E r d T F a b a A R R A K C C F N X e s 0 Animal Feed Science and Technology 221 (2016) 35–43 Contents lists available at ScienceDirect Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci ffects of dietary fibrolytic enzymes on chewing time, uminal fermentation, and performance of mid-lactating airy cows .H. Silvaa, C.S. Takiyaa, T.H.A. Vendraminia, E. Ferreira de Jesusb, F. Zanferari a, .P. Rennóa,∗ Department of Animal Nutrition and Production, University of Sao Paulo, Pirassununga, Brazil Department of Animal Science, UNESP—Universidade Estadual Paulista “Júlio de Mesquita Filho’, Jaboticabal, Brazil r t i c l e i n f o rticle history: eceived 10 February 2016 eceived in revised form 15 August 2016 ccepted 16 August 2016 eywords: ellulase hewing activity iber digestion eutral detergent fiber ylanase a b s t r a c t Exogenous fibrolytic enzymes (EFE) can increase ruminal digestion of neutral detergent fiber (NDF) and improve its fermentation for cattle. Twenty-four multiparous Holstein cows (33.72 ± 7.63 kg milk/d and 176 ± 82.27 days in milk – DIM, at the start of the experiment) were used in a replicated 4 × 4 Latin square experimental design. Increasing doses of a com- mercial product was used to evaluate the effect of xylanase activity (100 units per gram of product) on intake and total-tract digestion of nutrients, sorting index, chewing time, milk yield and composition, N use, and ruminal fermentation. Treatments consisted of 0, 8, 16 or 24 g/d per cow of EFE product mixed into the concentrate. Corn silage was used as forage source. The basal diet had a forage-to- concentrate ratio of 50:50 (dry matter basis). Even though EFE supplementation had a positive linear effect on dry matter and NDF intake, it had no impact on total-tract digestion of nutrients. Moreover, this enzyme promoted a linear increase of the intake of feed with larger particle size (>19 mm) and quadratically affected rumination and chewing activity (hours), reaching the highest values at interme- diate doses (8 g/d and 16 g/d). Treatments had no effect on milk yield and composition; however, the N use efficiency was linearly decreased by EFE, reducing thus the ability to synthesize milk and quadratically decreasing N retention. No treatment effect was found on ruminal pH, whereas a negative quadratic effect on ruminal NH3-N concentration was significant. Regarding ruminal parameters, this enzyme supplementation provided linear increased in acetate, propionate, butyrate, and total short-chain fatty acids synthesis. As result, EFE supplementation improved DM and NDF intake, increasing the time spent chew- ing and ruminating, leading to a greater total short-chain fatty acids production in rumen. Nonetheless, EFE did not improve milk yield and composition of mid-lactating dairy cows. © 2016 Elsevier B.V. All rights reserved. Abbreviations: aADF, acid detergent fiber; aNDF, neutral detergent fiber; BFCA, branched-chain fatty acids; CP, crude protein; DM, dry matter; EE, ether xtract; FCM, fat corrected milk; iADF, indigestible acid detergent fiber; N, nitrogen; NH3-N, ammonia nitrogen; SD, standard error of the mean; SCFA, hort-chain fatty acids. ∗ Corresponding author. E-mail address: francisco.renno@usp.br (F.P. Rennó). http://dx.doi.org/10.1016/j.anifeedsci.2016.08.013 377-8401/© 2016 Elsevier B.V. All rights reserved. dx.doi.org/10.1016/j.anifeedsci.2016.08.013 http://www.sciencedirect.com/science/journal/03778401 http://www.elsevier.com/locate/anifeedsci http://crossmark.crossref.org/dialog/?doi=10.1016/j.anifeedsci.2016.08.013&domain=pdf mailto:francisco.renno@usp.br dx.doi.org/10.1016/j.anifeedsci.2016.08.013 36 T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 1. Introduction Ruminants are able to use nutrients from the digestion of fibrous material, and convert them into products for human nutrition (Burns, 2008). However, forage digestion is lower and slower than the digestion of other carbohydrates, limiting nutrient intake and performance of dairy cows (Yang et al., 2000; Mertens and Huhtanen, 2007). In addition, forages produced in Brazil, notably corn silages, have higher neutral detergent fiber (NDF) content and lower digestibility whether compared to those from temperate climate countries (545 