Effects of exogenous β-glucanase on ileal digesta soluble β-glucan molecular weight, digestive tract characteristics, and performance of coccidiosis challenged broiler chickens fed hulless barley-based diets with and without medication

Limited use of medication in poultry feed led to the investigation of exogenous enzymes as antibiotic alternatives for controlling enteric disease. The objective of this study was to evaluate the effects of diet β-glucanase (BGase) and medication on β-glucan depolymerization, digestive tract characteristics, and performance of broilers. Broilers were fed hulless barley (HB) based diets with BGase (Econase GT 200P from AB Vista; 0 and 0.1%) and medication (Bacitracin and Salinomycin Na; with and without) arranged as a 2 × 2 factorial. In Experiment 1, 160 broilers were housed in cages from d 0 to 28. Each treatment was assigned to 10 cages. In Experiment 2, broilers (2376) were housed in floor pens and challenged with a coccidiosis vaccine on d 5. Each treatment was assigned to one floor pen in each of nine rooms. In Experiment 1, the soluble β-glucan weight average molecular weight (Mw) in the ileal digesta was lower with medication in the 0% BGase treatments. Peak molecular weight (Mp) and Mw were lower with BGase regardless of medication. The maximum molecular weight for the smallest 10% β-glucan (MW-10%) was lower with BGase. In Experiment 2, Mp was lower with medication in 0% BGase treatments. Beta-glucanase resulted in lower Mp regardless of medication, and the degree of response was lower with medication. The MW-10% was lower with BGase despite antibiotic addition. Body weight gain (BWG) and feed efficiency were higher with medication regardless of BGase use through-out the trial (except d 11-22 feed efficiency). Beta-glucanase resulted in higher BWG after d 11, and lower and higher feed efficiency before and after d 11, respectively, in unmedicated treatments. In conclusion, BGase and medication caused the depolymerization of soluble ileal β-glucan. Beta-glucanase appeared as a partial replacement for diet medication to increase coccidiosis challenged broiler performance.

performance.   Performance data collection 192 Body weight and feed intake (FI) were measured on a cage basis at d 7, 14, 21 and 28 in Beta-glucan molecular weight distribution 245 Ileal supernatant samples were boiled for 15 min and centrifuged at 17,013 × g for 10 246 min using a Beckman microfuge (Model E348720, Beckmann instruments, INC, Palo Alto, CA). 247 The sample was then analyzed for β-glucan molecular weight using size exclusion  valeric, caproic and lactic acids were used to make the standard solution. The digesta was thawed 262 and mixed with 25% phosphoric acid at 1:1 and kept at room temperature for 10 min with 263 occasional shaking. It was then centrifuged at 12,500 × g for 10 min. The supernatant (1 ml) was 264 mixed with 1 ml of the internal standard and centrifuged at 12,500 × g for 10 min. It was filtered 265 using a 0.45-micron nylon filter, and the filtrate was placed in a GC autosampler vial and injected into a Zebron Capillary Gas Chromatography column (length 30m, internal diameter  randomized complete block designs, and the battery cage level and room were considered as 286 blocks for Experiments 1 and 2, respectively. Treatments were replicated 10 times in Experiment nine different rooms). Differences were considered significant when P ≤ 0.05. Data were 289 checked for normality and analyzed using 2-way ANOVA. Tukey-Kramer test was used to detect 290 significant differences between means.

293
Ingredient nutrient composition 294 In Experiment 1, TDF, IDF, SDF and total β-glucan in HB were 29.0, 19.6, 9.6 and 295 8.70%, respectively, and the same parameters were 15.2, 13.7, 1.6 and 0.68%, respectively for 296 wheat. The content of total starch, CP, fat and ash were measured as 49.7, 16  Without medication, 0.1% β-glucanase (C) With medication, 0% β-glucanase  (Table 4). Similarly, caecal digesta SCFA concentrations and molar percentages 340 were also not affected by treatment (Table 5). Noteworthy, the interaction between medication 341 and BGase tended to be significant (P = 0.06-0.09) for the concentrations of total and individual 342 SCFA. In all cases, levels tended to decrease with enzyme use in the non-medicated diets and 343 increase with enzyme use in the medicated diets.

