A method for freezing and preserving bovine semen

Abstract

The present invention provides a method for freezing and preserving bovine semen, and relates to the technical field of semen freezing. The method for freezing and preserving bovine semen mainly comprises the following steps: feeding the breeding cattle with mixed folic acid, lycopene, anthocyanin, and anisene in the early stage, artificially collecting semen, adding caffeine, dendrobium phenol, chlorogenic acid, and pinoresinol diglucoside to a basic semen diluent, diluting the bovine semen, precooling the diluted bovine semen, and finally subjecting the precooled semen to low-temperature treatment and then freezing with liquid nitrogen. The present invention overcomes the shortcomings of the prior art, can effectively reduce the effect of freezing on the vitality of bovine semen, ensure the motility and plasma membrane integrity of bovine sperm after freezing, and provide a good foundation for cattle breeding.

Description

Technical Field

[0001] The invention relates to the technical field of semen freezing, and in particular to a method for freezing and preserving bovine semen. Background Art

[0002] Semen cryopreservation technology is a new reproductive technology discovered by Italian scientist Spallanzanie in 1787. The purpose of this technology is to prolong the survival time and lifespan of sperm in vitro, that is, to keep sperm in a relatively static state by lowering the environmental temperature of the sperm. This technology is conducive to the efficient use of the genetic resources of excellent breeding animals and greatly promotes the breeding and improvement process of livestock. At the same time, it improves the reproduction rate of animals, reduces the spread of diseases, improves animal health and biosafety, reduces the cost of animal husbandry, and improves economic benefits; the application of this technology also promotes the protection of endangered species and the development of human reproductive medicine.

[0003] In animal husbandry, the breed of breeding stock has a great impact on the benefits of production and breeding. In order to ensure the breeding and improvement of excellent breeds, artificial insemination is an important breeding method, and semen cryopreservation is a supporting process for artificial insemination, especially for cattle breeding. However, the existing cryopreservation of semen of breeding cattle is prone to a significant reduction in semen vitality and oxidative damage, which brings certain troubles to subsequent artificial insemination.

[0004] Generally speaking, the oxidative damage of bovine semen is mainly caused by the drastic change in temperature during the freezing process, which causes oxidative stress in sperm, lipidation of sperm membrane, acrosome damage, mitochondrial membrane depolarization and changes in plasma membrane permeability, causing irreversible damage to sperm, destroying sperm structure, integrity and genetic material, causing sperm to lose the ability to interact with the female reproductive tract and thus fertilization failure. This damage is one of the determining factors affecting the effect of sperm cryopreservation. Therefore, how to improve the freezing method of bovine semen and thus enhance the vitality of bovine semen after freezing is an important research direction at this stage. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a bovine semen freezing and preservation method, which can effectively reduce the impact of freezing on the activity of bovine semen, ensure the motility and plasma membrane integrity of bovine sperm after freezing, and provide a good foundation for cattle breeding.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0007] A method for freezing and preserving bovine semen, comprising the following steps:

[0008] S1. Feeding treatment of breeding cattle before semen collection: folic acid, lycopene, anthocyanin and anethole are added to the ordinary feed 15-45 days before semen collection of breeding cattle;

[0009] S2. Semen collection: artificial semen collection from the breeding cattle after breeding, and screening of semen for subsequent cryopreservation;

[0010] S3. Preparation of semen diluent: adding 15-30 µg / mL of caffeine, 8-12 µg / mL of dendrobium phenol, 20-30 µg / mL of chlorogenic acid, and 4-8 µg / mL of pinoresinol diglucoside to a basic semen diluent to prepare a semen diluent;

[0011] S4, semen dilution treatment: the freshly taken bovine semen is kept warm in a 37° C. water bath, and the semen diluent is preheated to 37° C., and then the semen diluent is added to the bovine semen for dilution treatment, and the diluted semen is obtained for standby use;

[0012] S5, precooling of diluted semen: precooling the diluted semen at 4°C, and then transferring it to an ice water bath for further precooling to obtain precooled semen for use;

[0013] S6, pre-cooled semen freezing treatment: the pre-cooled semen is placed in an automatic freezer to cool to -140°C - -150°C, and then quickly transferred to liquid nitrogen for freezing and preservation;

[0014] S7. Semen thawing treatment: Thaw the above-mentioned frozen semen in a 37°C water bath.

