Summary. Keywords: methanol, glycerin, intake, beef cattle. Introduction

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1 Effect of Methanol Infusion on Intake and Digestion of a Grain-based Diet by Beef Cattle K.N. Winsco, N.M. Kenney, R.O. Dittmar, III, J.A. Coverdale, J.E. Sawyer, and T.A. Wickersham Texas A & M University, College Station, TX Summary Our objective was to determine the effects of methanol concentration on intake and digestion in cattle consuming a grain-based diet. Four ruminally cannulated Holstein steers (BW = 399 ± 34 kg) were used in a 4 4 Latin square and provided ad libitum access to a grain-based diet (49% corn, 14.7% CP, 32.1% starch). Treatments consisted of 4 levels of methanol (0, 70, 140, and 210 g/d) infused directly into the rumen to prevent volatilization from feed. Experimental periods were 16 d, with 9 d of adaptation to treatment and 7 d of data collection. Determinations of intake were made on d 10 through 14 of each period to correspond with fecal grab samples collected from d 11 through 15. Titanium dioxide, dosed daily at 10 g/d, was used as an external marker to estimate fecal production. Ruminal fluid was collected on d 16. Methanol intake increased linearly (P < 0.01) from 0 to 6,563, 13,356, and 19,831 ppm of the diet for 0, 70, 140, and 210 g/d of methanol, respectively. Intake was not affected (P > 0.71) by methanol infusion (9.93, 9.93, 9.73, and 9.83 kg OM/d for 0, 70, 140, and 210 g/d of methanol). Methanol infusion did not affect (P > 0.12) OM or starch total tract digestion, which averaged 75.6 and 93.9%, respectively, across treatments. Methanol infusion did not significantly (P> 0.20) effect total volatile fatty acid concentration, ruminal ph, or the molar proportion of acetate. However, increasing methanol infusion tended to quadratically decrease (P = 0.07) the molar proportion of propionate while quadratically increasing butyrate (P = 0.03). Our results indicate that levels of methanol consumption, in excess of the current recommendation of 150 ppm, did not have adverse effects on intake, digestion, and ruminal fermentation in cattle. This data suggests that there is minimal risk in allowing ruminant diets to contain greater levels of methanol than nonruminant diets. Keywords: methanol, glycerin, intake, beef cattle Introduction Biodiesel production in the U.S. has increased from 112 million gallons in 2005 to 315 million gallons in Accordingly, crude glycerin production has increased from 78 million pounds to million pounds over the same time span. While glycerin has recently been increasingly evaluated as a feedstuff for both finishing and grazing cattle, relatively little work has been conducted to address the impact of methanol contamination on intake and digestion in beef cattle. Therefore, our objective was to determine the effect of methanol on intake, digestion, and ruminal fermentation in beef cattle.

2 Experimental Procedure This study was designed to determine the effects of increasing methanol infusion on the intake and digestion of a grain-based diet. Four ruminally cannulated Holstein steers (BW = 399 ±34 lbs.) were used in a 4 4 Latin square and provided ad libitum access to a grain based diet (Table 1) and water throughout the entire project. Treatments consisted of 4 levels of methanol (0, 70, 140, and 210 g/d) infused directly into the rumen. Methanol was ruminally infused to prevent volatilization of the methanol, ensuring complete provision of the specified level of methanol. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Texas A&M University. Experimental periods were 16 d long, with 10 d for adaptation and 6 d for sample collection and intake determination. Intake and digestion were determined from d 11 to 15. Feed samples were collected from d 11 to 14just prior to feeding, and orts were collected from d 12 to d 15at 0600h. Feed samples (500 g) and ort samples (200 g) were retained daily for subsequent analysis. Fecal samples were collected from d 12 to d 15. Fecal grab samples were collected every 8 h between and was advanced 2 h every day to gather representative samples from each even hour of the day, such that 12 samples were collected. On d 16, rumen fluid samples were collected by suction strainer just before treatments were administered (0 h) and at 0, 1, 2, 4, 6, 8, 10, 12, and 16 hours after infusion of the methanol. Ruminal ph was measured at each collection and 10 ml of rumen fluid was combined with 2 ml of 25% (wt/vol) metaphosphoric acid for volatile fatty acid analysis. Partial DM on feed, ort, and fecal samples was determined by drying samples at 60º C in a forced-air oven for 96 h, air-equilibrating them for 24 h, and weighing them. Dried samples were subsequently ground with a Wiley mill to pass a 1-mm screen. Feed samples collected during each period were composited across days on an equal weight basis. Ort and fecal samples were composited by steer across days for each period. Feed, ort, and fecal DM were determined by drying samples for 24 h at 105 C in a forced-air oven. Samples were then combusted at 450 C for 8 h in a muffle furnace for determination of OM. Nitrogen content was determined using Dumas combustion with crude protein calculated as N Feed, ort, and fecal samples were analyzed for NDF with a fiber analyzer (model 200, ANKOM Technology, Fairport, NY) with sodium sulfite and amylase omitted and without correction for residual ash. Total starch content of feed, ort, and fecal samples were determined using enzyme hydrolysis. Fecal titanium dioxide concentrations were determined using a colorimetric procedure. Ruminal volatile fatty acids were determined using GLC.

