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Background: Pathogen or diet-induced immune activation can partition energy and nutrients away from growth, but clear relationships between immune responses and the direction and magnitude of energy partitioning responses have yet to be elucidated. The objectives were to determine how β-mannanase supplementation and lipopolysaccharide (LPS) immune stimulation affect maintenance energy requirements (MEm) and to characterize immune parameters, digestibility, growth performance, and energy balance. Methods: In a randomized complete block design, 30 young weaned pigs were assigned to either the control treatment (CON; basal corn, soybean meal and soybean hulls diet), theenzymetreatment (ENZ;basal diet +0.056% β-mannanase), or the immune system stimulation treatment (ISS; basal diet + 0.056% β-mannanase, challenged with repeated increasing doses of Escherichia coli LPS). The experiment consisted of a 10-d adaptation period, 5-d digestibility and nitrogen balance measurement, 22 h of heat production (HP) measurements, and 12 h of fasting HP measurements in indirect calorimetry chambers. The immune challenge consisted of 4 injections of either LPS (ISS) or sterile saline (CON and ENZ), one every 48 h beginning on d 10. Blood was collected pre- and post-challenge for complete blood counts with differential, haptoglobin and mannan binding lectin, 12 cytokines, and glucose and insulin concentrations. Results: Beta-mannanase supplementation did not affect immune status, nutrient digestibility, growth performance, energy balance, or ME . The ISS treatment induced fever, elevated proinflammatory cytokines and decreased leukocyte concentrations (P < 0.05). The ISS treatment did not impact nitrogen balance or nutrient digestibility (P > 0.10), but increased total HP (21%) and ME (23%), resulting in decreased lipid deposition (−30%) and average daily gain (−18%) (P <0.05). Conclusions: This experiment provides novel data on β-mannanase supplementation effects on immune parameters and energy balance in pigs and is the first to directly relate decreased ADG to increased ME independent of changes in feed intake in immune challenged pigs. Immune stimulation increased energy partitioning to the immune system by 23% which limited lipid deposition and weight gain. Understanding energy and nutrient partitioning in immune-stressed pigs may provide insight into more effective feeding and management strategies. Keywords: Acute phase proteins, β-Mannan, Cytokines, Digestibility, Feed induced immune response, Heat production, Inflammation, Lipopolysaccharide, Nitrogen balance, Swine * Correspondence: email@example.com Department of Animal Science, Iowa State University, Ames, IA 50011, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 2 of 16 Background energy requirements and to characterize changes in The negative influence of an immune challenge on ani- immune parameters, nutrient digestibility, growth mal growth is well established. Pro-inflammatory cyto- performance, and energy balance. We hypothesized that kines orchestrate an immune response resulting in fever, innate immune stimulation would increase maintenance acute phase protein (APP) production, and leukocyte energy requirements by initiating a cytokine-driven proliferation, each of which requires additional energy febrile response and inflammatory state, and that and amino acids (AA). Therefore, a perceived immune β-mannanase supplementation would decrease mainten- challenge can theoretically partition energy and nutri- ance energy requirements through an energy sparing ents away from productive processes such as muscle effect of FIIR prevention. growth and negatively impact the efficiency and cost of meat production . Innate immune activation occurs Methods when pathogen-associated molecular patterns are de- All experimental procedures adhered to guidelines for tected such as the lipid-A component of lipopolysac- the ethical and humane use of animals for research and charide (LPS) from gram-negative bacteria . However, were reviewed and approved by the University of certain dietary components, such as β-mannan in soy- Manitoba Animal Care Committee. bean, copra, and palm kernel meals, mimic carbohydrate structures on pathogen surfaces  and have previously Animals and experimental design been shown to activate the innate immune system [4, 5], Thirty growing barrows [(Yorkshire × Landrace) × termed a feed-induced immune response (FIIR). Duroc] were acquired from the Glenlea Swine Research To inhibit a β-mannan derived FIIR, interest in Unit, University of Manitoba at an average body weight β-mannanase enzyme supplementation has increased. It is (BW) of 9.60 ± 2.00 kg. The experiment was conducted hypothesized that the hydrolyzed manno-oligosaccharides using a randomized complete block design. Pigs were can no longer crosslink and stimulate multiple mannose blocked by weight and randomly assigned to one of receptors, thus reducing immune stimulation and associ- three treatments (Table 1). A staggered time course was ated energy costs. Research in poultry demonstrated that utilized to accommodate the limited number of calorim- β-mannanase decreased plasma APP concentration and etry chambers available, whereby 10 blocks of three pigs improved growth performance and feed efficiency leading each (one pig per treatment) began the experiment 4 d to the conclusion that β-mannanase supplementation after the previous block. Day one BW was similar among spared energy through prevention of the FIIR [6, 7]. In treatments (10.27 ± 0.08 kg). pigs, performance responses to β-mannanase are less con- sistent than in poultry and reports on immune responses Experimental diets, treatments and procedures are limited and effects on energy partitioning have yet to All diets were formulated on the ratio of standardized be evaluated. ileal digestible lysine to metabolizable energy (ME) and Nutrient partitioning in pigs during a pathogen met or exceeded all specified nutrient requirements of challenge has received more attention, often utilizing a growing pigs from 11 to 25 kg . Pigs were fed at 2.5 LPS challenge model . Physiological responses to a times their maintenance ME requirements , once LPS challenge in pigs have been well characterized. Simi- daily at 08:00 h and had free access to water at all times. lar to disease challenges, LPS induces anorexia, fever, Pigs were fed a common pre-trial diet (Additional file 1: and nutrient repartitioning leading to decreased growth Table S1) that was corn-soybean meal-based. The ex- and efficiency [1, 2, 9]. Fever is an energetically expen- perimental basal diet (Table 2) was formulated with high sive process and its effects on sheep and human main- soybean meal and soybean hull inclusion levels to in- tenance energy requirements have been estimated . crease dietary β-mannan concentration. Immune system activation also significantly shifts glu- Due to the availability of three indirect calorimetry cose metabolism and glucose requirements during an chambers, three experimental treatments were evaluated LPS challenge in pigs have been estimated to be approxi- (Table 1). The control treatment (CON) received the 0.75 mately 1.1 g/(kg BW ·h) . Yet few studies have basal diet, while the enzyme treatment (ENZ) received addressed comprehensive changes in energy partitioning CON supplemented with 0.056% β-mannanase (Hemi- during an immune response and clear relationships cell™ HT-D, Elanco Animal Health, Guelph, ON, Canada; between measured immune responses and the direction endo-1,4-β-mannanase (160 × 10 units/kg) from Paeni- and magnitude of changes in energy partitioning have bacillus alvei). The third treatment was challenged with yet to be elucidated. repeated LPS immune system stimulation (ISS) and re- Therefore, the objectives of this experiment were to ceived the same diet as ENZ. This treatment design was