基于代谢组学技术筛选人工授精后未妊娠奶牛血浆潜在生物标志物的研究

doi:10.11843/j.issn.0366-6964.2025.07.019

摘要/Abstract

摘要：

旨在揭示未妊娠荷斯坦奶牛在人工授精当天到第28天的血浆代谢变化规律，并筛选指示奶牛未妊娠结局的生物标志物，为减少奶牛空怀时间、缩短产犊间隔及早期妊娠诊断提供新思路。本研究以宁夏某奶牛场健康荷斯坦奶牛(体重550±50 kg、3~4岁、2~3胎次)为试验对象。经同期发情处理后人工授精(AI)。分别于AI后第0天(A组)、第5天(B组)、第17天(C组)和第28天(D组)采集尾静脉血(晨饲前)，分离血浆并冻存于－80℃。在第32天经B超筛选出未妊娠且未返情的12头奶牛，对其4个时期(n=12)血浆样品进行单样本超高效液相色谱串联四级杆飞行时间质谱(UHPLC-QTOF-MS)非靶向代谢组学技术检测与分析。结果显示：1)人工授精后不同时期，未妊娠奶牛血浆代谢特征存在差异；2)在筛选出的32种差异代谢物中，8种差异代谢物具备较高灵敏度(AUC>0.7)，相比于人工授精当天，DL-香草扁桃酸和光黄素在授精第5天高表达，色氨酸-谷氨酸、异亮氨酸-精氨酸、甘氨脱氧胆酸和2-乙氧基乙醇在授精第17天高表达，而精氨酸-酪氨酸和硬脂酸在授精第28天高表达，且授精后第0天和第17天之间未妊娠奶牛血浆差异代谢物主要富集在精氨酸生物合成通路上。结果表明，精氨酸生物合成提示为影响奶牛妊娠发生的关键通路，DL-香草扁桃酸、光黄素等AUC值较高的8种关键代谢物提示为人工授精后早期指示奶牛未妊娠的生物标志物，结果可为提高奶牛繁殖效率提供新的思路。

关键词: 荷斯坦奶牛, 人工授精, 未妊娠, 代谢组学, 生物标志物

Abstract:

This study aimed to reveal the changes in plasma metabolism of non-pregnant Holstein dairy cows from the day of artificial insemination to day 28 and to screen biomarkers indicating non-pregnant dairy cows, to provide new ideas for reducing the idle time of dairy cows, shortening the calving interval and early pregnancy diagnosis. This study was conducted using healthy Holstein dairy cows (body weight: 550±50 kg, age: 3-4 years, parity: 2-3) from a dairy farm in Ningxia. Following estrus synchronization, artificial insemination (AI) was performed (designated as Day 0). Blood samples were collected from the tail vein (before morning feeding) at 4 time points: Day 0 (Group A), Day 5 (Group B), Day 17 (Group C), and Day 28 (Group D). Plasma was separated and stored at －80℃. On Day 32 post-AI, ultrasound examination confirmed 12 non-pregnant cows without return to estrus. Plasma samples from these cows (n=12) were analyzed using ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS)-based untargeted metabolomics. The results showed as follows: 1) The plasma metabolism characteristics of non-pregnant cows were different at different periods after artificial insemination; 2) Among the 32 differential metabolites screened, 8 had higher sensitivity (AUC> 0.7). Compared with the day of artificial insemination, DL-vanillyl mandelic acid and lumichrome were highly expressed on the 5th day of insemination, and Trp-Glu, Ile-Arg, glycodeoxycholic acid, and 2-ethoxyethanol were highly expressed on the 17th day of insemination. However, Arg-Tyr and pristanic acid were highly expressed on the 28th day of insemination, and the plasma differential metabolites between d 0 and d 17 after insemination were mainly concentrated in the arginine biosynthesis pathway. The results indicate that arginine biosynthesis may be a key pathway affecting the occurrence of pregnancy in dairy cows. The 8 key metabolites with high AUC values such as DL-vanillyl mandelic acid and lumichrome may be biomarkers indicating that dairy cows are not pregnant early after artificial insemination. The results can provide new ideas for improving the reproductive efficiency of dairy cows.

