Semen freezing liquid and its application

Abstract

The invention discloses a semen freezing liquid and its application. The semen freezing liquid of the invention comprises melatonin and N,N-dimethylformamide. The invention also discloses the application of melatonin in the cryopreservation of semen of poultry animals. The semen freezing liquid of the invention can significantly improve the indexes such as sperm motility rate, plasma membrane integrity, acrosome integrity, etc. after thawing, reduce the level of ROS in semen, improve the level of sperm mitochondrial membrane potential, and improve the hatching rate of artificial insemination of semen after thawing, and can be used in the fields of poultry semen cryopreservation, germplasm resource preservation and utilization.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a semen freezing liquid and an application thereof. Background Art

[0002] Beijing Oil Chicken is an excellent local chicken breed in China, with fine meat and rich egg flavor. It is a meat and egg dual-purpose chicken breed with a medium body and the "three yellow" characteristics of yellow feathers, yellow beak and yellow shanks. The preservation of Beijing Oil Chicken is particularly important for the genetic improvement of the breed and the diversity of genetic resources.

[0003] Semen freezing is a necessary means for animal conservation and gene bank establishment. Cryopreservation technology can preserve the genetic resources of rare animal breeds and poultry with excellent production performance. It can store and transport valuable genetic materials economically and labor-savingly, and it also has great advantages in reducing disease risks. Since 1949, when Polge's team successfully hatched chicks by adding glycerol as a cryoprotectant to chicken semen diluent, scholars from various countries have conducted many studies on technical aspects such as poultry semen diluent, cryoprotectant, freezing procedure, and thawing process.

[0004] Sperm is susceptible to oxidative stress in vitro and during freezing. This is because the cell membrane of sperm is rich in polyunsaturated fatty acids and is very sensitive to reactive oxygen species (ROS) produced during in vitro culture, leading to lipid peroxidation (LPO), followed by a chain reaction of cytotoxic aldehydes (such as malondialdehyde), which greatly affects the fertilization rate. Since the semen physiology and fertilization physiology of poultry and livestock are very different, and compared with mammalian sperm, poultry sperm is more sensitive to low-temperature freezing due to its unique cell morphology and physiological characteristics. Therefore, it is necessary to develop sperm cryopreservation fluid based on the characteristics of poultry sperm. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a semen freezing liquid and its application in semen cryopreservation. In the present invention, N,N-dimethylformamide is used as a cryoprotectant, and melatonin is added exogenously during the semen cryopreservation process, which can improve semen quality and increase the hatching rate after artificial insemination, especially providing a theoretical basis for poultry seed preservation and production practice.

[0006] The first aspect of the present invention provides the use of melatonin in the cryopreservation of semen of avian animals.

[0007] In some embodiments, the poultry includes pheasants and ducks, preferably selected from chickens, ducks, geese and quails. In some specific embodiments, the poultry is selected from Beijing oil chicken.

[0008] A second aspect of the present invention provides a semen freezing solution comprising melatonin and N,N-dimethylformamide.

[0009] In some embodiments, the freezing solution further comprises a basic diluent. In some embodiments, the basic diluent comprises glucose, sodium glutamate, magnesium acetate, polyvinyl pyrrolidone and potassium acetate.

[0010] In some embodiments, the concentration of melatonin is 10 -7 mol / L to 10 -2 mol / L, for example, 10 - 7 mol / L、10 -6 mol / L、10 -5 mol / L、10 -4 mol / L、10 -3 mol / L、10 -2 mol / L or any value therebetween. In some preferred embodiments, the concentration of melatonin is 10 -4 mol / L to 10 -2 mol / L, more preferably 10 -4 mol / L to 10 -3 mol / L, most preferably 10 -3 mol / L.