determine how β-mannanase supplementation and in- determined based on the hypothesis, supported by previ- nate immune stimulation each affect maintenance ous research, that β-mannanase would inhibit a FIIR if it Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 3 of 16 Table 1 Summary of experimental treatments Experimental treatment a b c CON ENZ ISS Diet Control Control + β-Mannanase Control + β-Mannanase β-mannanase inclusion No Yes Yes Challenge treatment Saline Saline E. coli LPS Control treatment (CON) = pigs fed basal diet with no LPS (Escherichia coli serotype O55:B5) injection Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with no LPS injection Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% β-mannanase) with LPS injection occurred in CON [6, 7]. In this way, the effect of an in- maintained a febrile response (rectal temperature ≥ nate immune stimulation by LPS could be evaluated in- 40 °C) at all four challenges while minimizing an- dependent of a FIIR. orexia and vomiting. Upon arrival and during the pre-trial period, pigs During the main experiment, pigs were injected intra- were housed individually in pens (1.83 m × 1.22 m) with muscularly at 10:00 h on d 10, 12, 14, and 16 with either plastic-covered expanded metal flooring in a sterile saline or LPS, following the previously described temperature-controlled room (26 ± 2 °C). Daily feed dosing regimen determined from the pilot study. Baseline allotment during the pre-trial period was adjusted rectal temperature was measured on d 5 and 8 at 14:00 h based on BW measured every 4 d. Pigs were maintained and at 4 h post-challenge (14:00 h) on d 10, 12, and 14. on the pre-trial diet for at least 4 d until initiation of Blood samples were then collected on d 8 (pre-challenge) the experiment for their respective block, at which time and d 10 (post-challenge) via jugular venipuncture into pigs received their assigned treatment diets. The ex- two 10-mL tubes for EDTA-whole blood and serum. periment consisted of a 10-d adaptation phase, a 5-d Whole blood samples were placed on ice pending trans- total feces and urine collection phase, and 34 h of heat portation to the laboratory for complete blood count production (HP) measurements. (CBC) analysis with white blood cell (WBC) manual dif- At trial initiation (d 1), pigs were individually housed ferential. Serum was separated by centrifugation (2,000×g in adjustable metabolism crates (1.80 m × 0.60 m) with for 15 min at 4 °C), collected and divided into three sub- smooth transparent plastic sides and plastic-covered ex- samples, and stored at − 80 °C until analyzed. panded metal flooring in a temperature controlled room (26 ± 2 °C). Body weight was measured on d 1, 5, 10, 16, Digestibility daily feed allotment was adjusted accordingly, and pigs On d 10, pigs received 5 g of ferric oxide as an indigest- were trained to consume the entire meal within 1 h of ible marker mixed with 100 g of feed; the remaining feeding at 08:00 h. Orts, if any, were measured to accur- allotted feed was offered after the marked feed was con- ately determine average daily feed intake (ADFI). sumed. Fecal collection commenced when the marker first appeared in the feces. On d 15, pigs were offered Immune challenge 100 g of marked feed as previously described, and fecal A low dose, repeated LPS challenge, following the modi- collection terminated when the marker appeared in the fied procedures described by Rakhshandeh and de Lange feces. Feces were weighed and stored at − 20 °C until fur- , was chosen to induce an inflammatory response ther processing. Total urine collection commenced at representative of sustained immune system stimulation 08:00 h on d 11 and terminated at 08:00 h on d 16. Urine in the ISS treatment. The challenge consisted of four was collected once daily into jugs containing 10 mL of repeated low-dose injections of Escherichia coli LPS 6 mol/L HCl. Urine was weighed, thoroughly mixed and serotype O55:B5 (Sigma–Aldrich, St. Louis, MO, USA) subsampled (10% of urine weight), strained through glass for pigs on treatment ISS, or a control injection of sterile wool, and stored at − 20 °C. Urine subsamples were saline for pigs in treatments CON and ENZ. The LPS pooled per pig throughout the collection period. was dissolved in sterile PBS so that an injection of 0.1 mL/kg of BW achieved the desired dosage . Heat production A pilot study with 12 pigs was conducted prior to ex- On d 16, within 30 min of consuming their daily feed al- periment initiation to discern the lowest appropriate ini- lotment, pigs were transferred to open-circuit indirect tial LPS dose and the subsequent dose increase regimen calorimetry chambers (1.22 m × 0.61 m × 0.91 m metallic required to limit LPS tolerance development. Results of box with a glass door on the front side, plastic-covered the pilot study (not reported herein) indicated that an expanded metal sheet flooring, and a valve at the bottom initial dose of 20 μg LPS/kg of BW with subsequent dose to collect urine; Columbus Instruments, Columbus, OH, increases of 20%, 30%, and 40% was the regimen that USA) for 34 h of calorimetric measurements. Pigs were Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 4 of 16 Table 2 Experimental diet ingredient and analyzed nutrient to 170 values over 34 h. The first 24 h following feeding composition (as-fed basis) was designated as the fed state and the last 12 h as the Item Control diet Enzyme diet fasting state [14, 15]. Urine voided during the fed and fasting periods was collected separately and processed as Ingredient, % of diet previously described. Personnel movement within the Corn 47.33 47.27 room was minimized during HP measurement to avoid Soybean meal, (dehulled, 38.40 38.40 any disturbance of the pigs. The system was validated solvent extracted) using the alcohol combustion method described by Soybean hulls 10.00 10.00 Aulick et al.  and the O and CO sensors were 2 2 Soybean oil 1.85 1.85 calibrated prior to each block of the experiment. The Limestone 1.04 1.04 chambers were air-conditioned to maintain a constant Monocalcium phosphate 0.60 0.60 temperature (23 ± 1 °C). Pig BW on d 16 was similar Vitamin premix 0.33 0.33 across all treatments (14.1 ± 0.3 kg). Heat production was measured in only seven of the ten experimental Trace mineral premix 0.20 0.20 blocks because of equipment failure during three blocks; Salt 0.25 0.25 therefore, for HP data, n =7. Hemicell HT-D 0.00 0.06 Calculated composition, % of diet Analytical methods SID Lys 1.18 1.18 All diet, orts, and fecal samples were dried at 60 °C to a SID Met 0.32 0.32 constant weight and were ground to a particle size of 1 mm. Urine samples were thawed, sieved through cot- SID Thr 0.75 0.75 ton gauze, and filtered with glass wool. Urine, diet, and SID Trp 0.26 0.26 fecal samples were analyzed in duplicate for nitrogen (N; SID Cys + Met 0.62 0.62 method 990.03 ; TruMac®; LECO Corp., St. Joseph, β-mannan 1.33 1.33 MI, USA). An EDTA sample (9.56% N) was used as the Analyzed composition, % of diet standard for calibration and was determined to be (9.55 DM 86.75 87.17 ± 0.01)% N. Crude protein (CP) was calculated as N × 6.25. Diets were analyzed for mannan (Galactomannan GE, Mcal/kg 4.03 4.00 Assay Kit, Megazyme International, Wicklow, Ireland) CP 22.28 21.83 and β-mannanase concentration (colorimetric determin- EE 4.02 3.97 ation, Elanco Animal Health, Gaithersburg, MD). Starch 29.34 30.89 Diet and fecal samples were analyzed in duplicate for NDF 12.17 11.83 dry matter (DM; method 930.15), acid hydrolyzed ether ADF 6.97 6.79 extract (EE; method 2003.06), and starch (Total Starch f g Assay Kit, Megazyme International, Wicklow, Ireland, endo-1,4-β-mannanase , IU/kg Below detectable limit 150,000 a method 996.11) using standard methods ; and in Provided per kilogram of complete diet: 6,614 IU of vitamin A; 827 IU of vitamin D; 26 IU of vitamin E; 2.6 mg of vitamin K; 29.8 mg of niacin; 16.5 mg triplicate for neutral and acid detergent fiber compo- of pantothenic acid; 5.0 mg of riboflavin; 0.023 mg of vitamin B nents (NDF  and ADF , respectively). Hemicellu- Provided per kilogram of complete diet: Zn, 165 mg as ZnSO ; Fe, 165 mg as lose was calculated as the difference between NDF and FeSO ; Mn, 39 mg as MnSO ; Cu, 17 mg as CuSO ; I, 0.3 mg as Ca (IO ) ; and 4 4 4 3 2 Se, 0.3 mg as Na SeO 2 3 ADF concentrations. Gross energy (GE) was determined Hemicell™ HT-D, Elanco Animal Health, Guelph, ON, Canada; endo-1,4-β-mannanase 6 using a bomb calorimeter (model 6200; Parr Instrument (160 × 10 units/kg) from Paenibacillus alvei Acid hydrolyzed ether extract Co., Moline, IL). Benzoic acid (6,318 kcal GE/kg; Parr β-mannan concentration was calculated using values reported in Shastak Instrument Co.) was used as the standard for calibration et al.  and was determined to contain 6,325 ± 6.9 kcal GE/kg. endo-1,4-β-mannanase activity. 