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Influence of Tallow and Aspergillus oryzae Fermentation Extract in Dairy Cattle Rations1
Influence of Tallow and Aspergillus oryzae Fermentation Extract in Dairy Cattle Rations1 J. A. BERTRAND2,3 and L. W. GRIMES4 Clemson University, Clemson, SC 29634-0361
ABSTRACT Objectives were to determine the effects of adding 3 g/d of Aspergillus oryzae fermentation extract to diets with or without 5.6% added tallow. Twenty-eight Holstein cows ( X = 98 d of lactation) were assigned to a randomized block experiment in a 2 × 2 factorial arrangement of treatments. Treatments were the basal diet 1 ) without tallow or extract, 2 ) with extract but no tallow, 3 ) with tallow but no extract, and 4 ) with tallow and extract. Milk production, dry matter intake, 3.5% fat corrected milk, digestibility of neutral detergent fiber in the total tract were depressed for cows fed tallow. Addition of fermentation extract did not stimulate fiber digestion or milk production of cows fed diets with or without fat. Addition of extract did not overcome depression of fiber digestibility by cows fed tallow. ( Key words: dairy, Aspergillus oryzae, tallow)
Because cellulolytic bacteria are inhibited by unprotected fats, the addition of A. oryzae, which increases cellulolytic bacteria, should increase fiber digestion when additional fat is fed. Firkins et al. ( 7 ) fed Holstein steers animal-vegetable fat and A. oryzae, but A. oryzae had no effect on the diets containing fat. However, fiber digestibility was not depressed in that study when fat was fed, probably because diets were high in forage. Denigan et al. ( 4 ) reported that fiber digestibility tended to be enhanced by the addition of A. oryzae when the ration was low in fiber. The objectives of this study were 1 ) to determine whether the lactation response to dietary fat could be augmented with the addition of A. oryzae, 2 ) to determine whether reduced fiber digestibility from dietary fats could be alleviated by the addition of A. oryzae, and 3 ) to determine whether the addition of fat or A. oryzae to the diets of lactating dairy cows or the interaction of fat and A. oryzae would improve the response of cows to heat stress.
INTRODUCTION Inclusion of fats in the diets of high producing dairy cows has become a common method of increasing energy density. However, ruminal cellulolytic bacteria are inhibited by additional dietary fat, which depresses the fiber digestibility of diets containing fat that is not ruminally inert (25). Fiber digestibility in high concentrate rations of lactating cows has been increased by the addition of the fermentation extract of the fungus Aspergillus oryzae (10, 12, 29). Wiedmeier et al. ( 2 9 ) reported a 40% increase in cellulolytic bacteria when A. oryzae was fed. Milk production (11, 15) was also increased when A. oryzae was fed. Rectal temperatures of cows during heat stress were lower for cows fed diets containing Aspergillus oryzae (11, 13).
Received December 18, 1995. Accepted December 6, 1996. 1Technical Contribution Number 4109 of the South Carolina Agricultural Experiment Station. Partially supported by a grant from Diamond V Mills, Inc., Cedar Rapids, IA. 2Reprint requests. 3Department of Animal, Dairy, and Veterinary Science. 4Department of Experimental Statistics. 1997 J Dairy Sci 80:1179–1184
MATERIALS AND METHODS Twenty-eight Holstein cows ( X = 98 d of lactation) were used in an 84-d trial that was conducted from July to December 1993. Cows were introduced to the study in three groups as they became available, starting on July 7, August 11, and September 22, respectively. Cows were housed in four adjacent concrete pens with access to individual free stalls and electronic feeding gates (American Calan, Inc., Northwood, NH). Cooling fans were provided in each pen during periods of heat stress. Cows were adapted to the experimental area and trained to use the Calan gates for approximately 2 wk prior to the start of the experiment. Cows were fed a TMR once daily at 0800 h to ensure at least 10% orts. Two TMR, one containing tallow, were formulated (Table 1). The TMR were manipulated to maintain similar fiber and CP concentrations. Fatty acid composition of the tallow is shown in Table 2. Treatments were 1 ) control (the basal diet containing no extract or tallow), 2 ) the basal diet plus 3 g of A. oryzae fermentation extract, 3 ) the basal diet plus 5.6% tallow, and 4 ) the basal diet plus tallow and extract. The A. oryzae (Diamond V Mills,
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TABLE 1. Ration ingredients and dietary composition. Ration Composition
Control
Tallow
( % of DM) Ingredient Tallow Corn silage Alfalfa hay Corn grain Barley Soybean meal Cottonseed hulls Deflourinated rock phosphate Dynamate1 Trace-mineralized salt Sodium bicarbonate Composition DM CP NDF ADF Fat NEL,2 Mcal/kg Forage3:concentrate
0 36.1 10.6 21.9 7.1 18.8 2.7 1.4 0.5 0.3 0.6
5.6 37.1 10.9 10.1 9.6 20.9 3.0 1.5 0.6 0.3 0.5
46.9 16.6 39.7 21.0 2.4 1.71 49:51
47.2 16.5 39.8 22.8 8.3 1.83 51:49
1Contained 22% S, 18% K, and 11% Mg (Pitman-Moore, Inc., Mundelein, IL). 2Estimated from NRC (21). 3Includes cottonseed hulls.
