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Total tract nutrient digestion of steers consuming diets containing ammonium polyacrylate
Bioresource Technology 73 (2000) 81±85
Short communication
Total tract nutrient digestion of steers consuming diets containing ammonium polyacrylate K.G. Maciorowskia,*, M.J. Cecavab, A.L. Suttonc, J.A. Pattersonc b
a Department of Poultry Science, Texas A & M University, College Station, TX 77843-2472, USA Consolidated Nutrition, Research and Technology Center, P.O. Box 2508, Ft. Wayne, IN 46801, USA c Department of Animal Science, Purdue University, West Lafayette, IN 47906, USA
Received 18 April 1997; received in revised form 23 September 1999; accepted 30 September 1999
Abstract One of the potential factors that may limit the ruminant animalÕs ability to digest ®ber is the reduced retention and subsequent passage of cellulolytic microorganisms from the rumen. Bacterial passage may be decreased by an inert support. Ammonium polyacrylate is a hygroscopic organic polymer that is non-toxic to animals and environmentally degradable. A digestibility trial was conducted with six Holstein steers cannulated at ruminal, duodenal and ileal sites to determine if an ammonium polyacrylate gel (GEL: 20 g ammonium polyacrylate hydrated in 2 l tap water containing 20 g glucose animalÿ1 day ÿ1 ) would increase total tract nutrient digestion. Intake and digestion of organic matter were not aected p > 0:10 by GEL but NDF digestibility was decreased p < 0:06 when GEL was consumed. Although GEL appeared to be present in the rumen samples, higher concentrations may be needed to elicit a detectable in vivo response. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Ammonium polyacrylate; Steers; Fiber; Cellulolysis; Support
1. Introduction Polymers containing acrylamide and/or acrylate have been shown to be non-toxic to animals and humans and resistant to microbial attack (Seybold, 1994). The polymers have been investigated as soil additives to decrease erosion (Seybold, 1994), gelling agents in insect diets (Honda et al., 1996), coagulants for protein recovery in wastewater (Guerrero et al., 1998) and as promoters of fungal growth in vitro through the prevention of spore aggregation (Wainwright et al., 1993). Cellulolytic bacteria, fungi and protozoa in the rumen are sensitive to high ruminal passage rates, washout of ®ber containing attached microorganisms (Latham et al., 1979; Akin and Barton, 1983; Morris and Cole, 1987; Miron et al., 1989; Bhat et al., 1990) and decreases in ruminal pH below 6.0 (Smith et al., 1973; Mould and érskov, 1983; Hoover, 1986). A gel consisting of hydrated ammonium polyacrylate (GEL: 20 g ammonium polyacrylate hydrated in 2 l of tap water containing 20 g glucose) does not contain the neurotoxin acrylamide (Seybold, 1994) and may serve as an inert support to *
Corresponding author. Tel.: +409-845-3406; fax: +409-845-1921. E-mail address: [email protected] (K.G. Maciorowski).
trap cellulolytic bacteria within the rumen when dilution rates exceed growth rates (Haggstr om, 1983) or when ®ber levels are low (Prigge et al., 1990). If a decrease in cellulolytic numbers could result in a substantial lag time between ®ber intake and the onset of ®ber degradation on a particular diet, increased cellulolytic populations could colonize ingested feed more rapidly, decrease the time before the initiation of cellulolysis and increase overall ®ber digestion. GEL may also form a diusion barrier against a sudden decrease in pH in a method suggested by Haggstr om (1983) and may bind free hydrogen ions around the GEL with ammonium ions. As polyacrylamide has been shown to serve as the sole N source for soil bacteria (Kay-Shoemake et al., 1998), GEL may also provide a nitrogen source to attached bacteria. The objective of this experiment was to determine if the concentration of GEL increased the total tract nutrient digestion of steers. 2. Methods 2.1. Animals and diets Six Holstein steers with an average weight of 475 kg and ®tted with ruminal, duodenal and ileal cannulas
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 8 - 8
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K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
