Effects of Urea and Isoacids on in vitro Fermentation of Diets Containing Formaldehyde-Treated or Untreated Soybean Meal1

Effects of Urea and Isoacids on In Vitro Fermentation of Diets Containing Formaldehyde-Treated or Untreated Soybean Meal 1 G. A. V A R G A 2 , W . H. H O O V E R , L. L. J U N K I N S ~, and B. J. S H R I V E R 4 Dwision of Animal and Veterinary Sciences West Virginia University Morgantown 26506 ABSTRACT

wide ratio of nonstructural carbohydrate to degradable protein when the formaldehyde-treated protein was fed.

Effects on microbial fermentation were determined when buffer containing urea, branched-chain volatile fatty acids, a combination of these infusates or buffer only was infused into fermenters fed diets containing soybean meal treated with .3% formaldehyde or untreated. Fermentations were conducted at a solids retention time of 16 h and a liquid dilution rate of 12%/h. When buffer only was infused, the diet containing formaldehyde-treated soybean meal depressed digestibilities of NDF and ADF and fat-free organic matter compared with diets containing untreated soybean meal. In cultures fed treated soybean meal, digestibilities were partially restored by either infusion of urea or branched-chain acids and were increased to control levels by the combined infusates. Nitrogen digestion (35 vs. 69%) and microbial protein production (1.27 vs. 1.60 g/d) were depressed in fermenters fed the treated soybean meal, and neither was improved by the infusions. The data suggest microbial growth may have been impaired by a

INTRODUCTION

Received May 26 1987. Accepted October 28, 1987. ~Published with the approval of the Director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper Number 2075. This research was supported by funds provided by the Hatch Act and by Agway Inc., Syracuse, NY 132214933. 2Department of Dairy and Animal Science, The Pennsylvania Stare University, University Park, PA 16802. 3H. K, Webster Co., Lawrence, MA 01842. 4Department of Animal Science, The Ohio State University, Wooster 44691. 1988 J Dairy Sci 71:737--744

Inclusion of formaldehyde-treated soybean meal (SBMF) in the diets of lactating cattle generally has not increased milk production relative to an equal amount of untreated soybean meal (SBM) (4, 5, 7, 10). Junkins (14) compared fermentation of diets containing SBMF and SBM in dual flow continuous cultures. Digestibilities of organic matter (OM), NDF, ADF, and protein were decreased for diets containing SBMF. These continuous culture studies were conducted at 14 and 24-h solids retention times. Depressions in digestibility due to formaldehyde were more severe at the 14-h solids retention time than at 24 h. In addition, formaldehyde treatment decreased microbial N production at both solids retention times and concentrations of NH3, isobutyrate, and isovalerate were higher for the control diet than for the SBMF diet. Both NH3 and branched-chain VFA are important in the growth and metabolic activity of cellulolytic organisms (12). The purpose of this study was to determine if decreased availability of NH3 and branched chain fatty acids was associated with the digestibility depressions and reduced microbial N production observed when diets containing SBMF were fermented in continuous culture. MATERIALS AND METHODS Fermentation Conditions

The continuous culture system was that described by Hoover ".t al. (13) modified as described by Crawford et al. (6). Fermenters were fed 103 g DM of a pelleted diet given in eight equally spaced portions over

737

738

VARGA ET AL.

24 h. The pH was maintained at 6.5 -+ .1 by infusion of 5 N NaOH. Fermenters were infused with buffer containing branched-chain VFA (V), urea (U), a combination of V and U (V + U), or no additive (0). Buffer (21) containing .256, .301, and .301 g/L of isobutyrate, isovalerate, and 2-methylbutyrate, respectively, was infused in the V treatments, whereas U treatments received buffer with .17 g u r e a / L . The V + U treatments contained both the isoaeids and urea in the concentrations given, whereas the control buffer contained no isoacids or urea. Buffer was infused at a rate of 2.49 ml/min, which gave a liquid dilution rate of 12%/h. A mean solids retention time of 16 h was maintained in all fermentations by adjusting the ratio of the filtered:overflow effluent volumes. Fermenter operation and sampling were as described by Crawford et al. (6). The ingredient and chemical compositions of the diets are in Table 1. The mineral mixture was formulated to achieve Ca:P of 1.5:1, maintain a S:N of 1:10 and supply 1.0% trace-mineralized salt in the total diet. The TABLE 1. Ingredient and chemical composition of diets. 1 Diets Item

