Application of arginine in luteinization of replacement gilts

Abstract

The invention relates to the technical field of animal nutrition and reproduction, and discloses application of arginine in luteinization of replacement gilts. The arginine is used as a nutritional supplement for regulating and controlling the hormone level of the replacement gilts, the progesterone content can be effectively increased, and corpus luteum generation is promoted. Meanwhile, after the porcine ovarian granular cells are cultured, arginine can up-regulate expression of genes related to synthesis of progesterone and down-regulate expression of genes related to synthesis of estrogen, so that the progesterone is increased, the estrogen is reduced, and the effects of increasing the generation quantity of corpus luteum and maintaining a good pregnancy environment are achieved. According to the invention, 0.5% of arginine is directly added into the pig daily ration, so that the progesterone content in follicular fluid and serum can be remarkably increased, the estrogen content can be reduced, the quantity of small corpus luteum can be increased, and the pregnancy quality and success rate of the replacement gilts after mating can be improved. According to the invention, the problem of hormone level regulation can be solved, and a new nutrition regulation target is provided for increasing the luteum generation of the replacement gilts and improving the reproductive performance of the gilts.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and reproductive technology, and relates to the application of arginine in the formation of corpus luteum in gilts. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the fields of animal nutrition and reproductive technology, improving sow reproductive performance has always been a key research focus and challenge. Therefore, arginine is frequently used in medicine and animal husbandry to improve related physiological functions, such as enhancing animal reproductive performance and assisting in the treatment of cardiovascular diseases or immunodeficiency. Studies have shown that adding 1.3% arginine to the diet of sows during the first 1-14 days of gestation can improve total litter size and the number of live piglets per litter (Li J, 2015); metabolomic analysis of serum, follicular fluid, and urine from sows with low and normal fertility revealed that the arginine metabolic pathway was significantly enriched in the body fluids of low-fertility sows (Chen M, 2019).

[0004] Elevated LH levels are a prerequisite for corpus luteum formation, but not the sole determining factor. Whether the corpus luteum will successfully form ultimately requires a comprehensive assessment based on ovarian morphology. Previous studies have shown that dietary supplementation with arginine before mating can significantly affect the levels of luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) in rat plasma. However, whether this intervention can ultimately promote corpus luteum formation requires further verification through more research (Li Jie, 2015).

[0005] Furthermore, current research on the role of arginine in corpus luteum formation in mammals is insufficient, especially regarding the regulatory mechanisms of corpus luteum formation in the specific population of gilts, where relevant studies remain lacking. Although some studies have confirmed that arginine can promote progesterone secretion in pregnant sows, gilts are in a pre-pregnancy physiological stage, and their physiological state differs significantly from that of pregnant sows. Therefore, the specific effects and regulatory mechanisms of arginine on progesterone secretion in gilts remain unclear. Summary of the Invention

[0006] In response to the current status and existing problems in livestock farms, the purpose of this invention is to provide a product containing arginine that promotes corpus luteum formation in gilts. Based on previous research on the spatial metabolomics of porcine ovaries, this invention found that amino acids and their metabolites constitute the largest proportion of metabolites at different stages of follicle development, with arginine and various amino acids forming small peptides at relatively low levels. Therefore, this invention demonstrates that directly adding 0.5% arginine to the sow diet significantly increases the number of corpora lutea in gilts, revealing the important role of arginine in regulating sow hormone levels and providing a new genetic selection strategy for improving sow reproductive performance.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides the application of arginine in regulating corpus luteum formation in gilts.

[0008] A second aspect of the invention provides the application of arginine in improving the reproductive performance of sows.

[0009] A third aspect of the present invention provides a nutritional supplement for gilts, comprising: arginine.

[0010] In a fourth aspect, the present invention provides a gilt diet in which arginine is added at a level of 0.5% to 1%.

[0011] Beneficial effects of the present invention (1) This invention has significant practical application effects. Adding arginine to the diet of gilts can significantly increase the content of progesterone and lactic acid, thereby promoting the formation of corpus luteum.

[0012] (2) This invention reveals the important role of arginine in regulating the hormone levels of sows, and improves the reproductive performance of sows in the form of nutritional supplementation. It has the advantages of being economical, efficient, flexible, safe and widely applicable, and avoids potential problems such as veterinary drug residues and withdrawal periods in mammals such as pigs, thus ensuring the safety of mammal products.

