I n m E T a b c a A R R A K A A C C I P l a 0 Animal Feed Science and Technology 219 (2016) 59–67 Contents lists available at ScienceDirect Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci nfluence of a blend of functional oils or monensin on utrient intake and digestibility, ruminal fermentation and ilk production of dairy cows . Ferreira de Jesusb, T.A. Del Vallea, G.D. Calomenia, T.H. Silvaa, C.S. Takiyaa, .H.A. Vendraminia, P.G. Paivaa, G.G. Silvaa, A.S. Nettoc, F.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 Department of Animal Sciences, Faculty of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, Brazil r t i c l e i n f o rticle history: eceived 1 February 2016 eceived in revised form 31 May 2016 ccepted 1 June 2016 eywords: dditives ntimicrobial ashew nut shell liquid astor oil onophore henolic lipids a b s t r a c t Cashew nut shell liquid (CNSL) and castor oil (CO) are considered functional oils since they present antitumor, antioxidant, gastroprotective, and antibiotic properties. The objec- tive of this study was to evaluate the effects a commercial blend of functional oils (CNSL and CO) and monensin supplementation on nutrient intake and total tract apparent digestibility, ruminal fermentation, milk yield and composition, N utilization, microbial protein synthesis, and blood metabolites of dairy cows. Twenty-four multiparous Hol- stein cows (150.2 ± 61.4 days in milk, 619 ± 76 kg of BW and 29.1 ± 4.0 kg/d of milk yield, mean ± SD) were used in a replicated 3 × 3 Latin square experimental design, in which six ruminally cannulated cows were used to assess ruminal fermentation. The animals were randomly assigned to one of the following three treatments: control (CON; without addi- tive); 500 mg/kg DM of functional oil (FO; commercial blend of CNSL and CO), and 22 mg/kg DM of monensin sodium (MON). The treatments did not affect either nutrient intake or digestibility of diets. Both feed additives provided an increase in ruminal propionate molar proportion compared to CON. In addition, FO increased ruminal propionate concentration when compared to MON and CON. Although both additives increased (P < 0.01) milk and protein yields, MON had lower milk fat concentration compared to CON, not differing from FO. Monensin and FO increased milk nitrogen excretion. Neither rumen microbial N synthesis nor blood glucose concentration were changed by the supplements. Finally, FO decreased (P < 0.001) blood urea concentration compared to CON or MON, besides increasing milk yield without altering nutrient intake; thus, it might be an alternative to monensin in lactating cow diets. © 2016 Elsevier B.V. All rights reserved. Abbreviations: aADF, acid detergent fiber; aNDF, neutral detergent fiber; BW, body weight; BFCA, branched-chain fatty acids; CNSL, cashew nut shell iquid; CO, castor oil; CON, control; CP, crude protein; DM, dry matter; FCM, fat corrected milk; FO, functional oils; MON, monensin; N, nitrogen; NH3-N, mmonia nitrogen; standard error of the mean, SD; volatile fatty acid, VFA. ∗ Corresponding author. E-mail address: francisco.renno@usp.br (F.P. Rennó). http://dx.doi.org/10.1016/j.anifeedsci.2016.06.003 377-8401/© 2016 Elsevier B.V. All rights reserved. dx.doi.org/10.1016/j.anifeedsci.2016.06.003 http://www.sciencedirect.com/science/journal/03778401 http://www.elsevier.com/locate/anifeedsci http://crossmark.crossref.org/dialog/?doi=10.1016/j.anifeedsci.2016.06.003&domain=pdf mailto:francisco.renno@usp.br dx.doi.org/10.1016/j.anifeedsci.2016.06.003 60 E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 1. Introduction After the banning of monensin (MON) as a growth promoter by the European Union (Regulation 1831/2003/EC), natural compounds have been evaluated to replace MON in dairy cow diets. Functional oils (FO), including cashew nut shell liquid (CNSL) and castor oil (CO), are chemical substances extracted from various plants by distillation, compression or using solvents, providing health benefits besides their nutritive properties. Cashew nut shell liquid and CO have been considered potential rumen modulators because beyond antimicrobial effects, these oils have shown anti-inflamatory (Vieira et al., 2000), anti-oxidative (Trevisan et al., 2006), and gastroprotective (Hamad and Mubofu, 2015) properties which classify them as components of livestock functional feeds. Cashew nut shell liquid is a by-product of