Inc., Cedar Rapids, IA) was top-dressed and handmixed into the TMR for each cow. Amounts of feed offered and orts were recorded daily. Cows were milked twice daily at 0200 and 1400 h, and milk weights were recorded electronically. Milk samples were collected weekly from two consecutive milkings and were composited based on milk weights. Samples were analyzed for milk fat using the Babcock method ( 2 ) and for milk protein by a modified method of Kuboyama et al. (17). Rectal temperatures, recorded at 1300 h, and BW, recorded at 0800 h, were taken on 2 consecutive d, averaged, and recorded weekly. Data for rectal temperatures were analyzed only for periods when cows were under heat stress. Cows were determined to be under heat stress when the weekly temperaturehumidity index averaged at least 74 (18). Heat stress was measured according to the temperature-humidity index between July 7 and September 28, 1993, and the temperature-humidity index was calculated from daily high and low temperatures, assuming that the minimum temperature was equivalent to the minimum dew point (18). The daily high and low temperatures, collected less than 1 km from the experimental site, were obtained from the monthly climatological data for South Carolina from the National Climatic Data Center (Ashville, NC). Journal of Dairy Science Vol. 80, No. 6, 1997
Samples of ruminal fluid were collected via a stomach tube on d 42 and 84 at 0900 h. Five milliliters of ruminal fluid were mixed with 1 ml of 1.2N H2SO4 and centrifuged at 1700 × g for 20 min at 20°C. Internal standard (2-ethyl butyric acid) was added to the supernatant. Volatile fatty acids were determined by GLC (Hewlett Packard 5710A gas chromatograph; Hewlett Packard, Avondale, PA) using a 152- × 0.32-cm stainless steel column packed with 20% NPGS/2% H3PO4 on 60/80 mesh Chromosorb AW-DMCS. Oven temperatures were 118°C for the column and 200°C for the injector and detector. Nitrogen was the carrier gas. Gelatin capsules containing 10 g of Cr2O3 were orally administered as an indigestible reference marker at 0800 and 1700 h during each of the last 11 d of the trial. Fecal grab samples were taken twice per day during the last 4 d in a time sequence so that every 3rd h over 24 h was represented. Samples of feed, feces, and orts were dried at 60°C for 24 h in a forced-air oven and ground in a Wiley mill (2-mm screen; Arthur H. Thomas Co., Philadelphia, PA). Samples were analyzed for DM at 100°C, for NDF and ADF ( 9 ) , for Kjeldahl N ( 2 ) , and for fatty acids (28). Chromic oxide content of fecal samples was determined using the procedure of Fenton and Fenton ( 6 ) . Data for total tract digestibility and VFA were analyzed by ANOVA using the general linear models procedure of SAS (25). Sources of variation included in the model for digestibility were treatment, group, and the interaction of treatment and group. Sources of variation included in the model for VFA were treatment, cow within treatment, sampling period, and the interaction of treatment and sampling period. The effect of sampling period was significant for butyrate and approached significance for acetate ( P = 0.07).
TABLE 2. Fatty acid composition of tallow. Fatty acid1
14:0 16:0 16:1 18:0 18:1 18:2 Iodine value2 1Number
of carbons:number of double bonds. according to methods of the American Oil Chemists’
2Calculated
Society ( 1 ) .
(g/100 g of methyl esters) 3.2 28.7 2.3 26.2 37.7 1.4 36.9
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EFFECTS OF FAT AND ASPERGILLUS ORYZAE EXTRACT TABLE 3. Effects of Aspergillus oryzae fermentation extract with or without tallow on DMI, milk production, milk composition, BW, and rectal temperatures (RT). Ration Item
Control
DMI,a kg/d Milk,a kg/d 3.5% FCM,a kg/d Milk fat,a % Milk fat,a kg/d Milk protein, % Milk protein,a kg/d RT,1 °C BW, kg Initial Ending Change aTallow
20.4 30.4 30.7 3.63 1.08 3.69 1.10 38.8 608.4 645.3 36.8
Control plus A. oryzae Tallow 19.7 30.6 30.0 3.44 1.04 3.43 1.04 38.9 574.8 607.2 32.4
17.5 26.9 25.2 3.12 0.84 3.52 0.93 38.7 559.5 600.5 41.0
Tallow plus A. oryzae 17.6 27.2 25.6 3.19 0.86 3.50 0.94 38.9 561.6 593.0 31.4
SEM 0.63 1.10 1.16 0.11 0.05 0.11 0.03 0.16 30.3 29.6 7.2
effect ( P < 0.05). data when temperature-humidity index exceeded 74.
1Includes
However, the interaction of treatment and sampling period was not significant for any parameter, so data were pooled across the sampling periods. For intake, production variables, and rectal temperatures, restricted maximum likelihood ( 1 6 ) was used with sources of variation, including a 2-wk pretreatment milk weight as covariables, treatment, group, and the interaction of treatment and group. Linear contrasts were used to test the effects of tallow, A. oryzae, and the interaction of tallow and A. oryzae. RESULTS AND DISCUSSION Addition of tallow to the diet decreased production of milk and 3.5% FCM (Table 3), which was likely a result of decreased DMI. Response to added tallow has been variable. Elliott et al. ( 5 ) reported that DMI tended to decrease when tallow was increased from 2.5 to 5.0% in diets containing high oil corn grain. In that study ( 5 ) , however, production of milk and 4% FCM was not affected. Palmquist and Conrad ( 2 3 ) reported a tendency toward reduced DMI and a subsequent reduction in milk production when tallow was included at 10% of the grain diet and when grain constituted 50% of the dietary DM. Other studies, however, did not report reduced DMI when tallow was added. The DMI did not decrease when cows were fed 4% tallow in addition to 10% whole soybeans (26). Production and percentage of milk fat were also decreased by dietary tallow (Table 3), suggesting inhibited fiber fermentation. Wrenn et al. ( 3 0 ) also reported decreased milk fat percentage when 5% tal-
low was fed. In our study, milk protein production also decreased when fat was added, although milk protein percentage was not changed (Table 3). Most studies in which supplemental fat was fed reported decreased milk protein percentage ( 3 ) . Supplementation of both the control and tallow diets with A. oryzae did not affect DMI, milk production, or milk composition (Table 3), and no interactions of tallow and A. oryzae were detected. Response to A. oryzae has been variable (14). Kellems et al. ( 1 5 ) reported increased milk production during later lactation when A. oryzae was fed. Those researchers ( 1 5 ) hypothesized that A. oryzae had its greatest effect during the early stages of the lactation cycle and that the subsequently higher milk production was a result of higher initial production, which was reflected in increased persistency. Gomez-Alarcon et al. ( 1 1 ) reported that milk production, efficiency of milk production, and nutrient digestibilities were higher for early lactation cows fed a high concentrate diet, but midlactation cows fed a lower energy diet were less responsive to A. oryzae. Several studies (4, 12, 13, 27) reported that additional A. oryzae had no effect on DMI or milk production; effects on milk components have varied. Higginbotham et al. ( 1 2 ) reported increased percentages of milk protein and SNF. Sievert and Shaver ( 2 7 ) reported a decrease in milk fat percentage. Huber et al. ( 1 4 ) summarized reports from 14 lactation studies in which 3 g/d of A. oryzae were fed. The mean increase in milk production was 1.0 kg/d or 4%. Cows fed A. oryzae had significantly higher milk production in 6 studies and slightly higher milk production in 3 Journal of Dairy Science Vol. 80, No. 6, 1997
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BERTRAND AND GRIMES TABLE 4. Effects of Aspergillus oryzae fermentation extract with or without tallow on apparent total tract DM and nutrient digestibility. Ration Digestibility DM NDFa ADF CP Fatty acid aTallow
Control 75.7 67.3 60.9 78.9 80.1
Control plus A. oryzae
Tallow (%) 75.5 63.7 58.9 80.5 80.0
76.6 67.7 62.0 78.8 83.4
Tallow plus A. oryzae
SEM
75.0 61.7 56.2 80.6 80.5
1.44 2.36 2.96 1.03 1.82
effect ( P < 0.06).