[0015] Preferably, in step S1, 20-40 mg of folic acid, 30-40 mg of lycopene, 30-60 mg of anthocyanin and 20-40 mg of anethole are added to each kilogram of the general feeding diet.

[0016] Preferably, the screening criterion in step S2 is sperm motility ≥ 70%.

[0017] Preferably, the composition of the basic semen diluent in step S3 calculated based on 100 mL is: 1.354 g sodium citrate, 2.4224 g TRIS, 1.1 g glucose, 5.33 mL glycerol, 20 mL egg yolk, 0.587 mL double antibody, and 68.82 mL double distilled water.

[0018] Preferably, the volume ratio of bovine semen to semen diluent in the diluted semen in step S4 is 1:8-12.

[0019] Preferably, the precooling treatment time at 4° C. in step S5 is 1-2 hours, and the precooling treatment time in the ice water bath is 40-60 minutes.

[0020] Preferably, in step S5, the diluted semen is first packaged into EP tubes before pre-cooling treatment and then sealed for subsequent treatment.

[0021] Preferably, the time for cooling the temperature to -140°C to -150°C in the automatic freezer in step S6 is controlled within 5-10 minutes.

[0022] Preferably, the time of the water bath thawing treatment in step S7 is controlled within 30-40 seconds.

[0023] The present invention provides a method for freezing and preserving bovine semen, which has the following advantages compared with the prior art:

[0024] The present invention adopts folic acid, lycopene, anthocyanin and anethole for auxiliary feeding before semen collection of the breeding bulls, which effectively improves the sperm motility of the breeding bulls and provides a good basis for subsequent freezing of the semen of the breeding bulls. In addition, caffeine, dendrobium phenol, chlorogenic acid and pinoresinol diglucoside are subsequently used as auxiliary raw materials to be added to the semen diluent, which can effectively reduce the damage of the sperm of the breeding bulls under low-temperature freezing and ensure the motility of the sperm after low-temperature freezing and thawing, providing a good basis for subsequent artificial insemination. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of a field of view in the sperm activity detection of experimental group 6 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility rate in this field of view is 45.7%, the sperm rapid forward motility rate is 32.3%, the sperm slow forward motility rate is 6.5%, the sperm circular motility rate is 0.9%, the sperm in situ motility rate is 5.9%, and the sperm static rate is 54.3%;

[0026] Figure 2 This is a schematic diagram of a field of view in the sperm activity detection of experimental group 7 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility rate in this field of view is 52.0%, the sperm rapid forward motility rate is 33.2%, the sperm slow forward motility rate is 6.6%, the sperm circular motility rate is 0.7%, the sperm in situ motility rate is 11.5%, and the sperm immobility rate is 48.0%;

[0027] Figure 3 This is a schematic diagram of a field of view in the sperm activity detection of the experimental group 8 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility rate in this field of view is 29.1%, the sperm rapid forward motility rate is 17.3%, the sperm slow forward motility rate is 4.5%, the sperm circular motility rate is 0.5%, the sperm in situ motility rate is 6.8%, and the sperm immobility rate is 70.9%;

[0028] Figure 4This is a schematic diagram of a field of view in the sperm activity detection of the experimental group 9 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility rate in this field of view is 37.4%, the sperm rapid forward motility rate is 23.9%, the sperm slow forward motility rate is 5.8%, the sperm circular motility rate is 0.0%, the sperm in situ motility rate is 7.7%, and the sperm immobility rate is 62.6%;

