in high hatching rates and in high sterilization rates, and by which fish or shellfish bacterial diseases originated from the rotiferas can be reduced. <P>SOLUTION: This method for sterilizing the rotifera monogenital eggs is characterized by immersing the rotifera monogenital eggs in a glutar aldehyde solution to sterilize the rotifera monogenital eggs, preferably immersing the rotifera monogenital eggs in natural seawater or artificial seawater (concentration of about 60%) containing glutar aldehyde in a concentration of 1,000 to 2,000 mg/L. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for disinfecting a rotifer single reproductive egg. Specifically, it relates to a disinfection method for obtaining a high hatching rate and disinfection rate for a single reproductive egg of a rotifer. More specifically, the present invention relates to a method for disinfecting rotifer monozygotic eggs that is effective for preventing bacterial diseases of seafood caused by pathogens derived from rotifers.

  In Japan, 51 species of marine seafood were produced in fiscal 2001, and many species of rotifers such as Brachionus plicatilis (in the present invention these rotifers are referred to as “rotifers”). Feeding) is mandatory. On the other hand, the occurrence of bacterial diseases in which rotifers are suspected of being the source of infection has been reported, and the development of technology for controlling the occurrence of diseases in varieties that are derived from rotifers has become an urgent issue. To that end, it is necessary to reduce the level of contamination by the pathogens of rotifers supplied as feed.

In order to reduce the level of contamination by rotifer pathogens, it is necessary to disinfect the water, aquarium, equipment, food used for rotifer culture, and the original species of rotifer that is inoculated first. Techniques for disinfecting culture environments such as rotifer culture water, aquariums and tools have already been established. However, since feed and original species disinfection methods have not yet been studied, it is necessary to establish these disinfection techniques as soon as possible.

Japanese Patent Application Laid-Open No. 07-79659 JP-A-11-98965 Issued by Fisheries Agency / Japan Cultivation Fisheries Association 8 “Recent findings on culturing rotifers” (Kazuji Hirayama) Issued by the Fisheries Agency / Japan Cultivation Fisheries Association 1 “Mass culture of chlorella and its use in fisheries” (by Yakult Honsha, Tetsuo Fukada) Aquaculture Research (1997) vol.28, 559-565, Balompapueng et al. Aquaculture (1999) vol.176, 195-207, G. Rombaut et al.

  Patent Document 1 discloses a high-density mass culture method for rotifers. Patent Document 2 discloses a method for enhancing nutrition of high-density cultured rotifers. However, neither Patent Document 1 nor Patent Document 2 describes any method for disinfecting rotifer single reproductive eggs.

  In the following non-patent literature, methods for disinfecting rotifer eggs are frequently found. That is, Non-Patent Document 1 repeats several times an operation of collecting and washing the first laying eggs of a rotifer and immersing the solution containing an antibiotic-containing solution in a ratio of 2 to 10 with respect to sterilized seawater for about 3 hours. After that, a method for obtaining a sterile rotifer by further transferring to and leaving in sterile seawater is disclosed. Non-Patent Document 2 discloses that a rotifer single reproductive egg was washed three times with a medium supplemented with 0.1% Tween 80, then spun down and washed at 25 ° C. in a medium containing penicillin 500 μg / mL and streptomycin 625 μg / mL. And a method for obtaining a sterile rotifer by centrifuging for 24 hours and then centrifuging. However, these rotifer egg sterilization methods are not suitable for the purpose of obtaining a large amount of original species because the hatching rate is not clear and, as described above, it is necessary to repeat complicated operations. Moreover, the rotifer obtained by this operation is not necessarily sterilized.

  In addition, about the disinfection method of a rotifer durable egg, it is reported to the nonpatent literature 3 and the nonpatent literature 4. That is, in Non-Patent Document 3, by treating 60 mg of a rotifer durable egg with 1 mg / L (0.0001%) sodium hypochlorite, the hatching rate is improved as compared with the untreated egg. It has been reported that bacteria are no longer observed on the egg surface. However, since the toxicity of sodium hypochlorite to single reproductive eggs has been confirmed, this method is not helpful at all.

