Maternal N-Carbamylglutamate supply during early pregnancy

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Bioactive Constituents, Metabolites, and Functions

Maternal N-Carbamylglutamate supply during early pregnancy enhanced pregnancy outcomes in sows through modulations of targeted genes and metabolism pathways Shuang Cai, Jinlong Zhu, Xiangzhou Zeng, Qianhong Ye, Changchuan Ye, Xiangbing Mao, Shihai Zhang, Shiyan Qiao, and Xiangfang Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01637 • Publication Date (Web): 27 May 2018 Downloaded from http://pubs.acs.org on May 27, 2018

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Journal of Agricultural and Food Chemistry

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Maternal N-Carbamylglutamate supply during early pregnancy enhanced

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pregnancy outcomes in sows through modulations of targeted genes and

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metabolism pathways

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Shuang Cai,†,§ Jinlong Zhu,† Xiangzhou Zeng,† Qianhong Ye,† Changchuan Ye,†

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Xiangbing Mao,‡ Shihai Zhang,|| Shiyan Qiao,† and Xiangfang Zeng†,*

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†

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Centre, China Agricultural University, Beijing 100193, China;

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‡

State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry

Animal

Nutrition

Institute,

Sichuan

Agricultural

University,

No.

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Gongpinghuimin Road, Wenjiang District, Chengdu 611130, China;

11

||

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Animal Science, South China Agricultural University, Guangzhou 510642, China

211,

Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of

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ACS Paragon Plus Environment


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ABSTRACT: Reducing pregnancy loss is important for improving reproductive

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efficiency for both human and mammalian animals. Our previous study demonstrates

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that maternal N-Carbamylglutamate (NCG) supply during early pregnancy enhances

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embryonic survival in gilts. However whether maternal NCG supply improves the

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pregnancy outcomes is still not known. Here, we found maternal NCG supply during

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early pregnancy in sows significantly increased the numbers of total piglets born alive

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per litter (P < 0.05), and significantly changed the levels of metabolites in amniotic

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fluid and serum involved in metabolism of energy, lipid, and glutathione, and

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immunogical regulation. The expression of endometrial progesterone receptor

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membrane component 1 (PGRMC1) were significantly increased by NCG

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supplementation (P < 0.05), as well as the expression of PGRMC1, endothelial nitric

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oxide synthesases (eNOS), and lamin A/C in fetuses and placentae (P < 0.05). Among

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the NCG associated amino acids, arginine and glutamine markedly increased

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PGRMC1 and eNOS expression in porcine trophectoderm cells (P < 0.05). While

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glutamate could stimulate the expression of vimentin and lamin A/C in porcine

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trophectoderm (pTr) cells (P < 0.05), and proline stimulated lamin A/C expression (P

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< 0.05). Collecetively, these data reveal mechanisms of NCG in reducing early

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embryo loss. These findings have important implications that NCG has great potential

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to improve pregnancy outcomes in human and mammalian animals.

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KEYWORDS: embryo, metabolome, N-carbamylglutamate, pregnancy outcome,

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sows

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INTRODUCTION

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Embryo implantation is a critically important biological process in pregnancy in

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mammals, which, to a large degree, has a direct impact on the pregnancy outcome.1

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Early pregnancy loss, accounting for 30% - 50% in mammals, profoundly restricts

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reproductive performance.2 Embryo implantation failure is the major cause of these

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early pregnancy losses.1 In women, about 15% of all pregnancies end due to early

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pregnancy loss.3 Even though in vitro fertilization (IVF) has become a successful

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technology in treating infertility, the pregnancy rate of IVF is still relatively low at

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around 25% - 30%, because of the failure of the IVF embryo to implant in the uterus.4

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Therefore, reducing early embryo implantation failure is a crucially important issue in

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improvement of the reproductive efficiency for both humans and mammalian animals.

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Although numerous studies have demonstrated that nutrition plays important roles

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in fetal development and subsequent pregnancy outcomes, only limited studies have

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focused on the impacts and roles of specific nutrients or general nutritional status on

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embryo development and embryo implantation.5-7 Arginine is a conditionally essential

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amino acid for most mammals. Many studies have shown that arginine regulates

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embryo development and improves embryo quality subsequently enhancing embryo

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implantation.8-10 Arginine supplementation during early pregnancy is demonstrated to

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improve embryo implantation through the PI3K/PKB/mTOR/NO signaling pathway,

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ultimately enhancing the pregnancy outcome in rats.11,12 As an activator of

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endogenous arginine synthesis, NCG has been reported to improve early embryonic

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implantation and pregnancy outcome in rats.13,14 In gilts, maternal NCG supply during

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early pregnancy also improves embryonic survival and development through

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modulation of endometrial proteomes.15,16 However, whether maternal NCG

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supplementation during early pregnancy improves pregnancy outcomes in sows is still 3



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unknown, as well as the specific roles of NCG in regulation of targeted genes’

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expression in fetuses, placentae and endometriums, and the metabolic changes in

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serum and amniotic fluid. Additionally, pig, which shares high similarity with humans

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in anatomic and physiologic characteristics, becomes one of the best models for

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human reproduction studie.17 Therefore, approaches to enhance sows’ reproductive

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performance have important implication for improving human reproduction. The

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objective of this study was to investigate whether maternal NCG supplementation

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during early pregnancy improved pergancy outcomes in sows and to examine the

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targeted genes’ expression in fetuses, placentae andendometriums, as well as the

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metabolic changes in serum and amniotic fluid.

