Anthocyanins Alleviate Persistent Corpus Luteum and Ovarian Quiescence in Beef Cows by Modulating Gut Microbiota and Reducing Granulosa Cell Apoptosis

TL;DR

Anthocyanins improve ovarian function in beef cows by balancing gut bacteria and protecting ovarian cells from damage.

Contribution

This study demonstrates that anthocyanins can alleviate ovarian dysfunction in beef cows through gut microbiota modulation and reduced cell apoptosis.

Findings

High-dose anthocyanins increased large follicle numbers and reduced persistent corpus luteum in cows.

Anthocyanins altered gut microbiota by increasing beneficial bacteria and decreasing harmful ones.

Anthocyanins reduced oxidative stress and cell apoptosis in ovarian granulosa cells.

Abstract

Ovarian disorders like persistent corpus luteum and ovarian quiescence can reduce the reproductive ability of female beef cattle, negatively impacting the cattle industry. This study explored whether anthocyanins could help improve ovarian function. We fed anthocyanins to cows with ovarian problems and found that higher doses increased the number of growing follicles, lowered harmful hormone levels, and changed the types of bacteria in the gut. Anthocyanins also protected ovarian cells from damage caused by oxidative stress. These results suggest that anthocyanins could be a natural and safe way to support cattle reproduction by balancing gut bacteria and protecting ovarian cells. This research offers a promising approach to enhancing cattle breeding and supports the use of natural antioxidants in livestock management.

Persistent corpus luteum (PCL) and ovarian quiescence (OQ) are key manifestations of ovarian dysfunction (OD) that lead to reduced reproductive capacity in beef cattle, posing a serious challenge to the industry. Anthocyanins (ACNs) are known for their antioxidant properties. This study aimed to investigate the regulatory effects of ACNs on PCL and OQ and to explore the underlying mechanisms. Forty-eight beef cows diagnosed with both OQ and PCL were selected and continuously fed ACNs for 60 days. The results showed that the regulatory effects of ACNs were dose-dependent. A high dose of ACNs (ACNH) significantly increased the number of large follicles and reduced the occurrence of PCL. ACNH treatment significantly decreased serum progesterone (P4) levels and increased estradiol (E2) levels. Furthermore, ACNH reduced microbial diversity in OD cows but significantly increased the abundance of Patescibacteria, Actinobacteriota, Proteobacteria, and Chloroflexi, while decreasing the abundance of Desulfobactera, indicating that ACNs may affect ovarian function by regulating the gut microbial environment. In an ovarian granulosa cell model of oxidative damage, ACN intervention could reduce oxidative stress levels and mitigate oxidative damage. ACNs downregulated various pro-apoptotic genes, such as P53, Fas, and Bax, while upregulating anti-apoptotic genes Bcl-2 and Bcl-xL, suggesting that ACNs significantly inhibit cell apoptosis. To conclude, these results demonstrate that ACNs improve the ovarian function of beef cows by regulating gut microbiota and reducing oxidative stress-induced apoptosis in ovarian granulosa cells, thereby enhancing the reproductive capacity of beef cattle that show reproductive disorders. These findings provide a theoretical basis for the application of ACNs in the cattle industry and showcase their potential value as natural antioxidants.

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1. Introduction

The reproductive capacity of beef cattle is a critical determinant of the economic sustainability and efficiency of the beef industry [1,2]. Clinically, reproductive disorders in cattle are primarily characterized by ovarian dysfunction (OD), which manifests as ovarian quiescence (OQ) and persistent corpus luteum (PCL) [3]. PCL is defined by the prolonged presence of a functional corpus luteum in the ovary, leading to continuous progesterone secretion, suppression of follicular development, and disruption of the estrous cycle, ultimately causing infertility [4]. OQ is marked by a reduction in the number of antral follicles, decreased ovarian volume, and lower estradiol (E2) levels, resulting in ovulation disorders and reduced fertility [5,6]. These conditions severely impact the reproductive efficiency and economic viability of beef cattle operations. Specifically, anestrus resulting from OD is one of the most prevalent and costly reproductive disorders in modern dairy and beef production systems. It directly leads to extended calving intervals, an increased number of services per conception, and higher involuntary culling rates, which collectively impose substantial financial losses on farms [7,8,9]. Effectively managing cows with impaired estrus expression is therefore a critical focus of targeted reproductive management strategies aimed at ensuring the economic and productive sustainability of modern cattle operations [10,11].

