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/lp/springer-journals/dietary-supplementation-with-0-4-l-arginine-between-days-14-and-30-of-zB8TQixNPF
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- Springer Journals
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- Copyright © The Author(s) 2022
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- 2049-1891
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- 10.1186/s40104-022-00794-0
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Abstract
Background: Most embryonic loss in pigs occurs before d 30 of gestation. Dietary supplementation with L-arginine (Arg) during early gestation can enhance the survival and development of conceptuses (embryo/fetus and its extra- embryonic membranes) in gilts. However, the underlying mechanisms remain largely unknown. Methods: Between d 14 and 30 of gestation, each gilt was fed daily 2 kg of a corn- and soybean-meal based diet (12% crude protein) supplemented with either 0.4% Arg (as Arg-HCl) or an isonitrogenous amount of L-alanine (Control). There were 10 gilts per treatment group. On d 30 of gestation, gilts were fed either Arg-HCl or L-alanine 30 min before they were hysterectomized, followed by the collection of placentae, embryos, fetal membranes, and fetal fluids. Amniotic and allantoic fluids were analyzed for nitrite and nitrate [NOx; stable oxidation products of nitric oxide (NO)], polyamines, and amino acids. Placentae were analyzed for syntheses of NO and polyamines, water and amino acid transport, concentrations of amino acid-related metabolites, and the expression of angiogenic factors and aquaporins (AQPs). Results: Compared to the control group, Arg supplementation increased (P < 0.05) the number of viable fetuses by 1.9 per litter, the number and diameter of placental blood vessels (+ 25.9% and + 17.0% respectively), embryonic survival (+ 18.5%), total placental weight (+ 36.5%), the total weight of viable fetuses (+ 33.5%), fetal crown-to-rump length (+ 4.7%), and total allantoic and amniotic fluid volumes (+ 44.6% and + 75.5% respectively). Compared to con- trol gilts, Arg supplementation increased (P < 0.05) placental activities of GTP cyclohydrolase-1 (+ 33.1%) and ornithine decarboxylase (+ 29.3%); placental syntheses of NO (+ 26.2%) and polyamines (+ 28.9%); placental concentrations of NOx (+ 22.5%), tetrahydrobiopterin (+ 21.1%), polyamines (+ 20.4%), cAMP (+ 27.7%), and cGMP (+ 24.7%); total amounts of NOx (+ 61.7% to + 96.8%), polyamines (+ 60.7% to + 88.7%), amino acids (+ 39% to + 118%), glucose (+ 60.5% to + 62.6%), and fructose (+ 41.4% to + 57.0%) in fetal fluids; and the placental transport of water (+ 33.9%), Arg (+ 78.4%), glutamine (+ 89.9%), and glycine (+ 89.6%). Furthermore, Arg supplementation increased (P < 0.05) placental mRNA levels for angiogenic factors [VEGFA120 (+ 117%), VEGFR1 (+ 445%), VEGFR2 (+ 373%), PGF (+ 197%), and GCH1 (+ 126%)] and AQPs [AQP1 (+ 280%), AQP3 (+ 137%), AQP5 (+ 172%), AQP8 (+ 165%), and AQP9 (+ 127%)]. *Correspondence: g-wu@tamu.edu Department of Animal Science, Texas A&M University, College Station, TX 77843, USA Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 2 of 13 Conclusion: Supplementing 0.4% Arg to a conventional diet for gilts between d 14 and d 30 of gestation enhanced placental NO and polyamine syntheses, angiogenesis, and water and amino acid transport to improve conceptus development and survival. Keywords: Angiogenesis, Arginine, Fetus, Placenta, Reproduction Introduction to reductions in the number of corpora luteum (CL) and Pigs suffer high rates of embryonic mortality, especially concentrations of progesterone in the maternal plasma during the peri-implantation period of pregnancy [1, 2] because of excessive NO production [29], indicating the when embryos elongate rapidly, signal for pregnancy rec- importance of the timing of maternal Arg provision. ognition, and attach to the uterine wall [3, 4]. Maternal Water is transported rapidly across the placenta and nutrition plays an important role in the development and accumulates in allantoic and amniotic fluids during early subsequent survival of conceptuses (embryo/fetus and gestation to support conceptus growth, development, its extra-embryonic membranes), especially the maternal and survival in mammals, including swine [30, 31]. Aqua- dietary intake of amino acids (AAs) [5–7]. Both low and porins (AQPs) are plasma membrane proteins that allow high dietary protein intake can contribute to problems rapid water transport across membranes [32] and are also with fetal development and embryonic death due to defi - essential for placental development [33]. AQPs are acti- ciencies and excesses of AAs, respectively [8, 9]. Specifi - vated by multiple signaling pathways, including cGMP, cally, L-arginine (Arg) is a “conditionally essential AA” in cAMP, mitogen-activated protein kinases, protein kinase the diet that is important for optimal embryonic develop- C, and phosphatidylinositide 3-kinases/protein kinase B/ ment and survival by affecting placental growth [10– 12]. mechanistic target of rapamycin [34–36]. To date, 13 iso- Arg is the nitrogenous substrate for the synthesis of nitric forms of AQP have been discovered in mammals [32], 12 oxide (NO), which is essential for placental angiogenesis of which are expressed in the female reproductive tract (the sprouting of new blood vessels from existing ones) [37]. Pigs can potentially use AQP1, AQP5, AQP8, and [13, 14] and the regulation of cell metabolism [15–17]. AQP9 to transport water from the endometrial blood- Angiogenesis provides the physical conduit for utero-pla- stream to the allantoic bloodstream or allantoic fluid [38]. cental blood flow to enhance mother to fetus exchanges Although the timing and dose of Arg supplementation of water, AAs, and other nutrients, as well as gases and to pregnant gilts has been studied, the exact mechanism wastes [11]. Arg is also known to be a precursor for syn- through which it increases embryonic survival is not theses of polyamines, ornithine, creatine, agmatine, and fully understood [37, 39]. We conducted this study to homoarginine, each of which has enormous physiologi- test the hypothesis that supplementation of 0.4% Arg to cal significance [18– 21]. These biologically important gilts between d 14 and d 30 of gestation would increase substances are essential for conceptus growth and sur- embryonic survival and development by increasing the vival [22]. In support of this view, we found that dietary placental expression of angiogenic factors and AQP. We supplementation with 0.4% or 0.8% Arg to gilts between extended the period of Arg supplementation to d 30 of d 14 and 25 of gestation enhanced placental syntheses of