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The effect of lactose and a prototype Lactobacillus acidophilus fermentation product on digestibility, nitrogen balance, and intestinal function of weaned pigs

The effect of lactose and a prototype Lactobacillus acidophilus fermentation product on... Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 The effect of lactose and a prototype Lactobacillus acidophilus fermentation product on digestibility, nitrogen balance, and intestinal function of weaned pigs Jesus. A. Acosta, Nicholas. K. Gabler, and John. F. Patience Department of Animal Science, Iowa State University, Ames, IA 50011 ABSTRACT:  The objective of this study was average daily feed intake (ADFI; P  =  0.017), the to determine the effects of lactose (LA) and a ATTD of DM (P  =  0.014), the ATTD of GE prototype Lactobacillus acidophilus fermentation (P = 0.028), and N retention (P = 0.043) and tended product (FP) on growth performance, diet digest- to increase the butyric acid concentration in the ibility, nitrogen (N) balance, and intestinal func- colon (P = 0.062). The FP tended to increase the tion of weaned pigs. Twenty-eight newly weaned digestibility of N (P = 0.090). Neither LA nor the pigs [approximately 21 d of age; initial body weight FP affected intestinal barrier function or inflam- (BW) = 5.20 ± 0.15 kg] were housed in metabolism mation markers. The interaction between LA and crates and assigned to one of four treatments FP affected intestinal morphology: in the jejunum, (n = seven pigs per treatment) corresponding to a pigs fed LA+FP− had increased villus height com- 2 × 2 factorial design: with (LA+; 15% inclusion) pared with those fed LA+FP+ and LA−FP−, or without (LA−) LA and with (FP+) or without whereas LA+FP+ was intermediate (interaction (FP−) the prototype FP (1  g of FP per kilogram P  =  0.034). At the terminal ileum, pigs fed LA− of diet; Diamond V, Cedar Rapids, IA). Feed and FP+ and LA+FP− had increased villus height and water were provided ad libitum. At day 5, pigs villus: crypt compared with those fed LA−FP−, were orally given lactulose and mannitol to assess whereas LA+FP+ was intermediate (interaction small intestinal permeability. Fecal samples were P  =  0.007 and P  =  0.007, respectively). In con- collected on days 5–9 to determine the apparent clusion, the addition of LA brings important nu- total tract digestibility (ATTD) of dry matter tritional attributes to nursery diets by improving (DM), gross energy (GE), and N.  Total urine feed intake, digestibility of DM and GE, and the output and fecal samples were collected on days N retention of weaned pigs; however, the func- 10–13 to determine N retention. On day 15, all pigs tional capacity of LA to improve markers of in- were euthanized to collect intestinal lumen and testinal function is limited. On the other hand, the tissue samples. Data were analyzed for the main FP showed only a mild increase in the digestibility effects of LA and FP and their interaction using of N but a limited capacity to improve markers of the MIXED procedure of SAS. Lactose improved intestinal function. Key words: intestinal enzymes, intestinal morphology, intestinal permeability, nursery pigs, weaning stress. The authors thank Diamond V Mills for financial sup- port. Appreciation is also expressed to DSM nutritional products and Ajinomoto Heartland, Inc., for in-kind con- tributions to the Applied Swine Nutrition Program at Iowa State University. Corresponding author: jfp@iastate.edu Received October 25, 2019. Accepted April 15, 2020. 1 Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. © The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2020.4:1-14 doi: 10.1093/tas/txaa045 INTRODUCTION MATERIALS AND METHODS Weaning is one of the most difficult transi- All experimental procedures adhered to guide- tions in the life of a pig and greatly impacts the lines for the ethical and humane use of animals for productivity of swine operations. Exposure to research according to the Guide for the Care and human handling, a new physical environment, a Use of Laboratory Animals (FASS, 2010) and were different diet, and new social interactions can re- approved by the Institutional Animal Care and sult in reduced feed intake, decreased growth, and Use Committee at Iowa State University (number an increased incidence of disease (Jones et  al., 7-15-8049-S). 2012; McLamb et al., 2013). The response of the intestinal mucosa to weaning is a particularly im- Animals Housing and Experimental Design portant concern (Smith et al., 2010). As an imma- ture tissue in the young pig, it suffers some degree A total of 28 barrows [5.22  ± 0.15  kg body of inflammation and dysfunctionality in response weight (BW); the progeny of C22 or C29 sows × to the new antigens, dietary components, and so- 337 terminal sires; PIC Inc., Hendersonville, TN] cial stressors (Boudry et al., 2004; Pié et al., 2004; were blocked by initial BW (seven blocks) and ran- Li et al., 2019). domly assigned to individual metabolism crates Feed has tremendous potential to improve the within block. Crates were randomly assigned to weaning transition because it is the most practical one of four dietary treatments (n = 7 pigs per treat- means of delivering substances with beneficial ment) corresponding to a 2  × 2 factorial design: nutritional and functional properties to the pig. with (LA+; 15% inclusion) or without (LA−) LA Although dietary antibiotics have been effectively and with (FP+) or without (FP−) the prototype FP used to control and prevent diseases in nursery (1 g of FP per kilogram of diet; Diamond V, Cedar pigs, antibiotic resistance and consumer pressure Rapids, IA). have demanded a reduction in their use. Therefore, Diets were manufactured in mash form at the evaluation of products and dietary components the Swine Nutrition Farm feed mill (Iowa State with functional and nutritional properties is in- University; Ames, IA; Table  1). Dietary levels of creasingly important. amino acids, vitamins, and minerals were set to Lactose (LA) is a common carbohydrate in meet the nutrient requirements of nursery-aged nursery diets as it provides a familiar source of pigs (NRC, 2012). Titanium dioxide was added at available carbohydrate and induces the produc- 0.4% to all diets as an indigestible marker. tion of volatile fatty acids (VFA) in the large in- Pigs were housed in a controlled environment testine (Pierce et  al., 2006). On the other hand, facility. Each metabolism crate (0.53 × 0.71 m) was a prototype Lactobacillus Acidophilus fermenta- equipped with a fully slatted floor, a stainless-steel tion product (FP; Diamond V, Cedar Rapids, IA) feeder, and a cup drinker. Pigs had ad libitum ac- is believed to provide metabolites that enhance cess to feed and water during the entire experi- intestinal health and the establishment of com- mental period (15 d). mensal microorganisms. The objective of this study was to identify Sample Collection and characterize the beneficial effects of LA and the FP in nursery pig diets with an emphasis on To measure in vivo gut permeability, all pigs intestinal function. We hypothesized that LA were fasted for 6  h and then orally dosed on day and FP would ameliorate some of the adverse 5 with a solution of 0.300  g lactulose/kg BW and effects of the weaning transition by promoting 0.030 g mannitol/kg BW (Sigma-Aldrich, St Louis, digestion and decreasing markers of intestinal MO). Urine was collected for the following 12  h dysfunction. in a plastic jug located under the crate after the Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Table 1. Ingredient composition of the experimental diets* LA− LA+ Item FP− FP+ FP− FP+ Corn 65.73 65.63 50.58 50.48 Soybean meal 20.00 20.00 20.00 20.00 Fish meal 3.72 3.72 3.72 3.72 Casein 5.00 5.00 5.00 5.00 Lactose — — 15.00 15.00 Soybean oil 1.50 1.50 1.50 1.50 -Lys HCl 0.40 0.40 0.43 0.43 -Met 0.15 0.15 0.20 0.20 DL -Thr 0.15 0.15 0.18 0.18 -Trp 0.01 0.01 0.02 0.02 Monocalcium phosphate 21% 1.14 1.14 1.20 1.20 Limestone 0.90 0.90 0.88 0.88 NaCl 0.50 0.50 0.50 0.50 Vitamin premix 0.25 0.25 0.25 0.25 Trace mineral premix 0.15 0.15 0.15 0.15 Prototype FP — 0.10 — 0.10 Titanium dioxide 0.40 0.40 0.40 0.40 *LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of prototype FP added (1 g of FP per kilogram of diet; Diamond V Mills, Cedar Rapids, IA) added. Provided per kilogram of diet: 7,600 IU of vitamin A; 875 IU of vitamin D3; 62 IU of vitamin E; 3.7 mg of menadione (to provide vitamin K); 61 μg of vitamin B12; 14 mg of riboflavin; 34 mg of d-pantothenic acid; and 70 mg of niacin. Provided per kilogram of diet: 165 mg of Fe (ferrous sulfate); 165 mg of Zn (zinc sulfate); 39 mg of Mn (manganese sulfate); 2 mg of Cu (cooper sulfate); 0.3 ppm of I (calcium iodate); and 0.3 ppm of Se (sodium selenite). administration of the lactulose/mannitol solution. before each collection to minimize N losses due to Before urine collections, five drops of chlorhexidine volatilization of ammonia-N. Total urine output gluconate 20% (weight/volume; Sigma-Aldrich, St was weighed and stored at −20  °C. At the end of Louis, MO) were added to each jug to inhibit bac- the collection period, urine was homogenized and terial activity. After collection, total urine output filtered prior to N analysis. Urine samples were ali- was weighed, homogenized, and filtered using fiber quoted into 250-mL plastic bottles and stored at glass wool; a 3-mL aliquot of urine sample was col- −20  °C until chemical analyses were performed. lected and stored at −20 °C for later analysis. In the determination of N balance, only the days In support of determining digestibility and ni- 10–13 fecal samples were utilized to correspond trogen (N) balance, and to ensure the acquisition of with the urine samples as described above. samples fully representative of the total batch, 10 All pigs were euthanized on day 15 by captive feed samples were collected from the feed mill at the bolt stunning followed by exsanguination. After time of diet preparation; these samples were thor- euthanasia, each pig was dissected and the entire oughly homogenized, pooled into one subsample, intestinal tract was removed. Samples (~20  cm and ground through a 1-mm screen in a Retsch sections long) of the proximal jejunum (taken grinder (Model ZM1, Retsch Inc., Newton, PA). 100  cm from the pyloric sphincter), the distal Total fecal output was collected twice daily from day ileum (taken 30  cm from the ileocecal valve), and 5 to 8 and day 10 to 13 and stored at −20 °C. Once the mid colon were removed and gently flushed collected, fecal samples were thawed, homogenized, with ice-cold Krebs buffer. One segment of the subsampled, dried in an oven at 65  °C to constant jejunum and ileum samples were snap-frozen in weight (Jacobs et  al., 2011), and ground through liquid N and stored at –80  °C until further ana- a 1-mm screen in a Wiley grinder (Model ED-5, lysis. Additionally, subsamples of jejunum, ileum, Thomas Scientific Inc., Swedesboro, NJ). Ground and colon were fixed for 24 h in 10% neutral buf- fecal and feed samples were stored in plastic bags in fered formalin and then transferred to 75% alcohol desiccator cabinets until assays were completed. for later morphology and histochemistry analysis. For N balance, total urine output was collected Approximately 20  mL of intestinal contents from twice daily during days 10–13 (96 h) into plastic jugs the mid-section of the colon were snap-frozen in containing 5  mL of 6 N hydrochloric acid, added liquid N and stored at –80 °C. