Embryo culture method and embryo culture solution for improving quality of pig somatic cell nuclear transfer embryos

Abstract

The application discloses an embryo culture method and embryo culture solution for improving the quality of pig somatic cell nuclear transfer embryos, wherein a small molecule substance X1 capable of specifically combining with the RepA domain of Xist is added into a pig early embryo culture solution, so as to improve the development quality of pig SCNT embryos.

Description

Technical Field

[0001] This invention relates to porcine in vitro embryo culture, specifically to an early embryo culture medium supplemented with a small molecule substance X1 and a method for early embryo culture that improves the quality of porcine somatic cell nuclear transfer embryos. Background Technology

[0002] Somatic cell nuclear transfer (SCNT) technology allows highly differentiated somatic cells to acquire totipotency through reprogramming, thereby developing into new animal individuals. However, SCNT technology has long suffered from extremely low cloning efficiency, primarily manifested in high rates of embryonic developmental loss, gestational or perinatal mortality of cloned animals, and developmental abnormalities after birth. These issues severely hinder the widespread application of this technology. Currently, it is widely believed that abnormal histone modification, inadequate reprogramming of donor cells, and impaired zygotic genome activation are the main reasons for the low cloning efficiency of SCNT technology.

[0003] X-chromosome inactivation (XCI) refers to the process by which female mammals achieve gene expression levels nearly identical to those of male mammals by inactivating one X chromosome. XCI is primarily caused by… Xist (X-inactive specific transcript) expresses regulation. Xist Overexpression leads to downregulation of X-linked genes across the entire X chromosome, resulting in abnormal histone modifications and XCI abnormalities, such as in some blastomeres of SCNT female embryos. Xist High expression at all loci, and inactivation of both X chromosomes; also high expression of the only X chromosome in some blastomeres of male cloned embryos. Xist And it becomes inactive.

[0004] In practice, many cloned animals fail due to reprogramming errors. Xist X-linked gene expression abnormalities and XCI abnormalities. Abnormal XCI expression has been detected in stillborn cloned cattle and pigs. However, XCI abnormalities are not the only cause of abnormal embryonic development and reduced blastocyst numbers, thus affecting the quality and efficiency of cloned embryos. For example, in early embryo culture of pig cloned embryos (pig oocyte parthenogenetic embryos), there is a problem of poor blastocyst quality due to delayed X chromosome inactivation (see: CN118813526A).

[0005] Studies have shown (see: Targeting Xist with compounds that disrupt RNA structure and X inactivation) that the small molecule X1 can specifically bind to the RepA domain of Xist both in vitro and in vivo. The binding of X1 reduces the conformational space of RepA, displacing homologous interacting protein factors (PRC2 and SPEN), inhibiting histone H3K27me3, and suppressing the initiation of X chromosome inactivation. However, this study treated embryonic stem cells with X1, and the treatment affected the embryoid growth of female embryonic stem cells. Furthermore, it only focused on X chromosome inactivation itself and had no correlation with blastocyst rate (or early embryonic development quality). Summary of the Invention

[0006] The purpose of this invention is to provide an embryo culture method and embryo culture medium for improving the quality of porcine somatic cell nuclear transfer (SCNT) embryos. By adding a small molecule substance X1 to the in vitro early embryo culture medium, the developmental quality of porcine somatic cell nuclear transfer (SCNT) embryos is improved.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, an embryo culture method is provided, comprising the following steps:

[0009] Porcine SCNT embryos were constructed, and these embryos were transferred into porcine early embryo culture medium supplemented with 4–15 μmol·L⁻¹. -1 The small molecule substance X1 was then used to culture the porcine SCNT embryos to the blastocyst stage, wherein the porcine early embryo culture medium contained 1–2 mmol·L⁻¹. -1 (i.e., 1–2 mM) glutamine and 5–8 mmol·L -1 (i.e., 5-8 mM) taurine.

[0010] Preferably, the small molecule substance X1 is added to the porcine early embryo culture medium to a final concentration of 5–10 μmol·L⁻¹. -1 The small molecule X1 inhibited histone H3K27me3 in porcine SCNT embryos and reduced the occurrence of X chromosome inactivation abnormalities in porcine SCNT embryos, thereby improving the developmental quality of porcine SCNT embryos.

