Method for low-temperature preservation of sheep semen

Abstract

The invention relates to a low-temperature preservation method for sheep semen, and belongs to the technical field of animal reproduction. The invention provides a method for low-temperature preservation of sheep semen, which comprises the following steps: (1) collecting breeding male sheep semen by a false vagina method to obtain the collected sheep semen; (2) mixing the sheep semen collected in the step (1) with a semen diluent and fullerene to obtain a sheep semen diluent; and (3) preserving the sheep semen diluent under the condition of 2-8 DEG C. The sheep semen is preserved according to the method, the effective application time of sperm preservation can be remarkably prolonged, the blastocyst forming rate of in-vitro fertilization embryos can be remarkably increased, and the method is applied to reproduction technologies such as artificial insemination (AI) and in-vitro fertilization (IVF) of sheep, the utilization rate of high-quality breeding rams can be increased, and genetic resource protection and efficient breeding can be promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal reproductive technology, and particularly relates to a method for cryopreservation of sheep semen. BACKGROUND

[0002] Cryopreservation of sheep semen is a key link of artificial insemination (AI) and in vitro fertilization (IVF) technology, and is of great significance for improving the utilization rate of high-quality breeding rams, promoting genetic resource protection and efficient breeding, etc. At present, sheep sperm still faces many challenges in the process of cryopreservation, such as lipid peroxidation of sperm cell membrane, impaired mitochondrial function, DNA fragmentation, etc. caused by cryopreservation, which significantly reduces sperm motility, survival rate and fertilization capacity. Although traditional cryoprotectants can partially alleviate the damage caused by freezing, they cannot effectively inhibit the accumulation of reactive oxygen species (ROS), which intensifies oxidative stress, affects the functional integrity of sperm, and reduces the quality of sperm. The fertilization rate and blastocyst development potential of sperm combined with oocytes are often low, which limits the application effect of reproductive technology. At present, some studies have improved the effect of sperm cryopreservation by adding antioxidants (such as vitamin E, glutathione, etc.), but these substances have poor stability, single action, limited effect, and cannot completely alleviate the multiple damages caused by cryopreservation to sperm.

[0003] Based on this, the present application is proposed. SUMMARY

[0004] The purpose of the present application is to provide a method for cryopreservation of sheep semen, which solves the problems of poor stability, single action, limited effect and inability to completely alleviate the multiple damages caused by cryopreservation to sperm in the prior art.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a method for cryopreservation of sheep semen, comprising the following steps: (1) collecting semen of a breeding ram sheep by a false vagina method to obtain collected sheep semen; (2) mixing the collected sheep semen of step (1) with a semen diluent and fullerene to obtain a sheep semen diluent; (3) storing the sheep semen diluent at 2-8℃.

[0006] Preferably, the motility of the collected sheep semen in step (1) is greater than 0.8.

[0007] Preferably, the volume ratio of the collected sheep semen to the semen diluent mixed in step (2) is 1:2-4.

[0008] Preferably, the semen diluent in step (2) is composed of a base solution and a yolk solution. The volume ratio of the base liquid and the yolk liquid is 8-10:1.

[0009] Preferably, the base liquid comprises the following components in mass fraction: 3.04 parts of tris-hydroxymethyl aminomethane, 1.70 parts of citric acid, 1.50 parts of glucose, 0.06 parts of penicillin, 0.10 parts of streptomycin, and 100 parts of double distilled water.

[0010] Preferably, the final concentration of fullerene in the sheep semen diluent in step (2) is 0.01 µM-0.1 µM.

[0011] The application provides application of the method in sheep genetic resource protection.

[0012] The application provides application of the method in sheep efficient breeding.

[0013] The application has the following technical effects and advantages: According to the method, the application can significantly prolong the effective application time of sperm preservation, and significantly improve the blastocyst formation rate of in vitro fertilization embryos, and can be applied to sheep artificial insemination (AI) and in vitro fertilization (IVF) and other reproduction technologies, thereby improving the utilization rate of high-quality breeding rams, and promoting genetic resource protection and efficient breeding. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Apoptosis of cryopreserved sheep sperm in different treatment groups; Figure 2 Changes in mitochondrial membrane potential of cryopreserved sheep sperm in different treatment groups. DETAILED DESCRIPTION

[0015] The application provides a method for cryopreservation of sheep semen, comprising the following steps: (1) collecting semen of a breeding ram by a false vagina method to obtain collected sheep semen; (2) mixing the sheep semen collected in step (1) with a semen diluent and fullerene to obtain a sheep semen diluent; (3) storing the sheep semen diluent at 2-8 ℃, preferably 5 ℃.

