Effects of an exogenous enzyme, Roxazyme® G2, on intake, digestion and utilisation of sorghum and barley grain-based diets by beef steers

Available online at www.sciencedirect.com

Animal Feed Science and Technology 145 (2008) 159–181

Effects of an exogenous enzyme, Roxazyme® G2, on intake, digestion and utilisation of sorghum and barley grain-based diets by beef steers夽 D.R. Miller b,∗ , R. Elliott a , B.W. Norton b a

DSM Nutritional Products Australia Pty Ltd., 13 Princeton Court, Kenmore, Qld 4069, Australia b School of Animal Studies, University of Queensland, Qld 4072, Australia Accepted 14 May 2007

Abstract A series of experiments were undertaken to determine effects of a mixed xylanase and endoglucanase exogenous enzyme (EE) product, Roxazyme® G2, on nutrient intake, digestion and feed conversion in beef steers fed sorghum or barley grain-based diets. Sixteen Bos indicus crossbred steers (314.2 ± 26.07 kg) were allocated within stratified liveweight (LW) blocks to four treatments consisting of dry-rolled, sorghum or barley based (∼0.60) diets treated with concentrate applied EE at 0 or 4.43 ml/kg diet dry matter (DM). The EE supplementation occurred for 7 weeks with digestibility measurements 2 and 6 weeks after commencement. The EE treatment resulted in increased daily voluntary DM intakes (P<0.05) for steers fed the sorghum diet, but not for steers fed the barley Abbreviations: ADFom, acid detergent fibre; ADG, average daily gain; AUS-MEAT, Authority for Uniform Specification of Meat and Livestock; DM, dry matter; DMD, DM digestibility; DOM, digestible OM; DOMI, DOM intake; EDTA, Ethylene diamine tetra acetic acid; EE, exogenous enzyme; EMA, eye muscle area; EMNP, efficiency of microbial N production; FCE, feed conversion efficiency; FPR, fractional passage rate; FT, fat thickness; HG, high grain; HGP, hormonal growth promotant; HSCW, hot standard carcase weight; LG, low grain; LW, liveweight; ME, metabolisable energy; MSA, Meat Standards Australia; aNDFom, neutral detergent fibre; OM, organic matter; PD, Purine derivatives; RF, rumen fluid; RG2, Roxazyme® G2 Liquid (DSM Nutritional Products Pty Ltd., Basel, Switzerland); RT, retention time; US BCS, US body condition score; VFA, volatile fatty acid. 夽 This paper is part of a special issue entitled “Enzymes, Direct Fed Microbials and Plant Extracts in Ruminant Nutrition” guest edited by R. J. Wallace, D. Colombatto and P. H. Robinson. ∗ Corresponding author. Present address: Tasmanian Institute of Agricultural Research, P.O. Box 46, Kings Meadows, Tas 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.2007.05.045

160

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

diet. Daily LW gain increased numerically on both diets (920 g/d versus 740 g/d, P=0.138) with no changes in feed efficiency. The EE treatment had no effect on total tract OM or fibre digestibility, but interacted with diet (P<0.05) whereby sorghum starch digestibility at 6 weeks was reduced by EE treatment (0.68 versus 0.81 control) without change in barley starch digestion (0.96). The EE supplements also increased (P<0.05) urinary N excretion. In a second 4 × 4 Latin Square experiment with 24 d periods, ruminally cannulated B. indicus crossbred steers (364.3 ± 21.98 kg, n = 4) were fed sorghum grain diets, either as in the first experiment or at reduced grain levels (0.35 of diet DM), and untreated or treated with EE (4.18 ml/kg diet DM) as previously. Under these conditions, EE treatment had no effects on feed intake, total tract digestibility or ruminal fermentation measurements. A marker dilution technique indicated that EE treatment reduced (P<0.05) the fractional passage rate of a grain-associated marker when applied to the high grain diet, while increasing it on the low grain diet, but EE did not affect fluid or fibre marker flows from the rumen. Ruminal in sacco incubations of 3 mm ground pangola grass (Digitaria decumbens) or sorghum grain revealed a reduction (P<0.01) in the insoluble potentially degradable fraction of the grass with EE supplementation, likely due to reduced (P=0.058) anaerobic fungi colonisation, and a tendency (P=0.082) for increased extent of grain DM disappearance. A 70 d feedlot experiment used 96 Santa Gertrudis steers (351 ± 25.3 kg) allocated in balanced groups to one of four replicates of four levels of EE supplementation (i.e., 0, 1.08, 2.16, 4.33 l/tonne DM total ration) applied to a high quality, dry-rolled sorghum (0.72 of DM) finishing ration. The EE had no effect on DM intake (120 g/kg LW0.75 ), LW gain (1.92 kg/d), feed efficiency (5.72 kg DM/kg LW gain) or carcase attributes. A mixed activity EE product fed to beef cattle had dietary dependant (both type and composition) effects on feed intake, starch and N digestion, microbial efficiency, grain marker flow rates and the extent of in sacco degradation of a grass forage. However, production performance and carcase measures were not effected by adding the EE to a high quality feedlot diet fed to growing steers. © 2007 Elsevier B.V. All rights reserved. Keywords: Enzymes; Grain; Digestion; Feed intake; Weight gain; Efficiency

1. Introduction Increases in performance of ruminants eating forages supplemented with grain are due to improvements in organic matter (OM) digestibility (Mathers and Miller, 1981; Hoover and Stokes, 1991) and higher intakes of digestible OM and energy (Reis and Combs, 2000; Knowlton, 2001). However, inclusion of higher levels of grain has also been associated with declining rates of fibre digestion due to lower fibrolytic activity in the rumen (Martin and Michalet-Doreau, 1995; Noziere et al., 1996) and an apparent microbial preference for nonstructural carbohydrates (Mould and Ørskov, 1983). This may create conditions in which supplementary fibrolytic exogenous enzyme (EE) will have beneficial effects (Beauchemin et al., 2001). Roxazyme® G2 Liquid (RG2, DSM Nutritional Products Pty Ltd, Basel, Switzerland) is a mixed activity EE that has been shown to produce diet and application rate dependant increases in N balance and acid detergent fibre (ADFom) digestibility in growing lambs, but did not change neutral detergent fibre (aNDFom), and starch digestibilities or voluntary feed intake, liveweight (LW) gain or feed conversion efficiency (FCE, Miller et al., 2008). Van Soest (1994) indicated that when offered high quality forages, sheep usually have higher digestibility co-efficients than cattle, and Theurer (1986) reported that total tract

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

161

digestibility for non-processed grains in sheep (0.96) was higher than in cattle (0.91). Yang et al. (2000) reported that dry matter (DM) digestibility (DMD) of a 450 g/kg barley grain diet fed to dairy cattle (0.64) was increased by mixed activity EE supplements, but not for lambs achieving DM digestibilities of 0.76. This suggests that EE may help overcome limitations between actual and potential performance where some restriction to digestion exists (Beauchemin et al., 2003), and that responses may occur in cattle that do not occur in sheep. In vitro incubations of RG2 with ground barley grain, sorghum grain and pangola grass (Digitaria decumbens) chaff showed that reducing sugar release was highest from barley grain versus pangola grass and sorghum grain (Miller, D.R., unpublished data), indicating specificity of RG2 for different substrates. Responses to EE are known to depend on the degree of complementarity with dietary ingredients (Beauchemin et al., 2003) and LW gain responses can vary depending on grain inclusion rate (Wang et al., 2003). A series of three experiments was undertaken to investigate whether RG2 had positive effects on feed intake, fermentation, digestion and utilisation of grain-based (i.e., sorghum and barley) diets fed to growing beef cattle.