[0011] According to some embodiments of the present invention, exogenously added 10 -7 mol / L to 10 -3 Melatonin at a concentration of 10 mol / L can improve the viability and vitality of sperm after thawing. -3 M and 10 -5 The addition of melatonin to M can significantly improve sperm motility, -3 M, 10 -5 M and 10 -7 The addition of melatonin to M significantly increased sperm motility, but had no significant effect on sperm motility-related indicators. -3 Melatonin addition can significantly improve the plasma membrane integrity and acrosome integrity of sperm. In addition, melatonin can significantly reduce the level of sperm ROS and improve the level of sperm mitochondrial membrane potential, thereby significantly improving the hatching rate of sperm after artificial insemination. These results show that exogenous addition of melatonin during cryopreservation has a positive effect on various indicators of sperm after thawing.

[0012] In some embodiments, the volume concentration of the N,N-dimethylformamide is 10%-20%, for example, 10%, 12%, 14%, 16%, 18%, 20% and any value therebetween. In some embodiments, the volume concentration of the N,N-dimethylformamide is 10%-150%.

[0013] In some embodiments, based on the total weight of the basic diluent, the basic diluent includes 5-15 parts of glucose, 10-30 parts of sodium glutamate, 0.5-1.5 parts of magnesium acetate, 1-5 parts of polyvinyl pyrrolidone, 2-10 parts of potassium acetate and 1000 parts of water.

[0014] A third aspect of the present invention provides a method for cryopreservation of semen, the method comprising mixing the semen freezing liquid described in the second aspect with semen.

[0015] In some embodiments, the volume ratio of the semen freezing liquid to the semen is (1:2)-(2:1), for example, 1:1.

[0016] In some embodiments, the cryopreservation is performed in liquid nitrogen.

[0017] In some embodiments, cryopreservation is performed by freezing in straws.

[0018] In some embodiments, the method comprises the following steps:

[0019] (1) mixing semen with semen freezing solution for balancing;

[0020] (2) Seal the balanced mixed solution in a capillary tube;

[0021] (3) Freeze the tube in liquid nitrogen.

[0022] In some embodiments, the equilibration temperature is 1° C.-10° C., preferably 5° C. In some embodiments, the equilibration time is 10 min-60 min, preferably 30 min.

[0023] In some embodiments, the capillary is a 0.25 ml capillary.

[0024] In some embodiments, the inner diameter of the capillary is 2.5 mm.

[0025] In some embodiments, the freezing includes first fumigating the capillary tube at a distance of 10-20 cm from the liquid nitrogen surface for 2 min-10 min (freezing rate is about -14°C / min to -9°C / min), then slowly lowering the capillary tube to the surface of liquid nitrogen, fumigating it on the liquid nitrogen surface for 1 min-5 min (freezing rate is about -60°C / min to -25°C / min), and then completely immersing it in liquid nitrogen.

[0026] The fourth aspect of the present invention provides the use of the semen freezing solution described in the second aspect in the cryopreservation of animal semen.

[0027] In some embodiments, the application is one or more of the following applications:

[0028] (1) Improve the survival rate of sperm after thawing;

[0029] (2) Improve the motility of sperm after thawing;

[0030] (3) Improve the plasma membrane integrity of sperm after thawing;

[0031] (4) Improve the acrosome integrity of sperm after thawing;

[0032] (5) reduce the level of ROS in sperm after thawing;

[0033] (6) Improve the mitochondrial membrane potential level of sperm after thawing;

[0034] (7) Improve the hatching rate of sperm after artificial insemination.

[0035] In some embodiments, the animal is selected from poultry. In some preferred embodiments, the animal includes pheasants and ducks, preferably selected from chickens, ducks, geese and quails. In some specific embodiments, the poultry is selected from Beijing oil chicken.

[0036] In the semen freezing liquid of the present invention, melatonin and N,N-dimethylformamide are used as the effective ingredients of the cryopreservative, and the two cooperate with each other to well protect the physiological and biochemical activity of sperm during the cryopreservation of poultry semen. Melatonin, as a powerful antioxidant and anti-apoptotic agent, can directly remove toxic oxygen derivatives and reduce the formation of active oxygen. N,N-dimethylformamide has a weak toxic effect on sperm, and has the characteristics of easy operation and no need for centrifugation during artificial insemination.

