Application of MG53 in cryopreservation of goat semen and semen freezing diluent containing MG53

Abstract

The invention discloses application of MG53 in cryopreservation of goat semen and a semen freezing diluent containing the MG53. The invention firstly discloses application of MG53 in cryopreservation of goat semen. The invention further discloses a sperm freezing diluent for improving the quality of the frozen sperm of the goat. The invention further discloses a cryopreservation method of the goat semen. According to the invention, MG53 is used as an additive to be applied to a goat semen cryopreservation technology for the first time, so that the problem that sperm motility and fertilization ability are greatly reduced due to damage of plasma membranes and acrosomes of sperms in a goat semen cryopreservation process is solved.

Description

Technical Field

[0001] The present invention relates to the field of animal husbandry, and more specifically, to the application of MG53 in cryopreservation of goat semen and a sperm freezing diluent containing MG53. Background Art

[0002] Semen cryopreservation technology is an ultra-low temperature preservation method that heats up sperm when needed to restore sperm vitality and fertilization ability. Semen cryopreservation is a technology that processes fresh semen collected and then freezes it under liquid nitrogen (-196°C). These treatments usually include semen dilution, addition of cryoprotectants, antioxidants, and gradual cooling. The emergence of semen cryopreservation technology has greatly changed the limitations of traditional semen storage and use, allowing sperm storage time to be extended, thus breaking the time and space limitations of semen use. Through cryopreservation, the storage time of high-quality male livestock and poultry semen is greatly extended, and it can be thawed for artificial insemination when needed, thereby expanding the scope of use of high-quality male livestock and poultry and extending their service life.

[0003] At present, in terms of dairy cow breeding, more than 90% is achieved through artificial insemination using frozen semen. However, the application of frozen semen artificial insemination breeding technology in goats has not been widely promoted and applied. The main reason is that goat sperm is prone to damage to sperm membrane and acrosome during the operation of ultra-low temperature preservation, resulting in low pregnancy efficiency of frozen semen artificial insemination. Therefore, the application scale of cold matching technology (frozen semen artificial insemination breeding technology) in goats is relatively small, and most of them are achieved through laparoscopic deep uterine insemination to improve the purpose of pregnancy efficiency. However, ordinary farms lack relevant equipment and facilities and skilled technicians, which limits the promotion of this technology. Improving the quality of goat frozen semen and using conventional artificial insemination to obtain ideal pregnancy efficiency is a prerequisite for the large-scale promotion of cold matching technology in the goat breeding industry.

[0004] Therefore, it is necessary to provide a product for cryopreservation that can improve the quality of goat frozen semen to solve the above technical problems. Summary of the invention

[0005] One object of the present invention is to provide a new application of MG53 as an additive in the cryopreservation of goat semen, thereby solving the problem that during the cryopreservation of goat semen, the plasma membrane and acrosome of sperm are damaged, resulting in a significant reduction in sperm motility and fertilization ability.

[0006] The second object of the present invention is to provide a sperm freezing diluent for improving the quality of goat frozen semen.

[0007] The third object of the present invention is to provide a method for freezing and preserving goat semen.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] Mitsugumin53 (MG53) is a new muscle-specific member of the TRIM protein family and a membrane repair protein. MG53 maintains the integrity of myocardial and skeletal muscle cells by promoting cell membrane repair. MG53 participates in myocardial ischemic preconditioning and postconditioning protection by activating the PI3K-Akt-GSK3β and ERK1 / 2 cell survival signaling pathways. Moreover, exogenous administration of MG53 recombinant protein can protect multiple organs including the heart, skeletal muscle, lungs, kidneys and skin from ischemia / reperfusion and mechanical damage.

[0010] In the first aspect, the present invention proposes for the first time a new application of MG53 as an additive in cryopreservation of goat semen. The inventors found that adding MG53 to a sperm cryo-diluent to dilute goat semen and then cryopreserving the semen can effectively improve the plasma membrane integrity, acrosome integrity, mitochondrial activity and / or DNA integrity of goat sperm after cryopreservation and thawing. In the present invention, there is no need to limit the composition of the sperm cryo-diluent. It can be any sperm cryo-diluent in the prior art that can be used for semen dilution during cryopreservation of goat sperm. It can be purchased commercially or prepared according to the method reported in the literature. The performance of MG53 in improving the quality of goat frozen semen is not affected by the formula of the sperm cryo-diluent.

[0011] According to a specific embodiment of the present invention, the added concentration of MG53 is 0.4%-1.2%, specifically 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, etc.;

[0012] Preferably, the added concentration of MG53 is 0.8%.

