JP2023170612A - Cryopreservation solution and cryopreservation method for porcine germ cell - Google Patents

Abstract

To provide a cryopreservation solution or a cryopreservation method for porcine sperm and porcine embryos (blastocysts), which demonstrates practical cryopreservation effects applicable to production sites.SOLUTION: Cryopreservation of porcine sperm involves using a cryopreservation solution that includes DMGA-PLL (dimethylglytarate-modified polylysine) 0.2-0.3 w/w%, glycerol 2-4 w/w%, trehalose 5-15 w/w%, and yolk 15-25 w/w%. Cryopreservation of porcine embryos (blastocysts) involves using a cryopreservation solution that includes DMGA-PLL (dimethylglytarate-modified polylysine) or CPLL (succinate-modified polylysine) 8-12 w/w%, ethylene glycol 30-35 w/w%, polyethylene glycol 1-3 w/w%, trehalose 15-25 w/w%, and bovine fetal serum 15-25 w/w%.SELECTED DRAWING: Figure 2

Description

The present invention relates to a cryopreservation solution and cryopreservation method useful for cryopreservation of reproductive cells such as porcine sperm and embryos.

For long-term preservation of animal cells or tissues, cryopreservation methods at temperatures below 0°C are routinely used. However, animal cells or tissues contain water, and when frozen, water molecules crystallize while excluding the medium of solutes and contaminants, forming ice crystals consisting only of water molecules. It is known that solutes and contaminants diffuse non-uniformly in a material, resulting in freeze concentration.

In order to prevent such freeze-concentration, methods of adding various low-molecular-weight compounds have been used. For example, when cryopreserving cells, low-molecular-weight dimethyl sulfoxide, glycerol, etc. are added as cryoprotectants to minimize damage to cells due to intracellular crystallization that occurs during cryopreservation. method is being done.

In particular, in order to improve livestock productivity and preserve genetic resources, reproductive cells such as sperm of livestock are also cryopreserved. In particular, a cryopreservation solution containing 6 to 7% by weight of glycerin has been used to freeze bovine spermatozoa. Additionally, glycerin, ethylene glycol, and propane diol are known as cryoprotectants used for freezing bovine eggs and embryos.

However, cryopreservation of pig spermatozoa and embryos (blastocysts) is generally considered difficult because of the large damage caused during freezing (Non-patent Documents 1 and 2). The beginning of Non-Patent Document 1 states as follows: ``Pig frozen semen cannot be produced because the sperm functionality after thawing is impaired due to damage during freezing and the reproductive performance after artificial insemination is low.'' It is hardly used in the field."

Further, [0002] to [0003] of Patent Document 4 describes as follows.
[0002] Artificial insemination techniques for livestock other than cows are not yet fully established.
[0003]...Japan's pig farming industry mainly uses natural breeding, which is expensive as it is necessary to raise male pigs. Also, the work is dangerous. Furthermore, natural breeding requires a great deal of effort...

According to Non-Patent Documents 1 and 2 and Patent Document 4, a cryopreservation solution for pig sperm is ``NSF (Niwa and Sasaki freezing extender; 80%(v/ v), 0.31mol Lactose monohydrate, 20%(v/v) egg yolk, 1000U/ml penicillin G potassium, 1mg/ml streptomycin sulfate), with glycerin added to approximately 3% by weight, osmotic pressure 400mOsm. /kg solutions have mainly been studied.

On the other hand, in cryopreserving bovine spermatozoa, the applicant used carboxylated polylysine (particularly succinic anhydride-modified polylysine) at 0.3 to 0.9 w/w% and glycerin at 2 to 4 w/w% as ampholytic polyelectrolytes. Excellent effects were found with a very limited composition (Patent Document 1).

