Evaluation of various methods for protecting soya-bean protein from degradation by rumen bacteria

Animal Feed Science and Technology, 25 (1989) 111-122 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

111

Evaluation of Various Methods for Protecting Soya-Bean Protein from Degradation by Rumen Bacteria D.M. WALTZ* and M.D. STERN

Department o[ Animal Science, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Received 23 June 1988; accepted for publication 27 October 1988)

ABSTRACT Waltz, D.M. and Stern, M.D., 1989. Evaluation of various methods for protecting soya-bean protein from degradation by rumen bacteria. Anim. Feed Sci. Technol., 25: 111-122. A dual-flow continuous-culture system was used to study the effects of protection method on protein degradation of soya-bean meal (SBM) by rumen bacteria. Treatments included solvent extraction (control), sodium hydroxide, ethanol, formaldehyde, expeller processing, propionic acid, extrusion and lignosulfonate. Diets contained approximately 17.0% crude protein, with 50% of the crude protein coming from the respective treated SBM and were fed to rumen bacteria in fermenters at a rate of 75 g dry matter day- ~. Crude protein degradation of formaldehyde-treated, expeller processed, propionic acid-treated, extruded and lignosulfonate-treated SBM diets were lower (P < 0.05) than the control diet. Sodium hydroxide and ethanol treatments did not affect (P < 0.05 ) crude protein degradation. Total bacterial N output was lowest (P < 0.05) for SBM protected by formaldehyde, expeller processing and lignosulfonate treatments. Undegraded dietary N in the effluent was highest (P < 0.05) for SBM protected by formaldehyde, expeller processing, propionic acid and lignosulfonate treatments. Protection by formaldehyde, expeller processing, propionic acid and lignosulfonate treatments increased (P < 0.05 ) total amino-acid flow compared with the control. Formaldehyde and expeller processing treatments also increased (P < 0.05) essential amino-acid flow compared with the control. An in sacco study was also conducted which showed that treatment of SBM with formaldehyde, lignosulfonate, or expeller processing resulted in the greatest (P < 0.05) reduction in protein degradation.

INTRODUCTION High-producing or rapidly growing animals may require more high quality protein than that provided by rumen microorganisms. This protein can be supplied by increasing the amount of dietary protein escaping degradation in the rumen. Various chemical and physical methods of treating proteins have been *Present address: Division Animal Feeds, Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 20857, U.S.A.

0377-8401/89/$03.50

© 1989 Elsevier Science Publishers B.V.

112 used to reduce their degradation in the rumen. Chemical treatments can be divided into two categories; methods in which the chemicals actually combine with the protein and those in which the chemicals alter the protein structure by denaturization. Formaldehyde (Rooke et al., 1982 ) treatment is an example of the first category and ethanol (Lynch et al., 1987), sodium hydroxide (Mir et al., 1984) and propionic acid (Waltz and Loerch, 1986) are examples of the latter category. The most successful physical treatment has been heat, however, there has been a wide variation in application methods and amount of heat utilized. Extrusion (Stern et al., 1985) and expeller processing (Broderick, 1986) are two protection methods involving heat. Calcium lignosulfonate treatment utilizes wood sugars and heat to elicit the Maillard reaction (Windschitl and Stern, 1988). All of these methods have been investigated recently, but most researchers have concentrated on one or two protection methods and this has made comparisons between methods difficult. The objective of this experiment was to examine the effects of various protection methods on ruminal microbial degradation of soya-bean protein and influence on amino-acid flow using the dacron bag technique and a dual-flow continuous-culture system. MATERIALAND METHODS