1 IU = the amount of enzyme which generates 0.72 micrograms of reducing sugars per minute from a mannose-containing Urine GE was calculated as 192 plus 31 times the con- substrate at pH 7.0 and temperature of 40 °C centration of urinary N  and multiplied by a factor The lowest detectable limit was 15,000 IU/kg of 0.239 to convert the unit to kcal. randomly assigned to chambers to reduce the possibility Pre- and post-challenge whole blood samples and of a chamber bias. The first 2 h of HP (08:00–10:00 h), blood smears were analyzed for CBC performed by measured prior to pigs receiving the fourth and final Manitoba Veterinary Diagnostic Services (Winnipeg, challenge of either LPS or saline, were designated as ac- MB, Canada) using Advia 2120i (Siemens Healthcare climation and not included in HP calculations. Total Diagnostics, Tarrytown, NY, USA) with a manual differ- oxygen consumption (VO ) and carbon dioxide produc- ential. The main response variables of interest were total tion (VCO ) were measured every 12 min corresponding cell, RBC, and WBC (mature and immature neutrophils, 2 Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 5 of 16 eosinophils, basophils, lymphocytes, and monocytes) metabolic rate and not associated with feed consump- concentrations. tion, digestion, or physical activity [27, 28]. The respira- Serum was divided into three subsamples. One set was tory quotient (RQ) was calculated as VCO divided by analyzed for glucose and insulin concentrations by Animal VO during the fed (RQ ) and fasting (RQ ) states. 2 fed fast Health Laboratory (University of Guelph, ON, Canada). To best estimate components of HP not attributed to Glucose concentration was determined on an automated the basal metabolic rate, HP values for physical activity Roche Cobas C501 analyzer (GLUC3 application, Roche and the thermic effect of feeding (TEF) were calculated. Diagnostics, Indianapolis, IN, USA) and insulin concen- Activity heat production (AHP) was estimated utilizing tration was quantified using commercial RIA kits (PI-12 K, fed-state HP data measured over 10 h post-challenge to EMD Millipore, Billerica, MA, USA). The second serum represent normal post-feeding daytime behavior. The subset was analyzed for cytokine concentrations (granulo- difference between the average HP over this 10 h period cyte macrophage colony-stimulating factor (GM-CSF), (HP ) and the average of the 10 lowest HP values over tumor necrosis factor alpha (TNFα), Interleukin (IL)-o- the same time (HP ; representative of sedentary, rest- low ne-alpha (IL-1α), IL-1β, IL-one-receptor antagonist ing behavior; [27, 28]) was designated as AHP. The TEF (IL-1ra), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, and IL-18) by was calculated as the difference between HP and the a commercial multiplex assay using laser bead tech- sum of AHP and FHP, so that any HP in excess of basal nology (Eve Technologies, Calgary, AB, Canada). The metabolism and activity was partitioned toward TEF. third serum set was analyzed for concentration of APPs Heat increment (HI) was then calculated as the sum of haptoglobin and mannose-binding lectin A (MBL) using AHP and TEF. Using these data, the efficiency of utiliz- porcine-specific commercial ELISA kits (Immunology ing ME for maintenance and growth (k , %) was calcu- mg Consultants Laboratory, Inc., Portland, OR, USA; MyBio- lated as (1 – HI / ME intake) × 100 [25, 29]. To address Source, Inc., San Diego, CA, USA, respectively). the primary research question of how an immune chal- lenge and β-mannanase supplementation impact main- Calculations tenance energy requirements, metabolizable energy used Dry matter, GE, CP, EE, starch, hemicellulose, NDF, and for maintenance (ME ) was then calculated as FHP × ADF apparent total tract digestibility (ATTD; %) were 100/ k [25, 29]. mg calculated on a DM basis as [(nutrient intake – nutrient Together, dietary energy, N balance, and HP values output in feces)/nutrient intake] × 100. Digestible energy were utilized to characterize energy use and balance in (DE) content of the diet was calculated as GE × GE the pig. Retained energy (RE) was calculated by the dif- ATTD. Dietary ME was calculated according to the ference of ME intake and the total HP during the 24 h equation of Noblet et al. : ME = DE – [urine GE + fed-state (both pre- and post-challenge) to account for (0.4% of DE intake)]. Nitrogen retention (NR) was calcu- all energy not available for tissue accretion . Energy lated by the difference between N intake and N excreted retained as protein (RE ) was calculated from N balance in the feces and urine, and protein deposition (PD) was assuming a PD (g) energy value of 5.64 kcal/g . En- determined as NR × 6.25. ergy retained as lipid (RE ) was calculated as the differ- Heat production was calculated from respiratory gas ence between RE and RE . Lipid deposition (LD) exchanges and urinary N production according to the was then determined from RE assuming an energy con- equation of Brouwer : HP (kcal) = 3.87 × VO con- tent of 9.49 kcal/g of deposited lipid . Dietary net en- sumed (L) + 1.20 × VCO produced (L) – 1.43 × urinary ergy (NE; kcal/kg) was calculated as the sum of RE and N production (g). Methane production was not FHP divided by DMI . accounted for, but has been estimated to be very low in growing pigs (< 1% ). All HP parameters were nor- Statistical analyses malized to a period of 24 h, expressed as kcal of heat Data were analyzed as a randomized complete block de- 0.60 produced per kg of BW  and per kg of DM intake sign with pig as the experimental unit. The UNIVARI- (DMI) in order to remove known effects of variations in ATE procedure of SAS (Version 9.4, SAS Inst., Cary, BW and DMI [25, 26]. NC) was used to verify normality and homogeneity of Total heat production (HP ) was the average HP variances. Statistical outliers (> 3 SD away from the total during the 22 h of post-challenge, fed state measure- mean) were removed; therefore, one pig from the ISS ment. Total fasting heat production (FHP ) was the treatment was removed from HP data because of poor total average HP over the 12 h fasted-state. Because the sys- feed intake on the day of HP measurement. Immature tem was not equipped to quantify and separate HP due neutrophil, eosinophil, and basophil CBC data were log to physical activity, fasting heat production (FHP) was transformed to achieve a normal distribution. derived from the 10 lowest HP values over the The main effects of dietary treatment and block were fasted-state, reflecting energy metabolism due to basal analyzed using the MIXED procedure of SAS. The Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 6 of 16 staggered block experimental design resulted in varia- and ENZ treatments maintained normal rectal tempera- tions in time and body weight among blocks. This vari- tures (38.85 °C ± 0.15) throughout the experiment. ation was expected and resulted in statistical detection There was a significant interaction