KR102298287B1 - Effects of Cryopreservation Medium Composition and Treatment Methods on Spotted Halibut, Verasper variegatus - Google Patents

Abstract

The present invention provides a cryopreservation method of Verasper variegatus sperms, comprising the following steps of: collecting the cryopreservation solution of sperm and the semen of Verasper variegatus; and cryopreserving the cryopreservation solution containing the semen of Verasper variegatus obtained by mixing the collected semen of Verasper variegatus with the cryopreservation solution based on a volume ratio of 1 : 1 to 1 : 2 and by performing at a speed of -1℃·min^-1, -5℃·min^-1, thereby enabling the method to be used for artificial insemination in the field of seed production of Verasper variegatus.

Description

Methods for cryopreservation of flounder sperm {Effects of Cryopreservation Medium Composition and Treatment Methods on Spotted Halibut, Verasper variegatus}

According to the present invention, in the cryopreservation of sperm used in the production of flounder seeds, the sperm motility and viability are high during the freezing process of semen and even after freezing, so that successful cryopreservation of sperm is possible. It relates to a method for cryopreserving sperm flounder sperm, including

Seed production technology for marine life has already developed to a commercial level in Korea. One of the potential marine species in Korea and with a high market value, the verasper inhabits the East China Sea, the coasts of Japan and Korea, and lives on muddy-sand bottoms at a depth of 100 m.

It grows up to 60 cm in length and 4.0 kg in weight. According to the overview of the Korean seafood market (2019), flounder is the most popular species among the top 10 imported seafood, and because the aquaculture technology for flounder is developing very rapidly, especially China and Korea, where natural resources are rapidly decreasing, are Efforts are being made to increase aquaculture production.

Stable seed supply is very important for flounder culture, but there are many difficulties because seed production technology has not yet been established. Among them, the limited production of semen from the mature parent during the artificial seed production period is a problem.

Most fish have a distinct spawning season, and the spawning cycle is affected by various environmental factors, especially photoperiod and water temperature. Guided spawning is one of the most common methods for promoting ovulation in fish in hatcheries. However, when rearing in captivity, the maturation period of males and females often does not match, so cryopreservation of sperm can solve this problem.

Sperm cryopreservation is an effective method used in artificial seed production fields because it is useful for parent fish management and artificial insemination. The research history of cryopreservation in fish sperm is very long. Beginning with the study of Blaxter (1953), sperm from more than 200 freshwater and marine fish species were cryopreserved. Advantages of cryopreservation include synchronization in case of inconsistency in spawning time, preservation of genes, simplification of sperm transport, reduction of disease transmission, and reduction of maintenance and management costs of male brood fish.

One of the major factors for sperm cryopreservation is the composition of the cryoprotective agent (CPA) and the cryoprotective solution (diluent). CPA has the function of protecting cells from the harmful effects that occur during freezing and thawing. The diluent has the ability to keep sperm in a non-motile state and to sustain all metabolic activities, so it helps maintain sperm viability and motility after freezing and thawing. It is important to ensure that the diluent matches the intestinal properties of a particular species because fish sperm are highly species specific to the diluent. Therefore, a suitable diluent should be used according to the fish species.

Another essential factor for obtaining high-quality sperm in cryopreservation is the freezing rate that can be controlled by the program freezer, which can significantly affect sperm motility. Equilibrium time, among other factors, is related to the dilution ratio related to physical shock during dilution, cell penetration to equalize intracellular and intracellular concentrations, and, in the case of thawing temperature, a more rapid and It is associated with full recovery. Studies on the cryopreservation of fish sperm so far have been conducted in the direction of finding the optimal cryopreservation method, focusing on the results of the effects of antifreeze agents and diluents.

However, there are few studies on improving sperm cryopreservation technology suitable for flounder by considering the species specificity of various factors affecting cryopreservation. Accordingly, the present invention aims to provide a method capable of optimally cryopreserving sperm by identifying the optimal type and concentration of CPA and a diluent specific to the species of flounder.

