Feeding a High Concentrate Diet Down-Regulates Expression of ACACA, LPL and SCD and Modifies Milk Composition in Lactating Goats

High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase α (ACACA, ACCα) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.


Introduction
Diet plays an important role in the modulation of fatty acid composition in ruminant products. The types and proportion of fatty acids in food have different effects on human health maintenance and disease prevention [1]. Polyunsaturated fatty acids (PUFAs) are fatty acids containing two or more double bonds. In particular n-3 fatty acids, which first double bond is 3 carbons from the methyl end of the fatty acid chain, includes linolenic acid (LNA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3) and

Ethics statement
The Animal Care and Use Committee of Nanjing Agricultural University approved the experimental protocol, which was performed in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology (2006, Beijing, China).

Animals and experimental design
Eight healthy multiparous mid-lactating goats (average body weight, 43.8±2.6 kg) with rumen fistula were randomly assigned to two groups. Goats were housed in pens and fed total mixed rations that were formulated to meet nutritional requirements. Goats in control group were fed a low concentrate diet (LCD) with a forage-to-concentrate ratio (F:C) of 6:4. Goats in the HCD group were fed with a high concentrate diet (HCD) with an F:C of 4:6. Diet formulas are shown in Table 1. 0.4kg dry matter (DM) were offered to goats twice a day at 07:00 and 19:00, and goats were free access to fresh water. Dry matter intake (DMI) was measured. The feeding adaption period was 3 weeks. Goats were milked at 07:00 and 19:00 h daily, and milk yield was recorded.

Sample collection
Days were counted from day 1 to day 24 as the goats were adapted with the diets. Milk samples were taken from morning milking on day 22. One aliquot was stored at 4°C until it was analyzed for milk components, and a second aliquot was stored at -70°C until analyzed for fatty acid composition. Blood samples were collected 2 h after the morning feeding via jugular veins using vacuum tubes containing sodium heparin as anticoagulant on day 23. Blood samples were centrifuged at 1900 g, 4°C for 15 min, and plasma was harvested. Ruminal fluid samples were taken 2 h after morning feeding and strained through a double-layered muslin gauze on day 24 for fatty acids analysis. Mammary gland tissues were obtained by biopsy after 2 h after the morning feeding on day 25 of the experimental period. Local anesthesia (lidocaine hydrochloride) was administered into breast skin in a circular pattern surrounding the incision site, then 2cm incision was made and mammary gland tissue was dissected. Tissue samples (500-1000 mg) were rinsed with 0.9% saline, snap frozen in liquid nitrogen and subsequently stored at −70°C until RNA, DNA and protein extraction. Incisions were sutured, and antibiotics were administered intramuscularly to avoid infection. After slaughtered, longissimus muscle, Semitendinosus muscle and subcutaneous fat were collected for extraction of total lipids.

Measurement of rumen, plasma and milk parameters
Rumen pH was measured using a pH meter on day 22, and the duration of pH value lower than 5.6 and 6.0 was recorded. LPS content in rumen fluid and plasma was determined using a chromogenic endpoint assay (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, China) with a minimum detection limit of 0.01 EU/mL. These procedures were performed according to the manufacturer's instructions. Milk samples were taken to determine milk components (MilkoScan FT1, FOSS, Denmark) and somatic cell count (SCC) (Fossomatic 5000, Denmark).