according to procedures approved by the Purdue Animal Care and Use Committee were used in a modi®ed switchback design with three periods and two treatments (=ÿGEL). The animals were individually housed in free stalls at the Purdue University Beef Cattle Research Center and were allowed a full range of movement as well as continuous lighting, ventilation, trace mineral salt blocks and water. The steers consumed a diet consisting of 50% roughage and 50% concentrate on a dry matter basis and containing between 14.1% and 14.5% crude protein (Table 1). Mineral levels were within recommended NRC levels for 500 kg steers (NRC, 1984). 2.2. Feeding regimen Periods were 21 days long and consisted of 17 days for adjustment and 4 days for sample collection. Steers were oered diets at 0730 and 1930 and unconsumed feed was measured before the morning feeding. If unconsumed feed exceeded approximately 1.5 kg dayÿ1 during the adjustment period, the respective steerÕs feed was incrementally reduced by 0.1% or 0.2% units of body weight until feed refusals were less than 1.5 kg dayÿ1 . When feed refusals were consistently less than 1.5 kg dayÿ1 , the steerÕs daily ration was considered to be that steerÕs ad libitum intake. During collection periods, steers were oered rations at 90% of each steerÕs ad libitum intake. To form approximately 2 l of GEL, 20 g of ammonium polyacrylate was added to 2 l of tap water containing 20 g of glucose and allowed to hydrate overnight at 4°C. The morning ration of each treatment animal was top-dressed with approximately 2 l of GEL. The morning ration of each control animal was topdressed with 20 g of glucose (Sigma, St. Louis, MO). All diets were then manually mixed to insure even distri-
bution before consumption. In most cases, the animals consumed feed containing GEL or glucose within 1 h of feeding. 2.3. Feed and intestinal sampling During sampling periods, feed refusals and each feed component were sampled daily, composited by period and stored at 4°C until processed. Every 4 h, 300 ml of duodenal and 200 ml of ileal digesta were sampled and composited by steer. Fecal grab samples were taken every 6 h and also composited by animal. The duodenal, ileal and fecal sample collection times were advanced by 1 h each day to adjust for diurnal variation in gastrointestinal ¯ow rates. Samples were stored at )20°C between time of sampling and processing or analysis, unless otherwise noted. 2.4. Ruminal sampling procedures and assays On the fourth day of each sampling period, ruminal contents were sampled at 3, 6, 9 and 12 h after the morning feeding. The samples were obtained from four dierent areas of the reticulorumen using a core sampling device (Firkins et al., 1986) and strained through two layers of cheesecloth for a ®nal sample volume of 150 ml. The pH of each sample was measured immediately using an Orion pH meter (Orion Research, Boston, MA). Eight ml of ruminal ¯uid were added to scintillation vials containing 2 ml of 25% m-phosphoric acid and placed on ice for short chain fatty acid (SCFA) analysis by gas chromatography (Wachenheim and Patterson, 1992). Fifty ml of ruminal ¯uid were poured into bottles containing 3 ml of 6 N HCl, placed on ice and transported to the lab for the determination of
Table 1 Composition of diet fed to steers
a
Ingredients
% of DM
Chemical analysis of complete diet
% of DM
Alfalfa silage Chopped alfalfa hayc Cracked corn Soybean meal (48%) Trace mineralized saltd Dicalcium phosphate Se 200 premixe Vitamin ADEK premixf Vitamin E premixg
40.00 10.00 40.21 8.60 0.50 0.40 0.16 0.10 0.03
Crude proteina;b ADFg NDFg AIAg Calcium Phosphorus Potassium Magnesium Sulfur
14.30 19.30 31.53 0.38 0.75 0.36 1.42 0.21 0.22
Calculated as Kjeldahl N * 6.25. Acid detergent ®ber (ADF), neutral detergent ®ber (NDF) and acid insoluble ash (AIA) were calculated as the average of analyzed values of three periods. c Ground in a tub grinder to give an average particle length of 7 cm. d g/100 g: NaCl, 98; Zn, 0.35; Fe, 0.2; Mn, 0.2; Cu, 0.033; I, 0.007. e 0.2 g Se kgÿ1 premix. f kgÿ1 premix: vitamin A, 10 000 IU; vitamin D3 , 2502 IU; vitamin E, 1102 IU; menadione (vitamin K precursor), 734 mg. g 20 k IU of vitamin E kgÿ1 premix. b
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