Domestic Patent Publication No. 10-2018-0082275 relates to a cryopreservation solution for sardinosa spermatozoa for improving the preservation effect of marijuana sperm, and a method for cryopreserving marsupial spermatozoa using the same. Thus, the cryopreservation solution and the adult fish sperm using the cryopreservation solution can solve the problems of seedling production caused by the lack of spermatozoa, the inconsistent spawning time of the mackerel, or the imbalance in the sex ratio of the adult mackerel, as it has excellent refrigeration preservation ability of the sperm. Disclosed is a cryopreservation method of Domestic Registered Patent No. 10-1847707 describes the steps of stimulating mature male abalone at 25~30℃ for 2 hours, irradiating UV rays at 18~21℃ for 1 hour immediately after stimulation, and UV stimulation is finished. Abalone semen, comprising the steps of collecting sperm by pressing the gonad of male abalone and freezing the collected abalone sperm in a freezing solution containing 2.5 to 15 v/v% of dimethyl sulfoxide (DMSO) at -196 ° C. Cryopreservation method and step of thawing cryopreserved abalone sperm in a breeding tank at 40 ° C. Dilute thawed abalone sperm to 200,000 to 300,000/ml and mix it with 2000/ml abalone eggs at a volume ratio of 1:1 to artificially Disclosed is a method for producing artificial abalone seedlings using cryopreservation of abalone semen, further comprising the step of fertilization. According to the Korean Patent Registration No. 10-1399432, the viability of mini-pig sperm is improved and the acrosomes and genes of the sperm can be effectively protected from low-temperature shock, so the cryopreservation technology of mini-pig sperm is improved and freeze-thawed mini pig sperm. Disclosed is a cryopreservation method for mini-pig sperm that can contribute to the preservation of genetic resources and mass production of mini-pigs by increasing the fertilization rate of the mini-pigs. Korean Patent Registration No. 10-1109410 proposes an anti-freeze agent suitable for cryopreservation of Jabari sperm in order to maximize the effect of cryopreserving sperm, and proposes a species-specific diluent (artificial plant) by identifying the characteristics of Jabari semen. . An artificial suit for cryopreservation of Jabari sperm and an appropriate antifreeze agent for cryopreservation of spermatozoa are disclosed regarding an artificial suit for cryopreservation of Jabari sperm that can preserve sperm for a long time.

The present invention intends to provide a method for cryopreserving Sperm flounder sperm, including an appropriate anti-freeze agent, diluent, dilution ratio and freezing rate, etc.

In order to achieve the above object, the cryopreservation solution of Verasper variegatus sperm according to an embodiment of the present invention may contain 15 parts by volume of dimethyl sulfoxy active material based on 100 parts by volume of 300 mM sucrose.

According to another embodiment of the present invention, there is provided a method for freezing Beomfloat sperm; It may consist of a step of cryopreserving the cryopreservation solution containing the semen obtained by performing a mixing step of 1:1 to 1:2 based on the cryopreservation solution and the volume ratio of the collected flounder semen.

According to another embodiment of the present invention, there is provided a method for freezing Beomfloat sperm; A mixing step of mixing the collected flounder semen with the cryopreservation solution in a volume ratio of 1:1 to 1:2, and freezing rates -1, -5, -10, and -20℃·min ^-1; It may consist of a step of cryopreserving the cryopreservation solution containing the semen of flounder obtained by performing the mixing step.

The present invention is used for artificial insemination at the field of production of flounder seeds by providing a method for cryopreservation of sperm flounder sperm including the appropriate anti-freeze agent, diluent, equilibration time and dilution ratio, etc., which have the greatest influence on the success of cryopreservation of sperm possible effect.

1 shows the results of Movable sperm ratio (MSR) and sperm activity index (SAI) after thawing in Verasper variegatus sperm according to the type of antifreeze agent.
2 shows the results of movable sperm ratio (MSR) and sperm activity index (SAI) after thawing in Verasper variegatus sperm according to the type of diluent.
3 shows the Movable sperm ratio (MSR) and survival rate (%) after thawing in Verasper variegatus sperm according to concentrations of antifreeze agent (DMSO) diluted with 300 mM suucose.
4 shows the ratio of movable sperm ratio (MSR) and survival rate (%) of flounder sperm after freezing and thawing according to the ratio of semen: cryopreservation solution.
5 shows the movable sperm ratio (MSR) and survival rate (%) after freezing and thawing of flounder sperm according to the freezing rate.
6 shows the results of examining the correlation between the motility, viability, and DNA damage of sperm subjected to freezing and thawing.