The applicant had previously discovered that a cryopreservation solution containing, for example, 7 to 10 w/w% of this carboxylated polylysine has an excellent effect on the cryopreservation of cells and tissues such as fibroblasts and stem cells. discovered (Patent Document 2). On the other hand, it has also been proposed to add ethylene glycol and sucrose as a "vitrification solution" which is a partially improved cryopreservation solution for stem cells and the like (Patent Document 3). This partially improved "vitrification fluid" is specifically (in the examples) succinic anhydride-modified polylysine (CPLL) or dimethyl glitaric acid-modified polylysine (DMGA-PLL) as carboxylated polylysine. Contains 25mM (approximately 10% by weight; molecular weight approximately 4000), 6M ethylene glycol (approximately 37% by weight; molecular weight 62), and 0.5M sucrose (approximately 17% by weight; molecular weight 342). According to Example 8 of Patent Document 3 and FIG. 15 showing the results, the survival rate of mesenchymal stem cells was high when dimethylglitaric acid-modified polylysine was used.

Unlike bovine sperm, etc., the development of a practical cryopreservation solution or cryopreservation method for pig sperm and pig embryos (blastocysts) that can be introduced into production sites has been awaited.

PCT/JP2017/029979 (WO2018/038115A) PCT/JP2009/002941 (WO2009/157209A; Japanese Patent No. 5726525) Japanese Patent No. 6678931 JP2018-113936

Journal of the Japan Pig Society 49 (3) 128-132 (September 2012), “Effect of glycerol concentration on cryopreservation of Berkshire and Agu spermatozoa using high osmotic freezing dilution solution”, Tsukasa Chinen et al. https://www. jstage.jst.go.jp/article/youton/49/3/49_128/_pdfhttps://agriknowledge.affrc.go.jp/RN/2010833581 Journal of Reproduction and Development, 45(5) 345-350 (1999), “Reproduction in Pigs Using Frozen-Thawed Spermatozoa from Epididymis Stored at 4C”, Kazuhiro KIKUCHI et al. https://www.jstage.jst.go. jp/article/jrd/45/5/45_5_345/_pdf

Therefore, the present invention provides a cryopreservation composition and a cryopreservation method for pig sperm and pig embryos (blastocysts) at a practically acceptable level for the purpose of improving livestock productivity and preserving genetic resources. We provide products that provide a preservation effect.

As a result of intensive research to solve the above problems, we found that pig sperm and pig embryos (blastocysts) have good cryopreservation effects when the following very limited composition is adopted. The present invention has now been completed.

For cryopreservation of pig spermatozoa, DMGA-PLL (dimethylglitarate-modified polylysine) 0.2 with a proportion of substituted building blocks (carboxylated individual anhydride lysine units) ranging from 50 to 99 mol% is used. ~0.3w/w% (or more than 0.2w/w% but less than 0.3w/w%), glycerin 2~4w/w% (or 2~3w/w%), and trehalose 5~15w/w% ( or 5 to 13 w/w%) and egg yolk to 15 to 25 w/w% (or 17 to 23 w/w%).

For cryopreservation of pig embryos (blastocysts), use DMGA-PLL (dimethyl glutaric acid-modified polylysine) or CPLL (succinic acid-modified polylysine) 8-12w with a proportion of substituted structural units of 50-99 mol%. porcine embryo ( Use cryopreservation solution of blastocyst).

According to the present invention, productivity can be improved by cryopreserving pig sperm and pig embryos (blastocysts) and using them at sites such as child production.

It is a graph showing the influence of the concentration of DMGA-PLL on the survival index of pig sperm after thawing. 2 is a graph showing the influence of the concentration of DMGA-PLL on the in vitro developmental ability of in vitro fertilized eggs produced using thawed pig sperm. It is a graph showing the influence of the concentration of DMGA-PLL on the in vitro fertilization ability of pig sperm after thawing. 2 is a graph showing the influence of the concentration of CPLL on the survival index of pig sperm after thawing. 2 is a graph showing the influence of the concentration of CPLL on the in vitro developmental ability of in vitro fertilized eggs produced using thawed pig sperm.

The cryopreservation solution for pig germ cells according to the present invention contains succinic anhydride or dimethyl Contains carboxylated polylysine (hereinafter appropriately referred to as "antifreeze polyamino acid") that is blocked by carboxylation by reacting with glitaric anhydride (chemical formula II below). The degree of polymerization n of ε-poly-L-lysine of the following chemical formula I is typically 25 to 35 (number average molecular weight 3200 to 4500).

