Cryopreservation solution and cryopreservation method for porcine germ cells

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

[Technical Field]

[0001] This invention relates to a cryopreservation solution and a cryopreservation method useful for the cryopreservation of germ cells such as pig sperm and embryos. [Background technology]

[0002] Cryopreservation at temperatures below 0°C is routinely used to preserve animal cells or tissues for long periods. However, animal cells or tissues contain water, and when frozen, water molecules crystallize during freezing, excluding solutes and impurities, to form ice crystals composed solely of water molecules. This leads to uneven diffusion of solutes and impurities within the water-containing material, resulting in freeze-concentration.

[0003] To prevent such freeze-concentration, various methods are employed to add low-molecular-weight compounds. For example, when cryopreserving cells, low-molecular-weight compounds such as dimethyl sulfoxide or glycerol are added as cryoprotective agents to minimize damage to cells caused by crystallization during cryopreservation.

[0004] In particular, to improve the productivity of livestock offspring and to preserve genetic resources, the reproductive cells of livestock, such as sperm, are sometimes cryopreserved. For freezing bovine sperm, cryopreservation solutions containing 6-7% by weight of glycerin have been used. Glycerin, ethylene glycol, and propanediol are known cryoprotective agents used for freezing bovine oocytes and embryos.

[0005] However, the cryopreservation of pig sperm and embryos (blastocysts) is generally considered difficult due to the significant damage caused during freezing (Non-Patent Documents 1-2). Non-Patent Document 1 begins by stating: "Pig frozen semen is rarely used in production facilities because the sperm's functionality is impaired after thawing due to damage during freezing, resulting in poor reproductive performance after artificial insemination."

[0006] Furthermore, paragraphs

[0002] to

[0003] of Patent Document 4 describe the following:

[0002] ...For livestock other than cattle, artificial insemination technology is not yet fully established.

[0003] In Japan's pig farming industry, natural mating is the primary method, which incurs costs because it requires the raising of breeding boars. Furthermore, the work itself is dangerous. Moreover, natural mating requires a great deal of labor.

[0007] According to Non-Patent Documents 1-2 and Patent Document 4, the main solutions considered for the cryopreservation of pig sperm have been those with an osmotic pressure of 400 mOsm / kg, which are obtained by adding glycerin to "NSF (Niwa and Sasaki freezing extender; 80% (v / v), 0.31 mol Lactose monohydrate, 20% (v / v) egg yolk, 1000 U / ml penicillin G potassium, 1 mg / ml streptomycin sulfate)," a common diluent for artificial insemination of pigs.

[0008] On the other hand, the applicant has found excellent results in cryopreserving bovine sperm using a very limited composition of 0.3-0.9 w / w% carboxylated polylysine (particularly succinic anhydride-modified polylysine) and 2-4 w / w% glycerin as amphoteric polyelectrolytes (Patent Document 1).

[0009] The applicant had previously discovered that a cryopreservation solution containing, for example, 7-10 w / w% of this carboxylated polylysine was highly effective for the cryopreservation of cells and tissues such as fibroblasts and stem cells (Patent Document 2). On the other hand, it has also been proposed to add ethylene glycol and sucrose to a partially improved "vitrification solution" for such cryopreservation solutions for stem cells (Patent Document 3). Specifically (in the examples), this partially improved "vitrification solution" contains 25 mM (approximately 10% by weight; molecular weight approximately 4000) of succinic anhydride-modified polylysine (CPLL) or dimethyl glycerate-modified polylysine (DMGA-PLL) as carboxylated polylysine, 6 M (approximately 37% by weight; molecular weight 62), and 0.5 M (approximately 17% by weight; molecular weight 342). According to Example 8 of Patent Document 3 and Figure 15 showing the results, the survival rate of mesenchymal stem cells was high when dimethyl glycerate-modified polylysine was used.