[0013] (3) This invention verified that the expression levels of progesterone synthesis-related genes (such as CYP11A1 and HSD3B1) were increased in porcine ovarian granulosa cells, thereby increasing progesterone content.

[0014] (4) The method of the present invention is safe and harmless to the mother, and combines reproductive nutrition methods to provide new nutritional regulation targets and genetic selection strategies for improving the reproductive performance of sows. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The changes in various indicators of mice fed a diet supplemented with 1% arginine for 4 weeks in this invention are shown. (A) Experimental design. (B) HE-stained sections of ovarian tissue. Scale bar = 500 μm. (C) Number of corpora lutea. Data are expressed as mean ± standard error (n = 10). P <0.01. (DG) Serum estradiol and progesterone levels were detected by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard error (n = 10). * P <0.05,** P <0.01. (H) Representative Western blot images of steroidogenesis-related proteins (FSHR, LHR, CYP19A1, CYP17A1, CYP11A1, HSD3B1, STAR) in ovarian tissue. (I) Quantitative analysis of steroidogenesis-related proteins. Data are expressed as mean ± standard error of three independent experiments. * P <0.05,** P <0.01.

[0017] Figure 2 This study describes the changes in various indicators of gilts after 8 weeks of feeding with 0.5% arginine added to their diet. Serum hormone levels (including arginine, estrogen, progesterone, gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone) were detected using enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard error. The sample size was n = 7, and statistical significance was considered (*). P <0.05,** P <0.01). (GL) Hormone levels in follicular fluid (including arginine, estrogen, progesterone, gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone) were detected by enzyme-linked immunosorbent assay (ELISA). Data are derived from the mean ± standard error of at least three independent experiments, and statistical significance is required (*). P <0.05, *** P <0.001). (M) Hematoxylin-eosin stained sections of sow ovaries. Scale bar = 5000 μm. (N) Count of small corpora lutea. Data are expressed as mean ± standard error. Sample size (n = 3). Statistical significance is defined as (*). P <0.05).

[0018] Figure 3 This invention illustrates the changes in various parameters of porcine ovarian granulosa cells after arginine treatment. (A) Viability of granulosa cells after treatment with different concentrations of L-arginine. Data are expressed as the mean ± standard error of three independent experiments. * P<0.05. (B) Estrogen levels in the culture medium were detected by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard error (n = 3), ** P <0.01. (C) Progesterone levels in the culture medium were detected by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard error (n = 3), * P <0.05. (D) Western blot analysis of protein levels in steroid hormone synthesis-related genes (CYP19A1, STAR, CYP11A1, CYP17A1, HSD3B1, LHR, and FSHR). (E) Quantitative analysis of the Western blot results. Data are expressed as mean ± standard error (n = 3), * P <0.05. (F) Real-time quantitative PCR analysis of steroid hormone synthesis-related genes, including STAR, CYP19A1, CYP11A1, CYP17A1, HSD3B1, LHR, and FSHR. Data are expressed as mean ± standard error of at least three independent experiments. * P <0.05,** P <0.01, *** P <0.001. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or according to the product instructions. Similarly, unless otherwise specified, the test methods of this invention are performed in accordance with conventional methods in the art or industry-standard methods or criteria. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0021] This invention provides the application of arginine in regulating corpus luteum formation in gilts. By using arginine as a nutritional supplement to regulate hormone levels in gilts, this invention can effectively increase progesterone levels and promote corpus luteum formation.

[0022] In some implementations, progesterone levels are increased and estrogen levels are decreased in gilts. Culture of porcine ovarian granulosa cells revealed that arginine upregulates the expression of genes related to progesterone synthesis and downregulates the expression of genes related to estrogen synthesis, thereby increasing progesterone levels and decreasing estrogen levels. This increases the number of corpora lutea and maintains a favorable pregnancy environment.

[0023] In some implementations, the expression of genes related to progesterone synthesis is upregulated, while the expression of genes related to estrogen synthesis is downregulated.

[0024] In some embodiments, the arginine is added to the diet at a rate of 0.5% to 1%. Directly adding 0.5% arginine to the pig diet can significantly increase progesterone levels in follicular fluid and serum, decrease estrogen levels, and increase the number of corpora lutea, thus helping to improve the pregnancy quality and success rate of gilts after mating.

[0025] In some embodiments, hypothalamic gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and lactate levels are increased. Experiments have shown that after 8 weeks of feeding with a diet supplemented with 0.5% arginine, the levels of GnRH, LH, and progesterone in the body are significantly increased, as is lactate levels. This invention can solve the problem of hormone level regulation and provides a new nutritional regulatory target for increasing corpus luteum formation in gilts and improving sow reproductive performance.

