N-carbamylglutamate and L-arginine Supplementation Improve the Intestinal Oxidative Resistance of the Intrauterine Growth-retarded Ovine Fetuses

Background: The maternal under nutrition-induced intrauterine growth retardation (IUGR) is associated with intestinal oxidative injury in fetuses and neonates in various animal models. However, whether maternal dietary Arginine (Arg) and N-carbamylglutamate (NCG) supplementation during IUGR alters fetal small intestine redox status is unclear. Objective: The ovine model of IUGR was used to elucidate whether dietary supplementation of rumen-protected Arg (RP-Arg) and NCG modulates the fetal intestinal oxidative resistance via the nitric oxide (NO) -dependent pathway. Methods: On day 35 of gestation, 32 twin-bearing Hu ewes were randomly assigned into 4 treatment groups, 8 ewes each. The rst and second groups received 100% (Control, CON) and 50% (restricted, RES) of NRC-recommended pregnancy nutrient requirements, respectively. The third and fourth treatment groups included the RES ewes supplemented with 20 g/day of RP-Arg (RES+ARG) or 5 g/day of NCG (RES+NCG), respectively. On day 110 of gestation, fetal blood and intestinal specimens were collected and assayed for oxidative damage biomarkers. Results: The NCG or Arg-supplemented RES ewes elevated the fetal jejunal NO concentrations and NO synthase (NOS) activity, but decreased the fetal jejunal and plasma concentrations of interleukin (IL)-6 and tumor necrosis factor α (TNF-α) (P < 0.05) compared with those in the RES ewes. Further, the NCG or Arg treatment increased the contents of catalase (CAT), glutathione peroxidase 1 (GPx1), nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), heme oxygenase (HO-1), quinone oxidoreductase 1 (NQO1), claudin-1, zonula occludens-1 (ZO-1), epithelial NOS (eNOS) and inducible NOS (iNOS) in the fetal jejunum (P < 0.05). Conclusion: These results indicate that both NCG and Arg supplementation for RES ewes help maintain intestinal function in fetuses experiencing IUGR through modulating the oxidation status.


Introduction
Optimal maternal nutrition during pregnancy is crucial for the development of a functional small intestine in the fetus ( Cao et al., 2014;Meyer and Caton, 2016). The maternal undernutrition-induced intrauterine growth retardation (IUGR) is associated with abnormal gut development and function in the fetuses and neonates in various animal models ( Gao et al., 2008;Meyer et al., 2013). IUGR has been shown to adversely affect the pre-and post-natal development and function of the small intestine in various mammals (Meyer and Caton, 2016;Huang et al., 2019).
Newborn IUGR animals have remarkably decreased antioxidant abilities at birth and weaning (Biri et al., 1985a;Gupta et al., 2004). Oxidative stress (OS) results from the imbalance between the antioxidant capacity and the production of reactive oxygen species (ROS) (Yin et al., 2013). As intestinal lumen is in direct contact with oxidants either from microbiota or nutrient intake, it is likely to be affected by oxidative injury induced by the IUGR (Zheng et al., 2018;Marseglia et al., 2015). The IUGR has been shown also to adversely affect the protein abundance in the fetal small intestine at various stages of development . These changes collectively comprise the intestinal digestive and absorptive functions (Wang et al., 2005;Xu et al., 1994) which might be responsible in part-for the postnatal growth retardation of IUGR newborns (Hu et al., 2016;Zhang et al., 2019a;Zhang et al., 2019b).
Arginine (Arg), the essential amino acid among young mammals, particularly for animals that are subjected to stress conditions (Dasgupta et al., 2006), exerts a vital part among numerous metabolic pathways (Moncada and Higgs, 1993). N-carbamylglutamate (NCG) is the N-acetylglutamate (NAG) analogue with metabolic stability, which is suggested to elevate the plasma content of Arg as well as the synthesis of endogenous Arg through the activation of intestinal pyrroline-5-carboxylate synthase and carbamylphosphate synthase-1 (Wu et al., 2004). Another important function of arginine pertains to its role in mitochondrial biological function and biogenesis via the NO signal transduction pathway (Guo et al., 2018). Particularly, Arg enhances the synthesis of NO in normal conditions within the small intestine (Weckman et al., 2019), thus, it exerts a vital part in regulating the antioxidant defense (Liang et al., 2018). Recent evidence emphasized the importance of arginine/NO pathway during pregnancy as indicated by the increase in the circulating levels of arginine and its metabolites in feed-restricted ewes bearing multiple-fetuses (Berlinguer et al., 2020). However, whether maternal dietary Arg or NCG supplementation during IUGR alters fetal small intestine redox status is unclear.
We hypothesized that maternal dietary Arg or NCG supplementation would improve the development of the fetal intestine through reducing oxidant status in fetuses exposed to IUGR. Speci c objectives were to elucidate the bene ts of maternal dietary Arg or NCG supplementation on intestinal dysfunction in IUGR ovine fetuses and to offer a theoretical basis for using NCG or Arg as a functional component in the feeds of pregnant ewes at risk of undernutrition.

