Sheep semen antifreeze protectant and preparation method and use method thereof

Abstract

The present invention discloses a sheep semen cryoprotectant, which is composed of a first protective agent and a second protective agent. Per 100 mL of the first protective agent, it contains 46 mg-184 mg of empagliflozin, 2.5-3.5 g of glucose, 1.0-2.0 g of sodium citrate, 0.05-0.10 g of lincomycin, and 15-25 mL of egg yolk; per 100 mL of the second protective agent, it contains 80-90 mL of the first protective agent and 10-20 mL of glycerol. The protective agent introduces empagliflozin and utilizes its unique antioxidant and anti-inflammatory functions to effectively improve the cryopreservation effect of sheep semen. The present invention also discloses a preparation method and a method for using the above-mentioned cryoprotectant.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial insemination, and in particular to a sheep semen antifreeze protectant and a preparation method and a use method thereof. Background Art

[0002] Despite significant advances in sheep semen cryopreservation technology over the past few decades, only 40-60% of sperm retain motility after freezing, and only 20-30% of these remain biologically functional. One reason is that sheep sperm is sensitive to temperature fluctuations and susceptible to oxidative damage during the freezing and cooling process. Oxidative damage to the sperm membrane can cause it to lose fluidity, impairing sperm-egg recognition, membrane fusion, sperm-acrosome fusion, and sperm capacitation during fertilization. Oxidative damage to mitochondria can also lead to DNA fragmentation. Therefore, addressing oxidative damage in frozen sperm is crucial for improving freezing efficiency.

[0003] Empagliflozin is a novel glucose-lowering drug that enhances renal glucose excretion and reduces hyperglycemia in an insulin-independent manner, exerting cardiovascular protective effects and reducing the incidence of cardiovascular disease. Its mechanism of action is that empagliflozin selectively inhibits sodium-glucose cotransporter 2 (SGLT2), thereby reducing apoptosis, oxidative stress, inflammation, mitochondrial dysfunction, and maintaining ion homeostasis. Studies have shown that empagliflozin improves diabetic myocardial structure and function and reduces myocardial oxidative stress by inhibiting the TGF-β / SMAD pathway and activating the Nrf2 / ARE signaling pathway. Empagliflozin also significantly reduces the expression of endoplasmic reticulum stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, and Atf6, and activates autophagy by increasing AMPK phosphorylation, thereby reducing mTOR and increasing the expression of the autophagy-related protein LC3B. In terms of anti-inflammatory properties, empagliflozin can reduce pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor (TNF), monocyte chemoattractant protein 1 (MCP-1), and interferon gamma (IFN-γ). However, to date, empagliflozin has not been used in semen cryopreservation agents. Summary of the Invention

[0004] The purpose of the present invention is to provide a sheep semen cryoprotectant, which introduces empagliflozin and utilizes its unique antioxidant and anti-inflammatory functions to effectively improve the cryopreservation effect of sheep semen.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A sheep semen antifreeze protective agent, comprising a first protective agent and a second protective agent:

[0007] Every 100 mL of the first protective agent contains 46 mg-184 mg of empagliflozin, 2.5-3.5 g of glucose, 1.0-2.0 g of sodium citrate, 0.05-0.10 g of lincomycin, and 15-25 mL of egg yolk; every 100 mL of the second protective agent contains 80-90 mL of the first protective agent and 10-20 mL of glycerol.

[0008] Preferably, each 100 mL of the first protective agent contains 92 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, and 20 mL of egg yolk; each 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned sheep semen antifreeze protectant, comprising the following steps:

[0010] Step 1: Weigh the corresponding weights of glucose and sodium citrate into a sterile beaker, add 75-85 mL of double-distilled water to the beaker to fully dissolve the drugs, then filter with a sterile filter, add the corresponding weights of lincomycin and empagliflozin, and then add 15-25 mL of fresh egg yolk to make the total volume 100 mL. After mixing, the first protective agent is obtained;

[0011] Step 2: Take 80-90 mL of the first protective agent, add 10-20 mL of glycerol to make the total volume 100 mL, and mix well to obtain the second protective agent.

