Preparation method and application of geniposide diluent for low-temperature preservation of milk goat semen

Abstract

The preparation method comprises the following steps: step 1, preparing a basic diluent: respectively and accurately weighing 1.4 g of citric acid, 1g of fructose and 2.7 g of Tris by using an electronic analytical balance, dissolving in 100mL of ultrapure water, adjusting the pH value to 7, filtering by using a 0.22 mu m filter, then adding 2000IU / mL of penicillin and 2000IU / mL of streptomycin, and uniformly stirring to obtain the basic diluent; oscillating and uniformly mixing by using an ultrasonic oscillator, and storing in a refrigerator at 4 DEG C for later use after all the reagents are completely dissolved; according to the diluent for low-temperature preservation of the milk goat semen, the sperm motility rate and the acrosome integrity rate which are preserved for 5 days are correspondingly improved through detection, and compared with a low-temperature diluent of the milk goat semen without geniposide, the sperm motility rate and the acrosome integrity rate are remarkably different. Detection of antioxidant indexes (ROS) in semen shows that the low-temperature preservation diluent added with geniposide can obviously improve the antioxidant capacity of sperms.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic technology, and specifically to a preparation method and application of geniposide in a diluent for cryopreservation of dairy goat semen. Background Art

[0002] The present invention discloses a preparation method of geniposide (Geniposide, GNP) for preparing a diluent for cryopreservation of dairy goat semen. In the present invention, a geniposide solution is added to a basic diluent mixed with citric acid, fructose, Tris, penicillin, streptomycin, and water to obtain a diluent for cryopreservation of dairy goat semen. The diluent for cryopreservation of dairy goat semen of the present invention can effectively reduce the damage suffered during the preservation of dairy goat semen, significantly prolong the sperm survival time, improve the sperm motility rate and the integrity rate of the acrosome. By detecting the antioxidant index (ROS) in the semen, it can be known that the cryopreservation diluent added with geniposide can significantly improve the antioxidant capacity of sperm, achieving the purpose of improving fertilization and conception.

[0003] Therefore, we propose a preparation method and application of geniposide in a diluent for cryopreservation of dairy goat semen in order to solve the problems raised above. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of geniposide in a diluent for cryopreservation of dairy goat semen to solve the problems in the current market raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A preparation method of geniposide in a diluent for cryopreservation of dairy goat semen, including the following steps: Step 1: Preparation of the basic diluent: Weigh accurately 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris with an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter with a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, mix well with an ultrasonic oscillator, and store in a 4°C refrigerator for later use after all reagents are completely dissolved; Step 2: Preparation of the geniposide diluent: Purchase 20 mg of geniposide standard product, transfer it to a brown sample bottle, dilute it to 1 ml with the basic diluent to obtain a geniposide solution with a concentration of 20 mg / mL, invert the solution to completely dissolve and mix it evenly, and finally filter with a 0.22 μm filter to obtain 1 ml of 20 mg / mL geniposide solution, and store it sealed at 4°C. The preparation method of geniposide solutions with different concentrations is as follows: ① Take 10 μL of geniposide solution with a concentration of 20 mg / mL, add basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 20 μg / mL for standby. ② Take 20 μL of geniposide solution with a concentration of 20 mg / mL, add basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 40 μg / mL for standby. ③ Take 30 μL of geniposide solution with a concentration of 20 mg / mL, add basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 60 μg / mL for standby. ④ Take 40 μL of geniposide solution with a concentration of 20 mg / mL, add basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 80 μg / mL for standby. ⑤ Take 50 μL of geniposide solution with a concentration of 20 mg / mL, add basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 100 μg / mL for standby. Dispense the prepared basic diluent into 5 centrifuge tubes to obtain diluents for cryopreservation of goat semen with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Step 3. Semen examination and dilution: (1) Semen examination ① Appearance assessment: The appearance assessment includes ejaculate volume: After semen collection, transfer it to a graduated sterile centrifuge tube with a pipette to observe the ejaculate volume. Color and luster: No foreign matter, no abnormal odor, and the color and luster are milky white or milky yellow. Cloud-like movement: When observing freshly collected ram semen with the naked eye, a tumbling and rolling like a cloud caused by sperm motility can be seen. ② Viability detection: The viability detection is to take an appropriate amount of semen from the centrifuge tube with a pipette onto a glass slide, press the slide, and then examine it under a microscope. The sperm morphology is normal, the malformation rate is below 5%, the sperm motility is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent experiments. In this study, to eliminate individual differences, the semen passing the microscopic examination was evenly mixed in equal amounts at 37°C. (2) Semen dilution ① First dilution Select the semen passing the microscopic examination, add the diluent to the semen at a ratio of 1:2, balance at room temperature for 3 - 5 minutes. When diluting, pay attention to slowly adhering to the wall and rotating the tube wall. After each step of dilution, slowly invert it several times to mix well, and then wrap it with multiple layers of gauze and absorbent cotton and place it in a 4°C refrigerator to balance for 2 h.