gestation, so that the placentae of gilts could be success- NO and polyamines, as well as the placental expression of fully mounted into Ussing chambers to determine water angiogenic factors [15]. and AA transport. Previous studies demonstrated that dietary supplemen- tation with Arg to gilts during specific periods of gesta - Materials and methods tion improved the placental expression of antioxidative Experimental design genes [23], embryonic and fetal survival rates, as well as Twenty gilts (F1 crosses of Yorkshire × Landrace sows conceptus growth [5, 6, 24–26]. For example, Mateo et al. and Duroc × Hampshire boars) with a body weight of [26] reported that supplementation of 0.83% Arg between 100–125 kg were bred at the onset of the second estrus d 30 and d 114 of gestation increased litter size in gilts by and 12 h later. The day of breeding was recorded as d 0 2. In addition, Li et al. [24] found that dietary supplemen- of gestation. Following breeding, gilts were assigned ran- tation with 0.4% and 0.8% Arg between d 14 and d 25 of domly to 1 of 2 treatment groups, 0 (control) or 0.4% gestation increased the litter size in gilts by 2 and amni- Arg (as 0.484% Arg-HCl; Ajinomoto Co., Inc., Tokyo, otic fluid volume. Similar results were reported for rats Japan), with 10 gilts in each treatment group. An isoni- [27, 28]. Interestingly, dietary supplementation with 0.8% trogenous amount of 0.83% of L-alanine (Ajinomoto Co., Arg from d 0 to d 25 impaired embryonic survival due Inc., Tokyo, Japan) and 0.43% cornstarch were added to Her ring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 3 of 13 the 0 and 0.4% Arg diets, respectively, as top dressing. Arg), 5 mmol/L D-glucose, 100 units/mL penicillin, Each gilt was fed 1 kg of a corn- and soybean meal-based 100 µg/mL streptomycin and 0.25 µg/mL amphotericin diet containing 12% crude protein twice daily (0700 and B [42], preincubated at 37 °C for 0.5 h in 4 mL of fresh 1800 h) beginning on d 0 of gestation, with a feed intake oxygenated medium, and then incubated at 37 °C for of 2 kg/d [24]. The content of nutrients in the basal diet 6 h in 1 mL of fresh oxygenated medium that contained was the same as reported in our previous study [29], 5 mmol/L D-glucose, 0.2 mmol/L Arg, and concentra- which contained 12.0% crude protein (including 0.70% tions of other AAs found in the plasma of gilts [42]. At Arg and 0.57% lysine, as analyzed by high-performance the end of a 6-h incubation period, the medium was ana- liquid chromatography (HPLC) following acid hydroly- lyzed for nitrite plus nitrate (stable oxidation products of sis [40]) and 12.9 MJ metabolizable energy/kg. Either NO). In all experiments, the medium incubated without 0.4% Arg or the isonitrogenous amount of L-alanine was cells was analyzed as the blank. Nitrite and nitrate in cul- supplemented to the basal diet between d 14 and d 30 of ture medium were quantified by HPLC as we previously gestation. described [43, 44]. To determine effects of treatment on polyamine syn - Hysterectomy and tissue collection thesis, placentae were incubated as described above, On d 30 of gestation, gilts were fed either Arg-HCl or except that the medium contained 0.5 mmol/L L-[1- C] L-alanine, and hysterectomized and necropsied within ornithine [45]. C-labeled putrescine, spermidine, and 30 min of consuming the supplement. Gilts were anes- spermine were separated by HPLC and their radioactivi- thetized with an intramuscular injection of 10 mg Tela- ties were measured by a liquid scintillation counter, as we zol (Midwest Veterinary Supply, Lakeville, MN, USA) per described previously [46, 47]. The rates of the production kg of body weight that was followed by the inhalation of of putrescine, spermidine, and spermine were calculated 1%–5% isofluorane to achieve a surgical plane of anes - on the basis of their radioactivities and the specific radio - thesia [24]. Blood was collected from the uterine vein activity of L-[1- C]ornithine in the incubation medium. and artery before euthanasia, and after euthanasia gilts were hysterectomized to obtain uteri and conceptuses. Determination of NOx, polyamines, BH , cAMP, cGMP, Euthanasia was performed with an intracardiac injec- glucose, and fructose tion of saturated KCl. The number of CL, the number For the analysis of NOx (nitrite and nitrate) and poly- of live fetuses, placental weight, fetal body weight, fetal amines (the sum of putrescine, spermidine and sper- crown-to-rump length (the distance from the crown of mine), placentae (~ 50 mg) were homogenized in 1 mL of the head to the base of the tail), the volumes of amniotic 1.5 mol/L HClO , followed by neutralization with 0.5 mL and allantoic fluid in viable conceptuses, and the number of 2 mol/L K CO [45]. NOx (nitrite plus nitrate) and 2 3 and diameter of placental blood vessels were determined, polyamines were determined using our established HPLC as we described previously [22, 41]. Briefly, a picture was methods [43, 44]. For BH analysis, tissues (~ 50 mg) taken of each placenta. Three placentae (from the first, were homogenized in 0.5 mL of 0.1 mol/L phosphoric middle, and last fetuses within the left uterine horn) and acid containing 5 mmol/L dithioerythritol and 60 µL of three placentae (from the first, middle, and last fetuses 2 mol/L trichloacetic acid, and the tissue extract was used within the right uterine horn) from each gilt were evalu-for BH analysis by HPLC [13]. For the determination of ated to determine the total number of blood vessels per cGMP in placentae, the tissue (~ 100 mg) was homoge- 1 cm , and to measure the diameter of the central blood nized in 1 mL of 1.5 mol/L HClO , followed by the neu- vessel under a microscope (40 × objective). For each vari- tralization with 0.5 mL of 2 mol/L K CO . The extract 2 3 able, the mean of the six placental measurements was was analyzed for 3’-5’-cGMP using the cGMP Enzymeim- calculated to represent the value for the gilt. Samples of munoassay Biotrak System (GE Healthcare, Chalfont St placentae were snap-frozen in liquid nitrogen. For the Giles, Buckinghamshire, UK). cAMP was analyzed by an analyses of metabolites, allantoic or amniotic fluid from HPLC method involving the precolumn derivatization each viable conceptus of the same gilt was combined in with 2-chloroacetaldehyde and fluorescence detection, equal proportions. as we previously described [48]. Glucose, fructose, and glycerol were determined as described by He et al. [49], Determination of NO and polyamine syntheses Li et al. [24], and Jobgen et al. [50], respectively. by placentae Placental tissues (~ 200 mg) were rinsed three times with Determination of enzymatic activities 1 mL of oxygenated (95% O /5% CO ; v/v) custom-made Fresh placental tissue (~ 100 mg) was used to prepare the 2 2 Dulbecco’s-modified Eagle medium containing physi - cytosolic fraction for the assay of ornithine decarboxy- ological concentrations of AAs (including 0.2 mmol/L lase (ODC) activity with the use of 2 mmol/L L-[1- C] Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 4 of 13 ornithine (2500 dpm/nmol), as we described previously Embryonic survival rates were compared using the X [45]. The activities of constitutive NO synthase (cNOS), analysis [58]. Probability values ≤ 0.05 were considered and inducible NO synthase (iNOS) in frozen placental statistically significant. tissue were measured using L-[U- C]Arg [13]. The activ - ity of GTP-cyclohydrolase-1 [GTP-CH1; the key enzyme Results for BH synthesis) in frozen placental tissue was deter- Reproductive performance of gilts mined using desalted tissue extract and 2 mmol/L GTP, After dietary supplementation with 0 (control) or 0.4% as we described previously [51, 52]. Arg between d 14 and 30 of gestation, gilts were eutha- nized and hysterectomized to assess their reproductive Determination of placental transport of water and AAs performance. Maternal body weight and CL number Transport of H O was measured with the use of Ussing did not differ (P > 0.05) between control and Arg-sup- chambers (Physiologic Instruments, San Diego, CA, plemented gilts (Table 2). Embryonic survival rates were USA) containing 5 mL of oxygenated (95% O/5% CO ) determined as the number of live fetuses being divided 2 2 Krebs buffer as well as physiological concentrations by the number of CL present on the ovaries at the time of AAs and glucose, as we described [53, 54]. Pieces of of necropsy. Compared with the control group, dietary placental tissue (1 cm ) were mounted onto Ussing supplementation with 0.4% Arg enhanced (P < 0.05) the chambers, followed by the addition of H O (20 µL), number of viable fetuses by 1.9 per litter. The embryonic 14 14 0.2 mmol/L L-[U- C]Arg, 0.5 mmol/L L-[U- C]glu- survival rate of the Arg-supplemented gilts was 18.5% tamine, or 1 mmol/L [U- C]glycine (similar to physi- greater (P < 0.05) than that of the control group (P < 0.05) ological concentrations of AAs in the pig plasma) to the (Table 2). Compared to control gilts, Arg supplementa- “mucosal” side of each chamber. The specific radioactiv - tion increased (P < 0.05) total placental weight (36.5%), ity of H O on the “mucosal” side of the chamber was the total weight of viable fetuses (33.5%), fetal crown-to- 500 dpm/µL H O, whereas the specific radioactivities rump length (4.7%), total allantoic fluid volume (44.6%), 14 14 of 0.2 mmol/L L-[U- C]Arg, 0.5 mmol/L L-[U- C]glu- and total amniotic fluid volume (75.5%) (Table 2). tamine, and 1 mmol/L [U- C]glycine on the “mucosal” 4 4 3 side of the chamber were 3 × 10 , 1.2 × 10 , and 6 × 10 Eec ff ts of dietary Arg supplementation dpm/nmol, respectively. Thereafter, an aliquot of 20 on the concentrations of AAs and related metabolites µL solution was obtained from the “serosal” side of the in the maternal plasma and placenta, as well as their total chamber at 5, 10 and 15 min for the measurement of amounts in fetal fluids of gilts 3 14 H O and C radioactivity using a Packard liquid scintil- Compared to control gilts, Arg supplementation increased lation counter [47]. (P < 0.05) concentrations of Arg, ornithine, and proline in maternal plasma (37.2%, 29.6%, and 16.4%, respectively) and RNA extraction, reverse transcription and quantitative PCR placentae (14.4%, 11.7%, and 15.7%, respectively), but had Placental tissue (~ 100 mg) was homogenized with 1 mL no effect (P > 0.05) on those of other AAs (Table 3). Con- of TRIzol (Invitrogen, Waltham, MA, USA) and RNA centrations of glucose, fructose, and glycerol in the mater- was extracted with chloroform and precipitated with nal plasma and placentae did not differ (P > 0.05) between isopropanol [55–57]. RNA was washed with 75% etha- the control and Arg groups of gilts. By contrast, concentra- nol. Total RNA was measured using a NanoDrop ND tions of alanine in the maternal plasma and placentae were 1000 spectrophotometer. cDNA was synthesized using 98.4% and 11.1% greater (P < 0.05) in control gilts than in the SuperScript First Strand Synthesis System for RT- Arg-supplemented gilts. Compared to the control group, PCR (Invitrogen). RT-qPCR was performed using the Arg supplementation increased (P < 0.05) concentrations SYBR Green and the Applied Biosystems 7900HT Real of Arg (50.3%), glutamine (12.5%), glycine (33.3%), ornith- Time PCR system [56]. Sequences of primers, which were ine (25.7%), and serine (27.1%) in allantoic fluid, but had no designed using the Primer-BLAST software (http:// www. effect (P > 0.05) on those of other AAs, glucose and fructose ncbi. nlm. nih. gov/ tools/ primer- blast/) for the quantita- (Table 3). Concentrations of all measured AAs, glucose and tive RT-PCR analysis, are shown in Table 1. Tubulin α 1b fructose in amniotic fluid did not differ (P > 0.05) between (TUBA1B) was used as the housekeeping gene [55]. The the control and Arg-supplemented gilts. Dietary supple- relative expression values were calculated using the ΔΔCt mentation with Arg reduced (P < 0.01) concentrations of method [57]. glycerol in allantoic and amniotic fluids by 27.0% and 30.1% respectively, compared with control gilts. Due to increased Statistical analysis volumes of allantoic and amniotic fluids, total amounts of All data, except for embryonic survival rates, were all AAs, glucose and fructose in the fetal fluids were 31%– analyzed statistically using the unpaired t-test [58]. 117.8%, 60.5%–62.6%, and 41.4%–57.0% greater (P < 0.01) Her ring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 5 of 13 Table 1 Sequences of primers used for the quantitative RT-PCR analysis of genes in porcine placentae Gene Primer sequence Accession number NOS3Forward: 5’- ATC TTC AGC CCC AAA CGG AG -3’ NM_214295.1 Reverse: 5’- TTT CCA CCG AGA GGA CCG TA -3’ VEGF120Forward: 5’- AAG GCC AGC ACA TAG GAG AG -3’ KJ729036 Reverse: 5’- CCT CGG CTT GTC ACA TTT TT-3’ VEGF164Forward: 5’- GAG GCA AGA AAA TCC CTG TG -3’ NM214084 Reverse: 5’- TCA CAT CTG CAA GTA CGT TCG- 3’ VEGFR1Forward: 5’- CAC CCC GGA AAT CTA TCA GATC -3’ EU714325.1 Reverse: 5’- GAG TAC GTG AAG CCG CTG TTG -3’ VEGFR2Forward: 5’- GAA ATG GCT TCA TCC TCC AA -3’ AF513909.1 Reverse: 5’- CAA GGA AGA CTT GGC TCA GG -3’ GCH1Forward: 5’- AGT TCT TGG CCT CAG CAA AC -3’ XM_021102249.1 Reverse: 5’ TGC TTC AAC CAC TAC TCC GAC -3’ PGFForward: 5’- CAT CGT GTC TGT GTA CCC CA -3’ FJ177137.1 Reverse: 5’- TGA CAT TGA CCG TCT CCA CG -3’ FGF2Forward: 5’- GTG CAA ACC GTT ACC TTG CT -3’ NM_001001855.2 Reverse: 5’- ACT GCC CAG TTC GTT TCA GT -3’ AQP1Forward: 5’- TTG GGC TGA GCA TTG CCA CGC -3’ XM_021078524.1 Reverse: 5’- CAG CGA GTT CAG GCC AAG GGA GTT -3’ AQP2Forward: 5’- TCA ACC CTG CCG TGA CTG TAG -3’ EU636238.1 Reverse: 5’- GTT GTT GCT GAG GGC ATT GAC -3’ AQP3Forward: 5’- ACC CTT ATC CTC GTG ATG TTT -3’ HQ888860.1 Reverse: 5’- CAT TCG CAT CTA CTC CTT GTG -3’ AQP4Forward: 5’- TCT GGC TAT GCT TAT CTT TGTCC -3’ NM_001110423.1 Reverse: 5’- CGA TGC TAA TCT TCC TGG TGC -3’ AQP5Forward: 5’- TGA GTC CGA GGA GGA TTG GG -3’ NM_001110424.1 Reverse: 5’- GAG GCT TCG CTG TCA TCT GTTT -3’ AQP8Forward: 5’- GGT GCC ATC AAC AAG AAG ACG -3’ EU220426.1 Reverse: 5’- CCG ATA AAG AAC CTG ATG AGCC -3’ AQP9Forward: 5’- TTT GCT GAT GGA AAA CTG CTC -3’ NM_001112684.1 Reverse: 5’- CTC TGG TTT GTC CTC CGA TTGT -3’ AQP10Forward: 5’-TGG GCG TTA TAC TAG CCA TCTAC-3’ EU582021 Reverse: 5’-GGT TGG GCA CAG TTT ACT TCCT-3’ AQP11Forward: 5’- CGT CTT GGA GTT TCT GGC TACC -3’ EU220425 Reverse: 5’- CCT GTC CCT GAC GTG ATA CTTG -3’ TUBA1BForward: 5’- GCT GCC AAT AAC TAT GCC CG-3’ NM_001044544 Reverse: 5’- ACC AAG AAG CCC TGA AGA CC-3 respectively in Arg-supplemented than in control gilts. Total (60.7%–88.7%) in allantoic and amniotic fluids of gilt amounts of glycerol in these fluids did not differ (P > 0.05) (Table 5) due to their increased volumes. Total amounts between the two groups of gilts (Table 4). of the individual polyamines (putrescine, spermidine, and spermine) in allantoic and amniotic fluids also increased Eec ff ts of dietary Arg supplementation (P < 0.01) in response to dietary Arg supplementation. on the concentrations and total amounts of NOx and polyamines in fetal allantoic and amniotic fluids Eec ff ts of dietary Arg supplementation Concentrations of NOx and polyamines in allantoic and on the concentrations of metabolites (NOx, polyamines, amniotic fluids did not differ (P > 0.05) between the control BH , cAMP, and cGMP), NO and polyamine syntheses, and Arg groups of gilts. Compared with the control group, and enzyme activities in the placentae of gilts dietary supplementation with 0.4% Arg increased (P < 0.01) The data on the effects of dietary Arg supplementation total amounts of NOx (61.7%–96.8%) and polyamines on concentrations of NOx, polyamines, and BH , NO 4 Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 6 of 13 Table 2 Reproductive performance and placental angiogenesis were more developed and more abundant than those in of gilts fed diets supplemented with 0 (control) or 0.4% the allantois of placentae of conceptuses in control gilts L-arginine (Arg) between d 14 and d 30 of gestation (Panel A). Variable Control 0.4% Arg 3 14 Placental transport of H O and C‑AAs Maternal body weight at breeding, kg 119.7 ± 2.6 118.1 ± 3.4 Water and AAs were readily transported across the pla- Number of corpora lutea/littler, n 14.1 ± 0.6 13.9 ± 0.7 cental tissue from the “mucosal” side to the “serosal” side Number of live fetuses/litter, n 11.2 ± 0.7 13.1 ± 0.4 of Ussing chambers at a constant rate during the 15-min Embryonic survival rate, % 79.5 ± 3.5 94.2 ± 2.1 period of measurement (Table 7). Compared with control Weight of the viable fetus, g 1.75 ± 0.06 1.98 ± 0.08 gilts, dietary supplementation with 0.4% Arg increased Weight of total viable fetuses/litter, g 19.4 ± 0.9 25.9 ± 1.1 (P < 0.01) the rates of net transport of water (33.9%), Arg Fetal crown-to-rump length, mm 25.4 ± 0.35 26.6 ± 0.43 (78.4%), L-glutamine (89.9%), and glycine (89.6%) by pla- Weight of the placenta for the live fetus, g 31.6 ± 1.1 36.8 ± 1.7 centae (Table 7). Weight of total placentae/litter, g 353 ± 22 482 ± 26 Volume of allantoic fluid/viable fetus, mL 185 ± 11 229 ± 15 Expression of angiogenic factors and AQPs in the placenta Total volume of allantoic fluid/litter, mL 2,075 ± 179 3,001 ± 262 qPCR was performed on placental tissue at d 30 of ges- Volume of amniotic fluid/viable fetus, mL 1.31 ± 0.04 1.97 ± 0.17 tation from gilts supplemented with either 0 (control) or Total volume of amniotic fluid/litter, mL 14.7 ± 0.8 25.8 ± 2.5 0.4% Arg from d 14 to d 30 of gestation to analyze the 2 † Number of placental blood vessels, n/cm 9.53 ± 0.40 12.0 ± 0.95 mRNA expression of key factors associated with angio- Diameter of placental blood vessels, mm 7.18 ± 0.25 8.40 ± 0.32 genesis and water transport. Results are summarized in Data are mean values ± SEM, n = 10. Embryonic survival rate was calculated as Table 8. VEGFA120 and VEGFA164 are the two isoforms number of live fetuses per number of corpora lutea present on the ovaries at the of VEGF. The placental mRNA level for VEGFA120 was time of necropsy on d 30 of gestation 117% greater (P < 0.05) in the 0.4% Arg-supplemented P < 0.05 vs the control group gilts, compared to the control group, but the placen- P < 0.01 vs the control group tal mRNA level for VEGFA164 did not differ (P > 0.05) between these two groups of gilts. Placental mRNAs for and polyamine syntheses, and the activities of related VEGFR1 and VEGFR2, as well as PGF and GCH1 were enzymes in porcine placentae are summarized in Table 6. 445%, 373%, 197%, and 126% more abundant (P < 0.01), Compared with control gilts, dietary supplementation respectively, in the 0.4% Arg-supplemented gilts than with 0.4% Arg increased (P < 0.05) placental concentra- those in control gilts. By contrast, dietary supplemen- tions of NOx (22.5%), polyamines (putrescine + spermi- tation with 0.4% Arg did not affect (P > 0.05) placental dine + spermine; 20.4%), BH (21.1%), cAMP (27.7%), mRNA levels for NOS3 or FGF2. and cGMP (24.7%). The rates of placental syntheses AQPs 1, 2, 3, 4, 5, 8, 9, and 11 were expressed in the of NO and polyamines were 26.2% and 28.9% greater placentae of gilts on d 30 of gestation (Table 8). mRNA (P < 0.01), respectively, in Arg-supplemented gilts than in for AQP10 was not detected in the placentae from con- the control group. The concentrations and rates of syn - trol or Arg-supplemented gilts. Dietary supplementation theses of the individual polyamines (putrescine, spermi- with 0.4% Arg enhanced (P < 0.05) mRNA levels for AQP1 dine, and spermine) in placentae also increased (P < 0.01) (280%), AQP3 (137%), AQP5 (172%), AQP8 (165%), and in response to dietary Arg supplementation. Compared AQP9 (127%) in the porcine placentae (Table 7). There with the control group, dietary supplementation with was no difference (P > 0.05) in placental mRNA levels for 0.4% Arg increased (P < 0.05) the enzymatic activities of AQP2, AQP4, and AQP11 between the control and 0.4% GTP-CH1 (33.1%) and ODC (29.3%) in placentae but did Arg-supplemented gilts. not affect (P > 0.05) those of cNOS and iNOS. Discussion Placental angiogenesis Because swine experience high rates of embryonic mor- Angiogenesis of placentae was determined by counting tality during early gestation, a management practice