Effects of high-grain diet feeding on fatty acid profiles in milk, blood, muscle, and adipose tissue, and transcriptional expression of lipid-related genes in muscle and adipose tissue of dairy cows

doi:10.21203/rs.3.rs-2104340/v1

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Research Article

Effects of high-grain diet feeding on fatty acid profiles in milk, blood, muscle, and adipose tissue, and transcriptional expression of lipid-related genes in muscle and adipose tissue of dairy cows

https://doi.org/10.21203/rs.3.rs-2104340/v1

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Journal Publication

published 08 Apr, 2023

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Background

High-grain (HG) diets affect lipid metabolism in the liver and mammary tissue of dairy cows, but its effects on muscle and adipose tissue have not been wide evaluated. Thus, the aim of this study is to clarify this issue.

Methods

Twelve Holstein cows were randomly divided into two groups: conventional diet group (CON, n = 6) and the HG diet (n = 6). On day7 of week4, rumen fluid was sampled to measure pH, milk was sampled to measure components, and blood was sampled to measure biochemical parameters and fatty acid composition. After the experiment, cows were slaughtered to collect muscle and adipose tissue for fatty acid composition and transcriptome analysis.

Results

HG feeding decreased the ruminal pH, milk’s fat content and long-chain fatty acid proportion (P < 0.05) and increased the proportion of short- and medium-chain fatty acids in the milk (P < 0.05) as compared with CON diets. The concentrations of blood cholesterol, low-density lipoprotein, and polyunsaturated fatty acids in the HG cows were lower than those in CON cows (P < 0.05). In muscle tissue, HG feeding tended to increase the triacylglycerol (TG) concentration (P < 0.10). Transcriptome analysis revealed changes in the biosynthesis of the unsaturated fatty acids pathway, the regulation of lipolysis in the adipocytes pathway, and the PPAR signalling pathway. In adipose tissue, HG feeding increased the concentration of TG and monounsaturated fatty acids (P < 0.05). At the transcriptome level, the fatty acid biosynthesis pathway, linoleic acid metabolism pathway, and PPAR signalling pathway were activated.

Conclusion

HG feeding leads to subacute rumen acidosis (SARA) and a decreased milk fat content. The fatty acid profiles in the milk and plasma of dairy cows were changed by HG feeding. In muscle and adipose tissue, HG feeding increased TG concentration and up-regulated the expression of genes related to adipogenesis, while down-regulated the expression of genes related to lipid transport. These results complement our knowledge of the fatty acid composition of muscle and adipose tissue in dairy cows and expand our understanding of the mechanisms by which HG diets affect lipid metabolism in muscle and adipose tissue.

High-grain diets

muscle

adipose tissue

fatty acid composition

lipid transcriptional profiles

Diets rich in grain, often used for maintaining elevated energy levels in lactating cows, may be responsible for the occurrence of SARA, as pointed out by several authors^{[1, 2]}. SARA represents a disorder of the rumen and features intermittent and moderate drops in rumen pH ^[3]. High-grain (HG) diets containing excess non-structural carbohydrates and insufficient crude fibre allow the production of ruminal volatile fatty acids faster than their buffer, thereby reducing rumen pH and contributing to SARA development^{[2, 4]}. SARA is a widespread problem in mid-lactation cows and causes serious economic losses. Field studies in Wisconsin revealed that the prevalence of SARA in mid-lactating cows was 26% ^[5], and the economic cost associated with SARA per cow is about $400 annually^[6]. Grain-based SARA challenges are often accompanied by a number of typical complications, including reduced feed intake, milk fat depression, laminitis, and liver abscesses ^{[7, 8]}.

Milk fat is the principal nutritional component of milk. Fatty acids in milk originate from the de novo synthesis of mammary tissue and uptake from blood^[9]. The types and amounts of fatty acids taken from blood by the mammary tissue are affected by many factors, such as diet, the rumen fermentation pattern, and the lipid metabolism status of other tissues.^[8,10−12]. Blood serves as a lipid carrier between the mammary tissue and other tissues, thus assisting in milk fat synthesis. Triacylglycerol (TG) and cholesterol are the core components of blood lipids, which are transported by lipoproteins^[13]. Numerous studies have demonstrated that HG diets change the lipid profiles of milk and blood^[14–16], possibly by affecting the production of the milk fat precursor acetic acid, the expression of mammary gland fat synthesis genes, and the redistribution of liver fat^{[17, 18]}. Although muscle and adipose tissue play an indispensable role in the lipid metabolism of lactating ruminants, there is a lack of studies on the effects of grain-based SARA on the fatty acid profiles of muscle and adipose tissue.

Muscle is a major contributor to the body’s ability to adapt to physiological changes and maintain metabolic homeostasis during lactation^[19]. In early lactation cows, the lipid metabolism of muscle is enhanced to relieve the pressure on liver lipid metabolism^[20]. Adipose tissue acts as a fatty acid logistics warehouse in animals, capable of storing fatty acids in the form of triglycerides during periods of energy excess or mobilising adipocytes to release fatty acids during periods of energy deficit^[21]. One way in which the fatty acids released from adipose tissue are utilised is to synthesise milk fat. In humans, maternal adipose tissue contributes to the formation of 70% of the long-chain fatty acids in milk^[22]. However, unlike liver and mammary tissue, the effects of HG feeding on the lipid metabolism of muscle and adipose tissues of mid-lactation dairy cows have not been extensively studied.

Therefore, the objective of this study was to determine the effects of HG feeding on the fatty acid profiles of milk and blood and on the lipid metabolism in the muscle and adipose tissue of mid-lactation dairy cows. We hypothesised that HG feeding alters the fatty acid profiles of milk, blood, muscle, and adipose tissue and affects the transcriptional expression of lipid-related genes in the muscle and adipose tissue of dairy cows.