In Non-Patent Document 4, there is no difference in hatching rate from non-treated eggs by treating rotifer durable eggs with 0.05 μL / L (0.05 mg / L) glutaraldehyde for 6 hours. It is reported that it is no longer detected. That is, Table 2 of Non-Patent Document 5 (200 pages)
In the case of sterilizing a durable egg of a rotifer using methyl oleate or glutaraldehyde, when the glutaraldehyde solution is immersed in a very thin concentration of 0.05 to 0.25 μL / L and the durable egg is immersed for about 6 hours, 50 to 32 It is disclosed that a hatching rate of about% and bacteria were not detected.

  However, when the method described in Non-Patent Document 4 is applied to a rotifer single reproductive egg, the single reproductive egg is soaked in a disinfectant for 6 hours, so that most eggs will hatch during this time. That is, since it is very difficult to sterilize rotifer single reproductive eggs, there are eggs that hatch during sterilization, and there is a problem with sterilization for a long time. According to the knowledge of the present inventors, it is considered that the drug immersion of a single reproductive egg is limited to about 40 minutes. Moreover, rotifers hatched from single reproductive eggs are extremely vulnerable to chemicals, and this method causes many rotifers to die, resulting in a decrease in hatching rate.

  In other words, rotifer eggs are broadly classified into single reproductive eggs (more precisely, single-phase single reproductive eggs) and durable eggs. In contrast, durable eggs are produced by bisexual reproduction due to the appearance of males, resulting in dormancy. Durable eggs are much more resistant to disinfectants. The likelihood of bisexual reproduction depends on the rotifer strain.

  In this way, rotifers' single reproductive eggs and durable eggs have greatly different susceptibility to the disinfectant, so the durable egg disinfection method should be used as it is to disinfect eggs to obtain the original species of rotifer at the seedling production site. Is very dangerous. In other words, rotifer durable eggs have a thickened secondary egg membrane, so they are extremely resistant to changes in the external environment (so called "durable eggs"), and can be disinfected with, for example, chlorine. On the other hand, it is known that a rotifer single reproductive egg has only a primary egg membrane, so it is vulnerable to changes in the external environment and cannot withstand chlorine disinfection (Fisheries Series 44 “Shiomizutsubo” Rotifer-Production Science and Mass Culture ", page 22). In addition, rotifer durable eggs can be preserved for a long time, whereas rotifer monozygotic eggs can hardly be preserved. For example, durable eggs can be hatched even in dried eggs that have been washed with fresh water and stored for 3 years. In addition, durable eggs can be hatched even when stored for 8 years in dark seawater at 0-5 ° C., but single eggs have a hatching time of 15 ° C. for 1-2 days, and 20 ° C. for 1-1. 5 days, 0.5-1.0 days at 25 ° C., 3-18 hours at 30 ° C., hatched immediately and cannot be stored (Kunihiko Fukusho and Kazuharu Hirayama, “Initial Feed Biology-Shio” See "Vorticella". In this way, even with the same rotifer egg, the ecological difference between the durable egg and the single reproductive egg is extremely large, and the sensitivity to the disinfectant is different. It cannot be applied to single reproductive eggs as it is.

  On the other hand, rotifers currently cultivated in Japan's seafood seedling production organizations are selected based on their proliferative potential, so many institutions use strains that produce single reproductive eggs. It is estimated that there are very few institutions that use stocks that produce

  Therefore, it is necessary to establish a sterilization technique unique to a rotifer single reproductive egg irrespective of the technique for sterilizing a durable egg. In particular, it is necessary to develop a method for disinfecting rotifer eggs that is suitable for production of seafood seedlings that require large amounts of rotifers. However, a method for disinfection or sterilization of rotifer single reproductive eggs has not yet been established. As a result of repeated tests and examinations on the sensitivity and disinfection effect of rotifer single reproductive eggs to various disinfectants, the present inventors finally completed the present invention.

  In view of the above situation, the present invention provides a simple method for disinfecting a single rotifer, which can be easily applied to the field of seafood seedling production that requires a large amount of rotifer. Let it be 1 issue. Moreover, this invention makes it a 2nd subject to provide the disinfection method with a high hatching rate and a high disinfection rate about a rotifer single reproduction egg. Furthermore, the present invention provides a disinfecting method capable of reducing bacterial diseases of seafood derived from rotifers by increasing the number of rotifers hatched from eggs that have been disinfected by the disinfection method as the original species and feeding them as food for larvae This is a third problem.

