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Research| Volume 102, ISSUE 5, P4179-4189, May 2019

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Effects of exogenous fibrolytic and amylolytic enzymes on ruminal fermentation and performance of mid-lactation dairy cows

Open ArchivePublished:March 14, 2019DOI:https://doi.org/10.3168/jds.2018-14949

      ABSTRACT

      Lactation diets are composed mostly of carbohydrates that are not fully fermented by rumen microbes. The aim of this study was to evaluate exogenous fibrolytic (Fibrozyme, Alltech Inc., Nicholasville, KY) and amylolytic (Amaize, Alltech Inc.) enzymes on nutrient intake, sorting index, total-tract apparent digestibility, ruminal fermentation, nitrogen utilization, milk yield, and composition of dairy cows in mid-lactation. Thirty-two multiparous Holstein cows (181 ± 35 d in milk, 571 ± 72.7 kg of body weight, and 29.6 ± 5.24 kg/d of milk yield at the start of experiment) were blocked according to milk yield and randomly allocated to treatments in a 4 × 4 Latin square design. Treatments were (1) control, basal diet without exogenous enzymes; (2) fibrolytic enzyme (FIB), dietary supplementation of Fibrozyme at 12 g/d (51 IU of xylanase activity/kg of diet dry matter); (3) amylolytic enzyme (AMY), dietary supplementation of Amaize at 8 g/d (203 fungal amylase units/kg of diet dry matter); and (4) both fibrolytic and amylolytic enzymes (FIB+AMY) added at the same dose of the individual treatments. Enzyme products were added to the concentrate during its preparation (once a week). The supply of FIB and AMY had no effect on nutrient intake and digestibility. However, an interaction effect was observed on sorting index of feed particle size between 8 and 19 mm. Amylolytic enzyme increased the sorting for feed particles between 8 and 19 mm, only when fed without FIB. In addition, AMY decreased the sorting for feed with particle size greater than 19 mm. An interaction effect was observed between FIB and AMY for ruminal butyrate concentration and N excretion. Amylolytic enzyme increased ruminal butyrate concentration in cows treated with FIB. Further, FIB decreased milk protein production and feed efficiency only in cows not fed AMY. Amylolytic enzyme reduced urinary N excretion. Exogenous enzymes had no effect on milk production and composition of dairy cows. This study lacks evidence that fibrolytic and amylolytic enzymes can affect nutrient digestibility, ruminal fermentation, and performance of mid-lactation cows.

      Key words

      INTRODUCTION

      Ruminants use energy from structural polysaccharide digestion to produce meat and milk, which have a relatively high nutritive value. Chemical, physical, and biological methods are used to neutralize anti-nutritional factors and improve feed digestibility in livestock feedstuffs (
      • Sujani S.
      • Seresinhe R.T.
      Exogenous enzymes in ruminant nutrition: A review.
      ). The dietary supplementation with exogenous enzymes has attracted attention of researchers because it is a biological treatment method and generally recognized as safe. Exogenous enzymes have been used to improve nutrient utilization in several species, especially in the poultry and swine industries. Although experiments evaluating dietary enzymes for ruminants have been reported since 1960 (
      • Burroughs W.
      • Woods W.
      • Ewing S.A.
      • Greig J.
      • Theurer B.
      Enzyme additions to fattening cattle rations.
      ), the feasible utilization of dietary enzymes have started in this century, largely due to a decrease in fermentation/generation costs and new technologies to produce purified and more consistent enzyme preparations. A series of experiments to expand the knowledge of fibrolytic (
      • Beauchemin K.A.
      • Rode L.M.
      Use of feed enzymes in ruminant nutrition.
      ;
      • Beauchemin K.A.
      • Colombatto D.
      • Morgavi D.P.
      • Yang W.Z.
      Use of exogenous fibrolytic enzymes to improve feed utilization by ruminants.
      ;
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      ) and amylolytic (
      • Chen K.H.
      • Huber J.T.
      • Simas J.
      • Theurer C.B.
      • Yu P.
      • Chan S.C.
      • Santos F.
      • Wu Z.
      • Swingle R.S.
      Effect of enzyme treatment or steam-flaking of sorghum grain on lactation and digestion in dairy cows.
      ;
      • DiLorenzo N.
      • Smith D.R.
      • Quinn M.J.
      • May M.L.
      • Ponce C.H.
      • Steinberg W.
      • Engstrom M.A.
      • Galyean M.L.
      Effects of grain processing and supplementation with exogenous amylase on nutrient digestibility in feedlot diets.
      ;
      • McCarthy M.M.
      • Engstrom M.A.
      • Azem E.
      • Gressley T.F.
      The effect of an exogenous amylase on performance and total-tract digestibility in lactating dairy cows fed a high-byproduct diet.
      ) enzyme activities in the rumen has been performed in the last decade. These studies provided evidences that enzymes are somewhat resistant to rumen degradation and potentially increase the rate of ruminal carbohydrate degradation.
      Xylanases and cellulases are the most used fibrolytic enzymes in ruminant diets and have demonstrated positive effects on total-tract digestibility of DM and NDF in dairy cows (
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      ). Improvements in fiber digestibility suggest that ruminal endogenous enzyme secretion is limited/inefficient (
      • Beauchemin K.A.
      • Colombatto D.
      • Morgavi D.P.
      • Yang W.Z.
      Use of exogenous fibrolytic enzymes to improve feed utilization by ruminants.
      ).
      • Oba M.
      • Allen M.S.
      Evaluation of the importance of the digestibility of neutral detergent fiber from forage: effects on dry matter intake and milk yield of dairy cows.
      reported that 1-unit increase in forage NDF digestibility was associated with an increase of 0.17 and 0.25 kg/d in DMI and 4% FCM, respectively. Fibrolytic enzymes have increased milk yield, whereas the variation in response has been attributed to different sources and doses of exogenous enzymes (
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      ). Amylolytic enzymes have been used in ruminant diets to increase ruminal starch digestibility (
      • Nozière P.
      • Steinberg W.
      • Silberberg M.
      • Morgavi D.P.
      Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.
      ) and milk production (
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      ), but the most reported effects of amylolytic enzymes are increased NDF digestibility in lactating cows (
      • Klingerman C.M.
      • Hu W.
      • McDonell E.E.
      • DerBedrosian M.C.
      • Kung Jr., L.
      An evaluation of exogenous enzymes with amylolytic activity for dairy cows.
      ;
      • Gencoglu H.
      • Shaver R.D.
      • Steinberg W.
      • Ensink J.
      • Ferraretto L.F.
      • Bertics S.J.
      • Lopes J.C.
      • Akins M.S.
      Effect of feeding a reduced-starch diet with or without amylase addition on lactation performance in dairy cows.
      ;
      • Weiss W.P.
      • Steinberg W.
      • Engstrom M.A.
      Milk production and nutrient digestibility by dairy cows when fed exogenous amylase with coarsely ground dry corn.
      ) and improved energy balance in transition dairy cows (
      • DeFrain J.M.
      • Hippen A.R.
      • Kascheur K.F.
      • Tricarico J.M.
      Effects of dietary α-amylase on metabolism and performance of transition dairy cows.
      ).
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      proposed the cross-feeding mechanism to explain the improvements in digestibility of nutrients not targeted by enzymes: exogenous enzymes hydrolyze complex carbohydrates into different products (malto-, cello-, and xylo-oligosaccharides), which supports growth of fibrolytic microorganisms. Thus, a synergistic effect between fibrolytic and amylolytic enzymes could be expected.
      The combination of fibrolytic and amylolytic enzymes has been little explored in dairy cow nutrition, and to the best of our knowledge, only
      • Hristov A.N.
      • Basel C.E.
      • Melgar A.
      • Foley A.E.
      • Ropp J.K.
      • Hunt C.W.
      • Tricarico J.M.
      Effect of exogenous polysaccharide-degrading enzyme preparations on ruminal fermentation and digestibility of nutrients in dairy cows.
      evaluated the effects of dietary supplementation of enzyme products with both xylanase and α-amylase activity on nutrient digestibility in dairy cows. However, these authors did not evaluate animal performance and enzymes were provided intraruminally, using only 4 animals. As
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      reported no effect of DIM on response of dairy cows to fibrolytic enzymes and the most positive amylolytic enzyme effect on performance of cows was found in mid-lactation dairy cows (
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      ;
      • Andreazzi A.S.R.
      • Pereira M.N.
      • Reis R.B.
      • Pereira A.N.
      • Morais Junior, N.N.
      • Acedo A.S.
      • Hermes R.G.
      • Cortinhas C.S.
      Effect of exogenous amylase on lactation performance of dairy cows fed a high-starch diet.
      ), cows in this lactation phase were chosen and the trial was performed with a greater number of experimental units to enhance statistical power.
      In this context, we hypothesized that dietary supplementation with exogenous enzymes would increase nutrient digestibility (especially of NDF) and ruminal total VFA concentration, and improve milk production and composition of dairy cows. The aim of this study was to evaluate the effects of both fibrolytic and amylolytic enzymes on intake and apparent total-tract digestibility of nutrients, sorting index, ruminal fermentation, milk yield and composition, N utilization, and microbial protein synthesis of mid-lactation cows.