[0006] ② Second dilution After the first equilibration is completed, the semen is further diluted by adding an isothermal diluent at a ratio of 1:1. After thorough mixing, it is placed in a 4°C refrigerator for further equilibration for 2 h, and then wrapped with 12 layers of gauze and stored at 4°C.

[0007] The application of a diluent for low-temperature preservation of semen containing geniposide. The geniposide diluent prepared by any one of the methods described in claims 1 to 3 in the method for preparing a diluent for low-temperature preservation of dairy goat semen using the said geniposide is used for low-temperature preservation of animal semen.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for preparing a diluent for low-temperature preservation of dairy goat semen using the said geniposide and its application. For the diluent for low-temperature preservation of dairy goat semen of the present invention, the sperm motility rate and acrosome integrity rate detected after 5 days of preservation are correspondingly increased. Compared with the low-temperature diluent for dairy goat semen without adding geniposide, the differences in sperm motility rate and acrosome integrity rate are significant. By detecting the antioxidant index (ROS) in the semen, it can be known that the low-temperature preservation diluent added with geniposide can significantly improve the antioxidant ability of sperm. Description of the Drawings

[0009] Figure 1 It is a schematic diagram showing the effect of different concentrations of geniposide of the present invention on sperm motility rate; Figure 2 It is a schematic diagram showing the effect of different concentrations of geniposide of the present invention on sperm acrosome integrity rate; Figure 3 It is a schematic diagram showing the effect of different concentrations of geniposide of the present invention on the ROS content in semen on the 5th day of low-temperature preservation. Detailed Embodiments

[0010] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0011] The present invention provides a semen cryopreservation diluent added with geniposide, citric acid, fructose, Tris, penicillin, and streptomycin. Tris is a weak alkaline buffer solution, which, when used in combination with citric acid, can stabilize the pH and osmotic pressure of the reaction system. Penicillin and streptomycin, as common bacteriostatic agents, can inhibit the synthesis of peptidoglycan in the bacterial cell wall to achieve a bactericidal effect. Geniposide is a product extracted and refined from the dried ripe fruits of Gardenia jasminoides Ellis, a plant of the Rubiaceae family, and has many biological activities, such as antioxidant, antiviral, antibacterial, and anti-inflammatory effects. By intragastric administration of exogenous geniposide to mice with circadian rhythm disorders, it was found that the damage to male mouse fertility and spermatogenesis was reduced, and the levels of testosterone, dihydrotestosterone (DHT), and androstenedione (ASD) were normalized. When studying the inflammatory response and oxidative stress of rats with liver ischemia-reperfusion injury by geniposide, it was found that geniposide could improve the liver function of rats, reduce oxidative stress, inflammatory response, and apoptosis by activating the PI3K / Akt signaling pathway. Through the experimental summary of the inventor, it was found that adding geniposide to the semen cryopreservation diluent for dairy goats played a protective role on sperm during semen preservation.

[0012] In order to determine the role of geniposide and the basic dilution ratio in semen cryopreservation, a total of 5 groups were set, and the geniposide content in different groups increased in a gradient manner to further illustrate the beneficial effects of geniposide in the semen cryopreservation diluent for dairy goats.