Key words: Holstein dairy cows, artificial insemination, non-pregnant, metabolomics, biomarkers

中图分类号:

S823.3

郑浩, 罗芳, 宋承磊, 陶金忠. 基于代谢组学技术筛选人工授精后未妊娠奶牛血浆潜在生物标志物的研究[J]. 畜牧兽医学报, 2025, 56(7): 3252-3264.

ZHENG Hao, LUO Fang, SONG Chenglei, TAO Jinzhong. Screening Potential Plasma Biomarkers of Non-pregnant Dairy Cows after Artificial Insemination Based on Metabolomics Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3252-3264.

图/表 9

表 1

图 1

图 2

表 2

图 3

表 3

已鉴定差异代谢物"

代谢物 Metabolite	质荷比 m/z	保留时间/s RT	AUC	A vs. B			A vs. C			A vs. D
代谢物 Metabolite	质荷比 m/z	保留时间/s RT	AUC	VIP	P	FC	VIP	P	FC	VIP	P	FC
1. DL-香草扁桃酸 DL-vanillyl mandelic acid	240.078	323.702	0.806	2.074	0.007	2.538	-	-	-	-	-	-
2. 光黄素 Lumichrome	243.086	128.928	0.757	1.651	0.028	2.214	-	-	-	-	-	-
3. 组氨酸-苏氨酸 His-Thr	257.125	334.948	0.167	1.923	0.015	0.328	-	-	-	-	-	-
4. 硬脂酰胺 Stearamide	284.295	32.742	0.201	1.722	0.033	0.357	-	-	-	-	-	-
5. 二十碳五烯酸 Eicosapentaenoic acid	303.232	39.786	0.264	1.665	0.035	0.417
6. 苏氨酸-丝氨酸 Thr-Ser	248.125	332.097	0.292	1.755	0.029	0.376	-	-	-	-	-	-
7. 缬氨酸-苏氨酸 Val-Thr	201.125	198.098	0.174	2.088	0.008	0.458	-	-	-	-	-	-
8. 色氨酸-谷氨酸 Trp-Glu	334.140	326.883	0.813	-	-	-	1.794	0.015	2.225	-	-	-
9. 异亮氨酸-精氨酸 Ile-Arg	288.203	331.332	0.813	-	-	-	1.860	0.009	2.099	-	-	-
10. 甘氨脱氧胆酸 Glycodeoxycholic acid	450.322	200.131	0.771	-	-	-	1.555	0.035	2.060	-	-	-
11.2-乙氧基乙醇 2-Ethoxyethanol	151.097	63.058	1.000	-	-	-	2.591	0.000	2.049	-	-	-
12. 组氨酸-蛋氨酸 His-Met	304.145	218.511	0.028	-	-	-	2.399	0.000	0.265
13. 精氨酸琥珀酸 Argininosuccinic acid	290.130	237.428	0.076	-	-	-	2.370	0.000	0.281
14. 异胱氨酸 Allocystathionine	187.058	276.689	0.215	-	-	-	1.796	0.007	0.469	-	-	-
15. 三乙醇胺 Triethanolamine	150.113	154.899	0.014	-	-	-	2.770	0.000	0.204