<Chemical formula I>

<Chemical formula II>
Succinic anhydride (succinic anhydride) Dimethylglitaric anhydride

In the present invention, examples of ε-poly-L-lysine include ε-poly-L-lysine produced by microorganisms or enzymes and having a number average molecular weight of 1,000 to 20,000 or 1,000 to 10,000, particularly 3,000 to 5,000. can. ε-Poly-L-lysine is produced by actinomycetes belonging to the genus Streptomyces and is used as a food preservative. In addition to those with a degree of polymerization of 15 to 35 or 25 to 35, attempts have been made to produce those with a degree of polymerization of 20 or less (for example, JP-A No. 2003-171463 and JP-A No. 2005-318815). The number average molecular weight or number average degree of polymerization is measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using, for example, an electrophoresis device and a densitograph (model AE-6920V) manufactured by ATTO Co., Ltd. It can be easily measured using At this time, standard protein markers are used. Note that ε-poly-L-lysine can also be used with a molecular weight of 30,000 or more by increasing the molecular weight by heat treatment. However, from the viewpoint of preventing an increase in viscosity, etc., the above molecular weight range is preferable.

Carboxylated lysine as an "antifreeze polyamino acid" is preferably 50 to 99 mol%, particularly 50 to 93 mol%, more preferably 50 to 90 mol%, and It is preferably blocked by 55 to 80 mol%, most preferably 58 to 76 mol% by carboxylation.

Approximately 50 mol% of the amino groups of ε-poly-L-lysine are blocked by reacting 52 to 53 mol% of succinic anhydride or dimethylglitaric anhydride (chemical formula II above) to the amino groups of ε-poly-L-lysine. be able to. Furthermore, when 100 mol% of succinic anhydride or dimethylglitaric anhydride is reacted, 90 to 95 mol% of amino groups can be blocked under normal reaction conditions. If the block rate exceeds or falls below the above range, the cryopreservation effect will be reduced.

The cryopreservation solution for pig reproductive cells according to the present invention may be a physiological aqueous solution in which the above-mentioned antifreeze polyamino acid (carboxylated lysine) and additional components described below are dissolved. As the physiological aqueous solution, in addition to physiological saline, general culture solutions for various cells or tissues can be used. For example, Dulbecco's modified Eagle's MEM medium (DMEM) can be mentioned as preferred.

In the first embodiment of the present invention, the pig sperm cryopreservation solution according to the present invention contains 0.2 to 0.3 w/w% of DMGA-PLL (dimethyl glycolate modified polylysine), 2 to 4 w/w% of glycerin, Contains 5 to 15 w/w% trehalose and 15 to 25 w/w% egg yolk. The glycerin content is significantly reduced or halved from the amount conventionally used for cryopreservation of bovine sperm, etc., and the cryopreservation solution for pig sperm according to the present invention has low cytotoxicity.

A preferable cryopreservation solution according to the first embodiment includes a predetermined amount of DMGA-PLL (dimethyl glycolate modified polylysine) and glycerin in NSF (Niwa and Sasaki freezing extender), which is a diluent for pig semen during artificial insemination. only added. The NSF used here is a type of modified NSF that contains trehalose as a low-molecular polyalcohol component or low-molecular sugar component instead of lactose hydrate.

That is, NSF, which forms the base of the cryopreservation solution, contains trehalose as a low-molecular polyalcohol or low-molecular sugar compound (monosaccharide or disaccharide), egg yolk, and antibacterial and antiviral substances such as antibiotics. NSF particularly has an osmolality of about 400 mOsm/kg (eg 350-430 mOsm/kg). The NSF may be based on a Tris-citrate buffer (egg yolk-Tris sugar solution (ET)) to which low egg yolk has been added. Furthermore, the antibiotic may include amikacin, dibekacin, penicillin, streptomycin, and the like.

