Porcine oocyte in-vitro maturation culture solution additive and application thereof

Abstract

The invention provides a porcine oocyte in-vitro maturation culture solution additive and application thereof, and belongs to the field of embryo engineering, and the additive contains taurine. The additive also contains sodium pyruvate, cysteine, glucose, NaHCO3, fetal calf serum, estradiol, pregnant horse serum gonadotropin and human chorionic gonadotropin. When the porcine oocyte in-vitro maturation culture solution added with the additive is used for culturing porcine oocytes in vitro, the ROS content in the porcine oocytes can be remarkably reduced, the oxidation resistance of the porcine oocytes can be improved, and the mitochondrial distribution of the oocytes can be improved, so that the development of the oocytes can be improved; the maturation of porcine oocytes and the development of early embryos are promoted. When the technology is applied to the pig assisted reproduction technology, the success rate and quality of embryo engineering can be improved; the pig oocyte in-vitro maturation culture solution added with the additive can improve the premature embryo division rate in parthenogenetic activation of the pig oocytes.

Description

Technical Field

[0001] The invention belongs to the field of embryo engineering, and in particular is a pig oocyte in vitro maturation culture fluid additive and application thereof. Background Art

[0002] The reproductive performance of pigs is related to the scale and economic benefits of the pig farming industry. In order to improve the reproductive performance of pigs, the in vitro maturation technology of oocytes (IVM) has been rapidly developed. Although the in vitro maturation technology of oocytes has appeared in the last century, the low maturation rate has always troubled scientific research and production workers. IVM is a technology that induces the maturation of cumulus-oocyte complexes (COCs) to the M11 stage of oocytes in vitro. The purpose of this technology is to promote the in vitro maturation of immature oocytes. The IVM culture technology has been continuously improved during the development process, including culture schemes, culture medium improvements and culture environment optimization. It has developed a scheme with a higher oocyte maturation rate than conventional IVM culture and a variety of maturation fluid additives to improve the maturation rate of oocytes, and induce oocytes to develop into early embryos through parthenogenetic activation.

[0003] Parthenogenetic activation refers to the process in which oocytes in the M11 stage develop into early embryos without sperm fertilization, but under the stimulation of certain physical and chemical factors. This method does not require the combination of sperm and egg. At present, in vitro fertilization is still a conventional breeding method in animal reproduction, but the cost of purchasing frozen semen and various breeding devices is often high, and fertilization is not successful in one time, so this unique activation method brings new opportunities for animal production.

[0004] The in vitro maturation and parthenogenetic activation technology of oocytes are both embryo engineering technologies. This technology is conducive to the development of animal husbandry and has gradually matured, becoming an indispensable biotechnology for animal reproduction. This technology has a far-reaching impact on population expansion, shortening the breeding cycle of animals, accelerating variety improvement, and improving production efficiency. At present, the developmental capacity of oocytes obtained in slaughterhouses is lower than that of mature oocytes in vivo. The quality and level of these eggs are uneven, resulting in poor stability in the number of mature oocytes with division ability and the potential to develop into blastocysts. This is the main defect of this technology at present. If the in vitro developmental capacity of oocytes can be stably improved, the overall efficiency of the technology can be further improved, and the application level of the technology in animal production can be improved. Summary of the invention

[0005] In order to improve the maturation rate of oocytes, the present invention provides a porcine oocyte in vitro maturation culture fluid additive and application thereof, so as to promote the maturation of porcine oocytes.

[0006] (I) The present invention provides an additive for porcine oocyte in vitro maturation culture medium.

[0007] The additive contains taurine.

[0008] The additive also contains sodium pyruvate, cysteine, glucose, NaHCO3, fetal bovine serum, estradiol, pregnant mare serum gonadotropin and human chorionic gonadotropin (sodium pyruvate, glucose and fetal bovine serum supplement substrate energy for oocytes; cysteine ​​provides protein supplement; estradiol, pregnant mare serum gonadotropin and human chorionic gonadotropin supplement hormone requirements for oocyte development).

[0009] (ii) The present invention provides a porcine oocyte in vitro maturation culture medium containing the above-mentioned additives.

[0010] The culture medium contains 0.954 g of basal culture medium, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 3.05 mM glucose, 0.22 g of NaHCO3, 10% volume concentration of fetal bovine serum, 0.1% volume concentration of estradiol, 1% volume concentration of pregnant mare serum gonadotropin and 1% volume concentration of human chorionic gonadotropin per 100 mL, and the final concentration of taurine is 25-150 μg / mL.