Fatty acids composition analysis
Volatile fatty acids anylysis. Acetate, propionate, butyrate, isobutyrate, and isovalerate concentrations were determined by using gas chromatography (Agilent Technologies 7890A, GC system) with a flame ionization detector, based on the method with some modifications [23]. Crotonic acid was used as internal standard, and chloroform was served as extracting solvent. And the gas chromatography was performed using a 30 m×0.320 mm×0.50 μm fused silica capillary column (J&W Science 123-3233, USA). Helium was used as the carrier gas and hydrogen as a fuel gas at the flow rate of 40 mL/min with air as a combustion-supporting gas. The temperature of the column, the flame ionization detector and the injector were 155°C, 180°C and 180°C, respectively. The split ratio was 20:1 and 1 μL was injected. Crotonic acid was served as internal standard, and chloroform were used to extract VFA in ruminal fluid. Long-chain fatty acids analysis. Total lipids of ruminal fluid, plasma, muscle, subcutaneous fat and milk were extracted from samples using a mixture of polar and non-polar solvents according to Folch et al. [24]. Given no significance in total lipids of muscle and subcutaneous fat between two groups, fatty acids composition of them was not further detected. Fatty acid methyl esters (FAME) were prepared by esterification using sodium methoxid, followed by 14% borontrifluoride in methanol [25]. Heptadecanoic acid methyl ester served as an internal standard, and it was added to samples prior to extraction and methylation. FAME extracts were used for gas chromatographic analysis of total fatty acids. Fatty acid composition was determined using gas chromatography (GC) on a CP SIL 88, 100 m×0.25 mm×0.25 μm capillary column (Agilent J&W Advanced Capillary GC Columns, Netherlands) in an Agilent 7890A (Agilent Technologies, USA) with an auto sampler, flame ionization detector and split injection. The initial oven temperature was 150°C, which was held for 5 min then increased to 200°C at a rate of 2°C/min and held for 10 min, then increased to 220°C at 5°C/ min and held for 35 min. Helium was used as a carrier gas at a flow rate of 1 mL/min. The injector was set at 260°C, and the detector was set at 280°C. FAMEs were identified by comparisons with the retention times of the standards. The standards were FAME Mix C4-C24 Unsatures (Sigma 18919, Germany), Methyl trans-11 C18:1 (Sigma 46905, Germany) and Methyl cis-9, trans-11 CLA (Matreya1255, USA).

RNA extraction and analysis
Total RNA was extracted from 50 mg mammary gland tissues in RNAiso Plus reagent (Takara Co., Otsu, Japan) via homogenization on ice. The purity and concentration of RNA were measured using Eppendorf BioPhotometer Plus (Eppendorf AG, Hamburg, Germany). First-strand cDNA of each sample was synthesized using 500 ng of total RNA template in PrimeScript RT Master Mix Perfect Real Time (Takara Co., Otsu, Japan). All templates were performed in triplicate, and glyceraldehyde phosphate dehydrogenase (GAPDH) and β-actin served as internal control genes for normalization. Primers were designed using Primier 6.0 (Premier Biosoft International, USA) or online tool PrimerQuest of Integrated DNA technologies Co. Ltd (http://www.idtdna.com/Scitools/Applications/Primerquest/) based on known goat sequences (shown in Table 2). qPCR was performed using SYBR Premix Ex Taq (Takara Co., Otsu, Japan) and an ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the instruction manual's recommendations. Each sample was measured in triplicate. Data were analyzed using the 2 -ΔΔCt method using the geometric means of the selected housekeeping genes, GAPDH and β-actin, for normalization according to the strategy described previously [26].

DNA methylation assay of SCD
Total DNA from mammary gland tissues was extracted using a commercial kit (Invitrogen, California, USA), and sodium bisulfate and Hydroquinone solutions were added to the DNA for getting bisulfate-converted DNA according to Ogino's method [27]. DNA methylation was assayed using bisulfite sequencing PCR (BSP) (BBI, Canada). DNA was treated with bisulfate to convert unmethylated cytosines(C) to uracils(U) through deamination, but methylated cytosines remained unchanged during treatment. Uracils were eventually replaced by thymine (T) during PCR amplification. The modified target sequences were amplified by PCR with primers, 5'-GTAGYGGAAGGTTTYGAGTATAG-3' (forward) and 5'-TTCCCAACAAACTAAAAAC AATAC-3' (reverse) that specifically bind to the plus strand of the corresponding genomin regions. This causes the occurrence of C/T polymorphisms at these positions in subsequent sequencing reactions. PCR amplification made a mixture of molecules with either C or T bases at CpG (cytosine-phosphate-guanine) dinucleotides. PCR products were cloned and sequenced to determine DNA methylation levels of the SCD gene using BiQ Analyzer software by Shanghai Sangon Biotech Company (Shanghai, China).