      MATERIALS AND METHODS

      The experiment was conducted at the Dairy Cattle Research Laboratory of the Department of Animal Nutrition and Animal Production of the School of Veterinary Medicine and Animal Sciences, Pirassununga, São Paulo, Brazil. Experimental procedures were carried out under the approval of the Ethics Committee of the School of Veterinary Medicine and Animal Sciences of University of São Paulo (approval number: 2388290517).

      Animals and Treatments

      Thirty-two multiparous Holstein cows (181 ± 35.3 DIM, 571 ± 72.7 kg of BW, and 29.6 ± 5.24 kg/d of milk yield at the start of experiment; mean ± SD) were enrolled to the experiment. Cows were housed in a barn containing individual pens (17.5 m2 area), with sand bedding, feed bunks, and free access to water. Animals were distributed into 8 blocks according to their milk production, DIM, ruminal cannula presence, and BW. Animals within blocks were randomly allocated to a sequence of treatments in a 4 × 4 Latin square experimental design. Eight rumen-cannulated cows (within those 32 cows) were used to evaluate ruminal fermentation. Each experimental period lasted 21 d, where the first 14 d was allowed for treatment adaptation (
      • Machado M.G.
      • Detmann E.
      • Mantovani H.C.
      • Valadares Filho S.C.
      • Bento C.P.B.
      • Marcondes M.I.
      • Assunção A.S.
      Evaluation of the length of adaptation period for changeover and crossover nutritional experiments with cattle fed tropical forage-based diets.
      ) and the following 7 d was used for data sampling.
      Experimental diets were formulated according to the
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ; Table 1) recommendations to meet the nutrient requirements for a cow with 580 kg of BW, yielding 30 kg/d of milk, and 180 DIM. Treatments were obtained from a 2 × 2 factorial arrangement and cows were assigned to the following treatments: (1) control, basal diet without exogenous enzymes; (2) fibrolytic enzyme (FIB), dietary supplementation of Fibrozyme (Alltech Inc., Nicholasville, KY) at 12 g/d (51 IU of xylanase activity/kg of diet DM); (3) amylolytic enzyme (AMY), dietary supplementation of Amaize (Alltech Inc.) at 8 g/d [203 fungal amylase units (FAU)/kg of diet DM]; and (4) both fibrolytic and amylolytic enzymes (FIB+AMY) added at the same dose of the individual treatments. Enzyme products were added to the concentrate during its preparation (once a week) at a rate of 1.00 g/kg of DM of concentrate for FIB and 0.667 g/kg of DM of concentrate for AMY. According to the manufacturer, Fibrozyme is an extract from Trichoderma longibrachiatum fermentation (a dry mixture of inactive yeast, dry brewery yeast, and yucca extract) with a minimum of 100 IU of xylanase activity per gram of product. Amaize consists of an Aspergillus oryzae culture extract with known α-amylase activity (600 FAU/g of product). One FAU is defined as the amount of enzyme that degrades 1 g of dextrinized soluble starch per h at 30°C and pH of 4.8 (
      • Food Chemicals Codex
      ).
      Table 1Ingredients, chemical composition, and particle size distribution of experimental diet
      ItemValue
      Ingredient (g/kg of DM)
       Corn silage
      Chemical composition (DM basis): 36.4% DM, 7.8% CP, 51% NDF, and 21.6% starch.
      480
       Ground corn212
       Soybean meal (48% CP)149
       Citrus pulp67.3
       Whole raw soybean
      Chemical composition (DM basis): 91.5% DM, 37.9% CP, 25.6% NDF, 9.75% starch, and 21.7% ether extract.
      60.4
       Mineral premix
      Each kilogram contained 205 g of Ca, 60 g of P, 35 g of K, 70 g of Na, 20 g of S, 20 g of Mg, 2,500 mg of Zn; 1,600 mg of Mn, 700 mg of Cu, 700 mg of Fe, 40 mg of I, 19 mg of Se, 10 mg of Cr, 200,000 IU of vitamin A, 50,000 IU of vitamin D, and 1,500 IU of vitamin E.
      12.6
       Calcium bicarbonate8.55
       Urea3.68
       Limestone3.09
       Salt3.31
      Chemical composition (g/kg of DM)
       DM (g/kg as fed)524
       OM928
       NFC
      NFC = 1,000 – [(CP – neutral detergent insoluble protein) + ether extract + ash + NDF] according to Hall (2000); all values expressed as grams per kilogram of DM.
      408
       NDF329
       Starch285
       ADF235
       CP168
       Indigestible NDF120
       Ether extract38.0
       Lignin37.7
       Neutral detergent insoluble protein19.2
       Acid detergent insoluble protein16.0
       Total digestible nutrient (1×)
      Estimated at maintenance level of feed intake according to NRC (2001).
      728
       NEL (3×)
      Estimated at three times maintenance level according to NRC (2001).
      (Mcal/kg of DM)
      1.67
      Particle size distribution (g/kg as fed)
       >19 mm85.8
       19–8 mm366
       8–4 mm182
       <4 mm334
      1 Chemical composition (DM basis): 36.4% DM, 7.8% CP, 51% NDF, and 21.6% starch.
      2 Chemical composition (DM basis): 91.5% DM, 37.9% CP, 25.6% NDF, 9.75% starch, and 21.7% ether extract.
      3 Each kilogram contained 205 g of Ca, 60 g of P, 35 g of K, 70 g of Na, 20 g of S, 20 g of Mg, 2,500 mg of Zn; 1,600 mg of Mn, 700 mg of Cu, 700 mg of Fe, 40 mg of I, 19 mg of Se, 10 mg of Cr, 200,000 IU of vitamin A, 50,000 IU of vitamin D, and 1,500 IU of vitamin E.
      4 NFC = 1,000 – [(CP – neutral detergent insoluble protein) + ether extract + ash + NDF] according to
      • Hall M.B.
      Calculation of non structural carbohydrate content of feeds that contain non-protein nitrogen. Bulletin 339.
      ; all values expressed as grams per kilogram of DM.
      5 Estimated at maintenance level of feed intake according to
      • NRC
      Nutrient Requirements of Dairy Cattle.
      .
      6 Estimated at three times maintenance level according to
      • NRC
      Nutrient Requirements of Dairy Cattle.
      .
      Before the start of the experiment, samples of diet ingredients were shipped to Alltech Inc., and the company performed a dual-flow continuous in vitro trial to determine the appropriate doses of enzymes for the diet used in the current study.