ammonia concentration by the method of Chaney and Marbach (1962). 2.5. Exchange of ruminal contents Ruminal contents were transferred among animals of dierent treatments between sampling periods to minimize carryover eects. Feed was withheld on the morning when contents were transferred to lessen total ruminal bulk. Steers of approximately equal weight were paired between treatments and their rumens were evacuated using a wet/dry vacuum (Shop-Vac, Williamsport, PA and Irvine, CA). Ruminal contents were switched within the pairs and reintroduced into the rumen within 15 min of evacuation. 2.6. Intestinal analyses Duodenal and ileal digesta were later thawed and mixed. A homogenous 1000 g wet weight subsample was freeze dried (Unitop 600 L freeze-drier, The Virtis, Gardiner, NY 12525). Feed components, duodenal subsamples and fecal samples were analyzed for dry matter (DM) content and ground through a 1 mm screen (no. 1 Wiley Mill, Arthur H. Thomas, Philadelphia, PA). Organic matter analysis was performed by ashing the samples overnight at 600°C. Acid detergent ®ber content was determined by the method of Van Soest (1963), neutral detergent ®ber by the method of Van Soest et al. (1991) and Kjeldahl-nitrogen content as per AOAC (1984). 2.7. Flow rate determination Capsules containing chromium oxide were placed in the rumen of each steer twice daily (15 g dayÿ1 ) through X-shaped incisions in the ruminal cannulas for the determination of ¯ow rate. Freeze-dried duodenal digesta and fecal samples were analyzed for chromium content by the method of Williams et al. (1962) using a Perkin± Elmer 460 Atomic Absorption Spectrophotometer (Perkin±Elmer, Norwalk, CT). Acid insoluble ash content was determined by the method of Van Keulen and Young (1977). 2.8. Statistical analysis Data were analyzed by ANOVA using the general linear model procedure of SAS for an incomplete switchback design with two treatments (SAS, v. 6.03, Cary, NC, 1986). Sums of squares for the model were separated into steer, period and treatment eects. Analysis of ammonia, pH and SCFA concentrations also included the eect of sampling time and an interaction term to test for time by treatment interaction. Statistical signi®cance was de®ned as a probability level
83
of P < 0.05 and a trend towards a particular eect was de®ned as a probability level of P < 0.10. 3. Results and discussion No signi®cant dierences between steers fed GEL and those fed glucose were detected for intake or digestion of any feed fraction measured, total tract apparent digestibility, duodenal ¯ow, fecal excretion, ruminal pH or ruminal concentrations of ammonia, butyrate or isovalerate (Tables 2 and 3, P > 0:10). Nitrogen ¯ow and digestibility at the duodenum was unreasonably high, probably due to variability in duodenal ¯ow marker concentration. Acetate, propionate, isobutyrate and valerate concentrations signi®cantly changed over time P < 0:05, but did not vary between treatments P > 0:10. A trend towards greater fecal NDF excretion P < 0:06 and a decrease in NDF digestibility P 0:10 was detected. GEL may have inhibited digestion of the NDF fraction of the feed, but the decrease may also be due to diculties in the analysis of NDF in the presence of GEL. The distilled water used in the NDF assay may have hydrated GEL particles and prevented proper ®ltration. Since GEL was not present in control animals, incomplete ®ltration could produce a greater amount of apparent NDF residue and the appearance of decreased feed digestion in samples from treated animals. This eect was also noted in duodenal samples, which could not be analyzed for NDF due to an inability to ®lter the samples. GEL may have been inappropriate as a support for cellulolytic bacteria. Nylon bag studies (data not shown) indicated that a coccobacillus resembling Fibrobacter succinogenes was associated with GEL, but the attached bacteria may have been non-cellulolytic in vivo. The concentration of GEL used in this trial may not have provided sucient support to increase cellulolytic populations or aect rumen fermentation patterns. GEL swollen in tap water shrinks about 10-fold when immersed in a high-mineral solution such as ruminal ¯uid (preliminary data not shown) and may be sensitive to physical breakdown (Seybold, 1994). The diculties in ®ltering the NDF fraction suggest that GEL was present, but as GEL resembled the color of surrounding ruminal ¯uid when immersed in vitro, it was dicult to detect visually. Due to the limited number of cannulated and ®stulated steers available, funding restrictions and the limited time allotted for the study, though, only the concentration of GEL suggested by the manufacturer (Red Sea Construction) was evaluated. GEL may be eective at higher doses or with animals with a greater metabolic demand for energy, such as high producing dairy cattle. Future studies with in vitro continuous fermentors containing mixed ruminal microorganisms could investigate the eect of GEL concentration
