Feeding rumen-protected lysine prepartum alters placental metabolism at a transcriptional level

Rumen-protected Lys (RPL) fed to Holstein cows prepartum resulted in a greater intake and improved health of their calves during the first 6 wk of life. However, whether increased supply of Lys in late gestation can influence placental tissue and, if so, which pathways are affected remain to be investigated. Therefore, we hypothesize that feeding RPL during late gestation could modulate placental metabolism, allowing for improved passage of nutrients to the fetus and thus influencing the offspring development. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Health and Nutrition North America) prepartum (0.54% DM of TMR) on mRNA gene expression profiles of placental samples of Holstein cows. Seventy multiparous Holstein cows were randomly assigned to 1 of 2 dietary treatments, consisting of TMR top-dressed with RPL (PRE-L) or without (con-trol, CON), fed from 27 ± 5 d prepartum until calving. After natural delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to clean any dirtiness from the environment and weighed. Then, 3 placentomes were collected, one from each placental region (cranial, central, and caudal), combined and flash-frozen in liquid nitrogen to evaluate the expression of transcripts and proteins related to protein metabolism and inflammation. Placental weights did not differ from cows in PRE-L (15.5 ± 4.03 kg) and cows in CON (14.5 ± 4.03 kg). Feeding RPL prepartum downregulated the expression of NOS3 (nitric oxide synthase 3), involved in vasodilation processes, and SOD1 , which encodes the enzyme superoxide dismutase, involved in oxidative stress processes. Additionally, feeding RPL prepartum upregulated the expression of transcripts involved in energy metabolism ( SLC2A3 , glucose transporter 3; and PCK1 , phospho-enolpyruvate


INTRODUCTION
The dry period is critical in the production cycle of a dairy cow because it directly affects the future lactation performance (Drackley, 1999;Grummer and Rastani, 2004), and it coincides with the greatest fetal growth rate, which is primarily determined by nutrient availability (Jones et al., 2007).For instance, intake restriction during late gestation reduced calf birth weight in beef cattle, possibly due to limited nutrient availability to the fetus (LeMaster et al., 2017).Fetal nutrient availability is directly related to maternal nutrition and placental nutrient and oxygen transport (Jones et al., 2007).In bovine placenta, the placentomes are the areas of maternal-fetal interface (Schlafer et al., 2000).As a highly metabolically active tissue, cotyledon growth continues throughout gestation (Reynolds et al., 1990), with binuclear trophoblast giant cells migrating toward the caruncle epithelium, in a process that releases molecules of fetal origin into the maternal compartment (Wooding and Wathes, 1980;Polei et al., 2020).This process of trophoblast cells proliferation and migration requires resources, such as glucose for cellular energy Feeding rumen-protected lysine prepartum alters placental metabolism at a transcriptional level A. R. Guadagnin, 1 L. K. Fehlberg, 1 B. Thomas, 1 Y. Sugimoto, 2 I. Shinzato, 2 and F. C. Cardoso 1 * production and biosynthesis, and AA for protein synthesis (Nishitani et al., 2019).
Catabolism of Lys can play a role in biosynthesis processes, as described to happen in the mammary gland (Lapierre et al., 2009).The bovine placenta produces a range of proteins throughout the whole gestation period, and therefore requires an adequate supply of energy source and AA (Reynolds and Redmer, 1995).For instance, increased Met supply to dairy cows during late gestation resulted in increased expression of genes involved in neutral AA transport, glucose transport, and the mTOR pathway of the placenta (Batistel et al., 2017).Amino acids are also key nutrients for the fetus (Shennan and Boyd, 1987).In dairy cows, increasing the maternal supply of Met during the last month before calving enhances fetal growth in utero, as well as in the pre-and postweaning periods (Alharthi et al., 2018).These effects are attributed to modifications in the uteroplacental transport of glucose and AA, and modulation of genes involved in the mTOR (mechanistic target of rapamycin) pathway (Batistel et al., 2017).Therefore, ensuring adequate maternal nutritional during the dry period is of utmost importance to guarantee proper function of placental metabolism and fetal development.
Reports of the effects of increased intestinal availability of Lys on uteroplacental tissue metabolism are lacking.Greater metabolic protein and Lys intake during the pre-calving period are attributable to increased DMI postpartum (Girma et al., 2019;Fehlberg et al., 2020) by alleviating the deficiency of those components in the transition diets.Feeding rumen-protected Lys (RPL) to prepartum dairy cows tends to increase DMI and crude protein intake for their calves in their first 6 wk of life and tends to increase average daily gain during preweaning phase (Thomas et al., 2022).Additionally, calves born to cows fed with RPL during prepartum tended to have a greater percentage of phagocytic neutrophils than calves born to cows that are not fed with RPL (Thomas et al., 2022).However, the mechanisms by which increased prepartum supply of metabolizable Lys would affect offspring development and immunity remain unclear.Thus, we hypothesize that these effects are possible due to changes in placental metabolism, which allow for the improved passage of nutrients to the fetus, and improved immunity.This enhanced exchange of nutrients between the dam and the fetus, particularly AA, would allow for better use by the fetus but probably by the placenta itself.Therefore, we aim to determine the effects of maternal supplementation with RPL during late pregnancy on protein and gene expression of transcripts involved in the placental cell proliferation, AA transport system, protein metabolism, energy metabolism, and immune metabolism.