[0011] The final concentration of taurine in the culture medium is preferably 50 to 100 μg / mL, more preferably 50 μg / mL.

[0012] The basic culture medium is TCM199 culture medium purchased from GIBCO.

[0013] (III) The present invention discloses the use of the additive and culture medium in in vitro culture of pig oocytes.

[0014] The additive and the porcine oocyte in vitro maturation culture fluid are used to improve the maturation rate and quality of porcine oocytes in vitro culture; to reduce the internal ROS content of porcine oocytes and improve the anti-oxidation capacity of porcine oocytes; and to improve the distribution of mitochondria in oocytes.

[0015] The additive and culture medium are used in the preparation of drugs and feed additives for promoting the maturation of pig oocytes. Using the pig oocyte in vitro maturation culture medium added with 50 μg / mL taurine, morulae with higher quality and quantity can be obtained in the parthenogenetic activation of pig oocytes after maturation.

[0016] (IV) The present invention discloses that the additive and culture medium are used to improve the early embryonic division rate in parthenogenetic activation of pig oocytes.

[0017] The present invention discloses a method for improving the early embryonic division rate in parthenogenetic activation of pig oocytes, comprising the following steps:

[0018] Step (1): culturing porcine oocytes in the culture medium, removing cumulus cells, collecting porcine oocytes, and then placing mature porcine oocytes in a chemical activation solution, and using an electroporator to activate the oocytes to complete parthenogenetic activation;

[0019] Step (2): The porcine oocytes activated parthenogenetically in step (1) are cultured in embryo culture medium.

[0020] Compared with the prior art, the present invention has the following significant advantages:

[0021] (1) Adding the additive to the in vitro maturation culture medium of porcine oocytes for in vitro culture of porcine oocytes, taurine can significantly reduce the ROS content in porcine oocytes, improve the antioxidant capacity of the porcine oocytes, improve the mitochondrial distribution of the oocytes, thereby improving the development of the oocytes and promoting the maturation of porcine oocytes. This technology is applied to assisted reproductive technology of pigs to improve the success rate and quality of embryo development.

[0022] (2) The present invention can improve the early embryonic division rate after parthenogenetic activation of porcine oocytes. Adding taurine to the culture medium is beneficial to reducing the ROS content in porcine oocytes, and the oocytes are caused to divide after electrical activation; the porcine oocytes after parthenogenetic activation are subjected to embryonic development, thereby facilitating the realization of early embryonic development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The microscope pictures and oocyte maturation rate result diagram taken during the oocyte maturation detection in Example 2;

[0024] Figure 2 The microscope picture taken when the cumulus cell expansion degree is measured in Example 2 and the result diagram of the cumulus cell expansion degree;

[0025] Figure 3 The microscope pictures taken during the test of ROS content in oocytes in Example 2 and the result graph of ROS content in oocytes;

[0026] Figure 4 The microscope picture and mitochondrial distribution result diagram taken during the oocyte mitochondrial distribution detection in Example 2;

[0027] Figure 5 The microscope pictures taken for observing the cleavage of oocytes and the cleavage rate result diagram of oocytes in Example 3;

[0028] Figure 6 This is a graph showing the embryonic development rate results for each stage of early embryos in Example 3. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0030] Example 1

[0031] Configuration of porcine oocyte in vitro maturation culture medium:

[0032] (1) To every 100 mL of maturation solution, add 0.954 g of TCM-199 (GIBCO) powdered culture medium; 0.91 mM sodium pyruvate (P-5280, Sigma); 0.57 mM cysteine ​​(C7352, Sigma); 3.05 mM glucose (G-6152, Sigma); 0.22 g NaHCO3 (S-5761, Sigma); 10% of the volume of the maturation solution by fetal bovine serum (F8318, Sigma); 0.1% of the volume of the maturation solution by estradiol (E8875, Sigma); 1% of the volume of the maturation solution by pregnant mare serum gonadotropin (Ningbo Sansheng Biological); and 1% of the volume of the maturation solution by human chorionic gonadotropin (Ningbo Sansheng Biological).

[0033] (2) adding taurine so that the final mass concentration of taurine is 25, 50, 75, 100 and 150 μg / mL, respectively.

