1. Introduction
Albas white cashmere goat is a world famous local breed producing cashmere and meat, coming from the plateau region of Ordos, Inner Mongolian, China. In recent years, due to the limitation of natural grassland resources, degeneration of the grassland, and the increase of mutton market demand, the fattening pattern has changed from traditional pasture fattening to feedlot fattening, which can alleviate the pressure of grassland and increase the economic benefits. However, changes in fattening pattern mean the alteration of diet nutritional level, which can cause metabolic changes in livestock. For lambs fed an energy-restricted feeding sequence program to mimic the seasonal changes of the natural grasslands, the results indicated that sequentially restricting metabolizable energy intake resulted in a reduction of body weight, serum glucose (GLU) and triglyceride (TG) concentration [
1]. Feeding high-energy diets increased serum GLU and cholesterol (CHO) content in ewes [
2]. Chen et al. reported that the diet with low ratio of forage to concentrate (F:C = 50:50) had a higher concentration of serum high-density lipoprotein cholesterol (HDL-C) in Yak than the diet with high-forage (F:C = 70:30, F:C = 60:40) groups [
3]. Previous research reported an increase of plasma total lipids, CHO, TG, phospholipids, and non-esterified fatty acid (NEFA) in ruminant, which lead to lipid infiltration of the liver, and this favors the appearance of ketoacidosis, to the detriment of the health and reproductive performance of the animal [
4]. Therefore, the increasing studies ameliorating the lipid metabolism of ruminants in stall fattening are very necessary. Manipulating the dietary fat might be a feasible approach to regulate blood lipid metabolism, however, very little information on ruminants is available. Flaxseed contains 32% to 45% of its mass as oil, of which 51% to 55% is a-linolenic acid (ALA) and 15% to 18% is linoleic acid, and it includes the flax lignan complex [
5]. The main components of flaxseed oil are saturated fatty acid (SFA, 9%), monounsaturated fatty acid (18%), and ALA (57%) [
6]. ALA is the most important fatty acid in flaxseed oil and flaxseed, which are the most important functional phytochemicals in flaxseeds for human health [
7], and it is the precursor to the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The research observed that the addition of flaxseed could reduce the serum CHO concentration of cows [
8], and another study reported that dietary calcium ALA could significantly reduce serum CHO, TG, and low-density lipoprotein cholesterol (LDL-C) levels in high-fat-fed mice [
9]. Our previous study showed that flaxseed oil and flaxseed grain supplemented in diet showed different effects on increasing the concentration of ALA, EPA, DHA, and n-3 polyunsaturated fatty acids (n-3PUFA) in plasma and tissues in Albas cashmere goats, and flaxseed grain was more efficient [
10]. n-3 PUFAs reduced plasma TG by approximately 20–30% and decreased the substrate of oxidative LDL-C and remnant CHO taken up by macrophages [
11,
12]. These researches hinted that flaxseed and flaxseed oil rich in n-3 PUFA may regulate the blood lipid profiles and lipid deposition, and they probably have different influences, but limited data are available, and the mechanism is also unclear.