2. Materials and methods The University of Queensland Animal Ethics Committee (Experiments 1 and 2) and the Queensland Department of Primary Industries Central Queensland Animal Ethics Committee (Experiment 3) approved the experimental design and animal care provisions. The Australian Pesticides and Veterinary Medicines Authority approved the use of RG2 for Experiment 3. 2.1. Enzyme assay RG2 contains a Trichoderma longibrachiatum derived enzyme complex free of viable microbial cells, and the RG2 used in these experiments was from a single fermentation lot (803501). Just prior to each experiment, RG2 samples were analysed for enzymatic activity in the manufacturer’s laboratory using a 2-hydroxy-3,5-dinitrobenzoic acid colorimetric assay and RG2 application rates standardised among the three experiments based on measured xylanase activity levels. Samples were analysed in our laboratory for a wider range of enzymatic activity using the procedures of Colombatto and Beauchemin (2003). Protein content was determined using a Biuret method (Price, 1996) as described by Miller et al. (2008). Promote® (Biovance Technologies, Omaha, NE, USA) was included in the activity assay for enzymatic activity comparison purposes. 2.2. Experimental design and treatments – Experiment 1 Sixteen Bos indicus crossbred steers were weighed after 18 h off feed (314.2 ± 26.07 kg) 5 d before study commencement and stratified into four LW blocks for random allocation to treatment within each block group. The treatments consisted of two grain based diets (∼0.60 dry-rolled barley or sorghum; Table 1) with or without RG2 applied to the grain during concentrate mixing, which occurred every 7 d. RG2 was applied at 4.43 ml/kg diet

162

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

Table 1 Ingredients (g/kg DM) and chemical composition (g/kg DM) of barley (Experiment 1) and sorghum grain based diets (Experiments 1 and 2) fed to Brahman crossbred steers Experiment 1

Experiment 2

Barley

Sorghum

High grain

Low grain

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

620 296 51 11 20 2

580 236 153 14 15 2

601 246 123 14 14 2

349 494 144 11 0 2

Chemical composition Dry matterb Organic matter Crude protein Starch aNDFom ADFom Lignin (sa)

887 929 123 302 338 159 19

892 934 129 394 263 150 22

889 940 142 366 263 156 25

893 940 151 193 428 245 36

a b

Feedlot vitamin/mineral Premix® (Roche Vitamins Australia Pty Ltd., Frenchs Forest, NSW, Australia). Reported on an ‘As Fed’ basis.

DM diluted in water to 20 ml/kg diet DM, (water only for controls) ensuring the RG2 was on the grain for at least 24 h prior to feeding. Strict mixing sequences and thorough utensil washing was used to avoid RG2 transfer between batches. Diets were formulated using SCA (1990) recommendations to provide for the nutritional requirements of a 325 kg Brahman steer growing at 1 kg/d and were fed out at 1.05 to 1.10 of ad libitum levels (all chaff and half of concentrate at 08:00 h with the remainder at 14:00 h). After a 21 d grain adaptation period, RG2 supplementation was implemented over a 7-week feeding period at the full grain levels. The final unfasted steer average LW was 402.7 ± 31.95 kg. Steers were held in individual, shaded and floored pens and inspected daily and pre-feeding LW recorded on a weekly basis. One of the steers, fed the sorghum control diet, suffered a leg injury unrelated to the treatment conditions and was removed from the study 5 weeks after commencement. Metabolism crates inside an animal house were used to collect digestibility measurements over 7 d periods 2 and 6 weeks after RG2 treatments commenced to investigate changes in feed utilisation over time with EE supplementation. Intakes were restricted to 0.90 of ad libitum levels at those times. Due to a limited number of metabolism crates, steers were randomly allocated to one of two groups of eight steers with the first group commencing the trial 7 d prior to the second group, in order to maintain the timing of the digestibility measures. 2.3. Sampling and analytical procedures – Experiment 1 Prior to study commencement, a representative sample of the feed ingredients was collected to calculate initial dietary proportions and RG2 application rates. Daily chaff and

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

163

concentrate samples were collected, composited weekly and dried in a forced draught oven (60 ◦ C) to constant weight to measure weekly ration DM contents. Refusals were weighed daily and composited weekly before duplicate samples were dried for 6 d to determine voluntary DM intakes. Over the entire study, chaff was sampled weekly and the concentrate feed ingredients sampled at each mixing event, composited separately and stored at −20 ◦ C before drying to constant weight and grinding (1 mm screen) for nutrient analysis. During the digestibility studies, feed ingredient samples were collected, the amount of diet offered and refused was weighed daily and any refusals composited. Faeces were collected and weighed daily, a sample dried in a forced draught oven (60 ◦ C) to determine daily faecal DM production and a further 50 g/kg composited at −20 ◦ C for the period. This sample was thawed, mixed thoroughly and a sub-sample freeze dried (Cryodos 80 Freeze Dryer, Telstar Industrial, Terrassa, Spain) for N analysis. Further faecal sub-samples (duplicate) and the feed ingredient samples and refusals were dried in a forced air dehydrator (60 ◦ C) to constant weight, ground (1 mm screen) and analysed to determine nutrient digestibility and balances. DM content of the 1 mm ground feed, refusal and faecal samples was determined in duplicate by oven drying at 65 ◦ C to constant weight. OM content was determined by incineration of duplicate ground samples at 550 ◦ C for 4 h. Fibre, N and starch analysis of the feed, refusals and faeces samples was as described by Miller et al. (2008). Briefly, aNDFom and ADFom fibre 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 heat stable ␣-amylase in the digestion phase and heat stable ␣-amylase in the first and second rinses only. aNDFom and ADFom measurements were corrected for ash content. N 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 and using a Cassava leaf external standard. Starch content was determined using a twostep enzymatic method involving heat stable ␣-amylase followed by amyloglucosidase using Megazyme Total Starch Assay kit (Deltagen, Boronia, Vic., Australia) reagents and procedures. Urine excreted was collected daily during the digestibility period into a volume (approximately 200 ml) of 250 ml/l sulphuric acid sufficient to maintain the pH of the collection below 3 (Chen and Gomes, 1992). The total weight was measured daily and 50 g/kg composited at −20 ◦ C over the 7 d period before subsampling and storage at −20 ◦ C for analysis to determine N balance. N levels in 1 ml urine samples (on a weight basis) were determined using a LECO CNS analyser at 1100 ◦ C, calibrated with an EDTA internal standard. Purine derivative concentrations were also determined but are not reported due to inexplicably low values. Rumen fluid (RF, ∼300 ml) samples were collected by intubation under vacuum 4 h after the morning feeding on the day following the digestibility study and then again the following morning just prior to feeding (08:00 h) to provide an indication of the influence of enzyme treatment on rumen fermentation characteristics. Steers were fed sequentially to maintain the 4 h period between feeding and sampling. The RF sample was strained through nylon stocking and pH measured immediately. At each sampling time, duplicate RF samples were collected for NH3 -N determination using steam distillation, and a single sample collected

