Effect of dietary nitrogen content and ammonium phosphate inclusion on lysine requirement for nitrogen retention in growing pigs

Abstract Inclusion of a source of non-protein nitrogen (NPN) may improve essential amino acid (EAA) and nitrogen (N) utilization in N-limiting diets. Growing barrows (20.4 ± 0.5 kg) were randomly assigned to 1 of 10 dietary treatments (n = 9 pigs/treatment) in nine blocks. Diets contained no ammonium phosphate (NAP) or 1.7% ammonium phosphate (AP) to have an EAA-N:total N ratio of 0.36 and 0.33, respectively, with graded levels of dietary lysine (Lys; 0.8%, 0.9%, 1.0%, 1.1%, and 1.2% standardized ileal digestible (SID)). Following a 7-day dietary adaptation, a 4-day N-balance collection period was conducted. Blood samples were obtained on day 2 of the collection period. Nitrogen retention (NR) increased and urinary N output decreased with inclusion of NPN and increasing Lys (P < 0.01). Plasma urea N decreased with increasing Lys (P < 0.05). Total plasma EAA content was reduced with NPN supplementation (P < 0.05), while content of Arg, Asp, Gln, and Glu was increased (P < 0.01). The linear breakpoint model indicated that NR was maximized at 1.00% SID Lys in NAP-fed pigs and at 1.09% SID Lys in AP-fed pigs. These results indicate that diets deficient in dietary N reduce NR and Lys requirement, which were in turn increased with NPN supplementation.


Introduction
Current practice in swine prodlyuction is to feed reducedprotein diets, formulated to meet essential amino acid (EAA) requirements according to the ideal protein concept, which allows for a reduction in total dietary protein (Wang et al. 2018).Benefits of reducing dietary protein can include a reduction in diet cost and nitrogen (N) excretion (Tuitoek et al. 1997;Wang et al. 2018).However, pigs require both EAA and non-essential amino acids (NEAA) to ensure proper growth and productivity, and reduced protein diets assume that pigs can produce sufficient levels of NEAA to meet their requirements (Wu et al. 2013;Mansilla et al. 2015) with potentially limiting dietary N. When there is a decrease in dietary supply of NEAA-N, the necessary N may be sourced from the catabolism of amino acids (AA), including EAA, impacting the efficiency of EAA utilization for lean gain.Consequently, both EAA and NEAA, or a source of N for the synthesis of NEAA, should be provided.A practical method to optimize both EAA-N and total dietary N is the EAA-N:total N (E:T) ratio (Heger 2003), as this ratio gives an indication of sufficiency of both EAA and NEAA.Nitrogen retention (NR) is affected by E:T, with reduced efficiency observed at extreme ratios (Heger et al. 1998;Lenis et al. 1999).We have previously demonstrated that pigs fed diets with a lower E:T ratio, adjusted through supplementation of intact protein (i.e., soybean meal), had improved NR and an increased lysine (Lys) requirement compared to high ratio-fed pigs (Camiré et al. 2023).
Previous research has indicated that non-protein nitrogen (NPN) may be used as a source of N for the endogenous synthesis of NEAA, with positive responses observed in growth performance of pigs when NPN is included in diets that are deficient in NEAA-N (Mansilla et al. 2015(Mansilla et al. , 2017a(Mansilla et al. , 2017b)).For example, Mansilla et al. (2017a) demonstrated that feeding ammonia-N is as effective in maintaining growth performance as feeding synthetic AAs or intact protein to growing pigs that are fed diets limiting in NEAA-N.The objective of the present study was to determine the effect of including NPN in N-deficient diets (based on the E:T ratio) on EAA (i.e., Lys) requirement for NR in growing pigs.It was hypothesized that the EAA requirement for NR would be increased when a source of NPN was included in a diet (low E:T ratio) compared to an unsupplemented, N-limiting diet (high E:T ratio).

Materials and methods
The experimental protocol was reviewed and approved by the University of Saskatchewan's Animal Research Ethics Board (AUP # 20130054) and followed the Canadian Council on Animal Care guidelines (CCAC 2009).