[0037] The semen freezing liquid of the present invention also has the following beneficial effects:

[0038] (1) The use of the semen freezing solution of the present invention can significantly improve the indexes such as sperm motility, plasma membrane integrity, acrosome integrity, etc. after thawing, reduce the level of ROS in semen, improve the level of sperm mitochondrial membrane potential, and improve the hatching rate of artificial insemination of semen after thawing. It has no effect on the health of hens after insemination, and can be widely promoted.

[0039] (2) The semen freezing solution of the present invention is simple to prepare and operate, and can effectively improve the motility and hatching rate of frozen-thawed sperm, providing technical support for research in the fields of poultry semen cryopreservation, germplasm resource preservation and utilization.

[0040] (3) The semen freezing method of the present invention can improve the quality of semen after thawing and the hatching rate after artificial insemination. The operation is simple and fast, the equipment is simple, and the method is simple and effective.

[0041] (4) The semen freezing liquid of the present invention is mainly suitable for the cryopreservation of semen of poultry, especially Beijing oily chickens and roosters. It can effectively improve the quality of semen after thawing, increase the hatching rate after artificial insemination, greatly improve the utilization efficiency and seed preservation efficiency of breeding roosters, and greatly improve the breeding benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A graph showing the effects of different concentrations of melatonin on post-thawing sperm motility, sperm viability and sperm movement-related indicators according to some embodiments of the present invention.

[0043] Figure 2 The effect of melatonin on post-thaw sperm plasma membrane integrity and acrosome integrity according to some embodiments of the present invention is shown.

[0044] Figure 3 The effect of melatonin on ROS levels in thawed Beijing oil chicken sperm according to some embodiments of the present invention is shown.

[0045] Figure 4 The effect of melatonin according to some embodiments of the present invention on the mitochondrial membrane potential level of sperm of Beijing oil chicken after thawing is shown. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0047] The reagents and materials used in the following examples are all commercially available products. Melatonin (MT) used was purchased from Sigma.

[0048] Example

[0049] 1. Experimental Materials and Methods

[0050] 1.1 Experimental animals

[0051] The 12 Beijing Oil Chicken roosters and 50 Beijing Oil Chicken laying hens used in this study were all from the Poultry Genetic Resources and Breeding Experimental Base of China Agricultural University. The experimental chickens were raised under the same conditions, with free access to feed and water. The feeding management and immunization procedures were carried out in accordance with the normal production procedures of the experimental chicken farm of China Agricultural University.

[0052] 1.2 Semen collection and initial evaluation

[0053] The single-person semen collection method of dorsal and abdominal combined massage was used, and semen was collected every 48 hours. Before semen collection, the centrifuge tube containing the diluent was kept warm at about 37°C and placed in a constant temperature box to prevent temperature difference shock to the sperm. The collected semen was immediately diluted 1:1 and gently shaken and placed in an incubator. Each rooster took about 1 minute and was quickly brought back to the laboratory within 15 minutes. Sperm motility was subjectively estimated under a 400X microscope, and the semen volume was measured in microliters with a micropipette. Semen with >60% motility was pooled and divided into five equal parts for subsequent experiments.

[0054] 1.3 Solution preparation

[0055] Basic diluent: weigh 8.0 g of glucose, 19.2 g of sodium glutamate, 0.8 g of magnesium acetate compound, 3.0 g of polyvinyl pyrrolidone and 5.0 g of potassium acetate, dissolve in 1 L of distilled water, test the osmotic pressure (340-350 mOsm / kg) and pH (7.0-7.2), filter and sterilize, divide into 10 mL, store at -20 °C, and thaw at room temperature 1 h before use.

[0056] Melatonin concentrated stock solution: Accurately weigh 0.0232 g of melatonin and dissolve it in 1 mL of DMSO to obtain 10 -1 mol / L concentrated stock solution was obtained by dilution.

[0057] Dilution: No or different concentrations (10 -9 mol / L、10 -7 mol / L、10 -5 mol / L、10 -3 mol / L) of melatonin.