g/kg vs. 450 g/kg, respectively; Valadares Filho et al., 2016; NRC, 2001). The relatively low digestion of forage coupled with the NDF contents of Brazilian corn silages may impair milk production efficiency, thus being necessary to explore ways to enhance fiber digestion (Varga and Kolver, 1997; Vicini et al., 2003; Allen and Piantoni, 2014). The addition of exogenous fibrolytic enzymes (EFE) to dairy cow diets aims to increase ruminal enzyme activity and fiber digestion, to improve animal performance (Morgavi et al., 2000; Meale et al., 2014). Dehority and Tirabasso (1998) suggested that the enzyme binding sites in fiber substrates might not be fully occupied by ruminal microorganisms and, thus, the EFE supplementation could led to rise in NDF degradability. Beauchemin et al. (2003a,b) reviewed a dietary supplementation with EFE to dairy cows and observed increases in dry matter intake (1.0 ± 1.3 kg/d) and in milk yield (1.1 ± 1.5 kg/d). However, there have been reported inconsistent findings regarding the effect of such enzyme supplementation on nutrient digestion, metabolism and performance of dairy cows (Yang et al., 2000; Beauchemin et al., 2000; Reddish and Kung, 2007; Arriola et al., 2011). Given the variability of literature findings allied to the relatively low digestibility of corn silage produced in Brazilian dairy farms, this study aimed to evaluate increasing dietary levels of EFE on intake and total-tract digestion of nutrients, sorting index, chewing time, milk yield and composition, N use, and ruminal fermentation in mid-lactating cows. 2. Material and methods The experimental procedures were approved by the Bioethics Committee of the School of Veterinary Medicine and Animal Science, University of São Paulo (approval number: 7066120214). 2.1. Animals, experimental design and treatments Twenty-four multiparous Holstein cows (33.72 ± 7.63 kg milk/d and 176 ± 82.27 days in milk, mean ± SD) were dis- tributed into a replicated 4 × 4 Latin square experimental design in which two squares consisted of rumen-cannulated cows used for ruminal fermentation assessment, beyond the other parameters evaluated. The animals were housed in indi- vidual pens (17.5 m2), with sand bedding, feed bunks and forced ventilation. Experimental periods consisted of 21 d, within which the first 14 d were for treatment adaptation and the last 7 d for data collection. Animals were randomized to receive 0, 8, 16 or 24 g/d per cow of Fibrozyme (Alltech, Nicholasville, KY, USA; batch: 417990-2). This product is an extract from Trichoderma longibarachiatum fermentation with a minimum of 100 IU of xylanase activity per gram of product. According to the manufacture’s directions, Fibrozyme consists of a dry mixture of inactivated yeast, dry brewery yeast, yucca extract and soluble extract of Trichoderma longibarachiatum fermentation. The EFE was daily supplied in the form of a brown dry powder hand-mixed into the concentrate before morning feeding. Total Mixed Ration (TMR) was prepared twice daily and supplied to the animals in equal amounts at 0700 h and 1300 h. Table 1 shows the diet composition that was formulated according to the NRC (2001) recommendations. 2.2. Nutrient intake Feed offered and refusals were weighed daily to estimate feed intake and restrict orts to 50–100 g/kg of total offered food (on as-fed basis). Throughout the sampling period, ingredients were collected during the concentrate preparation (4 samples, one per period); silage and ort samples of each cow were taken daily to provide a composite sample. Immediately after collections, samples were stored at −20 ◦C and later dried in a forced air oven at 55 ◦C for 72 h and ground in 1-mm or 2-mm screen Willey mill (MA340, Marconi, Piracicaba, Brazil). Dry matter (DM, AOAC 950.15), ether extract (EE, AOAC 920.39) and total N (AOAC, 984.13) contents were analyzed in all samples according to the methods described by AOAC (2000). The contents of NDF and ADF were estimated according to the methods described by Van Soest and Mason (1991). The aNDF analysis was performed using �-amylase without sodium sulphide (TE-149 fiber analyzer, Tecnal Equipment for Laboratory Inc., Piracicaba, Brazil). Total digestible nutrient was calculated according to NRC (2001): TDN1X = tdNFC + tdCP + (tdEE × 2.25) + tdNDF − 7, wherein tdNFC is the total digestible non-fiber carbohydrate, tdCP is the total digestible crude protein, tdEE is the total digestible EE, and tdNDF is the total digestible NDF. Non-fiber carbohydrate was calculated according to Hall (2000) in which: NFC = 100 − (CP + NDF + EE + ash) and net energy of lactation (NEL) was calculated according to Weiss et al. (1992): NEL (Mcal/kg) = 0.0245 × TDN (%) − 0.12, using 1 as processing factor. T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 37 Table 1 Ingredients and chemical composition of basal diet (g/kg DM, otherwise stated). Item Diet Ingredient Corn silage 500 Ground corn 278 Soybean meal 144 Whole raw soybean 51.5 Urea 1.30 Ammonium sulfate 1.30 Limestone 7.40 Mineral and vitaminsa 16.2 Salt 0.40 Chemical composition Dry matter 617 Organic matter 935 Crude protein 164 PIDNb 15.3 PIDAc 8.90 Ether extract 46.5 Non fiber carbohydrated 416 Neutral detergent fiber (aNDF) 311 FDNcp e 286 Acid detergent fiber 163 iADFf 45.1 Lignin 40.0 Total digestible nutrientsg 717 Net energy of lactation (MJ/ kg DM)7 6.69 Particle size separation First (top) sieve (19-mm) 71.1 Second sieve (8-mm) 441 Third sieve (1.18-mm) 147 Collection pan 341 a Contained per kg: Ca, 88 g/kg; P, 42 g/kg; S, 18 g/kg; Mg, 45 g/kg; K, 20 g/kg; Na, 123 g/kg; Co, 14 mg/kg; Cu, 500 mg/kg; Cr, 20 mg/kg; Fe, 1050 mg/kg; I, 28 mg/kg; Mn, 1400 mg/kg; Se, 18 mg/kg; Zn, 2800 mg/kg; F, 420 mg/kg; Biotin, 80 mg/kg; vitamin A, 200,000 IU/kg; vitamin D3, 40,000 IU/kg; and vitamin E, 1200 IU/kg. b Insoluble crude protein of neutral detergent fiber content. c Insoluble crude protein of acid detergent fiber content. d According to NRC (2001): NFC = 100 − (CP + NDF + Ether Extract + ash). e Neutral detergent fiber corrected to crude protein content. f Indigestible acid detergent fiber. g + f 2 e w o D C n t i 2 P o a ( Total digestible nutrients were estimated according to NRC (2001) considering total digestibility (td) of nutrients: TDN1X = tdNFC + tdCP + (tdEE*2.25) tdNDF − 7, and net energy of lactation (NEL) was calculated according to Weiss et al. (1992): NEL (Mcal/kg) = 0.0245 × TDN (%) − 0.12, using 1 as processing actor. .3. Total-tract digestion Fecal samples were collected from each cow every 9 h from day 16 until day 18, in each experimental period, representing very 3 h of a 24 h period. After collections, samples were stored at −20 ◦C, and afterwards proportionally homogenized (on et-basis) to form composite samples, which were then dried in a forced air oven at 55 ◦C during 72 h. The dried samples f feed ingredients, orts and feces were ground in a 2-mm screen Willey mill (MA340, Marconi, Piracicaba—Brazil). Total M fecal excretion was determined based on indigestible ADF (iADF) intake and iADF concentration in feces according to asali et al. (2008). For analysis of iADF content, dried and ground samples of feed, orts, and feces were placed in bags of on-woven fabric tissue (pore size 50 �m, 100 g/m2) with dimensions of 5 × 5 cm. Samples were incubated in the rumen of wo cannulated dry cows receiving a similar diet of the current experiment. After 288 h incubation, samples were washed n running tap water, and analyzed for ADF, as previously described. .4. Sorting index Total mixed ration and refusals were analyzed for particle size distribution using a particle separator system (Penn State article Separator—Nasco, Fort Atkinson, WI, USA) as described by Kononoff and Buckmaster (2003), to determine the extent f sorting (Silveira et al., 2007). This equipment is able to detect four particle fractions (>19.0, >8.0, >1.18 and <1.18 mm). The sorting index was calculated as the ratio of actual intake to expected intake for each particle size (as-fed basis), ccording to Eqs. (1)–(3): 1) Expected intake = particle size distribution of the TMR (%, as fed basis) × actual as-fed intake; 38 T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 (2) Actual intake = (% particle size distribution in the TMR × amount of feed offered) (%, as-fed basis) − (% particle size distribution in the orts samples × amount feed refused) (%, as-fed basis); (3) Sorting Index = (actual intake/expected intake) (%, as-fed basis), which: 1 = no sorting; <1 sorting against, and >1 indicates sorting for, particles on the particular screen (Silveira et al., 2007). 