  Among the present inventions that can solve all of the above problems, the invention described in claims 1 and 2 is characterized in that rotifer unity is characterized by immersing a rotifer's single reproductive egg in a glutaraldehyde solution to disinfect it. This is a method of disinfecting a reproductive egg.

  The invention described in claim 2 is the method of disinfection according to claim 1, wherein a single reproductive egg of a rotifer is immersed in natural seawater or artificial seawater containing 1000 to 2000 mg / L of glutaraldehyde for 20 to 40 minutes. is there.

  The invention described in claim 3 is a method of immersing rotifer simple reproductive eggs in 60% concentration artificial seawater containing 1000 mg / L of glutaraldehyde for 30 to 40 minutes in the disinfection method according to claim 2. .

  The invention described in claim 4 is a method of immersing a rotifer single reproductive egg in 60% artificial seawater containing 1250 mg / L of glutaraldehyde for 20 to 40 minutes in the disinfection method according to claim 2. .

  The invention described in claim 5 is a method of immersing rotifer simple reproductive eggs in 60% concentration artificial seawater containing 1500 to 2000 mg / L of glutaraldehyde in the disinfection method according to claim 2 for 20 to 30 minutes. It is.

  Further, the invention described in claim 6 is a method for disinfecting a rotifer single reproductive egg according to any one of claims 1 to 5 for obtaining a rotifer to be fed to larvae of red sea bream, tiger puffer, flounder, sweetfish and prawn. Is the method.

  In addition, the invention described in claim 7 provides a single species of rotifer according to any one of claims 1 to 5 for obtaining an original species used for mass culture of a rotifer fed to a larva in a field of seafood seedling production. This is a method for disinfecting sexually reproduced eggs.

  As described above, the present invention is a method for disinfecting rotifer single reproductive eggs by a very simple method. Therefore, in the field of seafood seedling production that requires a large amount of rotifers of 1 billion to 10 billion individuals every day. It can be used easily. In addition, a rotifer single reproductive egg disinfected by the method of the present invention has a high hatching rate and a high disinfection rate. Therefore, bacteriologically safe rotifers can be cultured in large quantities using rotifers hatched from rotifer single reproductive eggs that have been disinfected using the method of the present invention as the original species. In addition, to prevent bacterial diseases of seafood by feeding the larvae immediately after carrying out fortification treatment of deficient nutrients after hatching rotifer single reproductive eggs that have been disinfected using the method of the present invention Can do.

  In consideration of the susceptibility of rotifer single reproductive eggs, the present inventors initially selected several drugs that are usually used as disinfectants in fish rearing and the like, and diluted these drugs to concentrations that are normally used. Tests were conducted to observe the state of disinfection and hatching by immersing single reproductive eggs in disinfecting water, but no favorable results could be obtained from any of the drugs. Next, a similar test was conducted using electrolytic seawater and oxidant seawater, which are known to have a disinfecting effect on breeding equipment at the site of seedling production of seafood, but no favorable results could be obtained from either. .

  Therefore, when the disinfectant was switched to glutaraldehyde and tested, almost favorable results were obtained.Furthermore, as a result of repeating the test by changing the concentration and application conditions of glutaraldehyde, the preferable concentration range and conditions were finally obtained. As a result, the present invention has been completed. Glutaraldehyde used in the present invention is an aldehyde-based disinfectant, and is known to have a bactericidal effect on a wide range of microorganisms such as general bacteria, tuberculosis bacteria, and molds, and is difficult to produce resistant bacteria.

  The present invention is characterized by sterilizing a rotifer single genital egg by immersing it in a glutaraldehyde solution. Specifically, in the present invention, it is preferable to adopt a method of disinfecting a rotifer simple reproductive egg by immersing it in natural seawater or artificial seawater containing 1000 to 2000 mg / L of glutaraldehyde for 20 to 40 minutes.

