Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Ruminal Fermentation of Beef Steers Consuming Bermudagrass Hay

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1 Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Ruminal Fermentation of Beef Steers Consuming Bermudagrass Hay F. M. Ciriaco 1, D. D. Henry 1, V. R. G. Mercadante 1, T. Schulmeister 1, M. Ruiz-Moreno 1, G. C. Lamb 1, and N. DiLorenzo 1 Synopsis Supplementation with energy and protein sources is crucial during critical periods of the year and molasses and crude glycerol combined in a liquid mixture have the potential of serving as an energy supplement for beef cattle consuming forage-based diets. Summary A study was conducted to evaluate the effects of molasses and crude glycerol in a 50:50 liquid mixture on ruminal fermentation of beef steers consuming Tifton 85 bermudagrass hay. Steers were provided Tifton 85 bermudagrass hay ad libitum and four increasing amounts of the 50:50 mixture were compared: 1) CTRL=0 lb/d; 2) SUP1=1 lb/d; 3) SUP3=3 lb/d; and 4) SUP5=5 lb/d. Eight ruminally cannulated Angus crossbred steers were used in a duplicated 4 4 Latin Square design. Steers were housed at the North Florida Research and Education Center (NFREC) Feed Efficiency Facility (FEF) for four periods of 28 d each and intake of hay was monitored using the GrowSafe system. The liquid supplement was weighed and offered daily to each steer. Ruminal fluid and blood were collected every 3 h postfeeding for 24 h for measurement of average ruminal ph, analyses of volatile fatty acids (VFA) profile, and ammonianitrogen (NH 3 -N) and blood urea nitrogen (BUN) concentrations. Concentrations of NH 3 -N and BUN were influenced by liquid supplementation, decreasing linearly as the amount of supplementation increased. Average ruminal ph was also influenced by liquid supplementation and decreased linearly as the amount of liquid supplement increased. A treatment time interaction was observed for ruminal ph. Molar proportions of branched chain VFA (BCVFA) and acetate linearly decreased whereas molar proportions of propionate, butyrate, and valerate linearly increased as the amount of liquid supplemented increased. Total VFA concentration was affected cubically by liquid supplementation where it decreased from CTRL to SUP1 and SUP5, however, SUP3 was not different when compared to the others. There was an effect of liquid supplementation on acetate to propionate ratio (A:P) where it decreased linearly as the amount of liquid supplemented increased. The liquid mixture of molasses and crude glycerol has the potential of improving animal performance due to increase in propionate production. Introduction The Southeastern region of the United States has several characteristics that make it unique when compared to other beef cattle regions. The states that represent the southeast and Gulf Coast regions account for 48% of all beef cows in the United States. In the current context of increasing feed input costs, the abundant forage production in the Southeastern U.S. provides an opportunity to decrease the cost of production, considering that feed is the largest cost in a cattle operation. However, the predominant forages in this region can be of limited nutritive and often not sufficient to support high levels of production. As a result, there are some critical periods during the year in which there is a need for supplementation with energy and/or protein in cow/calf operations in the Southeast (Hersom et al., 2011). Combined with the abundance of forages, another advantage of beef production in the Southeastern U.S. is the availability of several byproducts from diverse industries, which can have great nutritional value for cattle and can provide an excellent opportunity to correct nutritional imbalances 1 North Florida Research and Education Center, University of Florida, Marianna, FL

2 through strategic supplementation. The sugar industry is strong in the state of Florida and as a result, byproducts such as molasses have been fed to cattle for decades (Pate and Kunkle, 1989; Kunkle et al., 2000). Crude glycerin (or crude glycerol) is generally recognized as safe (GRAS) animal food ingredient and it can be produced as a coproduct of the soap making industry; however, more recently, the rapid expansion of the biodiesel industry has generated large amounts of crude glycerol as a byproduct, resulting in reduction of prices and increased concerns related to its disposal in the environment, which makes crude glycerol a potential high-energy feed source for cattle (Tan et al., 2013). The objective of this study was to evaluate the effects of different amounts of supplementation with a 50:50 mixture of molasses and crude glycerol on Tifton 85 bermudagrass hay intake and digestibility of nutrients in the total tract of beef heifers. Materials and Methods The study was initiated in October, 2013 and was conducted at the FEF located at the NFREC in Marianna, FL. Liquid supplement was provided by Westway Feed Products (New Orleans, LA). Eight ruminally cannulated Angus crossbred steers (712±93 lb BW) were used in a 4 4 duplicated Latin square design. In each of the four 28-d periods, d 0 to 14 were for adaptation to the diet, d 14 for collection of ruminal fluid, blood and ruminal ph, and d 21 to 28 for washout, when all the animals were consuming only hay. All procedures described were performed similarly for all four periods. Steers were randomly assigned to 1 of 4 treatments: 1. CTRL = no supplementation 2. SUP1 = 1 lb/d of the 50:50 molasses and crude glycerol mixture 3. SUP3 = 3 lb/d of the 50:50 molasses and crude glycerol mixture 4. SUP5 = 5 lb/d of the 50:50 molasses and crude glycerol mixture On d 0 steers were housed in individual pens at the FEF and had ad libitum access to water and Tifton 85 bermudagrass hay, which was chopped using a Tub Grinder (Haybuster, Jamestown, ND), placed in the feed bunk and sampled for nutrient analysis and further use in the in vitro experiment. The amount of liquid supplement corresponding to each treatment was weighed and offered daily to each animal in a plastic container inside the pen, separately from hay. Any unconsumed supplement was weighed and recorded for the first 7 d. By d 8, all steers were consuming the entire amount of liquid supplement daily, thus no orts recording was necessary. Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, Alberta, Canada) to record hay intake by weight change measured to the nearest gram. Ruminal fluid and blood were collected before liquid feeding (0 h) and every 3 h postfeeding for 24 h. Ruminal fluid was strained from a representative sample of digesta through four layers of cheesecloth and ph was immediately measured using a manual ph meter (Corning Pinnacle M530, Corning Inc., Corning, NY). A 10-mL sample was taken and 0.1 ml of a 20% H 2 SO 4 solution was added to stop fermentation. Ruminal fluid samples were stored frozen at -20 C for further analysis. Blood samples were collected from jugular venipuncture in 10-mL evacuated tubes containing sodium heparin, placed on ice following collection, and centrifuged for 15 min at 4,000 g at 4 C. After centrifugation, plasma was transferred