[0034] Example 2

[0035] 1. Pig Ovarian Collection and Oocyte Collection

[0036] Ovaries of three-way pigs were collected from the slaughterhouse, placed in an insulated bucket containing 38°C saline (the saline contained 100μg / mL penicillin and 100U / mL streptomycin by mass concentration), and immediately sent back to the laboratory for processing; washed with saline containing 100μg / mL penicillin and 100U / mL streptomycin by mass concentration, and excess tissue was cut off; washed three times with saline containing 100μg / mL penicillin and 100U / mL streptomycin by mass concentration; follicular fluid of 3-6mm follicles was extracted using a 5mL syringe and a 16G needle, collected in a 50mL centrifuge tube, and heated in a 38.5°C water bath for 20min.

[0037] 2. In vitro maturation of oocytes

[0038] Discard part of the follicular fluid and add egg washing solution to clarify the liquid. The formula of egg washing solution is 3.33165g NaCl, 0.1193g KCl, 0.084g NaHCO3, 0.0204g NaH2PO4, 0.05085g MgCl2.6H2O, 1.1915g 4-hydroxyethylpiperazineethanesulfonic acid, 0.0325g penicillin, 0.005g phenol red, 0.147g CaCl2.2H2O, 0.05g polyvinyl alcohol, 1.093g sorbitol, 0.0125g gentamicin, 0.011g sodium pyruvate and 0.934mL sodium lactate added to 500mL pure water. Under a stereo microscope, collect cumulus-oocyte complexes (COCs) with normal morphology, uniform cytoplasm and at least 3 layers of granulosa cells for use. The collected COCs were placed in in vitro maturation culture droplets (300 μL) containing different final mass concentrations of taurine and covered with mineral oil for 48 h. The culture conditions were 38.5°C, 5% CO2 and saturated humidity.

[0039] The in vitro maturation culture medium containing different final mass concentrations of taurine was prepared in Example 1. The in vitro maturation culture medium with final mass concentrations of taurine of 25, 50, 75, 100 and 150 μg / mL was used as the experimental group, and the in vitro maturation culture medium without taurine was used as the control group.

[0040] 3. Oocyte maturation detection

[0041] The COCs after 48 h of culture were transferred to fresh maturation solutions of the corresponding taurine concentrations in Example 1, and the cumulus cells were removed by continuous blowing with a 200 μL pipette. The oocytes were then transferred to maturation droplets of the corresponding taurine concentrations in Example 1 and washed three times, and the discharge of the first polar body was observed under an inverted microscope.

[0042] The maturation rate of porcine oocytes is shown in Figure 1 The first polar body extrusion rate in the control group was 51.93% ± 6.00%; in the 25 μg / mL taurine group, it was 53.60% ± 4.19%, which was not significantly different from the control group (P>0.05); in the 50 μg / mL taurine group, it was 66.87% ± 3.78%, which was significantly higher than the control group (P<0.001); in the 100 μg / mL taurine group, it was 41.80% ± 7.70%, which was significantly lower than the control group (P<0.01), indicating that 50 μg / mL taurine significantly promoted oocyte maturation. Therefore, 50 μg / mL taurine was selected for subsequent experiments.

[0043] 4. Determination of the expansion degree of cumulus cells

[0044] During the oocyte maturation process, the cumulus diffusion was detected at 0, 24 and 48 h of culture, and digital images were collected using a CCD camera. Each COC was measured from the digital images. S Image J software was used to measure the expansion of cumulus cells at different time periods.

[0045] The results of cumulus cell expansion are shown in Figure 2 At the beginning of culture (0h), the diameters of the cumulus in the control group and the 50μg / mL taurine group were 105.50μm±7.10μm and 102.66μm±9.35μm, respectively, with no significant difference between the two groups (P>0.05); after 24h of culture, the diameters of the cumulus in the control group and the 50μg / mL taurine group were 114.51μm±14.75μm and 192.33μm±15.35μm, respectively, and the degree of cumulus diffusion in the taurine group was significantly higher than that in the control group (P<0.001); after 48h of culture, the diameters of the cumulus in the control group and the 50μg / mL taurine group were 196.70μm±8.37μm and 340.79μm±20.90μm, respectively, and the degree of cumulus diffusion in the taurine group was significantly higher than that in the control group (P<0.001), indicating that taurine significantly promotes cumulus diffusion during in vitro oocyte maturation.