Recent studies have highlighted the role of reactive oxygen species (ROS) in ovarian physiological activities, such as the ovarian cycle and luteal maintenance. Excessive ROS production can induce oxidative stress, leading to ovarian dysfunction [12]. Additionally, the gut microbiota has emerged as a key factor influencing reproductive health by modulating the host’s endocrine and immune systems [13]. The decrease in probiotics and the increase in inflammatory bacteria in the gut microbiota of patients with ovarian cancer and premature ovarian failure also indicate that gut microbiota imbalance is an important factor contributing to OD [14]. Further evidence from germ-free mouse models demonstrates that microbial metabolites, particularly short-chain fatty acids (SCFAs), are essential for preventing follicular atresia and maintaining ovarian function [15]. Supporting this concept in livestock, a study in swine revealed that SCFA-producing bacteria (e.g., Bifidobacterium and Lactobacillus) enhance follicular maturation and litter size by activating the SCFA–leptin–PI3K/AKT signaling axis [16]. The use of dietary additives like resveratrol, rapamycin, and naringenin, which modulate gut microbiota, has shown promise in regulating ovarian function [17,18,19,20]. These findings suggest that interventions targeting oxidative stress and gut microbiota could potentially improve reproductive outcomes in beef cattle.

Anthocyanins (ACNs), as powerful antioxidants, are found in various fruits, such as blueberries [21], plums, grape seeds, and grape skins, and can be used to treat various diseases like cancer and heart disease by scavenging free radicals [22]. Notably, specific ACNs are also present in certain forage plants, such as napier grass [23]. However, their concentration in common natural pastures is typically very low (accounting for less than 0.01% of dry matter in grasses like ryegrass) and highly variable, making it impractical to achieve a consistent and therapeutically effective dose through forage intake alone [24]. In addition, ACNs also have anti-aging effects, and they can resist apoptosis of porcine ovarian granulosa cells by reducing oxidative stress damage [25,26]. ACNs can also ameliorate the disorder of the gut microbiota caused by obesity, regulating the content of Firmicutes/Bacteroide (Fir/Bac) and short-chain fatty acids (SCFAs) [27]. Given these properties, ACNs may offer a promising approach to improving ovarian dysfunction in beef cattle by regulating oxidative stress and gut microbiota.

The present study investigated the impact of ACNs on ovarian function in beef cows with OD, focusing on changes in serum reproductive hormone levels, gut microbiota composition, and cellular oxidative stress. By examining these factors, we aim to uncover the mechanisms through which ACNs exert their beneficial effects and enhance reproductive capacity in beef cattle. Our research adds to the expanding knowledge base on the role of natural antioxidants and microbiota modulation in improving livestock health and productivity.

2. Materials and Methods

2.1. Animal Ethics and Experimental Design

2.1.1. Institutional Animal Care and Use Approval

All experimental procedures were approved by the Ningxia Academy of Agricultural and Forestry Sciences Institutional Animal Care and Use Committee (IACUC Number: NXNKYKJLL-2024-5).

2.1.2. Experimental Design

In a large-scale beef cattle farm surrounding Yinchuan, the ovarian status of beef cows was monitored via transrectal ultrasonography to identify individuals with ovarian dysfunction. Cows were diagnosed with OQ if no corpus luteum (CL) or dominant follicle (>10 mm in diameter) was detected on the ovaries, which was coupled with a baseline serum progesterone (P4) concentration of <1 ng/mL. PCL was diagnosed when a static, fully developed CL (diameter > 20 mm) with homogeneous echogenicity persisted for more than 18 days in the absence of a pregnancy, accompanied by sustained elevated serum P4 (>2 ng/mL) (according to [28]).

Based on these criteria, a total of 48 multiparous beef cows (parity 2–4) that met both diagnostic criteria were selected and randomly divided into a control group (CON), a low-concentration ACN group (ACNL), and a high-concentration ACN group (ACNH), with 16 cows in each group.

