Effects of an exogenous enzyme, Roxazyme® G2 Liquid, on digestion and utilisation of barley and sorghum grain-based diets by ewe lambs

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Animal Feed Science and Technology 140 (2008) 90–109

Effects of an exogenous enzyme, Roxazyme® G2 Liquid, on digestion and utilisation of barley and sorghum grain-based diets by ewe lambs D.R. Miller a,c,∗ , R. Elliott b , B.W. Norton c a

Tasmanian Institute of Agricultural Research, P.O. Box 46, Kings Meadows, Tasmania 7249, Australia b DSM Nutritional Products Australia Pty Ltd., 13 Princeton Court, Kenmore, Qld 4069, Australia c School of Animal Studies, University of Queensland, Qld 4072, Australia Received 4 September 2006; received in revised form 15 February 2007; accepted 21 February 2007

Abstract A study was conducted to determine effects of a predominantly xylanase/endoglucanase exogenous enzyme (EE) product on digestion and production characteristics of growing lambs (25.3 kg) fed barley or sorghum grain-based diets. Dorset cross ewe lambs were allocated within four liveweight (LW) block groups to one of eight treatments (2 × 4 factorial design) comprising either whole barley or cracked sorghum grain diets (630 g/kg, DM basis) treated with one of four levels of a concentrate applied EE (0, 1.22, 4.88 and 9.76 ml/kg ration DM). Dietary digestibility was determined 4 and 8 weeks after EE treatments commenced and the lambs were fed for 84 d (until average LW > 40 kg). Compared with lambs fed barley-based diets, the lambs fed sorghum-based diets had superior (P<0.05) feed conversion to LW (7.13 and 5.80 kg as fed/kg, respectively) and daily wool growth, although average daily LW gain (172 g) was not affected by diet. Supplementing lambs with EE did not change

Abbreviations: ADFom, acid detergent fibre; CMC, carboxymethyl-cellulose; CP, crude protein; DM, dry matter; DOMD, digestible OM in DM; DOMR, digestible OM apparently fermented in the rumen; EDTA, ethylene diamine tetra acetic acid; EE, exogenous enzyme; EMNP, efficiency of microbial N production; FCE, feed conversion efficiency; LAP, liquid associated protozoa; lignin (sa), sulphuric acid lignin; LW, liveweight; ME, metabolisable energy; aNDFom, neutral detergent fibre; OM, organic matter; PD, purine derivatives; RG2, Roxazyme® G2 Liquid (DSM Nutritional Products Pty Ltd., Basel, Switzerland); RF, rumen fluid; RS, reducing sugar; VFA, volatile fatty acid ∗ Corresponding author at: Tasmanian Institute of Agricultural Research, P.O. Box 46, Kings Meadows, Tasmania 7249, Australia. Tel.: +61 3 6336 5340; fax: +61 3 6336 5395. E-mail address: [email protected] (D.R. Miller). 0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.02.008

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voluntary feed intakes or total tract digestibility of NDF and starch compared to the lambs fed EE untreated diets. Lambs fed the sorghum diet exhibited a linear increase in total tract ADF digestibility with increasing rate of EE treatment and N balance also increased linearly, potentially due to improved ruminal protein availability. However EE supplementation did not improve lamb performance in terms of LW gain, feed conversion efficiency or wool growth. © 2007 Elsevier B.V. All rights reserved. Keywords: Enzymes; Digestibility; Barley; Sorghum; Lambs; Growth

1. Introduction In order to achieve the genetic potential of the current generation of domestic animals, ruminant diets are becoming more nutrient dense, with inclusion of grain in diets to increase energy density. Hogan and Flinn (1999) reported that in 1990/1991, 1.3 million tonnes of cereal grain was eaten by ruminants in Australia, with barley and sorghum representing the most common grains fed. Sorghum grain has corneous endosperm areas characterised by starch granules embedded in a continuous protein matrix (McAllister and Cheng, 1996). This association lowers rumen starch degradability in sorghum to about 0.49, compared to 0.90 for barley grain-based diets (Herrera-Saldana et al., 1990). Barley grain is high in fibre (201 g NDF/kg DM, McDonald et al., 2002), and the outer hull, which contains about 0.96 of the total cellulose present (Newman and Newman, 1992), forms a barrier to microbial colonisation and digestion, particularly in whole grains (McAllister et al., 1990). As a biological grain-processing method, judicious application of an exogenous enzyme (EE) product containing fibrolytic enzyme activities has the potential to improve barley fibre degradation. Similarly, an EE product containing proteolytic enzyme activities may improve sorghum starch availability in the rumen. Improvements in N retention (McAllister et al., 2000; Titi and Tabbaa, 2004) and total tract fibre digestibilities (Titi and Tabbaa, 2004) have been reported for EE supplementation of lambs fed barley-based diets, and Mora et al. (2002) found increased ruminal starch digestion (average 0.85 versus 0.75 for control) in sheep and lambs fed a 50% sorghum grain diet with the application of bacterial and fungal amylases. However, responses in grain-fed sheep to EE supplementation have been variable with no changes in DM intake (Rojo et al., 2005) or total tract digestibility (McAllister et al., 1999) also reported and generally there have been no significant improvements in sheep liveweight (LW) or feed conversion efficiency (FCE) performance (McAllister et al., 2000; Mora et al., 2002). Rate of EE application has been implicated in producing some of the variability in EE research results (Beauchemin et al., 2003). For example, Beauchemin et al. (1995) applied a mixed cellulose/xylanase EE product at six incremental levels to diets of alfalfa hay, lucerne hay or barley silage and found that only low and moderate levels of EE increased steer weight gain for alfalfa hay, but only high levels increased weight gain on the timothy hay. No response was observed for the barley silage. These results suggest effective rates of EE application need to be determined for specific dietary and EE product combinations. This experiment investigated impacts of increasing levels of a mixed-activity EE product, Roxazyme® G2 Liquid (RG2, DSM Nutritional Products Pty Ltd., Basel, Switzerland) on

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digestion, utilisation and in vivo production responses (LW gain, FCE and wool production) of growing crossbred sheep fed high sorghum or barley grain diets.