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MATERIALS AND METHODS

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Ethics statement

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This study was carried out in strict accordance with the Chinese guidelines for animal

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welfare. Animal handling procedures reported herein were approved by the

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Institutional Animal Care and Use Committee of China Agricultural University.

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Animals and protocols

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In order to examine whether maternal NCG supplementation during early pregnancy

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improved pergancy outcomes in sows, a total of 80 sows (parity 3 or 4; Landrace ×

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Yorkshire) with an initial body weight of 210.00 ± 10.91 kg were used in this study.

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Sows were housed individually in gestation stalls (2.2 m × 0.65 m) with concrete

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floors. Sows were checked daily for estrus using direct boar exposure in the morning

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and artificially inseminated three times with fresh semen during estrus (10-12 h apart).

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Immediately after breeding, sows were allocated based on body weight, back fat, and

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parity into 4 groups. Sows in the control group were fed the basal diet for the entire

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gestation period. Sows in the three N-carbamylglutamate groups were fed the basal 4


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diet supplemented with 0.05% NCG (wt:wt) during d 1-8, d 9-28, or d 1-28.

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Thereafter sows were fed the basal diet until delivery (n = 20). Here, we designed

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three NCG supply phases to determine the optimal one. All sows were fed 1.25 kg

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diets during d 1-28 of pregnancy and 1.75 kg diets from d 29 of pregnancy to delivery

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twice daily at 06:00 h and 16:00 h. Sows had free access to water throughout the

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experiment. Diets were formulated to meet the recommended nutrient requirements of

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NRC (2012) for gestating sows (Table 1). NCG (purity, 97%) was obtained from

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National Feed Engineering Technology Research Center (Beijing, China) and 0.05%

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NCG level was based on results from our previous study.15 Litter size, live litter size,

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litter weight, number of stillborn and mummified piglets, and birth weight were

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recorded soon after birth.

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To investigate whether NCG improving pregnancy outcome was associated with

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the role of NCG in regulation of targeted genes’ expression in fetuses, placentae and

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endometriums, as well as the metabolic changes in serum and amniotic fluid, sixteen

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gilts (Duroc × Yorkshire × Landrace) with average initial body weight of 132.0 kg

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were randomly assigned to the the basal diet (control) or fed the basal diet

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supplemented with 0.05% NCG between d 1 and d 28 of pregnancy (0.05% NCG, n =

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8). This NCG supply phase was chosen based on the results of the first animal study.

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Diets were the same as in Exp. 1. On d 28 of gestation, blood samples were collected

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via jugular venepuncture into 5 mL tubes after overnight starvation before being

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centrifuged at 3,000 × g for 10 min at 4°C to obtain serum samples. Gilts were killed

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by electrocution to obtain uteri, which were transported on ice to the lab. A volume of

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5 mL amniotic fluid from each fetus was harvested with syringes. Placentae and

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endometriums were carefully isolated from the individual fetus. All the samples were

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immediately snap frozen in liquid nitrogen and stored at -80°C until further analysis. 5



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Chemicals and reagents

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HPLC-grade methanol and ACN were obtained from Fisher Scientific International

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(Hampton, NH, USA). HPLC-grade formic acid was purchased from Dikma

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Technology (RichmondHill, Ontario, Canada). Arginine, glutamine, glutamate, and

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proline were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Ultrapure

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water (Milli-Q, Millipore Corporation, Bedford, MA, USA) was used to prepare all

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aqueous solutions.

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Cell culture and treatment

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To further examine the contributions of arginine, glutamine, glutamate, and proline,

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the concentrations of which are changed in maternal serum in response to maternal

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NCG supply during early pregnancy, pTr cells were used as the in vitro model. Cells

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were isolated originally from d 12 conceptuses and was kindly provided by Dr.

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Zhenlong Wu from China Agricultural University. Cells were cultured in 6-well plates

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in DMEM/F-12 medium with 10% FBS, 1% insulin–transferrin–selenium (ITS,

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Sigma–Aldrich, St. Louis, Missouri, USA) until reaching 80% confluence. Cells were

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serum starved overnight in customized medium, deprived of either arginine, proline,

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glutamate or glutamine, and then treated with different doses (0, 0.25, 0.50, 1.00 mM)

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of Arg, Pro, Glu or Gln for 24 h. All experiments were repeated at least 2-3 times.

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Non-targeted metabolic fingerprinting analysis

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Metabolite extraction

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Methanol was used to extract both intracellular and extracellular metabolites. Serum

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and amniotic fluid samples (200 µL) were centrifuged at 14,000 × g for 20 min at 4°C.

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The supernatant were transferred and mixed with 4 volumes of methanol. The samples

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were incubated at -80°C for 2 h and then centrifuged at 14,000 × g for 10 min at 4°C.

