DFA-IV enhanced growth performance and relative organ weight of muscle

The average daily gain (ADG) was enhanced by DFA-IV (0.01, 0.05, and 0.1%) supplementation in feeding phases I and I + II compared with the control (Fig. 1A). DFA-IV supplementations caused no substantial differences in the average daily feed intake (ADFI) compared with the control (Fig. 1B). The 0.1% DFA-IV supplementation decreased the feed conversion ratio (FCR) in phase I compared with the control (Fig. 1C). Furthermore, 0.05 and 0.1% DFA-IV supplementation effectively increased the relative breast muscle weight compared with the control (Fig. 2). However, DFA-IV supplementation showed no marked differences in the relative liver weight compared with the control (Fig. 2).

Figure 1 Effect of dietary difructose anhydride (DFA)-IV supplementation on growth performance of broilers in vivo. Broilers were randomly allocated to four groups: CON, corn-soybean meal-based control; 0.01% DFA-IV, corn-soybean meal-based control plus 0.01% DFA-IV; 0.05% DFA-IV, corn-soybean meal-based control plus 0.05% DFA-IV; and 0.1% DFA-IV, corn-soybean meal-based control plus 0.1% DFA-IV. (A) ADG, (B) ADFI, and (C) FCR of broilers in four groups over time during experiments. Diets were fed in two phases (phase I and II from d 0 to 21, and d 21 to 35, respectively, and phase I + II from d 0 to 35). a,bp < 0.05, between treatments based on Duncan’s multiple range tests. Error bars indicate standard errors (SEs) of analyses (n = 12). ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio. Full size image

Figure 2 Effect of dietary difructose anhydride (DFA)-IV supplementation on relative breast muscle and liver weight of broilers in vivo. a,bp < 0.05, between treatments based on Duncan’s multiple range tests. Error bars indicate standard error (SE) of analyses (n = 10). Full size image

DFA-IV increased expression of genes related to growth and muscle development

To confirm whether DFA-IV enhanced growth performance and relative breast muscle weight, we examined its effects on the expression pattern of genes related to these phenomena in the liver and breast muscle respectively. DFA-IV significantly increased the gene expression of insulin growth factor 1 receptor (IGF1R, p < 0.01), IGF2R (p < 0.05), leptin receptor (LEPR, p < 0.05), and growth hormone receptor (GHR, p < 0.05) in the liver (Fig. 3A). DFA-IV significantly decreased the gene expression of myostatin (MSTN, p < 0.01) and increased that of myogenic differentiation 1 (MYOD1, p < 0.01), myogenin (MYOG, p < 0.05), and myogenic factor 5 (MYF5, p < 0.05) in breast muscle (Fig. 3B).

Figure 3 Quantitative gene expression of growth- and muscle-related genes following dietary difructose anhydride (DFA)-IV supplementation in broilers. (A) Expression pattern of growth-related genes (insulin-like growth factor receptor 1 [IGFR1], IGFR2, growth factor receptor [GHR], and leptin [LEPR]) in liver following feeding with DFA-IVs. (B) Expression pattern of muscle development-related genes (myostatin, [MSTN], myogenic differentiation 1 [MYOD1], myogenin [MYOG], and myogenic factor 4 [MYF4]) in muscle following feeding with DFA-IVs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised relative to the expression of the GAPDH as an endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. Significant differences between control and treatment groups were indicated as *p < 0.05 and **p < 0.01. Error bars indicate standard errors of triplicate analyses. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Full size image

DFA-IV improved digestibility and blood concentration of calcium and iron

To investigate the effect of DFA-IV on calcium and iron absorption, their concentration and digestibility were analysed. Supplementation with 0.1% DFA-IV markedly improved the digestibility of calcium compared with the untreated control (Fig. 4A). Additionally, 0.05 and 0.1% DFA-IV supplementation improved the digestibility of iron compared with the untreated control. Serum calcium concentration was increased by 0.1% DFA-IV supplementation on d 21 and 0.05 and 0.1% DFA-IV supplementation on d 35 (Fig. 4B). Supplementation with 0.1% DFA-IV markedly improved the serum iron concentration compared with the untreated control (Fig. 4C).