2. Materials and methods 2.1. Enzyme assay According to the manufacturer, RG2 contains a Trichoderma longibrachiatum derived enzyme complex and is not a source of viable microbial cells. The RG2 used in this experiment was from a single lot (803501), which was sub-sampled and analysed for enzymatic activity using a Nelson/Somogyi assay (McCleary, 2001) with the following modifications. Avicel® PH-101 (Fluka Biochemica Pty Ltd., Buchs, Switzerland – Product # 11365, Lot 398174), the medium viscosity sodium salt of carboxymethyl-cellulose (CMC, Sigma Chemical Co., St. Louis, MO, USA – C4888, Lot 20K0237), oats spelts xylan (Sigma – X0627, Lot 30K0707), purified wheat starch (Sigma – S2760, Lot 109H0040) and barley ␤glucan (Sigma – G6513, Lot 20K0023) were used for determination of exo-1,4-␤-glucanase (EC 3.2.1.91), endo-1,4-␤-glucanase (EC 3.2.1.4), endo-1,4-␤-xylanase (EC 3.2.1.8), ␣-amylase (EC 3.2.1.1) and ␤-d-glucanase, respectively. Substrate solutions/suspensions (20 g/l, 5 g/l for ␤-glucan) were produced in triple deionised water, including 50 ml/l 0.2 M sodium phosphate buffer (pH 6.0), and the pH adjusted to 6.0. Toluene was added to prevent microbial growth. Equal volumes (0.5 ml, 0.167 ml for ␤-glucan) of RG2 solution (0.1 ml/l 0.2 M sodium phosphate buffer) and pre-equilibrated substrate solution were incubated in an agitating waterbath at 39 ◦ C for 15 min (60 min for Avicel). Absorbance was measured at 520 nm against reaction blanks and 0–200 ␮g d-glucose or d-xylose (for xylanase) standard curves. The pH and temperature levels were selected to replicate rumen conditions under moderate levels of grain feeding (Beauchemin et al., 1999). In order to overcome some of the difficulties encountered when comparing results published by different research groups, Colombatto and Beauchemin (2003) suggested standardised assay procedures based on the methods of Miller (1959) and Wood and Bhat (1988). These procedures were also undertaken, with Promote® (Biovance Technologies, Omaha, NE, USA) included for comparison. The previously described substrates were used plus filter paper (50 mg Whatman #1, Lot B1074926), sulphanilamide-azocasein (Sigma A-2765, Lot 043K7021), pectin from citrus fruits (Sigma P9135, Lot 034K0223) and ␳-nitro-phenyl-␤-dglucopyranoside (Sigma N7006, Lot 062K1336). Due to turbidity, the pectinase assay tubes were cooled at 4 ◦ C for at least 10 min and centrifuged for 10 min at 1460 × g before reading. RG2 samples were analysed for protein concentration using a Biuret method (Price, 1996), and crude protein (CP) content was determined using a LECO CNS 2000 combustion analyser (Leco Corp., St. Joseph, MI, USA) set at 1100 ◦ C, calibrated with an ethylene diamine tetra acetic acid (EDTA) internal standard. 2.2. Animals and experimental design Thirty-two Dorset cross ewe lambs (11 months old) were shorn and drenched with 10 ml of cobalt chloride solution 3 weeks prior to commencement of the study, which was

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undertaken in the animal house at the University of Queensland Mt Cotton Research Farm (153.24E, −27.60S). Lambs had a subcutaneous booster vaccine (1 ml UltravacTM 5 in 1 Vaccine, CSL Ltd., Parkville, Vic., Australia) against clostridial diseases 5 d prior to entry into the experimental pens and were drenched with Cydectin® (1 g/l Moxidectin, 1 ml/5 kg LW, Fort Dodge Australia Pty Ltd., Baulkham Hills, NSW, Australia) at entry. The experiment was designed as a 2 (diet) × 4 (enzyme level) factorial experiment. At commencement, the lambs were weighed (25.3 ± 2.83 kg) after a 12 h overnight fast, allocated on LW into four blocks and randomly allocated to treatment within each block. Lambs were then randomly allocated to, and placed in, metabolism crates with clean drinking water available ad libitum. Digestibility measurements were taken 4 and 8 weeks after treatments commenced in order to determine changes in feed utilisation over time with EE supplementation. LW (pre-feeding) was measured weekly at 08:00 h until the average LW reached 40 kg, which occurred after 12 weeks on feed. The University of Queensland Animal Ethics Committee approved the experimental design and animal care provisions and a qualified veterinarian carried out routine health inspections. 2.3. Diets and treatments The lambs were adapted over a 16 d period to a grain-based (i.e., whole barley or dryrolled sorghum) ration including pangola grass chaff (Digitaria decumbens), cottonseed meal, limestone, salt and a mineral premix (Sheep Premix® without copper, Roche Vitamins Australia Pty Ltd., Frenchs Forest, NSW, Australia, Table 1—calculated from ingredient analysis). During the adaptation period, lambs were offered ad libitum chaff that was subTable 1 The ingredients (g/kg DM) and chemical composition (g/kg DM) of the grain-based diets fed to ewe lambs Barley based

Sorghum based

Ingredients Grain Pangola chaff Cottonseed meal Limestone Salt Mineral pre-mixa

629 246 98 13 13 1

629 215 130 15 10 1

Chemical composition DMb OM CP aNDFom ADFom ADL Starch

918 937 139 343 157 33 370

908 939 136 294 150 35 426

a

The mineral premix (Sheep Premix® without copper) provided the following active ingredients (per kg): magnesium (200 g), zinc (40 g), iron (30 g), ethoxyquin (20 g), cobalt (1 g), iodine (0.5 g), selenium (0.1 g), vitamin A (8 mIU), vitamin D (1.6 mIU) and vitamin E (10 g). b Reported on an ‘As Fed’ basis.

