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

Abstract

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

CLC Number:

S823.3

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.

Figures/Tables 9

Table 1

Fig. 1

Fig. 2

Table 2

Fig. 3

Table 3

The identified differential metabolites"

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

Table 3

Fig. 4

Fig. 5

Fig. 6

References 34

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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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Screening Potential Plasma Biomarkers of Non-pregnant Dairy Cows after Artificial Insemination Based on Metabolomics Technology

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进样时间/min Time	流速/(mL· min^-1) Flow velocity	流动相B比率/% Flow phase B ratio
0.0	0.5	95
0.5	0.5	95
7.0	0.5	65
8.0	0.5	40
9.0	0.5	40
9.1	0.5	95
12.0	0.5	95