Cryopreservation liquid for goat semen and application of cryopreservation liquid

Abstract

The invention provides a goat semen cryopreservation solution. The goat semen cryopreservation solution comprises melatonin and one or more of sodium citrate, fructose, yolk, glycerol, penicillin, streptomycin and tris (hydroxymethyl) aminomethane. The cryopreservation liquid is used for preserving goat semen and can improve the pregnancy rate of long-term infertility ewes.

Description

Technical Field

[0001] The invention relates to the field of agricultural animal husbandry and veterinary medicine, and in particular to a cryopreservation solution for goat semen and an application thereof. Background Art

[0002] Cryopreservation of goat semen can not only reduce the number of rams raised, improve the breeding efficiency of excellent breeding rams, make full use of hybrid advantages, and reduce labor intensity, but also realize the long-term storage and long-distance transportation of semen, thereby preserving preferred genes to a greater extent and making full use of excellent breeding ram resources. It is of great significance in improving the breeding and reproduction potential of breeding rams.

[0003] However, sperm itself is sensitive to temperature changes. During the freezing process of semen, sperm will suffer from oxidative damage, cold shock, osmotic damage and other reasons, which will reduce the quality of sperm after thawing, resulting in a low pregnancy rate after artificial insemination. It is mainly manifested in the excessive production of oxidative substances such as reactive oxygen, which breaks the original oxidation-reduction balance system, leads to an increase in the apoptosis rate, and damage to the sperm plasma membrane, acrosome, tail, mitochondria and other parts. During the freezing process, sperm mainly suffers oxidative damage. Solving the problem of oxidative damage to sperm has always been the focus of research in related fields. The content of polyunsaturated fatty acids in the plasma membrane of goat sperm is higher than that of other species, so it is more likely to suffer oxidative damage during the freezing-thawing process. Therefore, it is very important to supplement antioxidants exogenously to alleviate sperm oxidative damage during freezing. Summary of the invention

[0004] The purpose of the present disclosure is to provide a cryopreservation solution for goat semen and its application.

[0005] According to one aspect of the present disclosure, there is provided a cryopreservation solution for goat semen, comprising melatonin.

[0006] In some embodiments, the cryopreservation solution comprises 1×10 -6 mol / L~1×10 -3 mol / L of melatonin.

[0007] In some embodiments, the semen cryopreservation solution further includes one or more of sodium citrate, fructose, egg yolk, glycerol, antibiotics, and tris(hydroxymethylaminomethane).

[0008] In some embodiments, the semen cryopreservation solution includes 20-50 g / L sodium citrate, 60-100 g / L fructose, 120-180 mL / L egg yolk, 30-70 mL / L glycerol, 1900-2300 IU / L antibiotics, and / or 0.1-2 g / L tris(hydroxymethyl)aminomethane.

[0009] According to another aspect of the present disclosure, a method for cryopreserving goat semen using the cryopreservation solution is also provided, the method comprising:

[0010] S1, collect ram semen;

[0011] S2. Mix the goat semen cryopreservation solution disclosed herein and the semen collected in step S1 in a frozen sperm tube, and then store them in liquid nitrogen.

[0012] In some embodiments, step S2 further comprises placing the cryotube at a distance of 4 cm from the liquid nitrogen surface, fumigating for 10 minutes, and then rapidly placing the cryotube in liquid nitrogen for storage after the fumigation is completed.

[0013] According to another aspect of the present disclosure, there is also provided a use of the cryopreservation solution in cryopreservation of goat semen.

[0014] In some embodiments, the cryopreservation solution can improve the viability and vitality of goat sperm after freezing.

[0015] In some embodiments, the cryopreservation solution can improve the plasma membrane integrity of goat sperm after freezing.

[0016] In some embodiments, the cryopreservation solution can improve the acrosome integrity of goat sperm after freezing.

[0017] In some embodiments, the cryopreservation solution can improve the antioxidant capacity of goat sperm after freezing.

[0018] In some embodiments, the cryopreservation solution can increase the content of catalase in goat sperm after freezing.

[0019] In some embodiments, the cryopreservation solution can increase the content of superoxide dismutase in goat sperm after freezing.

[0020] In some embodiments, the cryopreservation solution can increase the survival time of goat frozen sperm after thawing.

[0021] In some embodiments, the cryopreservation solution can reduce the content of malondialdehyde in goat sperm after freezing.