between the effects of block as a significant main effect in most response of time (pre- or post-challenge) and treatment on WBC, variables. Therefore, block remained in the statistical mature neutrophil, lymphocyte, and monocyte counts and model, but block P-values are not reported herein. a trend for an interaction on RBC count (Additional file 2: Differences among treatments were determined using Table S2). In all four variables, treatments had similar cell ANOVA and means were separated using the least square counts at the pre-challenge time point (P ≥ 0.10). means statement and the PDIFF option. Immune and rectal Post-challenge, immune stimulation by LPS decreased temperature data were analyzed as repeated measures WBC, mature neutrophil, lymphocyte, and monocyte and covariance structures resulting in the lowest AIC counts compared to CON and ENZ (P ≤ 0.05; Fig. 2). values for each variable were applied. To further Therewerenodifferences among treatments ortimepe- evaluate β-mannanase effects on immune parameters riods for total cell, immature neutrophil, eosinophil, or pre-challenge, contrasts comparing CON versus ENZ basophil counts (P > 0.10; Additional file 2: Table S2). and ISS values were generated using the contrast Glucose, insulin, haptoglobin, and MBL serum con- statement of the MIXED procedure. Differences were centrations were not affected by the interaction of time considered significant if P was ≤0.05 and a trend if P and treatment (P > 0.10), but concentrations were higher was > 0.05 and ≤ 0.10. pre-challenge compared to post-challenge for glucose, haptoglobin, and MBL (Additional file 3: Table S3). Results Lipopolysaccharide challenge increased IL-1β, IL-1ra, Immune response parameters IL-6, IL-8, and TNFα concentrations post-challenge Immune system stimulation effects compared to ISS pre-challenge and both pre- and Pigs on the ISS treatment exhibited minimal vomiting, post-challenge concentrations in CON and ISS (Fig. 3). diarrhea and signs of lethargy and hyperventilation after All other cytokines were not significantly impacted by the first and to a lesser extent, the second LPS injection. the interaction or main effects of time and treatment After the third and fourth challenges, ISS pigs continued (P > 0.10; Additional file 3: Table S3). Interferon-gamma to demonstrate signs of lethargy and hyperventilation, but was not detected in any of the samples. Serum GM-CSF vomiting and diarrhea were not observed. No pigs died concentrations were not different (P > 0.10) among CON after any injection. The immune stimulation model suc- and ENZ pre-and post-challenge and ISS pre-challenge, cessfully induced a sustained febrile response (rectal while ISS post-challenge GM-CSF concentration was in- temperature ≥ 40 °C) in ISS pigs on d 10, 12, and 14 (treat- creased compared to the ISS pre-challenge value and ment by day interaction P < 0.0001; Fig. 1). Pigs in CON CON and ENZ post-challenge values (P ≤ 0.015; Fig. 3). Fig. 1 Effect of treatment on young weaned pig (n = 10 per treatment) rectal temperature (°C). Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with saline injection. Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with saline injection. Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% β-mannanase) with LPS (Escherichia coli serotype O55:B5) injection. The arrows indicate days on which either a saline (CON and ENZ) or LPS (ISS) injection were administered at 10:00 h. Rectal temperatures were measured 4 h post-challenge. Data points on d 5 and 8 represent average baseline pre-challenge temperature, and d 10, 12, and 14 represent post-challenge temperatures. Treatment by day interaction P < 0.0001; day P < 0.0001; treatment P < 0.0001; block P = 0.0015 Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 7 of 16 Fig. 2 Effect of treatment on complete blood count before (d 8) and after (d 10) challenge. Serum was collected at 14:00 h each day (4 h post- challenge). Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with saline injection. Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with saline injection. Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% β-mannanase) with a,b LPS (Escherichia coli serotype O55:B5) injection. n = 10 per treatment. Within a graph, bars without a common superscript differ, P < 0.05 β-mannanase effects Immune system simulation numerically decreased ADFI Contrasts comparing immune cell dynamics of pigs fed ei- and thus N intake compared to CON and ENZ (P = ther the control or β-mannanase diet prior to the first 0.021), resulting in decreased fecal N excretion on a g per challenge on d 10 detected no differences in CBC values d basis (P = 0.007). Urine N excretion during the challenge (P ≥ 0.10; Table 3). Similarly, serum glucose, insulin, MBL, period was similar among treatments (P = 0.045), but and cytokine concentrations (except IL-1α) did not differ retained N in the ISS treatment was less than that of because of β-mannanase supplementation (P ≥ 0.230; CON, with ENZ being intermediate (P = 0.045; Table 5). Table 4). Serum haptoglobin and IL-1α concentrations Partitioning of excreted N to either the feces or urine was were decreased in diets supplemented with β-mannanase not different among treatment (P = 0.78). When N excre- (P ≤ 0.05; Table 4). tion was expressed as a percent of N intake, the previously observed significant treatment effect on fecal N excretion was no longer evident (Table 5). Therewerenodifferences Pig growth performance, nitrogen balance, and diet among treatments in ATTD of any analyzed nutrient (P ≥ digestibility 0.120; Table 6) and all ATTD coefficients were within nor- Average initial BW was 10.27 ± 0.15 kg, d 16 average mal ranges for 10 to 15 kg pigs. BW was 15.12 ± 0.27 kg, and BW did not differ among treatments at either time point (P ≥ 0.471). Average daily Heat production, maintenance energy requirements, and gain (ADG) over the entire 16-d experiment was not dif- energy retention ferent among treatments (P = 0.13; Table 5), but ISS Day 16 ME intake was similar among treatments 0.60 ADG during the immune challenge (d 10–16) was less (759.4 ± 37.7 kcal/kg BW /kg DMI/d; P =0.92). Im- than CON and ENZ gain (P = 0.010; Table 7). mune system stimulation increased fed state HP total Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 8 of 16 Fig. 3 Effect of treatment on serum cytokine concentrations before (d 8) and after (d 10) challenge. Serum was collected at 14:00 h each day (4 h post- challenge). Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with saline injection. Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with saline injection. Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% β-mannanase) with LPS a,b (Escherichia coli serotype O55:B5) injection. n = 10 per treatment. Within a graph, bars without a common superscript differ, P <0.05 compared to CON and ENZ (P = 0.040; Table 7). In Immune system stimulation increased ME (kcal/kg 0.60 the fasting state, neither immune stimulation nor BW /kg DMI/d) compared to CON or ENZ pigs β-mannanase supplementation affected FHP or (P =0.045), but k among treatments did not differ total mg FHP compared to control (P ≥ 0.135). Treatment did (P =0.13; Table 7). When ME was expressed as not affect RQ in the fed and fasting states (P ≥ 0.23; kcal/d, the significant treatment effect was no longer Table 7). detected (P = 0.90). Beta-mannanase supplementation Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 9 of 16 Table 3 Complete blood count values in young weaned pigs Table 4 Effect of β-mannanase on pig serum glucose, insulin, a,b a,b fed a diet with or without β-mannanase acute phase protein, and cytokine concentrations c d c d Treatment Control diet β-mannanase diet Contrast Treatment Control diet Enzyme diet Contrast P-value P-value Estimate SEM Estimate SEM Estimate SEM Estimate SEM 9 e Cell type count, cells × 10 /L Glucose, mmol/L 7.70 0.46 7.41 