Gut microbiota may be involved in the regulation of serum lipid levels and lipid accumulation, because it plays important roles in host metabolism [
13]. Wen et al. reported that the genus
Methanobrevibacter and the species
Mucispirillum schaedleri were strongly associated with fat deposition in gut of chickens [
14]. The bacteria in cecum of pigs positively associating with LDL-C/HDL-C in serum belongs to Proteobacteria, which is associated with inflammation [
15]. An increase of serum LDL-C, very LDL-C and lipids concentration by dietary butyrate glycerides in broilers, not only significantly increased the
Bifdobacterium abundance but also boosted the species diversity, and the results suggested the potential contribution of intestinal bacteria to lipid metabolism/energy homeostasis in broilers [
16]. The previous research results of our team showed that compared to diet supplemented with linseed oil, added flaxseed increased linoleic acid biohydrogenation by reducing the relative abundance of
Ruminobacter and increasing the relative abundance of
Prevotellaceae_UCG-001 and
Fretibacterium in rumen, protected ALA away from biohydrogenation, and lead to more n-3 PUFAs entering the post-intestinal tract [
17]. The previous studies have shown that except for the rumen and large intestine, the ileum also serves as an indispensable fermentation site in goats [
18,
19]. Yan et al. reported that there is a larger number of cellulolytic bacteria in sheep ileum, particularly
Clostridium cluster IV [
20]. Therefore, we hypothesized that ALA-rich flaxseed oil and flaxseed grain supplements have different effects in ameliorating blood lipid profiles and lipid deposition in Albas cashmere goats, and the mechanism is probably involved in altering ileal microbial composition. The current study aimed to test whether supplementing flaxseed oil and flaxseed grain in diet has different regulation on lipid deposition and ileal microbiota community in Albas cashmere goats, and to explain the possible mechanism of flaxseed oil and flaxseed in ameliorating lipid deposition.
2. Materials and Methods
The experiment was conducted in the experimental farm of Inner Mongolia Agricultural University (Hohhot, China). All animal procedures were performed under the national standard Guidelines for Ethical Review of Animal Welfare (GB/T 35892-2018).
2.1. Experimental Design, Diet, and Feeding Management
A single-factor completely randomized design was used. Sixty 4-month-old castrated Albas cashmere male kid goats were selected from a farm, at Etuoke Town, Inner Mongolia, with an initial live weight of 18.6 ± 0.1 kg, and were randomly assigned to three treatments, with each treatment comprising 4 units of 5 goats. The control group (CON) was fed the basal diet with no supplementation. The experimental group was fed the flaxseed oil-supplemented diets (LNO) prepared by manually blending the oil thoroughly into the ground concentrate to ensure homogenous distribution of the oil in the ration. Another experimental group was fed the basal diet supplemented with heated flaxseed grain (HLS, the flaxseed contains about 36% oil and it was stir roasted at 120 °C for 10 min), which provided the same content of flaxseed oil as the LNO group. The nutrition levels of the diet could meet the needs of growing cashmere kids, according to the feeding standard of meat-producing sheep and goats (NY/T816, 2004 [
21];
Table 1). The trial consisted of a 14-day adaptation period and a 90-day treatment period, including early (1 to 30 days), medium (31 to 60 days), and late (61 to 90 days) fattening stages. The diet was offered to goats twice daily at 08:30 am and 16:30 pm as total mixed ration (concentrate to forage ratio of 50:50) and the goats were given free access to drinking water. To estimate feed intake for five goats in each pen, refusals were collected and weighed 30 min before each feeding, at 08:00 am daily. All goats were weighed (before feeding in the morning) on day 0 (initial body weight, IBW) and days 90 (final body weight, FBW) of the measurement periods to determine changes in body weight (total body weight gain, BWG).
2.2. Sample Collection
At the end of the experiment, 2 goats from each experimental unit (8 goats per treatment) were randomly selected for slaughtering by exsanguination. Before slaughter, the goats were prevented from consuming feed for 24 h and from drinking for 2 h. Jugular blood (20 mL) was sampled into Vacutainer tubes after the goats were prevented from consuming feed for 12 h. Blood was centrifuged at 3000× g for 15 min and serum was harvested, and at last stored at −20 °C for lipid profiles and enzymatic activity analysis. Immediately after death, the ileal digesta were collected, flash-frozen by liquid nitrogen, and stored at −80 °C for microbial diversity analysis.