[0058] N,N-dimethylformamide freezing solution: Add appropriate amount of N,N-dimethylformamide (Shanghai McLean, China) to the basic diluent to prepare N,N-dimethylformamide freezing solution with a volume concentration of 12%.

[0059] Semen freezing solution: No or different concentrations of melatonin were added to 12% N,N-dimethylformamide freezing solution.

[0060] 1.4 Sperm freezing and thawing

[0061] Balance: Place the diluted semen in a 5℃ refrigerator for 30 minutes, then use a pre-cooled pipette and gun tip to slowly rotate the pre-cooled semen freezer along the wall of the EP tube in a 1:1 ratio into the balanced chicken diluted semen in a 5℃ environment. Shake well to mix thoroughly, place in a 5℃ constant temperature box for 10 minutes, and the final concentration of N,N-dimethylformamide is 6%.

[0062] Freezing: The balanced semen was sucked into a 0.25 ml straw (purchased from IMV Technlogies) and sealed with sealing powder. The straw was fumigated for 4 minutes at a distance of 17 cm from the liquid nitrogen surface (freezing rate was about -14℃ / min to -9℃ / min), then the straw was slowly lowered to the liquid nitrogen surface, fumigated on the liquid nitrogen surface for 2 minutes (freezing rate was about -60℃ / min to -25℃ / min), and then completely immersed in liquid nitrogen.

[0063] Thawing: Use tweezers to remove the tube from liquid nitrogen and place it in a 40°C warm water bath to thaw for about 10 seconds. Quickly take it out, dry it, cut it with scissors and examine it under a microscope.

[0064] 1.5 Data Analysis

[0065] The data were processed by Excel software and SPSS 26.0 statistical software was used for one-way analysis of variance and significance test. P < 0.05 was considered significant.

[0066] Example 1: Effects of melatonin on sperm motility, viability, and motility-related indicators of thawed Beijing chicken sperm

[0067] Sperm quality assessment was performed using a computer-assisted semen analyzer (CASA), Tsinghua Tongfang Sperm Analyzer MX 7.0 version at 37°C. For each sample, the computer randomly selected five fields for analysis. The following variables were measured: sperm motility, sperm motility, mean curvilinear velocity (VCL) (μm / s), mean linear velocity (VSL) (μm / s), mean path velocity (VAP) (μm / s), mean lateral amplitude of sperm (ALH) (μm) \ linearity of movement (LIN) (%), oscillatory nature of movement (WOB) (%), forward direction of movement (SRT) (%), and mean angle of sperm movement (MAD) (degrees).

[0068] The data on the effects of different concentrations of melatonin on the movement of Beijing oil chicken sperm after dilution, cooling, equilibrium and freezing and thawing are shown in Figure 1 ,in Figure 1 A to C show the effects of melatonin on post-thaw semen vitality (A), semen motility (B) and sperm motility-related indices (C); the bars represent the mean ± SEM, and different letters above the bars indicate significant differences (P < 0.05).

[0069] Figure 1 The results of A showed that adding different concentrations of melatonin during the freezing process of chicken semen could improve the motility of semen after thawing, which was 10 times higher than that of the control group. -3 M and 10 -5 The effect of melatonin addition on semen motility was significantly different (P<0.05), and 10 -3M melatonin added to sperm motility.

[0070] Figure 1 The results of the study showed that adding different concentrations of melatonin during the freezing process of chicken semen could increase the motility of semen after thawing, and 10 -3 M, 10 -5 M and 10 -7 The effect of melatonin addition on semen motility in M ​​was significantly different from that in the control group.

[0071] Figure 1 The results in Figure C showed that the addition of melatonin had no significant effect on other sperm motility variables (ALH, MAD, VSL, VAP, VCL, LIN, WOB, and SRT) in thawed semen samples.

