Sheep in-vitro fertilization culture method based on serum substitute

Abstract

The invention discloses a sheep in-vitro fertilization culture method based on a serum substitute, and belongs to the technical field of biology. The method comprises the following steps that mature oocytes of sheep are cultured through in-vitro fertilization liquid, the in-vitro fertilization liquid does not contain oestrus sheep serum or fetal calf serum, and the in-vitro fertilization liquid contains serum substitutes. Experiments show that 10% XRS replaces 10% FBS, and an ideal oocyte maturation rate can be obtained; xRS replaces OSS, the cleavage rate can be increased, and therefore the XRS replacement scheme is feasible in the sheep germ cell in-vitro culture technology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a sheep in vitro fertilization culture method based on serum substitutes. Background Art

[0002] Sheep in vitro fertilization (IVF) is a biotechnology that uses artificial intervention to achieve in vitro fertilization of oocytes and sperm, and complete early embryonic development. This technology plays an irreplaceable role in genetic resource conservation, large-scale livestock production, and basic research in life sciences. By integrating gene editing technologies such as CRISPR-Cas9, targeted gene modification can be achieved during the embryonic development stage in vitro, thereby breeding transgenic sheep strains with specific genetic characteristics, such as disease-resistant sheep or bioreactor sheep. With breakthrough advances in gene editing technology and synthetic biology research, the application scope of sheep IVF technology will continue to expand, providing important technical support for the genetic improvement and sustainable development of animal husbandry.

[0003] The sheep in vitro fertilization technology system mainly includes the following key links: oocyte collection (Oocyte Collection), oocyte in vitro maturation (In Vitro Maturation, IVM), in vitro fertilization (IVF) and in vitro embryo culture (Embryo Culture).

[0004] In vitro oocyte maturation (IVM) culture systems, conventional culture medium formulations contain 10% fetal bovine serum (FBS) to provide essential nutrients and cytokines for oocyte maturation. However, the use of FBS not only involves the high ethical costs of fetal bovine blood collection and potential animal welfare issues, but also the heterogeneity of its composition from different sources can lead to decreased culture system stability, thereby affecting oocyte maturation efficiency and embryonic developmental potential.

[0005] In the sheep IVF technology system, estrus sheep serum (OSS) is a necessary additive for frozen-thawed semen fertilization (added at 5%-10%) and has a significant effect on the embryo cleavage rate. However, the OSS preparation process involves estrus cycle monitoring and blood collection operations, which has certain technical complexity and time costs. In addition, there are biosafety risks due to the hidden dangers of zoonotic diseases such as brucellosis. Therefore, the development of safe and efficient serum replacement solutions has become an important research direction in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an in vitro embryo culture method that does not use fetal bovine serum. The technical problem to be solved is not limited to the technical subject described above. Those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The invention provides a method for in vitro fertilization of sheep oocytes, comprising the following steps: culturing mature sheep oocytes with in vitro fertilization fluid, wherein the in vitro fertilization fluid does not contain estrus sheep serum or fetal bovine serum and contains a serum substitute.

[0009] In the above method, the volume percentage of the serum substitute in the in vitro fertilization fluid is 10%.

[0010] The in vitro fertilization solution consists of: 10% by volume of XRS, 1 mM sodium pyruvate, 16 μM norepinephrine, 2 U / mL heparin and 5 μg / mL gentamicin, and the remainder is basal culture medium.

[0011] The basal culture medium is a modified SOF solution having the following composition: NaCl 107.7 mM, NaHCO 3 25.07 mM, CaCl 2·2H 2 O 1.71 mM, KH 2 PO 4 1.19 mM, KCl 7.16 mM, MgCl 2·6H 2 O 0.49 mM, sodium DL-lactate 5.3 mM, and the balance being distilled water.

[0012] Composition of in vitro fertilization fluid of the control group:

[0013] The mature oocytes in the above method are obtained by culturing the oocytes with an in vitro maturation medium. The in vitro maturation medium does not contain fetal bovine serum, so the in vitro maturation medium contains a serum substitute.

[0014] The in vitro maturation medium consists of: 10% XRS (volume percentage), 50 μg / ml gentamicin, 10 μg / ml FSH, 5 μg / ml LH and 2 μg / ml estradiol, with the remainder being M199 culture medium (ThermoFish, 11150059).

[0015] In the above method, the volume percentage of the XRS standard serum substitute in the in vitro maturation fluid is 10%.

[0016] The present invention also provides the use of the serum substitute in preparing a product for maturing animal oocytes in vitro.

[0017] The present invention also provides the use of the serum substitute in preparing a product for in vitro fertilization of animal oocytes.