Materials And Methods
Animals, housing and management All procedures were carried out according to the protocols from the Guide for the Care and Use of Laboratory Animals formulated by the Ethics Committee of Yang Zhou University (SYXK2013-0057).
Forty-eight multiparous Hu ewes [with the mean body weight (BW) of 40.1±1.2 kg] that had similar body condition score (BCS; 2.55±0.18; range, 0 (emaciation) -5 (obesity) and similar age (18.5±0.5 months) (Russel et al., 1969) were chosen for the study. The animals were housed in an indoor barn at the Jiangyan Experimental Station in Taizhou of Jiangsu (China). The barn was equipped with heating radiators to maintain the environmental temperature at an average of 15.3±0.9℃. The barn lighting was automatically controlled for mimicking the normal environmental light cycle. The animals were drenched against parasites using ivermectin (0.2 mg kg -1 ). Afterward, the animals were subjected to estrus synchronization using the intravaginal progestogen sponge protocol (30mg; Pharmp) for 12 days. Starting 2 days following the removal of the sponge, the ewes were subjected to 3 vasectomized rams (at 08:00 and 16:00) for detecting the estrus behavior and arti cial insemination was performed accordingly.
The inseminated ewes (at day 0 of gestation) were then put into individual pens (1.05 m×1.60 m) for 35 days. On day 35 of gestation, the ewes were tested by ultrasonography (Asonics Microimager 1000 sec for scanning instrument; Ausonics) for pregnancy diagnosis and twins determination. Accordingly, 32 ewes carrying twin fetuses were selected for the present research. Diet composition (Table 1) was formulated to meet 100% of the nutrient requirements for pregnant ewes (NRC, 1985). From day 0 to day 35 of the study, each animal was given 100% of the nutrient requirements (NRC, 1985). Then, the animal was fed once every day at 08:00 and could drink clean water freely.

Experimental design
On day 35 of gestation, the animals were divided randomly into 4 treatment groups, 8 animals each, the rst group served as a control (CON) and received 100% of nutrient requirements. In the second group, the feeding allowance was restricted to 50% of nutrient requirements (restricted feeding; RES). The third and fourth treatment groups included the RES ewes supplemented with 20 g/day of rumen-protected (RP) Arg (RES+ARG) or 5 g/day of NCG (RES+NCG), respectively. The RP-Arg product (Beijing Feeding Feed Science Technology Co., Beijing, China) and the NCG product (Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, China) contain 50% Arg and NCG, respectively (Zhang et al., 2016a) and, thus actual supplemented amounts of RP-Arg and NCG were 10 g/day and 2.5 g/day, respectively. The RP-Arg product is made up of a matrix of glycerides and phospholipids prepared via the spray congealing and spray drying procedures (Eldem et al., 1991). The ruminal protection of Arg is estimated to be at ≥85%, whereas the intestinal release of Arg is estimated at ≥90% (Chacher et al., 2012). The Arg dose was determined based on prior research on pregnant ewes receiving parenteral Arg or RP-Arg supplementation (Mccoard et al., 2013;Zhang et al., 2018a). The NCG dose was determined based on prior research on piglets (Zeng et al., 2012), dairy cows (Chacher et al., 2014), andpregnant sheep (Zhang et al., 2016b). The restricted feeding could be attained by giving 50% of the total diet determined to satisfy the 100% of pregnancy nutrient requirements set by the NRC (1985). From the 35 th gestational day, BW was measured on 10-days intervals, and then the feed intake was adjusted accordingly. All diets were in the form of pellets and the experiment continued to day 110 of gestation.