2.5. Time spent eating, chewing and ruminating Feeding behavior data were recorded on days 14, 15 and 16 of each experimental period. Animals were observed for 72 h with visual observations made each 5-min intervals. Ruminating, feeding and chewing measurements (hours and hours/kg NDF) were carried out to ascertain if there was influence of treatments on masticatory activity. These data were used to calculate the time spent chewing per kg dietary dry matter and unit NDF intake (Armentano and Pereira, 1997). 2.6. Milk yield and composition Cows were mechanically milked twice daily (0600 h and 1600 h) and milk yield was electronically recorded (Alpro®, DeLaval, Tumba, Sweden). Milk samples were likewise collected from each cow (according to the individual milk yield in each milking) on days 15–17 of each experimental period. Immediately after collections, milk samples were analyzed for fat, protein and lactose contents by infrared analysis (Lactoscan®, Entelbra, Londrina, Brazil). Sub-samples (20 mL) were depro- teinized, according to Broderick and Clayton (1997), and analyzed for milk urea nitrogen using commercial kits (Bioclin®, Belo Horizonte, Brazil) by an enzymatic colorimetric endpoint method, and reading was performed by a semi-automatic biochemistry analyzer (SBA-200, CELM®, São Caetano do Sul, Brazil). Fat-corrected milk (FCM) was calculated according to Sklan et al. (1992) wherein: FCM (kg/d) = (0.432 + 0.1625 × Fat%) × kg milk. Body weights were measured using an electronic livestock scale for large animals, after milking and before the morning feeding on days 7 and 21 of each experimental period. 2.7. Nitrogen balance Nitrogen balance was calculated as the difference between N intake and the sum of fecal, urinary and milk N losses. Nitrogen excreted in milk was calculated based on milk CP concentration, milk yield, and using 6.38 as conversion factor. Nitrogen excreted in feces was estimated based on the following equation: fecal N = CP in feces (g/kg) × DM fecal excretion (kg/d) ÷ 6.25. To determine the total N excretion, urine samples were collected every 9 h from day 16 until day 18 of each experimental period, representing every 3 h of 24 h period. Each urine sample was filtered and an aliquot (20 mL) was imme- diately diluted with a sulfuric acid solution (80 mL, 0.036 N), being stored at −20 ◦C. Daily total urine output was estimated based on urinary creatinine concentration (Chizzotti et al., 2008), which was determined using commercial kits (Bioclin®) by kinetic colorimetric enzymatic reaction with readings taken in a semi-automatic biochemistry analyzer (SBA- 200, CELM®). Therefore, total urine volume was estimated dividing daily urinary creatinine excretion by creatinine concentrations in com- posite urine samples. Urinary daily excretion of creatinine was estimated based on the ratio of 24.05 mg/kg of animal body weight, as described by Chizzotti et al. (2008). Finally, total N in urine samples was determined according to the methods described by AOAC (method 984.13, AOAC, 2000). 2.8. Ruminal fermentation Ruminal fluid was collected from multiple sites (anterior dorsal, anterior ventral, medium ventral, posterior dorsal, and posterior ventral) within the rumen of cannulated cows, before morning feeding (time 0) and 2, 4, 6, 8, 10, 12, 14 and 16 h thereafter. The next step was measuring pH with a potentiometer (MB-10, Marte, Santa Rita do Sapucaí, Brazil). Then, aliquots (1600 �L) of ruminal fluid were mixed with methanoic acid (400 �L, p.a. H2CO2), being centrifuged at 7000 × g for 15 min at 4 ◦C, and the supernatant of each sample was stored at −20 ◦C for further short chain fatty acids (SCFA) analyses. In addition, other aliquots (2 mL) of ruminal fluid were mixed to sulfuric acid solution (1 mL, 0.5 Mol/L H2SO4) and stored at −20 ◦C for subsequent analysis of ammonia nitrogen (NH3-N) by the colorimetric phenol-hypochlorite method (Broderick and Kang, 1980). Ruminal SCFA were measured using a gas chromatograph (GC-2014, Shimadzu, Tokyo, Japan) equipped with a capillary column (Stabilwax, Restek, Bellefonte, USA). The gases used were helium as the carrier gas (8.01 mL/min flow), hydrogen as the fuel gas (pressure of 60 kPa), and synthetic air as oxidizer gas (pressure of 40 kPa). Steamer temperature was set at 220 ◦C; ionization detector flames at 250 ◦C; and separation column at 145 ◦C for 3 min, which was then raised to 10 ◦C/min until reaching 200 ◦C. Short chain fatty acids concentrations were corrected for the amount of liquid and solid mass in the rumen by equation proposed by Hall et al. (2015): SCFA (mol) = SCFA (mmol/L) × Volume ruminal liquid (L) × 1 (mol)/1000 (mmol). Ruminal contents were evacuated manually through the ruminal cannula at 0400 h (4 h before the morning feeding) on day 20 and at 1200 h (4 h after the morning feeding) on d 21 of each period, withdrawing aliquots (10% of total evacuated, on wet-basis). Total ruminal content mass and volume were determined and the sampled aliquots were squeezed through a nylon screen (pore size: 1 mm) for separation into primarily solid and liquid phases, weighing both to minimize sampling errors. Moreover, dry matter was determined by drying at 55 ◦C for more than 72 h in a forced-air oven. Thus, ruminal pool T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 39 Table 2 Effects of increasing doses of exogenous fibrolytic enzymes on nutrient intake and total tract digestion and sorting index of mid-lactating dairy cows. Item Treatmenta SEM P-value 0 8 16 24 Linear Quadratic Intake (kg/d) Dry matter 23.1 23.7 23.3 23.8 0.370 0.047 0.864 Total digestible nutrient 16.7 16.9 16.8 17.1 0.261 0.077 0.588 Non-fiber carbohydrate 9.91 9.97 9.93 10.1 0.153 0.237 0.590 Neutral detergent fiber 6.66 6.82 6.73 6.97 0.119 0.048 0.652 Crude protein 3.95 4.02 3.97 4.03 0.061 0.297 0.977 Ether extract 1.07 1.09 1.09 1.09 0.017 0.157 0.512 Total tract digestion (g/kg) Dry matter 744 736 742 741 0.640 0.948 0.705 Neutral detergent fiber 470 473 491 472 1.129 0.801 0.571 Crude protein 760 744 758 758 0.691 0.856 0.379 Ether extract 734 716 733 720 1.199 0.637 0.794 Sorting indexb 19 mm 0.701 0.743 0.781 0.840 0.023 0.001 0.722 19–8 mm 1.01 1.00 1.00 1.01 0.003 0.829 0.043 8–1.18 mm 1.02 1.02 1.02 1.02 0.002 0.702 0.101 s 2 2 A w t r M K m w e l w p w M l 3 T A b a t ( <1.18 mm 1.03 1.03 1.05 1.04 0.005 0.131 0.531 a Dietary addition of 0, 8, 16, and 24 g/cow per day of exogenous fibrolytic enzymes. b According to Silveira et al. (2007). ize (kg) of DM was determined by multiplying total mass of ruminal digesta by the DM of each sample (Oba and Allen, 003). And finally, ruminal liquid volume was determined by subtracting DM from total ruminal digesta. .9. Statistical analysis The obtained data were submitted to simple polynomial regression using SAS Proc Mixed SAS, version 9.4 (Statistical nalysis for Windows − Institute Inc., Cary, USA) according to the following model: yijkl = � + ˛i + ˇj + γl + cγkl + eijk herein: yijkl stands for the observation of animal k in treatment i at period j in squared l; �i represents the fixed effect of he ith treatment (i = 1–4); �j is the fixed effect of the jth period (j = 1–4); γl = is the fixed effect of the lth square (l = 1–6); cγkl epresents the random effect of animal within square (k = 1–24); and eijk the random error associated with each observation. eans were adjusted by the LSMEANS procedure, and degrees of freedom were calculated using the method detailed by enward and Roger (1997). Ruminal fermentation variables (0, 2, 4, 6, 8, 10, 12, 14 and 16 h after morning feeding) were analyzed as repeated easures, ordered by time, using the PROC MIXED procedure of SAS 9.4, and considering the following statistical model: yijkl = � + ˛i + ˇj + γl + cγkl + e(a)ijk l + ım + ˛ıim + e(b)ijklm herein: yijkl represents the observation for animal k in a given treatment i at a period j in squared l; �i stands for the fixed ffect of the ith treatment (i = 1–4); �j represents the fixed effect of the jth period (j = 1–4); γl expresses the fixed effect of the th square (l = 1–6); cγkl is the random effect of the animal within square (k = 1–24); eijk depicts the random error associated ith each observation of main plot (a); �m represents the fixed effect of the mth time (m = 0, 2, 4, 6, 8, 10, 12, 14 and 16); ��im lays the fixed effect of interaction between i treatment and m time; and