studies; in 5 studies, milk production was unchanged or slightly lower. As recommended by the manufacturer, supplementation rate for A. oryzae fermentation extract was 3 g/ d. Oellermann et al. ( 2 2 ) studied the addition of 0, 1, 2, 4, and 6 g of A. oryzae fermentation extract and reported no differences in digestibility of CP, ADF, and NDF. Cows fed 1 g/d of A. oryzae had lower ruminal NH3 N than did control cows and cows fed 2, 4, and 6 g/d. Ruminal proteolytic bacteria were lower in cows fed 2, 4, and 6 g/d than in control cows. The possibility that added fat alleviates heat stress by providing nonfermentative energy to cows is of interest and was discussed (14). In our study, responses to heat stress were determined by measuring rectal temperatures when the temperaturehumidity index exceeded 74 (18). Based on those observations, neither the addition of fat nor the addition of A. oryzae reduced the effects of heat stress (Table 3). No interactions of tallow and A. oryzae were found. Addition of A. oryzae was associated with decreased rectal temperatures for 4 of 10 weekly determinations
taken during summer in Arizona (11). In addition, inner ear temperatures for cows fed A. oryzae averaged about 0.3°C less than those for cows fed a control diet, except in early morning when ambient temperatures were lowest. Higginbotham et al. ( 1 3 ) reported that cows fed yeast plus A. oryzae had lower rectal temperatures in 7 of 17 weekly determinations. Differences were most evident during August when mean maximum ambient temperature was 37.5°C. The mechanism for this effect remains unclear. However, it has been suggested that fungal metabolites influence temperature control centers in cows (14, 20). Decreased body temperature has been observed in cows fed aflatoxin ( Aspergillus flavus) (19). In contrast, Denigan et al. ( 4 ) reported that cows fed A. oryzae did not have lower rectal temperatures in hot weather and actually had increased respiration rates. In our study, addition of tallow decreased ( P = 0.06) apparent total tract digestibility of NDF (Table 4). Addition of fat to ruminant diets commonly reduces fiber digestion (24). Addition of A. oryzae had no effect on DM or nutrient digestibilities.
TABLE 5. Effects of Aspergillus oryzae fermentation extract with or without tallow on ruminal measures. Ration Item
Control
Control plus A. oryzae Tallow
Tallow plus A. oryzae
SEM
62.3 22.1 1.3 10.9 2.2 1.3 2.9
0.71 0.69 0.09 0.40 0.10 0.30 0.12
(mol/100 mol) VFA Acetate ( A ) a Propionate ( P ) a Isobutyrate Butyratea Isovalerate Valerate A:Pa aTallow
effect ( P < 0.07).
Journal of Dairy Science Vol. 80, No. 6, 1997
62.9 20.3 1.3 12.2 2.1 1.5 3.1
63.8 19.2 1.2 12.4 2.0 1.9 3.3
61.6 22.2 1.4 11.1 2.3 1.4 2.8
EFFECTS OF FAT AND ASPERGILLUS ORYZAE EXTRACT
Addition of A. oryzae has been shown to change ruminal fermentation patterns, resulting in increased cellulolytic bacteria, which were often accompanied by increased fiber digestion. Wiedmeier et al. ( 2 9 ) reported that the percentage of cellulolytic bacteria increased 27% when A. oryzae was fed. Cellulolytic bacteria were increased nearly 40% with the addition of yeast and A. oryzae. Addition of A. oryzae increased the digestibility of DM, ADF, and hemicellulose. Gomez-Alarcon et al. ( 1 0 ) reported that in all but one trial containing a high forage diet, A. oryzae increased ruminal and total tract digestibilities of fiber. Ruminal VFA and NH3 were not affected by fungal cultures. In contrast, Fondevila et al. ( 8 ) reported increased total numbers of culturable bacteria with A. oryzae treatment, but numbers of cellulolytic bacteria, ciliate protozoa, and aerobic fungi were not significantly altered. Other studies did not show differences in total tract digestibilities of CP, ADF, or NDF (4, 22) or molar percentages of acetic, propionic, butyric, isovaleric, or valeric acids when A. oryzae was fed (13, 22). Oellermann et al. ( 2 2 ) suggested that the strong lignocellulose in the straw contained in the ration might have impaired the ability of the A. oryzae to improve digestibility. Molar percentages of acetate, propionate, and butyrate and the ratio of acetate to propionate were depressed when tallow was fed, indicating that ruminal fermentation was depressed (Table 5). Our hypothesis, that the decrease in fiber digestion from the addition of tallow would be compensated by the addition of A. oryzae, was not realized. In fact, fiber digestion was not stimulated by A. oryzae in the control diet, although acetate production was numerically but not statistically increased. In one other study analyzing the interactions between animalvegetable fat and A. oryzae, Firkins et al. ( 7 ) reported that fat decreased the ratio of acetate to propionate in the rumen and that A. oryzae tended to increase the ratio. However, few differences were found in ruminal or total tract digestibilities. In that study ( 7 ) , fat had fewer inhibitory effects on ruminal fiber degradation than would be expected if more highly unsaturated fats were fed. CONCLUSIONS Addition of tallow in this study negatively affected DMI, milk production, and the production of 3.5% FCM. Rectal temperatures were not affected by the addition of either tallow or A. oryzae during heat stress. We expected fiber digestion to be depressed with the addition of tallow, which did, in fact, occur