3 into polypropylene vials (12 mm 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at -20 C for further analysis. Data were analyzed as a duplicated 4 4 Latin square with repeated measures using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The model included the fixed effects of treatment, time, the treatment time interactions, square, period within square, and animal within square. Animal within period was the subject and the covariance structure used for all the parameters was unstructured, with the exception for total VFA, which was analyzed using compound symmetry. Unstructured and compound symmetry were the best covariance structures based upon the smallest Akaike Information Criterion (AIC) values. Orthogonal polynomial contrasts were conducted to determine the linear, quadratic, and cubic effects of supplementation level on animal performance or nutrient digestibility. Differences between treatment means were identified by Tukey s least squares means comparison and significance was declared at P<0.05 and tendencies considered when 0.05<P<0.10. Results Chemical composition of Tifton 85 bermudagrass hay and liquid mixture fed to heifers is presented in Table 1. Ruminal fermentation parameters and BUN are presented in Table 2. Concentrations of NH 3 -N and BUN were influenced by liquid supplementation, decreasing linearly (P<0.001) as the amount of supplementation increased. Average ruminal ph was also influenced by liquid supplementation and decreased linearly (P=0.03) as the amount of liquid supplement increased. A treatment time interaction (P=0.005) was observed for ruminal ph and is presented in Figure 2. The greatest drop in ruminal ph was observed at 3 h after supplementation for steers on SUP5 (from 6.81 at 0 h to 6.32 at 3 h postfeeding), followed by steers on SUP3 (from 6.81 at 0 h to 6.58). Ruminal VFA profile is presented in Table 3. Molar proportions of acetate (P<0.001) and branched chain VFA (BCVFA; P<0.001) linearly decreased whereas molar proportions of propionate (P<0.001), butyrate (P=0.007), and valerate (P=0.002) linearly increased as the amount of liquid being supplemented increased. Total VFA concentration was affected cubically (P=0.005) by liquid supplementation where it decreased from CTRL to SUP1 and SUP5, however, SUP3 was not different when compared to the others. There was an effect of liquid supplementation on A:P where it decreased linearly (P=0.004) as the amount of liquid supplemented increased which reflects the decrease in acetate and increase in propionate molar proportions. Liquid supplementation is clearly shifting VFA production in the rumen towards more propionate and butyrate production at the expense of acetate. Although ruminal ph was reduced with liquid supplementation in the current study, even with the greatest amount of supplementation (SUP5), ph never dropped below 6.0, which is the threshold known to affect fiber digestion. Thus, we conclude that providing the 50:50 liquid mixture of molasses and crude glycerol to beef steers consuming Tifton 85 bermudagrass hay might be beneficial to improve animal performance due to increase in propionate production.

4 Table 1. Analyzed 1 chemical composition of Tifton 85 bermudagrass hay and liquid supplement fed to beef steers. Item Hay Liquid Supplement 2 Dry matter (DM), % Organic matter, % DM Crude protein, % DM Neutral detergent fiber, % DM Acid detergent fiber, % DM Total digestible nutrients, % DM Calcium, % DM Phosphorus, % DM Magnesium, % DM Potassium, % DM Sodium, % DM Sulfur, % DM Methanol, ppm 3 - <100 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 2 50:50 mixture (as-fed) of molasses:crude glycerol (Westway Feed Products, New Orleans, LA.) 3 Analyzed by SDK Laboratories (Hutchinson, KS). Table 2. Effects of supplementing increasing levels of a 50:50 mixture of molasses:crude glycerol on daily average ruminal fermentation parameters and blood urea nitrogen (BUN) of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment 1 Item 2 CTRL SUP1 SUP3 SUP5 SEM 3 Contrast 4 NH 3 -N, mm L BUN, mg/dl L Ruminal ph L 1 All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTR: no supplementation; SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture. 2 Ruminal fluid and blood samples were collected every 3 h for 24 h. 3 SE of treatment means, n = 8 steers/treatment. 4 Orthogonal polynomial contrast: L = Linear effect of liquid feed supplementation amounts, P 0.05

5 ph Table 3. Effects of supplementing increasing amounts of a 50:50 mixture of molasses:crude glycerol on ruminal volatile fatty acid (VFA) profile of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment 1 Item CTRL SUP1 SUP3 SUP5 SEM 2 Contrast 3 VFA, mol/100 mol Acetate L Propionate L Butyrate L BCVFA L Valerate L Caproate NS Total VFA, mm C A:P L 1 All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTR: no supplementation; SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture. 2 SE of treatment means, n = 8 steers/treatment 3 Orthogonal polynomial contrasts: L or C = Linear or cubic effect of liquid feed supplementation amounts, P 0.05; NS = not significant, P> BCVFA = Branched chain VFAs: isobutyrate + isovalerate + 2 methylbutyrate CTRL SUP1 SUP3 SUP Time postfeeding, h Figure 2. Effects of supplementing increasing amounts of a 50:50 mixture of molasses:crude glycerol on ruminal ph of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment time interaction observed (P=0.005). All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTRL: no supplementation SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: + 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture (n=8 steers/treatment).