[0013] Example 1: This example provides a semen cryopreservation diluent containing added geniposide, citric acid, fructose, Tris, penicillin, and streptomycin. The specific preparation method of the semen cryopreservation diluent for dairy goats includes the following steps: (1) Preparation of the basic diluent Accurately weigh 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris with an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter with a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, and mix well with an ultrasonic oscillator. After all the reagents are completely dissolved, store them in a 4°C refrigerator for standby. Note that it should be prepared and used immediately.

[0014] (2) Preparation of the geniposide diluent Purchase 20 mg of geniposide standard product, transfer it to a brown sample bottle, dilute it to 1 ml with the basic diluent to obtain a geniposide solution with a concentration of 20 mg / mL, invert the solution to completely dissolve and mix it evenly, and finally filter it with a 0.22 μm filter to obtain 1 ml of 20 mg / mL geniposide solution, and store it sealed at 4°C. The preparation method of geniposide solutions with different concentrations is as follows: ① Take 10 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 20 μg / mL geniposide dilution solution for use; ② Take 20 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 40 μg / mL geniposide dilution solution for use; ③ Take 30 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 60 μg / mL geniposide dilution solution for use; ④ Take 40 μL of 20 mg / mL Gardenia glycoside solution, add basic diluent to dilute to 10 mL, and obtain 80 μg / mL Gardenia glycoside dilution solution for use; ⑤ Take 50 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 100 μg / mL geniposide dilution solution for use; The prepared basic diluent was dispensed into 5 centrifuge tubes to obtain diluents for cryopreservation of goat semen with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL.

[0015] Note: The diluent for cryopreservation of goat semen should be prepared and used immediately, and the diluted gardenia glycoside solution should be stored away from light. Gardenia glycoside standard can be weighed in multiple portions at one time, placed in sealed bottles and frozen for storage, and then mixed with the basic diluent before use.

[0016] (3) Semen examination ①Appearance assessment 1) Ejaculation volume: After semen collection, use a pipette to transfer it to a sterile centrifuge tube with a scale to observe the ejaculation volume; 2) Color: no foreign matter, no abnormal smell, milky white or creamy yellow in color; 3) Cloud movement: When observing freshly collected ram semen with the naked eye, one can see the tumbling and rolling of spermatozoa like clouds; ② Vitality detection Use a pipette to take an appropriate amount of semen from the centrifuge tube and place it on a glass slide. After pressing the slide, examine it under a microscope. Sperm morphology must be normal, the deformity rate is less than 5%, the sperm motility is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent experiments. In order to eliminate individual differences in this study, the semen that passed the microscopic examination was evenly mixed in equal amounts at 37°C.

[0017] (4) Semen dilution ①First dilution Select semen that has passed microscopic examination. Add diluent to the semen at a ratio of 1:2 and equilibrate at room temperature for 3 - 5 minutes. When diluting, pay attention to slowly adhere to the wall and rotate the tube wall. After each step of dilution, slowly invert several times to mix well. Then wrap it with multiple layers of gauze and absorbent cotton and place it in a 4°C refrigerator to equilibrate for 2 h.

[0018] ② Second dilution The semen after the first equilibration is further diluted by adding an isothermal diluent at a ratio of 1:1. After thorough mixing, place it in a 4°C refrigerator to continue equilibration for 2 h, and wrap it with 12 layers of gauze for low-temperature storage at 4°C.

[0019] (5) Detect sperm motility The diluted semen is stored at 4°C for 5 days, observed daily, and the sperm motility is detected using a computer-assisted semen analyzer (CASA).

[0020] Control 1: In this experiment, a cryopreservation diluent without geniposide will be detected. The specific method for preparing the cryopreservation diluent for dairy goat semen includes the following steps: (1) Preparation of basic diluent Weigh 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris accurately using an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter using a 0.22 μm filter, then add penicillin at 2000 IU / mL and streptomycin at 2000 IU / mL, and mix well using an ultrasonic oscillator. After all the reagents are completely dissolved, store it in a 4°C refrigerator for later use. Note that it should be prepared and used immediately.