to the number of their blood vessels and measuring their ameliorate such loss would be highly beneficial to both diameter. The number of blood vessels per cm and their the swine industry and researchers [3, 4, 6, 59, 60]. A diameter in the placentae of the 0.4% Arg-supplemented corn- and soybean meal-based diet containing 12% crude group were 25.9% and 17.0% greater (P < 0.05), respec- protein is considered optimal to provide most AAs and tively, compared with control gilts (Table 2). As shown prevent hyperammonemia (a major factor contributing in Fig. 1, the blood vessels of the allantois of placentae to embryonic death) in gestating pigs [8]. However, a ges- of conceptuses in 0.4% Arg-supplemented gilts (Panel B) tation diet containing 12% crude protein does not meet Her ring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 7 of 13 Table 3 Eec ff ts of dietary supplementation with 0 (control) or 0.4% L-arginine (Arg) between d 14 and d 30 of gestation on concentrations of amino acids and related metabolites in the maternal uterine arterial plasma and in fetal fluids of gilts Variable Maternal uterine plasma Placenta Allantoic fluid Amniotic fluid Control 0.4% Arg Control 0.4% Arg Control 0.4% Arg Control 0.4% Arg * † Ala 843 ± 55 425 ± 20 661 ± 20 595 ± 23 258 ± 16 268 ± 18 269 ± 15 254 ± 13 β-Alanine 7.2 ± 0.46 7.1 ± 0.39 16.3 ± 0.8 15.8 ± 1.1 9.4 ± 0.68 9.7 ± 0.84 8.1 ± 0.55 8.2 ± 0.42 * † * Arg 148 ± 6.5 203 ± 7.9 367 ± 12 420 ± 15 181 ± 7.4 272 ± 9.4 152 ± 10 155 ± 9.1 Asn 67.2 ± 4.4 62.0 ± 3.7 164 ± 7.6 157 ± 8.1 62.2 ± 3.0 66.7 ± 3.5 52.0 ± 2.2 53.3 ± 2.6 Asp 14.1 ± 0.7 12.6 ± 0.9 353 ± 16 346 ± 18 15.5 ± 0.65 16.9 ± 0.72 15.2 ± 0.71 15.6 ± 0.52 Citrulline 63.5 ± 2.6 62.3 ± 2.3 19.7 ± 1.0 19.3 ± 1.1 10.3 ± 0.54 10.6 ± 0.47 8.2 ± 0.46 8.5 ± 0.39 Cys 196 ± 7.8 204 ± 9.3 266 ± 9.4 269 ± 10 76.7 ± 3.7 78.0 ± 4.4 35.7 ± 1.8 35.3 ± 1.7 Glu 203 ± 9.2 178 ± 12 705 ± 22 657 ± 19 129 ± 5.9 143 ± 6.2 132 ± 7.4 125 ± 8.1 Gln 472 ± 20 459 ± 18 816 ± 25 805 ± 32 630 ± 23 709 ± 27 680 ± 18 692 ± 35 Gly 779 ± 34 738 ± 41 873 ± 40 883 ± 30 847 ± 38 1,129 ± 57 347 ± 11 339 ± 14 His 78.7 ± 4.9 80.1 ± 3.8 185 ± 7.3 173 ± 8.0 78.8 ± 5.4 82.1 ± 5.7 48.6 ± 3.0 49.4 ± 2.4 Hyp 21.9 ± 1.6 21.4 ± 1.8 102 ± 5.9 108 ± 7.4 51.6 ± 2.4 52.8 ± 3.0 35.2 ± 1.6 36.4 ± 2.0 Ile 121 ± 5.2 114 ± 6.5 177 ± 6.9 171 ± 5.8 29.8 ± 1.5 29.1 ± 2.3 51.5 ± 2.7 50.8 ± 3.2 Leu 207 ± 8.0 189 ± 7.2 225 ± 11 216 ± 8.9 65.2 ± 4.2 66.3 ± 3.4 133 ± 8.4 139 ± 7.0 Lys 176 ± 16 168 ± 13 410 ± 19 414 ± 16 358 ± 15 371 ± 17 204 ± 5.9 198 ± 8.3 Met 48.8 ± 2.4 46.3 ± 1.7 195 ± 9.0 174 ± 9.8 21.6 ± 1.6 21.3 ± 1.1 48.1 ± 2.0 47.4 ± 3.7 † † † Ornithine 81.0 ± 6.6 105 ± 7.6 163 ± 5.4 182 ± 6.0 113 ± 6.1 142 ± 6.5 104 ± 6.0 102 ± 6.7 Phe 75.6 ± 3.8 70.3 ± 4.2 175 ± 5.4 167 ± 6.9 37.7 ± 1.9 38.4 ± 2.6 70.5 ± 4.4 71.2 ± 3.7 * * Pro 238 ± 7.6 277 ± 9.5 383 ± 13 443 ± 16 256 ± 13 277 ± 8.5 104 ± 6.5 108 ± 8.6 Ser 124 ± 5.8 109 ± 6.5 486 ± 15 461 ± 19 569 ± 26 723 ± 28 365 ± 17 361 ± 15 Taurine 107 ± 7.5 114 ± 6.1 936 ± 37 963 ± 35 461 ± 20 458 ± 22 114 ± 7.5 110 ± 6.2 Thr 175 ± 8.2 166 ± 9.5 390 ± 13 376 ± 11 221 ± 11 215 ± 13 229 ± 12 224 ± 16 Trp 56.8 ± 1.9 55.4 ± 2.3 68.5 ± 3.7 73.4 ± 3.4 14.1 ± 1.0 14.5 ± 1.1 14.3 ± 0.86 13.6 ± 0.94 Tyr 97.5 ± 6.2 96.1 ± 7.5 198 ± 7.4 187 ± 8.1 46.9 ± 2.3 48.2 ± 2.6 53.5 ± 3.1 52.8 ± 2.9 Val 267 ± 10 254 ± 13 286 ± 8.8 108 ± 7.4 88.6 ± 6.1 90.7 ± 5.8 175 ± 8.8 169 ± 9.1 Glucose 5,341 ± 83 5,279 ± 74 294 ± 15 286 ± 17 1,394 ± 84 1,540 ± 61 1,496 ± 75 1,415 ± 64 Fructose 505 ± 31 508 ± 38 78.0 ± 3.2 80.5 ± 3.7 2,538 ± 169 2,467 ± 121 2,515 ± 107 2,305 ± 101 * * Glycerol 115 ± 6.3 121 ± 7.4 59.2 ± 3.4 61.3 ± 3.9 230 ± 9.5 168 ± 8.1 116 ± 9.4 81.1 ± 5.3 Values, expressed as nmol/mL for plasma and fetal fluid and as nmol/g tissue for placentae, are means ± SEM, n = 10 gilts/treatment group Cysteine + ½ cysteine; Hyp: 4-hydroxyproline P < 0.05 vs the corresponding control group P < 0.01 vs the corresponding control group dietary requirements for Arg [5, 12]. Thus, supplement - 0.4% and 0.8% Arg to gilts from d 14 to d 25 of gestation enhanced litter size by 2 conceptuses, as well as allantoic ing this deficient AA to the maternal diet is an effective and amniotic fluid volumes, when compared with control way to enhance the growth and development of the con- gilts. Of note, dietary supplementation with 1.075% Arg to ceptus without any detrimental effects associated with sows (parity ≥ 2; an average of approximately 4) from d 1 increasing dietary crude protein intake [5–7, 26, 61, 62]. to d 30 of gestation increased the number of piglets born Most embryonic loss in pigs occurs before d 30 of gesta- alive per litter by 1.63 [61]. The dose of Arg supplemen tion, making this time period an appropriate target for - improvement in the reproductive performance of gilts and tation is important to prevent an imbalance among basic sows [2–4, 24]. However, Li et al. [29] discovered that die- AAs in diets [62, 63]. Therefore, total dietary Arg should tary supplementation with 0.8% Arg between d 0 to d 25 be less than 2%, so that the ratio of Arg to lysine does not decreased the number of CL and, accordingly, litter size, exceed 3 to prevent competition for transport into cells between these two basic AAs [63]. We used a supple and concentrations of progesterone in maternal plasma - due to excessive NO generation. In a subsequent study, mental dose of 0.4% Arg in the present study, because this Li et al. [24] found that dietary supplementation with amount was determined to be sufficient for enhancing the Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 8 of 13 Table 4 Eec ff ts of dietary supplementation with 0 (control) or 0.4% L-arginine (Arg) between d 14 and d 30 of gestation on total amounts of amino acids and related metabolites in fetal fluids of gilts Variable Allantoic fluid Amniotic fluid Control 0.4% Arg % increase Control 0.4% Arg % increase * * Ala 537 ± 59 820 ± 110 52.7 3.95 ± 0.33 6.34 ± 0.43 60.5 * * β-Ala 18.6 ± 0.83 27.4 ± 0.81 47.3 0.12 ± 0.01 0.21 ± 0.02 75.0 * * Arg 376 ± 36 819 ± 76 117.8 2.20 ± 0.14 3.81 ± 0.19 73.2 * * Asn 126 ± 8.7 194 ± 12 54.0 0.76 ± 0.05 1.38 ± 0.15 81.6 * * Asp 32.2 ± 3.0 50.4 ± 4.8 56.5 0.22 ± 0.02 0.40 ± 0.04 81.8 * * Cit 21.3 ± 2.1 31.5 ± 2.8 47.9 0.12 ± 0.01 0.22 ± 0.03 83.3 1 * * Cys 157 ± 13 232 ± 22 47.8 0.53 ± 0.05 0.92 ± 0.11 73.6 * * Glu 266 ± 24 429 ± 41 61.3 1.91 ± 0.12 3.24 ± 0.40 69.6 * * Gln 1307 ± 129 2,129 ± 211 62.9 10.0 ± 0.68 17.2 ± 1.1 72.0 * * Gly 1720 ± 118 3,403 ± 359 97.8 5.02 ± 0.19 8.59 ± 0.80 71.1 * * His 160 ± 15 246 ± 27 53.8 0.70 ± 0.04 1.30 ± 0.18 85.7 * * Hyp 105 ± 7.5 156 ± 13 48.6 0.52 ± 0.04 0.91 ± 0.06 75.0 * * Ile 60.5 ± 4.3 84.1 ± 6.6 39.0 0.76 ± 0.07 1.32 ± 0.17 73.7 * * Leu 130 ± 6.9 194 ± 12 49.2 1.97 ± 