# Effects of dietary L-arginine or N-carbamylglutamate supplementation during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and eNOS expression in umbilical vein

X. D. X. D. Liu, X. X. Wu, Y. L. Y. L. Yin, Y. Q. Y. Q. Liu, M. M. M. M. Geng, H. S. H. S. Yang, Francois F. Blachier, G. Y. G. Y. Wu

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## ► To cite this version:

X. D. X. D. Liu, X. X. Wu, Y. L. Y. L. Yin, Y. Q. Y. Q. Liu, M. M. M. M. Geng, et al., Effects of dietary L-arginine or N-carbamylglutamate supplementation during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and eNOS expression in umbilical vein. Amino Acids, 2012, 42 (6), pp.2111 - 2119. 10.1007/s00726-011-0948-5. hal-01004190

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# Effects of dietary L-arginine or N-carbamylglutamate supplementation during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and eNOS expression in umbilical vein

X. D. Liu · X. Wu · Y. L. Yin · Y. Q. Liu · M. M. Geng · H. S. Yang · Francois Blachier · G. Y. Wu

Received: 3 April 2011 / Accepted: 4 May 2011 / Published online: 3 June 2011
© The Author(s) 2011. This article is published with open access at Springerlink.com

**Abstract** Placental vascular formation and blood flow are crucial for fetal survival, growth and development, and arginine regulates vascular development and function. This study determined the effects of dietary arginine or N-carbamylglutamate (NCG) supplementation during late gestation of sows on the microRNAs, vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) expression in umbilical vein. Twenty-seven landrace × large white sows at day (d) 90 of gestation were assigned randomly to three groups and fed the following diets: a control diet and the control diet supplemented with 1.0% L-arginine or 0.10% NCG. Umbilical vein of fetuses with body weight around 2.0 kg (oversized), 1.5 kg (normal) and 0.6 kg (intrauterine growth restriction, IUGR) were obtained immediately after farrowing for miR-15b, miR-16, miR-221, miR-222, VEGFA and eNOS real-time PCR analysis. Compared with the control diets, dietary Arg or NCG supplementation enhanced the reproductive performance of sows, significantly increased

(P < 0.05) plasma arginine and decreased plasma VEGF and eNOS (P < 0.05). The miR-15b expression in the umbilical vein was higher (P < 0.05) in the NCG-supplemented group than in the control group. There was a trend in that the miR-222 expression in the umbilical vein of the oversized fetuses was higher (0.05 < P < 0.1) than in the normal and IUGR fetuses. The expression of eNOS in both Arg-supplemented and NCG-supplemented group were lower (P < 0.05) than in the control group. The expression of VEGFA was higher (P < 0.05) in the NCG-supplemented group than in the Arg-supplemented and the control group. Meanwhile, the expression of VEGFA of the oversized fetuses was higher (P < 0.05) than the normal and IUGR fetuses. In conclusion, this study demonstrated that dietary Arg or NCG supplementation may affect microRNAs (miR-15b, miR-222) targeting VEGFA and eNOS gene expressions in umbilical vein, so as to regulate the function and volume of the umbilical vein, provide more nutrients and oxygen from the maternal to the fetus tissue for fetal development and survival, and enhance the reproductive performance of sows.

**Keywords** L-Arginine · N-Carbamylglutamate · Umbilical vein · VEGFA · ENOS · MicroRNAs

**Abbreviations**

<table><tr><td>Arg</td><td>L-Arginine</td></tr><tr><td>eNOS</td><td>Endothelial nitric oxide synthase</td></tr><tr><td>iNOS</td><td>Inducible nitric oxide synthase</td></tr><tr><td>IUGR</td><td>Intrauterine growth restriction</td></tr><tr><td>NAG</td><td>N-Acetylglutamate</td></tr><tr><td>NCG</td><td>N-Carbamylglutamate</td></tr><tr><td>NO</td><td>Nitric oxide</td></tr><tr><td>RT-PCR</td><td>Real-time polymerase chain reaction</td></tr><tr><td>VEGFA</td><td>Vascular endothelial growth factor A</td></tr></table>

<footnote>X. D. Liu · X. Wu (✉) · Y. L. Yin (✉) ·
Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan 410125, China
e-mail: wuxin@isa.ac.cn</footnote>

<footnote>Y. L. Yin
e-mail: yinyulong@isa.ac.cn</footnote>

<footnote>F. Blachier
INRA, CNRH-IdF, Paris, France</footnote>

<footnote>G. Y. Wu
Department of Animal Science, Texas A&M University, College Station, TX 77843, USA</footnote>
## Introduction

High embryonic loss and fetal deaths during gestation limited the number of piglets born at farrowing in sows (Pope 1994; Mateo et al. 2007). Higher fetal growth rates may require an increased provision of nutrients for supporting the metabolic needs of both the sow and her fetuses (Wu et al. 2004; Kim et al. 2005). Adequate vasculogenesis and angiogenesis of the maternal vasculature is important for providing adequate maternal oxygen/nutrients and blood flow to the placenta. Placental angiogenesis supports the required blood flow on the fetal side necessary for fetal growth and development. Therefore, vasculogenesis and angiogenesis are essential for proper placental development (Wu et al. 2004; Demir et al. 2007; Arroyo and Winn 2008). Umbilical venous blood flow is crucial for fetal growth and development (Barbera et al. 1999; Ferrazzi et al. 2000; Boiti et al. 2002).