SBM -

Ingredients Corn silage Soybean meal Corn starch Mineral mix2 Chemical analyses CP NDF ADF Lignin Soluble N, % of total N Total nonstructural carbohydrate Ash Ether extract

-

SBMF (%)

56.5 28.5 13.0 2.0

56.5 28.5 13.0 2.0

18.90 22.00 12.97 1.21 17.14

18.86 22.11 13,15 1.92 11.71

50.68 5.68 2.17

55.83 5.60 2.13

t Dry matter basis. SBM = Soybean meal; SBMF = SBM treated with formaldehyde. 2Percent of mix: dicalciurn phosphate, dibasic, 28.05 ; ground limestone, 8.85 ; sodium sulfate, 15.52; trace mineral salt, 47.58.

Journal of Dairy Science Vol. 71, No. 3, 1988

SBMF was obtained commercially (Hubbard Milling Co., Mankato, MN 56001). Diets contained 0 (SBM) or 3.0 g (SBMF) of formaldehyde per 100 g of air-dry soybean meal. The corn silage was dried at 55°C and ground through a 4-mm screen. During mixing, 15 ml of water were added per 100 g diet to facilitate subsequent pelleting. Inoculum was obtained from a ruminally cannulated steer maintained on a diet of 8.2 kg of corn silage and 1.1 kg soybean meal/d. Three hours after feeding, rumen contents were collected and squeezed through one layer of cheesecloth. Approximately 1045 ml of inoculum and 200 ml of buffer were placed in each fermenter. Following a 6-d equilibration period, saturated HgC12 was added to the filtrate and overflow collection containers at the rate of 1 ml HgC12/100 ml effluent. The effluents (filtrate and overflow) were combined, homogenized in a Waring blender and a 1-L aliquot retained. Three collections at 24-h intervals provided 3 L of composite effluent for analysis. Microbial Harvest

After completion of fermentation, microbes were harvested from the rumen fluid in the fermenters. The solid material within the fermenter was allowed to settle, and approximately 800 ml of the fluid were siphoned into a 1-L graduated cylinder. The solid portion was allowed to resettle and approximately 250 ml of rehively particle-free fluid was pipetted into each of two glass centrifuge bottles. The fluid was centrifuged at 150 × g for 10 min; the supernatant was transferred into clean centrifuge bottles and recentrifuged for 15 min at 150 x g. Microscopic examination of the supernatant verified virtually no contamination of bacteria with feed particles. The contents of the glass centrifuge bottles were transferred into eight 50-ml centrifuge tubes and spun at 18,000 × g for 30 min. The supernatant was poured off, and microbial pellets from all eight centrifuge tubes were rinsed into two 50-mI tubes using 20 to 30 ml of 5% trichloroacetic acid. These tubes then were centrifuged at 18,000 × g for 30 min, rinsed into polyethylene containers with distilled water, homogenized with a Virtis Model 45 homogenizer (Virtis Inc., Gardiner, NY 12525) and frozen at

UREA AND ISOACID INFUSIONS ON FERMENTATION --4°C. Samples were freeze-dried prior to total N and diaminopimelic acid (DAPA) analyses. Combined effluent samples were used for determination of fat-free organic matter (FFOM), NDF, ADF, total N, NH3N, total nonstructural carbohydrate (TNC), DAPA, and VFA. All analytical procedures and calculations were as described by Sbriver et al. (19). Statistical Analyses