[0022] In some embodiments, the cryopreservation solution can improve the pregnancy rate of long-term infertile female goats.

[0023] In some embodiments, the goat is a dairy goat. In some specific embodiments, the goat is a Saanen dairy goat. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The results of the effects of different concentrations of melatonin on sperm plasma membrane integrity according to one embodiment of the present disclosure are shown. Compared with the blank control group (0 μm / mL melatonin), different lowercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05), the same below.

[0025] Figure 2 The results of the effects of different concentrations of melatonin on sperm acrosome integrity according to one embodiment of the present disclosure are shown.

[0026] Figure 3 The results of the effects of different concentrations of melatonin on the total antioxidant capacity of sperm according to one embodiment of the present disclosure are shown.

[0027] Figure 4 The results of the effects of different concentrations of melatonin on the sperm catalase content according to one embodiment of the present disclosure are shown.

[0028] Figure 5 The results of the effects of different concentrations of melatonin on the superoxide dismutase content of sperm according to one embodiment of the present disclosure are shown.

[0029] Figure 6 The results of the effects of different concentrations of melatonin on the malondialdehyde content of sperm according to one embodiment of the present disclosure are shown.

[0030] Figure 7 The results of the effects of different concentrations of melatonin on the level of reactive oxygen species in sperm according to one embodiment of the present disclosure are shown.

[0031] Figure 8 The results of the effects of different concentrations of melatonin on the survival time (37° C.) of sperm after thawing according to one embodiment of the present disclosure are shown.

[0032] Fig. 9 The results of the effects of different concentrations of melatonin on pregnancy rate after insemination at the cervix of long-term infertile ewes according to one embodiment of the present disclosure are shown. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of 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.

[0034] definition

[0035] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0036] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0037] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0038] Goats are mammals of the genus Capra in the family Capra in the order Artiodactyla. There are more than 150 goat breeds in the world, including dairy goats, wool goats, etc.; there are more than 40 goat breeds in China.

[0039] Dairy goats are mainly used for milk production, with stable milk production performance and high milk production. Both male and female goats reach sexual maturity at 4-5 months of age, and are usually bred at 5-6 months of age. The estrus season is from September to November, with the peak in October, and the sexual cycle is 21 days. The gestation period of ewes is 150 days, and the average lambing rate is 178%. The breeding goats can be used for 5-7 years.

[0040] "Long-term infertility" refers to infertility after three or more consecutive matings due to persistent corpus luteum, thin endometrium, ovarian cysts, hidden abortion, being too fat or too thin, bacterial diseases and / or viral diseases.

[0041] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0042] Main instruments and supplies: Tsinghua Tongfang semen quality analysis system (model: CASAS-QH-Ⅲ).

[0043] Main reagents and drugs: melatonin (Sigma), sodium citrate (Shanghai Reagent Company), fructose (Sigma), glycerol (Solabo), Tris (Research reagents), Giemsa dye (Biosharp), fresh eggs, total antioxidant capacity (T-AOC) detection kit, superoxide dismutase (SOD) detection kit, malondialdehyde (MDA) detection kit were purchased from Solabo, and catalase (CAT) detection kit and reactive oxygen species (ROS) detection kit were purchased from Biotech.

[0044] Example 1: Preparation of goat semen cryopreservation solution

[0045] In this example, a goat semen cryopreservation solution was prepared.

[0046] Preparation of frozen liquid I: weigh 3.6g sodium citrate, 0.1g Tris and 8g fructose on a balance, add ultrapure water to dissolve, filter, add 10 g each of penicillin and streptomycin. 5 IU, so that the final volume of frozen solution I is 100mL. Frozen solution I is divided into 5 equal parts, and melatonin is added to each of the 5 frozen solutions I, so that the melatonin concentrations of the 5 frozen solutions I are 0, 10, and 10. -3 mol / L、10 -4 mol / L、10 -5 mol / L、10 -6 mol / L, shake well, seal, and store in a 4℃ refrigerator.

[0047] Preparation of frozen liquid II: Take 90 mL of frozen liquid I and add 10 mL of glycerol to obtain 100 mL of frozen liquid II. Divide the frozen liquid II into 5 equal parts and add melatonin to make the melatonin concentrations of the 5 frozen liquid II equal to 0, 10, and 10, respectively. -3 mol / L、10 -4 mol / L、10 - 5 mol / L、10 -6 mol / L, shake well, seal, and store in a 4℃ refrigerator.

