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Feeding Value of Wheat-Based Thin Stillage: In Vitro Protein Degradability and Effects on Ruminal Fermentation P. Iwanchysko*, J. J. McKinnon*,1, A. F. Mustafa*, D. A. Christensen*, and D. McCartney† *Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, S7N 5B5 and †Agriculture and Agri-Food Canada, Lacombe, AB ABSTRACT: Two experiments were conducted to evaluate the nutritive value of wheat-based thin stillage as a fluid source for ruminants. In vitro CP degradability of thin stillage was estimated relative to canola meal and heated canola meal in a completely randomized design. Four ruminally cannulated steers were used in a double cross-over design to determine the effects of consuming thin stillage or water as drinking sources on ruminal fermentation traits. The in vitro CP degradability of thin stillage (55.4%) was lower (P < .05) than that of canola meal (59.4%) and higher than that of heated canola meal (31.6%). Ruminal pH for steers consuming thin stillage was higher (P < .05) at 1000 and 1100 and lower (P < .05) at 1900 and 2000 than that for steers consuming water. Total VFA followed a pattern that was the reverse of that reported for pH. Ruminal NH3 N levels were higher (P < .05) for steers fed thin stillage than for water-fed steers through most of the collection period. Ruminal fluid and particulate matter passage rates were not affected by treatment and averaged .165 and .06 /h, respectively. The amount of thin stillage and water that did not equilibrate with the ruminal fluid and, thus, was considered to bypass the rumen was estimated to be 51.9 and 59.2% of total fluid consumed, respectively. Feeding wheat-based thin stillage had no adverse effects on ruminal metabolism. Key Words: Wheat, Ethanol, Rumen Metabolism 1999 American Society of Animal Science. All rights reserved. Introduction Thin stillage from corn-based ethanol production been shown to be an excellent source of nutrients for growing and finishing cattle (Aines et al., 1987; Ham et al., 1994). Wheat-based thin stillage was similarly shown to enhance growth of cattle grazing crested wheatgrass pastures (Ojowi et al., 1996). In addition to its nutrient profile, thin stillage may influence performance by altering the site of nutrient digestion. Fisher (1995) found that thin stillage fed as a fluid source improved total tract digestibility of DM, CP, and NDF of the total mixed diet. Ham et al. (1994) found similar improvements in digestibility when thin stillage was infused into the rumen and indicated that rumen pH and acetate:propionate ratio were reduced. Other rumen traits that may be affected by thin stillage feeding are ruminal fluid osmolality and ammonia N levels as well as fluid and particulate matter passage rates. In addition, commercial practices such as high grain feeding that lead to an increase in ruminal acid production can decrease the efficiency of microbial protein synthe- 1 To whom correspondence should be addressed. Received November 12, 1998. Accepted April 6, 1999. J. Anim. Sci. 1999. 77:2817–2823 sis (Sniffen et al., 1992). This pattern could be compounded by the low pH (i.e., 4) of thin stillage (Fisher, 1995). Larson et al. (1993) has shown that up to 50% of the fluid from thin stillage bypassed the rumen. Rumen bypass may result from inadequate mixing or activation of the esophageal-groove reflex in growing cattle (Ørskov, 1992). With respect to DM that is available for ruminal bacterial fermentation, little information exists on the degradability characteristics of thin stillage nutrients, particularly protein. Our objectives for this study were to define the chemical and in vitro degradability characteristics of thin stillage protein derived from wheat-based ethanol production and to determine the effects of consuming thin stillage as a fluid source on selected ruminal fermentation traits. A final objective was to measure the extent that thin stillage bypasses ruminal fermentation. Materials and Methods Composition and In Vitro Degradability of Thin Stillage Protein. Thin stillage samples (n = 10) were collected on a weekly basis from Pound-Maker Agventures