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because total tract apparent digestibility of NDF was lower for diets that included tallow ( P = 0.06). We hypothesized that A. oryzae would stimulate fiber digestion to compensate for reduced fiber digestion of tallow diets, but no interactions of tallow and A. oryzae were detected. In fact, A. oryzae did not stimulate fiber digestion in this study. ACKNOWLEDGMENTS Appreciation is expressed to Jack Garrett and Diamond V Mills, Inc. for partial support of this project. Appreciation is also extended to Mike Moore and coworkers at the LaMaster Dairy Center and to Sue Block for technical assistance. REFERENCES 1 American Oil Chemists’ Society. 1989. Official Methods and Recommended Practices. 4th ed. Am. Oil Chem. Soc., Champaign, IL. 2 Association of Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. AOAC, Washington, DC. 3 Coppock, C. E., and D. L. Wilks. 1991. Supplemental fat in high-energy rations for lactating cows: effects on intake, digestion, milk yield, and composition. J. Anim. Sci. 69:3826. 4 Denigan, M. E., J. T. Huber, G. Alhadhrami, and A. Al-Dehneh. 1992. Influence of feeding varying levels of Amaferm on performance of lactating dairy cows. J. Dairy Sci. 75:1616. 5 Elliott, J. P., J. K. Drackley, D. J. Schauff, and E. H. Jaster. 1993. Diets containing high oil corn and tallow for dairy cows during early lactation. J. Dairy Sci. 76:775. 6 Fenton, T. W., and M. Fenton. 1979. An improved procedure for the determination of chromic oxide in feed and feces. Can. J. Anim. Sci. 59:631. 7 Firkins, J. L., W. P. Weiss, M. L. Eastridge, and B. L. Hull. 1990. Effects of feeding fungal culture extract and animalvegetable fat on degradation of hemicellulose and on ruminal bacterial growth in heifers. J. Dairy Sci. 73:1812. 8 Fondevila, M., C. J. Newbold, P. M. Hotten, and E. R. Ørskov. 1990. A note on the effect of Aspergillus oryzae fermentation extract on the rumen fermentation of sheep given straw. Anim. Prod. 51:422. 9 Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 10 Gomez-Alarcon, R. A., C. Dudas, and J. T. Huber. 1990. Influence of cultures of Aspergillus oryzae on rumen and total tract digestibility of dietary components. J. Dairy Sci. 73:703. 11 Gomez-Alarcon, R. A., J. T. Huber, G. E. Higginbotham, F. Wiersma, D. Ammon, and B. Taylor. 1991. Influence of feeding Aspergillus oryzae fermentation extract on the milk yields, eating patterns and body temperatures of lactating cows. J. Anim. Sci. 69:1733. 12 Higginbotham, G. E., D. L. Bath, and L. J. Butler. 1993. Effect of feeding an Aspergillus oryzae extract on milk production and related responses in a commercial dairy herd. J. Dairy Sci. 76: 1484. 13 Higginbotham, G. E., C. A. Collar, M. S. Aseltine, and D. L. Bath. 1994. Effect of yeast culture and Aspergillus oryzae extract on milk yield in a commercial dairy herd. J. Dairy Sci. 77: 343. 14 Huber, J. T., G. Higginbotham, R. A. Gomez-Alarcon, R. B. Taylor, K. H. Chen, S. C. Chan, and Z. Wu. 1994. Heat stress interactions with protein, supplemental fat, and fungal cultures. J. Dairy Sci. 77:2080. Journal of Dairy Science Vol. 80, No. 6, 1997
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15 Kellems, R. O., A. Lagerstedt, and M. V. Wallentine. 1990. Effects of feeding a fermentation extract or Aspergillus oryzae plus yeast plus mineral and vitamin supplement on performance of Holstein cows during a complete lactation. J. Dairy Sci. 73:2922. 16 Koonce, K. L., ed. Applications of mixed models in agriculture and related disciplines. 1989. Southern Coop. Ser. Bull. 343. Louisiana Agric. Exp. Stn., Baton Rouge. 17 Kuboyama, M., G. A. Richardson, and J. O. Young. 1962. Adaptation of the milk protein dye-binding test to field condition. J. Dairy Sci. 44:2339.(Abstr.) 18 Linville, D. E., and F. E. Pardue. 1992. Heat stress and milk production in the South Carolina Coastal Plains. J. Dairy Sci. 75:2598. 19 Mertens, D. R. 1979. Biological effects of mycotoxins upon rumen function and lactating dairy cows. Page 118 in Interaction of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. 20 Meyers, R. D. 1974. Handbook of Drug and Chemical Stimulation of the Brain. Van Nostrand Reinhold Co., New York, NY. 21 National Research Council. 1989. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 22 Oellermann, S. O., M. J. Arambel, B. A. Kent, and J. L. Walters. 1990. Effect of graded amounts of Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility in cattle. J. Dairy Sci. 73:2413.
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23 Palmquist, D. L., and H. R. Conrad. 1980. High fat ration for dairy cows. Tallow and hydrolyzed blended fat at two intakes. J. Dairy Sci. 63:391. 24 Palmquist, D. L., and T. C. Jenkins. 1980. Fat in lactation rations: review. J. Dairy Sci. 63:1. 25 SAS User’s Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 26 Schauff, D. J., J. P. Elliott, J. H. Clark, and J. K. Drackley. 1992. Effects of feeding lactating dairy cows diets containing whole soybeans and tallow. J. Dairy Sci. 75:1923. 27 Sievert, S. J., and R. D. Shaver. 1993. Carbohydrate and Aspergillus oryzae effects on intake, digestion, and milk production by dairy cows. J. Dairy Sci. 76:245. 28 Sukhija, P. S., and D. L. Palmquist. 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Feed Chem. 36:1202. 29 Wiedmeier, R. D., M. J. Arambel, and J. L. Walters. 1987. Effect of yeast culture and Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility. J. Dairy Sci. 70:2063. 30 Wrenn, T. R., J. Bitman, R. A. Waterman, J. R. Weyant, D. L. Wood, L. L. Strozinski, and N. W. Hooven, Jr. 1978. Feeding protected and unprotected tallow to lactating cows. J. Dairy Sci. 61:49.
