Cryoprotectant for improving freezing tolerance of cattle and sheep blastocysts and freezing method

Abstract

The invention discloses a cryoprotectant for improving the freezing tolerance of cattle and sheep blastocysts and a freezing method, and the cryoprotectant for improving the freezing tolerance of the cattle and sheep blastocysts comprises cGMP and programmed freezing fluid. Immersing a blastocyst to be frozen in the cryoprotectant, placing the blastocyst on a heating table at 37 DEG C, and pretreating the blastocyst for 10 minutes; and freezing the embryo by adopting an embryo programmed freezing operation. According to the method, the blastocysts are soaked in the cryoprotectant containing cGMP for pretreatment, the content of lipid droplets in the blastocysts can be reduced, after the pretreated blastocysts are subjected to programmed cryopreservation treatment and unfreezing, the recovery rate and hatching rate of the cattle and sheep blastocysts are remarkably increased, better cellular morphology is shown, and the freezing tolerance of the blastocysts can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of embryo freezing, and in particular relates to a cryoprotectant and a freezing method for improving the freezing tolerance of bovine and sheep blastocysts. Background Art

[0002] During in vitro production (IVP), an unsuitable culture environment can cause abnormal lipid metabolism in the embryo, which may result in insufficient or excessive energy production, unstable membrane structure, physical damage caused by ice crystals during embryo freezing, cell dysfunction, and oxidative stress caused by lipid peroxide accumulation. These adverse effects greatly reduce the success rate and quality of embryo cryopreservation.

[0003] IVP embryos have a higher fat content than in vivo-derived (IVD) embryos. IVP embryos cultured in serum-containing medium exhibit abnormal accumulation of lipid droplets. Excessive lipid droplet accumulation in embryos can reduce their cryotolerance. Excessive lipid droplets alter the intracellular environment, and the presence of lipids can interfere with the normal formation of ice crystals, changing their speed and morphology, leading to irregular ice crystals and irreversible mechanical damage to cellular structures such as cell membranes and organelles.

[0004] Therefore, in order to improve the programmed freezing effect of cattle and sheep blastocysts and enhance the freezing tolerance of cattle and sheep blastocysts, there is an urgent need in the prior art for a cryoprotectant and a freezing method that can enhance the freezing tolerance of cattle and sheep blastocysts. Summary of the Invention

[0005] The first purpose of the present invention is to overcome the shortcomings of the existing technology and provide a cryoprotectant that improves the freezing tolerance of cattle and sheep blastocysts.

[0006] The first objective of the present invention is achieved through the following technical solution: a cryoprotectant for improving the freezing tolerance of bovine and sheep blastocysts, the cryoprotectant comprising cGMP (cyclic guanosine monophosphate) and programmed freezing fluid.

[0007] Preferably, in the cryoprotectant, the molar concentration of cGMP is 1 mmol / L.

[0008] Preferably, in the cryoprotectant, the molar concentration of cGMP is 0.5 mmol / L.

[0009] Preferably, the programmed freezing solution comprises ethylene glycol with a molar concentration of 1.5 mol / L and 4 mg / mL bovine serum albumin, which are dissolved in phosphate buffered saline (PBS).

[0010] Preferably, the blastocyst is a blastocyst obtained by in vitro culture.

[0011] Preferably, the blastocyst is a bovine or ovine blastocyst.

[0012] The second purpose of the present invention is to overcome the shortcomings of the existing technology and provide a freezing method that improves the freezing tolerance of cattle and sheep blastocysts.

[0013] The second object of the present invention is achieved through the following technical solution: a freezing method for improving the freezing tolerance of cattle and sheep blastocysts, characterized by comprising the following steps: 1) Immerse the blastocyst to be frozen in the cryoprotectant of any one of claims 1 to 4 and place it on a 37°C heating platform for 10 minutes; 2) Pre-cool the freezer. When using the freezer for the first time each day, fill the liquid nitrogen bath with 5 cm of liquid nitrogen and pre-cool the freezing chamber for 5 minutes. After 5 minutes, fill the liquid nitrogen bath with liquid nitrogen again. Then, close the lids of the liquid nitrogen bath and the freezing chamber to maintain the temperature in the freezing chamber at the starting temperature of the cooling program. The freezing program is the built-in cooling program 2 of the FREEZECONTROL® Systems (CL-8800i). 3) Mark the freezing date and embryo information on the frozen straw; 4) Tubing: Load the frozen straws with programmed freezing solution, air column, cryoprotectant containing embryos, air column, and programmed freezing solution in this order from the closed end to the open end. Seal the tubes with sealing powder. 5) Freezing: Load the frozen capillary tubes loaded in the previous step into the freezing chamber, and then start the freezing program to make the instrument run according to the selected cooling program; 6) Ice planting: When the temperature drops to -7℃, ice planting begins; 7) Add liquid nitrogen to the liquid nitrogen bath every 30-40 minutes to ensure the amount of liquid nitrogen in the freezing chamber; 8) When the freezing process is completed, remove the frozen tubes from the freezing chamber and quickly store them in liquid nitrogen.