Ltd., at Lanigan, Saskatchewan and analyzed for moisture, ash, ether extract, Kjeldahl nitrogen, ADF (AOAC, 1990), and NDF (Van Soest et al., 1991). Soluble 2817 2818 IWANCHYSKO ET AL. CP and nonprotein nitrogen content were determined according to the procedures of Licitra et al. (1996). Variability in CP degradability of the 10 thin stillage samples was determined by incubating duplicate samples (.2 g air-dry CP) in 10 mL of fresh protease enzyme (protease type XIV from Streptomyces griseus; Sigma Chemical Co., St. Louis, MO) at 39°C for 18 h (Roe et al., 1990). The insoluble residues were then filtered through Whatman no. 54 filter paper and residual N was determined using the Kjeldahl method (AOAC, 1990). To estimate rumen CP kinetic parameters and effective degradability of thin stillage, a composite sample was compiled by taking 20 g of each of the 10 samples. For comparison purposes, two additional protein sources were included: canola meal and heated (125°C for 10 min) canola meal. Canola meal is considered a good source of ruminally degraded protein (Mustafa et al., 1996), and heated canola meal is considered a good source of ruminally undegraded protein (McKinnon et al., 1995). The protein sources were ground through a 1-mm screen using a Retsch grinder. Duplicate samples of each protein source containing the equivalent of .2 g air-dry CP of thin stillage, canola meal, and heated canola meal were incubated in protease enzyme solution for 1, 2, 4, 6, 8, 12, 18, and 24 h as described previously. In vitro CP disappearance data were fitted to the equation of Ørskov and McDonald (1979): P = a + b × (1 − e−ct), where P is CP disappearance at t time, a is the soluble CP fraction (%), b is the slowly degradable CP fraction (%), and c is the rate of degradation (per hour) of the b fraction. The constants a, b, and c were estimated using an iterative least squares method applying the nonlinear regression analysis of SAS (1989). Effective degradability of CP (EDCP) was estimated according to the equation of Ørskov and McDonald (1979): EDCP = a + [(b × c)/(c + k)], where k is the ruminal outflow rate (.05/h), and a, b, and c are as defined above. Rumen Metabolism Study Animals and Dietary Treatments. Four ruminally cannulated steers (381 ± 5.1 kg) were used in a double cross-over design (Cochran and Cox, 1959) consisting of four 28-d periods. The animals were housed in individual pens (3.6 × 3.6 m) in the livestock research barn of the Department of Animal and Poultry Science at the University of Saskatchewan. The temperature was kept at approximately 18°C. Animals were cared for and procedures carried out according to the guidelines of the Canadian Council of Animal Care. Dietary treatments included thin stillage from wheat-based ethanol production or water as fluid sources. The thin stillage was obtained weekly from the same source and stored in a 2,000-L plastic container equipped with a recirculating pump. The fluid treatments were offered to cattle in 60-L containers. Fluid consumption was monitored daily by the use of a calibrated meter ruler. Residual fluid levels were measured Table 1. Ingredient and chemical composition of diet used in the metabolism and the rumen bypass trials Item % DM basis Ingredient composition, % DM basis Barley silage Alfalfa/brome hay Barley Tallow Limestone Mineral salta Vitamin premixb 33.7 15.4 49.1 .31 .46 .51 .65 Chemical composition, % DM basis CP Ash Ether extract NDF ADF 11.7 7.4 4.3 44.8 23.3 a 37.8% Na, 10,000 mg/kg Zn, 75 mg/kg I, 4,050 mg/kg Fe, 6,500 mg/kg Mn, 4,000 mg/kg Cu, and 30 mg/kg Co per kg of premix. b 440,000 IU vitamin A and 89,000 IU vitamin D per kg of premix. each morning prior to and following the filling of the containers. The cattle were fed a basal diet of 51% barley grain concentrate, 34% barley silage, and 15% alfalfa/brome hay (DM basis, Table 1) at 0900 and 1600 daily. During each period, the cattle had ad libitum access to the fluids and feed for the first 2 wk, but they were restricted to 90% of ad libitum intake during the 7 d prior to the collection period. Rumen Sampling Passage Rates. On d 22 of each period, each steer was dosed intraruminally with .5 g of Co-EDTA in 50 mL of distilled water and 100 g of chromium-mordanted hay at 3 h prior to feeding following procedures outlined by Uden et al. (1980). Ruminal contents were manually emptied, thoroughly hand-mixed with the markers, and returned to the rumen. During evacuation, ruminal contents were placed in plastic 20-L pails, covered with aluminum foil, immersed in a water bath at 39°C, and maintained under carbon dioxide. Ruminal fluid was sampled via the ruminal cannula from four sampling sites hourly for a total of 15 h after dosing. Following removal from the rumen, the fluid was squeezed through four layers of cheesecloth. Rumen pH was measured immediately using a Fisher pH meter (Model 825 MP; Fisher Scientific, Pittsburgh, PA). Aliquots of the filtrate (100 mL) were also acidified (1 mL of 50% H2SO4) and frozen for VFA and NH3 N analysis. Ruminal fluid samples (100 mL) for osmolality measurements were not acidified. Ruminal particulate and rectal fecal grab samples were collected 3, 6, 9, 12, 15, 18, 24, 27, 31, 34, 37, 41, 44, 47, 51, 54, 57, 60, 63, 69, and 73 h after dosing with the chromium-mordanted hay. Samples were mixed, covered, and frozen in aluminum foil containers. Ruminal fluid, particulate, and fecal samples were collected at 0500 for determination of baseline cobalt and chromium levels. 2819 THIN STILLAGE AND RUMEN METABOLISM Rumen Bypass Study. The protocol to determine the extent to which thin stillage or water bypasses the rumen was based on that of Larson et al. (1993). On d 27 of each period, the animals were restricted from drinking for 12 h. The animals were given access for 15 min to 53 L of their respective fluid treatment marked with Co-EDTA (.06 g/L). The fluid source was then withdrawn, and the rumen of each animal was manually emptied. Rumen evacuation was completed within 25 min. Ruminal particulate matter was separated from the fluid by squeezing through four layers of cheesecloth. Ruminal fluid volume was estimated from the volume of the separated ruminal fluid. Ruminal particulate matter and fluid were then weighed, subsampled, mixed together, and returned to the rumen. Subsamples were placed in plastic containers and frozen. Refused fluid marked with Co-EDTA was measured, subsampled, and frozen. This procedure was carried out for each animal in each of the four periods. Laboratory Analyses. Ruminal fluid samples were thawed at room temperature and centrifuged at 10,000 × g for 10 min. Ruminal ammonia concentration was determined using a Cole Parmer (Vernon Hills, IL) ammonium electrode (Model No. 27502-02). For VFA analysis, .2 mL of 25% metaphosphoric acid and .3 mL of 1% crotonic acid (internal standard) were added per milliliter of ruminal fluid. The samples were allowed to stand for 10 min and then centrifuged (14,000 × g for 12 min), and the supernatant was used for VFA analysis. Concentrations of VFA were determined with gas chromatography (Model 5790A; Hewlett Packard, Avondale, PA) with a Poropak Q column (Poropak, Framingham, PA). The oven temperature was 210°C, and the injector and detector block temperature was 220°C. Run time was 13 min. Cobalt concentration was determined with atomic absorption spectroscopy (Model 2380; Perkin-Elmer, Foster City, CA) on supernatant from ruminal fluid samples that were centrifuged at 3,000 × g for 10 min. Ruminal particulate matter and fecal samples were thawed at room temperature, dried at 60°C for 3 d, ground through a 1-mm screen, and analyzed for chromium concentration with atomic absorption spectroscopy (Model 2380, Perkin-Elmer) following nitric/perchloric acid digestion. Ruminal fluid osmolality was measured on supernatant from thawed, nonacidified ruminal fluid samples using a Westcor (Logan, UT) 5100C vapor pressure osmometer. Drinking fluids and ruminal contents collected during the rumen bypass study were analyzed for Co as described for ruminal fluid. The percentage of thin stillage or water that bypassed the rumen was calculated using the following equation (Larson et al., 1993): 100 − [(mg of Co measured in the rumen/mg of Co consumed) × 100]. Feed samples collected during the metabolism trial were analyzed for