the cashew industry containing phenolic compounds (eg anacardic acid, cardanol, and cardol) that inhibit Gram-positive bacteria growth and allow the proliferation of Gram-negative bacteria, thereby increasing ruminal propionate production (Van Nevel et al., 1971; Ipharraguerre and Clark, 2003). Watanabe et al. (2010) reported that CNSL linearly increased the total volatile fatty acid (VFA) concentrations and propionate production in an in vitro fermentation study. Castor oil, obtained from pressing castor seeds, is rich in ricinoleic acid which has shown antimicrobial properties (Ferreira et al., 2002). Additionally, Ramos Morales et al. (2012) reported a reduction in acetate and an increase in propionate for ruminal digestion in sheep supplemented with ricinoleic acid. A blend of CNSL and CO, marketed in Brazil (Essential®, Oligo Basics Agroindustry Ltda., Cascavel, Brazil) was primarily used as anticoccidial in broilers (Murakami et al., 2014) and more recently as a feed additive to ruminants. While some authors have shown that the blend of FO is able to improve dry matter (DM) digestibility (Cruz et al., 2014), feed efficiency (Valero et al., 2014), and meat quality (Prado et al., 2015a) of beef steers, such information is scarce for dairy cattle performance. The objective of this study was to compare the effects of a mixture of CSNL and CO with MON on nutrient intake and total tract apparent digestibility, ruminal fermentation, milk yield and composition, N utilization efficiency, microbial protein synthesis, and blood metabolites of lactating dairy cows. The hypothesis was that the FO blend could replace MON in dairy cow diets without reducing the productive performance. 2. Material and methods The experiment was approved by the Animal Research Ethics Committee of the School of Veterinary Medicine and Animal Science, University of São Paulo—USP (approval number: 6134130415). 2.1. Animals and experimental design The experiment was performed at the Dairy Cattle Research Laboratory, University of São Paulo in Pirassununga—SP, Brazil. Twenty-four multiparous Holstein cows (150.2 ± 61.4 days in milk, 619 ± 76 kg of BW and 29.1 ± 4.0 kg/d of milk yield at the start of the experiment, mean ± SD) were blocked by the milk yield, days in milk, and live weight in a replicated 3 × 3 Latin square design experiment. Six of the animals were ruminally fistulated to assess ruminal fermentation parameters. The three periods of the experiment were composed each of 14 days of adaptation to treatments and 7 days for sampling and data analysis. Cows were randomly assigned to receive one of the three treatments in the first period, and then diets were switched among cows for the second and third periods, according the Latin Square design. The three treatments were control (CON; without additive), 500 mg/kg DM of functional oil (FO; commercial blend of CNSL and CO—Essential®, Oligo Basics Agroindustrial Ltda.), and 22 mg/kg DM of monensin sodium (MON—Rumensin®, Elanco Animal Health, São Paulo, Brazil). The two dietary supplements (MON and FO) were added to a mineral mixture and then to a concentrate. The basal diet (Table 1) was formulated according to the NRC (2001) and was offered as a Total Mixed Ration at a 50:50 ratio between the morning and afternoon feedings (0700 h–1300 h). Refusals were restricted to 5–10% of feed offered (on wet-basis). Throughout the experiment, cows were housed in individual pens (17.5 m2 of area), with sand bedding, individual feed bunks and forced ventilation. The corn silage DM content was measured twice a week and diet adjustments were made when necessary. 2.2. Nutrient intake and total tract apparent digestibility Corn silage and ryegrass haylage samples of 0.3 kg were daily collected during each 7-d sampling period to form a composite sample (on wet-basis). Concentrate ingredients were sampled (0.5 kg) during blending (n = 4) and individual orts (12.5% of daily orts) were taken daily within each 7-d sampling period. Composite samples of feed, orts and feces were dried in a forced-air oven at 55 ◦C for 72 h, and ground in a Wiley mill (Arthur H. Thomas, Philadelphia, PA, USA) to pass through a 1 mm screen. The contents of DM (method 930.15; AOAC, 2000), organic matter—OM (DM—ash), crude protein—CP (N × 6.25, method 984.13; AOAC, 2000), and ether extract—EE (method 920.39, AOAC, 2000) were evaluated in all samples. The content