  In the present invention, the water for dissolving glutaraldehyde may be natural seawater or artificial seawater, but among them, artificial seawater having a salt concentration of 50 to 70% is preferably used, and more preferably adjusted to a salt concentration of 60%. The most preferred is the use of 60% Herbst artificial seawater. That is, using 60% Herbst artificial seawater, soak for 30-40 minutes in seawater containing glutaraldehyde 1000 mg / L, or soak for 20-40 minutes in seawater containing 1250 mg / L, or When the method of sterilizing by soaking in seawater containing 1500 to 2000 mg / L for 20 to 30 minutes is used, an optimal disinfection effect can be obtained for rotifer single reproductive eggs.

  In general, rotifers are broadly classified into L-type rotifers (Brachionus plicatilis) and S-type rotifers (Japanese name undecided: Brachionus rotundiformis). Applicable.

  The rotifer hatched from a single reproductive egg sterilized using the method of the present invention can be fed to any seafood. For example, it can be suitably used as a food for larvae such as red sea bream, tiger puffer fish, Japanese flounder, sweetfish, and prawns.

The rotifer hatched from the eggs sterilized by the method of the present invention is fed directly after the undernutrient enrichment treatment as a feed for the larvae of these seafood, or phytoplankton as the original species of rotifer mass culture. It can be fed as a larvae feed after strengthening the deficient nutrients after increasing the number by unisexual reproduction with orchid as a feed.

Hereinafter, the present invention will be described in more detail with test examples. In addition, including the following test examples, the hatching rate and disinfection rate of rotifers shown in the present invention are calculated using the following mathematical formulas. << Calculation formula of hatchability >>
Hatching rate = [(number of surviving individuals) / (number of surviving individuals + number of dead individuals + number of single reproductive eggs)] × 100
<< Calculation formula of disinfection rate >>
Disinfection rate = [1- (number of viable bacteria after immersion / number of viable bacteria before immersion)] × 100

Test example 1

<Disinfection water selection test>
(1) Cultured water and test rotifer In this test example, as the water to dissolve the disinfectant, the same center as the natural seawater pumped from the ground and filtered by sand at the National Fisheries Research Center and Notojima Cultivation Fisheries Center. The rotifer is cultured in “60% seawater” at 20-25 ° C. using culture water (hereinafter referred to as “60% seawater”) diluted to a salinity of about 20 psu using well water taken from the wells of Among them, the Shimizu Rotifer (L-type rotifer) Obama strain was used.
(2) Isolation of single reproductive eggs Stir the rotifer together with “60% seawater” with a home mixer to separate the eggs from the worm body, transfer this “60% seawater” to the measuring cylinder, and the eggs will sink. The supernatant containing the worm body was discarded. “60% seawater” was added to this precipitate, transferred to another graduated cylinder, and after waiting for the egg to sink again, the supernatant was discarded in the same manner. This operation was repeated several times to recover the settled eggs. (3) Disinfecting water As disinfecting water, a) purified water, b) 0.9% NaCl solution adjusted with purified water and sodium chloride, c) 60% Herbst artificial seawater, and d) “60 The following test was conducted using “% seawater”.

(4) Test method The test was performed in a 20 ° C. room using the above-mentioned disinfecting water left in a thermostat at 20 ° C. for one day or longer. That is, 1 L of each disinfecting water was put into a square water tank (210 × 270 × 40 mm), and about 1 g of a single reproductive egg was immersed for 30 minutes. During the immersion, a water tank was set on the stirrer and stirred at 100 rpm. After immersion, the eggs are filtered through a net, and several hundred eggs are put into a 50 mL test tube containing 30 mL of “60% seawater” containing 10 million cells / mL of freshwater chlorella (Chlorella Industries: raw chlorella V12), 100 rpm Then, the mixture was shaken for two days and nights, and under a stereomicroscope, worms with a green stomach in the chlorella were considered as living individuals, and those that did not show as dead individuals were counted.
(5) Test results Table 1 shows the hatching rate when eggs were immersed in each disinfecting water.