A preferred cryopreservation method according to the first embodiment includes (1) a pretreatment step of pretreating pig semen to obtain pig sperm (highly concentrated suspension of pig sperm in a pretreatment solution); (3) a first dilution step in which the treated pig spermatozoa (highly concentrated suspension) is diluted (first dilution) with the above modified NSF; ) and glycerin are prepared, and the second dilution step is performed by adding the second dilution solution to the suspension after the first dilution, and (4) for storage. After filling it into a straw, it is pre-frozen and then maintained at a temperature of -60°C or lower.

Note that from the pretreatment process to the time of filling the storage straw (tubular container for cryopreservation), the temperature ranges from around 37°C (e.g. 33 to 40°C) to around 5°C (e.g. 2 to 8°C). Lower the temperature step by step or gradually. Further, pre-freezing can be performed, for example, by placing the filled storage straw in an atmosphere of -5°C to -20°C.

Preferably, a straw for cryopreservation with a diameter of 5 mm or less is used, and after preliminary freezing, a slow freezing method using a program freezer or the like at a cooling rate of 100°C/min or less, or a cooling rate of 300°C by immersion in liquid nitrogen is used. Cool to below -140°C using the vitrification freezing method at a rate of at least °C/min.

In the second embodiment of the present invention, the cryopreservation solution for pig embryos (blastocysts) according to the present invention is a DMGA-PLL (dimethyl glutaric acid modified polylysine) or CPLL (succinic acid-modified polylysine) 8-12 w/w%, ethylene glycol 30-35 w/w%, polyethylene glycol 1-3 w/w%, trehalose 15-25 w/w%, and fetal bovine serum. Contains 15 to 25 w/w%. Examples of pig embryos to be cryopreserved include pig embryos developed for 4 to 8 days (preferably 5 to 7 days) by in vitro fertilization (IVF).

A preferred cryopreservation method according to the second embodiment includes the steps of sequentially transferring a pig embryo (blastocyst) into a first equilibrium solution, a second equilibrium solution, and the above-mentioned cryopreservation solution; The first equilibrium solution includes a step of maintaining the pig embryo in an atmosphere below -60°C without pre-freezing, and the first equilibrium solution is a cell culture medium containing 5 to 15% ethylene glycol and 15 to 25% FBS. (fetal bovine serum) and a solution of 3-7% DMGA-PLL and/or CPLL, and the second equilibrium solution is a cell culture medium containing 5-15% ethylene glycol, It was added to contain a solution of 0.5-2% polyethylene glycol, 0.2-0.4M trehalose, 15-25% FBS, and 7-13% DMGA-PLL and/or CPLL.

When using frozen porcine reproductive cells, frozen bovine cells can be thawed and used according to a general thawing method for various cells. Note that antifreeze polyamino acids have low cytotoxicity, and unlike dimethyl sulfoxide and the like, there is no need to remove them upon thawing.

Furthermore, the cryopreservation solution for pig germ cells according to the present invention can also be provided as a kit for cryopreservation of pig germ cells. In addition to the composition for cryopreservation, the kit can include, for example, a container used for cryopreservation, an instruction manual for the kit, and the like.

Hereinafter, the present invention will be explained in more detail using Examples, but the technical scope of the present invention is not limited to these Examples.

[Example 1] Cryopreservation of pig sperm using antifreeze polyamino acids 1-1. Preparation of dimethyl glutaric acid-modified polylysine (DMGA-PLL) Add 65% 3,3-dimethylglutaric anhydride (DMGA , Sigma-Aldrich) or succinic anhydride (Tokyo Kasei) to carboxylate 60 mol% of the amino groups in the ε-poly-L-lysine molecule to produce blocked antifreeze polyamino acids.

1-2. Preparation of primary dilution liquid and secondary dilution liquid First, as NSF (Niwa and Sasaki freezing extender), which is a dilution liquid for pig semen during artificial insemination, the conventional I modified it from the original and used it. NSF was obtained by mixing the composition shown below, stretching the mixture, and collecting the supernatant liquid.