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stituted with concentrate mix at 100 g every 2 d. Diets were formulated using SCA (1990) recommendations to provide for the nutritional requirements of a 30–45 kg crossbred ewe growing at 200 g/d, and to be equivalent in energy and protein levels. Lambs were fed to appetite with the chaff offered at 14:00 h daily including 2/3 of the daily concentrate allocation mixed through the chaff. The remainder of the concentrate mix was fed out in equal amounts at 07:00 and 10:00 h the following morning. Feed offered and refused was recorded throughout the experiment. After the grain-adaptation period, the diet was supplemented with RG2. The treatments included a control (no enzyme added, water only), Low RG2 level (1.22 ml RG2/kg DM total ration), Medium RG2 level (4× Low RG2 rate) and a High RG2 level (8× Low RG2 rate). The RG2 liquid was diluted with water to 20 ml/kg DM of total ration and applied evenly onto the grain portion of the concentrate during mixing in a paddle mixer. The enzyme treated grain was mixed for approximately 5 min prior to addition of the cottonseed meal, limestone, salt and mineral premix and mixing continued until ingredients were evenly distributed. Concentrate preparation occurred every 2 weeks and followed a set sequence—Control, Low RG2, Medium RG2 then High RG2 mixes, with thorough washing of equipment between the production of each treatment batch. 2.4. Sampling and analytical procedures Diet feed ingredients were sampled at each concentrate mixing prior to the addition of RG2 and composited for nutrient analysis. Four and 8 weeks after the RG2 treatments commenced, 7 d digestibility studies were undertaken with intakes restricted to 950 g/kg of ad libitum to minimise feed refusals. The amount of diet offered and refused was weighed daily and any refusals composited for subsequent analysis. Feed and refusal DM was determined by drying in a forced air dehydrator at 60 ◦ C to constant weight before grinding through a 1 mm screen for analysis. Faeces were collected and weighed during the digestibility period with 100 g/kg composited for the period and stored at −20 ◦ C. This sample was dried to constant weight in a force air dehydrator at 70 ◦ C and ground through a 1 mm screen for nutrient analysis to determine total tract digestibility of the nutrients in the diets. The DM content of the 1 mm ground feed, refusals and faecal samples was determined in duplicate by oven drying at 65 ◦ C to constant weight. Organic matter (OM) content was determined by ashing duplicate ground samples at 550 ◦ C for 4 h. Metabolisable energy (ME) content of the diets was calculated according to SCA (1990) using the following equation: ME MJ/kg DM = 18 × DOMD − 1.8, where DOMD is the digestible organic matter in DM, calculated as: DOMD = (Feed OM − Faeces OM)/Feed DM. Neutral detergent fibre (aNDFom), acid detergent fibre (ADFom) and sulphuric acid lignin (lignin (sa)) content was determined using an Ankom220 Fibre Analyser (Ankom Technology Corporation, Fairport, NY, USA) and the method of Van Soest et al. (1991), including sodium sulphite and a heat stable ␣-amylase in the digestion phase and heat stable ␣-amylase in the first and second rinses only. The aNDFom, ADFom and lignin (sa) measurements were corrected for ash content. N content of the ground feeds, refusals and faecal samples was determined using a LECO CNS 2000 combustion analyser set

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at 1100 ◦ C, calibrated with an EDTA internal standard and using a Cassava leaf external standard. Apparently digested N retained was calculated as: (N intake − N in faeces − N in urine)/(N intake − N in faeces) × 1000 g/kg. Starch content was determined using a two-step enzymatic method involving heat stable ␣-amylase followed by amyloglucosidase (AOAC Method 996.11, 1995). The enzymes, reagents and procedure used were supplied in the Megazyme Total Starch Assay kit (Deltagen, Boronia, Vic., Australia). The 1 mm ground samples were further reduced in a Janke and Kunkle A10 grinder for 30 s before analysis (corrected for final DM content). Samples were pre-treated with ethanol to remove any free glucose and faecal compounds (Rode et al., 1997) interfering with the colour reaction (B.V. McCleary, personal communication). Urine excreted was collected daily during the digestibility periods into 300 ml of water containing a volume of 2 M sulphuric acid sufficient to maintain the pH of the collection below 3 (Chen and Gomes, 1992). The total volume was measured daily, 100 ml/l composited over the 7 d period before sub-samples were collected and stored at −20 ◦ C. N levels were determined using a LECO CNS analyser at 1100 ◦ C, calibrated with an EDTA internal standard. For determination of purine derivatives (PD), 5 ml of acidified urine was added to 45 ml of 0.1 M ammonium phosphate (NH4 H2 PO4 ) buffer at pH 4.5. Samples were then filtered through a 0.22 ␮m Millex® -GV syringe driven filter unit (Millipore Corp., Bedford, MA, USA) and then through a Maxi-CleanTM C18 solid phase extraction cartridge (Alltech Associates Pty Ltd., Sydney, NSW, Australia). The filtered solution was run through a Waters High Performance Liquid Chromatograph using a 2× Activon Goldpak C18 10 ␮m reversed-phase column (150 mm × 3.9 mm i.d.) with an injection volume of 20 ␮l, flow rate of 0.8 ml/min and a wavelength of 205 nm. The excretion of total PD in urine was calculated according to the method of Chen and Gomes (1992). As the total PD excretion estimate fell below about 2 mmol/d, corresponding to lambs with low feed intakes, the Newton–Raphson iteration process returned a negative estimate of microbial purine absorption, and so the estimate of microbial protein production was not included in those instances (2 and 3 estimates at the first and second digestibility periods, respectively). Rumen fluid (RF) was collected by intubation on the third day following each digestibility period. Collection occurred 2 h after the PM feeding near the time of expected maximal synergy with endogenous enzymes (i.e., 1.5 h—Hristov et al., 1998) and the microbial population. The sample was strained through one layer of nylon stocking and pH measured immediately. Duplicated 4 ml RF samples were collected for NH3 -N determination into 4 ml of 0.2 M hydrochloric acid and placed on ice before subsequent storage at −20 ◦ C. The NH3 N content was determined using steam distillation (B¨uchi 321 Distillation Unit, B¨uchi AG, Flawil, Schweiz) with 40 ml saturated sodium tetraborate and a 4 min distillation period. The distillate was collected into 20 ml of 0.32 M boric acid and back titrated using standardised 0.01 M HCl. A 1 mg N/ml (NH4 )2 SO4 standard solution was used to determine recoveries. To determine volatile fatty acid (VFA) concentrations, 4 ml of RF was collected into 1 ml of 200 ml/l meta-phosphoric acid including an internal standard (600 mg iso-caproic acid per 250 ml 200 ml/l meta-phosphoric acid) and placed on ice before storage at −20 ◦ C. The samples were centrifuged (1200 × g, 15 min), filtered through a 0.45 ␮m Millex® -HA syringe driven filter unit (Millipore Corp., Bedford, MA, USA) and 0.5 ml diluted with 1.0 ml triple deionised water. This was analysed using gas chromatography (30 m DB-