3 Intake and digestion were analyzed using PROC MIXED (SAS Inst. Inc., Cary, NC). Terms in the model were treatment and period with steer included as the random term. Fermentation profile data was also analyzed in PROC MIXED. Terms in the model were treatment, period, hour, hour treatment with steer, and treatment period steer included in the random statement. Treatment steer served as the subject, and compound symmetry was used for the covariance structure. The LSMEANS option was used to calculate treatment means. LSMEANS were separated using linear, quadratic, and cubic contrast for the level of methanol infusion. Results Ruminal infusion of methanol, as per the design of the project, resulted in a linear increase (P < 0.01) in the added methanol concentration of diet from 0 ppm for 0 g/d to 19,831 ppm for 210 g/d. Methanol was infused directly into the rumen to ensure that each animal received the specified dose of methanol. Incorporation of methanol into the feed would have allowed the methanol to volatilize, making it difficult to determine methanol intake. Our objective was to determine how a specific level of methanol delivered to the rumen impacts intake, digestion, and ruminal fermentation. An additional study is required to determine the impact of methanol concentration in the feed on rate and extend of feed consumption in cattle consuming a grainbased diet. Intake of DM, OM, NDF, and starch was not significantly impacted (Table 2; P > 0.71) by increasing levels of methanol infusion. Dry matter intake was in excess of 2.6% of body weight which was expected for the diet provided. Dry matter digestion ranged from 76.4 to 78.0% with no significant effect (P > 0.59) of increasing methanol infusion. Starch digestion was not significantly affected (P > 0.12) by increasing methanol infusion and ranged from 94.6% with 0 g/d of methanol to 93.2% with 210 g/d of methanol. Ruminal ph, total VFA concentration, and molar proportion of acetate were not significantly affected (P > 0.20) by methanol infusion (Figures 1 and 3). A quadratic tendency (P = 0.07) for a reduction in the proportion of propionate was observed, and was likely the result of a quadratic (P = 0.03) increase in the proportion of butyrate. While these shifts were significant, the biological importance is relatively minor. Implications Observations from this study suggest that methanol, when infused at levels less than 19,000 ppm, does not significantly impact intake, digestion, or ruminal fermentation. Additionally, no adverse health or well-being effects were observed when methanol was infused. While additional work is required to determine how the presence of methanol in the feed impacts consumption, it appears as though methanol contained in crude glycerin does not present a challenge to the animal and could be safely fed.

4 Tables and Figures Table 1.Composition of basal diet and methanol Item Feed Methanol Corn 48.9 Cottonseed Meal 16.0 Cottonseed Hulls 15.6 Molasses 10.0 Rice Bran 7.5 Premix % As-Fed % of DM Organic Matter Crude Protein Starch Neutral Detergent Fiber

5 Table 2. Effects of methanol infusion on intake and digestion in cattle consuming a grain-based diet Methanol g/d Contrast P-value Item SEM Linear Quadratic Cubic No. of observations Dietary Methanol, ppm 0 6,563 13,356 19, < Intake, kg/d Dry Matter Organic Matter Starch Neutral Detergent Fiber Total tract digestion, % Dry Matter Organic Matter Starch Neutral Detergent Fiber

6 Total VFA, mm Ruminal ph Figure1. Effect of methanol infusion on total volatile fatty acid concentration and ruminal ph in cattle consuming a grain-based diet. No significant effects of methanol Methanol Level, g/d

7 Molar Proportion Figure 2.Effect of methanol infusion on molar proportions of acetate, propionate, and butyrate in cattle consuming a grain-based diet. Quadratic effect of methanol on propionate (P = 0.07) and butyrate (P = 0.03). 0.5 Acetate Propionate Butyrate Supplement, % CP