[0048] Example 2: Cryopreservation of goat semen

[0049] In this example, the semen cryopreservation solution prepared in Example 1 was used to cryopreserve goat semen.

[0050] Select a few fresh eggs, spray them with 75% alcohol, and then wipe the surface dry. Tap the middle of the egg to split the eggshell into two halves, drain the egg white, and keep the yolk and yolk membrane. Roll the yolk back and forth on sterile absorbent paper until the egg white and yolk membrane are completely removed, and absorb the remaining yolk for later use.

[0051] One day in advance, the pseudo vagina and the sperm collection cup were cleaned and disinfected with alcohol, and the residual alcohol was washed off with physiological saline and then naturally dried. 10 minutes before sperm collection, the pseudo vagina liner was filled with 38°C-40°C water. On the day of sperm collection, the thermostatic microscope was turned on and preheated, and the water bath was adjusted to 37°C. Egg yolk accounting for 15% of the total volume was added to the freezing liquid I and II prepared in Example 1, and the egg yolk was repeatedly inverted for 2-3 minutes until the egg yolk was evenly distributed. After standing, the floating foam was sucked off, and the tube containing the freezing preservation solution was placed in a 37°C water bath for preheating. The semen of 5 rams (Saanen goats) was collected by the pseudo vagina method. The color was milky white or light yellow, the sperm density was 1 billion / mL, and the semen with sperm motility greater than 60% could be mixed evenly and then frozen.

[0052] Mix the semen with frozen liquid I at a ratio of 1:9, then place the tubes containing liquid I and liquid II in a water bath in a container at 37°C, and place them together with the container in a 4°C refrigerator for 3 to 4 hours. When the temperature stabilizes and drops to 4°C, mix liquid I and liquid II at a ratio of 1:1. Aspirate the treated semen into a 250μL frozen sperm tube and seal it, keeping the tube 4cm away from the liquid nitrogen surface, and fumigate for 10 minutes. After the fumigation is completed, quickly put the frozen sperm tube into liquid nitrogen for storage. Thawing requires the frozen sperm tube to be bathed in 38°C water for 30 seconds.

[0053] Example 3: Effect of adding melatonin to semen cryopreservation solution on sperm motility and vitality after freezing

[0054] In this example, the viability and vitality of frozen sperm after adding different concentrations of melatonin were measured.

[0055] The thawed semen was tested using the Tsinghua Tongfang semen quality analysis system, and the sperm motility, sperm vitality, average curve movement speed, average linear movement speed, average path movement speed, average side swing amplitude, average whipping frequency, and average movement angle were calculated. The results are shown in Table 1. -4 mol / L and 1×10 -5 mol / L melatonin can significantly improve the viability, vitality and average curve movement speed of sperm after thawing. -4 mol / L melatonin can significantly increase the average linear motion speed of sperm after thawing. Adding 1×10 -3 mol / L、1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L melatonin can increase the average path movement speed after thawing.

[0056] Table 1. Evaluation of sperm motility and vitality of fresh and frozen goat semen

[0057]

[0058] Note: Data with different lowercase letters in the same row indicate significant differences (p<0.05), and the same lowercase letters indicate no significant differences (p>0.05). Abbreviations: VCL, mean curvilinear velocity; VSL, mean linear velocity; VAP, mean path velocity; ALH, mean lateral swing amplitude; BCF, mean whiplash frequency; MAD, mean movement angle.

[0059] Example 4: Effect of adding melatonin to semen cryopreservation solution on the integrity of sperm plasma membrane after freezing

[0060] In this example, the plasma membrane integrity of frozen sperm after adding different concentrations of melatonin was measured.

[0061] Dissolve 0.9g fructose and 0.49g sodium citrate in 100mL sterile enzyme-free water to prepare a hypotonic solution. Thaw the semen, take 0.1mL of semen, slowly drip it into 1mL of hypotonic solution along the tube wall, incubate in a 37℃ cell culture incubator for 30 minutes, mix well and observe the tail bending rate under a microscope. Count more than 200 sperm each time. The sperm with a bent tail means that the plasma membrane is intact.