[0029] Figure 5 This is a schematic diagram of a field of view in the sperm activity detection of the experimental group 10 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility rate in this field of view is 31.1%, the sperm rapid forward motility rate is 19.6%, the sperm slow forward motility rate is 6.3%, the sperm circular motility rate is 0.0%, the sperm in situ motility rate is 5.2%, and the sperm immobility rate is 68.9%;

[0030] Figure 6 This is a schematic diagram of a field of view in the sperm activity detection of the experimental group 11 after 14 days of frozen storage in Example 2 of the present invention. The sperm motility in this field of view is 19.6%, the rapid forward motility of sperm is 13.2%, the slow forward motility of sperm is 2.3%, the circular motility of sperm is 0.3%, the in situ motility of sperm is 3.8%, and the sperm immobility rate is 80.4%. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The formula of the common diet in the following examples is shown in Table 1:

[0033] Table 1

[0034]

[0035] The premix provides trace elements copper 10 mg, iron 65 mg, manganese 30 mg, zinc 25 mg, iodine 0.5 mg, selenium 0.1 mg, vitamin A 15000IU, vitamin D 5000IU, and vitamin E 50mg per kilogram of feed. Embodiment 1:

[0036] Feeding of breeding cattle and detection of sperm motility in semen:

[0037] Twenty Yunnan yellow cattle breeders aged 3 to 5 years old, in good physical condition and with good reproductive performance were selected as experimental breeders, and the breeders were randomly divided into 5 groups, namely experimental groups 1-5, with 4 breeders in each group, and on March 3, 2024, each group of breeders was fed with ordinary diets mixed with additives, and fed freely, and the amount of additives added to each group is shown in Table 2 below:

[0038] Table 2

[0039]

[0040] Artificial semen was collected from the breeding cattle of each group at 9:00-10:00 in the morning using the pseudo-vagina method on 0, 10, 15, 30, 45 and 50 days of feeding, and the sperm quality analyzer AndroVision was used to detect the sperm motility in the semen collected at different times of each group. The specific results are shown in Table 3 below:

[0041] Table 3

[0042]

[0043] It can be seen from the above table that the sperm motility in the semen of the breeding cattle using the feeding method of experimental group 1-2 is higher when feeding for 15d-45d. Embodiment 2:

[0044] Cryopreservation of semen of breeder bulls:

[0045] 1. Selection of semen for breeding bulls:

[0046] Artificial semen was collected from several breeding cows fed with the feeding method in the above experimental group 1 for 30 days, and the collected semen was evenly divided into 6 groups, which were designated as experimental groups 6-11;

[0047] 2. Semen freezing treatment:

[0048] 2.1. Preparation of semen diluent:

[0049] The following formula is used as the basic semen diluent (calculated based on 100 mL):

[0050] Sodium citrate 1.354 g, TRIS 2.4224 g, glucose 1.1 g, glycerol 5.33 mL, egg yolk 20 mL, double antibody 0.587 mL, double distilled water 68.82 mL;

[0051] Add adjuvants to the basic semen diluent according to Table 4 below to prepare different semen diluents:

[0052] Table 4

[0053]

[0054] 2.2. Dilution of bovine semen:

[0055] Part of the bovine semen from each group was taken as a sample for sperm activity detection. The specific results are shown in Table 5 below:

[0056] Table 5

[0057]

[0058] The remaining semen was kept warm in a 37°C water bath, and the semen diluent was preheated to 37°C, and then the semen diluent was added to the bovine semen (the volume ratio of bovine semen to semen diluent was 1:10) for dilution treatment to obtain diluted semen. The semen diluents used in each group of semen are shown in Table 6 below:

[0059] Table 6

[0060]

[0061] 2.3. Cryopreservation of bovine semen:

[0062] The diluted semen was divided into EP tubes and sealed (2-3 EP tubes for each group), then precooled at 4°C for 1.5 hours, and then transferred to an ice water bath for further precooling for 50 minutes. After the precooling, it was placed in an automatic freezer to cool to minus 145°C for 8 minutes, and then quickly transferred to liquid nitrogen for cryopreservation.