The vascular endothelial growth factor (VEGF) proteins are mostly known to regulate the processes of vasculogenesis and angiogenesis (Hanahan 1997; Otrock et al. 2007; Demir et al. 2007; Arroyo and Winn 2008; Yao et al. 2011). As a potent endothelial survival factor, VEGF induces vasodilation and facilitates blood flow by increasing nitric oxide (NO) production (Hood et al. 1998; Otrock et al. 2007). MicroRNAs (miRNAs), about 22-nucleotide, non-coding RNAs, have been shown to be involved in various biological processes in animals (Ambros 2004; Kloosterman and Plasterk 2006), including angiogenesis regulation (Kuehbacher et al. 2008; Anand et al. 2010). Recently, it is reported that miR-15b, miR-16, miR-221 and miR-222 target VEGFA (Hua et al. 2006; Karaa et al. 2009) and eNOS (Poliseno et al. 2006; Suárez et al. 2007) expressions in angiogenesis.

Arginine (Arg) can enhance the reproductive performance of pigs (Mateo et al. 2007; Wu et al. 2007) and also regulate angiogenesis (Raghavan and Dikshit 2004). In addition, N-carbamylglutamate (NCG) increases the endogenous synthesis of Arg (Frank et al. 2007; Wu et al. 2010).

Therefore, we hypothesized that dietary supplementation with Arg or NCG may enhance the reproductive performance of sows and the potential mechanisms are that microNRAs (miR-15b, miR-16, miR-221 and miR-222) target VEGFA and eNOS gene expression in fetal umbilical vein so as to regulate the function and volume of the umbilical vein, thereby providing more nutrients and oxygen from the maternal to the fetus tissue for fetal development and survival.

## Materials and methods

This study was performed in accordance with the Chinese guidelines for animal welfare and approved by the Animal

Care and Use Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences (Yin et al. 1993).

## Animals and experimental design

A total of 27 Large White × Landrace crossbred sows at d 90 of gestation with initial body weight (BW) of 187 ± 5 kg, parity of 3.2 ± 0.7 and similar reproductive performance at last parity were chosen and assigned to three groups randomly: a control group (fed a corn- and soybean meal-based diet) and two treatment groups (fed a corn- and soybean meal-based diet supplemented with 1.0% L-Arg·HCl (Arg) or 0.1% NCG) (Table 1). L-Arg·HCl was obtained from Ajinomoto Inc. (Tokyo, Japan) and NCG was provided by the Institute of Subtropical Agriculture, the Chinese Academy of Sciences.

The dose of Arg was based on previous study (Mateo et al. 2007), and the dose of NCG was based on our own study.

The sows were housed individually in gestation crates (2.0 × 0.6 m, concrete floor) and transferred to individual farrowing crates (2.2 × 1.5 m) at d 107 of gestation. The sows were provided 2 kg diet (on an as-fed basis) daily as two equal-sized meals (08:00 and 16:30 h) during the entire gestation period. All the diets provided 13.5 MJ metabolizable energy/kg and 14.7 crude protein (on as-fed basis). All the sows had free access to drinking water.

## Sample collection

Blood samples were collected 2 h after feeding via jugular venepuncture into heparinized tubes on d 110 of gestation. Samples were centrifuged at 2,000×g, 15 min at 4°C (Mateo et al. 2007; Yin et al. 2010). Plasma was transferred to 1.5 microcentrifuge tubes and stored at -20°C until analysis (Geng et al. 2011). The total number of piglets and their BW at birth were recorded. The piglets were classified as born alive or dead as previously described (Mateo et al. 2007). The number of mummified fetuses (early or middle gestation deaths) was neglected.

The umbilical veins of piglets with BW of about 2.0 kg (oversized), 1.5 kg (normal) and 0.6 kg (IUGR) were obtained immediately after farrowing. Samples were of length about 5 cm, 10 cm from the body, and washed with 4°C PBS (RNA free). Then the samples were collected into 1.5 microcentrifuge tubes (RNA free) with RNAlater (Applied Biosystems, Valencia, CA, USA) in it and stored at -20°C for RT-PCR analyses (Wu et al. 2010).