[0016] In the application, the survival rate of the collected sheep semen in step (1) is greater than 0.8.

[0017] In the application, the volume ratio of the collected sheep semen to the semen diluent mixed in step (2) is 1:2-4, preferably 1:3.

[0018] In the application, the semen diluent in step (2) is composed of a base liquid and a yolk liquid. The volume ratio of the base liquid and the egg yolk liquid is 8-10:1, preferably 9:1.

[0019] In the present application, the base liquid comprises the following components in mass fraction: 3.04 parts of tris-hydroxymethyl aminomethane, 1.70 parts of citric acid, 1.50 parts of glucose, 0.06 parts of penicillin, 0.10 parts of streptomycin, and 100 parts of double distilled water.

[0020] In the present application, the final concentration of fullerene in the sheep semen diluent in step (2) is 0.01 µM-0.1 µM, preferably 0.01 µM.

[0021] The present application provides the application of the method in the protection of sheep genetic resources.

[0022] The present application provides the application of the method in the efficient breeding of sheep.

[0023] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0024] Example 1: Fullerene-treated sheep semen cryopreservation experiment

[0025] 1. Preparation of semen diluent

[0026] (1) Preparation of base liquid

[0027] Accurately weigh 3.04 g of tris-hydroxymethyl aminomethane, 1.70 g of citric acid, 1.50 g of glucose, 0.06 g of penicillin, and 0.10 g of streptomycin, mix with 100 g of double distilled water to obtain the base liquid; (2) Preparation of egg yolk liquid Take fresh eggs from a disease-free chicken farm with clean and uncontaminated eggshells, carefully disinfect the surface of the eggshells with 75% alcohol, and after the alcohol evaporates, use a sterile forceps blunt end to gently knock open a 2 cm opening at the large end of the eggshell, then use a sterile forceps to tear open the eggshell membrane, pour out the egg white (try to remove it cleanly), and only leave the egg yolk, and transfer the egg yolk to a 50 mL sterile centrifuge tube, use a 20 mL sterile syringe without a needle to repeatedly suck and blow, so that the egg yolk is fully mixed and uniform, and the egg yolk liquid is obtained; (3) Take 90 mL of the base liquid and 10 mL of fresh egg yolk liquid, mix thoroughly to obtain the semen diluent, and store in a 32°C constant temperature incubator for standby.

[0028] 2. The experimental method is: (1) Take the estrus female sheep as the platform, collect the semen of the male sheep by the false vagina method, wipe the external genitalia of the male sheep with a clean towel dipped in warm water before semen collection, collect the semen into a 15 mL sterile centrifuge tube immediately, check the sperm density and viability under a microscope, and select the semen with a milky white or gray color, no odor, normal density and viability greater than 0.80.

[0029] (2) Mix the sheep semen obtained in step (1) with the semen diluent at a temperature of 32°C at a ratio of 1:3 to obtain a sheep semen diluent, divide the sheep semen diluent into 4 equal parts and divide them into 4 15 mL centrifuge tubes, and divide them into a control group, a 0.01 µM C60 treatment group, a 0.10 µM C60 treatment group, and a 1.00 µM C60 treatment group. The control group does not add fullerene (C60), i.e. the final concentration of C60 is 0, and the other groups add C60 (purchased from Sigma-Aldrich Company, shelf number: 379646). The 0.01 µM C60 group adds C60 with a final concentration of 0.01 µM, the 0.10 µM C60 group adds C60 with a final concentration of 0.10 µM, and the 1.00 µM C60 group adds C60 with a final concentration of 1.00 µM. Label them, mix them gently to obtain sheep semen diluents with different concentrations of C60, and do 3 repeats for each group. (3) Wrap 10 layers of gauze around the centrifuge tubes of the 4 groups of sheep semen diluents, tie the gauze with rubber bands, and transfer them to a 5°C refrigerator for storage.

[0030] Every 24 hours, mix the sheep semen diluents of different treatment groups, take a drop of semen and place it on a glass slide, cover it with a cover glass, and observe the viability of the sperm under a microscope (400x) at 37°C. The results are shown in Table 1, where D represents days, and “—” represents a viability of less than 0.10. Different lowercase letters in the same column indicate significant differences (P<0.05), and the same letters indicate no significant difference (P>0.05).