Hereinafter, the present invention will be described in more detail through specific experimental examples. These examples are only for explaining the present invention in more detail, and it is to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident

Experimental Example 1: Effect of cryopreservation of sperm flounder according to the type of antifreeze agent

Experimental Example 1 according to the present invention was carried out from October 2019 to February 2020. Seven males with an average length of 38.03±0.25 cm and a weight of 372.2±9.4 g were raised through a running tank at a farm in Yeosu, Jeollanam-do (water temperature: 10~13°C, salinity: 31.1±1.2 psu, dissolved oxygen ( DO): 8.1 mg L ^-1 pH: 7.9).

Two days before semen collection, Ovaprime (Syndel, Canada) was injected into male back muscles at a dose of 0.3 mL/kg to induce allocation. To collect semen, all males were anesthetized with 50 ppm 2-phenoxyethanol and collected by abdominal compression.

In Experimental Example 1 of the present invention, dimethyl sulfoxide (DMSO), glycerol, methanol, ethylene glycol, and propylene glycol were used in the first step to determine the effect of various types of antifreeze agents on sperm cryopreservation.

At this time, 300 mM glucose and sucrose were used as diluents, and each of the five anti-freeze agents was adjusted so that the final concentration of the anti-freeze agent was 10% when the semen and the diluent were mixed. Hereinafter, the mixed solution of the antifreeze agent and the diluent will be referred to as a cryopreservation solution in the present invention. The mixed solution of the antifreeze agent and the diluent was placed in a 15 mL tube, and the ratio of semen: cryopreservation solution was set to 1:1.

Equilibration time was not set separately and immediately frozen after sealing in straw. Freezing was performed by first freezing for 2 minutes in liquid nitrogen vapor (-76 ℃) and then immersing in liquid nitrogen (-196 ℃). At this time, the capacity of straw used was 0.5 mL. Three or more straws for all frozen sperm were used in the experiment, and the straws were thawed in distilled water at 10.5℃.

To investigate the sperm motility and viability, the movable sperm ration (MSR) was measured by diluting fresh control sperm or thawed sperm 500-fold in filtered seawater in a ×200-magnification microscope (CH30, Olympus, Japan). It was determined by the proportion of sperm moving in the middle.

The sperm activity index (SAI) was calculated according to the motility characteristics of the sperm, and after determining the proportion of sperm assigned to each score, it was calculated by referring to previous studies. When measuring sperm motility, the temperature of the filtered seawater was adjusted to the same temperature as the room temperature, and MSR and SAI were determined within 30 seconds of dilution in a microscope field.

The viability of sperm was measured using cell counting kit-8 (CCK-8 kit, Bimake, USA), and absorbance (Spectra Max 190, Molecular Devices, USA) was measured at a wavelength of 450 nm. The viability values were expressed as a percentage of fresh sperm viability was set as 100%.

Statistical processing is as follows. Measured values obtained through the experimental example of the present invention were expressed as mean and standard error (mean ±), and the presence or absence of a significant difference was tested by ANOVA and Duncan's multiple range test using SPSS-statistics program (version 23) (p<0.05). ).

1 shows the results of movable sperm ratio (MSR), sperm activity index (SAI), and survival rate (%) after thawing in Kareius bicoloratus sperm according to the type of antifreeze agent.

In FIG. 1, control denotes fresh milk, 10D denotes 10% dimethyl sulfoxide, 10PG denotes 10% propylene glycol, 10EG denotes 10% ethylene glycol, 10Gl denotes 10% glycerol, and 10M denotes 10% methanol.

As a result of the investigation of an antifreeze agent suitable for cryopreservation of flounder sperm, the MSR of 10% DMSO, propylene glycol, glycerol, methanol, and ethylene glycol in 300 mM sucrose was 56.67±1.67%, which was significantly higher than that of other CPAs. was observed (p<0.05), and the SAI was also the highest at 4 for both.

Antifreeze agents that are usually used to minimize physical and chemical damage during freezing and thawing of fish sperm should have a low molecular weight, neutrality, and hydrophilicity. And it should be able to be a solvent for electrolyte or organic acid, and it should have high permeability and low toxicity to cells.