0.25 0.585 Total cells 468.6 34.9 432.4 23.8 0.405 Insulin, pmol/L 88.92 10.99 83.35 6.06 0.664 WBC 24.50 1.43 21.51 0.98 0.104 Insulin:Glucose 11.56 1.21 11.08 0.67 0.734 Neut 6.47 0.94 6.83 0.64 0.758 Acute phase protein, mg/mL Bands 0.200 0.050 0.266 0.034 0.504 Haptoglobin 1.57 0.22 1.02 0.14 0.050 Eos 0.202 0.099 0.300 0.068 0.599 MBL 125.4 8.6 117.5 5.2 0.445 Baso 0.048 0.061 0.100 0.042 0.451 Cytokine, pg/mL Lymph 15.35 1.46 12.46 1.00 0.124 GM-CSF 17.01 13.01 14.56 7.85 0.874 Mono 0.727 0.181 0.709 0.123 0.934 IL-1α 35.01 6.81 7.50 4.27 0.004 RBC 7.44 0.14 7.55 0.09 0.547 IL-1β 1064 598 1138 361 0.917 Blood was collected on d 8 of the experiment 6 h post-feeding, prior to the IL-1ra 428.6 257.5 673.7 161.5 0.435 immune challenge beginning on d 10 IL-2 328.4 157.6 228.8 95.0 0.596 n = 10 pigs per treatment Control diet was a corn, soybean mean, and soy hulls based diet containing IL-4 995.5 545.6 711.6 329.0 0.662 1.33% β-mannans, and did not contain β-mannanase enzyme. Pigs on the control (CON) treatment were fed the control diet and estimates are IL-6 190.2 61.5 98.72 38.59 0.230 representative of the CON treatment only d IL-8 263.1 95.4 402.2 57.5 0.230 Enzyme diet was the control diet supplemented with 0.056% β-mannanase (Hemicell™ HT-D, Elanco Animal Health, Guelph, ON, Canada; endo-1,4-β- IL-10 499.4 159.1 332.9 95.9 0.383 mannanase (160 × 10 units/kg) from Paenibacillus alvei). Pigs on the enzyme (ENZ) and immune system stimulation (ISS) treatments were fed IL-12 1570 173 1730 104 0.439 the enzyme diet. Estimates are representative of the ENZ and ISS treatments prior IL-18 1994 546 1380 329 0.350 to immune stimulation Basophils (Baso); eosinophils (Eos); immature neutrophils (Bands); TNFα 24.29 39.90 52.30 25.02 0.563 lymphocytes (Lymph); mature neutrophils (Neut); monocytes (Mono); Blood was collected on d 8 of the experiment 6 h post-feeding, prior to the white blood cells (WBC) immune challenge beginning on d 10 n = 10 pigs per treatment Control diet was a corn, soybean mean, and soy hulls based diet containing did not change ME relative to CON whether m 1.33% β-mannans, and did not contain β-mannanase enzyme. Pigs on the 0.60 control (CON) treatment were fed the control diet and estimates are expressed as kcal/kg BW /kg DMI/d (P =0.98), representative of the CON treatment only kcal/kg BW/d (P =0.72), or kcal/d (P =0.77). d Enzyme diet was the control diet supplemented with 0.056% β-mannanase (Hemicell™ HT-D, Elanco Animal Health, Guelph, ON, Canada; endo-1,4-β- Absorbed energy not lost via urine, gases, heat in- mannanase (160 × 10 units/kg) from Paenibacillus alvei). Pigs on the enzyme crement, activity and TEF, or maintenance, is (ENZ) and immune system stimulation (ISS) treatments were fed the enzyme retained as either protein or lipid. Immune system diet. Estimates are representative of the ENZ and ISS treatments prior to immune stimulation stimulation decreased RE compared to CON and Mannose binding lectin A (MBL) ENZ (P = 0.046) but RE and total RE were not dif- Granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-1α (IL-1α); interleukin-1β (IL-1β); interleukin-1 receptor antagonist (IL-1ra); ferent among treatments (P > 0.32) when expressed as 0.60 interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-6 (IL-6); interleukin-8 (IL-8); kcal/kg BW /kg DMI/d (Table 7). When RE was interleukin-10 (IL-10); interleukin-12 (IL-12); interleukin-18 (IL-18); tumor expressed as a proportion of ME intake, similar treat- necrosis factor alpha (TNFα) ment effects were observed for RE and RE ,buta l p significant decrease in total RE was detected due to Discussion ISS (P = 0.033; Table 7). As less energy was retained During an immune challenge, pro-inflammatory cyto- as lipid, LD was decreased in the ISS treatment com- kines initiate a shift in nutrient partitioning away from paredto CON andENZ (P = 0.047) while no differ- tissue growth to support activation and maintenance of ences were observed in PD (P = 0.15; Table 7). an immune response [1, 11, 31]. The results of this ex- periment clearly demonstrated that a systemic inflam- matory response to LPS occurred, verified by increased Dietary energy values and efficiency concentrations of pro-inflammatory cytokines and ele- The ENZ and ISS treatments tended to decrease diet DE vated body temperature. To our knowledge, these data and ME values relative to CON (P ≤ 0.052; Table 8). Nei- are the first to directly relate decreased ADG to in- ther ISS nor β-mannanase supplementation (ENZ treat- creased ME independent of changes in feed intake dur- ment) affected dietary NE value (P = 0.75) or ME and ing an immune response. Additionally, this experiment NE efficiency (P ≥ 0.46). provides novel data on β-mannanase supplementation Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 10 of 16 Table 5 Growth performance and nitrogen balance in pigs on Table 7 Effect of treatment on energy balance, respiratory control, enzyme, or immune system stimulation treatment quotient, maintenance energy requirements, and nutrient d e f deposition Item CON ENZ ISS SEM Treatment P-value Treatment Treatment d e f P-value Body Weight, kg Item CON ENZ ISS SEM d 0 10.23 10.21 10.38 0.15 0.651 Day 16 BW, kg 14.37 14.19 13.77 0.26 0.313 d 16 15.25 15.26 14.86 0.27 0.471 Day 16 DMI, kg 0.51 0.51 0.46 0.03 0.348 0.60 ADG d 1–16, g/d 313.9 316.0 279.7 13.6 0.128 Energy balance, kcal/kg BW /kg DMI/d Nitrogen (N) balance, g/d ME intake 771.3 755.9 751.1 37.7 0.924 a a b Intake 21.02 20.93 17.38 0.95 0.021 Heat production b b a Excreted 6.21 7.08 6.24 0.45 0.318 HP 278.8 274.9 333.0 14.9 0.040 total a a b In feces 2.62 2.83 2.27 0.11 0.007 FHP 287.8 276.0 324.3 17.1 0.178 total In urine 3.59 4.25 3.97 0.47 0.624 FHP 207.8 206.6 243.3 12.9 0.135 a ab b Retained 14.81 13.85 11.14 0.99 0.045 Retained energy % of excreted N in feces 41.31 41.21 38.42 3.29 0.778 As protein 197.5 173.6 191.0 18.3 0.627 a a b % of excreted N in urine 58.69 58.79 61.58 3.29 0.778 As lipid 291.4 302.9 227.7 19.2 0.046 Nitrogen balance, % of intake Total 488.9 476.5 418.7 32.0 0.318 Excreted in feces 12.41 13.54 13.23 0.44 0.212 k , % 87.07 86.44 83.01 1.34 0.130 mg b b a Excreted in urine 17.38 21.40 24.56 4.14 0.495 Estimated ME 239.0 239.5 295.5 15.3 0.045 a,b Within a row, treatment means without a common superscript differ, P < 0.05 Retained energy, % of ME intake n = 10 pigs per treatment As protein 25.68 23.15 24.99 1.27 0.354 Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with saline injection a a b As lipid 37.77 40.07 29.81 2.02 0.013 Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with a a b saline injection Total 63.44 63.22 54.80 2.18 0.033 Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% Respiratory quotient β-mannanase) with LPS (Escherichia coli serotype O55:B5) injection Fed state 0.92 0.90 0.88 0.01 0.225 Fasting state 0.74 0.73 0.73 0.01 0.381 Nutrient deposition , g/d As protein 87.74 78.55 69.80 5.86 0.150 Table 6 Apparent total tract digestibility in pigs on the control, a a b As lipid 76.22 79.43 55.45 6.21 0.047 enzyme, or immune system stimulation treatment a a b ADG d 10–16, g/d 447.1 404.8 330.7 21.3 0.010 b c d Item CON ENZ ISS SEM Treatment a,b Within a row, treatment means without a common superscript differ, P < 0.05 P-value n = 7 pigs per treatment (CON and ENZ) and 6 pigs per treatment (ISS) ATTD ,% Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with saline injection DM 88.05 87.36 87.66 0.33 0.356 Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with saline injection GE 87.35 86.73 86.86 0.33 0.418 Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% CP 87.59 86.47 86.77 0.44 0.212 β-mannanase) with LPS (Escherichia coli serotype O55:B5) injection f Heat production (HP) = (3.87 × O consumption (L) + 1.20 × CO production (L) 2 2 EE 70.41 69.62 66.85 1.18 0.122 0.60 – 1.43 × urinary N)/BW (kg) ; Total HP (HP ) = avg. HP over 22 h fed total