鞣花酸及其代谢物对山羊卵巢颗粒细胞促增殖、抗氧化、抑凋亡的效果对比研究

doi:10.11843/j.issn.0366-6964.2025.12.024

Abstract

Abstract: This study aimed to compare the effects of ellagic acid (EA) and its metabolites urolithin A (UA), urolithin B (UB), and urolithin C (UC) on promoting proliferation, enhancing antioxidant activity, inhibiting apoptosis in Guizhou black goat ovarian granulosa cells (OGCs), in order to identify a superior alternative to EA.Ovarian tissues from healthy, sexually mature goats were collected to isolate and culture OGCs, which were validated for subsequent experiments. At the same time, based on previous research findings of the research group, 1 μmol·L^-1concentration of EA had the optimal biological effect on OGCs. Therefore,cells were divided into 5 groups: control (CON), EA, UA, UB, and UC, with treatment groups supplemented with 1 μmol·L^-1 of the corresponding compounds. Proliferation was assessed via CCK-8 assay at 0, 24, 36, and 48 h. Cell migration was evaluated using a scratch assay. Intracellular ROS levels were measured using ROS detection kits. RT-qPCR was employed to analyze mRNA expression of anti-apoptotic (Bcl-2), pro-apoptotic (Bax, Caspase-3, Caspase-9), inflammatory factors (IL-1β, IL-6, TNF-α), and reproduction-related genes (GDF9, BMPR-1B, CYP19A1, FSHβ). UA exhibited the strongest pro-proliferative effect on OGCs, followed by EA and UB, with UC showing the weakest activity. The scratch assay revealed that UA significantly enhanced cell migration, whereas UB inhibited migration; no significant difference was observed between EA and UA. ROS levels were markedly reduced in UA and EA groups. qRT-PCR results demonstrated that UA, UB, UC, and EA significantly downregulated pro-apoptotic genes (CASP3, CASP9, BAX) compared to the CON group. UA upregulated BCL-2 expression most prominently. Among inflammatory factors, UA, UC, and EA significantly suppressed IL-1β expression (with UA being the lowest), there was no significant differences between CON group and other groups, while TNF-α levels decreased across all groups. UA and EA significantly enhanced the expression of reproduction-related genes (GDF9, BMPR-1B, CYP19A1, FSHβ), with UA showing the highest efficacy. In this study, compared to EA and other metabolites, UA demonstrated superior effects in promoting OGCs proliferation and migration, reducing oxidative stress and apoptosis, and modulating reproductive-inflammatory gene expression. These findings highlight UA as a promising candidate for further research as a reproductive regulatory additive in mammals.

Key words: ellagic acid, ovarian granulosa cells, metabolites, comparative study of effects

CLC Number:

S827.3

CHEN Yi, LIU Bin, ZHANG Xilu, LU Qingmei, PAN Zhiren, SHI Xiaoli, LUO Zhijun, ZHAO Jiafu. A Comparative Study on the Effects of Ellagic Acid and Its Metabolites on Proliferation Promotion, Antioxidation, Apoptosis Inhibition of Goat Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6219-6231.