of neutral detergent fiber (aNDF), acid detergent fiber (aADF), and lignin (sa) of samples were measured according to the methods described by Van Soest et al. (1991), and the results were expressed with residual ash. The NDF analysis was determined using �-amylase without sodium sulfite (TE-149 fiber analyzer, Tecnal Equipment for Laboratory Inc., Piracicaba, Brazil). Total digestible nutrient was calculated according to NRC (2001) equations. Net energy of lactation was estimated E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 61 Table 1 Ingredients and chemical composition of the basal diet. Item Diet Ingredient (g/kg DM) Corn silage 368 Ryegrass haylage 112 Ground corn 219 Soybean meal 135 Soybean hulls 49.8 Wheat middling 40.0 Corn gluten meal 31.9 Mineral mixture1 18.0 Limestone 12.1 Sodium bicarbonate 8.10 Urea 3.10 Sodium chloride 1.80 Ammonium sulfate 0.90 Chemical (g/kg DM) Dry matter (g/kg natural matter) 641 Neutral detergent fiber 369 Acid detergent fiber 236 Crude protein 157 Ash 91.2 Lignin 35.5 Ether extract 27.9 Non-fiber carbohydrate2 355 Net energy of lactation3×3 (MJ/kg DM) 6.53 1 Contained per kilogram of the product: 88.0 g Ca, 42.0 g P, 18.0 g S, 45.0 g Mg, 123.0 Na, 0.22 mg Co, 22.0 mg Cu, 20.0 mg Cr, 35 mg Fe, 1.20 mg I, 28 mg Mn, 0 a N e d f a f ( c r w ( 2 a v r c S b A o .70 mg Se, 90.0 mg Zn, 80 mg Biotin, 7000 IU vitamin A, 2000 IU vitamin D, and 50 IU vitamin E. 2 NFC = 1000 − [(CP − CP of urea + %urea) + NDF + EE + ash], from Hall (2000). CP = crude protein, NDF = neutral detergent fiber, and EE = ether extract. 3 Estimated according to NRC (2001). s described by Weiss et al. (1992). Finally, non-fiber carbohydrate (NFC) was ascertained through equation of Hall (2000): FC = 100 − [(CP − CP from urea + urea) + NDF + EE + ash], with values expressed in percentage. Fecal samples (0.3 kg) were collected directly from the rectum of each cow every 9 h from day 16 until day 18 of each xperimental period, representing a collection every 3 h in a period of 24 h, to estimate the amount of excreted nutrient a ay. After collection, samples were stored at −20 ◦C, homogenizing them (wet basis) at the end of each evaluation period to orm composite samples for each animal. Afterwards, samples were dried in a forced-air oven at 55 ◦C for 72 h, and ground in Wiley mill (Arthur H. Thomas) to pass through a 2 mm screen. Ground samples (2-mm screen) of feed ingredients, orts and eces were placed in non-woven textile bags (5 × 5 cm) following the recommendation of a maximum of 20 mg of DM/cm2 Nocek, 1988). These samples were then incubated for 288 h in the rumen of two Holstein cows, previously adapted to a ontrol diet as described in the technique of Casali et al. (2008). After removal from the rumen, the bags were washed in unning tap water, dried at 55 ◦C in a forced-air oven for 72 h, and analyzed for ADF content as described above. Fecal samples ere evaluated for NDF, CP, and EE according to AOAC (2000) as previously described. The indigestible acid detergent fiber iADF) was used as an internal marker to analyze total tract digestion, being calculated by the following equations: Digestibility of DM = 100 − [ 100 × ( % iADF intake % iADF in feces )] Digestibility of nutrient = 100 − [ 100 × ( % iADF intake % iADF in feces ) × ( % nutrient in feces % nutrient intake )] .3. Ruminal fermentation Ruminal digesta were collected on day 20 of each experimental period, always by the same technician, withdrawing equal mounts of digesta from multiples sites within the rumen of cannulated cows (anterior dorsal, anterior ventral, medium entral, posterior dorsal, and posterior ventral), before the morning feeding (time 0) and 2, 4, 6, 8, 10, 12, 14, 16 and 24 h elative to the morning feeding. Immediately after each collection, samples were pooled and strained in four layers of heesecloth to obtain ruminal fluid (250 mL). The ruminal fluid pH was measured using a potentiometer (MB-10, Marte, anta Rita Sapucai, Brazil). Aliquots of 1600 �L of these samples were mixed with 400 �L methanoic acid (98–100% H2CO2), eing centrifuged at 7000g for 15 min at 4 ◦C, and the supernatant of each sample was stored at −20 ◦C for VFA analysis. lso, aliquots of 2 mL were mixed with 1 mL of sulfuric acid (0.5 Mol/L H2SO4) and stored at −20 ◦C for subsequent analysis f ammonia nitrogen (N-NH3) by the colorimetric phenol-hypochlorite method (Broderick and Kang, 1980). 