Application of demethylase alkbh5 as target in preparation of product for relieving function damage of granulosa cells of mother livestock caused by heat stress

Abstract

The application relates to the field of biological medical technology, and discloses application of a demethylation enzyme ALKBH5 as a target point in preparation of a product for relieving functional damage of female livestock ovary granulosa cells caused by heat stress. The product for relieving functional damage of female livestock ovary granulosa cells caused by heat stress is prepared by inhibiting ALKBH5 expression through transfection of ALKBH5 small interfering RNA, can relieve the increase of pro-apoptosis gene and protein expression of female livestock ovary granulosa cells caused by heat stress, can relieve the decrease of cell proliferation activity and estrogen synthesis of female livestock ovary granulosa cells caused by heat stress, and has the effect of protecting the reproductive performance of female livestock.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a demethylase ALKBH5 as a target in the preparation of products for alleviating heat stress-induced damage to the ovarian granulosa cells of female animals. Background Technology

[0002] Heat stress is a key factor restricting the reproductive performance of female livestock. With global warming and the development of high-density intensive farming, the impact of heat stress on the reproductive performance of female livestock is becoming increasingly severe. Heat stress interferes with the function of the hypothalamus-pituitary-ovarian axis, leading to gonadotropin secretion disorders, inhibited follicle development, decreased oocyte maturation rate, inducing cytoplasmic abnormalities, zona pellucida rupture, and mitochondrial dysfunction. It also reduces the incidence of estrus and disrupts the stability of the estrous cycle. At the same time, heat stress increases the accumulation of reactive oxygen species (ROS), leading to oxidative stress, which further impairs the fertilization capacity of oocytes, reduces the cleavage rate of fertilized eggs, decreases the blastocyst formation rate, and reduces the number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocyst, while increasing the proportion of apoptotic cells, significantly reducing the embryo implantation and survival potential.

[0003] Ovarian granulosa cells are the core components of follicles, forming a multi-layered structure around the oocyte and playing a crucial role in maintaining follicle development and oocyte maturation. Ovarian granulosa cells exchange substances and transmit signals with the oocyte through gap junctions, providing nutrients to the oocyte and secreting regulatory factors such as inhibin and activin, participating in the feedback regulation of the hypothalamus-pituitary-ovarian axis, and precisely regulating follicle recruitment, dominant selection, and atresia. As the main site of estrogen synthesis, ovarian granulosa cells, under the influence of gonadotropins, convert androgens into estrogens through aromatase, providing the necessary hormonal microenvironment for estrus, oocyte maturation, and the establishment of endometrial receptivity. Furthermore, ovarian granulosa cells secrete extracellular matrix components to stabilize follicle structure and participate in the regulation of follicle rupture and ovulation. Their functional status directly affects oocyte quality, fertilization capacity, and subsequent embryonic developmental potential, making them an important indicator of ovarian function and female reproductive performance.

[0004] Post-transcriptional modifications, as important regulators in various physiological processes, have received increasing attention in biological science research. N6-methyladenosine (m... 6 A) is a methylation modification occurring at the 6th N atom of adenine in RNA molecule, and it is also the most common internal modification in eukaryotic mRNA. Numerous studies have shown that m 6Methylation modification is closely related to the function and development of ovarian granulosa cells. The methyltransferase METTL3 can regulate the cell cycle of Heima goat granulosa cells by modulating the expression of CCND1 and AURKB. Similarly, the demethylase FTO can regulate cell cycle progression by controlling the expression of CCND1 in goat granulosa cells. 6 The A-reading protein YTHDF2 enhances the expression of cyclin-dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA), thereby promoting the proliferation of porcine ovarian granulosa cells. The demethylase FTO promotes proliferation by downregulating m... 6 A modification level regulates MNT expression, activating the AKT / Nrf2 (protein kinase B / nuclear factor E2-related factor 2) pathway to alleviate cadmium-induced bovine granulocyte apoptosis. Currently, m 6 The regulatory role of A-methylation modification in the function of granulosa cells in the ovaries of female animals under heat stress remains unclear. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of the demethylase ALKBH5 as a target in the preparation of products for alleviating heat stress-induced damage to the ovarian granulosa cells of female animals. This product can alleviate heat stress-induced damage to the ovarian granulosa cells of female animals and has the effect of protecting the reproductive performance of female animals.