moisture, ash, ether extract, Kjeldahl N, ADF, and NDF as previously described. Table 2. Chemical composition of thin stillage (n = 10) from wheat-based ethanol production Item DM Ash Ether extract ADF NDF CP Soluble protein, % of CP Nonprotein nitrogen, % of CP In vitro protein degradability, % of CP Level, % DM basis SD 6.3 9.4 6.9 2.0 38.4 46.6 19.9 1.0 1.1 1.1 .2 5.9 4.9 2.09 18.7 1.44 65.1 3.8 Passage Rate Calculations and Statistical Analysis. Ruminal fluid and particulate matter passage rates were calculated from the disappearance curves of cobalt and chromium, respectively. Fecal passage rate was calculated from the disappearance curve of chromium in fecal grab samples. Disappearance curves of markers were fitted to gamma age-dependent and age-independent two-compartment models (Pond and Ellis, 1988) using the NLIN procedure of SAS (1989) as described by Moore et al (1992). The order of gamma age dependency was two for cobalt and three for chromium. The two orders were selected because of differences in the degree of right skewness of the disappearance curves of markers; that degree was less pronounced for curves associated with particle markers than for curves associated with fluid markers (Luginbuhl et al., 1994). The data were analyzed using analysis of variance techniques for a double cross-over design including repeated measures analysis for ruminal fluid measurements using the GLM procedure of SAS (1989). Data from the in vitro CP degradability incubations were analyzed as a completely randomized design (three treatments and three replicates). Means separation was carried out where necessary (P < .05) using the Student-Newman-Keul procedure (Steel and Torrie, 1980). Results and Discussion Chemical Composition of Thin Stillage. The primary fermentation substrate used for ethanol production in this trial was wheat, which made up 60 to 91% of the feedstock for fermentation. The remaining feedstock consisted of varying proportions of triticale, corn, rye, and barley. Despite this variation in substrate composition, the nutrient profile of the thin stillage samples was relatively similar throughout the trial (Table 2). Furthermore the ash, ether extract, CP, NDF, and ADF values reported in the present study for thin stillage were similar to values reported for thin stillage derived from 100% wheat (Ojowi et al., 1996). The average CP and NDF values reported in this study for wheat-based thin stillage were higher than those of corn-based thin stillage as reported by Larson et al. (1993) and Ham et 2820 IWANCHYSKO ET AL. Table 3. In vitro CP kinetic-parameters and effective degradability of thin stillage relative to canola meal and heated canola meal Protein source Item Soluble fraction, % of CP Slowly degradable, % of CP Degradation rate, /h Effective degradability, % of CP Thin stillage Canola meal Heated canola meal 20.4c 39.9b 15.7d .18 46.9 39.3 44.5 3.13 .147b 55.4c .049c 59.4b .029 d 31.6d SEMa .005 .61 a Pooled standard error of the mean. Means within each row with different superscripts are different (P < .05). b,c,d al. (1994). Soluble CP and NPN of thin stillage averaged 19.9 and 18.7% of total CP, respectively. These results indicate that most of the soluble CP in thin stillage is in the form of nonprotein nitrogen. In Vitro CP Degradability of Thin Stillage. The extremely fine particle size of dried thin stillage made it difficult to estimate ruminal CP degradability using the nylon bag technique. Other researchers have used in vitro procedures based on protease enzymes to estimate CP degradability of different protein supplements (Assoumani et al., 1992; Susmel et al., 1993). The average in vitro CP disappearance of the 10 thin stillage samples was 65.1 ± 3.8% (Table 1). Due to the small differences between the thin stillage samples in terms of chemical composition and in vitro CP disappearance, the samples were pooled to form a composite sample to estimate CP kinetic parameters and effective degradability of thin stillage relative to canola meal and heated canola meal. The rapidly soluble (fraction a) CP content of thin stillage was lower (P < .05) than that of canola meal but higher (P < .05) than