Dietary grape seed proanthocyanidin extract supplementation improves antioxidant capacity and lipid metabolism in finishing pigs

Figures & data

Table 1. Feed ingredients and nutrient composition of the basal diet, % as fed basis (air-dry basis).

Item	Basal diet	Nutrient levels^c	Content
Corn	86.69	Digestible energy (MJ/kg)	14.23
Soybean meal	5.90	Crude protein (%)	10.30
Soybean oil	2.28	Calcium (%)	0.46
Wheat bran	2.65	Non-phytate phosphorus (%)	0.21
L-Lys·HCl (78%)	0.39	Digestible lysine (%)	0.61
DL-Met	0.08	Digestible Met + Cys (%)	0.36
L-Thr	0.15	Digestible Thr (%)	0.40
L-Trp (78%)	0.04	Digestible Trp (%)	0.11
Limestone	0.64
Dicalcium phosphate	0.50
NaCl	0.30
Chloride choline (50%)	0.15
Vitamin premix^a	0.03
Mineral premix^b	0.20
Total	100.00

^aVitamin premix provides the following per kg of complete diet: VA, 9000 IU; VB₁, 3 mg; VB₂, 7.5 mg; VB₁₂, 0.036 mg; VB₆, 3.6 mg; VD₃, 3000 IU; VE, 24 IU; VK₃, 3.0 mg; biotin, 0.15 mg; folic acid, 1.5 mg; calcium pantotenate, 15 mg; nicotinic acid, 30 mg.

^bMineral premix provides the following per kg of complete diet: Mn (MnSO₄), 2 mg；I (KI), 0.14 mg；Se (Na₂SeO₃), 0.15 mg Cu (CuSO₄.5H₂O), 3 mg；Fe (FeSO₄.H₂O), 40 mg； Zn (ZnSO₄.7H₂O), 50 mg.

^cData were calculated values.

Table 2. Primers used in real-time quantitative PCR.

Gene	Primer	Sequence (5’ → 3’)	Accession No.	Product size (bp)
SOD1	Forward	AGACCTGGGCAATGTGACTG	NM_001190422	102
	Reverse	GTGCGGCCAATGATGGAATG
SOD2	Forward	TGAACAACCTGAACGTCGTG	NM_214127	99
	Reverse	AGCGGTCAACTTCTCCTTGA
GST	Forward	CCAACCCAGAAGACTGCTCA	AB000884	102
	Reverse	CATTCAGGTGGGCTCTTCGT
GR	Forward	GTGAGCCGACTGAACACCAT	AY36827	141
	Reverse	CAGGATGTGAGGAGCTGTGT
CAT	Forward	CAGATGAAGCATTGGAAGGAGC	NM_214301	83
	Reverse	TTGTCTCCTATCGGATTCCCAG
NRF2	Forward	GCCCCTGGAAGCGTTAAAC	XM_003133500	67
	Reverse	GGACTGTATCCCCAGAAGGTTGT
CPT1a	Forward	CTACAAATTACGTGAGCGACT	NM_001129805.1	195
	Reverse	GTTTGATTTCCTCTCGGTCCA
CPT1b	Forward	TGACTCGAATGTTCCGGGAG	NM_001007191	118
	Reverse	AGATCTTGCAGGTCTGCTTTCA
PPARα	Forward	GAGTTCGCCAAGTCCATCC	NM_001044526	122
	Reverse	CCGTCCTTGTTCATCACAGAG
ACC	Forward	ACCGAATTGGTTCCTTTGGAC	AF175308	123
	Reverse	CCAGTCCGATTCTTGCTCCA
HSL	Forward	CCCATCCTCTCCATCGACT	NM_214315	83
	Reverse	CAGCAGTAGGCGTAGAAGCAC
FAS	Forward	ACACCTTCGTGCTGGCCTAC	NM_001099930	112
	Reverse	ATGTCGGTGAACTGCTGCAC
AMPK1	Forward	CGGCAAAGTGAAGGTTGG	NM_001167633	123
	Reverse	AGGTTCTGAATTTCTCTGCGG
AMPK2	Forward	TGAGGTCGATATCTGGAGCTG	NM_214266	151
	Reverse	AGTGGCAACAGAACGATTGAG
PGC-1α	Forward	GCCCTCATTTGATGCACTG	DQ437884	150
	Reverse	AGCTGAGTGTTGGCTGGTG
Sirt1	Forward	ACTCTCCCTCTTTTAGACCAAGC	NM_001145750	149
	Reverse	AAACCTGGACTCTCCATCGG
β-actin	Forward	ATATTGCTGCGCTCGTGGT	XM_021086047.1	147
	Reverse	TAGGAGTCCTTCTGGCCCAT

SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; GST: glutathione S-transferase; GR: glutathione reductase; CAT: catalase; HO-1: haem oxygenase-1; NQO1: NAD(P)H, quinone oxidoreductase 1, Nrf2: NF-E2-related nuclear factor 2,. CPT1a: Carnitine palmitoyl-transferase-1a;CPT1b: carnitine palmitoyl transferase-1b; PPARα:peroxisome proliferator-activated receptor α; PPARγ: peroxisome proliferator-activated receptor γ; ACC: acetyl-CoA carboxylase; HSL: hormone sensitive lipase; FAS: fatty acid synthase; AMPK: AMP-activated protein kinase; p-AMPK: phospho-AMP-activated protein kinase; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1 alpha; Sirt1: sirtuin 1.

