TY - JOUR
AU - McAllister, T. A.
AB - ABSTRACT The increasing availability of crude glycerin from the biodiesel industry has led to an interest in its use as an energy source in ruminant diets. However, its effects on ruminal fermentation patterns and methane (CH4) production are unclear, and there are no reports on the effect of its inclusion in the diet on wool production or growth of Merino sheep. Thus, the objectives of this study were to determine the effects of increasing levels of crude glycerin on in vitro ruminal fermentation and CH4 production and DMI, BW, feeding behavior, and wool growth and quality in Merino ewes. Crude glycerin (99.2% pure, colorless, odorless, viscous liquid) replaced whole wheat grain in completely pelleted diets at levels of 0%, 6%, and 12% DM in both in vitro and in vivo studies. For in vitro studies, diets were dried and ground through a 1-mm screen and incubated on 2 different days for 24 h. Modified McDougal's buffer and rumen liquor were mixed 3:1, and gas production and CH4 concentration was measured after 6, 12, and 24 h of incubation with pH and IVDMD measured at 24 h. Cumulative gas (mL/g DM) and methane (mL) production was similar (P ≥ 0.35) among dietary treatments. In vitro dry matter disappearance (%) increased (P < 0.01) with increasing concentrations of crude glycerin. For the in vivo study, 39 Merino ewes were randomly assigned to 3 treatments (n = 13 ewes/treatment). Pelleted diets were available continuously for a 10-wk period through the use of automatic feeders. Ewes were weighed every 7 d. Wool yield was determined on mid-side patches of 100 cm2 shorn at d 0 and d 70. Dye bands were used to determine wool growth and fiber length. Intake and ADG were similar among treatments (P = 0.59). Neither wool yield, length, spinning fineness, nor fiber diameter (μm) were affected after supplementation with crude glycerin (P ≥ 0.13). This study indicates the potential for crude glycerin to be included in the diets of Merino sheep at up to 12% DM without negatively affecting wool yield and quality. INTRODUCTION Australia is the largest exporter of wool in the world, accounting for almost one quarter of global wool exports (Australian Bureau of Statistics, 2010). Yet harsh environmental conditions necessitate dietary supplementation, as nutrients from native pasture are insufficient for the production of high-quality wool (NRC, 2007). Supplementary feeding, however, is becoming increasingly expensive because of drought and the redirection of grains from feed to fuel (Food and Agriculture Organization, 2008; Fung et al., 2010; Leng, 2010). The few studies that examined the effects of crude glycerin on CH4 production reported contrasting results. Lee et al. (2011) reported a reduction in in vitro CH4 per unit of DE. However, Avila et al. (2011) found no effect on either in vitro or in vivo CH4 production (g/kg digested DM) when glycerol replaced barley grain at up to 21% DM in the diet of finishing lambs (Avila et al., 2013). The use of high-grain diets in these studies may have lessened the impact of an increase in propionate production on CH4 production when compared with a forage-based diet. Additionally, the effects of including crude glycerin in the diets of Merino sheep on feeding behavior and wool production have not yet been examined. Strategies that have the potential to reduce methanogenesis without compromising animal performance are of increasing importance. We hypothesize that the inclusion of crude glycerin up to 12% in the diet DM fed to Merino ewes will affect neither animal performance nor wool characteristics. Thus, the objectives of this study were to examine the effects of crude glycerin on in vitro ruminal fermentation and CH4 production and to determine the in vivo effects of crude glycerin on DMI, body condition, feeding behavior, and wool production and quality in Merino ewes. MATERIALS AND METHODS All animals were cared for in accordance with the guidelines of The University of Sydney Research Integrity Animal Ethics Committee. In Vitro Study Substrates. A 24-h in vitro batch culture incubation was conducted using a completely randomized design. The 3 dietary treatments consisted of a wheat-based maintenance diet supplemented with 0%, 6%, or 12% crude glycerin (DM basis; Table 1). Representative samples of the diets were oven-dried at 55°C for 24 h and ground through a 1-mm sieve (Micro Hammer Cutter Mills, Glen Creston Limited, London, UK). On the day before the incubation, 0.5 g DM of each diet was weighed into an ANKOM bag (model F57, ANKOM Technology, Macedon, NY) with 3 technical replicates per treatment and sealed (Avila et al., 2011). On the day of incubation, each bag was placed into a 50-mL amber serum bottle with inoculum and sealed with a rubber stopper. The entire incubation procedure was repeated twice (i.e., 2 incubation runs × 3 replicates per treatment, resulting in a total of 6 replicate vials per treatment). Table 1. Ingredients and chemical composition of dietary treatments (n = 3; mean ± SEM) Item  Crude glycerin, % DM  Ryegrass hay  0  6  12  Ingredients, %      Crude glycerin1  0.00  6.00  12.00        Dry rolled wheat  49.95  40.35  30.00        Canola meal  7.00  8.50  10.65        Faba bean hulls  22.0  24.0  26.0        