KR100604262B1 - Artificial seminal plasma for cold preservation of sperm of the oliver flounder, Paralichthys oliveaceus - Google Patents

Abstract

The present invention relates to an artificial suit for refrigeration preservation of halibut sperm, and more particularly, to provide an artificial suit that can be stored refrigerated sperm of the halibut in vitro, and to preserve the sperm of the flounder for more than three weeks in the artificial suit provided It is possible to breed male and female flounders of excellent thoroughbred, so that not only good breeding can be obtained, but also economical benefit from extracting and transferring only the sperm without carrying the live flounder males in order to obtain high quality sperm. It relates to the artificial preservation of cold storage of halibut sperm to pursue.

The present invention is composed of 8.8 ~ 14.3wt% potassium chloride, 84.1 ~ 60.4wt% sodium chloride, 3.7 ~ 5.5wt% calcium chloride, 1.3 ~ 19.6wt% magnesium chloride, 2.0 ~ 0.2wt% glucose, the hydrogen ion concentration index It is characterized by keeping it at 7.9.

Flounder, suit, sperm, chilled, artificial, in vitro, fertilization

Description

Artificial seminal plasma for cold preservation of sperm of the oliver flounder, Paralichthys oliveaceus

1 is a graph showing the relationship between the storage temperature and the storage period of sperm movement speed of artificial spermatozoa and fresh sperm for storage of flounder sperm according to the present invention.

Figure 2 is a graph showing the motility relationship of sperm between the artificial suit for fresh preservation and fresh coat of the flounder sperm according to the present invention.

The present invention relates to an artificial suit for refrigeration preservation of halibut sperm, and more particularly, to provide an artificial suit that can be stored refrigerated sperm of the halibut in vitro, and to preserve the sperm of the flounder for more than three weeks in the artificial suit provided It is possible to breed male and female flounders of excellent thoroughbred, so that not only good breeding can be obtained, but also economical benefit from extracting and transferring only the sperm without carrying the live flounder males in order to obtain high quality sperm. It relates to the artificial preservation of cold storage of halibut sperm to pursue.

In general, when the sperm of an animal is released into the body, it cannot survive for a certain period of time. Therefore, in order to preserve the sperm of the animal in vitro for a certain period of time, the sperm do not exercise outside of the body to conserve the energy necessary to maintain sperm life. In particular, fish have a lot of problems in the preservation of sperm due to the peculiarity of aquatic environment, and because the species is very diverse, the characteristics of sperm and the preservation method are different. Known.

The necessity of preservation of fish sperm is known for the artificial insemination for the production of new artificial seedlings of fish that are difficult to breed. Especially, even though the maturity of fish is apparent, it is very necessary to develop eggs and sperm. In addition, the method of preserving and using sperm for breeding and hybrid production of the same species of fish is evaluated as economically very useful.

In particular, halibut is a large species that tastes good and has been a pronoun of high quality fish since ancient times. In the mid-1980s, artificial seedling production technology was developed, and it is a mass-produced fishery. It is one of the most competitive fish species in Korea, and its output is continuously increasing to 16,426 tons in 2001, 23,343 tons in 2002 and 34,533 tons in 2003.

However, at present, domestic fish farming in Korea has decreased the productivity of fish per unit area (ha) from 9.3 ton in 1997 to 5.4 ton in 2000 due to unplanned mother group management and deterioration due to inbreeding. to be. Currently, the development level of flounder breeding is at the level of selection breeding that selects and breeds individuals with good growth, and productivity improvement is not as expected.

Since such selective breeding requires a lot of time, fertilized eggs are obtained by breeding by genetically growing genetically improved or disease-resistant individuals. Because the sperm are often not obtained, artificial fertilization is most effective at the time of harvesting of matured eggs while preserving them from the flounder males.

Therefore, there is a need for a simple way of preserving the flounder sperm, generally stored in the refrigerated fish semen sperm can survive about 7 to 10 days, but the spawning rhythm of the fish is wolju (月 週) Because rhythms are common, they must be stored for at least 15 days before fertilization of ovulated eggs.

Currently, the sperm preservation method of fish is mainly focused on cryopreservation which can be preserved for a long time, and some researches on the kinetic environment of sperm have been conducted, but artificial suit development is required for some species for aquaculture industrialization.

According to the present invention, in order to meet the demand as described above, all animals' sperm survive in the testis, but because they die when released into the body, the substance surrounding the sperm in the testis (sperm consists of sperm and suit) Taking advantage of the fact that it is possible to preserve sperm, we have provided an in-process plant that can store the sperm of halibut refrigerated in vitro, and the sperm of the halibut can be preserved for more than 3 weeks in this artificial suit. The male and female flounders of the pedigree can be bred 1: 1 so that not only good breeding can be obtained, but also the economical benefits of extracting and transferring only the sperm without the need to carry the males of the flounder lively to obtain high quality sperm. It is technical task to be able to pursue.