[0010] Unlike bovine sperm, the development of practical cryopreservation solutions or methods for pig sperm and pig embryos (blastocysts) that could be introduced into production facilities was eagerly awaited. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] PCT / JP2017 / 029979(WO2018 / 038115A) [Patent Document 2] PCT / JP2009 / 002941 (WO2009 / 157209A; Japanese Patent No. 5726525) [Patent Document 3] Japanese Patent No. 6678931 [Patent Document 4] Japanese Patent Publication No. 2018-113936 [Non-patent literature]

[0012] [Non-Patent Document 1] Japanese Journal of Swine Science 49(3)128-132 (September 2012), "Effects of glycerol concentration on cryopreservation of Berkshire and Agu sperm using hyperosmolar cryodiluent," Tsukasa Chinen et al. https: / / www.jstage.jst.go.jp / article / youton / 49 / 3 / 49_128 / _pdf https: / / agriknowledge.affrc.go.jp / RN / 2010833581 [Non-Patent Document 2] 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 [Overview of the project] [Problems that the invention aims to solve]

[0013] Therefore, the present invention provides a composition and method for cryopreserving porcine sperm and porcine embryos (blastocysts) that can achieve a practically acceptable level of preservation effect, with the aim of improving the productivity of offspring and preserving genetic resources. [Means for solving the problem]

[0014] In order to solve the above problems, we conducted intensive research and found that when the following very limited composition is used for porcine sperm and porcine embryos (blastocysts), good cryopreservation effects are achieved, and thus the present invention was completed.

[0015] For cryopreservation of porcine sperm, a cryopreservation solution containing DMGA-PLL (dimethyl glutaric acid-modified polylysine) with a ratio of substituted structural units (carboxylated ones among individual lysine anhydride units) of 50 to 99 mol% at 0.2 to 0.3 w / w% (or 0.2 w / w% or more and less than 0.3 w / w%), glycerol at 2 to 4 w / w% (or 2 to 3 w / w%), trehalose at 5 to 15 w / w% (or 5 to 13 w / w%), and egg yolk at 15 to 25 w / w% (or 17 to 23 w / w%) is used.

[0016] For cryopreservation of porcine embryos (blastocysts), a cryopreservation solution for porcine embryos (blastocysts) containing DMGA-PLL (dimethyl glutaric acid-modified polylysine) or CPLL (succinic acid-modified polylysine) with a ratio of substituted structural units of 50 to 99 mol% at 8 to 12 w / w%, ethylene glycol at 30 to 35 w / w%, polyethylene glycol at 1 to 3 w / w%, trehalose at 15 to 25 w / w%, and fetal bovine serum at 15 to 25 w / w% is used.

Advantages of the Invention

[0017] According to the present invention, by cryopreserving porcine sperm and porcine embryos (blastocysts) and using them in the field of livestock production etc., productivity can be improved.

Brief Description of the Drawings

[0018] [Figure 1] It is a graph showing the influence of the concentration of DMGA-PLL on the survival index of porcine sperm after thawing. [Figure 2] It is a graph showing the influence of the concentration of DMGA-PLL on the in vitro development ability of in vitro fertilized eggs produced using porcine sperm after thawing. [Figure 3] It is a graph showing the influence of the concentration of DMGA-PLL on the in vitro fertilization ability of porcine sperm after thawing. [Figure 4] It is a graph showing the influence of the concentration of CPLL on the survival index of porcine sperm after thawing. [Figure 5]This graph shows the effect of CPLL concentration on the in vitro developmental potential of in vitro fertilized eggs produced using thawed pig sperm. [Modes for carrying out the invention]

[0019] The cryopreservation solution for porcine germ cells according to the present invention contains carboxylated polylysine (hereinafter referred to as "antifreeze polyamino acid"), which is obtained by blocking 50 to 99 mol% of the amino groups of ε-poly-L-lysine (Chemical Formula I below), which has a number average molecular weight of 1,000 to 20,000, by reacting it with succinic anhydride or dimethylglycaric anhydride (Chemical Formula II below) to carboxylate them. The degree of polymerization n of ε-poly-L-lysine of Chemical Formula I below is typically 25 to 35 (number average molecular weight 3200 to 4500).