2.1 Animals and experimental treatments

Twelve healthy multiparous mid-lactation Holstein cows (BW = 613 ± 58 kg, milk yield = 18.2 ± 2.7 kg/d, DIM = 233 ± 23 (mean ± SD)) with rumen fistulas were chosen. The cows were randomly divided into two groups of six: the conventional diet group (CON) and the HG diet group. The experiment lasted for four weeks, including one week for pre-feeding and three weeks for the trial period. During the pre-feeding period, both groups were fed a conventional diet (forage-to-concentrate 6:4, dry matter (DM) basis, Table 1). However, in the feeding trial, the cows in the CON group continued to receive a conventional diet, while the HG cows were switched to a HG diet (forage-to-concentrate 4:6, dry matter (DM) basis, Table 1). The dietary concentrate level of the HG group was increased gradually during the first two days (by approximately 10% each day, based on the CON diet). The diets had comparable CP and were formulated according to NRC (2001) to meet the energy and milk production requirements of a 620-kg Holstein cow yielding 25 kg/d of milk with 4.0% milk fat and 3.5% milk protein. All cows were fed twice a day at 08:00 and 20:00 (5–10% orts on an as-fed basis were allowed) and milked twice before feeding. All cows were healthy and had free access to fresh water throughout the experiment.

Table 1

Ingredients and nutritional composition of the conventional diet (CON) and the high-grain diet (HG)
Item	Diet
Item	CON	HG
Ingredient (% of DM)
Alfalfa hay	24.00		17.00
Oaten hay	24.00		17.00
Corn silage	12.00		6.00
Corn grain	19.40		24.92
Soybean	13.50		13.48
Barley	—		12.00
DDGS¹	3.80		5.91
CaCO₃	0.80		1.48
Ca (HCO₃)₂	1.10		0.92
NaCl	0.40		0.37
Premix²	1.00		0.92
Nutrient composition
DM (%)	46.77		48.03
CP (% of DM)	16.16		16.12
Crude Fat (% of DM)	3.05		3.05
NDF (% of DM)	36.14		29.92
NFC³ (% of DM)	35.39		42.34
Starch (% of DM)	17.96		27.82
Ash (% of DM)	5.97		4.87
Ca (% of DM)	1.14		1.18
P (% of DM)	0.52		0.51
NE_L⁴ (Mcal/kg of DM)	1.57		1.64
NFC/NDF	0.97		1.42
¹Dried distillers grains with solubles.
²Premix contained the following ingredients per kilogram of diet: vitamin A, 22.5 kIU; vitamin D3, 5.0 kIU; vitamin E, 37.5 IU; vitamin K3, 5.0 mg; Mn, 63.5 mg; Zn, 111.9 mg; Cu, 25.6 mg; and Fe, 159.3 mg.
³Calculated as NFC = 100 − (% NDF + % CP + % ether extract + % ash).
⁴Calculated based on Ministry of P. R. China recommendations (MOA, 2004).

2.2 Ruminal and diet sample collection and analysis

Rumen contents were collected through rumen fistulas at 0, 2, 4, 6, 8, 10, and 12 h after the morning feeding on day 7 of week 4. The samples were immediately filtered using four layers of sterile cheesecloth, and the rumen pH was measured using a portable pH metre (HI 99161; Hanna, Woonsocket, RI).

The feed and orts offered were measured and recorded daily to calculate feed intake. In the meantime, fresh feed and orts were sampled daily and stored at − 20°C until required for feed DM analysis by oven drying at 55°C for 72 h. The analytical DM content was determined by drying at 135°C for 3 h^[23].

2.3 Blood and milk sampling and analysis

At 4 h after the morning feeding on day 7 of week 4, blood samples were collected from the tail vein into Vacutainer tubes using heparin sodium as an anticoagulant. Plasma was separated by centrifuging the blood samples (10 min, 3,000 × g at 4°C) and immediately storing them at − 20°C until required for further analysis. Plasma biochemical parameters, such as glucose (Glu), total protein (TP), total cholesterol (TCHO), TG, total bile acid (TBA), high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were determined using an automatic biochemical analyser (Beckman AU5800). Another portion of the plasma was stored at − 20°C to be used for fatty acid composition analysis.

Milk production was recorded during the experiment. On day 7 of week 4, milk samples were collected during 2 consecutive milkings. The samples were mixed and divided into two parts: one was used to analyse milk composition by mid-infrared spectroscopy (Fossomatic 4000, Foss Electric A/S), and the other was stored at − 20°C until needed for fatty acid composition analysis.

2.4 Slaughter and tissue sample collection

At the end of the experimental period, the cows were humanely euthanised using a high-tension current stunning method. After slaughter, samples of the longissimus dorsi muscle and abdominal adipose tissue were collected from each cow. The samples were stored in liquid nitrogen for TG content, fatty acid composition, transcriptome, and qRT-PCR analysis.

2.5 Triacylglycerol concentration detection

The TG content in muscle and adipose tissue was measured using commercial kits (Jian-Cheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions.

2.6 Fatty acid composition analysis

The extraction of milk fat and the preparation of fatty acid methyl esters (FAMEs) were performed according to the procedure published by Nafikov et al.^[24]. The extraction of plasma fat and the preparation of FAMEs were performed as described by Xu et al.^[25]. The FAMEs were placed into gas chromatography vials and used for chromatographic analysis.

An Agilent 8890A gas chromatograph (Agilent Analytical Instruments, CA, USA) equipped with an auto-sampler, auto-injector, split/splitless injector, and flame ionisation detector was used to analyse the FAMEs using nitrogen as the carrier gas. One microlitre of FAMEs in hexane was injected into a fused silica capillary column (Agilent J&W Advanced Capillary GC Columns, Netherlands; 100 m × 0.25 mm i.d., with 0.2 µm film thickness) with the initial column temperature set at 150°C and held at this temperature for 5 min. Then, the column temperature was increased to 200°C at a rate of 2°C/min and held at this temperature for 10 mins, with a subsequent increase to 220°C at a rate of 5°C/min, where it was held for 35 mins^[25]. The injector and detector temperatures were set constant at 260°C and 280°C, respectively. The peak area was measured using OpenLab CDS (Agilent Analytical Instruments), and the peaks were identified by comparing the retention times with FAME standards (18919-1AMP; Sigma Chemicals, St. Louis, MO, USA). Fatty acid composition analysis of muscle and adipose tissue was performed under the same operating conditions for the plasma.