62 E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 Ruminal VFA 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 the oxidizer gas (pressure of 40 kPa). The steamer temperature was set at 220 ◦C, the ionization detector flames at 250 ◦C, and the separation column at 145 ◦C for 3 min, which was then raised 10 ◦C/min up to 200 ◦C. 2.4. Milk yield and composition Cows were milked daily at 0600 h and 1530 h and milk yield was recorded electronically (Alpro®, DeLaval, Tumba, Sweden). Based on milk yield, samples were collected from each cow on days 15, 16 and 17 of each experimental period, being directly analyzed for fat, protein and lactose content by infrared methodology (Lactoscan®, Entelbra, São Paulo, Brazil). Sub-samples of 20 mL were deproteinized according to Broderick and Clayton (1997) and subsequently analyzed for milk urea nitrogen by colorimetric commercial kits (Bioclin®, Belo Horizonte, Brazil) and readings were performed by a semi- automatic biochemistry analyzer (SBA-200, CELM®, São Caetano do Sul, Brazil). The 3.5% fat-corrected milk (FCM) was calculated according to Sklan et al. (1992): 3.5% FCM = (0.432 + 0.165 × milk fat) × milk yield (kg/d). 2.5. Nitrogen utilization and microbial N synthesis Nitrogen excreted in milk was calculated based on the following equation: milk N (g/d) = milk CP concentration (g/kg) × milk yield (kg/d) ÷ 6.38. Nitrogen excreted in feces was calculated based on the equation: fecal N (g/d) = CP in feces (g/kg) × DM fecal excretion (kg/d) ÷ 6.25. Samples of urine were also collected at the same time as feces as described previously. After being collected, urine samples were filtered and aliquots of 10 mL were diluted in 40 mL of 0.036 N sulfuric acid to avoid bacterial destruction of purine derivatives and uric acid precipitation, being stored at −20 ◦C. Non-diluted urine samples were also stored at −20 ◦C, for further assessment of nitrogen and creatinine concentrations. Total urine volume was estimated according to Chizzotti et al. (2008), and urine N was determined according to AOAC (method 984.13, AOAC, 2000); hence, urinary N excretion was calculated by multiplying urine N by urine volume. Daily urinary volume was estimated based on creatinine concentrations in urine. Creatinine and uric acid concentrations were determined using commercial kits (Bioclin®, Belo Horizonte, Brazil) by colorimetric method and readings were per- formed in a semi-automatic biochemistry analyzer (SBA-200, CELM®). The daily creatinine urinary excretion was estimated assuming that daily rate of creatinine excretion as a ratio to BW is fixed at 24.05 mg/kg BW (Chizzotti et al., 2008). Body weights were measured using an electronic livestock scale for large animals (DeLaval), after milking and before the morning feeding on days 7 and 21 of each experimental period. Microbial N synthesis was determined according to Chen and Gomes (1992). Allantoin in urine and in milk were ana- lyzed by a colorimetric method (Chen and Gomes, 1992). Milk samples of 10 mL were mixed with 5 mL of trichloroacetic acid (25%), rested for 5 min and then being filtered through paper filter (14-�m pore size) to obtain deproteinized milk samples. The total excretion of purine derivatives (PD, mmol/d) was calculated as the sum of allantoin and uric acid amounts excreted in urine and milk (Orellana Boero et al., 2001). The absorbed PD (PDabs, mmol/d) were calculated as follows: PDabs = (PD − 0.385 × BW0.75) ÷ 0.84; in which 0.84 represents the recovery of PDabs as PD and 0.385 × BW0.75 represents the endogenous excretion of PD (Chen and Gomes, 1992). The ruminal synthesis of nitrogen compounds (Nmic g of N/d) was calculated based on the PDabs, through the equation of Chen and Gomes (1992): Nmic = (70 × PDabs) ÷ (0.83 × 0.134 × 1000); in which 70 is the N purine derivative content (mg N/mol), 0.134 is the ratio between N purine derivatives and N microbial (Valadares et al., 1999), and 0.83 is the intestinal digestibility of microbial purines. 2.6. Blood profile On day 17 of each period, prior to the morning feeding, blood samples were collected from all cows by puncture of coccygeal vessels into sterile vacutainers without clot activator (BD Vacutainer systems, Franklin Lakes, NJ, USA). After clotting, samples were centrifuged for 15 min at 2000g and 4 ◦C; the supernatant serum was transferred into labeled plastic tubes and stored at −20 ◦C. Blood serum was analyzed for glucose, urea, aspartate-aminotransferase, and gamma-glutamyl transferase using colorimetric commercial kits (Bioclin®) and readings were performed by a semi-automatic biochemistry analyzer (SBA-200, CELM®). 