[0014] The third purpose of the present invention is to overcome the shortcomings of the existing technology and provide a use of cGMP for improving the freezing tolerance of cattle and sheep blastocysts.

[0015] The third object of the present invention is achieved through the following technical solution: Use of cGMP for improving the freezing tolerance of cattle and sheep blastocysts, including the following contents: 1) Using a cryoprotectant formulated with cGMP and programmed freezing fluid to improve the freezing tolerance of bovine and ovine blastocysts; immersing the blastocysts to be frozen in the cryoprotectant and placing them on a 37°C heating platform for 10 minutes; 2) Pre-cool the freezer. When using the freezer for the first time each day, fill the liquid nitrogen bath with 5 cm of liquid nitrogen and allow the freezing chamber to pre-cool for 5 minutes. After 5 minutes, fill the liquid nitrogen bath with liquid nitrogen again. Then, close the lids of the liquid nitrogen bath and the freezing chamber to maintain the temperature in the freezing chamber at the starting temperature of the cooling program. The freezing program is the built-in cooling program 2 of the FREEZECONTROL® Systems (CL-8800i). 3) Mark the freezing date and embryo information on the frozen straw; 4) Tubing: Load the frozen straws from the closed end to the open end in the order of programmed freezing solution, air column, cryoprotectant containing embryos, air column, programmed freezing solution, and then seal with sealing powder. 5) Freezing: Load the frozen capillary tubes loaded in the previous step into the freezing chamber, and then start the freezing program to make the instrument run according to the selected cooling program; 6) Ice planting: When the temperature drops to -7℃, ice planting begins; 7) Add liquid nitrogen to the liquid nitrogen bath every 30-40 minutes to ensure the amount of liquid nitrogen in the freezing chamber; 8) When the freezing process is completed, remove the frozen tubes from the freezing chamber and quickly store them in liquid nitrogen.

[0016] Preferably, in the cryoprotectant, the molar concentration of cGMP is 1 mmol / L.

[0017] Preferably, in the cryoprotectant, the molar concentration of cGMP is 0.5 mmol / L.

[0018] The beneficial effects of the present invention are as follows: In order to improve the programmed cryopreservation technology for bovine and ovine blastocysts, the present invention uses cGMP to reduce the lipid content in blastocysts. cGMP can change the permeability of calcium ion channels within cells. When the intracellular calcium ion concentration increases, it activates calcium-dependent lipases (such as hormone-sensitive lipase), promoting lipid degradation. Hormone-sensitive lipase (HSL) is a key lipase that is sensitive to changes in intracellular calcium ion concentration. Under normal physiological conditions, HSL is mainly present on the surface of lipid droplets. When the intracellular calcium ion concentration increases, calcium ions interact with calcium binding sites on HSL, or by activating calcium-dependent protein kinases (such as calcium / calmodulin-dependent protein kinase CaMK), HSL undergoes phosphorylation modification and participates in lipid degradation, thereby improving the programmed cryopreservation effect of blastocysts.

[0019] The present invention immerses the blastocyst in a cryoprotectant containing cGMP for pretreatment, which can reduce the fat droplet content in the blastocyst. The incubated blastocyst is subjected to a programmed cryopreservation treatment. After thawing, the recovery rate and hatching rate of the bovine and sheep blastocysts are significantly improved, and better cell morphology is exhibited. The present invention can improve the freezing tolerance of the blastocyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Microscopic images of programmed freezing and thawing of sheep blastocysts after pretreatment with 0.2-3 mM cGMP for 10 minutes; Figure 2 This is a statistical chart showing the recovery rate of sheep blastocysts after programmed freezing and thawing 24 hours after pretreatment with 0.2-3 mM cGMP for 10 minutes; Figure 3 This is a statistical chart showing the recovery rate of sheep blastocysts after programmed freezing and thawing 48 hours after pretreatment with 0.2-3 mM cGMP for 10 minutes; Figure 4 The expression levels of genes related to lipid degradation in sheep blastocysts after pretreatment with 0.2-3 mM cGMP for 10 min were statistically analyzed. Figure 1 ; Figure 5 The expression levels of genes related to lipid degradation in sheep blastocysts after pretreatment with 0.2-3 mM cGMP for 10 min were statistically analyzed. Figure 2 ; Figure 6 This is a fluorescence image of lipid droplet content in sheep blastocysts after pretreatment with 0.5 mM cGMP for 10 minutes; Figure 7 This is a statistical diagram of lipid droplet content in sheep blastocysts after pretreatment with 0.5 mM cGMP for 10 minutes; Figure 8 This is a fluorescence image of the phosphorylated-HSL protein expression in sheep blastocysts after pretreatment with 0.5 mM cGMP for 10 minutes; Figure 9 The statistical graph shows the expression level of phosphorylated-HSL protein in sheep blastocysts after pretreatment with 0.5 mM cGMP for 10 minutes. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings.