Animals, housing, diets, and experimental design
A total of 90 growing barrows (Camborough Plus × C3378; PIC Canada, Ltd., Winnipeg, MB) with an initial body weight (BW) of 20.4 ± 0.5 kg were used in an N-balance experiment at the Prairie Swine Centre, Inc. (Saskatoon, Canada).Pigs were housed individually in metabolism crates (1.4 m × 1.5 m) in a temperature-controlled room (22 ± 1 • C) and were randomly assigned to 1 of 10 dietary treatments arranged as a 2 × 5 factorial in a randomized complete block design over nine blocks (n = 9 pigs/treatment).The factors were NPN inclusion (no ammonium phosphate (NAP) or supplemented with ammonium phosphate at 1.7% (AP)) and graded levels of standardized ileal digestible (SID) Lys content (0.8%, 0.9%, 1.0%, 1.1%, or 1.2%).The NAP diet was formulated to have an E:T ratio of 0.36 (considered to be relatively deficient in N) and the AP diet to have a ratio of 0.33 (N supplemented).While these ratios are lower than the suggested optimum in swine (Heger et al. 1998), previous ratios were calculated using total AAs and only considering AA-N, ignoring the contribution of NPN (Heger et al. 1998;Heger 2003).As in our previous study (Camiré et al. 2023), the E:T ratio in the current study was calculated using SID EAA-N to the requirement (NRC 2012) and total dietary N content.As such, all excess EAA-N and all other sources of N are considered as potentially contributing to N supply.Lysine was chosen as the test EAA based on our previous study (Camiré et al. 2023) and as it is generally first-limiting in diets for swine and is first limiting for lean gain.Diets were formulated to meet or exceed nutrient requirements according to NRC (2012), except for Lys, and contained titanium dioxide as an indigestible marker (Table 1).Only the diets containing the lowest and highest Lys content were milled under standard commercial conditions and then mixed in appropriate proportions to obtain the 0.9%, 1.0%, and 1.1% SID Lys diets (Canadian Feed Research Centre, North Battleford, SK, Canada).Daily feed allowance was provided at 2.8× metabolizable energy requirements for maintenance (determined as 110 × BW 0.75 ) and was adjusted to BW at the start of adaptation and collection periods.Diets were fed in two equal meals per day at 0800 and 1500 h with ad libitum access to water.Feed refusals were collected for each pig daily and weighed to determine daily feed intake.

Nitrogen balance and sample collection
The experimental period totalled 11 days, consisting of a 7-day dietary and environmental adaptation period, followed by a 4-day N-balance collection period.During the collection period, fresh fecal samples were obtained daily and stored at −20 • C. At the end of each collection period, fecal samples were thawed and homogenized for each pig, and a subsample was stored at −20 • C until further analysis.Urine was collected quantitatively each day over a 24 h period for each pig using metal trays and containers placed under each crate.The containers contained a sufficient amount of 6 N HCl to maintain the pH below 3 to minimize N losses (de Lange et al. 2001).At the end of each collection, total urine collected was weighed, and a 15% sub-sample was obtained, pooled per pig, and stored at −20 • C until further analysis.On day 2 of the collection period, blood samples were obtained 2 h after the morning meal via jugular venipuncture into vacutainer tubes containing heparin, centrifuged at 3000 × g for 30 min at 4 • C, and plasma collected and stored at −20 • C until further analysis of plasma urea nitrogen (PUN) and AAs.