164

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

for volatile fatty acid (VFA) determination using gas chromatography as outlined by Miller et al. (2008). 2.4. Experimental design and treatments – Experiment 2 Four ruminally cannulated, B. indicus crossbred steers (18 months of age) were weighed (364.3 ± 21.98 kg LW), randomly allocated to treatments and placed into individual, shaded and floored pens. The cannulas (#4C, Bar Diamond Inc., Parma, ID, USA) had been inserted for a prior experiment. The experiment was arranged as a 4 × 4 crossover (Latin Square) design with treatments consisting of two diets (High Grain (HG) and low grain (LG) rations of 601 and 349 g/kg DM dry-rolled sorghum, respectively) with water (nil) or RG2 (4.18 ml/kg diet DM, diluted in water to 20 ml/kg diet DM) applied to the grain as in Experiment 1. Each experimental period consisted of 24 d comprising an initial 10 d dietary adaptation period, followed by a 7 d nutrient digestibility study and a 1 d rest period. This was followed by a 3 d rumen flow rate measurement period and a further 3 d rate of ruminal digestion in sacco incubation period. An additional 6 d prior to the start of period 1 were allocated to adapting the steers to the concentrate, with a 4 d adaptation used in later periods for steers going from the low to high grain diets. Steers were weighed at the beginning and end of each digestibility study and again prior to feeding on the morning of the first day of the next period. Diets (Table 1) were formulated using SCA (1990) recommendations for a 375 kg Brahman steer growing at 1 kg/d with the high grain (HG) diet composed to approximate the sorghum diet used in Experiment 1. The low grain (LG) diet used an increased proportion of pangola hay chaff to investigate how RG2 would interact with a grain diet of higher forage content. Diets were fed ad libitum (1.05 of intake), and were offered as described for Experiment 1. Following the discovery of mould in the LG mixes 2 weeks after commencement, the dilution rate for RG2 applied to the LG diets was reduced to 20 ml/kg DM of the concentrate mix rather than of the total ration DM. 2.5. Sampling and analytical procedures – Experiment 2 Samples of feed ingredients were collected at concentrate mixing for DM and chemical analysis. Chaff, concentrate mixes and feed refusal samples were collected regularly, and dried to determine DM intake levels. Average ad libitum DM intake levels were calculated using DM intake for the 3 d prior to the digestibility periods. The steers were moved into metabolism crates for digestibility studies, with the collection, sampling and analysis of feed, refusals, faeces and urine samples as described for Experiment 1. Purine derivatives (PD) were determined using high performance liquid chromatography as reported in Miller et al. (2008), with excretion of total PD in urine and microbial N flow calculated according to the method of Chen and Gomes (1992), with an endogenous purine excretion estimate developed for B. indicus cattle (0.190 mmol/kg LW0.75 , Bowen, 2003). During the 3 d rumen flow rate measurement period, a marker dilution technique was used to investigate fluid and particulate passage rates and mean retention time (RT). Chromium (Cr) labelled ground pangola chaff (Ud´en et al., 1980), ytterbium (Yb) labelled cracked

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

165

sorghum grain (Teeter et al., 1984) and cobalt (Co) as Co-Li/EDTA (Ud´en et al., 1980) was administered to trace the movement of the fibre, grain and liquid phases, respectively. To prepare the fibre marker, 1 mm ground (8 Christy Mill, Christy and Norris Ltd., Chelmsford, England) pangola chaff was mixed (1 kg/3 l) into a 500 ml/l ND solution (Van Soest et al., 1991) and soaked overnight. The chaff was then rinsed in triple de-ionised H2 O until clear of detergent, rinsed and soaked in acetone for 20 min before another acetone rinse and drying at 65 ◦ C for 48 h. The fibre residue (856 g/kg NDF) was mordanted with Cr equivalent to 20 g/kg. The marked fibre was delivered in three paper towel packets providing the equivalent of 1.94 g Cr/steer. Co-Li/EDTA was dosed into the rumen at a rate of 2.12 g Co in 300 ml solution per steer. The marked grain was delivered in two paper packets providing 0.34 g Yb/steer. Markers were administered to the rumen prior to the morning feeding. The marked fibre was first inserted and mixed by hand into the rumen contents below the dorsal raft in order to promote mixing. Marked grain was then inserted and mixed in, before the liquid marker was inserted without mixing into the rumen (five to six sites) using a syringe attached to infusion tubing held within a rigid plastic pipe, followed by a flush of 30 ml of water. Representative rumen contents samples were bulked from three to four locations below the rumen mat before RF samples were bulked (about 150 ml filtered through three layers of stocking) from three to four sites in the ventral rumen and the pH measured immediately. Samples were taken prior to dosing (background matrix, liquid samples in duplicate) and again at 4, 8, 12, 16, 20, 24, 28, 32, 36 and 48 h after dosing with marker analysis of the pre-dosing, 4, 8, 12, 16, 20, 24, and 48 h samples. The pH readings collected more than 24 h after dosing were averaged with the equivalent prior measurements to produce a 24 h cycle. Rumen content samples were weighed and dried to constant weight at 60 ◦ C before grinding (1 mm), digestion in 5 nitric:1 perchloric acid and determination of Cr and Yb concentrations using inductively coupled plasma atomic emission spectroscopy (ICP-AES Flame-P spectrometer, Spectro Co., Kleve, Germany). Fractional passage rate (FPR) was estimated as the slope of the linear regression between the natural log of marker concentration in either RF or rumen content DM and the time of sampling. Mean retention times were estimated as the reciprocal of the passage rates. Of the RF collected, 70 ml sub-samples were stored at −20 ◦ C for determination of Co and additional samples collected (pre-feeding and 4, 8, and 12 h after feeding) for NH3 -N and VFA measurements as for Experiment 1. Following marker sampling days, ruminal in sacco incubations were completed using undried, ground (3 mm screen) sorghum grain and pangola grass to investigate apparent rate of digestion differences between treatments. Procedures followed recommendations of SCA (1990). Seven days prior to each incubation, RG2 (0 and 4.0 ml/kg DM diluted to 20 ml/kg DM) was applied evenly using a liquid atomiser before the material was dried at room temperature for 48 h and stored in sealed plastic bags until incubated. Polyester bags (100 mm × 240 mm, 40 ␮m pore size; Allied Screen Fabrics, Sydney, Australia) were used to hold 3.3 g DM of sample (13.2 mg/cm2 ). The two substrates were incubated for 3, 6, 9, 12, 18, 24, 36 and 48 h with a 72 h incubation for the pangola chaff samples. Duplicate 0 h bags were dipped into the rumen contents to determine initial solubilisation levels and lag times. Bags were attached to a 1 kg, 600 mm length of chain suspended from the cannula plug by 750 mm of woven curtain rope. All bags were inserted together prior to the morning

166

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

feeding on the first day and removed sequentially into ice slurry to halt microbial activity. Bags were rinsed for 9 min in a commercial washing machine to remove residual rumen contents, non-adherent microbial biomass and small particles. The residue-containing bags were dried (60 ◦ C) in a forced draught oven to constant weight and weighed to determine apparent DM disappearance (uncorrected for microbial matter). The data was fitted to the model of McDonald (1981) where apparent DM disappearance is described as: DM disappearance = A (initial washing loss) up to the lag time T0 DM disappearance = a + b(1 − e−ct ) from lag time T0 onwards where ‘a’ denotes the ruminally soluble fraction, ‘b’ the insoluble potentially degradable fraction, ‘c’ the degradation constant for fraction ‘b’ and ‘t’ the time of incubation. The incubated sorghum values could not be fitted using the McDonald (1981) model and so the apparent rate of DM disappearance from 3 to 36 h is reported as a linear relationship between DM disappearance and time with the 0 h DM loss (initially soluble fraction) and extent of digestion at 48 h reported separately. Bags containing segments of barley straw (about 25 of 10 mm in length) were incubated in duplicate in the rumen for 24 h to qualitatively measure RG2 treatment effects on fungal populations using the procedure of Elliott et al. (1987). The recovered segments were stored in 100 ml/l formal saline. Fungal sporangia colonising the segments were stained with lactophenol cotton blue and an average count determined for the internal stalk surface (five random 3.14 mm2 fields of view per surface) of two segments at 100× magnification (Morrison et al., 1990). 2.6. Experimental design and treatments – Experiment 3 Ninety-six Santa Gertrudis steers (18 months old, implanted with a hormonal growth promotant (HGP); Compudose 100TM , Elanco Animal Health, West Ryde, NSW, Australia) were weighed (351 ± 24.7 kg) and assessed for US body condition score (US BCS). Steers were allocated into 16 groups (n = 6 steers/group) balanced on HGP implant history and LW, and groups randomly allocated to one of four replicates of four levels of RG2 supplementation. At the feedlot (149.75E, −24.80S), the replicates formed sequential blocks of 4 consecutive pens (25 m2 /steer) and within each replicate the four RG2 treatments were randomly allocated to the four pen groups. The RG2 treatments included a control (no enzyme added, tap water only), Low RG2 (1.08 DM total ration), Medium RG2 (2.16 l/tonne DM) and a High RG2 Level (4.33 l/tonne DM), all diluted with water to 20 l/tonne DM ration. Commencing the day after entry into the feedlot, steers were adapted over 11 d (6 d starter, 5 d intermediate) onto a dry-rolled sorghum grain based diet (Table 2) for the remainder of the 70 d feeding period. During the period of adaptation, RG2 application levels were half of those outlined above. Feed mixes were produced in a mixer/feeder wagon with RG2 treatments applied to the grain during mixing such that RG2 was on the feed for a minimum period of 30 min before feeding. Feed allocations were based on the ‘clean bunk at midday’ protocol of Lawrence (1998) with a single evening feeding. The study produced grain-finished steers to meet Australian domestic market specifications.