Since the antifreeze agent has a low eutectic point, it rapidly penetrates into the cell and prevents the colloidal state of the protoplasm from changing through inhibition of dehydration and reduction of ice crystals, thereby minimizing damage to sperm after freezing and thawing. As with the diluent, the anti-freeze agent has a strong species specificity, so an anti-freeze agent that can be applied to all fish to be frozen has not yet been developed. Looking at the results according to Experimental Example 1, DMSO showed the most excellent cryopreservation effect for cryopreservation of flounder sperm.

Experimental Example 2: Effect of cryopreservation of sperm flounder according to the type of diluent

Experimental Example 2 of the present invention is to determine the type of appropriate diluent, and the antifreeze agent was fixed with 10% DMSO, and a mixture of 300 mM glucose, 300 mM sucrose, SS (Stain'solution), and RS (Ringer'solution) was prepared. Experimental fish and settling, freezing and thawing processes were carried out in the same manner as in Experimental Example 1.

2 shows the results of movable sperm ratio (MSR), sperm activity index (SAI), and survival rate (%) after thawing in Verasper variegatus sperm according to the concentration of the diluent. Different lowercase letters indicate significance between dilutions of different concentrations. (P<0.05). Control stands for Fresh Milt.

In order to determine the effect of the dilution on the cryopreservation of Sperm flounder, four dilutions of 300 mM glucose, 300 mM sucrose, SS (Stain'solution), and RS (Ringer's solution) were irradiated with 10% DMSO. 10% DMSO+300mM sucrose showed significant differences in MSR (78.67±0.58%) and survival rate (93.97±1.30%) in thawed sperm when compared with other dilutions. SAI values were not significant for all dilutions with a value of 5. As a dilution, RS showed lower values for both MSR and survival, followed by SS. The effect of different concentrations of DMSO was investigated by mixing with 300 mM sucrose as a diluent.

In general, the concentration of the antifreeze agent in cryopreservation of flounder sperm was different depending on the type of diluent. This is thought to be because the effect of the antifreeze agent interacts with the diluent used together. Therefore, in order to determine the most effective antifreeze agent and concentration, the cryopreservation effect must be reviewed together with the diluent, so that the appropriate antifreeze agent and diluent can be selected together. Regardless of the type of the conventional antifreeze agent for flounder, the treatment concentration was between 10 and 15%.

According to Experimental Example 2 of the present invention, the sperm flounder also showed high motility and viability in 15% DMSO when 300 mM sucrose was used as a dilution solution. For cryopreservation of fish sperm, more than 15% of the antifreeze agent has a high osmolality, which can give an osmotic shock to the sperm and has a strong toxicity.

Experimental Example 3: Effect of cryopreservation of sperm flounder according to the concentration of antifreeze agent

In Experimental Example 3 of the present invention, the concentration was set to 5, 10, 15, or 20% in order to determine the treatment concentration suitable for cryopreservation of flounder sperm using DMSO, which is an antifreeze agent, which showed good cryopreservation effect in Experimental Example 1 above. was set to investigate the effect of cryopreservation. At this time, 300 mM sucrose was used as a diluent, and subsequent freezing and thawing processes were performed in the same manner as in Experimental Example 1.

3 shows the Movable sperm ratio (MSR) and the survival rate (%) after thawing in Verasper variegatus sperm according to the concentration of DMSO diluted with 300 mM sucrose.

When various concentrations of DMSO were used as antifreeze agents and 300 mM sucrose was used as a diluent, MSR at 15% DMSO showed significantly higher values than other concentrations (p<0.05). The survival rate of sperm was also high in 15% DMSO, showing no significant difference from the control group (p>0.05).

Experimental Example 4: Confirmation of freezing rate and mixing ratio of antifreeze agent and diluent

In order to determine the effect of the dilution ratio on the cryopreservation of sperm in 15% DMSO and 300 mM sucrose, which showed excellent cryopreservation effect according to Experimental Example 3, the ratio of the mixed solution of semen: cryopreservation solution was calculated based on the volume ratio. After setting the mixing ratio to 1:1, 1:2, 1:10, 1:100, 1:500, it was frozen. The equilibration time was not set separately, and the freezing and thawing methods were carried out in the same manner as in Experimental Example 1.