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The metabolites containing supernatant were transferred to new tubes on dry ice and 6


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then dried to pellets using speedvax/lyophilizer with no heat. The dried metabolite

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samples were stored at -80°C until further processing.

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HPLC methods for polar metabolites

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Ultimate 3000 UHPLC (Dionex) coupled with Q Exactive was used to perform LC

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separation. In positive mode, Waters Atlantis HILIC Silica column (2.1 × 100 nm)

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was used. Mobile phase A was prepared by dissolving 0.63 g of ammonium formate in

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50 mL of HPLC-grade water, then adding 950 mL of HPLC-grade acetonitrile and 1

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µL of formic acid. Mobile phase B was prepared by dissolving 0.63 g of ammonium

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formate in 500 mL of HPLC-grade water, followed by 500 mL of HPLC-grade

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acetonitrile and 1 uL of formic acid. Linear gradient was as follows: 0 min, 1% B; 2

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min, 1% B; 3.5 min, 20% B; 17 min, 80% B; 17.5 min, 99% B; 19 min, 99% B; 19.1

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min, 1% B; 22 min, 1% B. In negative mode, Waters BEH Amide column was applied

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(2.1 × 100 mm). Mobile phase A was prepared by dissolving 0.77 of ammonium

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acetate in 50 mL of HPLC-grade water, then 950 mL of HPLC-grade acetonitrile was

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added. pH was adjusted to 9.0 with ammonium hydroxide solution. Mobile phase B

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was prepared by dissolving 0.77 g of ammonium acetate in 500 mL of HPLC-grade

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water, subsequently, adding 500 mL of HPLC-grade acetonitrile and adjusting pH to

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9.0 with ammonium hydroxide solution. Linear gradient was as follows: 0 min, 5% B;

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2 min, 5% B; 4 min, 20% B; 18 min,85% B;19 min, 95% B; 21 min, 95% B; 21.1 min,

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5% B; 25 min, 5% B.

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MS methods for polar metabolites

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Polar metabolite analysis was performed on Q Exactive Orbitrap mass spectrometer.

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The detailed mass spectrometer parameters are as follows: spray voltage, 3.5 kV for

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positive and 2.5 kV for negative; capillary temperature, 275 °C for positive and 320°C 7



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for negative; sheath gas flow rate (arb), 35; aux gas flow rate (arb), 8; mass range

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(m/z), 70-1,050 for positive and 80-1,200 for negative; full MS resolution, 70,000;

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MS/MS resolution, 17,5000; topN, 10; NCE, 15/30/45; duty cycle, 1.2s.

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Data processing

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Two levels of identification were performed simultaneously using Tracefinder.

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Metabolites were first potentially assigned according to endogenous MS database by

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accurate masses. At the same time, those that can match with the spectra in fragment

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database were confirmed and potentially assigned metabolites were displayed in the

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results. 10 ppm and 15 ppm mass tolerance was applied for precursor and fragment

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matching. Still, 0.25 min retention time shift was allowed for quantitation.

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Western blot analysis

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The pTr cells, frozen placenta, endometrium and fetuses were separately homogenized

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in RIPA lysis buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate,

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0.1 % SDS, 50 mM Tris–HCl at pH 7.4) supplemented with a protease inhibitor

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cocktail (Apply gene, Beijing, China). After centrifugation at 1,4000 × g for 15 min at

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4oC, the protein concentration in the supernatant was determined using the BCA

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protein assay kit (Pierce, Rockford, IL, USA). Equal amount of proteins was

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electrophoresed (Bio-Rad, Richmond, CA, USA) on SDS polyacrylamide gels. Then

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proteins were electrotransferred to polyvinylidene difluoride membrane (Millipore,

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Bedford, MA, USA) and blocked with 5% Bovine Serum Albumin (BSA) overnight at

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4 °C or 1 h at room temperature. Prestained protein markers (Fermentas, Waltham,

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MA, USA) were used in each gel. Samples were incubated with primary antibodies

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(1:1,000 dilution) against Membrane-associated PGRMC1 (Santa Cruz Biotechnology,

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Santa Cruz, CA, USA), lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 8


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eNOS (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody

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(Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed with

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Tris-Tween 20 buffer (pH 7.4) for three times, membranes were incubated with the

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HRP-conjugated secondary antibody (Zhongshan Gold Bridge, Beijing, China) for 1 h

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at room temperature. The membrane was exposed by AlphaImager 2200 (Alpha

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Innotech, San Leandro, CA, USA) automatically. Band densities were detected with

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the Western Blot Luminance Reagent (Applygene, Beijing, China) and quantified

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using AlphaImager 2200 (Alpha Innotech, San Leandro, CA, USA).