[0021] (2) Semen examination ① Appearance assessment 1) Ejaculate volume: After semen collection, transfer it to a graduated sterile centrifuge tube using a pipette to observe the ejaculate volume; 2) Color and luster: No foreign matter, no abnormal odor, and the color is milky white or milky yellow; 3) Cloud-like movement: When observing freshly collected ram semen with the naked eye, a tumbling and rolling like a cloud caused by sperm motility can be seen; ② Motility detection Use a pipette to take an appropriate amount of semen from the centrifuge tube onto a glass slide. After pressing the slide, examine it under a microscope. The sperm morphology is normal, the malformation rate is below 5%, the sperm motility is higher than 0.8, and the density reaches over 1.5 billion / mL before it can be used for subsequent experiments. In this study, to eliminate individual differences, the semen that has passed microscopic examination is evenly mixed in equal amounts at 37°C.

[0022] (3) Semen dilution ① First dilution Select semen that has passed microscopic examination. Add the basic diluent to the semen at a ratio of 1:2, and equilibrate at room temperature for 3 - 5 minutes. When diluting, pay attention to slowly adhering to the wall and rotating the tube wall. After each step of dilution, slowly invert several times to fully mix. Then wrap it with multiple layers of gauze and absorbent cotton, and place it in a 4°C refrigerator to equilibrate for 2 h.

[0023] ② Second dilution Add the isothermal basic diluent to the semen after the first equilibration at a ratio of 1:1 for further dilution. After fully mixing, place it in a 4°C refrigerator to continue equilibration for 2 h, and wrap it with 12 layers of gauze for storage at 4°C.

[0024] (4)Detect sperm motility The diluted semen is stored at 4°C for 5 days, observed every day, and the sperm motility is detected with the help of a computer - assisted semen analyzer (CASA).

[0025] The experimental results are as Figure 1 shown, and the following conclusions can be drawn: For the low - temperature preservation diluent of dairy goat sperm added with geniposide, the sperm motility is significantly improved after 5 days of preservation. Among them, the sperm motility with a geniposide addition amount of 80 μg / mL is the best in the first three days, and the preservation effect with 60 μg / mL is the best on the 4th and 5th days.

[0026] Example 2: This example provides a low - temperature preservation diluent for semen containing geniposide, citric acid, fructose, Tris, penicillin, and streptomycin. The specific preparation method of the low - temperature preservation diluent for dairy goat semen includes the following steps: (1)Preparation of the basic diluent Weigh 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris accurately with an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter with a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, and mix well with an ultrasonic oscillator. After all the reagents are completely dissolved, place them in a 4°C refrigerator for storage and use as needed. Note that it should be prepared and used immediately.

[0027] (2)Preparation of the geniposide diluent Purchase 20 mg of geniposide standard product, transfer it to a brown sample bottle, dilute it to 1 ml with the basic diluent to obtain a geniposide solution with a concentration of 20 mg / mL. Invert the solution to completely dissolve and mix it evenly, and finally filter it with a 0.22 μm filter to obtain 1 ml of 20 mg / mL geniposide solution, which is sealed and stored at 4°C. The preparation method of geniposide solutions with different concentrations is as follows: ① Take 10 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a 20 μg / mL geniposide diluent for standby; ② Take 20 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 40 μg / mL for standby. ③ Take 30 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 60 μg / mL for standby. ④ Take 40 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 80 μg / mL for standby. ⑤ Take 50 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a geniposide diluent with a concentration of 100 μg / mL for standby. Dispense the prepared basic diluent into 5 centrifuge tubes to obtain diluents for cryopreservation of goat semen with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL.

[0028] Note: The diluent for cryopreservation of goat semen should be prepared and used immediately. The diluted geniposide solution should be stored in the dark. The geniposide standard can be weighed in multiple portions at one time, stored frozen in sealed bottles respectively, and mixed with the basic diluent when in use.

[0029] (3) Semen examination ① Appearance assessment 1) Ejaculate volume: After semen collection, transfer it to a graduated sterile centrifuge tube with a pipette to observe the ejaculate volume. 2) Color and luster: Without foreign matters, without abnormal odors, and the color and luster are milky white or milky yellow. 3) Cloud-like movement: When observing freshly collected ram semen with the naked eye, it can be seen that the sperm activity causes a tumbling and rolling like a cloud. ② Viability detection Take an appropriate amount of semen from the centrifuge tube with a pipette onto a glass slide. After making a smear, examine it under a microscope. The sperm morphology is normal, the malformation rate is below 5%, the sperm viability is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent experiments. In this study, to eliminate individual differences, the semen passing the microscopic examination was evenly mixed in equal amounts at 37°C.