(6) Findings From Table 1, the hatching rates when eggs were immersed in purified water and 0.9% NaCl solution for 30 minutes were about 11% and about 66%, respectively. On the other hand, hatching rates when eggs were immersed in 60% Herbst artificial seawater and “60% seawater” were about 97% and about 95%, respectively. Therefore, a significant difference was observed in the hatching rate when these four types of disinfecting water were used (p <0.05). Furthermore, when a multiple comparison test was performed, a significant difference was not observed between the 60% concentration Herbst artificial seawater and “60% seawater”, but a significant difference was recognized between the other disinfecting waters. Therefore, it was considered that purified water and 0.9% NaCl solution are not suitable as water for studying the effect of the disinfectant. On the other hand, natural seawater contains organic matter, which may lead to attenuation of the disinfectant, and it is expected that the components contained will differ depending on the season, which may affect the disinfection effect, As water for studying effective disinfectants, 60% Herbst artificial seawater, that is, “60% artificial seawater” was used.

Test example 2

<Disinfection selection test 1>
(1) Test method Rotifer single reproductive eggs separated and collected by the same method as in Test Example 1 are generally used for the “60% artificial seawater” (60% concentration Herbst artificial seawater) selected in Test Example 1 8 types of disinfectants (ethanol, isopropyl alcohol, n-propyl alcohol, sodium hypochlorite, phenol, cresol, benzalkonium chloride, formalin) are soaked in disinfecting water in their normal concentrations. (Immersion time is 5 to 40 minutes), after immersion, the disinfection rate and hatching rate of each were calculated, and the suitability as a disinfectant was examined. In addition, about sodium hypochlorite and formalin, it tested to the density | concentration in which hatching of a rotifer egg is recognized.
In addition, the hatching rate of the control group (eggs not immersed in the disinfectant) was 1st = 73.6%, 2nd = 57.1%, and 3rd = 62.7%.
(2) Determination method The test results are as shown in Tables 2 to 9.
The hatching rate in each table is ○ for the disinfection condition that gave the hatching result that can be judged to be equivalent to the control group, △ for the hatching rate slightly inferior to the control group, and a markedly bad hatching rate. The indicated condition was judged as x. In addition, the disinfection rate in each table shows a disinfection rate of 99.9% or more, ○ when the sufficient disinfection effect was obtained, Δ when the disinfection effect was slightly recognized, and no disinfection effect were observed. The condition was determined as x. Further, in the comprehensive judgment, a condition where both the hatching rate and the disinfection rate were evaluated as ◯, and a condition evaluated as Δ or x on the other hand was determined as ×. This determination method was also adopted for other test examples of the present invention.

(3) Test results a) For alcohols such as ethanol, isopropyl alcohol, and n-propyl alcohol (Tables 2 to 4), all were hatched by immersion for 5 to 15 minutes at a concentration of 30% to 70%. It had a bad influence on the rate and was rated as x.
B) For sodium hypochlorite (Table 9), △ evaluation was obtained at a concentration of 0.001% for hatching, but the disinfection rate at this concentration was × evaluation. On the other hand, when immersed in 0.1% and 0.2% for 30 minutes and in 0.5% for 15 minutes or more, the disinfection rate was evaluated as ◯, but the hatching rate was evaluated as x. If the concentration is less than 0.001%, there is a possibility that the hatching rate can be evaluated as ◯. However, the disinfection rate at this concentration has already been evaluated as x, and the purpose of “egg disinfection” is more than this. The test at a concentration of was considered meaningless and was therefore discontinued.
C) Immerse it in concentrations of 1%, 3%, 5% phenol, 1%, 2%, 3% cresol and 0.1% benzalkonium chloride for 5, 10, and 15 minutes, respectively. Although it was evaluation, the hatching rate became x evaluation (Table 5-Table 7).
D) For formalin (Table 8), a △ evaluation was obtained for the hatching rate by immersion for 20 minutes or less at a concentration of 0.2% or less, but the disinfection rate was evaluated as x. If the concentration is 0.1% or less, there is a possibility that a hatching rate of ○ evaluation can be obtained, but the disinfection rate at this concentration is already × evaluation, and for the purpose of “egg disinfection”, test at a concentration higher than this Was judged to be meaningless and the test was discontinued. Since formalin is an aqueous solution containing about 35 to 38% formaldehyde, 0.1% formalin corresponds to 350 mg / L formaldehyde. As shown in Test Example 5, glutaraldehyde, which is the same aldehyde, has been confirmed to be safe at a concentration of 2000 mg / L or less when immersed within 20 minutes. The bactericidal action of glutaraldehyde is said to be due to the fact that the aldehyde group in the molecule coagulates the bacterial surface and internal proteins and deactivates the enzyme. The same aldehyde group is considered to have a bactericidal effect in formaldehyde, but it was confirmed that there is a significant difference in safety against rotifer monozygotic eggs between formaldehyde and glutaraldehyde.