Animal Care and Housing, and Experimental Design
All experimental procedures were approved by the University of Illinois (Urbana-Champaign) Institutional Animal Care and Use Committee (#18157).Details of the animal handling, experimental design, and diets were previously described elsewhere (Fehlberg et al., 2020).Briefly, 89 multiparous Holstein cows were categorized into low, intermediate, or high mature-equivalent milk production based on the tertiles for mature-equivalent milk production, and a similar concept was used for BCS as well.Then, they were blocked by lactation number (3.3 ± 1.1), previous 305-d mature-equivalent milk production (11,363 ± 1,860 kg), expected calving date, and BCS during the far-off period (from dry-off date until 21 d before expected calving date; 3.76 ± 0.84).Each block had 4 cows in it, except for one block that had 6 cows.Cows were then assigned to 1 of 2 dietary treatments [TMR with or without RPL (AjiPro-L Generation 3; 42% l-Lys-HCl, Ajinomoto Health and Nutrition North America)].The number of cows per treatment was calculated to detect a minimum of 7% difference in postpartum DMI between groups, assuming a power of 0.9 and a 2-tailed α of 0.05.Additional calculations were made to ensure that the experimental design had the power to detect a minimum of 10% increase in transcript expression (mRNA) from placental samples of 2 different independent groups, assuming a power of 0.8 and a 2-tailed α of 0.05.This additional power analysis determined that, to detect such a difference among groups, it would be required a minimum of 32 cows per treatment group, which was met by the experimental design.Prepartum (−28 d to calving), cows were fed a diet with RPL [PRE-L (n = 35); 0.54% RPL of dietary DMI] or without RPL [CON (n = 35)] top-dressed in a carrier of 300 g of dried sugarcane molasses.The amount of RPL top-dressed was adjusted for each cow daily, using their individual DMI of the previous day.Cows that were not receiving RPL were top-dressed with 300 g of sugarcane molasses only.According to the manufacturer, there is 80% rumen bypass and 80% intestinal digestibility of this encapsulated RPL product, which would provide 1.4 g of intestinally available Lys prepartum per kilogram of DMI.Prepartum diet was formulated using AMTS.Cattle.Pro version 4.7 (2017, AMTS, LLC) to meet or exceed recommendations for dry cows at 694 kg of BW and a predicted DMI of 13 kg/d.According to AMTS.Cattle.Pro prediction, cows in CON received 1.17 kg/d of MP, resulting in 6.86% MP as Lys and 2.98% MP as Met with a Lys: Met ratio of 2.30, whereas cows in PRE-L received 1.19 kg/d of MP, resulting in 8.24% MP as Lys and 2.94% MP as Met with a Lys: Met ratio of 2.80.The exclusion criteria included calving with twins or not having consumed the treatment for at least 16 d during the prepartum period.Four cows were excluded due to twins (CON n = 2; PRE-L n = 2) and 1 cow was excluded for calving too early (PRE-L n = 1).Nine cows were excluded postpartum due to health problems (CON n = 2; PRE-L n = 7).A total of 75 cows concluded the study.Five cows had retained placenta (CON n = 3; PRE-L n = 2), therefore their placentas were not collected.