[0013] In a second aspect, the present invention provides a sperm freezing diluent for improving the quality of goat frozen semen, wherein the sperm freezing diluent comprises liquid A and liquid B, wherein:

[0014] Solution A includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibiotic 1200-1800 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%;

[0015] Solution B includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%, and glycerol 8%-10%.

[0016] Exemplarily, the sperm freezing diluent includes liquid A and liquid B, wherein:

[0017] Solution A includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibiotic 1500 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%;

[0018] Solution B includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%, and glycerol 8%.

[0019] In a third aspect, the present invention further provides a method for cryopreservation of goat semen, the method comprising the following steps:

[0020] The collected goat semen is diluted to the required concentration with sperm freezing diluent A solution, mixed and placed in a 4°C environment for equilibration for 2-4 hours to obtain semen-containing solution A, and then sperm freezing diluent B solution is added at least twice, the total volume of the B solution is equal to the volume of the semen-containing solution A, and then cryopreserved;

[0021] The sperm freezing diluent includes liquid A and liquid B, wherein:

[0022] Solution A includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibiotic 1200-1800 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%;

[0023] Solution B includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%, and glycerol 8-10%.

[0024] Further, the sperm freezing diluent includes liquid A and liquid B, wherein:

[0025] Solution A includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibiotic 1500 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%;

[0026] Solution B includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%, and glycerol 8%.

[0027] Furthermore, when adding Solution B in batches, there should be at least 15 minutes between each addition.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention is the first to apply MG53 as an additive to goat semen cryopreservation technology. By adding MG53 to sperm cryo-diluent, the quality indicators such as sperm plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity, and DNA integrity rate after freezing and thawing can be effectively improved. The improvement of frozen sperm quality will promote the improvement of artificial insemination conception rate. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. Similar components are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0031] In the following examples, MG53 was purchased from Ruibosai Biotechnology Co., Ltd., TP750051.

[0032] The goat sperm freezing diluent used in the embodiment includes liquid A and liquid B, wherein:

[0033] Solution A includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 1500 IU / L, glucose 6 g / L, egg yolk 20%, and different concentrations of MG53;

[0034] Solution B includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6 g / L, egg yolk 20%, different concentrations of MG53, and glycerol 8%.

[0035] In the present invention, unless otherwise specified, the percentage concentration (%) of each component refers to the volume percentage concentration (% (v / v)) of the component.

[0036] Example 1

[0037] MG53 was added to the goat sperm cryo-dilution buffer at concentrations of 0%, 0.4%, 0.8% and 1.2%.

[0038] The freezing method is as follows: the collected goat semen is diluted to the required concentration with sperm freezing diluent A solution, mixed and placed in a 4°C environment for equilibrium for 2-4 hours to obtain semen-containing solution A, and then sperm freezing diluent B solution is added twice (each time with an interval of 15 minutes), the total volume of the B solution is equal to the volume of semen-containing solution A, and the semen is divided into 0.25ml frozen sperm tubes using a filling machine and placed in a program freezer for freezing. After the program is completed, it is placed in liquid nitrogen for long-term storage.

[0039] The results showed that the 0.4%, 0.8% and 1.2% groups significantly increased the plasma membrane integrity rate of Saanen goat sperm after thawing (P < 0.05), and there was a significant difference compared with the control group (0% group) (P < 0.05). Among them, the plasma membrane integrity rate of sperm in the 0.8% group after thawing was significantly higher than that in the 0.4% group and the 1.2% group (P < 0.05); however, with the increase of the concentration of MG53 added, the plasma membrane integrity rate of sperm showed a downward trend; there was no significant difference in the plasma membrane integrity rate of sperm between the 0.4% and 1.2% groups (P > 0.05). According to the experimental results, it can be concluded that adding 0.4%-1.2% MG53 to conventional goat sperm cryo-dilution has a protective effect on the integrity of sperm plasma membrane, and the effect of adding 0.8% MG53 is the best.

[0040] Table 1 Effects of different concentrations of MG53 on the plasma membrane integrity of dairy goat sperm after freezing and thawing

[0041]

[0042] Note: First, the data were tested for normality, and then one-way ANOVA was performed if they met the normal distribution. The data in the table have different letters, which means the difference is significant (P < 0.05), and the same letters mean the difference is not significant (P > 0.05) (the same below).