[0020] <Chemical formula I> TIFF0007846893000001.tif42143

[0021] <Chemical formula II> Succinic anhydride (amber anhydride), Dimethyl glycerate anhydride TIFF0007846893000002.tif36136

[0022] In the present invention, ε-poly-L-lysine can be ε-poly-L-lysine produced by microorganisms or enzymes with a number-average molecular weight of 1,000 to 20,000 or 1,000 to 10,000, particularly 3,000 to 5,000. 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 also been made to produce those with a degree of polymerization of 20 or less (for example, Japanese Patent Publication No. 2003-171463 and Japanese Patent Publication No. 2005-318815). The number-average molecular weight or number-average degree of polymerization can be easily measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using, for example, an electrophoresis apparatus and densitograph (AE-6920V type) manufactured by ATTO Corporation. Standard protein markers are used at this time. Note that ε-poly-L-lysine can also be used with a molecular weight of 30,000 or more by heat treatment. However, the above molecular weight range is preferred from the viewpoint of preventing an increase in viscosity.

[0023] Carboxylated lysine as an "antifreeze polyamino acid" is obtained by blocking the amino group of ε-poly-L-lysine by carboxylation of preferably 50-99 mol%, particularly 50-93 mol%, more preferably 50-90 mol%, even more preferably 55-80 mol%, and most preferably 58-76 mol%.

[0024] Approximately 50 mol% of the amino groups of ε-poly-L-lysine can be blocked by reacting 52-53 mol% succinic anhydride or dimethylglycaric anhydride (chemical formula II above) with the amino groups of ε-poly-L-lysine. Furthermore, when reacted with 100 mol% succinic anhydride or dimethylglycaric anhydride, 90-95 mol% of the amino groups can be blocked under normal reaction conditions. Both exceeding and falling below the above blocking ranges will reduce the cryopreservation effect.

[0025] The cryopreservation solution for porcine germ 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 later are dissolved. As the physiological aqueous solution, in addition to physiological saline, various general culture media for cells or tissues can be used. For example, Dulbecco's modified Eagle MEM medium (DMEM) is a preferred example.

[0026] In the first embodiment of the present invention, the cryopreservation solution for porcine sperm according to the present invention contains 0.2-0.3 w / w% DMGA-PLL (dimethyl glycerate-modified polylysine), 2-4 w / w% glycerin, 5-15 w / w% trehalose, and 15-25 w / w% egg yolk. The glycerin content is significantly reduced or halved compared to the amount conventionally used for cryopreservation of bovine sperm, and the cryopreservation solution for porcine sperm according to the present invention has low cytotoxicity.

[0027] The preferred cryopreservation solution according to the first embodiment is obtained by adding predetermined amounts of DMGA-PLL (dimethyl glycerate-modified polylysine) and glycerin to NSF (Niwa and Sasaki freezing extender), which is a diluent for porcine semen used in artificial insemination. The NSF used here is a type of modified NSF that contains trehalose as a low-molecular-weight polyalcohol component or low-molecular-weight sugar component instead of lactose hydrate.

[0028] In other words, NSF, which forms the base of the cryopreservation solution, contains trehalose as a low molecular weight polyalcohol or low molecular weight sugar compound (monosaccharide or disaccharide), egg yolk, and antibacterial / antiviral substances such as antibiotics. NSF has an osmotic pressure of approximately 400 mOsm / kg (e.g., 350-430 mOsm / kg). NSF may also be based on Tris-citrate buffer (egg yolk Tris sugar solution (ET)) with added low egg yolk. Furthermore, it may contain antibiotics such as amikacin, dibekacin, penicillin, and streptomycin.

[0029] A preferred cryopreservation method according to the first embodiment includes: (1) a pretreatment step of pretreating porcine semen to obtain porcine sperm (a high-concentration suspension of porcine sperm in a pretreatment solution); (2) a primary dilution step of diluting the pretreated porcine sperm (high-concentration suspension) with the modified NSF (primary dilution); (3) a secondary dilution step of preparing a secondary dilution solution by adding DMGA-PLL (dimethyl glyceryl phosphate modified polylysine) and glycerin to the modified NSF, and performing secondary dilution by adding the secondary dilution solution to the suspension after primary dilution; and (4) a step of filling into a storage straw, pre-freezing, and then maintaining at a temperature of -60°C or lower.