<Table 1> Composition of NSF (when adjusting 100ml)

Based on the NSF in Table 1 above, a first dilution solution and a second dilution solution were prepared as follows.
・Primary dilution liquid: A mixture of NSF 99.26% and Orvus ES Paste 0.74% (volume ratio). Here, Orvus ES Paste is a surfactant used to uniformly dissolve components contained in egg yolk.
・Secondary dilution liquid: A mixed solution of NSF 91.26%, Orvus ES Paste 0.74%, glycerin 6%, and 2% (volume ratio) of a 25% by weight solution of DMGA-PLL or CPLL.

1-3. Cryopreservation of pig sperm To cryopreserve sperm from edible pigs (Duroc), after measuring the sperm concentration, dilute and equilibrate using a pretreatment solution (Modena solution), primary dilution solution, and secondary dilution solution using a standard method. At the same time, I gradually lowered the temperature. At this time, the same amounts of the primary dilution liquid and the secondary dilution liquid were used. Specifically, it was performed as follows.

(1) Pretreatment The collected semen was diluted with Modena solution warmed to 37°C, centrifuged (3000 rpm, 25°C, 15 minutes), and then precipitated using an aspirator connected to a Pasteur pipette. The supernatant was removed without aspirating any substances (sperm). Next, a pretreatment solution (Modena solution) was added and suspended so that the sperm concentration was 10.0×10 ⁸ sperm/ml. Here, the Modena liquid having the composition shown in Table 2 below was used.

<Table 2> Composition of Modena liquid (when adjusting 1000ml)

(2) Primary dilution The mixture was centrifuged again in the same manner as in the pretreatment, and the supernatant was removed. Then, a primary dilution solution was added and suspended so that the sperm concentration was 20.0×10 ⁸ sperm/ml. Then, it was placed in a 15°C incubator for 30 minutes, in a 10°C incubator for 1.5 hours, and in a 5°C incubator for 1 hour in this order.

(3) Secondary dilution The final concentration of spermatozoa can be reduced to 10.0x by adding the secondary dilution in several times in an equal volume to the primary dilution to the suspension obtained in the primary dilution above. The concentration was adjusted to ¹⁰⁸ animals/ml. Then, it was left standing in an incubator at 5°C for 20 minutes.

(4) Filling straws for storage and cryopreservation The diluted sperm solution prepared as described above was filled in 0.5 ml portions into straws for storage and sealed using a straw enclosing device. The straws were then pre-frozen by holding them 4 cm above the liquid nitrogen for 10 minutes, and then transferred into liquid nitrogen and stored.

(5) Thawing/artificial insemination Frozen straws were thawed in warm water, and artificial insemination was performed using a syringe.

1-4. Effects of adding 0.25% by weight of DMGA-PLL and CPLL on pregnancy and parturition status in pigs
By adding DMGA-PLL (dimethyl glitaric acid-modified polylysine) or CPLL (succinic acid-modified polylysine) to the above secondary dilution solution at a concentration of 0.5% by weight, it can be added to the cryopreservation solution at a concentration of 0.25% by weight. included. As described below, it was shown that the optimal concentration of both CPLL and DMGA-PLL to be added to NSF is 0.25% by weight.

A female (Landrace x Large Yorkshire) was artificially inseminated using the cryopreserved pig sperm according to the above procedure, and the pregnancy and parturition status were investigated. At the same time, artificial insemination was performed using spermatozoa cryopreserved in a solution without carboxylated lysine and spermatozoa preserved at low temperature (17°C) for 3 days. The results of an investigation into the effects of artificial insemination on the pregnancy and parturition status of pigs in this manner are summarized in Table 3 below.

Storage at low temperature (17°C) was carried out in accordance with the method commonly used in piglet production sites for artificial insemination of pigs. Specifically, after collecting semen from a male pig, dilute it with HIRO-SWINE B solution heated to 37℃, transport it in a light-shielding container while maintaining an environment of 25℃, and then gently heat it to 17℃. It was cooled and stored at ℃. Additionally, experiments using a separate sperm motility analysis system (SMAS) have shown that there are no signs of decline in sperm motility (resting motility rate and linear velocity) until storage at 15-17°C for 3 days. was not seen.