[0006] This invention provides the following technical solution: Application of demethylase ALKBH5 as a target in the preparation of products for alleviating heat stress-induced damage to the function of ovarian granulosa cells in female animals.

[0007] Furthermore, the effects of the product include: inhibiting the increased expression levels of pro-apoptotic genes caspase3, caspase9, BAX, and BAX / BCL-2 in ovarian granulosa cells of female animals caused by heat stress, as well as the increased expression levels of pro-apoptotic proteins caspase3 and BAX / BCL-2.

[0008] Furthermore, the product's effects include alleviating the decreased expression levels of proliferating cell nuclear genes and the protein PCNA in ovarian granulosa cells of female animals caused by heat stress, as well as the reduced cell viability.

[0009] Furthermore, the effects of the product include: alleviating the reduced expression levels of cellular steroid synthesis genes Cyp19a1, Cyp11a1, and Star in ovarian granulosa cells of female animals caused by heat stress, and reducing estradiol synthesis.

[0010] Furthermore, the application includes the preparation of products for alleviating heat stress-induced damage to the ovarian granulosa cells of female animals by inhibiting ALKBH5 expression.

[0011] Furthermore, the method for inhibiting ALKBH5 expression includes: inhibiting ALKBH5 expression by transfecting ALKBH5 small interfering RNA.

[0012] Furthermore, the sense strand of the small interfering RNA is shown in SEQ ID No. 1, and the antisense strand is shown in SEQ ID No. 2.

[0013] Furthermore, the justice chain is SEQ ID NO.1: CGGCGCAAGUCCUACGAGCTT; the antisense chain is SEQ ID NO.2: GCUCGUAGGACUUGCGCCGTT.

[0014] Furthermore, the method for transfecting ALKBH5 small interfering RNA includes: The target cells were seeded in a multi-well plate and allowed to grow to a density of 55%–65%. The transfection reagent was mixed with Opti-MEM medium to obtain solution A, and the siRNA solution was mixed with Opti-MEM medium to obtain solution B. Solutions A and B were mixed and incubated to obtain the transfection mixture. Discard the original culture medium in the multi-well plate, add new complete culture medium, and then add the transfection mixture.

[0015] Furthermore, solution A and solution B are mixed at a volume ratio of 1:1 and incubated for 8-15 minutes.

[0016] Furthermore, the female livestock includes Hu sheep.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention describes a product prepared by transfecting ALKBH5 small interfering RNA to inhibit ALKBH5 expression, which is used to alleviate heat stress-induced damage to the function of ovarian granulosa cells in female animals. It can alleviate the increased expression of pro-apoptotic genes and proteins, decreased cell proliferation and estrogen synthesis in ovarian granulosa cells of female animals (such as Hu sheep) caused by heat stress, thus protecting the reproductive performance of female animals. Attached Figure Description

[0018] Figure 1 This is a comparison of the expression levels of the demethylase ALKBH5 gene and protein in the ovarian granulosa cells of the control group and the ovarian granulosa cells of the Hu sheep after heat stress in Example 2 of the present invention. Figure 2 This is a comparison of the expression levels of apoptosis genes and proteins in the CON+siNC group, HS+siNC group, and HS+siALKBH5 group of granulosa cells from the ovaries of Hu sheep in Example 3 of the present invention. In this figure, A is a comparison of the expression levels of apoptosis genes, and B and C are comparisons of the expression levels of proteins. Figure 3 This is a comparison of the expression levels of cell proliferation nuclear gene and protein PCNA and cell viability in the CON+siNC group, HS+siNC group and HS+siALKBH5 group of granulosa cells of Hu sheep ovaries in Example 4 of the present invention. In this figure, A is a comparison of the expression levels of cell proliferation nuclear gene and protein PCNA, and B is a comparison of cell viability. Figure 4 This is a comparison of the expression levels of steroid synthesis genes and the concentration of estradiol in the cell culture medium of the CON+siNC, HS+siNC, and HS+siALKBH5 groups of ovarian granulosa cells from Hu sheep in Example 5 of the present invention. In this figure, A is a comparison of the expression levels of steroid synthesis genes, and B is a comparison of the concentration of estradiol. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and should not be construed as limiting the scope of protection of the present invention. All experimental reagents used in the embodiments were purchased from reagent suppliers.