2.7. Statistical analyses Data were analyzed with PROC MIXED (SAS Inst. Inc., Cary, NC) as a 3 × 3 Latin square design according to the statistical model below: Yijkl = � + Ti + Pj + Sk + Al(Si) + eijk Wherein: Yijkl = dependent variable; � = overall mean; Ti = fixed effect of treatment (i = 1 to 3); Pj = fixed effect of period (j = 1 to 3); Sk = fixed effect of square (k = 1 to 8); Al(Si) random effect of animal within square (l = 1 to 24) and eijkl = residual error. E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 63 Table 2 Influence of a blend of functional oils or monensin on nutrient intake and total tract apparent digestibility of lactating dairy cows. Treatment1 Item CON FO MON Mean SEM P-value Intake (kg/d) Dry matter 24.6 25.1 24.7 24.8 0.443 0.455 Neutral detergent fiber 8.40 8.65 8.52 8.52 0.161 0.271 Crude protein 3.93 4.02 3.96 3.97 0.071 0.395 Ether extract 0.70 0.72 0.71 0.71 0.013 0.144 Non-fiber carbohydrate2 9.56 9.82 9.76 9.71 0.175 0.257 Net energy of lactation3x (MJ/d)3 144 145 142 144 2.468 0.352 Intake (% live weight) Dry matter 3.89 3.97 3.90 3.93 0.066 0.302 Neutral detergent fiber 1.33 1.37 1.34 1.35 0.023 0.235 Coefficient of digestibility Dry matter 0.67 0.67 0.67 0.67 0.006 0.746 Neutral detergent fiber 0.63 0.62 0.62 0.62 0.006 0.299 Crude protein 0.69 0.69 0.71 0.70 0.006 0.142 Ether extract 0.85 0.84 0.83 0.84 0.005 0.441 1 Control (CON), no additive; Functional Oil (FO), dietary inclusion of 500 mg/kg DM of a blend of functional oils composed by cashew nut liquid and castor oil; and Monensin (MON), dietary inclusion of 22 mg/kg DM of monensin. 2 NFC = 1000 − [(CP − CP of urea + %urea) + NDF + EE + ash], from Hall (2000). CP = crude protein, NDF = neutral detergent fiber, and EE = ether extract. 3 Estimated according to NRC (2001). Table 3 Influence of a blend of functional oils or monensin on ruminal fermentation parameters of lactating dairy cows. Item Treatment1 Mean SEM P-value CON FO MON Treatment Time Treatment*Time pH 6.31 6.25 6.30 6.29 0.026 0.115 <0.001 0.943 NH3-N (mg/dL) 20.8 22.5 21.9 21.7 0.604 0.522 <0.001 0.740 Fatty acids (mmol/100 mmol) Acetate 61.1 60.7 61.1 61.0 0.170 0.970 <0.001 0.978 Propionate 19.9b 21.0a 20.7a 20.5 0.193 0.006 <0.001 0.999 Butyrate 13.8 13.3 13.4 13.5 0.100 0.525 0.014 0.956 Valerate 1.47 1.50 1.46 1.48 0.017 0.397 <0.001 0.960 Total BCFA2 3.52 3.68 3.31 3.50 0.052 0.277 0.024 0.778 Fatty acids (mmol/L) Acetate 84.2 83.4 79.0 82.4 0.931 0.366 <0.001 0.283 Propionate 27.3b 29.5a 27.4b 28.2 0.566 0.037 <0.001 0.919 Butyrate 19.1 17.9 17.4 18.2 0.238 0.203 <0.001 0.235 Valerate 2.05 2.09 1.93 2.02 0.041 0.319 <0.001 0.810 Total BCFA2 4.90a 5.03a 4.27b 4.73 0.095 0.004 <0.001 0.460 Total VFA3 138 138 130 135 1.728 0.419 <0.001 0.335 Acetate to propionate ratio 3.08 2.82 2.88 3.01 0.035 0.849 <0.001 0.969 a,bValues in the same row with a different superscript differ significantly. 1 Control (CON), no additive; Functional Oil (FO), dietary inclusion of 500 mg/kg DM of a blend of functional oils composed by cashew nut liquid and c m m s e t a 3 T ( F astor oil; and Monensin (MON), dietary inclusion of 22 mg/kg DM of monensin. 2 Total branched-chain fatty acids. 3 Total volatile fatty acids. Ruminal fermentation characteristics (VFA, pH, NH3-N) were analyzed as repeated measures by adding to the previous odel sampling time (0, 2, 4, 6, 8, 10, 12, 14, 16, and 24 h relative to the morning feeding) and its interaction with treat- ents as fixed effects. A first-order autoregressive covariance structure [AR (1)] was used for data analysis based on the mallest Akaike’s information criterion (AIC) values. Other covariance structures tested included compound symmetry, het- rogeneous compound symmetry, unstructured, autoregressive 1 and heterogeneous autoregressive 1. Differences among reatments were analyzed by the least significant difference test (PDIFF). The values in tables were adjusted by LSMEANS nd the significant level was set at P < 0.05. . Results Overall, treatments had no effect on nutrient intake (kg/d and% live weight) and total apparent digestibility (P ≥ 0.142, able 2), not altering (P ≥ 0.115) ruminal pH and NH3-N