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. ,† Table 2. Chemical composition of the experimental diets, as-fed basis* LA− LA+ Item FP− FP+ FP− FP+ Analyzed chemical composition DM, % 88.88 88.76 90.75 90.92 GE Mcal/kg 3.92 3.92 3.94 3.90 AEE, % 4.71 4.70 4.41 4.43 CP, % 21.00 21.06 20.03 20.28 Titanium dioxide, % 0.40 0.41 0.42 0.41 Calculated chemical composition SID of AA, % Lys 1.44 1.44 1.44 1.44 Thr 0.84 0.84 0.84 0.84 Met 0.55 0.55 0.57 0.57 Met + Cys 0.79 0.79 0.79 0.79 Trp 0.24 0.24 0.24 0.24 Ile 0.82 0.82 0.79 0.79 Val 0.96 0.96 0.92 0.92 Ca, % 0.82 0.82 0.82 0.82 Total P, % 0.70 0.70 0.67 0.67 STTD of P, % 0.42 0.42 0.42 0.42 AA, Amino acids; AEE, acid hydrolyzed ether extract; CP, crude protein; SID, standardized ileal digestibility; STTD, standardized total tract digestible. *LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. AA levels, phosphorus, STTD phosphorus, and calcium were calculated from NRC (2012); all other values were analyzed. Digestibility, N Balance Analysis, and Urinary mannitol concentrations were determined by High- Lactulose and Mannitol performance liquid chromatography with known standard solutions according to the method de- Feed, fecal, and urine samples were analyzed scribed by Kasangra et al. (2003). at the Monogastric Nutrition Laboratory (Iowa State University, Ames, IA). Feed (Table  2) and Intestinal Morphology and Histochemistry Analysis fecal samples were assayed for dry matter (DM; method 930.15; AOAC, 2007). Gross energy (GE) Fixed jejunum, ileum, and colon samples were was determined using a Parr model 6200 isoper- prepared and stained at the Veterinary Diagnostic ibolic bomb calorimeter (Parr Instrument Co., Laboratory (Iowa State University, Ames, IA). Each Moline, IL); benzoic acid (6,318 kcal GE/kg; Parr sample was sliced into 5-μm sections. The jejunum Instruments, Moline, IL) was used as a standard for and ileum sections were stained using the hema- calibration and was determined to contain 6,319 ± 1 toxylin and eosin procedure, whereas colon samples kcal GE/kg. Titanium dioxide was determined col- were stained with the alcian blue-periodic acid-Schiff orimetrically using a Synergy 4 spectrophotometer (AB-PAS) procedure (Mowry, 1963) to distinguish (BioTek, Winooski, VT) according to the method between neutral and acidic mucin types in colon of Leone (1973). Additionally, feed samples were crypts. The neutral mucin was stained magenta, and assayed for acid hydrolyzed ether extract (AEE; acidic mucin was stained blue. The mixture of neu- method 2003.06; AOAC International, 2007) using tral-acidic mucins showed purple, magenta-purple, a SoxCap SC 247 hydrolyzer and a Soxtec 255 sem- or blue-purple colors in goblet cells. iautomatic extractor (FOSS North America, Eden Images of intestinal mucosa were captured using Prairie, MN). The N content of the feed, fecal, a light microscope (DMI3000 B Inverted Microscope, and urine samples was determined by thermo- Leica Microsystems, Bannockburn, IL) with an at- combustion (method 990.03; AOAC International, tached camera (12-bit QICAM Fast 1394, QImaging, 2007; Leco TruMac N, LECO Corporation, St. Surrey, BC, Canada). Individual images of villi and Joseph, MI). The standard for calibration was crypts were taken using Q-capture Pro 6.0 soft- Ethylenediaminetetraacetic acid (9.56% N; Leco ware (QImaging, Surrey, BC) and measured using Corporation, St. Joseph, MI) and determined to Image-Pro Plus 7.0 (Media Cybernetics, Bethesda, contain 9.56  ± 0.01% N). Urinary lactulose and MD). Images were measured using Image J software, Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs version 1.48s (Rasband W, National Institutes of using a Nanodrop 1000 instrument (NanoDrop Health, MD). The villus height was measured from Technologies, Rockland, DE). The purity was as- the tip of the villus to the crypt–villus junction, and sessed by determining the ratio of the absorbance at the crypt depth was measured from the crypt–villus 260 and 280 nm (all samples had absorbance ratios junction to the base of the crypt. At least 10 well-de- above 1.8). One microgram of RNA was transcribed fined villi and associated crypts from each sample of in a reaction combining genomic DNA elimination each intestinal segment were measured; averages were using a commercially available kit (Quantitect re- then calculated and reported as one number per pig. verse transcription kit; Qiagen Inc., Valencia, CA). The mucin area was calculated as a percentage of the The resulting complementary DNA was quanti- total mucosal tissue area in each image. fied using a NanoDrop 1000 instrument and ap- plied to quantitative real-time PCR (qPCR). The qPCR was completed using a BioMark HD system Enzyme Activity and Secretory Immunoglobulin A (S-IgA) (Fluidigm Corporation, San Francisco, CA). No reverse transcriptase control samples were included Mucosal scrapings (~0.5  g) of the jejunum and in the extraction process to assure no genomic DNA ileum were added to 4.5  mL of Phosphate-buffered contamination. Complementary DNA was used saline buffer containing a protease inhibitor cocktail for specific target amplification using the TaqMan (SKU, P8340; Sigma-Aldrich, St. Louis, MO) and PreAmp Master Mix (Life Technologies, Carlsbad, triton (0.1%). The resulting solution was homogen- CA) and loaded onto Fluidigm’s Dynamic Array ized and centrifuged at 10,000 × g for 15 min at 4 °C. Integrated Fluidic Circuits according to Fluidigm’s The supernatant was extracted and stored in aliquots. EvaGreen DNA binding dye protocols. Gene sym- The total protein concentration of hydrolyzed mucosa bols and primer sequences are listed in Table 3. One scrapings was quantified using a Pierce Bicinchoninic 48.48 Dynamic Array, Integrated Fluidic Circuit Acid Protein Assay Kit (BCA; Thermo Scientific, plate, was used to analyze mRNA abundance of Woltham, MA). Disaccharidase activity was de- selected genes in porcine jejunal and ileal tissues. To termined according to Dahlqvist (1964) using LA, select an endogenous reference gene, ribosomal pro- maltose, and sucrose as substrates. Alkaline phos- tein L19 (RPL19) was included in the qPCR array. phatase activity was determined using a porcine al- kaline phosphatase assay kit (ABCAM, Cambridge, VFA Concentration MA). Enzyme activity was expressed as micromole of hydrolyzed substrate per minute per gram of tissue In preparation for VFA analysis, 1.0 g of colon protein. The concentration of secretory IgA was de- contents were mixed with 2.5  g of purified water termined using a porcine IgA ELISA kit (E101-102; and 1 mL was extracted from the homogenized so- Bethyl Laboratories, Inc., Montgomery, TX) fol- lution. Then 0.2  mL of 25% metaphosphoric acid lowing the manufacturer’s directions. (used to deprotonize the samples) and 0.1  mL of 4-methylvaleric acid (as an internal standard) were RNA Extraction and Real-Time PCR added to the extracted homogenate. These samples were centrifuged at 12,000 × g for 25 min, and the RNA was extracted from frozen jejunal and ileal supernatant was removed for analysis. Volatile fatty tissues according to the Trizol protocol (Invitrogen, acids were determined using a model 3900 gas chro- Grand Island, NY). The purity and quantity of matograph fitted with a CP 8400 automatic injector RNA were determined by spectrophotometry (Varian Analytical Instruments, Walnut Creek, Table 3. Primers used for quantitative reverse transcription-PCR Primer sequence, 5ʹ→3 ʹ Gene name Accession number Sense (forward) Anti-sense (reverse) RPL19 AF435591 AACTCCCGTCAGCAGATCC AGTACCCTTCCGCTTACCG OCLN NM_001163647 TCGTCCAACGGGAAAGTGAA ATCAGTGGAAGTTCCTGAACCA CLDN3 NM_001160075 TTGCATCCGAGACCAGTCC AGCTGGGGAGGGTGACA IL-6 AF518322 GGCTGTGCAGATTAGTACC CTGTGACTGCAGCTTATCC IL-10 L20001 TGGGTTGCCAAGCCTTGT GCCTTCGGCATTACGTCTTC IL-17a AB102693 CCAGACGGCCCTCAGATTAC CACTTGGCCTCCCAGATCAC IL-22 AY937228 AAGCAGGTCCTGAACTTCAC CACCCTTAATACGGCATTGG TNFα X54859 AACCCTCTGGCCCAAGGA GGCGACGGGCTTATCTGA Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. –ΔΔCt CA) using a 30 m, 0.25 mm, and 0.25 μm column calculated as 2 , in which ΔΔCt represents ΔCt (DB-FFAP, Agilent, Santa Clara, CA). Helium was sample − ΔCt calibrator (Livak and Schmittgen, used as carrier gas. Purified VFA samples (Sigma- 2001). Aldrich, St. Louis, MO) were used for the identifi- cation of VFA peaks. Statistical Analysis Data were analyzed using the following model: Calculations y = μ + τ + λ +(τλ) + δ + ijkl i j k ijkl ij Individual pig weights and feed disappear- where y represents the observed value, μ is the ance were measured on days 0 and 14 to calculate ijkl overall mean, τ represents the fixed effect of LA average daily gain (ADG), average daily feed intake [i = 1, 2 (LA− and LA+, respectively)], λ represents (ADFI), and gain to feed ratio (G:F). Apparent the fixed effect of FP [j = 1, 2 (FP− and FP+, re- total tract digestibility (ATTD) of DM, GE, and spectively)], τλ represents the interaction between N was calculated using the following equation LA and FP, δ represents the random effect of block (Oresanya et al., 2008): (k = 1–7), and ϵ is the random error associated with ATTD, % =[100 [100 × (% TiO2 in feed/% TiO2 in feces) y (l = 1–7) assuming δ ~Ν (0, ) and ϵ ~Ν (0, ). ijkl × (concentration of component in feces/ The pig was the experimental unit for all ana- concentration of component in feed)]] lyses. The UNIVARIATE procedure of SAS ver- sion 9.3 (SAS Inst., Inc., Cary, NC) was used to N intake (g/d) was calculated by multiplying the verify the normality and homogeneity of the re- percentage of N in the feed (DM basis) by DM sidual variance from the reported models. Jejunal feed intake (g/d); N excreted in the urine was calcu- and ileal mucosa S-IgA, mannitol, and lactulose re- lated by multiplying the average daily urine weight covery and lactulose:mannitol ratio variables were (kg/d) by the average urinary N concentration (%). analyzed and reported after log-transformation to Nitrogen excreted in feces (g/d) was calculated by satisfy normality. The models were analyzed using multiplying daily N intake minus the product of the MIXED procedure of SAS. Differences were multiplying N intake times the ATTD of N (%). considered significant with P-values ≤0.050, and Total N excretion (g/d) was calculated as the sum P-values between 0.050 and 0.100 were considered of daily N excreted in urine and feces. Finally, N re- trends. tention was calculated as daily N intake minus daily N excretion. Protein retention was calculated as N RESULTS retention (g/d) × 6.25 divided by the ADG (g/d). All N balance variables were calculated using data Growth Performance, Digestibility, and N Balance from samples collected on days 10–14. Evaluating the impact of dietary treatments on Lactulose and mannitol recoveries (%) were growth performance in metabolism crates is diffi- calculated as the amount of these sugars excreted cult due to the typically small number of animals (g/d) in the urine [calculated as total urine weight and the conditions that differ substantially from (g) times lactulose or mannitol concentration (%)] normal housing. Nonetheless, it is useful to do so to divided by the amount of lactulose or mannitol provide an understanding of the state of the animal given (g/d) orally times 100. Lactulose:mannitol under which other measurements were recorded. ratio was calculated by dividing lactulose recovery No interactions between LA and FP were ob- by mannitol recovery (Musa et al., 2019). served for initial BW, final BW, growth performance, Plates for PCR analysis were balanced so that digestibility, or N balance variables. Therefore, the an equal number of treatments were represented on results of these variables are presented as the main each plate. Additionally, a pooled control sample effects. By design, the initial BW was not affected representative of all treatment groups was run on by the addition of LA or FP (Table  4). Similarly, each gene as an internal control. Normalized ex- final BW (day 14)  and ADG were not affected by pression (ΔCt) for each sample was determined the addition of LA or the FP. Although there was using RPL19 as an endogenous housekeeping gene. no effect of FP on ADFI, pigs fed LA had increased The average normalized expression of the pooled ADFI (P = 0.017). control sample was used as the calibrator to calcu- The addition of LA improved the ATTD of late relative gene expression (Livak and Schmittgen, DM and GE (P = 0.014 and P = 0.028, respectively; 2001). For each sample, relative expression was Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Table 4.  Effect of LA and a prototype Lactobacillus acidophilus FP on growth performance of nursery ,† pigs* LA FP P-value Item − + − + SEM LA FP Day 0 BW, kg 5.23 5.23 5.22 5.23 0.06 0.744 0.334 Day 14 BW, kg 7.23 7.78 7.70 7.30 0.26 0.105 0.216 ADG, kg 0.148 0.189 0.184 0.153 0.018 0.102 0.205 ADFI, kg 0.180 0.229 0.218 0.190 0.016 0.017 0.150 G:F 0.787 0.809 0.822 0.774 0.043 0.731 0.445 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment). LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 5. Effect of LA and a prototype Lactobacillus acidophilus FP on ATTD of DM, GE, and N in nur- ,† sery pigs* LA FP P-value Item − + − + SEM LA FP ATTD of DM, % 88.1 89.2 88.4 88.9 0.4 0.014 0.167 ATTD of GE, % 86.4 87.7 86.7 87.4 0.5 0.028 0.206 ATTD of N, % 83.8 83.6 82.9 84.5 0.9 0.900 0.090 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment), diets contained titanium dioxide (0.4%) as an indigestible marker, and total fecal output was collected twice daily from day 5 to 8 and day 10 to 13. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of prototype FP (1 g of FP per kilogram of diet) added. Table  5). However, the addition of the FP did not N retained. The addition of LA increased the pro- affect the ATTD of DM or GE. There were no ef- tein retained as a percentage of ADG (P = 0.020), fects of LA on the ATTD of N. However, the add- whereas FP tended to increase the protein retained ition of the FP tended to increase the ATTD of N as a percentage of ADG (P = 0.053). The protein (P = 0.090). retained expressed as a percentage of ADG is a The addition of LA tended to increase N in- useful standard to validate N retention data—it take (P  =  0.080), whereas no effect of FP was should be in the range of 15–18% for pigs of this observed (Table  6). In contrast, the FP tended age—and is also a very good indicator of the lean to decrease N excretion (P = 0.060), whereas LA gain in the pigs. did not affect N excretion. When N excretion was partitioned into fecal or urinary components, Intestinal Morphology, Mucin Staining Area, and the addition of LA increased fecal N excretion Concentration of S-IgA (P  =  0.017), while FP decreased it (P  =  0.044). The addition of LA decreased urinary N and in- The morphology of the small intestine was af- creased the overall N retention (P  =  0.006 and fected by the interaction between LA and FP, so P  =  0.043, respectively). The addition of FP did individual treatment means are presented. In the not affect urinary N excretion or overall N reten- jejunum, pigs fed LA+FP− had increased villus tion, expressed as g/d. height compared with those fed LA+FP+ and LA− The addition of LA decreased the percentage FP−, whereas LA-FP+ was intermediate (Table 7; of N excreted. There was no effect of LA on the interaction P  =  0.034). In the terminal ileum, percentage of N excreted in the feces, but it de- pigs fed LA−FP+ and LA+FP− had increased creased the percentage of N excreted in the urine villus height compared with those fed LA−FP−, (P  =  0.002). As a result, the addition of LA in- whereas LA+FP+ was intermediate (interaction creased the percentage of N retained (P  =  0.043). P  =  0.007). Although neither LA nor FP altered The addition of FP had no effect on the total per- villus:crypt ratio in the jejunum, an interaction centage of N excreted or the percentage of N ex- between LA and FP affected villus:crypt ratio in creted in the urine and the feces. Consequently, the the ileum; pigs fed LA−FP+ and LA+FP− had addition of FP had no effect on the percentage of increased villus:crypt ratio compared with those Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. ,† Table 6. Effect of LA and a prototype Lactobacillus acidophilus FP on N balance of nursery pigs* LA FP P-value Item − + − + SEM LA FP N balance, g/d Intake 7.57 9.28 8.80 8.06 0.65 0.072 0.414 Excretion 2.85 2.88 3.09 2.64 0.19 0.901 0.060 Fecal 1.16 1.59 1.55 1.20 0.15 0.017 0.044 Urine 1.68 1.29 1.54 1.44 0.09 0.006 0.468 Retention 4.72 6.41 5.71 5.42 0.56 0.043 0.716 N balance % Excretion 38.7 32.1 35.8 35.0 2.1 0.043 0.804 Fecal 15.0 17.3 17.4 14.9 1.1 0.163 0.120 Urinary 23.7 14.8 18.3 20.2 1.7 0.002 0.445 Retention 61.3 67.9 64.2 65.0 2.1 0.043 0.804 Protein retained as % of ADG 15.0 17.0 15.2 16.8 0.6 0.020 0.053 *A total of 28 barrows (5.22 ± 0.15 kg BW) was assigned to individual metabolism crates for 15 d (seven pigs per treatment); total urine and fecal output was collected twice daily during days 10–13 (96 h). ADG was calculated using weights collected on days 10 and 14. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 7. Effect of LA and a prototype Lactobacillus acidophilus FP on intestinal morphology, S-IgA, and ,† mucins of nursery pigs* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Jejunum b ab a b Villus height, μm 275 320 390 307 28 0.089 0.511 0.034 Crypt depth, μm 313 338 343 326 21 0.677 0.848 0.321 Villus: crypt 0.88 1.00 1.15 0.95 0.11 0.321 0.682 0.160 a b b b S-IgA, (log) μg/mg of protein 3.04 2.41 2.59 2.64 0.14 0.637 0.065 0.005 Ileum b a a ab Villus height, μm 266 341 378 317 25 0.065 0.780 0.007 Crypt depth, μm 262 247 263 274 16 0.332 0.891 0.391 b a a ab Villus: crypt 1.02 1.43 1.48 1.19 0.12 0.351 0.591 0.007 S-IgA, (log) μg/g of protein 2.62 2.76 2.71 2.70 0.12 0.939 0.610 0.520 Colon Crypt depth, μm 435 395 440 420 24 0.502 0.189 0.645 Goblet cells/100 μm crypt 5.8 6.2 6.0 5.3 0.7 0.720 0.782 0.263 Acid mucins, % 11.4 11.0 12.8 7.3 1.4 0.437 0.053 0.087 Mixed mucins, % 12.7 10.7 13.7 11.8 2.0 0.597 0.318 0.979 a b ab a Neutral mucins, % 2.6 1.6 2.1 3.1 0.4 0.175 0.938 0.010 a,b Means with different superscripts significantly differ (P < 0.050). *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (7 pigs/treatment), intestinal tissues, and digesta were collected after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. fed LA−FP−, whereas LA+FP+ was intermediate percentage of neutral mucins compared with those (interaction P  =  0.007). Neither LA nor FP af- fed LA−FP+, whereas LA+FP− was intermediate fected crypt depth at the jejunum or at the ileum. (interaction P = 0.010). In the mid colon, there were no effects of LA The concentration of S-IgA was affected by the or FP on crypt depth, the number of goblet cells interaction of LA and FP. Pigs fed LA−FP− had per crypt, or the percentage of acid or mixed increased levels of mucosal S-IgA compared with mucins. However, there was an interaction between the other three treatments (interaction P  =  0.005). LA and FP on the percentage of neutral mucins: However, neither LA nor FP impacted the concen- pigs fed LA−FP− and LA+FP+ had an increased tration of S-IgA at the terminal ileum. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Small Intestine Permeability, Enzymatic Activity, increase total VFA (P  =  0.099), whereas the add- and Transcript Abundance of Tight Junction ition of FP decreased total VFA concentration in Proteins and Cytokines colonic contents (P = 0.048). When VFAs were looked at as proportions of There were no main effects or interactions of LA the total concentration, the percentages of acetic, or FP on mannitol recovery, lactulose recovery, or propionic, butyric, and valeric acid were not af- in the lactulose:mannitol ratio (Table 8). Similarly, fected by the addition of LA or FP. However, activities of lactase, sucrase, maltase, or alkaline LA and FP tended to interact for the percentages phosphatase were not affected by the addition of of branched-chain fatty acids; pigs fed LA−FP+ LA or FP (Table 9). Likewise, gene transcript abun- tended to have increased percentages of isobutyric dance of tight junction protein genes [occludin and isovaleric acid compared with LA−FP− and (OCLN) and claudin-3 (CLDN3); Table 10] in the LA+FP+, whereas LA+FP− was intermediate jejunum and terminal ileum and ileal cytokines (interaction P = 0.069 and P = 0.099, respectively). [tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), IL-10, IL-17, and IL-22] were not affected DISCUSSION by the addition of LA or FP. One of the most important strategies to ameli- orate the impact of weaning stress is the use of VFA Concentrations in the Colon specialty ingredients and feed additives targeted to possess nutritional, as well as functional, properties. Although LA had no effect, the addition of FP The primary target of these functional properties decreased the concentration of acetic acid in the is the gastrointestinal tract. The first experimental colon (Table 11; P = 0.022). Neither LA nor FP af- approach herein was to evaluate the effect of LA fected the concentration of propionic acid. Pigs fed and FP on digestibility and N retention while at LA tended to increase butyric acid concentration the same time monitoring feed intake and growth (P = 0.062), but there was no effect when adding FP. performance. These would represent key outcomes There were no effects of LA or FP on the concen- that would potentially be improved if negative ef- tration of branched-chain fatty acids (isobutyric, fects of weaning are reduced; more specifically, the isovaleric, and valeric acid). Pigs fed LA tended to Table 8. Effect of LA and a prototype Lactobacillus acidophilus FP on small intestinal permeability at day ,† 5 postweaning* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Mannitol recovery, (log) % 1.41 1.29 1.37 1.51 0.12 0.435 0.942 0.281 Lactulose recovery, (log) % −0.14 −0.16 −0.38 −0.17 0.16 0.432 0.553 0.479 Lactulose: mannitol (log) −1.54 −1.55 −1.66 −1.66 0.16 0.530 0.977 0.956 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment). Intestinal permeability was assessed by providing each pig a solution of 0.300 g lactulose/kg BW and 0.030 g mannitol/kg BW (Sigma-Aldrich, St Louis, MO). Urine was collected for the following 12 h after the administration of the lactulose/mannitol solution. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 9. Effect of LA and a prototype Lactobacillus acidophilus FP on the enzymatic activity at the jejunum* LA− LA+ Contrast P-value Item FP- FP+ FP- FP+ SEM LA FP LA × FP Activity, U/mg protein Lactase 16.6 10.6 15.7 14.7 3.6 0.976 0.335 0.222 Sucrase 20.9 25.5 31.2 24.3 5.1 0.387 0.847 0.511 Maltase 182.7 190.7 206.2 228.4 22.4 0.116 0.427 0.708 Alkaline phosphatase 60.7 60.6 65.4 53.1 6.4 0.833 0.340 0.348 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); jejunal tissue was col- lected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. Table 10. Effect of LA and a prototype Lactobacillus acidophilus FP on RNA abundance of tight junction ,†,‡ proteins and cytokines in the jejunum and ileum* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Jejunum OCLN 1.23 1.44 2.08 1.20 0.37 0.394 0.352 0.139 CLDN3 2.33 1.56 1.63 1.76 0.40 0.550 0.448 0.285 Ileum OCLN 1.67 1.49 1.10 1.09 0.36 0.223 0.892 0.569 CLDN3 1.59 1.48 0.87 1.28 0.36 0.565 0.873 0.353 TNFα 1.03 1.37 1.14 1.36 0.27 0.797 0.146 0.727 IL-6 0.61 0.79 1.17 0.99 0.24 0.102 0.839 0.324 IL-10 0.87 1.04 1.23 1.15 0.26 0.264 0.801 0.545 IL-17 0.80 1.03 1.15 0.85 0.18 0.618 0.872 0.137 IL-22 1.08 1.35 1.14 0.77 0.54 0.900 0.543 0.238 *All values indicate relative expression of genes. Normalized expression (ΔCt) for each sample was determined using RPL19 as an endogenous control gene. The average normalized expression of the pooled control sample was used as the calibrator to calculate relative gene expression. For −ΔΔCt each sample, relative expression was calculated as 2 , in which ΔΔCt represents ΔCt sample − ΔCt calibrator (Livak and Schmittgen, 2001). A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); intestinal tissue was collected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 11. Effect of LA and a prototype Lactobacillus acidophilus FP on volatile fatty acid concentrations ,† in colonic contents* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Concentration μM/g Acetic acid 63.77 49.76 71.53 58.17 6.07 0.292 0.022 0.767 Propionic acid 24.14 18.48 26.86 23.13 3.00 0.232 0.132 0.749 Butyric acid 15.16 14.56 20.60 20.19 2.81 0.062 0.858 0.974 Isobutyric acid 2.21 2.34 3.21 2.07 0.44 0.522 0.201 0.129 Isovaleric acid 3.46 3.69 4.97 3.23 0.71 0.468 0.303 0.184 Valeric acid 3.30 3.21 4.65 4.36 0.81 0.173 0.676 0.819 Total 115.1 92.1 131.8 111.2 10.3 0.099 0.048 0.910 Distribution, % Acetic acid 58.1 55.1 54.3 52.8 3.2 0.330 0.471 0.810 Propionic acid 20.8 19.6 20.3 20.7 1.5 0.850 0.804 0.591 Butyric acid 13.2 15.1 15.6 17.7 1.6 0.140 0.227 0.945 y x xy y Isobutyric acid 1.9 2.6 2.4 1.9 0.3 0.729 0.761 0.069 y x xy y Isovaleric acid 3.0 4.1 3.8 3.0 0.5 0.740 0.842 0.099 Valeric acid 2.9 3.5 3.7 3.9 0.7 0.660 0.405 0.559 x,y Means with different superscripts tend to differ (P < 0.100). *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); intestinal tissue was collected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. consumption of specialty ingredients may ameli- stimulate appetite are highly desirable. Lactose is orate stress on the intestinal tract. the primary dietary carbohydrate consumed be- Postweaning