[0030] Furthermore, from the pre-treatment step to filling the storage straws (thin tubular containers for cryopreservation), the temperature is gradually lowered from around 37°C (e.g., 33-40°C) to around 5°C (e.g., 2-8°C). Pre-freezing can be performed, for example, by placing the filled storage straws in an atmosphere of -5°C to -20°C.

[0031] Preferably, a straw for cryopreservation with a diameter of 5 mm or less is used, and after pre-freezing, the product is cooled to -140°C or below by a slow freezing method with a cooling rate of 100°C / min or less using a programmable freezer, or by a vitrification freezing method with a cooling rate of 300°C / min or more by immersion in liquid nitrogen.

[0032] In a second embodiment of the present invention, the cryopreservation solution for pig embryos (blastocysts) according to the present invention contains 8-12 w / w% of DMGA-PLL (dimethylglycaric acid-modified polylysine) or CPLL (succinic acid-modified polylysine) having a substituted component ratio of 50-99 mol%, 30-35 w / w% of ethylene glycol, 1-3 w / w% of polyethylene glycol, 15-25 w / w% of trehalose, and 15-25 w / w% of fetal bovine serum. Examples of pig embryos to be cryopreserved include pig embryos developed for 4-8 days (preferably 5-7 days) by in vitro fertilization (IVF).

[0033] A preferred cryopreservation method according to the second embodiment includes the steps of sequentially transferring a pig embryo (blastocyst) to a first equilibrium solution, a second equilibrium solution, and the above-mentioned cryopreservation solution, and thereafter maintaining the pig embryo in the cryopreservation solution in an atmosphere of -60°C or lower without pre-freezing, wherein the first equilibrium solution is prepared by adding 5-15% ethylene glycol, 15-25% FBS (fetal bovine serum), and 3-7% DMGA-PLL and / or CPLL solution to a cell culture medium, and the second equilibrium solution is prepared by adding 5-15% ethylene glycol, 0.5-2% polyethylene glycol, 0.2-0.4M trehalose, 15-25% FBS, and 7-13% DMGA-PLL and / or CPLL solution to a cell culture medium.

[0034] When using frozen porcine germ cells, the frozen bovine cells can be thawed and used in accordance with the general thawing methods for various types of cells. Note that antifreeze polyamino acids have low cytotoxicity and, unlike dimethyl sulfoxide and others, do not need to be removed during thawing.

[0035] Furthermore, the cryopreservation solution for porcine germ cells according to the present invention can also be provided as a kit for cryopreserving porcine germ cells. In addition to the cryopreservation composition, the kit may include, for example, a container for cryopreservation, instructions for use with the kit, and so on. [Examples]

[0036] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0037] [Example 1] Cryopreservation of porcine sperm using antifreeze polyamino acids 1-1. Preparation of dimethyl glycerate-modified polylysine (DMGA-PLL) A 25% aqueous solution of ε-poly-L-lysine (JNC, molecular weight 4000) was mixed with 65% mol% of 3,3-dimethylglutaric anhydride (DMGA, Sigma-Aldrich) or succinic anhydride (Tokyo Chemical Industries), and 60 mol% of the amino groups in the ε-poly-L-lysine molecule were carboxylated to create a blocked, antifreeze polyamino acid.

[0038] 1-2. Preparation of primary and secondary dilutions First, as a diluent for porcine semen used in artificial insemination, we used NSF (Niwa and Sasaki freezing extender), which was modified from the conventional formula as shown in Table 1 below. NSF was prepared by mixing the following components, then stretching the mixture and collecting the supernatant.

[0039] Composition of NSF (when prepared in 100 ml) JPEG0007846893000003.jpg41137

[0040] Based on the NSF listed in Table 1 above, the primary and secondary dilutions were prepared as follows. • Primary dilution: A mixture of 99.26% NSF and 0.74% Orvus ES Paste (by volume). Here, Orvus ES Paste is a surfactant used to uniformly dissolve the components contained in egg yolk. Secondary dilution: A mixture of 91.26% NSF, 0.74% Orvus ES Paste, 6% glycerin, and 2% (by volume) of a 25% wt% solution of DMGA-PLL or CPLL.