Placental Collection
Placentas were only collected after natural expel and there was no human intervention in the process.After natural at-term delivery (6.87 ± 3.32 h), placentas were rinsed with physiological saline (0.9% sodium chloride solution) to remove particulates from the environment and weighed.Then, 3 placentomes from each placenta were dissected and rinsed with physiological saline.Placentomes were collected from the cranial, central, and caudal regions of the placenta (Figure 1).A subset of each of the placentomes was combined and flashfrozen in liquid nitrogen.Samples were stored at −80°C for further RNA and protein extraction.

Placental RNA Extraction and Transcript Expression
RNA extraction was performed using the Direct-zol RNA Miniprep system (Zymo Research) was used following the manufacturer's protocols.Briefly, cells from 50 μg of placental tissue were lysed with TRI-Reagent and RNA was isolated using the Zymo-Spin IIC column kit.Concentration of RNA was measured in NanoDrop ND-1000 (NanoDrop Technologies).Complementary DNA was synthesized using 100 ng of RNA following a procedure reported in detail by Guadagnin et al. (2022).RNA integrity was measured using and Agilent 2100 Bioanalyzer (Agilent).The RNA Integrity Number for the placenta samples ranged from 4.70 to 6.3, with a median of 6.0.Messenger RNA expression was analyzed on BioMark 96.96 Dynamic Array platform (Fluidigm) using primers designed through Primer Express 2.0, with minimum amplicon size of 70 bp (when possible, amplicons of 100e120 bp were chosen) and limited 30 G þ C (Applied Biosystems).Primers were aligned against publicly available databases using BLASTN at NCBI (NCBI Resource Coordination, 2013) and UCSC's Cow (Bos taurus) Genome Browser Gateway.All evaluated genes and primers information are reported in the supplemental material (Supplemental Table S1; https: / / uofi .box.com/s/ x9snihxbe6e3ibnq8cqibaqv9b9vw1o3 ).To control analytical and tissue sampling variation, the final data were normalized to the geometric mean expression of GAPDH, ACTB, and H2AFZ, previously validated to be used in bovine placental tissue (Batistel et al., 2017).The cycle threshold (ΔCT) for each gene was calculated following guidelines reported by Schmittgen and Livak (2008), by subtracting the geometric mean of the CT of GAPDH, ACTB, and H2AFZ from the CT of the gene of interest.Fold change was calculated using the 2 −ΔΔCT method, as described by Schmittgen and Livak (2008).

Statistical Analysis
Statistical analysis was performed using SAS 9.4 (SAS Institute Inc.).The MIXED procedure of SAS was used to model the fixed effects of treatment and block using the following model: where Y jkl = the observations for dependent variables; μ = the overall mean; A j = the fixed effect of treatment (PRE); B k = the fixed effect of block; C l = the random effect of cow; and ε jkl = the random residual error.Cow was used as the experimental unit.Residual distribution was evaluated for normality and homoscedasticity variance in all analysis.The Kenward-Roger degrees of freedom approximation was used to determine the denominator degrees of freedom for tests of fixed effects.Statistical analysis of transcript expression was performed after the 2 −ΔCT transformation was calculated to obtain the mean ± standard deviation, following guidelines reported by Schmittgen and Livak (2008).Statistical significance was declared at P ≤ 0.05 and tendency at 0.05 > P ≤ 0.10.