anorexia has profound negative fore weaning (~5% in sow milk; Rosero et al., 2015) effects on the growth and health of nursery pigs and is believed to be highly palatable to the nursery (Spreeuwenberg et al., 2001) and is the main factor pig. Results of this experiment and other research affecting subsequent growth performance (Jones (Tokach et al., 1989; Kim et al., 2010; Tran et al., et  al., 2012). Therefore, dietary compounds that 2012; Pierce et al., 2006) support the use of LA in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs the starter diet to enhance feed intake during the improving the digestibility of DM and GE and postweaning period. N retention, as well as improving feed intake. The In addition to stimulating appetite, nursery addition of FP had a more modest impact on the diets are designed to be highly digestible. This is be- digestibility of N, with no detectable improvement cause feed intake and gut capacity of weaned pigs in growth. is limited (Dong and Pluske, 2007). Additionally, The second experimental approach was to ex- the digestibility of nutrients is a key determinant of plore the effects of LA and FP on specific markers the growth response of nursery pigs after weaning of intestinal function. Recovery from weaning (Jones and Patience, 2014). The addition of LA in- stress by the intestinal tissue has been described creased the digestibility of DM and GE. These same to occur in two phases: the first is an acute phase, results were reported by Jin et al. (1998) by adding lasting 2–4 d, in which the major dysfunctional 20% of LA to nursery pigs and by Pierce et al. (2005) changes occur, including the disruption of barrier feeding increased levels of LA (0–11%) to finishing function and immune activation (including inflam- pigs. Compared with corn, LA has a much simpler mation; Pié et  al., 2004; Smith et  al., 2010). The chemical and physical structure that is readily avail- subsequent adaptive phase, lasting about 2  wk able to be cleaved by lactase at the brush border of after the acute phase, is associated with tissue re- the small intestine and absorbed by the enterocytes covery (Montagne et  al. 2007). The length of the as galactose and glucose. Additionally, LA escap- acute phase and a proper recovery of the intes- ing digestion is rapidly utilized by bacterial popu- tinal tissue after weaning are key determinants lations in the distal small intestine, as well as in the of the performance and the health parameters of large intestine (Bach Knudsen, 2012). Thus, the re- nursery pigs and potentially their performance sults of this experiment confirm that LA contrib- in subsequent growth stages. Therefore, the add- utes to the objective of making nursery diets highly ition of functional products and specialty ingredi- digestible by providing readily usable energy and an ents targeted to ameliorate the weaning transition easily fermentable substrate. should help the pig recover from one or more of Protein deposition is the most valued compo- these dysfunctional changes. One of the most im- nent of the total weight gain of the nursery pig (de portant targets is the speed of barrier function res- Vries and Kanis, 1994; Colina et al., 2010). In this titution after weaning (Wijtten et  al., 2011). The experiment, the addition of LA increased overall results of this experiment suggest no effect of LA N retention, the percentage of N retained, and the and FP on the permeability of the small intestine protein retained as the percentage of the ADG. The at the end of the acute phase of weaning (day 5). increase in overall N retention can be explained Likewise, no effects on the RNA abundance of largely by the increase in feed intake in the LA-fed CLDN3 and OCLN—essential components of pigs since nutrient supply is the main factor limit- the tight junction complex (Saitou 1997; Niewold, ing protein deposition in nursery pigs (Van Milgen 2015)—or on the mRNA abundance of proinflam- et  al., 2000). Interestingly, pigs fed LA not only matory cytokines in the ileum (IL-6, IL-10, IL-22, had greater protein deposition but were more effi- and TNFα) were observed in the small intestine cient in using N toward protein deposition. These at the end of the adaptive phase of weaning (day results agree with those obtained by Pierce et  al. 14). Proinflammatory cytokines are expected to be (2005), who fed a LA supplement to finishing pigs. upregulated when immune cells are recruited (Renz Interestingly, they also observed that LA improves et  al., 2012). No effects of LA on these variables N utilization by decreasing the percentage of N ex- have been found in the literature, but the FP has creted in the urine and not through an increase in been shown to decrease proinflammatory medi- N digestibility. ators (such as TNFα, interferon gamma, and IL-6, Independent of the responses to LA, the add- IL-8, and IL-1-beta-1) under an LPS challenge ition of FP tended to increase the digestibility of N (Lee et  al., 2016). An alternative explanation of and the protein retained as a percentage of ADG; these results is that the weaning stress experienced this suggested that the mode of action of FP may by these pigs, not associated with clinical signs of be related to improving the ability of the pig to di- disease, was mild (Pié et al., 2004: Montagne et al., gest dietary protein. 2007), resulting in a limited window of chance for Overall, the results of the data on nutrient di- improvement of makers of barrier function and in- gestibility and N balance, within the context of flammation. However, this explanation is difficult growth performance, suggest that LA plays an to support since there was no measurement of the important role in nourishing the weaned pig by stress experienced by these pigs in the first place. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. Despite a lack of responses on the barrier func- improving feed intake, digestibility of DM and GE, tion and the inflammation parameters, the results and N retention of weaned pigs; however, the func- obtained in this experiment showed less S-IgA tional capacity of LA to improve markers of intes- concentration in the jejunum of pigs fed either tinal function is limited at least under the conditions LA or FP. The concentration of S-IgA in weaned of this study. On the other hand, the FP showed only pigs is the result of an adaptive immunological re- a mild increase in the digestibility of N but a limited sponse to the exposure to luminal immunogenic capacity to improve markers of intestinal function. molecules (Rey et  al., 2004; Suzuki and Fargasan, Conflict of interest statement. The authors de- 2008; Brandtzaeg, 2013). Additionally, the increase clare no real or perceived conflicts of interest. in S-IgA concentration can be the result of the normal development of the acquired immunity of the weaned pig (McGlone and Pond, 2003). Since LITERATURE CITED the decrease in the concentration of S-IgA in pigs AOAC. 2007. Official methods of analysis. 18th ed. AOAC Int., fed LA and FP was observed only in the jejunum, Arlington, VA. it is likely the result of a localized response of the Bach  Knudsen,  K.  E. 2012. Lactose in diet influences the degradation of mixed linked β(1-3;1-4)-D-glucan in the pigs; there probably was no significant impact of small intestine of pigs. J. Anim. Sci. 90(Suppl 4):125–127. weaning on the intestinal health or the develop- doi:10.2527/jas.53788. ment of the acquired immunity of these pigs. Thus, Boudry,  G., V.  Péron, I.  Le  Huërou-Luron, J.  P.  Lallès, and additional research is needed to determine if LA B.  Sève. 2004. Weaning induces both transient and and FP decrease the growth of antigenic-IgA trig- long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. J. Nutr. 134:2256– gering bacteria probably by inducing the growth of 2262. doi:10.1093/jn/134.9.2256. nonpathogenic microorganisms. Brandtzaeg, P. 2013. Secretory IgA: designed for anti-microbial de- Among the multiple changes in gastrointes- fense. Front. Immunol. 4:222. doi:10.3389/fimmu.2013.00222. tinal function in the weaning pig, the activity of Colina,  J.  J., P.  S.  Miller, A.  J.  Lewis, R.  L.  Fischer, and disaccharidases have been reported to shift during R.  M.  Diedrichsen. 2010. Growth, body chemical com- the weaning recovery; lactase activity decreases position, and tissue deposition rates of nursery pigs fed crystalline or protein- bound lysine. Prof. Anim. Sci. while maltase activity increases after weaning 26:230–238. doi:10.15232/S1080-7446(15)30584-2. (Montagne et al., 2007; Tsukahara et al., 2013). In DahlqvisT ,  A. 1964. Method for assay of intestinal disaccharidases. this experiment, lactase activity was expected to Anal. Biochem. 7:18–25. doi:10.1016/0003-2697(64)90115-0. be elevated on day 15 in pigs receiving LA due to de  Vries  A.  G., and E.  Kanis. 1994. Selection for efficiency substrate induction, the consequence of keeping of lean tissue deposition in pigs. In: D.  J.  A.  Cole, J.  Wiseman, and M.  A.  Varley, editors, Principles of pig LA in the diet. However, similar lactase activities science. Nottingham University Press, Loughborough, were observed between the pigs receiving the LA+ UK; p. 23–41. and LA− diets. To the knowledge of the authors, Dong, G. C., and J. R. Pluske. 2007. The low feed intake in new- there is no specific data reporting substrate induc- ly-weaned pigs: problems and possible solutions. Asian- tion for lactase activity in nursery pigs. Instead, Aust. J. Anim. Sci. 20:440–452. doi:10.5713/ajas.2007.440. the mentioned shift in disaccharidase activities FASS. 2010. Guide for the care and use of agricultural animals in research and teaching. 3rd ed. Federation of Animal has been reported to take place regardless of the Science Societies, Champaign, IL; p. 169. presence of LA in the diet (Hedemann et al., 2006; Hedemann,  M.  S., B.  B.  Jensen, and H.  D.  Poulsen. 2006. Montagne et al., 2007). Troelsen et al. (1992) and Influence of dietary zinc and copper on digestive enzyme Motohashi et  al. (1997) suggested that the de- activity and intestinal morphology in weaned pigs. J. cline in lactase activity after weaning is the result Anim. Sci. 84:3310–3320. doi:10.2527/jas.2005-701. Jacobs, B. M., J. F. Patience, W. A. Dozier III, K. J. Stalder, and of the decrease of the intestinal nuclear factor B.  J.  Kerr. 2011. Effects of drying methods on nitrogen [NF-LPH1] lactase promoter and the LA expres- and energy concentrations in pig feces and urine, and sion during the intestinal development process poultry excreta. J. Anim. Sci. 89:2624–2630. doi:10.2527/ after weaning rather than lack of LA induction jas.2010-3768. in the diet. The current experiment not only sup- Jin, C. F., J. H. Kim, H. K. Moon, W. T. Cho, and Y. K. Han. ports this rational but also suggests that even with 1998. Effects of various carbohydrate sources on the growth performances and nutrient utilization in pigs the reduction of lactase activity during the post- weaned at 21 days of age. Asian-Australas. J. Anim. Sci. weaning period, the weaned pig has enough lac- 11:285–292. doi:10.5713/ajas.1998.285. tase activity to effectively use LA− based on the Jones, C. K., and J. F. Patience. 2014. Variation in nutrient and digestibility and N balance results. energy digestibility and energy intake are key contribu- In conclusion, the addition of LA brings im- tors to differences in post-weaning growth performance. J. Anim. Sci. 92:2105–2115. doi:10.2527/jas2013-6335. portant nutritional attributes to nursery diets by Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Jones, C. K., N. K. Gabler, R. G. Main, and J. F. Patience. 2012. lactulose and mannitol with conventional five hours Characterizing growth and carcass differences in pigs with and shortened two hours urine collection by two dif- varying weaning weights and post-weaning performance. ferent methods: HPAE-PAD and LC-MSMS. Plos One J. Anim. Sci. 90:4072–4080. doi:10.2527/jas.2011–4793. 14:e0220397. doi:10.1371/journal.pone.0220397. 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T.  E.  Burkey, and P.  S.  Miller. 2012. Effects of lactose doi:10.1111/j.1740-0929.2012.01039.x. and yeast-dried milk on growth performance, fecal micro- Van  Milgen,  J., N.  Quiniou, and J.  Noblet. 2000. Modelling biota, and immune parameters of nursery pigs. J. Anim. the relation between energy intake and protein and lipid Sci. 90:3049–3059. doi:10.2527/jas.2011-4544. deposition in growing pigs. Anim. Sci. 71:119–130. doi: Troelsen,  J.  T., J.  Olsen, O.  Norén, and H.  Sjöström. 1992. A 10.1017/S1357729800054941. novel intestinal trans-factor (NF-LPH1) interacts with the Wijtten,  P.  J., J.  J.  Verstijnen, T.  A.  van  Kempen, lactase-phlorizin hydrolase promoter and co-varies with H.  B.  Perdok, G.  Gort, and M.  W.  Verstegen. 2011. the enzymatic activity. J. Biol. Chem. 267:20407–20411. Lactulose as a marker of intestinal barrier function Tsukahara,  T., E.  Kishino, R.  Inoue, N.  Nakanishi, in pigs after weaning. J. Anim. Sci. 89:1347–1357. 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The effect of lactose and a prototype Lactobacillus acidophilus fermentation product on digestibility, nitrogen balance, and intestinal function of weaned pigs