ABSTRACT Objectives were to determine the effects of adding 3 g/d of Aspergillus oryzae fermentation extract to diets with or without 5.6% added tallow. Twenty-eight Holstein cows ( X = 98 d of lactation) were assigned to a randomized block experiment in a 2 × 2 factorial arrangement of treatments. Treatments were the basal diet 1 ) without tallow or extract, 2 ) with extract but no tallow, 3 ) with tallow but no extract, and 4 ) with tallow and extract. Milk production, dry matter intake, 3.5% fat corrected milk, digestibility of neutral detergent fiber in the total tract were depressed for cows fed tallow. Addition of fermentation extract did not stimulate fiber digestion or milk production of cows fed diets with or without fat. Addition of extract did not overcome depression of fiber digestibility by cows fed tallow. ( Key words: dairy, Aspergillus oryzae, tallow)
Because cellulolytic bacteria are inhibited by unprotected fats, the addition of A. oryzae, which increases cellulolytic bacteria, should increase fiber digestion when additional fat is fed. Firkins et al. ( 7 ) fed Holstein steers animal-vegetable fat and A. oryzae, but A. oryzae had no effect on the diets containing fat. However, fiber digestibility was not depressed in that study when fat was fed, probably because diets were high in forage. Denigan et al. ( 4 ) reported that fiber digestibility tended to be enhanced by the addition of A. oryzae when the ration was low in fiber. The objectives of this study were 1 ) to determine whether the lactation response to dietary fat could be augmented with the addition of A. oryzae, 2 ) to determine whether reduced fiber digestibility from dietary fats could be alleviated by the addition of A. oryzae, and 3 ) to determine whether the addition of fat or A. oryzae to the diets of lactating dairy cows or the interaction of fat and A. oryzae would improve the response of cows to heat stress.
INTRODUCTION Inclusion of fats in the diets of high producing dairy cows has become a common method of increasing energy density. However, ruminal cellulolytic bacteria are inhibited by additional dietary fat, which depresses the fiber digestibility of diets containing fat that is not ruminally inert (25). Fiber digestibility in high concentrate rations of lactating cows has been increased by the addition of the fermentation extract of the fungus Aspergillus oryzae (10, 12, 29). Wiedmeier et al. ( 2 9 ) reported a 40% increase in cellulolytic bacteria when A. oryzae was fed. Milk production (11, 15) was also increased when A. oryzae was fed. Rectal temperatures of cows during heat stress were lower for cows fed diets containing Aspergillus oryzae (11, 13).
Received December 18, 1995. Accepted December 6, 1996. 1Technical Contribution Number 4109 of the South Carolina Agricultural Experiment Station. Partially supported by a grant from Diamond V Mills, Inc., Cedar Rapids, IA. 2Reprint requests. 3Department of Animal, Dairy, and Veterinary Science. 4Department of Experimental Statistics. 1997 J Dairy Sci 80:1179–1184
MATERIALS AND METHODS Twenty-eight Holstein cows ( X = 98 d of lactation) were used in an 84-d trial that was conducted from July to December 1993. Cows were introduced to the study in three groups as they became available, starting on July 7, August 11, and September 22, respectively. Cows were housed in four adjacent concrete pens with access to individual free stalls and electronic feeding gates (American Calan, Inc., Northwood, NH). Cooling fans were provided in each pen during periods of heat stress. Cows were adapted to the experimental area and trained to use the Calan gates for approximately 2 wk prior to the start of the experiment. Cows were fed a TMR once daily at 0800 h to ensure at least 10% orts. Two TMR, one containing tallow, were formulated (Table 1). The TMR were manipulated to maintain similar fiber and CP concentrations. Fatty acid composition of the tallow is shown in Table 2. Treatments were 1 ) control (the basal diet containing no extract or tallow), 2 ) the basal diet plus 3 g of A. oryzae fermentation extract, 3 ) the basal diet plus 5.6% tallow, and 4 ) the basal diet plus tallow and extract. The A. oryzae (Diamond V Mills,
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TABLE 1. Ration ingredients and dietary composition. Ration Composition
Control
Tallow
( % of DM) Ingredient Tallow Corn silage Alfalfa hay Corn grain Barley Soybean meal Cottonseed hulls Deflourinated rock phosphate Dynamate1 Trace-mineralized salt Sodium bicarbonate Composition DM CP NDF ADF Fat NEL,2 Mcal/kg Forage3:concentrate
0 36.1 10.6 21.9 7.1 18.8 2.7 1.4 0.5 0.3 0.6
5.6 37.1 10.9 10.1 9.6 20.9 3.0 1.5 0.6 0.3 0.5
46.9 16.6 39.7 21.0 2.4 1.71 49:51
47.2 16.5 39.8 22.8 8.3 1.83 51:49
1Contained 22% S, 18% K, and 11% Mg (Pitman-Moore, Inc., Mundelein, IL). 2Estimated from NRC (21). 3Includes cottonseed hulls.