## Chemical analyses

Plasma samples were assayed for biochemical indices using Beckman Coulter CX4 Pro. (Beckman, USA),
<table><caption>**Table 1** Composition of gestation diets, on an as-fed basis (%)</caption><thead><tr><th rowspan="2">Item</th><th colspan="3">Treatment</th></tr><tr><th>Control</th><th>Arg</th><th>NCG</th></tr></thead><tbody><tr><td><strong>Ingredient</strong></td><td></td><td></td><td></td></tr><tr><td>Corn</td><td>70.30</td><td>70.30</td><td>70.30</td></tr><tr><td>Soybean meal</td><td>12.00</td><td>12.00</td><td>12.00</td></tr><tr><td>Wheat middling</td><td>4.00</td><td>3.00</td><td>3.90</td></tr><tr><td>Rice bran meal</td><td>3.00</td><td>3.00</td><td>3.00</td></tr><tr><td>Puffed soybean</td><td>3.00</td><td>3.00</td><td>3.00</td></tr><tr><td>Salt</td><td>0.35</td><td>0.35</td><td>0.35</td></tr><tr><td>Potassium chloride</td><td>0.75</td><td>0.75</td><td>0.75</td></tr><tr><td>Vitamin-mineral premix<sup>a</sup></td><td>3.00</td><td>3.00</td><td>3.00</td></tr><tr><td>Dicalcium phosphate</td><td>2.20</td><td>2.20</td><td>2.20</td></tr><tr><td>Limestone</td><td>0.50</td><td>0.50</td><td>0.50</td></tr><tr><td>Soy oil</td><td>0.50</td><td>0.50</td><td>0.50</td></tr><tr><td>Mold-inhibitor</td><td>0.09</td><td>0.09</td><td>0.09</td></tr><tr><td>L-Lys-HCl</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>DL-Met</td><td>0.06</td><td>0.06</td><td>0.06</td></tr><tr><td>L-Thr</td><td>0.05</td><td>0.05</td><td>0.05</td></tr><tr><td>L-Arginine-HCl</td><td>—</td><td>1.00</td><td>—</td></tr><tr><td>NCG</td><td>—</td><td>—</td><td>0.10</td></tr><tr><td><strong>Nutrient composition</strong></td><td></td><td></td><td></td></tr><tr><td>DM (%)</td><td>88.2</td><td>88.2</td><td>88.2</td></tr><tr><td>ME (MJ/kg)</td><td>13.5</td><td>13.5</td><td>13.5</td></tr><tr><td>CP (%)</td><td>14.7</td><td>14.7</td><td>14.7</td></tr><tr><td>Lys (%)</td><td>0.78</td><td>0.78</td><td>0.78</td></tr><tr><td>Met + Cys (%)</td><td>0.69</td><td>0.69</td><td>0.69</td></tr><tr><td>Ca (%)</td><td>0.92</td><td>0.92</td><td>0.92</td></tr><tr><td>Total P (%)</td><td>0.67</td><td>0.67</td><td>0.67</td></tr><tr><td>Available P (%)</td><td>0.45</td><td>0.45</td><td>0.45</td></tr></tbody></table>

<sup>a</sup> The premix provided the following per kg of diet: Fe (FeSO₄·H₂O) 80 mg, Mn (MnSO₄·5H₂O) 45 mg, Zn (ZnO) 100 mg, Cu (CuSO₄·5H₂O) 20 mg, I (KI) 0.70 mg, Se (Na₂SeO₃·H₂O) 0.25 mg, vitamin A 10,000 IU, vitamin D₃ 2,500 IU, vitamin E 100 IU, vitamin K 10 IU, vitamin B₂ 10 mg, vitamin B₆ 1 mg, vitamin B₁₂ 50 µg, biotin 80 µg, folic acid 5 mg, nicotinic acid 15 mg, choline chloride 1,500 mg

standards obtained from Beckman (Beckman, USA) (Tang et al. 2005). Plasma concentrations of free amino acids were analyzed by Amino Acid Analyzer, Hitachi L8800 (Hitachi, Japan), and amino acid standards were obtained from Sigma Chemical (Kong et al. 2009). Plasma concentrations of VEGF and eNOS were analyzed using enzyme-linked immunosorbent assay (ELISA) from R & D system (Minneapolis, MN, USA) and an ELISA plate reader (BioTek, USA) (Deng et al. 2010). Concentrations of hormones were analyzed by radioimmunoassay (Jiuding, China).

## Real-time PCR analyses

### RT-PCR for VEGFA and eNOS in fetal umbilical vein

Total RNA was isolated by Trizol (Invitrogen, USA, Karaa et al. 2009) and treated with DNase. Reverse transcription was performed using AMV Reverse Transcriptase Kit (Promega, USA). mRNA levels for VEGFA and eNOS

were determined by a standard real-time polymerase chain reaction (RT-PCR) method. RT-PCR was performed with the total RNA using TaKaRa one-step RNA PCR Kit (TaKaRa Bio Inc, Japan). The primer pairs for VEGFA, eNOS and GAPDH are presented in Table 2. GAPDH was used as the housekeeping gene, whose mRNA levels in the fetal umbilical vein did not differ among the groups. The RT-PCR conditions were: 10 min pre-denaturation at 95°C, and then 15 s denaturation at 94°C, and 30 s annealing at 60°C for 40 cycles. The relative quantification of gene amplification by RT-PCR was performed using cycle threshold (C<sub>T</sub>) values. The comparative C<sub>T</sub> value method was employed to quantitate expression levels for VEGFA and eNOS relative to those for GAPDH. The final PCR product was visualized in a 2% agarose gel.

### RT-PCR for miR-15b, miR-16, miR-221 and miR-222

Expression of mature miRNAs was measured using mi-Script PCR System (Qiagen, Hilden, Germany) (Chen et al.
<table><caption>**Table 2** Primers for VEGFA, eNOS and GAPDH</caption><thead><tr><th>Target gene</th><th>Primer sequences</th><th>Products</th><th>Annealing temperature (°C)</th></tr></thead><tbody><tr><td rowspan="2">VEGFA</td><td>F: CAACGACGAAGGTCTGGAGTG</td><td rowspan="2">155</td><td rowspan="2">60</td></tr><tr><td>R: GCCTCGCTCTATCTTTCTTTGG</td></tr><tr><td rowspan="2">eNOS</td><td>F: ACGCCCGTCTTCCACCA</td><td rowspan="2">193</td><td rowspan="2">60</td></tr><tr><td>R: ACGCCTTCACTCGCTTCG</td></tr><tr><td rowspan="2">GAPDH</td><td>F: GTGCTGAGTATGTCGTGGAGTC</td><td rowspan="2">196</td><td rowspan="2">60</td></tr><tr><td>R: CAGTTGGTGGTACAGGAGGC</td></tr></tbody></table>

F forward, R reverse

2005). The miScript PCR System comprises the following components: miScript Reverse Transcription Kit, miScript SYBR Green PCR Kit, miScript Primer Assay.