2.3. Chemical Analysis
The concentrations of TG, CHO, LDL-C, and HDL-C in serum were analyzed in an automatic biochemical analyzer (L-8900) using commercially available kits (Lepu Diagnostics Co., Ltd., Beijing, China). The concentrations of NEFA, GLU, and β-hydroxybutyric acid (BHBA) in serum were analyzed through commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The quantity of acetyl-coa carboxylase (ACC, CK-E75273), fatty acid synthetase (FAS, CK-E75339), malic dehydrogenase (MDH, CK-E75029), lipoprotein lipase (LPL, CK-E75274), hormone-sensitive lipase (HSL, CK-E75402), and lipase (LPS, CK-E75263) were measured by enzyme-linked immunosorbent assay (ELISA) kits (QUANZHOU RUIXIN BIOTECHNOLOGY CO., LTD., Quanzhou, Fujian, China), and the experiments were performed strictly according to the manufacturer’s instructions. Meanwhile, the standard curves of enzymes were established. Briefly, 10 μL serum sample and 40 μL sample diluent were added to the coated plate, and 100 μL diluted enzyme-labeled antibody was added to the plate, incubated at 37 °C for 1 h, then washed five times with phosphate-buffered saline (PBS) (let stand for 1 min). 100 μL substrate Tetramethylbenzidine (TMB) was added and avoided light incubation at 37 °C for 15 min, the reaction was terminated with 50 μL 2 M H2SO4, and the optical density (OD) was measured at 450 nm.
2.4. 16S rRNA Gene Sequencing and Operational Taxonomic Units (OTUs) Picking
2.4.1. DNA Extraction and Checking
Five ileal digesta samples from each treatment group were selected to extract the microbial DNA using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the instruction manual. The final DNA concentration and OD (Optical Density) 260/280 values were determined by NanoDrop 2000 UV-vis (ultraviolet-visible) spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and DNA quality was checked through 1% agarose gel electrophoresis.
2.4.2. PCR (Polymerase Chain Reaction) Amplification and Checking
The amplified region is V3–V4 hypervariable regions of the bacteria 16S rRNA gene. The amplified primers are 338F (5′-ACCHOCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWCHOTAAT-3′), and are universal primers. The procedure of PCR reactions was as follows: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. The mixed volume of PCR reactions was 20 μL: 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs (Deoxynucleotide Triphosphates), 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA) according to the instruction manual.
2.4.3. Illumina MiSeq Sequencing
Purified amplicons were pooled on an Illumina MiSeq PE300 instrument (Illumina, San Diego, CA, USA) which were equimolar and paired-end sequenced (2 × 300) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
2.4.4. Bioinformatics
Raw data was filtered and analyzed using QIIME (Quantitative Insights into Microbial Ecology, version 1.9.1) software, quality-filtered by Trimmomatic and merged by FLASH (Fast Length Adjustment of Short Reads). Low-quality reads were removed with the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window, (ii) primers’ matching allowed 2-nucleotide mismatching, and reads containing ambiguous bases were removed, and (iii) sequences with an overlap longer than 10 bp were merged according to their overlap sequence. The assembled sequences were assigned to operational taxonomic units (OTUs) at 97% similarity cutoff using UPARSE (Highly Accurate OTU Sequences from Microbial Amplicon Reads, version 7.1,
http://drive5.com/uparse/, 30 September 2013) and chimeric sequences were identified and removed using UCHIME (Chimera Prediction for Amplicon Sequencing). OTUs were used for α-diversity (Coverage, Sobs, ACE, Chao, Shannon and Simpson) analysis. OTUs were taxonomically analyzed by the Ribosomal Database Project (RDP) Classifier algorithm (
http://rdp.cme.msu.edu/, 30 September 2016). The rarefaction curves’ analysis with Mothur v.1.21.1 was performed to reflect the sequence depth. Principal coordinate analysis (PCoA) was performed using the weighted Unifrac distance with R Language.
