KR102653056B1 - Method for encapsulating sperm and cryopreservation of encapsulated sperm - Google Patents

Abstract

The present invention relates to a method for encapsulating sperm and a method for cryopreservation of encapsulated sperm.
In the present invention, we have developed a method for encapsulation of sperm and cryopreservation of encapsulated sperm that can be stored for a long period of time by preventing frost damage during the cryopreservation process while extending the preservation time by storing it in a capsule in a liquid semen state. In addition, the viability of sperm preserved in the capsule, their motility when released from the capsule, and the sperm acrosome integrity are maintained, so the viability and vitality of the encapsulated sperm upon thawing are also excellent.

Description

Method for encapsulating sperm and cryopreservation of encapsulated sperm

The present invention relates to a method for encapsulating sperm and a method for cryopreservation of encapsulated sperm.

The role of artificial insemination in the modern livestock industry is very important in terms of improving the economic genetic ability of livestock by expanding the use of breeders with excellent genetic ability. Artificial insemination is essential in terms of improving livestock and reducing management costs, and it is advisable to actively utilize sperm preservation technology that can be easily used by sheep farmers.

Artificial insemination technology has undoubtedly become a powerful means of genetic improvement in livestock, but it is difficult to predict due to the various ovulation times for each livestock species and is sensitive to storage temperature due to different sperm physiology, so insemination must be performed multiple times per estrus period, so it needs to be resolved. Problems exist.

While it is relatively easy to freeze sperm in cows, dogs, sheep, and deer, in the case of these females, the time from estrus to ovulation is so long and varied that determining the right time for fertilization is the key to determining the conception rate. In addition, pigs are known to be sensitive to temperature due to their physiological characteristics and suffer from cold shock at temperatures below 15°C. As pig sperm is difficult to freeze-preserve, more than 95-97% of the world's artificial insemination is done with liquid semen, and frozen semen is used only for a limited 3-5%, which means the storage period of liquid semen is 3. This is because it only lasts about 5 days.

Meanwhile, the best advantage of sperm encapsulation is that the sperm stored in the capsule are released at various times as the capsule gradually melts (capsule thickness can be adjusted), so fertilization can be done only once regardless of ovulation time, and it can be reproduced using traditional artificial insemination methods. There is no difference in grades. Although various sperm encapsulation methods have been developed so far, conception rate is only guaranteed if the viability of sperm preserved in the capsule, motility when released from the capsule, and sperm acrosome integrity are maintained. The methods and technologies developed so far have been limited to simply trapping sperm in capsules, making it difficult to use them as actual sperm for artificial insemination.

Dilution solutions (diluents) are used to freeze-preserve sperm, and the loss of sperm cell membrane proteins that occurs during the sperm dilution process causes an acrosome reaction, which causes problems during fertilization. In addition, during the process of freezing and thawing sperm, the sperm cell membrane is stripped of the spermatozoa, the reorganization of the lipid component of the cell membrane (with lipids and proteins in the diluted preservative), and the imbalance of ion and water exchange, making it sensitive to stress. Sperm are easily killed by the stress of external environmental changes.

In order to overcome the shortcomings arising from these dilution and freezing processes, the regular release of sperm is controlled within the reproductive pathway of the female to preserve the external physiological extracellular environment, and sperm are released using polymers. It encapsulates or wraps sperm, where the polymer acts as a shield from various harmful environments outside the sperm or as an emitter at the fertilization site.

The main advantages of sperm release control are 1) sperm cells can reach the fertilization site while being protected from various harmful external environments, and 2) sperm cells of good vitality are continuously and slowly released by controlling the polymer membrane. Selection of an appropriate polymer is an essential step for a high fertilization rate after encapsulation. The conditions that the polymer must have are: 1) biological degradability, 2) adhesion to the uterine mucosa or other capsules to prevent semen reflux, and 3) It can be said to be non-toxic to sperm, 4) minimal environmental change to sperm, 5) minimal interaction with immune substances, and 6) reasonable price.