Total RNA was extracted as described above. After cDNA synthesis, the cDNA serves as the template for real-time PCR analysis using a miScript Primer Assay in combination with the miScript SYBR Green PCR Kit. Mature miRNAs are amplified using the miScript Universal Primer together with the miRNA-specific primer (the miScript Primer Assay). Primers for sus scrofa miR-15b, miR-16, miR-221 and miR-222 (miRBase, http://www.mirbase.org) were designed by Qiagen. 5S rRNA (forward primer: 5'-gcccgatctcgtctgatct-3', reverse primer: 5'-agcct acagcaccgggtatt-3') was used as the referent for miRNAs expression for its constant expression level across all samples and suitable size. The amplification protocol was as follows: 95°C for 5 min, 50 cycles of denaturation at 94°C/15 s, annealing temperature of 55°C/30 s, and extension at 70°C/30 s. Real-time analysis of PCR amplification was performed on an Applied Biosystems 7900HT Sequence Detection System and analyzed with an SDS 2.3 Software (Applied Biosystems).

The final PCR product was visualized in a 2% agarose gel. All the procedures above followed the instructions of each manufacturer.

## Statistical analyses

The relative quantification of gene amplification by RT-PCR was performed using cycle threshold ($C_T$) values. The comparative $C_T$ value method was employed to

quantitate expression levels for VEGFA and eNOS relative to those for GAPDH. The $ΔΔC_T$ method is used for relative quantification when working with the miScript PCR System. This comparative method relies on comparing the differences in $C_T$ values obtained with normal versus experimental samples. The threshold cycle ($C_T$) obtained with the miScript PCR Control (5S rRNA) is used to normalize the data.

Values are presented as the mean ± SEM. Data of gene and miRNAs expression were analyzed using the GLM and the others using the one-way ANOVA (SAS 9.1.3, SAS Inc., USA). In case of a $P$ value < 0.05, the result was regarded as statistically significant, while $0.05 ≤ P < 0.1$ was considered as a trend.

## Results

### Gestation performance

The reproductive performance of sows fed diets supplemented with Arg or NCG can be seen in Table 3. The total number of piglets born, birth weight of all piglets born or born alive, and litter birth weight of all piglets born did not differ between the three groups of sows. However, there was a trend ($0.05 < P < 0.1$) toward an increase in the number of piglets born alive for sows fed the Arg or NCG-supplemented diet compared with sows fed the control diet. The litter birth weight of all piglets born alive were 15% higher ($P < 0.05$) for Arg-supplemented sows and 14% ($P < 0.05$) higher for NCG-supplemented sows, both

<table><caption>**Table 3** Reproductive performance of sows</caption><thead><tr><th>Items</th><th>Control</th><th>Arg</th><th>NCG</th><th>SEM</th></tr></thead><tbody><tr><td>Total piglets born per litter (n)</td><td>11.22</td><td>11.38</td><td>11.33</td><td>0.26</td></tr><tr><td>Total piglets born alive per litter (n)</td><td>9.78</td><td>10.87</td><td>10.77</td><td>0.24</td></tr><tr><td>Birth weight of all piglets born (kg)</td><td>1.44</td><td>1.50</td><td>1.49</td><td>0.02</td></tr><tr><td>Birth weight of all piglets born alive (kg)</td><td>1.45</td><td>1.51</td><td>1.49</td><td>0.02</td></tr><tr><td>Litter birth weight of all piglets born (kg)</td><td>16.09</td><td>16.87</td><td>16.83</td><td>0.25</td></tr><tr><td>Litter birth weight of all piglets born alive (kg)</td><td>14.12b</td><td>16.26a</td><td>16.04a</td><td>0.29</td></tr><tr><td>Piglets born dead per litter (n)</td><td>1.44a</td><td>0.50b</td><td>0.56b</td><td>0.14</td></tr></tbody></table>

In the same row, values with different letter mean significant difference ($P < 0.05$)

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 4** Plasma biochemical indices</caption><thead><tr><th>Items</th><th>Control</th><th>Arg</th><th>NCG</th><th>SEM</th></tr></thead><tbody><tr><td>Glucose (mmol/L)</td><td>3.51</td><td>3.77</td><td>3.89</td><td>0.14</td></tr><tr><td>Ammonia (µmol/L)</td><td>85.42</td><td>85.09</td><td>85.73</td><td>0.16</td></tr><tr><td>Urea nitrogen (mmol/L)</td><td>5.83</td><td>5.19</td><td>5.41</td><td>0.14</td></tr><tr><td>Albumin (g/L)</td><td>26.29</td><td>28.03</td><td>26.87</td><td>0.61</td></tr><tr><td>Total protein (g/L)</td><td>62.09</td><td>65.96</td><td>67.12</td><td>1.59</td></tr><tr><td>Phosphorus (mmol/L)</td><td>1.52a</td><td>1.74b</td><td>1.76b</td><td>0.04</td></tr><tr><td>Ca<sup>2+</sup> (mmol/L)</td><td>2.09</td><td>2.28</td><td>2.19</td><td>0.05</td></tr><tr><td>Cu<sup>2+</sup> (µmol/L)</td><td>33.84</td><td>32.40</td><td>32.22</td><td>0.79</td></tr><tr><td>Mg<sup>2+</sup> (mmol/L)</td><td>1.73</td><td>1.71</td><td>1.65</td><td>0.20</td></tr><tr><td>Zn<sup>2+</sup> (mmol/L)</td><td>3.12a</td><td>4.94b</td><td>4.27b</td><td>0.26</td></tr></tbody></table>

In the same row, values with different letter mean significant difference (P < 0.05)

compared with the control group. The number of piglets born dead were 65% lower (P < 0.05) for the Arg-supplemented sows and 61% lower (P < 0.05) for the NCG-supplemented sows, both compared with the control group. The days from weaning to estrus of sows did not differ between the three groups (data not shown).