[0022] The concept and technical effects of the present invention are further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, the methods described are conventional experimental methods. Materials described are commercially available unless otherwise specified. Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, USA).

[0024] The results of the following examples were statistically analyzed using the following method: each example was repeated at least three times, and the data are expressed as mean ± standard deviation (SD). All data were statistically analyzed using GraphPad Prism V.8.0.2 (GraphPad Software, San Diego, California, USA). P < 0.05 indicated a significant difference.

[0025] Example 1 cGMP improves the slow freezing effect of bovine blastocysts 1.1 Obtaining bovine in vitro embryos Bovine oocytes from slaughterhouses underwent in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC). The completion of IVF was designated as day 1, and blastocysts (with a dense and compact inner cell mass and numerous trophoblasts closely surrounding the inner cell mass) were selected on day 7.5.

[0026] 1.2 Programmed freezing and thawing of bovine blastocysts Use a pipette to drop 100 μL of programmed freezing solution (control group) and cryoprotectant containing different concentrations of cGMP (0.2 mM, 0.5 mM, 1 mM, and 3 mM) into each culture dish. Transfer the blastocysts selected in step 1.1 into each culture dish and place them on a 37°C heating block for 10 minutes. Then, freeze the embryos using the programmed freezing procedure described in steps 2-8 of the present invention for improving the freezing tolerance of bovine and ovine blastocysts.

[0027] To thaw embryos, remove the frozen straw from the liquid nitrogen tank, thaw in air for 5 seconds, and then thaw in a 37°C water bath for 1 minute. After 1 minute, remove the frozen straw from the water bath and dry. Cut off the seal and allow the embryos to flow into a sterile culture dish. Then, under a microscope, wash the thawed embryos 2-3 times in prewarmed embryonic development medium containing SOF medium, 2% essential amino acids, 1% non-essential amino acids, and 3 mg / mL BSA (bovine serum albumin). After washing, transfer the embryos to embryonic development medium supplemented with 10% FBS (fetal bovine serum) for culture. The recovery rate (recovery rate = number of recovered blastocysts / total number of blastocysts) was calculated 24 hours after thawing, and the hatching rate (hatching rate = number of hatched blastocysts / number of recovered blastocysts) was calculated 48 hours after thawing.

[0028] The results are shown in Table 1. Compared with the control group, pretreatment with a cryoprotectant containing 1 mM cGMP for 10 min significantly increased the recovery rate of bovine embryos 24 hours after thawing (p<0.05). After pretreatment with a cryoprotectant containing 0.2 mM cGMP, the hatching rate of bovine embryos 48 hours after thawing was significantly lower than that of bovine embryos pretreated with a cryoprotectant containing 1 mM and 3 mM cGMP (p<0.05).

[0029]

[0030] Note: Data in the same group with different lowercase letters in the superscript indicate significant differences (P < 0.05), and data with the same lowercase letters in the superscript indicate no significant differences (P > 0.05).

[0031] Example 2 cGMP improves the effect of programmed freezing of sheep blastocysts 2.1 Obtaining sheep in vitro embryos Ovine oocytes from slaughterhouses underwent in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC). The date of completion of IVF was designated as day 1, and blastocysts (with a dense and compact inner cell mass and numerous trophoblasts closely surrounding the inner cell mass) were selected for cryopreservation on day 7.

[0032] 2.2 Programmed freezing and thawing of sheep blastocysts Use a pipette to drop 100 μL of programmed freezing solution (control group) and a protective agent containing different concentrations of cGMP (0.2mM, 0.5mM, 1mM, and 3mM) into each culture dish. Transfer the blastocysts selected in 1.1 to each culture dish and place them on a 37°C heating block for 10 minutes. Then, freeze the embryos using the programmed freezing procedure described in steps 2-8 of the present invention for improving the freezing tolerance of bovine and ovine blastocysts.