Data were analyzed as a completely randomized design with a 2 × 2 × 2 factorial arrangement of treatments with three replications per treatment. The General Linear Models program of the Statistical Analysis System (18) was used for computational analysis of the data. All data are presented as least square means. RESULTS

Effects of diets and infusates on nutrient digestibilities are in Table 2. When the buffer contained no additive (0), fermentation of the SBMF diet resulted in lower digestion of FFOM, NDF, ADF, and N than did the SBM diet. The addition of VFA (V) to the buffer improved the digestibilities (P<.O1) of all components except N in fermenters fed the SBMF diet, but values remained significantly lower than for those fed the SBM diet. Urea also was effective in increasing the digestion of FFOM, ADF, and NDF (P<.01) for the SBMFcontaining diet. Addition of V + U to the buffer restored the digestion of NDF and ADF to values similar to those of the control SBM diet. Interactions of diet with U and V suggested that U depressed the digestion of both NDF and ADF and V depressed NDF digestion when infused into fermentations of the diet with SBM. Although TNC digestion was significantly higher for the SBMF diet with no infusion and when U was infused, these increases were small, ranging from 1.1 to 1.6% of dietary TNC. Volatile fatty acid data are in Table 3. Both total VFA produced per day and VFA concentrations were reduced when SBMF was included in the diet. Infusion of VFA in the V and V + U treatments increased VFA (M/24 h) for both the SBM and SBMF diets, with the infusate accounting for 100% of the increase in the SBM diet and 86% in the SBMF diet. Acetate:propionate was decreased by SBMF,

739

due to a reduced proportion of acetate. When U was infused, either alone or with V, and interaction (P<.05) was observed; molar percent of acetate decreased in diets with SBM and increased in those with SBMF. Proportions of other VFA were not affected by diet or infusion, except that concentration of branched chain fatty acids were increased by infusions of V. Daily production of NH3N (P<.01) and microbial N (P<.05) were higher, whereas bypass N was lower (P<.05) for the SBM diet than for the SBMF diet (Table 4). Cultures responded to U infusion for both diets with almost a threefold increase in NH3N concentration, but there was no positive response in microbial N production due to either V or U infusions. In fact, V infusion appeared to depress microbial N in the cultures with SBMF. There were no significant dietary effects noted for microbial efficiency, but there was a trend for a decreased efficiency in all cultures receiving SBMF compared with cultures fed SBM. DISCUSSION

The observed decreases in NDF and ADF digestion when SBMF was fed (Table 2) are in agreements with previous reports (1, 4, 14). Results of the current study suggest these responses are due to limited supplies of both isoacids (V) and ammonia N (U), since NDF and ADF digestion was improved by either V or U, and restored to control values when V + U was infused. None of the infusions improved digestibilities of fiber when untreated SBM was fed, which agrees with the results of Klusmeyer (15) and Fieo et al. (9). In a summary of the results of several studies on NH3N requirements (12), for diets with >6% natural protein, microbial growth was optimized at 3.3 mg/dl of NH3N but 8.0 mg/dl was needed for optimum nutrient digestion. Results of the current study support this concept, since nutrient digestion, but not microbial growth, was improved when NH3N was increased above 5.9 mg/dl. Urea appeared to increase digestibility more than did V for the SBMF treatment versus the SBM diet. Although not significantly different from V or U, the combination of V + U tended to enhance fiber digestibility over that of the individual infusates. Journal of Dairy Science Vol. 71, No. 3, 1988

q~ 0

e~

o

TABLE 2. Effects of protein source and infusates on digestibility of diets containing formaldehyde-treated (SBMF) or untreated soybean meal (SBM) during continuous culture) 0 lnfusates z Z