[0012] A third object of the present invention is to provide a method for using the above-mentioned sheep semen cryoprotectant, comprising the following steps:

[0013] Step S1: taking qualified semen and mixing it with the first protective agent in a volume ratio of 1:4 to obtain diluted semen;

[0014] Step S2: taking the diluted semen into a first centrifuge tube, taking the second protective agent into a second centrifuge tube, and then refrigerating the first centrifuge tube and the second centrifuge tube in a refrigerator at 4°C, shaking the first centrifuge tube intermittently during the refrigeration to prevent the sperm from entering a state of suspended animation, and after cooling to 4°C, mixing the diluted semen and the second protective agent in equal volumes to obtain a mixed semen, testing the sperm motility, and freezing the mixed semen if the motility is greater than 65%;

[0015] Step S3: The mixed semen is placed in a frozen sperm tube, and the tube is placed in a 4°C balance cabinet for 0.5 h. The temperature is then lowered. After the frozen sperm tube is cooled to -145°C, it is quickly placed in liquid nitrogen for storage.

[0016] The beneficial effect of the present invention is that empagliflozin is used in the preparation of an antifreeze protectant for sheep semen, and cooperates with other components to produce a good antifreeze effect. Experiments show that when the concentration of empagliflozin in the sheep semen antifreeze protectant is 0.4-1.6 μM, it has the best sperm motility and linear velocity after thawing. At the same time, the deformity rate of frozen-thawed Hu sheep sperm is significantly reduced, the sperm acrosome integrity rate and sperm head plasma membrane integrity rate after thawing are significantly increased, and the survival time of frozen Hu sheep sperm after thawing reaches 5.5-6.7 hours, which is at least 25% higher than the 4.4 hours of the antifreeze protectant without empagliflozin. When the added concentration of empagliflozin is 0.8 μM, the pregnancy rate reaches 62.9%-65.7%, and the lambing rate reaches 195%-210%. DETAILED DESCRIPTION

[0017] The present invention will be further described below through specific embodiments:

[0018] 1. Formula and preparation of sheep semen antifreeze protectant

[0019] Example 1 Sheep semen antifreeze protectant formula 1

[0020] Each 100 mL of the first protective agent contains 46 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk;

[0021] Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0022] Example 2 Sheep semen antifreeze protectant formula 2

[0023] Each 100 mL of the first protective agent contains 92 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk;

[0024] Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0025] Example 3 Sheep semen antifreeze protectant formula 3

[0026] Each 100 mL of the first protective agent contains 184 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk;

[0027] Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0028] Example 4 Sheep semen antifreeze protectant formula 4

[0029] Each 100 mL of the first protective agent contains 46 mg of empagliflozin, 2.5 g of glucose, 1.0 g of sodium citrate, 0.05 g of lincomycin, 85 mL of double-distilled water, and 15 mL of egg yolk;

[0030] Every 100 mL of the second protective agent contains 80 mL of the first protective agent and 20 mL of glycerol.

[0031] Example 5 Sheep semen antifreeze protectant formula 5

[0032] Each 100 mL of the first protective agent contains 184 mg of empagliflozin, 3.5 g of fructose, 2.0 g of sodium citrate, 0.10 g of lincomycin, 75 mL of double-distilled water, and 25 mL of egg yolk;

[0033] Every 100 mL of the second protective agent contains 90 mL of the first protective agent and 10 mL of glycerol.

[0034] Comparative Example 1: Formula without Empagliflozin

[0035] Per 100 mL of the first protective agent, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk;

[0036] Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0037] Comparative Example 2: Formula with more empagliflozin

[0038] Each 100 mL of the first protective agent contains 368 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk;

[0039] Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

[0040] Example 6 Preparation method of sheep semen antifreeze protectant of Example 1-5

[0041] Step 1: Weigh the corresponding weights of glucose and sodium citrate into a sterile beaker, add 75-85 mL of double-distilled water to the beaker to fully dissolve the drugs, then filter with a sterile filter, take the dilution and add the corresponding weights of lincomycin and empagliflozin, then add 15-25 mL of fresh egg yolk to make the total volume 100 mL, and mix thoroughly to obtain the first protective agent;

[0042] Step 2: Take 80-90 mL of the first protective agent, add 10-20 mL of glycerol to make the total volume 100 mL, and mix well to obtain the second protective agent.

[0043] Comparative Examples 1 and 2 were prepared according to the above method.