2.7 Transcriptome sequencing and data analysis

The total RNA of the muscle of 12 dairy cows was extracted using a TRIzol reagent kit (Takara Bio, Otsu, Japan) following the manufacturer’s protocol. The concentration and integrity of the RNA were determined using a NanoDrop spectrophotometer (Thermo, Waltham, MA, USA) and 1.4% agarose formaldehyde gel electrophoresis, respectively. A total of 1 µg of RNA was used to construct a sequencing library using a NEBNext®Ultra™RNA Library Prep Kit (Illumina, CA, USA). The prepared library was sequenced using the Illumina HiSeq platform (HiSeqTM 2500). Trimmomatic was utilised to filter out the low-quality reads^[26]. The high-quality filtered sequences were mapped to the bovine Bowtie2 (V2.1.0) reference genome using Hisat2^[27], and the coverage of the sequencing results was evaluated in Qualimap (V2.2.1) ^[28]. The gene expression level was calculated by subread software using the FPKM value^[29], While the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using SIMCA (SIMCA v. 14.1, SIMCA Inc.). The DESeq R package (v1.18.0, 2012) was used to screen differentially expressed genes (DEGs) with a fold change ≥ 2 and FDR < 0.05^[30]. A KEGG pathway enrichment analysis of the DEGs was conducted using the R package clusterProfiler (v4.2.2). Transcriptome analysis of adipose tissue was performed under the same operating conditions.

2.8 Quantitative real-time PCR analysis

Total RNA was extracted from 200 mg muscle samples from 12 cows using TRIzol (Invitrogen, Carlsbad, CA). The cDNA was synthesised using PrimeScript RT Master Mix Perfect Real Time (Takara Co., Otsu, Japan). The primers were designed in Primer 5.0 (Premier Biosoft International, USA) and are presented in Table S1 in Additional File 1. The cDNA samples were amplified using SYBR Green (Takara Co., Otsu, Japan) on the QuantStudio 7Flex rapid real-time PCR system and a 20 µL amplification system containing 10 µL SYBR Premix Ex TaqTM, 0.8 µL primers, 0.4 µL ROX, 2 µL cDNA, and 6.8µL ddH2O. The protocol consisted of keeping the samples at 95°C for 30 s, 95°C for 5 s, and 60°C for 20 s, with 40 cycles. NADPH was used as the reference gene. The relative expression of genes was calculated using the 2 − ΔΔCt method ((Ct target gene − Ctβ-actin) treatment − (Ct target gene − Ctβ-actin) control)^[31]. The adipose tissue sequencing data were validated using the same method.

2.9 Statistical analysis

The data were analysed using the SPSS software package (SPSS v. 24, SPSS Inc.). The linear mixed-effects models (MIXED) procedure was used to analyse the pH data. Student’s t-test analysis was conducted on the data on the milk yield and components, fatty acid composition, biochemical indicators, and mRNA expression level. We considered that a P < 0.05 indicates a significant difference, and a P < 0.10 indicates a trend.

3.1 Rumen pH

The ruminal pH of the HG group significantly decreased (6.129 vs 5.618, P < 0.001) compared with that of the CON group and was lower than that of the CON group at each sampling time point (Fig. 1). The ruminal pH of the cows fed a HG diet was below 5.8 for more than 3 h within a day.

3.2 Milk Yield and Composition, DMI

As shown in Table 2, the milk fat content was lower (P = 0.019) in the HG group compared with the CON group. However, the milk yield (P = 0.338), DMI (P = 0.436), milk protein content (P = 0.169), milk lactose content (P = 0.821), total solid content (P = 0.292), somatic cell count (P = 0.829), and urea nitrogen content (P = 0.152) were unaffected by the HG diet.

Table 2

Effects of high-grain diet (HG) or conventional diet (CON) on milk yield and composition of dairy cows¹
Item	CON	HG	P-value
Milk yield (kg/d)	14.53 ± 2.99	17.91 ± 1.54	0.338
DMI (kg/d)	22.19 ± 1.17	23.69 ± 1.43	0.436
Milk fat (%)	4.49 ± 0.15	3.92 ± 0.14	0.019
Milk protein (%)	3.86 ± 0.17	4.13 ± 0.08	0.169
Milk lactose (%)	4.96 ± 0.05	4.94 ± 0.08	0.821
Total solid (%)	13.69 ± 0.25	13.48 ± 0.19	0.292
Somatic Cell Count (×10 cells/mL)	189.50 ± 78.87	212.58 ± 70.25	0.829
Urea nitrogen (mg/dL)	17.95 ± 0.34	18.82 ± 0.45	0.152
¹Data expressed as mean ± SEM, n = 6.

3.3 Plasma biochemical parameters

The cows on the HG diet had lower plasma TCHO and LDL concentrations (P = 0.031 and P = 0.015, respectively) than the cows on the CON diet (Figs. 2d and 2f, respectively). Furthermore, the HG diet tended to increase the plasma TBA concentration (P = 0.054, Fig. 2c) and decrease the plasma TG concentration (P = 0.081, Fig. 2e). No significant difference was observed in the concentrations of plasma GLU, TP, and HDL (P = 0.143, P = 0.650, and P = 0.605, respectively) between the two groups (Figs. 2a, 2b, and 2g, respectively).