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

167

Table 2 Ingredients (g/kg DM) and chemical composition (g/kg DM) of sorghum grain based diets fed to Santa Gertrudis steers under feedlot conditions (Experiment 3) Diet Starter

Intermediate

Final

Ingredient Sorghum Supplementa Cottonseed meal Molasses Mixed vegetable oil Whole cottonseed Wheat straw Sorghum silage Lucerne hay

536 28 42 130 – 75 71 22 96

633 40 38 85 18 87 61 38 –

719 52 – 42 24 98 20 45 –

Chemical composition Dry matterb Organic matter Crude protein Ether extract aNDFom ADFom Lignin (sa)

858 935 152 45 189 125 17

843 943 143 58 200 116 16

829 943 130 74 188 110 16

The rations were formulated according to NRC (1996) recommendations and to maximise dietary energy density. a Supplement (939 g DM/kg) contained (g/kg as fed basis): 475.5 cereal carrier, 355.6 limestone, 62.2 urea, 40.0 ammonium sulphate, 16.7 magnesium oxide, 37.8 salt, 7.8 ENC Beef-B and 4.5 Rumensin® (Elanco Animal Health, West Ryde, NSW, Australia). This was formulated to contain (g/kg DM): 324 crude protein, 142 calcium, 11.3 sulphur, 12.2 magnesium, 40.2 salt and 464 ppm Monensin. b Reported on an ‘As Fed’ basis.

All steers were weighed 4 and 9 weeks after entry to the feedlot (starting at 07:00 h) and assessed for US BCS. Ten weeks after feedlot entry, all steers were again weighed and US BCS assessed prior to a 9.5 h transport to the abattoir. Steers were fed without restriction on the day prior to loading and had access to water both before and after transport and prior to slaughter. Steers were held overnight (∼13 h) then slaughtered and dressed according to commercial procedures. Dressing proportion was calculated as the measured hot standard carcase weight (HSCW) as a proportion of the liveweight measured prior to transport. Fat depth at the P8 site was assessed using Authority for Uniform Specification of Meat and Livestock (AUS-MEAT) methods prior to entry into the chillers with carcase sides hung from the Sacro-Sciatic position. An ultimate temperature and pH reading was taken at carcase grading (about 20 h after slaughter) and assessments made by a certified Meat Standards Australia (MSA) grader of eye–muscle area (EMA – area of the Longissimus dorsi in square centimetres (cm) using the standard AUS-MEAT grid) and rib fat thickness (FT – subcutaneous fat excluding seam fat present three quarters of the distance ventrally on the rib–eye muscle at the quartering site, measured in mm). The quantity of marbling (intramuscular fat) was estimated using the standard AUS-MEAT (Aus Mb, 0–6 with 0.1 unit gradations) system by the MSA grader.

168

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

2.7. Sampling and analytical procedures – Experiment 3 Daily weights of ration offered were recorded and samples for each treatment collected (first and last trough of a feeding), bulked weekly and sub-samples dried at 100 ◦ C for 24 h to calculate average DM intake per steer (pen DM intake divided by six). FCE was calculated on a pen basis where the amount of DM consumed by a pen of steers was divided by the combined LW gain for the steers in that pen. The feed samples were stored at −20 ◦ C before bulking within ration and enzyme treatments for chemical analysis. The analytical methods used were as described for Experiment 1 and ether extract content was determined by Soxhlet extraction over 6.5 h. 2.8. Data analysis Unless otherwise indicated, all data was analysed using the MIXED procedure of the SAS/STAT® software (Version 8.02, 2001, SAS Institute Inc., Cary, NC, USA) with variance components estimated using the REML method and repeated measures analysed using the first order autoregressive (AR1) correlation structure (tested as being the most appropriate). Differences among means were analysed using a protected (P<0.05) t-test where significance was declared at P<0.05 and trends discussed at 0.05
D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

169

RG2 dose–response function. When data were collected on individual animals, treatment effects were tested using the between pen variation rather than the between animal term.

3. Results 3.1. Enzyme characterisation Results of the enzyme assays undertaken in our laboratory are in Table 3. Protein contents were 141 ± 0.9 and 98 ± 2.2 mg bovine serum albumin equivalents/ml for RG2 and Promote respectively. The manufacturer of RG2 reported that it contains approximately 180 mg/ml of enzyme complex. 3.2. Experiment 1 RG2 supplementation increased voluntary DM intakes (Fig. 1, P<0.05) for steers on the sorghum diet (116.6 g/kg LW0.75 versus 105.0 g/kg LW0.75 for control) but not for steers on the barley diet. RG2 supplementation numerically increased (P=0.138) ADG with 921 ± 72.0 g/d for treated versus 738 ± 81.7 g/d for steers fed control diets. Weekly LW measurements indicated that after 5 weeks of dietary RG2 application LW tended to be increased (P=0.097) versus the controls irrespective of diet (data not shown). FCE was unaffected by diet or RG2 treatment (Table 4). RG2 treatment had no effect on total tract apparent DM, OM or fibre digestibility. There was an interaction with diet (P<0.05) where RG2 treatment decreased starch digestibility on the sorghum diet, due to a extensive decline at the second digestibility period (0.684 versus 0.808 control), but RG2 did not affect starch digestion in steers fed the barley diet. RG2 supplementation of steers increased (P<0.05) urinary N excretion levels (66.0 g/d) Table 3 Average enzymatic activity of RG2 and Promote recorded using the 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 ␤-Glucosidasec Pectinased Proteasee Filter paper

RG2

Promote

18.7 2200 2.6 710 11.0 7.9 0.395 (8.0) ND

28.3 2890 3.9 340 11.7 11.3 1.00 (20.3) ND

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

170

Diet

S.E.M.

Barley

Average daily gain Feed efficiency

LW DOM intake DM digestibility OM digestibility aNDFom digestibility ADFom digestibility Faecal starch excretion Starch digestibility Urinary N excretion Daily N balance Apparent N digestibility Apparently digested N retained

P value

Sorghum

Control

RG2

Control

RG2

765 10.4

985 8.5

715 12.9

855 12.3

2 weeks

6 weeks

2 weeks

6 weeks

2 weeks

6 weeks

2 weeks

6 weeks

365 59.4 743 752 546 443 0.10 a 952 a 51.2 46.3 704 475

394 56.9 735 744 543 447 0.10 a 954 a 55.0 43.0 689 434

375 56.5 730 739 558 458 0.06 a 972 a 57.9 41.1 722 415

407 58.6 740 748 572 464 0.06 a 972 a 63.9 43.9 717 403

368 53.9 593 590 538 477 0.50 b 847 b 54.9 49.3 609 471

382 48.9 566 562 505 450 0.62 b 808 b 63.7 37.9 592 375

379 56.5 594 594 491 420 0.54 b 840 b 68.7 38.6 593 355

413 54.1 575 573 514 430 1.16 c 684 c 73.4 37.2 579 336

NS: not significant, P>0.10; § P<0.10; *P<0.05; **P<0.01. Row means without a common letter differ (P<0.05).