(4) Findings Based on the above results, ethanol, isopropyl alcohol, n-propyl alcohol, sodium hypochlorite, phenol, cresol, benzalkonium chloride, and formalin are all unsuitable as disinfectants for rotifer monozygotic eggs. It was confirmed that.

Test example 3

<Disinfection selection test 2>
(1) Test method Rotifer single reproductive eggs separated and collected by the same method as in Test Example 1 are put in a net, and the following two types of 100% seawater (water temperature 10-20 ° C) are made into flowing water at a flow rate of 3 L / min. The sample was immersed in a 25 L container while shaking for 50 to 60 minutes. After the immersion, the eggs were filtered off with a net, and a sufficient amount of sterile 2% peptone water (1.5% NaCl added) was added to stop the action of chlorine and oxidant.
A. Electrolyzed seawater that has been reported to have a disinfection effect on breeding equipment used in seedling production sites (effective chlorine concentration 0.5 to 4.8 mg / L: produced by Towa Denki Seisakusho prototype and seawater electrolyzer)
B. Oxidant seawater (Oxidant concentration 0.5 mg / L: manufactured by Ebara Jitsugyo Co., Ltd., ozone egg cleaning device (seawater ozone treatment device))

  About 0.1 g of eggs were placed in a sterilized plastic bag and weighed, and then 9 times the amount of sterilized artificial sea water of Herbst was added and homogenized. A 10-fold dilution series was prepared, smeared on the surface of a seawater agar plate, cultured aerobically at 20 ° C. for 5 days, and the viable cell count was calculated from the number of appearance colonies. In addition, it measured similarly about the viable count before immersing in the seawater which has bactericidal power. The disinfection rate was calculated by the above formula, and the disinfection effect was evaluated.

(2) Test results Table 10 shows the disinfection rate and hatching rate of rotifer single reproductive eggs immersed in electrolyzed or ozone-treated seawater.

(3) Findings From Table 10, no sterilizing effect was observed in oxidant seawater (ozone-treated seawater) containing 0.5 mg / L of oxidant even when rotifer single reproductive eggs were immersed for 60 minutes. On the other hand, in electrolyzed seawater, a decrease in the number of viable bacteria on the order of 1 order was recognized, but it was not considered that there was a sufficient disinfecting effect. Furthermore, it was found that when the chlorine concentration was about 3 mg / L or more, the hatching rate decreased to less than 10% even after immersion for only 5 minutes. Therefore, the electrolyzed seawater was hardly considered as an effective disinfectant for rotifer single reproductive eggs.

Test example 4

<Disinfectant selection test 3>
(1) Test method Four types of disinfectants (Isodine solution, Triton-X, Hydrogen peroxide, Glutaral for fisheries) were dissolved in “60% artificial seawater” used in Test Example 1, and the same method as Test Example 1 was used. The isolated and collected rotifer single reproductive eggs were immersed and measured, and the disinfection rate and the hatching rate were calculated in the same manner as in Test Example 2, respectively.

(2) Test results Table 11 shows the test results.
From Table 11, when treated with isodine solution at an effective iodine concentration of 50 mg / L for 10 minutes or more, a disinfection rate of 99% or more was obtained, but the hatching rate was about 2% and had an adverse effect on egg hatching. Was confirmed. When the effective iodine concentration was 50 mg / L and the immersion time was 5 minutes or less, conditions for obtaining a disinfection rate of 99% or more were not obtained.
Triton-X did not show a disinfecting effect.
When the pH and hydrogen peroxide were examined, it was confirmed that the number of viable cells decreased by 2 orders at pH 2 and 1 order by 10000 mg / L of hydrogen peroxide, but both hatching rates were as low as 0.5%. At pH 4 and pH 12, the disinfection rates were as low as 81% and 80%, respectively.
(3) Findings From the above results, isodine solution, Triton-X, pH and hydrogen peroxide were not considered to be effective disinfectants under the present test conditions.