Inc., Cedar Rapids, IA) was top-dressed and handmixed into the TMR for each cow. Amounts of feed offered and orts were recorded daily. Cows were milked twice daily at 0200 and 1400 h, and milk weights were recorded electronically. Milk samples were collected weekly from two consecutive milkings and were composited based on milk weights. Samples were analyzed for milk fat using the Babcock method ( 2 ) and for milk protein by a modified method of Kuboyama et al. (17). Rectal temperatures, recorded at 1300 h, and BW, recorded at 0800 h, were taken on 2 consecutive d, averaged, and recorded weekly. Data for rectal temperatures were analyzed only for periods when cows were under heat stress. Cows were determined to be under heat stress when the weekly temperaturehumidity index averaged at least 74 (18). Heat stress was measured according to the temperature-humidity index between July 7 and September 28, 1993, and the temperature-humidity index was calculated from daily high and low temperatures, assuming that the minimum temperature was equivalent to the minimum dew point (18). The daily high and low temperatures, collected less than 1 km from the experimental site, were obtained from the monthly climatological data for South Carolina from the National Climatic Data Center (Ashville, NC). Journal of Dairy Science Vol. 80, No. 6, 1997
Samples of ruminal fluid were collected via a stomach tube on d 42 and 84 at 0900 h. Five milliliters of ruminal fluid were mixed with 1 ml of 1.2N H2SO4 and centrifuged at 1700 × g for 20 min at 20°C. Internal standard (2-ethyl butyric acid) was added to the supernatant. Volatile fatty acids were determined by GLC (Hewlett Packard 5710A gas chromatograph; Hewlett Packard, Avondale, PA) using a 152- × 0.32-cm stainless steel column packed with 20% NPGS/2% H3PO4 on 60/80 mesh Chromosorb AW-DMCS. Oven temperatures were 118°C for the column and 200°C for the injector and detector. Nitrogen was the carrier gas. Gelatin capsules containing 10 g of Cr2O3 were orally administered as an indigestible reference marker at 0800 and 1700 h during each of the last 11 d of the trial. Fecal grab samples were taken twice per day during the last 4 d in a time sequence so that every 3rd h over 24 h was represented. Samples of feed, feces, and orts were dried at 60°C for 24 h in a forced-air oven and ground in a Wiley mill (2-mm screen; Arthur H. Thomas Co., Philadelphia, PA). Samples were analyzed for DM at 100°C, for NDF and ADF ( 9 ) , for Kjeldahl N ( 2 ) , and for fatty acids (28). Chromic oxide content of fecal samples was determined using the procedure of Fenton and Fenton ( 6 ) . Data for total tract digestibility and VFA were analyzed by ANOVA using the general linear models procedure of SAS (25). Sources of variation included in the model for digestibility were treatment, group, and the interaction of treatment and group. Sources of variation included in the model for VFA were treatment, cow within treatment, sampling period, and the interaction of treatment and sampling period. The effect of sampling period was significant for butyrate and approached significance for acetate ( P = 0.07).
TABLE 2. Fatty acid composition of tallow. Fatty acid1
14:0 16:0 16:1 18:0 18:1 18:2 Iodine value2 1Number
of carbons:number of double bonds. according to methods of the American Oil Chemists’
2Calculated
Society ( 1 ) .
(g/100 g of methyl esters) 3.2 28.7 2.3 26.2 37.7 1.4 36.9
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EFFECTS OF FAT AND ASPERGILLUS ORYZAE EXTRACT TABLE 3. Effects of Aspergillus oryzae fermentation extract with or without tallow on DMI, milk production, milk composition, BW, and rectal temperatures (RT). Ration Item
Control
DMI,a kg/d Milk,a kg/d 3.5% FCM,a kg/d Milk fat,a % Milk fat,a kg/d Milk protein, % Milk protein,a kg/d RT,1 °C BW, kg Initial Ending Change aTallow
20.4 30.4 30.7 3.63 1.08 3.69 1.10 38.8 608.4 645.3 36.8
Control plus A. oryzae Tallow 19.7 30.6 30.0 3.44 1.04 3.43 1.04 38.9 574.8 607.2 32.4
17.5 26.9 25.2 3.12 0.84 3.52 0.93 38.7 559.5 600.5 41.0
Tallow plus A. oryzae 17.6 27.2 25.6 3.19 0.86 3.50 0.94 38.9 561.6 593.0 31.4
SEM 0.63 1.10 1.16 0.11 0.05 0.11 0.03 0.16 30.3 29.6 7.2
effect ( P < 0.05). data when temperature-humidity index exceeded 74.
1Includes
However, the interaction of treatment and sampling period was not significant for any parameter, so data were pooled across the sampling periods. For intake, production variables, and rectal temperatures, restricted maximum likelihood ( 1 6 ) was used with sources of variation, including a 2-wk pretreatment milk weight as covariables, treatment, group, and the interaction of treatment and group. Linear contrasts were used to test the effects of tallow, A. oryzae, and the interaction of tallow and A. oryzae. RESULTS AND DISCUSSION Addition of tallow to the diet decreased production of milk and 3.5% FCM (Table 3), which was likely a result of decreased DMI. Response to added tallow has been variable. Elliott et al. ( 5 ) reported that DMI tended to decrease when tallow was increased from 2.5 to 5.0% in diets containing high oil corn grain. In that study ( 5 ) , however, production of milk and 4% FCM was not affected. Palmquist and Conrad ( 2 3 ) reported a tendency toward reduced DMI and a subsequent reduction in milk production when tallow was included at 10% of the grain diet and when grain constituted 50% of the dietary DM. Other studies, however, did not report reduced DMI when tallow was added. The DMI did not decrease when cows were fed 4% tallow in addition to 10% whole soybeans (26). Production and percentage of milk fat were also decreased by dietary tallow (Table 3), suggesting inhibited fiber fermentation. Wrenn et al. ( 3 0 ) also reported decreased milk fat percentage when 5% tal-
low was fed. In our study, milk protein production also decreased when fat was added, although milk protein percentage was not changed (Table 3). Most studies in which supplemental fat was fed reported decreased milk protein percentage ( 3 ) . Supplementation of both the control and tallow diets with A. oryzae did not affect DMI, milk production, or milk composition (Table 3), and no interactions of tallow and A. oryzae were detected. Response to A. oryzae has been variable (14). Kellems et al. ( 1 5 ) reported increased milk production during later lactation when A. oryzae was fed. Those researchers ( 1 5 ) hypothesized that A. oryzae had its greatest effect during the early stages of the lactation cycle and that the subsequently higher milk production was a result of higher initial production, which was reflected in increased persistency. Gomez-Alarcon et al. ( 1 1 ) reported that milk production, efficiency of milk production, and nutrient digestibilities were higher for early lactation cows fed a high concentrate diet, but midlactation cows fed a lower energy diet were less responsive to A. oryzae. Several studies (4, 12, 13, 27) reported that additional A. oryzae had no effect on DMI or milk production; effects on milk components have varied. Higginbotham et al. ( 1 2 ) reported increased percentages of milk protein and SNF. Sievert and Shaver ( 2 7 ) reported a decrease in milk fat percentage. Huber et al. ( 1 4 ) summarized reports from 14 lactation studies in which 3 g/d of A. oryzae were fed. The mean increase in milk production was 1.0 kg/d or 4%. Cows fed A. oryzae had significantly higher milk production in 6 studies and slightly higher milk production in 3 Journal of Dairy Science Vol. 80, No. 6, 1997
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BERTRAND AND GRIMES TABLE 4. Effects of Aspergillus oryzae fermentation extract with or without tallow on apparent total tract DM and nutrient digestibility. Ration Digestibility DM NDFa ADF CP Fatty acid aTallow
Control 75.7 67.3 60.9 78.9 80.1
Control plus A. oryzae
Tallow (%) 75.5 63.7 58.9 80.5 80.0
76.6 67.7 62.0 78.8 83.4
Tallow plus A. oryzae
SEM
75.0 61.7 56.2 80.6 80.5
1.44 2.36 2.96 1.03 1.82
effect ( P < 0.06).