Polymers commonly used to encapsulate sperm include sodium alginate, barium chloride, poly vinyl alcohol (PVA), chitosan, and polyurethane. While sperm are preserved within the polymer, the selective permeation of necessary nutrients, immune substances, etc. must be possible, and metabolites must be selectively exported. However, since these polymers themselves are not permeable to substances, these polymers cannot simply The original purpose of sperm cannot be achieved by encapsulating and preserving it.

Therefore, the present invention sought to develop a method for encapsulating sperm and cryopreservation of encapsulated sperm that can be stored for a long time by preventing frost damage during the cryopreservation process while extending the preservation time by storing it in a capsule in a liquid semen state.

In addition, we aim to develop an encapsulation method and a cryopreservation method for encapsulated sperm that can maintain the viability, motility, and acrosome integrity of sperm preserved in the capsule. did.

The object of the present invention is to provide a method for encapsulating sperm.

Another object of the present invention is to provide a method for cryopreservation of encapsulated sperm, comprising the step of freezing sperm encapsulated by the above encapsulation method.

Another object of the present invention is to provide sperm encapsulated by the above encapsulation method.

The present invention relates to a method for encapsulating sperm and a method for cryopreservation of encapsulated sperm.

In the present invention, we have developed a method for encapsulation of sperm and cryopreservation of encapsulated sperm that can be stored for a long time by preventing freeze damage during the cryopreservation process while extending the preservation time by storing it in a capsule in a liquid semen state. In addition, the viability of sperm preserved in the capsule, motility when released from the capsule, and acrosome integrity are maintained, so the viability and vitality of the encapsulated sperm upon thawing are also excellent.

Figure 1 shows a flowchart of the sperm encapsulation method of the present invention.
Figure 2 shows sperm stored in a bead-shaped capsule.
Figure 3 shows securing an air layer in a semen sample for freezing to prevent straw rupture due to volume expansion during the freezing process.
Figure 4 shows the results of observing pig sperm inside the encapsulated polymer.
Figure 5 shows the sperm survival rate and vitality after thawing of cryopreserved pig sperm.
Figure 6 shows the results of observing bovine sperm inside the encapsulated polymer.

Best mode for carrying out the invention

As an aspect for achieving the above object, the present invention includes the first step of preparing a semen suspension by mixing a diluent or gelling solution with livestock semen; Step 2, preparing a saline solution containing bentonite and alginate; Step 3 of mixing the semen suspension from step 1 with the physiological saline solution from step 2; and a fourth step of producing encapsulated semen by rotating the microgel formed in step four on a magnetic stirrer.

The sperm encapsulation method of the present invention includes the first step of mixing livestock semen with a diluent or gelling solution to prepare a semen suspension.

The first step is to prepare a semen suspension by mixing livestock semen or sperm contained in livestock semen with a diluent or gelling solution.

Semen suspension can be prepared by mixing the collected semen or sperm of livestock with a diluent at a ratio (w/w) of 1:1 and mixing the gelling solution.

The diluent is used as a liquid semen preservation solution or as a diluent for cryopreservation as a diluent for semen preservation. Additionally, diluents suitable for semen preservation are different depending on the type of livestock.

The diluent used to preserve pig semen is prepared by mixing glucose, sodium citrate hydrate, EDTA, sodium bicarbonate, potassium chloride, and gentamicin sulfate with purified water.

The diluent used to preserve semen from cattle or deer is prepared by mixing Lactose (80%) (w/v) with 20% (w/v) egg yolk, and then added to the prepared primary diluent. Mix 14% (w/v) of glycerol and prepare a secondary diluent. The primary diluent and the secondary diluent are mixed at a volume ratio (v/v) of 1:1, the osmotic pressure is adjusted to 290-310 mosm and the pH is 6.8-7.0 to prepare a final diluent for preservation, and stored at 4°C.

Additionally, the diluent for dog semen preservation is prepared by mixing Tris-fructose-citric acid (3.187 g tris; 1.781 g citric acid; 1.136 g fructose) and distilled water (80 ml).

The livestock semen may be one or more selected from the group consisting of pigs, cows, deer, dogs, sheep, horses, and goats.

