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Three studies involving 352 grower pigs were conducted to determine the effects of dietary supplementation with multistrain Bacillusspp. direct-fed microbial (DFM) and protease, alone or in combination, on growth performance, nutrient digestibility, blood characteristics, fecal microbiota, and noxious gas emissions, and to use a meta-analysis to increase the reliability of the findings. Treatments (n= 4) were set up as a 2 × 2 factorial design with 2 levels of protease (0 and 5.000/6.000 units/kilogram of feed [U/KG]) and 2 levels of DFM (0 and 1.5 × 105colony forming units/gram of feed [CFU/G]), plus a protease + DFM combination. Pigs were housed in groups of 3 or 4/pen with 8 replicate pens/treatment. Experimental diets were fed for 42 d and feed intake and BW were measured weekly. Fecal samples were collected at d 42 and analyzed to determine apparent total tract digestibility (ATTD). Fecal counts of Lactobacillusand coliforms, and noxious gas emissions were measured. Blood samples were taken by anterior vena cava puncture to measure blood urea nitrogen (BUN) and creatinine. Data from the 3 studies were pooled and analyzed as a 2 × 2 factorial using the Fit Model platform of JMP 11 (SAS Inst. Inc., Cary, NC). Means separation was determined using Tukey's honest significant difference test. The main effect of protease and DFM increased: BW at 42 d, overall ADG, and overall G:F compared to the control (P< 0.04). There were no interactions between protease and DFM (P> 0.05); however, the protease + DFM combination was the only treatment to improve ADG and G:F in all phases compared to the control. The main effect of protease increased ATTD of DM, nitrogen (N), and ADF (P< 0.04). The main effect of DFM increased ATTD of DM, N, GE, DE, ADF, and fat (P< 0.02). There was a trend for an interaction between protease and DFM for ATTD of GE and DE (P< 0.08) because the protease + DFM combination increased energy digestibility more than the additive effects of the protease and DFM alone. The main effects of protease and DFM decreased fecal ammonia emissions (P< 0.01), but the protease + DFM combination was the only treatment to decrease ammonia emissions compared to the control. In conclusion, the main effects of protease and DFM improved growth performance and nutrient digestibility compared to the control, but there was a greater additive effect of the protease + DFM combination on energy and N digestibility.

A trial was carried out to evaluate the effect of a multi-strain Bacillusspp. direct-feed microbial (DFM) in combination with a protease on growth performance and death loss of wean-finish pigs fed corn/soybean meal type diets under commercial conditions. Two treatments were tested (18 replicates per treatment and 31 pigs per replicate): 1) negative control (NC), and 2) NC + Bacillus-based DFM (Danisco Animal Nutrition, Marlborough, UK) and a Bacillus subtilisprotease (Danisco Animal Nutrition, Marlborough, UK). A corn/SBM/DDGs based NC diet was formulated to meet the nutrient requirements (NRC, 2012) with the exception of net energy which was reduced by 80, 25 and 15 kcal/kg in nursery (d 0-48), grower (d 48-104) and finisher (d 104-132), respectively. The DFM supplied three strains of Bacillusin equal proportions to provide 1.5 × 105cfu/g. The protease preparation supplied 5000 U/kg. All pigs in the trial were fed the mash diet with ad lib.access from weaning (d 0 post-weaning, BW=6.00±0.33 kg) to finish (d 132). Both diets were supplemented with 750 FTU/kg Buttiauxellasp. phytase. Performance, including body weight and feed intake by pen, was measured on d 0 and every subsequent 28 days until the end of the trial. Over the entire growth period, liveability was 87.5% in pigs fed NC and 89.9% in pigs fed NC+DFM+protease. Overall FCR (d 0-132) of pigs fed DFM+protease diet was 6 percentage points lower (2.38 vs 2.43 g/g, P <0.01), with numerically greater ADG (794 vs 777 g/d, P >0.05) and ADFI (1909 vs 1891 g/d, P >0.05) than those fed NC treatment. Responses of pigs to DFM+protease inclusion differed depending on growth phase. The ADG and ADFI were similar between pigs fed NC and NC+DFM+protease diets during nursery (average, 528 g/d) and grower (average, 979 g/d) phases (P >0.05). As a result, FCR was not affected by diet treatments, with an average of 1.54 and 2.39 g/g for nursery and grower, respectively (P >0.05). During finisher phase, even though there were only numerical improvements in ADG (874 vs 821 g/d; P =0.07) and ADFI (2,926 vs 2,793 g/d; P =0.07) due to DFM+protease inclusion, the resulting FCR was 15 percentage points lower as compared to those fed NC only diet (3.26 vs 3.41 g/g, P <0.01). In conclusion, the combination of a multi-strain Bacillusspp. DFM and a protease improved FCR of pigs fed reduced energy diet, which was most effective during the finisher phase.