In Table 3 below, the numbers in parentheses for "total number of litters" and "number of live litters" are the numbers per calving cow. The right end of Table 3 below shows the value obtained by dividing the "number of live offspring" by the "number of artificially inseminated animals." Note that the "number of live offspring" is the number of offspring that were alive at the time of delivery.

<Table 3> Effect of addition of CPLL and DMGA-PLL to sperm cryopreservation solution on pregnancy and parturition status of artificially inseminated pigs

^* Percentage of artificially inseminated animals
^** Percentage of pregnant animals
^*** Number of live offspring/number of artificial inseminations
^acThere is a significant difference between different signs in each column (P<0.01 or 0.05)

According to the results on the right side of Table 3, when using a specified cryopreservation solution containing 0.25% by weight of DMGA-PLL (dimethyl glitaric acid-modified polylysine), the temperature at a relatively low temperature (17°C) without freezing was observed. There was not much difference between the two cases and the case of short-term storage. From this, it was determined that the cryopreservation effect was achieved at a level applicable to child livestock production sites.

On the other hand, the effects of the concentration of DMGA-PLL (dimethyl glitaric acid-modified polylysine) and CPLL (succinic acid-modified polylysine) on the survival index, in vitro developmental ability, and in vitro fertilization ability of pig sperm after thawing are shown in Table 3 above. I researched this before the experiment. The results of this experiment are shown in FIGS. 1 to 6.

Using the above procedure, the above-mentioned modified NSF (trehalose-based) was prepared to contain 3.0% glycerin and 0, 0.125, 0.25, and 0.5% carboxylated lysine (DMGA-PLL or CPLL) as a cryopreservation solution. Sperm from edible pigs (Duroc) was cryopreserved. After 0, 3, 6, 9, and 12 hours of thawing, the sperm vitality was observed under a microscope, and the sperm viability index was calculated from the obtained results. Here, the sperm survival index is a numerical representation of the percentage of sperm determined to be motile and the strength of their motile force through microscopic observation.

In addition, in order to investigate the in vitro fertilization potential of cryopreserved pig sperm, we inseminated in vitro matured eggs recovered and produced from ovaries from meat centers with thawed sperm. Twelve hours after insemination, the eggs were fixed and stained and the fertilization status was observed.

Finally, we investigated the in vitro developmental potential of in vitro fertilized eggs produced using pig sperm cryopreserved in solutions containing various concentrations of CPLL. After 12 hours of insemination, the eggs were transferred to a developmental medium and cultured, and the cleavage status was observed after 2 days and the blastocyst formation status after 7 days.

Figures 1 to 3 show that when DMGA-PLL is used as carboxylated lysine, the concentration of DMGA-PLL is the survival index of pig sperm after thawing, and the survival index of in vitro fertilized eggs produced using pig sperm after thawing. The effects on the in vitro developmental ability and the in vitro fertilization ability of pig sperm after thawing will be shown. Figures 4 and 5 also show that when CPLL is used instead of DMGA-PLL as the carboxylated lysine, the concentration of CPLL is the survival index of pig sperm after thawing, and the concentration of CPLL produced using pig sperm after thawing. The effects on the in vitro developmental potential of the in vitro fertilized eggs are shown below.

When using DMGA-PLL, as shown in Figure 1, there was no significant difference in the sperm viability index between different concentrations after 0 to 9 hours of thawing, but there was no significant difference in the sperm viability index after 12 hours of thawing. The index was significantly higher in the plot to which 0.25% DMGA-PLL was added than in the plot without additive. Furthermore, as shown in FIG. 2, regarding the in vitro developmental potential, the blastocyst formation rate was significantly higher in the group to which 0.25% DMGA-PLL was added than in the group without addition. However, the difference in cleavage rate was not significant. In addition, as shown in FIG. 3, no significant difference was observed in in vitro fertilization ability due to the difference in concentration.