studies; in 5 studies, milk production was unchanged or slightly lower. As recommended by the manufacturer, supplementation rate for A. oryzae fermentation extract was 3 g/ d. Oellermann et al. ( 2 2 ) studied the addition of 0, 1, 2, 4, and 6 g of A. oryzae fermentation extract and reported no differences in digestibility of CP, ADF, and NDF. Cows fed 1 g/d of A. oryzae had lower ruminal NH3 N than did control cows and cows fed 2, 4, and 6 g/d. Ruminal proteolytic bacteria were lower in cows fed 2, 4, and 6 g/d than in control cows. The possibility that added fat alleviates heat stress by providing nonfermentative energy to cows is of interest and was discussed (14). In our study, responses to heat stress were determined by measuring rectal temperatures when the temperaturehumidity index exceeded 74 (18). Based on those observations, neither the addition of fat nor the addition of A. oryzae reduced the effects of heat stress (Table 3). No interactions of tallow and A. oryzae were found. Addition of A. oryzae was associated with decreased rectal temperatures for 4 of 10 weekly determinations
taken during summer in Arizona (11). In addition, inner ear temperatures for cows fed A. oryzae averaged about 0.3°C less than those for cows fed a control diet, except in early morning when ambient temperatures were lowest. Higginbotham et al. ( 1 3 ) reported that cows fed yeast plus A. oryzae had lower rectal temperatures in 7 of 17 weekly determinations. Differences were most evident during August when mean maximum ambient temperature was 37.5°C. The mechanism for this effect remains unclear. However, it has been suggested that fungal metabolites influence temperature control centers in cows (14, 20). Decreased body temperature has been observed in cows fed aflatoxin ( Aspergillus flavus) (19). In contrast, Denigan et al. ( 4 ) reported that cows fed A. oryzae did not have lower rectal temperatures in hot weather and actually had increased respiration rates. In our study, addition of tallow decreased ( P = 0.06) apparent total tract digestibility of NDF (Table 4). Addition of fat to ruminant diets commonly reduces fiber digestion (24). Addition of A. oryzae had no effect on DM or nutrient digestibilities.
TABLE 5. Effects of Aspergillus oryzae fermentation extract with or without tallow on ruminal measures. Ration Item
Control
Control plus A. oryzae Tallow
Tallow plus A. oryzae
SEM
62.3 22.1 1.3 10.9 2.2 1.3 2.9
0.71 0.69 0.09 0.40 0.10 0.30 0.12
(mol/100 mol) VFA Acetate ( A ) a Propionate ( P ) a Isobutyrate Butyratea Isovalerate Valerate A:Pa aTallow
effect ( P < 0.07).
Journal of Dairy Science Vol. 80, No. 6, 1997
62.9 20.3 1.3 12.2 2.1 1.5 3.1
63.8 19.2 1.2 12.4 2.0 1.9 3.3
61.6 22.2 1.4 11.1 2.3 1.4 2.8
EFFECTS OF FAT AND ASPERGILLUS ORYZAE EXTRACT
Addition of A. oryzae has been shown to change ruminal fermentation patterns, resulting in increased cellulolytic bacteria, which were often accompanied by increased fiber digestion. Wiedmeier et al. ( 2 9 ) reported that the percentage of cellulolytic bacteria increased 27% when A. oryzae was fed. Cellulolytic bacteria were increased nearly 40% with the addition of yeast and A. oryzae. Addition of A. oryzae increased the digestibility of DM, ADF, and hemicellulose. Gomez-Alarcon et al. ( 1 0 ) reported that in all but one trial containing a high forage diet, A. oryzae increased ruminal and total tract digestibilities of fiber. Ruminal VFA and NH3 were not affected by fungal cultures. In contrast, Fondevila et al. ( 8 ) reported increased total numbers of culturable bacteria with A. oryzae treatment, but numbers of cellulolytic bacteria, ciliate protozoa, and aerobic fungi were not significantly altered. Other studies did not show differences in total tract digestibilities of CP, ADF, or NDF (4, 22) or molar percentages of acetic, propionic, butyric, isovaleric, or valeric acids when A. oryzae was fed (13, 22). Oellermann et al. ( 2 2 ) suggested that the strong lignocellulose in the straw contained in the ration might have impaired the ability of the A. oryzae to improve digestibility. Molar percentages of acetate, propionate, and butyrate and the ratio of acetate to propionate were depressed when tallow was fed, indicating that ruminal fermentation was depressed (Table 5). Our hypothesis, that the decrease in fiber digestion from the addition of tallow would be compensated by the addition of A. oryzae, was not realized. In fact, fiber digestion was not stimulated by A. oryzae in the control diet, although acetate production was numerically but not statistically increased. In one other study analyzing the interactions between animalvegetable fat and A. oryzae, Firkins et al. ( 7 ) reported that fat decreased the ratio of acetate to propionate in the rumen and that A. oryzae tended to increase the ratio. However, few differences were found in ruminal or total tract digestibilities. In that study ( 7 ) , fat had fewer inhibitory effects on ruminal fiber degradation than would be expected if more highly unsaturated fats were fed. CONCLUSIONS Addition of tallow in this study negatively affected DMI, milk production, and the production of 3.5% FCM. Rectal temperatures were not affected by the addition of either tallow or A. oryzae during heat stress. We expected fiber digestion to be depressed with the addition of tallow, which did, in fact, occur