[0020] Example 1: Construction of a heat stress model of ovarian granulosa cells in Hu sheep Isolation of granulosa cells from the ovaries of Hu sheep: Healthy ewes around 2 years old were selected as experimental sheep. Fresh ovaries were collected from the ewes and immediately transported back to the laboratory after being placed in physiological saline at 37℃. The ovaries were rinsed three times each with 75% alcohol and physiological saline. Follicular fluid was aspirated from the follicles 3-5 mm in diameter on the surface of the ovary using a syringe and transferred to a 15 mL centrifuge tube. An appropriate amount of complete culture medium was added and mixed well. The tube was centrifuged at 1500 rpm for 5 min. The supernatant was discarded, PBS was added and mixed by pipetting, and the tube was centrifuged at 1500 rpm for 5 min. This process was repeated three times. The supernatant was discarded, complete culture medium was added, and the tubes were transferred to T25 cell culture flasks or six-well plates for culture.

[0021] Establishment of a heat stress model of ovarian granulosa cells in Hu sheep: Ovarian granulosa cells from Hu sheep were randomly divided into a control group (CON) and a heat stress group (HS), with three replicates in each group. The CON group was cultured in a 37℃ incubator, while the HS group was subjected to high-temperature treatment at 42℃ for 2 hours daily, followed by rewarming at 37℃. This treatment was repeated for 3 consecutive days. After the high-temperature treatment ended on the 3rd day, the cells were returned to the 37℃ incubator for 6 hours for rewarming. Total RNA and protein were then extracted from the cells for subsequent analysis.

[0022] Example 2: Effects of heat stress on the expression of ALKBH5 demethylase in granulosa cells of Hu sheep ovaries Total RNA extraction: Total RNA was extracted from cells using the Trizol method. The culture medium in the cell culture plate was discarded, and the cells were washed twice with PBS. 1 mL of Trizol lysis buffer was added to each well, and the cells were incubated for 15 min. Cells were then pipetted from the culture plate and transferred to 1.5 mL EP tubes. 200 μL of chloroform was added to each EP tube, and the mixture was vigorously shaken for a few seconds to ensure thorough mixing. The mixture was incubated for 3 min. The tubes were centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was transferred to a new 1.5 mL EP tube. An equal volume of isopropanol was added, and the mixture was inverted to mix. The tubes were incubated for 10 min. The tubes were centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was discarded, and the white precipitate at the bottom of the tube was RNA. 1 mL of 75% ethanol (prepared with DEPC water) was added to each EP tube, and the mixture was inverted to mix. The tubes were centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was discarded. The tubes were left exposed at room temperature for 10 min to allow the precipitate to dry. Add 15-20 μL of DEPC water according to the amount of RNA, dissolve the RNA thoroughly, and mix well by pipetting. Use a micro spectrophotometer to detect the RNA concentration and purity.

[0023] RNA was reverse transcribed into cDNA: Total RNA was reverse transcribed using the HiScript III RT SuperMix for qPCR Reverse Transcription Kit (Nanjing Novizan Biotechnology Co., Ltd.) and the procedure was performed on ice.

[0024] Primer design: Sheep-related gene sequence information was found through NCBI, primers were designed using Primer 5.0, and then validated using BLAST in NCBI to ensure primer specificity. The primer sequences were then synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0025] RT-qPCR reaction: Prepare the reaction system as shown in Table 1 below according to the instructions of ChamQ universal SYBR qPCR Master mix (Nanjing Novizan Biotechnology Co., Ltd.).

[0026] Table 1 Reaction System

[0027] After preparing the PCR plate, centrifuge to mix, and perform the following reaction using an ABI QuantStudio™ 5 real-time quantitative PCR instrument: preheat at 95℃ for 5 min, then cycle 40 times at 95℃ for 10 s and 60℃ for 30 s, and finally determine the melting curve using a program of 95℃ for 15 s, 60℃ for 60 s and 95℃ for 15 s.