16.3-羟基异戊酸 3-Hydroxyisovaleric acid	237.134	223.740	0.097	-	-	-	2.270	0.000	0.407	-	-	-
17.5′-O-甲基胸苷 5′-O-methylthymidine	256.105	296.654	0.215				1.797	0.020	0.468	-	-	-
18. (3-羧丙基)三甲基铵阳离子 (3-Carboxypropyl)trimethylammonium cation	191.086	249.592	0.111	-	-	-	-	-	-	1.861	0.009	0.429
19. 丙氨酸-亮氨酸 Ala-Leu	185.129	406.947	0.007	-	-	-	-	-	-	2.707	0.000	0.467
20. 丙戊酸 Valproic acid	183.078	276.612	0.153	-	-	-	-	-	-	1.986	0.004	0.334
21. 柠檬酸 Citramalic acid	207.052	298.551	0.174	-	-	-	-	-	-	1.910	0.006	0.333
代谢物 Metabolite	质荷比 m/z	保留时间/s RT	AUC	B vs. C			B vs. D			C vs. D
代谢物 Metabolite	质荷比 m/z	保留时间/s RT	AUC	VIP	P	FC	VIP	P	FC	VIP	P	FC
1. 色氨酸-谷氨酸 Trp-Glu	334.140	326.883	0.833	1.779	0.021	2.177	-	-	-	-	-	-
2.2-乙氧基乙醇 2-Ethoxyethanol	151.097	63.058	1.000	2.734	0.000	2.037	-	-	-	-	-	-
3. 前列腺素 I2 Prostaglandin I2	416.249	240.224	0.188	1.784	0.026	0.213
4. 中康酸 Mesaconic acid	191.049	224.975	0.153	1.958	0.012	0.471
5.1-甲基鸟苷 1-methylguanosine	298.103	282.209	0.285	1.570	0.035	0.488	-	-	-	-	-	-
6. 异亮氨酸-丙氨酸-精氨酸 Ile-Ala-Arg	358.245	49.318	0.118	2.222	0.004	0.340
7.3-羟基异戊酸 3-Hydroxyisovaleric acid	237.134	223.740	0.181	2.164	0.006	0.400	-	-	-	-	-	-
8. 己酸 Hexanoic acid	158.118	45.819	0.181	1.700	0.041	0.179	-	-	-	-	-	-
9. 精氨酸-酪氨酸 Arg-Tyr	337.173	460.582	0.813	-	-	-	1.457	0.048	2.075	-	-	-
13. 吡哆醇 Pyridoxine	339.156	198.852	0.188	-	-	-	1.802	0.016	0.449
11. 富马酸二羟基酯 Dihydroxyfumarate	148.076	233.057	0.208	-	-	-	1.977	0.008	0.485
10. 丙氨酸-亮氨酸 Ala-Leu	185.129	406.947	0.000	-	-	-	3.190	0.000	0.492	-	-	-
12. S-甲基-5′-硫腺苷 S-Methyl-5′-thioadenosine	298.098	71.609	0.208	-	-	-	1.620	0.017	0.466	-	-	-
14. 柠檬酸 Citramalic acid	207.052	298.551	0.125	-	-	-	2.545	0.002	0.405	-	-	-
15. 硬脂酸 Pristanic acid	297.280	38.254	0.910	-	-	-	-	-	-	2.037	0.025	2.227

