New use of an inhibitor of ferroptosis

Abstract

The application aims to provide a new application of an iron death inhibitor, Liproxstatin-1. The application studies the iron death phenomenon in the process of follicle atresia of laying poultry, finds that the iron death inhibitor Liproxstatin-1 can rescue the follicular granulosa cell death induced by tamoxifen (TMX), can inhibit the oxidative stress level and intracellular iron ion overload caused by TTMX treatment, can effectively inhibit the follicular iron death phenomenon induced by TTMX, and finally can restore the egg production function of livestock and poultry, and has the development into a drug for treating follicle atresia and used for treating follicle atresia of livestock and poultry.

Description

Technical Field

[0001] This invention relates to the field of poultry breeding technology, specifically to a new application of a ferritin inhibitor. Background Technology

[0002] The reproductive performance of laying poultry largely depends on the development of follicles. For example, a chicken is born with approximately 400,000 primordial follicles, but only 500-1000 develop normally to the ovulation stage. This indicates that the vast majority of follicles in the chicken ovary fail to reach the pre-ovulation stage and instead undergo atresia, gradually degenerating and disappearing. Follicular atresia is a physiological phenomenon of selective cell death regulated by multiple factors. Its pathological characteristics include smaller follicles, petechiae on the follicle surface, and cloudy color. Further research has revealed that animal follicular atresia is regulated by different forms of granulosa cell death. However, there is also considerable evidence suggesting that granulosa cell apoptosis is not the only factor leading to follicular atresia; other cell death mechanisms, such as ferroptosis, also play a crucial role in the process. This invention explores the regulatory role of ferroptosis in chicken follicular atresia, aiming to explore new methods to regulate the process of follicular atresia, thereby improving the reproductive performance of laying poultry, particularly chickens, and providing new insights into the mechanism of follicular atresia in poultry. Summary of the Invention

[0003] This invention investigates ferroptosis during follicular atresia in laying poultry and discovers that subcutaneous injection of Liproxstatin-1 (Lip-1), a ferroptosis inhibitor, can rescue tamoxifen (TMX)-induced granulosa cell death in follicles. This provides a novel application of ferroptosis inhibitors, aiming to reduce follicular atresia by promoting follicular development and maturation through modern molecular breeding techniques, thereby improving poultry reproductive performance, increasing farming efficiency and economic benefits, and promoting the sustainable development of the poultry farming industry. The technical solution of this invention is as follows:

[0004] In a first aspect, the present invention provides the use of a ferroptosis inhibitor in the preparation of a drug for treating follicular atresia and / or restoring egg production function, wherein the ferroptosis inhibitor is selected from Liproxstatin-1.

[0005] Preferably, the drug for treating follicular atresia and / or restoring egg production function is used in livestock and poultry, and preferably, the livestock and poultry include chickens, ducks, and geese.

[0006] More preferably, the dosage of ferroptosis inhibitor is 5–20 mg / kg of livestock and poultry, and more preferably 8–12 mg / kg of livestock and poultry.

[0007] In a second aspect, the present invention provides a medicament for treating follicular atresia and / or restoring egg production function, comprising a ferroptosis inhibitor selected from Liproxstatin-1.

[0008] Preferably, it also includes pharmaceutically acceptable excipients.

[0009] Preferably, the drug is used on livestock and poultry.

[0010] More preferably, the dosage of the ferroptosis inhibitor in the drug is 5-20 mg / kg of livestock and poultry, more preferably 5-8 mg / kg of livestock and poultry.

[0011] Thirdly, the present invention provides a method for promoting the proliferation and development of granulosa cells in livestock and poultry without diagnostic or therapeutic purposes, comprising: using a ferroptosis inhibitor on livestock and poultry, wherein the ferroptosis inhibitor is selected from Liproxstatin-1.

[0012] Preferably, the method of using iron mortality inhibitors for livestock and poultry includes injection.