[0028] Using GAPDH as an internal reference gene, and utilizing 2 —△△CT The method calculates the expression level of the target gene.

[0029] Protein extraction from cells: Discard the culture medium from the cell culture plate and wash twice with PBS. Add 120 μL of RIPA lysis buffer containing 1% PMSF (phenylmethylsulfonyl fluoride) to each well and incubate on ice for 30 min. Collect the mixed lysis buffer into a 1.5 mL EP tube, centrifuge at 15,000 rpm for 15 min at 4 °C, and transfer the supernatant to a new EP tube. Mix the protein sample with 5×SDS loading buffer at a ratio of 4:1 and incubate at 95 °C in a metal bath for 10 min to denature the protein.

[0030] Western blot analysis of protein expression levels: Protein samples were subjected to gel electrophoresis at 80V for 40 min and 150V for 50 min. After protein separation, a pre-cut PVDF (polyvinylidene fluoride) membrane was activated in methanol for 30 s, and then transferred in a semi-dry transfer apparatus using a sponge-filter paper-PVDF membrane-gel-filter paper-sponge configuration at 1.3 A and 25 V for 20 min. The transferred PVDF membrane was then immersed in 5% skim milk and blocked at room temperature for 2 h. After blocking, the PVDF membrane and primary antibody were placed in an incubation box and incubated overnight at 4°C. The primary antibody was recovered the next day, and the secondary antibody was incubated at room temperature for 1 h. After incubation, the secondary antibody was recovered and washed three times with 1×TBST for 10 min each time. ECL chemiluminescence solution was added to the membrane, and images were developed using a gel imaging system. Grayscale values ​​were analyzed using ImageJ software.

[0031] Experimental results are as follows Figure 1 As shown, compared with the CON group, the expression levels of ALKBH5 gene and protein in the HS group after heat stress treatment were significantly increased (P<0.05). This indicates that heat stress may regulate the function of granulosa cells in the ovaries of Hu sheep by increasing the expression of the demethylase ALKBH5.

[0032] Example 3: Detection of the effect of inhibiting ALKBH5 gene expression on apoptosis of ovarian granulosa cells in Hu sheep under heat stress. The granulosa cells of the ovarian ovaries isolated in Example 1 were divided into three groups: CON+si-NC group (transfected with si-NC, without heat stress treatment), HS+si-NC group (transfected with si-NC first, then subjected to heat stress treatment), and HS+si-ALKBH5 group (transfected with si-ALKBH5 first, then subjected to heat stress treatment). The heat stress treatment method was the same as in Example 1.

[0033] Transfection with si-ALKBH5 refers to the use of Lipofectamine 3000 to transfect small interfering RNA to inhibit ALKBH5 expression in cells. The method is as follows: Granulosa cells from each group of sheep ovaries were seeded in six-well plates. When the cells reached a density of 60%, transfection was performed. The transfection reagents were prepared according to the Lipofectamine 3000 instructions. The transfection volumes per well in the six-well plate were as follows: Solution A: 5 μL Lipofectamine 3000 + 250 μL opti-MEM medium; Solution B: 10 μL siRNA solution + 250 μL opti-MEM medium. The prepared solutions A and B were mixed in a 1:1 ratio and allowed to stand for 10 min to obtain the transfection mixture. The original medium in the six-well plate was discarded, and 1.5 mL of fresh complete medium was added to each well, followed by the prepared transfection mixture.

[0034] Total RNA and protein were extracted from cells in the CON+si-NC group, HS+si-NC group and HS+si-ALKBH5 group, respectively, and the expression of pro-apoptotic genes caspase3, caspase9, BAX and anti-apoptotic gene BCL-2 was detected.

[0035] The results are as follows Figure 2 As shown, the expression levels of pro-apoptotic genes caspase3, caspase9, BAX, and BAX / BCL-2 significantly increased after heat stress. P <0.05, the expression levels of pro-apoptotic proteins caspase3 and BAX / BCL-2 were significantly increased ( P <0.05. Inhibition of ALKBH5 gene expression under heat stress significantly decreased the expression levels of pro-apoptotic genes caspase3, caspase9, BAX, and BAX / BCL-2. P <0.05, the expression levels of pro-apoptotic proteins caspase3 and BAX / BCL-2 were significantly decreased ( P <0.05). The results indicate that inhibiting ALKBH5 gene expression under heat stress can alleviate the increased apoptosis rate of ovarian granulosa cells in Hu sheep induced by heat stress.