Fetal blood and intestinal sample collection
On day 110 of gestation, each animal was stunned using the captive bolt gun (Supercash Mark 2; Acceles and Shelvoke), followed by euthanasia by exsanguination. A 5 mL blood sample was extracted from the fetal hearts at 0900 hours. All blood samples were immediately placed in heparinized tubes on ice, followed by 15 min of centrifugation at 4 °C and 3000 × g, and then kept at −80 °C for subsequent analysis. Then, 10 g duodenal, jejunal, and ileal samples (rinsed with cold PBS) were collected, respectively, immediately frozen within liquid nitrogen, and then stored at -80°C for subsequent analyses .
Intestinal morphological analyses and counting of goblet cells The jejunal specimens that were xed within the 4% paraformaldehyde were washed using the gradient xylene and ethanol, and then the dried specimens were subjected to para n embedding. Then, 5 jejunal slides with 3 respective sections (5 μm in thickness) were prepared, and then the samples were rehydrated by gradient ethanol dilutions and depara nized with xylene (5 μm). To observe the intestinal morphology, each slide was stained using the hematoxylin and eosin (H&E) through 20 well-oriented crypts and villi in every section (Optimus software, version 6.5, Media Cybergenetics). Moreover, the ratio of villi to crypt (VCR) was determined, and the number of goblet cells in each villus was calculated (NIS-Elements BR 2.3, Nikon France SAS). Then, values were collected from 10 villi of every small intestine segment, which were then averaged (Zhang et al., 2018b;Zhang et al., 2019c).
Analyses of insulin-like growth factor 1 (IGF-1), insulin, nitric oxide (NO), nitric oxide synthase (NOS) and protein in plasma and jejunum The IGF-1 and insulin contents were determined by an ELISA micro-plate kit speci c to ewes (Mercodia, Guangzhou, China). Later, the NOS and NO contents were determined by the commercial kit (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) following manufacturer protocols (Ando et al., 2016). Protein content was determined by a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, U.S.A.).

Enzyme activity assays in jejunal tissues
Jejunal tissue was subjected to homogenization and centrifuged within the lysis buffer for 15 min at 4 ℃ and 15,000 ×g. Supernatants were extracted to determine the markers related to the OS as well as the activities of antioxidant enzymes. A bicinchoninic acid protein assay kit (Pierce, Rockford, IL, U.S.A.) was used to determine the protein content. Besides, the maleic dialdehyde (MDA) level, the total antioxidant capacity (T-AOC) degree, together with the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were determined in both homogenized jejunal tissue and plasma using respective commercial ELISA kits (Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).
mRNA levels determined through real-time PCR assay in jejunal tissues The total RNA was extracted by the RNA isolation kit (Qiagen, Frankfurt, Germany) following manufacturer protocol, and detected by real-time PCR according to the previous description (Zhang et al., 2018b). The RNA content was determined by spectrophotometry, and the RNA integrity was assessed using the agarose gel electrophoresis and spectrophotometry. Afterward, the reverse transcription was carried out using the PrimeScript RT reagent kit (TaKaRa) following the manufacturer protocol. Later, the qPCR was carried out using the FastStart Universal SYBR Green Master kit (Roche, Mannheim, Germany) in the Step One Plus Real-Time PCR System (Applied Biosystems, CA, USA). All primers were designed with the Primer 5.0 (Table 2), among which, β-actin was used to be a housekeeping normalizing gene. The reaction mixture (10 μL) contained freshly prepared SYBR Premix Ex Taq II (5 μL, Tli RNaseH Plus), RT products (1 μL), ROX Reference Dye II (0.2 μL, 50×), diethylpyrocarbonate-treated water (3 μL), and primers (0.8 μL). The PCR conditions were as follows: 30 s at 95 °C for 1 cycle; followed by 5 s at 95 °C and then 31 s at 60 °C for 40 cycles; and 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C for 1 cycle. The e ciency of ampli cation was determined according to the previously reported method (Zhang et al., 2019a). Each correlation coe cient (r) for the standard curve was ≥ 0.99, whereas the ampli cation e ciency value was 90-110%. Meanwhile, the ampli cation speci city was veri ed using the melting curves, and the mRNA expression was determined according to the 2 -△△Ct method. According to our results, the difference in the β-actin level was not statistically signi cant across the 4 treatments. Besides, the mRNA expression of a target gene within the CON group was used to normalize that in different treatment groups (namely, the fold-change) (Zhang et al., 2019a).

