Effects of Different Supplemental Levels of Eucommia ulmoides Leaf Extract in the Diet on Carcass Traits and Lipid Metabolism in Growing–Finishing Pigs

This study examined the effects of dietary Eucommia ulmoides leaf extract (ELE) supplements on carcass traits and lipid metabolism in growing–finishing pigs. A total of 144 crossbred (Duroc × Landrace × Yorkshire) piglets with an average initial weight of 10.11 ± 0.03 kg were randomly allotted to four treatment groups, each with six replicates and six piglets per replicate. Each group of pigs was fed a basal diet or a diet supplemented with increasing levels of ELE (0.1, 0.2, or 0.3%). The results showed that adding ELE had no negative effect on the growth performance of pigs. Dietary supplements of 0.1% ELE significantly increased carcass weight (p < 0.01), dressing percentage (p < 0.01), carcass length (p < 0.05), and eye muscle area (p < 0.05). Compared with the control group, a 0.2% ELE supplement significantly increased (p < 0.01) the levels of adiponectin, insulin-like growth factor 1, and hormone-sensitive lipase and lipoprotein lipase activity in the serum. Histological examination showed that ELE inhibited fat deposition in the backfat tissue. Lipid metabolism-related biochemical indices and mRNA expression levels were improved after supplementing diets with ELE. Moreover, all three levels of ELE dramatically upregulated (p < 0.05) the protein levels of p-AMPK-α and p-ACC. In summary, adding ELE to pig diets could improve the carcass traits of growing–finishing pigs and exert a lipid-lowering effect by activating the AMPK-ACC pathway and regulating mRNA expression levels related to lipid metabolism. Supplementing the diet with 0.1–0.2% ELE is the optimal range to reduce fat deposition in pig backfat tissue.


INTRODUCTION
Obesity is becoming one of the most important health problems in several countries, affecting scores of people, as it increases the risk of various diseases, such as fatty liver, diabetes, and coronary heart disease (1). In recent years, an antiobesity effect of Eucommia ulmoides has been supported by an increasing number of studies. Two prevenient studies have reported that Eucommia ulmoides improved hyperglycemia in diabetic rats (2) and type 2 diabetes patients (3). Moreover, Eucommia ulmoides promoted the recovery of lipid metabolism disorders caused by a high-fat diet in rats (4).
Eucommia ulmoides (Chinese: Duzhong), also known as Gutta-percha tree, Sixian, and Sizhong, is a perennial deciduous tree of the Eucommiaceae family (5). Eucommia ulmoides is widely distributed in China, with a high annual yield (6). Its medicinal history can be traced back thousands of years and is now widely used in clinics (5). Eucommia ulmoides is rich in lignans, iridoid terpenoids, flavonoids, polysaccharides, and other active components, with antihypertensive, hypoglycemic, anti-inflammatory, liver protection, antitumor, and other pharmacological effects (5,7,8). Studies into the potential of Eucommia ulmoides as a feed supplement in Chinese herbal medicine have been gradually developed. Previous studies focused on the effects of Eucommia ulmoides leaf and its extracts on growth performance and antioxidant activity in pigs (9)(10)(11). However, at present, there are few studies on the effect of Eucommia ulmoides leaf extracts (ELE) on lipid metabolism in growing-finishing pigs, and the optimal supplement level is also unknown. Since there are many similarities between pigs and humans in terms of structure and function, the effect of ELE as a dietary supplement in pigs can be used as a model for the study of human nutrition and metabolism (12).
Following from previous research, we added dietary supplements of 0, 0.1, 0.2, or 0.3% ELE to growing-finishing pig diets and recorded the effects on growth performance, carcass traits, and lipid metabolism. This provides a basis for the wider application of ELE in animal husbandry and reducing the incidence of human obesity.

Preparation of ELE
ELE were purchased from Zhangjiajie Hengxing Biotechnology Co., Ltd. (Zhangjiajie, China). Data provided by the company show that the main active ingredients include 5% chlorogenes, 8% EL flavonoids, and 20% EL polysaccharides.