      Sampling and Chemical Analysis

      Cows were fed twice daily (0800 and 1300 h), after the forage and concentrate were hand mixed in each feed bunk. Refusals from each cow were weighed daily to maintain orts between 5 and 10% (on an as-fed basis) of feed supplied on the previous day. During the sampling period, corn silage and ort samples were collected daily and pooled to form a composite sample per cow per period for chemical analysis. Concentrate ingredients were collected weekly during the concentrate mixing at the feed mill. Samples of TMR and orts from each cow were collected on d 16 and 17 of each period and assessed for particle size distribution using a particle separator system (Penn State Particle Separator, Nasco, Fort Atkinson, WI). Sorting index was calculated according to
      • Silveira C.
      • Oba M.
      • Yang W.Z.
      • Beauchemin K.A.
      Selection of barley grain affects ruminal fermentation, starch digestibility, and productivity of lactating dairy cows.
      .
      From d 17 to 19 of each experimental period, fecal and urine samples were collected every 9 h (starting at 0600 h on d 17) for estimation of total-tract nutrient digestibility, microbial protein synthesis, and nitrogen balance. After each sampling, fecal samples (100 g) were frozen and at the end of each experimental period, samples from each cow were thawed and pooled on a wet basis. Samples of feeds, orts, and feces were dried at 60°C in a forced-air oven for 72 h and ground in a Wiley mill (MA340, Marconi, Piracicaba, Brazil) through a 2-mm or a 1-mm sieve. Samples of feeds, orts, and feces (processed at 1 mm) were analyzed for DM (method 930.15;
      • AOAC International
      Official Methods of Analysis.
      ), CP (N × 6.25; Kjeldahl method 984.13;
      • AOAC International
      Official Methods of Analysis.
      ), ether extract (EE; method 920.39;
      • AOAC International
      Official Methods of Analysis.
      ), ADF, and lignin (method 973.18; AOAC International, 2000), ash (method 942.05;
      • AOAC International
      Official Methods of Analysis.
      ), and NDF using α-amylase (
      • Undersander D.
      • Mertens D.R.
      • Theix N.
      Forage Analysis.
      ) and without addition of sodium sulfite (TE-149 fiber analyzer, Tecnal Laboratory Equipment Inc., Piracicaba, Brazil). Starch content was determined by enzymatic degradation (Termamyl Amyloglucosidase AMG 300L, Novozymes, Bagsvaerd, Denmark) and absorbances were measured in a spectrophotometer (SBA 200, CELM) as described by
      • Hendrix D.L.
      Rapid extraction and analysis of nonstructural carbohydrates in plant tissues.
      .
      Indigestible neutral detergent fiber (iNDF) was used as an internal marker to estimate daily DM fecal excretion. Samples of feeds, orts, and feces (ground at 2 mm) were placed into bags of nonwoven fabric tissue (5 × 5 cm at 100 g of DM/m2;
      • Casali A.O.
      • Detmann E.
      • Valadares Filho S.C.
      • Pereira J.C.
      • Henriques L.T.
      • Freitas S.G.
      • Paulino M.F.
      Influence of incubation time and particles size on indigestible compounds contents in cattle feeds and feces obtained by in situ procedures.
      ) and incubated for 288 h in the rumen of 2 Holstein cows according to
      • Huhtanen P.
      • Kaustell K.
      • Jaakkola S.
      The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given six different diets.
      . After the incubation period, samples were washed in running tap water and analyzed for NDF content, as described earlier. Total apparent digestibility was calculated based on nutrient intake and nutrient excretion.
      Ruminal digesta was collected (5 different sites within the rumen) from cows with ruminal cannula (n = 8) on d 20 of each experimental period, before the morning feeding (time 0), and 2, 4, 6, 8, 10, 12, 14, and 16 h after. Digesta was squeezed into 4 cheesecloth layers to extract ruminal fluid and pH was measured using a digital potentiometer (MB-10, Marte Científica, Santa Rita do Sapucaí, Brazil). For NH3-N concentration, aliquots of ruminal fluid (2 mL) were mixed with sulfuric acid (1 mL at 1 N) and analyzed by the colorimetric phenol-hypochlorite method (
      • Broderick G.A.
      • Kang-Meznarich J.H.
      Automated simultaneous determination of ammonia and total amino-acids in ruminal fluid and in vitro media.
      ). Ruminal fluid was also mixed with formic acid (at a ratio of 4 to 1) for VFA analysis. Volatile fatty acids were determined according to
      • Ferreira E.M.
      • Ferraz Júnior, M.V.C.
      • Polizel D.M.
      • Urano F.S.
      • Susin I.
      • Gentil R.S.
      • Biehl M.V.
      • Biava J.S.
      • Pires A.V.
      Milk yield and composition from ewes fed raw soybeans and their lambs' performance.
      , using a gas chromatograph (Agilent 7890A) equipped with flame ionization detector (7683B) and a fused-silica capillary column (J & W 19091 F-112, Agilent Technologies, Santa Clara, CA), 25 m length and 320 µm internal diameter, containing 0.20 µM cyanopropyl polysiloxane. The total chromatographic run time was 16.5 min, divided into 3 heating cycles, as follows: 80°C (1 min), 120°C (20°C/min for 3 min), and 205°C (10°C/min for 2 min). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL/min, and the temperature of the injector and detector was 260°C. Nitrogen gas was used as the “make-up” gas at a rate of 30 mL/min. Volatile acid peaks and concentrations were determined using ChemStation software (Agilent Technologies).
      Cows were milked twice daily, at 0600 and 1600 h, and milk production was electronically recorded (Alpro, DeLaval, Tumba, Sweden). Milk samples were taken from every milking from d 16 to d 18 of each experimental period and analyzed for fat, lactose, and crude protein concentration using an infrared milk analyzer (Lactoscan, Entelbra, São Paulo, Brazil). Milk samples (20 mL) were deproteinized according to
      • Broderick G.A.
      • Clayton M.K.
      A statistical evaluation of animal and nutritional factors influencing concentrations of milk urea nitrogen.
      and analyzed for urea concentration using commercial kits (Bioclin, Belo Horizonte, Brazil) in a semi-automatic analyzer (SBA 200, CELM). Milk yields were corrected for 3.5% fat content according to
      • Sklan D.R.
      • Ashkenazi R.
      • Braun A.
      • Devorin A.
      • Tabori K.
      Fatty acids, calcium soaps of fatty acids, and cottonseeds fed to high yielding cows.
      .
      Nitrogen balance was calculated as the difference between N intake and the sum of fecal, urinary, and milk N. Nitrogen excreted in milk was calculated based on milk CP content (6.38 as conversion factor). Fecal excretion of N was calculated multiplying N content in feces (g/kg) by the estimated DM fecal excretion (kg/d). Nitrogen efficiency was considered as the ratio of milk N to N intake (g/g). Daily urine volume was estimated based on creatinine concentration (mg/L) in urine samples collected between d 17 and 19 of each experimental period. Urinary creatinine concentrations were analyzed using commercial kits (kinetic creatinine: catalog no. K-067, Bioclin) in a semi-automatic spectrophotometer (SBA 200, CELM). The ratio of daily creatinine excretion was assumed as 24.05 mg/kg of BW (
      • Chizzotti M.L.
      • Valadares Filho S.C.
      • Valadares R.F.D.
      • Chizzotti F.H.M.
      • Tedeschi L.O.
      Determination of creatinine excretion and evaluation of spot urine sampling in Holstein cattle.
      ).
      Microbial protein synthesis was calculated based on the excretion of purine derivatives in urine (uric acid and allantoin) and milk (allantoin), as described by
      • Chen X.B.
      • Gomes M.J.
      Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivate.
      . The ratio of purine N to microbial N was considered to be 0.116, and the intestinal digestibility of microbial purines was set at 0.83 (
      • Chen X.B.
      • Gomes M.J.
      Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivate.
      ). The urinary recovery of absorbed purines and the endogenous contribution of purines were considered to be 0.70 and 0.512 mmol/kg of BW0.75, respectively (
      • González-Ronquillo M.
      • Balcells J.
      • Guada J.A.
      • Vicente F.
      Purine derivate excretion in dairy cows: Endogenous excretion and the effect of exogenous nucleic acid supply.
      ). Uric acid concentration in urine was determined using a commercial kit (uric acid stable liquid: catalog no. K-052; Bioclin) and absorbance measured in a spectrophotometer (SBA 200, CELM). Urinary and milk allantoin concentrations were determined according to
      • Fujihara T.
      • Orskov E.R.
      • Reeds P.J.
      • Kyle D.J.
      The effect of protein infusion on urinary excretion of purine derivates in ruminants nourished by intragastric nutrition.
      .
      Blood samples were collected from all cows by puncture of coccygeal vein on d 16, 4 h after the morning feeding. Blood samples were collected in vacutainer tubes for serum and centrifuged (2,000 × g for 15 min at 25°C) after clotting. The analyses were performed using colorimetric kits (glucose: catalog no. K-082; urea: catalog no. K-056; Bioclin) and absorbance measured in a spectrophotometer (SBA 200, CELM).