[0026] This invention provides the application of arginine in improving the reproductive performance of sows. The selected gilts' reproductive systems are in a critical developmental stage, making them more sensitive to arginine supplementation and better able to respond to nutritional regulation.

[0027] In some implementations, pregnancy quality and success rate in gilts are improved.

[0028] This invention provides a diet for gilts, wherein the amount of arginine added to the diet is 0.5% - 1%.

[0029] In some embodiments, the diet further includes: corn, soybean meal, coarse bran, dicalcium phosphate, fine limestone powder, lysine sulfate, sodium chloride, threonine, Pharmaron PK200, 0.1% pig micronutrient premixed feed Z-801, Mite 20000 microgranules C7, choline chloride, Lubao fine salt, HF01, HF02, methionine, tryptophan, sandoquinone, and vitamin E.

[0030] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0031] In the following examples, arginine was purchased from Pusitang. The formulated gilt diet met the nutritional requirements for gilts set by the National Research Council (NRC, 2012), and its composition and nutrient levels are shown in Table 1. All ingredients in the diet were commercially available products.

[0032]

[0033] Example 1: Effect of arginine on corpus luteum formation in gilts This invention selected 14 healthy gilts, 115 days old and weighing (59.21 ± 1.71) kg, and divided them into two groups with 7 replicates per group and one replicate per gilt. The control group was fed a basal diet, while the experimental group was fed a diet containing 0.5% arginine. Both diets met the nutritional levels recommended by the National Research Council (NRC, 2012). Blood and ovarian samples were collected from the gilts at slaughter before mating, immediately frozen in liquid nitrogen, and subsequently stored at -80°C. ° C is used for subsequent RNA and protein blot analysis. The experiment lasted 53 days. This invention was conducted at the Animal Husbandry Teaching and Research Base of Northwest A&F University. The pigpen was 2.5 meters × 2.5 meters in size, with one sow per pen to ensure a consistent and suitable rearing environment. The sows were fed twice a day, 2.2 kg per day in the first week, and restricted to 2.6 kg per day in the second week, and vaccinated according to the immunization program for gilts.

[0034] Example 2: Effect of arginine on corpus luteum formation in mice 8-week-old C57BL / 6J mice were purchased from Shaanxi Rui Medical Biotechnology Co., Ltd. (Shaanxi, China, license number: SYXK[Shaan]2024–005). The mice were housed in the Experimental Animal Center of Northwest A&F University (license number: SYXK[Shaan]2022–03). The housing environment was a 12-hour light / 12-hour dark cycle, and the mice had free access to standard mouse feed and water until the experiment began. Twenty mice were randomly divided into a control group (10 mice) and an arginine group (10 mice), and each group was further divided into 2 cages for housing. The mice in the control group drank distilled water freely, while the mice in the arginine group drank water containing 1% arginine. The food intake, water intake, and body weight of the mice were recorded weekly. After 1 month of treatment, the mice were injected with 5 IU of pregnant mare serum gonadotropin (PMSG, Solarbio, Beijing, China, product number: P9970), and 48 hours later, 5 IU of human chorionic gonadotropin (HCG, AibeiBio, Nanjing, China) was injected. The mice were sacrificed 12 h later. The mice were anesthetized with isoflurane and sacrificed by cervical dislocation under deep anesthesia. Subsequently, the ovaries were collected: one ovary was fixed in 4% paraformaldehyde for follicle counting; the other side was stored at -80 ° °C for subsequent qPCR and Western blot analysis. Blood samples were centrifuged at 4 ° °C and 4000 rpm for 10 minutes to separate the serum, which was then stored at -80 ° °C for analysis.

[0035] Example 3 Collection of Porcine Ovarian Granulosa Cells The collection of porcine ovarian granulosa cells was carried out in accordance with the animal experiment procedures in accordance with the "Regulations on the Administration of Laboratory Animals" in China. Ovaries of 180-day-old Landrace pigs (n = 20, approximately 110 kg) were obtained from Benxiang Slaughterhouse. The ovaries were stored in physiological saline at 37 ° °C, which was supplemented with 100 IU / mL penicillin (Cytiva, Shanghai, China) and 100 μg / mL streptomycin (Cytiva, Shanghai, China), and transported to the laboratory within 2 h. Follicular fluid was collected from antral follicles with a diameter of 3 - 5 mm and centrifuged at 1000 rpm for 10 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended in F12 (Cytiva, Shanghai, China) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China), and then seeded into 12-well plates at a density of 1×10 5 cells / well and placed at 37 °C. Cultured in an incubator containing 5% CO2. After 24 hours, the cells were washed with PBS (Pricella, Wuhan, China) and then cultured in arginine-free F12 medium (Pricella, Wuhan, China) for another 24 hours. After 24 hours of starvation culture, the cells were treated with 8 mM arginine (Psaitong, Beijing, China, catalog number: 74-79-3) for 24 hours (arginine group was designated Arg-8, control group was designated Arg-0). Cell supernatant and cell samples were collected for subsequent experiments.