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FFAP 30 m × 0.53 mm × 1.0 ␮m polar capillary column, 0.6 ␮l injection volume) with the internal standardization method for calibration. Samples (5 ml) of RF were collected into 5 ml of 100 ml/l formal saline and stored at 4 ◦ C for liquid associated protozoa (LAP) counting to investigate the potential of RG2 to act as a defaunating agent. A sample of the preserved RF (0.25 ml) was diluted with 0.5 ml of 300 ml/l glycerol solution and stained with 0.25 ml of Lugol’s Iodine Solution (1.0 g iodine added to 2.0 g KI in 300 ml triple deionised water). Ciliate protozoa were counted in duplicate in an Improved Neubauer Bright-Line® Hemacytometer (Hausser Scientific, Horsham, PA, USA). LAP were differentiated into family Isotrichidae (as genus Isotricha or genus Dasytricha) or family Ophryoscolecidae (subfamily Entodiniinae, subfamily Diplodiniinae and subfamily Ophryoscolecinae as Epidinium spp.) according to Dehority (1993). Samples from the first digestibility period were accidentally stored at −20 ◦ C and thawing caused the protozoa to lyse, therefore these samples were discarded. Wool growth was measured using clip-patch and dye-banding techniques. The clippatch technique involved collecting wool from an 80-cm2 area on the right flank mid-side using Oster clippers (#30 blade). While the lamb was restrained in a standing position an outline of the shorn area was collected for area measurement using a Paton Electric Planimeter. Dye banding was carried out using the procedure of Wheeler et al. (1977) using dye prepared as described by McCloghry (1997). The sheep were shorn 20 d prior to the commencement of the adaptation period and application of the initial band. Further dye bands were applied when the sheep started and completed the RG2 treatments (36 and 104 d post-shearing, respectively), and collected 10 d after the end of the treatment period. Staple length between bands was measured as an average of three unstretched staples using a Vernier calliper (MWF-6X, NSK). Greasy wool production (mg/cm2 and mm/d) was calculated for both the adaptation and treatment periods. 2.5. Data analysis Digestibility, RF and microbial N data were analysed using the MIXED procedure of the SAS/STAT software (Version 8.02, 2001, SAS Institute Inc., Cary, NC, USA) with a model including LW block, RG2 level, diet and sampling run as main effects and the associated interaction terms for RG2 level, diet and run. Voluntary as fed intake and LW measurements repeated weekly over the course of the experiment were analysed using the first order autoregressive (AR1) correlation structure of the SAS MIXED procedure, but as there were no significant interactions between RG2 level and time, intake and LW summary data is presented in tables. Variance components for block, between animal and within animal were estimated using the REML method of SAS (Version 8.02, 2001). Protozoal population data and the summary data of wool growth, ADG and FCE for the RG2 treatment period were analysed using the GLM procedure of SAS (Version 8.02, 2001) with a model including LW block, RG2 level and diet as main effects and the associated interaction term, RG2 level × diet. Wool growth during the adaptation period was included as a covariate in the model for wool growth analyses. Linear and quadratic polynomial contrasts were used to define the RG2 dose–response function. Least square means were estimated for effects of RG2 level, diet and run where applicable. Differences among means were analysed using a protected (P<0.05) t-test where significance was declared at P<0.05.

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Table 2 Enzymatic activity of RG2 and Promote recorded using the Nelson/Somogyi assay and procedures of Colombatto and Beauchemin (2003) at pH 6.0 and 39 ◦ C

␣-Amylasea Endo-1,4-␤-xylanasea Exo-1,4-␤-glucanasea,b Endo-1,4-␤-glucanasea ␤-d-Glucanasea ␤-Glucosidasec Pectinased Proteasee Filter paper

Nelson/Somogyi

Colombatto and Beauchemin

RG2

RG2

Promote

5.7 558 13.9 546 246 – – – –

17.8 2172 2.5 695 – 11.0 8.0 40.7 (8.25) ND

28.3 2886 3.9 336 – 11.7 11.3 100 (20.25) ND

ND—not detected. a Expressed as ␮mol reducing sugar released min−1 ml−1 original product. b Sixty minutes incubation of Avicel® PH-101 for NS assay and 120 min incubation for CB assay. c Expressed as ␮mol ␳-nitrophenol min−1 ml−1 original product. d Expressed as ␮mol galacturonic acid released min−1 ml−1 original product. e Expressed as a % of Promote activity, values in brackets = 1 unit for each 0.001 increase in absorbance over the blank (McAllister et al., 1999).

3. Results 3.1. Enzyme characterisation The results of the characterisation assays are in Table 2. It was not possible to obtain the release of 2.0 mg of glucose equivalents from the filter paper. The RG2 solution contained 141 ± 0.9 mg bovine serum albumin equivalents/ml and 194 mg/ml CP. The manufacturer of RG2 reported that it contains approximately 180 mg/ml of enzyme complex. The Promote solution contained 98 ± 2.2 mg bovine serum albumin equivalents/ml. 3.2. Nutrient intake and digestibility The DM, OM and DOM intakes measured 4 and 8 weeks after the RG2 treatments commenced were not affected by grain type, but increasing RG2 application produced quadratic responses (P<0.05) in barley diet consumption and a linear increase (P<0.05) in DOM intakes of the sorghum diet (Table 3). DM and OM intakes also tended (P=0.071) to be linearly increased by increasing rates of RG2 supplementation on the barley diet. Apparent total tract DM, OM and aNDFom digestibilities were not affected by diet, RG2 treatment or time of sampling run. The calculated ME content of the diets was 10.7 ± 0.11 MJ ME/kg DM. Despite higher total tract starch digestibility for the barley diet (986 g/kg DM) versus the sorghum diet (961 g/kg DM), digestible starch intake remained higher (P<0.01) for the sorghum-based diet as a result of higher daily starch intakes (29.0 g/kg LW0.75 ) compared to the barley (23.2 g/kg LW0.75 ) based diets. Excretion of starch in the faeces for the sorghumbased diet increased (P<0.01) at the second digestibility measure (148 g/d) compared to the

98

Diet

S.E.M.