### Plasma biochemical assays

Concentrations of glucose, ammonia, albumin, total protein, Ca²⁺, Cu²⁺ and Mg²⁺ in plasma did not differ between the three groups (Table 4). Concentrations of phosphorus and Zn²⁺ were both higher (P < 0.05) in Arg or NCG-supplemented sows than in the control group of sows (Table 4). There was a trend (0.05 < P < 0.1) toward the decrease in the concentrations of urea nitrogen for Arg-supplemented sows compared with the control group of sows.

### Plasma-free amino acids concentration

Concentrations of the most measured free amino acids in plasma did not differ among the three groups of sows at d 110 of gestation. Compared with the control group, dietary supplementation with Arg (P < 0.01) or NCG (P < 0.05) increased the concentrations of arginine in the plasma of sows (Fig. 1). Compared with the control diet and Arg-supplemented diet, NCG increased (P < 0.05) the concentrations of aspartate in the plasma of sows and decreased (P < 0.05) the concentrations of proline (Fig. 1).

### Plasma hormone concentrations

Concentrations of estriol and progesterone did not differ among the three groups (Table 5). Plasma growth hormone in Arg or NCG-supplemented sows were higher (P < 0.05) compared with sows fed the control diets. Concentrations of estradiol were lower (P < 0.05) in NCG-supplemented

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Concentrations of free amino acids in plasma (µmol/L). For the same grayscale of the bar, values with different letter mean significant difference (P < 0.05), the same as given below</figcaption></figure>

sows than in the other two groups. In addition, there was a trend (0.05 < P < 0.1) for sows fed the NCG-supplemented diet to have increased hormone concentrations of insulin-like growth factor-1 compared with sows in the control group.

### Plasma concentrations of VEGF and eNOS

Protein concentrations of VEGF in plasma were 11 and 10% lower in Arg-supplemented sows (P < 0.05) and NCG-supplemented sows (P < 0.05) than in the control group of sows, respectively (Table 6). Protein concentrations of eNOS were 17 and 23% lower in Arg-supplemented sows (P < 0.05) and NCG-supplemented sows (P < 0.01) than in the control group of sows (Table 6).

### VEGFA and eNOS gene expression

The expression of eNOS in both Arg-supplemented and NCG-supplemented group was lower (P < 0.05) than in the control group (Fig. 2). The expression of VEGFA was higher (P < 0.05) in the NCG-supplemented group than in
<table><caption>**Table 5** Concentration of hormones in plasma</caption><thead><tr><th>Items</th><th>Control</th><th>Arg</th><th>NCG</th><th>SEM</th></tr></thead><tbody><tr><td>Insulin (µg/dl)</td><td>37.92</td><td>40.53</td><td>35.97</td><td>2.46</td></tr><tr><td>Growth hormone (ng/ml)</td><td>1.21a</td><td>1.70b</td><td>1.68b</td><td>0.10</td></tr><tr><td>Insulin-like growth factor-1 (ng/ml)</td><td>30.52</td><td>43.68</td><td>46.22</td><td>3.91</td></tr><tr><td>Estradiol (pg/ml)</td><td>135.72b</td><td>138.44b</td><td>67.02a</td><td>13.60</td></tr><tr><td>Estriol (pg/ml)</td><td>4.38</td><td>4.98</td><td>4.21</td><td>0.66</td></tr><tr><td>Progesterone (ng/ml)</td><td>2.74</td><td>2.48</td><td>2.11</td><td>0.18</td></tr></tbody></table>

In the same row, values with different letter mean significant difference (P < 0.05)

<table><caption>**Table 6** Plasma concentration of VEGF and eNOS</caption><thead><tr><th>Items</th><th>Control</th><th>Arg</th><th>NCG</th><th>SEM</th></tr></thead><tbody><tr><td>VEGF (mg/L)</td><td>177.38a</td><td>158.03b</td><td>159.57b</td><td>3.85</td></tr><tr><td>eNOS (U/L)</td><td>10.61a</td><td>8.78b</td><td>8.15b</td><td>0.37</td></tr></tbody></table>

In the same row, values with different letter mean significant difference (P < 0.05)

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Effects of Arg and NCG on the VEGFA and eNOS gene expression levels in the umbilical vein</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3** Effects of body weight on the VEGFA and eNOS gene expression levels in the umbilical vein</figcaption></figure>

the Arg-supplemented and the control group (Fig. 2). Meanwhile, the expression of VEGFA of the oversized fetuses was higher (P < 0.05) than the normal and IUGR fetuses (Fig. 3). There was no effect of the diet × BW interaction on VEGFA and eNOS gene expression.

## MiR-15b, miR-16, miR-221 and miR-222 expression

The miR-15b expression in the umbilical vein was higher (P < 0.05) in the NCG-supplemented group than in the control group (Fig. 4). There was a trend toward the miR-222 expression in the umbilical vein of the oversized fetuses being higher (0.05 < P < 0.1) than the normal and IUGR fetuses (Fig. 5). There was no effect of diet × BW interaction on these miRNAs expression.

