Effects of supplementing milk replacer with essential amino acids on blood metabolites, immune response, and nitrogen metabolism of Holstein calves exposed to an endotoxin

This study evaluated the effects of supplementing calf milk replacer with essential AA on immune responses, blood metabolites, and nitrogen metabolism of 32 Holstein bull calves [28 d of age, 44 ± 0.8 kg of body weight (BW)] exposed to lipopolysaccharide (LPS). Calves were bottle-fed a commercial milk replacer (20% crude protein and 20% fat, dry matter basis) twice daily along with a calf starter (19% crude protein, dry matter basis) for 45 d. The experiment was a randomized complete block design and treatments were a 2 × 2 factorial arrangement. Treatments were milk replacer (fed twice daily at 0.5 kg/d of powder) supplemented with or without 10 essential AA (+AA vs. −AA), and subcutaneous injection of sterile saline with or without LPS (+LPS vs. −LPS) at 3 h after the morning feeding on d 15 (4 µg LPS per kg of BW) and 17 (2 µg LPS per kg of BW). Calves also received a 2-mL subcutaneous injection of ovalbumin (6 mg of ovalbumin/mL) on d 16 and 30. Rectal temperature and blood samples were collected on d 15 before LPS injection and at h 4, 8, 12, and 24 thereafter. From d 15 to 19, total fecal and urinary output were collected, and feed refusals were documented. Rectal temperature was greater in +LPS than −LPS calves at h 4, 8, and 12 after LPS injection. Serum cortisol was greater for +LPS than −LPS at h 4 after LPS exposure. At d 28, serum antiovalbumin IgG level was greater in +LPS +AA calves compared with +LPS −AA. Serum glucose was lower for +LPS than −LPS at h 4 and 8. Serum insulin was greater in +LPS than −LPS calves. Plasma concentrations of Thr, Gly, Asn, Ser, and hydroxyproline were lower for +LPS versus −LPS calves. Plasma concentrations of Met, Leu, Phe, His, Ile, Trp, Thr, and Orn were greater in +AA calves than −AA calves. Plasma urea N and N retention were not different among LPS and AA treatments. The lower concentrations of AA in +LPS than −LPS calves indicate higher demand for AA in immu-no-compromised calves fed milk replacer. Additionally, higher concentration of ovalbumin-specific IgG level in +LPS calves supplemented with +AA compared with +LPS calves with −AA suggests that supplementing AA to immune-compromised calves might improve immune status.


INTRODUCTION
Exposure to infectious agents stimulates inflammatory processes and results in metabolic changes that alter protein and AA requirements in mammals (Kominsky et al., 2010;Ren et al., 2018). Absorbed AA are directed away from lean tissue growth during immunological stress due to greater demand for the synthesis of proteins and cells related to the immune response (Rauw, 2012). During inflammation, AA are used for the synthesis of acute-phase proteins, glucose precursors, plasma proteins, antibodies, free radical scavengers, metabolic cofactors, and hormones (Li et al., 2007). If an imbalance exists between the composition of AA obtained from tissue protein catabolism and the AA composition of acute-phase proteins, AA loss via nitrogen excretion will likely increase (Reeds and Jahoor, 2001). Our research (Waggoner et al., 2009a;Löest et al., 2018) demonstrated that plasma concentrations of multiple EAA (Met, Lys, His, Leu, Ile, Val, Phe, Thr, Trp) decreased in growing steers in response to lipopolysaccharide (LPS), which suggests that providing cattle with these potentially limiting AA may be beneficial during periods of immune system activation and stress.
Previous research (Kanjanapruthipong, 1998;Hill et al., 2008;Morrison et al., 2017;Vasquez et al., 2017) shows that AA, such as Met, Lys, Thr, and Ile, may be limiting for young calves fed milk replacer. However, the effects of AA supplementation on performance of milk replacer-fed calves have been variable and are currently not well-defined (Silva et al., 2021). We hypothesized that calves with an activated immune system have increased needs for multiple EAA, and that supplementation of milk replacer with 10 EAA will increase the supply of potentially limiting AA for the immune response and thus alleviate decreases in nitrogen retention associated with excess protein degradation and AA catabolism of immune-challenged calves. The objectives were to evaluate the effects of supplementing milk replacer with EAA on immune responses, blood metabolites, and nitrogen metabolism of Holstein calves exposed to LPS.

