Dietary grape pomace mediates jejunum epithelial apoptosis through antioxidative activity in ram lambs

Figures & data

Table 1. Nutrient composition of wine grape pomace (air-dry matter basis, %).

Items	%
Dry matter, DM	92.37
Crude protein, CP	11.50
Ether extract, EE	6.53
Crude ash, ash	8.12
Neutral detergent fibre, NDF	43.83
Acid detergent fibre, ADF	35.12
Calcium	1.20
Phosphorus	0.05
Total phenols, TP	7.20

Table 2. Dietary ingredients and chemical composition of diets (air-dry matter basis, %).

Dietary ingredient	Control	5% WGP^a	10% WGP
Corn, %	29.00	27.00	24.95
Soybean meal %, 44% crude protein	9.00	8.60	8.20
Wheat bran, %	4.00	4.00	4.00
Oil cake of flax seed, %	5.00	5.00	5.00
Mineral/vitamin premix, %	5.00	5.00	5.00
WGP, %	0	5.00	10.00
Naked oats straw, %	35.00	34.65	34.25
Potato rattan, %	13.00	10.75	8.60
Total	100.00	100.00	100.00
Chemical composition
Dry matter, %	88.43	88.57	88.51
Digestible energy, MJ/kg	10.60	10.53	10.47
Metabolise energy, MJ/kg	8.74	8.68	8.63
Crude protein, %	11.73	11.90	12.21
Ether extract, EE	2.18	3.17	3.35
Neutral detergent fibre, %	42.85	43.20	43.38
Calcium, %	0.40	0.40	0.39
Phosphorus, %	0.25	0.26	0.26
Total phenols, TP, %	0.41	0.87	1.14

^aWine grape pomace.

Figure 1. Reactive oxygen species (ROS) and malondialdehyde (MDA) concentration in jejunum epithelium of ram lambs fed with the control (□), 5% WGP (

) and 10% Wine grape pomace (WGP) diet (■). (A) ROS content in jejunum epithelium was reduced in both 5% and 10% WGP supplementation groups. (B) MDA content was decreased when lambs were fed with 10% WGP (mean ± SEM; n = 6). WGP: wine grape pomace

Figure 2. Jejunum morphology among control (□), 5% WGP (

) and 10% WGP (■) supplementation groups. (A) Images of H&E staining of jejunums from different groups. (B) The villus height and crypt depth, data showed that 10% dietary WGP reduced villus height, and 5% WGP was sufficient to decrease crypt depth. (C) Ratio of the villus height to crypt depth was increased in both 5% and 10% supplementation groups (mean ± SEM; n = 6; Scar bar = 100 μm). WGP: wine grape pomace

Figure 3. Jejunum epithelial apoptosis among control (□), 5% WGP (

) and 10% WGP (■) supplementation groups. (A) Tunnel staining showed decreased apoptotic cell numbers in WGP supplementation groups. (B) Immunohistochemistry staining showed that caspase 3 positive cell numbers were decreased in jejunum of lambs fed the WGP containing diet (mean ± SEM; n = 6). WGP: wine grape pomace

Figure 4. The apoptotic related protein abundance among different groups. (A) Phosphorylated ATM content. (B) Bax abundance. (C) Bcl-2 content. (D) Cleaved-caspase 9 content. (E) Cleaved-caspase 8 content. (F) Cleaved-caspase 3 content. Data show that WGP supplementation altered apoptotic related protein abundance (mean ± SEM; n = 6). WGP: wine grape pomace

Table 3. Effect of dietary wine grape pomace (WGP) supplementation on antioxidative enzyme activities in jejunum epithelium of ram lambs.

Item	Control	5% WGP	10% WGP	SEM	p Value
T-AOC, U/mg^c	0.87^b	0.97^b	1.35^a	0.12	.03
Catalase, U/mg	5.07^b	6.33^a	6.62^a	0.21	.02
GPx4, U/mg^d	75.92^b	92.52^a	98.45^a	5.45	.03
SOD, U/mg^e	5.75^b	6.15^ab	6.37^a	0.13	.02

^a,bThe values within a row with different superscripts are significantly different.

^cTotal antioxidant capacity.

^dGlutathione peroxidase.

^eSuperoxide dismutase.

Figure 5. Antioxidative enzymes abundance in jejunum epithelium of ram lambs fed with control (□), 5% WGP (

) and 10% WGP diet (■). (A) Measurements of superoxide dismutase (SOD) protein abundance. (B) Glutathione peroxidase 4 (GPx4) protein abundance. (C) Catalase protein abundance. (D) Nuclear factor-like-2 factor (Nrf2) content. Data showed that SOD, catalase and GPx4 protein abundance were increased when lambs were fed the WGP-containing diet (mean ± SEM; n = 6). WGP: wine grape pomace