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because total tract apparent digestibility of NDF was lower for diets that included tallow ( P = 0.06). We hypothesized that A. oryzae would stimulate fiber digestion to compensate for reduced fiber digestion of tallow diets, but no interactions of tallow and A. oryzae were detected. In fact, A. oryzae did not stimulate fiber digestion in this study. ACKNOWLEDGMENTS Appreciation is expressed to Jack Garrett and Diamond V Mills, Inc. for partial support of this project. Appreciation is also extended to Mike Moore and coworkers at the LaMaster Dairy Center and to Sue Block for technical assistance. REFERENCES 1 American Oil Chemists’ Society. 1989. Official Methods and Recommended Practices. 4th ed. Am. Oil Chem. Soc., Champaign, IL. 2 Association of Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. AOAC, Washington, DC. 3 Coppock, C. E., and D. L. Wilks. 1991. Supplemental fat in high-energy rations for lactating cows: effects on intake, digestion, milk yield, and composition. J. Anim. Sci. 69:3826. 4 Denigan, M. E., J. T. Huber, G. Alhadhrami, and A. Al-Dehneh. 1992. Influence of feeding varying levels of Amaferm on performance of lactating dairy cows. J. Dairy Sci. 75:1616. 5 Elliott, J. P., J. K. Drackley, D. J. Schauff, and E. H. Jaster. 1993. Diets containing high oil corn and tallow for dairy cows during early lactation. J. Dairy Sci. 76:775. 6 Fenton, T. W., and M. Fenton. 1979. An improved procedure for the determination of chromic oxide in feed and feces. Can. J. Anim. Sci. 59:631. 7 Firkins, J. L., W. P. Weiss, M. L. Eastridge, and B. L. Hull. 1990. Effects of feeding fungal culture extract and animalvegetable fat on degradation of hemicellulose and on ruminal bacterial growth in heifers. J. Dairy Sci. 73:1812. 8 Fondevila, M., C. J. Newbold, P. M. Hotten, and E. R. Ørskov. 1990. A note on the effect of Aspergillus oryzae fermentation extract on the rumen fermentation of sheep given straw. Anim. Prod. 51:422. 9 Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 10 Gomez-Alarcon, R. A., C. Dudas, and J. T. Huber. 1990. Influence of cultures of Aspergillus oryzae on rumen and total tract digestibility of dietary components. J. Dairy Sci. 73:703. 11 Gomez-Alarcon, R. A., J. T. Huber, G. E. Higginbotham, F. Wiersma, D. Ammon, and B. Taylor. 1991. Influence of feeding Aspergillus oryzae fermentation extract on the milk yields, eating patterns and body temperatures of lactating cows. J. Anim. Sci. 69:1733. 12 Higginbotham, G. E., D. L. Bath, and L. J. Butler. 1993. Effect of feeding an Aspergillus oryzae extract on milk production and related responses in a commercial dairy herd. J. Dairy Sci. 76: 1484. 13 Higginbotham, G. E., C. A. Collar, M. S. Aseltine, and D. L. Bath. 1994. Effect of yeast culture and Aspergillus oryzae extract on milk yield in a commercial dairy herd. J. Dairy Sci. 77: 343. 14 Huber, J. T., G. Higginbotham, R. A. Gomez-Alarcon, R. B. Taylor, K. H. Chen, S. C. Chan, and Z. Wu. 1994. Heat stress interactions with protein, supplemental fat, and fungal cultures. J. Dairy Sci. 77:2080. Journal of Dairy Science Vol. 80, No. 6, 1997
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15 Kellems, R. O., A. Lagerstedt, and M. V. Wallentine. 1990. Effects of feeding a fermentation extract or Aspergillus oryzae plus yeast plus mineral and vitamin supplement on performance of Holstein cows during a complete lactation. J. Dairy Sci. 73:2922. 16 Koonce, K. L., ed. Applications of mixed models in agriculture and related disciplines. 1989. Southern Coop. Ser. Bull. 343. Louisiana Agric. Exp. Stn., Baton Rouge. 17 Kuboyama, M., G. A. Richardson, and J. O. Young. 1962. Adaptation of the milk protein dye-binding test to field condition. J. Dairy Sci. 44:2339.(Abstr.) 18 Linville, D. E., and F. E. Pardue. 1992. Heat stress and milk production in the South Carolina Coastal Plains. J. Dairy Sci. 75:2598. 19 Mertens, D. R. 1979. Biological effects of mycotoxins upon rumen function and lactating dairy cows. Page 118 in Interaction of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. 20 Meyers, R. D. 1974. Handbook of Drug and Chemical Stimulation of the Brain. Van Nostrand Reinhold Co., New York, NY. 21 National Research Council. 1989. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 22 Oellermann, S. O., M. J. Arambel, B. A. Kent, and J. L. Walters. 1990. Effect of graded amounts of Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility in cattle. J. Dairy Sci. 73:2413.
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23 Palmquist, D. L., and H. R. Conrad. 1980. High fat ration for dairy cows. Tallow and hydrolyzed blended fat at two intakes. J. Dairy Sci. 63:391. 24 Palmquist, D. L., and T. C. Jenkins. 1980. Fat in lactation rations: review. J. Dairy Sci. 63:1. 25 SAS User’s Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 26 Schauff, D. J., J. P. Elliott, J. H. Clark, and J. K. Drackley. 1992. Effects of feeding lactating dairy cows diets containing whole soybeans and tallow. J. Dairy Sci. 75:1923. 27 Sievert, S. J., and R. D. Shaver. 1993. Carbohydrate and Aspergillus oryzae effects on intake, digestion, and milk production by dairy cows. J. Dairy Sci. 76:245. 28 Sukhija, P. S., and D. L. Palmquist. 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Feed Chem. 36:1202. 29 Wiedmeier, R. D., M. J. Arambel, and J. L. Walters. 1987. Effect of yeast culture and Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility. J. Dairy Sci. 70:2063. 30 Wrenn, T. R., J. Bitman, R. A. Waterman, J. R. Weyant, D. L. Wood, L. L. Strozinski, and N. W. Hooven, Jr. 1978. Feeding protected and unprotected tallow to lactating cows. J. Dairy Sci. 61:49.