The control group was fed a basic diet (Supplementary Table S1). For the treatment groups, ACN powder was first uniformly mixed into a small portion of ground corn meal as an inert carrier, and then this premix was thoroughly blended into the basic diet. Consequently, the low- and high-concentration ACN groups received the diet supplemented with 200 and 400 mg of ACNs per kg of diet (mg/kg diet), respectively, for a continuous period of 60 days. The CON group received an equal amount of the corn meal carrier without ACNs to serve as the vehicle control. This dosage range was selected based on prior animal studies, where supplementation at 200 and 400 mg/kg diet improved antioxidant capacity and gut health without adverse effects [29], and aligns with the dose–response relationship observed for ACN efficacy in other models [30]. The supplemented doses are substantially higher than the trace amounts naturally found in pasture [24] or typical concentrations in grapes [31]. ACNs were provided by Nanjing Xinhou Biotechnology Co., Ltd., (Nanjing, China) with a purity of ≥95%. After the start of the experiment and the administration of ACNs, blood was collected from the tail vein of the cows in the three groups every 6 days. Serum was collected and stored at −80 °C for subsequent measurement of serum reproductive hormone indicators. At the end of the experiment, fecal samples were collected from the three groups of cows via rectal sampling.

2.2. Isolation and Culture of Ovarian Granulosa Cells

Ovarian granulosa cells were isolated from the ovaries of healthy, multiparous cows (2 to 4 parities) without OD, sourced from slaughterhouses in Ningxia. Healthy antral follicles with an estimated diameter of 10 ± 3 mm were selected for cell isolation based on visual inspection and caliper measurement. Under a dissecting microscope, follicles were punctured with a 1 mL syringe needle to release the granulosa cells into PBS, followed by mixing and filtration through a 200-mesh cell strainer. The cells were then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the granulosa cells were obtained. The cells were cultured in complete growth medium (DMEM supplemented with 10% FBS and 1% penicillin–streptomycin (exogenous steroidogenic precursors free), all from Gibco, (New York, NY, USA)) at 37 °C with 5% CO_2_. When the primary cells reached approximately 60% confluence, the collected cells were divided into four groups, each with six replicates: blank control group (Blank), hydrogen peroxide group (oxidative damage, H_2_O_2_), ACN control group (ACN), and H_2_O_2_-ACN group. The Blank group received no treatment. Cells in the H_2_O_2_ and H_2_O_2_ + ACN groups were first exposed to 100 μM H_2_O_2_ for 1 h to induce oxidative damage, a concentration consistent with established in vitro stress models [32]. Following medium replacement, the ACN and H_2_O_2_ + ACN groups were then treated with 150 μM ACNs, a dose within the bioactive range for polyphenolic compounds [33]. All cells were subsequently incubated at 37 °C with 5% CO_2_ for 24 h prior to transcriptome sequencing.

ACNs were initially dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution, which was filter-sterilized (0.22 μm), aliquoted, and stored at −80 °C in the dark. For cell treatments, this stock solution was diluted in complete DMEM medium to the final working concentration of 150 μmol/L. The final concentration of DMSO in all culture media, including control groups, was maintained at 0.15% (v/v) or less, a level verified in preliminary tests to have no detectable effect on cell viability.

2.3. Ovarian Morphology Observation

Ovarian follicle and corpus luteum development in beef cows were performed using an ultrasound system (Honda Electronics Co., Ltd., Toyohashi, Japan) equipped with a 7.5 MHz linear array transducer (HLV-875M) (HONDA ELECTRONICS Co., Ltd., Toyohashi, Aichi, Japan). Follicles were classified based on diameter and ultrasonographic appearance: primary follicles (2–5 mm) appeared as small, round anechoic structures with thin walls; growing follicles (5–10 mm) appeared as medium-sized anechoic structures with clearly defined walls; and large follicles (>10 mm) appeared as distinctly larger anechoic structures with thick, well-defined walls, representing mature preovulatory follicles. The corpus luteum appeared as a heterogeneous, semi-echogenic structure with distinct borders, and its diameter (mm) was measured at the maximum cross-sectional dimension.

2.4. Serum Reproductive Hormone Level Detection

Serum concentrations of estradiol (E2) and progesterone (P4) were measured using commercial ELISA kits (Cloud-Clone Corp., Wuhan, China) with the following performance characteristics: P4 assay range 1–16 ng/mL, sensitivity <0.1 ng/mL, and intra-/inter-assay CV < 10%/15%; E2 assay range 1–32 pg/mL, sensitivity <0.1 pg/mL, and intra-/inter-assay CV < 10%/15%. All samples were analyzed in duplicate.