0

O

tao

00 00

FFOM 3 NDF ADF TNC N

V

U

V+U

SBM

SBMF

SBM

SBMF

SBM

SBMF

SBM

SBMF

SE

71.0 42.6 42.3 86.9 67.9

60.5* 16.7"* 19.8" * 88.0* 37.3**

70.2 40.6 40.7 88.1 72.8

65.7* 29.4** 29.2* * 88.8 38.0**

71.1 38.5 37.5 88.5 73.4

64.3** 33.7 34.7 90.1" * 29.7**

70.3 41.3 41.2 89.0 63.5

66.1 37.9 43.2 89.6 35.1"*

1.82 a 2.49 b 3.27 c .36 d 10.72 e

aDiet, P<.01; Diet X V, P < . 0 7 . bDiet, V, U, P<.01; Diet × V, Diet × U, P<.05. CDiet, V, U, P<.05; Diet × U, P<.01. dDiet, U, P<.01. eDiet, P<.O1. 1 Each value is a mean of three fermentations. 20 = No additive, V = fermenter infused with VFA, U = fermenter infused with urea. 3 FFOM, fat-free organic matter; TNC, total nonstructural carbohydrate. Different between diets within an infusate (P<.05). Different between diets within an infusate (P<.01).

< ) > >

TABLE meal. l

3. E f f e c t s o f p r o t e i n s o u r c e a n d i n f u s a t e s o n V F A in c o n t i n u o u s c u l t u r e s o f d i e t s c o n t a i n i n g f o r m a l d e h y d e - t r e a t e d

( S B M F ) o r u n t r e a t e d (SBM) s o y b e a n

> Z ~J

Infusates 2

0 SBM Total VFA: M/24 h

gM/ml

V SBMF

.47 131

A:P a

3.6

SBM

.41 114 3.0

U SBMF

.48 132 3.6

SBM

.46 126 2.3

aDiet, P < . 0 5 ; V, P < . 0 6 .

V+U

SBMF

.45 125 3.1

SBM

.44 120 2.8

SBMF

.50 138 2.8

.46 128 3.6

SE

© > 63

.02a 6.3 b .35 c

© z © Z

b D i e t , P < . 0 6 ; V, P < . 0 8 . t~

CDiet × U, P < . 0 5 . E a c h value is t h e m e a n o f t h r e e f e r m e n t a t i o n s . ,<

2 0 = No additive, V = fermenter infused with VFA, U = fermenter infused with urea. a Acetate: propionate.

z >

5 Z

< 9-

Z

O

-ix

.q t~

TABLE 4. Effects of protein source and infusates on N fractions of effluents from diets containing formaldehyde-treated (SBMF) or untreated soybean meal (SBM) during continuous cultures.

Infusates ~ O

O SBM

Z

V SBMF

SBM

U SBMF

SBM

V+U SBMF

SBM

SBMF

SE

O

,g oo 0o

Effluents N, g/d NH 3 N, g/d Microbial N, g/d Bypass N

3.27 .60 1.67 1.00

3.38 .22** 1.32 1.95"

3.27 .51 1.90 .85

3.25 .17"* 1.15"* 1.93" *

4.00 1.54 1.40 1.06

4.12 .60** 1.33 2.19" *

4.07 1.56 1.43 1.13

4,00 .67** 1.30 2.02* *

NH 3 N, mg/dl

16.58

5.92**

13.84

4.60**

42.35

16.56"*

42.43

18.49"*

2.11 e

Microbial efficiency, g N/kg DOM 3

29.8

27.9

24.5

27.6

23.3

3.30 f

25.7

28.6

23.3

au, P<.01, Diet × U, P<.01. bDiet, U, P<.01; Diet × U, P<.01. CDiet, P < . 0 5 . dDiet, P<.01. eDiet, U P<.01 ; Diet × U, P<.01. fNonsignificant. 1Each value is a mean of three fermentations. 20 = No additive, V = fermenter infused with VFA, U = fermenter infused with urea. 3Corrected for microbial dry matter. Different between diets within an infusate (P<.05). Different between diets within an infusate (P<.O1).