Barley

Average LW DM intake DM digestibility OM intake OM digestibility DOM intake Starch intake Starch digestibility aNDFom Digestibility ADFom Digestibility

Sorghum

P value Diet

Control

Low

Medium

High

Control

Low

Medium

High

35.1 56.2 720 52.8 735 39.3 21.8 993

34.9 60.9 731 57.0 744 42.6 23.3 987

32.0 51.5 741 48.3 755 36.5 20.0 991

37.8 71.5 720 67.0 733 49.3 27.5 974

35.6 61.3 721 57.5 732 42.2 26.7 964

37.2 59.7 716 56.1 728 40.8 26.3 957

37.9 75.8 724 71.1 734 52.0 33.1 958

38.3 69.1 750 64.9 762 49.4 30.1 964

2.19 4.99 12.2 4.65 12.3 3.54 2.03 9.0

518

522

530

502

507

496

502

553

21.6

NS

328

364

327

353

338

326

357

435

26.8

NS

NS—not significant, P>0.05; * P<0.05; ** P<0.01. a Intake based on an offer of 95% of ad libitum during digestibility runs. b Polynomial contrasts, B—barley diet, S—sorghum diet, L—linear effect and Q—quadratic effect.

RG2b BL

NS NS NS NS NS NS ** **

NS NS NS NS NS NS NS NS

BQ

SL

SQ

NS

NS

NS NS NS NS NS

*

*

*

NS

NS NS

NS NS NS NS NS NS NS NS

NS

NS

NS

NS

NS

NS

**

*

*

NS *

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Table 3 Average live weight (kg), daily average DM, OM, DOM and starch intakesa (g/kg LW0.75 ), total tract apparent DM, apparent OM and actual starch and fibre digestibilities (g/kg) recorded at 4 and 8 weeks post-commencement of RG2 supplementation at 0 ml/kg (Control), 1.22 ml/kg (Low), 4.88 ml/kg (Medium) or 9.76 ml/kg (High) ration DM of crossbred ewe lambs fed barley and sorghum grain-based diets

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first (99 g/d). There was no effect of RG2 on total tract starch digestibility for either diet, but ADFom total tract digestibility of the sorghum diet increased (P<0.05) at the higher levels of RG2 application. Apparent total tract N digestibility was higher (P<0.01) for the barley diet (743 g/kg DM) versus the sorghum diet (666 g/kg DM, Table 4). Although faecal N excretion was higher (P<0.01) for the sorghum diet, urinary N excretion was higher (P<0.05) for the barley diet such that N balance was higher (P<0.05) on the sorghum diet (7.8 g/d) versus the barley diet (5.6 g/d). Apparently digested N retained was higher (P<0.05) for the sorghum diet (481 g/kg) versus the barley diet (338 g/kg). RG2 treatment had no effect on apparent total tract N digestibility, but increasing RG2 application linearly increased (P<0.05) N balance for lambs consuming the sorghum diet. 3.3. Rumen fermentation characteristics RF pH was lower (P<0.01) for lambs on the cracked sorghum-based diet (6.39) versus the whole barley-based diet (6.62) 2 h post-feeding (Table 5) and increased (P<0.05) at the second sampling. Rumen NH3 -N levels were lower (P<0.01) for the sorghumbased diet (134 mg/l) versus the barley-based diet (169 mg/l), and linearly increased with RG2 treatment (P < 0.05) of the sorghum diet. Rumen NH3 -N levels were also higher at the second sampling (179 mg/l versus 124 mg/l). Total VFA and acetate concentrations were unaffected by diet, but RG2 treatment produced a quadratic response in those measures (P<0.05) when applied to the barley diet. There was an RG2 × run interaction (P<0.05) whereby on the Medium RG2 level the propionate concentration increased (22.6 mM versus 15.8 mM) at the second sampling time compared to a 7% decrease for the other RG2 levels. Propionate concentration and molar proportions 2 h post-feeding were higher (21.5 mM and 27.5 mol/100 mol) on the cracked sorghumbased diet compared to the whole barley-based diet (17.7 mM and 24.1 mol/100 mol). Butyrate concentrations and molar proportions were lower on the sorghum-based diet versus the barley-based diet and increased (P<0.05) for the barley-based diet with increasing RG2 supplementation levels. The proportion of branch chain VFA in RF was increased (P<0.05) at the High RG2 treatment level in lambs fed the sorghum-based diet. 3.4. Microbial N production and protozoa populations Total estimated purine derivative excretion, microbial N flows and efficiency of microbial N production were unaffected by diet or RG2 application (Table 6), and increased (P<0.05) at the second sampling. LAP total counts per milliliter RF and representation of the different protozoal groups in the overall population 8 weeks after commencement of RG2 supplementation were generally unaffected by dietary composition or RG2 supplementation. The Holotrichs (as Dasytricha because Isotricha were not observed in the RF from either diet) were more prevalent (P<0.05) for the barley-based diet than the sorghum-based diet and the counts of Epidinium sp. and Diplodiniinae were reduced (P<0.05) in a quadratic manner by application of increasing levels of RG2 to the sorghum- and barley-based diets, respectively.

100

Diet

S.E.M.