Molasses dried  2.50  2.50  2.50        Oat hulls  15.9  15.9  15.9        Canola oil  0.00  0.10  0.20        Calcium carbonate  1.10  1.10  1.10        Sheep mineral  1.20  1.20  1.20        Ammonium chloride  0.30  0.30  0.40        Vitamin ADE complex  0.02  0.02  0.02    Chemical composition      DM, %  90.1 ± 0.4  90.3 ± 0.4  91.3 ± 1.1  90.5 ± 0.8      CP, % DM  12.6 ±1.6  11.5 ± 0.1  11.9 ± 0.4  5.29 ± 0.9      NDF, % DM  33.6 ± 2.4  28.6 ± 1.8  28.0 ± 0.4  74.6 ± 2.9      Ether extract, % DM  4.19 ± 1.9  4.80 ± 0.7  4.67 ± 0.2  3.42 ± 2.6      NFC,2 % DM  42.1 ± 1.7  47.8 ± 2.4  48.9 ± 0.7  11.0 ± 3.8      Ash, % DM  7.53 ± 0.7  7.36 ± 1.1  6.65 ± 0.3  5.69 ± 1.7  Item  Crude glycerin, % DM  Ryegrass hay  0  6  12  Ingredients, %      Crude glycerin1  0.00  6.00  12.00        Dry rolled wheat  49.95  40.35  30.00        Canola meal  7.00  8.50  10.65        Faba bean hulls  22.0  24.0  26.0        Molasses dried  2.50  2.50  2.50        Oat hulls  15.9  15.9  15.9        Canola oil  0.00  0.10  0.20        Calcium carbonate  1.10  1.10  1.10        Sheep mineral  1.20  1.20  1.20        Ammonium chloride  0.30  0.30  0.40        Vitamin ADE complex  0.02  0.02  0.02    Chemical composition      DM, %  90.1 ± 0.4  90.3 ± 0.4  91.3 ± 1.1  90.5 ± 0.8      CP, % DM  12.6 ±1.6  11.5 ± 0.1  11.9 ± 0.4  5.29 ± 0.9      NDF, % DM  33.6 ± 2.4  28.6 ± 1.8  28.0 ± 0.4  74.6 ± 2.9      Ether extract, % DM  4.19 ± 1.9  4.80 ± 0.7  4.67 ± 0.2  3.42 ± 2.6      NFC,2 % DM  42.1 ± 1.7  47.8 ± 2.4  48.9 ± 0.7  11.0 ± 3.8      Ash, % DM  7.53 ± 0.7  7.36 ± 1.1  6.65 ± 0.3  5.69 ± 1.7  1Colorless, odorless, viscous liquid obtained from Biodiesel Producers, Barnawartha, Victoria, Australia. 2NFC, nonfibrous carbohydrates [NFC = 100 − (CP + NDF + EE + ash)]. View Large Table 1. Ingredients and chemical composition of dietary treatments (n = 3; mean ± SEM) Item  Crude glycerin, % DM  Ryegrass hay  0  6  12  Ingredients, %      Crude glycerin1  0.00  6.00  12.00        Dry rolled wheat  49.95  40.35  30.00        Canola meal  7.00  8.50  10.65        Faba bean hulls  22.0  24.0  26.0        Molasses dried  2.50  2.50  2.50        Oat hulls  15.9  15.9  15.9        Canola oil  0.00  0.10  0.20        Calcium carbonate  1.10  1.10  1.10        Sheep mineral  1.20  1.20  1.20        Ammonium chloride  0.30  0.30  0.40        Vitamin ADE complex  0.02  0.02  0.02    Chemical composition      DM, %  90.1 ± 0.4  90.3 ± 0.4  91.3 ± 1.1  90.5 ± 0.8      CP, % DM  12.6 ±1.6  11.5 ± 0.1  11.9 ± 0.4  5.29 ± 0.9      NDF, % DM  33.6 ± 2.4  28.6 ± 1.8  28.0 ± 0.4  74.6 ± 2.9      Ether extract, % DM  4.19 ± 1.9  4.80 ± 0.7  4.67 ± 0.2  3.42 ± 2.6      NFC,2 % DM  42.1 ± 1.7  47.8 ± 2.4  48.9 ± 0.7  11.0 ± 3.8      Ash, % DM  7.53 ± 0.7  7.36 ± 1.1  6.65 ± 0.3  5.69 ± 1.7  Item  Crude glycerin, % DM  Ryegrass hay  0  6  12  Ingredients, %      Crude glycerin1  0.00  6.00  12.00        Dry rolled wheat  49.95  40.35  30.00        Canola meal  7.00  8.50  10.65        Faba bean hulls  22.0  24.0  26.0        Molasses dried  2.50  2.50  2.50        Oat hulls  15.9  15.9  15.9        Canola oil  0.00  0.10  0.20        Calcium carbonate  1.10  1.10  1.10        Sheep mineral  1.20  1.20  1.20        Ammonium chloride  0.30  0.30  0.40        Vitamin ADE complex  0.02  0.02  0.02    Chemical composition      DM, %  90.1 ± 0.4  90.3 ± 0.4  91.3 ± 1.1  90.5 ± 0.8      CP, % DM  12.6 ±1.6  11.5 ± 0.1  11.9 ± 0.4  5.29 ± 0.9      NDF, % DM  33.6 ± 2.4  28.6 ± 1.8  28.0 ± 0.4  74.6 ± 2.9      Ether extract, % DM  4.19 ± 1.9  4.80 ± 0.7  4.67 ± 0.2  3.42 ± 2.6      NFC,2 % DM  42.1 ± 1.7  47.8 ± 2.4  48.9 ± 0.7  11.0 ± 3.8      Ash, % DM  7.53 ± 0.7  7.36 ± 1.1  6.65 ± 0.3  5.69 ± 1.7  1Colorless, odorless, viscous liquid obtained from Biodiesel Producers, Barnawartha, Victoria, Australia. 2NFC, nonfibrous carbohydrates [NFC = 100 − (CP + NDF + EE + ash)]. View Large Inoculum. Rumen liquor was collected from 3 lactating ruminally fistulated Holstein Friesian dairy cows maintained on pasture (perennial ryegrass, kikuyu, oats, and forage rape at 70%, 10%, 10%, and 10% DM basis, respectively), supplemented with 6 to 8 kg DM of corn silage and 6.5 kg protein (18% CP/kg DM) pellets. Rumen fluid was collected 2 h after feeding from three different sites within the rumen and pooled across the 3 cows. The composite rumen sample was filtered through 4 layers of cheesecloth into a prewarmed Thermos (Thermos PTY Limited, Seven Hills, NSW, Australia) flask and transported immediately to the laboratory. Inoculum was prepared by mixing rumen fluid and a mineral buffer with 0.5 mL of cysteine sulfide solution as a reducing agent (Chaves et al., 2006) in a ratio of 1:3. Inoculum (25 mL) was then transferred into preloaded, prewarmed (39°C) serum bottles under a stream of O2-free N gas. Serum bottles were sealed and placed on an orbital shaker (120 oscillations/min) in an incubator set at 39°C. Six bottles without substrate were also prepared for each time point to serve as blanks. Determination of Total Gas, Methane Concentration, and IVDMD. After 6, 12, and 24 h of incubation, bottles were removed from the incubator for measurement of gas production using the water displacement technique (Fedorak and Hrudey, 1983). Immediately before gas measurement, 