[0013] More preferably, the injection includes subcutaneous injection or intraperitoneal injection.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention constructs a follicular atresia model through TMX induction, and further uses this model to verify that Lip-1, a ferroptosis inhibitor, can rescue TMX-induced follicular granulosa cell death, inhibit TMX-induced oxidative stress and intracellular iron overload, and effectively suppress TMX-induced follicular ferroptosis. Therefore, it has the potential to be developed into a drug for the treatment of follicular atresia in livestock and poultry. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0017] Figure 1In Example 1 of this invention, ferroptosis levels significantly increased during TMX-induced follicular atresia. Figures AC and D show the egg production rate, ovarian morphology, and ovarian weight of chickens (n=12) treated with TMX and a control (ethanol). Figure D shows the plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in chickens treated with TMX for 9 days (n=12). Figure EG shows the expression levels of ACSL4, FTH1, and GPX4 mRNA in chicken follicles during TMX treatment (n=6) as determined by qPCR analysis. Figure H shows the protein levels of ACSL4, FTH1, and GPX4 in chicken follicles during TMX treatment (n=3), with GAPDH used as an internal control. Figure IL shows the relative concentrations of GSH, MDA, iron, and GSSG in chicken follicles during TMX treatment detected by ELISA (n=6). Error is expressed as mean ± SD; *P<0.05 indicates significant difference, and **P<0.01 indicates extremely significant difference.

[0018] Figure 2 This is an example of TMX treatment inducing ferroptosis in granulosa cells (GCs) in Example 2 of the present invention. Figure A shows the cell viability of GCs treated with TMX or Erastin, determined by the CCK-8 assay (n=6). Figure B shows the cell viability of GCs treated with TMX or Erastin, determined by propidium iodide (PI) staining (n=6). Figures CF show the relative concentrations of GSH, MDA, iron, and GSSG in GCs treated with TMX or Erastin (n=6). Figure G shows the lipid peroxidation level in GCs treated with TMX or Erastin, analyzed by flow cytometry (n=6). Figure H shows the levels of ACSL4, FTH1, and GPX4 proteins in Erastin-treated GCs, analyzed by Western blot with GAPDH as a standard control (n=3).

[0019] Figure 3To illustrate the effects of Liproxstatin-1 treatment on ferroptosis and its influence on the recovery of chicken follicle function after TMX treatment in Example 3 of this invention, chickens were subjected to intraperitoneal injections of TMX (6 mg / kg), TMX + liproxstatin-1 (5 mg / kg), or ethanol (control group) for 9 days. Figure A shows the results of Western blot analysis detecting protein levels associated with ferroptosis in chicken follicles after injection of TMX or TMX + liproxstatin-1 (n=3). Figure BD shows the results of qPCR determination of mRNA expression levels of genes related to ferroptosis in chicken follicles after TMX or Lip-1 treatment (n=9). Figure EN shows the relative values ​​of reactive oxygen species, GSH, MDA, iron ions, and GSSG concentrations in chicken follicles after TMX or Lip-1 treatment (n=6). Figure OQ shows the egg production rate, ovarian morphology, and ovarian weight of hens after TMX or Lip-1 treatment (n=12). Errors are expressed as mean ± SD. A t-test was used, with *P < 0.05 indicating a significant difference and **P < 0.01 indicating an extremely significant difference.

[0020] Figure 4 To illustrate the effect of Fer-1 treatment on TMX-induced follicular atresia in Example 3 of this invention, hens at peak egg production underwent intraperitoneal injection of either TMX (6 mg / kg) or TMX + Fer-1 (10 mg / kg) for 13 days. Figure A shows the protein expression levels of ACSL4, FTH1, and GPX4 in the two groups (n=12) detected by Western blot. Figures B and D show the mRNA expression levels of ACSL4, FTH1, and GPX4 detected by qPCR (n=9). Figure E shows the ROS levels of follicles in the two treatment groups (n=6) detected by flow cytometry. Figure FI shows the levels of GSH, MDA, and Fe in follicles of different treatment groups detected using an ELISA kit. 2+ The concentrations of GSSG were measured (n=6). Figure J shows the egg production rate of hens in the different treatment groups (n=12). Figure K shows the morphology of the ovaries in the different treatment groups (n=12). Figure L shows the weight of the ovaries in the different treatment groups (n=12). Detailed Implementation

[0021] The animal experiments in the embodiments of this invention were conducted in accordance with the research protocol approved by the Animal Ethics Committee of Sichuan Agricultural University (Approval No. 80153 / 2022). The experimental animals used were commercial Lohmann pink-shelled chickens, obtained from the poultry farm of Sichuan Agricultural University. The experimental chickens were housed individually in cages with free access to water and feed, and the light cycle consisted of 14 hours of light and 10 hours of darkness.