concentration (Table 3). Both MON and FO supplementation increased P = 0.006) ruminal propionate proportion compared to CON (20.9 ± 0.387 vs. 19.9 ± 0.550 mmol/100 mmol, respectively). urthermore, cows fed FO exhibited the highest propionate concentration (P = 0.037), and the ones fed MON showed similar 64 E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 Fig. 1. Influence of a blend of functional oils or monensin on ruminal propionate concentration of lactating dairy cows. Control, no additive; Functional Oil (FO), dietary inclusion of 500 mg/kg DM of an oil composed by cashew nut liquid and castor oil; and Monensin (MON), dietary inclusion of 22 mg/kg DM of sodic monensin. Different superscripts in treatments differ significantly. Table 4 Influence of a blend of functional oils or monensin on milk yield and composition of dairy cows. Item Treatment1 Mean SEM P-value CON FO MON Yield (kg/d) Milk 25.9b 27.1a 27.2a 26.7 0.659 <0.001 FCM 26.5 27.4 27.0 26.9 0.683 0.188 Fat 0.94 0.97 0.94 0.95 0.027 0.400 Protein 0.78b 0.81a 0.81a 0.80 0.020 0.006 Lactose 1.17b 1.22a 1.22a 1.20 0.030 0.003 Efficiency2 1.07 1.08 1.10 1.08 0.020 0.296 Proportion (g/kg) Fat 36.6a 36.0a,b 34.6b 35.7 0.673 0.025 Protein 30.1 30.1 30.0 30.0 0.132 0.475 Lactose 45.1a,b 45.2a 44.8b 45.0 0.331 0.046 Ureic N (mg/dL) 8.12 8.25 8.46 8.29 0.168 0.545 a,bValues in the same row with a different superscript differ significantly. 1 Control (CON), no additive; Functional Oil (FO), dietary inclusion of 500 mg/kg DM of a blend of functional oils composed by cashew nut liquid and castor oil; and Monensin (MON), dietary inclusion of 22 mg/kg DM of monensin. propionate concentration those fed CON (Fig. 1). Total branched-fatty acids was lower (P = 0.004) in the rumen of cows fed MON whether compared to FO or CON. Despite the differences found in ruminal propionate, the ratio acetate to propionate was not altered (P = 0.849) by treatments; and no interaction between diet and time was found. Although milk production was increased (P < 0.001) by FO or MON supplementations (Table 4), the FCM yield was not affected (P = 0.188) by the treatments. Milk protein and lactose yield were also increased (P ≤ 0.006) with FO and MON addi- tion. Milk fat concentration of cows fed MON and FO were similar; however, MON-fed cows had lower milk fat concentration than the CON-fed ones. Interestingly, FO promoted higher milk lactose concentration compared to MON, but not differing from CON. It is noteworthy mention that treatments had no effect on milk urea nitrogen. Supplementation with FO or MON increased (P = 0.006) milk N excretion in comparison with CON (Table 5). However, microbial protein synthesis was not affected by any of treatments. It is interesting to highlight that FO reduced (P < 0.001) the amount of urea in blood in relation to both CON and MON. Besides, the blood markers of liver damage such as aspartate- aminotransferase and gamma-glutamyl transferase were not altered by dietary treatments. 4. Discussion Nutrient intake and total apparent digestibility were not altered by treatments evaluated. The assessed feed intake mechanisms are related to a short-term regulation, especially with respect to hepatic fuel oxidation (Allen et al., 2009). This process together with ATP production, over a few minutes, may greatly fluctuate and indirectly affect satiety centers, thus increasing or decreasing feed intake (Allen, 2000). Since no differences were observed on nutrient digestibility, neither interactions of diet and time were reported on ruminal fermentation; liver fuel oxidation might have been similar. Some studies have also reported the lack of effects of MON on DM intake and digestibility of dairy cows (Akins et al., 2014; Prado et al., 2015b; Vendramini et al., 2016). Besides of that, data of FO effects on dairy cow performance are still scarce in E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 65 Table 5 Influence of a blend of functional oils or monensin on nitrogen utilization, microbial protein synthesis, and blood profile of lactating dairy cows. Item Treatment1 Mean SEM P-value CON FO MON N utilization (g/d) N intake 629 643 633 635 11.30 0.391 Urinary N 264 287 286 279 12.14 0.575 Fecal N 190 186 181 185 4.82 0.476 Milk N 122b 127a 127a 125 3.12 0.006 Microbial N (g/d) 351 379 397 376 23.30 0.608 Microbial N synthesis efficiency2 133 140 153 142 8.98 0.492 Blood profile Glucose (mg/dL) 43.2 43.9 44.5 43.9 1.09 0.830 Urea (mg/dL) 37.3a 32.4b 39.2a 37.1 0.90 <0.001 Aspartate-aminotransferase (U/L) 66.9 70.8 68.6 68.8 1.17 0.173 Gamma-glutamyl transferase (U/L) 29.1 29.5 30.6 28.6 2.41 0.417 a,bValues in the same row with a different superscript differ significantly. 