Application of adding carotenoids and / or zinc oxide in basal diet in regulating reproductive reserve of weaned female piglets

Abstract

The present application relates to the technical field of poultry feed, and particularly relates to application of adding carotenoids and / or zinc oxide in basal diet in regulating reproductive reserve of weaned female piglets. The present application adds carotenoids and zinc oxide in basal diet, can significantly up-regulate GDF9 protein expression of key marker of follicle development, follicle lumen expansion is most significant, and the proportion of up-regulated genes in transcriptome reaches 45.4%, which is better than that of single treatment group. The present application provides basis for development of green product for regulating reproductive reserve of weaned female piglets.

Description

Technical Field

[0001] This invention relates to the field of poultry and livestock feed technology, and in particular to the application of adding carotenoids and / or zinc oxide to the basal diet in regulating the reproductive reserves of weaned female piglets. Background Technology

[0002] Weaning is a critical stress window for piglets' growth and development. Weaning stress can cause oxidative imbalance and metabolic disorders in piglets, inhibiting growth performance and damaging the early development of the female piglet's reproductive system, thus disrupting the construction of ovarian reproductive reserve. Reproductive reserve refers to the potential physiological capacity of an animal's reproductive system before sexual maturity or during the reproductive cycle, including the structural integrity of reproductive organs, the quantity and quality of germ cells, hormone secretion levels, and the homeostasis of related regulatory networks. The construction of reproductive reserve in weaned female piglets directly affects the sow's subsequent estrus, ovulation, and lifelong reproductive performance.

[0003] Zinc oxide is a widely used pharmacological additive in the industry for controlling diarrhea in weaned piglets. However, long-term high-dose zinc oxide feeding has many drawbacks: First, zinc is excreted in feces, causing heavy metal pollution in soil and water. Second, high zinc levels can interfere with the absorption of minerals such as iron, copper, and selenium, leading to nutritional antagonism. Third, long-term high zinc exposure can cause toxic damage to ovarian granulosa cells and follicles, inhibiting follicle cell proliferation and destroying follicle structure. Fourth, it can induce the development of drug resistance genes in intestinal microorganisms. Currently, both domestic and international policies restrict the use of high-dose zinc oxide in feed, making the development of zinc-reducing and zinc-alternative nutritional regulation programs an urgent need for the pig industry.

[0004] Carotenoids (mainly β-carotene) possess antioxidant and anti-inflammatory biological activities, which can alleviate stress experienced by piglets during weaning. β-carotene, an orange-yellow, fat-soluble pigment, is a precursor to vitamin A and can be converted into active vitamin A in the body as needed. Simultaneously, β-carotene has strong antioxidant capabilities, effectively scavenging free radicals in the body. However, current research on the effects of carotenoids in weaned female piglets remains somewhat inconsistent, and their spatiotemporal metabolic patterns in reproductive tissues and downstream signaling regulatory networks have not yet been systematically elucidated.

[0005] Therefore, developing a feed additive that can effectively promote the construction of reproductive reserves in weaned female piglets while reducing the amount of zinc oxide used is of great practical value for improving the lifetime reproductive efficiency of sows and promoting the green development of the pig industry. Summary of the Invention

[0006] The purpose of this invention is to provide the application of adding carotenoids and / or zinc oxide to the basal diet in regulating the reproductive reserves of weaned female piglets. This aims to address the following problems: (1) the problem of hindered ovarian development and insufficient reproductive reserve construction in female piglets due to weaning stress; (2) the environmental emissions and animal health risks caused by long-term use of high-dose zinc oxide; and (3) the problem of unclear optimal dosage and time window of action of carotenoids in regulating the reproductive function of weaned female piglets.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of adding carotenoids and / or zinc oxide to the basal diet in regulating the reproductive reserves of weaned female piglets.

[0008] Preferably, 1-3g of carotenoids are added per kilogram of basal diet; Add 1-3g of zinc oxide per kilogram of basal diet.

[0009] Preferably, the carotenoids contain 0.8-1.2% β-carotene.

[0010] This invention also provides the application of carotenoids and / or zinc oxide in the preparation of products that regulate the reproductive reserves of weaned female piglets.

[0011] Preferably, the product includes feed additives, feed, or medicine.

[0012] Preferably, the reproductive reserve of the weaned female piglets includes any one or more of the following: 1) Promotes follicle development; 2) Upregulates the expression of GDF9 protein during follicle development; 3) Downregulate the expression of c-fos, a gene associated with follicular atresia; 4) Regulates the expression of ferroptosis-related proteins; 5) Remodeling the ovarian metabolic network; 6) Alleviate oxidative damage.

[0013] The present invention also provides a feed additive for regulating the reproductive reserves of weaned female piglets, including carotenoids and / or zinc oxide; When feed additives include carotenoids and zinc oxide, the mass ratio of the two is 1~3:1~3.

[0014] The present invention also provides the application of the aforementioned feed additive in regulating the reproductive reserves of weaned female piglets.

[0015] This invention also provides a feeding method for regulating the reproductive reserves of weaned female piglets, comprising the following steps: (1) During the first 1 to 14 days after weaning, add carotenoids and zinc oxide to the basic diet and feed the baby. (2) From the 15th to the 28th day after weaning, add carotenoids to the basic diet for feeding.

[0016] Preferably, the amount of carotenoids added in steps (1) and (2) is 1~3g / kg of basal diet; In step (1), the amount of zinc oxide added is 1~3g / kg of basal diet.

[0017] Beneficial effects

[0018] (1) The combined use of carotenoids and zinc oxide has a synergistic effect: Experiments showed that the combined use of carotenoids and zinc oxide resulted in better follicle development indicators than the two individual treatment groups, with the upregulated gene ratio in the ovarian transcriptome reaching 45.4%, significantly higher than the carotenoid group (16.9%) and the zinc oxide group (16.2%). Differential metabolites were mainly enriched in reproductive development-related pathways such as arginine and proline metabolism, pentose phosphate pathway, and ABC transporter. GDF9 protein is associated with follicle development, and the expression level of GDF9 protein in the combined treatment group was significantly higher than that in the two individual treatment groups.

[0019] (2) Avoiding the risk of long-term high zinc exposure: The present invention adopts staged feeding (adding zinc oxide on days 1 to 14 and removing it on days 15 to 28), which not only utilizes the beneficial effect of zinc oxide in relieving weaning stress, but also avoids the potential harm of long-term high zinc exposure to animal health and the ecological environment.

[0020] (3) Revealing that carotenoids protect ovarian cells by regulating ferroptosis pathway: Changes in the expression of ferroptosis-related proteins GPX4 and SLC40A1 were detected in the ovarian tissue of weaned female piglets. The experimental results showed that carotenoids can maintain ovarian iron homeostasis and alleviate zinc oxide-induced GPX4 inhibition by upregulating the iron export protein SLC40A1, thereby inhibiting ovarian cell ferroptosis. This discovery provides a new molecular perspective for understanding the regulation of reproductive reserve by carotenoids in weaned female piglets.

[0021] (4) Raw material safety: Carotenoids are natural pigments with advantages such as natural safety, environmental friendliness, and reasonable cost. This invention can replace additives such as high-dose zinc oxide, which have potential environmental and health hazards, and is in line with the industrial direction of green farming and sustainable development. Attached Figure Description

[0022] Figure 1 The results of H&E staining of ovarian tissue from weaned female piglets in different treatment groups; Figure 2Electron microscopy results of ovarian tissue from weaned female piglets in different treatment groups (scale bar: 2 μm). Figure 3 Differential metabolites in the blood of weaned female piglets in the control group and carotenoid group; Figure 4 Differential metabolites in the blood of weaned female piglets in the control group and zinc oxide group; Figure 5 Differential metabolites in the blood of weaned female piglets in the control group and the combined group; Figure 6 For the classification and pathway enrichment of differential metabolites; Figure 7 Distribution of differentially expressed genes (blue indicates total differentially expressed genes, green indicates downregulated genes, and orange indicates upregulated genes); Figure 8 Gene Ontology (GO) enrichment analysis results of differentially expressed genes in the ovarian tissues of weaned female piglets in the control group and carotenoid group; Figure 9 The results of gene ontology (GO) enrichment analysis of differentially expressed genes in the ovarian tissues of weaned female piglets in the control group and zinc oxide group; Figure 10 The results of gene ontology (GO) enrichment analysis of differentially expressed genes in the ovarian tissues of weaned female piglets in the control and combined groups; Figure 11 The distribution of differential metabolites in the ovarian tissue of weaned female piglets (left figure shows differential metabolites between the control and carotenoid groups, middle figure shows differential metabolites between the control and zinc oxide groups, and right figure shows differential metabolites between the control and combined groups). Figure 12 Classification of differential metabolites in the ovarian tissue of weaned female piglets; Figure 13 KEGG pathway enrichment analysis of ovarian tissue from weaned female piglets in the control group and carotenoid group; Figure 14 KEGG pathway enrichment analysis of ovarian tissue from weaned female piglets in the control and zinc oxide groups; Figure 15 KEGG pathway enrichment analysis of ovarian tissue from weaned female piglets in the control and combined groups; Figure 16 Results of KEGG pathway enrichment for differential metabolites; Figure 17 Principal component analysis of transcriptome-metabolome combined in the ovarian tissues of weaned female piglets in the control group and carotenoid group; Figure 18 Principal component analysis of transcriptome-metabolome combined in the ovarian tissues of weaned female piglets in the control group and zinc oxide group; Figure 19Principal component analysis of transcriptome-metabolome of ovarian tissue from weaned female piglets in the control and combined groups; Figure 20 Transcriptome-metabolome weighted gene co-expression network analysis of ovarian tissues from weaned female piglets in the control and carotenoid groups; Figure 21 Transcriptome-metabolome weighted gene co-expression network analysis of ovarian tissues from weaned female piglets in the control and zinc oxide groups; Figure 22 Transcriptome-metabolome weighted gene co-expression network analysis of ovarian tissues from weaned female piglets in the control and combined groups; Figure 23 Venn diagram of differentially expressed genes and metabolite pathways in the ovarian tissues of weaned female piglets in the control group and carotenoid group; Figure 24 Venn diagram of differentially expressed genes and metabolite pathways in ovarian tissues of weaned female piglets in the control group and zinc oxide group; Figure 25 Venn diagram of differentially expressed genes and metabolite pathways in ovarian tissues of weaned female piglets in the control group and combined group; Figure 26 The expression trend of GDF9 protein in the ovarian tissue of weaned female piglets; Figure 27 The expression trend of HSP70 protein in the ovarian tissue of weaned female piglets; Figure 28 The expression trend of c-fos protein in the ovarian tissue of weaned female piglets; Figure 29 The expression trend of GPX4 protein in the ovarian tissue of weaned female piglets; Figure 30 The expression trend of SLC40A1 protein in the ovarian tissue of weaned female piglets. Detailed Implementation