Animals, Facilities, and Experimental Design
The Institutional Animal Care and Use Committee at New Mexico State University approved all procedures and the sample size for this study. Thirty-two Holstein bull calves (28 d of age, 44 ± 0.8 kg of BW) were obtained from a single-source commercial dairy. Calves had previously received colostrum, milk replacer (20% CP, 20% fat; Central Supply), and ad libitum calf starter (DairyWay Calf Starter, Cargill Incorporation). Upon arrival, blood samples were collected from each calf via jugular venipuncture into vacuum tubes (10-mL Corvac serum separator, Kendall). Serum samples were analyzed for IgG concentrations by the New Mexico Department of Agriculture Veterinary Diagnostic Services. All calves had serum IgG concentrations greater than 5 g/L, which suggest that the calves for this study had acquired at least marginal passive immunity according to Quigley et al. (2006). Calves were vaccinated with Clostridium Chauvoei-Septicum-Hemolyticum-Novyi-Sordellii-Perfringens types C and D Bacterin-Toxoid (Vision-8 with SPUR; Intervet/Schering-Plough Animal Health), Moraxella Bovis Bacterin (Piliguard pinkeye triview; Intervet/ Schering-Plough Animal Health), and Pyramid 10 Bovine Rhinotracheitis-Virus Diarrhea-Parainfluenza 3-Respiratory Syncytial Virus Vaccine (Fort Dodge Animal Health). Calves received primary and secondary inoculations using the aforementioned vaccines at 25 d of age (at the commercial dairy) and 56 d of age (at the research facility), respectively. Calves were weighed upon arrival and placed in individual pens (1.2 × 2.4 m) in a temperature-controlled (22.5 ± 2.7°C) animal metabolism facility where they had free access to fresh water. Bedding for individual pens was wood shavings (Premium Pine Shavings, Tractor Supply Co.) that was inspected daily and replaced if soiled or wet. Calves were fed reconstituted milk replacer (Table 1; Calf Start Premium 20/20, Central Supply) twice daily via 2-L nursing bottles at 10% of initial average BW per day. The milk replacer was reconstituted according to label specifications, which was to thoroughly mix 0.454 kg of powder with 3.8 L of warm (49°C) drinking water. Calves were also fed (once daily) 0.454 kg of a medicated calf starter ( Animal Health) according to label recommendations, and calves with apparent morbidity (based on visual appraisal described above and a rectal temperature >39.7°C) were treated with florfenicol (Nuflor; Intervet/Schering-Plough) according to label instructions. The experiment was a randomized complete block design with 2 blocks (periods) of 16 calves (blocked because of limited housing capacity of the research facility). Each block of calves remained on the experiment for 45 d (from 28 d of age to 73 d of age). For the first 12 d, calves were allowed to adapt to their diet and individual pens of the indoor metabolism facility. On d 12, calves were weighed and moved to ruminant metabolism crates (0.75 × 1.5 m; fully adjustable for each animal) for an adaptation period of 3 d before beginning a 6-d sample collection period. The 6-d collection period consisted of 1 d for blood collection and 5 d for fecal and urine collections. After completing the collection period in the metabolism facility, calves were moved to outdoor soil-surfaced pens with partial shade for 24 d for the collection of a blood sample at 14 and 28 d after the first collection day (d 15).
Treatments were arranged as a 2 × 2 factorial, and calves were allocated to the treatments randomly by creating random numbers. The 2 × 2 factorial treatments were milk replacer supplemented with or without 10 EAA (+AA vs. −AA), and subcutaneous injection of sterile saline with or without LPS (+LPS vs.

−LPS).