3.4 Fatty acid composition of milk, plasma, muscle, and adipose tissue

The effect of HG feeding on the fatty acid composition of milk is shown in Table 3. The majority of fatty acids in dairy cow milk were saturated fatty acids (SFAs) (average 65.68%, SEM = 1.33%) followed by unsaturated fatty acids (UFAs) (average 34.32%, SEM = 1.33%), which were primarily composed of monounsaturated fatty acids (MUFAs) (average 26.67%, SEM = 1.42%). The average proportion of polyunsaturated fatty acids (PUFAs) in milk was 7.65% (SEM = 0.53%). The proportions of milk C12:0 (P = 0.013) and C ≤ 16 (P = 0.048) in the HG group were higher than those in the CON group, but the proportions of milk C17:0 (P = 0.036) and C > 16 (P = 0.048) were lower than those in the CON group. In addition, HG feeding tended to increase the proportions of milk C10:0 (P = 0.064), C11:0 (P = 0.064) and C16:1 cis-9 (P = 0.094), while the opposite trend was observed for the proportion of C18:0 (P = 0.091).

Table 3

Effects of high-grain diet (HG) or conventional diet (CON) on milk fatty acid proportions¹
Item	CON	HG	SEM	P-value	Item	CON	HG	SEM	P-value
C4:0	1.13	1.40	0.12	0.263	C18:1 trans9	0.50	0.44	0.07	0.663
C6:0	1.26	1.46	0.10	0.354	C18:1 cis9	19.90	14.00	1.71	0.105
C8:0	0.84	0.95	0.06	0.433	C18:2 trans9,12	0.16	0.23	0.02	0.195
C10:0	3.13	3.67	0.14	0.064	C18:2 cis9,12	5.65	7.06	0.44	0.122
C11:0	0.74	1.04	0.08	0.064	C20:0	0.60	0.49	0.06	0.353
C12:0	4.33	5.59	0.27	0.013	C20 cis11	0.85	0.87	0.05	0.895
C13:0	0.60	0.88	0.09	0.132	C20:3 cis8,11,14	0.35	0.35	0.03	0.983
C14:0	12.92	14.42	0.55	0.186	C20:3 cis11,14,17	0.64	0.85	0.07	0.161
C14:1 cis9	2.22	3.06	0.29	0.150	SFA	64.45	66.91	1.33	0.387
C15:0	3.15	3.94	0.28	0.185	UFA	35.55	33.09	1.33	0.387
C16:0	22.29	24.04	0.83	0.315	MUFA	28.74	24.60	1.42	0.172
C16:1 cis-9	4.02	4.85	0.24	0.094	PUFA	6.81	8.49	0.53	0.123
C17:0	1.79	1.25	0.13	0.036	C ≤ 16	56.62	65.29	2.26	0.048
C17:1 trans10	1.25	1.39	0.10	0.546	C > 16	43.38	34.71	2.26	0.048
C18:0	11.67	7.79	1.14	0.091
¹g/100g of total fatty acid.
SEM = Standard error of the mean between the two treatments; SFA = saturated fatty acid; UFA = unsaturated fatty acid; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.

The effect of HG feeding on the fatty acid composition of plasma is shown in Table 4. UFAs accounted for the largest proportion of plasma fatty acids (average 60.19%, SEM = 1.29%) and consisted of PUFAs (average 42.46%, SEM = 1.54%) and MUFAs (average 17.73%, SEM = 0.51%). The average proportion of plasma SFAs was 39.82% (SEM = 1.29%). The cows on the HG diet had higher proportions of plasma C4:0 (P = 0.029), C6:0 (P = 0.020), and C17:1 trans-10 (P = 0.041) but lower proportions of plasma C16:1 cis-9 (P = 0.032), C18:2 cis-9,12 (P = 0.033) and PUFAs (P = 0.026) than the cows fed a CON diet. HG feeding showed a tendency for higher proportions of plasma SFAs (P = 0.051) and C ≤ 16 (P = 0.058), as well as a trend towards lower proportions of plasma C20:3 cis-8,11,14 (P = 0.093), UFAs (P = 0.051), and C > 16 (P = 0.058).

Table 4

Effects of high-grain diet (HG) or conventional diet (CON) on plasma fatty acid proportions¹
Item	CON	HG	SEM	P-value	Item	CON	HG	SEM	P-value
C4:0	3.16	4.68	0.35	0.029	C18:1 trans9	2.48	1.78	0.24	0.169
C6:0	2.81	3.61	0.17	0.020	C18:2 trans9,12	0.52	0.58	0.03	0.321
C8:0	0.73	1.02	0.09	0.125	C18:2 cis9,12	38.89	33.10	1.40	0.033
C10:0	0.49	0.63	0.05	0.201	C20:0	0.54	0.64	0.05	0.342
C11:0	0.41	0.46	0.04	0.566	C18:3 cis9,12,15	0.89	0.92	0.09	0.894
C12:0	0.64	0.72	0.04	0.293	C20 cis11	1.81	1.61	0.18	0.598
C13:0	0.57	0.59	0.04	0.852	C20:3 cis8,11,14	2.80	2.49	0.09	0.093
C14:0	1.00	1.14	0.34	0.848	C20:3 cis11,14,17	2.66	2.06	0.19	0.120
C14:1 cis9	0.55	0.68	0.07	0.377	SFA	37.34	42.29	1.29	0.051
C15:0	0.66	0.55	0.09	0.573	UFA	62.66	57.71	1.29	0.051
C16:0	11.32	12.62	0.57	0.279	MUFA	16.89	18.57	0.51	0.116
C16:1 cis-9	0.89	0.64	0.06	0.032	PUFA	45.77	39.14	1.54	0.026
C17:0	0.70	0.61	0.05	0.375	C ≤ 16	23.23	27.33	1.10	0.058
C17:1 trans10	11.16	13.86	0.67	0.041	C > 16	76.77	72.67	1.10	0.058
C18:0	14.31	15.03	0.26	0.181
¹g/100g of total fatty acid.
SEM = Standard error of the mean between the two treatments; SFA = saturated fatty acid; UFA = unsaturated fatty acid; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.