84
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
Table 2 Eect of GEL on nutrient digestion in steers GEL ())a
(+)b
SEMc
P-value
Item n 6 DM OM NDFd ADFe N
9240 8600 2920 1800 212
9270 8630 2910 1790 212
440 410 130 80 10
0.97 0.96 0.96 0.93 0.98
Flow at duodenum (g dayÿ1 ) DM OM
4460 3960
3540 3000
410 490
0.14 0.18
Fecal excretion (g dayÿ1 ) DM OM NDF ADF N
2080 1860 1040 590 53
2330 2080 1290 730 60
180 160 80 70 4
0.35 0.39 0.06 0.16 0.25
Nutrients digested (g dayÿ1 ) DM OM
7160 6740
6940 6550
430 400
0.72 0.76
Apparent preduodenal digestibility (% of intake) DM 50.8 OM 53.0
61.5 65.1
4.9 5.4
0.15 0.15
Apparent total tract digestibility (% of intake) DM 77.3 OM 78.9 NDF 64.3 ADF 66.8 N 74.9
74.9 75.9 56.3 60.2 71.8
2.2 2.2 3.0 3.1 2.3
0.46 0.51 0.10 0.16 0.38
a
20 g glucose top dressed at 0730 feeding. Two l of GEL (20 g ammonium polyacrylate added to 2 l tap water containing 20 g glucose and allowed to hydrate overnight at 4°C) top dressed at 0730 feeding. c Standard error of the mean. d Neutral detergent ®ber residue. e Acid detergent ®ber residue. b
Table 3 Eect of GEL on ruminal characteristics of steers Hours post feeding parameter (n 6)
(+GEL)b
SEMc
3
6
9
12
3
6
9
12
NH3 ±N (mg dl ) pH Acetate (mM) Propionate (mM) Isobutyrate (mM) Butyrate (mM) Isovalerate (mM) Valerate (mM)
9.8 6.1 61.6 15.9 1.0 10.4 0.8 1.3
9.4 6.2 58.6 15.4 1.0 10.3 0.8 1.3
7.3 6.3 54.6 13.4 0.9 8.6 0.6 1.0
9.9 6.3 53.7 14.5 0.9 9.4 0.8 1.0
9.4 6.2 62.3 16.2 1.0 10.4 0.8 1.3
9.5 6.2 64.0 16.5 1.1 10.7 0.8 1.3
9.1 6.2 56.6 14.4 0.9 9.4 0.6 1.1
9.3 6.3 55.3 14.9 0.9 9.0 0.8 1.0
0.9 0.1 3.0 0.8 0.1 0.7 0.1 0.1
Total SCFA (mM)
91.0
87.4
79.1
80.3
92.0
93.4
83.0
81.9
4.5
ÿ1
a
()GEL)a
20 g glucose top dressed at 0730 feeding. Two l of GEL (20 g ammonium polyacrylate added to 2 l tap water containing 20 g glucose and allowed to hydrate overnight at 4°C) top dressed at 0730 feeding. c Standard error of the mean. b
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
without having to rely upon expensive and labor intensive in vivo trials. Acknowledgements This work was supported by Purdue University Department of Animal Science Hatch funds and the support of Red Sea Construction The authors would like to thank P. Boccazzi, P.A. Jaynes, D. Mcintyre and R. Pender for technical assistance. References Akin, D.E., Barton II, F.E., 1983. Rumen microbial attachment and degradation of plant cell walls. Fed. Proc. 42, 114±121. Association of Ocial Analytical Chemists, 1984. Ocial Methods of Analysis. 13th ed. Assoc. Oc. Analyt. Chem. Washington, DC. Bhat, S., Wallace, R.J., érskov, E.R., 1990. Adhesion of cellulolytic ruminal bacteria to barley straw. Appl. Environ. Microbiol. 56, 2698±2703. Chaney, A.L., Marbach, E.P., 1962. Modi®ed reagents for determination of urea and ammonia. Clin. Chem. 8, 130±132. Firkins, J.L., Berger, L.L., Merchen, N.R., Fahey Jr., G.C., 1986. Eects of forage particle size, level of feed intake and supplemental protein degradability on microbial protein synthesis and site of nutrient digestion in steers. J. Anim. Sci. 62, 1081±1094. Guerrero, L., Omil, F., Mendez, R., Lema, J.M., 1998. Protein recovery during the overall treatment of wastewaters from ®shmeal factories. Biores. Technol. 63, 221±229. Haggstr om, L., 1983. Fermentations by immobilized strict anaerobes. In: Mattiasson, B. (Ed.), Immobilized Cells and Organelles, vol. 2. CRC Press, Boca Raton, FL, pp. 61±77. Honda, J., Han, M.-W., Leppa, N.C., 1996. Sodium polyacrylamide polyacrylate, a gelling agent in diets for cabbage looper, omnivorous looper and western avocado leafroller. Entomol. Exper. Appl. 81, 175±180. Hoover, W.H., 1986. Chemical factors involved in ruminal ®ber digestion. J. Dairy Sci. 69, 2755±2766. Kay-Shoemake, J.L., Watwood, M.E., Lentz, R.D., Sojka, R.E., 1998. Polyacrylamide as an organic nitrogen source for soil microorgan-
85