[0030] (4) Semen dilution ① First dilution Select the semen passing the microscopic examination, add the diluent to the semen at a ratio of 1:2, balance at room temperature for 3 - 5 minutes. When diluting, it should be noted to slowly adhere to the wall and rotate the tube wall. After each step of dilution, slowly invert it several times to fully mix. Then wrap it with multiple layers of gauze and absorbent cotton and place it in a 4°C refrigerator to balance for 2 h.

[0031] ②Second dilution The semen after the first equilibration was further diluted with an equal volume of isothermal diluent, thoroughly mixed, placed in a 4 °C refrigerator for continued equilibration for 2 h, and then stored at 4 °C wrapped with 12 layers of gauze.

[0032] (5)Detection of sperm acrosome integrity ① Pipette 50 μL of the frozen-thawed semen with geniposide at 20, 40, 60, 80, and 100 μg / mL onto a clean glass slide and spread evenly. Allow to air dry naturally.

[0033] ② After air drying, fix the sample with absolute methanol for 10 min and then air dry again.

[0034] ③ Cover the sample with 30 μL of FITC-PNA staining solution, place at 37 °C, and incubate in the dark for 30 min.

[0035] ④ After incubation, rinse the sample with PBS 2 - 3 times, air dry naturally, seal with colorless nail polish, and then observe under a fluorescence microscope.

[0036] ⑤ For the detection of acrosome integrity, select more than 3 clear fields of view, and ensure that the number of sperm in each field of view reaches more than 200.

[0037] Use fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining method and operate in the dark throughout the process.

[0038] The acrosome is a specialized vesicle at the tip of the sperm head. It is a specialized lysosome developed from Golgi vesicles. The acrosome surrounds the front part of the sperm nucleus. Through the reaction of the sperm acrosome with the egg, the sperm can penetrate through the outer membranes of the egg and enter the egg. The contents of the acrosome are surrounded by the acrosomal membrane, which can be further divided into two sub-domains. The inner acrosomal membrane is closely opposed to the nuclear membrane, while the outer acrosomal membrane is exactly located beneath the plasma membrane. The acrosome reaction is an important physiological process that occurs after sperm capacitation and is a prerequisite for fertilization. Only sperm that have completed the acrosome reaction can fuse with the oocyte to achieve fertilization.

[0039] Control Example 2: In this experiment, the cryopreserved diluent without geniposide was detected, and the acrosome integrity of sperm cryopreserved for 5 days was detected. The specific preparation method of the cryopreserved diluent for dairy goat semen includes the following steps: (1)Preparation of the basic diluent Weigh 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris accurately using an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter through a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, mix well using an ultrasonic oscillator, and store in a 4 °C refrigerator for later use after all reagents are completely dissolved. Note that it should be prepared and used immediately.

[0040] (2)Semen examination ①Appearance assessment 1) Ejaculate volume: After semen collection, transfer it to a graduated sterile centrifuge tube using a pipette to observe the ejaculate volume; 2) Color and luster: Without foreign objects, without abnormal odor, the color is milky white or milky yellow; 3) Cloud-like movement: When observing freshly collected ram semen with the naked eye, a tumbling and rolling like a cloud caused by sperm motility can be seen; ②Motility detection Use a pipette to take an appropriate amount of semen from the centrifuge tube onto a glass slide. After making a smear, examine it under a microscope. The sperm morphology is normal, the malformation rate is below 5%, the sperm motility is higher than 0.8, and the density reaches over 1.5 billion / mL before it can be used for subsequent experiments. In this study, to eliminate individual differences, the semen with qualified microscopy was evenly mixed in equal amounts at 37 °C.

[0041] (3)Semen dilution ①First dilution Select semen with qualified microscopy, add the basic diluent to the semen at a ratio of 1:2, balance at room temperature for 3 - 5 minutes. When diluting, pay attention to slowly sticking to the wall and rotating the tube wall. After each step of dilution, slowly invert it several times to mix well, then wrap it with multiple layers of gauze and absorbent cotton, and place it in a 4 °C refrigerator to balance for 2 h.