[0036] Example 4: Detection of the effect of inhibiting ALKBH5 gene expression under heat stress on the proliferation and viability of ovarian granulosa cells in Hu sheep. The granulosa cells of the ovarian ovaries isolated in Example 1 were divided into three groups: CON+si-NC group (transfected with si-NC, without heat stress treatment), HS+si-NC group (transfected with si-NC first, then subjected to heat stress treatment), and HS+si-ALKBH5 group (transfected with si-ALKBH5 first, then subjected to heat stress treatment). The heat stress treatment method was the same as in Example 1.

[0037] The method for transfecting si-ALKBH5 cells was as follows: Cells were seeded in 96-well plates, and transfection was performed when the cell density reached 60%. The transfection reagents were prepared according to the Lipofectamine 3000 instructions. The transfection volumes per well in the 96-well plate were as follows: Solution A: 0.2 μL Lipofectamine 3000 + 10 μL opti-MEM medium; Solution B: 0.4 μL siRNA solution + 10 μL opti-MEM medium. Solution A and Solution B were mixed in a 1:1 ratio and allowed to stand for 10 min to obtain the transfection mixture. The original medium in the 96-well plate was discarded, and 75 μL of fresh complete medium was added to each well, followed by the transfection mixture.

[0038] Cell viability was assessed in the CON+si-NC group at 37℃, while in the HS+si-NC and HS+si-ALKBH5 groups, cell viability was assessed after daily heat stress followed by 6 hours of rewarming. The specific protocol was as follows: old culture medium was discarded, and the cells were washed with PBS. 100 μL of fresh complete culture medium was added to each well, followed by 10 μL of CCK-8 reagent. Cells were incubated at 37℃ for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The formula was: Cell viability = [OD(experimental group) - OD(blank group)] / [OD(control group) - OD(blank group)] × 100%.

[0039] The expression levels of proliferating cell nuclear genes and protein PCNA were detected using total RNA and protein extracted from cells in Example 3.

[0040] The results are as follows Figure 3 As shown, the expression levels of nuclear genes and the protein PCNA in proliferating cells were significantly decreased after heat stress, while the expression levels of nuclear genes and the protein PCNA in proliferating cells were significantly increased after inhibiting ALKBH5 expression under heat stress (P<0.05). Cell viability was significantly decreased after heat stress, while cell viability was significantly increased after inhibiting ALKBH5 expression under heat stress (P<0.05). The results indicate that inhibiting ALKBH5 gene expression under heat stress can alleviate the decrease in proliferation and viability of granulosa cells in the ovaries of Hu sheep caused by heat stress.

[0041] Example 5: Detection of the effect of inhibiting ALKBH5 gene expression under heat stress on estrogen synthesis in granulosa cells of Hu sheep ovaries. The expression levels of steroid synthesis genes Cyp19a1, Cyp11a1, and Star were detected using total RNA extracted from cells in Example 3. Cell culture medium from the cell treatment in Example 3 was collected, centrifuged at 4000 rpm for 20 min, cell particles were removed, and the supernatant was retained.

[0042] Estradiol (E2) Quantitative Assay Kit (ELISA) was placed at room temperature for two hours to equilibrate. The concentrated wash buffer was diluted 1:20 with distilled water according to the number of assays, and mixed thoroughly. The pre-coated plate was removed from the sealed bag. One blank control well was prepared without any liquid. Two wells were prepared for each calibrator, with 15 μL of the corresponding calibrator added to each well. 15 μL of supernatant was added to each of the remaining assay wells. 100 μL of Asaay Diluent was added to all wells except the blank well, mixed thoroughly, and sealed with a sealing film. The plate was incubated at 37°C for 60 min. After incubation, the plate was manually washed; the liquid in the wells was discarded, and the wash buffer was filled into each well. The plate was allowed to stand for 10 seconds and then patted dry. This process was repeated three times. 100 μL of SA-HRP was added to each well (except the blank control well), and the plate was sealed with a sealing film. The plate was incubated at 37°C for 30 min. The manual washing process was repeated after incubation. Add 100 μL of TMB to each well, incubate at 37°C in the dark for 15 min, and then add 100 μL of stop solution to each well. Detect using a microplate reader at a wavelength of 450 nm. First, zero the instrument using a blank control well, and then measure the absorbance (OD value) of each well.