0.18 3.52 ± 0.33 78.7 * * Lys 735 ± 59 1,109 ± 109 50.9 2.98 ± 0.17 4.99 ± 0.38 67.4 * * Met 43.6 ± 3.5 62.0 ± 3.7 42.2 0.70 ± 0.03 1.16 ± 0.08 65.7 * * Ornithine 231 ± 20 418 ± 29 81.0 1.51 ± 0.09 2.54 ± 0.18 68.2 * * Phe 78.1 ± 8.0 113 ± 9.9 44.7 1.03 ± 0.07 1.79 ± 0.14 73.8 * * Pro 529 ± 49 837 ± 87 58.2 1.54 ± 0.16 2.72 ± 0.26 76.6 * * Ser 1175 ± 108 2,137 ± 161 81.9 5.25 ± 0.19 9.34 ± 1.1 77.9 * * Taurine 945 ± 75 1,353 ± 104 43.2 1.65 ± 0.11 2.70 ± 0.15 63.6 * * Thr 450 ± 35 633 ± 50 40.7 3.31 ± 0.19 5.56 ± 0.43 68.0 * * Trp 28.7 ± 2.2 41.6 ± 2.5 44.9 0.21 ± 0.02 0.35 ± 0.04 66.7 * * Tyr 94.8 ± 5.7 141 ± 10 48.7 0.77 ± 0.05 1.31 ± 0.09 70.1 * * Val 176 ± 8.7 263 ± 17 49.4 2.58 ± 0.23 4.33 ± 0.45 67.8 * * Glucose 2866 ± 265 4,600 ± 413 60.5 21.9 ± 1.6 35.6 ± 2.7 62.6 * * Fructose 5120 ± 338 7,238 ± 505 41.4 37.2 ± 3.0 58.4 ± 4.9 57.0 Glycerol 473 ± 41 506 ± 55 NC 1.72 ± 0.19 2.08 ± 0.21 NC Values, expressed as µmol/litter, are means ± SEM, n = 10 gilts/treatment group Cysteine + ½ cysteine; Hyp: 4-hydroxyproline; NC: no change P < 0.05 vs. the corresponding control group P < 0.01 vs. the corresponding control group survival and development of conceptuses in gestating gilts VEGFR1, VEGFR2, PGF, and GCH1 (Table 8). VEGFA is [24]. considered as the conventional form of VEGF and acts NO is a potent vasodilator and also stimulates placen- on endothelial cells to induce their migration and pro- tal angiogenesis [13, 14, 22]. Specifically, NO enhances liferation along with increasing the endothelial produc- blood flow through inducing the dilation of the blood tion of NO [13]. VEGFA120 and VEGFA164 are splice vessels and increasing vascular density via a cGMP- variants of VEGFA expressed in the porcine placenta to dependent mechanism [19, 22]. The placental vasculature increase vascular permeability [65]. VEGFA binds to the is responsible for the delivery of nutrients and gases for VEGF receptors 1 and 2, thereby exerting its physiologi- exchange across the utero-placental interface between cal function [66, 67]. PGF is also part of the VEGF family mother and fetus, as well as for the removal of fetal that acts in synergy with VEGF to promote angiogenesis metabolic wastes [64, 65]. Our results suggest that Arg [67]. As noted previously, eNOS converts Arg to NO in increases placental angiogenesis by increasing the expres- endothelial cells [19] and GTP-CH1 is a rate-controlling sion of genes for angiogenic factors, such as VEGFA120, enzyme in the production of BH [51, 52], which is an 4 Her ring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 9 of 13 Table 5 Eec ff ts of dietary supplementation with 0 (control) or 0.4% L-arginine (Arg) between d 14 and d 30 of gestation on the concentrations and total amounts of NOx and polyamines in allantoic and amniotic fluids of gilts Variable Allantoic fluid Amniotic fluid Control 0.4% Arg Control 0.4% Arg Concentrations, nmol/mL NOx 69.1 ± 2.4 78.0 ± 3.0 45.6 ± 1.8 52.8 ± 2.5 Putrescine 1.64 ± 0.08 1.84 ± 0.14 0.44 ± 0.04 0.48 ± 0.05 Spermidine 2.32 ± 0.15 2.57 ± 0.15 0.71 ± 0.05 0.80 ± 0.06 Spermine 2.54 ± 0.18 2.77 ± 0.16 0.74 ± 0.07 0.85 ± 0.06 Total polyamines 6.50 ± 0.41 7.18 ± 0.43 1.89 ± 0.14 2.12 ± 0.15 Total amounts per litter * * NOx, µmol 141 ± 9.5 228 ± 12 664 ± 37 1307 ± 70 * * Putrescine, nmol 3,308 ± 195 5,376 ± 478 6.41 ± 0.77 11.4 ± 0.54 * * Spermidine, nmol 4,685 ± 345 7,536 ± 543 10.3 ± 0.74 19.6 ± 1.5 * * Spermine, nmol 5,114 ± 409 8,145 ± 614 10.8 ± 1.1 20.9 ± 1.7 * * Total polyamines, nmol 13,106 ± 929 21,056 ± 1,568 27.5 ± 2.4 51.9 ± 3.1 Data are means ± SEM, n = 10 gilts/treatment group. NOx: oxidation end products (nitrite plus nitrate) of NO; total polyamines: putrescine + spermidine + spermine P < 0.01 vs. the corresponding control group Table 6 Eec ff ts of dietary supplementation with 0 (control) or 0.4% L-arginine (Arg) between d 14 and d 30 of gestation on the concentrations of NOx and polyamines, NO and polyamine syntheses, and enzyme activities in the placentae of gilts Variable Control 0.4% Arg Placental concentrations NOx, nmol/g tissue 46.6 ± 2.5 57.1 ± 2.9 Putrescine, nmol/g tissue 49.1 ± 2.0 60.5 ± 1.8 Spermidine, nmol/g tissue 99.4 ± 4.1 115 ± 3.0 Spermine, nmol/g tissue 101 ± 4.6 125 ± 4.1 Total polyamines, nmol/g tissue 250 ± 10 301 ± 8.2 BH , pmol/g tissue 346 ± 15 419 ± 18 cAMP, pmol/g tissue 202 ± 9.4 258 ± 11 cGMP, pmol/g tissue 15.4 ± 0.8 19.2 ± 0.9 Placental synthesis NO, nmol/g tissue/h 12.2 ± 0.48 15.4 ± 0.68 Putrescine, nmol/g tissue/h 0.58 ± 0.02 0.71 ± 0.03 Spermidine, nmol/g tissue/h 0.92 ± 0.05 1.16 ± 0.06 Spermine, nmol/g tissue/h 0.91 ± 0.06 1.26 ± 0.07 Fig. 1 Placental blood vessels in the allantois on d 30 of gestation Total polyamines, nmol/g tissue/h 2.42 ± 0.11 3.12 ± 0.13 in gilts supplemented with either 0 (control) or 0.4% L-arginine. Placental enzyme activity Placental blood vessels are shown in the allantois of gilts without cNOS, nmol/g tissue/h 1.30 ± 0.08 1.42 ± 0.09 L-arginine supplementation (control; Panel A) and gilts receiving dietary supplementation with 0.4% L-arginine (Panel B) iNOS, nmol/g tissue/h 0.43 ± 0.02 0.45 ± 0.03 GTP-CH1, nmol/g tissue/h 1.57 ± 0.10 2.09 ± 0.14 ODC, nmol/g tissue/h 8.66 ± 0.57 11.2 ± 0.80 Data are means ± SEM, n = 10 gilts/treatment group. cNOS: constitutive essential cofactor of all NOS isoforms for NO synthesis NO synthase; GTP-CH1: GTP cyclohydrolase-I; iNOS: inducible NO synthase; [17]. As reported for endothelial cells in both normal NOx: oxidation end products (nitrite plus nitrate) of NO; ODC: ornithine decarboxylase; total polyamines: putrescine + spermidine + spermine and diabetic rats [51, 52], Arg increases the synthesis and P < 0.05 vs. the control group bioavailability of BH , thereby increasing the generation P < 0.01 vs. the control group of NO by the porcine placentae (Table 6). In addition to NO, Arg supplementation augmented placental ODC Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 10 of 13 Table 7 Rates of the net transport of water and amino acids by placentae from gilts fed diets supplemented with 0 (control) or 0.4% L-arginine (Arg) between d 14 and d 30 of gestation Time, min Net transport of water, nL/mg Net transport of Arg, pmol/mg Net transport of Gln, pmol/mg Net transport of Gly, pmol/ tissue/min tissue/min tissue/min mg tissue/min Control 0.4% Arg Control 0.4% Arg Control 0.4% Arg Control 0.4% Arg * * * * 5 356 ± 14 475 ± 20 32.1 ± 2.2 58.8 ± 3.6 33.4 ± 2.5 64.1 ± 4.2 65.9 ± 3.3 123.8 ± 6.6 * * * * 10 352 ± 17 473 ± 21 32.5 ± 2.0 57.3 ± 3.2 33.0 ± 2.2 62.8 ± 3.6 65.4 ± 3.8 125.2 ± 6.2 * * * * 15 358 ± 15 479 ± 22 31.9 ± 1.7 56.1 ± 3.9 33.7 ± 2.4 63.2 ± 4.0 65.6 ± 3.6 124.4 ± 6.9 Values are means ± SEM, n = 10 gilts/treatment group. On d 30 of gestation, pieces of placental