[0041] 1-3. Cryopreservation of pig sperm When cryopreserving sperm from edible pigs (Duroc), the sperm concentration was measured, and then the sperm was gradually diluted and equilibrated using a standard method with a pretreatment solution (Modena solution), a primary diluent, and a secondary diluent, while the temperature was slowly lowered. In this process, the primary and secondary diluents were used in equal amounts. Specifically, the procedure was as follows:

[0042] (1) Pretreatment The collected semen was diluted with Modena solution warmed to 37°C, centrifuged (3000 rpm, 25°C, 15 minutes), and then the supernatant was removed using an aspirator connected to a Pasteur pipette, taking care not to aspirate the precipitate (sperm). Subsequently, the sperm concentration was 10.0 × 10⁶. 8 The pretreatment solution (Modena solution) was added to achieve a concentration of insects / ml and the mixture was suspended. The Modena solution used had the composition shown in Table 2 below.

[0043] Composition of Modena solution (when preparing 1000 ml) TIFF0007846893000004.tif62134

[0044] (2) Primary Dilution The sperm was again subjected to centrifugation, as in the pretreatment, and the supernatant was removed. The sperm concentration was then 20.0 × 10⁶. 8 The primary diluent was added to achieve a concentration of insects / ml and the mixture was suspended. The mixture was then left to stand in the following order: 30 minutes in a 15°C incubator, 1.5 hours in a 10°C incubator, and 1 hour in a 5°C incubator.

[0045] (3) Secondary Dilution By adding an equal volume of the primary dilution to the suspension obtained from the primary dilution in several stages, the final sperm concentration reached 10.0 × 10⁶. 8 The sample was adjusted to a concentration of animals / ml. It was then left to stand in a 5°C incubator for 20 minutes.

[0046] (4) Filling straws for storage and freezing The sperm diluent prepared as described above was filled into 0.5 ml portions into storage straws and sealed using a straw sealing device. Next, the straws were pre-frozen by holding them 4 cm above the surface of liquid nitrogen for 10 minutes, and then transferred to liquid nitrogen for storage.

[0047] (5) Thawing and artificial insemination Frozen straws were thawed in warm water, and artificial insemination was performed using an injector.

[0048] 1-4. Effects of adding 0.25% by weight of DMGA-PLL and CPLL on pregnancy and farrowing status in pigs. DMGA-PLL (dimethylglycerate-modified polylysine) or CPLL (succinate-modified polylysine) was added to the above secondary dilution solution to a concentration of 0.5% by weight, resulting in a concentration of 0.25% by weight in the cryopreservation solution. As described later, it was shown that the optimal addition concentration to NSF for both CPLL and DMGA-PLL is 0.25% by weight.

[0049] Following the procedure described above, we artificially inseminated female pigs (Landrace x Large Yorkshire) with cryopreserved pig sperm and investigated pregnancy and farrowing status. Simultaneously, we also performed artificial insemination using sperm cryopreserved in a solution without carboxylated lysine, and sperm stored at a low temperature (17°C) for 3 days. The results of our investigation into the effects of these methods on pregnancy and farrowing status in the pigs are summarized in Table 3 below.

[0050] Storage at low temperatures (17°C) was carried out according to the method commonly used in livestock production for artificial insemination of pigs. Specifically, after sperm collection from boars, the sperm was diluted with HIRO-SWINE B solution heated to 37°C, transported in a light-shielding container while maintaining an environment of 25°C, and then slowly cooled to 17°C for storage. Furthermore, experiments using a separate sperm motility analysis system (SMAS) showed no signs of decrease in sperm motility (resting motility and linear velocity) up to 3 days of storage at 15-17°C.

[0051] In Table 3 below, the numbers in parentheses for "Total number of offspring" and "Number of surviving offspring" represent the number per litter. The rightmost column of Table 3 below shows the value obtained by dividing "Number of surviving offspring" by "Number of artificially inseminated offspring." Note that "Number of surviving offspring" refers to the number of offspring that survived at the time of birth.