  On the other hand, in glutarar for fisheries (glutaraldehyde solution), the disinfection rate when immersed for 15 minutes at a concentration of 1250 mg / L as glutaraldehyde is 99.9% or more, and 99.99% or more when immersed for 30 minutes, A decrease in the number of viable bacteria was observed. The hatching rate was 59 to 74% when immersed for 15 minutes, and 66 to 96% when immersed for 30 minutes. Thus, since glutaraldehyde provides a high disinfection rate and has a high hatching rate, it is recognized that the use of a glutaraldehyde solution is useful for disinfection of rotifer single embryonated eggs.

  In addition, when the rotifer single reproductive egg isolate | separated and extract | collected by the same method as Test Example 1 was used for the hatching test without disinfecting, the hatching rate was 60 to 90%. As a result of statistical tests, no significant difference was found between the hatching rate of eggs not immersed in disinfectant, the hatching rate of eggs immersed in glutaraldehyde solution for 15 minutes, and the hatching rate of eggs immersed in glutaraldehyde solution for 30 minutes. (P <0.05).

Test Example 5

<Confirmation of appropriate concentration range of glutaraldehyde>
Glutaraldehyde is dissolved in various concentrations in the "60% artificial seawater" used in Test Example 1, and the number of viable bacteria is measured by immersing the rotifer single reproductive eggs separated and collected in the same manner as in Test Example 1. The disinfection rate and hatching rate were calculated in the same manner as in Test Example 2, respectively.

(2) Test results Table 12 shows the test results.
From Table 12, the hatching rate was evaluated as ◯ except that it was evaluated as Δ when immersed in glutaraldehyde of 1500 mg / L or more for 40 minutes.
The disinfection rate was evaluated as x for any time when immersed in glutaraldehyde of 500 mg / L or less. (Triangle | delta) evaluation was a case where it was immersed in 1000 mg / L for 20 minutes. In the case of being immersed in 1000 mg / L for 30 minutes or more and in the case of being immersed in 1250 mg / L or more, both were evaluated as good.
(3) Findings Rotifer single reproductive eggs, unlike durable eggs, have a fast hatching time and cannot be stored, so an immersion time of about 40 minutes is considered appropriate.
Table 12 shows that glutaraldehyde solution is immersed in rotifer by immersion for 30-40 minutes at a concentration of 1000 mg / L, 20-40 minutes at a concentration of 1250 mg / L, and 20-30 minutes at a concentration of 1500-2000 mg / L. It has been confirmed that eggs can be suitably sterilized.

To sum up the above tests, in order to obtain bacteriologically safe rotifers at the seafood seed and seedling production site, 60% concentration containing 1000 mg / L of glutaraldehyde was used to disinfect rotifer single reproductive eggs. Soaked in artificial seawater for 30-40 minutes, or soaked in artificial seawater at 60% concentration containing 1250 mg / L of glutaraldehyde, or 60% concentration containing 1500-2000 mg / L of glutaraldehyde It is considered that the method of immersing in the artificial seawater for 20 to 30 minutes is the most effective.

Test Example 6

<Feeding test using flounder>
(1) Outline of Test Method For each flounder larvae in the following two test sections, a breeding test was conducted by feeding rotifers hatched from single reproductive eggs.
Control group = group that feeds rotifers hatched from non-sterilized single reproductive eggs Disinfection group = group that feeds rotifers hatched from single reproductive eggs that have been sterilized During the test period, the temperature of the tank is 16.2 The temperature was 19.8 ° C. and the pH was 7.9 to 8.6. A total of 341.5 to 378.5 mL of chlorella was added to the breeding water, and a total of 168.5 million to 1972 million rotifers were fed.
(2) Donated flounder In this test example, flounder larvae hatched from fertilized eggs handed over from May 25, 2003 from the Himi Cultivation Fisheries Center, Toyama Prefectural Agriculture, Forestry and Fisheries Corporation were used. Hatching began on May 28 and housed 1000 hatched larvae counted by volume method per aquarium on 29th. The test started on May 30 when the larval opening was confirmed.