4 shows sperm activity after freezing and thawing of Sperm flounder sperm according to the ratio of the mixed solution of semen and cryopreservation solution. Each small lowercase letter indicates the significance between each dilution ratio. (p<0.05).

Sperm motility showed no significant difference in the mixed solution of semen: cryopreservation solution in a ratio of 1:1 and 1:2 (p>0.05). However, the MSR decreased significantly from 1:10, and almost no motility was observed at 1:100 and 1:500. The survival rate of sperm after thawing showed the highest value of 80.67 ± 0.67% in the 1:1 experimental group, and decreased with increasing dilution ratio.

Dilution at a high magnification may cause the dilution itself to be a negative factor in the survival of sperm. When the diluent mixed with the antifreeze agent is added more than the intestinal tract, the sperm is suddenly placed in a new environment. In this study, as the dilution ratio increased, the activity of thawed sperm decreased.

Since the survival rate and activity of sperm showed an inverse relationship with the degree of DNA damage according to Experimental Example 5, it is thought that the higher the dilution ratio of the sperm, the lower the MSR and the survival rate are closely related to the degree of DNA damage. The decrease in sperm activity is thought to be due to osmotic shock and the dilution itself being a physical shock to the sperm.

5 is a program freezer using a mixture of semen: cryopreservation solution to determine the effect of the freezing rate on the cryopreservation of sperm in 15% DMSO and 300 mM sucrose, which showed excellent cryopreservation effect according to Experimental Example 3; -1, -5, -10, and -20℃·min ^-1 were set and then frozen. The equilibration time was not set separately, and the freezing and thawing methods were carried out in the same manner as in Experimental Example 1.

The MSR had a high freezing rate of -1℃·min ^-1 (81.67 ±1.67%), and there was no significant difference between the two methods in the ^{freezing rate of -5℃·min -1 (79.83 ±1.04%).} At the same freezing rate, the survival rate of sperm after thawing was increased. SAI values were not significant for all dilutions of value 5.

According to the above results, the freezing rate during cryopreservation of flounder sperm ^{did not affect the motility of the sperm thawed to -5℃·min -1} , but the motility decreased as the freezing time decreased.

Optimal freezing and thawing is highly variable between species, and its efficacy relates to dehydration processes during freezing and prevention of intracellular ice crystal formation and recrystallization during thawing. According to the above results, it can be seen that the cells of the sperm were well protected from the formation of intracellular ice crystals during the cryopreservation process. This is because it provides an opportunity for intracellular dehydration to occur in a balanced manner when crystallization is slow.

Experimental Example 5: Degree of DNA damage in sperm during freezing and thawing

In order to identify genetic defects of cryopreserved sperm and prevent abnormal development of embryos after fertilization, it is important to evaluate DNA damage. Although research results have been reported that the sperm nucleus is a stable component that is not affected by freezing and thawing, cryopreservation of sperm from European bass (Dicentrarchus labrax), rainbow trout (Oncorhynchus mykiss) and gilthead sea bream ( Sparus aurata ) sperm known to cause DNA damage. Although the DNA of fish sperm shows differences due to structural differences in species or chromosomes, and the presence or absence of diluents and antifreeze agents, it is clear that they are damaged during freezing and thawing.

After searching for appropriate antifreeze agents and diluents through the above experimental examples, the degree of DNA damage to sperm during freezing and thawing was investigated using ^{Single Cell Gel Electrophoresis Assay (Comet assay) kit (CometAssay ⓡ} Trevigen ^{ⓡ, USA).}

After electrophoresis, the sperm were stained with SYBR Gold (Dilution x 10,000 DMSO) for 30 minutes and observed with a fluorescence microscope (DM 2500 microscope, Leica, Germany). Head intensity (DNA _h ), tail intensity, and tail migration measurements were obtained using Comet assay IV Lite System (Insterm, USA). % Tail DNA (DNA _t ) was calculated by applying the formula _{100× c} - DNA _h )/DNA _c.