[0022] In a specific embodiment of the present invention, the PI staining procedure is as follows: First, the culture medium of each group of cells is aspirated, adherent granular cells are washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and washed three times with PBS. Next, the cells are incubated with 0.1% Triton X-100 at room temperature for 10 min, and washed three times with PBS. Then, PI staining solution (5 μg / mL PI + 100 μg / mL RNase A, prepared with PBS) is added, and the cells are incubated in the dark for 15 min, followed by three washes with PBS. Finally, under a fluorescence microscope (Ex / Em 535 / 617nm), the nuclei of dead cells or late apoptotic cells show red fluorescence, and the number of dead cells is counted using ImagJ software.

[0023] In a specific embodiment of the present invention, RNA isolation and real-time quantitative PCR include: extracting total RNA from follicular cells using a total RNA isolation kit (Foregene, Chengdu, China), TaKaRa PrimeScript. TM The RT kit (TaKaRa, Tokyo, Japan) was used for cDNA synthesis; the extraction steps and methods were performed according to the kit instructions. Real-time quantitative PCR (qPCR) analysis was performed using Premix Ex TaqⅡ (TaKaRa). To standardize the cycle threshold, each experiment was performed in triplicate, and the qPCR results were analyzed using the ΔCt method. The oligonucleotide primers used in this invention are listed in Table 1.

[0024] Table 1. Primers used in this invention

[0025]

[0026] The reaction steps for quantitative real-time PCR were performed according to the kit instructions, and the annealing temperature of the reaction system was screened and optimized: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; X℃ annealing for 30s (X is related to the target gene and was screened by temperature gradient); 72℃ extension for 10s; a total of 42 cycles; the temperature during the melting curve generation stage was increased from 65℃ to 95℃ per second, with an increase of 0.5℃ per second.

[0027] The reaction system is shown in Table 2 below:

[0028] Table 2 qPCR system

[0029]

[0030] Following the above reaction system and conditions, using GAPDH as an internal reference gene, quantitative real-time PCR analysis was performed on a real-time PCR instrument, with three technical replicates for all samples.

[0031] In a specific embodiment of the present invention, the protein immunoblotting process includes: adding a phosphatase inhibitor (Sigma-Aldrich) and a protease inhibitor (Promega Madison, Wisconsin, USA) to cell lysis buffer (Cell Signaling Technology) to extract total protein from follicular cells. Protein concentration is quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA). Approximately 20 μg of protein is electrophoresed on an SDS-PAGE gel and then transferred to a PVDF membrane. Molecular weight is determined using a protein marker (Sigma-Aldrich). After blocking the PVDF membrane with 5% milk, it is incubated overnight at 4°C with primary antibody. The next day, it is incubated with HRP-labeled secondary antibody for 1 hour, followed by exposure and imaging with ECL luminescence solution (Millipore, Bedford, MA, USA). Band density is analyzed using ImageJ.

[0032] In a specific embodiment of the present invention, the determination of lipid peroxidation, GSH, and iron includes: culturing cells in 12-well plates, performing specific treatment, and centrifuging to obtain cell lysates. Following the manufacturer's instructions, the levels of lipid peroxidation products in the cell lysates are detected using an MDA (cat.no.ab118970, Abcam) or GSSG (cat.no.ab141393, Abcam) lipid peroxidation assay kit; the concentration level of GSH is quantified using a GSH assay kit (cat.no.CS0260; Sigma-Aldrich); and the ferrous iron concentration is determined using an iron assay kit (cat.no.ab83366, Abcam).