Analytical procedures
Fecal samples were freeze dried (Labconco Freeze Dry System, 18L; Kansas City, MO, USA) and then ground in a centrifugal mill (Grinder Retsch ZM 200 GmbH & Co. Rheinische Straße, Germany) through a 1 mm sieve.The dry matter (DM) content of the fecal samples and diets was determined in duplicate by oven drying at 135 • C for 2 h (forced air ovens, Thermo Fisher Scientific Isotemp 750F, Waltham, MA, USA).Nitrogen content in diet, urine, and fecal samples was determined using an automatic analyzer (LECO FP 528; LECO FP 828; MI, USA; Method 990.03;AOAC 2007).Titanium dioxide was determined as described by Myers et al. (2004).Diet samples were analyzed for AA composition (Table 2) at Central Testing Laboratories (Winnipeg, MB, Canada).Plasma urea nitrogen was analyzed using a commercially available kit according to manufacturer instructions (Invitrogen Urea Nitrogen Colorimetric Detection Kit #EIABUN (BUN), Thermo Fisher Scientific, Waltham, MA, USA).
Plasma AA analysis was conducted at Agriculture and Agri-Food Canada (Sherbrooke, QC, Canada) as described by Calder et al. (1999).Briefly, a mixture of standard isotopes (200 μL) was added to the samples.A solution of 100 μL of DLdithiothreitol (15.4 mg/mL of water) was added to the sample and let stand for 30 min at room temperature.Afterwards, the samples were passed through columns (Poly-Prep 731-1550; Bio-Rad, Brossard, QC, Canada) prepared with 0.8 cm (0.4 mL) of resin (Dowex 50WX8-200 ion exchange resin; Sigma-Aldrich, Oakville, ON, Canada).The columns were rinsed twice with 2 mL of ultra-pure water.Amino acids were recovered by adding 2 mL of NH 4 OH 2 N to the columns.The columns were rinsed with 1 mL of ultra-pure water and left to drain into vials.The vials were covered with Parafilm and vortexed.The samples were frozen at −80 • C and lyophilized.The vials were rinsed with 250 μL of ultra-pure water, and the contents were transferred to a reaction vial (Pierce 13,221; Reacti-Vial, Thermo Fisher Scientific, Waltham, MA, USA).The contents of the reaction vials were dried with nitrogen at 90 • C for about 20 min, and 20 μL of DL-dithiothreitol (15.4 mg/mL) and 80 μL of NH 4 OH 2 N were added to the samples.The samples were left to stand for 30 min at room temperature and were then dried with nitrogen at 90 • C for 20 min before being derived with 60 μL of MTBSTFA:DMF 1:1 (MTBSTFA: Aldrich 394,882, DMF: Aldrich 27.054-7; Oakville, ON, Canada).The samples were heated at 90 § E:T ratios of 0.33 (AP diets) and 0.36 (NAP diets) reflect the amount of nitrogen in the diets coming from essential amino acids (EAA-N) and from the other components (T), with the higher ratio having a larger contribution of nitrogen from EAA.
Canada).All AA samples were measured by gas chromatography coupled to mass spectrometry (Agilent Technologies 7890B GC System coupled to an Agilent Technologies 5977A MSD).

Calculations
Apparent total tract digestibility (ATTD) of N was determined using the indicator method according to the following equation: where TiO 2D and TiO 2F are the titanium dioxide concentrations in the diet and feces, respectively.N D and N F are the N concentration in the diet and feces, respectively.NR was determined using the following equation: N retained (g/day) = N intake (g/day) − (fecal N output (g/day) + urinary N output (g/day)) Protein deposition (g/day) was calculated as N retained (g/day) × 6.25 The marginal efficiency of utilizing SID N (K nitrogen ) or Lys (K lysine ) intake above maintenance was calculated using the following equations: where SID N and Lys maintenance requirement include endogenous gut losses and losses with skin and hair and were estimated according to NRC (2012).Body protein was assumed to contain 7.10% Lys and the efficiency of SID Lys utilization for maintenance was assumed to be 0.75 (NRC 2012).The SID N and Lys intake were based on calculated diet content and determined feed intake.Nitrogen retained in body protein and body protein deposition were based on analyzed values.

Statistical analysis
Data were verified for normality and outliers identified using the UNIVARIATE procedure and outliers identified using the studentized residual analysis (SAS version 9.4; SAS Institute, Inc., Cary, NC).Data were analyzed using the MIXED procedure of SAS with dietary Lys, dietary NPN content, and their interactions as fixed effects and block as a random effect.Orthogonal polynomial contrast statements were defined to determine the linear and quadratic effects of dietary Lys content on NR, K lysine , and K nitrogen in the NAP and AP diets.Linear and quadratic breakpoint modeling was used to estimate Lys requirement for maximum NR and minimum PUN (PROC NLIN).The regression model (PROC REG) was used to determine Lys utilization efficiency for NR.Differences between least-square means were assessed using the Tukey-Kramer mean separation test.The significance level was defined as P ≤ 0.05.