[0031] Table 1 Effect of different concentrations of C60 on the viability of cryopreserved sheep sperm

[0032] The storage time of sperm motility above 0.30 is the effective storage time of artificial insemination, i.e. the effective time of sperm. According to Table 1, the motility of sperm gradually decreased with the extension of storage time, but the 0.01 µM C60 treatment group showed the best protective effect. The motility of this group maintained at a high level (0.85±0.03 and 0.81±0.05) at D1 and D2, and the motility at D3 (0.62±0.07) was significantly higher than that of the control group (0.44±0.03), and the effective survival time of sperm was extended to D6 (0.19±0.05). In contrast, the motility of the control group continuously decreased from 0.83±0.04 at D1 to 0.16±0.03 at D5, and the protective effect of the 0.10 µM C60 treatment group and the 1.00 µM C60 treatment group was poor, especially the motility of the 1.00 µM C60 treatment group decreased more rapidly, which was only 0.43±0.04 at D3, and the motility could not be detected after D4. The above results showed that the addition of C60 with a final concentration of 0.01 µM in the semen diluent could significantly improve the motility of sheep sperm during cryopreservation, and the effective time of sperm motility above 0.3 after cryopreservation was 5 days, while the effective time of sperm motility after cryopreservation without C60 and with a final concentration of 0.10 µM and 1.00 µM in the semen diluent was 3 days.

[0033] 3. Apoptosis of cryopreserved sheep sperm in different treatment groups

[0034] At 3d of storage, 20 μL of semen was taken from each treatment group, and the apoptosis of cryopreserved sheep sperm in different treatment groups was detected by one-step TUNEL cell apoptosis detection kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., product number: C1086). The results are shown in Figure 1 , where A represents the TUNEL staining immunofluorescence of cryopreserved sheep sperm in different treatment groups, and B represents the TUNEL fluorescence intensity column chart of cryopreserved sheep sperm in different treatment groups. The abscissa represents the final concentration of C60, and the ordinate represents the TUNEL fluorescence intensity.

[0035] According to Figure 1 A, the green fluorescence signal (apoptotic positive cells) of the 0.01 µM C60 treatment group was significantly weaker than that of the other groups, while the control group (0.00 µM) and the 0.10 µM C60 treatment group and the 1.00 µM C60 treatment group showed dense green fluorescence aggregation, indicating an increase in the proportion of apoptotic cells. According to Figure 1 B, the relative fluorescence intensity of the 0.01 µM C60 treatment group was significantly lower than that of the control group, with a decrease of 45.1%; there was also a significant difference compared with the 0.10 µM C60 treatment group and the 1.00 µM C60 treatment group. These results showed that the 0.01 µM C60 treatment group reduced the apoptosis of cryopreserved sperm, which was conducive to the survival of sperm.

[0036] 4. Changes in mitochondrial membrane potential of cryopreserved sheep sperm in different treatment groups

[0037] At 3 days of preservation, 20 μL of semen was taken from each treatment group, and the changes in mitochondrial membrane potential of cryopreserved sheep sperm in different treatment groups were detected using an enhanced mitochondrial membrane potential detection kit (JC-1) (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., item number: C2003S). The results are shown in Figure 2 Figure A shows JC-1 staining immunofluorescence images of cryopreserved sperm in different treatment groups, and Figure B shows JC-1 fluorescence intensity bar charts of cryopreserved sperm in different treatment groups. The horizontal axis represents the final concentration of C60, and the vertical axis represents the JC-1 fluorescence intensity.

[0038] According to Figure 2 A, it was found that the proportion of green fluorescence (monomeric JC-1, indicating loss of membrane potential) in the control group (0.00 μM) and the 0.1 μM C60 treatment group (0.10 μM) and the 1.00 μM C60 treatment group (1.00 μM) was significantly higher, while the 0.01 μM C60 treatment group (0.01 μM) showed obvious red fluorescence aggregation, indicating that its mitochondrial function integrity was best. According to Figure 2 B, the relative fluorescence intensity of the 0.01 μM C60 treatment group (0.01 μM) was significantly higher than that of the control group and the 0.10 μM C60 treatment group (0.10 μM) and the 1.00 μM C60 treatment group (1.00 μM), while there was no significant difference between the other groups. These results suggest that the 0.01 μM C60 treatment group may prolong the preservation time of sperm by enhancing the mitochondrial function integrity of sperm and reducing sperm apoptosis.

[0039] In summary, the best method for cryopreservation of sheep semen is as follows: (1) Collect the semen of male sheep using the false vaginal method to obtain sheep semen with normal density and a viability of greater than 0.8; (2) Mix the sheep semen with the prepared semen diluent at a volume ratio of 1:3, then add C60 to obtain a sheep semen diluent with a final C60 concentration of 0.01 μM; (3) Store the sheep semen diluent at 5°C.

[0040] Example 2: Sheep sperm in vitro fertilization experiment after cryopreservation

[0041] (1) Ovary collection and transportation: Collect the ovaries of Tan sheep from a slaughterhouse and immediately place them in a thermos flask containing 35°C physiological saline (containing 1% penicillin-streptomycin). Transport back to the laboratory within 3 hours.