e(b)ijklm stands for the random error associated ith each observation of subplot (b). Covariance matrix was estimated using the lowest value found by Akaike’s criterion. oreover, linear and quadratic contrasts were performed to evaluate the dose-response effect of EFE addition. Significance evel was set at 5% (P ≤ 0.05) and tendency towards significance at 0.05 < P < 0.10. . Results The use of EFEs promoted linear increases (P ≤ 0.048) of DM and NDF intake and tended to increase linearly (P = 0.077) DN intake (Table 2). However, EFE did not significant effect (P ≥ 0.379) on total-tract digestion of nutrients of dairy cows. dditionally, EFE supplementation provided a linear increase (P = 0.001) in the intake of feed with particle sizes >19 mm, esides a quadratic effect (P = 0.043) for intake of particles sizes 19–8 mm particle size. Overall, treatments quadratically ffected ruminating (P = 0.005) and chewing activity (P = 0.002) of animals (Table 3). Milk yield and composition were similar among the treatments (Table 4). Nonetheless, enzymatic supplementation ended to have a quadratic effect on milk CP concentration (P = 0.074) and a linear effect increasing body weight gain P = 0.012) of cows. Exogenous fibrolytics enzyme supply caused also a linear reduction of the efficiency of N use for milk 40 T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 Table 3 Effects of increasing doses of exogenous fibrolytic enzymes on time eating, ruminating and chewing of mid-lactating dairy cows. Item Treatmenta SEM P-value 0 8 16 24 Linear Quadratic Activities (h/kg DM) Eating 4.54 4.92 4.92 4.89 0.079 0.111 0.157 Ruminating 7.64 8.55 8.46 8.33 0.095 0.015 0.005 Chewing 12.2 13.6 13.4 13.2 0.095 0.006 0.002 Activities (h/kg NDFb) Eating time 0.640 0.661 0.671 0.652 0.013 0.554 0.349 Ruminating time 1.08 1.13 1.16 1.13 0.021 0.245 0.250 Chewing time 1.70 1.78 1.79 1.79 0.032 0.100 0.250 a Dietary addition of 0, 8, 16, and 24 g/cow per day of exogenous fibrolytic enzymes. b NDF = neutral detergent fiber. Table 4 Effects of increasing doses of exogenous fibrolytic enzymes on milk yield and composition of mid-lactating dairy cows. Item Treatmenta SEM P-value 0 8 16 24 Linear Quadratic Yield (kg/d) Milk 31.2 30.9 31.2 30.8 0.781 0.590 0.749 FCMb 31.7 32.2 31.9 31.6 0.800 0.743 0.451 Fat 1.10 1.14 1.11 1.10 0.032 0.640 0.250 Crude protein 0.934 0.933 0.942 0.921 0.028 0.537 0.456 Lactose 1.40 1.40 1.41 1.38 0.042 0.570 0.463 Composition Fat (g/kg) 36.9 38.4 37.4 37.0 0.056 0.762 0.156 Crude protein (g/kg) 30.9 31.0 30.9 30.8 0.010 0.114 0.074 Lactose (g/kg) 46.3 46.5 46.3 46.2 0.015 0.327 0.196 Milk urea nitrogen (mg/dL) 12.6 12.9 12.7 12.7 0.269 0.822 0.478 Body weight gain (kg/d) 0.08 0.17 0.41 0.42 0.076 0.012 0.531 a Dietary addition of 0, 8, 16, and 24 g/cow per day of exogenous fibrolytic enzymes. b 3.5% Fat corrected milk, FCM = (0.432 + 0.165 × percentage of fat) x kg of milk, from Sklan et al. (1992). Table 5 Effects of increasing doses of exogenous fibrolytic enzymes on nitrogen utilization of mid-lactating dairy cows. Item Treatmenta SEM P-value 0 8 16 24 Linear Quadratic N intake (g/d) 628 644 644 643 9.89 0.179 0.251 N fecal excretion (g/d) 153 166 158 157 5.55 0.886 0.302 N urinary excretion (g/d) 298 239 261 289 12.35 0.934 0.017 N milk secretion (g/d) 146 146 147 144 4.40 0.548 0.431 N retention (g/d) 36.4 92.3 95.8 59.4 11.13 0.360 0.011 Milk N (g/100 g N intake) 23.2 22.8 22.7 22.4 0.60 0.048 0.756 a Dietary addition of 0, 8, 16, and 24 g/cow per day of exogenous fibrolytic enzymes. production (P = 0.048). Furthermore, EFE supplementation exerted a quadratic effect on urinary excretion (P = 0.017) and retention (P = 0.011, Table 5) of N in cows. Moreover, EFE doses showed a quadratic behavior (P = 0.024) regarding ruminal NH3-N concentration. In contrast, concentrations of acetate (P < 0.001), propionate (P = 0.036), butyrate (P = 0.004), valerate (P = 0,017), and total SCFA (P = 0.001) were linearly by EFE supply. As well, the acetate-to-propionate ratio (P = 0.054), the proportion of propionate (P = 0.012) and butyrate (P = 0.046) in rumen were quadratically decreased by EFE supply doses. Finally, no interaction effects were found for ruminal fluid parameters (Table 6). 