Run

Diet

RG2

Diet × RG2

122.5 1.65

– –

NS

§

NS NS

NS NS

8.0 3.10 11.3 11.4 26.6 33.6 0.078 20.4 4.01 4.77 11.4 27.4

** 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 145 (2008) 159–181

Table 4 Average daily LW gain (g/d) and feed conversion efficiency (kg DM/kg LW gain) over the RG2 supplementation period and live weight (kg), daily average digestible OM intake (DOMI, g/kg LW0.75 ), total tract apparent DM, OM and N digestibility (g/kg), total tract aNDFom, ADFom and starch digestibility (g/kg), starch excretion (kg/d), N excretion and balance (g/d) and apparently digested N retained (g/kg) of steers fed barley and sorghum grain based diets recorded at 2 and 6 weeks post commencement of RG2 supplementation at 0 (Control) or 4.43 ml/kg dietary DM (RG2)

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

171

Fig. 1. Daily average voluntary DM intake of steers (g/100 g of LW) fed high barley or sorghum based diets with (RG2) or without (control) RG2 supplementation.

compared with unsupplemented steers (56.2 g/d) and tended (P=0.077) to increase overall N digestibility in steers fed the barley diet while depressing it for the sorghum diet. The proportion of apparently digested N retained was lower (P<0.05) on the sorghum diet versus the barley diet (384 g/kg versus 432 g/kg) as a result of a reduction (P<0.05) in retention with RG2 treatment. The pH of RF collected from RG2 supplemented steers fed the barley diet tended (P=0.081) to be lower (pH 6.8) versus unsupplemented steers (pH 7.1), but there was no effect of RG2 on the pH of RF from steers on the sorghum diets (Table 5). Diet interacted with RG2 whereby NH3 -N concentration among the samplings was higher (P<0.05) in RF from RG2 supplemented steers (89 mg/L) versus unsupplemented steers (51 mg/l) on the barley diet did not differ on the sorghum diet. Total VFA concentrations in RF were unaffected by diet, but were higher (P<0.05) at the second sampling through an increase in VFA concentration with RG2 supplementation (82.1 mM versus 68.5 mM control). Total VFA concentration was higher (P<0.05) 4 h postfeeding for RG2 supplementation versus the control. Acetate concentrations were slightly higher with RG2 treatment (P=0.097) as a result of concentration increases at the second sampling event (52.0 mM versus 42.3 mM control period 2). RG2 supplementation increased (P<0.05) butyrate concentrations in RF from steers fed the barley diet, and decreased them for steers fed sorghum. 3.3. Experiment 2 Steers used in the experiment gained LW over the entire experimental period at 0.76 kg/d and RG2 treatment had no effect on feed intake or digestibility (Table 6). aNDFom digestibility was higher (P<0.05) for steers on the LG diet versus the HG diet (629 and 567 g/kg, respectively). Analysis of PD in urine indicated that diet and RG2 treatment had no effect

172

Sampling time

Barley diet

Sorghum diet

Control 2 weeks 7.3 6.7

RG2 6 weeks 7.5 6.8

2 weeks 7.0 6.6

Control 6 weeks

Pre-feed 4 h post

NH3 -N

Pre-feed 4 h post

67 26

77 32

89 60

Total VFA

Pre-feed 4 h post

64.4 71.0

62.9 71.7

68.9 80.3

77.0 94.4

74.3 60.9

Acetate

Pre-feed 4 h post

41.4 46.0

39.8 44.7

42.7 49.6

48.0 57.4

Propionate

Pre-feed 4 h post

11.9 14.0

14.7 17.7

15.3 19.2

Butyrate

Pre-feed 4 h post

7.5 8.4

5.6 7.0

7.5 8.6

NS: not significant, P>0.10; § P<0.10; *P<0.05; **P<0.01.

7.1 6.6

2 weeks

pH

129 77

6.8 7.0 85 86

S.E.M. RG2

6 weeks 7.2 6.9 100 83

2 weeks 7.3 6.7

P value Run

Time

Diet

RG2

Diet × RG2

NS

**

NS

NS

§

*

**

NS

NS

*

6 weeks 7.2 6.8

0.12

45 49

78 87

14.2

70.3 70.3

48.5 65.3

75.1 81.9

5.67

*

*

NS

NS

NS

46.0 37.9

42.1 43.4

30.7 40.7

49.3 53.6

3.41

*

*

NS

§

NS

14.1 21.7

15.4 13.9

17.9 17.6

10.6 16.4

16.9 18.4

2.54

*

**

NS

NS

NS

11.3 12.0

9.2 6.5

6.3 6.3

4.8 6.1

5.6 7.4

0.93

NS

NS

*

NS

*

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

Table 5 pH, NH3 -N concentration (mg/L) and total VFA, acetate, propionate and butyrate concentrations (mM) in RF sampled before and 4 h after feeding from steers fed barley and sorghum grain diets recorded 2 and 6 weeks after RG2 supplementation started (0 (control) or 4.43 ml/kg diet DM (RG2))

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

173

Table 6 Ad libitum average DM intakes (g/kg LW0.75 /d) prior to and measurements taken during the digestibility periods of average live weight (kg), total tract apparent DM, OM and N digestibility (g/kg), digestible OM intakes (g/kg LW0.75 /d), faecal starch excretion (kg/d), and total tract starch, aNDFom and ADFom digestibilities (g/kg), faecal and urinary N excretion (g/d), N balance (g/d), apparently digested N retained (g/kg), total purine derivative (PD) excretion (mmol/d), calculated microbial N flow (g/d), efficiency of microbial N production (EMNP, g/kg DOMI) and fungal populations (average sporangia count per mm2 on barley stalk surfaces following a 24 h in sacco incubation in the rumen) of Bos indicus crossbred steers fed high and low sorghum-based diets supplemented with RG2 at 0 (control) or 4.18 ml/kg dietary DM (RG2) Diet

S.E.M.

High grain

Low grain

Control

RG2

Control

RG2

Ad libitum DM intake

106.3

106.7

100.9

101.3

1.85

Average LW DM digestibility OM digestibility Digestible OM intake Faecal starch excretion Starch digestibility aNDFom digestibility ADFom digestibility Apparent N digestibility Faecal N excretion Urinary N excretion N balance Apparent digested N retained

414.1 616 618 54.8 0.78 763 569 531 584 82.3 72.9 42.2 365

412.8 617 616 51.7 0.71 760 565 496 596 76.4 83.7 27.6 250

417.4 619 626 52.9 0.46 725 632 574 638 73.7 83.7 46.8 352

414.1 636 643 55.4 0.44 733 626 576 646 72.8 83.3 48.8 367

1.70 9.3 9.3 1.67 0.075 17.7 22.7 28.8 11.8 3.06 3.75 4.88 32.4

Total PD excretion Microbial N flow EMNP Fungal sporangia count

104.6 74.5 15.0 10.91

125.6 92.5 19.4 5.32

113.3 81.9 16.8 15.98

103.2 73.4 14.4 12.34

8.66 7.45 1.506 2.383

P value Diet

RG2

Diet × RG2

*

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: not significant, P>0.10; § P<0.10; *P<0.05; **P<0.01.

on estimated microbial N flows, although the efficiency of microbial N production tended (P=0.065) to be increased by RG2 on the HG diet but decreased on the LG diet. The number of fungal sporangia on the inner surface of incubated straw segments showed that RG2 treatment tended to reduce (P=0.058) sporangia numbers. The RG2 treated HG diet had a higher (P≤0.05) RF pH level 20 h post-feeding versus the untreated HG diet (Fig. 2). RG2 supplementation reduced (P<0.05) the fractional passage rate of the Yb marker when applied to the HG diet while increasing it on the LG diet (Table 7), but RG2 had no effects on the RT of the Cr or Co markers. FPR of the Co, Yb and Cr markers were higher (P<0.05, P<0.10 and P<0.01, respectively) for the LG diet versus the HG diet, with corresponding shifts in mean marker RT. Applying RG2 directly to feed samples incubated in sacco had no effect on apparent DM disappearance. The potentially degradable fraction of insoluble pangola chaff was reduced (P<0.01) by dietary RG2 application and was lower (P<0.01) on the HG diet versus the LG diet (Table 7). Dietary RG2 supplementation did not affect the appar-

174

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

Fig. 2. Average rumen fluid pH measured over two 24 h feeding cycles in steers fed high or low sorghum grain diets, untreated (control) or treated (RG2) with RG2. SEM were 0.071 (0, 8, 16 and 20 h), 0.060 (4 and 24 h) and 0.061 (12 h).

ent rate of pangola chaff DM disappearance but tended to increase extent (P=0.082) of sorghum grain DM disappearance to 48 h of incubation, independent of dietary grain content. 3.4. Experiment 3 The DM intake (11.1 ± 0.17 kg DM) and FCE (5.72 ± 0.061 kg DMI/kg LW gain) were not affected by RG2 treatment (Table 8). The LW and US BCS and carcase attributes were also unaffected by RG2 treatment, although there tended (P<0.10) to be a linear increase in dressing proportion with increasing RG2 application.