The +AA solution was prepared by dissolving 4 g of l-Leu, 2 g of l-Ile, 2 g of l-Val, 2 g of dl-Met, 2 g of l-Phe, 2 g of l-His, 2 g of l-Lys, 2 g of l-Thr, 6 g of l-Arg, and 1 g of l-Trp in 500 g of deionized water containing 4 g of 6 M hydrochloric acid, and then adjusting the pH to 4.0 with sodium hydroxide. The sources of AA were Glanbia Nutritionals (l-Leu, l-Ile, l-Phe, and l-His), Ajinomoto Heartland Inc. (l-Val, l-Lys, l-Thr, and l-Trp), and Degussa Corporation (dl-Met). The +AA solution (500 g/calf daily) was divided into 2 equal portions that were thoroughly mixed with milk replacer before feeding at 0600 and 1800 h from d 1 to d 21 of the experiment. These amounts of EAA for the +AA solution were 40% of that supplemented to calves by Löest et al. (2018), and were estimated to increase the EAA supply of the calf milk replacer (Table 1) by 25% or more. Because Met is often a limiting AA for young calves fed milk replacer (Hill et al., 2008), the amount of dl-Met in the +AA solution increased the calf milk replacer Met supply by 100%. We assumed that the suckling reflex from bottle-feeding would allow the majority of the supplemental AA to bypass the rumen for absorption in the small intestine (Hegland et al., 1957). The +LPS was prepared by dissolving 1 mg of LPS (Escherichia coli O55:B5; Sigma Chem. Co.) in 100 mL of sterile saline. At 3 h after the morning feeding on the first and third day of collection (d 15 and 17), +LPS calves were injected subcutaneously (Ballou et al., 2008) with 4 and 2 µg of LPS per kg of BW, respectively. Simultaneously, −LPS calves were subcutaneously injected with an equal volume of sterile saline. Additionally, each calf was inoculated with ovalbumin by subcutaneous injection on d 16 of the experiment and again 14 d later (d 30 of the experiment). The inoculation consisted of a single site injection of 2 mL of a 1:1 solution of commercially prepared aluminum hydroxide adjuvant solution (Anhydrogel, Cat. No. A1090 S; Accurate Chemical Corp.) and sterile saline containing suspended ovalbumin (Cat. No. A5503; Sigma-Aldrich). This vaccine, which contained 6 mg of ovalbumin per mL, was prepared by first dissolving the ovalbumin in sterile saline with gentle stirring to avoid foaming and then adding the adjuvant. The vaccine was stored in sterile bottles and refrigerated (4°C) until use.
From d 15 to 19, milk replacer and calf starter intake was measured, total fecal output was collected daily into fecal collection pans, and total urinary output was collected into 20-L buckets containing 100 mL of 6 M hydrochloric acid to minimize ammonia loss. The weight of total fecal and urinary output was recorded daily. All feces and a representative sample (1%) of urine were stored at −20°C and later composited for each calf for analysis. Also, dietary samples and calf  starter refusals were collected, composited for each calf, and frozen at −20°C. On d 15, rectal temperatures were measured (Cooper TM99A digital thermometer, Cooper Atkins Corp.) and blood samples were collected via jugular venipuncture into vacuum tubes (10-mL Corvac serum separator and 9-mL Monoject Sodium Heparin, Kendall) before LPS injection and at 4, 8, 12, and 24 h thereafter. Blood samples were also collected for the analysis of serum ovalbumin-specific IgG concentration immediately before the subcutaneous injection of ovalbumin on d 16, and then 14 and 28 d later (d 30 and 44 of the experiment, respectively). Blood samples obtained for the collection of serum were allowed to coagulate at room temperature for 30 min, whereas samples obtained for plasma were immediately placed on ice. All blood samples were centrifuged (Sorvall RT600B, Thermo Electron Corp.) at 1,500 × g for 20 min at 10°C. Serum and plasma samples were immediately decanted into 1.5-mL and 7-mL vials and frozen at −70°C for later analysis. Final BW of calves were measured on d 45 of the experiment.

Laboratory Analyses
Composite samples of grain starter, feed refusals, and feces were dried at 55°C for 72 h in a forced-air oven (Blue M Electric Company), allowed to air-equilibrate for 48 h, weighed to determine moisture loss, and then ground to pass a 2-mm screen (Wiley Model 4, Thomas Scientific). Ground samples were analyzed for DM (105°C for 24 h) in a convection oven (Model 845, Precision Scientific Group). Concentrations of nitrogen in milk, grain starter, feed refusals, feces, and urine were determined by measuring N 2 from nitrogen-combustion products with a thermo-conductivity cell (Leco FP-528, Leco Corp.). Total nitrogen intake was calculated as the sum of nitrogen intake from calf starter and milk replacer (with and without added AA).