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Statistical analysis

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For results of reproductive performance, PGRMC1, eNOS, vimentin, and lamin A/C

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expression of gilts and pTr cells, data were analyzed using MIXED procedures of

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SAS (version 9.0; SAS Institute Inc., Cary, NC) following a randomized complete

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block design. Sow was considered the experimental unit. Data for the number of

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stillborn and mummified, and embryonic mortality were analyzed using the chi-square

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test of SAS. A value of P < 0.05 was considered statistically significant. Statistical

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analysis for metabolics results were performed with identified polar metabolites. The

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student’s t test was evaluated by SAS. Principal component analysis (PCA) and

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pathway analysis was completed using Metaboanalyst. Compared with the control

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group, metabolites with a threshold of > 1.50- or < 0.55-fold, values of P < 0.1, and

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VIP > 1.35 were considered differentially metabolites.

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RESULTS

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The reproductive performance of sows and serum concentration of amino acids

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As shown in Table 2, dietary NCG supplementation during d 1-28 of pregnancy in

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sows increased the number of total piglets born per litter and total piglets born alive 9



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per litter, compared with the control diet and NCG supplementation during d 1-8 or d

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9-28 of pregnancy (P < 0.05). However, dietary NCG supplementation during three

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different stages of early pregnancy had no effect on sows’ weight gain, number of

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stillborn and mummified piglets or birth weight of piglets.

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The metabolomic profiles in serum and amniotic fluid

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To identify possible relationships within classes of data, PCA was employed.

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Performing PCA on the statistics-filtered data set, distinct differences was observed

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between the NCG group and control group, showing that NCG highly influences the

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serum and amniotic fluid metabolome. Data were visualized by constructing PC

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scores, where each point on the plot represented a single sample. In serum, PC1 and

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PC2 represented 19.4% and 13.1% of the variation, respectively. The first two PCs

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explained 32.5% of the total variances within the data (Fig. 1A). And in amniotic fluid,

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PC1 and PC2 represented 8.2% and 8% of variation, respectively (Fig. 1B).

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Metabolomes in serum from NCG and control groups were differentially

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expressed (Table 3). To identify which variables account for such a significant

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separation, we calculated the P value, VIP score (variable in project, the higher the

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score, the more important it is) and fold change (FC), defined as the ratio of the NCG

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group to the control group for a given expression profile. Taking the three aspects into

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account, 19 metabolites are selectively shown in Table 3. Fifteen of the metabolites

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detected were found to be up-regulated in NCG group, while 4 were down-regulated.

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The detailed analysis of the most relevant pathways was performed by

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MetaboAnalysis. The results showed that maternal NCG supplementation

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up-regulated metabolites mainly involved in fatty acid metabolism, energy

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metabolism and immunological regulation. 10


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For amniotic fluid, 14 metabolites are selectively shown in Table 4. Ten of the

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metabolites detected were found to be up-regulated in NCG group, while 4 were

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down-regulated. The up-regulated metabolites are mainly involved in fatty acid

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metabolism, energy metabolism and glutathione metabolism while down-regulated

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metabolites are involved in cysteine metabolism and absorption of vitamins.

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The protein abundance of PGRMC1, lamin A/C, and eNOS in the endometrium,

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fetuses, and placentaeplacentae of gilts on d 28 of pregnancy

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The protein abundance of PGRMC1, eNOS, and lamin A/C in placentae were

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significantly increased (P < 0.05) in gilts fed the NCG supplemented diet on d 28 of

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pregnancy, compared with those in gilts fed the control diet (Fig. 2A). Similarly, in

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fetuses, dietary NCG supplementation significantly increased (P < 0.05) the

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expression of PGRMC1, eNOS, and lamin A/C on d 28 of pregnancy, compared with

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the control diet (Fig. 2B). In endometrium, NCG supplementation also dramatically

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enhanced (P < 0.05) the expression of PGRMC1 and eNOS on d 28 of pregnancy,

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compared with the control diet (Fig. 2C), while the expression of lamin A/C was not

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affected.

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The expression levels of PGRMC1, lamin A/C, eNOS, and vimentin in pTr cells

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in the presence of arginine, proline, glutamine, or glutamate

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Western blot analysis revealed that compared to the control treatment, a 24-h arginine

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treatment markedly increased PGRMC1 expression in a dose dependent manner in

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pTr cells, which peaked at 0.5 mM. Similarly, the expression levels of eNOS were

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also dose-dependently increased in pTr cells, with the peak at 0.5 mM (Fig. 3A).

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However, arginine had no effect on vimentin and lamin A/C expression level in pTr

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cells (Fig. 3A). 11



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Glutamine also dose-dependently stimulated the expression levels of PGRMC1

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and eNOS in pTr cells, in comparison with the control treatment, with the peak at 0.5

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mM for PGRMC1, and 1.0 mM for eNOS (Fig. 3B). Glutamine treatment had no

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obvious effect on the expression of lamin A/C and Vimentin (Fig. 3B).

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Compared with the control treatment, glutamate stimulated the expression of

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vimentin and lamin A/C in pTr cells, peaking at 0.25 mM and 0.50 mM, respectively

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(Fig. 3C). However, glutamate had no effects on the expression of PGRMC1 and

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eNOS in pTr cells (Fig. 3C).

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A dose-dependent augmentation of lamin A/C was also observed in pTr cells

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treated with proline (Fig. 3D). Proline markedly increased the expression levels of

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lamin A/C at 0.25 mM treatment in pTr cells, compared to the control treatment (Fig.