<figure><img src="image_7.png" /><figcaption>**Fig. 4** Effects of Arg and NCG on the miR-15b, 16, 221 and 222 expression levels in the umbilical vein</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5** Effects of body weight on the miR-15b, 16, 221 and 222 expression levels in the umbilical vein</figcaption></figure>
Discussion

Maternal nutrition and oxygen play a key role in regulating fetal survival, growth and development (Wu et al. 2004). Malnutrition is known to be a major cause of pregnancy complications, such as intrauterine growth restriction (IUGR) or even worse, such as embryonic loss and fetal deaths during gestation. Thus, providing the pregnant dam with proper nutrition is vital for the fetus (Snoeck et al. 1990; Hoet and Hanson 1999; McPherson et al. 2004). Various evidences have substantiated the importance of arginine in the survival, growth, and development of fetal pigs (Wu et al. 2004, 2007). Furthermore, amino acid malnutrition in gestating sows results in lower concentrations of arginine in the placenta and fetal plasma (Wu et al. 1998), as well as reduced the synthesis of NO (the endothelium-derived relaxing factor) from L-arginine (Wu et al. 2009) and the synthesis of polyamines (Pegg 1986). Impaired placental synthesis of both NO and polyamines is considered a major factor contributing to IUGR (Wu et al. 2004, 2006). Additionally, previous studies showed that uterine uptake of arginine may not be sufficient to meet fetal growth requirements during late gestation in pigs (Wu et al. 1999). NCG is a safe and metabolically stable analog of NAG (Wu et al. 2009; Gessler et al. 2010) and increases the endogenous synthesis of Arg (Frank et al. 2007; Wu et al. 2009).

The results of this study showed that Arg or NCG supplementation to gestation diets for late pregnant sows improved pregnancy outcome, decreased plasma urea concentrations and increased the plasma concentrations of free arginine of sows at d 110 of gestation. Mateo et al. (2007) also reported the similar results. This suggested that both Arg and NCG supplementation provided better nutrients to sows, and therefore probably improved the uterine environment for fetal growth and development. Additionally, arginine is not only required for protein synthesis and ammonia detoxification, but is also a precursor of many metabolically important molecules, including proline, ornithine, polyamines and NO (Wu and Morris 1998; Kim et al. 2007).

However, proper nutrition for sows cannot guarantee good reproductive efficiency. The placenta is responsible for the exchange of nutrients and oxygen from the mother to the fetus. Adequate vasculogenesis and angiogenesis of the maternal vasculature are important for providing adequate maternal nutrients/oxygen and blood flow to the placenta. Placental vascular formation and function are important for fetal growth and development. Proper development of the placenta is critical for a successful pregnancy, mediates important steps, such as maternal blood flow to the placenta and delivery of nutrients to the fetus, and ensures the exchange of nutrients/oxygen and

blood flow necessary for fetal growth (Arroyo and Winn 2008).

Also, umbilical venous blood flow is crucial for fetal growth and development (Barbera et al. 1999; Ferrazzi et al. 2000; Boiti et al. 2002). Pathologic umbilical vein leads to pregnancy complications too (Klaritsch et al. 2008; Koech et al. 2008). Vascular growth is necessary to increase placental fetal blood flow over gestation. Poor vascular development is known to cause intrauterine embryonic death characterized by low vascular density in the placental villi along with fibrosis and other deficiencies. The VEGF proteins are the most studied family of growth factors known to regulate the processes of vasculogenesis and angiogenesis. VEGFA (also known as VEGF), aside from being a potent endothelial survival factor, is also known to induce vasodilation by increasing nitric oxide (NO) production, another function which facilitates blood flow. ENOS is critical in the regulation of vascular function (Lu et al. 2011) and can generate both nitric oxide (NO) and superoxide ($O_2^−$), which are key mediators of cellular signaling (Chen et al. 2010).

MicroRNAs (miRNAs), about 22-nucleotide, non-coding RNAs, have come into focus as a powerful mechanism to regulate angiogenesis (Dews et al. 2006; Urbich et al. 2008). It has been demonstrated that miR-221 and miR-222 block endothelial cell migration, proliferation and angiogenesis and indirectly regulate the expression of endothelial nitric oxide synthase (Poliseno et al. 2006). In addition, miR-221 and miR-222 inhibit cell proliferation and reduce the expression of c-Kit in hematopoietic progenitor cells—a process that can contribute to vessel growth (Kuehbacher 2007). Two other miRNAs that might be involved in angiogenesis are miR-15b and miR-16. miR-15b and miR-16 have been shown to control the expression of VEGF (Hua et al. 2006). Data indicate that hypoxia-induced reduction of miR-15b and miR-16 contributes to an increase in VEGF. Some other miRNAs also regulate angiogenesis and vascular function (Anand et al. 2010).

In this study, the levels of gene expression of VEGFA and eNOS in umbilical vein and decrease in the plasma concentrations of VEGFA and eNOS in both the Arg- and NCG-supplemented groups may be a feedback regulatory mechanism of arginine-produced NO in the fetal umbilical vein and placenta compared with the control group. This is supported by high expression of miR-15b and miR-222 in the umbilical vein of dietary Arg- or NCG-supplemented groups.