[0023] In this invention, the basal diet comprises the following components in the following mass ratios: 60-65 parts corn, 17-17.5 parts soybean meal, 4-6 parts whey powder, 4-6 parts fish meal, 2-4 parts wheat bran, 1-2 parts soybean oil, 0.7-0.75 parts limestone powder, 1-1.05 parts calcium dihydrogen phosphate, 0.2-0.4 parts salt, 0.55-0.6 parts lysine, 0.1-0.15 parts methionine, 0.18-0.25 parts threonine, 0.04-0.06 parts tryptophan, 0.15-0.19 parts valine, 0.09-0.13 parts isoleucine, 0.8-1.2 parts premix, and 0.8-1.2 parts choline chloride.

[0024] In this invention, each kilogram of basal diet includes the following premixes: Vitamin A: 2200 IU, Vitamin D3: 220 IU, Vitamin E: 16 IU, Vitamin K: 0.5 mg, Vitamin B1: 1 mg, Vitamin B2: 3.5 mg, Vitamin B6: 7 mg, Vitamin B12: 17.5 µg, Calcium pantothenate: 10 mg, Folic acid: 0.3 mg, Niacin: 30 mg, Biotin: 0.05 mg; Minerals: Cu: 6 mg, Iodine: 0.14 mg, Iron: 100 mg, Mn: 4 mg, Se: 0.3 mg, Zn: 100 mg.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] The carotenoids in this embodiment of the invention were purchased from Hunan Longsen Biotechnology Co., Ltd. HPLC analysis showed that the β-carotene content was 1%, and the remaining components were mainly a mixture of carotenoids (such as lutein, zeaxanthin, etc.).

[0027] Example 1

[0028] 1. Experimental Materials

[0029] 1.1 Experimental Animals and Grouping

[0030] The experiment was conducted at the Animal Husbandry Teaching Base of Northwest A&F University. Twenty-eight-day-old three-way crossbred weaned female piglets were randomly divided into four treatments, with 12 replicates per treatment, for a total of 48 piglets. This experiment was based on a basal diet. A 2×2 two-factor experimental design was used, with the two factors being the zinc oxide supplementation group and the carotenoid supplementation group. The groups were: 1. Blank control group (control group) (basal diet); 2. Zinc oxide group (basal diet + 2 g / kg zinc oxide (ZnO) – for a long time, to alleviate the negative effects of weaning stress, the feed industry has commonly used 2-3 g / kg pharmacological doses of zinc oxide as a basal diet additive)); 3. Carotenoid group (basal diet + 2 g / kg carotenoids (simulating the antioxidant and anti-stress effects of pharmacological doses of zinc oxide)); 4. 2 g / kg carotenoids + 2 g / kg zinc oxide group (combined group) (Table 1). The experimental period was 28 days. From day 15 to 28, the zinc oxide was removed from the diet. The specific feeding methods are shown in Table 1. Feeding was based on the daily nutritional requirements of healthy weaned piglets. No antibiotics were administered throughout the trial. The additions of carotenoids and zinc oxide were based on the basal diet.

[0031] Table 1. Experimental Groups and Feeding Methods

[0032] Note: During the first 14 days of the experiment, the diet was fed according to the above diet; during the 15th to 28th days, zinc oxide was discontinued in both the zinc oxide group and the combined group.

[0033] 1.2 Basic Diet

[0034] The basic diet was formulated with reference to the NRC (2012) corn-soybean meal type diet (Table 2) to ensure the nutritional needs of weaned piglets without adding any antibiotics.

[0035] Table 2 Basic Diet

[0036] The premixed feed contained per kilogram of basal diet includes: Vitamin A: 2200 IU, Vitamin D3: 220 IU, Vitamin E: 16 IU, Vitamin K: 0.5 mg, Vitamin B1: 1 mg, Vitamin B2: 3.5 mg, Vitamin B6: 7 mg, Vitamin B12: 17.5 µg, Calcium pantothenate: 10 mg, Folic acid: 0.3 mg, Niacin: 30 mg, Biotin: 0.05 mg; Minerals: Cu: 6 mg, Iodine: 0.14 mg, Iron: 100 mg, Mn: 4 mg, Se: 0.3 mg, Zn: 100 mg.

[0037] 2. Experimental Methods

[0038] 2.1 Feeding of weaned female piglets

[0039] Piglets were grouped and fed according to 1.1. Throughout the process, their mental state, fecal characteristics, and the occurrence of diarrhea were monitored and recorded completely to ensure that the feeding environment and management conditions of each group remained consistent.

[0040] 2.2 Collection and processing of samples from weaned female piglets

[0041] (1) Blood Sample Collection and Pretreatment: All sampling operations strictly followed the guidelines for the use of experimental animals approved by the Animal Ethics Committee of Beijing Agricultural College (License No.: BUA612409125). Sampling was conducted on the 14th and 28th days after the start of the experiment, with blood drawn from healthy weaned piglets at the Animal Husbandry Teaching and Experiment Base of Northwest A&F University. At each time point, two experimental individuals were randomly selected from each group, and three arterial blood samples were collected using vacuum blood collection tubes. After blood collection, the individual number, collection time, and processing information were immediately recorded using waterproof labels. The collected blood samples were immediately placed in dry ice for low-temperature transport and delivered to the laboratory within 48 hours for serum / plasma separation and storage at -80℃ for testing.

[0042] (2) Ovarian tissue collection and fixation: After euthanasia, the experimental pigs were fixed in a supine position. The skin and linea alba were incised along the midline of the abdomen from the anterior edge of the pubis to open the abdominal cavity. The uterine horn was located behind the kidneys and at the pelvic inlet. The uterine horn was traced distally to the distal end, and the ovarian sac and surrounding adipose tissue were separated to expose the ovary. The ovaries of weaned piglets were about the size of a mung bean to a soybean. The ovarian capsule was gently peeled off using tissue forceps and a scalpel to expose the mulberry-like ovarian parenchyma. After severing the ovarian mesentery, both ovaries were completely removed and longitudinally cut with a scalpel. The cut tissues were placed into 50 mL centrifuge tubes containing 4% paraformaldehyde and glutaraldehyde fixative. All containers were labeled with waterproof labels indicating the individual number, tissue location, and collection time.

[0043] 2.3 Preparation of paraffin sections of ovarian samples from weaned female piglets

[0044] (1) Trimming and washing: Take out the fixed tissue and place it in a fume hood. Use sterile scissors or scalpel to cut a 0.5cm2 ovarian tissue block. In order to remove 4% paraformaldehyde from the tissue, rinse the fixed tissue block twice, 8 minutes each time, and put it into a dehydration box.

[0045] (2) After the tissue samples were dehydrated by gradient ethanol, they were permeabilized with xylene and impregnated with paraffin in sequence. The order of operation is shown in Table 3.

[0046] Table 3. Alcohol gradient dehydration, permeation, and wax impregnation

[0047] (3) Tissue embedding: The specimen that has been infiltrated with paraffin is placed into the tissue embedding instrument. Then, molten paraffin is injected into the preheated mold. During the semi-solidification stage of the paraffin, the specimen is precisely oriented and placed in the center of the mold with the help of preheated tweezers. The mold is then transferred to the freezing platform for rapid cooling. After the paraffin has completely solidified, the tissue block is removed and trimmed.

[0048] (4) Section preparation: Fix the trimmed tissue block in the sample clamp of the paraffin microtome and adjust the section thickness to 5 μm for continuous sectioning. Transfer the sections to a 45℃ constant temperature slide spreader to flatten them on the water surface, and use a slide to retrieve the tissue sections. Then place the slides in a 65℃ constant temperature oven for baking for 2 hours, and finally store them at room temperature for later use.

[0049] 2.4 H&E staining of paraffin sections of ovaries from weaned female piglets.

[0050] (1) Oven treatment: Place the slices in the oven for 30 minutes until the wax is completely melted.

[0051] (2) Dewaxing and hydration treatment: The treatment sequence is shown in Table 4.

[0052] Table 4 Dewaxing and Hydration

[0053] (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 8 minutes or longer (adjust according to the observation under the microscope).

[0054] (4) Wash with water: Rinse with ddH2O for 2 minutes.

[0055] (5) Differentiation: Place the slices in hydrochloric acid ethanol rapid differentiation solution for 8 seconds.

[0056] (6) Blueing: Soak in ddH2O for 5 minutes, shake off the water and rinse with running water for 2 minutes.