Statistical methods
Statistical comparisons among the 4 groups of ewes killed on d 110 of gestation were completed using a 1-way ANOVA. Results were presented as least squares means ± SEM. Data analyses among different groups of fetuses were carried out by the one-way ANOVA following the SAS (version 9.2) PROC GLM procedure. The sex of the fetus was incorporated into the initial model, and no signi cance was found (P>0.05). Therefore, it was eliminated from that eventual model that only included the nutrition treatments in the ewes. The difference among different groups was determined using Duncan's multiple range test. A difference of P≤0.05 declared signi cance.

Results
Maternal BW, fetal BW andsmall intestinal development We previously reported that maternal BW in CON ewes was greater compared with RES, RES + ARG, and RES + NCG ewes ( Fig.1)

Fetal intestinal morphology and density of goblet cells
Relative to the CON group, the number of goblet cells per villus, villus height, and VCR were reduced (P < 0.05) in the jejunum the fetuses of the RES group ( Figure 2). In contrast, these variables were increased (P < 0.05) in the jejunum of the fetuses of the RES ewes subjected to dietary NCG or Arg supplementation compared with the RES group.

Insulin content, NOS activity and NO level in fetal plasma
Relative to the CON group, insulin content, NOS activity and NO level in fetal plasma of RES ewes were reduced (P < 0.05) (Figure 3), but these parameters were increased (P < 0.05) in RES + ARG or NCG group relative to those in the RES group.
Protein, NO, IGF-1, insulin and NOS contents in fetal jejunum Compared with the CON, the RES feeding resulted in a reduction of the concentrations of protein, IGF-1, insulin, NOS, and NO in the fetal jejunum (P < 0.05) ( Figure 4). Relative to RES group, a marked increase in protein, IGF-1, insulin, NOS, and NO contents were observed in the jejunum of fetuses of RES ewes supplemented with Arg or NCG (P < 0.05).

Fetal jejunal and plasma levels of cytokines
Compared with the CON, the RES group showed an increase of the fetal jejunal and plasma IL-N and TNFα contents (P < 0.05) ( Figure 5). By contrast, RES + NCG or ARG reduced the fetal jejunal and plasma IL-6 and TNF-α as well as the fetal plasma IL-1 contents (P < 0.05) compared with the RES group.

Jejunal and plasma antioxidant enzyme activities in fetuses
Relative to the CON group, the fetal jejunal and plasma T-AOC activities were reduced (P < 0.05) in the RES group ( Figure 6); nonetheless, ewes subjected to NCG or Arg treatment showed an increase (P < 0.05) in the fetal jejunal and plasma T-AOC activities compared with the RES ewes. The NCG or Arg treatment resulted in a reduction in the fetal jejunal and plasma MDA concentrations (P < 0.05) relative to those in the RES group. The SOD and GSH-Px activities within fetal jejunum and the plasma of the RES group were reduced relative to those in the CON group (P < 0.05). The NCG or Arg treatment resulted in increasing the SOD activity of the fetal jejunum and plasma (P < 0.05) relative to those in the RES group.