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3D). Proline treatment had no effects on the expression of PGRMC1, Vimentin, and

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eNOS in pTr cells (Fig. 3D).

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DISCUSSION

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Our results showed that NCG supply during early pregnancy improved the pregnancy

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outcome in sows, which indicated that the beneficial effects of NCG on embryonic

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survival during early pregnancy subsequently improved pregnancy outcome in sows.

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NCG supplementation had a similar response compared with dietary arginine

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supplementation during early pregnancy in sows.18 However, NCG has unique

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advantages over arginine because: (1) there is no impact on intestinal absorption of

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dietary tryptophan, histidine, or lysine; (2) a low dose is highly effectivein stimulating

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endogenous arginine synthesis; (3) it has a relatively long half-life in vivo (perhaps

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8-10 h); and (4) substantially reduced cost.19-21 Therefore, dietary NCG

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supplementation perhaps is a better choice than arginine to improve pregnancy

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outcomes in mammals. 12


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To further explore the mechanism of NCG in improving pregnancy outcomes,

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metabolomic analysis in amniotic fluid and serum was conducted. The results

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indicated that maternal NCG supplementation increased levels of metabolites in

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amniotic fluids and serum primarily involved in energy metabolism, lipid metabolism,

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glutathione metabolism, and immunological regulation. This is consistent with our

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previous proteome results, which indicates that maternal NCG supplementation

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modulates endometrial proteins associated with energy metabolism, lipid metabolism,

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protein metabolism, antioxidative stress, and immune response.15 Energy metabolism

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is crucially important to embryo growth, differentiation, and viability.22 The

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reinforcement in energy metabolism probably provides more energy for embryonic

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and fetal development. During pregnancy, oxidative stress has negative impacts on

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embryo development, such as a decrease in the percentage of two-cell and blastocyst

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stage embryos. Reactive oxygen species are generated from embryo metabolism

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and/or exist in embryo surroundings, which cause embryonic growth retardation and

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embryonic dysmorphogenesis.23,24 Glutathione status reflects the antioxidant capacity

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of developing embryos. GSH reduces the formation of free oxygen radical species,

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ultimately protecting embryos against oxidative stress.24,25 Our data indicated that

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beta-citryl-L-glutamic acid level in amniotic fluids was increased which indicated that

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glutathione metabolism was enhanced in NCG supplemented sows. This was

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beneficial for embryo growth and protecting embryos against damage from free

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radical oxygen species. Another notable finding of the metabolomics data in amniotic

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fluids and serum was that immunological regulation pathways were up-regulated in

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NCG supplemented sows during early pregnancy. During pregnancy, the maternal

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uterus has tolerance to the semiallogenic fetus, and many immune cells (such as

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macrophages, dendritic cells, and regulatory T cells) and molecules (such as 13



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Leukemia Inhibitory Factor and heme oxygenase-1) are involved in this process.26

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Platelet-activating factor (PAF) may be used as an indicator of embryo viability and

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pregnancy outcome prediction. It has been reported that PAF level is highly correlated

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with pregnancy outcomes in human.27 PAF initiates early pregnancy factor synthesis,

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which is recognized as an immunosuppressive factor and whose amount is highest on

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d 30 of pregnancy in gilts.28,29 In this study, the improvement in pregnancy outcome

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induced by maternal NCG supply could be associated with the increase in serum PAF

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level. Collectively, alternations in levels of amniotic fluid and serum metabolites

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involved in energy metabolism, lipid metabolism, glutathione metabolism, and

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immunological regulation by maternal NCG supply were beneficial to embryonic

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development, which ultimately enhances pregnancy outcomes in sows.

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Our previous proteome data indicates that maternal NCG supplementation during

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early pregnancy up-regulates factors related to embryo implantation and development.

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15

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targeted genes to validate the modulation of NCG on the expression of these genes in

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vivo and in vitro. A recent study demonstrates that PGRMC1 is localized in oocytes,

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embryos/fetuses, and uteri.30 PGRMC1 is different from classical nuclear PGRs

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receptors. PGRMC1 and serpine 1 mRNA binding protein (SERBP1) form a complex

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to bind progesterone, leading to the increase in cAMP levels and activation of protein

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kinase G,31 to mediate anti-apoptosis and to modulate steroidogenesis and cellular

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homeostasis.32,33 In bovine endometrium, PGRMC1 is co-expressed with SERBP1

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and nuclear progesterone receptor (PGR) during the first trimester of pregnancy,

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indicating PGRMC1 plays an important role in early pregnancy possesses.34

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PGRMC1 is also expressed in uterus and placenta in mice and humans indicating its

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potentially important role in cellular differentiation, modulation of apoptosis and

In the present study, PGRMC1, NOS, vimentin, and lamin A/C were chosen as

14


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steroidogenesis during early pregnancy.35 In the current study, the enhanced

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expression of PGRMC1 observed in endometrium, fetuses, and placentae in NCG

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supplemented gilts on d 28 of pregnancy indicates that maternal NCG supply might

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improve the development of endometrium, fetuses, and placentae through modulation

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of local PGRMC1 expression. The in vitro results showed only arginine and

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glutamine but not glutamate and proline remarkably induced PGRMC1 expression in

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pTr cells which indicates that arginine and glutamine are the key metabolites related

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to PGRMC1 stimulation by maternal NCG supply.