4. Discussion 4.1. Enzyme activity characterisation In comparison with 23 EE products previously characterised under similar assay conditions (Colombatto et al., 2003), 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. In vitro incubations (39 ◦ C, pH 6.5, up to 4 h) of RG2 with 1 mm ground barley grain, sorghum grain and pangola grass chaff showed the greatest reducing sugar release from barley grain then pangola grass and sorghum grain, although the relative rate of release did not differ (Miller, unpublished data). This RG2/feed specificity, relating to the balance of activities supplied, is the likely

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

175

Table 7 Fractional passage rate (FPR, g/100 g/h) and retention time (RT, h) of Co, Yb and Cr markers and in sacco DM disappearance of 3 mm ground pangola chaff and sorghum grain incubated at various times up to 48 (sorghum) or 72 h (pangola chaff) in Bos indicus crossbred steers fed high and low sorghum-based diets supplemented with RG2 at 0 (control) or 4.18 ml/kg dietary DM (RG2) Diet

S.E.M.

High grain

Low grain

P value Diet

RG2

Diet × RG2

Control

RG2

Control

RG2

6.99 14.5

6.65 15.1

7.86 12.8

8.18 12.5

0.339 0.50

* *

NS NS

NS NS

Grain (Yb) FPR RT

2.61 ab 39.3 a

2.16 b 48.0 b

2.56 ab 40.9 ab

2.89 a 34.7 a

0.146 2.72

§ §

NS NS

* *

Fibre (Cr) FPR RT

1.69 60.6

1.59 65.5

2.09 48.7

2.13 47.1

0.053 1.74

** **

NS NS

NS NS

15.6 11.9 63.3 0.040 1.56

15.6 11.8 59.7 0.039 1.31

15.9 11.2 65.0 0.047 1.56

15.7 11.5 63.5 0.050 1.40

0.22 0.77 0.83 0.0028 0.298

NS NS ** * NS

NS NS ** NS NS

NS NS NS NS NS

15.9 1.78 94.9

15.9 1.85 95.5

16.4 1.91 95.9

16.1 1.96 96.5

0.44 0.039 0.33

NS * *

NS NS

NS NS NS

Marker flows Liquid (Co) FPR RT

In sacco incubations Pangola chaffa Washing loss (A) a (g/100g) b (g/100g) c (h−1 ) Lag time (h) Sorghum grainb Initial washing loss Rate of disappearance Extent DMD 48 h

§

NS: not significant, P>0.10; § P<0.10; *P<0.05; **P<0.01. Row means without a common letter differ (P<0.05). a From the equation of McDonald (1981) where DMD = a + b(1 − e−ct ), A = initial washing loss (g/100g) and lag time is calculated from the fitted equation, i.e. the value of t when DMD = A. b Rate of disappearance is the slope of a linear regression of DM disappearance against time of incubation (h). Initial washing loss and extent figures refer to mean DM disappearance (%) at t = 0 and 48 h, respectively.

reason why effects of RG2 application were not consistent among the different grain diets (Experiment 1) or sorghum diet compositions (Experiment 2). 4.2. Nutrient intake and RG2 treatment In Experiment 1, RG2 resulted in an increase in voluntary DM intake for steers fed the sorghum diet, but not for steers fed the barley diet. Voluntary digestible OM (DOM) intakes were estimated from dietary DOM contents and voluntary DM intake as 68.5 and 67.6 g/kg LW0.75 for the unsupplemented and RG2 applied barley diets respectively and 60.5 and 68.1 g/kg LW0.75 for the unsupplemented and RG2 applied sorghum diets. Calculated metabolisable energy (ME) concentrations of the diets (SCA, 1990) were higher

176

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

Table 8 Average liveweight (kg) and US body condition score (BCS) changes, dry matter intake (kg/d) and feed conversion efficiency (FCE, kg DMI/kg LW gain) during a 70-d feeding period of Santa Gertrudis steers fed sorghum grain based diets treated with Roxazyme® G2 at 0× (control), 1× (low), 2× (medium) and 4× (high) 1.08 l/tonne DM total ration and the hot standard carcase weight (HSCW, kg), dressing proportion, P8 and rib fat thickness (FT, mm), ultimate loin pH, eye muscle area (EMA, cm2 ) and marbling score (Aus-Meat) of their carcases Overall mean

Diet Control

± ± ± ± ±

S.E.M. Low RG2

Medium RG2

High RG2

P value RG2a Lin

Quad

Initial LW Final LW ADG Initial BCS BCS change

351.8 488.2 1.92 4.5 1.5

2.52 3.44 0.029 0.05 0.05

353.7 494.1 1.98 4.6 1.5

351.1 483.1 1.86 4.6 1.4

350.0 490.5 1.98 4.4 1.6

352.6 485.1 1.87 4.4 1.6

5.19 7.03 0.058 0.10 0.10

NS NS NS NS NS

NS NS NS NS NS

DM intake FCE

11.11 ± 0.062 5.72 ± 0.061

11.25 5.62

10.91 5.79

11.23 5.61

11.06 5.84

0.171 0.098

NS NS

NS NS

HSCW Dressing proportion P8 FT Rib FT Ult. Loin pH EMA Marbling score

243.3 0.498 8.6 4.9 5.45 69.2 0.6

± ± ± ± ± ± ±

240.5 0.493 8.2 5.1 5.43 70.2 0.6

240.6 0.498 9.0 4.6 5.43 68.6 0.6

244.3 0.500 8.7 5.6 5.42 71.3 0.6

241.9 0.501 8.4 4.9 5.44 70.1 0.6

2.12 0.029 0.71 0.85 0.030 1.38 0.05

NS

NS NS NS NS NS NS NS

a

1.86 0.014 0.36 0.25 0.008 0.47 0.02

§

NS NS NS NS NS

Polynomial contrasts, Lin: linear effect and Quad: quadratic effect. NS: not significant; § P<0.10.

for the barley versus the sorghum diet (10.7 ± 0.12 MJ ME/kg DM versus 8.0 ± 0.13 MJ ME/kg DM, respectively), as a result of lower sorghum diet OM digestibility. Estimated voluntary ME intakes were 84.4 and 83.7 MJ ME/d for the steers fed the control and RG2 treated barley diets and 72.3 and 80.3 MJ ME/d for the control and RG2 treated sorghum diets respectively. RG2 supplementation increased voluntary intakes of the sorghum diet up to a point where ME intakes were similar to that of steers fed the barley diet, overcoming the ME intake restriction of lower OM digestibility. This finding lends support to the proposal that some dietary limitation to potential animal performance is required to enable the expression of EE responses (Yang et al., 2000; Beauchemin et al., 2001, 2003). In contrast, feed intake was unaffected by increasing levels of RG2 during the feedlot study. The average daily DM intake of the feedlot steers was 11.1 kg DM, which for an average 420 kg steer is 119.6 g/kg LW0.75 . This intake level is relatively high (NRC, 1996) for grain-fed steers. For example, Axe et al. (1987) reported an average DM intake of 99.5 g/kg LW0.75 for 52 Charolais cross steers (avg. 411 kg) fed an 800 g/kg high-moisture sorghum grain diet. In Experiment 1, the daily voluntary DM intake of RG2 untreated diet was almost 14% lower (105 g/kg BW0.75 ) and increased to 117 g/kg BW0.75 with RG2 supplements, which approximates these feedlot intake levels. The feedlot diet also had a higher energy density (11.9 MJ ME/kg; NRC, 1996; tabulated values) with steers performing at close to maximal levels (1.92 kg/d LW