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To a large extent, dietary treatment did not affect ileal digesta SCFA of 11d old broilers 345 in Experiment 2 ( Table 6). The exception was a significant interaction between medication and 346 BGase for valeric acid. Without medication, levels of valeric acid decreased with enzyme use, 347 while levels increased with enzyme use when the medication was included in the diet. A similar 348 trend (P = 0.10) was noted for isovaleric acid. Levels of caproic acid decreased with enzyme use.

349
Interactions between the main effects were found for the molar percentages of valeric, isovaleric 350 (P = 0.06), and caproic acids. In diets without medication, BGase did not affect acid 351 concentration. When the medication was used, BGase increased acid levels. Dietary treatment 352 interactions were also noted for the proportional levels of propionic and lactic acids. All mean 353 differences were small and often not significant, but values tended to increase and decrease with 354 BGase use in nonmedicated and medicated diets, respectively.

355
The interactions between medication and BGase use at 11 d were significant for total and 356 individual caecal digesta SCFA (Table 7). The concentrations were higher with 0.1 compared to 357 0% BGase in the birds given diets without medication. However, BGase did not affect SCFA 358 concentrations in the treatments with medication. Concentrations for birds fed medicated diets were lower than those fed un-medicated diets for the treatments with BGase. The molar 360 percentages of propionic and isobutyric acids were decreased by medication, while enzyme use 361 decreased and increased the proportions of acetic and butyric acids, and valeric acid, 362 respectively. The interaction between main effects was significant for the proportional isovaleric 363 levels, with enzyme tending to decrease levels in unmedicated diets and increase levels in 364 medicated diets. Although the above effects were significant, differences were small.

365
Medication and the interactions between medication and BGase did not affect the 366 concentrations and molar percentages of ileal SCFA at d 33 (Table 8). All ileal SCFA 367 concentrations except butyric acid were higher because of BGase use. In addition, the 368 percentages of valeric and isovaleric acids were higher for the 0.1 compared to the 0% BGase 369 treatment. In contrast, the lactic acid percentage was slightly lower with enzyme use.

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Main effect interactions were not found for the concentrations and molar percentages of 371 caecal digesta SCFA at d 33 (Table 9). However, the concentrations of total SCFA and acetic  Except for the duodenum, medication, BGase, and their interactions did not affect the 378 digestive tract pH in Experiment 1 (Table 10). Enzyme use increased duodenal pH from 6.08 to 379 6.20. Main effect interactions were not found for the digestive tract pH, except for caecal pH at d 380 11 in Experiment 2 (Table 11); pH was lower with the enzyme use, but only in the diets without 381 medication. Medication resulted in higher pH in the crop at d 11, and the ileum at both d 11 and 33. Duodenal and ileal pH was higher with the use of BGase at d 11. Gizzard and caecal pH were 383 lower with the enzyme, and ileal pH was higher with the addition of diet BGase at d 33.   1 BGase -β-glucanase; SCFA -short chain fatty acids; Ace -Acetic acid; Pro -Propionic acid; But -Butyric acid; Isob -Isobutyric acid; Val -Valeric acid; Isov -Isovaleric acid; Cap -Caproic acid. 2 SEM -pooled standard error of mean (n=20 birds per treatment).  1 BGase -β-glucanase; SCFA -short chain fatty acids; Ace -Acetic acid; Pro -Propionic acid; But -Butyric acid; Isob -Isobutyric acid; Val -Valeric acid; Isov -Isovaleric acid; Cap -Caproic acid. 2 SEM -pooled standard error of mean (n=12 birds per treatment).  1 BGase -β-glucanase; SCFA -short chain fatty acids; Ace -Acetic acid; Pro -Propionic acid; But -Butyric acid; Isob -Isobutyric acid; Val -Valeric acid; Isov -Isovaleric acid; Cap -Caproic acid. 2 SEM -pooled standard error of mean (n=12 birds per treatment).  1 BGase -β-glucanase, SCFA -short chain fatty acids; Ace -Acetic acid; Pro -Propionic acid; But -Butyric acid; Isob -Isobutyric acid; Val -Valeric acid; Isov -Isovaleric acid; Cap -Caproic acid. 2 SEM -pooled standard error of mean (n=18 birds per treatment).  1 BGase -β-glucanase; SCFA -short chain fatty acids; Ace -Acetic acid; Pro -Propionic acid; But -Butyric acid; Isob -Isobutyric acid; Val -Valeric acid; Isov -Isovaleric acid; Cap -Caproic acid. 2 SEM -pooled standard error of mean (n=18 birds per treatment).   414 Gastrointestinal wall histology was examined only in Experiment 2 (Table 12). 415 Treatment effects were not prevalent nor consistent between ages. At d 11, medication decreased 416 the crypt depth, while β-glucanase decreased villi width. At 33 d, medication increased the 417 number of acidic and decreased the number of mixed goblet cells per villus. The medication also 418 increased the villi height to crypt depth ratio.  422 In Experiment 1, interactions were not found between BGase and medication for empty 423 weights and lengths of the digestive tract sections, except for crop weight (Table 13). Crop 424 weight was lower with enzyme use when the birds were fed a non-medicated diet, but the 425 enzyme had no effect when the diets were medicated. Both ileum and colon weights were lower 426 when the enzyme was fed. Crop content weight was higher, and duodenal and ileal content 427 weights were lower when 0.1% BGase was fed (Table 14). Interactions were found for the 428 content weights of the gizzard, jejunum and small intestine. The gizzard content weight tended to 429 be higher and lower with enzyme use in birds fed non-medicated and medicated diets, 430 respectively. Beta-glucanase resulted in lower jejunal and small intestinal content weights in the 431 absence of dietary antibiotics but had no effect when the medication was used.