[0057] (7) Dehydration and permeation: The treatment sequence is shown in Table 5.

[0058] Table 5 Dehydration and Permeation

[0059] (8) Mounting: Add an appropriate amount of neutral resin mounting medium to the surface of the tissue section, and slowly cover it with a coverslip at a 45° angle to avoid air bubbles, and complete the mounting.

[0060] 2.5 Transmission electron microscopy of ovarian tissue from weaned female piglets

[0061] (1) Sampling and fixation: In glutaraldehyde fixative, the ovarian cortex was cut into small pieces of about 1 mm3 with a sharp blade below the liquid surface and fixed at 4°C for 3 hours.

[0062] (2) Rinsing and fixing followed by dehydration: The processing order is shown in Table 6.

[0063] Table 6 Rinsing, Fixing and Dehydration

[0064] (3) Impregnation: The mixture of embedding agent (such as Epon812 or Spurr resin) and anhydrous acetone is used for stepwise replacement: embedding agent: acetone = 1:3 (1 hour), 1:1 (2 hours), 3:1 (2 hours), and finally impregnated in pure embedding agent overnight (20 hours) at room temperature or 37°C.

[0065] (4) Embedding and polymerization: The impregnated tissue block is placed into capsules or embedding plates, fresh embedding agent is added, and the mixture is placed in an oven for polymerization. Polymerization procedure: 35℃ for 12 hours → 45℃ for 12 hours → 60℃ for 36 hours to allow the resin to fully harden.

[0066] (5) Ultrathin sectioning: After trimming the embedded block, use an ultrathin slicer to cut it into ultrathin sections with a thickness of 60 nanometers. Use tweezers to pick up the sections and place them on a copper grid to air dry naturally.

[0067] (6) Electronic staining: The processing order is shown in Table 7.

[0068] Table 7 Electronic staining

[0069] 2.6 Protein extraction and Western blot detection

[0070] (1) Extraction of tissue or cell proteins: Pre-cool PBS on ice, transfer tissue samples stored at -80℃ to ice for equilibration, and precisely cut 40mg of tissue sample (the size of a mung bean) using pre-cooled sterilized scissors and place it into a labeled 1.5mL EP tube. Add 400μL of pre-cooled 1× PBS to the EP tube containing the tissue, mince the tissue with scissors, briefly centrifuge with a small centrifuge and discard the supernatant, repeating the step 3 times. Add 1mL of lysis buffer (RIPA: 4× loading buffer:PMSF=300:100:3; for phosphorylated protein samples, phosphatase inhibitors need to be added, with the volume consistent with the PMSF volume ratio) to the EP tube, and repeatedly freeze and thaw 3 times in a -80℃ freezer (for cell samples, add lysis buffer directly to the well plate at a rate of 1mL / well for freeze and thaw). Preheat the metal bath to 100℃, boil the sample for 10min, cool to room temperature, and then store in a -80℃ freezer.

[0071] (2) Gel preparation: Align the bottom edges of the long and short glass plates, with the short plate facing the experimenter, and clamp it on the plate holder to ensure that the bottom edges are aligned to prevent gel leakage; prepare the separating gel, i.e., the lower gel (prepare according to the instructions of Novizan's One-Step PAGE Gel Fast Preparation Kit), shake the gel solution well and pour it into the glass plate; prepare the stacking gel, i.e., the upper gel (prepare according to the instructions of Novizan's One-Step PAGE Gel Fast Preparation Kit), slowly pour the well mixed stacking gel solution into the pre-assembled glass plate interlayer, and after the liquid level reaches the predetermined height, immediately insert the comb that has been cleaned and dried with pure water, being careful not to have air bubbles, and place it in a hybridization oven at 37°C for 25 minutes to gel.

[0072] (3) SDS-PAGE electrophoresis separation: Remove the polymerized gel glass plate from the gel casting rack, with the short glass plate facing inward, and fix its upper end in the green slot of the electrophoresis tank electrode plate. Pour pre-cooled 1×SDS-PAGE electrophoresis buffer (3g Tris base, 14.4g glycine, and 1g SDS dissolved in 1L pure water) into the electrophoresis tank. First, slowly pour the buffer between the two glass plates. After confirming that there is no leakage, vertically pull out the sample comb. Reserve protein marker wells and accurately add 15μL of sample to each well using a micropipette. First stage: 65V constant voltage electrophoresis for 35min to concentrate the sample to the separating gel interface; Second stage: 100V constant voltage electrophoresis. Adjust the electrophoresis time according to the molecular weight of the target protein: Small molecular weight protein (50kDa): Continue electrophoresis until the bromophenol blue indicator migrates to the bottom green line, which takes about 1 hour.

[0073] (4) Transfer: Prepare transfer buffer (13g Tris base + 14.4g glycine + 270mL methanol + 730mL pure water), cut the PVDF membrane, and soak the PVDF membrane in methanol for 10min with tweezers. During the transfer operation, the glass plate should be removed carefully. First, gently pry the glass plate to loosen it, then remove the small plate and carefully scrape off the stacking gel. Be careful to avoid damaging the separating gel. Place the transfer clamp in the tray containing the transfer buffer, with the negative electrode facing down. Place the pre-wetted sponge pad, filter paper, gel, PVDF membrane, filter paper and sponge pad in sequence. After ensuring that there are no air bubbles between the layers, fix the transfer clamp. Place the assembled transfer clamp correctly into the transfer tank (note the electrode direction, clamp facing up). Place an ice box in the tank to maintain a low temperature environment. Perform the transfer at a constant current of 200mA for 1.5h (the specific time can be adjusted appropriately according to the molecular weight of the target protein).

[0074] (5) Blocking: Commonly used blocking solution is 5% BSA or skim milk powder (50mL 1×TBST+2.5g BSA or skim milk powder). Block the membrane on a shaker at the slowest speed for 1 hour.

[0075] (6) Primary antibody incubation: Place the sealed membrane in PBST and wash it three times with rapid shaking, each time for 5-10 min. Dilute the primary antibody according to the instructions (the diluent can be 5% BSA or skim milk powder), seal the membrane, and then place it in a 4°C environment and shake it at the slowest speed overnight (>8 h). Then wash the membrane five times with rapid shaking in PBST, each time for 10 min.

[0076] (7) Secondary antibody incubation: Prepare the working solution of the secondary antibody using 5% bovine serum albumin (BSA) or 5% skim milk powder as a diluent according to the recommended ratio in the instructions. Transfer the membrane to the secondary antibody solution and incubate for 1 hour at the slowest shaking speed (20 rpm). After incubation, wash the membrane five times rapidly on a shaker for 8 minutes each time with PBST buffer.

[0077] (8) Development: Prepare the developer solution (A solution: B solution = 1:1), put the film into the developer, drop the developer solution onto the PVDF film for development, and record the data.

[0078] 2.7 Western blot data analysis

[0079] Following the Western blot experiment, ImageJ 2.0 image analysis software was used to perform quantitative analysis of the grayscale values ​​of the Western blot bands to obtain the relative expression levels of each experimental group. Subsequently, GraphPad Prism 8.0 statistical software was used for data analysis, employing a two-way ANOVA method for inter-group comparisons and generating corresponding statistical charts to visually display the experimental results and analyze the expression differences of various proteins in the ovarian tissue of weaned female piglets.

[0080] 3. Results and Analysis

[0081] 3.1 Observation of H&E staining on ovarian tissue of weaned female piglets

[0082] Figure 1 The study revealed the differences in ovarian tissue morphology and follicle development process in weaned female piglets after ingesting different feed additives.

[0083] From the perspective of time-varying changes, by day 14 of feeding, the follicles in the blank control group had regular morphology, small follicular volume, and tightly arranged granulosa cell layers. The follicular cavities in the carotenoid and zinc oxide groups showed an expansion trend compared to the blank control group, with intact granulosa cell structures. The combined group showed a greater expansion of the follicular cavity than the two single-treatment groups, demonstrating a synergistic trend in promoting follicular development. By day 28 of feeding, the differences between groups further widened. The follicular cavity in the blank control group remained at a low level, and the overall follicular development process was slow. The follicular cavity volume in the carotenoid and zinc oxide groups was significantly larger than that in the blank control group, indicating improved follicular maturity, thicker granulosa cell layers, and good proliferation. The combined group had the largest follicular cavity volume among all groups, with a significantly greater expansion than the single-treatment groups. The overall follicular morphology was closer to that of a mature follicle, with clear and orderly arrangement of granulosa cell layers.

[0084] In summary, preliminary histological observations indicate that, based on the experimental conditions, carotenoids, zinc oxide, and their combined treatments can all promote the development of follicular cavities in weaned female piglets, and this effect is time-dependent. Among them, the combined treatment of carotenoids and zinc oxide showed a significant synergistic promoting effect, which was better than the effect of using either additive alone.

[0085] Scale bar = 100 μm.

[0086] 3.2 Transmission electron microscopy observation of ovarian tissue in weaned female piglets

[0087] Figure 2 The results of ultrastructural observation of ovarian tissue in weaned female piglets using transmission electron microscopy are presented. Significant differences were found in the ultrastructure of ovarian cells in each treatment group at two time points: day 14 and day 28 after feeding.

[0088] As the feeding period was extended from 14 days to 28 days, the ultrastructure of ovarian cells in the experimental animals of all four treatment groups showed a positive trend of enhanced structural regularity and improved integrity: specifically, the cell outlines became more distinct, the cytoplasm was significantly more full, the arrangement of various organelles was more orderly, and the morphology of the nuclei also became more regular. These results suggest that extending the feeding period is an important factor in promoting ovarian tissue development and maintaining intracellular homeostasis in weaned female piglets, and that each treatment can synergistically enhance this positive effect to some extent.