12 mL of headspace gas was collected from each bottle with a 12-mL syringe and immediately transferred into a 5.9-mL evacuated Exetainer (Labco Ltd., High Wycombe, Buckinghamshire, UK), which was then analyzed for CH4 concentration by gas chromatography (model 5890, Hewlett Parkard, Little Falls, DE) as described by Chaves et al. (2006). Methane was expressed as milligrams of CH4/g DM and milligrams of CH4/g of disappeared incubated DM, and total net gas production was expressed as mL/g of incubated DM. After gas was sampled for CH4 and gas production was measured at 24 h of incubation, the fermentation bottles were opened, and pH was measured using a pH meter (Orion Model 260A, Fisher Scientific, Toronto, ON, Canada) calibrated at 39°C. The ANKOM bags with diet residues were removed from the bottles, rinsed thoroughly with distilled water, dried at 55°C for 48 h to a constant weight, and weighed to estimate IVDMD. Determination of VFA. At the beginning of the incubation (0 h) and at 24 h, two subsamples (1.0 mL) of the culture media from each bottle were transferred to 1.6-mL microcentrifuge tubes containing 200 μL of metaphosphoric acid (0.20%; wt/vol), and centrifuged at 14,000×g for 10 min at 4°C (Spectrafuse 16M, National Labnet Co., Edison, NJ). The supernatant was frozen at −20°C until analyzed for VFA concentrations using liquid gas chromatography (Agilent 6890, Series GC with HP injector; hydrogen carrier at 6.2 mL per minute; initial oven temperature was 100°C for 1 min, final temperature was 240°C, hold 1 min; injector 260°C, detector 265°C; split injection 5:1). In Vivo Study Dietary Treatments. The 3 dietary treatments, which were formulated to meet maintenance requirements of Merino ewes according to the Small Ruminant Nutrition System Version 1.8.18 (Cannas et al., 2004), were the same as those used in the in vitro experiment (Table 1). Crude glycerin (99.2% pure) was acquired from a tallow-based biodiesel production facility (Biodiesel Producers Ltd, Barnawartha, VIC, Australia). The 3 diets were formulated to be isonitrogenous and isolipidic. The diets were mixed and completely pelleted in batches of 2000 kg in advance for the entire experiment and stored in bags at room temperature until feeding. An additional 100 g ryegrass hay per head per day was provided to meet the daily minimum requirement for physical effective fiber (peNDF > 1.18; 22% of ration DM to maintain a ruminal pH of 6.0; Mertens, 1997). Animals, Feeding, and Sampling. Thirty-nine Merino ewes (initial BW 37.9 ± 4.41 kg; BCS 2.5 ± 0.41) approximately 3 to 4 yr old were used in a 70-d trial from mid-April to June conducted on the University of Sydney, Camden campus. All ewes were fitted with Allflex electronic half duplex identification ear tags (Dallas, TX) to facilitate the use of automatic feeders, model ARF (Agricultural Requirements, a division of Lockyer Investment Co. Pty Ltd., Gatton, Queensland, 4343). The ewes were given a 7-d adjustment period, during which the ewes were acclimatized to the automatic feeders. All ewes were fed the control diet throughout this period. A 7-d pretreatment period followed, in which ewes were stratified by BW and randomly assigned to one of 3 covered pens, approximately 60 m2 in size, with concrete flooring on half of the pen (i.e., 13 ewes/pen). Diets were available ad libitum for a 10-wk period through the use of 9 automatic feeders. Continuous access to ad libitum water was available. Feeders were suspended on load cells and recorded intake, feeding intervals and duration for each individual ewe in each of the 3 pens. A TIRIS tag reader in each feeder identified individual sheep through recognition of the ear tags. Data obtained from the feeders were interpreted using Adroit 5 Release 4.0, 2002 (Adroit Technologies, Johannesburg, South Africa), which received the data from the ASCII Basic Module PLC. This allowed recording and calculations of individual feed intake per visit (g), daily feeding time (min/d), eating rate (g/min), frequency of feeder visits (visits/d), and the average duration of each visit to the feeder (min/visit). Daily DMI was calculated from the total amount of DM corrected feed released by the automatic feeders in all feeding sessions for each ewe. Daily eating rate was calculated by dividing daily feed intake by the daily duration that each ewe spent at the feeder. Individual BW was recorded manually each week. Average daily gains were calculated by dividing BW gain of the sheep during the experiment (final BW minus initial BW) by the duration of the trial in days (i.e., 70 d). Wool Samples and Measurements. Wool samples were sent to Riverina Wool Testers Pty Ltd (Wagga Wagga, NSW, Australia) for analysis. A mid-side patch of approximately 100 cm2 was shorn on the left side of each sheep with clippers (Oster clippers, No. 40 cutting head; Hattersheim am Main, Germany) at the beginning of the treatment period (d 0). At the same time, a 5-cm dye band, using Schwarzkopf (Igora-Roya—NF1 Black; Frenchs Forest, NSW, Australia) hair dye, was applied to the right mid-side of each sheep. Schwarzkopf hair dye cream was mixed with the activator (Oxigenta 6%) immediately before application and applied in a thin line at skin level using the applicator provided. At the