When using CPLL, as shown in Figure 4, there was no significant difference in the sperm viability index between different concentrations after 0 to 3 hours of thawing, but the sperm survival index after 6 to 12 hours of thawing was not significantly different. The index was significantly higher in the plots with 0.125 to 0.25% CPLL added than in the plots without additives. Furthermore, as shown in FIG. 5, regarding the in vitro developmental potential, the blastocyst formation rate was significantly higher in the group to which 0.25% CPLL was added than in the group without addition. However, the difference in cleavage rate was not significant.

[Example 2] Cryopreservation of pig embryos (blastocysts) using antifreeze polyamino acids In vitro fertilization was performed using sperm from edible pigs (Duroc), and the early blastocysts obtained 6 days later were The cells were stored frozen for ~14 days, and the survival status was examined 1 day and 2 days after thawing. Specifically, it was performed as follows.

(1) Preparation of basal medium, first and second equilibrium solutions, and cryopreservation solution - Basal medium: HEPES-PZM-3 shown in Table 4 below.
- First equilibrium solution: Added to the basal medium to contain 10% ethylene glycol, 20% FBS (fetal bovine serum), and 5% CPLL solution.
-Second equilibrium solution: Added to the basal medium to contain 10% ethylene glycol, 1% polyethylene glycol, 0.3M trehalose, 20% FBS, and 10% CPLL solution.

- Cryopreservation solution: Add to the basal medium to contain 33% ethylene glycol, 2% polyethylene glycol, 0.6M trehalose, 20% FBS, and 20% CPLL solution.

<Table 4> Composition of basal medium (when adjusted to 100ml)

(2) Series of operations for cryopreservation ・Embryo preparation: Recover blastocysts from mated females, or recover blastocysts from eggs cultured in vitro after in vitro fertilization.
- Treatment with equilibrium solution and freezing solution: Pig embryos are dehydrated by sequentially transferring them into a first equilibrium solution, a second equilibrium solution, and a cryopreservation solution.
- Cryopreservation: Place the treated embryo on the tip of a cryotop, immerse it in liquid nitrogen, and preserve it for 3 to 14 days.

(2) Melting - Preparation of melt solution: Add 10% ethylene glycol, 0.3M trehalose, and 20% FBS to the basal medium.
- Melting operation: The tip of the cryotop taken out of liquid nitrogen is immersed in the melting solution to thaw the embryo. The embryos are equilibrated in the lysate and then transferred to regular culture medium.

The results of examining the survival status 1 day and 2 days after thawing are shown in Tables 5 to 10 below. In the following, expanded blastocysts were obtained 6 days after in vitro fertilization.

<Table 5> Effect of CPLL concentration in the cryopreservation solution on the survival status of early blastocysts derived from thawed pig IVF eggs

Number of experiments: 5 times
There is a significant difference between ^ab different signs (P<0.01)

<Table 6> Effect of CPLL concentration in the cryopreservation solution on the survival status of expanded blastocysts derived from thawed pig IVF eggs

Number of experiments: 5 times
There is a significant difference between ^ab different signs (P<0.05)

As shown in Table 5, when early blastocysts obtained 6 days after in vitro fertilization were cryopreserved, there was no significant difference in the survival rate after 1 day of thawing between different concentrations; The survival rate after 10 days was significantly higher in the group to which 10% CPLL was added than in the group without addition. In addition, as shown in Table 6, when expanded blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate after 1 day of thawing was higher in the group with 10% CPLL added than in the non-additive group. However, no significant difference was observed in the survival rate 2 days after thawing between the different concentrations.

As shown in Tables 5 and 6, when CPLL was used as the carboxylated polylysine and the concentration of CPLL was approximately 10% by weight, cryopreservation effects were observed for early blastocysts and expanded blastocysts.

<Table 7> Effect of DMGA-PLL concentration in cryopreservation solution on survival status of early blastocysts derived from thawed pig IVF eggs

Number of experiments: 5 times
There is a significant difference between ^ab different signs (P<0.01 or 0.05)

<Table 8> Effect of DMGA-PLL concentration in the cryopreservation solution on the survival status of expanded blastocysts derived from thawed pig IVF eggs

Number of experiments: 5 times
There is a significant difference between ^ab and opposite signs (P<0.01 or 0.05)

As shown in Table 7, when early blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate after 1 day of thawing was significantly higher in the group to which 10% DMGA-PLL was added than in the group without addition. It got expensive. In addition, as shown in Table 8, when expanded blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate after 2 days of thawing was higher than in the group with 10% DMGA-PLL added. It was significantly higher than the ward.