[0043] Example 2

[0044] MG53 was added to goat sperm cryo-dilution solution at concentrations of 0%, 0.4%, 0.8% and 1.2% respectively. The freezing method was the same as in Example 1. The results showed that the 0.8% group and the 1.2% group significantly increased the acrosome integrity rate of sperm compared with the control group (0% group) (P < 0.05), and there was no significant difference in the acrosome integrity rate between the 0.8% group and the 1.2% group (P > 0.05); with the increase in the concentration of MG53 added, the sperm acrosome integrity rate showed a downward trend; the acrosome integrity rate of the 0.4% group was slightly higher than that of the control group, but there was no significant difference between the two groups (P > 0.05); there was no significant difference in the acrosome integrity rate between the 0.4% group and the 1.2% group (P > 0.05). According to the experimental results, it can be concluded that adding 0.8% MG53 to conventional goat sperm cryo-dilution solution has the best effect on protecting the integrity of the sperm plasma membrane.

[0045] Table 2 Effects of different concentrations of MG53 on the acrosome integrity rate of dairy goat sperm after freezing and thawing

[0046]

[0047] Example 3

[0048] MG53 was added to goat sperm cryo-dilution solution at concentrations of 0%, 0.4%, 0.8% and 1.2% respectively. The freezing method was the same as in Example 1. The results showed that the 0.8% group significantly increased the mitochondrial activity of dairy goat sperm after thawing compared with the control group (0%) (P < 0.05), among which the 0.4% group and the 1.2% group were slightly improved compared with the control group, but there was no significant difference in mitochondrial activity among the three groups (P > 0.05). According to the experimental results, it can be concluded that adding 0.8% MG53 to conventional goat sperm cryo-dilution solution has the best effect on improving sperm mitochondrial activity.

[0049] Table 3 Effects of different concentrations of MG53 on mitochondrial activity of dairy goat sperm after freezing and thawing

[0050]

[0051] Example 4

[0052] MG53 was added to goat sperm freezing diluent at concentrations of 0%, 0.4%, 0.8% and 1.2%, respectively. The freezing method was the same as in Example 1. The results showed that the 0.8% group and the 1.2% group significantly improved the DNA integrity rate of sperm compared with the control group (0%) (P < 0.05), and there was no significant difference in the DNA integrity rate between the 0.8% group and the 1.2% group (P > 0.05); with the increase in the concentration of MG53 added, the sperm DNA integrity rate showed a downward trend; the acrosome integrity rate of the 0.4% group was slightly higher than that of the control group, but there was no significant difference between the two groups (P > 0.05). According to the experimental results, it can be concluded that adding 0.8% MG53 to conventional goat sperm freezing diluent has the best protective effect on sperm DNA integrity.

[0053] Table 4 Effects of different concentrations of MG53 on sperm DNA integrity rate after freezing and thawing of dairy goat sperm

[0054]

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of MG53 in cryopreservation of goat semen.

2. The use according to claim 1, It is characterized in that MG53 was added to sperm cryo-dilution solution for semen dilution before cryopreservation of goat semen.

3. The use according to claim 1, It is characterized in that The added concentration of MG53 is 0.4%-1.2%.

4. The use according to claim 1, It is characterized in that The added concentration of MG53 is 0.8%.

5. The use according to claim 1, It is characterized in that The MG53 can improve the plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity and / or DNA integrity rate of goat sperm after freezing and thawing.

6. A sperm freezing diluent for improving the quality of goat frozen semen, It is characterized in that The sperm freezing diluent includes liquid A and liquid B, wherein: Solution A includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibiotic 1200-1800 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%; Solution B includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%, and glycerol 8-10%.

7. The sperm freezing diluent according to claim 6, It is characterized in that The sperm freezing diluent includes liquid A and liquid B, wherein: Solution A includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 1500 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%; Solution B includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%, and glycerol 8%.

8. A method for freezing and preserving goat semen, It is characterized in that The method comprises the following steps: The collected goat semen is diluted to the required concentration with sperm freezing diluent A solution, mixed and placed in a 4°C environment for equilibration for 2-4 hours to obtain semen-containing solution A, and then sperm freezing diluent B solution is added at least twice, the total volume of the B solution is equal to the volume of the semen-containing solution A, and then cryopreserved; The sperm freezing diluent includes liquid A and liquid B, wherein: Solution A includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibiotic 1200-1800 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%; Solution B includes the following components: Tris 25-38 g / L, citric acid 12-22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6-15 g / L, egg yolk 20%-23%, MG53 0.4%-1.2%, and glycerol 8%-10%.

9. The cryopreservation method according to claim 8, It is characterized in that The sperm freezing diluent includes liquid A and liquid B, wherein: Solution A includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 1500 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%; Solution B includes the following components: Tris 38 g / L, citric acid 22 g / L, penicillin-streptomycin dual antibody 5 IU / L, glucose 6 g / L, egg yolk 20%, MG53 0.8%, and glycerol 8%.

10. The cryopreservation method according to claim 8, It is characterized in that When adding Solution B in batches, there should be at least 15 minutes between each addition.

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