Table 3. Effect of dietary GSPE supplementation on antioxidant enzyme activities and MDA content of finishing pigs.

Items	Control	GSPE 100	GSPE 200	SEM	p-value
Serum
MDA(nmol/mL)	1.14^a	0.92^ab	0.86^b	0.095	0.023
T-AOC(U/mL)	1.25^b	1.92^ab	2.12^a	0.263	0.013
T-SOD(U/mL)	135.36^b	148.01^ab	164.89^a	6.453	0.001
GSH-Px(U/mL)	1269.93^b	1270.93^b	1271.93^a	93.629	0.014
CAT(U/mL)	1.75^b	2.81^a	2.51^ab	0.339	0.020
GSH(U/mL)	4.56^b	4.01^b	6.60^a	0.783	0.012
LD Muscle
MDA(nmol/mg prot)	0.74^a	0.50^b	0.43^b	0.067	0.001
T-AOC(U/mg prot)	0.14^b	0.20^a	0.23^a	0.019	0.002
T-SOD(U/mg prot)	24.72^b	30.06^a	28.59^ab	1.909	0.036
GSH-Px(U/mg prot)	0.47^b	0.65^ab	0.82^a	0.094	0.008
CAT(U/mg prot)	3.02	5.00	3.98	0.743	0.055
GSH(U/mg prot)	3.68^b	4.86^ab	5.13^a	0.456	0.015
Liver
MDA(nmol/mg prot)	2.09^a	1.52^ab	1.18^b	0.245	0.007
T-AOC(U/mg prot)	0.88^b	1.14^a	1.14^a	0.102	0.031
T-SOD(U/mg prot)	595.41^b	691.18^ab	731.02^a	44.765	0.015
GSH-Px(U/mg prot)	12.03^b	15.56^a	15.89^a	0.998	0.002
CAT(U/mg prot)	29.46^b	36.00^ab	59.29^a	10.400	0.029
GSH(U/mg prot)	21.42^b	28.24^ab	34.14^a	3.299	0.006

MDA: malonaldehyde; T-AOC: total antioxidant capacity; T-SOD: total superoxide dismutase; GSH-Px: glutathione peroxidase; CAT: catalase; GSH: glutathione; prot: protein.

Data are presented as means and SEM (n = 6). Values with different letters within the same line indicated a significant difference (p < 0.05).

Figure 1. Effects of GSPE on antioxidant enzyme genes in LD muscle and liver. (A) The mRNA expression of antioxidant enzyme genes in LD muscle. (B) The mRNA expression of antioxidant enzyme genes in liver. Data are presented as means and SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001. SOD1: superoxide dismutase 1; SOD2 : superoxide dismutase 2; GR: glutathione reductase; CAT: catalase; GST: glutathione S-transferase.

Figure 2. Effects of GSPE on expression of Nrf2 pathway genes. (A) NQO1, HO-1and Nrf2 protein levels in the liver were determined by Western blot analysis. (B) NQO1, HO-1and Nrf2 protein levels in LD muscle were determined by Western blot analysis. Equal loading was monitored with an anti-β-actin antibody. (C) NQO1, HO-1and Nrf2 mRNA levels in liver. (D) NQO1, HO-1and Nrf2 mRNA levels in LD muscle. Western blot analysis data are presented as means and SEM (n = 3). Real-time quantitative PCR data are presented as means and SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001. NQO1: NAD(P)H, quinone oxidoreductase 1; Nrf2: NF-E2-related nuclear factor 2; HO-1: haem oxygenase-1.

Table 4. Effect of dietary GSPE supplementation on serum lipid metabolism of finishing pigs.

Items	Control	GSPE 100	GSPE 200	SEM	p-value
TG (mmol/L)	0.30^a	0.25^ab	0.21^b	0.027	0.023
T-CHO (mmol/L)	2.38^a	2.03^b	2.03^b	0.089	0.002
HDL-C (mmol/L)	1.45^b	1.84^a	1.86^a	0.101	0.002
LDL-C (mmol/L)	1.94	1.82	1.65	0.192	0.353

T-CHO: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein-cholesterol; HDL-C: high-density lipoprotein cholesterol.

High-density lipoprotein-cholesterol.

Data are presented as means and SEM (n = 6). Values with different letters within the same line indicated a significant difference (p < 0.05).

Figure 3. Effects of GSPE on lipid metabolism gene expression in LD muscle and liver. (A) The mRNA expression of lipid metabolism genes in LD muscle, (B) The mRNA expression of lipid metabolism genes in liver. Data are presented as means and SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001. CPT1a: carnitine palmitoyl-transferase-1a;CPT1b: carnitine palmitoyl transferase-1b; PPARα: peroxisome proliferator-activated receptor α; PPARγ: peroxisome proliferator-activated receptor γ; ACC: Acetyl-CoA carboxylase; HSL: hormone sensitive lipase; FAS: fatty acid synthase; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1 alpha; Sirt1: sirtuin 1.

Figure 4. Effect of GSPE on p-AMPK expression in LD muscle and liver. (A) The p-AMPK protein expression in LD muscle. (B) The p-AMPK protein expression in liver. The p-AMPK protein level was monitored with AMPK protein. Data are presented as means and SEM (n = 3). *p < 0.05 and **p < 0.01. AMPK: AMP-activated protein kinase; p-AMPK: phospho-AMP-activated protein kinase.