RESULTS
Cow performance data are reported elsewhere (Fehlberg et al., 2020(Fehlberg et al., , 2023)).Based on the metabolizable protein and AA predicted by AMTS, cows in PRE-L consumed on average 98 g/d of Lys in the prepartum, and cows in CON consumed 80 g/d of Lys in the same period.There was no difference in DMI (PRE-L 12.1 ± 0.21 kg; CON 11.8 ± 0.21 kg; P = 0.80) or BW (PRE-L 808 ± 2 kg; CON 803 ± 2 kg; P = 0.12) prepartum due to dietary treatment.Plasma concentrations of Lys prepartum (69.8 ± 1.8 μM) increased for cows consuming RPL compared with those that did not (62.46 ± 1.3 μM).Plasma concentrations of total AA, total dispensable AA, total branched-chain AA, total sulfur AA, and total urea cycle AA decreased, and total indispensable AA tended to decrease prepartum for cows in PRE-L (Fehlberg et al., 2020).An increase in hepatic protein abundance of SLC7A7 (solute carrier family 7 member 7) indicated increased uptake of Lys by the liver of cows in PRE-L (Fehlberg et al., 2023).Moreover, cows that consumed RPL had an improved liver functionality due to decreased inflammation, indicated by greater negative acute-phase proteins (albumin and globulin) and cholesterol, and decreased haptoglobin and aspartate aminotransferase concentrations prepartum (Fehlberg et al., 2023).Additionally, downregulation of hepatic expression of interleukin 1-β (IL-1β) for cows in PRE-L further supports the hypothesis of a decreased inflammatory response upon RPL consumption (Fehlberg et al., 2023).Occurrence of retained placenta did not differ between dietary treatments (Fehlberg et al., 2020).Placental weight did not differ between dietary treatments (PRE-L = 15.54 ± 4.03 kg and CON = 14.54 ± 4.03 kg, P = 0.43).