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© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.
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Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 The effect of lactose and a prototype Lactobacillus acidophilus fermentation product on digestibility, nitrogen balance, and intestinal function of weaned pigs Jesus. A. Acosta, Nicholas. K. Gabler, and John. F. Patience Department of Animal Science, Iowa State University, Ames, IA 50011 ABSTRACT:  The objective of this study was average daily feed intake (ADFI; P  =  0.017), the to determine the effects of lactose (LA) and a ATTD of DM (P  =  0.014), the ATTD of GE prototype Lactobacillus acidophilus fermentation (P = 0.028), and N retention (P = 0.043) and tended product (FP) on growth performance, diet digest- to increase the butyric acid concentration in the ibility, nitrogen (N) balance, and intestinal func- colon (P = 0.062). The FP tended to increase the tion of weaned pigs. Twenty-eight newly weaned digestibility of N (P = 0.090). Neither LA nor the pigs [approximately 21 d of age; initial body weight FP affected intestinal barrier function or inflam- (BW) = 5.20 ± 0.15 kg] were housed in metabolism mation markers. The interaction between LA and crates and assigned to one of four treatments FP affected intestinal morphology: in the jejunum, (n = seven pigs per treatment) corresponding to a pigs fed LA+FP− had increased villus height com- 2 × 2 factorial design: with (LA+; 15% inclusion) pared with those fed LA+FP+ and LA−FP−, or without (LA−) LA and with (FP+) or without whereas LA+FP+ was intermediate (interaction (FP−) the prototype FP (1  g of FP per kilogram P  =  0.034). At the terminal ileum, pigs fed LA− of diet; Diamond V, Cedar Rapids, IA). Feed and FP+ and LA+FP− had increased villus height and water were provided ad libitum. At day 5, pigs villus: crypt compared with those fed LA−FP−, were orally given lactulose and mannitol to assess whereas LA+FP+ was intermediate (interaction small intestinal permeability. Fecal samples were P  =  0.007 and P  =  0.007, respectively). In con- collected on days 5–9 to determine the apparent clusion, the addition of LA brings important nu- total tract digestibility (ATTD) of dry matter tritional attributes to nursery diets by improving (DM), gross energy (GE), and N.  Total urine feed intake, digestibility of DM and GE, and the output and fecal samples were collected on days N retention of weaned pigs; however, the func- 10–13 to determine N retention. On day 15, all pigs tional capacity of LA to improve markers of in- were euthanized to collect intestinal lumen and testinal function is limited. On the other hand, the tissue samples. Data were analyzed for the main FP showed only a mild increase in the digestibility effects of LA and FP and their interaction using of N but a limited capacity to improve markers of the MIXED procedure of SAS. Lactose improved intestinal function. Key words: intestinal enzymes, intestinal morphology, intestinal permeability, nursery pigs, weaning stress. The authors thank Diamond V Mills for financial sup- port. Appreciation is also expressed to DSM nutritional products and Ajinomoto Heartland, Inc., for in-kind con- tributions to the Applied Swine Nutrition Program at Iowa State University. Corresponding author: jfp@iastate.edu Received October 25, 2019. Accepted April 15, 2020. 1 Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. © The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2020.4:1-14 doi: 10.1093/tas/txaa045 INTRODUCTION MATERIALS AND METHODS Weaning is one of the most difficult transi- All experimental procedures adhered to guide- tions in the life of a pig and greatly impacts the lines for the ethical and humane use of animals for productivity of swine operations. Exposure to research according to the Guide for the Care and human handling, a new physical environment, a Use of Laboratory Animals (FASS, 2010) and were different diet, and new social interactions can re- approved by the Institutional Animal Care and sult in reduced feed intake, decreased growth, and Use Committee at Iowa State University (number an increased incidence of disease (Jones et  al., 7-15-8049-S). 2012; McLamb et al., 2013). The response of the intestinal mucosa to weaning is a particularly im- Animals Housing and Experimental Design portant concern (Smith et al., 2010). As an imma- ture tissue in the young pig, it suffers some degree A total of 28 barrows [5.22  ± 0.15  kg body of inflammation and dysfunctionality in response weight (BW); the progeny of C22 or C29 sows × to the new antigens, dietary components, and so- 337 terminal sires; PIC Inc., Hendersonville, TN] cial stressors (Boudry et al., 2004; Pié et al., 2004; were blocked by initial BW (seven blocks) and ran- Li et al., 2019). domly assigned to individual metabolism crates Feed has tremendous potential to improve the within block. Crates were randomly assigned to weaning transition because it is the most practical one of four dietary treatments (n = 7 pigs per treat- means of delivering substances with beneficial ment) corresponding to a 2  × 2 factorial design: nutritional and functional properties to the pig. with (LA+; 15% inclusion) or without (LA−) LA Although dietary antibiotics have been effectively and with (FP+) or without (FP−) the prototype FP used to control and prevent diseases in nursery (1 g of FP per kilogram of diet; Diamond V, Cedar pigs, antibiotic resistance and consumer pressure Rapids, IA). have demanded a reduction in their use. Therefore, Diets were manufactured in mash form at the evaluation of products and dietary components the Swine Nutrition Farm feed mill (Iowa State with functional and nutritional properties is in- University; Ames, IA; Table  1). Dietary levels of creasingly important. amino acids, vitamins, and minerals were set to Lactose (LA) is a common carbohydrate in meet the nutrient requirements of nursery-aged nursery diets as it provides a familiar source of pigs (NRC, 2012). Titanium dioxide was added at available carbohydrate and induces the produc- 0.4% to all diets as an indigestible marker. tion of volatile fatty acids (VFA) in the large in- Pigs were housed in a controlled environment testine (Pierce et  al., 2006). On the other hand, facility. Each metabolism crate (0.53 × 0.71 m) was a prototype Lactobacillus Acidophilus fermenta- equipped with a fully slatted floor, a stainless-steel tion product (FP; Diamond V, Cedar Rapids, IA) feeder, and a cup drinker. Pigs had ad libitum ac- is believed to provide metabolites that enhance cess to feed and water during the entire experi- intestinal health and the establishment of com- mental period (15 d). mensal microorganisms. The objective of this study was to identify Sample Collection and characterize the beneficial effects of LA and the FP in nursery pig diets with an emphasis on To measure in vivo gut permeability, all pigs intestinal function. We hypothesized that LA were fasted for 6  h and then orally dosed on day and FP would ameliorate some of the adverse 5 with a solution of 0.300  g lactulose/kg BW and effects of the weaning transition by promoting 0.030 g mannitol/kg BW (Sigma-Aldrich, St Louis, digestion and decreasing markers of intestinal MO). Urine was collected for the following 12  h dysfunction. in a plastic jug located under the crate after the Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Table 1. Ingredient composition of the experimental diets* LA− LA+ Item FP− FP+ FP− FP+ Corn 65.73 65.63 50.58 50.48 Soybean meal 20.00 20.00 20.00 20.00 Fish meal 3.72 3.72 3.72 3.72 Casein 5.00 5.00 5.00 5.00 Lactose — — 15.00 15.00 Soybean oil 1.50 1.50 1.50 1.50 -Lys HCl 0.40 0.40 0.43 0.43 -Met 0.15 0.15 0.20 0.20 DL -Thr 0.15 0.15 0.18 0.18 -Trp 0.01 0.01 0.02 0.02 Monocalcium phosphate 21% 1.14 1.14 1.20 1.20 Limestone 0.90 0.90 0.88 0.88 NaCl 0.50 0.50 0.50 0.50 Vitamin premix 0.25 0.25 0.25 0.25 Trace mineral premix 0.15 0.15 0.15 0.15 Prototype FP — 0.10 — 0.10 Titanium dioxide 0.40 0.40 0.40 0.40 *LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of prototype FP added (1 g of FP per kilogram of diet; Diamond V Mills, Cedar Rapids, IA) added. Provided per kilogram of diet: 7,600 IU of vitamin A; 875 IU of vitamin D3; 62 IU of vitamin E; 3.7 mg of menadione (to provide vitamin K); 61 μg of vitamin B12; 14 mg of riboflavin; 34 mg of d-pantothenic acid; and 70 mg of niacin. Provided per kilogram of diet: 165 mg of Fe (ferrous sulfate); 165 mg of Zn (zinc sulfate); 39 mg of Mn (manganese sulfate); 2 mg of Cu (cooper sulfate); 0.3 ppm of I (calcium iodate); and 0.3 ppm of Se (sodium selenite). administration of the lactulose/mannitol solution. before each collection to minimize N losses due to Before urine collections, five drops of chlorhexidine volatilization of ammonia-N. Total urine output gluconate 20% (weight/volume; Sigma-Aldrich, St was weighed and stored at −20  °C. At the end of Louis, MO) were added to each jug to inhibit bac- the collection period, urine was homogenized and terial activity. After collection, total urine output filtered prior to N analysis. Urine samples were ali- was weighed, homogenized, and filtered using fiber quoted into 250-mL plastic bottles and stored at glass wool; a 3-mL aliquot of urine sample was col- −20  °C until chemical analyses were performed. lected and stored at −20 °C for later analysis. In the determination of N balance, only the days In support of determining digestibility and ni- 10–13 fecal samples were utilized to correspond trogen (N) balance, and to ensure the acquisition of with the urine samples as described above. samples fully representative of the total batch, 10 All pigs were euthanized on day 15 by captive feed samples were collected from the feed mill at the bolt stunning followed by exsanguination. After time of diet preparation; these samples were thor- euthanasia, each pig was dissected and the entire oughly homogenized, pooled into one subsample, intestinal tract was removed. Samples (~20  cm and ground through a 1-mm screen in a Retsch sections long) of the proximal jejunum (taken grinder (Model ZM1, Retsch Inc., Newton, PA). 100  cm from the pyloric sphincter), the distal Total fecal output was collected twice daily from day ileum (taken 30  cm from the ileocecal valve), and 5 to 8 and day 10 to 13 and stored at −20 °C. Once the mid colon were removed and gently flushed collected, fecal samples were thawed, homogenized, with ice-cold Krebs buffer. One segment of the subsampled, dried in an oven at 65  °C to constant jejunum and ileum samples were snap-frozen in weight (Jacobs et  al., 2011), and ground through liquid N and stored at –80  °C until further ana- a 1-mm screen in a Wiley grinder (Model ED-5, lysis. Additionally, subsamples of jejunum, ileum, Thomas Scientific Inc., Swedesboro, NJ). Ground and colon were fixed for 24 h in 10% neutral buf- fecal and feed samples were stored in plastic bags in fered formalin and then transferred to 75% alcohol desiccator cabinets until assays were completed. for later morphology and histochemistry analysis. For N balance, total urine output was collected Approximately 20  mL of intestinal contents from twice daily during days 10–13 (96 h) into plastic jugs the mid-section of the colon were snap-frozen in containing 5  mL of 6 N hydrochloric acid, added liquid N and stored at –80 °C. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. ,† Table 2. Chemical composition of the experimental diets, as-fed basis* LA− LA+ Item FP− FP+ FP− FP+ Analyzed chemical composition DM, % 88.88 88.76 90.75 90.92 GE Mcal/kg 3.92 3.92 3.94 3.90 AEE, % 4.71 4.70 4.41 4.43 CP, % 21.00 21.06 20.03 20.28 Titanium dioxide, % 0.40 0.41 0.42 0.41 Calculated chemical composition SID of AA, % Lys 1.44 1.44 1.44 1.44 Thr 0.84 0.84 0.84 0.84 Met 0.55 0.55 0.57 0.57 Met + Cys 0.79 0.79 0.79 0.79 Trp 0.24 0.24 0.24 0.24 Ile 0.82 0.82 0.79 0.79 Val 0.96 0.96 0.92 0.92 Ca, % 0.82 0.82 0.82 0.82 Total P, % 0.70 0.70 0.67 0.67 STTD of P, % 0.42 0.42 0.42 0.42 AA, Amino acids; AEE, acid hydrolyzed ether extract; CP, crude protein; SID, standardized ileal digestibility; STTD, standardized total tract digestible. *LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. AA levels, phosphorus, STTD phosphorus, and calcium were calculated from NRC (2012); all other values were analyzed. Digestibility, N Balance Analysis, and Urinary mannitol concentrations were determined by High- Lactulose and Mannitol performance liquid chromatography with known standard solutions according to the method de- Feed, fecal, and urine samples were analyzed scribed by Kasangra et al. (2003). at the Monogastric Nutrition Laboratory (Iowa State University, Ames, IA). Feed (Table  2) and Intestinal Morphology and Histochemistry Analysis fecal samples were assayed for dry matter (DM; method 930.15; AOAC, 2007). Gross energy (GE) Fixed jejunum, ileum, and colon samples were was determined using a Parr model 6200 isoper- prepared and stained at the Veterinary Diagnostic ibolic bomb calorimeter (Parr Instrument Co., Laboratory (Iowa State University, Ames, IA). Each Moline, IL); benzoic acid (6,318 kcal GE/kg; Parr sample was sliced into 5-μm sections. The jejunum Instruments, Moline, IL) was used as a standard for and ileum sections were stained using the hema- calibration and was determined to contain 6,319 ± 1 toxylin and eosin procedure, whereas colon samples kcal GE/kg. Titanium dioxide was determined col- were stained with the alcian blue-periodic acid-Schiff orimetrically using a Synergy 4 spectrophotometer (AB-PAS) procedure (Mowry, 1963) to distinguish (BioTek, Winooski, VT) according to the method between neutral and acidic mucin types in colon of Leone (1973). Additionally, feed samples were crypts. The neutral mucin was stained magenta, and assayed for acid hydrolyzed ether extract (AEE; acidic mucin was stained blue. The mixture of neu- method 2003.06; AOAC International, 2007) using tral-acidic mucins showed purple, magenta-purple, a SoxCap SC 247 hydrolyzer and a Soxtec 255 sem- or blue-purple colors in goblet cells. iautomatic extractor (FOSS North America, Eden Images of intestinal mucosa were captured using Prairie, MN). The N content of the feed, fecal, a light microscope (DMI3000 B Inverted Microscope, and urine samples was determined by thermo- Leica Microsystems, Bannockburn, IL) with an at- combustion (method 990.03; AOAC International, tached camera (12-bit QICAM Fast 1394, QImaging, 2007; Leco TruMac N, LECO Corporation, St. Surrey, BC, Canada). Individual images of villi and Joseph, MI). The standard for calibration was crypts were taken using Q-capture Pro 6.0 soft- Ethylenediaminetetraacetic acid (9.56% N; Leco ware (QImaging, Surrey, BC) and measured using Corporation, St. Joseph, MI) and determined to Image-Pro Plus 7.0 (Media Cybernetics, Bethesda, contain 9.56  ± 0.01% N). Urinary lactulose and MD). Images were measured using Image J software, Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs version 1.48s (Rasband W, National Institutes of using a Nanodrop 1000 instrument (NanoDrop Health, MD). The villus height was measured from Technologies, Rockland, DE). The purity was as- the tip of the villus to the crypt–villus junction, and sessed by determining the ratio of the absorbance at the crypt depth was measured from the crypt–villus 260 and 280 nm (all samples had absorbance ratios junction to the base of the crypt. At least 10 well-de- above 1.8). One microgram of RNA was transcribed fined villi and associated crypts from each sample of in a reaction combining genomic DNA elimination each intestinal segment were measured; averages were using a commercially available kit (Quantitect re- then calculated and reported as one number per pig. verse transcription kit; Qiagen Inc., Valencia, CA). The mucin area was calculated as a percentage of the The resulting complementary DNA was quanti- total mucosal tissue area in each image. fied using a NanoDrop 1000 instrument and ap- plied to quantitative real-time PCR (qPCR). The qPCR was completed using a BioMark HD system Enzyme Activity and Secretory Immunoglobulin A (S-IgA) (Fluidigm Corporation, San Francisco, CA). No reverse transcriptase control samples were included Mucosal scrapings (~0.5  g) of the jejunum and in the extraction process to assure no genomic DNA ileum were added to 4.5  mL of Phosphate-buffered contamination. Complementary DNA was used saline buffer containing a protease inhibitor cocktail for specific target amplification using the TaqMan (SKU, P8340; Sigma-Aldrich, St. Louis, MO) and PreAmp Master Mix (Life Technologies, Carlsbad, triton (0.1%). The resulting solution was homogen- CA) and loaded onto Fluidigm’s Dynamic Array ized and centrifuged at 10,000 × g for 15 min at 4 °C. Integrated Fluidic Circuits according to Fluidigm’s The supernatant was extracted and stored in aliquots. EvaGreen DNA binding dye protocols. Gene sym- The total protein concentration of hydrolyzed mucosa bols and primer sequences are listed in Table 3. One scrapings was quantified using a Pierce Bicinchoninic 48.48 Dynamic Array, Integrated Fluidic Circuit Acid Protein Assay Kit (BCA; Thermo Scientific, plate, was used to analyze mRNA abundance of Woltham, MA). Disaccharidase activity was de- selected genes in porcine jejunal and ileal tissues. To termined according to Dahlqvist (1964) using LA, select an endogenous reference gene, ribosomal pro- maltose, and sucrose as substrates. Alkaline phos- tein L19 (RPL19) was included in the qPCR array. phatase activity was determined using a porcine al- kaline phosphatase assay kit (ABCAM, Cambridge, VFA Concentration MA). Enzyme activity was expressed as micromole of hydrolyzed substrate per minute per gram of tissue In preparation for VFA analysis, 1.0 g of colon protein. The concentration of secretory IgA was de- contents were mixed with 2.5  g of purified water termined using a porcine IgA ELISA kit (E101-102; and 1 mL was extracted from the homogenized so- Bethyl Laboratories, Inc., Montgomery, TX) fol- lution. Then 0.2  mL of 25% metaphosphoric acid lowing the manufacturer’s directions. (used to deprotonize the samples) and 0.1  mL of 4-methylvaleric acid (as an internal standard) were RNA Extraction and Real-Time PCR added to the extracted homogenate. These samples were centrifuged at 12,000 × g for 25 min, and the RNA was extracted from frozen jejunal and ileal supernatant was removed for analysis. Volatile fatty tissues according to the Trizol protocol (Invitrogen, acids were determined using a model 3900 gas chro- Grand Island, NY). The purity and quantity of matograph fitted with a CP 8400 automatic injector RNA were determined by spectrophotometry (Varian Analytical Instruments, Walnut Creek, Table 3. Primers used for quantitative reverse transcription-PCR Primer sequence, 5ʹ→3 ʹ Gene name Accession number Sense (forward) Anti-sense (reverse) RPL19 AF435591 AACTCCCGTCAGCAGATCC AGTACCCTTCCGCTTACCG OCLN NM_001163647 TCGTCCAACGGGAAAGTGAA ATCAGTGGAAGTTCCTGAACCA CLDN3 NM_001160075 TTGCATCCGAGACCAGTCC AGCTGGGGAGGGTGACA IL-6 AF518322 GGCTGTGCAGATTAGTACC CTGTGACTGCAGCTTATCC IL-10 L20001 TGGGTTGCCAAGCCTTGT GCCTTCGGCATTACGTCTTC IL-17a AB102693 CCAGACGGCCCTCAGATTAC CACTTGGCCTCCCAGATCAC IL-22 AY937228 AAGCAGGTCCTGAACTTCAC CACCCTTAATACGGCATTGG TNFα X54859 AACCCTCTGGCCCAAGGA GGCGACGGGCTTATCTGA Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. –ΔΔCt CA) using a 30 m, 0.25 mm, and 0.25 μm column calculated as 2 , in which ΔΔCt represents ΔCt (DB-FFAP, Agilent, Santa Clara, CA). Helium was sample − ΔCt calibrator (Livak and Schmittgen, used as carrier gas. Purified VFA samples (Sigma- 2001). Aldrich, St. Louis, MO) were used for the identifi- cation of VFA peaks. Statistical Analysis Data were analyzed using the following model: Calculations y = μ + τ + λ +(τλ) + δ + ijkl i j k ijkl ij Individual pig weights and feed disappear- where y represents the observed value, μ is the ance were measured on days 0 and 14 to calculate ijkl overall mean, τ represents the fixed effect of LA average daily gain (ADG), average daily feed intake [i = 1, 2 (LA− and LA+, respectively)], λ represents (ADFI), and gain to feed ratio (G:F). Apparent the fixed effect of FP [j = 1, 2 (FP− and FP+, re- total tract digestibility (ATTD) of DM, GE, and spectively)], τλ represents the interaction between N was calculated using the following equation LA and FP, δ represents the random effect of block (Oresanya et al., 2008): (k = 1–7), and ϵ is the random error associated with ATTD, % =[100 [100 × (% TiO2 in feed/% TiO2 in feces) y (l = 1–7) assuming δ ~Ν (0, ) and ϵ ~Ν (0, ). ijkl × (concentration of component in feces/ The pig was the experimental unit for all ana- concentration of component in feed)]] lyses. The UNIVARIATE procedure of SAS ver- sion 9.3 (SAS Inst., Inc., Cary, NC) was used to N intake (g/d) was calculated by multiplying the verify the normality and homogeneity of the re- percentage of N in the feed (DM basis) by DM sidual variance from the reported models. Jejunal feed intake (g/d); N excreted in the urine was calcu- and ileal mucosa S-IgA, mannitol, and lactulose re- lated by multiplying the average daily urine weight covery and lactulose:mannitol ratio variables were (kg/d) by the average urinary N concentration (%). analyzed and reported after log-transformation to Nitrogen excreted in feces (g/d) was calculated by satisfy normality. The models were analyzed using multiplying daily N intake minus the product of the MIXED procedure of SAS. Differences were multiplying N intake times the ATTD of N (%). considered significant with P-values ≤0.050, and Total N excretion (g/d) was calculated as the sum P-values between 0.050 and 0.100 were considered of daily N excreted in urine and feces. Finally, N re- trends. tention was calculated as daily N intake minus daily N excretion. Protein retention was calculated as N RESULTS retention (g/d) × 6.25 divided by the ADG (g/d). All N balance variables were calculated using data Growth Performance, Digestibility, and N Balance from samples collected on days 10–14. Evaluating the impact of dietary treatments on Lactulose and mannitol recoveries (%) were growth performance in metabolism crates is diffi- calculated as the amount of these sugars excreted cult due to the typically small number of animals (g/d) in the urine [calculated as total urine weight and the conditions that differ substantially from (g) times lactulose or mannitol concentration (%)] normal housing. Nonetheless, it is useful to do so to divided by the amount of lactulose or mannitol provide an understanding of the state of the animal given (g/d) orally times 100. Lactulose:mannitol under which other measurements were recorded. ratio was calculated by dividing lactulose recovery No interactions between LA and FP were ob- by mannitol recovery (Musa et al., 2019). served for initial BW, final BW, growth performance, Plates for PCR analysis were balanced so that digestibility, or N balance variables. Therefore, the an equal number of treatments were represented on results of these variables are presented as the main each plate. Additionally, a pooled control sample effects. By design, the initial BW was not affected representative of all treatment groups was run on by the addition of LA or FP (Table  4). Similarly, each gene as an internal control. Normalized ex- final BW (day 14)  and ADG were not affected by pression (ΔCt) for each sample was determined the addition of LA or the FP. Although there was using RPL19 as an endogenous housekeeping gene. no effect of FP on ADFI, pigs fed LA had increased The average normalized expression of the pooled ADFI (P = 0.017). control sample was used as the calibrator to calcu- The addition of LA improved the ATTD of late relative gene expression (Livak and Schmittgen, DM and GE (P = 0.014 and P = 0.028, respectively; 2001). For each sample, relative expression was Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Table 4.  