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Three nitric oxide synthesases (NOS), including neuronal nitric oxide

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synthesases (nNOS), eNOS, and inducible nitric oxide synthesases (iNOS) are

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involved in nitro oxide synthesis from arginine in the body.36 NO modulates embryo

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implantation through remodeling of the extracellular matrix,37 and plays critical roles

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in placental vascular development.38 NOS expression in the uterus is beneficial for

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endometrial decidualization and embryo implantation.39 Low expression of eNOS

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increases embryo loss and decreases embryo implantation sites in the eNOS-knockout

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mouse model.40 In contrast, high expression of eNOS in fetuses and placentae is

345

benefical for nutrient transfer from mother to fetus and efficient utilization of these

346

nutrients.41 Our results showed that arginine and glutamine supplementation increased

347

eNOS expression in pTr cells. We speculate that dietary NCG supplementation during

348

early pregnancy might improve embryo implantation through up-regulation of eNOS

349

expression induced by arginine and glutamine in pTr cells. NOSs, also highly

350

expressed in placenta, influence the placental vasculogenesis and angiogenesis

351

through modulation of VEGF and angiopoietin signaling pathways.38 We found that

352

eNOS expression was differentially modulated in uterus, fetus, and placenta in

353

response to maternal NCG supply in early pregnancy, with relatively stronger 15



354

stimulation in the placenta and fetus than that in the uterus. These data indicate that

355

maternal NCG supply exerted a stronger influence on the development of fetuses and

356

placentae, compared with the uterus.

357

Vimentin, one of the intermediate filament proteins, plays pivotal roles in cell

358

migration.42 Trophoblastic giant cells (TGC) exert critical roles in embryo and fetal

359

development through interaction with decidual cells and the extracellular matrix.43-45

360

Vimentin is found to be expressed in mice trophoblastic giant cells, which is benefical

361

for development of the chorioallantoic placenta.46,47 Our data indicated that glutamate

362

enhanced vimentin expression in pTr cells. Serum glutamate concentration was

363

reported to increase by NCG supplementation.15 Therefore, maternal NCG supply

364

appears to benefit embryo and fetal development partially through modulation of

365

vimentin expression in pTr cells. Lamin A/C is expressed in mouse embryonic stem

366

cells and developing blastocyst.48 Lamin A/C mediates cell mechanics, polarization,

367

and migration 49 and is important for determination of cell fate.50 The increased lamin

368

A/C expression in fetuses and placentae could be benefical for the early development

369

of embryos in response to maternal NCG supply.

370

In conclusion, maternal NCG supply during early pregnancy enchanced

371

pregnancy outcomes in sows through differential modulation of factors related to

372

embryo implantation and development in endometrium, fetuses, and placentae, as well

373

as the regulation of metabolites in amniotic fluids and serum, which are primarily

374

involved in energy metabolism, lipid metabolism, glutathione metabolism, and

375

immunological regulation. These findings imply that NCG has great potential to

376

improve pregnancy outcomes in humans and mammalian animals.

16


Page 16 of 36

Page 17 of 36


377

AUTHOR INFORMATION

378

Corresponding Authors

379

*(X.Z.)

380

[email protected].

381

Finding

382

This work was supported by National Natural Science Foundation of the P. R of China

383

(NO. 31301980 and NO. 31772614).

384

Notes

385

The authors declare no competing financial interest.

Phone:

+86

10-62733588.

Fax:

+86

10-62733688.

E-mail:

386 387

ABBREVIATIONS USED

388

eNOS, endothelial nitric oxide synthesases; FC, fold change; iNOS, inducible nitric

389

oxide synthesases; ITS, insulin–transferrin–selenium; IVF, in vitro fertilization; NCG,

390

N-carbamylglutamate; nNOS, neuronal nitric oxide synthesases; NOS, nitric oxide

391

synthesases; PAF, Platelet-activating factor; PCA, Principal component analysis; PGR,

392

progesterone receptor; PGRMC1, progesterone receptor membrane component 1; pTr,

393

porcine trophectoderm; SERBP1, serpine 1 mRNA binding protein; TGC,

394

Trophoblastic giant cells; VIP, variable in project

17



395

Refercences

396

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of embryo viability. Hum. Reprod. 2002, 17, 1306-1310.

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Vivas, A. B. Immunohistochemical distribution of early pregnancy factor in ovary,

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oviduct and placenta of pregnant gilts. Biotech. Histochem. 2015, 90, 14-24.

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platelet-activating factor in mice. J. Reprod. Fertil. 1997, 109, 187-191.

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receptor membrane component 1 expression and putative function in bovine oocyte

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maturation, fertilization, and early embryonic development. Reproduction. 2010, 140,

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receptor membrane component 1): a targetable protein with multiple functions in

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steroid signaling, P450 activation and drug binding. Pharmacol. Ther. 2009, 121,

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immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic

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acid treatment and functional analysis of PGRMC1 mutations. Endocrinology. 2008,

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partner serpine 1 mRNA binding protein in uterine and placental tissues of the mouse

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and human. Mol. Cell. Endocrinol. 2008, 287, 81-89.