completion of the treatment period (d 70), both the mid-side patch and the fleece containing the dye bands were removed at skin level, using clippers and stored in plastic bags for analysis. Wool samples from the mid-side patch were weighed to determine the total wool weight (greasy wool weight) and placed into nylon filter bags. Samples were then washed in a washing machine using hot water (90°C) and detergent (BD30, Huntington Professional Products, St-Paul, MN) and rinsed twice in cold water to remove any impurities and wool grease before being dried in an oven for 4 h at 110°C. The samples were then reweighed at a humidity of 65% to determine clean wool weight. Wool yield was then calculated by determining the percentage of clean wool weight relative to greasy fleece weight. Methodologies of wool fiber diameter (FD; μm), the SD, CV, number of fibers >30 μm, comfort factor (percentage of fibers below 30.5 μm), spinning fineness, and curvature (deg/mm) values of the wool samples are described in detail by Charles et al. (2012). Chemical Analysis. Chemical analyses were performed on each sample in duplicate, and where the CV for the replicate analysis was <0.05, the analysis was repeated (Table 1). Feed samples were collected on d 0, 14, 28, 42, 56, and 70. Dry matter content of feed samples was determined by oven-drying at 100°C for 24 h [method 967.03; Association of Official Analytical Chemists (AOAC), 1990]. Dried samples were ground to pass a 1-mm screen and analyzed for NDF and ADF using Van Soest et al.'s (1991) procedures modified for an Ankom 200/220 Fiber Analyzer (Ankom Technol. Corp.), with both heat-stable α-amylase and sodium sulfite included. Concentrations of NDF and ADF were expressed inclusive of residual ash. Ash content was determined after 2 h of oxidation at 600°C in a muffle furnace (method 942.05; AOAC, 1990). Crude protein was calculated as N × 6.25. Ether extract (EE) content was determined by extraction with diethyl ether (method 920.39; AOAC, 1990) with the procedure modified for an Ankom XT10 Extraction System (Ankom Technol. Corp.). Nonfibrous carbohydrate (NFC;Mertens, 1997) was calculated as  Analytical DM was determined by drying the ground samples at 100°C for 2 h (method 930.05; AOAC, 1990). All samples were weighed using a hot weighing technique. Statistical Analysis. For the in vitro study, the 3 replicate bags were averaged before statistical analysis. The data were analyzed as a completely randomized design using PROC MIXED (SAS Inst. Inc., Cary, NC) with treatment in the model as fixed effects. The run by treatment interaction was used as the error term to test the treatment effect. The univariate procedure of SAS was used to test for normal distribution of the in vitro data. In the in vivo study, the raw data obtained from the automatic feeders were refined to remove significant outliers greater than 2 SD from the mean and measurement errors. Data were then summarized to total daily feed intake (g), average daily feed intake (g), frequency of feeder visits (visits per day), intake per visit (g), eating time (min/d), eating rate (g/min), and duration of visit (s). Intake data and behavioral parameters were analyzed as a completely randomized design using the PROC MIXED procedure of SAS. Means were compared using the LSMEANS/DIFF with treatment, week, and the interaction of treatment × week as fixed terms; lambs nested within treatment as a random effect and week as a repeated measure. Compound symmetry was used as the covariance structure for repeated measures analysis because it demonstrated the minimum values of Akaike's information criterion. The REML method was used for estimating the variance components, and degrees of freedom were adjusted using the Kenward-Roger option. Wool data, ADG, and initial and final BW were analyzed using a model similar to that described above, but excluding week as a repeated measure. Initial sheep BW was used as a covariate. For both in vitro and in vivo studies, orthogonal polynomial contrasts were used to determine linear and quadratic responses to increasing concentrations of crude glycerin incorporation. Unless otherwise specified, treatment effects were declared significant when P ≤ 0.05, and a trend was considered when 0.05 < P < 0.10. RESULTS AND DISCUSSION In Vitro Study Gas Production and DM Disappearance. Increasing concentrations of crude glycerin in the diet had no effect on gas production (mL) at any time point or cumulative gas production (mL/g DM) at 24 h (P ≥ 0.41; Table 2). This is similar to a study by Avila et al. (2011) where glycerol concentrations up to 21% DM had no effect on cumulative gas production (mL/g DM). Conversely, the addition of pure glycerol to alfalfa or corn has been shown to reduce gas production (Ferraro et al., 2009; Lee et al., 2011). The authors suggest this may be a result of the preferential production of propionate as an end product of glycerol fermentation, as propionate produces less gas than acetate (Blümmel et al., 1997). In contrast, the inclusion of glycerol to alfalfa hay at up to 40% DM resulted in a linear increase in in vitro gas production (Krueger et al., 2010), potentially a result of the contrasting chemical composition between forages and grain. Table 2. Effect of glycerin