Hereinafter, the artificial suit for refrigeration preservation of halibut sperm according to the present invention will be described in detail by the accompanying drawings.

As a current technique, methods for preserving sperm include cryopreservation and refrigeration preservation. Among them, refrigeration preservation includes a method of preserving semen as it is, and a method of diluting the semen in a preservation liquid.

Generally, the method of preserving semen is used to use it for a short period of time within one week. However, since it is impossible to apply the semen, the preservation liquid was prepared to solve such problems. Preservation was done using Ringer's solution, but no satisfactory results were obtained.

Therefore, in the present invention, after analyzing the semen obtained by pressing the abdomen of the male flounder in breeding, based on this (applied to the molecular structure of the substance), the actual amount of chemicals that can be combined (commercially available chemicals, not sold in ionic state) The sperm preservation solution was prepared (hereinafter referred to as 'in-process plant'), and the pH value was corrected by adding HEPES to create the same environment as the hydrogen ion concentration index (pH) of the flounder suit. According to the preservation of sperm, it was found that the preservation performance of halibut artificial suit was better than that of the semen as it was refrigerated.

The artificial suit for cold preservation of halibut sperm of the present invention is potassium chloride (KCl) 8.8 ~ 14.3wt%, sodium chloride (NaCl) 84.1 ~ 60.4wt%, calcium chloride (CaCl ₂ ) 3.7 ~ 5.5wt%, magnesium chloride (MgCl ₂ ) 1.3 ~ 19.6wt%, glucose (Glucose) 2.0 ~ 0.2wt%, hydrogen ion index (pH) is to maintain 7.9.

Here, potassium (K), calcium (Ca) and magnesium (Mg) ions in potassium chloride, sodium chloride, calcium chloride, magnesium chloride and glucose are known to affect the initiation and inhibition of sperm movement. When less than 3.7 wt% of calcium chloride and 1.3 wt% of magnesium chloride are added, sperm movement is excessively suppressed, and the storage period of sperm is markedly shortened. Also, more than 14.3 wt% of potassium chloride and 5.5 wt% of calcium chloride When 19.6 wt% or more of magnesium chloride was added, sperm motility became excessively active, and the storage period of sperm was also significantly reduced.

Therefore, in order to preserve the spawning flounder semen in various states, it is desirable to formulate within the range of 8.8 to 14.3 wt% of potassium chloride (KCl), 3.7 to 5.5 wt% of calcium chloride (CaCl ₂ ), and magnesium chloride (MgCl ₂ ) of 1.3 to 19.6 wt%. Experimental results showed that.

In addition, the hydrogen ion concentration index (pH) is a very important factor in the conservation of sperm, so even if it is low or high affects the movement of sperm, according to the investigation so far the actual hydrogen ion concentration index (pH) of flounder semen is around 7.9 Since it is confirmed that the retention period of sperm is maintained for the longest, it is preferable to maintain the pH value of 7.9.

In addition, the sodium chloride (NaCl) and glucose (Glucose) was found to be high in the sperm preservation effect of adding in the range of 84.1 ~ 60.4wt%, 2.0 ~ 0.2wt%, respectively.

In particular, in the present invention, which of potassium chloride, sodium chloride, calcium chloride, magnesium chloride, and glucose components constituting the artificial suit prevents sperm conservation, ie, sperm movement (because sperm should not exercise outside the fish if possible for a long time). It is possible to use a number of artificial suits without sodium chloride (NaCl), artificial suits without potassium chloride (KCl) and artificial suits without calcium chloride (CaCl ₂₎ for the purpose of evaluating whether Experimental results showed little conservation effect.

Therefore, it is a task to be investigated more specifically through the study of how each component of the suit affects the preservation of halibut sperm.

Hereinafter, with reference to Figs. 1 and 2 attached to the artificial suit for refrigeration of halibut sperm according to the present invention will be described in detail.

1 is a graph showing the relationship between the storage temperature and the storage speed of the sperm according to the present invention for the storage of artificial flounder sperm according to the present invention, Figure 2 is a fresh preservation of the halibut sperm according to the present invention This graph shows the sperm motility relationship with the suit.

First, the artificial suit developed in the present invention, after calculating the respective composition values based on the results obtained by analyzing the semen of the flounder, prepared artificial suits I, II.

That is, artificial suit I is composed of 1.55 g / L potassium chloride, 6.52 g / L sodium chloride, 0.4 g / L calcium chloride, 2.12 g / L magnesium chloride, and 0.24 g / L glucose, and the hydrogen ion concentration index. (pH) was maintained at 7.9.

Artificial suit II is composed of 0.87 g / L potassium chloride, 8.24 g / L sodium chloride, 0.54 g / L calcium chloride, 0.13 g / L magnesium chloride, and 0.02 g / L glucose. (pH) was maintained at 7.9.

Table 1 below shows the components of artificial suits I and II.