Dacron bag technique An in sacco study was conducted to determine N disappearance of 8 soyabean sources from dacron polyester bags suspended in the rumen. Treatments included: solvent-extracted soya-bean meal (SBM) (control), SBM treated with sodium hydroxide at 5% of dry matter (NaOH), SBM treated with formaldehyde at 0.8% of the crude protein (CH20), SBM treated with propionic acid at 5% of the dry matter (PROP), calcium lignosulfonate-treated SBM (LS03) (Reed Lignin, Rothschild, WI), ethanol-treated soya flakes (EtOH) ( Land O Lakes, Minneapolis, MN ), extruded SBM (EXTR) (Triple "F" Feeds, Des Moines, IA), and expeller processed SBM (EXPL) (West Central Coop, Ralston, IA). Soya-bean meal treated by the first 5 methods came from the same source. Soya-bean meals in the latter 3 treatments each came from different sources. Dacron polyester bags (Erlanger, Blumgart and Co., New York) bags measured 6 × 10 cm and had an average pore size of 52 + 16/~m. Soya bean samples were ground through a 1-mm screen and 0.5 g was weighed into each bag. Bags were tied securely with silk thread, and attached near the end of a 60-cm nylon cord anchored by a 70-g stainless-steel weight. An empty bag (blank) and one bag for each of the soya-bean sources was attached to each nylon line. Seven lines were used daily with one line for each of the sampling times; 0, 2, 4, 8, 12, 16 and 24 h. Lines were placed in a styrofoam bucket containing warm water and transported to the dairy barn. All lines except the 0-h line were secured to

113 the cannula plug and suspended in the ventral sac of the rumen of a lactating Holstein cow, approximately 2 h after feeding. The cow was fed a diet consisting of 28% maize silage, 30% alfalfa pellets and 42% grain mix. Bags were removed from the rumen at 2, 4, 8, 12, 16 and 24 h. Bags were washed under warm tap water until rinse water was clear and free of rumen matter. Bags were dried at 65 ° C for 48 h and weighed. This was done on 2 consecutive days to obtain duplicate measurements. The entire bag, including the remaining contents, was analysed for N by macro-Kjedahl (A.O.A.C., 1975). Disappearance of dry matter (DM) and N from dacron bags was expressed as a percentage of original DM and N weighed into the bags.

Continuous-culture system The dual-flow continuous-culture system utilized in this study was designed to allow for differential solid-liquid removal rates as described by Hoover et al. (1976) and modified as described by Hannah et al. (1986). A buffer solution (Weller and Pilgrim, 1974), with urea added at 0.4 g 1-1 was infused at 1.72 ml m i n - 1to provide a liquid dilution rate of 0.10 h - 1. Retention time of solids was maintained at 18 h by adjusting the ratio of the filtrate: overflow effluent. The pH was maintained at 6.30 + 0.05 by infusion of 3 N HC1 or 5 N NaOH. Inoculum was obtained from a ruminally cannulated Holstein cow and was strained through 4 layers of cheesecloth. The cow was fed a total mixed diet consisting of 35% maize silage, 15% alfalfa cubes, and 50% concentrate mix on a DM basis. The concentrate mix contained 51.1% ground maize, 25% ground oats, 15% soya-bean meal, 5% dried molasses, 1.5% limestone, 1.0% monoammonium phosphate, 0.9% trace mineralized salt, 0.3% vitamin (A, D ), premix and 0.2% magnesium oxide (DM basis). Fermenters were provided with 75 g DM of a pelleted diet (Table 1 ), given in 8 equally spaced portions over a 24-h period. Fifty percent of the crude protein in the total diet was provided by the respective soya-bean products identified in the dacron bag study. Diets were formulated to be isonitrogenous and chemical analysis of the diets showed that crude protein ranged from 16.617.2% (DM basis). Components of each diet were ground through a 2-mm screen, mixed and pelleted (6 mm × 10 mm long) to facilitate automatic feeding. The experiment consisted of 4 experimental periods, with 5 days of adjustment followed by 3 days of sampling in each period. Fermenters were randomly assigned to treatments. Effluent collection vessels were maintained in a 2 °C water bath during the 3 days of sampling to retard microbial growth. Solid and liquid effluents were combined, homogenized with a Brinkmann Polytron (Brinkman Instruments, Westbury, NY) for 5 min and a 600-ml sample was removed daily during the 3 days of sampling. The three samples from each fermenter were composited and a portion was freeze-dried for each period.