[0033] In a specific embodiment of the present invention, cell viability assay includes: determining cell viability by staining with propidium iodide (PI) (cat.no. S6874, Selleck) followed by fluorescence microscopy, according to the manufacturer's instructions. Cells are seeded into 96-well plates after specific treatment, according to the instructions for the CCK-8 cell counting kit. 10 μL of CCK-8 is added to each well and incubated for 2 hours, and cell viability is measured. Live cell counting is performed by measuring the absorbance at 570 nm using a Varioskan LUX ELISA (Thermo Fisher, Waltham, MA, USA).

[0034] In the specific embodiments of the present invention, the statistical analysis method adopted is as follows: The results are expressed as mean ± S.D. The comparison of the means of two groups is performed using the t-test, and the comparison of more than two groups is performed using one-way analysis of variance (ANOVA). When significant results are obtained from the analysis of variance, the least significant difference test is used for post hoc comparison of the differences between groups. The statistical analysis is performed using GraphPad Prism 6.0 software. A P-value less than 0.05 indicates significant differences, a P-value less than 0.01 indicates extremely significant differences, and non-significant differences are indicated by the symbol "n.s.".

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] This example studied whether ferroptosis occurs during the follicular atresia process in chickens, as follows:

[0038] (1) Experimental procedure:

[0039] The experimental animals were Roman brown-egg laying hens, with a number of 24. They were randomly divided into a control group (n = 12) and an experimental group (n = 12). The experimental group was subcutaneously injected with tamoxifen (TMX) (an estrogen inhibitor) at a dose of 6 mg / kg body weight dissolved in 0.3 ml of ethanol, and the control group was subcutaneously injected with an equal amount of ethanol. Until the hens in the experimental group stopped laying eggs, the number of eggs laid by the hens in the experimental group and the control group was counted every day. Subsequently, all the hens were sacrificed by cervical bleeding, and the hierarchical follicles F3 - F1 (F3 < F2 < F1) were quickly isolated from the ovaries, and the granulosa layer was separated. For the hens in the experimental group, due to follicular atresia, the granulosa layer and theca layer could not be separated, so the follicular wall was collected as the granulosa layer. The collected tissue samples were stored at -80 °C for Western Blot, qPCR, and ELISA detection, and the results are as Figure 1 shown.

[0040] (2) Experimental results:

[0041] In this experiment, a follicular atresia model was established using TMX induction. The results showed that the hens treated with TMX completely stopped laying eggs on the 9th day ( Figure 1 Figure A in Figure 1In Figures B and C, Figure B shows the results of one sample in the control group and the experimental group (the same applies to those not specifically described in the embodiments of the present invention). ELISA detection found that the levels of follicle-stimulating hormone and luteinizing hormone in hens treated with TMX were significantly reduced ( Figure 1 in Figure D). Subsequently, it was detected whether ferroptosis occurred during follicular atresia. The qPCR results showed that the injection of TMX increased the mRNA expression level of ACSL4, but decreased the mRNA expression levels of GPX4 and FTH1 ( Figure 1 in Figures E - H). In addition, with the extension of the TMX treatment time, ELISA detection also found that the content of GSH was significantly reduced, while the contents of MDA, GSSG, and iron concentration were significantly increased ( Figure 1 in Figures I - L). These results indicate that ferroptosis occurred during the follicular atresia induced by TMX treatment.

[0042] Example 2

[0043] This example studied the effect of TMX regulating ferroptosis of follicular granulosa cells on follicular atresia process, specifically as follows:

[0044] The method for culturing granulosa cells is as follows: Six Roman commercial laying hens at 300 - day - old peak laying period were selected. After the hens were sacrificed by cervical bleeding, the whole ovaries were quickly taken out and placed in physiological saline. The hierarchical follicles F3 - F1 (26 - 35 mm; F3 < F2 < F1) were separated from the ovaries. The hierarchical follicles were cut with a blade to drain the egg yolk. The follicles were clamped with forceps and the granulosa cell layer was gently shaken out in PBS, washed clean with PBS, and placed in a small beaker containing PBS. The granulosa cell layer was cut into pieces with ophthalmic scissors, and then the mixture was added to a centrifuge tube and centrifuged (1000 rpm, 8 min), and the supernatant was discarded. 5 - 10 mL of 0.1% type II collagenase was added, and it was placed in a 37 °C water bath for digestion for 5 - 10 min. An equal volume of complete medium containing 10% FBS (fetal bovine serum) was added to terminate digestion; centrifuged (1000 rpm, 8 min), the liquid was carefully poured out, and the cells were resuspended with 20 ml of complete medium (DMEM + 10% FBS + 1% double antibody), filtered through a 200 - mesh (70 μm) cell sieve, and the filtrate was collected. Centrifuged (1000 rpm, 8 min), and the cells were resuspended with medium; the cell suspension was inoculated into a 24 - well culture plate at a cell density of 1×10 6 cells per well and cultured in an incubator at 37 °C, 5% CO2, 95% air, and saturated humidity.

[0045] Granulosa cells play a crucial role in maintaining follicular development and atresia by secreting estrogen, follicular fluid, and nutrients. To investigate whether TMX induces follicular atresia through ferroptosis in chicken granulosa cells, granulosa cells were cultured to 80% concentration and then treated with different concentrations of TMX (1 μM, 2 μM, 3 μM) for 24 h, while simultaneously treated with the classic ferroptosis inducer Erastin (positive control, 5 μM) for 24 h. The resulting granulosa cells were analyzed by CCK8, PI, and Western blot. The results are as follows: Figure 2 As shown. CCK8 and PI assays revealed that granulocytes treated with Erastin or TMX had significantly reduced cell viability. Figure 2 (Figures A and B) In addition, changes in the relative concentrations of GSH, MDA, iron, and GSSG in GC were observed, indicating that TMX treatment, like Erastin, resulted in GSH depletion, lipid peroxidation accumulation, and iron overload. Figure 2 (See CG image). Western blot analysis revealed that treatment with TMX or Erastin increased the expression level of ACSL4 protein, but decreased the expression levels of GPX4 and FTH1 proteins. Figure 2 (See Figure H). The above results indicate that TMX can induce granulosa cell death in follicles via the ferroptosis pathway.

[0046] Example 3

[0047] This embodiment focuses on a reagent that inhibits ferroptosis in granulosa cells to promote the recovery of follicular function, as detailed below:

[0048] This embodiment screened two ferroptosis inhibitors: Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1), both of which are potent and specific ferroptosis inhibitors.

[0049] (1) Can Lip-1 (5 mg / kg) rescue the process of follicular atresia by inhibiting TMX-induced ferritinization of follicular granulosa cells?

[0050] In vivo experiment: Commercial Lohmann pink-shelled laying hens at peak egg production (300 days old) were selected and randomly assigned to two groups (control group, experimental group 1, and experimental group 2), with 12 hens in each group. Hens in experimental group 1 were injected with TMX (dissolved in 0.2 mL of 0.9% physiological saline containing 0.05% fetal bovine serum albumin) at a dose of 6 mg / kg, injected into the abdominal region below the sternum. Hens in experimental group 2 were simultaneously injected with Lip-1 (5 mg / kg); the control group received an equal volume of the solvent (physiological saline). Egg production was recorded daily. After the hens in the experimental groups completely stopped laying, ovarian tissue was rapidly collected to detect lipid peroxidation levels, glutathione metabolism, iron metabolism, and the expression levels of key genes related to ferritin death in the follicles.

[0051] In vitro experiment: Large follicles (F1-F3 grade follicles) from healthy hens were collected, and granulosa cells were isolated. First, the follicles were aseptically removed, washed with PBS, and then the follicles were punctured to release granulosa cell clusters. The cell clusters were placed in collagenase (37℃, 20-30 minutes) and centrifuged (1000 rpm, 5 minutes) to collect the cells. The cells were washed 2-3 times with PBS or culture medium to remove oocytes and residual tissue. The cells were then processed at 1×10⁻⁶. 5 ~5×10 5 Cells were seeded at a density of 100 cells / mL in culture plates and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell concentration reached 80%, granulocytes were treated with PBS (control group) and TMX (5 μM, experimental group) for 24 h, respectively. Cell viability, lipid peroxidation level, glutathione metabolism, iron metabolism, and expression levels of key genes for ferroptosis were then measured.