[0062] The results are as follows Figure 1 As shown, 1×10 -4 mol / L melatonin can significantly improve the plasma membrane integrity of goat sperm after thawing, which is higher than that of other treatment groups; adding 1×10 -5 mol / L and 1×10 -6 There was no significant difference in mol / L melatonin compared with the control group.

[0063] Example 5: Effect of adding melatonin to semen cryopreservation solution on the integrity of sperm acrosome after freezing

[0064] In this example, the acrosome integrity of frozen sperm after adding different concentrations of melatonin was measured.

[0065] Take 100 μL of semen, add 1 mL of PBS buffer, centrifuge at 800 rpm for 3 minutes, centrifuge 3-5 times, completely remove impurities and mix the sperm. Take 20 μL of semen, evenly spread on a slide, air-dry and fix with methanol for 2-3 minutes, air-dry again and add a few drops of Giemsa staining solution, add an equal amount of phosphate buffer after 20 seconds, mix, rinse with water after 3-5 minutes, dry and examine under a microscope, observe each group of samples 4 times, observe 5 different fields of view each time, and count more than 200 sperm in each field of view on average.

[0066] The results are as follows Figure 2 As shown, 1×10 -3 , 1×10-4 and 1×10 -5 mol / L melatonin can significantly improve the acrosome integrity of sperm after thawing, among which 1×10 -4 mol / L melatonin has the best effect; adding 1×10 -6 There was no significant difference in the acrosome integrity of sperm after thawing between 100 mol / L melatonin and 200 mol / L melatonin.

[0067] Example 6: Effect of adding melatonin to semen cryopreservation solution on the antioxidant capacity of frozen sperm

[0068] In this example, the antioxidant capacity of frozen sperm after adding different concentrations of melatonin was measured.

[0069] The total antioxidant capacity (T-AOC) test kit was used to test the total antioxidant capacity of sperm according to the method in the instructions, and the test was repeated 4 times. Figure 3 As shown in the figure, compared with the control group (Control), adding different concentrations of melatonin to the cryopreservation solution can significantly improve the total antioxidant capacity of sperm after thawing; adding 1×10 -3 mol / L melatonin, the total antioxidant capacity of sperm after thawing was the highest, and was significantly different from that of other groups.

[0070] Example 7: Effect of adding melatonin to semen cryopreservation solution on the catalase (CAT) content of sperm after freezing

[0071] In this example, the catalase (CAT) content of frozen sperm after adding different concentrations of melatonin was measured.

[0072] The malondialdehyde (MDA) content detection kit was used to detect the malondialdehyde content according to the method in the instructions, and the test was repeated 4 times. Figure 4 Compared with the control group (Control), 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L melatonin can significantly increase the catalase content of sperm after thawing; adding 1×10 - 4 mol / L melatonin, the catalase of sperm after thawing was the highest; when 1×10 -3 There was no significant difference between the control group and the control group.

[0073] Example 8: Effect of adding melatonin to semen cryopreservation solution on the superoxide dismutase (SOD) content of sperm after freezing

[0074] In this example, the superoxide dismutase (SOD) content of frozen sperm after adding different concentrations of melatonin was measured.

[0075] The catalase (CAT) detection kit was used to detect the catalase content according to the method in the instructions, and the test was repeated 4 times. Figure 5 Compared with the control group (Control), 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L melatonin can significantly increase the superoxide dismutase content of sperm after thawing, and the addition of 1×10 -5 mol / L melatonin, the superoxide dismutase content of sperm after thawing was the highest; adding 1×10 -3 mol / L had no significant difference compared with the control group; adding 1×10 -5 mol / L melatonin and 1×10 -6 There was a significant difference in the superoxide dismutase content between the two after thawing of 100 mol / L melatonin.

[0076] Example 9: Effect of adding melatonin to semen cryopreservation solution on the malondialdehyde (MDA) content of sperm after freezing

[0077] In this example, the malondialdehyde (MDA) content of frozen sperm after adding different concentrations of melatonin was measured.

[0078] The superoxide dismutase (SOD) detection kit was used to detect the superoxide dismutase content according to the method in the instructions, and the detection was repeated 4 times. Figure 6 As shown in the figure, compared with the control group (Control), adding different concentrations of melatonin to the cryopreservation solution can significantly reduce the malondialdehyde content of sperm after thawing; among them, adding 1×10 -4 mol / L melatonin, the malondialdehyde content of sperm after thawing was the lowest, and it was significantly different from the other groups; -5 mol / L melatonin and 1×10 -6 mol / L melatonin, there was no significant difference in the malondialdehyde content between the two groups after thawing, while there were significant differences among the other groups.