state, post- challenge; Total fasting HP (FHP ) = avg. HP over 12 h fasted Starch 99.41 99.50 99.56 0.10 0.565 total state; Fasting HP (FHP) = avg. of 10 lowest HP values over the 12 h fasted state NDF 68.81 66.11 70.23 1.55 0.178 [27, 28]; HP = avg. HP over 10 h post-challenge (10:00 h – 20:00 h), fed state; HP = avg. of 10 lowest HP values over 10 h post-challenge (10:00 h – low ADF 71.65 68.04 74.13 2.26 0.175 20:00 h), fed state; Activity HP (AHP) = HP -HP ; Thermic effect of feeding 10 low Hemicellulose 65.00 63.51 64.99 0.92 0.429 (TEF) = HP - AHP – FHP; Heat increment (HI) = AHP + TEF; ME efficiency for maintenance and growth (k )=(1 – HI) × 100 ; ME used for maintenance mg n = 10 pigs per treatment (ME ) = FHP × 100/k  b m mg Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with Retained energy (RE) = ME intake – total fed-state HP, pre-and post-challenge saline injection 0.60 ; RE as protein (RE ) = [PD (g) × 5.66 (kcal/g)]/ BW /DMI ; RE as lipid c p Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with (RE ) = RE - RE  l p saline injection Protein deposition = nitrogen retention (g) × 6.25; Lipid deposition = RE (kcal) d f Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% / 9.49 (kcal/g)  β-mannanase) with LPS (Escherichia coli serotype O55:B5) injection ATTD, % = [(nutrient intake (kg) – fecal nutrient output (kg)) / nutrient intake (kg)] × 100 Acid hydrolyzed ether extract Hemicellulose = NDF - ADF Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 11 of 16 Table 8 Dietary energy values and efficiency in pigs on control, receptor and secreted MBL. Therefore, serum MBL con- enzyme, or immune system stimulation treatment centrations were measured to determine if β-mannanase Treatment Treatment supplementation decreased circulating MBL, theoretic- b c d P-value ally by removing the substrate for activation and synthe- Item CON ENZ ISS SEM sis. Serum MBL concentrations were not affected by Dietary energy value , Mcal/kg DM β-mannanase. This may indicate that the β-mannan con- GE 4.65 4.59 4.59 centration in the intestinal lumen was not high enough DE 4.07 3.99 4.00 0.02 0.051 to either interact with MBL, MBL-dietary β-mannan ME 3.96 3.86 3.89 0.03 0.052 interaction was not affected by β-mannanase supple- NE 3.29 3.30 3.11 0.19 0.748 mentation, or this interaction is not a mechanism ME/DE efficiency, % 97.31 96.92 97.35 0.37 0.457 through which β-mannans are sensed by the innate im- mune system. NE/ME efficiency, % 83.09 85.31 80.03 4.42 0.701 a Two significant differences in serum parameters n = 7 pigs per treatment (CON and ENZ) and 6 pigs per treatment (ISS) Control treatment (CON) = pigs fed basal diet (0.0% β-mannanase) with were detected when contrasts were applied to com- saline injection pare pre-challenge values between control pigs (no Enzyme treatment (ENZ) = pigs fed enzyme diet (0.056% β-mannanase) with β-mannanase, CON treatment) and β-mannanase saline injection Immune system stimulation treatment (ISS) = pigs fed enzyme diet (0.056% supplemented pigs (ENZ and ISS treatments). β-mannanase) with LPS (Escherichia coli serotype O55:B5) injection e Beta-mannanase supplementation decreased serum Gross energy (GE) analyzed via bomb calorimetry; digestible energy (DE) = GE apparent total tract digestibility coefficient × diet GE; metabolizable energy haptoglobin and IL-1α concentrations. In poultry, (ME) = DE – (urinary energy + 0.4% of DE intake); net energy (NE) = (retained decreased haptoglobin has been proposed as evidence of energy + fasting heat production)/DMI immune stress alleviation due to β-mannanase supplemen- effects on immune parameters and energy balance in tation . However, this response occurred in conjunction pigs. with growth performance and feed efficiency improvements which were not observed in this study. Beta-mannanase β-mannanase effects on IL-1α concentrations have not been previously As a constituent of hemicellulose, β-mannan is not reported. Interleukin-1-alpha can be involved in inflamma- digested by mammalian endogenous enzymes . Thus, tion initiation, but the relationship between serum concen- intact β-mannans are available to bind carbohydrate rec- tration and magnitude of immune challenge is not as clear ognition domains of pattern recognition receptors on in- as the implication of its counterpart, IL-1β on systemic in- nate immune cells surveying the intestinal epithelium flammation . Interleukin-1-beta concentrations were for potential pathogens [3, 33]. In this way, β-mannans not affected by β-mannanase supplementation in this study. are hypothesized to be capable of stimulating innate im- In total, decreased serum IL-1α and haptoglobin con- mune cells resulting in a nonproductive, energy draining centrations are not strong enough evidence of an allevi- immune response [4–6]. ated systemic FIIR when taken in context with the lack Commonly, only growth and feed efficiency responses of all other measured inflammatory-type variables. Im- have been measured from β-mannanase and reported in portantly, no differences were observed in HP, ME , and the animal nutrition literature. Reduced feed efficiency growth performance. It is possible that a localized re- and ADG have been reported with increasing dietary sponse may have occurred at the intestinal level yet went β-mannan concentrations . Therefore, there is interest undetected systemically. However, if this occurred, whole in β-mannanase supplementation to alleviate these nega- body nutrient and energy partitioning were still un- tive effects by enzymatic hydrolysis of β-mannan polysac- affected. The hypothesis that β-mannanase supplementa- charides. The FIIR was alleviated through β-mannanase tion would decrease ME was not supported. Pigs fed supplementation in poultry [6, 35]; however, β-mannanase diets supplemented with β-mannanase had similar WBC supplementation responses in swine have been inconsist- counts, cytokine concentrations, nutrient digestibility, ent. This experiment demonstrated no β-mannanase effect ADG, N and energy balance, PD, LD, and ME com- on the ATTD of DM, GE, CP, EE, or hemicellulose. pared to CON pigs. Growth performance responses are similarly inconsistent with positive results in some studies [36–38] but no Immune stimulation β-mannanase effect in others [39–42]. In this experiment, Innate immune stimulation was successfully induced in ENZ did not improve ADG, protein, or lipid deposition. pigs using sequential, increasing doses of E. coli LPS. El- Dietary β-mannans are proposed to stimulate the in- evated rectal temperature, increased pro-inflammatory nate immune system through direct interactions with cytokine concentrations, and altered nutrient and energy the carbohydrate binding domains of mannose recogni- partitioning are all hallmarks of a chronic immune chal- tion receptors such as the membrane bound mannose lenge  and were observed in ISS pigs in this study. Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 12 of 16 One limitation of this study was the number of calorim- caloric requirements. This value is higher than the previ- etry chambers available which limited the experiment to ously described range and may indicate that the major- a total of three treatments. Due to this limitation, we ity, but not all of the increase in maintenance caloric were unable to evaluate the interaction of β-mannanase requirement is to support the febrile response. The re- supplementation with LPS immune stimulation. Thus, mainder may be partially explained by an increase in im- interpretation of ISS effects has been made in compari- mune cell glucose requirements . son to the ENZ treatment. However, as discussed above, there were no differences between the CON and ENZ Cytokines treatments in nutrient digestibility, ADG, or N and en- Key pro-inflammatory cytokines include TNFα, IL-6, ergy balance. The major finding of this research indi- and IL-1β  and ISS pigs had increased serum con- cates that the innate immune challenge increased young centrations of all three after the first LPS challenge. pig maintenance energy requirements by 23.3% which Pro-inflammatory cytokines shift metabolism away from translated into a 18.3% decrease in ADG. anabolic processes toward a more catabolic state to gen- Unique to this study, decreased ADG could be attrib- erate AAs and energy necessary to support fever, in- uted primarily toward increased ME in ISS pigs as op- crease immune cell proliferation, and APP synthesis [50, posed to decreased feed intake or effects on nutrient 51]. In this study, the pro-inflammatory cytokine profile digestibility. Anorexia is a well-established response to of ISS pigs clearly shifted metabolism toward a lipolytic systemic immune stimulation [2, 9, 44] induced by state and this resulted in significantly less energy pro-inflammatory cytokine actions (especially IL-1β)in retained as lipid and decreased lipid deposition com- the brain and modulation of metabolism and hormone pared to non-immunologically challenged pigs. release . In this study, a numerical but not statisti- cally significant decrease in ADFI was observed in ISS Complete blood count pigs during the challenge period even though IL-1β in- Immune stimulation decreased WBC counts, specifically creased. It is likely that a stronger ADFI decrease was neutrophils, lymphocytes, and monocytes. This is similar not observed as a consequence of challenging the pigs to other instances of leukopenia observed due to LPS ad- 2 h post-feeding and limit feeding to 2.5 times mainten- ministration [11, 52]. However, WBC distribution drastic- ance energy requirements . This feeding level was ally changed following LPS administration and circulating designed to achieve similar ADFI for pigs on all treat- concentrations are dependent upon the time of sampling ments because of the known effect of previous feeding relative to immune challenge [52, 53]. Thus, variable re- level on HP . To further ensure HP results were sep- sponses in WBC counts have been reported due to LPS arated from feed intake and BW effects, all energy bal- immune stimulation. Rakhshandeh and de Lang observed 0.60 ance calculations were conducted on a kcal/BW / 1.6 times greater WBC  in one study, but in a second, DMI/d basis. Just as feed intake did not influence the WBC count decreased by 9% . At the time of sampling observed decrease in ADG of ISS pigs, nutrient digest- in this study, leukocyte extravasation into the LPS injec- ibility was not different across treatments. This is in tion site and into immunologically important tissues likely agreement with other studies reporting ATTD during a explains the observed leukopenia. chronic LPS challenge [46, 47]. Acute phase proteins Febrile response In addition to increased pro-inflammatory cytokine pro- Before the challenge period, rectal temperatures and duction and leukocyte migration that occur during infec- blood immune parameters in ISS pigs were not different tion, the acute phase response typically includes from those on the CON and ENZ treatments. This con- increased APP synthesis by the liver. However, in this firmed that prior to the challenge all pigs were in good study, ISS APP concentrations did not differ compared health and of similar immune status. Therefore, any sub- to CON. This was an unexpected result because LPS has sequent differences during the challenge were attributed been demonstrated to increase APPs such as haptoglo- to LPS immune stimulation. Elevated rectal tempera- bin [8, 46] and C-reactive protein  in pigs. A less re- tures (> 40 °C) post-challenge on d 10, 12, and 14 indi- sponsive APP, MBL has been demonstrated to attenuate cated a febrile response in ISS pigs. LPS-induced pro-inflammatory cytokine production  Fever is energetically expensive with increased caloric and inhibit T-lymphocyte activation . However, in requirement estimates ranging from 7 to 15% for each this study it did not appear that LPS induced greater 1 °C increase in body temperature . Utilizing the MBL or haptoglobin production. average rectal temperature of CON and ENZ pigs and Although a MBL response was not necessarily ex- the post-challenge temperature of ISS pigs on day 14, an pected, a haptoglobin response was. Haptoglobin is a increase of 1.2 °C resulted in a 23.6% increase in ME primary APP in pigs and is synthesized in the liver when m Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 13 of 16 activated by IL-6 and to a lesser extent IL-1 , However, it is clear that energy partitioning between both of which were significantly elevated in ISS pigs maintenance and growth was affected by ISS. A 23.3% post-challenge. Similar to our results, Koopmans et al. increase in ME was detected due to ISS. As caloric re-  discussed unpublished data which showed no quirements for maintenance increased to support the LPS effect on haptoglobin concentrations even though immune system, less dietary energy was retained for there were clear increases in plasma cortisol, TNFα, growth. This manifested as less RE resulting in a 30.2% IL-6, and C-reactive protein over a 24-h period after decrease in lipid deposition. LPS challenge. One possible explanation for a lack of Previous studies across all species have related in- haptoglobin response could be time related. Serum creased caloric requirements with fever [48, 63], but few samples in this study were collected 4 h after the first have directly related a chronic immune challenge with challenge and haptoglobin may be a better indicator increased ME . In vitro studies with isolated mitochon- of chronic inflammation . dria from rats stimulated with TNFα or IL-1 showed up to 30% increases in respiration rate . Demas et al.  reported that mice injected with a mild antigen had Nitrogen balance limited immune activation that resulted in significantly Disease is associated with decreased growth performance more O consumption than control mice injected with and changes in nutrient partitioning. Often, N metabol- saline. Interleukin-six infusions in humans increased ism is affected because of increasing AA requirements resting metabolic rates by 25% . for immune cell proliferation and APP synthesis . In In pigs, the direct relationship between immune this study, only numerical decreases in protein depos- stimulation and increased energy requirements has not ition were measured in ISS pigs compared to CON and previously been demonstrated. Some studies reported ENZ. If protein catabolism had increased to provide that immune system stimulation did not impact growth, AAs for APPs, an increase in urinary N would have been efficiency, or energy balance measurements [66, 67] expected because APPs have a distinctly different AA However, Moon et al.  reported fibroblast formation profile than skeletal muscle [50, 60]. However, due to at the injection site which encapsulated the immunogen the high dietary CP concentration, it is possible that and prevented systemic delivery. Williams et al.  these excessive dietary amino acids may have provided used the comparative slaughter technique and reported the additional amino acids required for APP synthesis no differences in the energetic costs of maintenance, PD, and prevented the typically observed increase in skeletal and LD between pigs raised in environments encour- muscle protein catabolism. aging high or low chronic immune activation. Conversely, Labussière et al.  and Campos et al. Energy balance  reported decreased HP in pigs during inflammatory Disease is well known to be detrimental to pig efficiency challenges. Labussière et al.  administered a single and productivity. A considerable amount of research has injection of complete Freund’s adjuvant to young focused on products to mitigate the drop in performance weaned pigs but did not measured HP until the day after [13, 61] or prevent initial disease onset . Yet few challenge and only re-entered the