Analysis of fatty acid composition and miRNA profile in adipose tissue after application of Allium mongolicum Regel extract in diet of sheep

Figures & data

Table 1. Information for constituents in basal diet and nutritional value.

Ingredients	Content (dry matter basis) %
Alfalfa	27.78
Whole corn silage	15.52
Gourd seed skin	12.15
Corn	19.25
Wheat bran	4.29
DDGS^a	4.33
Flax seed meal	4.62
Sunflower seed meal	6.09
Red dates	1.68
Limestone	1.48
Calcium hydrogen phosphate	0.69
Sodium chloride	0.72
Premix^b	1.40
Total	100.00
Nutrient levels (dry matter basis)^c
Digestible energy (MJ/kg)	16.83
Crude protein (g/kg)	153.22
Ether extract (g/kg)	30.91
Neutral detergent fibre (g/kg)	509.80
Acid detergent fibre (g/kg)	297.00
Calcium (g/kg)	10.17
Phosphorus (g/kg)	5.53

^aDistillers Dried Grains with Soluble.

^bThe premix provided the following per kg of diets: Iron (as FeSO₄) 25 mg, Zinc (as ZnSO₄) 29 mg, Copper (as CuSO₄) 8 mg, Manganese (as MnSO₄) 30 mg, Iodine (as KI) 0.04 mg, Cobalt (as CoSO₄) 0.1 mg, Vitamin A 3200 IU, Vitamin D 1200 IU, Vitamin E 20 IU.

^cDigestible energy was a calculated value, while other indicators were actual measured values.

Table 2. Fatty acid profile in subcutaneous adipose tissue with inclusion of WEA (%).

Fatty acids	Diet		SEM	p
	sCON	sWEA
C10:0	1.38	1.02	0.38	.452
C14:0	2.62	2.15	0.36	.014
C15:0	0.91	0.38	0.32	.398
C16:0	24.25	20.33	0.98	.027
C17:0	2.05	0.86	0.56	.435
C18:0	10.33	8.73	0.73	.869
C21:0	0.19	0.22	0.09	.431
C16:1	2.54	3.30	0.43	.674
C17:1	1.12	1.62	0.33	.328
C18:1n9t	6.95	6.80	1.05	.383
C18:1n9c	37.92	43.77	2.14	.457
C18:2n6c	5.00	5.31	0.74	.081
C18:3n3	1.57	2.05	0.23	.047
C18:3n6	0.02	0.04	0.00	.184
C20:1	0.98	1.01	0.43	.547
C20:2	0.76	0.83	0.25	.048
C20:3n3	0.01	0.02	0.00	.351
C20:4n6	0.06	0.07	0.01	.098
C20:5n3	0.02	0.03	0.00	.136
C22:1	0.54	0.66	0.27	.835
C22:2n6	0.28	0.49	0.09	.034
C22:6n3	0.03	0.04	0.00	.243
SFA	42.23	33.79	0.95	.049
MUFA	50.02	57.43	1.32	.033
PUFA	7.75	8.88	1.47	.046

WEA is the abbreviation of water extract of Allium mongolicum Regel; sCON: samples from subcutaneous adipose tissue of sheep receiving diet without WEA supplementation; sWEA: samples from subcutaneous adipose tissue of sheep with WEA supplementation in diet; SEM: standard error of the mean.

SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid.

Table 3. Fatty acid profile in visceral adipose tissue with inclusion of WEA (%).

Fatty acids	Diet		SEM	p
	pCON	pWEA
C10:0	2.73	1.12	0.35	.984
C14:0	2.01	1.45	0.22	.321
C15:0	2.52	1.58	0.29	.017
C16:0	19.65	19.28	0.20	.955
C17:0	0.42	0.75	0.16	.303
C18:0	29.54	27.72	0.73	.258
C20:0	0.24	0.18	0.02	.323
C21:0	0.28	0.30	0.04	.451
C23:0	0.31	0.32	0.03	.977
C16:1	0.40	1.09	0.13	.024
C18:1n9t	3.02	3.20	0.74	.172
C18:1n9c	30.16	34.26	1.05	.035
C18:2n6c	6.28	6.06	0.30	.143
C18:3n3	1.93	2.14	0.23	.844
C18:3n6	0.01	0.03	0.00	.186
C20:3n3	0.05	0.03	0.00	.196
C20:4n6	0.06	0.07	0.03	.988
C22:6n3	0.04	0.06	0.00	.374
SFA	57.70	52.70	1..03	.045
MUFA	33.58	38.55	0.85	.031
PUFA	8.73	8.75	0.49	.086

WEA is the abbreviation of water extract of Allium mongolicum Regel; pCON: samples from visceral adipose tissue of sheep receiving diet without WEA supplementation; pWEA: samples from visceral adipose tissue of sheep with WEA supplementation in diet; SEM: standard error of the mean.

Table 4. Top 10 expressed DE-miRNAs between sCON group and sWEA group.