[0089] During the 14-day feeding period, the control group had a smaller follicular cavity volume, fewer organelles (such as mitochondria and endoplasmic reticulum) in follicular cells, and some structures were not clearly defined. The follicular cavities in the carotenoid group and zinc oxide group showed an expansion trend, with an increase in the number of mitochondria in follicular cells and a gradually clearer endoplasmic reticulum structure. The expansion of the follicular cavity in the combined group was significantly higher than that in the single treatment group. The nuclei of follicular cells were regular in shape, with clear nucleoli. The organelles such as mitochondria and rough endoplasmic reticulum were abundant and arranged in an orderly manner, and no obvious organelle damage or vacuolation was observed.

[0090] When the feeding period was extended to 28 days, the differences in ultrastructure between the groups further widened. In the control group, the follicular cavity remained at a low level, and some follicular cells showed degenerative changes such as organelle swelling and vacuolation. The follicular cavity volume of the carotenoid group and the zinc oxide group increased significantly, the number of organelles in the follicular cells increased, and the structure tended to be more complete. The combined group showed the most significant expansion of the follicular cavity, and the ultrastructure of the follicular cells showed a typical state of active proliferation and secretion: the nucleus was large and round, the nucleoplasm was uniform, and the nucleolus was obvious; there were many mitochondria with clear cristae structure, the rough endoplasmic reticulum was well developed, the ribosomes were abundant, and the organelles were arranged in an orderly manner.

[0091] The above results indicate that treatment with carotenoids and zinc oxide can significantly improve the ultrastructure of follicular cells in weaned female piglets and promote follicular cavity expansion; the combined treatment has the best effect, maintaining the integrity and functional activity of follicular organelles and providing ultrastructural evidence for normal follicular development.

[0092] 3.3 Analysis of Blood Metabolomics Data from Weaned Female Piglets

[0093] 3.3.1 Distribution of Differential Metabolites

[0094] Figures 3-5This study presents the results of a systematic analysis of differentially expressed metabolites in the blood of weaned female piglets in the control group (Con), carotenoid group (Car), zinc oxide group (ZnO), and their combined treatment group (Z+C). Volcano plots visually demonstrate the magnitude and statistical significance of metabolite expression changes within each comparison group.

[0095] (1) Overall metabolic disturbance characteristics

[0096] From the perspective of the overall perturbation of the metabolic profile, the proportion of metabolites that did not show significant differences in expression exceeded 96% in all three comparison combinations (Con vs Car: 98.6%, Con vs ZnO: 96.7%, Con vs Z+C: 97.3%). This result indicates that the overall regulatory effect of adding carotenoids alone, adding zinc oxide alone, or using both in combination on the blood metabolic profile of weaned female piglets was relatively mild, consistent with the characteristics of physiological metabolic dynamic regulation under nutritional intervention, and did not induce severe or disordered metabolic disturbances. This suggests that each treatment regimen possesses good biosafety and regulatory rationality at the metabolic level.

[0097] Regarding the expression direction and composition of differentially regulated metabolites, the proportion of upregulated metabolites showed a clear gradient increasing trend, in the order of Con vs Car (0.3%) < Con vs ZnO (0.9%) < Con vs Z+C (1.5%). The proportion of upregulated metabolites in the combined treatment group was significantly higher than that in other combinations, suggesting that the combined administration of carotenoids and zinc oxide can more broadly activate potential metabolic pathways in the blood, thereby achieving systemic regulation of the metabolic network. The proportion of downregulated metabolites showed a differential distribution: the proportion of downregulated metabolites in the Con vs ZnO group (2.4%) was significantly higher than that in the other two groups, while the proportions in the Con vs Z+C group (1.2%) and the Con vs Car group (1.1%) were similar, both remaining at a low level. This phenomenon indicates that the combined treatment can effectively antagonize the excessive downregulation effect of metabolites caused by zinc oxide alone, significantly improve the metabolic inhibition state caused by single treatment, and play an important regulatory role in maintaining blood metabolic homeostasis.

[0098] (2) Analysis of key differential metabolites and enrichment pathways

[0099] In-depth analysis of the core differential metabolites and the metabolic pathways they participate in revealed that each comparative combination exhibited significant pathway specificity and regulatory characteristics: The Con vs Car combination revealed differential metabolites primarily concentrated in lipid metabolism pathways (such as N-oleoylglycine and triglyceride DG (16:0 / 18:0 / 0:0)) and vitamin metabolism pathways (menaquinone, i.e., vitamin K2). This result suggests that carotenoid treatment regulates the blood metabolic status of weaned female piglets mainly through targeting core pathways such as lipid synthesis and degradation, and vitamin metabolism. The regulatory pathways are well-defined and the effects are mild, allowing for precise optimization of lipid and vitamin metabolism levels in the blood.

[0100] The Con vs ZnO combination showed that the differentially regulated metabolites were mainly phospholipid metabolism-related molecules (such as phosphatidylcholine PC (20:4 (5Z,8Z,11Z,14Z) / P-18:1 (11Z)) and PC (O-14:0 / 16:0)) and short-chain fatty acid metabolites, with the number of downregulated metabolites being the highest among all control groups. This phenomenon reflects that zinc oxide treatment alone has a more significant interference effect on lipid metabolism and small molecule metabolism in the blood of weaned female piglets, possibly by excessively inhibiting key metabolic processes such as phospholipid synthesis and short-chain fatty acid production, thereby disrupting the body's original metabolic balance and adversely affecting the blood metabolic microenvironment.

[0101] The Con vs Z+C combination exhibits a wide distribution of differentially regulated metabolites across multiple metabolic pathways, including glucose metabolism (D-galactosamine hydrochloride), nucleotide metabolism (adenosine), and lipid metabolism (ceramide Cer (d18:1 / 16:0)), demonstrating a significant broad-spectrum pathway characteristic. Furthermore, this combination shows the highest proportion of upregulated metabolites, while the proportion of downregulated metabolites remains at a reasonably low level, fully demonstrating the synergistic and balanced effects of the combined treatment in metabolic regulation. Its core regulatory value lies in its ability to efficiently activate multiple metabolic pathways closely related to growth, development, and nutrient supply, comprehensively optimizing blood metabolite composition, while effectively avoiding the metabolic imbalance risks associated with single treatments. This provides a systematic and precise regulatory approach for maintaining blood metabolic homeostasis in weaned female piglets, thereby laying a solid metabolic foundation for improved ovarian tissue development and reproductive performance.

[0102] 3.3.2 Classification of differential metabolites and pathway enrichment

[0103] Figure 6As shown, this study classified and enriched pathways of differentially expressed metabolites in the blood of weaned female piglets, revealing the core characteristics and key regulatory pathways of blood metabolism. At the metabolite classification level, lipid metabolites accounted for the highest proportion (33.41%), followed by amino acids (20.66%) and organic acids (15.35%), while the proportions of carbohydrates, ketones / aldehydes / esters were relatively balanced. This distribution pattern closely matches the physiological state of weaned female piglets: lipid metabolites are core precursors for energy reserves and steroid hormone synthesis, providing the material basis for piglet growth and development and ovarian maturation; amino acid metabolites participate in protein synthesis, cell proliferation and differentiation, and immune regulation, serving as key nutrient substrates for maintaining homeostasis and reproductive readiness. Together, they constitute the core framework of blood metabolism.

[0104] Metabolic pathway enrichment analysis revealed that the ABC transporter pathway (enriched 32), D-amino acid metabolism (22), cancer-centric carbon metabolism (19), protein digestion and absorption (18), and amino acid acyl-tRNA biosynthesis (16) were the most significantly enriched core pathways. Among them, the ABC transporter pathway had the highest enrichment level, suggesting that this pathway plays a core regulatory role in the transmembrane transport of blood metabolites, nutrient distribution, and signal transduction, ensuring the metabolic needs and signal communication of various tissues and organs (including the ovary). The enrichment of D-amino acid metabolism, central carbon metabolism, and protein digestion and absorption pathways reflects the active energy metabolism and nutrient utilization status of weaned female piglets, providing sufficient energy and biosynthetic raw materials for body growth and ovarian development. Further focusing on amino acid metabolism pathways, multiple pathways such as phenylalanine metabolism (15), glycine, serine and threonine metabolism (13), and arginine and proline metabolism (10) were highly enriched, indicating that amino acid metabolism is not only a core link in blood metabolism, but also plays a synergistic role in regulating the overall metabolic homeostasis and reproductive physiological processes of weaned female piglets by participating in energy supply, antioxidant defense and signal molecule synthesis.

[0105] Figure 6The left diagram shows the enriched pathways, and the right diagram shows the classification of differential metabolites. Left diagram: Orange: ABC transporters; Yellow: D-Amino acid metabolism; Blue: Central carbon metabolism in cancer; Purple (18): Protein digestion and absorption; Light green: Aminoacyl-tRNA biosynthesis; Red (15): Phenylalanine metabolism; Red (13): Glycine, serine and threonine metabolism; Red (10): Arginine and proline metabolism. Right diagram: Gray: Lipid; Dark green: Amino acids; Light green: Organic acid.

[0106] 3.4 Transcriptomic analysis of ovarian tissue from weaned female piglets

[0107] (1) Detection and analysis: Ovarian tissue samples from partially weaned female piglets were flash-frozen in liquid nitrogen and stored at -80℃ and sent to Beijing Biomarker Biotechnology Co., Ltd. for transcriptomics detection.