Animals and Diets
The animal experiments were approved by the Committee on Animal Care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. A total of 144 crossbred barrows (Duroc × Landrace × Yorkshire, DLY, 10.11 ± 0.03 kg) were randomly divided into four treatments, six replicates in each treatment, and six pigs in each replicate. The experimental diets were as follows: (1) control diet; (2) control diet + 0.1% ELE; (3) control diet + 0.2% ELE; (4) control diet + 0.3% ELE. All the growing-finishing pigs were raised in pens and had ad libitum access to diets and clean drinking water. All pigs were weighed when the pigs in the control group weigh 10, 30, 70, and 115 kg, and feed intake was recorded every week to calculate the average daily gain (ADG), average daily feed intake (ADFI), and the ratio of feed to gain (F/G). The experiment used a corn-soybean meal diet referred to NRC (1998NRC ( , 2012. The ingredients and nutritional composition of basal diet are shown in Table 1.

Sample Collection
At the end of the trial, all the pigs were fasted overnight (12 h), and one or two pigs of each replicate with average final body weight was selected (8 pigs/treatment) to slaughter by electrical stunning in a commercial abattoir. Before slaughter, blood samples were collected into a plain tube and placed at room temperature for 30 min, then centrifuged at 3,000 × g for 10 min at 4 • C. Serum was collected and stored at −80 • C for further analysis (13). The backfat samples were immediately excised and stored at −20 • C for determination of the chemical composition or placed in liquid N 2 and then stored at −80 • C for the analysis of quantitative realtime PCR. Fresh samples of backfat (1 cm 3 ) were fixed in paraformaldehyde fixative for paraffin sections and hematoxylin and eosin staining.

Carcass Trait Analysis
At slaughter, the carcass and the left side of carcass were weighted so that slaughter rate could be calculated. Other carcass traits including carcass length (carcass straight length and carcass slant length), average backfat thickness (the 3rd−4th lumbar spine, the 10th−11th lumbar spine, and the last rib), and loin-eye area were measured from the left side of the carcass. The left side of the carcass was split up into skeletal muscle and fat as previously described (14). The fat mass rate percentage and lean mass percentage were weighed and calculated.

Backfat Tissue Histological Analysis
The mean cross-sectional area and quantity of adipocyte in backfat tissue were measured by classic hematoxylin and eosin staining. Serial tissue sections of 4 µm were sliced using a paraffin slicer (RM 2016, Shanghai Leica Instrument Co., Ltd., Shanghai, China). The slices were dyed with hematoxylin dye solution for 3-5 min, washed with tap water and dehydrated with 85 and 95% gradient alcohol for 5 min, respectively, then dyed with eosin dye solution for 5 min, dehydrated with absolute ethanol, and finally sealed with neutral gum. The stained slides are scanned with a panoramic slice scanner of Pannoramic DESK/MIDI/250/1000 (3DHISTECH, Budapest, Hungary), the scanned slices are opened with CaseViewer 2.4 software (3DHISTECH, Hungary), the field of view is intercepted, and Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) is used for calculation and analysis.

Total RNA Isolation and Quantitative Real-Time PCR Analysis
Total RNA isolation and real-time quantitative PCR were conducted as previously described (15). In brief, total RNA was extracted from backfat tissue samples using TRIzol Reagent (Hunan Aikerui Bioengineering Co., Ltd., Changsha, China). The purity of the total RNA was verified using a NanoDrop ND2000 (

Western Blot Analysis
An appropriate amount of backfat tissue sample was weighed and added to RIPA lysate for ice lysis, and then BCA protein assay kits (Beyotime Biotechnology, Shanghai, China) were used to measure the protein concentration. Next, SDS-PAGE electrophoresis was carried out. Firstly, the glass plate was cleaned, and then the glue with an appropriate concentration was prepared according to the protein concentration of the sample. The loading amount was calculated, and β-mercaptoethanol was added to the equal-volume buffer and 1/10-volume buffer, mixed well, put into the Mastercycler nexus PCR instrument (Eppendorf, Hamburg, Germany), and mixed well. After adding samples, electrophoresis was carried out, and then the membrane was transferred. After sealing the membrane, the primary antibody and secondary antibody were incubated for color development.