[0042] ②Second dilution Add the isothermal basic diluent to the semen after the first balance at a ratio of 1:1 for further dilution. After mixing well, put it in a 4 °C refrigerator to continue balancing for 2 h, and wrap it with 12 layers of gauze for low-temperature storage at 4 °C.

[0043] (4)Detect the acrosome integrity rate of sperm ①Aspirate 50 μL of the semen added with the basic diluent after freezing - thawing and evenly smear it on a clean glass slide, and let it air dry naturally.

[0044] ②After the sample is air - dried, fix it with absolute methanol for 10 min and then air - dry it.

[0045] ③Take 30 μL of FITC - PNA staining solution to cover the sample, place it at 37 °C, and incubate in the dark for 30 min.

[0046] ④ After the incubation, rinse the samples with PBS 2-3 times, air-dry them naturally, mount them with colorless nail polish, and then observe them under a fluorescence microscope.

[0047] ⑤ For the detection of acrosome integrity rate, select more than 3 clear fields of view, and ensure that the number of spermatozoa in each field of view reaches more than 200.

[0048] Use fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining method and operate under complete darkness throughout the process.

[0049] Obtain the results as Figure 2 shown below, and thus draw the following conclusion: For the low-temperature preservation diluent of dairy goat sperm added with geniposide, the acrosome integrity rate of sperm has been significantly improved after 5 days of preservation. Among them, the acrosome integrity rate of sperm is the best when the geniposide addition amount is 60 μg / mL.

[0050] Example 3: This example provides a low-temperature preservation diluent for semen containing added geniposide, citric acid, fructose, Tris, penicillin, and streptomycin. The specific preparation method of the low-temperature preservation diluent for dairy goat semen includes the following steps: (1) Preparation of the basic diluent Weigh accurately 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris with an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter with a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, mix well with an ultrasonic oscillator, and store in a 4°C refrigerator for standby after all the reagents are completely dissolved. Note that it should be prepared and used immediately.

[0051] (2) Preparation of the geniposide diluent Purchase 20 mg of geniposide standard product, transfer it to a brown sample bottle, dilute it to 1 ml with the basic diluent to obtain a geniposide solution with a concentration of 20 mg / mL, invert the solution to completely dissolve and mix it evenly, and finally filter it with a 0.22 μm filter to obtain 1 ml of 20 mg / mL geniposide solution, which is stored sealed at 4°C. The preparation methods of geniposide solutions with different concentrations are as follows: ① Take 10 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a 20 μg / mL geniposide diluent for standby; ② Take 20 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a 40 μg / mL geniposide diluent for standby; ③ Take 30 μL of the geniposide solution with a concentration of 20 mg / mL, add the basic diluent and dilute to a volume of 10 mL to obtain a 60 μg / mL geniposide diluent for standby; ④ Take 40 μL of 20 mg / mL Gardenia glycoside solution, add basic diluent to dilute to 10 mL, and obtain 80 μg / mL Gardenia glycoside dilution solution for use; ⑤ Take 50 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 100 μg / mL geniposide dilution solution for use; The prepared basic diluent was dispensed into 5 centrifuge tubes to obtain diluents for cryopreservation of goat semen with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL.

[0052] Note: The diluent for cryopreservation of goat semen should be prepared and used immediately, and the diluted gardenia glycoside solution should be stored away from light. Gardenia glycoside standard can be weighed in multiple portions at one time, placed in sealed bottles and frozen for storage, and then mixed with the basic diluent before use.

[0053] (3) Semen examination ①Appearance assessment 1) Ejaculation volume: After semen collection, use a pipette to transfer it to a sterile centrifuge tube with a scale to observe the ejaculation volume; 2) Color: no foreign matter, no abnormal smell, milky white or creamy yellow in color; 3) Cloud movement: When observing freshly collected ram semen with the naked eye, one can see the tumbling and rolling of spermatozoa like clouds; ② Vitality detection Use a pipette to take an appropriate amount of semen from the centrifuge tube and place it on a glass slide. After pressing the slide, examine it under a microscope. Sperm morphology must be normal, the deformity rate is less than 5%, the sperm motility is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent experiments. In order to eliminate individual differences in this study, the semen that passed the microscopic examination was evenly mixed in equal amounts at 37°C.