114 TABLE 1 Ingredient and chemical composition ( % of dry matter) of complete mixed diets provided to fermenters Item

Diet 1 Control

NaOH

EtOH ~

CH20

EXPL 2

PROP

EXTR 2

LSO,~

Ingredient Ground maize Corn silage Alfalfa Soybean meal Molasses Trace mineralized salt

40.0 20.7 16.0 19.3 2.0 2.0

40.0 20.7 16.0 19.3 2.0 2.0

40.0 23.9 15.0 17.1 2.0 2.0

40.0 20.7 16.0 19.3 2.0 2.0

40.0 20.0 16.2 19.8 2.0 2.0

40.0 20.7 16.0 19.3 2.0 2.0

40.0 20.7 16.0 19.3 2.0 2.0

40.0 20.7 16.0 19.0 2.0 2.0

Chemical Organic matter Crude protein Neutral detergent fiber Acid detergent fiber

87.2 16.9 30.1 13.9

87.0 16.7 28.2 13.0

87.0 16.7 32.3 13.9

87.2 16.6 31.2 14.8

86.8 16.7 30.2 14.0

86.9 16.9 31.4 14.1

86.8 17.2 30.9 15.0

87.1 16.6 30.1 14.4

1Control, solvent-extracted SBM; NaOH, SBM treated with sodium hydroxide at 5% of dry matter; EtOH, ethanol-treated soya flakes; CH20, SBM treated with formaldehyde at 0.8% of the crude protein; EXPL, expeller processed SBM; PROP, SBM treated with propionic acid at 5% of dry matter; EXTR, extruded SBM, LSO~, lignosulfonate-treated SBM. 2Different SBM source than the control. -~-- cm~rol

100

- - 8 - - NIIOH EtOH

80

~

c~o

6

0 c" ¢}

03

z

0

4

8

12

16

20

24

Rumen exposure, h

Fig. 1. Nitrogen disappearance from dacron bags suspended in the rumen containing solventextracted SBM {control), sodium hydroxide-treated SBM (NaOH), ethanol-treated soya flakes ( E t O H ) , formaldehyde -treated SBM (CH20), expeller processed S B M ( E X P L ) , propionic acidtreated SBM ( P R O P ) , extruded S B M ( E X T R ) and lignosulfonate-treated SBM (LSO3).

115

Sample analyses All analyses were performed on composite samples. Fresh composite samples were used for total N, NH3-N, and volatile fatty acid (VFA) analyses. Freeze-dried samples were ground through a 1-mm screen and used for all other analyses. Bacteria were harvested from fermenter contents remaining on the last day of each period by straining through two layers of cheesecloth. Strained contents were centrifuged at 1000 g for 10 min to remove feed particles. Bacteria were separated from the supernatant by centrifuging at 20 000 g for 20 min and then freeze-dried. Ammonia-N was determined by steam distillation (Bremner and Keeney, 1965). Volatile fatty-acid concentrations were determined using a Hewlett Packard 5880A gas chromatograph (Hewlett Packard, Palo Alto, CA), with a 80/120 Carbopack DA/4% Carbowax 20M column. Samples were prepared for analysis by the method of Erwin et al. (1961). Nitrogen contents of diets, effluent and bacteria were determined by macro-Kjeldahl procedure (A.O.A.C., 1975). Fibre composition was determined by the detergent system, using the sequential analysis method of van Soest and Robertson (1980). Total nonstructural carbohydrate (TNC) was determined as described by Smith (1969) modified to use ferricyanide as a colorimetric agent. Diaminopimelic acid (DAPA) concentrations were determined for effluent and bacterial cells by the procedure of Czerkawski (1974). Diet and effluent samples were prepared for amino-acid analysis using a modified procedure of Moore et al. (1958). Amino-acid concentrations were determined on a Beckman 119CL amino-acid analyzer (Beckman, Palo Alto, CA). Effluent DM was determined by weighing 500-g samples of homogenized effluent, freeze-drying and reweighing. One-gram samples of freeze-dried effluent were weighed, dried at 105 °C for 24 h and absolute DM was determined. Dried samples were ashed at 550°C for 12 h in a muffle furnace to measure organic matter (OM) content. DM and OM digestibilities were calculated as described by Crawford et al. (1980), with modifications described by Hannah and Stern (1985).