[0108] (2) Differential expression analysis: The DESeq2 (version 1.34.0) software was used to analyze the differential expression in RNA-seq data based on the negative binomial distribution.

[0109] Gene expression levels were calculated between different groups by processing discrete and biological replicates. Genes with significant differential expression (DEGs) were screened by setting significance thresholds (FDR < 0.05 and |log2FoldChange| > 1).

[0110] (3) Functional enrichment analysis: GO analysis and KEGG pathway enrichment analysis were carried out for differentially expressed genes, and the analysis results were presented intuitively in a variety of visualization methods such as volcano diagram and bubble diagram.

[0111] 3.4.1 Distribution of differentially expressed genes

[0112] Figure 7The results showed that sequencing analysis revealed the number and expression direction of differentially expressed genes (DEGs) in the ovarian tissues of weaned female piglets from the control group (Con), carotenoid group (Car), zinc oxide group (ZnO), and combined treatment group (Z+C). The bar chart visually presented the perturbation characteristics of the ovarian transcriptome by different treatments.

[0113] In terms of the total number of differentially expressed genes, the Con_vs_ZnO group had the most total DEGs (308), which was significantly higher than the Con_vs_Car group (148) and the Con_vs_Z-C group (130). This suggests that the addition of zinc oxide alone had the greatest impact on gene expression in ovarian tissue, while the transcriptome changes from treatment with carotenoids alone or in combination were relatively mild and closer to the physiological regulatory level. From the perspective of expression direction, all three treatments showed that the number of downregulated genes was greater than the number of upregulated genes, but the intensity of regulation differed significantly: the Con_vs_Car group had a high downregulated gene ratio of 83.1% (123 / 148), with only 25 genes upregulated, indicating that carotenoid treatment alone mainly inhibited the expression of ovarian-related genes, with a weak activation effect; the Con_vs_ZnO group further increased the downregulated gene ratio to 83.8% (258 / 308), and had the highest total number of DEGs, suggesting that zinc oxide treatment alone had a more severe inhibitory effect on the ovarian transcriptome, which may excessively interfere with the normal expression of ovarian function-related genes; while the Con_vs_Z-C group had a more balanced ratio of downregulated genes (71) to upregulated genes (59), with the upregulated gene ratio increasing to 45.4%, indicating that the combined treatment of carotenoids and zinc oxide can effectively alleviate the excessive inhibitory effect of single treatments, while maintaining mild transcriptome perturbation and activating the expression of more functional genes, which is more conducive to the maintenance of ovarian tissue physiological homeostasis and functional development.

[0114] 3.4.2 GO enrichment results analysis

[0115] Figures 8-10 The results showed that GO (Gene Ontology) functional enrichment analysis of ovarian tissue from weaned female piglets analyzed the differences in ovarian gene function between the control group (Con) and the carotenoid group (Car), zinc oxide group (ZnO), and combined treatment group (Z+C) from three dimensions: biological process (BP), cellular component (CC), and molecular function (MF).

[0116] From the overall enrichment characteristics, the core enriched items in all three groups focused on key aspects of ovarian tissue development and function maintenance, with the combined treatment group (Z+C) exhibiting a more comprehensive functional regulatory advantage. At the biological process (BP) level, all three groups showed core enriched items related to cellular processes, metabolic processes, biological regulation, and developmental processes. The Con vs Z+C group showed significant enrichment of genes related to immune processes and stress responses, and HSP70, a classic stress-protective molecule in the HSP cell stress family, was also observed. Furthermore, the number of enriched genes in the Con vs Z+C group was significantly higher than in the Con vs Car and Con vs ZnO groups, indicating that the combined treatment can more systematically regulate ovarian cell proliferation, differentiation, metabolism, and growth and development-related pathways. GDF9 is a key marker of follicle development, while c-fos, as an immediate early gene in follicle development and reproductive endocrine regulation, has a profound impact on ovarian development. The enrichment of genes in the Con vs Car group was relatively mild, while the enrichment features in the Con vs ZnO group were more prominent in immune processes and stress response-related processes, reflecting that zinc oxide treatment alone has a more significant effect on the immune and metabolic regulation of ovarian tissue.

[0117] At the cellular component (CC) level, all three groups were highly enriched in cellular anatomical entities, intracellular structures, and protein-binding complexes. The Con vs Z+C group had the highest number of enriched genes under the cellular anatomical entity category, suggesting that the combined treatment significantly optimizes the structural composition of ovarian cells and the formation of intracellular functional complexes, laying the foundation for cell structural stability and functional execution.

[0118] At the molecular function (MF) level, catalytic activity, binding and transport activity were the three common core enrichment items. The Con vsZ+C group had a much higher number of genes under catalytic activity and binding-related items than the single treatment group, indicating that the combined treatment can more broadly regulate molecular functions such as enzymatic reactions and signal molecule binding in ovarian cells, and help to precisely regulate metabolic pathways and signal transduction.

[0119] In summary, comparing the differences in GO enrichment among the three groups reveals that individual treatments with carotenoids and zinc oxide regulate ovarian gene function from dimensions such as metabolic regulation and immune stress, respectively. However, the combined treatment achieves a "superposition and optimization" of functional enrichment: not only is the number of enriched genes more comprehensive across the three GO categories, but the coverage of core functional items is also more balanced, without significant over-enrichment in any single dimension. This result indicates that the combined use of carotenoids and zinc oxide can reshape the gene functional network of ovarian tissue, more efficiently coordinating key processes such as cell proliferation, metabolic homeostasis, and structural maintenance, providing transcriptome-level functional evidence for improved ovarian development and subsequent reproductive performance in weaned female piglets.

[0120] 3.5 Non-targeted metabolomics analysis of ovarian tissue from weaned female piglets

[0121] 3.5.1 Differential Metabolite Distribution

[0122] Figure 11 As shown, non-targeted metabolomics technology was used to analyze the differential metabolite characteristics of ovarian tissues from weaned female piglets in the control group (Con), carotenoid group (Car), zinc oxide group (ZnO), and combined treatment group (Z+C). Volcano plots visually presented the magnitude, significance, and importance (VIP value, where the size of the dot represents the VIP value) of the expression changes of metabolites in each group.

[0123] From the perspective of overall metabolic disturbance, the vast majority of metabolites showing no significant difference were found in all three groups (Con vs Car: 1056, approximately 97%; Con vs ZnO: 1005, approximately 92%; Con vs Z+C: 954, approximately 88%), indicating that all three treatments were physiological interventions and did not induce severe metabolic disturbances in ovarian tissue, consistent with the physiological regulatory characteristics of ovarian development in weaned female piglets. Regarding the quantity and expression direction of differentially expressed metabolites: (1) Con vs Car group: A total of 32 significantly different metabolites were identified (14 upregulated and 18 downregulated). The number of upregulated and downregulated metabolites was close and the perturbation amplitude was the smallest, indicating that carotenoid treatment alone regulates ovarian metabolism in a mild and balanced manner, which may maintain ovarian homeostasis by regulating antioxidant and lipid metabolism pathways.

[0124] (2) Con vs ZnO group: A total of 83 significantly different metabolites were identified (26 upregulated and 57 downregulated). The number of downregulated metabolites was more than twice that of upregulated metabolites, suggesting that zinc oxide treatment alone had a more significant inhibitory effect on ovarian metabolism, with a greater perturbation amplitude than the carotenoid group, which may have excessively inhibited some metabolic pathways related to ovarian energy supply and hormone synthesis.

[0125] (3) Con vs Z+C group: A total of 134 significantly differentially expressed metabolites were identified (28 upregulated and 106 downregulated), which is the largest number of differentially expressed metabolites. The distribution of differential points with high VIP values ​​(key metabolites) is also more extensive, indicating that the combined treatment can more comprehensively regulate ovarian metabolic pathways. At the same time, the number of upregulated metabolites in the combined group is similar to that in the Con vs ZnO group, avoiding the excessive inhibition tendency of zinc oxide treatment alone. This reflects the synergistic regulatory effect of carotenoids and zinc oxide, which can not only widely activate metabolic pathways related to ovarian development and follicle maturation, but also maintain metabolic homeostasis, providing a better material basis for the maturation of ovarian function in weaned female piglets.

[0126] Figure 11The horizontal axis represents the fold change in gene expression (log2(FC)), and the vertical axis represents statistical significance (-log10(FC)). P (value). Red dots indicate significantly upregulated metabolites, blue dots indicate significantly downregulated metabolites, and gray dots indicate metabolites with no significant change in expression.

[0127] 3.5.2 KEGG pathway enrichment analysis

[0128] Differential metabolites in the ovarian tissue of weaned female piglets were classified and enriched using the KEGG pathway, revealing the material composition characteristics and core regulatory pathways of the ovarian metabolome.

[0129] like Figure 12 As shown, the metabolite classification results indicate that lipid metabolites are absolutely dominant, with phospholipids, eicosanoids, and fatty acids being the main components. These are the core precursors for the synthesis of ovarian steroid hormones, providing a crucial material basis for follicle development, ovulation, and hormone secretion. Amino acids are the second most abundant metabolites, participating in ovarian cell protein synthesis, energy metabolism, and signal molecule generation. They are the core nutrient substrates for maintaining cell proliferation, differentiation, and ovarian structural homeostasis. Carboxylic acids, vitamins and cofactors, nucleic acid-related metabolites, and hormones / neurotransmitters are present in relatively balanced proportions, collectively forming the functional network of ovarian metabolism and participating in physiological processes such as energy supply, antioxidant defense, and signal transduction. Furthermore, the presence of metabolites such as steroids (different carbon number subtypes) and peptides further demonstrates the metabolic basis of endocrine function and intercellular signaling in ovarian tissue.