References

[1] 周明帅. COL1A1调控卵巢颗粒细胞生物学功能影响贵州黑山羊产羔性状的研究[D]. 贵阳：贵州大学, 2023.
ZHOU M S,COL1A1 regulates the biological function ofovarian granulosa cells and affects the kiddingtraits of guizhou black goats Abstract[D]. Guiyang:Guizhou University, 2023.(in Chinese)
[2] ZHANG F L, KONG L, ZHAO A H, et al. Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary[J]. Environ Res, 2021,198:111225.
[3] YANG H, XIE Y, YANG D, et al. Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma[J]. Oncotarget, 2017,8(15):25310-25322.
[4] XUE H, HU Z, LIU S, et al. The mechanism of NF-kappaB-TERT feedback regulation of granulosa cell apoptosis in PCOS rats[J]. PLoS One, 2024,19(10):e312115.
[5] LANDETE J M. Ellagitannins, ellagic acid and their derived metabolites: a review about source, metabolism, functions and health[J]. Food Res Int, 2011,44:1150-1160.
[6] SHARIFI-RAD J, QUISPE C, CASTILLO C, et al. Ellagic acid: a review on its natural sources, chemical stability, and therapeutic potential[J]. Oxid Med Cell Longev, 2022,2022:e848084.
[7] WEN X, ZHOU M, LU Q, et al. Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of guizhou black goat[J]. Anim Prod Sci, 2024,64(1):1-11.
[8] CECI C, GRAZIANI G, FARAONI I, et al. Strategies to improve ellagic acid bioavailability: from natural or semisynthetic derivatives to nanotechnological approaches based on innovative carriers[J]. Nanotechnology, 2020,31(38):382001.
[9] GARCÍA-VILLALBA R, ESPÍN J C, TOMÁS-BARBERÁN F A. Chromatographic and spectroscopic characterization of urolithins for their determination in biological samples after the intake of foods containing ellagitannins and ellagic acid[J]. J Chromatogr A, 2016,1428:162-175.
[10] LUDWIG I A, MENA P, CALANI L, et al. New insights into the bioavailability of red raspberry anthocyanins and ellagitannins[J]. Free Radic Biol Med, 2015,89:758-769.
[11] XIAN W, YANG S, DENG Y, et al. Distribution of urolithins metabotypes in healthy chinese youth: difference in gut microbiota and predicted metabolic pathways[J]. J Agric Food Chem, 2021,69(44):13055-13065.
[12] 石莹, 杜凤, 王玥, 等. 尿石素A的药理作用及机制研究进展[J]. 中南药学, 2022,20(1):113-120.
SHI Y, DU F, WANG Y, et al. Research progress in pharmacological effect and mechanism of urolithin A[J]. Central South Pharmacy, 2022,20(1):113-120.(in Chinese)
[13] ANDREUX P A, BLANCO-BOSE W, RYU D, et al. The mitophagy activator urolithin a is safe and induces a molecular signature of improved mitochondrial and cellular health in humans[J]. Nat Metab, 2019,1(6):595-603.
[14] ROGOVSKII V S, MATYUSHIN A I, SHIMANOVSKII N L. Influence of urolithin a on cytokine production by various cancer cell lines[J]. Pharm Chem J, 2023,57:481-485.
[15] GARCÍA-VILLALBA R, GIMÉNEZ-BASTIDA J A, CORTÉS-MARTÍN A, et al. Urolithins: a comprehensive update on their metabolism, bioactivity, and associated gut microbiota[J]. Mol Nutr Food Res, 2022,66(21):e2101019.
[16] MA M, WANG Y, FAN S, et al. Urolithin a alleviates colitis in mice by improving gut microbiota dysbiosis, modulating microbial tryptophan metabolism, and triggering AhR activation[J]. J Agric Food Chem, 2023,71(20):7710-7722.
[17] CHEN P, GUO Z, CHEN F, et al. Recent advances and perspectives on the health benefits of urolithin b, a bioactive natural product derived from ellagitannins[J]. Front Pharmacol, 2022,13:917266.
[18] KANG I, KIM Y, TOMÁS-BARBERÁN F A, et al. Urolithin a, c, and d, but not iso-urolithin a and urolithin b, attenuate triglyceride accumulation in human cultures of adipocytes and hepatocytes[J]. Mol Nutr Food Res, 2016,60(5):1129-1138.
[19] SAVI M, BOCCHI L, MENA P, et al. In vivo administration of urolithin a and b prevents the occurrence of cardiac dysfunction in streptozotocin-induced diabetic rats[J]. Cardiovasc Diabetol, 2017,16(1):80.
[20] HU J, MESNAGE R, TUOHY K, et al. (Poly)phenol-related gut metabotypes and human health: an update[J]. Food Funct, 2024,15(6):2814-2835.
[21] ZHANG M, CUI S, MAO B, et al. Ellagic acid and intestinal microflora metabolite urolithin a: a review on its sources, metabolic distribution, health benefits, and biotransformation[J]. Crit Rev Food Sci Nutr, 2023,63(24):6900-6922.