Table 1 below shows the results of examining the degree of DNA damage according to the concentration of DMSO determined as an appropriate anti-freeze agent in the cryopreservation of flounder sperm according to Experimental Example 3. Table 1 shows the results of investigation of DNA damage by concentration of DMSO diluted with 300 mM sucrose.

DNA damage by concentration of DMSO Control DMSO Concentration (%) 5 10 15 20 Head Length 46.09 ±0.69 ^c 40.63 ±1.15 ^e 42.90 ±0.93 ^d 51 ±0.68 ^a 48.34 ±0.84 ^b Tail Length 21.44 ± 0.38 ^d 46.34 ±1.62 ^c 51.99 ±1.81 ^b 29.46 ±0.4 ^b 60.63 ±1.27 ^a Head Intensity 98.08 ±0.27 ^a 77.52 ±1.76 ^d 81.72 ±1.48 ^c 91.23 ±0.67 ^b 74.38 ±0.93 ^e Tail Intensity 1.92 ±0.27 ^e 22.48 ±1.76 ^b 18.27 ±1.48 ^c 8.77 ±0.67 ^d 24.95 ±0.92 ^a % Tail DNA 0.36 ±0.06 ^c 5.65 ±0.48 ^a 4.54 ±0.31 ^a 2.05 ±0.16 ^b 6.22 ±0.21 ^a Tail Migration 0.82 ±0.15 ^e 26.10 ± 1.69 ^c 30.57 ±1.9 ^b 4.63 ±0.39 ^d 36.46 ±1.30 ^a

In Table 1 above, based on the 300 mM sucrose dilution, the DNA damage degree of flounder sperm after freezing and thawing by DMSO concentration was 91.23 ± 0.67 in 15% DMSO, which was lower than that of fresh sperm, but significantly higher than that of other experimental groups. The resulting values are shown (p<0.05). Tail intensity, tail moment and tail migration were also lower in 15% DMSO than fresh sperm, but higher than in other experimental groups (p<0.05).

6 shows the results of examining the correlation between the motility, viability, and DNA damage of sperm subjected to freezing and thawing. The correlation between MSR and survival rate was positive, and as the survival rate of sperm increased, the proportion of moving sperm also tended to increase.

The correlation between MSR and % Tail DNA was negative (y=-7.6283x+94.39, r ² =0.9823), indicating that the higher the DNA damage of sperm, the lower the ratio of moving sperm. seemed Similarly, the correlation between survival rate and % Tail DNA was also negative (y=-6.7196x+106.16, r ² =0.9137), and as the degree of DNA damage increased, the survival rate of sperm decreased.

According to the experimental example of the present invention, the DNA of cryopreserved sperm was damaged compared to fresh sperm, and the degree of DNA damage and sperm motility and survival rate were inversely proportional.

This means that the motility, viability, and DNA damage of sperm that have undergone freezing and thawing are closely related to each other. Also, since comet assay measured values were different depending on the treatment concentration of DMSO used as a cryoprotectant, it is considered that a cryoprotectant such as DMSO has a close relationship with DNA damage of sperm.

The present invention provides a method for cryopreserving sperm with excellent survival rate and sperm motility in the production of artificial seeds of flounder, which is a fish species that has been tried for aquaculture due to high demand in the market, but for which brood management technology for complete aquaculture has not yet been developed. By increasing the possibility of artificial seed production, it helps to increase the profits of related fishermen, and thus has industrial application.

Claims (4)

delete delete A collection step of collecting the semen of the flounder;
A mixing step of mixing the collected flounder semen in a volume ratio of 1:1 to 1:2 with the cryopreservation solution; The cryopreservation solution contains 15 parts by volume of dimethyl sulfoxy active material based on 100 parts by volume of 300 mM sucrose,
Put the cryopreservation solution containing the semen of flounder semen obtained by performing the mixing step into a 0.5ml straw tube and seal,
cryopreservation solution containing flounder semen -1℃ min ^-1, or A step of freezing preservation at a rate of -5 ° C min ^-1 or a two-step freezing method of immersing in liquid nitrogen (-196 ° C) after primary freezing in liquid nitrogen vapor (-76 ° C) for 2 minutes. A method of freezing pan-float sperm, characterized in that it consists of The method according to claim 3, further comprising thawing the frozen straw in distilled water at 10.5°C.

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