Nitrogen balance
Nitrogen intake was greater in the AP-fed pigs when compared to the NAP-fed pigs and increased with increasing Lys content (Table 3; P < 0.01).There was no impact of inclusion of NPN on fecal N output, but was reduced with increasing Lys content (P < 0.05).Urinary N output and NR (g/day) were greater with NPN supplementation and increasing Lys content (P < 0.01).Apparent total tract digestibility of N was greater in the pigs fed NPN (P < 0.05).Nitrogen retention (% of intake) increased with increasing Lys content (P < 0.01) and was greater in NAP-fed pigs (P < 0.05).The PUN content was not affected by NPN content of the diets, but as dietary Lys increased, PUN concentration was reduced (P < 0.001).As the dietary Lys content increased, NR increased linearly and quadratically in the NAP-fed pigs (Table 4; P < 0.001), § Marginal efficiency of N intake above maintenance calculated as: (nitrogen retained in body protein)/(standardized ileal digestible nitrogen intake --maintenance nitrogen requirements).Standardized ileal digestible nitrogen intake and maintenance nitrogen requirement according to NRC (2012).Marginal efficiency of Lys intake above maintenance calculated as: (protein deposition × Lys % of body protein)/(standardized ileal digestible Lys intake--maintenance Lys requirements/efficiency of SID Lys utilization for maintenance).Standardized ileal digestible Lys intake and maintenance Lys requirements were calculated according to NRC (2012).Body protein was assumed to contain 7.10% Lys (NRC 2012).

Impact of NPN on efficiency of Lys and N intake
Efficiency of Lys utilization for NR was not impacted by NPN inclusion (Fig. 1).Inclusion of NPN increased the marginal efficiency of utilizing SID Lys (K lysine ; P < 0.01) and reduced the marginal efficiency of utilizing SID N (K nitrogen ; P < 0.01) for NR.These parameters were also impacted by Lys content, where K nitrogen was increased with greater Lys content and K lysine was reduced with increased Lys content (P < 0.01; Table 3).As the dietary Lys content increased, K lysine and K nitrogen increased both linearly and quadratically in both NAP-and AP-fed pigs (Table 4; P < 0.01).

Estimate of Lys requirement
Linear breakpoint analysis determined a Lys requirement of 1.00% SID Lys at 15.6 g/day NR in NAP-fed pigs and 1.09% Fig. 1.Regression analysis showing the efficiency of Lys intake on nitrogen (N) retention in pigs fed no ammonium phosphate (NAP) or supplemental ammonium phosphate at 1.7% (AP).The AP diet is represented in the figure with short dashes (---), while the NAP is presented in the figure with a solid line ( ----).Data of the regression analysis using the one slope model: Y = a + b(x), where Y is the N retention (g/day), a represents the intercept (extrapolating the maintenance requirement), b is the slope representing the efficiency of standardized ileal digestible (SID) Lys utilization for N retention, and x represents the SID Lys intake (g/day).The equations are as follows: AP diet (Y = 9.72 + 6.37x; R 2 = 0.70); NAP diet (Y = 8.69 + 6.18x; R 2 = 0.57).The intercept P = 0.88 and slope P = 0.79.SID Lys at 16.4 g/d NR in AP-fed pigs (Fig. 2).Quadratic breakpoint analysis determined a Lys requirement of 1.14% SID Lys at 2.67 mg/dL PUN in NAP-fed pigs, while the linear model analyses indicated a breakpoint of 1.12% SID Lys at 1.91 mg/dL PUN in the AP-fed pigs (Fig. 3).

Impact of NPN on efficiency of Lys and N intake
Efficiency of Lys utilization for NR was not impacted by NPN inclusion (Fig. 3).Inclusion of NPN increased the marginal efficiency of utilizing SID lysine (K lysine ; P < 0.01) and reduced the marginal efficiency of utilizing SID N (K nitrogen ; P < 0.01) for NR.These parameters were also impacted by Lys content, where K nitrogen was increased with greater Lys content and K lysine was reduced with increased Lys content (P < 0.01; Table 3).