supplementation (0%, 6%, and 12% DM) on in vitro ruminal fermentation characteristics and methane production Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Gas production, mL      6 h  23.7  24.8  23.9  6.28  0.41  0.80  0.24      12 h  19.0  19.2  19.8  2.05  0.82  0.59  0.88      24 h  19.2  19.5  19.5  0.76  0.92  0.75  0.86  Cumulative gas, mL  61.9  63.5  63.2  3.91  0.63  0.49  0.56  Cumulative gas production, mL/g DM  121.2  125.8  124.9  7.36  0.35  0.28  0.34  IVDMD, %  66.8  73.8  78.3  2.50  <0.01  <0.01  0.20  pH  5.59  5.45  5.41  0.131  0.07  0.04  0.27  Total VFA, mM  127.1  132.4  132.9  10.07  0.27  0.17  0.42  Proportion of individual VFA, %      Acetate  46.9  45.3  42.9  2.68  0.24  0.13  0.78      Propionate  30.1  32.3  33.4  2.28  0.10  0.05  0.48      Butyrate  15.6  14.9  15.6  1.13  0.77  0.99  0.52      BCVFA2  3.4  3.2  3.2  0.15  0.63  0.49  0.57      Valerate  3.4  3.6  4.2  1.03  0.20  0.11  0.59      Caproic acid  0.6  0.5  0.7  0.15  0.47  0.34  0.49      Acetate:propionate  1.56  1.40  1.28  0.180  0.09  0.04  0.64  Methane, mL      6 h  1.3  1.1  0.8  1.10  0.51  0.30  0.96      12 h  1.5  1.4  1.3  0.61  0.55  0.33  0.92      24 h  2.1  2.0  2.0  0.54  0.42  0.24  0.76  Cumulative methane, mL  4.8  4.5  4.1  0.46  0.66  0.42  0.98  Cumulative methane, mL/g DM  9.4  8.9  8.2  0.85  0.66  0.42  0.93  Cumulative methane, mL/g disappeared incubated DM  14.1  12.1  10.1  1.68  0.39  0.22  0.98  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Gas production, mL      6 h  23.7  24.8  23.9  6.28  0.41  0.80  0.24      12 h  19.0  19.2  19.8  2.05  0.82  0.59  0.88      24 h  19.2  19.5  19.5  0.76  0.92  0.75  0.86  Cumulative gas, mL  61.9  63.5  63.2  3.91  0.63  0.49  0.56  Cumulative gas production, mL/g DM  121.2  125.8  124.9  7.36  0.35  0.28  0.34  IVDMD, %  66.8  73.8  78.3  2.50  <0.01  <0.01  0.20  pH  5.59  5.45  5.41  0.131  0.07  0.04  0.27  Total VFA, mM  127.1  132.4  132.9  10.07  0.27  0.17  0.42  Proportion of individual VFA, %      Acetate  46.9  45.3  42.9  2.68  0.24  0.13  0.78      Propionate  30.1  32.3  33.4  2.28  0.10  0.05  0.48      Butyrate  15.6  14.9  15.6  1.13  0.77  0.99  0.52      BCVFA2  3.4  3.2  3.2  0.15  0.63  0.49  0.57      Valerate  3.4  3.6  4.2  1.03  0.20  0.11  0.59      Caproic acid  0.6  0.5  0.7  0.15  0.47  0.34  0.49      Acetate:propionate  1.56  1.40  1.28  0.180  0.09  0.04  0.64  Methane, mL      6 h  1.3  1.1  0.8  1.10  0.51  0.30  0.96      12 h  1.5  1.4  1.3  0.61  0.55  0.33  0.92      24 h  2.1  2.0  2.0  0.54  0.42  0.24  0.76  Cumulative methane, mL  4.8  4.5  4.1  0.46  0.66  0.42  0.98  Cumulative methane, mL/g DM  9.4  8.9  8.2  0.85  0.66  0.42  0.93  Cumulative methane, mL/g disappeared incubated DM  14.1  12.1  10.1  1.68  0.39  0.22  0.98  1Contrasts: Lin = linear; Q = quadratic. 2BCVFA, branched-chain volatile fatty acids (isovalerate + isobutyrate). View Large Table 2. Effect of glycerin supplementation (0%, 6%, and 12% DM) on in vitro ruminal fermentation characteristics and methane production Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Gas production, mL      6 h  23.7  24.8  23.9  6.28  0.41  0.80  0.24      12 h  19.0  19.2  19.8  2.05  0.82  0.59  0.88      24 h  19.2  19.5  19.5  0.76  0.92  0.75  0.86  Cumulative gas, mL  61.9  63.5  63.2  3.91  0.63  0.49  0.56  Cumulative gas production, mL/g DM  121.2  125.8  124.9  7.36  0.35  0.28  0.34  IVDMD, %  66.8  73.8  78.3  2.50  <0.01  <0.01  0.20  pH  5.59  5.45  5.41  0.131  0.07  0.04  0.27  Total VFA, mM  127.1  132.4  132.9  10.07  0.27  0.17  0.42  Proportion of individual VFA, %      Acetate  46.9  45.3  42.9  2.68  0.24  0.13  0.78      Propionate  30.1  32.3  33.4  2.28  0.10  0.05  0.48      Butyrate  15.6  14.9  15.6  1.13  0.77  0.99  0.52      BCVFA2  3.4  3.2  3.2  0.15  0.63  0.49  0.57      Valerate  3.4  3.6  4.2  1.03  0.20  0.11  0.59      Caproic acid  0.6  0.5  0.7  0.15  0.47  0.34  0.49      Acetate:propionate  1.56  1.40  1.28  0.180  0.09  0.04  0.64  Methane, mL      6 h  1.3  1.1  0.8  1.10  0.51  0.30  0.96      12 h  1.5  1.4  1.3  0.61  0.55  0.33  0.92      24 h  2.1  2.0  2.0  0.54  0.42  0.24  0.76  Cumulative methane, mL  4.8  4.5  4.1  0.46  0.66  0.42  0.98  Cumulative methane, mL/g DM  9.4  8.9  8.2  0.85  0.66  0.42  0.93  Cumulative methane, mL/g disappeared incubated DM  14.1  12.1  10.1  1.68  0.39  0.22  0.98  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Gas production, mL      6 h  23.7  24.8  23.9  6.28  0.41  0.80  0.24      12 h  19.0  19.2  19.8  2.05  0.82  0.59  0.88      24 h  19.2  19.5  19.5  0.76  0.92  0.75  0.86  Cumulative gas, mL  61.9  63.5  63.2  3.91  0.63  0.49  0.56  Cumulative gas production, mL/g DM  121.2  125.8  124.9  7.36  0.35  0.28  0.34  IVDMD, %  66.8  73.8  78.3  2.50  <0.01  <0.01  0.20  pH  5.59  5.45  5.41  0.131  0.07  0.04  0.27  Total VFA, mM  127.1  132.4  132.9  10.07  0.27  0.17  0.42  Proportion of individual VFA, %      Acetate  46.9  45.3  42.9  2.68  0.24  0.13  0.78      Propionate  30.1  32.3  33.4  2.28  0.10  0.05  0.48      Butyrate  15.6  14.9  15.6  1.13  0.77  0.99  0.52      BCVFA2  3.4  3.2  3.2  0.15  0.63  0.49  0.57      Valerate  3.4  3.6  4.2  1.03  0.20  0.11  0.59      Caproic acid  0.6  0.5  0.7  0.15  0.47  0.34  0.49      Acetate:propionate  1.56  1.40  1.28  0.180  0.09  0.04  0.64  Methane, mL      6 h  1.3  1.1  0.8  1.10  0.51  0.30  0.96      12 h  1.5  1.4  1.3  0.61  0.55  0.33  0.92      24 h  2.1  2.0  2.0  0.54  0.42  0.24  0.76  Cumulative methane, mL  4.8  4.5  4.1  0.46  0.66  0.42  0.98  Cumulative methane, mL/g DM  9.4  8.9  8.2  0.85  0.66  0.42  0.93  Cumulative methane, mL/g disappeared incubated DM  14.1  12.1  10.1  1.68  0.39  0.22  0.98  1Contrasts: Lin = linear; Q = quadratic. 