[0011] Preferably, the small molecule substance X1 is added to the porcine early embryo culture medium to a final concentration of 5–8 μmol·L⁻¹. -1 Adding small molecule substance X1 can increase the number of blastocyst cells and the blastocyst rate.

[0012] Preferably, the porcine early embryo culture medium is prepared with 1–2 mmol·L⁻¹ added. -1 Glutamine and 5–8 mmol·L -1 PZM-3 of taurine.

[0013] Preferably, the culture conditions for the porcine SCNT embryos further include: 38.0–38.5°C, 4.8%–5.2% CO2, 94.8%–95.2% N2, and saturated humidity; or, 38.0–38.5°C, 4.5%–5.5% CO2, 94.5%–95.5% N2, and saturated humidity.

[0014] Preferably, the construction of porcine SCNT embryos specifically includes the following steps: culturing porcine oocytes in an in vitro maturation culture medium for 44-46 hours, then forming a reconstructed embryo by nuclear transfer with porcine somatic cells, fusing the reconstructed embryo to obtain a porcine SCNT embryo to be cultured (for example, the reconstructed embryo can be transferred into a porcine early embryo culture medium after electrofusion and recovery).

[0015] Preferably, the porcine somatic cells are selected from porcine fibroblasts that have been starved in advance (usually, the culture medium of porcine fibroblasts is replaced with serum-free cell culture medium 22-24 hours before somatic cell nuclear transfer, thereby starving them).

[0016] On the other hand, a porcine SCNT embryo culture medium is provided, the embryo culture medium comprising a basal culture medium and a final concentration of 4–15 μmol·L⁻¹ added to the basal culture medium. -1 Small molecule substance X1.

[0017] Preferably, the embryo culture medium comprises a basal culture medium and a final concentration of 5–10 μmol·L⁻¹ added to the basal culture medium. -1 Small molecule substance X1.

[0018] Preferably, the embryo culture medium comprises a basal culture medium and a final concentration of 5–8 μmol·L⁻¹ added to the basal culture medium. -1 Small molecule substance X1.

[0019] Preferably, the basal culture medium comprises the following components: 10⁷–10⁹ mmol·L⁻¹ -1 NaCl, 9–11 mmol·L -1 KCl, 0.33–0.36 mmol·L -1 KH2PO4, 0.35–0.45 mmol·L -1 MgSO4, 25–26 mmol·L - 1NaHCO3, 0.18–0.22 mmol·L -1 Sodium pyruvate, 1.8–2.2 mmol·L -1 Calcium lactate pentahydrate, 1.8%–2.1% (v / v) essential amino acid solution (BME), 0.9%–1.1% (v / v) non-essential amino acid solution (MEM), 2.8–3.2 g·L -1 BSA and 9.5–10.5 μg·mL -1 Gentamicin.

[0020] Preferably, the basal culture medium is selected from the following porcine early embryo culture medium: PZM-3, or modified PZM-3.

[0021] Preferably, the modified PZM-3 contains 1–2 mmol·L⁻¹ -1 Glutamine and 5–8 mmol·L -1 PZM-3 of taurine.

[0022] The beneficial effects of this invention are reflected in:

[0023] This invention uses a small molecule substance X1 (known to have specific binding activity to the RepA domain of Xist) added to a certain concentration range in the culture medium of early porcine embryos to effectively prevent abnormal X chromosome inactivation in porcine SCNT embryos and improve the developmental quality of porcine SCNT embryos. Attached Figure Description

[0024] Figure 1 The structural formula of small molecule X1 is given.

[0025] Figure 2A The results of immunofluorescence staining observation of cell count in porcine somatic cell nuclear transfer blastocysts (one embryo randomly selected on day 7) in the control group are shown.

[0026] Figure 2B 5 μmol·L -1 Immunofluorescence staining results of cell count in pig somatic cell nuclear transfer blastocysts (one embryo was randomly selected on day 7) in group X1.

[0027] Figure 2C 10 μmol·L -1 Immunofluorescence staining results of cell count in pig somatic cell nuclear transfer blastocysts (one embryo was randomly selected on day 7) in group X1. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0029] (a) Source and preparation of reagents

[0030] (1) Embryos were treated with PBS

[0031] Formula: 0.203 mg / mL -1 KCl, 8.05 mg·mL -1 NaCl, 0.121 mg·mL -1 MgCl2, 0.036 mg·mL -1 Sodium pyruvate, 1 mg / mL -1 D-glucose, 1.153 mg / mL -1 Na2HPO4, 0.2 mg·mL -1 KH2PO4, 0.132 mg·mL -1 CaCl2·2H2O, 0.121 mg·mL -1 MgCl2·6H2O and 3% (v / v) FBS.