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the role of free fetal hemoglobin. Front. Physiol. 2015, 5, 516.

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vascular development and function. Placenta. 2011, 32, 797-805.

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implantation in rats. J. Reprod. fertil. 1998, 114, 157-161.

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T. J.; Flores, J. M.; Gonzalez, B. A. Disruption of the endothelial nitric oxide synthase

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gene affects ovulation, fertilization and early embryo survival in a knockout mouse

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model. Reproduction. 2008, 136, 573-579.

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intrauterine growth retardation: implications for the animal sciences. J. Anim. Sci.

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migration. Ontogenez. 2013, 44, 186-202.

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cytochemical and ultrastructural study. Biocell. 2005, 29, 261-270.

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giant cells in acute fasted mice examined by electron microscopy. Tissue. Cell. 1995,

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537

synthesized by mouse vascular trophoblast giant cells from embryonic day 7.5

538

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539

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540

trophoblastic giant cells. Tissue. Cell. 2001, 33, 40-45.

541

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542

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543

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Stewart, C. L.; Hodzic, D.; Wirtz, D. Nuclear lamin A/C deficiency induces defects in

545

cell mechanics, polarization, and migration. Biophys. J. 2007, 93, 2542-2552

546

(50) Rober, R. A.; Weber, K.; Osborn, M. Differential timing of nuclear lamin A/C

547

expression in the various organs of the mouse embryo and the young animal: a

548

developmental study. Development. 1989, 105, 365-378.

24


Page 24 of 36

Page 25 of 36


549

Table 1 Composition and nutrient composition of the basal diet for gestating sows

550

(as-fed basis) Item

Content

Ingredients, % Corn grain

66.10

Wheat bran

19.50

Soybean meal, 43%

10.00

Soybean oil

0.50

Limestone

0.60

Dicalcium phosphate

2.10

Salt

0.40

L-Lysine hydrochloride

0.10

DL-Methionine

0.05

Choline chloride

0.15

Vitamin mineral premix1

0.50

Nutrient levels, % Dry matter2

89.60

Digestible energy3, Mcal/kg

3.07

Metabolizable energy3, Mcal/kg

2.93

Crude protein2

13.10

Calcium2

0.79

Available phosphorus3

0.49

Total phosphorus3

0.69

Lysine3

0.64

Arginine3

0.66

Methionine + cystine3

0.50

551

1

552

cholecalciferol, 0.04; d-α-tocopheryl acetate, 10.10; menadione sodium bisulfate, 1.60;

553

thiamine, 1.50; riboflavin, 3.00; vitamin B-6, 1.50; vitamin B-12, 0.01; niacin, 22.50;

Provided the following (mg/kg of the basal diet): retinyl acetate, 3.47;

25



554

calcium pantothenate, 15.00; folic acid, 2.50; biotin, 0.20; manganese, 40.00; iron,

555

85.00; zinc, 75.00; copper, 1.50; iodine, 0.09; and selenium, 0.03.

556

2

Analyzed values.

557

3

Calculated values.

26


Page 26 of 36

Page 27 of 36


558

Table 2 Effects of N-Carbamylglutamate (NCG) supplementation on reproductive

559

performance in sows Treatment1

SEM2 P value

Items Weight at breeding (kg)

Control

NCG 1 NCG 2 NCG 3

216.78

215.00

204.72

207.88

10.91

0.85

225.17

222.81

213.11

217.24

10.93

0.87

8.39

7.81

8.39

9.35

0.62

0.40

4.00

3.88

4.00

3.83

0.46

0.99

11.31ab 11.67ab 12.18a

0.46

0.04

10.70a

0.27

0.02

Weight at day 28 of gestation (kg) Weight gain through d 28 of gestaion(kg) Parity Total piglets born per litter Total piglets born alive per

11.11b 9.61b

9.81ab 10.39a

1.50

1.50

1.33

1.53

0.20

0.89

13.32

13.13

11.30

12.43

1.58

0.80

13.73

14.38

15.15

15.43

0.62

0.23

1.44

1.45

1.43

1.40

0.05

0.94

litter Stillborn and mummified (n) Fetal mortality (%)3 Litter birth weight of all piglets born alive (kg) Weight of piglets (kg) 560

Note: 1the numbers of sows for Control group, NCG 1, NCG 2 and NCG 3 were 18,

561

18, 16, and 17, respectively. a, b within a row, significant difference at P < 0.05.

562

2

SEM, Standard error of the mean.

563

3

Fetal mortality = number of stillborn and mummified piglets/number of total

564

piglets born per litter.