[0130] like Figures 13-15 As shown, KEGG pathway enrichment analysis was performed on the differential metabolites of carotenoids (Car), zinc oxide (ZnO), and the combination of the two (Z+C) compared with the control group (Con), aiming to reveal the key metabolic pathways regulating ovarian function in weaned female piglets under different treatments. The results are as follows: Figure 13 Carotenoid group vs. control group (Car_vs_Con): The differential metabolites in the carotenoid treatment group were mainly enriched in the following key pathways: Biosynthesis of cofactors: This pathway showed the highest enrichment (highest RichFactor, lowest P value), suggesting that carotenoid treatment significantly regulated the synthesis of key cofactors such as vitamins and coenzymes. These cofactors are important coenzymes for follicle development and hormone synthesis-related enzymatic reactions, providing a material basis for ovarian function recovery.

[0131] Steroid hormone biosynthesis: This pathway was significantly enriched, and combined with previous ovarian tissue morphological observations, this suggests that carotenoids significantly affect the steroid hormone biosynthesis pathway. Carotenoids can promote the synthesis of reproductive hormones such as estrogen and progesterone in the ovary by regulating the steroid hormone synthesis pathway, thereby promoting follicular development in weaned female piglets. GDF9, as a core follicular development marker secreted by oocytes, directly reflects the follicular development status through changes in its expression.

[0132] Figure 14 Zinc oxide group vs. control group (ZnO vs. Con): The differential metabolites in the zinc oxide treatment group were mainly enriched in the following pathways: Fcepsilon RI signaling pathway: This pathway is closely related to inflammatory response and immune regulation. Zinc oxide treatment significantly modulates this pathway, suggesting that it can alleviate inflammatory damage to ovarian tissue caused by weaning stress by inhibiting excessive inflammatory response.

[0133] Parathyroid hormone synthesis, secretion and action: This pathway was significantly enriched, suggesting that zinc oxide can improve the local microenvironment homeostasis of the ovary by regulating calcium and phosphorus metabolism-related hormones, thus providing suitable conditions for follicle development.

[0134] Choline metabolism in cancer: The enrichment of this pathway reflects the regulatory role of zinc oxide in ovarian cell proliferation and metabolism. Choline metabolism is an important basis for cell membrane synthesis and cell proliferation, suggesting that zinc oxide can support the normal physiological activities of ovarian cells by regulating choline metabolism.

[0135] Figure 15 Combined treatment group vs. control group (Z+C vs. Con): The differential metabolite enrichment pathways in the combined treatment group of carotenoids and zinc oxide showed a significant synergistic effect, mainly enriching in: Fatty acid degradation: Enrichment of this pathway suggests that combined treatment can provide energy to ovarian tissue by regulating fatty acid metabolism, while reducing the accumulation of lipid peroxidation products and alleviating oxidative stress damage.

[0136] Phenylalanine metabolism: Phenylalanine is a precursor for the synthesis of neurotransmitters and hormones. The significant enrichment of this pathway suggests that combined treatment can indirectly regulate the local neuroendocrine function of the ovary by regulating amino acid metabolism.

[0137] Serotonergic synapse: Enrichment of this pathway suggests that combined treatment can alleviate the neuroendocrine effects of weaning stress on piglets and indirectly improve ovarian function by regulating central and peripheral serotonin signaling pathways.

[0138] Figure 16 KEGG pathway enrichment of differentially metabolites revealed enrichment in pathways involving arginine and proline metabolism, linoleic acid metabolism, pentose phosphate pathway, ABC transporter, and glutathione metabolism. Among these, the ABC transporter and glutathione metabolism pathways are related to iron metabolism. Furthermore, considering that zinc oxide has been reported to damage cells by inducing ferroptosis, and that carotenoids have antioxidant protective effects, this study considered detecting changes in the expression of key regulatory proteins of ferroptosis, GPX4 and SLC40A1.

[0139] In the graph, the horizontal axis represents the enrichment factor, with a higher value indicating a stronger enrichment. The vertical axis represents the pathway name. The color represents the significance p-value.

[0140] 3.6 Combined omics analysis of ovarian tissue from weaned female piglets

[0141] 3.6.1 Combined Transcriptome-Metabolic Principal Component Analysis (PCA)

[0142] Figures 17-19 Scatter plots of PCA on ovarian tissue transcriptome (red) and metabolome (blue) for the carotenoid group (Car vs Con), zinc oxide group (ZnO vs Con), and carotenoid + zinc oxide combined group (ZC vs Con); circles represent control group (Con) samples, triangles represent biological replicates of each treatment group, ellipses represent 95% confidence intervals, and PC1 and PC2 represent the two principal component dimensions with the highest contribution.

[0143] Figure 17 Carotenoids treated separately: At the transcriptome level: Con samples were concentrated in the high PC1 and medium-high PC2 regions, while Car samples clustered in the low PC1 and low PC2 regions. The red confidence ellipses were completely separated, proving that carotenoid feeding altered the ovarian gene expression pattern at the global transcriptional level, causing a wide range of regulatory perturbations on transcripts related to follicle development, antioxidation, and steroid synthesis.

[0144] At the metabolomics level: The Con and Car samples within the blue ellipse are also clearly separated along the PC1–PC2 axis, with Con located in the upper right and Car in the lower middle, confirming that carotenoids significantly remodel the overall metabolic profile of ovarian lipids, amino acids, and cofactors; the red ellipse in the transcriptome covers a larger area than the blue ellipse in the metabolome, indicating that the global fluctuation of gene transcription is higher than the changes in metabolic end products, and there are differences in hierarchical regulation such as post-transcriptional modification and enzyme activity buffering.

[0145] Figure 18 Zinc oxide treated separately: Metabolome (blue): Con and ZnO samples were completely separated along the PC1 axis. The control group was in the high negative range of PC1, while the zinc oxide group was concentrated in the more negative range of PC1. There was no sample crossover, indicating that zinc oxide has a strong remodeling effect on ovarian metabolic homeostasis and significantly changes the abundance of calcium, phosphorus, choline and inflammation-related metabolites. Transcriptome (red): Con and ZnO confidence ellipses are largely separated with only minimal contact. Zinc oxide intervention induces ovarian inflammation, apoptosis, and overall shifts in the expression of genes involved in ion transport pathways, which corroborates the anti-inflammatory phenotype of significant downregulation of c-fos protein in the previous period. The zinc oxide transcriptome ellipses have a larger vertical span, suggesting that their regulation of cellular stress and immune-related genes fluctuates more dramatically.

[0146] Figure 19 Co-treatment group: Co-treatment exhibits global molecular remodeling characteristics that differ from single addition: Metabolome (blue): Con and ZC samples were completely partitioned along the PC1 axis. The control group was located at the high left of PC1, while the combined group clustered in the lower negative range of PC1. There was no overlap between the groups, indicating that the synergistic effect of the two on the remodeling of the ovarian metabolic network was no weaker than that of zinc oxide treatment alone. The fatty acid degradation, phenylalanine metabolism, and neurotransmitter metabolism pathways were shifted as a whole. Transcriptome (red): The confidence ellipses of Con and ZC are completely independent. The distribution interval of the samples in the combined treatment group does not overlap with the positions of the Car and ZnO groups alone, which proves that the combined intervention produces a unique transcriptional regulation pattern that is different from that of a single additive, rather than a simple additive effect. Synergistic characteristics of multiple omics: The three groups of PCA comparison showed that the combined group had the highest overall separation between transcriptional and metabolic omics, which confirmed that carotenoids and zinc oxide have a synergistic regulatory effect and can simultaneously repair the molecular disorder of ovarian ovarian stress caused by weaning stress at both the transcriptional and metabolic levels. This is completely consistent with the previous morphological observation and transcriptomic analysis results of weaned female piglet ovarian tissue.

[0147] 3.6.2 Transcriptome-Metabome Weighted Gene Co-expression Network Analysis (WGCNA)

[0148] Figures 20-22 Pearson correlation heatmap of ovarian transcriptome gene co-expression module and metabolome metabolite co-expression module in three comparison groups; Figure 20 Control group vs. carotenoid group (Con vs Car) Figure 21 Control group vs. zinc oxide group (Con vs ZnO) Figure 22 Control group vs. carotenoid + zinc oxide combined group (Con vs. Z+C). The vertical column represents the transcriptional WGCNA gene module, and the horizontal column represents the WGCNA metabolite module; red indicates positive correlation between modules, and green indicates negative correlation, with color intensity corresponding to the absolute value of the correlation coefficient; the side-by-side dendrograms show the module hierarchical clustering results. This stage of multi-omics association analysis, based on the WGCNA module, identified the co-expression clusters of core candidate genes GDF9, c-fos, GPX4, and SLC40A1, constructing a "module-target gene-metabolite" regulatory chain to provide predictive basis for subsequent Western blot protein validation.

[0149] Figure 20 Con vs Car (carotenoid treatment alone) module association and target gene linkage: The large, deep red, strongly positively correlated paired modules in the half-region contain the reproductive development gene cluster belonging to GDF9. This gene module is highly positively correlated with steroid hormones and cofactor biosynthetic metabolites. Carotenoid intervention upregulates the overall module expression of core genes for follicle development, such as GDF9, and simultaneously increases the abundance of cholesterol, tocopherol, and estrogen precursor metabolites. WGCNA association results predict that carotenoids can reshape the steroid metabolic network by activating the GDF9 transcriptional module, thereby enhancing ovarian follicle development potential. This provides important omics support for subsequent quantitative validation of GDF9 protein.