4. Discussion We hypothesized that EFE supplementation would improve fiber digestion, ruminal fermentation, and increase milk yield. Although we found increasing DM and NDF intake, which raised ruminal production of SCFA, milk yield and composition was not altered. In view of this, Beauchemin et al. (2003a) reported potential increases in feed voluntary intake due to improvements on ruminal fiber digestion, increasing feed passage rate through the digestive tract, by EFE supplementation. In a review of twenty papers by Beauchemin et al. (2003a), EFE supplementing showed to increase in 1.0 kg/d the DM intake of dairy cows. In the current experiment, we associated such DM intake growth (up to 0.7 kg/d) with rumination since it is essential to break down feed particles enabling passage though reticulo-omasal orifice (Welch, 1982). T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 41 Table 6 Effects of increasing doses of exogenous fibrolytic enzymes on ruminal fermentation of mid-lactating dairy cows. Item Treatmenta SEM P-valueb 0 8 16 24 Time Linear Quadratic pH 6.11 6.09 6.06 6.11 0.022 <0.001 0.890 0.408 NH3-N (mg/dL) 18.1 19.1 20.1 17.8 0.365 <0.001 0.865 0.024 Concentration (mmol/100 mmol) Acetate 62.6 62.2 62.2 62.8 0.002 <0.001 0.798 0.158 Propionate 22.6 23.4 23.3 22.5 0.002 <0.001 0.795 0.012 Butyrate 14.8 14.4 14.5 14.8 0.009 <0.001 0.967 0.046 Production (mol) Acetate 6.45 6.69 6.78 7.14 0.008 <0.001 <0.001 0.653 Propionate 2.39 2.54 2.58 2.61 0.052 <0.001 0.036 0.457 Butyrate 1.53 1.56 1.57 1.70 0.023 <0.001 0.004 0.235 SCFAc 10.9 11.4 11.5 12.0 0.151 <0.001 0.001 0.829 A:Pd 2.86 2.81 2.78 2.90 0.037 0.001 0.689 0.054 a Dietary addition of 0, 8, 16, and 24 g/cow per day of exogenous fibrolytic enzymes. b K a i 2 a t m d ( s o w w A E d a t y y a n ( a t i h a d E e e w c t Variable were tested for time*treatment interaction but were not significant. c Total short-chain fatty acids. d Acetate to propionate ratio. Total-tract digestion of nutrients was similar among the treatments, likewise reported in other studies (Peters et al., 2010; nowlton et al., 2002). Therefore, these results suggest that digestion time has no influence from EFE supply, but rates are ffected and showing a positive effect on DM intake (Beauchemin et al., 2003a,b). Conversely, several studies have shown ncreases of DM, NDF and ADF total tract digestion when supplying fibrolytic enzymes to lactating dairy cows (Bowman et al., 002; Arriola et al., 2011). Several factors may influence responses to enzyme supplementation, including its preparation nd amounts (Beauchemin et al., 2003a), supply method (Schingoethe et al., 1999; Kung et al., 2002), to which fraction of he diet it is added (Bowman et al., 2002), animal lactation stage (Knowlton et al., 2002), and limited knowledge of its action echanism (Beauchemin and Holtshausen, 2010). It was expected that fiber intake growth would lead to a NDF digestibility ecline, but our findings demonstrated lack of fiber digestibility problems and thus proving the effectiveness of EFE. Studies have shown that whether fed TMR dairy cows sort rations against long particles and in favor of smaller ones Leonardi and Armentano, 2003; DeVries et al., 2007). However, this behavior may bring problems once cows, while feeding maller particle sizes, NDF intake is reduced because TMR larger particles contain higher amoutns of NDF if compared to the ther fractions (Leonardi and Armentano, 2003). The current experiment demonstrated an increase of longer fiber intake hen supplying EFE for cows, which must be related to an increased ruminating time and NDF intake of cows supplemented ith EFE. Similarly, Krause et al. (2002) reported positive associations of intake of larger particle sizes and ruminating time. s well, Leonardi et al. (2005) observed a correlation between longer fibers and NDF intakes. Adding EFE to feedstuffs may cause release of reducing sugars and partially degrade NDF and ADF (Krause et al., 1998). ven though fiber hydrolysis prior to ingestion could decrease both chewing and ruminating time, we observed longer urations for these processes, due to higher DM and NDF intakes provided by EFE supplement. Notwithstanding, Armentano nd Pereira (1997) noted a negative correlation between