27



Page 28 of 36

565

Table 3 Serum metabolites that differed between control and N-Carbamylglutamate

566

groups Metabolite

Fold

VIP

P

change1

score

value

3.31

2.90

0.02

Related pathway Cysteine and methionine

Indoleacrylic acid

metabolism Isohyodeoxycholic acid

3.23

2.42

0.00

Platelet activating factor

2.75

1.72

0.05

Lipid metabolism Immunological regulation

Isofucosterol 3-O-6-O-9,12-octadecadi 2.69

2.27

0.01

Lipid metabolism

2.64

1.94

0.01

Hydrolysis of xenobiotic

enoyl -b-d-glucopyranoside Diisobutyl phthalate

Energy metabolism and Retapamulin

2.42

2.77

0.02

immunological regulation

Polysorbate 60

2.25

1.99

0.01

Fatty acid metabolism

Oleamide

2.20

1.65

0.02

Energy metabolism

Benzyl isothiocyanate

2.05

1.74

0.04

Glucosinolate breakdown

Pyridostigmine

2.09

1.70

0.03

Cholinesterase Inhibitors

1.89

1.35

0.04

Energy metabolism

1.88

1.56

0.02

3-cis-hydroxy-b,e-carote n-3'-one 3-hydroxyphenylpyruvic

Tyrosine catabolism

acid

pathway Phenylalanine

Tiropramide

1.84

1.73

0.00 metabolism

Cholesteryl acetate

1.53

1.47

0.04

Steroid biosynthesis

3alpha-acetomethoxy-11

1.60

1.52

0.04

Energy metabolism

28


Page 29 of 36


alpha-oxo-12-ursen-24-o ic acid Dilauryl 0.41

1.89

0.04

Redox reaction

0.32

1.76

0.04

Redox reaction

0.43

2.85

0.02

Purine metabolism

3,3'-thiodipropionate O-tyrosine Guanosine hexaphosphate adenosine 567

Note: 1Fold change, which was based on the normalized data, was defined as the fold

568

difference in the observed concentrations between NCG group and control group.

569

2

570

markers for distinguishing the two groups. The higher the score is, the more

571

important the metabolite is.

VIP, variable in project, was used to select distinct variables as potential

29



Page 30 of 36

572

Table 4 Metabolites that differed in amniotic fluid between control and

573

N-Carbamylglutamate groups Fold

VIP

P

change1

score

value

12- ketodeoxycholic acid

6.15

3.67

0.00


Diisobutyl phthalate

4.30

2.70

0.00

Hydrolysis of xenobiotic

Beta-citryl-l-glutamic acid

3.14

2.57

0.03

Glutathione metabolism

Cladribine

2.92

2.00

0.02

Metabolite name

Related pathway

Immunological suppression Ethyl aconitate

2.41

2.20

0.01

Pristanal

2.15

2.05

0.01

Tricarboxylic acid cycle Oxidation of branched chain fatty acids

Lysopc100

1.94

1.94

0.03

Energy metabolism

9-hexadecenoylcholine

1.77

1.60

0.03


1.75

2.76

0.04

1-Pyrroline-4-hydroxy-2-car

Arginine and proline

boxylate

metabolism

Polyoxyethylene 600 1.54

1.50

0.02

Energy metabolism

Benzyl thiocyanate

0.49

1.88

0.02

Cysteine metabolism

Pyroglutamic acid

0.49

2.26

0.02

Glutathione metabolism

Umbelliferone

0.55

1.89

0.03

monoricinoleate

Biosynthesis of phenylpropanoids Metabolism of 2-Nonadecanone

0.45

2.12

0.05 carbohydrates

1

574

Note: Fold change, which was based on the normalized data, was defined as the fold

575

difference in the observed concentrations between NCG group and control group.

576

2

VIP, variable in project, was used to select distinct variables as potential

30


Page 31 of 36


577

markers for distinguishing the two groups. The higher the score is, the more

578

important the metabolite is.

31



579

Figure legends

580

Figure1. Principal component analysis (PCA). Each point represents an individual

581

sample. (A) PCA of the profiling data from the serum metabolome. (B) PCA of the

582

profiling data from amniotic fluid metabolome. Gilts were fed the control or 0.05%

583

N-Carbamylglutamate supplemented diets during d 1-28 of pregnancy.

584

Figure 2. The protein abundance of PGRMC1, eNOS, and lamin A/C expression in

585

placentae (A), fetuses (B), and endometriums (C) of gilts on d 28 of pregnancy. Gilts

586

were fed the control or 0.05% N-Carbamylglutamate supplemented diets during d

587

1-28 of pregnancy. On day 28 of gestation, the placenta and endometrium were

588

carefully isolated from the uterus of individual fetuses. All tissue samples were

589

immediately snap frozen in liquid nitrogen and stored at -80℃.

590

Figure 3. The protein abundance of PGRMC1, eNOS, lamin A/C, and vimentin in

591

poecine Tr cells treated with arginine (A), glutamine (B), glutamate (C), and proline

592

(D). Cells were serum starved overnight in customized medium, deprived of either

593

arginine, proline, glutamate or glutamine, and then treated with different doses (0,

594

0.25, 0.50, 1.00 mM) of Arg, Pro, Glu or Gln for 24 h. All experiments were repeated

595

at least 2-3 times.

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Figure 1

597

33



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Figure 2

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Page 35 of 36


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Figure 3

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600

TOC Graphic

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Maternal N-Carbamylglutamate supply during early pregnancy

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