The addition of direct-fed microbials (DFM) or protease to grower–finisher pig diets has not consistently led to growth performance improvements; however, new research suggests that there may be greater and more consistent effects of feeding protease and DFM in combination. This in vitro study aimed to investigate the effect of a protease (PRO) and Bacillusspp. DFM (DFM) on the structure and protein solubilization of different feed ingredients fed to growing pigs. Eight ileal cannulated barrows (30 kg initial BW) were fed 1 of 8 semipurified diets in an 8 × 8 Latin square design. The diets consisted of mostly corn–soy (19% CP), corn (6% CP), wheat (10% CP), corn distiller's dried grains with solubles (DDGS; 16% CP), SBM 46 (17% CP), SBM 48 (19% CP), canola meal (15% CP), or meat and bone meal (MBM; 14% CP) with chromic oxide. Diets were fed for 7 d with 5 d for adaptation and 2 d for ileal collection. The AID of CP was calculated. The corn–soy, wheat and SBM diets had higher AID of CP than the corn diet (P< 0.001), whereas the corn DDGS and MBM diets were intermediate. The AID of CP was affected by pig (P= 0.034) but not by period. Ileal samples from corn, wheat, and SBM 46 diets were selected from the pig with AID of CP closest to the population average and used for the in vitro study. The ileal digesta samples were incubated with no additive (control) or PRO, DFM, or PRO+DFM in a buffer solution at 40°C for 2 h. Centrifugation was used to separate the supernatant, which was analyzed in duplicate for total protein, and data was analyzed using the Fit Model platform in JMP 11. The pellet was analyzed by confocal laser scanning microscopy and scanning electron microscopy to determine internal and external microstructures, respectively. The PRO+DFM treatment increased protein solubilization of SBM compared with the control (P= 0.02; 1,635 vs. 730 ng/μL), but PRO and DFM alone did not. Confocal laser and scanning electron microscopy revealed that PRO+DFM was effective at degrading resistant starch in corn and aggregates of protein, lipid, and fiber in wheat and reducing the size of cell wall fragments in SBM. The PRO+DFM treatment was more effective than the individual additives at fragmenting the fibrous complexes in all 3 ingredients. In conclusion, the enzyme and DFM combination showed the greatest structural breakdown of all ingredients tested and solubilized more protein from SBM than the individual additives alone.

The addition of direct-fed microbials (DFM) or protease individually to grower–finisher pig diets has not consistently led to nutrient digestibility improvements; however, new research suggests that there may be greater and more consistent effects of feeding protease and DFM in combination. The objective of these studies was to investigate the effects on nutrient digestibility of feeding a protease and multistrain Bacillusspp. DFM combination (PRO+DFM) at different doses to growing pigs. A combined analysis of 2 trials involving 64 ileal cannulated barrows (25 ± 2 kg BW) was conducted. Treatments included 1) corn–soybean meal–based (control) diet with 20% coproducts; 2) Dose A, control plus PRO+DFM (2,500 units/kg protease, 7.5 × 104cfu/g DFM); 3) Dose B, control plus PRO+DFM (5,000 units/kg protease, 1.5 × 105cfu/g DFM); and 4) Dose C, control plus PRO+DFM (7,500 units/kg protease, 2.25 × 105cfu/g DFM). Pigs were randomly assigned to treatments, surgically equipped with a T-cannula at the distal ileum, and individually housed with 8 replicates/treatment in a randomized complete block design. Diets were fed for 14 d at 3 × maintenance energy requirement with 10 d of adaptation followed by 2 d of both fecal and ileal digesta collection. Duplicate samples were analyzed for chromic oxide and nutrients for the calculation of apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD). Data from the 2 trials were pooled and analyzed using the Fit Model platform of JMP11. Means separation was determined using Tukey's honest significant difference test. A regression analysis was performed to determine the correlation between the indigestible AA fraction and the uplift in AA digestibility for each combination dose. All PRO+DFM treatments (Dose A, B, and C) increased ATTD of N compared with the control (86.8, 86.8, and 86.6 vs. 84.5%, respectively; P= 0.01). Dose A also increased AID of starch (90.6 vs. 87.9%; P= 0.02), arginine (85.5 vs. 82.9%; P= 0.01), valine (77.4 vs. 73.8%; P= 0.02), isoleucine (80.8 vs. 77.1%; P= 0.01), and tryptophan (73.6 vs. 65.4%; P< 0.02) compared with the control. Dose B and C increased the AID of tryptophan compared with the control (P= 0.001). The regressions demonstrated that Dose A and B resulted in an average uplift in AID of AA of 33 and 9%, respectively, compared with the control and that the uplift was positively correlated with the indigestible fraction (P< 0.001). No AA uplift was found with Dose C. In conclusion, the dietary supplementation of a PRO+DFM combination to growing pigs resulted in improvements in ATTD of N and AID starch and key AA in a dose-dependent manner compared with the control.