that of heated canola meal (Table 3). Canola meal has been shown to have high soluble CP (Mustafa et al., 1996), and heating is known to reduce this protein fraction (Mustafa et al., 1997). Although not significantly different, the slowly degradable CP (fraction b) followed the order thin stillage > heated canola meal > canola meal. The rate of degradation of fraction b was highest (P < .05) for thin stillage, intermediate for canola meal, and lowest (P < .05) for heated canola meal. The results for canola meal and heated canola meal are in agreement with those found by Mustafa et al. (1997). The effective CP degradability (Table 3) of the thin stillage was higher (P < .05) than that of heated canola meal but lower (P < .05) than that of canola meal. The relatively high effective CP degradability of thin stillage CP is due to its high degradation rate (Table 3). Because the effective CP degradability of thin stillage is considerably closer to that of canola meal, one would classify thin stillage as a relatively degradable protein source for ruminants. The kinetics of thin stillage are somewhat different from those of most proteins. Usually proteins that are insoluble are degraded rather slowly (Mustafa et al., 1997). Thin stillage protein is relatively insoluble but has a rapid rate of degradation. This would explain the increased NH3 N levels found with thin stillage feeding (Figure 1). However, if thin stillage has a short residence time in the rumen, the insoluble nature of this protein could result in a considerable portion of thin stillage DM bypassing the rumen for absorption in the small intestine. Fluid Intake, Rumen pH, VFA, Osmolality and NH3 N Levels. Total fluid intake was 31.5 ± 8.4 L and 33.5 ± 6.4 L (P > .05) for water and thin stillage treatments, respectively. Rumen pH averaged 6.3 ± .05 throughout the day with no effect of treatment. There was a time effect: pH dropped (P < .01) throughout the day (Figure 1). A significant time × treatment interaction indicates that, when thin stillage was offered as the fluid source, ruminal fluid pH tended to be lower during the first 12 h following feeding, with significant differences (P < .05) noted at 1000 and 1100 (Figure 1). Thin stillage in the present study had a pH of 3.8 and as such would be expected to stress the rumen buffer systems when consumed. Differences (P < .05) were also noted at 1900 and 2000; however, the effects were reversed, and the thin stillage treatment resulted in a higher pH. These differences may be because, during the collection period, the animals had consumed all of the stillage by this time each day, and the effect of thin stillage on rumen pH was no longer evident. Total VFA concentrations followed an inverse pattern to that of rumen pH, with levels increasing (P < .01) over time. Even though no treatment effects were observed when the values were averaged over the course of the day, a time × treatment interaction indicated higher VFA levels at 0800, 1100, and 1400 in thin stillage-fed animals and at 2000 in water-fed animals (Figure 1). The negative inverse relationship between rumen pH and total VFA concentrations is well documented (Burrin and Britton, 1986; Lana et al., 1998). No differences were noted in the molar proportions of individual VFA or in the acetate:propionate ratio (data not shown). Weigand et al. (1974) noted that dietary regimen may greatly influence molar proportions and ratios of VFA as well as alter pH in the rumen of mature animals. Therefore, with the relatively minor differences shown in rumen pH, no differences would be expected in the proportions of acetate and propionate. Rumen osmolality was not effected by treatment, although there was an increase (P < .01) over time (Figure 1). Ruminal NH3 N concentrations were found to be higher (P < .05) for steers offered thin stillage than for steers offered water throughout most of the collection period (Figure 1). This indicates that thin stillage protein is highly degradable in the rumen, confirming the results of the in vitro protease incubations. Concentra- THIN STILLAGE AND RUMEN METABOLISM tions of NH3 N in this study are similar to those noted by