Statistical Analysis
All experimental data were analyzed using one-way analysis of variance (ANOVA) of SPSS (version 26.0, SPSS Inc., Chicago, IL, USA), and then the Duncan multiple-comparison test was performed. Results were expressed as mean and SEM, p < 0.05 was considered significant, and 0.05 ≤ p < 0.10 was considered as trend. Table 3 shows that from 10 to 30 kg, there was no significant difference (p > 0.05) in ADG, ADFI, or F/G with increasing levels of ELE supplements. At the 10-70-kg stage, ADG was higher in the group supplemented with 0.1% ELE (p < 0.05) than in the other groups. F/G was lower in the 0.1% ELE group (p < 0.05) than in the other groups, but there was no dramatic discrepancy (p > 0.05) compared with the control group. Adding 0.2 or 0.3% ELE to the diet could markedly improve ADFI (p < 0.05). Over the whole period of the experiment, ADFI was higher (p < 0.05) in the 0.1% ELE group than in the 0.3% group, but there were no observable change in ADG or F/G among the different treatments.  Table 4 shows that carcass weight (p < 0.05), slaughter rate (p < 0.01), and carcass straight length (p < 0.05) in the 0.1% ELE group were markedly higher than those in the other three groups. Meanwhile, compared with the control group, dietary supplemented with 0.1% ELE observably aggrandized the loineye area (p < 0.05) of growing-finishing pigs. In addition, adding 0.3% ELE notably increased the lean meat rate (p < 0.01) of growing-finishing pigs compared with the other three groups.

Carcass Trait
Serum Biochemical Indexes Table 5 shows that adding ELE in the diet memorably descended the content of TP (p < 0.05), TG (p < 0.05), and VLDL-C levels (p < 0.01) compared with the control group; meanwhile, it increased serum ALB (p < 0.01) and HDL-C levels (p < 0.01).
Serum Cytokine Levels Table 6 shows that dietary supplementation with 0.2% ELE could signally elevate the levels of ADPN (p < 0.01) and IGF-1 (p < 0.01) in serum. Serum leptin and insulin levels were not changed dramatically (p > 0.05) among the groups.

Activity of Enzymes Related to Lipid Metabolism
The activities of HSL, LPL, and ACAT first increased and then decreased as the level of ELE increased in the diet Frontiers in Veterinary Science | www.frontiersin.org  ( Table 7). ACC activity was lower (p < 0.01) at 0.1% ELE than at other levels. Compared with the control group, diet supplemented with 0.1% and 0.2% ELE notably enhanced (p < 0.01) HSL and LPL activities. Besides, the ATGL activity of the 0.1% ELE group exceeded (p < 0.05) that of the other two treatment groups, but there was no marked difference compared with the control group. In  addition, ACAT activity was no marked discrepancy among the four groups. Figure 1 shows that all the supplementary levels of ELE significantly decreased (p < 0.05) the mean cross-sectional area of adipocytes and increased (p < 0.05) the total number of adipocytes in backfat tissue. Figure 2 shows that dietary supplementation with 0.1% and 0.2% ELE could downregulate (p < 0.05) the mRNA expression levels of adipogenic genes such as ACC, FAS and SREBPS1c (Figure 2A) and upregulate (p < 0.05) the mRNA expression levels of lipid-lowering genes, such as HSL, ATGL and SREBP1c, but there was no dramatic variation (p > 0.05) in LPL in this study (Figures 2A-C). In the 0.2% ELE group, the mRNA expression levels of CPT1 and AMPK-α increased significantly (p < 0.05), but that of PPARγ did not change dramatically (p > 0.05) (Figures 2B,C). In addition, compared with the control group, supplementing with ELE markedly increased (p < 0.05) the mRNA expression levels of FAT/CD36 and FABP4, and 0.2% ELE decreased the mRNA expression level of FATP1 ( Figure 2D).

Western Blotting Analysis
Relative protein expression levels for AMPK-α, p-AMPK-α, ACC, and p-ACC were determined by using Western blotting.
The results showed that all three different levels of ELE upregulated (p < 0.05) the relative protein expression levels of p-AMPK-α and p-ACC (Figures 3A,B).