Statistical analyses Results of the in sacco studies were analyzed statistically, as a completely randomized design, by the general linear model of the statistical analysis system (SAS, 1979). Duncan's multiple range test (Duncan, 1955) was used to compare treatment means with significant F values. The continuous-culture experiment consisted of 4 replications (4 experimental periods). Eight fermenters were used in each period. Data were analyzed using the general linear model of the statistical analysis system (SAS, 1979 ). The experiment was analyzed as a randomized complete block design and Duncan's multiple range test (Duncan, 1955) was used to compare means.

116

RESULTS

Dacron

AND DISCUSSION

bag experiment

Disappearance of N from soya-bean products placed in dacron bags suspended in the rumen is shown in Fig. 1 and Table 2. Using this method, formaldehyde-treated and lignosulfonate-treated SBM had the lowest rate and extent of protein degradation (P < 0.05), followed by expeller processed SBM. Sodium hydroxide and EtOH-treated SBM had the highest (P < 0.05) proportion of readily available N at 0 h, however, extent of protein degradation was similar ( P < 0.05 ) to that of the control. Continuous-culture

experiment

The influence of SBM source on DM, OM and carbohydrate digestibilities is shown in Table 3. True OM digestibility was lowest (P < 0.05) for diets containing expeller processed SBM. Diets containing formaldehyde and lignosulfonate-treated SBM had a tendency towards lower true OM digestibilities, but these differences were not significant ( P > 0.05). True DM digestibilities followed similar trends. Soya-bean source did not affect (P > 0.05) apparent OM or total non-structural carbohydrate digestibilities. Differences ( P < 0.05 ) were observed among diets containing treated soya-bean sources in neutral deterTABLE2 In situ evaluation of SBM sources 1 Item

Protein degradation4 (%) N disappearance at 0 h '~ (%) N remaining at 24 h s (%) Rate of N disappearance~(h -1)

Treatment 2

SE3

Control N a O H

EtOH

CH20

EXPL

PROP

EXTR

LS02

55.5 g7

55.1 g

59.9 g

26.2 i

36.45i

45.95

49.0 gh

30.5 ~

6.6

6.75

23.5 g

17.7 g

1.45

1.45

3.35

8.15

4.55

3.4

22.5 i

35.0 ~

26.2 ~

71.4 g

51.65

38.35~

38.951

53.15

7.1

0.056 g

0.0365

0.054 g

0.017 i

0.0285i

0.0405

0.041 h

0.019 i 0.005

1Values are the means of single observations on 2 consecutive days. 2Control, solvent-extracted SBM; NaOH, S B M treated with sodium hydroxide at 5% of dry matter; E t O H , ethanol-treated soya flakes; CH20, S B M treated with formaldehyde at 0.8% of the crude protein; E X P L , expeller processed SBM; P R O P , S B M treated with propionic acid at 5% of dry matter; EXTR, extruded SBM; LS03, lignosulfonate-treated SBM. 3Standard error of the mean. 4Determined according to the equation of Mathers and Miller ( 1981 ) at kr= 0.05 h - 1. "~Expressed as a % of the original amount placed into the bag. 6Estimated as the slope of the regression of the natural log % remaining vs. time. 7Means in the same row not having a common superscript (g, h, i) differ ( P < 0 . 0 5 ) .

117 TABLE 3 Influence of S B M source on dry m a t t e r , organic m a t t e r a n d c a r b o h y d r a t e digestibilities {% of dry m a t t e r ) in continuous-culture f e r m e n t e r s ~ Digestibility

T r u e dry m a t t e r :~ A p p a r e n t organic m a t t e r T r u e organic m a t t e r :~ Total nonstructural carbohydrate Neutral detergent fibre Acid detergent fibre Hemicellulose Cellulose

Diet

SE ~

Control

NaOH

EtOH

CH20

EXPL

EXTR

PROP

LSO:~

63.6 e4 34.8 61.0 ~

65.2 ~ 37.1 63.8"