[0054] (4) Semen dilution ①First dilution Select the semen that passes the microscopic examination, add diluent to the semen in a ratio of 1:2, and equilibrate at room temperature for 3-5 minutes. When diluting, pay attention to slowly sticking to the wall and rotating the tube wall. After each dilution, slowly invert several times to mix thoroughly, then wrap it with multiple layers of gauze and absorbent cotton, and place it in a 4℃ refrigerator to equilibrate for 2 hours.

[0055] ② Second dilution After the first equilibration, the semen was further diluted with isothermal diluent at a ratio of 1:1. After thorough mixing, it was placed in a 4°C refrigerator for further equilibration for 2 h and wrapped with 12 layers of gauze and stored at 4°C.

[0056] (5) Detection of sperm ROS indicators The diluted semen was stored at 4 °C for 5 days, and the antioxidant capacity of sperm was measured using the Beijing Solarbio ROS kit.

[0057] Control Example 3: In this experiment, a cryopreservation diluent without geniposide was detected. The specific preparation method of the cryopreservation diluent for dairy goat semen includes the following steps: (1) Preparation of the basic diluent Weigh 1.4 g of citric acid, 1 g of fructose, and 2.7 g of Tris accurately using an electronic analytical balance, dissolve them in 100 mL of ultrapure water, adjust the pH to 7, filter using a 0.22 μm filter, then add penicillin at 2000 IU / mL and streptomycin at 2000 IU / mL, mix well using an ultrasonic oscillator, and store in a 4 °C refrigerator for later use after all reagents are completely dissolved. Note that it should be prepared and used immediately.

[0058] (2) Semen examination ① Appearance assessment 1) Ejaculate volume: After semen collection, transfer it to a graduated sterile centrifuge tube using a pipette to observe the ejaculate volume; 2) Color and luster: Without foreign matter, without abnormal odor, and the color is milky white or milky yellow; 3) Cloudy movement: When observing freshly collected ram semen with the naked eye, a tumbling and rolling like a cloud caused by sperm motility can be seen; ② Viability detection Take an appropriate amount of semen from the centrifuge tube using a pipette onto a glass slide. After pressing the slide, examine it under a microscope. The sperm morphology is normal, the malformation rate is below 5%, the sperm motility is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent experiments. In this study, to eliminate individual differences, the semen that passed the microscopic examination was evenly mixed in equal amounts at 37 °C.

[0059] (3) Semen dilution ① First dilution Select the semen that passed the microscopic examination, add the basic diluent to the semen at a ratio of 1:2, balance at room temperature for 3 - 5 minutes. When diluting, pay attention to slowly adhering to the wall and rotating the tube wall, and slowly invert it several times after each dilution to mix well. Then wrap it with multiple layers of gauze and absorbent cotton and place it in a 4 °C refrigerator to balance for 2 h.

[0060] ② Second dilution Add the isothermal basic diluent to the semen after the first balance at a ratio of 1:1 for further dilution. After mixing well, put it in a 4 °C refrigerator to continue balancing for 2 h, and wrap it with 12 layers of gauze for cryopreservation at 4 °C.

[0061] (4) Detection of sperm ROS index The diluted semen was stored at 4°C for 5 days, and the antioxidant capacity of sperm was measured using the ROS kit from Solarbio Science & Technology Co., Ltd. in Beijing.

[0062] The experimental results are as Figure 3 shown, and the following conclusions can be drawn: When the semen of dairy goats was stored at low temperature for 5 days, the relative ROS levels in the semen of each geniposide treatment group were significantly lower than those of the control group (P < 0.05). Among them, the ROS level of the 60 μg / mL treatment group reached 32.7, which was significantly lower than that of other treatment groups, but there was no significant difference compared with the 40 μg / mL and 80 μg / mL treatment groups (P > 0.05). The difference in ROS levels between the 40 μg / mL treatment group and the 100 μg / mL treatment group was small (P < 0.05). The results indicate that adding 60 μg / mL of geniposide to the diluent can effectively reduce the ROS level in semen.