DISCUSSION
Our objectives were to determine the effects of feeding RPL to prepartum dairy cows on protein and gene expression of transcripts involved in placental AA transport system, protein metabolism, energy metabolism, and immune metabolism.
Though histological evaluation was not performed, the greater transcript and protein abundance of FGF2 and the greater expression of FGFR2 could indicate a more significant cell differentiation and, consequently, greater metabolic activity in the placenta of cows consuming RPL during late gestation.Fibroblast growth factor-2 is mainly involved in stimulating trophoblasts' migratory activity and binucleation (Taniguchi et al., 1998), and serves as an angiogenic factor for vascular endothelial cells (Zhang et al., 1997).Additionally, FGF2 also affects the production of hormones by the binucleate trophoblast giant cells, as demonstrated by in vitro studies with stabilizing the endocrine phenotype of PC12 cells (catecholamine cell line; Grothe et al., 1998).Placental growth factor is involved in the proliferation and migration of cell, due to its strong angiogenic and mitogenic properties (Maglione et al., 1993;Cao et al., 1997).Thus, the upregulation of PGF expression in placenta of cows that were fed RPL is also likely involved in the proliferation and migration of trophoblasts.Furthermore, a stimulus of uterine cells proliferation in response to RPL supplementation was suggested in a derived study (Guadagnin et al., 2022), with a tendency for a greater number of glandular epithelial cells at 28 DIM in cows fed RPL.The exact mechanism resulting in this cell proliferation stimulus by increased intestinal availability of Lys is not yet elucidated.
As previously mentioned, binucleate trophoblast giant cells proliferation and migration process requires energy sources and synthesis of protein (Nishitani et al., 2019).Glucose is used as an energy resource by the trophoblast cells and is crucial for cell viability and proliferation processes in the placenta (Han et al., 2015) and glucose transporters are rate-limiting for overall glucose disposal in vivo (Fink et al., 1992).The greater expression of GLUT3 in placentomes of cows in PRE-L could indicate increased glucose uptake capacity by the placenta, to be used either for cell proliferation processes or to be transferred to the fetus.Unfortunately, the concentration of glucose or metabolites were not measured in fetal blood, fluids, or in placental tissue in the present study.However, Ticiani et al. (2020) as- sociated a greater expression of glucose transporters in bovine placentomes with greater glucose uptake by the conceptus, when investigating placental metabolism of bovine pregnancies established after superovulation, in vitro fertilization, and cloning by nuclear transfer.The fetal-glucose requirements are met by an increase in the transplacental glucose gradient and a concomitant increase in placental glucose transfer capacity (Ward et al., 2004;Hay, 2006;Desoye and Nolan, 2016).It is possible that the increased expression of GLUT3 in the placenta from cows consuming RPL could be an indicative of the increased glucose uptake to be transferred to the fetus.Thus, the increase in PCK1 expression could be resulting in either increased gluconeogenesis (Chakravarty et al., 2005) or energy generation through recycling carbon skeletons of AA back into the tricarboxylic acid cycle (Yang et al., 2009).However, the decarboxylation of AA for oxidative metabolism is more likely to happen in situations of hypoglycemia (Limesand et al., 2009), which was not the case for neither the cows in PRE-L nor cows in CON (Fehlberg et al., 2023).Thus, the concomitantly upregulation of PCK1 and GLUT3 could be indicating that the placenta relies on gluconeogenic processes while the fetus takes up glucose from the maternal circulation.Wang et al. (2019) reported increased cell proliferation as a response to increased glucose availability, whereas decreased Lys: Met ratio had no effect on cell metabolism (Wang et al., 2019).The increased hepatic protein expression of SLC7A7 of cows consuming RPL in prepartum (Fehlberg et al., 2023), combined with no differences in transcripts related to Lys-metabolism in uterine tissue (Guadagnin et al., 2022) nor in placental tissue (present study), indicates a "preference" for hepatic uptake of Lys, most likely for protein synthesis.In corroboration with this hypothesis, Fehlberg et al. ( 2023) reported enhanced liver function for cows that were fed RPL.Thus, it is likely that the increase in liver functionality during prepartum resulting from increased Lys availability allowed for greater fetal uptake of glucose, demonstrated by the increased transcription of GLUT3 in placentomes.Moreover, the increased protein abundance of LRP1 is likely related to the increased GLUT3 expression.LRP1 is necessary for GLUT3 translocation to the plasma membrane in neurons and hepatocytes upon insulin signaling (Dato and Chiabrando, 2018).However, GLUT3 is an insulinindependent transporter of glucose (Mora and Pessin, 2013).Additionally, LRP1 could be concomitantly serving as a sensor of the placental nutritional status, especially lipid composition (Au et al., 2017), possibly linked to the greater maternal circulation of cholesterol in cows that consumed PRE-L.As additional support 1 AASS = aminoadipate-semialdehyde synthase; ANGPT1 = angiopoietin 1; ANGPT2 = angiopoietin 2; FGF10 = fibroblast growth factor 10; FGF2 = fibroblast growth factor 2; FGF2R = fibroblast growth factor 2 receptor; G6PC1 = glucose-6-phosphatase catalytic subunit 1; GLUT3 = glucose transporter 3; GLUT5 = glucose transporter 5; HGF = hepatocyte growth factor; IGF1 = insulin-like growth factor 1; IGF1R = insulin-like growth factor 1 receptor; IGF2 = insulin-like growth factor 2; IGF2R = insulin-like growth factor 2 receptor; of no peripheral oxidation of Lys, Lobley et al. (2003) reported no catabolism of Lys across the gastrointestinal tract of sheep.Wang et al. (2019) also demonstrated that extrahepatic cells, particularly bovine mammary epithelial cells, do not respond to different Lys: Met ratios but increase cell proliferation upon glucose supplementation.Thus, the effects on extrahepatic tissues are probably an indirect effect of improved liver functionality arising from RPL supplementation.
Although the increased availability of intestinal Lys during late gestation modulated placental gene expression and protein abundance, this did not translate in differences in calf's birth weight (Thomas et al., 2022).The lack of difference in calves' birth weight is probably linked to the lack of difference in their dams' DMI or BW prepartum (Fehlberg et al., 2020).However, calves from PRE-L dams tended to have a greater dry matter and crude protein intakes than calves from CON dams, which resulted in greater ADG during the preweaning phase (Thomas et al., 2022).Bovine placenta has a high metabolic activity, with approximately 60% of the uterine glucose uptake and about 40% of the α-amino N being used by the uteroplacental tissue throughout gestation (Reynolds et al., 1986).Thus, we speculate that the modulation of placental energetic metabolism occurs in response to the stimulus of cell proliferation by the increased availability of intestinal Lys.
Additionally, RPL supplementation resulted in different utilization of other AA, as denoted by the upregulation of MAT2A in placental tissue of cows in PRE-L.Methionine adenosyltransferase 2A upregulation potentially increases supply of S-adenosylmethionine, linking Met metabolism to mTOR through SAMTOR protein (Coleman et al., 2020).As previously mentioned, mTOR is an intermediate in a translational control pathway that regulates the cell proliferation (Raught et al., 2001), which is a continuous process linked to trophoblast giant cells migration and metabolism.Although the exact mechanism explaining how increased supply of Lys can affect Met metabolism is not clear, this could be a result of increasing the supply of a limiting AA resulting in increased utilization of other AA.Within the theory of limiting AA, protein synthesis depends on the availability and efficiency of use of the most limiting AA in a diet; thus, when a limiting AA is not provided at sufficient amounts, protein synthesis is limited to the rate at which this limiting AA is available (Wolfe, 2017).Furthermore, Lys methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to Lys residues, resulting in different forms of methylated Lys (Smith and Denu, 2009).These methylated Lys act as regulators of different process, including the transcription factor tumor suppressor protein p53, thus being involved in the regulation of cell-cycle processes and apoptosis, for instance (Scoumanne and Chen, 2008).Upon fusing of the binuclear trophoblast giant cells with the caruncular epithelial cells, the formed short-lived fetomaternal hybrid cells undergo apoptosis after degranulation (Wooding, 1992).As so, the increased migration and proliferation process could result in greater apoptosis.