表 3

图 4

图 5

图 6

参考文献 34

1	EALY A D , SEEKFORD Z K . Symposium review: predicting pregnancy loss in dairy cattle[J]. J Dairy Sci, 2019, 102 (12): 11798- 11804.
2	GUI L S , DAI T S , GUO X R , et al. Recent advances in early pregnancy loss diagnosis in dairy cows: New approaches[J]. Reprod Domest Anim, 2024, 59 (4): e14566.
3	LEUNG K Y . Applications of advanced ultrasound technology in obstetrics[J]. Diagnostics (Basel), 2021, 11 (7): 1217.
4	JIANG C , LI G F . Sensitive fluorescence analysis of pregnancy plasma associated protein A in amniotic fluid based on the plasma superstructure enhanced carbon polymer dots[J]. Sci Adv Mater, 2022, 14 (7): 1151- 1158.
5	张馨蕊, 付予, 杨卓, 等. 奶牛早期妊娠诊断蛋白的研究[J]. 畜牧兽医学报, 2024, 55 (2): 451- 460. doi: 10.11843/j.issn.0366-6964.2024.02.004
	ZHANG X R , FU Y , YANG Z , et al. Study on early pregnancy diagnostic protein of dairy cows[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55 (2): 451- 460. doi: 10.11843/j.issn.0366-6964.2024.02.004
6	HAN A , QAMAR A Y , BANG S , et al. Effect of extracellular vesicles derived from oviductal and uterine fluid on the development of porcine preimplantation embryos[J]. Theriogenology, 2025, 234, 216- 224.
7	VASUDEVAN S , KAMAT M M , WALUSIMBI S S , et al. Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers^†[J]. Biol Reprod, 2017, 97 (1): 104- 118.
8	DE OLIVEIRA E B , MONTEIRO H F , PEREIRA J M V , et al. Changes in uterine metabolome associated with metritis development and cure in lactating Holstein cows[J]. Metabolites, 2023, 13 (11): 1156.
9	JIANG H B , BAO J , XING Y N , et al. Metabolomic and metagenomic analyses of the Chinese mitten crab Eriocheir sinensis after challenge with Metschnikowia bicuspidata[J]. Front Microbiol, 2022, 13, 990737.
10	BOYLE J G , DAVIDSON D F , PERRY C G , et al. Comparison of diagnostic accuracy of urinary free metanephrines, vanillyl mandelic Acid, and catecholamines and plasma catecholamines for diagnosis of pheochromocytoma[J]. J Clin Endocrinol Metab, 2007, 92 (12): 4602- 4608.
11	YANG R H , LIU B J , YANG M Y , et al. Lumiflavin reduces cisplatin resistance in cancer stem-like cells of OVCAR-3 cell line by inducing differentiation[J]. Front Oncol, 2022, 12, 859275.
12	KALE H , HARIKUMAR P , KULKARNI S B , et al. Assessment of the genotoxic potential of riboflavin and lumiflavin. A. Effect of light[J]. Mutat Res, 1992, 298 (1): 17- 23.
13	MARTINEZ C A , RIZOS D , RODRIGUEZ-MARTINEZ H , et al. Oocyte-cumulus cells crosstalk: New comparative insights[J]. Theriogenology, 2023, 205, 87- 93.
14	GAO L , ZHANG C , ZHENG Y , et al. Glycine regulates lipid peroxidation promoting porcine oocyte maturation and early embryonic development[J]. J Anim Sci, 2023, 101, skac425.
15	WU G , BAZER F W , SATTERFIELD M C , et al. Impacts of arginine nutrition on embryonic and fetal development in mammals[J]. Amino Acids, 2013, 45 (2): 241- 256.
16	LI X , BAZER F W , GAO H , et al. Amino acids and gaseous signaling[J]. Amino Acids, 2009, 37 (1): 65- 78.
17	黄祝. 小鼠围着床期子宫中精氨酸琥珀酸合成酶1的表达调节和功能[D]. 厦门: 厦门大学, 2012.
	HUANG Z. Expression regulation and function of argininosuccinate synthetase 1 in mouse uterus during peri implantation[D]. Xiamen: Xiamen University, 2012. (in Chinese)
18	BALERCIA G , MORETTI S , VIGNINI A , et al. Role of nitric oxide concentrations on human sperm motility[J]. J Androl, 2004, 25 (2): 245- 249.
19	DUTTA S , SENGUPTA P . The role of nitric oxide on male and female reproduction[J]. Malays J Med Sci, 2022, 29 (2): 18- 30.
20	LEFÈVRE PAVINE L C , MARIE-FRANCE P , MURPHY B D . Polyamines on the reproductive landscape[J]. Endocr Rev, 2011 (5): 694- 712.