Effect of LA and a prototype Lactobacillus acidophilus FP on growth performance of nursery ,† pigs* LA FP P-value Item − + − + SEM LA FP Day 0 BW, kg 5.23 5.23 5.22 5.23 0.06 0.744 0.334 Day 14 BW, kg 7.23 7.78 7.70 7.30 0.26 0.105 0.216 ADG, kg 0.148 0.189 0.184 0.153 0.018 0.102 0.205 ADFI, kg 0.180 0.229 0.218 0.190 0.016 0.017 0.150 G:F 0.787 0.809 0.822 0.774 0.043 0.731 0.445 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment). LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 5. Effect of LA and a prototype Lactobacillus acidophilus FP on ATTD of DM, GE, and N in nur- ,† sery pigs* LA FP P-value Item − + − + SEM LA FP ATTD of DM, % 88.1 89.2 88.4 88.9 0.4 0.014 0.167 ATTD of GE, % 86.4 87.7 86.7 87.4 0.5 0.028 0.206 ATTD of N, % 83.8 83.6 82.9 84.5 0.9 0.900 0.090 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment), diets contained titanium dioxide (0.4%) as an indigestible marker, and total fecal output was collected twice daily from day 5 to 8 and day 10 to 13. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of prototype FP (1 g of FP per kilogram of diet) added. Table  5). However, the addition of the FP did not N retained. The addition of LA increased the pro- affect the ATTD of DM or GE. There were no ef- tein retained as a percentage of ADG (P = 0.020), fects of LA on the ATTD of N. However, the add- whereas FP tended to increase the protein retained ition of the FP tended to increase the ATTD of N as a percentage of ADG (P = 0.053). The protein (P = 0.090). retained expressed as a percentage of ADG is a The addition of LA tended to increase N in- useful standard to validate N retention data—it take (P  =  0.080), whereas no effect of FP was should be in the range of 15–18% for pigs of this observed (Table  6). In contrast, the FP tended age—and is also a very good indicator of the lean to decrease N excretion (P = 0.060), whereas LA gain in the pigs. did not affect N excretion. When N excretion was partitioned into fecal or urinary components, Intestinal Morphology, Mucin Staining Area, and the addition of LA increased fecal N excretion Concentration of S-IgA (P  =  0.017), while FP decreased it (P  =  0.044). The addition of LA decreased urinary N and in- The morphology of the small intestine was af- creased the overall N retention (P  =  0.006 and fected by the interaction between LA and FP, so P  =  0.043, respectively). The addition of FP did individual treatment means are presented. In the not affect urinary N excretion or overall N reten- jejunum, pigs fed LA+FP− had increased villus tion, expressed as g/d. height compared with those fed LA+FP+ and LA− The addition of LA decreased the percentage FP−, whereas LA-FP+ was intermediate (Table 7; of N excreted. There was no effect of LA on the interaction P  =  0.034). In the terminal ileum, percentage of N excreted in the feces, but it de- pigs fed LA−FP+ and LA+FP− had increased creased the percentage of N excreted in the urine villus height compared with those fed LA−FP−, (P  =  0.002). As a result, the addition of LA in- whereas LA+FP+ was intermediate (interaction creased the percentage of N retained (P  =  0.043). P  =  0.007). Although neither LA nor FP altered The addition of FP had no effect on the total per- villus:crypt ratio in the jejunum, an interaction centage of N excreted or the percentage of N ex- between LA and FP affected villus:crypt ratio in creted in the urine and the feces. Consequently, the the ileum; pigs fed LA−FP+ and LA+FP− had addition of FP had no effect on the percentage of increased villus:crypt ratio compared with those Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. ,† Table 6. Effect of LA and a prototype Lactobacillus acidophilus FP on N balance of nursery pigs* LA FP P-value Item − + − + SEM LA FP N balance, g/d Intake 7.57 9.28 8.80 8.06 0.65 0.072 0.414 Excretion 2.85 2.88 3.09 2.64 0.19 0.901 0.060 Fecal 1.16 1.59 1.55 1.20 0.15 0.017 0.044 Urine 1.68 1.29 1.54 1.44 0.09 0.006 0.468 Retention 4.72 6.41 5.71 5.42 0.56 0.043 0.716 N balance % Excretion 38.7 32.1 35.8 35.0 2.1 0.043 0.804 Fecal 15.0 17.3 17.4 14.9 1.1 0.163 0.120 Urinary 23.7 14.8 18.3 20.2 1.7 0.002 0.445 Retention 61.3 67.9 64.2 65.0 2.1 0.043 0.804 Protein retained as % of ADG 15.0 17.0 15.2 16.8 0.6 0.020 0.053 *A total of 28 barrows (5.22 ± 0.15 kg BW) was assigned to individual metabolism crates for 15 d (seven pigs per treatment); total urine and fecal output was collected twice daily during days 10–13 (96 h). ADG was calculated using weights collected on days 10 and 14. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 7. Effect of LA and a prototype Lactobacillus acidophilus FP on intestinal morphology, S-IgA, and ,† mucins of nursery pigs* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Jejunum b ab a b Villus height, μm 275 320 390 307 28 0.089 0.511 0.034 Crypt depth, μm 313 338 343 326 21 0.677 0.848 0.321 Villus: crypt 0.88 1.00 1.15 0.95 0.11 0.321 0.682 0.160 a b b b S-IgA, (log) μg/mg of protein 3.04 2.41 2.59 2.64 0.14 0.637 0.065 0.005 Ileum b a a ab Villus height, μm 266 341 378 317 25 0.065 0.780 0.007 Crypt depth, μm 262 247 263 274 16 0.332 0.891 0.391 b a a ab Villus: crypt 1.02 1.43 1.48 1.19 0.12 0.351 0.591 0.007 S-IgA, (log) μg/g of protein 2.62 2.76 2.71 2.70 0.12 0.939 0.610 0.520 Colon Crypt depth, μm 435 395 440 420 24 0.502 0.189 0.645 Goblet cells/100 μm crypt 5.8 6.2 6.0 5.3 0.7 0.720 0.782 0.263 Acid mucins, % 11.4 11.0 12.8 7.3 1.4 0.437 0.053 0.087 Mixed mucins, % 12.7 10.7 13.7 11.8 2.0 0.597 0.318 0.979 a b ab a Neutral mucins, % 2.6 1.6 2.1 3.1 0.4 0.175 0.938 0.010 a,b Means with different superscripts significantly differ (P < 0.050). *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (7 pigs/treatment), intestinal tissues, and digesta were collected after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. fed LA−FP−, whereas LA+FP+ was intermediate percentage of neutral mucins compared with those (interaction P  =  0.007). Neither LA nor FP af- fed LA−FP+, whereas LA+FP− was intermediate fected crypt depth at the jejunum or at the ileum. (interaction P = 0.010). In the mid colon, there were no effects of LA The concentration of S-IgA was affected by the or FP on crypt depth, the number of goblet cells interaction of LA and FP. Pigs fed LA−FP− had per crypt, or the percentage of acid or mixed increased levels of mucosal S-IgA compared with mucins. However, there was an interaction between the other three treatments (interaction P  =  0.005). LA and FP on the percentage of neutral mucins: However, neither LA nor FP impacted the concen- pigs fed LA−FP− and LA+FP+ had an increased tration of S-IgA at the terminal ileum. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs Small Intestine Permeability, Enzymatic Activity, increase total VFA (P  =  0.099), whereas the add- and Transcript Abundance of Tight Junction ition of FP decreased total VFA concentration in Proteins and Cytokines colonic contents (P = 0.048). When VFAs were looked at as proportions of There were no main effects or interactions of LA the total concentration, the percentages of acetic, or FP on mannitol recovery, lactulose recovery, or propionic, butyric, and valeric acid were not af- in the lactulose:mannitol ratio (Table 8). Similarly, fected by the addition of LA or FP. However, activities of lactase, sucrase, maltase, or alkaline LA and FP tended to interact for the percentages phosphatase were not affected by the addition of of branched-chain fatty acids; pigs fed LA−FP+ LA or FP (Table 9). Likewise, gene transcript abun- tended to have increased percentages of isobutyric dance of tight junction protein genes [occludin and isovaleric acid compared with LA−FP− and (OCLN) and claudin-3 (CLDN3); Table 10] in the LA+FP+, whereas LA+FP− was intermediate jejunum and terminal ileum and ileal cytokines (interaction P = 0.069 and P = 0.099, respectively). [tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), IL-10, IL-17, and IL-22] were not affected DISCUSSION by the addition of LA or FP. One of the most important strategies to ameli- orate the impact of weaning stress is the use of VFA Concentrations in the Colon specialty ingredients and feed additives targeted to possess nutritional, as well as functional, properties. Although LA had no effect, the addition of FP The primary target of these functional properties decreased the concentration of acetic acid in the is the gastrointestinal tract. The first experimental colon (Table 11; P = 0.022). Neither LA nor FP af- approach herein was to evaluate the effect of LA fected the concentration of propionic acid. Pigs fed and FP on digestibility and N retention while at LA tended to increase butyric acid concentration the same time monitoring feed intake and growth (P = 0.062), but there was no effect when adding FP. performance. These would represent key outcomes There were no effects of LA or FP on the concen- that would potentially be improved if negative ef- tration of branched-chain fatty acids (isobutyric, fects of weaning are reduced; more specifically, the isovaleric, and valeric acid). Pigs fed LA tended to Table 8. Effect of LA and a prototype Lactobacillus acidophilus FP on small intestinal permeability at day ,† 5 postweaning* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Mannitol recovery, (log) % 1.41 1.29 1.37 1.51 0.12 0.435 0.942 0.281 Lactulose recovery, (log) % −0.14 −0.16 −0.38 −0.17 0.16 0.432 0.553 0.479 Lactulose: mannitol (log) −1.54 −1.55 −1.66 −1.66 0.16 0.530 0.977 0.956 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment). Intestinal permeability was assessed by providing each pig a solution of 0.300 g lactulose/kg BW and 0.030 g mannitol/kg BW (Sigma-Aldrich, St Louis, MO). Urine was collected for the following 12 h after the administration of the lactulose/mannitol solution. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 9. Effect of LA and a prototype Lactobacillus acidophilus FP on the enzymatic activity at the jejunum* LA− LA+ Contrast P-value Item FP- FP+ FP- FP+ SEM LA FP LA × FP Activity, U/mg protein Lactase 16.6 10.6 15.7 14.7 3.6 0.976 0.335 0.222 Sucrase 20.9 25.5 31.2 24.3 5.1 0.387 0.847 0.511 Maltase 182.7 190.7 206.2 228.4 22.4 0.116 0.427 0.708 Alkaline phosphatase 60.7 60.6 65.4 53.1 6.4 0.833 0.340 0.348 *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); jejunal tissue was col- lected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. Table 10. Effect of LA and a prototype Lactobacillus acidophilus FP on RNA abundance of tight junction ,†,‡ proteins and cytokines in the jejunum and ileum* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Jejunum OCLN 1.23 1.44 2.08 1.20 0.37 0.394 0.352 0.139 CLDN3 2.33 1.56 1.63 1.76 0.40 0.550 0.448 0.285 Ileum OCLN 1.67 1.49 1.10 1.09 0.36 0.223 0.892 0.569 CLDN3 1.59 1.48 0.87 1.28 0.36 0.565 0.873 0.353 TNFα 1.03 1.37 1.14 1.36 0.27 0.797 0.146 0.727 IL-6 0.61 0.79 1.17 0.99 0.24 0.102 0.839 0.324 IL-10 0.87 1.04 1.23 1.15 0.26 0.264 0.801 0.545 IL-17 0.80 1.03 1.15 0.85 0.18 0.618 0.872 0.137 IL-22 1.08 1.35 1.14 0.77 0.54 0.900 0.543 0.238 *All values indicate relative expression of genes. Normalized expression (ΔCt) for each sample was determined using RPL19 as an endogenous control gene. The average normalized expression of the pooled control sample was used as the calibrator to calculate relative gene expression. For −ΔΔCt each sample, relative expression was calculated as 2 , in which ΔΔCt represents ΔCt sample − ΔCt calibrator (Livak and Schmittgen, 2001). A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); intestinal tissue was collected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. Table 11. Effect of LA and a prototype Lactobacillus acidophilus FP on volatile fatty acid concentrations ,† in colonic contents* LA− LA+ P-value Item FP− FP+ FP− FP+ SEM LA FP LA × FP Concentration μM/g Acetic acid 63.77 49.76 71.53 58.17 6.07 0.292 0.022 0.767 Propionic acid 24.14 18.48 26.86 23.13 3.00 0.232 0.132 0.749 Butyric acid 15.16 14.56 20.60 20.19 2.81 0.062 0.858 0.974 Isobutyric acid 2.21 2.34 3.21 2.07 0.44 0.522 0.201 0.129 Isovaleric acid 3.46 3.69 4.97 3.23 0.71 0.468 0.303 0.184 Valeric acid 3.30 3.21 4.65 4.36 0.81 0.173 0.676 0.819 Total 115.1 92.1 131.8 111.2 10.3 0.099 0.048 0.910 