Figure 4 Effect of dietary difructose anhydride (DFA)-IVs supplementation on (A) digestibility and (B and C) blood concentration of calcium and iron of broiler at 21 and 35d in vivo. a,b,cp < 0.05, between treatments based on Duncan’s multiple range tests. Error bars indicate standard error (SE) of analyses (n = 10). Full size image

DFA-IV treatment increased expression of genes related to calcium absorption in small intestine

To confirm whether DFA-IV supplementation increased calcium and iron absorption, we examined its effects on the expression pattern of genes related to calcium transport, ATPase, and calcium channels in the duodenum, jejunum, and ileum. DFA-IV significantly increased the expression of solute carrier family 8 member 1 (SLC8A1, SLC8B1 [both p < 0.05], and SLC24A1 [p < 0.01]) in the duodenum (Fig. 5A) while SLC24A1 expression was also increased in the ileum (p < 0.05).

Figure 5 Quantitative gene expression of calcium absorption related genes by dietary 0.1% difructose anhydride (DFA)-IV supplementation on broilers. (A) Expression pattern of calcium carrier proteins (solute carrier family 8 sodium/calcium exchanger member 1 [SLC8A1, SLC8B1, and SLC24A1]) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. (B) Expression pattern of ATPase (ATPase, Ca++ transporting, cardiac muscle fast twitch 1 [ATP2A1, ATP2B1, and ATP13A4]) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. (C) Expression pattern of calcium-sensing receptor 1, CASP in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised relative to the expression of the GAPDH as an endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. Significant differences between control and treatment groups were indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate the standard errors of triplicate analyses. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Full size image

DFA-IV significantly increased the gene expression of ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 (APT2A1, p < 0.01) in the jejunum and ileum (Fig. 5B). ATP2B1 expression was increased in the duodenum, jejunum (both p < 0.001), and ileum (p < 0.05) following DFA-IV supplementation. DFA-IV also significantly increased the expression of APT13A4 in the duodenum (p < 0.05), jejunum, and ileum (both p < 0.001). The calcium sensing receptor (CARR, p < 0.001) expression was increased in the duodenum, jejunum, and ileum by DFA-IV supplementation (Fig. 5C).

DFA-IV significantly increased the expression of calcium channel, voltage-dependent, P/Q type, alpha 1 A subunit (CACNA1A, p < 0.05) and CACNB1 (p < 0.001) in the duodenum (Fig. 6). The CAC, gamma subunit 1 (CACNG1) expression was increased in the duodenum (p < 0.001) and jejunum (p < 0.01) by DFA-IV supplementation. DFA-IV significantly increased the expression of the two pore segment channel 1 (TPCN1) in the duodenum (p < 0.05), jejunum (p < 0.01), and ileum (p < 0.05), as well as TPCN2 expression in the duodenum, jejunum (both p < 0.001), and ileum (p < 0.05) while TPCN3 expression also increased the in the ileum (p < 0.01).

Figure 6 Quantitative gene expression of calcium channel-related genes following dietary 0.1% difructose anhydride (DFA-IV) supplementation in broilers. (A) Expression pattern of calcium channels (calcium channel, voltage-dependent, P/Q type, alpha 1 A subunit [CACNA14, CACNB1, and CACNG1]) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. (B) Expression pattern of two-pore calcium channels (two-pore calcium channel 3, [TPCN1, TPCN2, and TPCN3]) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised relative to the expression of the GAPDH as an endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. Significant differences between control and treatment groups were indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate the standard errors of triplicate analyses. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Full size image

DFA-IV treatment improved intestinal wound healing

To investigate whether the DFA-IV enhances intestinal wound healing, we analysed the expression pattern of related genes that modulate cell migration, proliferation, and differentiation. The effect of DFA-IV supplementation on the expression of genes related to migration in the small intestine is shown in Fig. 7A. DFA-IV significantly increased the expression of matrix metallopeptidase 2 (MMP2, p < 0.01) in the duodenum and jejunum and MMP9 (p < 0.05) in the jejunum and ileum. Cadherin 1 (CDH1) expression was decreased in the duodenum (p < 0.01), jejunum, and ileum (both p < 0.05) following DFA-IV supplementation.