2BCVFA, branched-chain volatile fatty acids (isovalerate + isobutyrate). View Large In vitro DMD increased linearly (P < 0.01) with increasing concentrations of dietary crude glycerin. An increase in digestibility was expected as glycerol, a completely digestible compound, replaced wheat, which is not entirely digestibly. Similarly, glycerol provides less ATP from microbial growth than carbohydrates, and therefore, the amount of microbial residues may have also been smaller in comparison with the control diet. It is noted that the difference in IVDMD observed in the current study may also be a reflection of the ability of crude glycerin to penetrate through the pores of the nylon bags, rather than a measurement of its digestion; however, the methods used were similar to that of Avila et al. (2011), who reported no such effects. Comparatively, a lack of effect of glycerol on total-tract digestibility of DM, OM, N, and NDF (Khalili et al., 1997), as well as in vitro (Avila et al., 2011) and in vivo (Schröder and Südekum, 1999; Krueger et al., 2010) nutrient digestibility, has been reported. Similarly, Rémond et al. (1993) found no differences in OM digestibility when glycerol was added to a wheat starch substrate but found that digestibility increased when the substrate was cellulose, indicating that the inclusion of glycerol in cellulose-based diets will most likely enhance digestibility, whereas the replacement of starch with glycerol, appears to have no effect on potential digestibly. Fermentation Characteristics and Methane Production. In contrast to previous studies (Mach et al., 2009; Wang et al., 2009), replacing increasing concentrations of wheat grain with crude glycerin in the current study had a tendency to decrease pH (P = 0.07) or total VFA (mM; P = 0.905). Although ruminal fermentation of carbohydrates favors the production of propionate, there is no obligation for glycerin to be preferentially fermented to propionate, as a carbon can be oxidized and subsequent conversion to acetate can occur. This was evident in the current study, where no effect was observed on either the molar proportion of acetate or propionate at any level of crude glycerin inclusion (P ≥ 0.10; Table 2). Likewise, no effect on cumulative CH4 production (mL) or CH4 production expressed as either milliliters per gram DM or milliliters per gram disappeared incubated DM was observed (P ≥ 0.42). Avila et al. (2013) also reported no effect of glycerol inclusion at up to 21% DM on CH4 emissions (expressed as mL/g digested DM) from lambs and concluded that the low NDF concentration of the diets may have influenced this result. Conversely, Lee et al. (2011) reported a reduction of the A:P ratio, with an associated reduction in in vitro CH4 production after the supplementation of alfalfa hay and corn grain with glycerin, suggesting that although the fermentation of glycerin does not necessarily result in the formation of a H2 sink, its ability to promote a shift in carbohydrate fermentation from the production of acetate to propionate may affect the overall electron balance in the rumen and reduce the availability of hydrogen for methane formation. The lack of effect on the proportion of butyrate is consistent with other in vitro (Krueger et al., 2010) and in vivo (DeFrain et al., 2004; Mach et al., 2009) studies. Conversely, Rémond et al. (1993) indicated that the molar proportion of butyrate is greater in fermenters fed starch-based diets vs. those fed cellulose, indicating that the effect of glycerin on the end product of fermentation is largely determined by dietary composition (Khalili et al., 1997; Trabue et al., 2007). In Vivo Study Production Performance. There was no effect of dietary treatment on DMI or ADG (P = 0.59; Table 3). Similarly, neither initial BW nor final BW differed (P ≥ 0.82). The lack of effect on performance characteristics in the current study is likely because Merino sheep are selectively bred for wool production rather than growth performance. Thus, it was expected that changes in available ME and protein of the diet would have been utilized for the development of wool fibers rather than growth, and therefore, large variations in traditional production performance parameters were not observed (NRC, 2007). Similarly, the ewes in the current study were at a mature age, and it was not expected that large variations in growth or ADG would be observed. However, a 22% to 25% increase in BW was observed across all groups, this could be attributed to the shift in diet from grazing pretrial to a concentrate-based trial diet. According to the estimation given by the Small Ruminant Nutrition System (SRNS) Version 1.8.11 report (data not presented), all dietary treatments provided ME approximately 25% above maintenance, which is in