Distribution, % Acetic acid 58.1 55.1 54.3 52.8 3.2 0.330 0.471 0.810 Propionic acid 20.8 19.6 20.3 20.7 1.5 0.850 0.804 0.591 Butyric acid 13.2 15.1 15.6 17.7 1.6 0.140 0.227 0.945 y x xy y Isobutyric acid 1.9 2.6 2.4 1.9 0.3 0.729 0.761 0.069 y x xy y Isovaleric acid 3.0 4.1 3.8 3.0 0.5 0.740 0.842 0.099 Valeric acid 2.9 3.5 3.7 3.9 0.7 0.660 0.405 0.559 x,y Means with different superscripts tend to differ (P < 0.100). *A total of 28 barrows (5.22 ± 0.15 kg BW) assigned to individual metabolism crates for 15 d (seven pigs per treatment); intestinal tissue was collected at day 15 after euthanasia. LA−: diets without lactose added; LA+: diets with 15% of lactose added; FP−: diets without the prototype FP (Diamond V Mills, Cedar Rapids, IA) added; FP+: diets with 0.1% of the prototype FP (1 g of FP per kilogram of diet) added. consumption of specialty ingredients may ameli- stimulate appetite are highly desirable. Lactose is orate stress on the intestinal tract. the primary dietary carbohydrate consumed be- Postweaning anorexia has profound negative fore weaning (~5% in sow milk; Rosero et al., 2015) effects on the growth and health of nursery pigs and is believed to be highly palatable to the nursery (Spreeuwenberg et al., 2001) and is the main factor pig. Results of this experiment and other research affecting subsequent growth performance (Jones (Tokach et al., 1989; Kim et al., 2010; Tran et al., et  al., 2012). Therefore, dietary compounds that 2012; Pierce et al., 2006) support the use of LA in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Specialty ingredients for weaned pigs the starter diet to enhance feed intake during the improving the digestibility of DM and GE and postweaning period. N retention, as well as improving feed intake. The In addition to stimulating appetite, nursery addition of FP had a more modest impact on the diets are designed to be highly digestible. This is be- digestibility of N, with no detectable improvement cause feed intake and gut capacity of weaned pigs in growth. is limited (Dong and Pluske, 2007). Additionally, The second experimental approach was to ex- the digestibility of nutrients is a key determinant of plore the effects of LA and FP on specific markers the growth response of nursery pigs after weaning of intestinal function. Recovery from weaning (Jones and Patience, 2014). The addition of LA in- stress by the intestinal tissue has been described creased the digestibility of DM and GE. These same to occur in two phases: the first is an acute phase, results were reported by Jin et al. (1998) by adding lasting 2–4 d, in which the major dysfunctional 20% of LA to nursery pigs and by Pierce et al. (2005) changes occur, including the disruption of barrier feeding increased levels of LA (0–11%) to finishing function and immune activation (including inflam- pigs. Compared with corn, LA has a much simpler mation; Pié et  al., 2004; Smith et  al., 2010). The chemical and physical structure that is readily avail- subsequent adaptive phase, lasting about 2  wk able to be cleaved by lactase at the brush border of after the acute phase, is associated with tissue re- the small intestine and absorbed by the enterocytes covery (Montagne et  al. 2007). The length of the as galactose and glucose. Additionally, LA escap- acute phase and a proper recovery of the intes- ing digestion is rapidly utilized by bacterial popu- tinal tissue after weaning are key determinants lations in the distal small intestine, as well as in the of the performance and the health parameters of large intestine (Bach Knudsen, 2012). Thus, the re- nursery pigs and potentially their performance sults of this experiment confirm that LA contrib- in subsequent growth stages. Therefore, the add- utes to the objective of making nursery diets highly ition of functional products and specialty ingredi- digestible by providing readily usable energy and an ents targeted to ameliorate the weaning transition easily fermentable substrate. should help the pig recover from one or more of Protein deposition is the most valued compo- these dysfunctional changes. One of the most im- nent of the total weight gain of the nursery pig (de portant targets is the speed of barrier function res- Vries and Kanis, 1994; Colina et al., 2010). In this titution after weaning (Wijtten et  al., 2011). The experiment, the addition of LA increased overall results of this experiment suggest no effect of LA N retention, the percentage of N retained, and the and FP on the permeability of the small intestine protein retained as the percentage of the ADG. The at the end of the acute phase of weaning (day 5). increase in overall N retention can be explained Likewise, no effects on the RNA abundance of largely by the increase in feed intake in the LA-fed CLDN3 and OCLN—essential components of pigs since nutrient supply is the main factor limit- the tight junction complex (Saitou 1997; Niewold, ing protein deposition in nursery pigs (Van Milgen 2015)—or on the mRNA abundance of proinflam- et  al., 2000). Interestingly, pigs fed LA not only matory cytokines in the ileum (IL-6, IL-10, IL-22, had greater protein deposition but were more effi- and TNFα) were observed in the small intestine cient in using N toward protein deposition. These at the end of the adaptive phase of weaning (day results agree with those obtained by Pierce et  al. 14). Proinflammatory cytokines are expected to be (2005), who fed a LA supplement to finishing pigs. upregulated when immune cells are recruited (Renz Interestingly, they also observed that LA improves et  al., 2012). No effects of LA on these variables N utilization by decreasing the percentage of N ex- have been found in the literature, but the FP has creted in the urine and not through an increase in been shown to decrease proinflammatory medi- N digestibility. ators (such as TNFα, interferon gamma, and IL-6, Independent of the responses to LA, the add- IL-8, and IL-1-beta-1) under an LPS challenge ition of FP tended to increase the digestibility of N (Lee et  al., 2016). An alternative explanation of and the protein retained as a percentage of ADG; these results is that the weaning stress experienced this suggested that the mode of action of FP may by these pigs, not associated with clinical signs of be related to improving the ability of the pig to di- disease, was mild (Pié et al., 2004: Montagne et al., gest dietary protein. 2007), resulting in a limited window of chance for Overall, the results of the data on nutrient di- improvement of makers of barrier function and in- gestibility and N balance, within the context of flammation. However, this explanation is difficult growth performance, suggest that LA plays an to support since there was no measurement of the important role in nourishing the weaned pig by stress experienced by these pigs in the first place. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa045/5821188 by DeepDyve user on 02 June 2020 Acosta et al. Despite a lack of responses on the barrier func- improving feed intake, digestibility of DM and GE, tion and the inflammation parameters, the results and N retention of weaned pigs; however, the func- obtained in this experiment showed less S-IgA tional capacity of LA to improve markers of intes- concentration in the jejunum of pigs fed either tinal function is limited at least under the conditions LA or FP. The concentration of S-IgA in weaned of this study. On the other hand, the FP showed only pigs is the result of an adaptive immunological re- a mild increase in the digestibility of N but a limited sponse to the exposure to luminal immunogenic capacity to improve markers of intestinal function. molecules (Rey et  al., 2004; Suzuki and Fargasan, Conflict of interest statement. The authors de- 2008; Brandtzaeg, 2013). Additionally, the increase clare no real or perceived conflicts of interest. in S-IgA concentration can be the result of the normal development of the acquired immunity of the weaned pig (McGlone and Pond, 2003). Since LITERATURE CITED the decrease in the concentration of S-IgA in pigs AOAC. 2007. Official methods of analysis. 18th ed. AOAC Int., fed LA and FP was observed only in the jejunum, Arlington, VA. it is likely the result of a localized response of the Bach  Knudsen,  K.  E. 2012. Lactose in diet influences the degradation of mixed linked β(1-3;1-4)-D-glucan in the pigs; there probably was no significant impact of small intestine of pigs. J. Anim. Sci. 90(Suppl 4):125–127. weaning on the intestinal health or the develop- doi:10.2527/jas.53788. ment of the acquired immunity of these pigs. Thus, Boudry,  G., V.  Péron, I.  Le  Huërou-Luron, J.  P.  Lallès, and additional research is needed to determine if LA B.  Sève. 2004. Weaning induces both transient and and FP decrease the growth of antigenic-IgA trig- long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. J. Nutr. 134:2256– gering bacteria probably by inducing the growth of 2262. doi:10.1093/jn/134.9.2256. nonpathogenic microorganisms. Brandtzaeg, P. 2013. Secretory IgA: designed for anti-microbial de- Among the multiple changes in gastrointes- fense. Front. Immunol. 4:222. doi:10.3389/fimmu.2013.00222. tinal function in the weaning pig, the activity of Colina,  J.  J., P.  S.  Miller, A.  J.  Lewis, R.  L.  Fischer, and disaccharidases have been reported to shift during R.  M.  Diedrichsen. 2010. Growth, body chemical com- the weaning recovery; lactase activity decreases position, and tissue deposition rates of nursery pigs fed crystalline or protein- bound lysine. Prof. Anim. Sci. while maltase activity increases after weaning 26:230–238. doi:10.15232/S1080-7446(15)30584-2. (Montagne et al., 2007; Tsukahara et al., 2013). In DahlqvisT ,  A. 1964. Method for assay of intestinal disaccharidases. this experiment, lactase activity was expected to Anal. Biochem. 7:18–25. doi:10.1016/0003-2697(64)90115-0. be elevated on day 15 in pigs receiving LA due to de  Vries  A.  G., and E.  Kanis. 1994. Selection for efficiency substrate induction, the consequence of keeping of lean tissue deposition in pigs. In: D.  J.  A.  Cole, J.  Wiseman, and M.  A.  Varley, editors, Principles of pig LA in the diet. However, similar lactase activities science. Nottingham University Press, Loughborough, were observed between the pigs receiving the LA+ UK; p. 23–41. and LA− diets. To the knowledge of the authors, Dong, G. C., and J. R. Pluske. 2007. The low feed intake in new- there is no specific data reporting substrate induc- ly-weaned pigs: problems and possible solutions. Asian- tion for lactase activity in nursery pigs. Instead, Aust. J. Anim. Sci. 20:440–452. doi:10.5713/ajas.2007.440. the mentioned shift in disaccharidase activities FASS. 2010. Guide for the care and use of agricultural animals in research and teaching. 3rd ed. Federation of Animal has been reported to take place regardless of the Science Societies, Champaign, IL; p. 169. presence of LA in the diet (Hedemann et al., 2006; Hedemann,  M.  S., B.  B.  Jensen, and H.  D.  Poulsen. 2006. Montagne et al., 2007). Troelsen et al. (1992) and Influence of dietary zinc and copper on digestive enzyme Motohashi et  al. (1997) suggested that the de- activity and intestinal morphology in weaned pigs. J. cline in lactase activity after weaning is the result Anim. Sci. 84:3310–3320. doi:10.2527/jas.2005-701. Jacobs, B. M., J. F. Patience, W. A. Dozier III, K. J. Stalder, and of the decrease of the intestinal nuclear factor B.  J.  Kerr. 2011. Effects of drying methods on nitrogen [NF-LPH1] lactase promoter and the LA expres- and energy concentrations in pig feces and urine, and sion during the intestinal development process poultry excreta. J. Anim. Sci. 89:2624–2630. doi:10.2527/ after weaning rather than lack of LA induction jas.2010-3768. in the diet. The current experiment not only sup- Jin, C. F., J. H. Kim, H. K. Moon, W. T. Cho, and Y. K. Han. ports this rational but also suggests that even with 1998. Effects of various carbohydrate sources on the growth performances and nutrient utilization in pigs the reduction of lactase activity during the post- weaned at 21 days of age. Asian-Australas. J. Anim. Sci. weaning period, the weaned pig has enough lac- 11:285–292. doi:10.5713/ajas.1998.285. tase activity to effectively use LA− based on the Jones, C. K., and J. F. Patience. 2014. 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