2.5. Statistical Analysis
The data of growth performance, organ weight, blood parameters, and enzymatic activity were analyzed in a completely randomized design using the Proc Mixed procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The model was Yij = μ + Ti + Pj + Ti × Pj + εij, where Yij was the dependent, continuous variable, μ was the overall mean, Ti was the fixed effect of diet treatment (i = basal diet, flaxseed oil, or flaxseed grain), Pj was the random effect of pen (j = 1, 2, 3, and 4), Ti × Pj was the fixed effect of the interaction between diet and pen, and εij was the residual error. The mixed model included fixed effects of diet and random effects of pen. Pen was considered the experimental unit and the repeated measurement. The model used to analyze body weight gain considers 4 replicates (number of pens), each with 5 observations (number of goats): organ weight, blood parameters, and enzymatic activity consider 4 replicates (number of pens), each with 2 observations (number of goats), for each treatment. Specifically, the model used to study feed intake considers 4 replicates (number of pens), each with 1 observation. The results are presented as the mean values and standard error of the mean (SEM). Data means significance was declared at p ≤ 0.05 and tendencies were considered at 0.05 < p ≤ 0.10.
For evaluation of bacterial diversity indexes, the bacteria community structure at phylum and genus level, a one-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out in SAS (SAS Inst. Inc., Cary, NC, USA). The results are presented as the mean values and SEM. Spearman correlation was used to correlate the growth performance, lipid deposition, blood lipid profiles, and blood lipid-related enzyme activity with the top 20 most relatively abundant bacterial genera through R Language (pheatmap package). Correlations with p ≤ 0.05 for the linear model were considered as significant.
4. Discussion
Our previous study has shown that, compared with the addition of flaxseed oil, flaxseed could protect ALA from the hydrogenation in rumen, increase the ALA content in blood, and then increase the ALA accumulation in muscle of goats, which is beneficial to human health [
10,
17]. However, the comparison effects of flaxseed oil and flaxseed grain on lipid deposition and ileum microbiota profiles have not been investigated. The present study indicated that flaxseed is more effective than flaxseed oil in reducing lipid deposition, which may work by modulating the ileal microbiota composition.
Candyrine et al. reported that 4% oil supplementation to diet significantly enhanced the growth performance in fattening sheep and goats [
22]. In the current study, compared with the CON group, the dietary flaxseed oil supplementation significantly increased the FBW and BWG, which agrees with the above report. Compared with the CON treatment, flaxseed oil significantly promoted the FBW, BWG, and lipid accumulation in goats, but the dietary flaxseed supplementation had no effect on the growth performance of goats. However, compared with the LNO group, the dietary flaxseed supplementation reduced the adipose tissue weight, indicating that flaxseed efficiently reduced the lipid deposition of goats. Blood biochemical parameters can reflect the physiological and metabolic state of animals, which is closely related to nutritional status. The present results showed that the dietary flaxseed grain supplementation significantly decreased the content of TG, CHO, and LDL-C in serum, indicating that flaxseed could reduce the blood lipid deposition, which is consistent with the results of changes in adipose tissue weight. In agreement with these findings, the majority of previous studies showed that flaxseed consumption reduced serum content of CHO [
23] and LDL-C [
24] levels. In the current study, compared with the CON and HLS groups, the flaxseed oil had a tendency to increase the serum GLU content and significantly reduced the serum BHBA content, which suggested that the addition of flaxseed oil to the diet reduced lipid mobilization, resulting in an increase of lipid deposition. Compared with the LNO group, flaxseed significantly increased the serum BHBA and NEFA content, which indicated that cashmere goats would mobilize lipid to meet growth needs, so they produced a large amount of BHBA and NEFA. ACC and FAS are the key enzymes involved in de novo FA synthesis and play a key role in animal lipid synthesis [
10]. MDH is closely involved in the trans hydrogenation during the conversion of carbohydrates to fatty acids [