The gelation solution contains one or more ions selected from the group consisting of calcium, sodium, barium, or magnesium. Specifically, the gelation solution may contain barium ions.

Additionally, the gelling solution may use barium salt.

Additionally, when a semen suspension is prepared by mixing a diluent or barium salt with the livestock semen, the final concentration of the barium salt in the semen suspension may be 40 to 60 mM, specifically 50mM.

The gelling solution can be used to form particles of alginate, for example to achieve gelation of alginate. Alginate forms a gel in the presence of divalent and multivalent ions. Gelation of alginate is induced by the use of barium ions, specifically barium chloride.

In the present invention, the term “suspension” means that the suspended substance is dispersed in a liquid.

The sperm encapsulation method of the present invention includes two steps: preparing a physiological saline solution containing bentonite and alginate.

The alginate used in the present invention is a polymer with medium viscosity, a polysaccharide extracted from brown seaweed, and has properties such as biodegradability, non-toxicity and biocompatibility, so it can be used in medicines and cosmetics such as wound dressings and drug-releasing preparations. , It is widely used as an additive in food. Alginate is composed of guluronic acid (G) and mannuronic acid (M), and their basic structures are made of homopolymers of -GGGG- or -MMMM-, respectively, G-blocks or They are called M-blocks. Also, when -GMGMGMGM- is formed with a heteropolymer, it is called GM-blocks. G- block acts as a molecular pocket and can hold moisture by combining with divalent and trivalent cations (e.g. Ca ²⁺ , Sr ²⁺ , Ba ²⁺ , Al ³⁺ ) to form a three-dimensional network. there is. The hardening and softening of capsule gel can be controlled by the ratio of G/M. If the G block is high, it becomes hard, and if the M block is high, it becomes soft.

However, when forming a capsule with alginate alone, there is a disadvantage in that the capsule quickly collapses during the release of sperm within the capsule, causing the sperm to be released rapidly, making it difficult to continuously and gradually release the sperm. Accordingly, when montmorillonite, a mineral of the bentonite series, is added to the alginate solution together with sperm and then dispersed with a magnetic stirrer, the montmorillonite structure has cations adsorbed on the surface of the layer. The thin plate-shaped Si-Al-Si structural units overlap in a honeycomb shape, resulting in excellent ion exchange ability, adsorption, swelling, and plasticity.

The bentonite may be montmorillonite, beidellite, or nontronite.

The montmorillonite is composed of a silicate layer and an aluminum layer in a ratio of 2:1, so water can freely flow between crystal units, so expansion and contraction can occur depending on the water content, and it is a mineral with a large surface area and high cation substitution ability. Accordingly, the montmorillonite can be used as a polymer-inorganic mixed bead that can improve the problems of alginate-only capsules.

The physiological saline solution contains 1 to 3 wt% (w/v) of alginate and 2 to 6 wt% (w/v) of bentonite, specifically 1.5 wt% (w/v) of alginate and 4 wt% of bentonite. Contains %(w/v).

The sperm encapsulation method of the present invention includes three steps of mixing the semen suspension of step 1 with the physiological saline solution of step 2.

As the semen suspension of step 1 is mixed with the physiological saline solution of step 2, the concentration of alginate is diluted so that the mixture of step 3 contains 0.5 to 1.5% by weight (w/v) of alginate, specifically 1.0. It may be included in weight % (w/v).

More specifically, the semen suspension was mixed in a physiological saline solution containing 1.5% (w/v) sodium alginate and 4% (w/v) bentonite at a ratio (w/v) of 2:1 to obtain the final solution. Adjust to a concentration of 1% by weight (w/v) sodium alginate.

The semen suspension from step 1 is slowly added to the physiological saline solution containing sodium alginate and bentonite from step 2 using a syringe.

When the semen suspension from step 1 is slowly added to the physiological saline solution containing sodium alginate and bentonite from step 2, barium salt beads are immediately formed, and when the solution is continuously stirred, monoginate and the barium salt inside react. While doing this, the mixture of alginate and bentonite forms a microgel on the outer membrane of the barium salt bead. The thickness of the gel wall can be adjusted by varying the reaction time of the two solutions, a saline solution containing sodium alginate and bentonite, and a semen suspension.