[0043] Results Calculation: After the test was completed, the concentration of the standard was used as the x-axis and the absorbance (OD value) was used as the y-axis. Using the computer software ELISACalc, a standard curve equation was created by fitting a four-parameter logistic curve. The concentration of estradiol in the supernatant was calculated using the equation based on the sample OD value.

[0044] The results are as follows Figure 4 As shown, the expression levels of steroid synthesis genes Cyp19a1, Cyp11a1, and Star significantly decreased after heat stress, while the expression levels of these genes significantly increased after inhibiting ALKBH5 expression under heat stress (P<0.05). The concentration of estradiol in the cell culture medium significantly decreased after heat stress, while the concentration of estradiol significantly increased after inhibiting ALKBH5 expression under heat stress (P<0.05). This indicates that inhibiting ALKBH5 gene expression under heat stress can alleviate the decrease in estrogen synthesis in granulosa cells of the ovary of Hu sheep induced by heat stress.

[0045] In summary, this invention, by transfecting ALKBH5 small interfering RNA to inhibit ALKBH5 expression, can alleviate the increased expression of pro-apoptotic genes and proteins, decreased cell proliferation activity, and reduced estrogen synthesis in ovarian granulosa cells of female animals (such as Hu sheep) caused by heat stress. It can alleviate heat stress-induced damage to ovarian granulosa cells in female animals and has the effect of protecting the reproductive performance of female animals. It can be applied to the preparation of drugs and other products that alleviate heat stress-induced damage to the function of ovarian granulosa cells in female animals.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of demethylase ALKBH5 as a target in the preparation of products for alleviating heat stress-induced damage to ovarian granulosa cell function in female animals.

2. The application according to claim 1, characterized in that, The effects of the product include: inhibiting the increased expression levels of pro-apoptotic genes caspase3, caspase9, BAX, and BAX / BCL-2 in ovarian granulosa cells of female animals caused by heat stress, as well as the increased expression levels of pro-apoptotic proteins caspase3 and BAX / BCL-2.

3. The application according to claim 1, characterized in that, The product's effects include alleviating the decreased expression levels of proliferating cell nuclear genes and the protein PCNA in ovarian granulosa cells of female animals caused by heat stress, as well as the reduced cell viability.

4. The application according to claim 1, characterized in that, The product's effects include alleviating the reduced expression levels of cellular steroid synthesis genes Cyp19a1, Cyp11a1, and Star in the ovarian granulosa cells of female animals caused by heat stress, as well as reducing estradiol synthesis.

5. The application according to claim 1, characterized in that, The applications include the preparation of products for alleviating heat stress-induced damage to the ovarian granulosa cells of female animals by inhibiting ALKBH5 expression.

6. The application according to claim 5, characterized in that, The method for inhibiting ALKBH5 expression includes: inhibiting ALKBH5 expression by transfecting ALKBH5 small interfering RNA.

7. The application according to claim 6, characterized in that, The sense strand of the small interfering RNA is shown in SEQ ID No. 1, and the antisense strand is shown in SEQ ID No.

2.

8. The application according to claim 6, characterized in that, The method for transfecting ALKBH5 small interfering RNA includes: The target cells were seeded in a multi-well plate and allowed to grow to a density of 55%–65%. The transfection reagent was mixed with Opti-MEM medium to obtain solution A, and the siRNA solution was mixed with Opti-MEM medium to obtain solution B. Solutions A and B were mixed and incubated to obtain the transfection mixture. Discard the original culture medium in the multi-well plate, add new complete culture medium, and then add the transfection mixture.

9. The application according to claim 8, characterized in that, The A solution and B solution are mixed at a volume ratio of 1:1 and incubated for 8-15 minutes.

10. The application according to claim 1, characterized in that, The female livestock mentioned include Hu sheep.

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