25]. HSL is the key enzyme and rate-limiting enzyme that initially mobilized lipolysis, it gradually hydrolyzes the fat stored in fat cells into free FA and glycerin and releases them into the blood [
26]. In the current study, compared with the LNO treatment, the flaxseed grain significantly decreased the quantity of FAS, ACC, and MDH, but remarkably promoted the HSL quantity. These results suggested that adding flaxseed grain to the diet could reduce the quantity of enzymes related to lipid de novo synthesis and increase the quantity of enzymes related to lipolysis, thereby reducing the blood TG content and TG accumulation in the adipose tissue, which further explained the above-mentioned change of growth performance and lipid deposition. Liu et al. reported that the diets containing unsaturated fatty acids (UFAs) could reduce serum CHO, TG, and LDL-C content, but increase serum HDL-C content [
27]. Egert et al. observed that enrichment of the diet with EPA or DHA decreased fasting serum TG concentration by 15% and 31% in humans, suggesting that DHA is more effective in reducing TG levels in the blood [
28]. Morin et al. reported a lower serum CHO and LDL-C levels of rats in the DHA monoglycerides treatment than the high-fat/high-carbohydrate diet [
29]. In our previous study, the flaxseed grain group had a higher plasma concentration of DHA than the control and supplemented flaxseed oil groups [
10], but in the present study, had a lower serum TG, CHO, and LDL-C content, hinting that flaxseed grain is more efficient to goats in reducing the blood TG, CHO, and LDL-C level because of the decreased plasma DHA. Sawada et al. reported that the EPA significantly improved the HDL-C concentration in blood of humans [
30]. HDL-C removes cholesterol from the bloodstream and carries it back to the liver for recycling [
31], which is beneficial to the health of livestock. In the present study, the diet supplemented with flaxseed oil and flaxseed grain had a tendency to increase the serum HDL-C concentration compared to the CON group, which probably related to the increase of plasma EPA concentration [
10]. Some researches indicated that the lipid-lowering effect of flaxseed is owing to its high content of lignans and soluble fibers [
32,
33]. Lignans are one of the vital groups of phytoestrogens, which inhibit acyl-coenzyme A activity: CHO acyltransferase involved in the formation of cholesteryl ester in tissues [
34,
35]. Soluble fibers can reduce intestinal absorption of dietary CHO and increase the production of bile acid [
33]. In conclusion, supplementation of flaxseed significantly alleviated the lipid accumulation in goats compared to the addition of flaxseed oil, indicating that flaxseed (enriched ALA) may play an important role in regulating lipid metabolism, and therefore it is beneficial to the goats.
In recent years, the gut microbiota has been shown to affect lipid levels and lipid metabolism in blood. In the present study, the dietary flaxseed supplementation significantly reduced the relative abundance of
Firmicutes,
Proteobacteria, and
Tenericutes, while it remarkably increased the
Actinobacteria relative abundance.
Firmicutes is known as obesity-related bacterial phylum, which accelerates degradation of food components to supply energy for the host [
36]. A high abundance of
Firmicutes goes with a low abundance of
Bacteroidetes, which leads to accelerate energy harvest from food and promote energy storage in adipose tissue of the host [
37] and would further suppress the fasting-induced adipose factor (FIAF) produced. Meanwhile, a higher TG was stored in adipose tissue and lower satiety hormones were released by the suppression of FIAF [
38]. In the current study, the dietary flaxseed significantly decreased the
Firmicutes relative abundance compared to the CON and LNO groups, so we speculated that adding flaxseed to the diet promoted the production of FIAF by reducing the
Firmicutes relative abundance, thereby releasing more satiety hormones, reducing the absorption of energy from the diet through ileum, which further explained the decrease in blood TG and fat tissue weight.
Proteobacteria is a major phylum of Gram-negative bacteria with a wide range of pathogenic microorganisms, and a high ratio of
Proteobacteria to
Firmicutes + Bacteroidetes was confirmed as a good indicator for rumen dysbiosis [
39]. The addition of flaxseed decreased the Proteobacteria relative abundance in ileum of goats, which may be caused by competitive relationships between the gut microbiota and pathogenic bacteria in the intestine [
40], indicating that the flaxseed is beneficial to the colonization of probiotic in the goats’ ileum.