Effect of Lys content and NPN inclusion on plasma AAs
Increasing Lys content increased plasma Lys concentration (Table 5; P < 0.001).There was also an interactive effect of NPN inclusion and Lys content on plasma concentration of Lys (P < 0.01), with greater Lys concentration in NPN-supplemented pigs fed 0.8%, 1.1%, and 1.2% SID Lys and greater Lys in unsupplemented pigs fed 0.9% and 1.0% SID Lys (P < 0.05).Increasing Lys content decreased plasma concentrations of several other EAAs including His, Ile, Phe, Thr, Tyr, Val, and total EAA (P < 0.01).Interactive effects of NPN and Lys inclusion were also observed for His and Val, with AP di-Fig.2. The linear breakpoint estimates of Lys requirement for nitrogen retention (NR; g/day) in pigs fed no ammonium phosphate (NAP) and ammonium phosphate (AP).The analyses indicated a breakpoint of 1.00% with maximum NR at 15.6 g/day in pigs fed the NAP diet (A).A breakpoint of 1.09% with maximum NR at 16.4 g/day was achieved in pigs fed the AP diet (B).SID, standardized ileal digestible.ets having greater concentration of His and Val at low dietary Lys and lower concentrations at high dietary Lys than NAPfed pigs (P < 0.05).Plasma Met concentration was reduced as Lys content increased (P < 0.01), while the opposite effect was observed for Trp (P < 0.01).Inclusion of AP reduced Leu, Phe, Val, and total EAA concentrations (P < 0.05), but no other EAAs were impacted by NPN supplementation.
Increasing Lys content increased plasma concentrations of the NEAA Ala, Asp, Glu, Gly, and total NEAA (P < 0.01), while Arg decreased (P < 0.05).Inclusion of NPN increased concentrations of the NEAA Arg, Asp, Gln, and Glu (P < 0.01), while Cys concentration was decreased (P < 0.01).An interactive effect of N and Lys content on Gln concentration was observed, where Gln concentration was greater in AP-fed pigs at the lowest dietary Lys content and above NRC (2012) Lys requirement (P < 0.01).The NEAA Pro and Asn were not impacted by either NPN inclusion or Lys content.