The effect of HG feeding on the fatty acid composition of the muscle is shown in Table 5. UFAs had the largest proportion of fatty acids in the muscle tissue of dairy cows (average 65.02%, SEM = 1.00%) and were primarily composed of MUFAs (average 39.24%, SEM = 1.57%). The average proportions of PUFAs and SFAs in muscle tissue were 9.62% (SEM = 1.58%) and 34.98% (SEM = 1.00%), respectively. Furthermore, different diets did not influence the fatty acid proportions in muscle.

Table 5

Effects of high-grain diet (HG) or conventional diet (CON) on muscle fatty acid proportions¹
Item	CON	HG	SEM	P-value	Item	CON	HG	SEM	P-value
C4:0	2.27	1.98	0.22	0.532	C18:1 cis9	28.34	28.65	1.07	0.893
C6:0	0.86	0.76	0.09	0.614	C18:2 trans9,12	1.42	3.31	1.19	0.469
C10:0	0.08	0.08	0.02	0.982	C18:2 cis9,12	17.15	15.54	1.96	0.705
C11:0	0.12	0.10	0.01	0.457	C20:1 cis11	1.15	1.10	0.07	0.761
C14:0	1.41	1.50	0.11	0.722	C20:3 cis8,11,14	1.65	2.19	0.21	0.239
C14:1 cis9	0.96	1.07	0.09	0.568	C20:3 cis11,14,17	5.19	5.13	0.35	0.937
C15:0	0.44	0.50	0.04	0.442	SFA	35.69	34.27	1.00	0.504
C16:0	14.39	14.06	0.57	0.786	UFA	64.31	65.73	1.00	0.504
C16:1 cis-9	5.34	5.84	0.48	0.627	MUFA	38.90	39.57	1.57	0.842
C17:0	1.36	1.45	0.07	0.520	PUFA	8.26	10.62	1.58	0.490
C17:1 trans10	1.95	1.60	0.28	0.549	C ≤ 16	25.87	25.89	0.79	0.991
C18:0	14.76	13.84	0.51	0.401	C > 16	74.13	74.11	0.79	0.991
C18:1 trans9	1.17	1.31	0.11	0.545
¹g/100g of total fatty acid.
SEM = Standard error of the mean between the two treatments; SFA = saturated fatty acid; UFA = unsaturated fatty acid; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.

The effect of HG feeding on the fatty acid composition of adipose tissue is shown in Table 6. UFAs were the main component of fatty acids in the adipose tissue of dairy cows (average 73.69%, SEM = 3.60%) and were mainly composed of MUFAs (average 47.16%, SEM = 1.57%). The average proportions of PUFAs and SFAs in muscle tissue were 26.46% (SEM = 1.33%) and 26.31% (SEM = 3.06%), respectively. The proportion of C18:1 cis-9 (P = 0.041) in the HG group was lower than in the CON group. Moreover, HG feeding tended to increase the proportions of C16:1 cis-9 (P = 0.052) and MUFAs (P = 0.084) in adipose tissue.

Table 6

Effects of high-grain diet (HG) or conventional diet (CON) on adipose tissue fatty acid proportions¹
Item	CON	HG	SEM	P-value	Item	CON	HG	SEM	P-value
C4:0	3.32	3.05	0.18	0.486	C18:2 trans9,12	2.96	2.73	0.13	0.396
C6:0	1.64	1.27	0.12	0.115	C18:2 cis9,12	19.62	19.33	1.00	0.891
C10:0	0.22	0.15	0.03	0.230	C18:3 cis6,9,12	2.77	2.80	0.15	0.943
C11:0	0.57	0.42	0.04	0.101	C20 cis11	2.47	2.14	0.13	0.232
C12:0	0.58	0.56	0.10	0.934	C20:3 cis8,11,14	1.32	1.46	0.11	0.570
C14:0	13.50	6.31	2.93	0.237	SFA	31.95	20.67	3.60	0.123
C14:1 cis9	8.82	16.42	2.71	0.171	UFA	68.05	79.33	3.60	0.123
C15:0	3.10	2.94	0.14	0.595	MUFA	41.37	53.02	3.39	0.084
C16:1 cis-9	19.76	25.98	1.62	0.052	PUFA	26.68	26.31	1.13	0.879
C17:0	9.02	5.98	1.57	0.359	C ≤ 16	51.50	57.10	2.08	0.197
C17:1 trans10	3.77	4.41	0.25	0.206	C > 16	48.50	42.90	2.08	0.197
C18:1 cis9	6.56	4.06	0.55	0.014
¹g/100g of total fatty acid.
SEM = Standard error of the mean between the two treatments; SFA = saturated fatty acid; UFA = unsaturated fatty acid; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.

3.5 Triacylglycerol concentration of muscle and adipose tissue

HG feeding led to an increase in TG concentration in muscle (P = 0.068) compared with the CON group (Fig. 2h). In addition, the TG concentration in the adipose tissue (P = 0.007) of the HG group was markedly higher than that in the CON group (Fig. 2i).

3.6 Transcriptome profile analysis of the muscle tissue

Transcriptome sequencing data on muscle tissue were utilised to perform the PCA, and the results indicated that Axes 1 and 2 explained 24.1% and 13.2% of the total variation, respectively (Fig. 3a). Likewise, Axes 1 and 2 of PLS-DA explained 15.2% and 16.3% of the total variation, respectively (Fig. 3b). A total of 218 DEGs; fold change ≥ 2 and FDR < 0.05 were identified from the 22,586 genes in the muscle transcriptome data, including 121 up-regulated genes and 97 down-regulated genes in the HG group. The pathway analysis of DEGs was conducted using the clusterProfiler R package (v4.2.2) according to the KEGG pathway database. A total of 36 significantly changed pathways were identified, as shown in Fig. 3c (P < 0.05). Among them, three pathways are related to lipid metabolism: the regulation of lipolysis in adipocytes, the biosynthesis of UFAs, and the PPAR signalling pathway. Compared with the CON group, a total of eight DEGs were identified among these pathways in the HG group, of which two genes were significantly up-regulated and six genes were significantly down-regulated (Table 7). In the regulation of lipolysis in the adipocytes pathway, CGA expression in the muscle tissue of the cows fed a HG diet was up-regulated, while the gene expressions of FABP4, PTGER3, and CIDEC were down-regulated (Figure S1 in Additional File 1). In the biosynthesis of the UFA pathway, HG feeding down-regulated the gene expressions of SCD5 and ELOVL6 in the muscle tissue (Figure S2 in Additional File 1). In the PPAR signalling pathway, the gene expressions of FABP4, ADIPOQ, and SCD5 in muscle tissue were down-regulated by HG feeding, while the gene expression of APOA2 were up-regulated (Figure S3 in Additional File 1).