[0072] Example 2: Effect of melatonin on sperm plasma membrane integrity and sperm acrosome integrity after thawing

[0073] The effect of melatonin addition on the plasma membrane integrity of thawed semen was evaluated by the hypoosmotic swelling test (HOST). 10 μL of thawed semen was added to a hypoosmotic solution composed of (sodium citrate 1.9 mM, fructose 5.0 mM) and then incubated at 37°C for 30 min. After that, a drop of semen was placed on a preheated slide and then covered with a coverslip and then observed under a microscope (Olympus, Tokyo, Japan), a total of 200 sperm were counted, and the percentage of sperm with curved and enlarged flagella was recorded.

[0074] The effect of adding melatonin on the acrosome integrity of thawed semen was evaluated by the PNA-FITC method. Fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) fluorescent dye was used for staining to evaluate the acrosome status of sperm. The dye was purchased from Sigma. 10 μL of the diluted sample was mixed with PNA working solution (1 μg / mL; final concentration was 0.02 μg / mL) and incubated at room temperature in the dark for 10 minutes. Using a flow cytometer, the FITC channel was selected to measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0075] Example 1 Discovery 10 -3 M melatonin has a significant effect on improving semen vitality and viability. -3 M melatonin was used as the experimental group (hereinafter referred to as the melatonin group) to evaluate the effect of melatonin on the plasma membrane integrity and acrosome integrity of Beijing oil chicken semen after thawing. Figure 2 shown. Figure 2In the figure, A is the analysis of sperm plasma membrane integrity after thawing, B is the analysis of sperm acrosome fluorescence intensity after thawing, CE is the flow cytometry analysis of sperm acrosome fluorescence intensity measured by PNA-FITC method, the bars represent mean ± SEM, n = 8, different letters above the bars indicate significant differences (P < 0.05).

[0076] Figure 2 The results of A show that adding 10 -3 Melatonin from M can significantly improve the plasma membrane integrity of semen (P<0.05). Figure 2 The results of BE showed that 10 -3 M-melatonin can significantly improve the acrosome integrity of semen (P<0.05). Specifically, the fluorescence intensity of the melatonin group after dye staining was significantly higher than that of the control group.

[0077] Example 3: Effect of melatonin on reactive oxygen species (ROS) levels in sperm after thawing

[0078] The reactive oxygen species detection kit (Solarbio-D6470) was used to detect the level of reactive oxygen species in cells using the fluorescent probe DCFH-DA. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. The semen and working solution were incubated in a cell culture incubator at 37°C for 20 minutes. Invert and mix every 3 to 5 minutes to allow the probe and sperm to fully contact. The sperm was washed three times with PBS to fully remove the DCFH-DA that did not enter the sperm. The flow cytometer was used to select the FITC channel to measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0079] Effect of melatonin on ROS levels in thawed Beijing chicken sperm Figure 3 As shown, A is a flow cytometry analysis of ROS levels in sperm after thawing measured by DCFH-DA fluorescent probe, the fluorescence intensity of the FITC channel represents the ROS level in sperm, and B is a statistical analysis of ROS levels. The bars represent mean ± SEM, n = 10000, and different letters above the bars indicate significant differences (P < 0.05). Figure 3 The results showed that the addition of melatonin can significantly reduce the ROS level of sperm, thereby improving semen quality.

[0080] Example 4: Effect of melatonin on sperm mitochondrial membrane potential level

[0081] The changes in mitochondrial membrane potential of pig sperm during room temperature storage were detected by combining the JC-10 method with flow cytometry. The mitochondrial membrane potential detection kit (Solarbio-CA1310) was used for determination. The specific operation is as follows: Take 100,000 to 600,000 sperm after thawing, resuspend them in 0.5 mL of basic diluent, add 0.5 mL of JC-10 staining working solution, and invert several times to mix. Incubate at 37°C in a cell culture incubator for 20 minutes; after the incubation at 37°C, centrifuge at 600g at 4°C for 3 to 4 minutes to precipitate the sperm. Discard the supernatant, and be careful not to aspirate the sperm as much as possible; wash twice with JC-10 staining buffer (1×), resuspend with an appropriate amount of JC-10 staining buffer (1×), and detect and analyze with a flow cytometer. The results are as follows Figure 4 As shown, A and B are flow cytometric graphs of sperm mitochondrial membrane potential levels in the control group and melatonin group after thawing measured by JC-10 staining method, respectively, and C is a statistical analysis graph of mitochondrial membrane potential levels, V = (Q1 + Q2) / (Q2 + Q3), bars represent mean ± SEM, n = 10000.