[0018] The present invention also provides the use of the serum substitute in preparing a product for improving the cleavage rate of embryos during in vitro culture.

[0019] The present invention also provides the use of a serum substitute in preparing a product for improving the blastocyst rate of embryos during in vitro culture.

[0020] The serum replacement is a product used to replace animal serum.

[0021] In a specific embodiment of the present invention, the serum substitute is an XRS standard serum substitute.

[0022] In a specific embodiment of the present invention, the XRS standard serum substitute is the product with the product number XRSS10-500 of Shanghai Zhongbin Technology Co., Ltd.

[0023] This study systematically evaluated the suitability of XRS as a replacement for FBS / OSS in ovine IVM / IVF. The results demonstrated that: ① Substituting 10% XRS for 10% FBS in oocyte maturation fluid resulted in optimal oocyte maturation rates; ② Substituting XRS for OSS in fertilization fluid, and co-incubating treated frozen-thawed sperm with oocytes for IVF, increased the cleavage rate to 89.50±0.78%, significantly higher than the 67.23±3.27% in the OSS group; and ③ Throughout the IVM / IVF / IVC process, the dual XRS substitution strategy resulted in a blastocyst development rate of 21.77±1.59%, a 10.97% improvement compared to the traditional FBS+OSS regimen (10.80±1.12%). These data confirm the feasibility of the XRS substitution strategy for ovine germ cell in vitro culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram showing the effects of different serotype combinations on sheep oocyte IVM and IVF. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0027] The data in the following examples were processed using SPSS statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference.

[0028] Example 1: XRS replaces FBS and estrous sheep serum to improve cleavage and blastocyst rates after IVF

[0029] XRS standard serum substitute was purchased from Shanghai Zhongbin Technology Co., Ltd. with the product number of XRSS10-500.

[0030] The experiment was repeated 3 times, and each repetition was as follows:

[0031] 1. Experimental methods

[0032] 1.1 Oocyte Collection

[0033] Fresh ovaries were obtained from a local slaughterhouse and transported at 30°C. Attached tissue was removed and the ovaries were rinsed three times with 0.9% (w / v) NaCl saline (GB / T 601-2016). HM199 basal solution (Punosai, PM150612) containing 2% FBS, 50 μg / mL gentamicin, and 2 U / mL heparin sodium was prepared. Ovarian follicles (3-8 mm) on the surface of the ovaries were mechanically dissected using a sterile scalpel blade. Cumulus-oocyte complexes (COCs) with intact granulosa cells were selected under a stereomicroscope and washed twice in oocyte collection fluid and twice in maturation medium.

[0034] 1.2 In Vitro Maturation (IVM)

[0035] The composition of the in vitro maturation medium of the control group was as follows: 10% FBS (volume percentage), 50 μg / ml gentamicin, 10 μg / ml FSH, 5 μg / ml LH and 2 μg / ml estradiol, and the remainder was M199 culture medium (ThermoFish, 11150059).

[0036] The composition of the in vitro maturation medium of the experimental group was as follows: 10% XRS (volume percentage), 50 μg / ml gentamicin, 10 μg / ml FSH, 5 μg / ml LH and 2 μg / ml estradiol, with the remainder being M199 medium.

[0037] Select COCs with uniform cytoplasm and three or more layers of granulosa cells for IVM. Spread 50-100 COCs evenly in a 4-well culture plate (containing 800 μL of maturation solution (control group maturation solution or experimental group maturation solution) 354110), placed in a constant temperature incubator (38.5°C, 5% CO2, 95% saturated humidity) and cultured for 24 h to obtain granulosa cell-ovum complexes (COCs) treated with in vitro maturation (IVM).

[0038] 1.3 In Vitro Fertilization (IVF)

[0039] The sheep in vitro fertilization experiment used a 30 μL microdroplet culture system covered with mineral oil, and the basal culture medium was a modified SOF solution (NaCl 107.7 mM, NaHCO3 25.07 mM, CaCl2·2H2O 1.71 mM, KH2PO4 1.19 mM, KCl 7.16 mM, MgCl2·6H2O 0.49 mM, sodium DL-lactate 5.3 mM, and the balance was distilled water).

[0040] The composition of the in vitro fertilization fluid of the control group was as follows: 10% volume percentage of estrus sheep serum (OSS), 1 mM sodium pyruvate, 16 μM norepinephrine, 2 U / mL heparin and 5 μg / mL gentamicin, and the remainder was basal culture medium.