Barley Control N intake Faecal N Urinary N N balance Apparent N digestibility Apparently digested N retained

Sorghum Low

Medium

High

Control

P value Diet

Low

Medium

High

1.3 4.6 8.2 5.5 739

1.4 5.2 9.3 5.7 740

1.2 3.8 8.0 3.8 757

1.6 6.5 10.8 7.4 736

1.3 7.0 6.7 6.1 641

1.3 7.2 7.5 5.5 652

1.7 8.1 7.3 10.2 684

1.5 7.4 7.0 9.2 686

0.12 0.88 0.98 1.22 18.6

NS

278

370

299

404

386

387

582

568

83.8

*

NS—not significant; * P<0.05; ** P<0.01. a Intake based on an offer of 950 g/kg ad libitum during digestibility runs. b Polynomial contrasts, B—barley diet, S—sorghum diet, L—linear effect and Q—quadratic effect.

** * * **

RG2b BL

BQ

SL

SQ

NS NS NS NS NS

*

NS NS NS NS

NS NS NS NS NS

NS

NS

NS

NS

NS NS NS NS

*

D.R. Miller et al. / Animal Feed Science and Technology 140 (2008) 90–109

Table 4 Daily average dietary N intakea (g/kg LW0.75 ), faecal and urinary N excretion (g/d), N balance (g/d), total tract apparent N digestibility (g/kg) and apparently digested N retained (g/kg) recorded 4 and 8 weeks post-commencement of RG2 supplementation at 0 ml/kg (Control), 1.22 ml/kg (Low), 4.88 ml/kg (Medium) or 9.76 ml/kg (High) ration DM of crossbred ewe lambs fed barley and sorghum grain-based diets

Diet

S.E.M.

Barley Control pH NH3 -N

Sorghum Low

Medium

High

Control

P value Diet

Low

Medium

High

6.67 163

6.53 158

6.70 168

6.60 189

6.42 118

6.31 123

6.40 133

6.44 162

0.078 13.1

Concentration Total VFA Acetate Propionateb,c Butyrate

72.4 45.6 16.6 7.9

75.3 45.7 21.4 6.2

66.5 41.5 14.4 8.7

81.3 50.2 18.3 10.6

74.0 47.2 17.8 7.0

81.7 51.2 22.8 5.8

78.7 47.2 24.1 5.9

76.6 46.5 21.1 6.7

3.62 2.53 1.86 0.89

Proportion Acetate Propionate Butyratec BCVFAc A:P ratio A + B:P

62.5 23.6 11.1 2.9 2.8 3.3

60.5 28.7 8.3 2.5 2.1 2.4

62.3 21.5 13.3 2.9 3.0 3.7

61.6 22.5 13.1 2.8 2.9 3.5

63.7 24.5 9.4 2.4 2.7 3.1

62.4 28.4 7.0 2.2 2.4 2.7

60.0 30.3 7.6 2.0 2.0 2.3

60.9 26.6 9.4 3.2 2.5 2.9

1.36 2.29 1.03 0.27 0.30 0.37

NS—not significant; * P<0.05; ** P<0.01. a Polynomial contrasts, B—barley diet, S—sorghum diet, L—linear effect and Q—quadratic effect. b Enz × Run interaction (P<0.05) c Run effect not significant (P>0.05).

RG2a BL

BQ

SL

SQ

NS NS

NS NS

NS *

NS NS

NS NS NS

*

*

**

*

NS NS NS NS

NS NS NS NS

NS *

NS NS

**

*

NS NS NS

NS NS NS

NS NS NS

NS NS NS

NS NS NS NS NS NS

*

**

NS NS

NS NS

** **

NS NS *

*

NS

D.R. Miller et al. / Animal Feed Science and Technology 140 (2008) 90–109

Table 5 Rumen fluid pH, NH3 -N (mg/l), VFA concentration (mM), molar proportions of VFA (mol/100 mol) and ratios of acetate to propionate and acetate plus butyrate to propionate 2 h post-feeding in crossbred ewe lambs fed barley and sorghum grain-based diets, recorded 4 and 8 weeks after commencement of RG2 supplementation at 0 ml/kg (Control), 1.22 ml/kg (Low), 4.88 ml/kg (Medium) or 9.76 ml/kg (High) ration DM

101

102

Diet

S.E.M.

Barley Control PD excretion Microbial N flow EMNP Protozoa Total count Entodiniinae Epidinium sp. Diplodiniinae Dasytricha Total count (%) Entodiniinae Epidinium sp. Diplodiniinae Holotrichids

4.7 4.6 10.9 209 174 29 7 1 84 12 4 0.2

Sorghum Low 4.7 3.3 8.5 163 147 14 0.7 1 82 17 1 0.6

Medium 4.5 4.3 12.4 218 157 57 2 3 73 26 1 0.7

High 6.4 5.1 10.1 230 192 34 3 1 85 14 1 0.5

P valueb Diet

Control

Low

Medium

High

5.1 3.8 9.2

6.2 4.8 10.9

5.1 3.9 7.4

6.5 5.2 10.3

153 122 31 0.4 ND

172 142 27 3 ND

115 98 16 0.0 1

156 137 18 0.3 ND

80 19 0.4 ND

83 15 3 ND

90 10 0.3 0.2

82 18 0.1 ND

RG2c BL

BQ

SL

SQ

NS NS NS

NS NS NS

NS NS NS

NS NS NS

NS NS NS

52.1 46.4 13.6 2.0 0.9

NS NS NS NS NS

NS NS NS NS NS

NS NS NS

NS NS *

NS

NS NS NS NS NS

8.1 7.8 1.5 0.2

NS NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

1.08 1.00 1.81

Microbial N calculations based on the formula of Chen and Gomes (1992). NS—not significant; * P<0.05. ND—not detected. a DOMR = 0.65 × digestible OM intake (Chen and Gomes, 1992). b Statistical analysis completed on log -transformed data for protozoa population count data. 10 c Polynomial contrasts, B—barley diet, S—sorghum diet, L—linear effect and Q—quadratic effect.