[0052] Effects of adding CPLL and DMGA-PLL to sperm cryopreservation solution on pregnancy and farrowing in artificially inseminated pigs TIFF0007846893000005.tif58144

[0053] * Percentage of artificial insemination cases ** Percentage of pregnant animals *** Number of live offspring / Number of artificially inseminated offspring a-c There is a significant difference between the different signs of each column (P<0.01 or 0.05).

[0054] According to the results on the far right of Table 3, there was little difference between using a predetermined cryopreservation solution containing 0.25% by weight of DMGA-PLL (dimethyl glitaric acid-modified polylysine) and storing the product at a relatively low temperature (17°C) for a short period without freezing. From this, it was determined that a level of cryopreservation effect applicable to livestock production sites was obtained.

[0055] On the other hand, the effects of the concentrations of DMGA-PLL (dimethylglycerate-modified polylysine) and CPLL (succinate-modified polylysine) on the viability index, in vitro developmental capacity, and in vitro fertilization capacity of thawed pig sperm were investigated prior to the experiments shown in Table 3 above. The results of this experiment are shown in Figures 1 to 6.

[0056] Following the procedure described above, sperm from edible pigs (Duroc) were cryopreserved using a cryopreservation solution prepared by adding 3.0% glycerin and 0, 0.125, 0.25, and 0.5% carboxylated lysine (DMGA-PLL or CPLL) to the modified NSF (trehalose-based) solution. Sperm motility was observed under a microscope 0, 3, 6, 9, and 12 hours after thawing, and the sperm viability index was calculated from the results. Here, the sperm viability index is a numerical representation of the percentage of sperm judged to be motile by microscopic observation and the strength of their motility.

[0057] Furthermore, to investigate the in vitro fertilization ability of cryopreserved pig sperm, thawed sperm were inseminated into in vitro matured eggs recovered and prepared from ovaries derived from a meat processing center. The eggs were fixed and stained 12 hours after insemination to observe the fertilization status.

[0058] Finally, we investigated the in vitro developmental potential of in vitro fertilized eggs produced using porcine sperm cryopreserved in solutions containing various concentrations of CPLL. Oocytes were transferred to developmental culture medium 12 hours after insemination and cultured for a period of time. Cleavage was observed after 2 days, and blastocyst formation after 7 days.

[0059] Figures 1-3 show the effects of DMGA-PLL concentration on the viability index of thawed pig sperm, the in vitro developmental potential of in vitro fertilized eggs produced using thawed pig sperm, and the in vitro fertilization potential of thawed pig sperm, respectively, when DMGA-PLL is used as the carboxylated lysine. Figures 4-5 show the effects of CPLL concentration on the viability index of thawed pig sperm and the in vitro developmental potential of in vitro fertilized eggs produced using thawed pig sperm, respectively, when CPLL is used instead of DMGA-PLL as the carboxylated lysine.

[0060] When using DMGA-PLL, as shown in Figure 1, there was no significant difference in sperm viability index between different concentrations 0-9 hours after thawing. However, the sperm viability index 12 hours after thawing was significantly higher in the group treated with 0.25% DMGA-PLL than in the group without DMGA-PLL. Furthermore, as shown in Figure 2, regarding in vitro developmental potential, the blastocyst formation rate was significantly higher in the group treated with 0.25% DMGA-PLL than in the group without DMGA-PLL. However, the difference in cleavage rate was not significant. Finally, as shown in Figure 3, no significant difference in in vitro fertilization potential was observed with different concentrations.

[0061] When CPLL was used, as shown in Figure 4, there was no significant difference in sperm viability index between different concentrations 0-3 hours after thawing. However, the sperm viability index 6-12 hours after thawing was significantly higher in the groups treated with 0.125-0.25% CPLL than in the untreated group. Furthermore, as shown in Figure 5, regarding in vitro developmental potential, the blastocyst formation rate was significantly higher in the group treated with 0.25% CPLL than in the untreated group. However, the difference in cleavage rate was not significant.