[0041] The composition of the in vitro fertilization fluid in the experimental group was as follows: 10% XRS fetal bovine serum substitute (Shanghai Zhongbin Technology Co., Ltd.) was used to replace OSS, and the other components were the same as the culture medium of the control group.

[0042] After the granulosa cell-egg complexes (COCs) treated with in vitro maturation (IVM) were rinsed with semen three times, 10-30 high-quality COCs were selected and transferred to the pre-equilibrated fertilization droplets. The sperm float tubes were prepared using 2mL polypropylene round-bottom centrifuge tubes (Sangong, F600619). 1.2mL of fertilization solution was added to each tube, covered with tin foil, and equilibrated in a 38.5℃, 5% CO2 incubator for ≥2h. The laboratory-made sheep straw frozen semen was quickly thawed in a 37℃ water bath (completed within 10s), and then diluted with pre-warmed fertilization solution at a volume ratio of 1:1. 200μL of semen was placed at the bottom of the float tube and incubated in a 38.5℃, 5% CO2 saturated humidity environment for 40-60min for sperm selection. The upper suspension was then aspirated and transferred to a 15mL conical centrifuge tube (Eppendorf Plus), centrifuged at 400 × g for 6 min, discarded the supernatant and retained 200 μL of the precipitated sperm suspension, which was resuspended and counted and adjusted to a density of 1 × 106 / mL. During the fertilization phase, the sperm and eggs were incubated (38.5°C, 5% CO2, saturated humidity) for 8 h, and the embryos were washed with the pre-equilibrated control group in vitro fertilization solution or the experimental group in vitro fertilization solution. The embryos were then separated using a 200 μL micropipette (Eppendorf Plus) mechanically peel off the granulosa cells and finally obtain the degranulated fertilized zygotes.

[0043] 1.4 Embryo culture

[0044] Sheep embryos were cultured using a modified SOFaa culture medium (component system: NaCl 107.7 mM, NaHCO3 25.07 mM, CaCl2·2H2O 1.71 mM, KH2PO4 1.19 mM, KCl 7.16 mM, MgCl2·6H2O 0.49 mM, sodium DL-lactate 5.3 mM, inositol 2.77 mM, Glutamax TM 0.5× [200 mM stock solution] (Gibco, 35050061), sodium pyruvate 0.4 mM, MEM non-essential amino acid solution [1:100 dilution] (Gibco, 11140050), MEM essential amino acid solution [1:100 dilution] (Gibco, 11130051), gentamicin 10 μg / mL, bovine serum albumin 8 mg / mL). The SOFaa culture medium system was sterile filtered and used in a 3.5 cm Nunc TM EasYDish TM Petri dishes (Thermo Scientific TM 150460) to prepare 50 μL mineral oil overlay droplets (Ovoil TM ,Vitrolife), and equilibrated in an incubator at 38.5±0.2℃ and 5% CO2 saturated humidity for ≥2h before use.

[0045] High-quality embryos selected after in vitro fertilization (IVF) and cleavage stage were rinsed three times with SOFaa medium and transferred to pre-equilibrated embryo culture microdroplets at a density of 20-25 embryos per droplet. The culture system was incubated in a 38.5°C, 5% CO2 incubator for 36 hours. The cleavage rate was monitored (Nikon SMZ800). After removing non-cleavage embryos, the embryo density was adjusted to 20-25 embryos per droplet and culture continued until Day 7 (D7). The number of blastocysts was counted under a stereomicroscope.

[0046] 1.5 Experimental Design

[0047] A 2×2 factorial experimental design was used. Chi-square test and two-way ANOVA were used for data statistics.

[0048] Table 1

[0049] Experimental group IVM serum IVF serum Number of experimental repetitions (number of samples) 1 group FBS OSS 3 times (≥40 oocytes per group) 2 groups FBS XRS 3 times (≥40 oocytes per group) 3 groups XRS OSS 3 times (≥40 oocytes per group) 4 groups XRS XRS 3 times (≥40 oocytes per group)

[0050] 2. Experimental results

[0051] The experimental data and analysis graphs of different serum type combinations of sheep oocyte IVM-IVF show that sheep oocytes exhibit different developmental potential in different IVM and IVF culture systems. The data show that the cleavage rate (89.50%) and blastocyst rate (21.77%) of the XRS+XRS combination are significantly better than those of other groups, showing the best embryo development effect. The FBS+XRS group shows moderate development level. The development potential of the OSS-containing groups (XRS+OSS and FBS+OSS) is relatively low, among which the FBS+OSS traditional group shows the weakest.