isms with potential eects on inorganic soil nitrogen in agricultural soil. Soil Biol. Biochem. 30, 1045±1052. Latham, M.J., Hobbes, D.G., Harris, P.J., 1979. Adhesion of rumen bacteria to alkali-treated plant stems. Ann. Rech. Vet. 10, 244±245. Miron, J., Yokoyama, M.T., Lamed, R., 1989. Bacterial cell surface structures involved in lucerne cell wall degradation by pure cultures of cellulolytic rumen bacteria. Appl. Microbiol. Biotechnol. 32, 218±222. Morris, E.J., Cole, O.J., 1987. Relationship between cellulolytic activity and adhesion to cellulose in Ruminococcus albus. J. Gen. Microbiol. 133, 1023±1032. Mould, F.L., érskov, E.R., 1983. Manipulation of rumen ¯uid pH and its in¯uence on cellulolysis in sacco, dry matter degradation and the rumen micro¯ora of sheep oered either hay or concentrate. Anim. Feed Sci. Technol. 10, 1±14. National Research Council, 1984. Nutrient Requirements of Beef Cattle. sixth revised ed., National Academy Press, Washington DC. Prigge, E.C., Stuthers, B.A., Jacquemet, N.A., 1990. In¯uence of forage diets on ruminal particle size, passage of digesta, feed intake and digestibility by steers. J. Anim. Sci. 68, 4352±4360. Seybold, C.A., 1994. Polyacrylamide review: soil conditioning and environmental fate. Commun. Soil Sci. Plant Anal. 25, 2171±2185. Smith, W.R., Yu, I., Hungate, R.E., 1973. Factors aecting cellulolysis by Ruminococcus albus. J. Bacteriol. 114, 729±737. Van Keulen, J., Young, B.A., 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44, 282±287. Van Soest, P.J., 1963. Use of detergents in the analysis of ®brous feeds. II. A rapid method for the determination of ®ber and lignin. J. Assoc. Oc. Analyt. Chem. 46, 829±835. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary ®ber, neutral detergent ®ber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583±3597. Wachenheim, D.E., Patterson, J.A., 1992. Anaerobic production of extracellular polysaccharide by Butyrivibrio ®brisolvens nyx. Appl. Environ. Microbiol. 58, 385±391. Wainwright, M.P., Trinci, A.P.J., Moore, D., 1993. Aggregation of spores and biomass of Phanerochaete chrysosporium in liquid culture and the eect of anionic polymers on this process. Mycol. Res. 97, 801±806. Williams, C.H., David, D.J., Iismaa, O., 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci. 59, 381±385.
Short communication
Total tract nutrient digestion of steers consuming diets containing ammonium polyacrylate K.G. Maciorowskia,*, M.J. Cecavab, A.L. Suttonc, J.A. Pattersonc b
a Department of Poultry Science, Texas A & M University, College Station, TX 77843-2472, USA Consolidated Nutrition, Research and Technology Center, P.O. Box 2508, Ft. Wayne, IN 46801, USA c Department of Animal Science, Purdue University, West Lafayette, IN 47906, USA
Received 18 April 1997; received in revised form 23 September 1999; accepted 30 September 1999
Abstract One of the potential factors that may limit the ruminant animalÕs ability to digest ®ber is the reduced retention and subsequent passage of cellulolytic microorganisms from the rumen. Bacterial passage may be decreased by an inert support. Ammonium polyacrylate is a hygroscopic organic polymer that is non-toxic to animals and environmentally degradable. A digestibility trial was conducted with six Holstein steers cannulated at ruminal, duodenal and ileal sites to determine if an ammonium polyacrylate gel (GEL: 20 g ammonium polyacrylate hydrated in 2 l tap water containing 20 g glucose animalÿ1 day ÿ1 ) would increase total tract nutrient digestion. Intake and digestion of organic matter were not aected p > 0:10 by GEL but NDF digestibility was decreased p < 0:06 when GEL was consumed. Although GEL appeared to be present in the rumen samples, higher concentrations may be needed to elicit a detectable in vivo response. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Ammonium polyacrylate; Steers; Fiber; Cellulolysis; Support
1. Introduction Polymers containing acrylamide and/or acrylate have been shown to be non-toxic to animals and humans and resistant to microbial attack (Seybold, 1994). The polymers have been investigated as soil additives to decrease erosion (Seybold, 1994), gelling agents in insect diets (Honda et al., 1996), coagulants for protein recovery in wastewater (Guerrero et al., 1998) and as promoters of fungal growth in vitro through the prevention of spore aggregation (Wainwright et al., 1993). Cellulolytic bacteria, fungi and protozoa in the rumen are sensitive to high ruminal passage rates, washout of ®ber containing attached microorganisms (Latham et al., 1979; Akin and Barton, 1983; Morris and Cole, 1987; Miron et al., 1989; Bhat et al., 1990) and decreases in ruminal pH below 6.0 (Smith et al., 1973; Mould and érskov, 1983; Hoover, 1986). A gel consisting of hydrated ammonium polyacrylate (GEL: 20 g ammonium polyacrylate hydrated in 2 l of tap water containing 20 g glucose) does not contain the neurotoxin acrylamide (Seybold, 1994) and may serve as an inert support to *
Corresponding author. Tel.: +409-845-3406; fax: +409-845-1921. E-mail address: [email protected] (K.G. Maciorowski).