*

*

NS NS

D.R. Miller et al. / Animal Feed Science and Technology 140 (2008) 90–109

Table 6 Total purine derivative excretion (mmol/d), calculated microbial N flows (g/d) and efficiency of microbial N production (EMNP, g/kg digestible OM apparently fermented in the rumen, DOMRa ) recorded 4 and 8 weeks after commencement of RG2 supplementation, and total counts (units of 10,000 ml−1 ) and proportions of liquid associated protozoal populations (% of total count) in rumen fluid sampled 2 h post-feeding 8 weeks after RG2 treatment commenced at 0 ml/kg (Control), 1.22 ml/kg (Low), 4.88 ml/kg (Medium) or 9.76 ml/kg (High) ration DM for crossbred ewe lambs fed barley and sorghum grain-based diets

Diet

S.E.M.

Barley

Voluntary feed intake LW gain FCE Wool growth Mid-side Dye band

Sorghum

P value Diet

Control

Low

Medium

High

Control

Low

Medium

High

2.81 163 6.96

2.89 154 7.99

2.73 141 7.32

3.21 196 6.23

2.80 183 5.96

2.84 166 5.53

3.28 203 5.96

3.00 183 5.77

0.181 31.1 0.787

1.15 0.28

1.00 0.28

1.01 0.29

1.00 0.32

1.24 0.28

1.25 0.29

1.13 0.29

1.25 0.29

0.118 0.014

NS—not significant; * P<0.05. a Polynomial contrasts, B—barley diet, S—sorghum diet, L—linear effect and Q—quadratic effect.

RG2a BL

BQ

SL

SQ

NS NS NS

NS NS NS

NS NS NS

NS NS NS

*

NS

NS

*

NS NS

NS NS

NS NS

NS NS *

D.R. Miller et al. / Animal Feed Science and Technology 140 (2008) 90–109

Table 7 Average daily voluntary feed intake (% LW), liveweight gain (g/d) and feed conversion efficiency (kg feed/kg LWG) for the entire experiment, and daily wool growth during the RG2 treatment period measured using the mid-side patch (mg/cm2 ) and dyeband (mm) techniques, for ewe lambs fed barley or sorghum grain-based diets treated with RG2 applied at 0 ml/kg (Control), 1.22 ml/kg (Low), 4.88 ml/kg (Medium) or 9.76 ml/kg (High) ration DM

103

104

D.R. Miller et al. / Animal Feed Science and Technology 140 (2008) 90–109

3.5. Voluntary feed intake, LW gain, feed conversion efficiency and wool growth Voluntary feed intake (Table 7) was variable and unaffected by diet or RG2 level. Average daily LW gain data was not affected by either diet or RG2 level. FCE on an as fed basis for the lambs fed the barley-based diet (7.13 kg feed/kg gain) were lower (P<0.05) than for lambs fed the sorghum-based diet (5.80 kg feed/kg gain). FCE was not affected by RG2 supplementation. Lambs fed the sorghum-based diet produced more (P<0.05) wool than lambs fed the barley-based diet (1.22 ± 0.058 and 1.04 ± 0.058 mg/cm2 /d, respectively), with no effect due to RG2. When measuring linear wool growth using the dyeband technique, increasing RG2 supplementation linearly increased (P<0.05) wool growth of lambs on the barley diet. The dyeband technique had a lower coefficient of variation (9.3%) compared to the mid-side clip-patch technique (20.5%). 4. Discussion 4.1. Enzyme activity characterisation Under similar assay conditions to those reported here, Colombatto et al. (2003) used a Nelson/Somogyi procedure (pH 6.0 and 39 ◦ C for 5–60 min) to analyse 23 EE products and recorded averages of 831 ␮mol (range 28–3228), 325 ␮mol (0–1047), 44 ␮mol (0–181), 340 ␮mol (5–1591) and 283 ␮mol (2.1–1892) RS equivalents released/min/g or ml for xylanase (oat spelt), endoglucanase, exoglucanase (Sigmacell 50), ␤-glucanase, and ␣-amylase, respectively. The average for ␤-glucosidase activity was 8.6 ␮mol (0–58) ␳nitrophenol equivalents released/min/g or ml and average protein content of the 23 products was 229 g/kg (37–795 g/kg). Therefore, in comparison with those results, RG2 contains lower than average protein concentrations, amylase and exoglucanase activities, with average ␤-glucanase and ␤-glucosidase activities and above average levels of xylanase and endoglucanase. The inability of RG2 to degrade the filter paper indicates that it does not contain a complete system of cellulose-degrading enzymes, or that negative feedback inhibition was occurring. The low level of activity observed against Avicel is more likely suggestive of the presence of endo-␤-1,4-glucanases that can act against this substrate (Buchholz et al., 1984; Wood and Bhat, 1988) given that Avicel can contain up to 300 g/kg amorphous cellulose (Buchholz et al., 1984). Despite their low concentrations in RG2, the secondary activities of amylase, pectinase and protease, and other unmeasured activities such as esterases, may have had an effect on substrate degradation by removing rate-limiting barriers to microbial digestion. EE development as a feed technology is yet to advance to the point where biochemical characterisation and in vitro assays can predict animal responses (Beauchemin et al., 2004). Therefore until the rate-limiting EE activities can be identified for specific animal and dietary combinations there will be a continuing need for in vivo evaluations of EE. 4.2. Nutrient intake, RG2 treatment and grain type Variability in voluntary feed intake was approximately twice that observed for other experiments with sheep fed high concentrate diets (Yang et al., 2000; McAllister et al.,