Western blotting analysis
Protein was extracted after homogenizing mammary gland tissue, and the concentrate was assayed using the Bradford method [28]. Proteins were separated using SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, USA). The membrane that included proteins <50 kDa was incubated with a specific primary antibody, polyclonal antibody raised in goat (sc-23016, Santa Cruz, USA), and the membrane that included proteins >50 kDa was normalized against β-tubulin and incubated with a polyclonal goat antibody (sc-9935, Santa Cruz, USA). Membranes were incubated overnight at 4°C and re-probed with a horseradish peroxidase-conjugated Affinipure rabbit anti-goat secondary antibody (E030130-01, Earthox LLC, San Francisco, CA). Protein bands were visualized using a chemiluminescent agent (BeyoECL plus, Shanghai, China). Membranes were scanned using a chemiluminescence imager, Image Quant LAS 4000 (GE, USA), and band intensities were densitometrically evaluated using Quantity One software (Bio-Rad, USA). Signal intensities of the samples are expressed as a percentage of the reference sample.

Statistical Analysis
The data were analyzed using general linear model (GLM) with random goat effect and the fixed factor of diets (SAS Systems, SAS Institute Inc., Cary, NC), and adjusted means were compared with a Student's t-test. Data were presented as least squares mean (LSM) and the standard error (SE), and all statistical tests were performed for a significance level P<0.05.

Rumen, plasma and milk parameters
Different diets had no influence on DMI of goats, milk yield, total lipids in muscle and subcutaneous fat ( Table 3). The pH value of ruminal fluid in the HCD group was significantly lower than in the LCD group (P<0.05). The pH was less than 5.6 for more than 3 h one day (212 ±29.15 min, shown in Table 3), which indicated that the high concentrate diet induced SARA status in goats in the HCD group. Milk component assays showed that milk fat in the HCD group was significantly declined (P<0.05). Somatic cell count tended to increase in the HCD group, but this increase was not significant. LPS content in ruminal fluid tended to be elevated in the HCD group, but, it was significantly increased in plasma (P<0.05) ( Table 3).

Fatty acid composition analysis
VFA compositions of ruminal fluid are shown in Table 4. Acetate concentration in HCD group was lower than that in control group (P<0.05), and propionate higher than that in control group (P<0.05) Total long chain fatty acid compositions of ruminal fluid, plasma and milk are shown in Table 5. Diet had no effect on VA content in ruminal fluid, but cis-9, trans-11 CLA content decreased significantly in the HCD group compared to the control group (P<0.01). The contents of C18:0 (P<0.05) and C20:1 (P<0.05) in ruminal fluid were higher in the HCD group than the control group. The content of C18:3n3 (P<0.05), C22:0 (P<0.01) and C24:1 (P<0.01) in ruminal fluid was lower in the HCD group than the control group.

RNA extraction and analysis
The effects of feeding different concentrate diets on mRNA expression levels of ACACA, SCD, LPL, FASN and SREBP1c are shown in Fig 1. The high concentrate diet in the HCD group significantly down-regulated transcription levels of ACACA, SCD and LPL mRNA compared to the control group (P<0.05). The expression of transcription factor SREBP1c tended to decrease

DNA methylation analysis of SCD gene
DNA methylation of the SCD gene, which encodes a stearoyl-CoA desaturase in mammary glands that is involved in cis-9, trans-11CLA synthesis, was significantly increased in goats fed a concentrate diet compared to a low concentrate diet (Fig 2).

Protein expression of SCD in mammary glands
Representative blots of SCD protein are shown in Fig 3. Expression levels of SCD protein in goat mammary glands was remarkably decreased in the HCD group compared to the control group fed a low concentrate diet. There was a significant difference between these two groups (P<0.05).