The arginine treatment may enhance placental angiogenesis and growth during early-to mid-gestation, thereby promoting an optimal intrauterine environment throughout pregnancy (Wu et al. 2004). Therefore, it is possible that dietary supplementation with arginine increases the synthesis of NO in the placenta and fetus, as reported for adult
rats (Wu and Morris 1998; Kohli et al. 2004). The outcome would be to enhance placental angiogenesis and growth (including vascular growth), utero-placental blood flow, the transfer of nutrients from mother to fetus, and, therefore, fetal survival, growth and development (Kwon et al. 2004; Wu et al. 2004, 2006).

Although the majority of the conceptus loss occurs during the peri-implantation period, there is evidence that significant losses also occur during later gestation (Wilson 2002). Piglets born dead per litter were significantly decreased both in Arg-supplemented and NCG-supplemented groups in this study, which is similar to the study reported previously (Mateo et al. 2007). This may suggest that dietary Arg or NCG supplementation also regulated placenta vascular functions.

Notably, we found that plasma concentrations of phosphorus and $Zn^{2+}$ were higher both in sows of the Arg-supplemented and NCG-supplemented groups, indicating that Arg or NCG supplementation increases protein synthesis of fetus (Castillo-Durán and Weisstaub 2003; Frank et al. 2007). This is in agreement with the findings reported previously (Mateo et al. 2007). This study showed that dietary Arg or NCG supplementation increased litter piglets born alive and litter birth weight of all piglets born alive, while there were not differences in the average birth weights of all piglets born or of piglets born alive between groups. Furthermore, plasma concentrations of growth hormone were higher in sows of Arg-supplemented and NCG-supplemented groups than in sows of the control group.

In summary, supplementing dietary Arg or NCG during late gestation enhanced the reproductive performance of sows. Also, Arg or NCG treatment improved efficiency in the utilization of dietary nutrients; we propose that Arg or NCG treatment may effect the expression of miRNA-15b and miRNA-222, thereby controlling its target, VEGFA and eNOS, respectively, gene expression in the umbilical vein. Thus, Arg may regulate angiogenesis and vascular development and functions of umbilical vein and placenta, providing more nutrients and oxygen from mother to fetuses for fetal survival, growth and development. However, it is necessary to determine how arginine regulate fetal survival, growth and development through microRNAs.

**Acknowledgments** This research was jointly supported by grants from the National Basic Research Program of China (2009CB118806), the Chinese Academy of Sciences and Knowledge Innovation Project (KZCX2-EW-412, KZCX2-EW-QN411), NSFC (30901040, 30901041, 30928018, 30828025, 30771558).

**Open Access** This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

## References

Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355

Anand S, Majeti BK, Acevedo LM et al (2010) MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat Med 16(8):909–914

Arroyo JA, Winn VD (2008) Vasculogenesis and angiogenesis in the IUGR placenta. Semin Perinatol 32:172–177

Barbera A, Galan HL, Ferrazzi E et al (1999) Relationship of umbilical vein blood flow to growth parameters in the human fetus. Am J Obstet Gynecol 181:174–179

Boiti S, Struijk PC, Ursem NTC et al (2002) Umbilical venous volume flow in the normally developing and growth-restricted human fetus. Ultrasound Obstet Gynecol 19:344–349

Castillo-Durán C, Weisstaub G (2003) Zinc supplementation and growth of the fetus and low birth weight infant. J Nutr 133:1494S–1497S

Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179

Chen CA, Wang TY, Varadharaj S et al (2010) S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468:1115–1118

Demir R, Seval Y, Huppertz B (2007) Vasculogenesis and angiogenesis in the early human placenta. Acta Histochem 109:257–265

Deng J, Wu X, Li TJ et al (2010) Dietary amylose and amylopectin ratio and resistant starch content affects plasma glucose, lactic acid, hormone levels and protein synthesis in splanchnic tissues. J Anim Physiol Anim Nutr 94:220–226

Dews M, Homayouni A, Yu DN et al (2006) Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet 38:1060–1065

Ferrazzi E, Rigano S, Bozzo M et al (2000) Umbilical vein blood flow in growth-restricted fetuses. Ultrasound Obstet Gynecol 16:432–438

Frank J, Escobar J, Nguyen HV et al (2007) Oral N-carbamylglutamate supplementation increases protein synthesis in skeletal muscle of piglets. J Nutr 137:315–319

Geng M, Li T, Kong X et al (2011) Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates. Amino Acids 40:1513–1522

Gessler P, Buchal P, Schwenk HU et al (2010) Favourable long-term outcome after immediate treatment of neonatal hyperammonemia due to N-acetylglutamate synthase deficiency. Eur J Pediatr 169:197–199

Hanahan D (1997) Signaling vascular morphogenesis and maintenance. Science 277:48–50

Hoet JJ, Hanson M (1999) Intrauterine nutrition: its importance during critical periods for cardiovascular and endocrine development. J Physiol 514:617–627

Hood JD, Meininger CJ, Ziche M et al (1998) VEGF upregulates eNOS message, protein, and NO production in human endothelial cells. Am J Physiol 274:H1054–H1058

Hua Z, Lv Q, Ye WB et al (2006) MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS ONE 1(1):e116

Karaa ZS, Iacovoni JS, Bastide A et al (2009) The VEGF IRESes are differentially susceptible to translation inhibition by miR-16. RNA 15:249–254

Kim SW, Wu G, Baker DH (2005) Amino acid nutrition of breeding sows during gestation and lactation. Pigs News Inform 26:N89–N99
Kim SW, RD Mateo, Y-L Yin et al (2007) Functional amino acids and fatty acids for enhancing production performance of sows and piglets. Asian-Aust J Anim Sci 20:295-306