Concentrations of serum glucose and plasma urea nitrogen were determined using colorimetric assays as described by Waggoner et al. (2009a;2009b). The glucose assay was modified by adding 4 µL (vs. 2 µL) of serum sample to improve accuracy. Samples were analyzed in duplicate, and samples with coefficient of variation (CV) ≥10% were re-analyzed. Serum concentrations of cortisol and insulin were determined in duplicate by solid-phase RIA, using components of commercial kits (Siemens Diagnostic) and protocols validated by Kiyma et al. (2004) and Reimers et al. (1982), respectively. These kits used antibody-coated tube technology, and assays were performed without prior extraction of the individual hormones from serum. Within-assay CV for cortisol and insulin were 6 and 8%, respectively. Serum samples were analyzed for haptoglobin by the Kansas State University Veterinary Diagnostic Lab as described by Smith et al. (1998). Additionally, serum samples were analyzed for ovalbuminspecific IgG concentration using ELISA adapted from the procedure described by Rivera et al. (2002). For our procedure, 20 µL of each sample were pooled within day of collection and were serially diluted from 1:400 to 1:51,200. From the data analysis of this dilution range, a dilution (1:3,200) was chosen which was most central to the linear portion of the dilution curve. Each sample was subsequently analyzed at the 1:3,200 dilution and the resulting data were used for statistical analysis. Pooled serum samples from all calves on d 14 (of the antiovalbumin IgG analysis collection period), diluted 1:1,600 in PBS (Sigma-Aldrich), were used as a positive control. Samples were analyzed in duplicate, and samples with CV ≥15% were re-analyzed. Plasma AA concentrations were analyzed by gas chromatography (CP-3800, Varian) using a commercially available kit (EZ: FAAST No. KGO-7165, Phenomenex) as described by Waggoner et al. (2009a). Intra-and interassay CV for plasma AA were less than 15%.

Statistical Analysis
A power analysis using residual errors from nitrogen retention as the primary response variable from previous AA research in Holstein calves (Löest et al., 2002) showed that 8 replicates per treatment were required to detect a 15% difference from control with α = 0.05 and β = 0.80. There were 16 metabolism crates in the facility, thus data collection occurred over 2 periods. The experiment was blocked (16 calves in block 1, and 16 calves in block 2) by the collection date. Two calves were removed from the experiment because of health concerns, and all data collected from these calves were excluded from the statistical analysis.
The experiment was a randomized complete block design, and all data were analyzed using the GLIMMIX procedure of SAS 9.4. The statistical model included the main effects of LPS, AA, and LPS × AA interaction for all dietary measures where block and calf were random effects. Rectal temperature and blood metabolites were analyzed as repeated measures (autoregressive order one covariance structure). The model for rectal temperature and blood metabolites (except ovalbumin-specific IgG concentration) included all possible interactions of LPS, AA, and hour. The model for ovalbumin-specific IgG concentration included all possible interactions of LPS, AA, and day. All response variables were tested for normality by using a Shapiro-Wilk test in the UNIVARIATE procedure of SAS 9.4 (SAS Inst. Inc.). Residuals of all response variables were normally distributed, except for serum cortisol, insulin, haptoglobin, and plasma asparagine. For serum cortisol, insulin, haptoglobin, and plasma asparagine, log-transformation was used to correct the distribution of the residuals. Untransformed least squares means were reported for manuscript readability, as P-values were not different between the untransformed and logtransformed means. The experimental unit was calf, data were presented as least squares means, and differences were considered significant at P ≤ 0.05.

RESULTS
There was a tendency for a LPS × AA × day interaction (P = 0.068) for serum antiovalbumin IgG ( Figure  1). Serum antiovalbumin IgG tended to increase on d 28 after initial inoculation for +LPS calves receiving AA supplementation compared with all other treatment groups. No LPS × AA × hour interactions (P ≥ 0.18) and no LPS × AA interactions (P ≥ 0.08) were observed for rectal temperature, serum cortisol, haptoglobin, insulin, and glucose concentrations, and plasma AA concentrations.
Rectal temperature (Figure 2) increased and was greater for +LPS than −LPS calves at 4 (peak), 8, and 12 h, but was not different at 24 h after LPS injection (LPS × hour interaction, P < 0.05). Serum cortisol (Figure 3) increased and was greater in +LPS than −LPS calves at 4 h after LPS injection, but was not different at 8, 12, and 24 h after LPS injection (LPS × hour interaction, P < 0.05). Serum haptoglobin ( Figure  3) was not different at 0, 4, and 8 h, but was greater for +LPS than −LPS calves at 12 and 24 h after LPS infusion (LPS × hour interaction, P < 0.05). Serum glucose concentration (Figure 4) decreased and was lower for +LPS than −LPS calves at 4 and 8 h, but was not different at 12 and 24 h after LPS injection (LPS × hour interaction, P < 0.05). A tendency for an LPS × hour interaction (P = 0.06) occurred for serum insulin; serum insulin was higher for +LPS than −LPS calves at 4 and 24 h, however not different at other times ( Figure 4). Plasma urea nitrogen (Table 3) was not affected by LPS administration (P = 0.24).