Discussion
In the present study, high concentrate diet induced SARA status, as demonstrated by the pH value of ruminal fluid, which remained lower than 5.6 for more than 3 h in the HCD group. Volatile fatty acids account for 60-70% metabolizeble energy supply in ruminants [29], and of the three main VFA, acetate and butyrate are essential components for synthesis of milk fat, while propionate, the only glucogenic VFA in lactating ruminants, is substrate for lactose synthesis [30]. In our study, acetate concentration in HCD group was lower than that in control group (P<0.05), and propionate higher than control group (P<0.05), which could finally Expression of ACACA, LPL and SCD and Milk Composition Modification influence the fat (P<0.01) and lactose (P<0.05) in milk. However, it was found that butyrate had greater influenced on milk yield than acetate and there was a positive regression relationship between A/P and milk fat [31], and in our study, the butyrate and milk yield were no significant between two groups. Goats suffered from SARA with low rumianl pH value may caused by high concentration of lactate, which was proved by our recent study in cows [32].
The supply of PUFA available for absorption is determined by both the amounts of PUFA in the diet and their metabolism in the rumen. In present study, the high concentrate diet had no effect on PUFA concentration in the ruminal fluid, which could result in no significant difference between the two groups in the milk PUFA. In grazing cow, increases the proportion of concentrate in diet from 30-350 g/kg dry matter resulted an increase C18:2n6 concentration but a reduction in C18:1n9c, c9t11CLA and C18:3n3 concentration in milk [33], and in the present study, it was found that high concentrate diet increased C18:2n-6 concentration (P<0.05) and reduced VA, c9t11 CLA and C18:3n3 concentration(P<0.05), and also reduced C18:1n9c, but without significance.
Previous studies showed that the quantity of biohydrogenation intermediates that are produced in the rumen affected their concentrations in milk [34]. VA is the precursor of cis-9, trans-11 CLA synthesis in the mammary glands of ruminants [35]. Changes in the cis-9, trans-11 CLA content in ruminant milk depend both on the processes involved in the biohydrogenation of diet-derived unsaturated fatty acids and on the availability of VA in the rumen. Cis-9, trans-11 CLA is a transient intermediate in the rumen, whereas VA is an intermediate that accumulates when certain diets are consumed, which results in a lower conversion of trans Expression of ACACA, LPL and SCD and Milk Composition Modification C18:1 to C18:0. Goats in the HCD group suffering from SARA produced less cis-9, trans11 CLA than goats in control group because the biohydrogenation of VA might be affected by the low pH value of ruminal fluid [36,37,38]. However, diet does not affect the concentration of cis-9, trans-11 CLA in the rumen [39].
Plasma is the transportation system of nutritional ingredients, and its fatty acids status reflects the overall long-term lipid metabolism that is influenced by diet and rumen microbial activity. The profile of fatty acids in plasma represents the profile of fatty acids that are available to the mammary gland. The VA content in the plasma was significantly lower in the HCD group in present experiment, which might be caused by less absorption and more tissue utilization. Feeding pasture-based diets to German Holstein and German Simmental bulls' results in significantly higher VA concentrations in plasma compared to concentrate-fed bulls [39]. High concentrate diet feeding in our study reduced VA concentration in plasma, and cis-9, trans-11 CLA concentration in plasma was also lower in the HCD group, which might be due to lower absorption from the rumen.
The content of cis-9, trans-11 CLA in milk was significantly lower in the HCD group, which might have resulted from less precursor and lower SCD expression in the mammary gland. The methylation rate of the SCD DNA promoter in the HCD group was up-regulated, which could down-regulated the expression of SCD, as a matter of fact the expression of SCD protein was significantly reduced in the HCD group than the control group, which indicated that less VA was desaturated into cis-9, trans-11 CLA in the HCD group. The feeding of lactating goats with a high concentrate diet led to a low expression of SCD protein in mammary glands. These results suggest that the low expression of SCD protein in mammary glands leads to a low content of cis-9, trans-11 CLA.
The administration of a high concentrate diet might improve high milk yield or rapid weight gain in the short term. However, long-term consumption of high concentrate diet is associated with disorders, such as SARA. This study demonstrated that a high concentrate diet decreased milk fat content and some milk fatty acid composition, which were good for human health.