[0044] 2. Use and Effect Comparison of Sheep Semen Antifreeze Protectants

[0045] 1. Semen collection and freezing

[0046] Semen was collected using the pseudo-vaginal method. The collected semen was then quality-tested. If milky white and ≥75% motility were acceptable, 250 μL of the qualified semen was diluted with 1 mL of the first protective agent at a 1:4 volume ratio. The semen was then placed in a 4°C refrigerated cabinet along with the second protective agent to cool. A thermometer was used to monitor the cooling process, and gentle shaking was performed every 0.5 h to prevent the sperm from entering a state of suspended animation. After cooling to 4°C, 250 μL of semen was mixed with 250 μL of the second protective agent in equal volumes. The mixed semen was then dispensed into 0.25 mL frozen sperm tubes and placed in a 4°C refrigerated cabinet for another 0.5 h. The frozen semen tubes were then frozen using a programmed automatic freezer with a set cooling curve. Once the frozen sperm tubes had cooled to -145°C, they were quickly placed in liquid nitrogen for storage. After 7 days, the following efficacy test was performed.

[0047] Parallel tests were conducted using the sheep semen cryoprotectants of Examples 1-3 and Comparative Examples 1 and 2, respectively, according to the above method. Before freezing, the final concentrations of empagliflozin were calculated to be 0.4, 0.8, 1.6, 0, and 3.2 μM (micromoles / liter), respectively.

[0048] 2. Effect of the cryoprotectant containing empagliflozin on the motility and motility parameters of frozen-thawed sheep semen

[0049] Frozen sperm tubes were removed from liquid nitrogen and incubated in a 37°C water bath for 30 seconds. Four volumes of the first protective agent were then added (at this point, the sperm was diluted 50-fold compared to the original semen). Four μL of semen was then aspirated and analyzed using a computer-assisted sperm analysis system for sperm motility (TM), linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN), wobble (WOB), forward motion (STR), beat frequency (BCF), average angle of motion (MAD), and amplitude of head lateral sway (ALH). Five randomly selected fields of view were analyzed each time, with at least 200 sperm in each field. The results were averaged and repeated three times.

[0050] As can be seen from Table 1 below, the cryoprotectants of Examples 1-3 can improve the motility of sperm after thawing of semen. When the concentration of empagliflozin is 0.4-1.6 μM, the motility, linear velocity and other indicators of sperm after thawing are all good. When the concentration of empagliflozin is 0.8 μM, the motility, linear velocity and other indicators of sperm after thawing are all the best.

[0051] Table 1 Effects of empagliflozin on sheep semen motility and motility parameters

[0052]

[0053] Note: Different letters indicate significant differences between groups (P<0.05), the same below

[0054] 3. Effect of cryoprotectants containing empagliflozin on sperm deformity in sheep

[0055] (1) Prepare a semen smear by diluting the thawed semen 5 times, then take 20 μL and drop it on one end of a clean glass slide. Use another glass slide to contact the semen and push it forward steadily at a 30° angle to spread the semen evenly on the slide and let it air dry naturally.

[0056] (2) Add 95% alcohol and fix for 5 minutes, rinse and dry.

[0057] (3) Place in eosin solution and stain for 5-10 minutes.

[0058] (4) Rinse with distilled water until the color on the slide is almost invisible to the naked eye.

[0059] (5) After the stained slides were naturally air-dried, they were tested using a sperm computer-assisted analysis system. Five fields of view were randomly selected each time, and the number of sperm in each field was more than 200. The sperm motility was tested and the average value was taken. The experiment was repeated three times. The results are shown in Table 2.

[0060] As can be seen from Table 2, when the concentration of empagliflozin was 0.4-1.6 μM, the sperm deformity rate after thawing was 15.26%-17.59%, and when the concentration of empagliflozin was 0.8 μM, the sperm deformity rate after thawing was the lowest, at 15.26%.

[0061] Table 2 Effect of empagliflozin on sheep sperm deformity rate

[0062]

[0063] 4. Effect of cryoprotectants containing empagliflozin on the acrosome integrity of sheep sperm

[0064] The sperm acrosome integrity rate was determined using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining. The sperm nuclei were also stained with DAPI fluorescent dye to eliminate interference from other factors and improve test accuracy.

[0065] (1) Cell collection: 0.5 mL of thawed semen was placed in a centrifuge tube and centrifuged at 300 rpm. -1 Centrifuge for 5 minutes and discard the supernatant.

[0066] (2) Wash twice with PBS, each time at 300 r·min -1 Centrifuge for 5 minutes and discard the supernatant.

[0067] (3) Pipette 100 μL of PBS to resuspend the sperm cells, add 2 μL (1 mg / mL) FITC-PNA and react at 4°C in the dark for 10 min.

[0068] (4) Add 2 μL (1 mg / mL) of PI, mix gently, and incubate at 4°C for 5 min in the dark. Place the sample on ice and examine it under a fluorescence microscope within 1 h.