agreement with the increase in ADG. Previous studies have indicated that including up to 15% DM crude glycerol in the diets of finishing wethers can improve feedlot performance (DMI, G:F, and ADG), particularly in the first 14 d, but feeding in excess of 30% negatively effects both growth and carcass quality (Gunn et al., 2010a,b), and feeding up to 45% DM causes a linear decrease in DMI (Musselman et al., 2008; Gunn et al., 2010b). Table 3. Effect of increasing concentrations of glycerin in the diet on Merino ewe performance (n = 13) Item  Experimental diets (crude glycerin content, % DM)  SEM  P-Value  Treatment  Contrast1  0  6  12  Lin  Q  Initial BW, kg  35.7  36.5  35.6  1.32  0.90  0.97  0.65  Final BW, kg  43.6  44.7  44.4  1.72  0.82  0.53  0.91  DMI, g/d  1058  994  972  61.4  0.59  0.32  0.77  ADG, g  114  118  137  16.7  0.59  0.34  0.72  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-Value  Treatment  Contrast1  0  6  12  Lin  Q  Initial BW, kg  35.7  36.5  35.6  1.32  0.90  0.97  0.65  Final BW, kg  43.6  44.7  44.4  1.72  0.82  0.53  0.91  DMI, g/d  1058  994  972  61.4  0.59  0.32  0.77  ADG, g  114  118  137  16.7  0.59  0.34  0.72  1Contrasts: Lin = linear; Q = quadratic. View Large Table 3. Effect of increasing concentrations of glycerin in the diet on Merino ewe performance (n = 13) Item  Experimental diets (crude glycerin content, % DM)  SEM  P-Value  Treatment  Contrast1  0  6  12  Lin  Q  Initial BW, kg  35.7  36.5  35.6  1.32  0.90  0.97  0.65  Final BW, kg  43.6  44.7  44.4  1.72  0.82  0.53  0.91  DMI, g/d  1058  994  972  61.4  0.59  0.32  0.77  ADG, g  114  118  137  16.7  0.59  0.34  0.72  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-Value  Treatment  Contrast1  0  6  12  Lin  Q  Initial BW, kg  35.7  36.5  35.6  1.32  0.90  0.97  0.65  Final BW, kg  43.6  44.7  44.4  1.72  0.82  0.53  0.91  DMI, g/d  1058  994  972  61.4  0.59  0.32  0.77  ADG, g  114  118  137  16.7  0.59  0.34  0.72  1Contrasts: Lin = linear; Q = quadratic. View Large Feeding Behavior. Daily eating time (min/d) decreased (P = 0.04) with the inclusion of crude glycerin. There was a tendency (P = 0.07) for eating rate (g/min) to increase with crude glycerin supplementation. However, the number of daily visits to the feeder, the duration of each visit (s), and intake per visit were similar among treatments (P ≥ 0.35; Table 4). Animals supplemented with crude glycerin spent less time eating than the control group; however, these ewes did not consume less as a result. There is a paucity of information regarding sheep feeding behavior when supplemented with crude glycerin, yet previous studies that examined DMI reported no changes when crude glycerol replaced rapidly fermentable starch in diets fed to dairy cattle and sheep up to 10% DM (Schröder and Südekum, 1999) or dry-rolled corn up to 20% DM in lamb diets (Gunn et al., 2010b). Table 4. Effect of increasing crude glycerin concentration (0%, 6%, and 12% DM) in the diet on the feeding behavior of Merino ewes Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Eating time, min/d  49.4  42.1  37.0  3.14  0.04  0.01  0.67  Eating rate, g/min  22.3  25.2  27.3  1.65  0.07  0.06  0.17  Visits per day  82.0  71.8  69.9  6.71  0.35  0.51  0.21  Duration of visit, s  45.0  47.4  43.6  7.99  0.94  0.91  0.75  Intake per visit, g  14.4  17.1  16.2  1.40  0.37  0.36  0.28  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Eating time, min/d  49.4  42.1  37.0  3.14  0.04  0.01  0.67  Eating rate, g/min  22.3  25.2  27.3  1.65  0.07  0.06  0.17  Visits per day  82.0  71.8  69.9  6.71  0.35  0.51  0.21  Duration of visit, s  45.0  47.4  43.6  7.99  0.94  0.91  0.75  Intake per visit, g  14.4  17.1  16.2  1.40  0.37  0.36  0.28  1Contrasts: Lin = linear; Q = quadratic. View Large Table 4. Effect of increasing crude glycerin concentration (0%, 6%, and 12% DM) in the diet on the feeding behavior of Merino ewes Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Eating time, min/d  49.4  42.1  37.0  3.14  0.04  0.01  0.67  Eating rate, g/min  22.3  25.2  27.3  1.65  0.07  0.06  0.17  Visits per day  82.0  71.8  69.9  6.71  0.35  0.51  0.21  Duration of visit, s  45.0  47.4  43.6  7.99  0.94  0.91  0.75  Intake per visit, g  14.4  17.1  16.2  1.40  0.37  0.36  0.28  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Eating time, min/d  49.4  42.1  37.0  3.14  0.04  0.01  0.67  Eating rate, g/min  22.3  25.2  27.3  1.65  0.07  0.06  0.17  Visits per day  82.0  71.8  69.9  6.71  0.35  0.51  0.21  Duration of visit, s  45.0  47.4  43.6  7.99  0.94  0.91  0.75  Intake per visit, g  14.4  17.1  16.2  1.40  0.37  0.36  0.28  1Contrasts: Lin = linear; Q = quadratic. View Large Wool Production. A strong positive, linear relationship exists between DMI and wool growth rates in sheep (Hynd and Masters, 2002; Rangel and Gardiner, 2009). The nature of this relationship is largely dependent on the supply of AA and energy substrates to the wool follicle, the genetic potential of the animals, and the methodology used to measure wool growth (Hynd and Masters, 2002). In the current study, similar DMI and chemical composition of the diets may account for the uniformity observed in fiber length across treatments (P = 0.82; Table 5). Similarly, wool yield, curvature, and comfort