[0079] Example 10: Effect of adding melatonin to semen cryopreservation solution on the content of reactive oxygen species (ROS) in sperm after freezing

[0080] In this example, the reactive oxygen species (ROS) content of frozen sperm after adding different concentrations of melatonin was measured.

[0081] The reactive oxygen species (ROS) detection kit was used to detect the reactive oxygen species content according to the method in the instruction manual and the detection was repeated 4 times. Figure 7As shown in the figure, compared with the control group (Control), adding different concentrations of melatonin to the cryopreservation solution can significantly reduce the content of reactive oxygen species in sperm after thawing; among them, adding 1×10 -5 mol / L melatonin, the level of reactive oxygen species in sperm after thawing was the lowest; when 1×10 -3 mol / L melatonin and 1×10 -5 mol / L melatonin, there was a significant difference in the level of reactive oxygen species between the two after thawing, and there was no significant difference between the other treatment groups.

[0082] Example 11: Effect of adding melatonin to semen cryopreservation solution on sperm survival time after thawing

[0083] In this example, the survival time (37° C.) of sperm after thawing was measured after adding different concentrations of melatonin.

[0084] The thawed semen was incubated in a 37°C incubator. The sperm motility and vitality were tested every 2 hours during the first 6 hours. After 6 hours, the above indicators were tested every hour until the sperm died completely. Figure 8 Compared with the control group (Control), 1×10 -3 mol / L、1×10 -4 mol / L、1×10 -5 mol / L melatonin can significantly increase the survival time of sperm after thawing. -4 mol / L melatonin has the most significant effect.

[0085] Example 12: Effect of adding melatonin to semen cryopreservation solution on the pregnancy rate of long-term infertile ewes

[0086] This example shows the effect of adding different concentrations of melatonin on the pregnancy rate of long-term infertile ewes.

[0087] The thawed semen was used to inseminate 150 long-term infertile ewes (Saanen goats) through the cervix, and ultrasound pregnancy tests were performed on the ewes 40-50 days after insemination. Fig. 9 Compared with the control group, 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L melatonin can significantly improve the pregnancy rate of insemination at the cervix of long-term infertile ewes after thawing (p<0.05). -3 mol / L melatonin could not significantly improve the pregnancy rate (p>0.05).

[0088] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A cryopreservation solution for goat semen, characterized in that: The cryopreservation solution includes melatonin.

2. The cryopreservation solution according to claim 1, characterized in that The cryopreservation solution includes 1×10 -6 mol / L~1×10 -3 mol / L of melatonin.

3. The cryopreservation solution according to claim 1 or 2, characterized in that The semen freezing preservation solution also includes one or more of sodium citrate, fructose, egg yolk, glycerol, antibiotics, and tris(hydroxymethyl)aminomethane.

4. The cryopreservation solution according to claim 3, characterized in that The semen freezing preservation solution includes 20-50 g / L sodium citrate, 60-100 g / L fructose, 120-180 mL / L egg yolk, 30-70 mL / L glycerol, 1900-2300 IU / L antibiotics, and / or 0.1-2 g / L tris(hydroxymethyl)aminomethane.

5. A method for cryopreserving goat semen using the cryopreservation solution according to any one of claims 1 to 4, the method comprising: S1. Collect ram semen; S2. Mix the cryopreservation solution described in any one of claims 1 to 4 and the semen collected in step S1 in a frozen sperm tube, and then store them in liquid nitrogen.

6. Use of the cryopreservation solution according to any one of claims 1 to 4 in cryopreservation of goat semen.

7. The use according to claim 6, characterized in that: The cryopreservation solution can improve the sperm viability and vitality, plasma membrane integrity, acrosome integrity, antioxidant capacity, catalase content, superoxide dismutase content and / or post-thawing survival time of goats after freezing.

8. The use according to claim 6, characterized in that: The cryopreservation solution can reduce the malondialdehyde content of goat sperm after freezing.

9. The use according to claim 6, characterized in that: The cryopreservation solution can improve the pregnancy rate of long-term infertile female goats.

10. The use according to any one of claims 6 to 9, characterized in that: The goats are dairy goats.

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