[22] GARCÍA-VILLALBA R, GIMÉNEZ-BASTIDA J A, CORTÉS-MARTÍN A, et al. Urolithins: a comprehensive update on their metabolism, bioactivity, and associated gut microbiota[J]. Mol Nutr Food Res, 2022,66(21):e2101019.
[23] 汪鸽, 韩延华. 早发性卵巢功能不全的颗粒细胞凋亡机制及中医药干预研究进展[J]. 中医药信息, 2022,39(2):83-88.
WANG G，HAN Y H. Research progress in mechanism of granulosa cell apoptosis in POI and TCM intervention[J].Information on Traditional Chinese Medicine, 2022, 39(2): 83-88.(in Chinese)
[24] 徐保阳, 秦文峡, 晏向华. 母猪卵泡发育质量的营养调控研究进展[J]. 动物营养学报, 2022,34(10):6334-6342.
XU B Y,QIN W X,YAN X H. Advances in nutritional regulation of follicular development quality of sows[J].Chinese Journal of Animal Nutrition,2022,34(10): 6334-6342.(in Chinese)
[25] HE Y, DENG H, JIANG Z, et al. Effects of melatonin on follicular atresia and granulosa cell apoptosis in the porcine[J]. Mol Reprod Dev, 2016,83(8):692-700.
[26] GONZÁLEZ-SARRÍAS A, NÚÑEZ-SÁNCHEZ M Á, GARCÍA-VILLALBA R, et al. Antiproliferative activity of the ellagic acid-derived gut microbiota isourolithin A and comparison with its urolithin a isomer: the role of cell metabolism[J]. Eur J Nutr, 2017,56(2):831-841.
[27] CHEN H, BAI M, ZHANG T, et al. Ellagic acid induces cell cycle arrest and apoptosis through TGF-β/Smad3 signaling pathway in human breast cancer MCF-7 cells[J]. Int J Oncol, 2015,46(4):1730-1738.
[28] MALIK A, AFAQ S, SHAHID M, et al. Influence of ellagic acid on prostate cancer cell proliferation: a caspase-dependent pathway[J]. Asian Pac J Trop Med, 2011,4(7):550-555.
[29] 张颖, 陈茜, 石华, 等. 尿石素A对肾癌786-O细胞的作用机制研究[J]. 湖南师范大学学报(医学版), 2024,21(1):20-25.
ZHANG Y,CHEN Q,SHI H,et al.Biological effects of urolithin a on renal carcinoma 786-O cells[J].Journal of Hunan Normal University(Medical Sciences),2024,21(1):20-25.(in Chinese)
[30] 陈婷婷, 吴宿慧, 李根林, 等. 鞣花酸类成分的肠道菌代谢过程及其药理作用变化研究进展[J]. 中国现代应用药学, 2024,41(21):2982-2989.
CHEN T T,WU S H, LI G L,et al.Research progress on cut microbiota metabolism process and pharmacological effects of ellagic acid components[J].Chinese Journal of Modern Applied Pharmacy 2024,41(21):2982-2989.(in Chinese)
[31] ZHU H, YAN Y, JIANG Y, et al. Ellagic acid and its anti-aging effects on central nervous system[J]. Int J Mol Sci, 2022,23(18):10937.
[32] UMESALMA S, SUDHANDIRAN G. Differential inhibitory effects of the polyphenol ellagic acid on inflammatory mediators NF-kappaB, iNOS, COX-2, TNF-alpha, and IL-6 in 1,2-dimethylhydrazine-induced rat colon carcinogenesis[J]. Basic Clin Physiol Pharmacol, 2010,107(2):650-655.
[33] 张建伟. 板栗壳斗鞣花单宁及其代谢产物鞣花酸和尿石素的生物活性研究[D].北京：北京林业大学, 2015.
ZHANG J W.Studies on biological activities of euclidean tannins from burso of castanea mollissima. and their metabolic products ellagic acid and urolithin[D].Beijing:Beijing Forestry University,2015.(in Chinese)
[34] 温晓艳. 鞣花酸对玉米赤霉烯酮诱导贵州黑山羊卵巢颗粒细胞损伤的改善作用[D]. 贵阳：贵州大学, 2024.
WEN X Y.The improvement effect of ellagic acid on zearalenone-induced ovarian granulosa cell damage in guizhou black goats [D]. Guiyang:Guizhou University, 2024.(in Chinese)
[35] ZHEN X, WU B, WANG J, et al. Increased incidence of mitochondrial cytochrome c oxidase 1 gene mutations in patients with primary ovarian insufficiency[J]. PLoS One, 2015,10(7):e132610.
[36] DEVINE P J, PERREAULT S D, LUDERER U. Roles of reactive oxygen species and antioxidants in ovarian toxicity[J]. Biol Reprod, 2012,86(2):27.
[37] ESPÍN J C, LARROSA M, GARCÍA-CONESA M T, et al. Biological significance of urolithins, the gut microbial ellagic acid-derived metabolites: the evidence so far[J]. Evid Based Complement Alternat Med, 2013,2013:270418.
[38] AHN D, PUTT D, KRESTY L, et al. The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes[J]. Carcinogenesis, 1996,17(4):821-828.
[39] GALANO A, FRANCISCO M M, PÉREZ-GONZÁLEZ A. Ellagic acid: an unusually versatile protector against oxidative stress[J]. Chem Res Toxicol, 2014,27(5):904-918.
[40] SHAFIE B, POURAHMAD J, REZAEI M. N-acetylcysteine is more effective than ellagic acid in preventing acrolein induced dysfunction in mitochondria isolated from rat liver[J]. J Food Biochem, 2021,45(7):e13775.