As shown in Tables 7 and 8, when DMGA-PLL is used as the carboxylated polylysine and the concentration of DMGA-PLL is approximately 10% by weight, cryopreservation effects are seen for early blastocysts and expanded blastocysts. Ta.

<Table 9> Effect of cryoprotectant on the survival status of early blastocysts derived from thawed pig IVF eggs

Number of experiments: 8 times
There is a significant difference between ^ab different signs (P<0.01 or 0.05)

<Table 10> Effect of cryoprotectant on the survival status of expanded blastocysts derived from thawed pig IVF eggs

Number of experiments: 8 times
There is a significant difference between ^ab different signs (P<0.01 or 0.05)

Tables 9 and 10 show a comparison of the cryopreservation effects of CPLL and DMGA-PLL. Regarding the number of viable embryos 2 days after thawing of expanded blastocysts, the effect of DMGA-PLL was significantly higher than that of CPLL.

Claims (6)

0.2-0.3% by weight of dimethylglitaric acid-modified polylysine with a proportion of substituted structural units of 50-99% by mole, 2-4% by weight of glycerin, 5-15% by weight of trehalose, and 15-25% by weight of egg yolk. A cryopreservation solution for pig sperm containing. The cryopreservation solution for pig sperm according to claim 1, wherein the content of dimethyl glitaric acid-modified polylysine is less than 0.3% by weight, and the content of glycerin is 3% by weight or less. A method for cryopreservation of pig sperm using the cryopreservation solution according to claim 1 or 2,
A pretreatment step of pretreating pig semen to obtain pig sperm;
a primary dilution step of diluting the pretreated pig sperm with a diluent for pig semen;
A secondary dilution solution is prepared by adding dimethyl glitaric acid-modified polylysine and glycerin to a dilution solution for pig semen, and by adding the second dilution solution to the suspension after the first dilution, the cryopreservation solution is a secondary dilution step in which pig spermatozoa are suspended;
After filling it into a storage straw, pre-freezing it, and then maintaining it at a temperature of -60°C or less,
A method for freezing and preserving pig sperm, wherein the diluent for pig semen contains the trehalose and the egg yolk. 4. The method for cryopreservation of pig sperm according to claim 3, characterized in that the cryopreservation straw is cooled to −140° C. or lower using a cryopreservation straw with a diameter of 5 mm or less. 8 to 12% by weight of dimethylglitaric acid-modified polylysine or succinic acid-modified polylysine in which the proportion of substituted structural units is 50 to 99 mol%, 30 to 35% by weight of ethylene glycol, and 1 to 3% by weight of polyethylene glycol. , a cryopreservation solution for pig embryos containing 15 to 25% by weight of trehalose and 15 to 25% by weight of fetal bovine serum. A method for cryopreservation of pig embryos using the cryopreservation solution according to claim 5,
A step of sequentially transferring the pig embryo into a first equilibrium solution, a second equilibrium solution, and the above-mentioned cryopreservation solution;
After this, the step of holding the pig embryo in the cryopreservation solution in an atmosphere of -60°C or lower without pre-freezing,
The first equilibrium solution contains a solution of 5-15% ethylene glycol, 15-25% fetal bovine serum, and 3-7% dimethyl glycolate-modified polylysine or succinate-modified polylysine in cell culture medium. It is added to
The second equilibrium solution consists of cell culture medium containing 5-15% ethylene glycol, 0.5-2% polyethylene glycol, 0.2-0.4M trehalose, 15-25% fetal bovine serum, and 7-13% dimethyl glycol. A method for cryopreservation of pig embryos, which comprises adding a solution of tar acid-modified polylysine or succinic acid-modified polylysine.