[0042] (2) After the ovary was sent back to the laboratory from the slaughterhouse, the excess adipose tissue and connective tissue around the ovary was cut off, and then washed with 75% alcohol and physiological saline with double antibodies for 3 times. The ovary was placed in a 10mm culture dish and the oocyte collection solution was poured in. The follicles with a diameter of 2-6mm on the ovary were cut using a cutting method, and the cumulus oocyte complex (COCs) with normal morphology, uniform cytoplasm and more than one layer of granulosa cells was collected using a capillary tube under a body microscope. The oocyte collection solution was prepared as follows: TCM199+2%FCS+10μg / mL heparin sodium+1% penicillin-streptomycin; (3) In vitro maturation of oocytes: the collected COCs were incubated in a commercial in vitro maturation medium (purchased from IVF Bioscience Company, item number: 71001) for 3h in advance for 24h. The culture conditions were the same, and were 38.5℃, 5%CO2 and saturated humidity.

[0043] (4) In vitro fertilization: 200uL of sperm in the control group and 0.01μM C60 treatment group in Example 1 were taken respectively, and after centrifugal washing with a commercial IVF solution (purchased from IVF Bioscience Company, item number: 71004), the final concentration of sperm was adjusted to 3.0×10 6 6 / mL, and was transferred into a 4-well plate (400uL per well), and then 40-50 in vitro matured oocytes were put in, and the sperm and oocytes were incubated together at 38.5℃, 5%CO2 and saturated humidity for 20h.

[0044] Early embryo in vitro culture: at 20h of fertilization, the presumptive fertilized oocytes were washed and transferred into a commercial IVC early embryo development solution (purchased from IVF Bioscience Company, item number: 71005), and were cultured at 38.5℃, 5%CO2 and saturated humidity. At 48h and 7d of culture, the cleavage and blastocyst formation were observed under a stereomicroscope, the blastocyst rate was counted, and the effect of different treatment of cryopreserved sperm on the development of in vitro fertilized embryos was determined, and the results are shown in Table 2.

[0045] Table 2 Effect of different treatment of cryopreserved sperm on the development of in vitro fertilized embryos

[0046] According to Table 2, the cleavage rate of the fertilized eggs in the 0.01 μM C60 treatment group (70.17±7.12%) was significantly higher than that in the control group (61.39±6.79%, P<0.05). More importantly, the blastocyst formation rate reached 18.33±3.01%, which was nearly 3 times higher than that in the control group (6.19±1.98%, P<0.05), and the blastocyst rates of the two groups were significantly different. The results show that the 0.01 μM C60 treatment group not only prolongs the effective time of the cryopreserved sperm of sheep, but also directly improves the quality of the sperm, which is beneficial to the formation of the in vitro fertilization blastocyst.

[0047] It can be known from the above examples that the method for cryopreserving sheep semen can significantly prolong the effective application time of sperm preservation and significantly improve the blastocyst formation rate of in vitro fertilization embryos, and is applied to artificial insemination (AI) and in vitro fertilization (IVF) and other reproductive technologies of sheep, which can improve the utilization rate of high-quality breeding ewes, promote genetic resource protection and efficient breeding.

[0048] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for cryopreservation of sheep semen, characterized in that, It comprises the following steps: (1) collecting ram semen of sheep by false vagina method to obtain collected sheep semen; (2) mixing the sheep semen collected in step (1) with semen diluent and fullerene to obtain sheep semen diluent; (3) storing the sheep semen diluent at 2~8℃.

2. The method of claim 1, wherein, The motility of the collected sheep semen in step (1) is greater than 0.

8.

3. The method of claim 1, wherein, The volume ratio of the collected sheep semen to the semen diluent mixed in step (2) is 1:2~4.

4. The method of claim 1, wherein, The semen diluent in step (2) is composed of base solution and yolk solution. The volume ratio of the base solution to the yolk solution is 8~10:

1.

5. The method of claim 4, wherein, The base solution comprises the following components by mass fraction: 3.04 parts of tris-hydroxymethyl aminomethane, 1.70 parts of citric acid, 1.50 parts of glucose, 0.06 parts of penicillin, 0.10 parts of streptomycin, and 100 parts of double distilled water.

6. The method of claim 1, wherein, The final concentration of fullerene in the sheep semen diluent in step (2) is 0.01 µM~0.1 µM.

7. The method of any one of claims 1~6 for use in the protection of sheep genetic resources.

8. The method of any one of claims 1~6 for use in the efficient breeding of sheep.

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