2.5. Intestinal Microbiota Detection and Bioinformatics Analysis

An E.Z.N.A.^®^ Soil DNA Kit DNA extraction kit (Omega Bio-tek, Inc. (Norcross, GA, USA) was used to extract total microbial DNA from fecal samples. The purity and concentration of DNA were then assessed using 1% agarose gel electrophoresis. Subsequently, the V3~V4 region of the 16S rRNA was amplified from individual metagenome DNA samples using PCR. After amplification, the PCR products were quantified using the QuantiFluor™-ST blue fluorescence quantification system (Promega Corporation, Madison, WI, USA). Based on the required sequencing depth for each sample, they were pooled in appropriate ratios. A TruSeq™ DNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA) was used to complete Novaseq library construction, and after the library was qualified, the amplified products were sequenced on the NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA). The PE reads obtained from Novaseq sequencing were first assembled based on the overlap relationship, and the sequence quality was controlled and filtered to distinguish samples, followed by OTU clustering analysis and species taxonomic analysis.

2.6. RNA-Sequencing

Total RNA was extracted from the cells (as described in Section 2.2) using the Trizol method, and mRNA libraries were prepared following the Shanghai Meji Biological Company’s kit instructions. These were sequenced on the Illumina NovaSeq 6000 platform with a read length of 2 × 150 bp. Clean data, with an average base error rate of less than 0.1%, were utilized for transcriptome analysis. Gene read counts were normalized to transcripts per million (TPM) using RSEM to generate normalized gene expression levels. DESeq2 package (version 1.42.0) was employed to identify differentially expressed genes (DEGs).

2.7. RT-qPCR Validation

Five differentially expressed genes (P53, Fas, Bcl-xl, Bcl-2, Bax) were selected for validation based on their established roles in apoptotic pathways, and the RT-qPCR method was employed to verify the accuracy of the transcriptome sequencing results. GAPDH was used as the internal reference gene. The primers of these genes were synthesized by Biotechnology Engineering (Shanghai) Co., Ltd., Shanghai, China and are listed in Table 1. SYBR qPCR Master Mix was used for validation (SYBR qPCR Master Mix kit, MedChemExpress, Monmouth Junction, NJ, USA). The 2^−ΔΔCT^ method was used to calculate the relative expression of the experimental groups in comparison with the control group.

2.8. Reactive Oxygen Species (ROS) Level Detection

After 24 h of treatment, cells from the four groups were stained with the ROS indicator SOX, and nuclei were counterstained with DAPI. Fluorescence intensity was analyzed using ImageJ (version 1.53, NIH, Bethesda, MD, USA) to quantify the extent of oxidative damage.

2.9. Statistical Methods

Data are presented as mean ± standard deviation. Statistical significance was determined using SPSS software (version 26.0). For in vivo comparisons among the three dietary groups, a one-way analysis of variance (ANOVA) was used. For in vitro data from the factorial experiment (factors: H_2_O_2_ and ACN), a two-way ANOVA was applied to assess main effects and interactions. A p-value of <0.05 was considered statistically significant.

2.10. Integrated Microbiome–Transcriptome Analysis

To explore interactions between gut microbiota and host gene expression, we performed correlation analyses at both the pathway and individual gene levels. For microbial functional potential, PICRUSt2 was used to predict KEGG pathways from 16S rRNA data of differential microbes between the ACNH and control groups. For host transcriptional response, KEGG pathway enrichment analysis was performed on differential genes between the H_2_O_2_-ACN and H_2_O_2_ groups. Spearman correlation coefficients were calculated between microbial pathway abundances and host pathway enrichment scores, visualized as a heatmap. Additionally, pairwise correlations between differential microbial taxa and differential genes were analyzed, with significant associations (|r| > 0.5, p < 0.05) displayed in heatmaps. All analyses were performed in R (version 4.2.1).