[0062] [Example 2] Cryopreservation of pig embryos (blastocysts) using antifreeze polyamino acids In vitro fertilization was performed using sperm from edible pigs (Duroc), and the resulting early blastocysts were cryopreserved for 3 to 14 days after thawing. The viability of the embryos was then examined one and two days after thawing. Specifically, the procedure was as follows:

[0063] (1) Preparation of basal culture medium, first and second equilibrium solutions, and cryopreservation solution • Basal culture medium: HEPES-PZM-3 as shown in Table 4 below. • First equilibrium solution: Add 10% ethylene glycol, 20% FBS (fetal bovine serum), and 5% CPLL solution to the basal medium. • Second equilibrium solution: Add 10% ethylene glycol, 1% polyethylene glycol, 0.3 M trehalose, 20% FBS, and 10% CPLL solution to the basal medium.

[0064] • Cryopreservation solution: Add to the basal culture medium to contain 33% ethylene glycol, 2% polyethylene glycol, 0.6 M trehalose, 20% FBS, and 20% CPLL solution.

[0065] Composition of basal culture medium (when prepared in 100 ml) TIFF0007846893000006.tif102131

[0066] (2) A series of operations for cryopreservation · Preparation of embryos: Collect blastocysts from mated females or collect those developed from in vitro - cultured eggs after in vitro fertilization. · Treatment with equilibration solution and cryopreservation solution: Dehydrate porcine embryos by sequentially transferring them into the first equilibration solution, the second equilibration solution, and the cryopreservation solution. · Cryopreservation: Place the treated embryos on the tip of a cryotop, immerse them in liquid nitrogen, and store for 3 - 14 days.

[0067] (2) Thawing · Preparation of thawing solution: Add to the basal medium so that it contains 10% ethylene glycol, 0.3 M trehalose, and 20% FBS. · Thawing operation: Immerse the tip of the cryotop taken out from liquid nitrogen into the thawing solution to thaw the embryos. After equilibrating the embryos in the thawing solution, transfer them to the normal medium.

[0068] The results of examining the survival status 1 day and 2 days after thawing are shown in Tables 5 - 10 below. Herein, the expanded blastocysts were obtained 6 days after in vitro fertilization.

[0069] Influence of CPLL concentration in cryopreservation solution on the survival status of thawed early blastocysts derived from porcine in vitro - fertilized eggs TIFF0007846893000007.tif44138

[0070] Number of experiments: 5 times a-b There is a significant difference between different signs (P < 0.01)

[0071] Influence of CPLL concentration in cryopreservation solution on the survival status of thawed expanded blastocysts derived from porcine in vitro - fertilized eggs TIFF0007846893000008.tif45137

[0072] Number of experiments: 5 times a-b There is a significant difference between different signs (P < 0.05)

[0073] As shown in Table 5, when early blastocysts obtained 6 days after in vitro fertilization were cryopreserved, there was no significant difference in survival rate 1 day after thawing between different concentrations. However, the survival rate 2 days after thawing was significantly higher in the group treated with 10% CPLL than in the group treated without CPLL. Similarly, as shown in Table 6, when expanded blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate 1 day after thawing was significantly higher in the group treated with 10% CPLL than in the group treated without CPLL. However, there was no significant difference in survival rate 2 days after thawing between different concentrations.

[0074] As shown in Tables 5-6, when CPLL was used as carboxylated polylysine and the CPLL concentration was approximately 10% by weight, cryopreservation effects were observed for early blastocysts and expanded blastocysts.

[0075] Effect of DMGA-PLL concentration in cryopreservation solution on the viability of thawed porcine in vitro fertilized egg-derived early blastocysts TIFF0007846893000009.tif37137

[0076] Number of experiments: 5 a-b There is a statistically significant difference between the opposite signs (P<0.01 or 0.05).

[0077] Effect of DMGA-PLL concentration in cryopreservation solution on the viability of thawed expanded blastocysts derived from porcine in vitro fertilized eggs TIFF0007846893000010.tif38138

[0078] Number of experiments: 5 a-b There is a statistically significant difference between the opposite signs (P<0.01 or 0.05).