trap cellulolytic bacteria within the rumen when dilution rates exceed growth rates (Haggstr om, 1983) or when ®ber levels are low (Prigge et al., 1990). If a decrease in cellulolytic numbers could result in a substantial lag time between ®ber intake and the onset of ®ber degradation on a particular diet, increased cellulolytic populations could colonize ingested feed more rapidly, decrease the time before the initiation of cellulolysis and increase overall ®ber digestion. GEL may also form a diusion barrier against a sudden decrease in pH in a method suggested by Haggstr om (1983) and may bind free hydrogen ions around the GEL with ammonium ions. As polyacrylamide has been shown to serve as the sole N source for soil bacteria (Kay-Shoemake et al., 1998), GEL may also provide a nitrogen source to attached bacteria. The objective of this experiment was to determine if the concentration of GEL increased the total tract nutrient digestion of steers. 2. Methods 2.1. Animals and diets Six Holstein steers with an average weight of 475 kg and ®tted with ruminal, duodenal and ileal cannulas
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 8 - 8
82
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
according to procedures approved by the Purdue Animal Care and Use Committee were used in a modi®ed switchback design with three periods and two treatments (=ÿGEL). The animals were individually housed in free stalls at the Purdue University Beef Cattle Research Center and were allowed a full range of movement as well as continuous lighting, ventilation, trace mineral salt blocks and water. The steers consumed a diet consisting of 50% roughage and 50% concentrate on a dry matter basis and containing between 14.1% and 14.5% crude protein (Table 1). Mineral levels were within recommended NRC levels for 500 kg steers (NRC, 1984). 2.2. Feeding regimen Periods were 21 days long and consisted of 17 days for adjustment and 4 days for sample collection. Steers were oered diets at 0730 and 1930 and unconsumed feed was measured before the morning feeding. If unconsumed feed exceeded approximately 1.5 kg dayÿ1 during the adjustment period, the respective steerÕs feed was incrementally reduced by 0.1% or 0.2% units of body weight until feed refusals were less than 1.5 kg dayÿ1 . When feed refusals were consistently less than 1.5 kg dayÿ1 , the steerÕs daily ration was considered to be that steerÕs ad libitum intake. During collection periods, steers were oered rations at 90% of each steerÕs ad libitum intake. To form approximately 2 l of GEL, 20 g of ammonium polyacrylate was added to 2 l of tap water containing 20 g of glucose and allowed to hydrate overnight at 4°C. The morning ration of each treatment animal was top-dressed with approximately 2 l of GEL. The morning ration of each control animal was topdressed with 20 g of glucose (Sigma, St. Louis, MO). All diets were then manually mixed to insure even distri-
bution before consumption. In most cases, the animals consumed feed containing GEL or glucose within 1 h of feeding. 2.3. Feed and intestinal sampling During sampling periods, feed refusals and each feed component were sampled daily, composited by period and stored at 4°C until processed. Every 4 h, 300 ml of duodenal and 200 ml of ileal digesta were sampled and composited by steer. Fecal grab samples were taken every 6 h and also composited by animal. The duodenal, ileal and fecal sample collection times were advanced by 1 h each day to adjust for diurnal variation in gastrointestinal ¯ow rates. Samples were stored at )20°C between time of sampling and processing or analysis, unless otherwise noted. 2.4. Ruminal sampling procedures and assays On the fourth day of each sampling period, ruminal contents were sampled at 3, 6, 9 and 12 h after the morning feeding. The samples were obtained from four dierent areas of the reticulorumen using a core sampling device (Firkins et al., 1986) and strained through two layers of cheesecloth for a ®nal sample volume of 150 ml. The pH of each sample was measured immediately using an Orion pH meter (Orion Research, Boston, MA). Eight ml of ruminal ¯uid were added to scintillation vials containing 2 ml of 25% m-phosphoric acid and placed on ice for short chain fatty acid (SCFA) analysis by gas chromatography (Wachenheim and Patterson, 1992). Fifty ml of ruminal ¯uid were poured into bottles containing 3 ml of 6 N HCl, placed on ice and transported to the lab for the determination of