The sperm encapsulation method of the present invention includes the fourth step of producing encapsulated semen by rotating the microgel formed in step 3 above on a magnetic stirrer.

The microgel formed as described above is rotated at 150 rpm on a magnetic stirrer for 120 seconds to achieve ion exchange between barium and alginate, thereby completing the encapsulation of the sperm in the form of beads.

Sperm encapsulated in the form of beads can be cryopreserved by washing them three times in physiological saline and then mixing them with a semen preservation diluent suitable for each livestock species.

The composition of the pig semen preservation solution (diluent), cow or deer semen preservation solution (diluent), and dog semen preservation solution (diluent) are as described above.

Figure 2 shows sperm preserved in a bead-shaped capsule through the above process.

In general, artificial insemination is used as an important means for the propagation of various individuals such as cows, pigs, sheep, etc. with excellent genetic characteristics. Collected semen is mainly cryopreserved by immersing it in liquid nitrogen at -196℃. This method can be stored semi-permanently, blocks the introduction of diseases, allows artificial insemination in emergency situations (foot-and-mouth disease, African swine fever, avian influenza, etc.), and can be actively used for practical use for livestock improvement and preservation of genetic resources. When semen is used for artificial insemination in liquid form, the preservation period is only about 3 to 5 days, so the utilization of excellent genetic resources is limited. Therefore, sperm cryopreservation is essential for breed improvement and selection using the genes of excellent breeders. .

According to the sperm encapsulation method of the present invention, the sperm is stored in a capsule in a liquid semen state, has a high survival rate upon fusion, has a low acrosome damage rate, and has excellent vitality, so it can be said to be a technology that can maximize the utilization efficiency of these genetic resources. .

In the encapsulated sperm of the present invention, in the female's birth canal, the barium-alginate polymer wall swells with moisture and divalent ions are naturally replaced with sodium. This process allows sperm to be released, and the release time varies depending on the type and concentration of ions.

In another aspect, the present invention provides a method for cryopreservation of encapsulated sperm, comprising the step of freezing sperm encapsulated by the sperm encapsulation method.

In the present invention, the term “sperm encapsulation method” is as described above.

The present invention freezes sperm encapsulated by the above method, enabling cryopreservation of the encapsulated sperm.

In addition, a step of cooling the encapsulated semen may be further included before the freezing step, and the encapsulated semen may be precooled at 4 to 5° C. for 50 minutes to 5 hours.

In addition, the sperm encapsulated in the form of beads can be cryopreserved by washing them three times in physiological saline and then mixing them with a semen diluent suitable for the semen species (semen according to the type of livestock).

When cryopreserving, semen can be frozen and preserved in liquid nitrogen using an automatic semen freezing device (computer controlled rate freezer) or using a Styrofoam box. Additionally, cryopreserved encapsulated sperm can be thawed using a constant temperature water bath and used for fertilization. Figure 5 shows the properties of encapsulated pig sperm after cryopreservation and thawing. As a result, it can be seen that the survival rate was high and vitality was excellent.

In another aspect, the present invention provides sperm encapsulated by the sperm encapsulation method.

In the present invention, the term “sperm encapsulation method” is as described above, and sperm encapsulated by the above encapsulation method can be provided.

Forms for practicing the invention

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

Experimental Example 1. Cow/deer sperm encapsulation and cryopreservation of encapsulated sperm.

1-1. Manufacture of thinner for preservation (for cattle and deer)

The first diluent was prepared by mixing 20% (w/v) of egg yolk with lactose (80%) (w/v). 14% (w/v) of glycerol was mixed with the first diluent to prepare a second diluent. The first and second diluents were mixed at a volume ratio (v/v) of 1:1, the osmotic pressure was adjusted to 290-310 mosm and the pH was 6.8-7.0 to prepare a diluent for sperm preservation, and stored at 4°C.

1-2. Encapsulation and cryopreservation of cow/deer semen

Encapsulation and cryopreservation of bovine/deer semen was performed according to the following procedures.