[0063] 2.4. Remelting treatment:

[0064] The bovine semen frozen for 7 days (168 hours) and 14 days (336 hours) in each group was taken out and thawed in a 37°C water bath for 35 seconds (one tube was thawed each time in each experimental group).

[0065] 3. Semen activity test:

[0066] The sperm motility of the above-mentioned semen groups before freezing and after thawing was tested (using stallion sperm analyzer Minitube-AndroVision for analysis and testing, and the system automatically divides the samples into several fields of view for statistical calculation according to the sample conditions during the analysis). The specific results are shown in Tables 7 and 8 below:

[0067] Table 7

[0068]

[0069] Table 8

[0070]

[0071] As can be seen from the table above, the bovine semen of experimental group 6-7 frozen and preserved still has a high sperm motility after 14 days of freezing. The sperm activity diagram of experimental group 6-7 frozen for 14 days is shown in the figure below. Figure 1-6 ,and Figure 1-6 This is the statistical data of one field of view of each group of samples when tested and analyzed by the stallion sperm analyzer Minitube-AndroVision. The sperm activity of the total samples is shown in the table above.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for freezing and preserving bovine semen, characterized in that: The bovine semen cryopreservation method comprises the following steps: S1. Feeding treatment of cattle before semen collection: 15-45 days before semen collection, folic acid, lycopene, anthocyanidin and anethole are added to the common feed for feeding; and 20-40 mg of folic acid, 30-40 mg of lycopene, 30-60 mg of anthocyanidin and 20-40 mg of anethole are added to each kilogram of the common feed; S2. Semen collection: artificial semen collection from the breeding cattle, and screening of semen for subsequent cryopreservation; S3. Preparation of semen diluent: adding 15-30 µg / mL of caffeine, 8-12 µg / mL of dendrobium phenol, 20-30 µg / mL of chlorogenic acid, and 4-8 µg / mL of pinoresinol diglucoside to a basic semen diluent to prepare a semen diluent; S4, semen dilution treatment: the freshly taken bovine semen is kept warm in a 37° C. water bath, and the semen diluent is preheated to 37° C., and then the semen diluent is added to the bovine semen for dilution treatment, and the diluted semen is obtained for standby use; S5, precooling of diluted semen: precooling the diluted semen at 4°C, and then transferring it to an ice water bath for further precooling to obtain precooled semen for use; S6, pre-cooled semen freezing treatment: the pre-cooled semen is placed in an automatic freezer to cool to -140°C - -150°C, and then quickly transferred to liquid nitrogen for freezing and preservation; S7. Semen thawing treatment: Thaw the above-mentioned frozen semen in a 37°C water bath.

2. The method for freezing and preserving bovine semen according to claim 1, characterized in that: The screening criterion in step S2 is sperm motility ≥ 70%.

3. The method for freezing and preserving bovine semen according to claim 1, characterized in that: The composition of the basic semen diluent in step S3 calculated based on 100 mL is: 1.354 g sodium citrate, 2.4224 g TRIS, 1.1 g glucose, 5.33 mL glycerol, 20 mL egg yolk, 0.587 mL double antibody, and 68.82 mL double distilled water.

4. The method for freezing and preserving bovine semen according to claim 1, characterized in that: In the step S4, the volume ratio of the bovine semen to the semen diluent in the diluted semen is 1:8-12.

5. The method for freezing and preserving bovine semen according to claim 1, characterized in that: In step S5, the precooling treatment time at 4° C. is 1-2 hours, and the precooling treatment time in the ice water bath is 40-60 minutes.

6. The method for freezing and preserving bovine semen according to claim 1, characterized in that: In step S5, the diluted semen is first packaged into EP tubes before pre-cooling and then sealed for subsequent processing.

7. The method for freezing and preserving bovine semen according to claim 1, characterized in that: In step S6, the time for cooling the temperature to -140°C to -150°C in the automatic freezer is controlled to be 5-10 minutes.

8. The method for freezing and preserving bovine semen according to claim 1, characterized in that: The time of the water bath thawing treatment in step S7 is controlled to be 30-40s.

Images