[0033] Take out the frozen straw from the liquid nitrogen tank, thaw in the air for 5 seconds, and then put it into a 37℃ water bath to thaw for 1 minute. After 1 minute, take out the frozen straw from the water bath and dry it. Cut off the seal to allow the embryo to flow into a sterile culture dish. Then, under a microscope, use preheated embryo development fluid to wash the thawed embryo 2-3 times. The embryo development fluid includes SOF culture medium, 2% essential amino acids + 1% non-essential amino acids and 3 mg / mL BSA (bovine serum albumin). After washing, transfer it into embryo development fluid supplemented with 10% FBS (fetal bovine serum) for culture. The recovery rate (recovery rate = number of recovered blastocysts / total number of blastocysts) was calculated 24 hours after thawing, and the hatching rate (hatching rate = number of hatched blastocysts / number of recovered blastocysts) was calculated 48 hours after thawing. The results are as follows. Figure 1 、 Figure 2 、 Figure 3 As shown in the results, pretreatment with a cryoprotectant containing 0.5 mM cGMP for 10 min can significantly improve the recovery rate 24 h after thawing and the hatching rate 48 h after thawing, and show better cell morphology, thereby improving the freezing tolerance of sheep blastocysts.

[0034] Example 3 cGMP participates in lipid degradation in blastocysts 3.1 Effect of 10 min cGMP incubation on the expression levels of lipid degradation-related genes in sheep blastocysts According to the method in Example 2, sheep blastocysts were pretreated for 10 minutes using programmed freezing solution (control group) and cryoprotectants containing different concentrations of cGMP (0.2 mM, 0.5 mM, 1 mM, and 3 mM), respectively. The incubated blastocysts were collected and the desired cDNA was obtained by single-cell amplification according to the instructions of the Single Cell Squence Specific Amplification Kit (purchased from Novozymes: P621). qPCR detection was then performed according to the instructions of TB Green® Premix Ex Taq™ II (TaKaRa: RR820A). The primer sequences are shown in Table 2:

[0035] The results are as follows Figure 4 、 Figure 5 As shown. In this example, a total of seven genes involved in lipid degradation were detected: ACSL4, HMGCR, HMGCS1, LIPE, LPL, LIPF, and PLIN2. The results showed that after 10 minutes of pretreatment in a cryoprotectant containing cGMP, except for the ACSL4 gene, whose expression level did not change significantly compared with the control group, the expression levels of the remaining genes increased. In addition, after 10 minutes of pretreatment with a cryoprotectant containing 0.5 mM cGMP, the expression level of the LIPE gene encoding the HSL protein increased the most, increasing by 27.10 times compared with the control group. This indicates that cGMP reduces the lipid droplet content in blastocyst cells through the lipid degradation pathway involving the LIPE gene, thereby improving the success rate of programmed freezing of blastocysts.

[0036] 3.2 Detection of lipid droplet content in blastocysts Following the method described in Example 1, ovine day 7 blastocysts were pretreated with a cryoprotectant containing 0.5 mM cGMP for 10 minutes and collected after incubation. The Nile Red Lipid Droplet Red Fluorescence Detection Kit (purchased from Beyotime Biotechnology, C2051S) was used according to the manufacturer's instructions. Observation was performed using a laser confocal microscope, and fluorescence intensity was measured using Image J software.

[0037] The results are as follows Figure 6 、 Figure 7 Pretreatment with a cryoprotectant containing 0.5 mM cGMP for 10 min significantly reduced the lipid droplet content in blastocysts (p<0.0001).

[0038] 3.3 Blastocyst Immunofluorescence According to the method in Example 2, sheep blastocysts on the 7th day were pretreated for 10 minutes using a cryoprotectant containing 0.5 mM cGMP, and the pretreated blastocysts were collected. Use 4% paraformaldehyde to fix at room temperature for 30 minutes, followed by treatment with cell immunofluorescence permeabilization solution for 1 hour. Then block with PBS solution containing 5% BSA for 30 minutes. Next, use P-HSL antibody (1:200, Affbiotech: AF2350) to incubate at 4°C overnight, and then use goat anti-rabbit IgG (H+L) fluor-488 conjugated antibody to incubate at 37°C for 40 minutes. DNA staining was performed using DAPI. Observation was performed using a laser confocal microscope, and fluorescence intensity was measured using Image J software.