MiRNA ID	Regulated	Log2 fold change	p Value	Conserved
miR-214	down	−1.14	.044066	conserved
miR-197	down	−2.16	.002085	conserved
novel_20_33380	up	1.90	.008023	–
novel_20_34738	down	−3.16	.001355	–
miR-218a	down	−1.31	.004932	conserved
miR-574-3p	down	−1.04	.049532	conserved
miR-6123	down	−1.42	.016324	conserved
novel_14_26842	down	−1.23	.013474	–
miR-338-3p	up	1.12	.032724	conserved
miR-877-3p	down	−2.09	.017377	conserved

DE-miRNAs: differentially expressed miRNAs; WEA is the abbreviation of water extract of Allium mongolicum Regel; sCON: samples from subcutaneous adipose tissue of sheep receiving diet without WEA supplementation; sWEA: samples from subcutaneous adipose tissue of sheep with WEA supplementation in diet; novel_: novel miRNA candidates; fold change: sWEA group/sCON group; a p Value < .05 is considered significantly different.

Table 5. Top 10 expressed DE-miRNAs between pCON group and pWEA group.

MiRNA ID	Regulated	Log2 fold change	p Value	Conserved
miR-150	down	−0.82	.011248	conserved
miR-133	up	1.07	.000111	conserved
miR-340-5p	up	1.56	.037451	conserved
miR-758-3p	down	−1.03	.02597	conserved
miR-200b	up	2.01	.002469	conserved
let-7e-5p	up	1.87	.007941	conserved
miR-6123	down	−1.75	.04597	conserved
miR-200a	up	2.40	.00041	conserved
miR-3956-5p	down	−1.08	.020836	conserved
miR-377-5p	down	−1.08	.019452	conserved

DE-miRNAs: differentially expressed miRNAs; WEA is the abbreviation of water extract of Allium mongolicum Regel; pCON: samples from visceral adipose tissue of sheep receiving diet without WEA supplementation; pWEA: samples from visceral adipose tissue of sheep with WEA supplementation in diet; fold change: pWEA group/pCON group; a p Value <.05 is considered significantly different.

Table 6. Top 10 expressed DE-miRNAs between sCON group and pCON group.

MiRNA ID	Regulated	Log2 fold change	p Value	Conserved
miR-23a	down	−1.16	9.50E-06	conserved
let-7b	down	−1.32	.002817	conserved
miR-22-3p	down	−1.08	.000220	conserved
miR-29a	down	−1.17	3.64E-09	conserved
miR-221	down	−2.31	3.22E-12	conserved
miR-376c-3p	up	1.08	.012608	conserved
miR-369-3p	up	1.13	.018888	conserved
miR-495-3p	up	1.15	9.95E-06	conserved
miR-3959-5p	up	1.12	.030175	conserved
miR-218a	up	1.75	4.04E-07	conserved

DE-miRNAs: differentially expressed miRNAs; WEA is the abbreviation of water extract of Allium mongolicum Regel; sCON: samples from subcutaneous adipose tissue of sheep receiving diet without WEA supplementation; pCON: samples from visceral adipose tissue of sheep receiving diet without WEA supplementation; fold change: pCON group/sCON group; p Value < .05 is considered significantly different.

Figure 1. Cluster heatmap of differentially expressed mRNAs (DE-mRNAs) regarding lipid metabolism between different groups. (a) Lipid-related DE-mRNAs in subcutaneous adipose tissue between control (sCON) group and water extract of Allium mongolicum Regel (sWEA) group. (b) Lipid-related DE-mRNAs in visceral adipose tissue between control (pCON) group and water extract of Allium mongolicum Regel (pWEA) group. (c) Lipid-related DE-mRNAs between subcutaneous (sCON) and visceral (pCON) adipose tissue without WEA supplementation.

Figure 2. QRT-PCR results of randomly selected differentially expressed miRNAs (DE-miRNAs) and differentially expressed mRNAs (DE-mRNAs) in contrast to results of sequencing. (a) QRT-PCR and sequencing results in subcutaneous adipose tissue between control (CON) group and water extract of Allium mongolicum Regel (WEA) group. (b) QRT-PCR and sequencing results in visceral adipose tissue between CON group and WEA group, * indicates a significant difference with a p value less than 0.05.

Figure 3. The top 50 enriched GO terms for intersection genes from target genes of differentially expressed miRNAs (DE-miRNAs) and actual differentially expressed mRNAs (DE-mRNAs). (a) GO terms for intersection genes between sCON group and sWEA group. (b) GO terms for intersection genes between pCON group and pWEA group. (c) GO terms for intersection genes between sCON group and pCON group.

Figure 4. The top 20 enriched KEGG pathways for intersection genes from target genes of DE-miRNAs and actual DE-mRNAs. (a) Enriched KEGG pathways for intersection genes between sCON group and sWEA group. (b) Enriched KEGG pathways for intersection genes between pCON group and pWEA group. (c) Enriched KEGG pathways for intersection genes between sCON group and pCON group.

Figure 5. MiRNA-mRNA network with regard to lipid metabolism. (a) Differential miRNA-mRNA network diagram between sCON group and sWEA group. (b) Differential miRNA-mRNA network diagram between pCON group and pWEA group. (c) Differential miRNA-mRNA network diagram between sCON group and pCON group. Pink represents up-regulation, green represents down-regulation.

Data availability statement

Raw data for sequencing can be accessed by NCBI BioProject database with identifer PRJNA894801.