LPL is an enzyme that hydrolyzes triacylglycerol (TAGs) to form fatty acids and glycerol. Long-chain fatty acids are taken up by mammary glands through the action of LPL, which markedly increases throughout lactation [40]. LPL hydrolyzes lipoprotein-associated triglycerides prior to fatty acid absorption by tissues, which could provide more fatty acids during milk fat depression [41]. In present study, the LPL mRNA expression was lower in mammary gland of goats fed on HCD, which indicated that less fatty acids were provided for milk fat synthesis in HCD condition, even more fat was in the diet. Our previous study demonstrated that the fat content in the milk was lower with a high concentrate diet, which is consistent with the abundant of LPL mRNA expression in LCD.
FASN, a complex multifunctional enzyme, plays an important role in energy homeostasis by converting excessively consumed food into lipids for storage and energy stimulation in βoxidation [42]. FASN is also required for the production of milk lipids during lactation [43]. Palmitate (C16:0) is the major saturated constituent of lipids in animal tissues, and it is partially synthesized de novo by FASN [44]. C16:0 was lower in plasma in HCD group than that in control group (P<0.05), but had no difference in milk in two groups, which was consistent with the expression of FASN in mammary gland, expression of FASN was higher in HCD group, but without significance. FASN regulates lipid storage when high energy is consumed [42]. The HCD contained more fat than the low concentrate diet, which could affect FASN expression in the mammary gland of goats in HCD group. A previous study in lactating mice demonstrated that high-fat diets in reduce lipogenesis in mammary glands [45]. Opstvedt et al. reported that a high concentrate diet inhibited FASN activation in mammary gland [46], which is not consistent with our result, where a high concentrate diet increased, although not significantly, FASN expression in goat mammary glands, but consistent with Dong et al.'s founds that high concentrate diet increased FASN expression [22]. Further study on this issue in a more representative goat sample is required.
ACC is an enzyme that mediates the incorporation of acetate carbon into fatty acids [47]. There are two ACC isomers, ACCα (ACACA) and ACCβ, and the former is the main isomer in the liver, adipose tissue and mammary gland. Fatty acids from C4-14 are synthesized de novo in the mammary gland, whereas C16 arises from diet and de novo synthesis, which is performed by ACACA and FASN utilizing acetyl-CoA and butyryl-CoA [48]. Other long-chain fatty acids are predominantly derived from the diet, depending on the amount of fat in the diet [44]. Inhibition of ACACA activity does not decrease the rate of de novo lipogenesis, but it does attenuate the synthesis of all long chain fatty acids [49]. This is in agreement with the present study, where a high concentrate diet decreased the expression of ACACA and the concentration of long chain fatty acids in milk, such as C22:0, C20:3n3, C20:5n3 and C22:6n3. Therefore, a high concentrate diet decreased elongation by depressing the activity of ACACA.
Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate the activation of genes involved in lipogenesis and fatty acid synthesis [50]. SREBP1c is one member of this family, and it regulates many genes involved in lipid synthesis and deposition, such as FASN, LPL and SCD [51], influencing fatty acids synthesis in white adipose tissue, liver, skeletal muscle and other tissues [52]. Low availability of dietary fatty acids activates SREBP1c expression and processing, and a high fat diet decreases the expression of many genes, including LPL, FASN and SCD. Furthermore, SREBP1c-null mice fed a low-fat diet have nearly a 30% reduction in milk lipid concentration [53]. SREBP1c also plays a vital role in the adaptation of mammary lipid synthesis to low-fat diet conditions. Our high concentrate diet contained more fat, reduced the abundance of SREBP1c, although not significantly, but resulted in the low expression of genes involved in fatty acid synthesis, as demonstrated for ACACA and LPL.
In conclusion, goats fed on a high concentrate diet would suffer from SARA, which could finally affect the products of goats. A high concentrate diet had no effects on SFA, MUFA and PUFA, but reduced gene expression involved in lipids metabolism, such as ACACA, LPL, and SCD, and consequently some FA, in particular cis-9, trans-11CLA content in the milk of lactating goats, and SCD expression, which is involved in the catalysis of VA to cis-9, trans-11CLA, was significantly down-regulated. The DNA methylation rate of SCD was increased and the expression of SCD mRNA and protein was down-regulated in the mammary glands of lactating goats fed a high concentrate diet, which also reduced cis-9, trans-11 CLA content in milk.