[0046] 5. Determination of ROS Content

[0047] The DCFH-DA storage solution (S0033S, Bio-Tech) was diluted to a 10 μM working solution at a ratio of 1:1000 using serum-free oocyte maturation solution. Thirty oocytes were randomly selected and added to a culture dish containing the DCFH-DA working solution and incubated in a 37°C incubator in the dark for 20 min. After staining, the oocytes were washed 3 times with PBS. Laser confocal microscopy was used for observation and photography.

[0048] The ROS (reactive oxygen) content in oocytes is shown in Figure 3 The ROS fluorescence intensity in the control group was 9.20±0.50, and that in the 50μg / mL taurine group was 4.69±1.26, which was significantly lower than that in the control group (P<0.001), indicating that taurine significantly improved the antioxidant capacity during in vitro oocyte maturation.

[0049] 6. Mitochondrial Distribution Detection

[0050] The Mito Tracker Red (C1035, Biyuntian) storage solution was diluted to 200 nM working solution using the oocyte maturation solution in Example 1 without serum. 30 oocytes were randomly selected and added to a culture dish containing Mito Tracker Red working solution, and incubated in a 37 ° C incubator in the dark for 30 minutes. After staining, the oocytes were washed 3 times with PBS; then the oocytes were fixed with 4% paraformaldehyde for 15 minutes, washed 3 times with PBS and sealed; laser confocal microscopy was used for observation and photography.

[0051] The distribution of mitochondria in oocytes is shown in Figure 4 . The figure shows the uniform and non-uniform distribution of oocyte mitochondria. After 48 hours of culture, the uniform distribution of mitochondria in the control group oocytes was 27%, and the proportion in the 50μg / mL taurine group was 69%, which was significantly higher than the control (P<0.05); the non-uniform distribution of mitochondria in the control group oocytes was 73%, and the proportion in the 50μg / mL taurine group was 31%, which was significantly lower than the control (P<0.05), indicating that taurine significantly improves the distribution of mitochondria in porcine oocytes during porcine oocyte maturation.

[0052] Example 3

[0053] 1. Electrochemical activation solution configuration

[0054] 0.11915 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.0012037 g of MgSO4, 0.00111 g of CaCl2, 5.4651 g of mannitol and 0.001 g of polyvinyl alcohol were added to 100 mL of ultrapure water.

[0055] 2. Preparation of Embryonic Fluid

[0056] In 100 mL of ultrapure water, add 0.6264 g NaCl, 0.07452 g KCl, 0.00476 g K2HPO4, 0.00984 g MgSO4·7H2O, 0.2106 g NaHCO3, 0.0022 g sodium pyruvate, 0.06166 g calcium dialaninate pentahydrate, 0.0146 g L-glutamine, 0.05458 g taurine, 2 mL β-mercaptoethanol, 1 mL MEM medium, 0.005 g gentamicin and 0.3 g bovine serum albumin.

[0057] 3. Parthenogenetic Activation

[0058] The cumulus cells were removed from the cumulus-oocyte complex after being cultured in the culture medium with different taurine concentrations for 48 h in Example 1, and the oocytes were collected. The mature oocytes were washed three times with an electrochemical activation solution, and then transferred into a 1 mm electrofusion tank in a row, activated twice with a voltage of 120 V, and washed three times with embryonic fluid, and then transferred into different embryonic fluids for culture in groups.

[0059] 4. Statistics of early embryo cleavage rate

[0060] After the parthenogenetically activated oocytes were cultured in the above embryonic fluid for 72 hours, the oocyte cleavage was observed under a stereomicroscope and the cleavage rate was calculated.

[0061] The cleavage rate of oocytes is Figure 5 . After 72 hours of parthenogenetic activation, the cleavage rate of oocytes in the control group was 47.27%±6.11%; that in the 50μg / mL taurine group was 60.14%±5.04%, which was significantly higher than that in the control group (P<0.01), indicating that taurine promotes early embryonic development of mature oocytes.

[0062] 5. Statistics of embryo development at each stage:

[0063] The experiment was repeated three times. The numbers of oocytes activated in the control group were 87, 84 and 89 respectively, and the numbers of oocytes activated in the 50 μg / mL taurine group were 107, 105 and 98 respectively. 96 hours after parthenogenetic activation, the development of early embryos at various stages was observed and counted.