Discussion
The objective of the present study was to determine the effect of altering the E:T ratio with NPN inclusion on the utiliza-Fig.3. The quadratic breakpoint and linear model estimate Lys requirement for plasma urea nitrogen (PUN; mg/dL) in pigs fed no ammonium phosphate (NAP) and ammonium phosphate (AP).The analyses indicated a breakpoint at 1.14% standardized ileal digestible (SID) Lys in pigs fed the NAP diets at a PUN of 2.67 mg/dL (A).A breakpoint was achieved at 1.12% SID Lys in pigs fed the AP diets at a PUN of 1.91 mg/dL (B).tion of N and requirement of Lys for NR in growing pigs.To accomplish this, an N-balance study was conducted wherein pigs were fed diets containing no supplemental NPN (high E:T ratio) or supplemented with NPN as AP (low E:T ratio).
As anticipated, increased N intake was observed in AP-fed pigs due to the greater N content in those diets.While this did not result in an increase in fecal N output, increased dietary N through NPN inclusion resulted in greater urinary N output, which is consistent with previous research (Wehrbein et al. 1970;Mansilla et al. 2015) and demonstrates the inherent inefficiency of N utilization.Similar results were observed in our previous study (Camiré et al. 2023) in which E:T ratio in diets was altered through supplementation of N as intact protein (i.e., soybean meal).However, in that study, both fecal and urinary N were increased with inclusion of supplemental N. The greater digestibility of NPN, based on fecal N output and previous studies (Mansilla et al. 2017a), indicates that it may be a superior source of N versus intact protein if attempting to improve N supply without increase in fecal N output.Urinary N excretion was greater in AP-fed pigs and decreased with increased dietary Lys, which is also consistent with past work (Wehrbein et al. 1970;Zhou et al. 2019) and with Lys being the first-limiting EAA for growth in swine and in the diets in the current study.Overall, these results demonstrate that N supplied as NPN is highly available to pigs and contributes positively to NR and improves marginal efficiency of Lys.
The efficiency with which N and Lys were utilized in the current study was dependent on NPN and Lys inclusion in the diet.The overall reduction in N utilization in pigs fed supplemental N, as NPN or intact protein, likely reflects the inherent inefficiency of N utilization in pigs (NRC 2012).This inefficiency was further demonstrated by the reduction in marginal efficiency (K nitrogen ) in pigs fed NPN, and is consistent with previous work (Camiré et al. 2023) where additional dietary N reduced K nitrogen .Improvements in N utilization efficiency for NR, as % of N intake and as % retained above maintenance (K nitrogen ), were expected with increasing Lys content as AA utilization for lean gain will increase as AA content approaches the requirement (Pencharz and Ball 2003).Furthermore, total N utilization has been shown to improve with increasing Lys content, attributed to increased protein deposition with increased Lys (O'Connell et al. 2006).
Likewise, the marginal efficiency of Lys (K lysnie ) was relatively constant until the estimated requirement breakpoint was reached, as further increases in Lys are not utilized for lean gain.Interestingly, the efficiency of total SID Lys use for NR was not affected by N supplementation in the current study, unlike our previous work (Camiré et al. 2023) where efficiency of Lys utilization for NR was improved in diets with supplemental intact protein (low E:T ratio) compared to unsupplemented diets (high E:T ratio).However, an increase in the marginal efficiency of SID Lys intake above maintenance (K lysine ) for protein synthesis was observed with NPN inclusion.Interestingly, when pigs were previously fed either high or low E:T ratio diets, K lysine was not impacted by ratio (Camiré et al. 2023).This indicates that NPN inclusion, not a reduced E:T ratio, may be the main contributor to improved marginal efficiency of SID Lys for NR.This result may be attributed to improved AA utilization compared to the NAP-fed pigs, as Lys and other EAAs were potentially catabolized at a reduced rate for the synthesis of NEAA when additional dietary N was provided in the form of NPN.This may indicate important differences in how supplemental N is utilized and dependent on the form in which it is provided.For instance, while N from NPN may be more readily available for NEAA synthesis, intact protein supplies pre-formed NEAA that can be utilized directly for lean gain.
Evidence for an improvement in EAA and N utilization due to supplemental NPN is further provided by the decrease in total plasma EAA, an indication of improved EAA incorporation into body protein and decreased EAA catabolism.The decrease in total plasma EAA was likely due to increased use for lean gain or production of secondary metabolites, as indicated by the improved NR observed in AP-fed pigs and has been previously shown by Mansilla et al. (2017a).The increase in NEAA Arg, Ala, Gln, and Glu provides further evidence that the supplemented NPN was being incorporated into AAs, and particularly those NEAAs that play key roles in N metabolism (Groff and Gropper 2000).While the present study and previous research have indicated that ammonia-N is an efficient source of N in diets limiting in NEAA-N (Mansilla et al. 2017a), it is often reported that absorbed ammonia is used primarily for ureagenesis (Columbus et al. 2014).More recently, data have demonstrated that while ammonia-N is partially used for urea production, it is not used effectively (Mansilla et al. 2017b), indicating that NPN is used in a variety of metabolic reactions.In pigs fed diets deficient in NEAA-N, dietary ammonia-N was poorly utilized for urea production across splanchnic organs (PDV and liver; Mansilla et al. 2017b).In work done by Mansilla et al. (2017aMansilla et al. ( , 2017b)), pigs were fed diets with high EAA-N:TN ratios supplemented with ammonium citrate at low and high inclusion levels (2% and 4%, Mansilla et al. 2017b;1.8% and 3.9%, Mansilla et al. 2017a).Net urea output did not increase with the low-ammonia diet across the splanchnic, bed but tended to increase with the high-ammonia diet, suggesting a limit to ammonia-N used in other metabolic reactions before the excess is excreted.Furthermore, urea appearance in the portal vein did not increase with the ammonia-N supply, and the authors suggested that the gastrointestinal tissue used this N supply to synthesize nitrogenous metabolites, including the synthesis of NEAA (Mansilla et al. 2017b).
While NR as % of N intake showed a similar trend as for NR (g/day), the % of N intake retained was higher in pigs fed the high E:T ratio.Similar results were observed previously, when the E:T ratio was decreased using intact protein instead of NPN (Camiré et al. 2023).This suggests that while N may be limiting the NR in high E:T ratio diets, the utilization of N is not affected by N source.This further supports the study by Mansilla et al. (2017a) in which it was shown that N from ammonia was as efficient as intact protein (i.e., soybean meal) in N-deficient diets.In particular, the increased concentrations of Arg, Gln, and Glu suggests that ammonium-N was utilized for NEAA synthesis as opposed to ureagenesis, as there was no impact of NPN inclusion on PUN, which was also observed by Mansilla et al. (2017b).This challenges the notion that swine cannot utilize NPN for growth and suggests the importance of N to growing pigs.
The current study estimated improved NR and a greater Lys requirement when pigs were fed a low E:T ratio diet supplemented NPN compared to pigs fed a high E:T ratio diet.This suggests that N may be limiting in diets even when sufficient EAAs have been supplied.This also provides evidence that the NRC (2012) requirements for EAAs may need to be adjusted as they are based on a lower total dietary N content (i.e., Lys:crude protein ratio) than what is indicated in the current study as resulting in an increase in NR.Based on the estimated Lys requirement and dietary protein content examined in the current study, the required Lys:crude protein ratio would be 6.62% and 6.64% for NAP-and AP-fed pigs, respectively, which is lower than the NRC (2012) recommendation of 7.45% Lys:crude protein ratio.While NR and N utilization have been shown to be affected at extreme E:T ratios (Heger et al. 1998), in the majority of studies, the effects of E:T ratio have been determined in diets where both EAA and NEAA fractions have been altered, resulting in diets that differ in both EAA and total N content (Heger 2003).This is unlikely to be the case in commercial practice, where EAAs are likely to be formulated to meet requirements while total dietary protein is adjusted.As in the present study, Lenis et al. (1999) observed an improvement in NR (g/day) when EAA-N was kept constant and the total N fraction was increased (i.e., decreasing E:T ratio).Overall, this suggests that both EAA and N recommendations need to be re-examined while considering the E:T ratio as an indicator of N sufficiency.These results provide further evidence that pigs can utilize sources of NPN to meet their AA requirements.This is consistent with previous research in which inclusion of NPN (e.g., urea, ammonium-N) has been shown to improve NR in pigs fed diets limiting in EAA (i.e., valine;Columbus et al. 2014) or NEAA (Mansilla et al. 2017a).