Klaritsch P, Haeusler M, Karpf E et al (2008) Spontaneous intrauterine umbilical artery thrombosis leading to severe fetal growth restriction. Placenta 29:374-377

Kloosterman WP, Plasterk R (2006) The diverse functions of MicroRNAs in animal development and disease. Dev Cell 11:441-450

Koech A, Ndungu B, Gichangi P (2008) Structural changes in umbilical vessels in pregnancy induced hypertension. Placenta 29:210-214

Kohli R, Meininger CJ, Haynes TE et al (2004) Dietary L-arginine supplementation enhances endothelial nitric oxide synthesis in streptozotocin-induced diabetic rats. J Nutr 134:600-608

Kong XF, Wu GY, Yin YL et al (2007) Dietary supplementation with Chinese herbal ultra-fine powder enhances cellular and humoral immunity in early weaned piglets. Livest Sci 108:94-98

Kong XF, Yin YL, He QH et al (2009) Diety supplementation with Chinese herbal powder enhances ileal digestibilities and serum concentrations of amino acids in young pigs. Amino Acids 37:573-582

Kuehbacher A, Urbich C, Dimmeler S (2008) Targeting microRNA expression to regulate angiogenesis. Trends Pharmacol Sci 29(1):12-15

Kwon H, Wu G, Meininger CJ et al (2004) Developmental changes in nitric oxide synthesis in the ovine placenta. Biol Reprod 70:679-686

Lu Y, Xiong Y, Huo YQ et al (2011) Grb-2-associated binder 1 (Gab1) regulates postnatal ischemic and VEGF-induced angiogenesis through the protein kinase A-endothelial NOS pathway. PNAS 108(7):2957-2962

Mateo RD, Wu G, Fuller W et al (2007) Dietary L-Arginine supplementation enhances the reproductive performance of gilts. J Nutr 137:652-656

McPherson RL, Ji F, Wu G et al (2004) Growth and compositional changes of fetal tissues in pigs. J Anim Sci 82:2534-2540

Otrock ZK, Makarem JA, Shamseddine AI (2007) Vascular endothelial growth factor family of ligands and receptors: review. Blood Cells Mol Dis 38:258-268

Pegg AE (1986) Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 234:249-262

Poliseno L, Tuccoli A, Mariani L et al (2006) MicroRNAs modulate the angiogenic properties of HUVECs. Blood 108:3068-3071

Pope WF (1994) Embryonic mortality in swine. In: Geisert RD (ed). Embryonic mortality in domestic species. CRC Press Boca Raton, pp 53-78

Raghavan S, Dikshit M (2004) Vascular regulation by the L-arginine metabolites, nitric oxide and agmatine. Pharmacol Res 49:397-414

Snoeck A, Remacle C, Reusens B et al (1990) Effect of a low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol Neonate 57:107-118

Suárez Y, Fernández-Hernando C, Pober JS et al (2007) Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells. Circ Res 100:1164-1173

Tang ZR, Yin LY, Nyachoti CM et al (2005) Effect of dietary supplementation of chitosan and galacto-mannan-oligosaccharide on serum parameters and the insulin-like growth factor-I mRNA expression in early-weaned piglets. Domest Anim Endocrinol 28:430-441

Urbich C, Kuehbacher A, Dimmeler S (2008) Role of microRNAs in vascular diseases, inflammation, and angiogenesis. Cardiovasc Res 79(4):581-588

Wilson ME (2002) Role of placental function in mediating conceptus growth and survival. J Anim Sci 80(Suppl 2):E195-E201

Wu G, Morris SM (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336:1-17

Wu G, Pond WG, Ott T et al (1998) Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs. J Nutr 128(5):894-902

Wu G, Ott TL, Knabe DA et al (1999) Amino acid composition of the fetal pig. J Nutr 129:1031-1038

Wu G, Bazer FW, Cudd TA et al (2004) Maternal nutrition and fetal development. J Nutr 134:2169-2172

Wu G, Bazer FW, Wallace JM et al (2006) Intrauterine growth retardation: implications for the animal sciences. J Anim Sci 84:2316-2337

Wu G, Bazer FW, Davis TA et al (2007) Important roles for the arginine family of amino acids in swine nutrition and production. Livest Sci 112:8-22

Wu G, Bazer FW, Davis TA et al (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37:153-168

Wu X, Ruan Z, Gao YL et al (2010) Dietary supplementation with L-arginine or N-carbamylglutamate enhances intestinal growth and heat shock protein-70 expression in weanling pigs fed a corn- and soybean meal-based diet. Amino Acids 39:831-839

Wu X, Yin YL, Li TJ et al (2010) Dietary protein, energy and arginine affect LAT1 expression in forebrain white matter differently. Animal 4:1518-1521

Yao K, Guan S, Li TJ et al (2011) Dietary L-arginine supplementation enhances intestinal development and expression of vascular endothelial growth factor in weanling piglets. Bri J Nutr 105:703-709

Yin YL, Zhong HY, Huang RL et al (1993) Nutritive value of feedstuffs and diets for pigs. I. Chemical composition, apparent ileal and fecal digestibility. Anim Feed Sci Technol 44:1-27

Yin YL, Yao K, Liu ZJ et al (2010) Supplementing L-leucine to a low-protein diet increases tissue protein synthesis in weanling pigs. Amino Acids 39:1477-1486