[0150] The dark green, strongly negatively correlated regions in the lower half correspond to the c-fos-mediated inflammation and apoptosis gene module and the GPX4-dominated antioxidant gene module. The c-fos stress gene cluster is significantly negatively correlated with the glutathione and polyunsaturated fatty acid antioxidant metabolite modules; the GPX4 gene is embedded in the antioxidant protection module and is strongly positively correlated with protective metabolites. Omics results predict that carotenoids will inhibit the overall expression of the c-fos stress module, activate the GPX4 antioxidant module, and simultaneously increase the level of glutathione metabolites, thus alleviating ovarian oxidative stress damage at the transcriptional-metabolic level.

[0151] SLC40A1 iron homeostasis gene: It belongs to the ion transport submodule and is moderately positively correlated with iron-bound amino acid metabolites, suggesting that carotenoids can balance cellular iron ion metabolism and reduce the risk of ferroptosis by upregulating SLC40A1.

[0152] Figure 21 Con vs ZnO (zinc oxide treatment alone) module association and target gene linkage: The ZnO group heatmap showed stronger contrast in negatively correlated color blocks, and the inflammatory response gene module containing c-fos formed a very strong negative correlation with the anti-inflammatory lipid and choline metabolite modules. Zinc oxide intervention significantly suppressed the stress-induced inflammatory gene cluster dominated by c-fos, while greatly upregulating the abundance of choline and anti-inflammatory phospholipid metabolites, confirming the classic anti-inflammatory physiological function of zinc ions. Compared with carotenoids, zinc oxide showed a stronger association with the inhibitory module of the c-fos inflammatory module, indicating a more prominent anti-inflammatory stress response advantage.

[0153] Although the reproductive module containing GDF9 is still positively correlated with steroid metabolites, the correlation coefficient is lower than that of the carotenoid group, indicating that zinc oxide has a limited direct activation effect on the transcription of follicle development genes. Its main regulatory focus is on immune ion homeostasis rather than directly promoting follicle development.

[0154] GPX4 and SLC40A1 ferroptosis axis: Under zinc oxide treatment, the positive correlation between the GPX4 gene module and glutathione metabolites was weak, while the SLC40A1 iron transport module was slightly upregulated. Omics suggests that long-term intervention with zinc oxide alone is insufficient to activate the GPX4 antioxidant system and poses a potential risk of lipid peroxidation accumulation, providing a basis for subsequent experiments to add antagonistic damage.

[0155] Figure 22 Con vs Z+C (co-treatment) synergistic module association and target gene synergistic regulation

[0156] The combined treatment group showed the highest positive correlation coefficient between the reproductive development gene module containing GDF9 and the steroid and cofactor metabolite modules among the three groups. Carotenoids primarily activate GDF9 transcription, while zinc oxide amplifies the GDF9 module effect by optimizing the ovarian calcium-phosphorus and phospholipid microenvironment. At the metabolite level, the hormone-promoting effects of the two types of additives are simultaneously superimposed. The WGCNA interaction network demonstrated that the combined treatment has a dual synergistic effect of transcription and metabolism on the GDF9 pathway, predicting that its protein expression enhancement is superior to that of any single group.

[0157] The combined treatment simultaneously achieves two major protective regulatory axes: ① The c-fos inflammation-apoptosis module and the antioxidant metabolite module are strongly negatively correlated (inheriting the potent anti-inflammatory properties of zinc oxide); ② The antioxidant iron homeostasis module, containing GPX4 and SLC40A1, is extremely positively correlated with glutathione and fatty acid metabolites (amplifying the antioxidant and ferroptosis control advantages of carotenoids). Single treatment can only focus on one pathway, while the combined treatment's two protective modules work in sync with high intensity, comprehensively blocking weaning stress-induced ovarian cell damage.

[0158] Unique interaction network verifies non-additive effect: The combined group shows a novel module pairing (phenylalanine metabolism, fatty acid degradation metabolite module) that does not exist in the individual groups. At the target gene level, SLC40A1 and GPX4 are synergistically upregulated at a higher level than when they are added alone. This confirms the molecular synergy between the two at the multi-omics level, and is not a simple additive effect.

[0159] 3.6.3 Venn diagram analysis of differentially expressed genes and metabolites from the transcriptome to the metabolome

[0160] Figures 23-25 This is a Venn diagram showing the intersection of differentially expressed gene and metabolite pathways in three comparison groups.

[0161] Figure 23 Con vs Car (Carotenoids treated separately): Transcriptome screening yielded 114 significantly different pathways, while metabolome screening yielded 4 significantly different pathways. The two sets of pathways had no overlap.

[0162] Transcriptional pathway perturbations were far greater than metabolic ones: Carotenoid feeding triggered extensive gene transcriptional remodeling in the ovary, with the number of differentially expressed pathways being 28.5 times that of the metabolome, confirming the hierarchical regulatory pattern in the PCA results mentioned earlier, where the overall fluctuation amplitude of the transcriptome was higher than that of the metabolome. Many gene pathways underwent transcriptional changes, but only a few ultimately resulted in alterations in metabolite abundance. Buffering mechanisms such as post-transcriptional modification, enzyme activity regulation, and substrate transport weakened the efficiency of transmitting transcriptional fluctuations to metabolic endpoints.

[0163] A limited number of metabolic pathways precisely respond to core reproductive functions: Only four differentially expressed metabolic pathways are concentrated in two core functions: steroid hormone biosynthesis and cofactor metabolism. This precisely matches the predicted strong positive correlation between the reproductive module (containing GDF9) and the steroid metabolite module in the WGCNA heatmap. This indicates that although carotenoids perturb a vast number of transcriptional pathways, they only directionally drive substantial changes in follicle development and antioxidant-related metabolic networks. Other transcriptional fluctuations are merely adaptive fine-tuning in response to stress, with no downstream metabolic phenotypic output.

[0164] The absence of a common pathway suggests a hierarchical regulatory logic: broad-spectrum regulation at the transcriptional level and targeted convergence at the metabolic level. Subsequently, upstream differentially expressed genes corresponding to four metabolic differential pathways were selected as candidates (including GDF9 and GPX4), which significantly narrowed the candidate range for Western blotting validation.

[0165] Figure 24 Con vs ZnO (zinc oxide treated alone): There were 161 differentially expressed gene pathways and 4 differentially expressed metabolic pathways, with no overlap between the two groups; and the total number of differentially expressed gene pathways was higher than that in the Car group.

[0166] Zinc oxide exhibits stronger perturbation effects on transcriptional networks: 161 differentially expressed gene pathways showed three groups of transcriptional perturbation peaks, corresponding to the higher contrast between positive and negative correlations in the ZnO group modules in the PCA and WGCNA results. Zinc ions broadly regulate multiple transcriptional pathways, including ion transport, inflammation and immunity, apoptosis, and choline metabolism, with diverse regulatory targets.

[0167] Metabolic differential pathways are biased towards anti-inflammatory homeostasis: four metabolic differential pathways are concentrated in parathyroid hormone calcium and phosphorus metabolism and inflammatory choline metabolism, matching the strong negative correlation between the c-fos inflammation module and anti-inflammatory metabolites in WGCNA. Zinc oxide extensively activates / inhibits genes related to inflammation and ion transport, but only calcium homeostasis and anti-inflammatory phospholipid pathways show changes in metabolite abundance; c-fos, as an inflammatory hub gene, belongs to a broad-spectrum differential transcription pathway set and is a core target of zinc oxide transcriptional regulation.

[0168] Comparing the patterns in the Car group: the number of metabolic pathways with differences between the two groups was the same (4 in each group), indicating that the two single additives ultimately only changed a few key metabolic terminals; however, zinc oxide had a wider range of transcriptional perturbations, suggesting that its transcriptional regulation of ovarian immunity and ion homeostasis has a much wider coverage than the specific reproductive endocrine regulation of carotenoids.

[0169] Figure 25 Con vs Z+C (joint processing): There were 141 differentially expressed gene pathways and 0 differentially expressed metabolic pathways. There was no overlap between transcriptional and metabolic pathways and no significant perturbation at the metabolic level, exhibiting unique regulatory characteristics.

[0170] The combined treatment resulted in a moderate level of transcriptional perturbation: 141 differentially expressed gene pathways were between Car (114) and ZnO (161), proving that the combined intervention was not a simple superposition of transcriptional perturbations, but rather neutralized the excessively broad-spectrum transcriptional fluctuations caused by zinc oxide, achieving precise regulation of the transcriptional network, which is consistent with the conclusion in WGCNA that the combined group formed a new synergistic module network.

[0171] The absence of significant differences in metabolic pathways indicates a homeostatic repair effect: individual additives can induce changes in four metabolic pathways, while the combined treatment resulted in a generally stable ovarian metabolic profile with no significant differences in metabolic pathways. This suggests that the synergistic effect of carotenoids and zinc oxide can repair metabolic disorders caused by weaning stress, restoring ovarian metabolic homeostasis to near-healthy baseline levels, achieving physiological protection without drastically altering metabolite abundance; and significantly enhancing homeostatic buffering capacity at the metabolic level.

[0172] Transcription continues to play a regulatory role: Even without significant differences in metabolic pathways, 141 differentially expressed gene pathways still exist. These genes are mainly responsible for maintaining the homeostatic balance of GDF9 promoting follicle development, c-fos suppressing inflammation, and GPX4 / SLC40A1 inhibiting ferrooxidative death. Continuous fine-tuning at the gene level to stabilize the metabolic internal environment is an important molecular feature that makes the combined group superior to the single group, providing strong omics predictive evidence for subsequent Western blotting to validate the four core genes.