Table 1 Composition of diet fed to steers
a
Ingredients
% of DM
Chemical analysis of complete diet
% of DM
Alfalfa silage Chopped alfalfa hayc Cracked corn Soybean meal (48%) Trace mineralized saltd Dicalcium phosphate Se 200 premixe Vitamin ADEK premixf Vitamin E premixg
40.00 10.00 40.21 8.60 0.50 0.40 0.16 0.10 0.03
Crude proteina;b ADFg NDFg AIAg Calcium Phosphorus Potassium Magnesium Sulfur
14.30 19.30 31.53 0.38 0.75 0.36 1.42 0.21 0.22
Calculated as Kjeldahl N * 6.25. Acid detergent ®ber (ADF), neutral detergent ®ber (NDF) and acid insoluble ash (AIA) were calculated as the average of analyzed values of three periods. c Ground in a tub grinder to give an average particle length of 7 cm. d g/100 g: NaCl, 98; Zn, 0.35; Fe, 0.2; Mn, 0.2; Cu, 0.033; I, 0.007. e 0.2 g Se kgÿ1 premix. f kgÿ1 premix: vitamin A, 10 000 IU; vitamin D3 , 2502 IU; vitamin E, 1102 IU; menadione (vitamin K precursor), 734 mg. g 20 k IU of vitamin E kgÿ1 premix. b
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
ammonia concentration by the method of Chaney and Marbach (1962). 2.5. Exchange of ruminal contents Ruminal contents were transferred among animals of dierent treatments between sampling periods to minimize carryover eects. Feed was withheld on the morning when contents were transferred to lessen total ruminal bulk. Steers of approximately equal weight were paired between treatments and their rumens were evacuated using a wet/dry vacuum (Shop-Vac, Williamsport, PA and Irvine, CA). Ruminal contents were switched within the pairs and reintroduced into the rumen within 15 min of evacuation. 2.6. Intestinal analyses Duodenal and ileal digesta were later thawed and mixed. A homogenous 1000 g wet weight subsample was freeze dried (Unitop 600 L freeze-drier, The Virtis, Gardiner, NY 12525). Feed components, duodenal subsamples and fecal samples were analyzed for dry matter (DM) content and ground through a 1 mm screen (no. 1 Wiley Mill, Arthur H. Thomas, Philadelphia, PA). Organic matter analysis was performed by ashing the samples overnight at 600°C. Acid detergent ®ber content was determined by the method of Van Soest (1963), neutral detergent ®ber by the method of Van Soest et al. (1991) and Kjeldahl-nitrogen content as per AOAC (1984). 2.7. Flow rate determination Capsules containing chromium oxide were placed in the rumen of each steer twice daily (15 g dayÿ1 ) through X-shaped incisions in the ruminal cannulas for the determination of ¯ow rate. Freeze-dried duodenal digesta and fecal samples were analyzed for chromium content by the method of Williams et al. (1962) using a Perkin± Elmer 460 Atomic Absorption Spectrophotometer (Perkin±Elmer, Norwalk, CT). Acid insoluble ash content was determined by the method of Van Keulen and Young (1977). 2.8. Statistical analysis Data were analyzed by ANOVA using the general linear model procedure of SAS for an incomplete switchback design with two treatments (SAS, v. 6.03, Cary, NC, 1986). Sums of squares for the model were separated into steer, period and treatment eects. Analysis of ammonia, pH and SCFA concentrations also included the eect of sampling time and an interaction term to test for time by treatment interaction. Statistical signi®cance was de®ned as a probability level
83