CONCLUSIONS
We confirmed our hypothesis of increased uteroplacental exchange of nutrients at least in part through changes in the transcription of genes (GLUT3 and PCK1) and expression of proteins (LRP1) involved in placental glucose uptake and metabolism.Increased transport of AA was not confirmed, as there were no differences in the AA transport systems evaluated.However, there was an increased expression of MAT2A in the placental tissue of cows consuming RPL, indicating increased Met catabolism.Placental immune metabolism was modestly affected by the maternal supplementation of RPL, with the main effects being the downregulation of transcription of NOS3 and SOD1.We suggest minimal biological significance to the latter findings due to the slight level of downregulation.The increased expression of FGF2, FGF2R, PGF, and IGF2R transcripts, along increased protein abundance of FGF2, indicate enhanced placental metabolic activity, possibly linked to trophoblast proliferation and migration processes.

Figure 1 .
Figure 1.Schematic figure depicting placenta collection after natural delivery.Black arrows indicate the placentomes, which were harvested and dissected for collection.

Figure 2 .
Figure 2. Western blot images obtained from Image Lab Software by ChemiDoc after 3 min incubation in western enhanced chemiluminescence substrate.Placenta samples derived from cows consuming dietary treatments included in top dress with rumen-protected Lys prepartum (PRE-L) or without rumen-protected Lys prepartum (CON) in a carrier of 300 g of dried molasses.Protein abundance of fibroblast growth factor 2 (FGF2) and LDL-receptor-related protein 1 (LRP1) was greater in placental samples from cows in PRE-L than from cows in CON (P = 0.03).Graph bars represent LSM ± SEM.

Table 1 .
Guadagnin et al.:RUMEN-PROTECTED LYSINE AND IMMUNITY Placental expression of transcripts (LSM of delta-Ct, normalized to internal control) from Holstein cows fed with rumenprotected Lys (PRE-L) during peripartal period or not(CON)