① Semen from cows/deer was collected hygienically. The average sperm count per collection was 6 to 1.5 billion/ml, and the semen volume was 5 cc.

The number of sperm was calculated through a semen test, sperm vitality and survival rate were tested, and the semen and the preservation diluent prepared in Experimental Example 1-1 were mixed in a ratio of 1:1 (v/v), and the final concentration was A semen suspension was prepared by mixing barium chloride to a concentration of 50mM.

② The semen suspension in ① was slowly dropped into a physiological saline solution containing 1.5% (w/v) sodium alginate and 4% (w/v) bentonite solution through a 24-gauge needle attached to a 2mL syringe. At this time, the physiological saline solution and the semen suspension of ① were mixed at a ratio (w/v) of 2:1 and adjusted to obtain a final concentration of sodium alginate of 1% (w/v).

③ When the semen suspension in ① is slowly dropped into a physiological saline solution containing sodium alginate and bentonite, it comes into contact with the barium chloride solution and solidifies to form a microgel.

The microgel was rotated at 150 rpm on a magnetic stirrer for 120 seconds to achieve ion exchange between barium and alginate, completing bead-shaped capsule coating. Figure 6 shows the results of observing bovine sperm inside the encapsulated polymer. Encapsulation did not reduce sperm viability and vitality.

④ The encapsulated semen was pre-cooled in a refrigerator at 4~5℃ for 4 hours.

⑤ The encapsulated semen was filled into a 0.5ml straw (German Mini Straw) using a syringe, and dispensed into a 0.5ml straw so that the number of sperm per straw was 25 to 30 million.

⑥ Sperm cryopreservation (styrofoam box freezing)

Freezing was performed using an automatic semen freezing device (computer controlled rate freezer) or a Styrofoam Box freezing system.

* Automatic semen freezing device: Freeze according to the freezing program below

5℃ → -5℃ (cooling rate 6℃/min) → -80℃ (40℃/min) → -150℃ (60℃/min)

* Freeze Styrofoam Box

A steel test tube rack was placed in a Styrofoam box, filled with an appropriate amount of liquid nitrogen, a straw was placed 5 cm above the surface of the liquid nitrogen, and the tube was left in the nitrogen gas for about 10 minutes to be frozen. Afterwards, the straw was immersed in liquid nitrogen at -196°C and completely frozen.

⑦ Melting of semen (using constant temperature water bath)

A 0.5 ml freeze-dried straw was thawed at 37°C for 30 seconds and tested for acrosome damage, sperm survival and vitality, and malformation rate. As a result, better results were obtained in terms of acrosome damage rate, sperm survival rate, vitality, and malformation rate compared to the case where non-capsulated sperm were cryopreserved and thawed.

Experimental Example 2. Dog sperm encapsulation and cryopreservation of encapsulated sperm.

2-1. Preservation diluent manufacturing (for dogs)

In the case of dogs, a diluent for cryopreservation was prepared by mixing Tris-fructose-citric acid (3.187 g tris; 1.781 g citric acid; 1.136 g fructose) and distilled water (80 ml).

2-2. Encapsulation and cryopreservation of dog semen

Encapsulation and cryopreservation of dog semen were performed according to the following procedures.

① Semen was collected from a mature beagle. Only the fraction with high sperm concentration (sperm rich fraction) was collected using a cup covered with sterile gauze. A semen suspension was prepared by mixing dog semen with the preservation diluent prepared in Experimental Example 2-1 at a ratio (v/v) of 1:1 and then mixing barium chloride to a final concentration of 50mM.

In the same way as the cow/deer semen encapsulation process ② and ③, the dog semen suspension ① was dropped into a physiological saline solution containing alginate and bentonite with a syringe, and sperm were encapsulated as beads were formed. Encapsulation did not reduce sperm viability and vitality.

④ The encapsulated sperm were pre-cooled in a refrigerator at 4~5℃ for 60 minutes.

⑤ The encapsulated micro gel had a diameter of 0.1~0.2mm, was washed three times with physiological saline, and then filled into a 0.25ml straw (German Mini Straw) using a syringe.