[0079] As shown in Table 7, when early blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate 1 day after thawing was significantly higher in the group treated with 10% DMGA-PLL than in the group without DMGA-PLL. Furthermore, as shown in Table 8, when expanded blastocysts obtained 6 days after in vitro fertilization were cryopreserved, the survival rate 2 days after thawing was significantly higher in the group treated with 10% DMGA-PLL than in the group without DMGA-PLL.

[0080] As shown in Tables 7-8, when DMGA-PLL was used as carboxylated polylysine and the concentration of DMGA-PLL was approximately 10% by weight, cryopreservation effects were observed for early blastocysts and expanded blastocysts.

[0081] Effects of cryoprotective agents on the survival status of thawed early blastocysts derived from in vitro fertilized pig eggs. JPEG0007846893000011.jpg37136

[0082] Number of experiments: 8 a-b There is a statistically significant difference between the opposite signs (P<0.01 or 0.05).

[0083] Effects of cryoprotection agents on the survival status of thawed expanded blastocysts derived from porcine in vitro fertilized eggs. TIFF0007846893000012.tif38137

[0084] Number of experiments: 8 a-b There is a statistically significant difference between the opposite signs (P<0.01 or 0.05).

[0085] Tables 9-10 show a comparison of the cryopreservation effects of CPLL and DMGA-PLL. The number of surviving embryos two days after thawing of expanded blastocysts was significantly higher with DMGA-PLL than with CPLL.

Claims

1. A cryopreservation solution for porcine sperm, comprising 0.2-0.3% by weight of dimethylglycaric acid-modified polylysine having a substituted component ratio of 50-99 mol%, 2-4% by weight of glycerin, 5-15% by weight of trehalose, and 15-25% by weight of egg yolk.

2. The cryopreservation solution for porcine sperm according to claim 1, wherein the content of dimethylglycaric acid-modified polylysine is less than 0.3% by weight and the content of glycerin is 3% by weight or less.

3. A method for cryopreserving porcine sperm using the cryopreservation solution of claim 1 or 2, A pre-treatment process to obtain pig sperm by pre-treating pig semen, A primary dilution step in which pre-treated pig sperm is diluted with a diluent for pig semen, A secondary dilution step is performed in which a secondary dilution solution is prepared by adding dimethyl glyceryl-modified polylysine and glycerin to the diluent for pig semen, and the secondary dilution solution is added to the suspension after the primary dilution so that the pig sperm is suspended in the cryopreservation solution. This process includes filling storage straws, pre-freezing them, and then maintaining them at a temperature of -60°C or lower. A method for cryopreserving pig sperm, wherein the diluent for pig semen contains the trehalose and the egg yolk.

4. The method for cryopreserving pig sperm according to claim 3, characterized by using a cryopreservation straw with a diameter of 5 mm or less and cooling to -140°C or below.

5. A cryopreservation solution for pig embryos containing 8-12% by weight of dimethylglycaric acid-modified polylysine or succinic acid-modified polylysine, in which the proportion of substituted constituent units is 50-99 mol%, 30-35% by weight of ethylene glycol, 1-3% by weight of polyethylene glycol, 15-25% by weight of trehalose, and 15-25% by weight of fetal bovine serum.

6. A method for cryopreserving pig embryos using the cryopreservation solution of claim 5, The process involves sequentially transferring the pig embryos to the first equilibrium solution, the second equilibrium solution, and the above-mentioned cryopreservation solution, This is followed by a step of maintaining the pig embryos in the cryopreservation solution in an atmosphere at -60°C or below without pre-freezing. The first equilibrium solution is prepared by adding a solution of 5-15% ethylene glycol, 15-25% fetal bovine serum, and 3-7% dimethylglycarate-modified polylysine or succinate-modified polylysine to the cell culture medium. A method for cryopreserving pig embryos, wherein the second equilibrium solution is prepared by adding a solution of 5-15% ethylene glycol, 0.5-2% polyethylene glycol, 0.2-0.4 M trehalose, 15-25% fetal bovine serum, and 7-13% dimethylglycarate-modified polylysine or succinate-modified polylysine to the cell culture medium.

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