chewing time and milk yield. Beauchemin et al. (2003b) observed hat physically effective NDF of diets is positively correlated to chewing time, which could impair the efficiency of milk ield. Leonardi et al. (2005), studying the geometric of food particles, found that increasing sizes caused declines in milk ield and raises in time spent chewing, fueling the demand for dairy herd maintenance. Herein, intermediate doses (8 g/d nd 16 g/d) resulted in higher NDF intake and chewing time, which might have affected animal energy use. Additionally, itrogen urinary excretion was exacerbated when the diet was supplemented with highest EFE dose. Likewise, Arndt et al. 2015) associated N retention depletions with increasing N excretion via the urine for animals receiveing diets with high mounts of degradable protein. Cows used in this experiment were after the peak of lactation, when milk yield falls and animals gain weight over the ime. After that, body architecture and physiological changes favor carcass deposition due to a decrease in peripheral tissue nsulin resistance (observed during early lactation) leading to weight gain (Allen and Piantoni, 2014). Furthermore, studies ave reported an increasing trend of milk yield in early lactating cows (Rode et al., 1999; Yang et al., 1999). Interestingly, lthough Beauchemin et al. (2000) observed no effects of EFE on milk production of mid-lactation cows, these authors escribed weight gain of animals. Given the above, we may infer that lactation stage interfere with animal response to FE, as described by Knowlton et al. (2002). Besides of that, the effects of EFE supplementation seem to increase if ruminal ndogenous capacity to degrade fiber is declined, which occurs during the early lactation (Knowlton et al., 2002). Elwakeel t al. (2007) found a quadratic effect on body weight gain of animals supplemented with increasing doses of EFE, among hich, intermediate doses promoted the lowest gains. These authors suggested changes in distribution of nutrients were aused by enzyme supplementation, increasing carcass deposition by adding high doses of it. Regarding ruminal parameters, EFE supplementation had a quadratic effect on ammonia N, which may partially explain he high N retention using intermediate doses of EFE. Similar results were found in cattle and sheep, due to a greater 42 T.H. Silva et al. / Animal Feed Science and Technology 221 (2016) 35–43 ruminal digestibility of diets supplemented with EFE compared to control diets (Hristov et al., 2000; Bhasker et al., 2013). In addition, acetate, propionate, and butyrate ruminal production linearly increased, which might have occurred because of improvements in ruminal enzyme activity and, consequently, greater availability of fermentable soluble carbohydrates (Hristov et al., 2000; Pinos-Rodriguez et al., 2002; Bhasker et al., 2013). Metabolites released from the cell-wall hydrolysis by the first colonizing microorganisms may stimulate secondary colonizers adherence to feed particles, increasing fermentation rates (Wang et al., 2001). Furthermore, ruminating time lasted longer with EFE supplementation ensuring greater access to feed particles for bacteria during fermentation, thus increasing total SCFA production. Apart from that, propionate production increased while butyrate decreased with added EFE, suggesting a shift in fermentative pathway regarding the synthesis of gluconeogenic precursors, indicating a diverse ruminal bacterial population compared to untreated diets (Giraldo et al., 2008). 5. Conclusion Exogenous fibrolytic enzymes increased DM and NDF intake, increasing both chewing and ruminating time of cows. Although EFE supplementation increased the total production of SCFA in rumen, no alterations were observed for milk yield and composition of mid-lactating dairy cows. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgments The authors acknowledge Dairy Cattle Research Laboratory of University of Sao Paulo, for providing the infrastructure and staff necessary for this study. Authors also acknowledge the Sao Paulo Research Foundation (FAPESP) for providing the scholarship of the first author (grant #2014/13202-3). References AOAC, 2000. 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