432
Interactions were found between medication and BGase for the empty proportional 433 weights of the duodenum, jejunum, small intestine and caeca at d 11 (Table 15). For all  (Table 17). Dietary BGase resulted in lower empty weights for the crop, 447 ileum and small intestine; enzyme also reduced the lengths of the duodenum and ileum.

448
Interactions between the main effects were found for the empty jejunum weight, and the lengths 449 of the jejunum and small intestine. For the interactions, enzyme use resulted in smaller tissues 450 when non-medicated diets were fed, but had no effect when diets contained medication.

451
Medication resulted in smaller digestive tract segments in these interactions.

452
The content weights of the duodenum and colon decreased with the use of BGase at d 33 453 (Table 18) 1 BGase -β-glucanase; Proven -proventriculus; Duo -duodenum; SI -small intestine. 2 SEM -pooled standard error of mean (n=18 birds per treatment). Means within a main effect or interaction not sharing a common superscript are significantly different (P ≤ 0.05). 1 BGase -β-glucanase; SI -small intestine 2 SEM -pooled standard error of mean (n=18 birds per treatment).

Performance parameters 486
Interactions between medication and BGase were significant or nearly significant for 487 BWG and FI from 0-7 d, 7-14 d (P = 0.06) and 0-28 d (P = 0.06-0.07), and F:G from 0-7 d in 488 Experiment 1 (Table 19). Body weight gain and FI followed a similar response to treatments. In 489 birds fed diets without medication, the addition of BGase tended to reduce gain or feed 490 consumption, however in those fed diets with medication, enzyme either did not affect or 491 increased these response criteria. For the 0-7 d F:G ratio interaction, enzyme decreased and 492 increased feed efficiency in unmedicated diets and medicated diets, respectively.

493
In Experiment 2, interactions between main effects were significant for BWG for all 494 periods (Table 20) BGase tended to decrease FI when medication was not fed and increase FI when it was.

504
Interactions were found between medication and BGase for F:G in all periods.

505
Medication increased feed efficiency throughout the trial, but as was the case for BWG, the 506 nature of the interaction with enzyme use changed with bird age. During the 0-11 d period, F:G 507 increased with enzyme use when birds were fed non-medicated diets, but had no effect when the medication was used. For the remainder of the periods, including the total trial, enzyme 509 decreased F:G in birds fed non-medicated diets, but had no effect in broilers consuming 510 medicated diets.

511
The total mortality of the trials was 3.8 and 3.9% in Experiment 1 and 2, respectively, 512 and not affected by HB or BGase. In Experiment 2, the mortality attributed to coccidiosis (by 513 necropsy) was identified as 4.3% of the total mortality. However, 46.7% of the total mortality   Means within a main effect or interaction not sharing a common superscript are significantly different (P ≤ 0.05). 1 BGase -β-glucanase; BWG -body weight gain; FI -feed intake; F:G -feed to gain ratio. 2 SEM -pooled standard error of mean (n=9 pens per treatment).

527
With minor exceptions, all three molecular weight parameters for soluble ileal digesta β-528 glucan were lower with the enzyme use, which confirms exogenous BGase mediates the 529 depolymerization of HB β-glucan in broiler chickens. In addition, the reduction of MW-10%