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

177

gain and 5.72 kg DMI/kg LW gain). Therefore, the combination of high voluntary feed intakes and dietary quality may have removed the potential for a response to EE supplementation. 4.3. Effect of RG2 on fibre, starch and N digestion RG2 supplementation had no effect on total tract DM, OM or fibre digestibility in either Experiment 1 or 2. Other experiments have been undertaken with moderate to high sorghum diets in steers and dairy cows supplemented with a sorghum grain specific EE product and reported no changes in DMI (Boyles et al., 1992) or fibre and starch digestibility (Chen et al., 1995). While the barley and sorghum diets had similar fibre digestibility in Experiment 1, Experiment 2 showed a decline in total tract fibre digestion with increasing grain inclusion. This could be expected to provide the conditions where a response to EE would be seen. However RG2 did not affect total tract fibre digestibility under these conditions, although the dietary adaptation period may not have been sufficiently long to see responses in light of the time taken to observe responses in Experiment 1. While RG2 application did not change the lag time or rate of pangola chaff (856 g/kg NDF) digestion in sacco, it decreased the potentially degradable fraction of the chaff for both diets. This was possibly a result of decreased colonisation by ruminal anaerobic fungi, causing a reduction in the degradation of the more digestion-resistant plant structures, either by enzymatic or physical means. This shift in fungal populations was more pronounced on the HG diet, which had lower initial fungal populations. It is unclear how RG2 application would influence fungal colonisation as FPR of fibre bound marker was unaffected by RG2 application so it is likely removal of fungi from the rumen was not increased. These rumen level changes did not result in changes in total tract fibre digestion. The total tract digestion of starch for the RG2 untreated diets in Experiment 1 was similar to other published values. Huntington (1997) reported that dry rolled barley grain had a starch digestibility of 0.943 and dry rolled sorghum of 0.872. However, in the present experiment, RG2 treatment lowered starch digestibility on the sorghum diet due to an extensive decline later in the supplementation period, without affecting total tract starch digestion in steers fed the barley diet. This would likely relate to an increase in grain flow rates, but the effect of RG2 was to increase grain marker RT in the rumen on high sorghum diets in Experiment 2, and only decreased RT at a lower sorghum grain inclusion rate. It should be noted that the increases in DM intake in Experiment 1 did not occur in Experiment 2, and so these effects may not be directly comparable. The reduction in starch digestion suggests that RG2 may depress feed utilisation efficiency when used with high sorghum diets for longer periods, although this was not observed in the subsequent feedlot experiment. In the initial experiment, RG2 supplementation of the diets increased urinary N excretion, suggesting that additional N was made available during digestion and explains why apparently digested N retention was decreased by treatment. NRC (1996) predictions indicated that the supplied protein was sufficient for LW gains of more than 1.70 and 1.03 kg/d for the sorghum and barley diets respectively, suggesting that N intakes were not limiting production and so the daily N balances were unaffected by RG2. Similar shifts in apparently digested N retained and urinary excretion with RG2 treatment were seen on the high

178

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

sorghum diet in Experiment 2, although these were not accompanied by changes in rumen fermentation characteristics. 4.4. Microbial population and fermentation Generally, RG2 had minimal effects on microbial N flows or protozoal populations (data not shown) in these experiments, consistent with previous observations of RG2 supplementation in sheep fed barley and sorghum based diets (Miller et al., 2008). Zinn (1993) reported that Holstein steers fed 740 g/kg dry rolled barley diets produced 19.1 g microbial N/kg OM fermented, consistent with the levels reported for the HG diet in Experiment 2. The effects of RG2 supplementation on LW gain, starch digestion, N excretion and VFA concentrations in Experiment 1 occurred later in the feeding period, so microbial responses to RG2 may require some time to be expressed. The increased pre-feeding RF NH3 -N and butyrate levels for the RG2 treated barley diet compared with a decrease for the sorghum diet with supplementation likely relates to changes in N availability and/or the synchrony between the release of protein and energy across the 24 h feeding cycle. In Experiment 2, the RG2 treated HG diet had a higher RF pH level 20 h post-feeding and an increased grain marker RT versus the untreated diet, suggesting reduced ruminal energy availability with RG2 later in the digestion cycle, maybe as a result of a reduction in the potentially degradable fraction of the pangola chaff. RG2 had no effects on RF NH3 -N and VFA levels in Experiment 2 (data not shown). 4.5. RG2 supplementation and animal performance In Experiment 1, improvements in LW gain of 24.8% occurred with RG2 across barley and sorghum diets, with LW increases after 5 weeks of dietary RG2 application. There were no changes in FCE despite decreasing starch digestibility after 6 weeks of supplementation. RG2 treatment was thought to provide an opportunity for improving animal performance on sorghum grain based diets if, under commercial feedlot conditions, FCE was not compromised. However, our large scale feedlot experiment revealed no increases in DM intake, LW gain, FCE or carcase characteristics. Dressing proportion tended to linearly increase with RG2 supplementation, although the dressing proportion (0.498) recorded was lower than could be expected (Rowan and Taylor, 1994; McAllister et al., 1999; Wang et al., 2003). Other experiments have been undertaken with medium to high sorghum diets supplemented with EE products and found no changes in DM intake (Boyles et al., 1992), or fibre and starch digestibility (Chen et al., 1995) but did improve ADG and FCE (Boyles et al., 1992), albeit at lower performance levels than those achieved here. Generally, EE supplementation has not resulted in changes in carcase characteristics of feedlot cattle (Boyles et al., 1992; Beauchemin et al., 1997; McAllister et al., 1999; ZoBell et al., 2000; Wang et al., 2003).

5. Conclusions RG2 enzyme supplementation resulted in increases in intake of a sorghum grain diet up to the level of a higher digestibility barley grain-based diet, and it was this discrepancy in

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

179

nutrient availability that seemingly provided the potential for a response to RG2. Similar intake responses did not occur in HGP treated steers with high feed intakes, LW gain and FCE performance consuming a high energy, sorghum feedlot ration. RG2 supplementation had no major effects on total tract OM or fibre digestibility, but sorghum starch digestibility was reduced 6 weeks after supplementation commenced. Similar effects did not occur in steers fed the barley diet, and this was considered a reflection of RG2/feed specificity. RG2 application also decreased the potentially degradable fraction of pangola chaff, possibly a result of inhibition of colonisation by fungal sporangia, and RG2 supplements increased urinary N excretion, suggesting increased ruminal N availability. These changes could have adverse consequences for efficiency of nutrient utilisation. RG2 supplementation did not improve animal performance under high grain feeding conditions where diets have been formulated for maximum productivity, and appears to have no substantive role in the improvement of productivity of cattle raised under feedlot conditions.

Acknowledgements The authors acknowledge the financial support of Roche Vitamins Australia and DSM Nutritional Products Pty Ltd. and thank Michael Nielsen and the Analytical Services staff of the University of Queensland’s School of Land and Food Sciences for technical assistance. Andrew Gibbon, Les Gardiner and Ted Hodby provided animal care during Experiments 1 and 2. Allan Lisle was generous in providing statistical advice and data analysis. The authors also acknowledge Ian Loxton of Beef Support Services, David Reid and Brigalow Research Station staff from the Queensland Department of Primary Industries, and John Doyle and Rob Lawrence of Integrated Animal Production for their efforts in implementing the feedlot study.