21	LI Z , YUE Z , AO Z , et al. Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows[J]. Anim Reprod Sci, 2018, 196, 211- 218.
22	PETRUJKI B T , EFER D S , JOVANOVI I B , et al. Effects of commercial selenium products on glutathione peroxidase activity and semen quality in stud boars[J]. Anim Feed Sci Technol, 2014, 197, 194- 205.
23	LIU N , SUN S , WANG P , et al. The Mechanism of Secretion and Metabolism of Gut-Derived 5-Hydroxytryptamine[J]. Int J Mol Sci, 2021, 22 (15): 7931.
24	WANG G , YAO S , LIANG X , et al. Detection of the metabolites of human plasma and follicular fluid in IVF-ET with microextraction and LC-TOF-MS[J]. Technol Health Care, 2015, 1 (s1): 29- 36.
25	DOE J E . Ethylene glycol monoethyl ether (2-ethoxyethanol)[J]. Environ Health Perspect, 1984, 57, 33- 41.
26	PEREZ M J , VELASCO E , MONTE M J , et al. Maternal ethanol consumption during pregnancy enhances bile acid-induced oxidative stress and apoptosis in fetal rat liver[J]. Toxicology, 2006, 225 (2-3): 183- 194.
27	LEROY J L M R , VANHOLDER T , MATEUSEN B , et al. Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro[J]. Reproduction, 2005, 130 (4): 485- 495.
28	MIRABI P , CHAICHI M J , ESMAEILZADEH S , et al. The role of fatty acids on ICSI outcomes: a prospective cohort study[J]. Lipids Health Dis, 2017, 16 (1): 18.
29	LIU S , SHARP A , VILLANUEVA E , et al. Breast Milk Iodine Concentration (BMIC) as a biomarker of iodine status in lactating women and children < 2 years of age: a systematic review[J]. Nutrients, 2022, 14 (9): 1691.
30	PATEL A , PATEL S , PATEL P , et al. Salivary exosomal miRNA-1307-5p predicts disease aggressiveness and poor prognosis in oral squamous cell carcinoma patients[J]. Int J Mol Sci, 2022, 23 (18): 10639.
31	王梦晓, 罗珂珂, 高文雅, 等. 基于非靶向尿液代谢组学技术的毛蕊花糖苷治疗嘌呤霉素氨基核苷肾病幼龄大鼠相关生物标志物研究[J]. 中国中药杂志, 2023, 48 (21): 5898- 5907.
	WANG M X , LUO K K , GAO W Y , et al. Study on biomarkers of verbascoside in the treatment of puromycin aminonucleoside nephropathy in young rats based on non targeted urine metabolomics technology[J]. China Journal of Chinese Materia Medica, 2023, 48 (21): 5898- 5907.
32	闫坤, 高黎明, 高正兴, 等. 唾液小细胞外囊泡novel-100作为母猪早期妊娠诊断候选生物标志物的研究[J]. 中国畜牧杂志, 2025, 61 (1): 299-304, 310.
	YAN K , GAO L M , GAO Z X , et al. Study on salivary small extracellular vesicle novel-100 as a candidate biomarker for early pregnancy diagnosis in sows[J]. Chinese Journal of Animal Science, 2025, 61 (1): 299-304, 310.
33	VELEK K, MICHAUD S, BOUCHER K, 等. 爱德士牛怀孕检测[C]//中国奶业协会第26次繁殖学术年会暨国家肉牛牦牛/奶牛产业技术体系第3届全国牛病防治学术研讨会论文集. 兰州, 2011: 140-143.
	VELEK K, MICHAUD S, BOUCHER K, et al. IDEXX cattle pregnancy test[C]//Proceedings of the26th Annual Conference of Breeding of China Dairy Association and the 3rd National Symposium on Cattle Disease Prevention and Control of National Beef Cattle, Yak/Dairy Cattle Industry Technology System. Lanzhou, 2011: 140-143. (in Chinese)
34	KUMAR R , ALI S A , SINGH S K , et al. Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties[J]. Sci Rep, 2021, 11 (1): 12427.

模式 Model	组别 Group	PCA	OPLS-DA		OPLS-DA置换检验 OPLS-DA permutation test
模式 Model	组别 Group	R²X	R²Y	Q²	Q² intercept
正离子模式 POS	A vs. B	0.502	0.997	0.563	－0.186
	A vs. C	0.510	0.980	0.683	－0.348
	A vs. D	0.474	0.974	0.631	－0.315
	B vs. C	0.449	0.988	0.584	－0.279
	B vs. D	0.432	0.990	0.635	－0.244
	C vs. D	0.491	1	0.428	－0.082 1
负离子模式 NEG	A vs. B	0.567	0.999	0.554	－0.234
	A vs. C	0.502	0.986	0.485	－0.283
	A vs. D	0.475	0.998	0.624	－0.271
	B vs. C	0.439	1	0.223	－0.131
	B vs. D	0.497	0.996	0.374	－0.263
	C vs. D	0.494	0.888	0.215	－0.267

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