factor (percentage of fibers below 30.5 μm) were not affected by dietary treatment (P ≥ 0.23). Table 5. Effects of increasing concentrations of glycerin in the diet on Merino wool yield and quality characteristics (n = 13) Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Yield, %  76.8  75.5  75.9  0.94  0.23  0.94  0.09  Length, cm  1.6  1.7  1.7  0.07  0.82  0.56  0.80  FD,2 μm  17.1  16.5  16.9  0.24  0.13  0.07  0.35  FDSD,3 μm  2.8  2.5  2.6  0.08  0.28  0.12  0.73  FDCV,4 %  16.1  15.3  15.5  0.41  0.83  0.55  0.98  Fibers > 30.5 mm, %  0.3  0.3  0.3  0.04  0.54  0.27  0.96  Comfort factor, %  99.7  99.7  99.7  0.04  0.54  0.27  0.96  Curvature, deg/mm  133.9  134.7  127.9  3.50  0.70  0.70  0.45  Spinning fineness, μm  16.0  15.4  15.7  0.23  0.09  0.05  0.33  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Yield, %  76.8  75.5  75.9  0.94  0.23  0.94  0.09  Length, cm  1.6  1.7  1.7  0.07  0.82  0.56  0.80  FD,2 μm  17.1  16.5  16.9  0.24  0.13  0.07  0.35  FDSD,3 μm  2.8  2.5  2.6  0.08  0.28  0.12  0.73  FDCV,4 %  16.1  15.3  15.5  0.41  0.83  0.55  0.98  Fibers > 30.5 mm, %  0.3  0.3  0.3  0.04  0.54  0.27  0.96  Comfort factor, %  99.7  99.7  99.7  0.04  0.54  0.27  0.96  Curvature, deg/mm  133.9  134.7  127.9  3.50  0.70  0.70  0.45  Spinning fineness, μm  16.0  15.4  15.7  0.23  0.09  0.05  0.33  1Contrasts: Lin = linear; Q = quadratic. 2FD = fiber diameter. 3FDSD = fiber diameter SD. 4FDCV = fiber diameter CV. View Large Table 5. Effects of increasing concentrations of glycerin in the diet on Merino wool yield and quality characteristics (n = 13) Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Yield, %  76.8  75.5  75.9  0.94  0.23  0.94  0.09  Length, cm  1.6  1.7  1.7  0.07  0.82  0.56  0.80  FD,2 μm  17.1  16.5  16.9  0.24  0.13  0.07  0.35  FDSD,3 μm  2.8  2.5  2.6  0.08  0.28  0.12  0.73  FDCV,4 %  16.1  15.3  15.5  0.41  0.83  0.55  0.98  Fibers > 30.5 mm, %  0.3  0.3  0.3  0.04  0.54  0.27  0.96  Comfort factor, %  99.7  99.7  99.7  0.04  0.54  0.27  0.96  Curvature, deg/mm  133.9  134.7  127.9  3.50  0.70  0.70  0.45  Spinning fineness, μm  16.0  15.4  15.7  0.23  0.09  0.05  0.33  Item  Experimental diets (crude glycerin content, % DM)  SEM  P-value  Treatment  Contrast1  0  6  12  Lin  Q  Yield, %  76.8  75.5  75.9  0.94  0.23  0.94  0.09  Length, cm  1.6  1.7  1.7  0.07  0.82  0.56  0.80  FD,2 μm  17.1  16.5  16.9  0.24  0.13  0.07  0.35  FDSD,3 μm  2.8  2.5  2.6  0.08  0.28  0.12  0.73  FDCV,4 %  16.1  15.3  15.5  0.41  0.83  0.55  0.98  Fibers > 30.5 mm, %  0.3  0.3  0.3  0.04  0.54  0.27  0.96  Comfort factor, %  99.7  99.7  99.7  0.04  0.54  0.27  0.96  Curvature, deg/mm  133.9  134.7  127.9  3.50  0.70  0.70  0.45  Spinning fineness, μm  16.0  15.4  15.7  0.23  0.09  0.05  0.33  1Contrasts: Lin = linear; Q = quadratic. 2FD = fiber diameter. 3FDSD = fiber diameter SD. 4FDCV = fiber diameter CV. View Large The predominant energy substrates available to the follicle appear to be glucose and glutamine, whereas acetate fails to maintain fiber growth (Hynd and Masters, 2002). As glycerol can be fermented to propionate, a gluconeogenic precursor (Donkin et al., 2009), there is the potential for glycerol to increase energy availability to wool follicles. However, no effects on wool length were observed after the dietary supplementation of crude glycerin in the current study, considered to be a result of the isonitrogenous and isolipidic nature of the dietary treatments. Similarly, a study by Kempton et al. (1978) found no effects on the rate of wool growth when lambs were given access to glucose (at up to 80 g/d) through a suckling bottle. Aitchison et al. (1989a) supplemented diets of mature Merino wethers with avoparcin and lasalocid and observed a similar shift toward the preferential production of propionate. However, no effects on wool production or BW change were reported. Wool produced in the current study was classed as superfine wool, as the FD was between 15 and 18.75 μm (Australian Wool Corporation, 1990) and did not differ across dietary treatments (P = 0.13). Fiber diameter is a major determinant of wool quality and price and is usually influenced by nutrition in a similar manner to fiber length, such that the ratio of fiber length to FD is generally constant in sheep housed in pens as they are provided with a consistent supply of the same diet (Aitchison et al., 1989b; Schlink et al., 1999). As such, the lack of effect of crude glycerin on fiber diameter is consistent with prior notions of the relationship between DMI and wool production. Conclusion Substituting wheat grain with crude glycerin at 12% DM in the diets of Merino ewes had no effect on in vitro CH4 production expressed as either milliliters per gram DM or milliliters per gram disappeared incubated DM, yet a linear increase in IVDMD was observed with crude glycerin inclusion. 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TI - Effects of crude glycerin supplementation on wool production, feeding behavior, and body condition of Merino ewes
JF - Journal of Animal Science
DO - 10.2527/jas.2012-5791
DA - 2013-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/effects-of-crude-glycerin-supplementation-on-wool-production-feeding-NHk70p013a
SP - 878
EP - 885
VL - 91
IS - 2
DP - DeepDyve
ER -