of P < 0.05 and a trend towards a particular eect was de®ned as a probability level of P < 0.10. 3. Results and discussion No signi®cant dierences between steers fed GEL and those fed glucose were detected for intake or digestion of any feed fraction measured, total tract apparent digestibility, duodenal ¯ow, fecal excretion, ruminal pH or ruminal concentrations of ammonia, butyrate or isovalerate (Tables 2 and 3, P > 0:10). Nitrogen ¯ow and digestibility at the duodenum was unreasonably high, probably due to variability in duodenal ¯ow marker concentration. Acetate, propionate, isobutyrate and valerate concentrations signi®cantly changed over time P < 0:05, but did not vary between treatments P > 0:10. A trend towards greater fecal NDF excretion P < 0:06 and a decrease in NDF digestibility P 0:10 was detected. GEL may have inhibited digestion of the NDF fraction of the feed, but the decrease may also be due to diculties in the analysis of NDF in the presence of GEL. The distilled water used in the NDF assay may have hydrated GEL particles and prevented proper ®ltration. Since GEL was not present in control animals, incomplete ®ltration could produce a greater amount of apparent NDF residue and the appearance of decreased feed digestion in samples from treated animals. This eect was also noted in duodenal samples, which could not be analyzed for NDF due to an inability to ®lter the samples. GEL may have been inappropriate as a support for cellulolytic bacteria. Nylon bag studies (data not shown) indicated that a coccobacillus resembling Fibrobacter succinogenes was associated with GEL, but the attached bacteria may have been non-cellulolytic in vivo. The concentration of GEL used in this trial may not have provided sucient support to increase cellulolytic populations or aect rumen fermentation patterns. GEL swollen in tap water shrinks about 10-fold when immersed in a high-mineral solution such as ruminal ¯uid (preliminary data not shown) and may be sensitive to physical breakdown (Seybold, 1994). The diculties in ®ltering the NDF fraction suggest that GEL was present, but as GEL resembled the color of surrounding ruminal ¯uid when immersed in vitro, it was dicult to detect visually. Due to the limited number of cannulated and ®stulated steers available, funding restrictions and the limited time allotted for the study, though, only the concentration of GEL suggested by the manufacturer (Red Sea Construction) was evaluated. GEL may be eective at higher doses or with animals with a greater metabolic demand for energy, such as high producing dairy cattle. Future studies with in vitro continuous fermentors containing mixed ruminal microorganisms could investigate the eect of GEL concentration
84
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
Table 2 Eect of GEL on nutrient digestion in steers GEL ())a
(+)b
SEMc
P-value
Item n 6 DM OM NDFd ADFe N
9240 8600 2920 1800 212
9270 8630 2910 1790 212
440 410 130 80 10
0.97 0.96 0.96 0.93 0.98
Flow at duodenum (g dayÿ1 ) DM OM
4460 3960
3540 3000
410 490
0.14 0.18
Fecal excretion (g dayÿ1 ) DM OM NDF ADF N
2080 1860 1040 590 53
2330 2080 1290 730 60
180 160 80 70 4
0.35 0.39 0.06 0.16 0.25
Nutrients digested (g dayÿ1 ) DM OM
7160 6740
6940 6550
430 400
0.72 0.76
Apparent preduodenal digestibility (% of intake) DM 50.8 OM 53.0
61.5 65.1
4.9 5.4
0.15 0.15
Apparent total tract digestibility (% of intake) DM 77.3 OM 78.9 NDF 64.3 ADF 66.8 N 74.9
74.9 75.9 56.3 60.2 71.8
2.2 2.2 3.0 3.1 2.3
0.46 0.51 0.10 0.16 0.38
a
20 g glucose top dressed at 0730 feeding. Two l of GEL (20 g ammonium polyacrylate added to 2 l tap water containing 20 g glucose and allowed to hydrate overnight at 4°C) top dressed at 0730 feeding. c Standard error of the mean. d Neutral detergent ®ber residue. e Acid detergent ®ber residue. b
Table 3 Eect of GEL on ruminal characteristics of steers Hours post feeding parameter (n 6)
(+GEL)b
SEMc
3
6
9
12
3
6
9
12
NH3 ±N (mg dl ) pH Acetate (mM) Propionate (mM) Isobutyrate (mM) Butyrate (mM) Isovalerate (mM) Valerate (mM)
9.8 6.1 61.6 15.9 1.0 10.4 0.8 1.3
9.4 6.2 58.6 15.4 1.0 10.3 0.8 1.3
7.3 6.3 54.6 13.4 0.9 8.6 0.6 1.0
9.9 6.3 53.7 14.5 0.9 9.4 0.8 1.0
9.4 6.2 62.3 16.2 1.0 10.4 0.8 1.3
9.5 6.2 64.0 16.5 1.1 10.7 0.8 1.3
9.1 6.2 56.6 14.4 0.9 9.4 0.6 1.1
9.3 6.3 55.3 14.9 0.9 9.0 0.8 1.0
0.9 0.1 3.0 0.8 0.1 0.7 0.1 0.1
Total SCFA (mM)
91.0
87.4
79.1
80.3
92.0
93.4
83.0
81.9
4.5
ÿ1
a
()GEL)a
20 g glucose top dressed at 0730 feeding. Two l of GEL (20 g ammonium polyacrylate added to 2 l tap water containing 20 g glucose and allowed to hydrate overnight at 4°C) top dressed at 0730 feeding. c Standard error of the mean. b
K.G. Maciorowski et al. / Bioresource Technology 73 (2000) 81±85
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