[0036] Example 4: CCK-8 Detection CCK-8 assay was performed using a commercial kit (APExBIO, USA) following the manufacturer's instructions. Granulosa cells were cultured at 2 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells / well in 96-well plates and treated with arginine for 24 h. After incubation at 37°C with 10 μL of CCK-8 reagent for 3 h, the absorbance at 450 nm was measured to assess cell viability.

[0037] Example 5 Enzyme-linked immunosorbent assay The intermediate metabolites of steroid hormone synthesis and estrogen concentrations were detected using an ELISA kit (Mlbio, Shanghai, China). Relative hormone concentrations were determined by recording absorbance at 450 nm using a Multiskan™ FC microplate reader (Thermo Fisher Scientific, Shanghai, China). This ELISA kit is coated with monoclonal antibodies to ensure extremely low cross-reactivity.

[0038] Example 6 H&E staining Animal ovarian tissue was completely extracted and fixed with 4% paraformaldehyde. The fixed ovaries were dehydrated and then embedded in paraffin; 5 μm thick sections were prepared and subjected to H&E staining. The H&E-stained samples underwent dewaxing, hydration, hematoxylin staining, differentiation and blueing, eosin staining, dehydration, and then air-drying and mounting. Finally, the number of corpora lutea was counted.

[0039] Example 7 Real-time Polymerase Chain Reaction Total RNA was extracted from ovarian tissue and cultured granulosa cells using TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer's instructions. RNA concentration and purity were assessed by measuring the A260 / A280 absorbance ratio. -∆∆Ct Gene expression levels were calculated using this method. The primer sequences used for RT-qPCR are shown in SEQ ID No. 1-16 and are listed in Table 2.

[0040]

[0041] Example 8: Protein Blotting Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). 120 μL of RIPA was added to each well of a 6-well plate, supplemented with 1% protease inhibitor (CWBIO, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Massachusetts, USA). A 1:4 volume ratio of 5× loading buffer (Ncmbio, Suzhou, China) was added to the supernatant, and the sample was boiled for 10 minutes. 15–20 μg of protein was separated by 10%–15% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane (Vazyme, Nanjing, China) at 250 mA for 2.5 h. The membrane was blocked at room temperature for 2 h in blocking buffer containing 5% skim milk powder (Beyotime, Shanghai, China). Subsequently, the membrane was incubated overnight at 4°C with primary antibodies (1:1000 dilution) against the following proteins: STAR, CYP19A1, CYP17A1, CYP11A1 (Abways, Shanghai, China), FSHR, LHR (Affinity, Jiangsu, China), and HSD3B1 (Santa Cruz, California, USA). The following day, the membrane was incubated for 1 hour at 4°C with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution) (Boster, Wuhan, China). Protein signals were detected using a chemiluminescent substrate (Santa Cruz, California, USA) and analyzed using ImageLab software (Bio-Rad, Berkeley, California, USA). All experiments were repeated at least three times, and the average value was calculated.

[0042] To verify that arginine promotes luteinization of granulosa cells and thus promotes corpus luteum formation, this invention designed a mouse experiment using 8-week-old female C57BL / 6 mice to investigate the effect of 1% Arg (arginine) on ovarian function. To synchronize the estrous cycle and ensure consistent mouse condition, exogenous gonadotropins (i.e., arginine treatment) were injected before mating. Blood was collected from the tail vein one month later, followed by injection of PMSG, then HCG 48 hours later, and finally, the mice were slaughtered 12 hours later. Serum and ovarian tissue were collected. Figure 1 (A). Ovarian tissue sections showed a significant increase in the number of corpora lutea in the 1% Arg group ( Figure 1ELISA results showed that, compared with the control group, the 1% Arg group had decreased estrogen and increased progesterone in the slaughter serum; while there was no difference in serum estrogen before injection, but progesterone levels increased significantly. Therefore, arginine treatment can increase progesterone levels. Figure 1 (DG). Western blot results showed that the 1% Arg group decreased the levels of FSHR and CYP19A1 estrogen synthesis proteins, and increased the levels of CYP11A1, LHR, and HSD3B1 progesterone synthesis proteins. Figure 1 (Middle HI). Therefore, 1% arginine can regulate the expression of proteins related to steroid hormone synthesis, thereby inhibiting estrogen secretion, promoting progesterone, and ultimately promoting the formation of corpus luteum in mouse ovaries, providing a practical basis for the role of arginine in female reproductive physiology.