.050 a .077b .231 c .230 d

< ;>

UREA AND ISOACID INFUSIONS ON FERMENTATION Digestion o f SBMF N (Table 2) by ruminal microorganisms was reduced 45% compared w i t h SBM N. Infusates did not alleviate the depression in N digestion. Crooker et al. (7) and Spears et al. (20) observed apparent protein digestion was depressed when lactating cows were fed SBMF compared with those fed SBM. However, digestibility was n o t depressed to the same e x t e n t as observed in this in vitro experiment. This may reflect the difference b e t w e e n w h o l e tract digestion c o m p a r e d with ruminal digestion o f dietary components. In a defined microbial growth medium, isoacids increased microbial cell yield and efficiency (3, 17). C u m m i n s and Papas (8) also d e m o n s t r a t e d that mixtures o f a m m o n i u m salts o f isocarbon-4 and isocarbon-5 f a t t y acids resulted in increased microbial growth in vitro. In the present study, microbial N p r o d u c t i o n was decreased by SBMF, and microbial N synthesis was not enhanced by addition of isoacids (Table 4). Several authors (2, 14, 16) have r e p o r t e d decreases in ruminal microbial N p r o d u c t i o n w h e n feedstuffs were treated with formaldehyde. Mahadevan et al. (16) concluded that the effects of f o r m a l d e h y d e t r e a t m e n t on microbial growth were caused by inadequate N H 3 N , which was less than 5 mg/dl. In the current study, microbial N p r o d u c t i o n was m o s t depressed by SBMF w h e n N H 3 N was less than 5 m g / d l (V infusion, Table 4). However, feeding o f SBMF was consistently associated w i t h numerically lower microbial g r o w t h and efficiency, regardless o f infusions. Hoover (11) reported that, in diets with high TNC, the ratio o f TNC to ruminally degradable crude protein (RDCP) m a y affect microbial efficiency. It was reported that high microbial efficiencies were associated with T N C : R D C P o f 2.5 to 3.5; efficiency declined as the ratio increased. In the current study, the T N C : R D C P was 3.4 o r less for all SBM diets and 6.0 or greater for the SBMF diets. Average corresponding microbial efficiencies were 28.5 and 24.2. These data suggest that a wide TNC: RDCP m a y have been responsible for the reduced microbial growth on the SBMF diets. In this continuous culture study, SBMF depressed fiber and protein digestion, V F A and microbial protein p r o d u c t i o n . Nitrogen provided as urea and carbon skeletons as isoacids restored fiber digestion. Protein digestion

743

remained depressed, and microbial protein p r o d u c t i o n was n o t increased by any o f the infusates. Increased a m o u n t s o f dietary N, particularly amino acids or peptides, m a y have been required by the microorganisms provided SBMF, and this needs to be evaluated. The low degradable protein m a y have caused u n c o u p l e d f e r m e n t a t i o n , because fiber digestion was restored to levels observed for the SBM diet, whereas microbial g r o w t h remained unchanged. REFERENCES