1 Control (CON), no additive; Functional Oil (FO), dietary inclusion of 500 mg/kg DM of a blend of functional oils composed by cashew nut liquid and castor oil; and Monensin (MON), dietary inclusion of 22 mg/kg DM of monensin. l t i d 2 b r R w t o c ( c a a a q p w C i a o r a r t o m f p D t 3 e 2 Synthesis of microbial crude protein (g)/total digestible nutrient intake (kg). iterature. Purevjav (2011) fed beef steers with FO at 250 mg/kg DM and did not find differences on DM intake compared o those animals fed MON or CON. Similarly, supplementing FO at 3 g/d in diets of finishing beef cattle had no effect on DM ntake (Cruz et al., 2014) and nutrient digestibility (Valero et al., 2014). Additionally, other studies that evaluated CNSL in iets of dairy cows also described no differences on DM intake and nutrient digestibility (Shinaki et al., 2012; Coutinho et al., 014; Branco et al., 2015). When supplemented with MON and FO, lactating cows had increased propionate concentration in rumen. Such result has een reported for MON-fed cattle since 70′ (Raun et al., 1974); emphasizing that these results have been consistently achieved egardless which diet was adopted or evaluated lactation stage (Prado et al., 2015a,b; Vendramini et al., 2016). Regarding ussell (1996), this increase in ruminal propionate concentration is attributed to an inhibition of Gram-positive bacteria hich affects acetate, butyrate and ammonia productions in rumen, thus favoring Gram-negative bacteria growth in addition o propionate production. It should be stressed that treatment effects on ruminal microbial population could change the ratio f purines and microbial protein (Fujihara and Shem, 2011). Nevertheless, microbial N synthesis had no differences which ould due to setting a fixed ratio between purines and microbial protein during microbial protein production calculations 0.134 N PD/N total microbial; Valadares et al., 1999). Few studies have approached ruminal fermentation when supplementing with similar FO mixtures to lactating dairy ows. On the other hand, in vitro fermentation studies have shown increased production of propionate using CSNL or CO s supplement. Seradj et al. (2015) performed an in vitro evaluation with the same FO of the current study and reported n increase in the molar proportion of propionate and a decline of NH3-N concentration. Watanabe et al. (2010) evalu- ted increasing doses of raw CNSL on a pure bacterial culture cultivated in rumen fluid-containing medium and found a uadratic positive response, in which propionate is enhanced by increasing the proportions of propionate- and succinate- roducing rumen bacteriaa. Stasiuk and Kozubek (2010) suggested that phenolic lipids (as cardanol and cardol) interact ith membranes and DNA structures promoting cytotoxic activities responsible for antimicrobial activity. The cardol (a SNL derivative) has been shown to inhibit the growth of Gram-positive bacteria (Himejima and Kubo, 1991), demonstrat- ng similar activity as a rumen modulator of MON. Shinaki et al. (2012) studied the use of CNSL in diets of non-lactating cows nd reported an increase in molar proportion of propionate without altering ruminal pH and ammonia concentration. More- ver, Ramos Morales et al. (2012) evaluated the addition of ricinoleic acid (a CO component) on bacterial cultures from sheep uminal digesta and notified an increase of propionate, and a reduction of acetate, butyrate and isovalarate concentration fter 24 h of fermentation. Both MON and FO increased milk yield without changing nutrient digestibility; therefore, this effect is may be related to uminal propionate increase. Propionate is the most important substrate for hepatic gluconeogenesis (accounting for 60–74% otal substrate), which is highly associate with milk yielding in cows (Aschenbach et al., 2010; Hammon et al., 2010). Besides f that, glucose is a precursor of lactose, an osmotic constituent of milk, which increases water secretion and consequently ilk volume (Miglior et al., 2006). Increasing protein yields have also been observed by high milk production derived rom