(3) Method of disinfection of rotifer Egg rotifer single reproductive egg (L-type rotifer Obama strain) separated and collected by the same method as in Test Example 1 is placed in “60% artificial seawater” and milk mixer MX-40 (Hanagi Seisakusho) The egg and insect body were separated by stirring and collected only for use.
This rotifer egg is immersed in Glutaral for fisheries (Mitaka Pharmaceutical Co., Ltd.) diluted 200-fold with “60% artificial seawater”, disinfected by shaking at 100 rpm for 30 minutes using a shaker, and contains 6500 ppm of sodium bisulfite. Neutralized with 60% artificial seawater.
(3) Breeding of flounder The 100 L aquarium containing the flounder managed the water temperature using a 2 kL FRP circular aquarium as a water bath. In order to maintain the environment, filtered seawater was poured into the breeding aquarium so that the water exchange rate was 2 revolutions / day. The water temperature and pH of the breeding water were measured for 14 days, and only the rotifer immediately after hatching was fed for breeding. The survival rate of Japanese flounder was investigated by counting the total number at the end of breeding. Super raw chlorella V12 was added so that the chlorella density of the breeding water was counted twice in the morning and evening and reached 500 to 2 million cells / mL. The density of the rotifer in the breeding water was counted twice in the morning and evening and fed to give 5 to 10 individuals / mL. The total length of the flounder larvae was magnified with a universal projector on the 7th day and the 14th day of the test, and measured using calipers.

(4) Test results The test results are as shown in Table 13 and Table 14.
From Table 14, the survival rate in the disinfecting group on the 14th day of breeding was 68.7-78.9%, which was slightly higher in the control group, but no significant difference was observed.
There was no difference in the growth of larvae between the sanitized area and the control area.
(5) Findings As described above, even when feeding rotifers hatched from sterilized rotifer single reproductive eggs, there is a large difference in growth and survival compared to feeding rotifers hatched from non-sterilized eggs. Was not recognized. Therefore, it was confirmed that rotifers hatched from sterilized rotifer single reproductive eggs were not harmed by sterilization even if they were fed, and could be used without problems as feed for larvae.

Industrial application fields

  As described above in detail, the present invention is a method for disinfecting rotifer monozygous eggs by a very simple method, and is a disinfection method suitable for implementation in the field of seafood seedling production. By increasing the number of rotifers hatched from single reproductive eggs sterilized by the method of the present invention as the original species and strengthening the necessary nutrients, and feeding them to marine seafood larvae, it is possible to appropriately control the bacterial diseases of seafood. Can be prevented. Thus, the present invention is an epoch-making method that can be used in the field of seedling production of marine seafood.

Claims (7)

  A method for disinfecting a rotifer single reproductive egg, which comprises disinfecting a single reproductive egg of a rotifer by dipping in a glutaraldehyde solution.   The disinfection method according to claim 1, wherein a single reproductive egg of a rotifer is immersed in natural seawater or artificial seawater containing 1000 to 2000 mg / L of glutaraldehyde for 20 to 40 minutes.   The disinfection method according to claim 2, wherein a single reproductive egg of a rotifer is immersed for 30 to 40 minutes in 60% artificial seawater containing 1000 mg / L of glutaraldehyde.   The disinfection method according to claim 2, wherein a single reproductive egg of a rotifer is immersed in artificial seawater of 60% concentration containing 1250 mg / L of glutaraldehyde for 20 to 40 minutes.   The disinfection method according to claim 2, wherein a single reproductive egg of a rotifer is immersed in artificial seawater with a concentration of 60% containing glutaraldehyde of 1500 to 2000 mg / L for 20 to 30 minutes.   The method for disinfecting a rotifer single reproductive egg according to any one of claims 1 to 5, wherein a rotifer to be fed to larvae of red sea bream, tiger puffer, flounder, sweetfish and prawn is obtained. The method for disinfecting a rotifer single reproductive egg according to any one of claims 1 to 5, for obtaining an original species to be used for mass culture of a rotifer fed to a larva in a seafood seedling production site.