mRNA level in fetal jejunum
Relative to the CON, the TLR-2, MyD88, TLR-9, TLR-4, IL-1β, IL-6, TNF-α, and NF-κB mRNA levels were upregulated (P < 0.05) in the fetal jejunum of the RES group (Figure 7). The NCG or Arg treatment resulted in downregulation (P < 0.05) of the above genes relative to those in the RES group. Relative to the CON, GPx1, eNOS, CAT, Nrf2, SOD2, NQO1, HO-1, claudin-1, ZO-1, iNOS, and occludin levels were downregulated (P < 0.05) in the fetal jejunum of the RES group. Supplementation of the RES group with NCG or Arg resulted in upregulation (P < 0.05) of the above-mentioned genes relative to those in the RES group.

Discussion
The development of the intestine is of crucial importance to neonate growth and survival. At the advanced gestation, fetal intestine develops more rapidly (Zhu et al., 2018). The integrity of the intestine is necessary to maintain appropriate digestive functions (Choksi et al., 2018), as a result, this re ects a crucial intestinal health aspect. Relative to the CON, the IUGR fetuses had reduced jejunal VCR and villus height, which was consistent with prior studies (Su et al., 2018;Zhu et al., 2018). Improvements in the IUGR fetal intestinal morphology of RES ewes under NCG or Arg treatment are related to the promoted enterocyte proliferation and maturation (Yang et al., 2013). Thus, these results might explain the damaged integrity of the intestine of the IUGR ovine fetus, which also strengthened the role of NCG or Arg in protection against fetal intestinal injury induced by IUGR.
Arginine is identi ed to be the potent factor that stimulates the release of insulin (Fisker et al., 1998).
Consequently, the increased serum concentration of this anabolic hormone enhances the utilization e ciency of nutrients through enhancing tissue protein synthesis (Yao et al., 2011). The fetal jejunal protein concentration and jejunal and plasma insulin concentrations of RES ewes under Arg or NCG treatment were in line with the above-mentioned results.
IUGR animals are more susceptible to OS, an effect that is probably associated with the dysregulation between prooxidants and antioxidants. Besides, excess OS affects cell homeostasis and could predispose to cell injuries (Biri et al., 2007;Celik et al., 2019). Such dysregulation leads to cell damage, which may even result in cell death as well as irreversible oxidative damage if it remains to be out of control, nally negatively affecting organism survival and function (Yin et al., 2013b). The OS has been identi ed as the critical factor contributing to bowel injury within animals a icted by IUGR (Su et al., 2017). Consequently, the markedly reduced jejunal T-AOC content and antioxidant enzyme activities, together with the remarkably increased MDA content of IUGR ovine fetuses emphasize the occurrence of oxidative injury. The above adaptations may result in bowel-related disorders through inducing oxidative injury to lipids, proteins, and DNA, as well as increase the membrane permeability (Murphy, 2009). The increased TNF-α, IL-6, and IL-1 contents in the jejunum of IUGR ovine fetuses compared with those of the CON group con rmed that the OS was positively correlated with pro-in ammatory cytokines (Artimani et al., 2018). The TNF-α serves as the pro-in ammatory cytokine, which participates in the systemic in ammation . Besides, the TNF-α is known to result in the increased intestinal epithelial barrier permeability (Lee, 2015). Therefore, the reduction in the oxidative stress along with the lower TNFα and IL-1β protein and mRNA contents in the fetal jejunum due to supplementing with Arg or NCG supports the view of a protective effect of these compounds.