      Calculations and Statistical Analysis

      The nutrient intake (NI, kg/d) was calculated accounting for DM offered (OF, kg/d) and the concentration of the nutrient in OF (OFc, % DM), and DM refused (RF, kg/d) and concentration of the nutrient in DM refused feed (RFc, % DM):
      NI(kg/d)=(OF×OFc100)(RF×RFc100).


      The sorting index (
      • Silveira C.
      • Oba M.
      • Yang W.Z.
      • Beauchemin K.A.
      Selection of barley grain affects ruminal fermentation, starch digestibility, and productivity of lactating dairy cows.
      ) was estimated as the intake corresponding to each sieve (Px; × = 1 to 4) expressed as the percentage of the total predicted intake of animals, in which the predicted intake of Px fraction is equal to the ratio between the ingested material (as-fed basis) and the Px fraction of the TMR (as-fed basis) according to Equations [1], [2], and [3]:
      Predicted intake(kg/d)=PxTMR(kg/kg)×consumed(kg/d),
      [1]


      Observed intake(kg/d)=[amount of feed offered(kg/d)×PxTMR(kg/kg)][amount of feed refused(kg/d)×Pxrefused(kg/kg)],
      [2]


      Sorting index=(Observed intake(kg/d)predicted intake(kg/d)).
      [3]


      The fecal excretion (FE, kg/d) was calculated dividing iNDF intake (iNDFi, kg/d) by the fecal excretion of iNDF (iNDFf, kg/kg), as follows:
      FE(kg/d)=iNDFi(kg/d)iNDFf(kg/kg).


      Apparent total-tract digestibility (ATD, g/kg) of DM, OM, CP, EE, NDF, and starch were calculated based on fecal excretion (FE, kg/d), nutrient concentration in feces (NCF, g/kg), and the corresponding nutrient intake (NI, kg/d):
      ATD(g/kg)=NI(FE×NCF)NI×100.


      The 3.5% FCM was calculated according
      • Sklan D.R.
      • Ashkenazi R.
      • Braun A.
      • Devorin A.
      • Tabori K.
      Fatty acids, calcium soaps of fatty acids, and cottonseeds fed to high yielding cows.
      :
      3.5%FCM=[0.432+0.163×fat in milk(%)]×milk yield(kg/d).


      Feed efficiency was expressed as milk yield (kg/d) to DMI (kg/d) ratio. Retained N was calculated subtracting N intake by N excretion (fecal, urinary, and milk N).
      Data were analyzed using the MIXED procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC) according to the following model:
      Yijklm=μ+Li+cj:i+Pk+Fl+Am+F×Alm+eijklm,


      with cj:iN(0;σc2)andeijklmN(0;σe2), where Yijklm = dependent variable of interest; µ = overall mean; Li = fixed effect of Latin square (i = 1 to 8); cj:i = random effect of cow nested in Latin square (j = 1 to 32); Pk = fixed effect of period (k = 1 to 4); Fl = fixed effect of fibrolytic enzyme (l = 1 to 2); Am = fixed effect of amylolytic enzyme (m = 1 to 2); F × Alm = fixed interaction effect between fibrolytic and amylolytic enzymes; eijklm = random error; N = normal distribution; σc2 and σe2 are variances associated with cow and residual effect, respectively.
      Ruminal fermentation variables were analyzed by repeated-measures analysis using the MIXED procedure of SAS (version 9.3) according to the following model:
      Yijklmn=μ+Li+cj:i+Pk+Fl+Am+F×Alm+ωijklm+Tn+T×Fnl+T×Anm+T×F×Anlm+eijklmn,


      with cj:iN(0;σc2), ωijklmN(0;σω2) and eijklmnMVN(0;R), where Yijklm = dependent variable of interest; µ = overall mean; Li = fixed effect of Latin square (i = 1 to 2); cj:i = random effect of cow nested in Latin square (j = 1 to 32); Pk = fixed effect of period (k = 1 to 4); Fl = fixed effect of fibrolytic enzyme (l = 1 to 2); Am = fixed effect of amylolytic enzyme (m = 1 to 2); F × Alm = fixed interaction effect between fibrolytic and amylolytic enzymes; ωijklm = is the random error associated with experimental units; Tn = fixed effect of time (n = 1 to 9); T × Fnl; T × Anm and T × F × Anlm are interaction terms; eijklmn = residual error; N indicates Gaussian distribution; σc2 = variance associated with animal; σω2 = variance associated with random effect of animal; MVN = indicates multivariate normal, and R is the variance-covariance matrix of residuals due to the repeated measurements. The following variance-covariance matrices were tested: compound symmetry (CS), heterogeneous CS, autoregressive (1), heterogeneous autoregressive (1), Toeplitz (TOEP), heterogeneous TOEP, unstructured, factor analytic (1), and ante-dependence (1). The matrix was selected using the corrected Akaike's information criterion (AICC) method. Means were adjusted by the LSMEANS procedure and interaction effect Fl × Am was decomposed using Fisher's protected least significant difference means test. Statistical differences were declared at P ≤ 0.05 and trends toward significance were considered when 0.05 < P ≤ 0.10.