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105

2000) and this variability was especially noticeable for lambs fed the Medium RG2 level barley-based diet. The linear trend suggests intake increased at the High RG2 application level compared to the controls. Use of EE supplementation has reduced feed intake variability on barley grain diets in feedlot cattle (Beauchemin et al., 1999) but this was not observed in the current experiment. The increased DOM intakes during the digestibility studies (950 g/kg ad libitum feed offer) with higher levels of RG2 supplementation were not accompanied by changes in total tract nutrient digestibility, or voluntary ad libitum feed intake, FCE or LW gain during the treatment period. A larger-scale, production feeding trial may have clarified the potential growth responses of lambs to these higher levels of RG2 supplements. McAllister et al. (2000) fed 21 kg Dorset cross lambs 750–810 g/kg whole barley grainbased rations supplemented with mixed-activity EE products and reported no increases in DM and OM intake with no change in total tract digestibilities. Two studies conducted by Yang et al. (2000) investigated the application of a high xylanase/low cellulase EE applied (0.05 g/kg total mixed ration DM) to the concentrate portion of a diet containing steam rolled barley grain (450 g/kg) fed to two groups of Dorset wether lambs. DM intake for the EE treatment groups did not differ from the control groups and digestibility was also not affected. In contrast, Titi and Tabbaa (2004) fed a 600 g/kg barley grain diet to 35 kg Awassi lambs supplemented with a Trichoderma derived cellulase (Maxicel 200® , George A. Jefferys, Salem, VA, USA; 0.15 g/kg forage consumed) and reported increased DM (0.663 versus 0.636), NDF (0.425 versus 0.400) and CP (0.735 versus 0.721) digestibilities. That response may have been related to the lower DM digestibility of the basal diet compared to the diets used here (>0.7 DM digestibility), and by McAllister et al. (2000) and Yang et al. (2000). The potential for EE supplements to improve the extent of total tract digestion in sheep seems to be limited where dietary digestibility is already high. The trend to increased nutrient intakes, such as digestible starch, and a higher N balance and FCE for sheep fed the sorghum-based diet resulted in improved wool growth (measured by the mid-side patch technique) versus sheep fed the barley diet. The dyeband technique was not as sensitive in detecting dietary differences in wool growth compared to the midside clip-patch technique over the relatively short time period measured, probably relating to the more open staple structure of crossbred sheep. RG2 supplementation did not increase the weight of wool produced. 4.3. Fibre, starch and protein digestion For the sorghum diet, increasing the rate of RG2 application resulted in a linear increase in total tract ADFom digestibility, potentially moderating some of the well documented decline in fibre digestion that is often observed under high grain feeding conditions (Noziere et al., 1996). Apart from some shifts in butyrate production on the barley diet, VFA concentrations and proportions were generally unaffected by RG2 treatment, indicating that the rate of fibre digestion was probably not enhanced by pre-feeding contact with the enzyme. The quadratic effects of RG2 supplementation on concentrations of VFA in lambs fed the barley diet likely relate to the feed intake responses previously described. RF collection occurred near the time of expected maximal synergy with the endogenous enzymes

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(Hristov et al., 1998), however this single sample time may not have enabled full expression of any RG2 effect to be recorded. In most studies using sheep there has been no effects of EE on rumen fluid characteristics such as pH, ammonia and VFA concentrations, molar proportions of VFA, ruminal enzyme activity or numbers of cellulolytic bacteria (Judkins and Stobart, 1988; McAllister et al., 1999, 2000; Mora et al., 2002; Pinos-Rodr´ıguez et al., 2002). It is unclear why the propionate concentration increased at the second sampling for the Medium RG2 level treatment, whereas concentrations decreased for the other RG2 levels. Total tract starch digestibility in our experiment for the barley (0.986) and the sorghumbased diets (0.961) were consistent with previous observations (Rowe et al., 1999) and unaffected by RG2, possibly due to the low levels of amylase and protease present in the product or because the high values left little potential for an increase. Lower RF pH and increased propionate concentrations and contribution to total VFA indicated that the starch in cracked sorghum was more ruminally available 2 h post-feeding than the starch in whole barley grain. Feeding of whole barley to sheep is recommended by Ørskov (1986) to reduce the rate of fermentation (Ørskov and Fraser, 1975), and therefore digestive and metabolic problems, and the grain forms (i.e., whole barley and cracked sorghum) used in this experiment reflect commercial feedlot practices in Australia. Lower NH3 -N concentrations in RF from lambs on the sorghum-based compared to the barley-based diet 2 h post-feeding may be a consequence of more active microbial uptake of NH3 -N in response to a more readily fermentable energy source. Alternatively, protein availability in the rumen was higher for the barley-based diet compared to the sorghumbased diet as indicated by higher NH3 -N concentrations, increased urinary N excretion compared to higher faecal excretion in sorghum fed sheep and increased apparent total tract N digestibility. Increased efficiency of apparently digested N retained on the sorghum diet for the same levels of N intake resulted in the N balance for sorghum-based diets being higher than with the barley-based diets. The responses of increasing NH3 -N concentrations and proportional contribution of branch chain VFA to increasing RG2 supplementation of the sorghum diet indicates that ruminal protein degradation increased at higher levels of EE. This may have contributed to the improved N balance responses on that diet. Overall these NH3 -N concentrations (>100 mg N/l) are not expected to have limited rumen microbial growth. 4.4. Microbial population The protozoal population size and composition are typical of a high starch diet fed to sheep (Jouany, 1989) and RG2 supplementation had little effect on the measured microbial populations. Nsereko et al. (2002) postulated that rumen microbial population changes observed with EE application may have been due to a defaunating action by EE preparations. Our results with RG2 do not support that hypothesis of EE action. McAllister et al. (2000) used PD to estimate microbial N flows and recorded microbial N flows (9 g/d) about twice those recorded in this experiment (4.4 g/d) and at a slightly higher efficiency (13 g/kg versus 10 g/kg DOMR) and the sheep in that experiment were gaining LW at over 300 g/d, compared to our barley diet average of 160 g/d. RG2 supplements did not appear to improve the amount, or efficiency, of microbial N production under our experimental conditions.

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5. Conclusions Growing lambs fed cracked sorghum-based diets had superior FCE, digestible starch intakes, N balance and wool growth versus lambs fed whole barley-based diets, although feed intakes were variable and LW gain was unaffected by diet. Supplementing lambs fed barley and sorghum grain-based diets with three levels of a mixed-activity enzyme product, RG2, produced some diet- and application rate-dependant increases in DOM intake, N balance and total tract ADF digestibility, but did not change total tract digestibility of NDF or starch compared to the control lambs fed untreated diets. Supplementation with this EE did not improve overall voluntary feed intakes or performance in terms of LW gain, FCE or wool production.

Acknowledgements The authors would like to acknowledge the financial support of DSM Nutritional Products Pty Ltd. and thank Michael Nielsen for his technical assistance, Andrew Gibbon and Les Gardiner for animal care and Allan Lisle for his generous statistical assistance.

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