[41] XIAO Y, HUANG R, WANG N, et al. Ellagic acid alleviates oxidative stress by mediating Nrf2 signaling pathways and protects against paraquat-induced intestinal injury in piglets[J]. Antioxidants (Basel), 2022,11(2):252.
[42] LARROSA M, GONZÁLEZ-SARRÍAS A, YÁÑEZ-GASCÓN M J, et al. Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism[J]. J Nutr Biochem, 2010,21(8):717-725.
[43] HARPER P. A review of the dietary intake, bioavailability and health benefits of ellagic acid (EA) with a primary focus on its anti-cancer properties[J]. Cureus, 2023,15(8):e43156.
[44] HOU Y, CHU X, PARK J, et al. Urolithin a improves alzheimer's disease cognition and restores mitophagy and lysosomal functions[J]. Alzheimers Dement, 2024,20(6):4212-4233.
[45] CHEN P, WU L, LEI J, et al. The ellagitannin metabolite urolithin c attenuated cognitive impairment by inhibiting neuroinflammation via downregulation of MAPK/NF-kB signaling pathways in aging mice[J]. Int Immunopharmacol, 2024,142(Pt B):113151.
[46] 贾晓燕, 胡朋朋, 王佩欣, 等. 覆盆子单宁富集组分消化稳定性及对肠道菌群的调节作用[J]. 食品科学, 2023,44(9):104-113.
JIA X Y,HU P P,WANG P X,et al. Digestive stability of tannin-enriched fraction of rubus chingii hu fruits and its regulatory effect on the intestinal microflora[J]. Food Science,2023,44(9):104-113.(in Chinese)
[47] HUO Y, LI Q, YANG L, et al. SDNOR, a Novel antioxidative lncRNA, is essential for maintaining the normal state and function of porcine follicular granulosa cells[J]. Antioxidants (Basel), 2023,12(4):799.
[48] ELGEBALY M M, HAZAA A B M, AMER H A, et al. L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9[J]. Reprod Domest Anim, 2022,57(7):734-742.
[49] SHI X, JIN X, LIN J, et al. Idebenone relieves the damage of heat stress on the maturation and developmental competence of porcine oocytes[J]. Reprod Domest Anim, 2022,57(4):418-428.
[50] AZAMI S H, NAZARIAN H, ABDOLLAHIFAR M A, et al. The antioxidant curcumin postpones ovarian aging in young and middle-aged mice[J]. Reprod Fertil Dev, 2020,32(3):292-303.
[51] SRI R, SASANGKA P, JANTJE S, et al. Luteinizing hormone effect on the GDF-9 and BMPR-1a expression of bovine granulosa cells culture[J]. IOP Conf Ser: Mater Sci Eng, 2019,546(6):62021.
[52] CHRISTENSON L K, DEVOTO L. Cholesterol transport and steroidogenesis by the corpus luteum[J]. Reprod Biol Endocrinol, 2003,1:90.
[53] MA X, YI H. BMP15 regulates FSHR through TGF-β receptor II and SMAD4 signaling in prepubertal ovary of Rongchang pigs[J]. Res Vet Sci, 2022,143:66-73.
[54] BANG Y, KWON Y, KIM M, et al. Ursolic acid enhances autophagic clearance and ameliorates motor and non-motor symptoms in parkinson's disease mice model[J]. Acta Pharmacol Sin, 2023,44(4):752-765.
[55] WANG Y, REN F, LI B, et al. Ellagic acid exerts antitumor effects via the PI3K signaling pathway in endometrial cancer[J]. J Cancer, 2019,10(15):3303-3314.
[56] SHEN M, LIU Z, LI B, et al. Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells[J]. Cell Death Dis, 2014,5(10):e1475.
[57] HUNZICKER-DUNN M E, LOPEZ-BILADEAU B, LAW N C, et al. PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells[J]. Proc Natl Acad Sci U S A, 2012,109(44):E2979-E2988.
[58] CHO J, RHO O, JUNCO J, et al. Effect of combined treatment with ursolic acid and resveratrol on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate[J]. Cancer Prev Res (Phila), 2015,8(9):817-825.
[59] CASTREJON-JIMENEZ N S, LEYVA-PAREDES K, BALTIERRA-URIBE S L, et al. Ursolic and oleanolic acids induce mitophagy in A549 human lung cancer cells[J]. Molecules, 2019,24(19):3444.
[60] LI L, ZHANG X, CUI L, et al. Ursolic acid promotes the neuroprotection by activating Nrf2 pathway after cerebral ischemia in mice[J]. Brain Res, 2013,1497:32-39.
[61] GARCIA-VILLALBA R, GIMENEZ-BASTIDA J A, CORTES-MARTIN A, et al. Urolithins: a comprehensive update on their metabolism, bioactivity, and associated gut microbiota[J]. Mol Nutr Food Res, 2022,66(21):e2101019.
[62] SINGH R, CHANDRASHEKHARAPPA S, VEMULA P K, et al. Microbial metabolite urolithin b inhibits recombinant human monoamine oxidase a enzyme[J]. Metabolites, 2020,10(6):258.