The barrier of the intestinal epithelium is comprised of a variety of junctional complexes, which ensures the close epithelial cell binding as well as the appropriate epithelial barrier maintenance (Zihni et al., 2016). Goblet cells can produce trefoil peptides and mucins to generate the physical barrier on the intestinal mucosal surface (Wang et al., 2007). The tight junction proteins (TJP) are considered to modulate the paracellular intestinal epithelial barrier function (Tokuda and Yu, 2019). The levels of the fetal intestinal TJP, including ZO-1 and claudin-1, were increased in the RES supplemented with NCG and Arg, and the numbers of goblet cells were also increased, which emphasized the favorable effects on the barrier function of the fetal intestine. The results of this study are consistent with the previous studies which indicated that the Arg treatment boosted the intestinal morphology of the weaned as well as the IUGR suckling piglets (He et al., 2011;Wang et al., 2012). These ndings imply that NCG or Arg could enhance the antioxidative defense in IUGR animals by reducing the pro-in ammatory cytokines. Nuclear factor erythroid 2-related factor (Nrf2) is of crucial importance to improve our understanding towards the underlying mechanism of the association with the antioxidant responsive element (ARE)-activated antioxidation and detoxi cation under the anti-oxidative state, which suggests that oxidation resistance may serve as the synergistic action regulated via different antioxidative enzymes, instead of via an antioxidative reaction alone (Suzuki and Yamamoto, 2015;Liang et al., 2018). Arg has the key effect of up-regulating Nrf2, the antioxidant transcriptional factor, as well as the phase II metabolizing enzymes (such as heme oxygenase (HO-1) and quinone oxidoreductase 1 (NQO1)), together with the antioxidant enzymes (like catalase (CAT), glutathione peroxidase 1 (GPx1), as well as superoxide dismutase2 (SOD2) (Zhang et al., 2019a). In our experimental conditions, Arg or NCG treatment markedly increased the mRNA abundance of antioxidant/detoxi cation genes (HO-1, NQO1, SOD2, CAT, GPx1) by activating Nrf2, which participated in the endogenous defense response of antioxidant. Overall, it alleviates oxidative injury and protects the intestinal epithelial cells (IOECs) against apoptosis in the presence of bowel in ammatory challenge (Zhang et al., 2019d). The NCG or Arg supplementation positively affected the Nrf2 protein and mRNA levels, elevated the SOD2, CAT, GPx1, and phase II metabolizing enzymes (including HO-1 and NQO1) contents in fetal jejunum, but reduced the MDA content within IUGR ovine fetuses, which con rmed that the above-mentioned compounds had positive effects on the intestinal functions. As far as we know, this is the rst study to report that Arg or NCG can activate and upregulate ARE-induced antioxidant and detoxi cation genes controlled by the Nrf2 signaling pathway within the fetal intestine from animals exposed to IUGR.
Nitric oxide, produced from arginine metabolism, can serve as the essential messenger, which plays various roles, including regulating cell proliferation and survival, immunologic function, as well as the cellular redox status (Thomas et al., 2008). The NO has been shown to play a role as a scavenger of free radicals (Az-Ma et al., 1996). In this study, maternal dietary supplementation with Arg or NCG had favorable action through promoting the jejunal oxidation resistance of IUGR ovine fetuses, which was ascribed to the fact that Arg exerted a role as the precursor of NO (El-Hattab et al., 2012).
To study whether the NO pathway was responsible for the Arg or NCG-enhanced fetal antioxidant capacity in the jejunum, both iNOS and eNOS protein and mRNA levels, NO content, and NOS activity were determined. In this study, maternal NCG or Arg treatment signi cantly increased the jejunal NOS protein and mRNA levels, NO level, or NOS activity in IUGR ovine fetuses. According to Dhar et al. (2012), Arg treatment decreased the OS induced by high glucose via the eNOS-independent signaling pathway. The NCG or Arg treatment had positive action on the intestinal health of IUGR fetuses in a NO pathwaydependent manner, which was partly ascribed to the enhanced jejunal oxidation resistance.