[0052] Table 2

[0053]

[0054] Note: Superscripts a, b represent significant difference from the conventional FBS-OSS group p≤0.05;

[0055] Superscript c represents extremely significant difference from the conventional FBS-OSS group p≤0.01.

[0056] The cleavage rate is the number of oocytes / COCs; the blastocyst rate is the number of blastocysts / COCs.

[0057] These results show that the use of XRS completely replacing FBS in sheep IVM and OSS in IVF to establish a double-added culture system (XRS+XRS) significantly improves the in vitro maturation quality of oocytes and the developmental potential of embryos. This finding provides important experimental basis for optimizing the sheep in vitro embryo production system. It also provides a theoretical basis for the application of XRS in sheep reproductive cell culture.

[0058] Since pig, cattle, buffalo, sheep, goat and other livestock oocytes are also matured in vitro using FBS, the addition of FBS in mammalian oocyte maturation medium can use XRS instead.

[0059] In the field of cell culture, XRS has been studied as a substitute for FBS, which exhibits good biocompatibility in a variety of mammalian cell lines (including primary cells), and has advantages such as maintaining cell proliferation efficiency, improving batch stability, and significantly reducing costs. However, the application of XRS in the field of reproductive biology is not simply an extension from the field of cell culture, but needs to overcome a series of technical difficulties and obstacles.

[0060] 1. Differences in cell types and functions: Germ cells (such as sperm, eggs, and embryonic cells) have unique physiological functions and metabolic properties that differ significantly from those of conventional cell lines. For example, germ cells differ from conventional cell lines in their nutrient requirements, sensitivity to oxidative stress, and cell cycle regulation. Therefore, the application of XRS in germ cell culture requires optimization and adjustment to these specific requirements to ensure that it can provide a suitable microenvironment to support normal development and function of germ cells.

[0061] 2. Complexity of hormones and signaling pathways: The reproductive process is meticulously regulated by multiple hormones and cell signaling pathways. In germ cell culture, precise simulation of in vivo hormone levels and signaling processes is required to ensure the smooth progress of key processes such as germ cell maturation and fertilization. As an alternative, XRS requires innovative formulations to ensure it provides hormones and signaling molecules similar to or superior to traditional FBS, thereby maintaining the normal physiological function of germ cells.

[0062] 3. Higher safety requirements: Germ cell culture and manipulation involve reproductive health and offspring development, necessitating extremely high safety requirements. Before XRS is applied in reproductive biology, rigorous safety assessments are required, including the detection and elimination of potentially harmful components and research into their long-term effects. This requires the establishment of more stringent quality control standards and testing methods to ensure the safety and reliability of XRS in germ cell culture.

[0063] 4. Challenges of batch consistency: Although XRS has demonstrated good batch stability in the field of cell culture, in the field of reproductive biology, due to the higher sensitivity of germ cells to culture conditions, the requirements for batch consistency are more stringent. Therefore, it is necessary to further optimize the production process and quality control system of XRS to ensure that it can provide highly consistent performance in germ cell culture, thereby ensuring the reliability and repeatability of experimental results. Through in-depth research and innovation, this patent successfully solved the above-mentioned technical difficulties and obstacles and developed an XRS substitute suitable for the field of reproductive biology. This substitute not only inherits the advantages of XRS in the field of cell culture, such as maintaining cell proliferation efficiency, improving batch stability and reducing costs, but also is optimized for the special needs of germ cells, so that it can play a similar or better role in germ cell culture than traditional FBS, providing a safe, efficient and economical alternative for reproductive biology research and related applications.

[0064] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for in vitro fertilization of sheep oocytes, characterized in that: The method comprises the following steps: culturing mature sheep oocytes using in vitro fertilization fluid, wherein the in vitro fertilization fluid does not contain estrus sheep serum or fetal bovine serum and contains serum substitute.

2. The method according to claim 1, wherein The volume percentage of the serum replacement in the in vitro fertilization fluid is 10%.

3. The method according to claim 1 or 2, characterized in that The mature oocytes are obtained by culturing the oocytes with an in vitro maturation medium. The in vitro maturation medium does not contain fetal bovine serum or estrus sheep serum, so the in vitro maturation medium contains serum substitute.

4. The method according to claim 3, characterized in that The volume percentage of serum replacement in the in vitro maturation fluid is 10%.

5. The use of serum substitutes in the preparation of products for in vitro maturation of animal oocytes.

6. Use of serum substitutes in the preparation of products for in vitro fertilization of animal oocytes.

7. Use of serum substitutes in the preparation of products for improving the cleavage rate of embryos during in vitro culture.

8. Use of serum substitutes in the preparation of products for improving the blastocyst rate of embryos during in vitro culture.

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