      RESULTS

      Treatments had no effect (P ≥ 0.111) on DM and nutrient intake. However, an interaction effect (P = 0.024) was observed between fibrolytic and amylolytic enzymes for sorting index of feed particles with size between 8 and 19 mm (Table 2). Amylolytic enzyme addition increased (P ≤ 0.05) sorting index of feed particles from 8 to 19 mm of size, only for cows that did not receive FIB. Furthermore, a main effect was observed for AMY to decrease (P = 0.031) preference for feed particles with size higher than 19 mm. Enzyme addition showed no effect (P ≥ 0.186) on DM, OM, NDF, CP, EE, and starch digestibility.
      Table 2Effects of fibrolytic and amylolytic enzymes on nutrient intake, sorting index, and apparent total-tract digestibility of lactating cows
      ItemTreatment
      Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      SEMP-value
      Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).
      CONFIBAMYFIB+AMYFIBAMYFIB × AMY
      Intake (kg/d)
       DM23.823.523.723.70.240.3380.7860.406
       OM22.522.222.422.40.230.3560.7540.398
       NFC10.510.310.410.40.110.5150.9970.111
       NDF7.397.317.417.310.0780.1430.8490.852
       Starch6.956.866.916.960.0700.7450.6330.212
       CP4.144.094.124.140.0410.6040.7170.275
       Ether extract0.930.910.930.930.0090.4770.4890.131
      Sorting index
       >19 mm0.8340.8670.8250.8190.00950.2760.0310.137
       19–8 mm0.983
      Fisher means test (LSD) at 0.05 of probability.
      0.986
      Fisher means test (LSD) at 0.05 of probability.
      0.990
      Fisher means test (LSD) at 0.05 of probability.
      0.983
      Fisher means test (LSD) at 0.05 of probability.
      0.00120.2840.3670.024
       8–4 mm1.011.011.021.010.0010.5040.1540.287
       <4 mm1.041.041.041.050.0010.1640.5570.404
      Apparent total-tract digestibility (g/kg)
       DM6516546526563.00.5190.7420.999
       OM6636686656683.20.5330.8360.879
       NDF3963943963974.90.9360.8900.880
       CP6896936966993.30.5510.2940.889
       Ether extract8017978108035.70.6090.5290.901
       Starch9309389319311.60.2730.3800.186
      a,b Fisher means test (LSD) at 0.05 of probability.
      1 Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      2 Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).
      Enzymes had no effect (P ≥ 0.112) on ruminal pH and ruminal concentrations of NH3-N, acetate, propionate, branched-chain fatty acids, and VFA (Table 3). An interaction effect (P = 0.043) was observed between FIB and AMY for ruminal butyric acid concentration. The addition of AMY increased (P ≤ 0.05) ruminal butyrate concentration only in cows not treated with FIB. However, FIB had no effect (P > 0.05) on butyric acid concentration in the rumen of animals fed diets either with or without AMY.
      Table 3Effects of fibrolytic and amylolytic enzymes on ruminal fermentation of lactating cows
      ItemTreatment
      Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      SEMP-value
      Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibroytic and amylolytic enzymes (FIB × AMY).
      CONFIBAMYFIB+AMYFIBAMYFIB × AMY
      pH6.066.076.066.020.0330.5890.4880.536
      NH3-N (mg/dL)18.719.719.120.40.450.1120.4390.827
      Acetate (mmol/100 mmol)54.353.854.554.30.290.4090.3690.865
      Propionate (mmol/100 mmol)23.523.822.723.70.310.2600.4370.589
      Butyrate (mmol/100 mmol)16.4
      Means within a row with different superscripts differ.
      16.9
      Means within a row with different superscripts differ.
      17.6
      Means within a row with different superscripts differ.
      16.9
      Means within a row with different superscripts differ.
      0.260.8460.0940.043
      Total BCFA
      Branched-chain fatty acids (BCFA).
      (mmol/100 mmol)
      5.275.15.215.160.0490.9900.2950.584
      Total VFA (mM)94.794.994.894.90.050.9460.2780.744
      Acetate:propionate2.282.442.292.560.0350.4040.1550.332
      Acetate (mM)51.551.751.151.50.260.3140.3300.751
      Propionate (mM)22.321.522.622.50.300.2680.4760.605
      Butyrate (mM)15.5
      Means within a row with different superscripts differ.
      16.1
      Means within a row with different superscripts differ.
      16.7
      Means within a row with different superscripts differ.
      16.0
      Means within a row with different superscripts differ.
      0.240.8420.0770.038
      a,b Means within a row with different superscripts differ.
      1 Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      2 Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibroytic and amylolytic enzymes (FIB × AMY).
      3 Branched-chain fatty acids (BCFA).
      Fibrolytic and amylolytic enzymes had no effect (P ≥ 0.372) on yields of milk, FCM, and milk fat (Table 4). However, FIB tended to decrease (P = 0.099) milk protein concentration, regardless of AMY. In addition, an interaction effect (P ≤ 0.053) was observed between FIB and AMY for lactose and protein production, and feed efficiency. The FIB decreased (P ≤ 0.05) protein production and feed efficiency only in animals that did not receive AMY. Furthermore, AMY decreased (P = 0.044) urinary N excretion of cows, without affecting (P ≥ 0.194) N balance (Table 5). Enzymes showed no effect (P ≥ 0.112) on N intake and fecal excretion, microbial protein synthesis, serum glucose, and urea concentration. A tendency (P = 0.094) was observed for the interaction effect between fibrolytic and amylolytic enzymes on milk N secretion and N intake ratio. Fibrolytic enzyme decreased (P ≤ 0.05) milk N secretion only in animals not treated with AMY.
      Table 4Effects of fibrolytic and amylolytic enzymes on performance of lactating cows
      ItemTreatment
      Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      SEMP-value
      Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).
      CONFIBAMYFIB+AMYFIBAMYFIB × AMY
      Milk (kg/d)
       Yield30.129.529.529.80.370.5950.6910.084
       3.5% FCM
      Calculated according to Sklan et al. (1992).
      28.628.328.128.40.380.9680.6240.372
       Lactose1.38
      Means within a row with different superscripts differ.
      1.35
      Means within a row with different superscripts differ.
      1.35
      Means within a row with different superscripts differ.
      1.36
      Means within a row with different superscripts differ.
      0.0160.4980.5490.017
       Fat0.9800.9680.9570.9670.01530.9530.4280.487
       Protein0.928
      Means within a row with different superscripts differ.
      0.898
      Means within a row with different superscripts differ.
      0.898
      Means within a row with different superscripts differ.
      0.903
      Means within a row with different superscripts differ.
      0.01080.1600.1760.053
      Milk composition (%)
       Lactose4.604.594.594.570.0120.1120.0570.626
       Fat3.313.293.283.280.0440.8710.5270.855
       Protein3.063.053.063.040.0080.0990.5580.718
       MUN (mg/dL)12.512.613.112.70.320.7410.4800.549
       Feed efficiency (milk/DMI)1.29
      Means within a row with different superscripts differ.
      1.25
      Means within a row with different superscripts differ.
      1.25
      Means within a row with different superscripts differ.
      1.26
      Means within a row with different superscripts differ.
      0.0130.1210.0480.025
      a,b Means within a row with different superscripts differ.
      1 Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      2 Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).
      3 Calculated according to
      • Sklan D.R.
      • Ashkenazi R.
      • Braun A.
      • Devorin A.
      • Tabori K.
      Fatty acids, calcium soaps of fatty acids, and cottonseeds fed to high yielding cows.
      .
      Table 5Effects of fibrolytic and amylolytic enzymes on nitrogen utilization, microbial protein synthesis, blood metabolites, and BW of lactating cows
      ItemTreatment
      Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      SEMP-value
      Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).
      CONFIBAMYFIB+AMYFIBAMYFIB × AMY
      N Intake (g/d)6616546616616.60.6040.7170.275
      Fecal N (g/g N intake)0.3690.3730.3740.3610.00460.5840.6620.279
      Urinary N (g/g N intake)0.2280.2120.2030.2000.00450.3220.0440.431
      Milk N (g/g N intake)0.221a0.213b0.214b0.214b0.00200.0460.1570.094
      N balance (g/g N intake)0.1920.2010.2100.2180.00640.5130.1940.957
      Microbial CP (kg/d)1.221.101.201.090.0320.5300.7940.984
      Blood glucose (mg/dL)64.564.763.666.90.810.2180.6380.257
      Blood urea (mg/dL)37.336.236.436.50.810.4760.6330.440
      BW (kg)5825805805795.70.1710.3770.974
      BW change (kg/d)0.3340.5170.3200.3310.05920.3970.3820.454
      1 Control (CON), no exogenous enzyme supplementation; fibrolytic enzyme (FIB), Fibrozyme (Alltech Inc., Nicholasville, KY) provided at 12 g/d (1 g/kg of concentrate on an as-fed basis); amylolytic enzyme (AMY), Amaize (Alltech Inc.) provided at 8 g/d (0.66 g/kg of concentrate on an as-fed basis); and FIB + AMY (1 g of Fibrozyme and 0.66 g of Amaize per kg of concentrate on an as-fed basis).
      2 Probabilities for fibrolytic enzyme effect (FIB), amylolytic enzyme effect (AMY), and interaction effect of fibrolytic and amylolytic enzymes (FIB × AMY).