tissue (1 cm ) were mounted onto Ussing chambers, followed by the 3 14 14 14 addition of either H O (20 µL), 0.2 mmol/L L-arginine (Arg) plus L-[U- C]Arg, 0.5 mmol/L L-glutamine (Gln) plus L-[U- C]Gln, or 1 mmol/L glycine (Gly) plus [U- C]Gly to the “mucosal” side of the Ussing chambers for their net transport into the “serosal side” of the Ussing chambers P < 0.01 vs. the corresponding control group activity as well as the availability of both Arg and proline by the embryo/fetus and for storage in both allantoic and (the major sources of ornithine in the porcine placenta amniotic fluids (Tables 2 and 4). [45]) due to enhanced AA transport (Table 7)] for synthe- The 12 AQPs (AQPs 1–12) expressed in the female ses of NO and polyamines (Table 6) that also stimulate reproductive tract can be classified into three differ - angiogenesis [21, 68, 69]. Elevated expression of these ent subgroups [33, 70, 71]. AQPs 1, 2, 4, 5, 6 and 8 are angiogenic factors increases angiogenic activity (includ- classical aquaporins that are highly selective for water ing the proliferation of endothelial cells) in the placentae, transport. AQPs 3, 7, 9 and 10 are aquaglyceroporins resulting in a more highly developed placental vascula- that transport urea, glycerol, and other small solutes in addition to water. AQPs 11 and 12 are superaquaporins. ture (Table 2 and Fig. 1). Therefore, more water and AAs As reported by Zhu et al. [33] for porcine placentae on can be transported across the placenta (Table 7) for use d 25 of gestation, the placentae of gilts expressed AQP1, AQP2, AQP3, AQP4, AQP5, AQP8, AQP9, and AQP11, but not AQP10, on d 30 of gestation (Table 8). Most Table 8 Relative expression of mRNAs for angiogenic factors recently, McLendon et al. [38] localized AQP 1, 5, 8, and and AQPs in the placentae of gilts fed a diet supplemented with 9 proteins to specific cell types within both the endome 0.4% L-arginine (Arg) versus 0 Arg between d 14 and d 30 of trium and placenta, suggesting that pigs can use AQP1, gestation AQP5, AQP8, and AQP9 to transport water from the Gene Fold change P‑ value endometrial bloodstream to the allantoic bloodstream and allantoic fluid. Much evidence shows that AQPs are VEGFA120 1.17 0.031 essential for maintaining the accumulation and reabsorp VEGFA164 0.99 0.948 tion of allantoic and amniotic fluids for optimal embry - VEGFR1 4.45 0.008 onic growth [37]. Of note, dietary supplementation with VEGFR2 3.73 < 0.001 Arg enhanced the expression of genes for AQP1, AQP3, NOS3 1.14 0.145 AQP5, AQP8, and AQP9 in the placenta (Table 8) and PGF 1.97 < 0.001 functionally the placental transport of water (Table 7). GCH1 1.26 0.001 This finding is consistent with the report that Arg FGF2 0.96 0.144 enhanced the expression of AQP3 in porcine trophecto AQP1 2.80 0.002 derm cells [36] and our additional observation that allan- AQP2 1.09 0.763 toic and amniotic fluid volumes in fetal pigs were much AQP3 1.37 0.046 greater in gilts receiving dietary supplementation with AQP4 1.05 0.148 0.4% Arg as compared with control gilts (Table 2). There AQP5 1.72 0.047 is clear evidence that volumes of these fetal fluids are AQP8 1.65 0.004 positively correlated with embryonic growth and survival AQP9 1.27 0.020 in mammals, including pigs [30, 31, 59]. AQP11 1.14 0.806 As reported for endothelial cells and skeletal mus Values are the relative expression of genes in the placentae of gilts cle of Arg-supplemented rats [69], dietary Arg supple- supplemented with 0.4% L-arginine (9 gilts), compared to gilts supplemented mentation increased the concentrations of both cAMP with 0 L-arginine (control; 7 gilts) between d 14 and d 30 of gestation. The abundances of mRNAs for the genes were measured by qPCR using SYBR Green and cGMP in porcine placentae (Table 6). Some AQPs Her ring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 11 of 13 Fig. 2 Proposed mechanisms responsible for beneficial effects of dietary L-arginine supplementation in improving embryonic/fetal growth and survival in gestating swine. L-Arginine stimulates the synthesis of tetrahydrobiopterin [BH , a required co-factor for nitric oxide (NO) synthase)] from GTP via the GTP cyclohydrolase-I (GCH1) pathway, thereby augmenting NO production by placental tissue. L-Arginine also increases the activity of ornithine decarboxylase (a key enzyme for the synthesis of polyamines). Both NO and polyamines, as well as growth factors (such as placental growth factor, vascular endothelial growth factor A120, and vascular endothelial growth factor receptors 1 and 2) promote placental angiogenesis and growth (including vascular growth) to increase rates of transfer of non-water nutrients [including amino acids (AAs)] and oxygen across the placenta from mother to embryo/fetus. In addition, L-arginine elevates the concentrations of both cGMP and cAMP in the placenta to increase the expression of aquaporins (AQPs) to promote the placental transport of water from mother to embryo/fetus. Ultimately, the coordinate actions of L-arginine result in improvements in the growth and survival of embryos/fetuses (e.g., AQPs 1 and 5) are cGMP-gated transmembrane by 38% [72]. These results support the previous conclu - channels [70, 71]. In addition, AQPs are activated by sion from in vitro studies [36] that the NO-cGMP and cAMP-dependent protein kinase A [32, 37]. Thus, cAMP-dependent pathways play an important role in cGMP and cAMP cell signaling can up-regulate water promoting water transport by the placentae of Arg-sup- transport across the cell membrane. In support of this plemented gilts to increase the volumes of allantoic and view, AQP3 expression was enhanced by Forskolin (a amniotic fluids of the conceptuses (Fig. 2). Arg is truly cell-permeable activator of adenylate cyclase) but inhib- a functional AA for successful pregnancy outcomes in ited by H-89 (an inhibitor of cAMP-dependent protein mammals (including swine) and must be included ade- kinase) in porcine conceptus trophectoderm cells [36]. quately in their diets [73, 74]. Furthermore, the addition of a membrane-permeable cGMP analog (i.e., para-chlorophenylthio-cGMP) to Conclusions culture medium stimulated water transport across the Results of the present study revealed new insights into human pigmented retinal epithelium [71] and porcine the mechanisms whereby dietary supplementation conceptus trophectoderm cells [36]. Likewise, addi- with 0.4% Arg to gilts between d 14 and d 30 of gesta- tion of diethylenetriamine-nitric oxide adduct (DETA- tion enhances embryonic survival, as well as the volumes NO; an NO donor; 15 µmol/L) to the “mucosal side” of of allantoic and amniotic fluids in the conceptuses. In Ussing chambers rapidly enhanced water transport by addition, Arg