calorimetry chamber studies have evaluated the energetic cost of an immune after visual recovery ; and this likely biased the challenge in order to generate more effective dietary in- response. Campos et al.  reported a 14% decrease in 0.60 terventions. In this study, total HP increased by 21.1% in total HP (kcal/BW /d) in response to a repeated LPS ISS pigs compared to the ENZ treatment. challenge in growing pigs even though typical Campos et al.  also evaluated HP components inflammatory-type and febrile responses were observed. during an immune response and reported significant Decreased HP was mainly attributed to lower TEF which decreases in ADFI leading to decreased TEF compared reflected the effect of feed intake depression on HP. Ac- to baseline values. In this study DMI did not differ, po- cording to the relationship reported by Labussière et al. tential feed intake effects on TEF were removed by inter- , lower ADFI should have decreased ME by 0.60 preting the data after normalizing to a constant feed 24 kcal/BW /d. Because this drop in ME did not intake, and TEF values were not affected by ISS. There- occur, the authors reasoned that the immune stimulation fore, both experiments indicate that a chronic inflamma- did in fact increase ME relative to baseline . This tory response did not increase HP through increased supports our experimental model of limit feeding to en- TEF. This is supported by the lack of treatment differ- courage similar feed intake and to evaluate energy bal- 0.60 ences in diet digestibility and further supports our sup- ance on a kcal/BW /DMI/d basis. Feed intake clearly position that the impact of immune stimulation on influences and can bias HP results and interpretations. energy balance in this study is not through influences on Interpretation of our results in context with the previ- diet digestion or nutrient uptake. ously discussed reports suggests that an inflammatory Huntley et al. Journal of Animal Science and Biotechnology (2018) 9:47 Page 14 of 16 response does increase ME relative to healthy control P-values, time P-values, and treatment P-values, as well as means comparisons animals, but in some experiments this response may be results for complete blood count response variables. (DOCX 20 kb) masked by decreased HP related to decreased feed in- Additional file 3: Table S3. Effect of treatment on serum glucose, insulin, acute phase protein, and cytokine concentrations. Table provides take. This may mean that during an immune response LS means, time by treatment P-values, time P-values, and treatment P- the total caloric requirement may not drastically change values, as well as means comparisons results for serum glucose, insulin, because of decreased feed intake, but how those calories acute phase protein, and cytokine response variables. (DOCX 26 kb) are partitioned does change; and this results in growth and feed efficiency depressions commonly observed dur- Abbreviations ing disease challenges. IL-1α: Interleukin-1-alpha; TNFα: Tumor necrosis factor-alpha; AA: Amino acid; ADF: Acid detergent fiber; ADFI: Average daily feed intake; ADG: Average These results supported our hypothesis that energy daily gain; AHP: Activity heat production; APP: Acute phase protein; partitioning shifts to allocate more energy for initiation ATTD: Apparent total tract digestibility; BW: Body weight; CBC: Complete and maintenance of immune functions and less toward blood count; CON: Control treatment; CP: Crude protein; DE: Digestible energy; DM: Dry matter; DMI: Dry matter intake; EE: Acid hydrolyzed ether nutrient deposition. Other research would support extract; ENZ: Enzyme treatment; FHP: Fasting heat production; FHP : Total total changes in N metabolism [46, 67, 69] whereas our data fasting heat production; FIIR: Feed-induced immune response; GE: Gross suggest that less energy was allocated for LD. Both result energy; GM-CSF: Granulocyte macrophage colony-stimulating factor; HI: Heat increment; HP: Heat production; HP : Heat production over 10 h post- in decreased ADG and efficiency losses in pork produc- challenge; HP : Average of 10 lowest HP values over 10 h post-challenge; low tion, yet these effects are generally given little consider- HP : Total heat production; IL: Interleukin; IL-1ra: Interleukin-1-receptor total ation in commercial swine feeding practices. antagonist; ISS: Immune system stimulation treatment; k : Energy efficiency mg for maintenance and growth; LD: Lipid deposition; LPS: Lipopolysaccharide; MBL: Mannose binding lectin; ME: Metabolizable energy; ME : Metabolizable energy used for maintenance; N: Nitrogen; NDF: Neutral detergent fiber; Conclusions NE: Net energy; NR: Nitrogen retention; PD: Protein deposition; RE: Retained This experiment provides novel data on β-mannanase energy; RE : Retained energy as lipid; RE : Retained energy as protein; l p supplementation effects on immune parameters and en- RFI: Residual feed intake; RQ: Respiratory quotient; RQ : Respiratory quotient fast during the fasting period of heat production measurements; ergy balance in pigs. Beta-mannanase supplementation RQ : Respiratory quotient during the fed period of heat production fed did not benefit immune status, nutrient digestibility, measurements; TEF: Thermic effect of feeding; VCO : Volume of carbon growth performance, energy balance, or ME in young m dioxide produced; VO : Volume of oxygen consumed; WBC: White blood cell pigs fed a corn, soybean meal, and soybean hulls-based Acknowledgments diet. More research is needed to determine how The authors would like to thank Deepak Velayundhan and Atta Agyekum for β-mannanase functions in pigs and in which environ- their technical assistance, Elanco® for financial support of this research, and ments and diets it might be effective. These novel data dir- National Pork Board for financial support of Nichole Huntley’sgraduate program. ectly relate decreased ADG to increased ME independent of changes in feed intake in immune chal- Funding lenged pigs. An innate immune challenge increased proin- Financial support of NH graduate program provided by the National Pork flammatory cytokine concentrations which induced a Board. Financial and in-kind support provided by Elanco, Greenfield, IN, USA. Neither funding agency had a role in the design, analysis, or writing of this febrile response and elevated HP and ME by 23.3%. In- article. creased energy partitioning toward the immune response limited LD by 30.2% leading to a 18.3% decrease in ADG Availability of data and materials during the immune challenge. These data expand upon All data generated or analyzed during this study are included in this published article and its additional information files. the available literature to describe the magnitude of in- crease in ME in immune challenged pigs relative to Authors’ contributions healthy control animals. Understanding the extent to NH and JP designed the study and had primary responsibility for the final which energy requirements and nutrient deposition content of the manuscript; CMN provided essential equipment and materials; NH conducted the research with the assistance of CMN graduate students; change in pigs experiencing sustained immune stress may and NH analyzed data and wrote the manuscript. All authors have read and help develop more effective feeding strategies for health approved the final manuscript. challenged herds and encourage appreciation for the eco- nomic benefits of maintaining high health populations. Ethics approval All experimental procedures adhered to guidelines for the ethical and humane use of animals for research and were reviewed and approved Additional files by the University of Manitoba Animal Care Committee. Competing interests Additional file 1: Table S1. 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Journal of Animal Science and Biotechnology – Springer Journals
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