Table 1 Composition of Artificial Suit of Olive Flounder (Unit: g / L)

KCl NaCl CaCl2 MgCl2 Glucose pH Artificial suit Ⅰ 1.55 6.52 0.4 2.12 0.24 7.9 Artificial suit II 0.87 8.24 0.54 0.13 0.02 7.9

After the artificial suits I and II are prepared, they can be stored and used in a refrigerator at 0 ° C. to 4 ° C. The semen collected from the flounder males is diluted in the artificial suits and fertilized with eggs from the females while preserving the refrigerator.

When explaining the embodiment of the present invention by the artificial suits I, II as follows.

(Example 1) Measurement of the ion composition, osmotic pressure, and pH of the flounder suit

In Example 1, in the preparation of the artificial flatfish of the artificial flatfish I and II, the abdominal male abdomen was lightly pressed to collect semen, and the semen was centrifuged at 12,000 rpm for 15 minutes, and then the supernatant (suit) was removed. Osmotic pressure, pH, electrolyte was stored in -80 ℃ ultra-low temperature freezer until analysis, with the stored suit was analyzed the ion composition, osmotic pressure, pH as shown in Table 2 below.

Table 2 Ion Composition, Osmotic Pressure, pH of Artificial Suits I and II

Ion composition Osmotic pressure (mOsm / kg) pH K ⁺ (mM / L) Na ⁺ (mM / L) Cl ^- (mM / L) Ca ²⁺ (mg / 100mL) Mg ²⁺ (mg / 100mL) glucose (mg / 100mL) Suit I 20.80 111.5 118.8 7.20 54.0 23.8 334.3 7.9 Suit II 11.64 141.0 123.0 9.71 3.33 2.38 294.7 7.79

Example 2 Sperm Refrigeration Preservation Test of Artificial Suits I and II

In Example 2, sperm preservation effect experiments were performed for each of the refrigeration temperatures of artificial suits I and II. The sperm cold storage effect experiment was carried out by diluting the collected flounder in fresh state and diluted 10-fold in artificial suits I and II, and then put them in a storage container and stored in an incubator at 0 ° C, 2 ° C and 4 ° C, respectively.

Judgment of the preservation effect was carried out by diluting the sperm in the refrigerated container with artificial seawater and dropping 4 µl onto the observation slide glass, and recording the sperm activity in the microscope under a microscope with a CCTV device. The preservation status of sperm's locomotor activity was determined by showing the sperm's movement speed and the number of sperm's movement.

As a result of refrigeration preservation experiments of artificial suits I and II, the sperm stored in the artificial flatfish as shown in Fig. 1 and 2 were able to be stored for 30 days at storage temperature 0 ℃, 25 days at 2 ℃ and 4 ℃, When the semen was stored as it is, sperm motility was preserved only for 15 days at 0 ° C and for 10 days at 2 ° C and 4 ° C.

Example 3 Fertilization Test of Artificial Suits I and II

The sperm cold storage ability test of artificial suits I and Ⅱ resulted in a very satisfactory result. Therefore, the fertilization test was carried out to correct the actual eggs. The fertilization rate of sperm stored in, II was compared in Table 3 below.

(Table 3) Fertilization rate of sperm stored in semen and artificial suit (mean ± standard error)

% Modification Fresh amount Artificial suit Ⅰ Artificial suit Ⅱ 0 ℃ 1.61 ± 8.39 3.35 ± 4.96 5.08 ± 0.86 2 ℃ 6.01 ± 1.82 44.17 ± 2.76 5.05 ± 4.72 4 ℃ 4.64 ± 3.08 30.12 ± 3.86 38.36 ± 3.44

As a result of the experiment, fertilization ability of sperm preserved by artificial suits I and Ⅱ was similar to fresh sperm, and when preserved at 0 ℃, fertilization rate was slightly higher than that of fresh sperm. It was high in the artificial suit for the preservation of flounder sperm.

As described above, the present invention focuses on the fact that all the sperm of the animal survive in the testis, but because they die when released into the body, the use of a substance called a suit surrounding the sperm in the testis can preserve the sperm. The artificial suit to provide preservation of the flounder sperm outside the body was provided, and the artificial suit can be preserved for more than three weeks in the sperm of the flounder can be mixed 1: 1 male and female flounder of excellent bloodline. Not only can you obtain good breeding, but you do not have to carry halibut males in the live state to obtain high quality sperm.

Claims (1)

8.8 to 14.3 wt% of potassium chloride (KCl), 84.1 to 60.4 wt% of sodium chloride (NaCl), 3.7 to 5.5 wt% of calcium chloride (CaCl ₂ ), 1.3 to 19.6 wt% of magnesium chloride (MgCl ₂ ), and glucose (Glucose) 2.0 ~ 0.2wt%, the artificial suit for cold preservation of flounder sperm, characterized in that to maintain a hydrogen ion concentration index (pH) of 7.9.

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