[0032] (2) Oocyte maturation culture medium OM

[0033] Formula: 3.05 mmol·L -1 Glucose, 0.91 mmol·L -1 Sodium pyruvate, 0.57 mmol·L -1 L-cysteine, 1% (w / v) ITS-G, 0.1% (w / v) PVA, 10 IU·mL -1 eCG, 10 IU·mL -1 hCG, 2.5 IU·mL -1 FSH, 40 ng / mL -1 FGF2, 20 ng·mL -1 IGF1, 20 ng·mL -1 LIF, 10 ng·mL -1 EGF and 10 μg·mL -1 Gentamicin.

[0034] (3) 0.1% hyaluronidase

[0035] Dissolve hyaluronidase powder in embryo-grade PBS at a concentration of 0.1% (w / v), filter, aliquot, and store at 4 °C.

[0036] (4) Porcine early embryo culture medium

[0037] After preparing the solution according to the PZM-3 formula, 1 mM glutamine and 5 mM taurine were added (to the final concentration) to create the modified PZM-3. The glutamine in the modified PZM-3 enhances the embryo's ability to develop to the blastocyst stage, while the taurine has antioxidant properties, overcoming the oxidative effects of embryonic metabolism during development and thus preventing developmental arrest.

[0038] Modified PZM-3 formulation: 108 mmol·L -1 NaCl, 10 mmol·L -1 KCl, 0.35 mmol·L -1 KH2PO4, 0.4 mmol·L -1 MgSO4, 25.07 mmol·L -1 NaHCO3, 0.2 mmol·L -1 Sodium pyruvate, 2 mmol·L -1 Calcium lactate pentahydrate, 2% (v / v) essential amino acid solution BME, 1% (v / v) non-essential amino acid solution MEM, 3 g·L -1 BSA, 10 μg·mL -1 Gentamicin, 1 mmol·L -1 Glutamine and 5 mmol·L -1 Taurine.

[0039] The non-essential amino acid solution MEM was purchased from Sigma, catalog number M7145 (w / v), and the formulation is as follows: 0.89 g·L⁻¹ -1 L-alanine, 1.5 g·L -1 L-Asparagine, 1.33 g·L -1 L-Aspartic acid, 1.47 g·L -1 L-glutamic acid, 0.75 g·L -1 Glycine, 1.15 g·L -1 L-proline and 1.05 g·L -1 L-Serine

[0040] The essential amino acid solution BME was purchased from Sigma, catalog number B6766 (w / v), and the following is the formulation: 1.05 g·L⁻¹ -1 L-arginine, 0.6 g·L -1 L-cysteine, 0.4 g·L -1 L-histidine, 1.3 g·L -1 L-Isoleucine, 1.3 g·L -1 L-Leucine, 1.849 g·L -1L-Lysine, 0.375 g·L -1 L-methionine, 0.825 g·L -1 L-Phenylalanine, 1.2 g·L -1 L-Threonine, 0.2 g·L -1 L-Tryptophan, 0.9 g·L -1 L-tyrosine and 1.175 g·L -1 L-valine.

[0041] (5) Small molecule substance X1 (SPEN-IN-1)

[0042] The structural formula of the small molecule X1 is as follows: Figure 1 As shown, the product was purchased from Taoshu Biotechnology, catalog number T60044. It was diluted with DMSO and stored at a concentration of 1 mmol·L⁻¹. -1 The 1 mmol·L -1 The storage solution of small molecule substance X1 was diluted to 5 μmol·L⁻¹. -1 and 10 μmol·L -1 That is, to prepare a solution containing 5 μmol·L -1 and 10 μmol·L -1 The culture medium for small molecule substance X1 was used as the subsequent 5 μmol·L⁻¹ culture medium. -1 Group X1 and 10 μmol·L -1 The porcine SCNT embryo culture experiment in group X1 (specifically, 5 μmol·L⁻¹ was added to the modified PZM-3 solution as described above) -1 and 10 μmol·L -1 Small molecule substance X1).