Conclusions
The inclusion of NPN, as AP, improved NR and increased the Lys requirement.These results indicate that diets with a high E:T ratio may be limiting in dietary N, even when providing sufficient EAA.The study further demonstrates that swine can utilize AP as a source of dietary N to improve N utilization when fed diets lacking in NEAA-N.The E:T ratio may be utilized as a tool during diet formulation to consider the sufficiency of dietary NEAA and N.
retained in body protein) / (SID N intake − maintenance N requirement) K lysine = (protein deposition × Lys % of body protein) / (SID Lys intake − maintenance Lys requirements/ efficiency of SID Lys utilization for maintenance)

Table 1 .
Ingredients and calculated nutrient composition (% as-fed basis) of experimental diets * .
Note: CP, crude protein; DM, dry matter; E:T, essential amino acid-nitrogen:total nitrogen ratio; ME, metabolizable energy; NE, net energy; SID, standardized ileal digestible; TN, total nitrogen; AP, ammonium phosphate; NAP, no ammonium phosphate.*The lowest and highest Lys diets were blended in appropriate proportions to achieve diets containing the other graded levels of Lys (not shown).†Supplied per kilogram of complete feed: vitamin A, 4000 IU; vitamin D, 0.019 mg; vitamin E, 15 IU; ‡ Nutrient content of diets based on estimated nutrient contents of feed ingredients according to NRC (2012).

Table 3 .
Nitrogen balance in pigs fed NAP or AP diets with increasing SID Lys content * .

Table 4 .
Linear and quadratic relationship of dietary Lys and non-protein nitrogen inclusion on nitrogen retention * .

Table 5 .
Plasma amino acid concentrations (μmol/L) in pigs fed no ammonium phosphate (NAP) or ammonium phosphate (AP) diets with graded levels of Lys * ., ammonium phosphate; NAP, no ammonium phosphate; SEM, standard error of the mean; TAA, total amino acids; TEAA, total essential amino acids; TNEAA, total non-essential amino acids.
* Data presented are least-square means (n = 9 pigs/treatment); samples were obtained 2 h after the morning meal (0800 h) on day 2 of the nitrogen-balance collection.AP† Pooled SEM.