3. Results

3.1. ACNs’ Regulatory Effect on Beef Cows with Ovarian Dysfunction

We evaluated the impact of dietary ACN supplementation on ovarian function in beef cows with PCL and ovarian quiescence. Cows were fed a basal diet supplemented with a carrier (control, CON) or with ACNs at 200 mg/kg (ACNL) or 400 mg/kg (ACNH) for 60 days (Figure 1A). Serum hormone analysis showed distinct temporal profiles (Figure 1B,C). Progesterone (P4) levels decreased significantly earlier in the ACNH group (day 36) than in the ACNL group (day 42) (p < 0.05), with no change in CON. Concurrently, estradiol (E2) levels increased progressively in the ACNH group, reaching significance by day 60 (p < 0.05), while remaining unchanged in CON. The effects of ACNL and ACNH treatment on follicular development and corpus luteum morphology were evaluated over a 60-day time course (Figure 1D–G, Supplementary Figure S1). Both treatments promoted follicular development across all follicle classes, with ACNH showing earlier and more pronounced effects than ACNL. Large follicles (>10 mm) emerged earlier in the treated groups, appearing at Day 24 in ACNH and Day 36 in ACNL, while remaining nearly absent in the controls until the end of the study (Figure 1D). Growing follicles (5–10 mm) and primary follicles (2–5 mm) increased progressively in all groups, with ACNH consistently exhibiting the highest counts throughout the experimental period, followed by ACNL and then the controls (Figure 1E,F). Conversely, corpus luteum diameter remained stable in the controls but declined markedly in both treatment groups after Day 30, with ACNH showing the most pronounced reduction (Figure 1G). The coordinated reduction in P4 levels and luteal structural regression confirms functional luteolysis in ACNH-treated cows. These results demonstrate that high-dose ACNs (400 mg/kg diet) effectively restore ovarian activity in beef cows with OD, primarily by enhancing follicular growth and correcting the aberrant progesterone–estrogen balance associated with PCL.

3.2. ACNs’ Regulatory Effect on the Rectal Microbiota of Beef Cows with Ovarian Dysfunction

Microbiota plays a regulatory role in the function of beef cattle. To investigate the regulatory effect of ACNs on the rectal microbiota of beef cows with OD, we conducted 16S rRNA sequencing. A total of 2010 OTUs were obtained from the three groups, with the ACNH group having 1691, with 40 unique OTUs, the ACNL group having 1833, with 39 unique OTUs, and the CON group having 1827, with 95 unique OTUs, and 1505 shared OTUs among the three groups, indicating that ACNH can reduce microbial richness (Figure 2A). The PCoA plot showed that ACNH was completely separated from CON, and ACNL was scattered between ACNH and CON, indicating that ACN can change the rectal microbiota composition of beef cows with ovarian dysfunction, with ACNH contributing more to the change in microbial composition than ACNL (Figure 2B). Further analysis through PLS-DA also found significant differences in microbial communities among the three groups (Figure 2C), further indicating that ACNs can significantly alter the gut microbiota composition of beef cows. Shannon and Simpson index analyses showed that the Shannon index of ACNH was significantly higher than that of ACNL and CON (Figure 2D), while the Simpson index was lower than that of ACNL and CON, indicating that ACNH reduced the rectal microbiota diversity of beef cows with ovarian dysfunction (Figure 2E). ACE and Chao also showed that ACNH was lower than ACNL and CON (p < 0.05) (Figure 2F,G), further proving that ACNH reduced rectal microbiota richness. On this basis, we further analyzed how the top 15 bacterial communities changed at the phylum level. In the microbial community bar plot, we can see that the dominant bacteria among the three groups are similar, mainly including Firmicutes, Bacteroidota, Patescibacteria, Actinobacteriota, Spirochaetota, Verrucomicrobiota, and Proteobacteria. In these three groups, we can see the common point that the proportion of Firmicutes and Bacteroidota is ranked 1 and 2, and then the proportion of dominant bacteria in ACNH is inconsistent with CON, but the top four dominant bacteria still have some similarity with ACNL (Figure 2H–J). This indicates that the proportion of these dominant bacteria (Patescibacteria, Actinobacteriota, Spirochaetota, Verrucomicrobiota, Proteobacteria) may be regulated by ACNs (Figure 2I). We further examined the differences in bacterial communities at the phylum and genus levels among the three groups through one-way ANOVA. The richness of Patescibacteria, Actinobacteriota, Proteobacteria, and Chloroflexi in ACNH was significantly higher than in CON, and Desulfobactera was lower than in CON (Figure 3A). Compared to ACNL, CON had higher Verrucomicrobiota and Desulfobacterota (Figure 3B). Comparing the two groups, the Student’s t-test bar plot at the phylum level showed that Proteobacteria and Chloroflexi were higher in ACNL compared to ACNH (Figure 3C). At the genus level, the following bacteria in CON were significantly higher than in ACNH and ACNL: norank_f UCG-010, Monoglobus, Romboutsia, norank f Eubacterium c, Paeniclostridium, Bacteroides, NK4A214_group, and norank_f_Ruminococcaceae. In contrast, Lachnospiraceae NK4A136_group and Candidatus Saccharimonas were lower in ACNH and ACNL (Figure 3D).