[0043] (6) Reagents such as cytochalasin B (CB), PBS-PVA and BTX solution were all purchased from Sigma.

[0044] (II) Collection and culture of porcine oocytes

[0045] Pig ovaries were obtained from a local slaughterhouse in Yangling (from April to August 2023), placed in physiological saline at 30-35°C with added penicillin and streptomycin, and delivered to the laboratory within 2 hours. 75% alcohol and physiological saline were preheated to 38.5°C. The ovaries were first washed with 75% alcohol to remove blood stains, then rinsed 3-4 times with physiological saline, and finally kept warm in a 38.5°C water bath. Using a negative pressure pump connected to a blood collection tube fitted with a No. 9 syringe needle, follicular fluid from follicles with a diameter of 3-8 mm on the ovary was collected in a 15 mL centrifuge tube and centrifuged at 50 g for 1 min. The supernatant was discarded until 2-3 mL of precipitate and follicular fluid remained in the tube. 2 mL of the resuspended follicular fluid was added to a 60 mm culture dish. Under a stereomicroscope, cumulus-oocyte complexes with three or more layers of granulosa cells and uniformly black cytoplasm were selected and placed in embryo-grade PBS. After cleaning, the complexes were washed once in OM (Ovum Oxygenation Medium) and cultured in OM that had been equilibrated for at least 2 hours and covered with paraffin oil. The incubator conditions were 38.5°C, 5% CO2, and saturated humidity.

[0046] (III) Somatic cell nuclear transfer

[0047] After culturing oocytes (specifically, the cumulus-oocyte complex) for 46 hours, they can be removed and placed under a stereomicroscope with a heated stage. At this point, a suitable micropipette is used, the tip is inserted, and the tip is sterilized and blunted with a flame before being pipetted. Once most of the granulosa cells surrounding the oocytes are exposed, the oocytes are aspirated using a retrieval needle and transferred to 0.1% hyaluronidase (Hy). The oocytes are repeatedly pipetted for no more than 5 minutes before being picked up with the retrieval needle. They are then placed in pre-equilibrated embryo-grade PBS. Under the stereomicroscope, the refractive angle is adjusted (to enhance the three-dimensional structure and visualize the first polar body). The oocytes are gently moved with the retrieval needle until the dorsal polar body is visible. The oocytes with polar bodies are collected and placed in a pre-equilibrated OM (Oocyte Oocyte) container for further processing. One droplet of cytochalasin B (CB) processing solution and one droplet of PBS-PVA processing solution are equilibrated in a 35 mm culture dish, and then covered with paraffin oil. Oocytes were placed in microdroplets of CB manipulation solution, then held on a fixation needle. An enucleation needle was used to expose the polar bodies to the right side of the field of view. The process and polar bodies were then aspirated through the zona pellucida. Pre-starved porcine fibroblasts (separated from female and male cells, isolated from the ear margin; collected in Rongchang District, Chongqing in August 2023) were resuspended in PBS-PVA. The porcine fibroblasts were then transferred into microdroplets of PBS-PVA manipulation solution using a micropipette. Appropriately sized porcine fibroblasts were then injected between the cytoplasm and zona pellucida of the oocytes using an enucleation needle. After nuclear transfer, the reconstructed embryos were placed in pre-equilibrated amniotic fluid (OM) for approximately 30 minutes. The pre-equilibrated BTX solution was then removed, made into microdroplets, and coated with oil. The reconstructed embryos were transferred into the BTX microdroplets and fused using a microelectrode method. The electrofusion instrument had an electric field strength of 2.0 kV / cm, a single-shot frequency, and a duration of 30 μs. After electrofusion, the embryos were placed in pre-equilibrated OM for 1 hour to recover.

[0048] (iv) Early embryo culture

[0049] One hour after reconstruction, embryos with fused somatic cells (i.e., porcine SCNT embryos) were selected under a microscope and transferred to culture medium supplemented with small molecule substance X1 for treatment (i.e., in vitro embryo culture experiment). One embryo was cultured with 10 μL of culture medium, and 50 embryos were cultured in 500 μL of culture medium in a culture dish, with a control group included. The experiment was specifically divided into three groups: a control group (cultured using modified PZM-3), a control group (cultured using 5 μmol·L⁻¹), and a control group (cultured using 5 μmol·L⁻¹). -1 Group X1 (using modified PZM-3 with an additional 5 μmol·L⁻¹) -1 Small molecule substance X1 (and cultured) and 10 μmol·L -1 Group X1 (using modified PZM-3 with an additional 10 μmol·L⁻¹) -1Small molecule substance X1 was cultured. The incubator conditions were 38.5 °C, 5% CO2, 95% N2, and saturated humidity.