      DISCUSSION

      Refuting our initial hypothesis, this study showed no effects of enzyme supplements on DMI, ruminal fermentation, and lactation performance. Previous studies of our research group demonstrated that different doses of both amylolytic and fibrolytic enzyme products had no effect on milk production and composition of mid-lactation cows (
      • Silva T.H.
      • Takiya C.S.
      • Vendramini T.H.A.
      • Ferreira de Jesus E.
      • Zanferari F.
      • Rennó F.P.
      Effects of dietary fibrolytic enzymes on chewing time, ruminal fermentation, and performance of mid-lactating dairy cows.
      ;
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      ). No differences in DMI of cows fed exogenous enzymes have been reported in a meta-analysis evaluating fibrolytic enzymes (
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      ) and in studies using amylase (
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      ;
      • Vargas-Rodriguez C.F.
      • Engstrom M.
      • Azem E.
      • Bradford B.J.
      Effects of dietary amylase and sucrose on productivity of cows fed low-starch diets.
      ;
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      ). The single study that evaluated the combination of fibrolytic and amylolytic enzymes (
      • Hristov A.N.
      • Basel C.E.
      • Melgar A.
      • Foley A.E.
      • Ropp J.K.
      • Hunt C.W.
      • Tricarico J.M.
      Effect of exogenous polysaccharide-degrading enzyme preparations on ruminal fermentation and digestibility of nutrients in dairy cows.
      ) also did not report effects of enzymes on DMI and ruminal fermentation of dairy cows.
      Although
      • Yang W.Z.
      • Beauchemin K.A.
      • Rode L.M.
      A comparison of methods of adding fibrolytic enzymes to lactating cow diets.
      observed no effect of fibrolytic enzyme on nutrient intake, the enzyme increased the DM, OM, and CP digestibility in lactating cows.
      • Beauchemin K.A.
      • Colombatto D.
      • Morgavi D.P.
      • Yang W.Z.
      Use of exogenous fibrolytic enzymes to improve feed utilization by ruminants.
      found an increase in DMI of dairy cattle fed a fibrolytic enzyme, which was attributed to improved ruminal particulate passage rate and fiber digestion. Furthermore, increased DM and NDF digestibility without affecting DMI when providing fibrolytic enzyme products to ruminants have been reported in the literature (
      • Beauchemin K.A.
      • Rode L.M.
      • Sewalt V.J.
      Fibrolytic enzymes increase fiber digestibility and growth rate of steers fed dry forages.
      ;
      • Rode L.M.
      • Yang W.Z.
      • Beauchemin K.A.
      Fibrolytic enzyme supplements for dairy cows in early lactation.
      ). The increase in nutrient digestibility is related to the mode of action of the enzyme; when applied before feeding, the enzyme alters the structure of the feed, making it more susceptible to degradation (
      • Dean D.B.
      • Staples C.R.
      • Littell R.C.
      • Kim S.
      • Adesogan A.T.
      Effect of method of adding a fibrolytic enzyme to dairy cow diets on feed intake digestibility, milk production, ruminal fermentation, and blood metabolites.
      ). In addition, the synergism between endogenous and exogenous enzymes increases overall ruminal enzymatic activity and hydrolysis (
      • Morgavi D.P.
      • Beauchemin K.A.
      • Nsereko V.L.
      • Rode L.M.
      • Iwaasa A.D.
      • Yang W.Z.
      • McAllister T.A.
      • Wang Y.
      Synergy between ruminal fibrolytic enzymes and enzymes from Trichoderma Longibrachiatum..
      ), improving the diet digestibility. However, none of these effects were observed in the present study. The response to enzyme supplementation would likely be greater when cows experience a limited DMI; for example, during the transition period or when cows are fed diets with low energy density (
      • Gencoglu H.
      • Shaver R.D.
      • Steinberg W.
      • Ensink J.
      • Ferraretto L.F.
      • Bertics S.J.
      • Lopes J.C.
      • Akins M.S.
      Effect of feeding a reduced-starch diet with or without amylase addition on lactation performance in dairy cows.
      ). In situations that DMI is not limited, enzymes are expected to increase the rate of degradation but not the extension, resulting in no improvements of animal performance. However, the magnitude of enzyme effects on ruminal degradation was possibly lower than expected in the present study. In addition, we could speculate that increased NFC hydrolysis could reduce microbial adhesion (
      • Miron J.
      • Ben-Ghedalla D.
      • Morrison M.
      Invited review: Adhesion mechanisms of rumen cellulolytic bacteria.
      ), thus having a negative effect on ruminal degradation instead.
      Agreeing with the current study, amylolytic enzymes have not affected DMI, DM digestibility, and milk production (
      • Ferraretto L.F.
      • Shaver R.D.
      • Espineira M.
      • Gencoglu H.
      • Bertic S.J.
      Influence of a reduced-starch diet with or without exogenous amylase on lactation performance by dairy cows.
      ;
      • Nozière P.
      • Steinberg W.
      • Silberberg M.
      • Morgavi D.P.
      Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.
      ).
      • Chen K.H.
      • Huber J.T.
      • Simas J.
      • Theurer C.B.
      • Yu P.
      • Chan S.C.
      • Santos F.
      • Wu Z.
      • Swingle R.S.
      Effect of enzyme treatment or steam-flaking of sorghum grain on lactation and digestion in dairy cows.
      did not observe effects on DMI and starch digestibility but reported an increase in CP digestibility when feeding α-amylase to dairy cows. Similarly,
      • Gencoglu H.
      • Shaver R.D.
      • Steinberg W.
      • Ensink J.
      • Ferraretto L.F.
      • Bertics S.J.
      • Lopes J.C.
      • Akins M.S.
      Effect of feeding a reduced-starch diet with or without amylase addition on lactation performance in dairy cows.
      found an increase in DM, CP, and NDF digestibility when cows were fed a low starch diet supplemented with exogenous amylase. The increase in DM, OM, CP, and NDF digestibility reported by
      • Chen K.H.
      • Huber J.T.
      • Simas J.
      • Theurer C.B.
      • Yu P.
      • Chan S.C.
      • Santos F.
      • Wu Z.
      • Swingle R.S.
      Effect of enzyme treatment or steam-flaking of sorghum grain on lactation and digestion in dairy cows.
      and
      • Gencoglu H.
      • Shaver R.D.
      • Steinberg W.
      • Ensink J.
      • Ferraretto L.F.
      • Bertics S.J.
      • Lopes J.C.
      • Akins M.S.
      Effect of feeding a reduced-starch diet with or without amylase addition on lactation performance in dairy cows.
      can be supported by the findings of