[0063] From the above examples and experimental examples, it can be seen that in the present invention, geniposide is used in the diluent for low-temperature preservation of dairy goat semen. The sperm motility, acrosome integrity rate, etc. after 5 days of low-temperature preservation are significantly improved, and the antioxidant capacity of sperm is effectively enhanced. The diluent for low-temperature preservation of dairy goat semen added with geniposide in the present invention can effectively preserve dairy goat semen for a long time. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a diluent for low-temperature storage of dairy goat semen using gardenia glycoside, characterized in that: The steps include: Step 1. Preparation of basic diluent: Use an electronic analytical balance to accurately weigh 1.4 g citric acid, 1 g fructose, and 2.7 g Tris, respectively, and dissolve them in 100 mL of ultrapure water. Adjust the pH to 7, filter with a 0.22 μm filter, then add 2000 IU / mL of penicillin and 2000 IU / mL of streptomycin, shake and mix with an ultrasonic oscillator, and store in a 4° refrigerator until all reagents are completely dissolved for later use; Step 2: Preparation of Gardenia glycoside dilution solution: Purchase 20 mg of Gardenia glycoside standard, transfer it to a brown sample bottle, dilute it to 1 ml with basic diluent to obtain a 20 mg / mL Gardenia glycoside solution, invert the solution to make it completely dissolved and mix evenly, and finally filter it with a 0.22 μm filter to obtain 1 ml of 20 mg / mL Gardenia glycoside solution, and store it in a sealed container at 4°C. The preparation methods of Gardenia glycoside solutions of different concentrations are as follows: ① Take 10 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 20 μg / mL geniposide dilution solution for use; ② Take 20 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 40 μg / mL geniposide dilution solution for use; ③ Take 30 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 60 μg / mL geniposide dilution solution for use; ④ Take 40 μL of 20 mg / mL Gardenia glycoside solution, add basic diluent to dilute to 10 mL, and obtain 80 μg / mL Gardenia glycoside dilution solution for use; ⑤ Take 50 μL of 20 mg / mL geniposide solution, add basic diluent to dilute to 10 mL, and obtain 100 μg / mL geniposide dilution solution for use; The prepared basic diluent was dispensed into 5 centrifuge tubes to obtain diluents for cryopreservation of goat semen with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL; Step 3: Semen examination and dilution: (1) Semen examination ①Appearance assessment; ② Vitality detection; (2) Semen dilution ①First dilution Select the semen that has passed the microscopic examination, add the diluent to the semen in a ratio of 1:2, and equilibrate at room temperature for 3-5 minutes. When diluting, pay attention to slowly sticking to the wall and rotating the tube wall. After each dilution, slowly invert it several times to fully mix, then wrap it with multiple layers of gauze and absorbent cotton, and place it in a 4℃ refrigerator for 2 hours. ② Second dilution After the first equilibration, the semen was further diluted with isothermal diluent at a ratio of 1:

1. After thorough mixing, it was placed in a 4°C refrigerator for further equilibration for 2 h and wrapped with 12 layers of gauze and stored at 4°C.

2. The method for preparing the diluent for low temperature storage of dairy goat semen containing geniposide according to claim 1 and its application, characterized in that: The appearance assessment includes the ejaculation volume: after the semen is collected, the ejaculation volume is transferred to a sterile centrifuge tube with a scale using a pipette and observed; Color: No foreign matter, no unusual smell, milky white or creamy yellow in color; Cloud movement: When observing freshly collected ram semen with the naked eye, one can see the churning and rolling cloud-like motion caused by sperm activity.

3. The method for preparing the diluent for low temperature storage of dairy goat semen containing geniposide according to claim 1 and its application, characterized in that: The vitality test is to take a proper amount of semen from a centrifuge tube on a glass slide with a pipette, press the slide and examine it under a microscope. The sperm morphology is normal, the deformity rate is below 5%, the sperm motility is higher than 0.8, and the density reaches more than 1.5 billion / mL before it can be used for subsequent tests. In order to eliminate individual differences in this study, equal amounts of semen that passed the microscopic examination were evenly mixed at 37°C.

4. An application of a diluent for low temperature storage of geniposide semen, characterized in that: The gardenia glycoside diluent prepared by the method described in any one of claims 1 to 3 in the method for preparing the gardenia glycoside diluent for low-temperature storage of dairy goat semen is used for low-temperature storage of animal semen.

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