[0050] (v) Immunofluorescence staining of blastocyst H3K27me3

[0051] Seven-day-old porcine SCNT embryos (blastocysts) were fixed overnight at 4°C with immunofluorescence staining fixative, washed with PBS-PVA for 5 min, and repeated three times. After washing away the fixative, the embryos were permeabilized in 0.1% Triton X-100 at room temperature for 30 min. The washing steps were repeated, and after washing away the permeabilization solution, the embryos were blocked in immunofluorescence staining blocking solution at room temperature for 2–3 h. After washing away the blocking solution, the embryos were incubated overnight at 4°C with primary antibody. After washing away the primary antibody, the embryos were incubated with secondary antibody at room temperature for 2 h, and then washed away the secondary antibody. Finally, the embryos were stained with DAPI for 8 min. A square was drawn on a glass slide with Vaseline, and 2 μL of anti-fluorescence quencher was added. The cleaned embryos were placed in the anti-fluorescence quencher drop, covered with a coverslip, and sealed with clear nail polish. The results were observed under a fluorescence microscope.

[0052] (vi) Cell count of blastocysts and immunofluorescence staining

[0053] Seven-day-old porcine SCNT embryos (blastocysts) were fixed overnight at 4°C with immunofluorescence staining fixative, washed with PBS-PVA for 5 min, and repeated 3 times. After washing off the fixative, the embryos were stained with DAPI for 8 min. A square was drawn on a slide with Vaseline, and 2 μL of anti-fluorescence quencher was added. The cleaned embryos were placed in the anti-fluorescence quencher drop, covered with a coverslip, and sealed with clear nail polish. The results were observed under a fluorescence microscope.

[0054] (vii) Experimental Results and Analysis

[0055] 1. Cleavage rate and blastocyst rate of porcine somatic cell nuclear transfer embryos

[0056] After 1 day of in vitro culture, the cleavage rate was calculated; on day 7, the blastocyst development rate (i.e., blastocyst rate) was calculated.

[0057] Table 1. Effects of adding small molecule substance X1 on the development of nuclear transfer embryos

[0058]

[0059] Note: In Table 1 * The difference was statistically significant (P < 0.05) compared with the control group.

[0060] As shown in Table 1, there was no significant difference in cleavage rate between the treatment groups with added small molecule substance X1 and the control group (P > 0.05). (5 μmol·L) -1 The blastocyst rate of group X1 was significantly different from that of the control group (P < 0.05), 10 μmol·L-1 The blastocyst rate in group X1 was not significantly different from that in the control group (P > 0.05). The results indicate that the small molecule X1 has no effect on cleavage in early porcine embryos, and the addition of 5 μmol·L⁻¹ further reduces the blastocyst rate. -1 Small molecule X1 can improve the blastocyst rate of nuclear transfer embryos.

[0061] 2. Cell number in porcine somatic cell nuclear transfer blastocysts

[0062] Table 2. Cell number analysis of porcine somatic cell nuclear transfer blastocysts

[0063]

[0064] Note: In Table 2 * The difference was statistically significant (P < 0.05) compared with the control group.

[0065] The results (see Table 2) show that 5 μmol·L -1 Group X1 (i.e., adding 5 μmol·L⁻¹) -1 Small molecule X1 can significantly increase the number of cells in porcine somatic cell nuclear transfer blastocysts (see also...). Figure 2A , Figure 2B , Figure 2C ).

[0066] 3. Porcine somatic cell nuclear transfer of blastocysts H3K27me3 and XCI

[0067] H3K27me3 staining was performed on female and male somatic cell nuclear transfer embryos (blastocysts) cultured to day 7 to determine the X chromosome inactivation status. For female embryos, one H3K27me3 staining spot on the nucleus indicated normal XCI, while two staining spots indicated abnormal XCI. For male embryos, one H3K27me3 staining spot indicated abnormal XCI.