Figure 7 Quantitative gene expression of intestinal wound healing related genes following dietary 0.1% difructose anhydride (DFA)-IV supplementation in broilers. (A) Expression pattern of migration-related genes (MMP2, MMP9, and CDH1) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. (B) Expression pattern of proliferation-related genes (CDX1, PCNA, and TP53) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. (C) Expression pattern of the differentiation-related gene (RND3) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised to expression of GAPDH as the endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. Significant differences between control and treatment groups were indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate the standard errors of triplicate analyses. MMP, matrix metallopeptidase; CDH1, cadherin 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase, CDX, caudal type homeobox; PCNA, proliferating cell nuclear antigen; TP53, tumour protein p53. Full size image

The effect of DFA-IV supplementation on the expression of genes related to small intestinal cell proliferation is shown in Fig. 7B. DFA-IV significantly increased the expression of caudal type homeobox 2 (CDX2) in the duodenum (p < 0.01) and jejunum (p < 0.05), and proliferating cell nuclear antigen (PCNA, p < 0.01) in the duodenum and ileum. The tumour protein p53 (TP53, p < 0.01) expression was increased in the duodenum and ileum by DFA-IV supplementation. The effect of DFA-IV supplementation on the expression of genes related to small intestinal cell differentiation is shown in Fig. 7C. DFA-IV significantly increased the expression of Rho family GTPase 3 (RND3) in the duodenum (p < 0.001), jejunum (p < 0.05), and ileum (p < 0.001).

To investigate whether DFA-IV treatment affects intestinal wound healing in vitro, we determined the appropriate dose of DFA-IV in IPEC-J2 cells by monitoring their viability. Pre-treatment with 100 and 200 µM DFA-IV for 24 h decreased the viability of IPEC-J2 cells (Fig. 8A). On the basis of these results, DFA-IV at 50 µM was considered safe and used for subsequent experiments. Furthermore, DFA-IV treatment stimulated IPEC-J2 cell growth in a time-dependent manner and significantly reduced wound width in scratch assays in IPEC-J2 cells (Fig. 8B and C). In the LPS-challenged IPEC-J2 cells, the expression of MMP2, MMP4, CDH1, and RND3 was significantly lower than that in the control cells. DFA-IV treatment significantly increased the expression of MMP2, MMP4, CDH1, and RND3 in LPS-challenged IPEC-J2 cells (p < 0.05, Fig. 8D).

Figure 8 Difructose anhydride (DFA)-IV regulated intestinal wound healing in vitro. (A) Cytotoxicity of DFA-IV in IPEC-J2 cells. Cell viability was determined using MTT assays. IPEC-J2 cells were incubated with DFA-IV (0–200 µM) for 24 h. a,bp < 0.05, between treatments based on Duncan’s multiple range tests. Error bars indicate standard errors (SEs) of analyses (n = 3). (B) The number of viable cells was determined 12, 24, 36, and 48 h after treatment with DFA-IV (50 μM) using WST-1 assays (n = 5). Significant differences between control and treatment groups are indicated as **P < 0.01 and *P < 0.05. (C) Effects of DFA-IV (50 μM) on migration in IPEC-J2 cells at 24 h (n = 3). Significant differences between control and treatment groups are indicated as *P < 0.05. (D) Expression of wound healing-related genes, such as MMP2, MMP9, CDH1, and RND3, analysed with or without DFA-IV (50 μM) after lipopolysaccharide (LPS) challenge in IPEC-J2 cells using real-time polymerase chain reaction (PCR). Quantitative reverse transcription (qRT)-PCR data were normalised to expression of GAPDH as an endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. a,bp < 0.05 between treatments based on Duncan’s multiple range tests. Error bars indicate standard error (SE, n = 3). MMP, matrix metallopeptidase; CDH1, cadherin 1; RND3, Rho family GTPase 3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; WTS, water-soluble tetrazolium. Full size image

DFA-IV treatment improved intestinal barrier function

To investigate whether the DFA-IV enhances the intestinal barrier function, we evaluated the expression pattern of genes related to tight junctions in the small intestine. The tight junction protein 1 (TJP1) and TJP2 (p < 0.01 and p < 0.05) expression was increased in the duodenum and jejunum, respectively by DFA-IV supplementation (Fig. 9). DFA-IV increased the expression of TJP3 (p < 0.05) in the duodenum, jejunum, and ileum while that of occludin1 (OCLN, p < 0.01) was increased in the jejunum and ileum by DFA-IV supplementation.