⑥ Sperm cryopreservation (styrofoam box freezing)

A steel test tube rack was placed in a Styrofoam box, filled with liquid nitrogen, and a straw was placed 5 cm above the surface of the liquid nitrogen. Freezing was performed by leaving it in nitrogen gas for about 10 minutes. Afterwards, the frozen straw was immersed in liquid nitrogen at -196°C and completely frozen.

⑦ Melting of semen (using constant temperature water bath)

A 0.25 ml freeze-dried straw was thawed at 37°C for 10 seconds and tested for acrosome damage, sperm survival and vitality, and deformation rate. As a result, better results were obtained in terms of acrosome damage rate, sperm survival rate, vitality, and malformation rate compared to the case where non-capsulated sperm were cryopreserved and thawed.

Experimental Example 3. Encapsulation, cryopreservation and thawing process of pig sperm

3-1. Manufacture of thinner for preservation (for pigs)

Glucose (440g), Sodium Citrate Hydrate (71.5g), EDTA (14.5g), Sodium Bicarbonate (13.75g), Potassium Chloride (8.8g), Gentamicin Gentamicin Sulfate (22g) was mixed with purified water to make 1 liter.

3-2. Encapsulation of pig sperm

① Semen collection and encapsulation of pig semen

Semen collected from a boar was diluted 1:1 using the pig preservation diluent (liquid semen preservation solution) prepared in Experimental Example 3-1.

Afterwards, a semen suspension was prepared by mixing it with barium salt, and then dropped into a physiological saline solution containing alginate and bentonite with a syringe as in Experimental Example 1-2, and beads were immediately formed to encapsulate the sperm. Figure 4 shows the results of observing pig sperm inside the encapsulated polymer. Encapsulation did not reduce sperm viability and vitality. Unlike other animals, pig sperm is very sensitive to temperature and difficult to freeze, so it was encapsulated and stored in a liquid state below 15~17℃.

3-3. Cryopreservation of pig semen

① Calculation of semen volume (scaling cylinder), sperm concentration (blood count plate), sperm vitality (CASA), and effective sperm count

After calculating the semen volume, sperm concentration, and vitality of the stored semen, the effective sperm count was calculated according to the formula below. In order to cryopreserve individuals with excellent genetic traits and use them semi-permanently, sperm vitality and effective sperm count were evaluated. Pig semen is difficult to freeze-preserve, so if it is encapsulated and frozen, it is much less susceptible to frost damage.

* Effective sperm count: Collected semen volume (ml) × sperm concentration per 1 ml of semen × sperm motility (%)

② Centrifugation of semen

The diluted semen was dispensed into 50 ml centrifuge tubes and centrifuged at room temperature at 800 g for 15 minutes. The seminal fluid in the supernatant was removed and the sperm pellet was used.

③ Calculate the quantity of frozen straws and diluent amount to be manufactured.

During centrifugation of semen, the number of 0.5 ml frozen straws was calculated based on the effective sperm count, and the dilution amount of the preservation diluent was calculated in advance.

* Effective sperm count: 2.5-30 million/0.5ml straw

④ Removal of supernatant

At the end of centrifugation, the supernatant, excluding the sperm pellet, was removed as much as possible.

⑤ First dilution (carried out at room temperature)

Pig semen preservation diluent was added to the sperm pellet, and the sperm pellet was released and collected into one tube. Considering the volume of the sperm pellet, a diluent for pig semen preservation was added to make up 2/3 of the total volume.

* Later, in the second dilution step, 1/3 of the total volume of secondary pig semen preservation diluent is added.

⑥ Preliminary cooling (stored at 5℃)

The semen sample in ⑤ was cooled at 5°C for 90 minutes.

* Straw labeling was performed during incubation, including dilution type, individual number, and date.

⑦ Second dilution (carried out at 5℃)

At 10-minute intervals, 1/3 of the total volume of the diluent for pig semen preservation was added three times, divided in a ratio of 1:1:2 (v/v).

⑧ Straw encapsulation of semen samples for freezing (carried out at 5℃)

5㎖ straw: Using an 18-21 gauge syringe, the second diluted semen from ⑦ was encapsulated and injected as above, and both ends were sealed with iron beads.