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      . Diet supplementation with α-amylase increased the production of maltodextrins, a substrate for a variety of ruminal bacteria, including amylolytic and nonamylolytic bacteria (
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      ), ultimately increasing the overall nutrient digestibility. As suggested by
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      , the lack of α-amylase effect on starch total-tract digestibility could be associated with increased postruminal compensatory starch digestion, in response to increased ruminal degradation; lack of enzyme effect on ruminal starch degradation; or starch fermentation in another digestive compartment.
      According to
      • Leonardi C.
      • Armentano L.E.
      Effect of quantity, quality and length of alfalfa hay on selective consumption by dairy cows.
      , dairy cows avoid larger particles and prefer smaller particles when fed TMR. Positive effects of fibrolytic (
      • Rode L.M.
      • Yang W.Z.
      • Beauchemin K.A.
      Fibrolytic enzyme supplements for dairy cows in early lactation.
      ) and amylolytic (
      • Gencoglu H.
      • Shaver R.D.
      • Steinberg W.
      • Ensink J.
      • Ferraretto L.F.
      • Bertics S.J.
      • Lopes J.C.
      • Akins M.S.
      Effect of feeding a reduced-starch diet with or without amylase addition on lactation performance in dairy cows.
      ;
      • Weiss W.P.
      • Steinberg W.
      • Engstrom M.A.
      Milk production and nutrient digestibility by dairy cows when fed exogenous amylase with coarsely ground dry corn.
      ) enzymes on NDF digestibility could be associated with animal preference for larger feed particles that contain higher NDF content. However, in the present study, AMY decreased the intake of larger feed particles (>19 mm), and increased intake of feed particles between 8 and 19 mm, without major effects either on starch and NDF digestibility or total feed intake. A relatively low intake of physically effective fiber by animals can be associated with the lack of enzyme effects on nutrient digestibility and decreased feed efficiency. Preference for feed particles with higher energy is expected when fiber requirement (longer particles) is achieved (
      • Miller-Cushon E.K.
      • DeVries T.J.
      Feed sorting in dairy cattle: Causes, consequences, and management.
      ).
      • DeVries T.J.
      • Dohme F.
      • Beauchemin K.A.
      Repeated ruminal acidosis challenges in lactating dairy cows at high and low risk for developing acidosis: Feed sorting.
      observed that lactating cows select few longer feed particles and prefer intermediate-size feed particles in diets with >60% of forage, and the opposite happens in diets with less than 50% of forage. According to
      • Keunen J.E.
      • Plaizier J.C.
      • Kyriazakis L.
      • Duffield T.F.
      • Widowski T.M.
      • Lindinger M.I.
      • McBride B.W.
      Effects of a subacute ruminal acidosis model on the diet selection of dairy cows.
      , dairy cows would increase their dietary preference for a feed with longer particle size when they present low ruminal pH. Although positive effect was observed on intermediate particle intake and butyrate ruminal concentration, AMY showed no effect on ruminal pH in the present study.
      Results of the current study agree with the small effects of enzyme effects on ruminal fermentation reported in the literature (
      • Bowman G.R.
      • Beauchemin K.A.
      • Shelford J.A.
      The proportion of the diet to which fibrolytic enzymes are added affects nutrient digestion by lactating dairy cows.
      ;
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      ;
      • Silva T.H.
      • Takiya C.S.
      • Vendramini T.H.A.
      • Ferreira de Jesus E.
      • Zanferari F.
      • Rennó F.P.
      Effects of dietary fibrolytic enzymes on chewing time, ruminal fermentation, and performance of mid-lactating dairy cows.
      ;
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      ).
      • Bowman G.R.
      • Beauchemin K.A.
      • Shelford J.A.
      The proportion of the diet to which fibrolytic enzymes are added affects nutrient digestion by lactating dairy cows.
      found an increase in ruminal propionate concentration and a decrease in acetate to propionate ratio with fibrolytic enzyme dietary supplementation. In contrast with the current study, these authors observed improvements in NDF digestibility and milk production, which might have occurred because of the increased ruminal enzyme activity and greater availability of fermentable soluble carbohydrates.
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      suggested that the increased milk production in cows receiving α-amylase was apparently mediated by changes in molar proportions of ruminal VFA without affecting ruminal starch digestibility. These authors reported changes in microbial populations and increased ruminal butyrate concentration in cows supplied α-amylase (Aspergillus oryzae extract) cows. The increase in ruminal butyrate concentration in cows fed amylolytic enzyme, as described by
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      , did not reflect in changes of ruminal propionate concentration or milk yield. The increased ruminal butyrate concentration that occurred in the present study could be associated with the growth of nonamylolytic bacteria. Bacteria such as Butyrivibrio fibrisolvens D1, Selenomonas ruminantium GA192, and Megasphaera elsdenii T81 grow faster in cultures with starch and addition of amylase (
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      ). Furthermore, these bacteria degrade fiber (Butyrivibrio fibrisolvens D1) and present high proteolytic activity (Megasphaera elsdenii T81), whereas the main product of their fermentation is butyrate.
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase.
      suggested that the mode of action of α-amylase involves the production of oligosaccharides from amylose and amylopectin. These oligosaccharides can be used by amylolytic and nonamylolytic bacteria in cross-feeding mechanisms that modify the products of fermentation in the rumen. However, as observed in this study,
      • Hristov A.N.
      • Basel C.E.
      • Melgar A.
      • Foley A.E.
      • Ropp J.K.
      • Hunt C.W.
      • Tricarico J.M.
      Effect of exogenous polysaccharide-degrading enzyme preparations on ruminal fermentation and digestibility of nutrients in dairy cows.
      found no effect on the ruminal pH VFA profile in lactating cows fed both xylanase and α-amylase.
      In the present study, amylolytic enzymes decreased urinary N excretion. However, other authors reported no effect of amylolytic enzymes on N excretion (
      • Nozière P.
      • Steinberg W.
      • Silberberg M.
      • Morgavi D.P.
      Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.
      ;