DISCUSSION
Previous studies have shown that ELE is rich in amino acids, minerals, and other nutrients. The essential amino acid content in ELE is high, of which leucine is the highest, followed by valine (5). In addition, iridoids, phenols, and flavonoids are abundant in ELE, which reduces blood lipids (17,18) and improves diabetes (19) and antioxidation (7). In recent years, Eucommia ulmoides is considered to be a very useful feed additive in healthy livestock and poultry breeding. This study compared the effects of different supplementary levels of ELE in the diet on growth performance, carcass traits, and lipid metabolism in pigs. Growth performance directly affects the meat growth performance of growing-finishing pigs, thus affecting the economic return. These results showed that supplementing pig diets with different levels of ELE had no significant effect on ADG, ADFI, or F/G in piglets, which was consistent with a previous study (9). This might have been due to the strong aromatic compounds in E. ulmoides leaf, which might have affected the palatability of the feed. However, our results differ from some previous studies (10,11), which might have been due to different processing technologies and amounts of ELE supplements used, while ELE supplements had no negative effects on growth performance. Human consumption of meat products containing a large amount of fat may pose a threat to health; long-term consumption may induce cardiovascular diseases and obesity (20). Compared with the control group, the average backfat thickness of the three treatment groups decreased by 2.7, 4.6, and 7.4%, respectively. Meanwhile, fat mass percentage in the three treatment groups decreased by 5.1, 3.2, and 1.2%, respectively. Unfortunately, none of them reached a significant level. However, the histomorphological analysis of backfat tissue showed that ELE significantly reduced the average cross-sectional area of adipocyte; the more mature a fat cell is, the larger it is (21), indicating that ELE effectively inhibited the growth and maturation of adipose cells. In conclusion, ELE had a potential inhibitory effect on fat accumulation in back fat tissue; this might be related to chlorogenic acid, the most important active ingredient in ELE. Dietary supplementation with 0.5% and 1% chlorogenic acid was previously reported to reduce the accumulation of visceral fat and lipid content in rats (22). Moreover, dietary supplementation with 0.2% ELE significantly increased carcass weight, slaughter rate, carcass straight length, and loin-eye area, which was consistent with our expectations, indicating that low-dose ELE could improve the carcass traits of growing-finishing pigs.
The changes in serum biochemical indexes can affect the metabolism and nutrient deposition of animals and are affected by the growth stage, endocrine status, and dietary nutrient level (23). We examined the indexes related to lipid and nitrogen metabolism in serum of growing-finishing pigs. Our results showed that adding ELE to the diet increased the serum ALB content and decreased the TP content in growing-finishing pigs, indicating that ELE was beneficial to the overall health of pigs. HDL, a "vascular scavenger, " has an anti-atherosclerosis function (24) and can prevent coronary heart disease (25). VLDL is known as an atherogenic factor. It is reported that Eucommia ulmoides reduce the levels of triglycerides and total cholesterol in the plasma of finishing pigs (9). In this study, ELE markedly improved HDL content and decreased the levels of VLDL and TC, which indicated that ELE effectively improved lipid metabolism and cardiovascular health in growing-finishing pigs and that the chlorogenic acid and geniposidic acid contained in ELE could play a vital role in antiobesity. Studies have shown that both chlorogenic acid and geniposidic acid from ELE reduce serum TG and TC in obese mice (17,18).
Adiponectin is secreted by adipocytes and has insulinsensitizing, anti-atherosclerotic, and anti-inflammatory effects (26). Previous studies have shown that adiponectin promotes the oxidation of fatty acids in muscle and adipose tissue (27). Adiponectin can increase HDL levels and decrease TG levels (28). IGF-1 is a hormone that is closely related to metabolic syndrome and is mainly secreted by the liver cells. This is related to carbohydrate and lipid metabolism (29). Recombinant IGF-1 enhances the lipolysis of adipose tissue, increases the rate of lipid oxidation (30), and promotes the use of free fatty acids in muscle (29). In this study, supplementation with ELE boosted the concentrations of adiponectin and IGF-1 in circulation, which indicates that it plays a lipid-lowering role by regulating hormone levels in growing-finishing pigs.