Conclusion
The fetal intestinal development dysfunction, together with the decreased oxidation resistance in jejunum induced by IUGR, is attenuated through the dietary NCG or Arg treatment. The above compounds enhance the intestinal barrier function partially through modulating the oxidation resistance through the NOdependent pathway. The ndings of this study expound the underlying mechanisms regarding the dysfunction in the small intestine of ovine fetuses suffering from IUGR.

Declarations
Ethics approval and consent to participate All animal experiments, which had gained approval from the Animal Care and Use Committee of the Yangzhou University, were carried out in accordance with the practical animal protection law and the Guide for the Care and Use of Laboratory Animals formulated by the National Research Council.

Consent for publication
Not applicable.

Availability of data and material
The datasets generated or analyzed during current study are available from the corresponding author by request. All data that support the ndings in of this study are included in the article.
Zhang H, Sun L, Wang Z, Deng M, Zhang G, Guo R, Ma T, Wang F. (2016). Dietary N-carbamylglutamate and rumen-protected L-arginine supplementation ameliorate fetal growth restriction in undernourished ewes. Journal of Animal Science, 94 (5)       Values are means, with standard errors represented by vertical bars (n = 8/group for ewes, n = 16/group for the fetus). Labeled means without a common letter differ, P < 0.05.  recommendations and supplemented with 20 g per day RP-Arg; RES NCG, ewes fed 50% of NRC (1985) recommendations and supplemented with 5 g per day NCG. Values are means, with standard errors represented by vertical bars (n = 8/group for ewes, n = 16/group for the fetus). Labeled means without a common letter differ, P < 0.05.

Figure 7
The mRNA abundance of genes in the jejunum of the fetus in control and RES ewes and in RES ewes supplemented with Arg or NCG on day 110 of gestation. The mRNA abundance of antioxidant related genes (A), immune function-related genes (B), intestinal integrity-related genes (C), and nitric oxidedependent pathway-related genes (D) were determined. CAT, catalase; GPx1, glutathione peroxidase 1; SOD2, superoxide dismutase2; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; . Labeled means without a common letter differ, P < 0.05.

Figure 9
Fetal jejunal immune function related protein expression in control and RES ewes and in RES ewes supplemented with Arg or NCG on day 110 of gestation. Representative charts of Western blot results (A) and TNF-α, IL-1β, p65 and p-p65 (B) were determined. IL, interleukin; TNF-α, tumor necrosis factor α; NF-κB, nuclear factor kappa B (p65); CON, ewes fed 100% of the National Research Council (NRC 1985)recommended pregnancy nutrient requirements; RES, ewes fed 50% of NRC (1985) recommendations for pregnancy; RES ARG, ewes fed 50% of NRC (1985) recommendations and supplemented with 20 g per day RP-Arg; RES NCG, ewes fed 50% of NRC (1985) recommendations and supplemented with 5 g per day NCG. Values are means, with standard errors represented by vertical bars (n = 8/group for ewes, n = 16/group for the fetus). Labeled means without a common letter differ, P < 0.05.

Figure 10
Fetal jejunal integrity-related protein expression in control and RES ewes and in RES ewes supplemented with Arg or NCG on day 110 of gestation. Representative charts of Western blot results (A) and ZO-1 and claudin-1 (B) were determined. ZO-1, zonula occludens-1; CON, ewes fed 100% of the National Research Council (NRC 1985)-recommended pregnancy nutrient requirements; RES, ewes fed 50% of NRC (1985) recommendations for pregnancy; RES ARG, ewes fed 50% of NRC (1985) recommendations and supplemented with 20 g per day RP-Arg; RES NCG, ewes fed 50% of NRC (1985) recommendations and supplemented with 5 g per day NCG. Values are means, with standard errors represented by vertical bars (n = 8/group for ewes, n = 16/group for the fetus). Labeled means without a common letter differ, P < 0.05.

Figure 11
Fetal jejunal nitric oxide-dependent pathway-related protein expression in control and RES ewes and in RES ewes supplemented with Arg or NCG on day 110 of gestation. Representative charts of Western blot results (A) and iNOS and eNOS (B) were determined. iNOS, inducible NO synthase; eNOS, epithelial NO synthase; CON, ewes fed 100% of the National Research Council (NRC 1985)-recommended pregnancy nutrient requirements; RES, ewes fed 50% of NRC (1985) recommendations for pregnancy; RES ARG, ewes fed 50% of NRC (1985) recommendations and supplemented with 20 g per day RP-Arg; RES NCG, ewes fed 50% of NRC (1985) recommendations and supplemented with 5 g per day NCG. Values are means, with standard errors represented by vertical bars (n = 8/group for ewes, n = 16/group for the fetus). Labeled means without a common letter differ, P < 0.05.