[0039] The results are as follows Figure 8 、 Figure 9 As shown in Figure 3 . Pretreatment with a cryoprotectant containing 0.5 mM cGMP for 10 minutes significantly increased the content of P-HSL protein in blastocysts (p < 0.0001). This is consistent with the PCR results, indicating that the cryoprotectant containing 0.5 mM cGMP can phosphorylate HSL, activate its lipid degradation activity, and ultimately improve the freezing tolerance of ovine blastocysts.

[0040] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A cryoprotectant for improving the freezing tolerance of cattle and sheep blastocysts, characterized by: The cryoprotectants include cGMP and programmed freezing fluids.

2. The cryoprotectant for improving the freezing tolerance of bovine blastocysts according to claim 1, characterized in that: In the cryoprotectant, the molar concentration of cGMP is 1 mmol / L.

3. The cryoprotectant for improving the freezing tolerance of sheep blastocysts according to claim 1, characterized in that: In the cryoprotectant, the molar concentration of cGMP is 0.5 mmol / L.

4. The cryoprotectant for improving the freezing tolerance of sheep blastocysts according to claim 1, characterized in that: The programmed freezing solution includes ethylene glycol with a volume molar concentration of 1.5 mol / L and 4 mg / mL bovine serum albumin, which are dissolved in a phosphate buffer solution.

5. The cryoprotectant for improving the freezing tolerance of cattle and sheep blastocysts according to claim 2 or 3, characterized in that: The blastocyst is a blastocyst obtained by in vitro culture.

6. A freezing method for improving the freezing tolerance of cattle and sheep blastocysts, characterized in that The steps include: 1) Immerse the blastocyst to be frozen in the cryoprotectant of any one of claims 1 to 4 and place it on a 37°C heating platform for 10 minutes; 2) Pre-cool the freezer. When using the freezer for the first time each day, fill the liquid nitrogen bath with 5 cm of liquid nitrogen and pre-cool the freezing chamber for 5 minutes. After 5 minutes, fill the liquid nitrogen bath with liquid nitrogen again. Then, close the lids of the liquid nitrogen bath and the freezing chamber to keep the temperature inside the freezing chamber at the starting temperature of the cooling program. 3) Mark the freezing date and embryo information on the frozen straw; 4) Tubing: Load the frozen straws from the closed end to the open end in the order of programmed freezing solution, air column, cryoprotectant containing embryos, air column, and programmed freezing solution. Once loaded, seal the tubes with sealing powder. 5) Freezing: Load the frozen capillary tubes loaded in the previous step into the freezing chamber, and then start the freezing program to make the instrument run according to the selected cooling program; 6) Ice planting: When the temperature drops to -7℃, ice planting begins; 7) Add liquid nitrogen to the liquid nitrogen bath every 30-40 minutes to ensure the amount of liquid nitrogen in the freezing chamber; 8) When the freezing process is completed, remove the frozen tubes from the freezing chamber and quickly store them in liquid nitrogen.

7. Use of cGMP for improving the freezing tolerance of cattle and sheep blastocysts, characterized in that Includes the following: 1) Using a cryoprotectant formulated with cGMP and programmed freezing fluid to improve the freezing tolerance of bovine and ovine blastocysts; immersing the blastocysts to be frozen in the cryoprotectant and placing them on a 37°C heating platform for 10 minutes; 2) Pre-cool the freezer. When using the freezer for the first time each day, fill the liquid nitrogen bath with 5 cm of liquid nitrogen and allow the freezing chamber to pre-cool for 5 minutes. After 5 minutes, fill the liquid nitrogen bath with liquid nitrogen again. Then, close the lids of the liquid nitrogen bath and the freezing chamber. Select the freezer cooling program and keep the temperature in the freezing chamber at the starting temperature of the cooling program. 3) Mark the freezing date and embryo information on the frozen straw; 4) Tubing: Load the frozen straws from the closed end to the open end in the order of programmed freezing solution, air column, cryoprotectant containing embryos, air column, programmed freezing solution, and then seal with sealing powder. 5) Freezing: Load the frozen capillary tubes loaded in the previous step into the freezing chamber, and then start the freezing program to make the instrument run according to the selected cooling program; 6) Ice planting: When the temperature drops to -7℃, ice planting begins; 7) Add liquid nitrogen to the liquid nitrogen bath every 30-40 minutes to ensure the amount of liquid nitrogen in the freezing chamber; 8) When the freezing process is completed, remove the frozen tubes from the freezing chamber and quickly store them in liquid nitrogen.

8. The use according to claim 7, characterized in that: In the cryoprotectant, the molar concentration of cGMP is 0.5 mmol / L.

9. The use according to claim 7, characterized in that: In the cryoprotectant, the molar concentration of cGMP is 1 mmol / L.

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