[0052] Western blot and qPCR assays revealed that, compared with the TMX-treated group, Lip-1 treatment significantly promoted the expression of FTH1 and GPX4 and inhibited the expression of ACSL4. Figure 3 (AD figure). ELISA analysis revealed that Lip-1 treatment could inhibit the decrease in glutathione, increase in reactive oxygen species, increase in oxidative stress, and iron overload induced by TMX treatment. Figure 3 (See EN diagram). The above results indicate that Lip-1 treatment significantly inhibited TMX-induced follicular ferroptosis. Furthermore, while hens completely stopped laying eggs on day 9 after TMX treatment, hens treated with Lip-1 maintained an egg production rate of over 50%. Figure 3 (Figure O). Morphological observation revealed that Lip-1 treatment significantly increased ovarian weight, clearly showed the distribution of yellow-grade follicles, and exhibited no follicular atrophy or atresia. Figure 3 (P, Q plots). The above results indicate that TMX can induce follicular atresia by promoting ferroptosis in follicular granulosa cells, while inhibiting ferroptosis can promote the functional recovery of follicles.

[0053] (2) Ferrostatin-1 (Fer-1, 10 mg / kg) was used as an inhibitor of ferroptosis. Following the in vivo experiments in (1) and the in vitro experiments in (2), commercial Lohmann pink-shelled chickens at 300 days of age at peak egg production were selected and randomly divided into a control group and an experimental group, with 12 chickens in each group. The control group received TMX (dissolved in 0.2 mL of 0.9% physiological saline containing 0.05% fetal bovine serum albumin) at a dose of 6 mg / kg, injected into the abdominal region below the sternum. The experimental group received Fer-1 (10 mg / kg) simultaneously with the TMX-injected hens. Egg production was recorded daily. After the experimental group hens completely stopped laying eggs, ovarian tissue was collected for relevant testing and analysis. The results showed that by simultaneously injecting TMX and Fer-1 subcutaneously into peak-laying hens, Fer-1 treatment significantly inhibited TMX-induced follicular ferroptosis (Fer-1). Figure 4 (AI diagram). Experimental results show that although Fer-1 treatment can delay the TMX-induced decrease in egg production to some extent ( Figure 4 J), but on day 13 of treatment, all experimental group hens still showed complete cessation of egg production. Although morphological observation showed improvements in follicle morphology and weight parameters (J), Figure 4 While Fer-1 (K, 4L) was used in the treatment group, its rescue effect on follicular atresia was relatively limited compared to the Lip-1 treatment group, and it did not have a positive effect on egg production, thus lacking further research and development potential. Currently, only Lip-1, a ferroptosis inhibitor, has been found to have a good effect on follicular atresia and has the potential to be developed into a related drug.

[0054] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. The application of a ferroptosis inhibitor in the preparation of a drug for treating follicular atresia and / or restoring egg production function, characterized in that, The ferroptosis inhibitor is Liproxstatin-1.

2. The application according to claim 1, characterized in that, The drugs for treating follicular atresia and / or restoring egg production function are used in livestock and poultry.

3. The application according to claim 2, characterized in that, The livestock and poultry mentioned are chickens, ducks, and geese.

4. The application according to claim 2 or 3, characterized in that, The dosage of ferromoricide inhibitors for livestock and poultry is 5-20 mg / kg.

5. The application according to claim 4, characterized in that, The dosage of ferromoribolin inhibitors for livestock and poultry is 5-8 mg / kg.

6. A method for promoting the proliferation and development of granulosa cells in livestock and poultry without diagnostic or therapeutic purposes, characterized in that, include: For livestock and poultry, a ferritin inhibitor is used, namely Liproxstatin-1.

7. The method according to claim 6, characterized in that, Iron-degrading inhibitors for livestock and poultry can be administered via injection.

8. The method according to claim 7, characterized in that, The injection is either subcutaneous or intraperitoneal.

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