[0064] The number of embryonic development stages is shown in Figure 6The average proportion of two-cell embryos in the control group was 4.23%±0.69%, and that in the 50μg / mL taurine group was 6.13%±1.48%, with no significant difference between the two groups (P>0.05); the average proportion of three-cell and four-cell embryos in the control group was 7.27%±2.22% and 3.45%±1.11%, and that in the 50μg / mL taurine group was 6.75%±1.46% and 2.57%±0.46%, with no significant difference between the two groups (P>0.05); the average proportion of eight-cell / sixteen-cell embryos in the control group was 10.77%±0.69%, and that in the 50μg / mL taurine group was The average proportion of morula in the control group was 10.77%±0.69%, while that in the 50μg / mL taurine group was 18.82%±2.02%, which was significantly higher than that in the control group (P<0.001). The average proportion of undeveloped oocytes in the control group was 55.45%±4.68%, while that in the 50μg / mL taurine group was 36.31%±8.79%, which was significantly lower than that in the control group (P<0.05). This indicates that taurine can significantly improve the quality of porcine oocytes and promote early embryonic development after inducing porcine oocyte maturation.

[0065] Through the above embodiments and experimental examples, it is shown that taurine can significantly improve the maturation rate and quality of porcine oocytes, significantly reduce the ROS content in porcine oocytes, promote the maturation of porcine oocytes, and improve the early embryo cleavage rate after parthenogenetic activation of oocytes. Based on this, taurine can be used to prepare porcine oocyte in vitro maturation culture medium additives or porcine oocyte in vitro maturation culture medium. And it is preferred that the final concentration of taurine added is 25-150 μg / mL. Too low or too high a final concentration of taurine added is not conducive to improving the maturation rate and quality of porcine oocytes. More preferably, the final concentration of taurine added is 50-100 μg / mL. Most preferably, the final concentration of taurine added is 50 μg / mL. The maturation rate of porcine oocytes cultured in vitro with the addition of 50 μg / mL taurine is higher.

[0066] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. A porcine oocyte in vitro maturation culture medium additive, characterized in that: The additive contains taurine.

2. The additive according to claim 1, characterized in that The additive also contains sodium pyruvate, cysteine, glucose, NaHCO3, fetal bovine serum, estradiol, pregnant mare serum gonadotropin and human chorionic gonadotropin.

3. A porcine oocyte in vitro maturation culture medium containing the additive according to claim 2, characterized in that: The culture medium contains 0.954 g of basal culture medium, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 3.05 mM glucose, 0.22 g of NaHCO3, 10% volume concentration of fetal bovine serum, 0.1% volume concentration of estradiol, 1% volume concentration of pregnant mare serum gonadotropin and 1% volume concentration of human chorionic gonadotropin per 100 mL, and the final concentration of taurine is 25-150 μg / mL.

4. The culture solution according to claim 3, characterized in that The basic culture medium is TCM199 culture medium.

5. Use of the additive according to claim 1 or claim 2 in in vitro culture of pig oocytes, characterized in that: The additive is used for improving the maturation rate of pig oocytes cultured in vitro, for reducing the internal ROS content of pig oocytes, or for improving the distribution of mitochondria in oocytes.

6. Use of the culture medium according to claim 3 or claim 4 in culturing porcine oocytes in vitro, characterized in that: The additive is used for improving the maturation rate of pig oocytes cultured in vitro, for reducing the internal ROS content of pig oocytes, or for improving the distribution of mitochondria in oocytes.

7. Use of the additive according to claim 1 or claim 2 in improving the early embryo cleavage rate in parthenogenetic activation of porcine oocytes.

8. Use of the culture solution according to claim 3 or claim 4 in improving the early embryonic cleavage rate in parthenogenetic activation of porcine oocytes.

9. Use of the culture medium according to claim 3 or claim 4 in improving the early embryonic cleavage rate in parthenogenetic activation of porcine oocytes, characterized in that: The steps include: Step (1): culturing porcine oocytes in the culture medium for a period of time, removing cumulus cells, collecting porcine oocytes, and then placing mature porcine oocytes in a chemical activation solution, and using an electroporator to activate the oocytes to complete parthenogenetic activation; Step (2): The porcine oocytes activated parthenogenetically in step (1) are cultured in embryo culture medium.

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