No LPS × AA interactions (P ≥ 0.13) or LPS effects (P ≥ 0.19) were observed for BW, DMI, N intake, fecal DM, fecal N, grams of digested DM, grams of digested N, DMD, N digestibility (as a percentage of total intake), urinary N excretion, grams of retained N, and N retention as a percentage of total N intake (Table 4). Average daily gain from d 12 to 45 tended to be less (P = 0.08) for +LPS than −LPS calves (Table 4). Calves supplemented with AA had greater (P < 0.05) total N intake and grams of digested N, and tended to  have greater (P = 0.07) urinary N excretion compared with calves receiving no supplemental AA (Table 4).

Endotoxin Challenge
Lipopolysaccharide was used as a noninfectious means to activate the innate immune response of calves so that we could evaluate if calves with an activated immune system have increased needs for multiple EAA. Our hypothesis was that supplementation of milk replacer with 10 EAA will increase the supply of EAA potentially needed by the immune response and thus alleviate decreases in nitrogen retention associated with excess protein degradation and AA catabolism. Use of LPS in cattle has been shown to produce the metabolic effects associated with stress and disease, including increased body temperature, blood cortisol, cytokines, and acute-phase proteins, as well as altered protein and energy metabolism (Steiger et al., 1999). In the current study, an inflammatory response to LPS injection was evident by increases in rectal temperature, cortisol, insulin, and haptoglobin and decreases in glucose in the LPS-exposed calves. These responses are similar to those previously reported for cattle exposed to LPS (Steiger et al., 1999;Waggoner et al., 2009a;Löest et al., 2018).
The observed decrease in serum glucose at 4 h after Holstein calves were injected with LPS is consistent with results from Waggoner et al. (2009a) and Löest et al. (2018). However, Waggoner et al. (2009a) and Löest et al. (2018) also observed an initial increase (at 2 h) before the glucose concentrations began to decrease at 4 h following LPS infusion. We would have anticipated a similar response if we had collected a blood sample 2 h after LPS injection; blood samples were collected at 4-h intervals for the first 12 h after LPS injection to minimize potential stress associated with jugular venipuncture. According to Steiger et al. (1999), the initial increase followed by a decrease in blood glucose concentrations in animals exposed to LPS are explained by increased glucose production via glycogenolysis and gluconeogenesis followed by increased glucose utilization by peripheral tissues that exceeds the capacity to synthesize glucose during LPS exposure. According to Steiger et al. (1999) and   Löest et al. (2018), LPS has a delayed effect of inhibiting hepatic gluconeogenesis. Additionally, Spurlock (1997) stated that LPS causes insulin resistance and therefore a decrease in glucose uptake by peripheral tissues. In the current study, serum insulin concentra-tions increased in LPS-challenged calves at 24 h after the LPS injection.
Decreases in plasma concentrations of Thr, Met, Gly, Ser, Pro, Hyp, Asn, Asp, Glu, Gln, and total NEAA in calves exposed to LPS might suggest either a shift in  Effect of LPS × AA × day (P = 0.068; see Figure 1). OD = optical density. 4 Effect of LPS × hour (P < 0.05; see Figure 2).
AA utilization toward the synthesis of proteins related to the immune response or increased AA catabolism (Reeds and Jahoor, 2001;Li et al., 2007). However, Hoskin et al. (2016) explained that simple changes in plasma concentrations of specific AA may not be directly indicative of a change in the metabolic demand for those AA, because multiple factors contribute to the entry and removal of AA from the plasma pool. Nevertheless, possible metabolic mechanisms that could explain the observed shifts in specific plasma AA in response to LPS are described below.