[0082] Figure 4 The results showed that the mitochondrial membrane potential level of sperm tended to increase after adding melatonin compared with the control group, and the difference was not significant. This indicates that after adding melatonin, ROS is reduced, thereby promoting the increase of mitochondrial membrane potential, restoring mitochondrial damage, and thus improving sperm apoptosis.

[0083] Example 5: Effect of melatonin on hatching rate

[0084] After the semen is thawed, 10 laying hens in each group are used for single-person insemination experiments. The insemination time is generally after laying eggs at 4 pm. The specific operation is: use a glass rubber-tipped dropper for deep vaginal insemination. Grab the chicken with your right hand, clamp it with your feet, and gently massage the back of your left hand 2-3 times. After the concentric circles of the hen's cloaca are exposed, slowly insert the glass tube into the chicken's vagina about 4cm with your right hand to inject the thawed semen. Insemination is performed twice in a row for the first time, and once every 2 days thereafter.

[0085] The eggs are collected from the third day after insemination, and stopped on the fifth day after the last insemination. The eggs are taken back to the laboratory and disinfected with 70% alcohol spray before incubation. The incubation temperature is 38.5℃ and the humidity is 65%-70% for 1-18 days, and 37.5℃ and the humidity is 70%-75% for 19-21 days. The eggs are candled on the seventh day of incubation.

[0086] Table 1 lists the hatching rates of frozen Beijing chicken sperm in the control group and melatonin group. The values ​​with different superscripts in the column are significantly different (P<0.05), n=10000. The results in Table 1 show that the addition of different concentrations of melatonin will increase the hatching rate after artificial insemination after thawing, among which 10 -3The hatching rate of artificial insemination with frozen semen in the M melatonin group was significantly higher than that in the control group, indicating that the addition of melatonin has a positive effect on the hatching rate.

[0087] Table 1

[0088]

[0089] The above description is only a general description of the present invention and a description of a specific embodiment, and does not limit the present invention in other forms. Any person skilled in the art can modify or modify the present invention based on the technical content disclosed in the present invention to an equivalent embodiment. Without departing from the concept and spirit of the present invention, any modification or modification of the present invention belongs to the protection scope of the technical solution of the present invention.

Claims

1. A semen freezing solution, comprising melatonin, N,N-dimethylformamide and a basic diluent; The basic diluent includes glucose, sodium glutamate, magnesium acetate, polyvinyl pyrrolidone and potassium acetate; The concentration of melatonin is 10 -3 mol / L; The volume concentration of the N,N-dimethylformamide is 12%; Based on the total weight of the basic diluent, the basic diluent includes 5-15 parts of glucose, 10-30 parts of sodium glutamate, 0.5-1.5 parts of magnesium acetate, 1-5 parts of polyvinyl pyrrolidone, 2-10 parts of potassium acetate and 1000 parts of water.

2. A method for cryopreservation of semen, comprising mixing the semen freezing liquid described in 1 with semen.

3. The method according to claim 2, characterized in that The volume ratio of the semen freezing liquid to the semen is (1:2)-(2:1).

4. The method according to claim 2, characterized in that: The cryopreservation was performed in liquid nitrogen.

5. Use of the semen freezing solution according to claim 1 in the cryopreservation of animal semen.

6. The use according to claim 5, characterized in that: The animal is selected from avian animals.

7. The use according to claim 6, characterized in that: The avian animals include pheasants and ducks.

8. The use according to claim 6, characterized in that: The poultry animals are selected from the group consisting of chickens, ducks, geese and quails.

Images