[0069] (5) The sample was placed on ice and examined using a fluorescence microscope within 1 hour.

[0070] The experimental results are shown in Table 3. As shown in Table 3, the cryoprotectant containing empagliflozin significantly improved the acrosome integrity rate of frozen-thawed sperm. The acrosome integrity rate of 0.4-3.2 μM empagliflozin-treated groups was significantly higher than that of the control group (p < 0.05). At an empagliflozin concentration of 0.8 μM, the acrosome integrity rate reached 77.58%.

[0071] Table 3 Effects of cryoprotectants containing empagliflozin on the acrosome integrity rate of sheep sperm

[0072]

[0073] 5. Effect of cryoprotectants containing empagliflozin on the integrity of the plasma membrane of sheep sperm heads

[0074] (1) Cell collection: 0.5 mL of thawed semen was transferred to a 37°C incubator and incubated for 30 minutes at 600 rpm. -1 Centrifuge for 5 minutes and discard the supernatant.

[0075] (2) Add 0.5 mL of Reagent A preservation solution, mix the pellet, count the sperm cells on a hemocytometer, and adjust the concentration to 2 × 10 7 Sperm cells / mL.

[0076] (3) Transfer 100 μL of sperm cells to a new 1.5 mL centrifuge tube and add 0.5 μL of Reagent B staining solution. Incubate at 37°C for 10 minutes, avoiding light exposure.

[0077] (4) Carefully add 2.5 μL of Reagent E staining solution and incubate at 37°C for 10 minutes. Avoid exposure to light. Transfer 50 μL onto a glass slide, carefully remove the staining solution, cover with a coverslip, and immediately observe and count under a (confocal) fluorescence microscope (note: count 200 sperm in each field of view).

[0078] (5) Observation of green fluorescence: filter excitation wavelength 490nm, emission wavelength 520nm; observation of red fluorescence: filter excitation wavelength: 488nm; emission wavelength: 630nm.

[0079] The experimental results are shown in Table 4. As shown in Table 4, the cryoprotectant containing empagliflozin significantly increased the rate of plasma membrane integrity in frozen-thawed sperm heads. The plasma membrane integrity rate of sperm heads in the cryoprotectant-treated group containing 0.8-1.6 μM empagliflozin after thawing was significantly higher than that in the control group and other treatment groups (P < 0.05), reaching a maximum of 51.01%.

[0080] Table 4 Effects of cryoprotectants containing empagliflozin on the integrity of the plasma membrane of sheep sperm heads

[0081]

[0082] 6. Effect of cryoprotectants containing empagliflozin on the plasma membrane integrity of sheep sperm tails

[0083] The hypotonic swelling test (HOST) was performed by weighing 0.25g sodium citrate and 0.45g fructose and dissolving them in 50mL of double-distilled water to prepare a hypotonic solution. Using a pipette, aspirate 20μL of thawed semen and add it to 180μL of the hypotonic solution. Incubate at 37°C for 30 minutes. At the end of the incubation period, a 30μL sample was pipetted and examined under a 400x optical microscope. Five fields of view were examined, with at least 200 sperm counts in each field. The proportion of sperm with curved tails relative to the total sperm count was calculated.

[0084] The experimental results are shown in Table 5. As shown in Table 5, the cryoprotectant containing empagliflozin can improve the plasma membrane integrity rate of sperm tails during freeze-thaw. The sperm tail integrity rate in the 0.4-3.2 μM empagliflozin-treated group after thawing was 43.17% to 48.13%, significantly higher than that in the control group (P < 0.05), with the highest sperm tail integrity rate reaching 48.13%.

[0085] Table 5 Effects of cryoprotectants containing empagliflozin on the integrity of the plasma membrane of sheep sperm tails

[0086]

[0087] 7. Survival time of frozen Huyang sperm after thawing

[0088] Quickly remove frozen semen straws from the same batch from the liquid nitrogen container, performing three replicates per group. Immediately place the straw in a 38°C water bath and gently shake for 10 seconds. Remove the straw, wipe the outside of the straw with gauze, cut the ends of the straw with scissors, and place a small drop of semen on a glass slide. Cover with a coverslip and examine the motility under a microscope at 400x magnification, with the microscope stage temperature maintained at 38°C. Simultaneously, place the straw flat on a holder, mark it, and store it in a 37°C incubator. Place a drop of semen on a compressed slide every 0.5 hours for microscopic examination until no viable sperm remain. Observe the motility of the sperm and record the survival time. Thaw the sperm sequentially according to the experimental temperature and record the data.