[0173] Transcriptional perturbation intensity ranking: ZnO (161) > Z+C (141) > Car (114); metabolic perturbation intensity: Car = ZnO (4 items) > Z+C (0 items). Combined treatment achieves the optimal balance of "precise transcriptional regulation and metabolic homeostasis." No common transcriptional-metabolic pathways were found in any of the three groups, confirming the existence of multi-level buffered regulation in the ovary: gene transcription fluctuates widely, but metabolic terminals selectively respond to core physiological pathways; this pattern perfectly corresponds to the results of PCA showing a larger span of the transcriptome ellipse and strong / weak differentiation correlation coefficients between WGCNA modules.

[0174] 3.7 Validation of candidate protein expression based on omics analysis

[0175] Based on the transcriptomics (3.4) and metabolomics (3.5) analyses described above, this study screened and validated proteins related to follicle development and cellular stress protection (GDF9, c-fos, HSP70). Furthermore, since differentially expressed metabolites were enriched in pathways such as ABC transporters and glutathione metabolism, and considering that zinc oxide has been reported to damage cells by inducing ferroptosis, while carotenoids have antioxidant protective effects, this study further examined the expression changes of key ferroptosis regulatory proteins GPX4 and SLC40A1 to explore whether carotenoids can alleviate zinc oxide-induced ovarian damage by inhibiting ferroptosis.

[0176] 3.7.1 Expression trend of GDF9 protein in the ovarian tissue of weaned female piglets

[0177] like Figure 26 As shown, Western blot analysis was used to detect... GDF9 The expression of the gene in the ovarian tissue of weaned female piglets was investigated. The results showed that the GDF9 protein band was located at approximately 70 kDa, and the internal reference protein β-actin band was located at 42 kDa. The bands were clear and there were no obvious impurities, indicating that the experimental results were reliable.

[0178] At 14 days of treatment: compared with the control group, the expression level of GDF9 protein in the carotenoid group was significantly increased ( P <0.001), although the expression level in the zinc oxide group increased, the difference was not significant. P > 0.05); the expression level of GDF9 protein in the combined treatment group was significantly higher than that in the control group ( P The value was < 0.01, and was higher than that of the two individual treatment groups, suggesting that the combined treatment has shown a trend of upregulating GDF9 expression in the short term.

[0179] At 28 days of treatment: the differences in GDF9 protein expression between groups further increased. Compared with the control group, the GDF9 protein expression levels in the carotenoid group, zinc oxide group, and combined treatment group were all significantly increased ( P < 0.01 or P <0.001); among them, the combined treatment group had the highest GDF9 protein expression level, which was significantly higher than that of the two individual treatment groups ( P <0.001), showing a clear synergistic upregulation effect.

[0180] The combined results from 14 days and 28 days show that the upregulation effect of each treatment group on GDF9 protein expression gradually increased with the extension of treatment time. Among them, the combined treatment of carotenoids and zinc oxide had the most significant effect at 28 days, indicating that the combination of the two can promote the recovery of ovarian function and follicle development in weaned female piglets by upregulating the expression of GDF9 protein in ovarian tissue, and this effect is time-dependent.

[0181] 3.7.2 Expression trend of HSP70 protein in the ovarian tissue of weaned female piglets

[0182] like Figure 27 As shown, the expression level of HSP70 protein in the ovarian tissue of weaned female piglets at 14 and 28 days of treatment was detected by Western blot. The results showed that at 14 days of treatment, the expression of HSP70 in the carotenoid group, zinc oxide group, and combined group was slightly lower than that in the control group, but the differences between the groups were not statistically significant (ns, P >0.05); at 28 days of treatment, the band characteristics were consistent with those at 14 days. Quantitative analysis showed that HSP70 expression was slightly upregulated in the carotenoid group, zinc oxide group, and combined group (especially the combined group) compared with the control group, but the difference between groups was still not statistically significant (ns, P >0.05). With prolonged treatment time, the expression trend of HSP70 in each group changed from a slight downregulation at 14 days to a slight upregulation at 28 days, but none of these changes reached statistical significance. As a key molecule involved in cellular stress protection and follicle development, HSP70 protein expression was not significantly altered by carotenoids, zinc oxide, or their combined treatments at 14 and 28 days in this study. This may be related to insufficient treatment time or dosage, or the relatively stable expression of HSP70 itself.

[0183] 3.7.3 Expression trend of c-fos protein in the ovarian tissue of weaned female piglets

[0184] like Figure 28 As shown in the Western blot results, after 14 days of treatment, compared with the control group, the expression levels of c-fos protein in the carotenoid group, zinc oxide group and combined treatment group were significantly reduced (P < 0.001), with the combined treatment group showing the largest downregulation. This indicates that each treatment can rapidly inhibit the expression of c-fos protein, and the short-term inhibitory effect of the combined treatment is more prominent.

[0185] At 28 days of treatment, the downregulation of c-fos protein persisted, and the differences between groups remained highly significant (P < 0.001). Compared with the control group, the expression levels of c-fos protein in the carotenoid group, zinc oxide group, and combined treatment group were significantly reduced, with the expression level in the combined treatment group remaining the lowest among all groups. Meanwhile, the downregulation effect in the zinc oxide group slightly increased over time.

[0186] The combined results from 14 and 28 days show that treatment with carotenoids and zinc oxide, whether used alone or in combination, significantly downregulated the expression of c-fos protein in the ovarian tissue of weaned female piglets, and this inhibitory effect remained stable at 28 days. The downregulation effect of carotenoid treatment alone and in combination showed highly significant differences throughout the observation period, with the inhibitory effect at 28 days being particularly stable and prominent. This suggests that this treatment can alleviate the adverse effects of weaning stress on ovarian tissue by inhibiting the overexpression of c-fos protein, creating favorable conditions for the recovery of ovarian function.

[0187] 3.7.4 Expression trend of GPX4 protein in the ovarian tissue of weaned female piglets

[0188] like Figure 29 As shown, after 14 days of treatment, GPX4 expression in the carotenoid group was significantly downregulated compared to the control group. , P <0.01%, while the zinc oxide group and the combined group showed significantly higher upregulation than the carotenoid group ( , P <0.01), and there was no statistically significant difference between the latter two groups (ns). At day 28 of treatment, GPX4 expression in the carotenoid group was slightly lower than that in the control group, but the difference was not statistically significant (ns), while expression in the zinc oxide group was significantly downregulated compared to both the control and carotenoid groups (ns). , P <0.01, the combined group expressed higher levels than the zinc oxide group, but still significantly lower than the control group ( , P <0.05).

[0189] 3.7.5 Expression trend of SLC40A1 protein in the ovarian tissue of weaned female piglets

[0190] like Figure 30 As shown, after 14 days of treatment, SLC40A1 expression in the carotenoid group was significantly upregulated compared to the control group. , P <0.05, the expression levels in the zinc oxide group and the combined group were close to those in the control group, and there was no statistically significant difference between the latter two groups (ns). The carotenoid group was significantly higher than both the zinc oxide group and the combined group. ,P <0.05); After 28 days of treatment, SLC40A1 expression in each treatment group was slightly upregulated compared with the control group, but the difference between the groups was not statistically significant (ns).

[0191] This invention, by supplementing weaned piglets with carotenoids and zinc oxide in their basal diet, significantly upregulated the expression of GDF9 protein, a key marker of follicular development, resulting in the most significant follicular cavity expansion. The upregulated gene ratio in the transcriptome reached 45.4%, superior to the single-treatment group. Carotenoids protect ovarian cells by inhibiting ferroptosis. Carotenoids upregulated the iron export protein SLC40A1, downregulated GPX4 expression, and antagonized zinc oxide-induced GPX4 downregulation, maintaining ovarian cell iron homeostasis and antioxidant capacity, and mitigating ferroptosis damage. This aligns with the industrial direction of green farming and sustainable development.

[0192] 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 principle 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 adding carotenoids and / or zinc oxide to the basal diet in regulating the reproductive reserves of weaned female piglets.

2. The application according to claim 1, characterized in that, Add 1-3g of carotenoids per kilogram of basal diet; Add 1-3g of zinc oxide per kilogram of basal diet.

3. The application according to claim 1 or 2, characterized in that, The carotenoids contain 0.8-1.2% β-carotene.

4. Application of carotenoids and / or zinc oxide in the preparation of products that regulate the reproductive reserves of weaned female piglets.

5. The application according to claim 4, characterized in that, The products include feed additives, feed, or medicines.

6. The application according to claim 1 or 4, characterized in that, The reproductive reserve of weaned female piglets includes any one or more of the following: 1) Promotes follicle development; 2) Upregulates the expression of GDF9 protein during follicle development; 3) Downregulate the expression of c-fos, a gene associated with follicular atresia; 4) Regulates the expression of ferroptosis-related proteins; 5) Remodeling the ovarian metabolic network; 6) Alleviate oxidative damage.

7. A feed additive for regulating the reproductive reserves of weaned female piglets, characterized in that, Including carotenoids and / or zinc oxide; When feed additives include carotenoids and zinc oxide, the mass ratio of the two is 1~3:1~3.

8. The use of the feed additive of claim 7 in regulating the reproductive reserves of weaned female piglets.

9. A feeding method for regulating the reproductive reserves of weaned female piglets, characterized in that, Includes the following steps: (1) During the first 1 to 14 days after weaning, add carotenoids and zinc oxide to the basic diet and feed the baby. (2) From the 15th to the 28th day after weaning, add carotenoids to the basic diet for feeding.

10. The feeding method according to claim 9, characterized in that, In steps (1) and (2), the amount of carotenoids added is 1~3g / kg of basal diet; In step (1), the amount of zinc oxide added is 1~3g / kg of basal diet.

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