0.5㎖ straw: By suctioning the side with the filter, the second diluted semen in ⑦ was encapsulated and injected as above, and sealed with straw sealing powder.

* In order to prevent straw rupture due to volume expansion during the freezing process, an air layer was secured in the middle of the straw as shown in Figure 3 (Figure 3).

⑨ Glycerol equilibrium

The packaged straws were subjected to glycerol equilibration at 5°C for 60 to 90 minutes. Since glycerin is cytotoxic, we proceeded to the next step by giving sufficient time for the sperm cells to adapt.

⑩ Cryopreservation

Freezing was performed using an automatic semen freezing device (computer controlled rate freezer) or a Styrofoam Box freezing system.

* Automatic semen freezing device: Freeze according to the freezing program below

5℃ → -5℃ (cooling rate 6℃/min) → -80℃ (40℃/min) → -150℃ (60℃/min)

* Freezing of Styrofoam Box (-196℃)

After placing a steel test tube rack in a Styrofoam box and filling it with an appropriate amount of liquid nitrogen, the straw was placed 7 to 9 cm above the surface of the liquid nitrogen and left in the nitrogen gas for about 12 to 15 minutes to freeze. Afterwards, the straw was immersed in liquid nitrogen and completely frozen.

⑪ Melting of semen (using constant temperature water bath)

The freeze-dried 5 ml straw and 0.5 ml straw were melted using a constant temperature water bath as shown in Table 1 below.

Experimental Example 4. Results of sperm encapsulation

In Experimental Example 3-2, the encapsulated pig sperm had a significantly lower acrosome damage rate compared to the control non-capsulated sperm when measured 4 hours after preservation (capsulated sperm 7.5% vs. non-capsulated sperm 12.8%), and the vitality was The rate of non-capsule sperm was slightly higher (77% for non-capsule sperm vs. 70% for capsule sperm), and there was no difference in the malformation rate (11.8% for non-capsule sperm vs. 10.2% for capsule sperm).

In addition, the survival rate of pig sperm measured after 72 hours of storage at 15-17°C tended to be slightly higher for non-capsule sperm (65.4% for non-capsule sperm vs. 58.2% for capsule sperm), but capsule semen is used for artificial insemination. It can be said that it is of great quality in terms of use.

Figure 5 shows the sperm survival rate and vitality after thawing of cryopreserved pig sperm in Experimental Example 3-3. As a result, the sperm survival rate was high even after thawing, and the vitality was also excellent.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (9)

Step 1 of preparing a semen suspension by mixing livestock semen with a gelling solution containing a diluent and barium ions;
Step 2, preparing a saline solution containing bentonite and alginate;
Step 3 of mixing the semen suspension from step 1 with the physiological saline solution from step 2; and
It includes a fourth step of producing encapsulated semen by rotating the microgel formed in step three above on a magnetic stirrer,
Thinners for bovine and deer semen preservation include lactose, egg yolk, and glycerol;
Diluents for canine semen preservation include tris, citric acid, and fructose;
A method of encapsulating sperm, wherein the diluent for preserving pig semen includes glucose, sodium citrate hydrate, EDTA, sodium bicarbonate, potassium chloride, and gentamicin sulfate. According to paragraph 1,
A method of encapsulating sperm, wherein the semen of the livestock is one or more selected from the group consisting of pigs, cows, deer, dogs, sheep, horses and goats. According to paragraph 1,
The method of encapsulating sperm, wherein the physiological saline solution contains 1 to 3% by weight (w/v) of alginate and 2 to 6% by weight (w/v) of bentonite. The method of claim 1, wherein the bentonite is montmorillonite, beidellite, or nontronite. delete The method of encapsulating sperm according to claim 1, wherein the mixture of the three steps contains 0.5 to 1.5% by weight (w/v) of alginate. A method of cryopreservation of encapsulated sperm comprising the step of freezing the encapsulated sperm by the method of claim 1. In clause 7,
A method for cryopreservation of encapsulated sperm, further comprising cooling the encapsulated sperm before the freezing step. Sperm encapsulated by the method of claim 1.

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