[0043] To investigate the effect of arginine on the reproductive performance of gilts, this invention selected 115-day-old gilts with an average weight of (59.21 ± 1.71) kg and randomly divided them into two groups (7 replicates per group, 1 gilt per replicate). The control group was fed a basal diet, while the arginine-treated group was fed a diet supplemented with 0.5% arginine. The gilts were slaughtered before estrus, and serum and ovarian tissue were collected for subsequent analysis. Enzyme-linked immunosorbent assay (ELISA) results showed that, compared with the control group, the arginine group had lower serum estrogen levels, while the levels of progesterone, arginine, gonadotropin-releasing hormone (GnRH), and luteinizing hormone (LH) were significantly higher. Figure 2 (AL). Histological analysis of ovarian tissue using H&E staining revealed that the number of corpora lutea in the arginine group was significantly higher than that in the control group, especially small corpus luteum cells (AL). Figure 2 These results indicate that arginine can regulate hormone levels and promote luteinization, which may have a positive impact on the reproductive performance of gilts.

[0044] The main function of ovarian granulosa cells is to produce steroid hormones, among which estrogen is related to estrus, and progesterone is related to luteinization of granulosa cells and pregnancy after ovulation. To study the effect of arginine on porcine granulosa cells, cell viability was measured using CCK8 assay after 24 hours of starvation in arginine-free F12 medium followed by the addition of different concentrations of arginine. The results showed that cells treated with 8 mM arginine were in good condition and had significantly higher viability than the control group. Figure 3 (A), therefore, the concentration of arginine added in subsequent experiments was 8 mM. ELISA results showed that arginine treatment decreased estrogen and increased progesterone ( Figure 3(BC). Furthermore, the expression of genes involved in the regulation of steroid production was examined, including reproductive hormone receptor genes (FSHR and LHCGR) and steroid production-related genes (STAR, CYP11A1, CYP17A1, HSD3B1, and CYP19A1). In the arginine group, the mRNA levels of FSHR, CYP19A1, STAR, and CYP17A1 genes were decreased, while the mRNA levels of LHCGR, CYP11A1, and HSD3B1 genes were significantly increased; the protein levels of FSHR, CYP19A1, and CYP17A1 were decreased, while the protein levels of LHCGR, CYP11A1, and HSD3B1 were significantly increased. Figure 3 (D-F). These results indicate that arginine reduces the expression of genes associated with estrogen synthesis and increases the expression of genes associated with progesterone synthesis, ultimately leading to decreased estrogen and increased progesterone.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of arginine in regulating corpus luteum formation in gilts.

2. The application as described in claim 1, characterized in that, The progesterone levels of gilts are increased, while the estrogen levels are decreased.

3. The application as described in claim 1, characterized in that, It upregulates the expression of genes related to progesterone synthesis and downregulates the expression of genes related to estrogen synthesis.

4. The application as described in claim 1, characterized in that, The arginine is added to the diet at a rate of 0.5% - 1%.

5. The application as described in claim 1, characterized in that, The levels of hypothalamic gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and lactate were elevated.

6. Application of arginine in improving the reproductive performance of sows.

7. The application as described in claim 6, characterized in that, Improved pregnancy quality and success rate in gilts.

8. A nutritional supplement for gilts, characterized in that, include: Arginine.

9. A diet for replacement gilts, characterized in that, The diet contains 0.5% - 1% arginine.

10. The gilt diet as described in claim 9, characterized in that, The diet also includes: corn, soybean meal, coarse bran, dicalcium phosphate, fine limestone powder, lysine sulfate, sodium chloride, threonine, Baolaikang PK200, 0.1% pig trace element premixed feed Z-801, Mite 20000 micro-particle C7, choline chloride, Lubao fine salt, HF01, HF02, methionine, tryptophan, sandoquinone, and vitamin E.

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