To explore whether ELE had a similar effect on enzymes related to lipid metabolism, we measured the activities of enzymes related to lipid metabolism in the serum of fattening pigs. AAC is a well-known rate-limiting enzyme (31)(32)(33). HSL and ATGL are two important lipases in the animal body. HSL can hydrolyze TG, diglyceride, monoglyceride, cholesterol ester, retinol ester, and other lipids and produce glycerol and free fatty acids (34,35). ATGL is highly expressed in adipose tissue and is highly specific for TG (36). Moreover, LPL is a rate-limiting enzyme for the degradation of blood triglycerides to glycerol and free fatty acids (37). In addition, ACAT is the only enzyme in the body that can catalyze cholesterol to produce cholesterol esters. Excessive cholesterol esters may lead to atherosclerosis (38). In the present study, although ELE did not dramatically change ACAT activity, dietary supplementation with 0.1% ELE increased HSL, LPL, and ATGL activities while decreasing ACC activity, enlightening the effect of ELE on reducing serum TG which could be achieved by regulating the activities of these enzymes. In addition, we also analyzed the histomorphology of backfat, and the results showed that the addition of ELE markedly reduced the mean cross-sectional area of adipocyte and increased the number of backfat adipocytes. It is proved that ELE can inhibit fat deposition.
We further examined the mRNA expression levels of enzymes and cytokines related to lipogenesis (ACC and FAS), lipolysis (HSL, LPL, and ATGL), fatty acid oxidation (CPT1B, AMPK α), fatty acid transportation (FATP1, FAT/CD36, and FABP4), and lipid deposition (SREBP-1c, PPARγ) to determine the molecular mechanism by which ELE regulates lipid metabolism in growing-finishing pigs. In this study, all three levels of ELE downregulated the mRNA expression of ACC and FAS, and 0.2% ELE significantly downregulated the expressions of SREBP1c and FATP1. The level of PPARγ mRNA showed a downward trend but did not reach a memorable level. These results showed that 0.1 and 0.2% ELE could effectively reduce the mRNA expression levels of adipogenesis genes. In addition, supplementing with 0.1 and 0.2% ELE upregulated the mRNA level expressions of HSL, ATGL, CPT1B, AMPK, FAT/CD36, and FABP4. Compared with the control group, the 0.3% ELE supplement also significantly upregulated the expressions of FAT/CD36 and FABP4 mRNA. Additionally, the 0.3% ELE supplement downregulated the mRNA expression level of HSL, suggesting that the lipid-lowering effect of ELE may decrease when the dosage exceeds 0.2%. The mRNA expression levels of HSL and ATGL were consistent with the observations that ELE increased HSL and ATGL enzyme activities in growing-finishing pigs. These results also reveal that ELE can exert a lipid-lowering effect by downregulating the mRNA expression levels of lipidproducing genes and upregulating the mRNA expression levels of lipid-lowering genes.
AMPK and ACC are not only the key links in their metabolic regulation but also closely related to each other, which can form upstream and downstream signal pathways in cells. The AMPK-ACC signaling pathway formed by AMPK and its downstream target ACC has important physiological significance in the process of fat synthesis and oxidation (39). When activated by adiponectin, AMPK phosphorylation inactivates ACC phosphorylation, which catalyzes the formation of malonyl-CoA. Malonyl-CoA is the substrate for fatty acid biosynthesis, which inhibits fatty acid oxidation (40). Therefore, we speculate that ELE has a lipid-lowering effect through the AMPK-ACC pathway. We measured the protein expression level of AMPKα and ACC and the expression level of phosphorylated proteins. As expected, all the three levels of ELE remarkably improved the protein levels of p-AMPK-α and p-ACC and showed a downward trend with the increase in dosage.

CONCLUSIONS
The addition of ELE < 0.3% in growing-finishing pigs could partially improve the carcass traits of growing-finishing pigs and had no adverse effect on growth performance. The 0.1% ELE supplement improved carcass traits, and the 0.1 and 0.2% ELE supplement can reduce the level of TG in serum and increase the level of hormones and enzyme activity that promote fat catabolism. The mRNA and protein expression levels of the key genes related to lipid metabolism showed that the lipid-lowering mechanism of ELE may be through the activation of the AMPK-ACC pathway to inhibit fat deposition in backfat tissue, and the lipid-lowering effect of the 0.1 and 0.2% ELE supplement was the best. However, when the supplemental level was 0.3%, there was no significant effect on carcass traits and lipid metabolism of growing-finishing pigs, and some indexes even had negative effects. In conclusion, the supplemental range of 0.1 to 0.2% ELE is the optimal addition. ELE contains a variety of bioactive components; which component plays a leading role needs our further study using the cell culture model.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ETHICS STATEMENT
The animal study was reviewed and approved by Committee on Animal Care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences.

Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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