1 Amos, H. E., D. Burdick, and T. H. Huber. 1974. Effects of formaldehyde treatment of sunflower and soybean meal on nitrogen balance. J. Anim Sci. 38:702. 2 Beever, D. E., D. J. Thomson, S. B. Cammel, and D. G. Harrison. 1977. The digestion by sheep of silages made with and without the addition of formaldehyde. J. Agric. Sci., Camb. 88:61. 3 Chalupa, W., and B. Bloch. 1982. Responses of rumen bacteria sustained in semicontinuous culture to amino nitrogen and to branched chain fatty acids. J. Dairy Sci. 66(Suppl. 1): 152. (Abstr.) 4 Clark, J. H., C. L. Davis, and E. E. Hatfield. 1974. Effects of formaldehyde treated soybean meal on nutrient use, milk yield and composition, and free amino acids in the lactating bovine. J. Dairy Sci. 57:1031. 5 Crawford, R. J., Jr., and W. H. Hoover. 1984. Effects of particle size and formaldehyde treatment of soybean meal on milk production and composition for dairy cows. J. Dairy Sci. 67:1945. 6 Crawford, R. J., Jr., B. J. Shriver, G. A. Varga, and W. H. Hoover. 1983. Buffer requirements for maintenance of pH during fermentation of individual feeds in continuous cultures. J. Dairy Sci. 66: 1881. 7 Crooker, B. A., J. H. Clark, and R. D. Shanks. 1983. Effects of formaldehyde treated soybean meal on milk yield, milk composition and nutrient digestibility in the dairy cow. J. Dairy Sci. 66:492. 8 Cummins, K. A., and A. H. Papas. 1985. Effect of isocarbon #5 volatile fatty acids on microbial protein synthesis and dry matter digestibility in vitro. J. Dairy Sci. 68:2588 9 Fieo, A. G., T. F. Sweeney, R. S. Kensinger, and L. D. Muller. 1984. Metabolic and digestion affects of the addition of ammonium salts of volatile fatty acids to the diets of cows in early lactation. J. Dairy Sci. 67(Suppl. 1):117. (Abstr.) 10 Folman, Y., H. Neumark, M. Kaim, and W. Kaufman. 1981. Performance, tureen and blood metabolites in high-yieldng cows fed varying protein percents and protected soybean. J. Dairy Sci. 64:759. 11 Hoover, W. H. 1987. Potential for managing rumen fermentation. Page 52 in Proc. Cornell Nutr. Conf., Ithaca, NY. 12 Hoover, W. H. 1986. Chemical factors involved in ruminal fiber digestion. J. Dairy Sci. 69: 2755. Journal of Dairy Science Vol. 71, No. 3, 1988

744

V A R G A ET AL.

13 Hoover, W. H., B. A. Crooker, and C. J. Sniffen. 1976. Effects of differential solid-liquid removal rates on protozoa n u m b e r s in continuous cultures o f r u m e n contents. J. Anita. Sci. 43:518. 14 Junkins, L. L. 1981. Use of a formaldehyde treated protein by r u m e n microorganisms. M. S. Thesis, West Virginia Univ., Morgantown. 15 Klusmeyer, T. H. 1985. Effects of feeding or infusing a m m o n i u m salts o f volatile fatty acids on ruminal fermentation, plasma parameters, and milk production o f dairy cows. M. S. Thesis, Dep. Dairy Sci., Univ. Illinois, Urbana. 16 Mahadevan, S., R. M. Teather, J. D. Erfle, and F. D. Sauer. 1983. Effect o f formaldehyde t r e a t m e n t o f soybean meal on rates o f protein degradation and microbial protein concentration in the bovine rumen. Can. J. Anim. Sci. 63:1181.

Journal o f Dairy Science Vol. 71, No. 3, 1988

17 Russell, J. B. 1984. Effects o f C 4 and C 5 volatile fatty acids on the growth o f m i x e d r u m e n bacteria in vitro. J. Dairy Sci. 67:987. 18 SAS User's Guide. 1979. SAS, Inc., Cary, NC. 19 Shriver, B. J., W. H. Hoover, J. P. Sargeant, R. J. Crawford, Jr., and W. V. Thayne. 1986. Ferm e n t a t i o n of a high carbohydrate diet as affected by ruminal pH and digesta flow. J. Dairy Sci. 69:413. 20 Spears, J. W., J. H. Clark, and E. E. Hatfield. 1979. Nitrogen utilization by steers fed f o r m a l d e h y d e treated soybean meal. J. Anita. Sci. 48 (Suppl. 1):407. (Abstr.) 21 Weller, R. A., and A. F. Pilgrim. 1974. Passage o f protozoa and volatile fatty acids from the r u m e n o f the sheep and from a c o n t i n u o u s in vitro ferm e n t a t i o n system. Br. J. Nutr. 32: 341.