The observed decreases in plasma Thr concentrations in calves receiving LPS are consistent with previous research (Waggoner et al., 2009a,b;Löest et al., 2018), and could be explained by a greater demand for the synthesis of mucosal proteins and acute-phase proteins during the inflammatory response (Faure et al., 2007;Hoskin et al., 2016). However, Hoskin et al. (2016) reported an increase in the activity of hepatic serine-threonine dehydratase in LPS-exposed lambs, which may indicate an increase in Thr catabolism. In the current study, calves exposed to LPS had lower  plasma Met concentrations, which is also consistent with previous research (Waggoner et al., 2009a,b;Carter et al., 2010;Löest et al., 2018). Metabolic mechanisms that could explain decreased plasma Met concentrations in LPS-exposed calves include increased need for S-adenosylmethionine in polyamine synthesis for immune cell replication (Grimble and Grimble, 1998) and(or) increased transulfuration of Met to Cys during the inflammatory process (Malmezat et al., 2000;McGilvray et al., 2019). Glutathione, which is a tripeptide of Cys, Gly, and Glu, has multiple roles in the immune response, including antioxidant defense, cell proliferation, and cytokine production (Newsholme et al., 2003;Wu et al., 2004). Although not measured in the current study, glutathione production likely increased in response to LPS exposure, which might explain, in part, the observed decreases in Met (because Cys is a product of Met metabolism), Gly, Glu, as well as Ser (because Gly is a product of Ser metabolism). Observed decreases in plasma Asn, Asp, Gln, and Glu in LPS-challenged calves may be related to their direct or indirect role as energy substrates for immune cells and small intestinal enterocytes (Newsholme et al., 2003;Wang et al., 2015). Chen et al. (2016) also demonstrated that Asn improves intestinal integrity of LPS-challenged pigs. In the current study, calves injected with LPS had decreased plasma concentrations of Pro and Hyp (a metabolite of Pro), perhaps because Pro is a major substrate for polyamine synthesis in intestinal epithelial cells and(or) because of its role in antioxidant defense (Wu et al., 2000). Kang et al. (2014) demonstrated that dietary supplementation of Pro increased superoxide dismutase activity and gastrointestinal digestibility of LPS-challenged pigs.
Exposing calves to LPS did not significantly alter plasma concentrations of the branched-chain AA (Leu, Ile, and Val), Lys, Phe, Trp, Ala, Orn, and Tyr. This lack of a response was surprising, because these results are in contrast to previous research (Waggoner et al., 2009a,b;Carter et al., 2010;Löest et al., 2018) and it has been documented that several of these EAA are necessary to support an activated immune system (Reeds and Jahoor, 2001). For example, decreases in plasma branched-chain AA reported by Waggoner et al., (2009a,b), Carter et al. (2010), and Löest et al. (2018) were attributed to increased uptake by prolifer-  ating lymphocytes and(or) by peripheral tissues for Gln synthesis (Newsholme and Calder, 1997;Monirujjaman and Ferdouse, 2014). In the current study, the only branched-chain AA that tended to decrease in LPSchallenged calves was Ile. Subcutaneous LPS injection also did not alter DMI, DM digestibility, or nitrogen retention of calves in the current study, whereas previous research (Waggoner et al., 2009a,b;Carter et al., 2010;Löest et al., 2018) demonstrated that LPS-challenged cattle have decreased DMI, decreased nitrogen intake, increased urinary nitrogen excretion and decreased (often negative) nitrogen retention. Waggoner et al. (2009a,b), Carter et al. (2010), andLöest et al. (2018) attributed the decreases in plasma AA concentrations and decreases in nitrogen retention to increased tissue protein degradation to support the AA demand of an activated immune response. In the current study, the dose of LPS sufficiently elicited an inflammatory response (increased rectal temperatures, cortisol, and haptoglobin), affected energy metabolism (decreased blood glucose and increased insulin), and altered plasma concentrations of most NEAA. However, the subcutaneous injections of LPS (4 µg/kg of BW on d 15 and 2 µg/kg of BW on d 17) did not alter plasma concentrations of EAA (except Thr and Met) and nitrogen retention (i.e., lean tissue accretion). Nevertheless, ADG of +LPS calves tended to be 20% less than -LPS calves from d 12 to 45 despite no differences in DMI of calf milk replacer and calf starter during the collection period (d 15 to 19

Amino Acid Supplementation