3.3. Transcriptional Regulatory Effects of ACNs on Oxidatively Damaged Granulosa Cells

Granulosa cells are essential for ovarian function. To further understand the regulatory effects of ACNs on oxidatively damaged granulosa cells, we conducted transcriptome sequencing analysis. The PCA analysis showed that there was a significant distance in gene expression between the four groups, with samples from ACNs and Blank being close, and samples from ACN-H_2_O_2_ and H_2_O_2_ being close, but overall still discrete (Figure 4A), indicating that oxidative damage and ACNs may have a certain effect on oxidative stress. Statistical analysis of differentially expressed genes was performed for ACN-H_2_O_2_ vs. H_2_O_2_ (up 27, down 27), H_2_O_2_-ACN vs. Blank (up 639, down 1211), H_2_O_2_ vs. Blank (up 728, down 1121) (Figure 4B), H_2_O_2_-ACN vs. ACN (395 up and 1098 down), ACN vs. Blank (17 up and 26 down), and ACN vs. H_2_O_2_ (1221 up and 746 down) (Figure 4C), indicating that oxidative stress affects the transcriptional regulation of granulosa cells, and the addition of ACNs may also have a certain transcriptional regulatory effect. To further clarify the life processes enriched by differential genes, we performed GO analysis and found that upregulated genes in H_2_O_2_ vs. Blank were enriched in protein binding, binding, biological regulation, regulation of metabolic process (Figure 4D); downregulated genes were enriched in developmental process, positive regulation of biological process, negative regulation of cellular process, and negative regulation of biological process (Figure 4E). Compared with H_2_O_2_, downregulated genes in H_2_O_2_-ACN were mainly enriched in receptor ligand activity signaling receptor activator activity, alkaline phosphatase activity, receptor regulator activity (Figure 4F), peroxidase activity, and oxidoreductase activity. Upregulated genes were most enriched in cell–cell adhesion, with the lowest Padjust being collagen trimer (Figure 4G). KEGG enrichment analysis found that upregulated genes in H_2_O_2_ vs. Blank were enriched in axon guidance, ECM–receptor interaction, Alzheimer’s disease, and pathways in cancer (Figure 5A). Downregulated genes were enriched in cytokine–cytokine receptor interaction and the MAPK signaling pathway (Figure 5B). Compared with H_2_O_2_, upregulated genes in H_2_O_2_-ACN were mainly enriched in pyrimidine metabolism, purine metabolism, and neuroactive ligand–receptor (Figure 5C); downregulated genes were mainly enriched in glutathione metabolism, the B cell receptor signaling pathway, metabolism of xenobiotics, hepatocellular carcinoma, and the MAPK signaling pathway (Figure 5D). We further analyzed the activity of apoptosis, which has the greatest impact on cells, and found that the related genes were mainly enriched in pathways in cancer, the MAPK signaling pathway, the PI3K-Akt signaling pathway, and the Rap1 signaling pathway (Figure 5E). The above KEGG pathway enrichment results reveal that multiple signaling pathways are involved in regulating cell proliferation and apoptosis, such as MAPK, PI3K-AKT, FoxO, P53, etc. Therefore, our anthocyanin intervention may reduce apoptosis of ovarian granulosa cells caused by oxidative damage. Our further RT-PCR results revealed that anthocyanin intervention can significantly downregulate several apoptotic genes, such as P53, Fas, and Bax, and upregulate anti-apoptotic genes, such as Bcl2 and Bcl-xL (Figure 5F).

3.4. Correlation Analysis Between Gut Microbiota and Ovarian Granulosa Cell Transcriptome

Pathway-level correlation analysis revealed distinct clusters of positively and negatively correlated pathways between microbiome functional potential and host transcriptomic activities (Figure 6A). Notably, the oxidative phosphorylation pathway showed a strong positive correlation, suggesting coordinated energy metabolism between microbiota and host. Microbe–gene correlation analysis identified specific associations between microbial phyla and host genes (Figure 6B). Firmicutes and Bacteroidota exhibited contrasting correlation patterns with multiple genes. Proteobacteria and Actinobacteriota showed strong positive correlations with a cluster of genes including VGLL3 and MAGI1 (p < 0.001). Patescibacteria, despite low abundance, demonstrated the strongest positive correlations with SAMD5, COL11A1 and PDGFD, indicating potential functional importance of rare taxa. These results provide candidate microbe–gene pairs for further mechanistic studies.