Table 7

Differentially expressed genes in muscle tissue due to high-grain diet feeding¹
Pathway	Gene symbol²	FDR	Log₂^FC	Gene-ID
Regulation of lipolysis in adipocytes pathway	CGA	< 0.001	1.438	12373
	FABP4	< 0.001	-1.391	18034
	PTGER3	< 0.001	-1.822	4070
	CIDEC	< 0.001	-1.066	27073
Biosynthesis of unsaturated fatty acids	SCD5	< 0.001	-1.005	8601
Biosynthesis of unsaturated fatty acids	ELOVL6	< 0.001	-1.001	7957
PPAR signalling pathway	APOA2	< 0.001	2.225	2917
	FABP4	< 0.001	-1.391	18034
	ADIPOQ	< 0.001	-1.602	622
	SCD5	< 0.001	-1.005	8601
¹ Results based on RNA sequencing, n = 6. The gene expressions were considered to be significantly altered when FC ≥ 2 and FDR < 0.05.
²CGA: glycoprotein hormones alpha chain precursor; FABP4: fatty acid-binding protein 4; PTGER3: prostaglandin E2 receptor EP3 subtype; CIDEC: cell death activator; SCD5: stearoyl-CoA desaturase 5; ELOVL6: elongation of very long chain fatty acids protein 6; ADIPOQ: adiponectin; APOA2: apolipoprotein A-2.

3.7 Transcriptome profile analysis of adipose tissue

The entire high-quality dataset of transcriptome sequencing was used to perform the PCA, and the results indicated that Axes 1 and 2 explained 22.8% and 12.1% of the total variation, respectively (Fig. 4a). The PLS-DA results showed that the samples of the CON and HG groups were divided into two clusters (PLS1 = 19.7% and PLS2 = 11.4%, Fig. 4b), which indicates significant differences between the two groups at the transcriptome level. Compared with the CON group, a total of 1,061 DEGs (fold change ≥ 2 and FDR < 0.05) were identified from the 23,097 genes in the HG group, including 504 up-regulated genes and 557 down-regulated genes. Figure 4c shows 38 significantly changed pathways (P < 0.05). Among these, three pathways are related to lipid metabolism: fatty acid biosynthesis, linoleic acid metabolism, and the PPAR signalling pathway. A total of 13 DEGs were identified in these three pathways, 8 of which were significantly up-regulated and 5 were significantly down-regulated in the HG group compared with the CON group (Table 8). In the fatty acid synthesis pathway, HG feeding increased the gene expressions of FASN, ACACA, and ACSBG2 (Figure S4 in Additional File 1). In the linoleic acid metabolism pathway, the gene expressions of CYP3A24, CYP2E1, HRAS-3, and PLA2G2D1 were up-regulated by feeding the cows HG diets (Figure S5 in Additional File 1). In the PPAR signalling pathway, the cows on a HG diet had up-regulated gene expressions of UCP1 and ACSBG2 in adipose tissue, while the expression levels of APO-A1, OLR1, APO-C3, SLC27A2, and ANGPTL4 were down-regulated (Figure S6 in Additional File 1).

Table 8

Differentially expressed genes in abdominal adipose tissue due to high-grain diet feeding¹
Pathway	Gene symbol²		FDR		Log₂^FC		Gene-ID
Fatty acid synthesis	FASN		< 0.001		1.375		24950
	ACACA		< 0.001		1.011		23708
	ACSBG2		0.037		1.683		9568
Linoleic acid metabolism	PLA2G2D1		< 0.001		2.239		2731
	CYP3A24		< 0.001		4.498		30249
	CYP2E1		< 0.001		13.169		30981
	HRAS-3		< 0.001		14.152		32537
PPAR signalling pathway	UCP1		< 0.001		3.011		21041
	ACSBG2	0.037		1.683		9568
	APO-A1	< 0.001		-1.230		18573
	OLR1	0.009		-1.145		7294
	APO-C3	0.002		-1.003		18572
	SLC27A2	0.047		-11.379		13784
	ANGPTL4	< 0.001		-1.228		9509
¹ Results based on RNA sequencing, n = 6. The gene expressions were considered to be significantly altered when fold change ≥ 2 and FDR < 0.05.
²FASN: fatty acid synthetase; ACACA: acetyl-CoA carboxylase; ACSBG2: long-chain-fatty-acid–CoA ligase ACSBG2 isoform X1; CYP3A24: cytochrome P450 3A24; CYP2E1: cytochrome P450 2E1; HRAS-3: HRAS-like suppressor 3; PLA2G2D1: calcium-dependent phospholipase A2 PLA2G2D1 precursor; UCP1: mitochondrial brown fat uncoupling protein 1; APO-A1: apolipoprotein A-I preproprotein; OLR1: oxidized low-density lipoprotein receptor 1; APO-C3: apolipoprotein C-III precursor; SLC27A2: very long-chain acyl-CoA synthetase; ANGPTL4: angiopoietin-related protein 4 precursor.

3.8 qPCR validation of the RNA-seq

Quantitative real-time PCR was conducted to verify the transcriptome results. The expression patterns of FASN, ACACA, ACSBG2, FABP4, ADIPOQ, and SCD5 were consistent with the transcriptome data. Briefly, in the HG group, the gene expressions of FABP4, ADIPOQ, and SCD5 were down-regulated (P < 0.05) in the muscle tissue (Fig. 5a), while the gene expressions of FASN, ACACA, and ACSBG2 were up-regulated (P < 0.05) in the adipose tissue (Fig. 5b).