We hypothesized that supplementation of milk replacer with 10 EAA will increase the supply of potentially limiting AA for the immune response and thus alleviate decreases in nitrogen retention associated with excess protein degradation and AA catabolism. The supplementation of calf milk replacer with EAA increased the EAA supply of the AA-fortified calf milk replacer by 25% (for Lys) to 100% (for Met). We added Met to the milk replacer to increase the Met supply by 100% because we speculated that Met might be the most limiting AA for our young growing Holstein calves (Greenwood and Titgemeyer, 2000;Hill et al., 2008). Greater plasma concentrations of Met, Leu, Ile, Val, His, Phe, Thr, Trp, and Orn (a metabolite of Arg) in +AA calves compared with -AA calves indicated that the 10 supplemental EAA in milk replacer escaped rumen degradation through the suckling response and were effectively absorbed. No differences in plasma Lys in response to +AA supplementation suggests that the 25% additional Lys added to the milk replacer was either insufficient to alter plasma Lys concentrations, or that Lys was the most limiting AA in these calves fed milk replacer, or both. Hill et al. (2007Hill et al. ( , 2008 reported that supplementing Lys with a 20% CP milk replacer increased ADG of calves, and Wang et al. (2012) observed that Lys was the first limiting AA in calves provided with milk replacer containing soy protein.   A 12% increase in total nitrogen intake, but no increase in fecal nitrogen excretion, for +AA versus −AA calves indicated that the individual crystalline AA dissolved with the calf milk replacer were efficiently absorbed (possibly near 100%), which is consistent with previous research (Löest et al., 2001;Nolte et al., 2008). Therefore, the 15% increase in apparent nitrogen digested by +AA calves compared with -AA calves is a result of efficient absorption of the individual crystalline AA dissolved with milk replacer, and not likely because of an increase in the nitrogen digestibility of the milk replacer or calf starter. A tendency for a 13% increase in urinary nitrogen excretion, but no increase in nitrogen retention, for +AA versus −AA calves demonstrate that the EAA supplemented with the calf milk replacer were not needed to increase protein accretion. Similarly, supplementing calf milk replacer with EAA from d 1 to 21 did not affect calf ADG during the 45-d study. The nitrogen balance and ADG responses are consistent with observed increases in the plasma concentrations of all the EAA (except Lys) and Orn (a metabolite of Arg) in +AA calves compared with -AA calves. Typically, concentration of an EAA in plasma will increase when supply exceeds requirements (Campbell et al., 1997). Therefore, the combined effect of increased plasma AA with no increase in nitrogen retention and ADG for AA-supplemented calves indicate that the metabolizable supply of EAA from both the calf milk replacer and calf starter were likely in excess of the calf's AA requirements to support the metabolizable energy-allowable performance.
During an inflammatory response, AA are used for the synthesis of immune system proteins (Le Floc'h et al., 2004;Li et al., 2007), and our previous research (Waggoner et al., 2009a;Löest et al., 2018) demonstrated that plasma concentrations of multiple EAA decreased in LPS-challenged steers. Therefore, we speculated that supplementing calf milk replacer with these potentially limiting AA may be beneficial for LPSchallenged calves. However, no significant interactions between LPS administration and AA supplementation for all the response variables measure in this study suggests that supplementing milk replacer with 10 EAA does not alter the effects of a low-dose endotoxin on the inflammatory response, blood metabolites, and nitrogen retention of young Holstein calves. Nevertheless, the observed tendency for an LPS × AA × day interaction for serum antiovalbumin IgG demonstrates that LPS-challenged calves receiving supplemental AA had greater concentrations of circulating antiovalbumin IgG than those of calves on other treatments. This increase indicates that supplementing calf milk replacer with EAA improved the adaptive immune response when calves were exposed to an immune challenge. This observation is consistent with previous research (Carter et al., 2011) that demonstrated increased blood concentrations of antiovalbumin IgG when newly received feedlot calves were supplemented with rumen-protected branched-chain AA.

CONCLUSIONS
Higher rectal temperature, cortisol, insulin, and haptoglobin and lower glucose in the +LPS than − LPS calves indicate that LPS activated the immune response. Despite these observations, exposure to LPS did not affect nitrogen retention and tended to decrease growth performance of calves. Lower plasma AA in calves exposed to LPS may indicate an increased need for AA during periods of immunological stress. However, the combined effect of increased plasma AA with no increase in nitrogen retention and ADG for calves supplemented with 10 EAA indicate that the basal diet (calf milk replacer and calf starter) fed to calves in the current study was not limiting in one or more EAA. Nevertheless, increased circulating ovalbumin-specific IgG levels in LPS-challenged calves supplemented with 10 EAA suggest that AA supplementation may improve the adaptive immune response.