3.5. Anthocyanin Intervention Can Reduce Oxidative Damage in Granulosa Cells

To investigate the impact of anthocyanin (ACN) intervention on oxidative stress in granulosa cells, we measured ROS levels using SOX fluorescence staining (Figure 7A). H_2_O_2_ treatment induced a significant increase in ROS levels compared to the control group (3.10 ± 0.10 vs. 1.00 ± 0.00, p < 0.001; Figure 7B). Two-way ANOVA revealed a significant H_2_O_2_ × ACN interaction (p < 0.05). Co-treatment with ACN attenuated H_2_O_2_-induced ROS accumulation by 45.2% (Figure 7C), with statistical analysis confirming significance (2.15 ± 0.15 vs. 3.10 ± 0.10, p < 0.05; Figure 7D). This reduction in ROS levels correlated with decreased granulosa cell apoptosis, suggesting that ACNs’ antioxidant properties contribute to cytoprotection against H_2_O_2_-induced oxidative damage.

4. Discussion

This study explored the role of ACNs in improving the reproductive capacity of beef cows with ovarian dysfunction and their potential mechanisms. The results indicated that ACNs could regulate the intestinal microbiota, adjust hormone levels, and reduce apoptosis of ovarian granulosa cells through their antioxidant effects.

Follicular enlargement depends on low concentrations of progesterone (P4), and the increase in follicle size indicates the production of higher-quality mature oocytes and sufficient estrogen. In contrast, persistent corpus luteum secretes progesterone, which inhibits the estrous cycle, preventing normal estrus and ovulation in female livestock, leading to infertility. Furthermore, in the ACNH group, serum progesterone (P4) levels significantly decreased, while estrogen (E2) levels significantly increased from the 42nd day, suggesting that ACNs may promote follicular development and ovulation by regulating hormonal balance [34]. This hormonal modulation by ACNs finds support in animal studies; for instance, anthocyanin-rich extracts have been shown to exhibit phytoestrogenic activities in ovariectomized rat models, influencing hormone-sensitive pathways [35]. In this study, the decrease in P4 levels, the enlargement of follicles, the decrease in estrogen levels on the 36th day, and the reduction in persistent corpus luteum in the ACNH group all indicate that ACNH can improve ovarian dysfunction in cows [36].

In the results of 16S rRNA sequencing analysis, ACNH increased the Shannon index and decreased the Simpson index. The decrease in ACE and Chao in this study further proves that ACNH reduces the richness of the rectal microbiota. This is similar to the results of E. coli Nissle 1917 intervention in polycystic ovary syndrome [37]. Compared with the ACNL group, ACNH significantly altered the gut microbiota structure of beef cows with ovarian dysfunction, leading to significant differences in microbial communities at both the phylum and genus levels. The richness of Patescibacteria, Actinobacteriota, Proteobacteria, and Chloroflexi was significantly higher than in the CON group, while Desulfobactera was lower than in CON. Patescibacteria, which are enriched in animals adapted to hypoxic high-altitude conditions, are important for the digestion and nutrition of ruminants [38]. Actinobacteriota, Proteobacteria, and Chloroflexi play a positive role in response to anticancer therapy [39], and an increase in Desulfobactera also indicates dysbiosis in cattle, as Desulfobactera may be associated with the production of inflammation and is related to hormone production [40,41]. Adding ACNs can reverse this trend, especially ACNH, indicating that ACNH regulates ovarian dysfunction by increasing the richness of Patescibacteria, Actinobacteriota, Proteobacteria, and Chloroflexi while decreasing the richness of Desulfobactera. At the genus level, Lachnospiraceae NK4A136_group has anti-inflammatory properties in colitis [42], and the abundance of Candidatus Saccharimonas is positively correlated with current CD4^+^ T cells, suggesting that it may play an important role in the immune recovery of immunodeficient patients [43]. This illustrates that ACNH can also regulate ovarian function by enhancing these immune-related microbial communities.