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      ). Additionally, fibrolytic enzymes decreased milk N secretion in accordance with the trend for lower milk protein concentration.
      • Silva T.H.
      • Takiya C.S.
      • Vendramini T.H.A.
      • Ferreira de Jesus E.
      • Zanferari F.
      • Rennó F.P.
      Effects of dietary fibrolytic enzymes on chewing time, ruminal fermentation, and performance of mid-lactating dairy cows.
      reported a linear reduction in milk N secretion with increasing doses of fibrolytic enzyme. As previously discussed, the lower intake of physically effective NDF and increased activity of microorganisms with high proteolytic activity could be related to the decreased N utilization efficiency of AMY-treated animals. In this study, fibrolytic enzyme had no effect on urinary N excretion or milk urea concentration of dairy cows.
      • Tricarico J.M.
      • Johnston J.D.
      • Dawson K.A.
      • Hanson K.C.
      • McLeod K.R.
      • Harmon D.L.
      The effects of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in lactating Holstein cows.
      and
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      showed no differences in blood urea and glucose concentration when supplementing diets for lactating cows with amylolytic enzymes. The results from the current study are similar to those reported by
      • Peters A.
      • Ulrich M.
      • Danicke S.
      Effect of exogenous fibrolytic enzymes on performance and blood profile in early and mid-lactation cows.
      and
      • Silva T.H.
      • Takiya C.S.
      • Vendramini T.H.A.
      • Ferreira de Jesus E.
      • Zanferari F.
      • Rennó F.P.
      Effects of dietary fibrolytic enzymes on chewing time, ruminal fermentation, and performance of mid-lactating dairy cows.
      , which also found no effects of fibrolytic enzymes on serum glucose and urea concentrations.
      The current study showed no improvements in milk yield and composition of cows fed enzymes. Indeed, regardless of the microbial origin (bacterial or fungal), enzyme products have not altered milk yield and composition of mid-lactating cows, neither fibrolytic (
      • Dean D.B.
      • Staples C.R.
      • Littell R.C.
      • Kim S.
      • Adesogan A.T.
      Effect of method of adding a fibrolytic enzyme to dairy cow diets on feed intake digestibility, milk production, ruminal fermentation, and blood metabolites.
      ;
      • Peters A.
      • Ulrich M.
      • Danicke S.
      Effect of exogenous fibrolytic enzymes on performance and blood profile in early and mid-lactation cows.
      ;
      • Silva T.H.
      • Takiya C.S.
      • Vendramini T.H.A.
      • Ferreira de Jesus E.
      • Zanferari F.
      • Rennó F.P.
      Effects of dietary fibrolytic enzymes on chewing time, ruminal fermentation, and performance of mid-lactating dairy cows.
      ) nor amylolytic enzymes (
      • Ferraretto L.F.
      • Shaver R.D.
      • Espineira M.
      • Gencoglu H.
      • Bertic S.J.
      Influence of a reduced-starch diet with or without exogenous amylase on lactation performance by dairy cows.
      ;
      • Vargas-Rodriguez C.F.
      • Engstrom M.
      • Azem E.
      • Bradford B.J.
      Effects of dietary amylase and sucrose on productivity of cows fed low-starch diets.
      ;
      • Takiya C.S.
      • Calomeni G.D.
      • Silva T.H.
      • Vendramini T.H.A.
      • Silva G.G.
      • Consentini C.E.C.
      • Bertoni J.C.
      • Zilio E.M.C.
      • Rennó F.P.
      Increasing dietary doses of an Aspergillus oryzae extract with alpha-amylase activity on nutrient digestibility and ruminal fermentation of lactating dairy cows.
      ). Recently,
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      performed a meta-analysis of 15 studies that evaluated effects of supplementing fibrolytic enzymes on performance of dairy cows and they reported a numerically but not significantly increase in milk production response relative to several enzyme preparations. However, according to
      • Beauchemin K.A.
      • Holtshausen L.
      Developments in enzyme usage in ruminants.
      the stage of lactation and energy status appear to be critical in terms of positive response to enzyme additives.
      Several factors may influence animal response to enzyme supplementation, such as the product preparation, dose, enzymatic activity (
      • Arriola K.G.
      • Oliveira A.S.
      • Ma X.Z.
      • Lean I.J.
      • Giurcanu M.C.
      • Adesogan A.T.
      A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.
      ), supply method (
      • Kung Jr., L.
      • Cohen M.A.
      • Rode L.M.
      • Treacher R.J.
      The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed ration to lactating dairy cows.
      ), diet fraction which enzyme is added (concentrate, TMR, or silage;
      • Bowman G.R.
      • Beauchemin K.A.
      • Shelford J.A.
      The proportion of the diet to which fibrolytic enzymes are added affects nutrient digestion by lactating dairy cows.
      ), stability of the enzymes in the rumen (
      • McAllister T.A.
      • Hristov A.N.
      • Beauchemin K.A.
      • Rode L.M.
      • Cheng K.J.
      Enzymes in ruminant diets.
      ), mechanism of action (
      • Beauchemin K.A.
      • Holtshausen L.
      Developments in enzyme usage in ruminants.
      ), and basal diet composition (
      • Gandra J.R.
      • Miranda G.A.
      • Goes R.H.T.B.
      • Takiya C.S.
      • Del Valle T.A.
      • Oliveira E.R.
      • Freitas Junior., J.E.
      • Gandra E.R.S.
      • Araki H.M.C.
      • Santos A.L.A.V.
      Fibrolytic enzyme supplementation through ruminal bolus on eating behavior, nutrient digestibility and ruminal fermentation in Jersey heifers fed either corn silage- or sugarcane silage-based diets.
      ;
      • Tirado-González D.N.
      • Miranda-Romero L.A.
      • Ruíz-Flores A.
      • Medina-Cuéllar S.E.
      • Ramírez-Valverde R.
      • Tirado-Estrada G.
      Meta-analysis: Effects of exogenous fibrolytic enzymes in ruminant diets.
      ). It is still a challenge for ruminant nutritionists to recommend enzyme supplements because results invariably have small magnitude and are inconsistent in the literature. In addition, the most positive effects of enzyme supplements have been reported in specific conditions such as diets with low starch content, and forage sources with low degradability. This study showed no evidence that the combination of amylolytic and fibrolytic enzymes can improve the performance of mid-lactation cows.

      ACKNOWLEDGMENTS

      The authors thank Alltech Inc. (Nicholasville, KY) for providing funding and assistance to carry out this work. Authors appreciate São Paulo Research Foundation (FAPESP, São Paulo, Brazil) for the fellowship provided to E. M. C. Zilio (grant #2016/15711-8).

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