Figure 9 Quantitative gene expression of intestinal barrier function related genes following dietary 0.1% difructose anhydride (DFA)-IV supplementation on broilers. Expression pattern of tight junction protein (TJP1, TJP2, TJP3, and OCLN) in duodenum, jejunum, and ileum following feeding with 0.1% DFA-IV. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised to expression of GAPDH as an endogenous control and calculated using the 2ΔΔCt method, where ΔΔCt = (Ct of the target gene − Ct of GAPDH) treatment − (Ct of the target gene − Ct of GAPDH) control. Significant differences between control and treatment groups were indicated as *p < 0.05 and **p < 0.01. Error bars indicate the standard errors of triplicate analyses. OCLN, occludin. Full size image

To determine whether DFA-IV treatment affects the intestinal barrier function in vitro, the TEER and permeability of FD-4 were examined in IPEC-J2 cells. The permeability of FD-4 in LPS-challenged IPEC-J2 cells was significantly higher than that of the control cells (p < 0.05, Fig. 10A). Pre-treatment with DFA-IV substantially reduced the permeability of FD-4 in LPS-challenged IPEC-J2 cells. The TEER in LPS-challenged IPEC-J2 cells was significantly lower than that of the control cells was (p < 0.05, Fig. 10B). Pre-treatment with DFA-IV markedly enhanced the TEER in LPS-challenged IPEC-J2 cells. Next, we investigated whether DFA-IV affects tight junction complexes in LPS-challenged IPEC-J2 cells. The results of immunocytochemistry (Fig. 10C) and western blotting assay (Fig. 10D) demonstrated that TJP1expression level was significantly decreased in IPEC-J2 cells treated with LPS compared with that in control cells and that treatment with DFA-IV in LPS-challenged cells preserved the expression of TJP1. The mRNA expression levels of tight junction protein-1 (TJP-1) and OCLN were significantly lower in LPS-challenged IPEC-J2 cells than they were in the control cells (p < 0.05, Fig. 10E). Pre-treatment with DFA-IV significantly increased the expression of TJP-1and OCLN in LPS-challenged IPEC-J2 cells (p < 0.05).

Figure 10 Protective effects of difructose anhydride (DFA)-IV on intestinal barrier function following lipopolysaccharide (LPS) challenge. (A) Treatment with DFA-IV (50 μM) increased transepithelial-electrical resistance (TEER) following LPS challenge in IPEC-J2 cells (n = 3). (B) Treatment with DFA-IV (50 μM) decreased permeability of fluorescein isothiocyanate-labelled dextrans of 4 kDa (FD-4) following LPS challenge in IPEC-J2 cells (n = 3). Immunofluorescence staining (C) and western blotting (D) showing the effects of DFA-IV on the expression of tight junction protein 1 (TJP1) in LPS-challenged IPEC-J2 cells. Nuclei were stained with DAPI and arrowhead indicates TJP1 in IPEC-J2 cells. (E) Relative quantitative expression of genes encoding tight junctions (TJP1 and OCLN) for DFA-IV (50 μM) treatment after LPS treatment in IPEC-J2 cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalised to expression of GAPDH as an endogenous control gene and calculated using the 2−ΔΔCt method. Error bars indicate the standard error of the mean (n = 3); p < 0.05, statistical significance. (F) Supplementation of DFA-IV (0.1%) decreased permeability of fluorescein isothiocyanate-labelled dextrans of 4 kDa (FD-4) following LPS challenge in serum of broiler (n = 5).a,bSignificant differences between treatments using Duncan’s multiple range test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TJP1, tight junction protein-1; OCLN, occludin. Full size image

To confirm whether DFA-IV treatment affects the intestinal barrier function in vivo, the permeability of FD-4 was examined by determining its levels in the serum of broilers. The permeability of FD-4 based on its serum levels in LPS-challenged broiler was significantly higher than that of the control (p < 0.05, Fig. 10F). Supplementation with DFA-IV did not affect the permeability of FD-4 in LPS-challenged broiler.