Actinobacteria is the dominant bacterial in most mammals [
41]. In the long-term co-evolution process of animal intestinal
Actinobacteria and the host, by producing a variety of bioactive substances, such as antibiotics, immunosuppressants, vitamins, and enzymes, they participate in the metabolism of the host and maintain the intestinal microecological balance of the host and a series of important physiological activities [
42]. Claus et al. reported that the
Actinobacteria was negatively associated with serum GLU level in the microbial colonization process [
43]. The current study observed that, compared to added flaxseed oil, dietary flaxseed supplementation increased the
Actinobacteria relative abundance and had a tendency to reduce the serum GLU content, which is similar to the above-mentioned reports.
[E.] cop. is a CHO-reducing bacterium, and on the one hand, converting CHO to coprostanol could decrease the CHO absorption [
44]. On the other hand, the bacterium might interrupt the enterohepatic circulation of billary CHO so that the liver would partition more CHO into the bile and less CHO into the blood [
45]. In the current study,
[E.] cop. group (16.92%),
Aeriscardovia (14.95%), and
Uncl. Pep. (13.13%) were the 3 most abundant genera in the goats’ ileum of the HLS group, which showed that the flaxseed significantly increased the
[E.] cop. group relative abundance and remarkably decreased the blood CHO content, and that the
[E.] cop. group was positively correlated with the content of HSL and LPL, illustrating that the flaxseed can reduce the absorption of CHO in ileum and increase the degradation of lipid by increasing the relative abundance of
[E.] cop. group. Whon et al. reported that the castrated male cattle harbor distinct ileum microbiota which were dominated by the family
Peptostreptococcaceae, with increased extra- and intra-muscular fat storage [
46]. A previous study revealed that the
Uncl. Pep. might play an important role in feed digestion [
47]. In the current study,
Uncl. Pep. (31.22%),
Intestinibacter (12.01%), and
Ruminococcus_2 (10.61%) were the 3 most abundant genera in the goats’ ileum of the LNO group,
Uncl. Pep. (35.14%) and
Intestinibacter (13.73%) were the 2 most abundant genera in the goats’ ileum of the CON group, the relative abundance of
Uncl. Pep. and
Intestinibacter are significantly higher in the CON and LNO groups than in the HLS group, and the blood TG content showed a consistent trend of change. Meanwhile, the current results showed that the
Uncl. Pep. was negatively associated with the quantity of HSL and LPL, and the
Intestinibacter was negatively associated with the content of BHBA and NEFA. These results hinted that the increased absorption of nutrients in ileum and the reduced mobilization of body fat in goats of CON and LNO groups was due to increasing the relative abundance of
Uncl. Pep. and
Intestinibacter. Jiang et al. reported that the increase of
Ruminococcus relative abundance can increase the digestibility of DM and NDF in vivo and the production performance of cows [
48]. The dietary supplementation of flaxseed oil significantly elevated the
Ruminococcus_2 relative abundance and increased the FBW and BWG of goats, and the
Ruminococcus_2 genus was positively associated with FBW, BWG, Omental Fat, GLU, CHO, LDL-C, ACC, and MDH, but negatively correlated with BHBA, NEFA, and HSL. Therefore, these results hinted that the flaxseed oil can promote the absorption of nutrients and reduce lipid mobilization, leading to much more GLU and TG flowed into blood, more lipid deposition in adipose tissue, and increasing the FBW of goats by increasing the
Ruminococcus_2 relative abundance.
The present study investigated the shift among blood lipid profiles in cashmere goats in response to dietary supplementation of flaxseed or flaxseed oil, which probably related to the change of ileal microbial composition, but the mechanism is unclear. Therefore, the ileal metabolites of goats fed a diet containing flaxseed or flaxseed oil needed to be measured to further elucidate the mechanism in the future. The [E.] cop. is the most relatively abundant genus in the ileum of HLS goats and the Ruminococcus_2 is the third top genus in the ileum of LNO goats, and they are strongly associated with host lipid metabolism, so the function of [E.] cop. and Ruminococcus_2 on lipid metabolism needed to be verified in vitro. In addition, further study is needed to test other intestinal microbes (duodenum, jejunum, cecum, and colon), to better interpret the microbiota compositions and the shift of microbiota in response to dietary supplementation of flaxseed or flaxseed oil.