Maternal N-carbamylglutamate and L-arginine supplementations improve foetal jejunal oxidation resistance, integrity and immune function in malnutrition sheep during pregnancy

Figures & data

Figure 1. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in villus morphology of foetal jejunum in underfed Hu ewes at d110 of gestation. Villous height and crypt depth (A), and VCR and number per villus (B) were determined. VCR: villous height: crypt depth ratio; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/d NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no identical letters suggest significant differences, p < .05.

Table 1. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in protein, IGF-1, insulin, NO levels, and NOS activities within foetal jejunum of underfed Hu ewes at 110 gestational days.

Items1	CON	RES	RES + ARG	RES + NCG	SEM	p value
Protein concentration, mg/g wet wt	24.600^a	13.500^c	17.800^b	18.100^b	1.440	.018
Insulin, pmol/g of protein	4.480^a	1.890^c	2.950^b	3.120^b	0.580	.026
NO, μmol/g of protein	3.830^a	1.690^c	2.570^b	2.830^b	0.390	.009
IGF-1, pmol/g of protein	193.700^a	112.500^c	148.300^b	151.600^b	6.210	.005
NOS, U/g of protein	612.600^a	408.700^c	523.600^b	531.400^b	9.640	.015

NO: nitric oxide; IGF-1: insulin-like growth factor 1; NRC: National Research Council; NOS: NO synthase; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-Arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/d NCG.

¹ Data represent means and pooled SEMs (n = 8/group for ewes, n = 16/group for the foetus).

^a–c Means within each row with superscripts with no identical letter are different (p < .05).

Figure 2. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diets in cytokine levels within foetal jejunum of underfed Hu ewes at 110 gestational days. IL, interleukin; TNF-α, tumour necrosis factor α; NRC, National Research Council; CON/RES, ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/d NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no identical letters represent statistical differences, p < .05.

Table 2. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in antioxidant activities within foetal jejunum of underfed Hu ewes at d110 of gestation.

Items1	CON	RES	RES + ARG	RES + NCG	SEM	p value
T-AOC, U/mg of protein	1.360^a	0.680^c	1.120^b	1.090^b	0.310	.013
MDA, nmol/mg of protein	0.340^c	0.660^a	0.450^b	0.410^b	0.090	.021
GPX, U/mg of protein	10.230^a	8.560^b	9.130^ab	9.390^ab	1.320	.036
SOD, U/mg of protein	94.100^a	63.500^b	88.300^a	89.600^a	7.160	.009

MDA: malondialdehyde; T-AOC: total antioxidant capacity; GPX: glutathione peroxidase; SOD: superoxide dismutase; NRC: National Research Council; CON: ewes fed 100% of NRC (2007) recommendations for pregnancy; RES: ewes fed 50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations and supplemented with 20 g/d RP-Arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations and supplemented with 5 g/d NCG.

¹ Data represent means and pooled SEMs (n = 8/group for ewes, n = 16/group for the foetus).

^a–c Means in a row with superscripts with no identical letter are different (p < .05).

Figure 3. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in gene mRNA levels within foetal jejunum in underfed Hu ewes at 110 gestational days. mRNA expression of anti-oxidation-associated (A), immune-associated (B), intestinal integrity-associated (C), and NO-dependent pathway-associated (D) genes were determined. GPX1: glutathione peroxidase 1; CAT: catalase; SOD2: superoxide dismutase2; HO-1: haem oxygenase-1; NRF2: nuclear factor erythroid 2-related factor 2; MYD88: myeloid differentiation factor 88; NQO1: quinone oxidoreductase 1; TLR: toll-like receptor; TRAF-6: tumour necrosis factor receptor-associated factor 6; TNF-α: tumour necrosis factor α; IL: interleukin; ZO-1: zonula occludens-1; NF-κB: nuclear factor kappa B (p65); NO: nitric oxide; eNOS: epithelial NO synthase; iNOS: inducible NO synthase; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/day NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no common letter represent significant differences, p < .05.

Figure 4. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in antioxidant defence-associated protein levels in foetal jejunum of underfed Hu ewes at 110 gestational days. Typical images showing WB analysis (A) and GPX1, CAT, NRF2, SOD2, NQO-1 and HO-1 levels (B) were measured. GPX1: glutathione peroxidase 1; CAT: catalase; NRF2: nuclear factor erythroid 2-related factor 2; SOD2: superoxide dismutase2; NQO1: quinone oxidoreductase 1; HO-1: haem oxygenase-1; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/day NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no identical letters are different, p < .05.

Figure 5. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in immune function related protein expression in foetal jejunum in underfed Hu ewes at 110 gestational days. Characteristic images demonstrating the Western blotting assay (A) and p65, p-p65, IL-1β, and TNF-α expression (B) was measured. IL: interleukin; NF-κB: nuclear factor kappa B (p65); TNF-α: tumour necrosis factor α; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/day NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with on common letter stand for significant differences, p < .05.

Figure 6. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in integrity-related protein levels in foetal jejunum of underfed Hu ewes at 110 gestational days. Typical images showing the Western blotting analysis (A) and claudin-1 and ZO-1 (B) were analysed. ZO-1: zonula occludens-1; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/day RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/day NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no identical letters are different, p < .05.

Figure 7. Roles of rumen-protected L-arginine (RP-arg) or N-carbamylglutamate (NCG) supplemented in diet in NO-dependent pathway-associated protein levels in foetal jejunum of underfed Hu ewes at 110 gestational days. Typical images showing the Western blotting analysis (A) and eNOS and iNOS levels (B) were determined. NO: nitric oxide; NOS: inducible NO synthase; eNOS: epithelial NO synthase; NRC: National Research Council; CON/RES: ewes fed 100%/50% of NRC (2007) recommendations for pregnancy; RES + ARG, ewes fed 50% of NRC (2007) recommendations with supplementation of 20 g/d RP-arg; RES + NCG, ewes fed 50% of NRC (2007) recommendations with supplementation of 5 g/d NCG. Data represent means, and standard errors are shown in vertical bars (n = 8/group for ewes, n = 16/group for the foetus). Labelled means with no common letters stand for significant differences, p < .05.

National Research Council. 2007. Nutrient requirements of small ruminants: sheep, goats, cervids and new world camelids. Washington, DC: National Academies Press.

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Data availability statement

Data used in the present work can be obtained from this manuscript and the supplementary materials.