[0089] Table 6 Survival time of frozen Hu sheep sperm after thawing

[0090]

[0091] 8. Effect of cryoprotectants containing empagliflozin on the efficacy of sperm transfusion after thawing

[0092] Thawed semen supplemented with empagliflozin (experimental group) and unsupplemented with empagliflozin (control group) were used in three batches of ewes for laparoscopic insemination. Of the 93 ewes in the control group, 66 became pregnant, for a pregnancy rate of 71%. Of the 59 ewes that farrowed, 118 lambs were born, for a lambing rate of 200%. Of the 103 ewes in the experimental group, 88 became pregnant, for a pregnancy rate of 85.4%, a significant improvement (p < 0.05). Of the 81 ewes that farrowed, 160 lambs were born, for a lambing rate of 197.5%.

[0093] Table 7 Effect of adding empagliflozin cryoprotectant on the insemination effect after thawing of frozen semen

[0094]

[0095] Note: Different lowercase letters on the shoulder indicate significant differences (p < 0.05)

[0096] It can be seen from the above examples that the sheep semen cryoprotectant of the present invention has a good antifreeze effect on sheep semen because of the addition of empagliflozin.

[0097] The above embodiments are merely illustrative of the concept and implementation of the present invention, and are not intended to limit the same. Under the concept of the present invention, technical solutions that have not been substantially changed are still within the scope of protection.

Claims

1. The use of empagliflozin in the preparation of an antifreeze protectant for sheep semen is characterized in that The sheep semen antifreeze protectant consists of a first protectant and a second protectant: Each 100 mL of the first protective agent contains 92 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk; Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol; Take 250 μL of qualified semen and dilute it with 1 mL of the first protective agent at a volume ratio of 1:4, then place it in a 4°C balance cabinet for refrigeration and cooling together with the second protective agent. Gently shake it every 0.5 h to prevent the sperm from entering a state of suspended animation. After cooling to 4°C, take 250 μL of semen and mix it with 250 μL of the second protective agent in equal volumes. Then, the mixed semen is packaged into 0.25 mL frozen sperm tubes, and continues to be placed in a 4°C balance cabinet for 0.5 h. Use a programmed automatic freezer to freeze, set the cooling curve, and after the frozen sperm tubes are cooled to -145°C, quickly place them in liquid nitrogen for storage.

2. A sheep semen antifreeze protectant, comprising a first protectant and a second protectant, characterized in that: Each 100 mL of the first protective agent contains 92 mg of empagliflozin, 3.1 g of glucose, 1.4 g of sodium citrate, 0.06 g of lincomycin, 80 mL of double-distilled water, and 20 mL of egg yolk; Every 100 mL of the second protective agent contains 86 mL of the first protective agent and 14 mL of glycerol.

3. The method for preparing the sheep semen antifreeze protectant according to claim 2, wherein The steps include: Step 1: Weigh the corresponding weights of glucose and sodium citrate into a sterile beaker, add 80 mL of double-distilled water to the beaker to fully dissolve the drugs, then filter with a sterile filter, add the corresponding weights of lincomycin and empagliflozin, and then add 20 mL of fresh egg yolk to make the total volume 100 mL. After mixing, the first protective agent is obtained; Step 2: Take 86 mL of the first protective agent, add 14 mL of glycerol to make the total volume 100 mL, and mix well to obtain the second protective agent.

4. The method for using the sheep semen cryoprotectant according to claim 2, wherein The steps include: Step S1: taking qualified semen and mixing it with the first protective agent in a volume ratio of 1:4 to obtain diluted semen; Step S2: taking the diluted semen into a first centrifuge tube, taking the second protective agent into a second centrifuge tube, and then refrigerating the first centrifuge tube and the second centrifuge tube in a refrigerator at 4°C, shaking the first centrifuge tube intermittently during the period to prevent the sperm from entering a state of suspended animation, and after cooling to 4°C, mixing the diluted semen and the second protective agent in equal volumes to obtain a mixed semen, and measuring the sperm motility. If the motility rate is greater than 65%, the mixed semen can be frozen; Step S3: The mixed semen is placed in a frozen sperm tube, and is placed in a 4°C balance cabinet for 0.5 h. The temperature is then lowered. After the frozen sperm tube is cooled to -145°C, it is quickly placed in liquid nitrogen for storage.

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