References Axe, D.E., Bolsen, K.K., Harmon, D.L., Lee, R.W., Milliken, G.A., Avery, T.B., 1987. Effect of wheat and highmoisture sorghum grain fed singly and in combination on ruminal fermentation, solid and liquid flow, site and extent of digestion and feeding performance of cattle. J. Anim. Sci. 64, 897–906. Beauchemin, K.A., Jones, S.D.M., Rode, L.M., Sewalt, V.J.H., 1997. Effects of fibrolytic enzymes in corn or barley diets on performance and carcass characteristics of feedlot cattle. Can. J. Anim. Sci. 77, 645–653. Beauchemin, K.A., Morgavi, D.P., McAllister, T.A., Yang, W.Z., Rode, L.M., 2001. The use of enzymes in ruminant diets. In: Garnsworthy, P.C., Wiseman, J. (Eds.), Recent Advances in Animal Nutrition. Nottingham University Press, Loughborough, UK, pp. 297–322. Beauchemin, K.A., Colombatto, D., Morgavi, D.P., Yang, W.Z., 2003. Use of exogenous fibrolytic enzymes to improve feed utilization by ruminants. J. Anim. Sci. 81 (E. Suppl. 2), E37–E47. Bowen, M.K., 2003. Efficiency of microbial protein production in cattle grazing tropical pastures. Ph.D. Thesis, University of Queensland, Australia. Boyles, D.W., Richardson, C.R., Robinson, K.D., Cobb, C.W., 1992. Feedlot performance of steers fed steam-flaked grain sorghum with added enzymes. Proc. Western Section Am. Soc. Anim. Sci. 43, 502–505. Chen, X.B., Gomes, M.J., 1992. Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives – an overview of the technical details. Occasional Publication 1992 (revised 1995). Rowett Research Institute, Aberdeen, UK.

180

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

Chen, K.H., Huber, J.T., Simas, J., Theurer, C.B., Yu, P., Chan, S.C., Santos, F., Wu, Z., Swingle, R.S., DePeters, E.J., 1995. Effect of enzyme treatment or steam-flaking of sorghum grain on lactation and digestion in dairy cows. J. Dairy Sci. 78, 1721–1727. Colombatto, D., Beauchemin, K.A., 2003. A proposed methodology to standardize the determination of enzymic activities present in enzyme additives used in ruminant diets. Can. J. Anim. Sci. 83, 559–568. Colombatto, D., Morgavi, D.P., Furtado, A.F., Beauchemin, K.A., 2003. Screening of exogenous enzymes for ruminant diets: Relationship between biochemical characteristics and in vitro ruminal degradation. J. Anim. Sci. 81, 2628–2638. Elliott, R., Ash, A.J., Calderon-Cortes, F., Norton, B.W., Bauchop, T., 1987. The influence of anaerobic fungi on rumen volatile fatty acid concentrations in vivo. J. Agric. Sci., Camb. 109, 13–17. Hoover, W.H., Stokes, S.R., 1991. Balancing carbohydrates and proteins for optimum rumen microbial yield. J. Dairy Sci. 74, 3630–3644. Huntington, G.B., 1997. Starch utilization by ruminants: from basics to the bunk. J. Anim. Sci. 75, 852–867. Knowlton, K.F., 2001. High grain diets for dairy cattle. In: Corbett, J.L. (Ed.), Recent Advances in Animal Nutrition in Australia, vol. 13. Animal Science. University of New England, Armidale, Australia, pp. 19–28. Lawrence, R.J., 1998. A comparison of feedlot bunk management strategies and their influence on cattle performance and health. Proc. Aust. Soc. Anim. Prod. 22, 177–180. Martin, C., Michalet-Doreau, B., 1995. Variations in mass and enzyme activity of rumen microorganisms: effect of barley and buffer supplements. J. Sci. Food Agric. 67, 407–413. Mathers, J.C., Miller, E.L., 1981. Quantitative studies of food protein degradation and the energetic efficiency of microbial protein synthesis in the rumen of sheep given chopped lucerne and rolled barley. Br. J. Nutr. 45, 587–604. McAllister, T.A., Oosting, S.J., Popp, J.D., Mir, Z., Yanke, L.J., Hristov, A.N., Treacher, R.J., Cheng, K.–J., 1999. Effect of exogenous enzymes on digestibility of barley silage and growth performance of feedlot cattle. Can. J. Anim. Sci. 79, 353–360. McDonald, I., 1981. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci., Camb. 96, 251–252. Miller, D.R., Elliott, R., Norton, B.W., 2008. Effects of an exogenous enzyme, Roxazyme® G2 Liquid, on digestion and utilisation of barley and sorghum grain-based diets by ewe lambs. Anim. Feed Sci. Technol. 140, 90–109. Morrison, M., Murray, R.M., Boniface, A.N., 1990. Nutrient metabolism and rumen microorganisms in sheep fed a poor-quality tropical grass hay supplemented with sulfate. J. Agric. Sci. 115, 269–275. Mould, F.L., Ørskov, E.R., 1983. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anim. Feed Sci. Technol. 10, 1–14. Noziere, P., Besle, J.M., Martin, C., Michalet-Doreau, B., 1996. Effect of barley supplement on microbial fibrolytic enzyme activities and cell wall degradation rate in the rumen. J. Sci. Food Agric. 72, 235–242. NRC (National Research Council), 1996. Nutrient Requirements of Beef Cattle, seventh ed. (revised). National Academy Press, Washington, DC, USA. Price, N.C., 1996. The determination of protein concentration. In: Engel, P.C. (Ed.), Enzymology Labfax. Bios Scientific Publishers Ltd., Oxford, UK, pp. 34–41. Reis, R.B., Combs, D.K., 2000. Effects of increasing levels of grain supplementation on rumen environment and lactation performance of dairy cows grazing grass-legume pastures. J. Dairy Sci. 83, 2888–2898. Rowan, K.J., Taylor, D.G., 1994. The effect of sex and genotype in cattle on feedlot performance, carcase characteristics and meat quality. Proc. Aust. Soc. Anim. Prod. 20, 108–111. SCA (Standing Committee on Agriculture), 1990. Feeding standards for Australian livestock – Ruminants. Ruminants Subcommittee, Australian Agricultural Council. CSIRO Publications, East Melbourne, Australia. Teeter, R.G., Owens, F.N., Mader, T.L., 1984. Ytterbium chloride as a marker for particulate matter in the rumen. J. Anim. Sci. 58, 465–473. Theurer, C.B., 1986. Grain processing effects on starch utilization by ruminants. J. Anim. Sci. 63, 1649– 1662. Ud´en, P., Colucci, P.E., Van Soest, P.J., 1980. Investigation of chromium, cerium and cobalt as markers in digesta. Rate of passage studies. J. Sci. Food Agric. 31, 625–632. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, second ed. Cornell University Press, Ithaca, NY, USA.

D.R. Miller et al. / Animal Feed Science and Technology 145 (2008) 159–181

181

Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wang, Y., McAllister, T.A., Baah, J., Wilde, R., Beauchemin, K.A., Rode, L.M., Shelford, J.A., Kamande, G.M., Cheng, K.-J., 2003. Effects of Tween 80 on in vitro fermentation of silages and interactive effects of Tween 80, Monensin and exogenous fibrolytic enzymes on growth performance by feedlot cattle. Asian-Aust. J. Anim. Sci. 16, 968–978. Yang, W.Z., Beauchemin, K.A., Rode, L.M., 2000. A comparison of methods of adding fibrolytic enzymes to lactating cow diets. J. Dairy Sci. 83, 2512–2520. Zinn, R.A., 1993. Influence of processing on the comparative feeding value of barley for feedlot cattle. J. Anim. Sci. 71, 3–10. ZoBell, D.R., Weidmeier, R.D., Olson, K.C., Treacher, R., 2000. The effect of an exogenous enzyme treatment on production and carcass characteristics of growing and finishing steers. Anim. Feed Sci. Technol. 87, 279–285.