supplementation increased the synthe- placentae from gilts on d 60 of gestation (i.e., 36% and ses of NO and polyamines by placentae, the expression 86% at 2 and 10 min, respectively, compared with the of angiogenic factors and angiogenesis in placentae (as absence of DETA-NO) [72]. Conversely, inhibition of indicated by increases in the number of placental blood NO synthesis reduced water transport by porcine pla- vessels and their diameters), placental growth and AQP cental cells [36]. Because NO stimulates the production expression, and the placental transport of water and of cGMP from GTP by guanylate cyclase in cells [37], AAs. These results advance the understanding of mecha - dietary supplementation with 0.4% Arg enhanced the nisms whereby dietary Arg supplementation beneficially concentration of cGMP in porcine placentae by 25% improves embryonic/fetal growth and survival. Our (Table 6). Similarly, increasing the extracellular concen- findings have important nutritional implications for tration of Arg from 0.1 to 0.25 mmol/L augmented the increasing reproductive performance in swine and other concentration of cGMP in porcine trophectoderm cells mammalian species. Herring et al. Journal of Animal Science and Biotechnology (2022) 13:134 Page 12 of 13 Abbreviations 5. Moreira RHR, Pérez Palencia JY, Moita VHC, Caputo LSS, Saraiva A, Fer- AA: Amino acid; AQP: Aquaporin; BH : Tetrahydrobiopterin; CL: Corpora reira RA, et al. Variability of piglet birth weights: A systematic review luteum; cNOS: Constitutive nitric oxide synthase; eNOS (NOS3): Endothelial and meta-analysis. J Anim Physiol Anim Nutr (Berl). 2020;104:657–66. nitric oxide synthase; FGF2: Fibroblast growth factor 2; GTP-CH1 (or GCH1): 6. Palencia JYP, Lemes MAG, Garbossa CAP, Abreu MLT, Pereira LJ, Zan- GTP cyclohydrolase-1; HPLC: High-performance liquid chromatography; iNOS: geronimo MG. Arginine for gestating sows and foetal development: a Inducible nitric oxide synthase; NO: Nitric oxide; NOx: Stable oxidation prod- systematic review. J Anim Physiol Anim Nutr (Berl). 2008;102:204–13. ucts of NO (nitrite plus nitrate); ODC: Ornithine decarboxylase; PGF: Placental 7. Wu G, Bazer FW, Satterfield MC, Li X, Wang X, Johnson GA, et al. growth factor; TUBA1B: Tubulin α 1b; VEGFA: Vascular endothelial growth Impacts of arginine nutrition on embryonic and fetal development in factor A; VEGFR: Vascular endothelial growth factor receptor. mammals. Amino Acids. 2013;45:241–56. 8. Herring CM, Bazer FW, Johnson GA, Wu G. Impacts of maternal dietary Acknowledgements protein intake on fetal survival, growth and development. Exp Biol We thank Gayan I. Nawaratna, Xilong Li, Erin Posey, Avery Kramer, and Bryan Med. 2018;243:525–33. McLendon for technical assistance. The current address of Mohammed 9. Ji Y, Wu Z, Dai Z, Wang X, Li J, Wang B, et al. Fetal and neonatal pro- Elmetwally is Department of Theriogenology, Faculty of Veterinary Medicine, gramming of postnatal growth and feed efficiency in swine. J Anim Sci Mansoura University, 35516 Mansoura, Egypt. Biotech. 2017;8:42. 10. Hou YQ, Yin YL, Wu G. Dietary essentiality of “nutritionally non- Authors’ contributions essential amino acids” for animals and humans. Exp Biol Med. GW, FBW and GAJ designed and supervised the study. CMH, FWB, GAJ, 2015;240:997–1007. HS, SH, MH, WH, DBL, and GW performed the experiment. CMH, HS, MH, 11. Wu G, Bazer FW, Burghardt RC, Johnson GA, Kim SW, Li XL, et al. and GW statistically analyzed and summarized results. CMH and GW wrote Impacts of amino acid nutrition on pregnancy outcome in pigs: the manuscript. CMH, FBW, GAJ, HS, SH, MH, and GW contributed to data mechanisms and implications for swine production. J Anim Sci. interpretation and manuscript revisions. All authors read and approved the 2010;88:195–204. final manuscript. 12. Wu G, Bazer FW, Johnson GA, Hou Y. Arginine nutrition and metabolism in growing, gestating, and lactating swine. J Anim Sci. 2018;96:5035–51. Funding 13. Meininger CJ, Wu G. Regulation of endothelial cell proliferation by nitric This research was supported by Agriculture and Food Research Initiative oxide. Methods Enzymol. 2022;352:280–95. Competitive Grant no. 2014–05142 from the USDA National Institute of Food 14. Chen DB, Zheng J. Regulation of placental angiogenesis. Microcirc. and Agriculture. 2014;21:15–25. 15. Elmetwally MA, Li XL, Johnson GA, Burghardt RC, Herring CM, Kramer Availability of data and materials AC, et al. Dietary supplementation with L-arginine between Days 14 All data generated or analyzed during this study are available from the cor- and 25 of gestation enhances NO and polyamine syntheses and the responding author upon reasonable request. expression of angiogenic proteins in porcine placentae. Amino Acids. 2022;54:193–204. 16. Blachier F, Davila AM, Benamouzig R, Tome D. Channelling of arginine in Declarations NO and polyamine pathways in colonocytes and consequences. Front Biosci (Landmark Ed). 2011;16:1331–43. Ethics approval and consent to participate 17. Jobgen WS, Fried SK, Fu WJ, Meininger CJ, Wu G. Regulatory role for the This study was approved by The Institutional Animal Care and Use Committee arginine-nitric oxide pathway metabolism of energy substrates. J Nutr of Texas A&M University. Biochem. 2006;17:571–88. 18. Agostinelli E. Biochemical and pathophysiological properties of polyam- Consent for publication ines. Amino Acids. 2020;52:111–7. Not applicable. 19. Durante W. Amino acids in circulatory function and health. Adv Exp Med Biol. 2020;1265:39–56. Competing interests 20. Hou YQ, Hu SD, Jia SC, Nawaratna G, Che DS, Wang FL, et al. Whole- All authors declare that the research was conducted in the absence of any body synthesis of L-homoarginine in pigs and rats supplemented with commercial and financial relationships that could be construed as a potential L-arginine. Amino Acids. 2016;48:993–1001. conflict of interest. 21. Li H, Meininger CJ, Hawker JR Jr, Haynes TE, Kepka-Lenhart D, Mistry SK, et al. Regulatory role of arginase I and II in nitric oxide, polyamine, and Author details proline syntheses in endothelial cells. Am J Physiol. 2001;280:E75-82. Department of Animal Science, Texas A&M University, College Station, TX 22. Wu G, Bazer FW, Johnson GA, Herring C, Seo H, Dai Z, et al. Functional 77843, USA. Department of Veterinary Integrative Biosciences, Texas A&M amino acids in the development of the pig placenta. Mol Reprod Dev. University, College Station, TX 77843, USA. 2017;84:870–82. 23. Li XL, Johnson GA, Zhou HJ, Burghardt RC, Bazer FW, Wu G. 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Journal
Journal of Animal Science and Biotechnology – Springer Journals
Published: Dec 8, 2022
Keywords: Angiogenesis; Arginine; Fetus; Placenta; Reproduction