Additionally, RNA-sequencing analysis has revealed that ACNs can regulate the transcription of ovarian granulosa cells. Oxidative damage typically exacerbates cellular injury by inducing the expression of related genes. ACNs can reduce oxidative stress and exert anti-inflammatory and anticancer effects [44] while also enhancing the antioxidant capacity of granulosa cells by regulating the expression of antioxidant-related genes [45]. Based on this study, ACNH intervention can significantly downregulate the expression of pro-apoptotic genes (such as P53, Fas, and Bax). P53 can activate downstream genes, such as Bax, promoting cell death. It can also directly interact with mitochondria, leading to the release of cytochrome C, which activates the caspase cascade and triggers apoptosis [46]. Fas, a member of the tumor necrosis factor (TNF) receptor family, can initiate apoptosis when it binds to its ligand FasL [47] and upregulate the expression of anti-apoptotic genes (such as Bcl-2, Bcl-xL) [48]. Bcl-2 interacts with pro-apoptotic proteins such as Bax and Bak, preventing their activation during the apoptosis process. Bcl-xL also interacts with pro-apoptotic proteins, inhibiting the formation of pores and thereby protecting mitochondria from damage [49]. This further supports the mechanism by which ACNs reduce apoptosis in OS granulosa cells. Moreover, KEGG pathway analysis shows that multiple key signaling pathways (such as the MAPK, PI3K-AKT, P53, and FoxO pathways) are involved in the regulation of granulosa cells by ACNs, revealing that ACNs may protect ovarian cells from damage caused by oxidative stress through multiple pathways [50].

Based on both the apparent increase in follicle size and changes in hormone levels, as well as alterations in the gut microbiota of beef cattle, our results indicate that the alleviation of ovarian dysfunction by ACNs is dose-dependent. We observed that higher doses were more effective than lower ones. This provides dosing guidance for production. But also, considering the cost in relation to the body weight of beef cattle, we conducted further investigations. We found that the extraction of ACNs has been industrialized and that they are widely sourced, making their prices more suitable for use in the beef cattle industry [51,52]. Although precise pricing varies by region and supplier, market analyses in China indicate that anthocyanin extracts for agricultural use are commercially available at competitive prices that support their implementation in cattle production systems. It is worth noting that we also studied the dose–effect of ACNs, and the results indicated that high concentrations of ACNs have a more significant effect on improving ovarian function, suggesting that the bioactivity of ACNs may be dose-dependent. This finding is of great reference value for optimizing the dosage of ACNs in practical applications. Although this study has achieved meaningful results, there are still some limitations. Despite the use of 16S rRNA sequencing and RNA-sequencing technologies, the omics data in this study still need further verification through proteomics and metabolomics to reveal a more comprehensive molecular mechanism of the effects of ACNs [53].

In this study, we performed an integrated analysis to explore the potential interactions between gut microbiota and host gene expression in response to ACN treatment. The pathway-level correlation analysis revealed distinct clusters of positively and negatively correlated pathways, suggesting that microbial functional potential and host transcriptomic activities are coordinately regulated. Notably, the strong positive correlation observed in the oxidative phosphorylation pathway indicates a potential coupling of energy metabolism between the microbiota and host, which may contribute to the metabolic effects of ACN.

The microbe–gene correlation analysis provided more granular insights into specific associations. The contrasting correlation patterns of dominant phyla Firmicutes and Bacteroidota with multiple host genes suggest their differential roles in mediating host responses. Interestingly, Patescibacteria, despite its low relative abundance, exhibited the strongest positive correlations with genes including SAMD5, COL11A1 and PDGFD, highlighting that even rare taxa may exert significant functional impacts. These findings provide candidate microbe–gene pairs for understanding the mechanisms underlying ACNs’ effects.

In summary, our research offers a scientific foundation for utilizing ACNs to bolster the reproductive capabilities of female beef cattle, uncovering the mechanisms behind their antioxidant properties and modulation of gut microbiota. In the future, research should further optimize the dosage and use strategy of ACNs and verify them in environments closer to actual production conditions to enhance the practicality and promotional value of the research results. As natural antioxidants, ACNs have a promising application prospect, but further research is still needed to overcome the existing limitations and ensure their effectiveness and safety in animal husbandry.

5. Conclusions

The study establishes a foundation for using anthocyanins to boost reproductive performance in female beef cattle, showing their effects on persistent corpus luteum and ovarian quiescence via antioxidants and gut microbiota regulation. Future work should refine anthocyanin dosing and strategies, test under practical conditions, and ensure the approach is effective and safe in livestock.