The reductions in rumen pH due to the increases in grain feeding in our study agree with previous research ^[32]. SARA induced by increasing easily fermentable carbohydrates and reducing the fibre intake in the HG diet results in the accumulation of short-chain fatty acids in the rumen, as well as a reduction in chewing capacity, salivary buffer supply, and rumen motility^[7]. A rumen pH below 5.8 for more than 180 min/day has been commonly used to diagnose SARA ^{[6, 33]}. In the present study, the rumen pH of the cows in the HG group satisfied this condition, indicating that feeding cows HG diets induced SARA.

Milk fat content is a crucial index for measuring the performance of dairy cows. In agreement with our work, several studies have shown that feeding cows HG diets affects the fat content and fatty acid composition of milk ^{[34, 35]}. Peterson et al.^[36] demonstrated that HG diets reduced milk fat percentage by 25% and yield by 27% but had no influence on dietary intake or the milk protein and lactose concentrations. In this study, HG feeding reduced milk fat content but did not affect milk yield and other milk characteristics, which was consistent with the findings of Bauman et al.^[37]. Data from this research showed that DMI was not different between the two dietary treatments, which concurred with previous results ^{[38, 39]}. Paradoxically, several researchers have reported that excessive grain feeding decreased the DMI^{[40, 41]}. The effects of HG diets on DMI were inconsistent, and the causes were probably the variance in diet formula, the length of the experimental period, or the individual differences between the cows. Most short- and medium-chain fatty acids (C ≤ 16) in milk come from de novo synthesis in mammary tissue ^[9]. The analysis of milk fatty acid composition in our study showed that adding high amounts of grain to a cow’s diet increases the proportion of short- and medium-chain fatty acids (C10:0, C11:0, C12:0, and C16:1 cis-9) in its milk. These changes suggest shifts in the de novo synthesis of milk fat and are analogous to the findings of Han et al.^[42] Furthermore, we also found HG feeding decreased the proportion of long-chain fatty acids (C17:0 and C18:0) in the milk. It is well accepted that most long-chain fatty acids (C > 16) in milk are absorbed from the blood ^[43]. Thus, we speculate that the finding in this study may be associated with an alteration of the blood fatty acid composition of dairy cows fed HG diets. In addition, HG feeding had no significant effect on the SFA, UFA, MUFA, and PUFA concentrations in the milk, which is in agreement with previous reports ^{[44, 45]}

The concentration of plasma TG is a commonly used biomarker for lipid metabolism ^[46]. In the present study, switching to a HG diet tended to decrease the plasma TG concentration, which was also observed by Wang et al.^[16]. The causes of this result are probably the decreased lipolysis and ruminal biohydrogenation of dietary fatty acids under the low ruminal pH ^[47] or the insufficient supply of endogenous fatty acids from liver and adipose tissue^{[48, 49]}. Previous studies have shown that grain-based SARA increases ruminal and peripheral bacterial endotoxin concentrations ^[50]. Zebeli Q et al.^[51] found that ruminal endotoxin concentration was negatively correlated with plasma TCHO concentration. In the present study, the decreased plasma TCHO level induced by feeding the cows a HG diet may be related to systemic inflammation triggered by the release of ruminal endotoxin into the peripheral circulation^[52]. On the other hand, LDL, as an essential lipoprotein primarily responsible for cholesterol transport ^{[53, 54]}, had a correspondingly reduced concentration in the plasma of the cows on the HG diet in the present study. Another key finding of our study was that excessive grain feeding significantly altered the plasma fatty acid composition of dairy cows. The HG group had higher proportions of C4, C6, C17:1 trans-10, and C ≤ 16 and lower proportions of C16:1 cis-9, C18:2 cis-9,12, C20:3 cis-8,11,14, and C > 16 compared with the CON group. Interestingly, for the cows in the HG group, the variation trends of C ≤ 16 and C > 16 in the plasma were consistent with those in the milk. The PUFA status of ruminants is particularly precarious due to the hydrogenation of UFAs by rumen microbes ^[55]. PUFAs in blood originate from the uptake of pre-formed fatty acids from the rumen ^[56]. Our results suggest that HG feeding decreases the blood PUFA proportion in dairy cattle, presumably due to alterations in rumen biohydrogenation.

Muscle is a key regulatory tissue that helps the body respond to changes in energy and lipid metabolism during lactation ^[57]. Studies on the effects of diet on muscle tissue have focused on beef cows ^[58]; however, the literature on dairy cows is scarce. Different dietary regimes have been shown to influence the fatty acid profile of beef ^[59]. Scollan et al.^[60] reported that forage-based beef cows had higher concentrations of n − 3 PUFAs in their muscle tissue than grain-based beef cows. Nevertheless, in the current study, a HG diet had no impact on the fatty acid composition in the muscle tissue of dairy cows, possibly because of the short experimental period or species differences in this experiment. Transcriptomic data from muscle tissue in the present study indicate that switching to a HG diet altered the UFA biosynthesis pathway by down-regulating the gene expressions of SCD5 and ELOVL6. The synthesis of UFAs requires the coordination of multiple related genes^[61]. In this study, there was no difference in the UFA concentration in muscle tissue between the two groups; one explanation could be the absence of differences in other related genes, especially those from the SCD and ELOVL families. Another key finding of the current study is that gene expression of FABP4 and ADIPOQ in the muscle of HG group was lower than the CON group. FABP4 is associated with the absorption, transport, and metabolism of fatty acids and mediates the transfer of fatty acids during lipolysis ^{[62, 63]}. ADIPOQ is one of the most abundant circulating adipokines and is negatively correlated with fat mass ^[64]. In the present study, we found that HG feeding tended to raise the TG concentration in the muscle tissue of dairy cows. Despite the lack of statistical differences in the expression of fat synthesis genes (e.g. FASN and ACACA), at least in part, the lower expression levels of FABP4 and ADIPOQ in the HG group is suggestive of decreased fatty acid transport capacity during lipolysis and increased fat mass in muscle tissue.