A Comparative Study on the Effects of Ellagic Acid and Its Metabolites on Proliferation Promotion, Antioxidation, Apoptosis Inhibition of Goat Ovarian Granulosa Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Ying, ZHANG Jiaojiao, WANG Xianzhong, QUAN Fusheng. Advances in Autophagy of Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1508-1517.
[2]	LI Xiaowei, TIAN Wei, LIU Yuan, LI Huixia. Study on the Difference of m⁶A Methylation Modification in Ovarian Granulosa Cells of Hu Sheep under Heat Stress [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1712-1721.
[3]	HE Yu, WANG Xiangyu, DI Ran, CHU Mingxing, LIANG Chen. BMP4/SMAD4 Downregulates GJA1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 679-688.
[4]	YI Yanyan, SUN Panpan, SUN Na, LI Hongquan. Proteomic Profiling Unveils the Protective Mechanism of Chlorogenic Acid against Zearalenone-induced Ovarian Granulosa Cell Death [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6477-6486.
[5]	ZHAO Xiaoyi, ZHU Longlong, LIU Hui, ZHANG Dongyan, CAI Long, WANG Yalei, WANG Jing, ZHAO Junxing, CHEN Meixia. Effects of L-Malic Acid on the Proliferation and Transcriptional Profile of Porcine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5563-5574.
[6]	SHI Xinqi, MA Mengmeng, GAO Tengyun, LIU Shenhe. Research Progress on the Regulation of Semen Quality by Intestinal Microorganisms in Animals [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 26-35.
[7]	LIU Yangguang, ZHANG Huibin, WEN Haoyu, XIE Fan, ZHAO Shiming, DING Yueyun, ZHENG Xianrui, YIN Zongjun, ZHANG Xiaodong. SNP/Indel Screening Analysis of Porcine Ovarian Granulosa Cells Treated with Follicular Fluid Exosomes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 576-586.
[8]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[9]	WANG Ruiling, WANG Xueyan, WANG Feifei, KONG Weiyi, MAO Yongxia, LIU Xin, DING Hui, XU Lihua, GUO Yansheng. Study on the Changes of Blood Oxidized Lipid Group in Postpartum Dairy Cows with Acute Endometritis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 373-387.
[10]	HU Yamei, SONG Xiangrong, HUANG Liang, ZHANG Lutong, GAO Lei, PANG Weijun, YANG Gongshe, CHU Guiyan. FGF21 Enhances Mitochondrial Function and Inhibits Apoptosis of Porcine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1034-1045.
[11]	GONG Haoyang, WU Jiaxin, YANG Xiaoyu, XIE Weichun, WANG Xueying, LI Jiaxuan, JIANG Yanping, CUI Wen, LI Yijing, TANG Lijie. Research Progress in Antiviral Mechanism of Intestinal Microflora [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4910-4919.
[12]	YUAN Tong, HUANG Liang, YANG Lin, WANG Wence, ZHU Yongwen. Regulation of Mitochondrial Function by Gut Microbiota and Their Metabolites in Animal [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 48-57.
[13]	ZHANG Deming, HUANG Jiahe, LI Jinshu, ZHENG Hongmei, WANG Shaoying, YANG Gongshe, SHI Xin’e. Research Progress of Gut Microbiota, Metabolites and Gut Barrier in Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1334-1344.
[14]	WANG Lei, HE Lina, TANG Xue, LI Bijun, HUANG Siyi, WANG Yukun, XU Dejun, ZHAO Zhongquan. Effects of miR-495-3p on Ovarian Granulosa Cell Functions in Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 436-446.
[15]	LI Ziqian, CHEN Zhuo, REN Qianglin, MA Yicheng, YE Qianwen, XIA Ruiyang, WANG Jinquan, ZHAO Hongqiong, MA Zhongxiang, LI Hongbo, YAO Gang. The Relationship between the Malnutritional Metabolism and Gut Microbiota Changes in Pregnant Angus Beef Cows with PICA [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 188-199.