[0068] Table 3. H3K27me3 staining analysis of porcine female somatic cell nuclear transfer blastocysts

[0069]

[0070] Table 4. H3K27me3 staining analysis of male porcine somatic cell nuclear transfer blastocysts

[0071]

[0072] Note: In Table 3 * The difference was statistically significant (P < 0.05). Compared with the control group, apart from the percentages of 1 H3K27me3 and 2 H3K27me3, the remaining percentage not shown was 0 H3K27me3; Table 4 *The difference was statistically significant (P < 0.05). Compared with the control group, apart from the percentage of 1 H3K27me3, the remaining percentage of undisplayed H3K27me3 was 0.

[0073] As shown in Tables 3 and 4, 5 μmol·L -1 Group X1 and 10 μmol·L -1 The X1 group significantly reduced X chromosome inactivation abnormalities in both male and female somatic cell nuclear transfer embryos.

[0074] In summary, this invention, through in vitro culture experiments of porcine SCNT embryos with the addition of small molecule X1 to the early embryo culture medium and combined with immunofluorescence staining, demonstrates that small molecule X1 increases the blastocyst rate and total number of blastocyst cells in porcine somatic cell nuclear transfer embryos, and reduces XCI abnormalities in porcine somatic cell nuclear transfer embryos. Therefore, adding small molecule X1 to the porcine early embryo culture medium plays an important role in improving the developmental quality of porcine somatic cell nuclear transfer embryos and mitigating XCI abnormalities.

Claims

1. A method for embryo culture, characterized in that: Includes the following steps: Porcine SCNT embryos were transferred into porcine early embryo culture medium and 4–15 μmol·L⁻¹ was added. -1 The small molecule substance X1, the porcine early embryo culture medium contains 1-2 mmol·L⁻¹ -1 Glutamine and 5–8 mmol·L -1 Taurine, and then the porcine SCNT embryos were cultured to the blastocyst stage; the small molecule X1 was SPEN-IN-1.

2. The embryo culture method according to claim 1, characterized in that: The small molecule substance X1 was added to the porcine early embryo culture medium to a final concentration of 5–10 μmol·L⁻¹. -1 .

3. The embryo culture method according to claim 1, characterized in that: The small molecule substance X1 was added to the porcine early embryo culture medium to a final concentration of 5–8 μmol·L⁻¹. -1 .

4. The embryo culture method according to claim 1, characterized in that: The porcine early embryo culture medium was supplemented with 1–2 mmol·L⁻¹ -1 Glutamine and 5–8 mmol·L -1 PZM-3 of taurine.

5. The embryo culture method according to claim 1, characterized in that: The construction of the porcine SCNT embryo specifically includes the following steps: porcine oocytes are cultured in an in vitro maturation medium for 44-46 hours, and then reconstructed embryos are formed by nuclear transfer with porcine somatic cells. The reconstructed embryos are then fused to obtain porcine SCNT embryos to be cultured.

6. A porcine SCNT embryo culture medium, characterized in that: The embryo culture medium includes a basal culture medium and a final concentration of 4–15 μmol·L⁻¹ added to the basal culture medium. -1 The small molecule substance X1; the small molecule substance X1 is SPEN-IN-1.

7. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The embryo culture medium includes a basal culture medium and a final concentration of 5–10 μmol·L⁻¹ added to the basal culture medium. -1 Small molecule substance X1.

8. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The embryo culture medium includes a basal culture medium and a final concentration of 5–8 μmol·L⁻¹ added to the basal culture medium. -1 Small molecule substance X1.

9. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The basal culture medium comprises the following components: 10 7 ~10 9 mmol·L -1 NaCl, 9–11 mmol·L -1 KCl, 0.33–0.36 mmol·L -1 KH2PO4, 0.35–0.45 mmol·L -1 MgSO4, 25–26 mmol·L -1 NaHCO3, 0.18–0.22 mmol·L -1 Sodium pyruvate, 1.8–2.2 mmol·L -1 Calcium lactate pentahydrate, 1.8%–2.1% essential amino acid solution (BME), 0.9%–1.1% non-essential amino acid solution (MEM), 2.8–3.2 g·L -1 BSA and 9.5–10.5 μg·mL -1 Gentamicin.

10. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The basal culture medium was selected from PZM-3 or a modified PZM-3, wherein the modified PZM-3 was supplemented with 1–2 mmol·L⁻¹. -1 Glutamine and 5–8 mmol·L -1 PZM-3 of taurine.

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