In line with our hypothesis, we found that the fatty acid composition of adipose tissue was affected by HG feeding. Overall, the cows on the HG diet showed a tendency for a higher proportion of C16:1 cis-9 and MUFAs and a lower proportion of C18:1 cis-9 in their adipose tissue compared with cows on the CON diet. Studies in humans clearly show that MUFAs regulate lipid metabolism through modifications in the composition of cell membranes^[65] ; however, the effects of MUFAs on ruminants need to be further studied. Because of the increased TG concentration and activated fatty acid synthesis pathways in the adipose tissue of cows on a HG diet, we suggest that HG intake enhances lipid synthesis capacity in the adipose tissue of dairy cows. FASN, ACACA, and ACSBG2 are genes involved mainly in the fatty acid synthesis pathway, which encodes key enzymes linked to fatty acid synthesis^[66–69]. In the present study, the up-regulated expressions of FASN, ACACA, and ACSBG2 in the adipose tissue of cows on a HG diet provide transcriptional evidence for the theory of Baldwin et al.^[70], which states that the activity of enzymes involved in fatty acid synthesis in adipose tissue increases when diets contain high amounts of grain. Additionally, it has been noted that FASN also participates in the regulation of TG synthesis^[71]. The results of this study showing that the up-regulation of FASN expression is accompanied by an increase in TG content seem to support this view. Furthermore, Gaynor et al.^[72] proposed that HG diets transfer nutrients from milk fat synthesis to body fat synthesis, which is in agreement with our study. In addition, we showed that the linoleic acid metabolism pathway in adipose tissue was also activated by HG feeding. The higher gene expression levels of CYP3A24, CYP2E1, HRAS-3, and PLA2G2D1 may lead to the conversion of more lecithin to linoleic acid and subsequently to epoxyoctadecanoic acid. However, linoleate is an important component of lipoproteins and is involved in the transport of lipids^{[73, 74]}. Thus, our results suggest that feeding cows HG diets may lead to a decrease in the lecithin content of their adipose tissue, which in turn leads to a decrease in lipid transport capacity. The PPAR signalling pathway consists of interrelated genes that encode transcription factors, enzymes, and downstream targets that coordinately act to regulate lipid uptake, synthesis, and transport^{[75, 76]}. Interestingly, in the current study, the DEGs of both muscle and adipose tissue marked this pathway, indicating that HG feeding affects the lipid metabolism of muscle and adipose tissue by regulating gene expression in the PPAR pathway.

In conclusion, HG feeding induced SARA, significantly decreased the milk fat content, and altered the milk fatty acid composition of dairy cows. The HG diet reduced the TG content in the blood and changed the fatty acid composition of the cows’ plasma. In muscle tissue, HG feeding tended to increase TG concentration but did not affect fatty acid composition. However, in adipose tissue, the HG diet significantly increased the TG concentration and changed the fatty acid composition. Finally, transcriptome analysis of muscle and adipose tissue showed that HG diets could increase lipid synthesis and decrease lipid transport.

BW: Body weight; DMI: Dry matter intake; DEGs: Differentially expressed genes; FC: Fold change; FDR: false discovery rate; F/G: Feed/gain ratio; FPKM: Fragments per kilobase per million mapped fragments; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; DMI: dry matter intake.

Acknowledgments

Not applicable.

Funding

This research was supported by the Natural Science Foundation of China (31872384).

Availability of data and materials

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Authors’ contributions

SM designed and supervised the study. QC, LL, and ZL performed the experiments. QC analyzed the data and wrote the paper. LL and ZL revised it critically for important content. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the Ethical Committee of Nanjing Agricultural University (Nanjing, China).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Additionalfile1.docx
Additional file 1: Table S1. Primer sequences are used for real-timePCR analysis. Figure S1: Regulation of lipolysis in adipocytes signaling KEGG pathway in muscle tissue. Figure S2: Biosynthesis of unsaturated fatty acids signaling KEGG pathway in muscle tissue. Figure S3: PPAR signaling KEGG pathway in muscle tissue. Figure S4: Fatty acid biosynthesis signaling KEGG pathway in adipose tissue. Figure S5: Linoleic acid metabolism signaling KEGG pathway in adipose tissue. Figure S6: PPAR signaling KEGG pathway in adipose tissue.

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published 08 Apr, 2023

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Editorial decision: Major revision
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Reviewers invited by journal
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Editor assigned by journal
27 Sep, 2022
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Effects of high-grain diet feeding on fatty acid profiles in milk, blood, muscle, and adipose tissue, and transcriptional expression of lipid-related genes in muscle and adipose tissue of dairy cows

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Journal Publication

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Background

2. Materials And Methods

2.1 Animals and experimental treatments

2.2 Ruminal and diet sample collection and analysis

2.3 Blood and milk sampling and analysis

2.4 Slaughter and tissue sample collection

2.5 Triacylglycerol concentration detection

2.6 Fatty acid composition analysis

2.7 Transcriptome sequencing and data analysis

2.8 Quantitative real-time PCR analysis

2.9 Statistical analysis

3. Results

3.1 Rumen pH

3.2 Milk Yield and Composition, DMI

3.3 Plasma biochemical parameters

3.4 Fatty acid composition of milk, plasma, muscle, and adipose tissue

3.5 Triacylglycerol concentration of muscle and adipose tissue

3.6 Transcriptome profile analysis of the muscle tissue

3.7 Transcriptome profile analysis of adipose tissue

3.8 qPCR validation of the RNA-seq

4. Discussion

5. Conclusion

Abbreviations

Declarations

Acknowledgments

Funding

Availability of data and materials

Authors’ contributions

Ethics approval and consent to participate

Consent for publication

Competing interests

References

Supplementary Files

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