MON and FO addition, without altering milk protein proportion compared to CON. Interestingly, MON decreased the roportion of milk fat compared to CON. Lately, MON has been associated with milk fat depression (McGuffey et al., 2001; uffield et al., 2003), since it reduces ruminal production of acetate and butyrate (Van Der Werf et al., 1998). Duffield et al. (2008) performed a meta-analysis of production responses of 77 trials supplementing dairy cows with MON; hey found an average increase of 2% in milk and protein yields, while the current experiment showed raises of 5.0% and .8% in milk and protein yields, respectively. To our knowledge, this is the first experiment assessing CNSL and CO mixture ffects on dairy cattle production; however, these compounds have been already evaluated separately. According to certain 66 E. Ferreira de Jesus et al. / Animal Feed Science and Technology 219 (2016) 59–67 authors, CNSL supplementation has not effect on milk production of dairy cows (Coutinho et al., 2014; Branco et al., 2015). Gandra et al. (2014) supplemented dairy cows with ricinoleic acid (a CO component) and found an increase of milk yield. Nitrogen intake and excretion (N in feces and urine) were not affected by treatments, since even N intake and digestibility were similar among all treatments. It is noteworthy that daily urinary volume was based on a fixed creatinine excretion rate (24.05 mg/kg BW), assuming that body tissue breakdown was unaffected by neither FO nor MON. Urinary creatinine excretion is little affected by dietary changes, varying only with animal growth rate (Chizzotti et al., 2008). The secretion of N increased with milk production in cows supplemented with MON or FO compared to CON. Nonetheless, the synthesis of microbial N was not affected by the treatments, agreeing with Branco et al. (2015), who supplemented lactating cows with CNSL and did not find any differences on N utilization, microbial N synthesis, and blood glucose concentration. Similarly, Vendramini et al. (2016) supplied diets of lactating cows with MON and did not observe differences on microbial N synthesis and blood profile, including glucose, urea, aspartate-aminotransferase and gamma-glutamyl transferase. Interestingly, blood urea concentration decreased in cows fed FO, suggesting a greater utilization of absorbed CP or a reduction of endogenous protein turnover. 5. Conclusion The mixture of functional oils evaluated in this study had no influence on nutrient intake and digestibility, however, shifted ruminal fermentation towards energetically most efficient routes, increasing propionate production and conse- quently improving milk yield whether compared to CON. This mixture reduced blood urea concentration; therefore, it may replace MON supplementation in dairy cow diets, without impairing productive performance. Conflict of interest None of the authors of the above manuscript has declared any conflict of interest. Acknowledgments The authors acknowledge the Dairy Cattle Research Laboratory of University of Sao Paulo, for providing the infrastructure and staff necessary for this study. We appreciate the support on the experiment provided by Oligo Basics Agroindustry. In addition, the authors express appreciation to the Brazilian funding agency CAPES (‘Coordenaç ão de Aperfeiç oamento de Pessoal de Nível Superior’) for providing the scholarship to E. Ferreira de Jesus. References AOAC, 2000. Official Methods of Analysis, 17th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Akins, M.S., Perfield, K.L., Green, H.B., Bertics, S.J., Shaver, R.D., 2014. 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http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 http://refhub.elsevier.com/S0377-8401(16)30209-7/sbref0225 dx.doi.org/10.1016/j.anifeedsci.2016.01.015 dx.doi.org/10.1080/09629350020025737 dx.doi.org/10.3168/jds.2009-2754 dx.doi.org/10.1016/0377-8401(92)90034-4 Influence of a blend of functional oils or monensin on nutrient intake and digestibility, ruminal fermentation and milk pr... 1 Introduction 2 Material and methods 2.1 Animals and experimental design 2.2 Nutrient intake and total tract apparent digestibility 2.3 Ruminal fermentation 2.4 Milk yield and composition 2.5 Nitrogen utilization and microbial N synthesis 2.6 Blood profile 2.7 Statistical analyses 3 Results 4 Discussion 5 Conclusion Conflict of interest Acknowledgments References