Ham et al. (1994), who reported that animals fed corn by-products such as wet corn gluten feed had a ruminal NH3 N value of 14.27 mg/dL. The ruminal NH3 N values reported in this study are greater than concentrations (2 to 5 mg/dL) considered to be optimal for microbial growth (Satter and Slyter, 1974) and are higher than those reported by Kang-Meznarich and Broderick (1980) for optimal fermentation (3.3 to 8.5 mg/dL). The ruminal NH3 N concentrations found in this study would indicate that all microbial N requirements are being met with thin stillage feeding. Therefore, with a readily available carbohydrate source and supply of appropriate precursors, microbial fermentation and protein synthesis should occur at a maximum rate. Ruminal Fluid and Particulate Matter Passage Rates. Ruminal fluid passage rates were not influenced by treatment, averaging .165/h (Table 3). The values reported in this study for ruminal fluid passage rates are within the range reported in the literature (Goetsch et 2821 al., 1987; Malcolm and Kiesling, 1990; Poore et al., 1990). The lack of any impact of thin stillage on fluid passage rate is somewhat surprising. Ojowi et al. (1996) reported a 43% increase in thin stillage consumption relative to water when consumed under grazing conditions. This large intake of fluid would be expected to increase ruminal fluid turnover. However, as previously reported, no differences in fluid consumption were observed in this study, in which the steers were fed in confinement. This may explain why no effect of thin stillage feeding was seen on fluid passage rates from the rumen. Particulate passage rates were not affected (P > .05) by fluid source (Table 4) and are similar to those reported by others (Okine et al., 1989; Poore et al., 1990; Bruining and Bosch, 1992). Fecal passage rates were also unaffected (P > .05) by dietary treatment (Table 4). The passage rate of particulate material from the rumen was similar to that of the fecal matter. Bypass Value of Thin Stillage. Within the 15-min period, steers consumed 17.1 ± .7 L and 26.2 ± 1.0 L of the 53 L of Co-EDTA marked thin stillage and water, Figure 1. Effects of thin stillage and water as fluid source on ruminal metabolism. *Means (± SD) are different (P < .05). 2822 IWANCHYSKO ET AL. Table 4. Effects of fluid source on ruminal fluid and particulate matter and total tract passage rates Fluid source Passage rate, /h Ruminal fluid Ruminal particulate matter Fecal Thin stillage Water SEMa .169 .055 .058 .161 .061 .062 .013 .004 .005 a Pooled standard error of the mean. respectively. Dietary treatment had no effect on rumen bypass value. The estimated rumen bypass value for water and fluid in thin stillage was 59.2 ± 22.9 and 51.9 ± 19.6%, respectively. Similar results were found by Larson et al. (1993), who reported that over 50% of the corn-based thin stillage consumed had bypassed ruminal fermentation. Similarly, Woodford et al. (1984) reported that a high proportion of water bypassed the rumen. These workers stated that not all of the drinking fluids should be assumed to equilibrate with the ruminal fluid. Others have also determined that a portion of the liquid ingested by drinking bypasses the rumen (Garza and Owens, 1989; Zorrilla-Rios et al., 1990). Bypass could occur by failure of thin stillage to equilibrate with rumen contents or via closure of the esophageal groove, which has been shown to function in certain circumstances in young adults (Mikhail et al., 1988; Ørskov, 1992). These results and those of Larson et al. (1993) suggest that, despite considerable variations in rumen bypass values, a considerable proportion of thin stillage bypasses the rumen and is available for digestive processes in the lower gastrointestinal tract. Implications Feeding wheat-based thin stillage as a fluid source does not seem to adversely affect rumen metabolism. Approximately 52% of thin stillage may bypass the rumen and, thus, become available for intestinal enzymatic digestion. However, 48% of thin stillage consumed should enter the rumen and can be considered as a good source of ruminal degradable protein. 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