64.3 ~ 38.8 62.6 ~

56.0 d~ 33.6 53.1 d~

51.5 d 38.2 47.8 d

61.9 d~ 38.4 59.3 ~

63.7 ~ 32.7 60.3 ~

57.4 d~ 36.4 54.8 d~

1.9 1.4 2.2

85.6 49.5 ~e 49.5 ~ 42.7 ~ 56.4

89.7 53.7 ~ 52.2 ~ 48.6 ~ 59.5

87.3 50.8 d~ 47.8 d~ 43.4 d~ 55.7

85.1 47.5 d 45.2 d 39.6 d 55.8

85.1 48.6 d~ 46.8 d~ 41.0 d 57.1

86.5 49.3 d~ 47.0 de 43.2 d~ 56.9

84.2 49.1 d~ 48.4 d~ 41.8 d~ 57.7

85.2 47.7 ~ 45.0 41.3 d 55.3

1.9 2.7 2.3 2.0 3.1

~Each value is the m e a n of 4 observations. ~Standard error of the t r e a t m e n t m e a n . :~True digestibility was corrected for bacterial m a t t e r . ~Means in the s a m e row not h a v i n g a c o m m o n superscript (d, e ) differ ( P < 0.05 ).

TABLE 4 Influence of S B M source on volatile fatty-acid c o n c e n t r a t i o n s in continuous-culture f e r m e n t e r s ~ Item

SE ~

Diet Control

NaOH

EtOH

CH20

EXPL

PROP

EXTR

LSO:~

Total VFA ( m M )

116.4

105.5

119.1

115.7

113.4

117.3

108.8

108.3

3.2

Individual VFA ( m M ) Acetate Propionate Isobutyrate Butyrate 2-Methyl b u t y r a t e

67.3 ":* 28.9 ca 1.0 12.8 2.1 a

59.2 d 25.5 c 0.8 13.6 2.3 de

67.2" 30.9 ca 0.9 13.8 2.2 d

57.8 d 36.9 d 0.7 14.5 1.2 c

67.P 26.8" 1.0 12.4 2.1 d

66.3 c 31.7 ~a 0.9 12.6 1.7 "d

62.8 "a 26.T 0.9 11.9 2.7 e

61.4 "d 26.T" 0.9 13.2 2.1 d

1.4 1.6 0.1 0.7 0.3

1.3 ~ 3.if"

1.0"d 3.1'

1.1 ':d 3.0 c

0.8' 3.8 a

1.0 cd 3.1'

1.0 cd 2.8 c

1.0 c~ 3.0 ('

0.1 0.2

Isovalerate Valerate

1.1 cd 2.9"

~Each value is the m e a n of 4 observations. '-'Standard error of the t r e a t m e n t m e a n . :~Means in the same row not h a v i n g a c o m m o n superscript (c, d, e) differ ( P < 0 . 0 5 ) .

gent fiber, acid detergent fiber and hemicellulose digestibilities but none of these were different from the control diet. Cellulose digestibilities were not affected ( P > 0.05) by soya-bean treatment. Total VFA (Table 4) was not affected ( P > 0.05) by diet. Diets containing formaldehyde and sodium hydroxide-treated SBM had lower (P < 0.05) acetate concentrations than the control diets. Propionate concentration had a tendency to be higher for the diet containing formaldehyde-treated SBM but

118

this was not significant (P>0.05). Soya-bean treatment had no effect (P > 0.05 ) on butyrate or isobutyrate. The diet containing formaldehyde-treated SBM had lower ( P < 0.05 ) 2-methyl butyrate, isovalerate and higher (P < 0.05 ) valerate concentrations than the control diet. This reduction in branched-chain VFA is probably a result of lower protein degradation of formaldehyde-treated SBM because branched-chain VFA are produced by deamination of branchedchain amino acids. The diet containing extruded SBM had a higher ( P < 0.05 ) 2-methyl butyrate concentration than the control diet.

Nitrogen metabolism The diet containing formaldehyde-treated SBM resulted in lower (P < 0.05 ) crude protein degradation, ammonia N concentration, bacterial N flow and efficiency of bacterial synthesis and higher (P < 0.05) non-ammonia and dietary N flows than diets containing control SBM (Table 5). This is consistent with results from several researchers (Tamminga, 1979; Rooke et al., 1982; Mir et al., 1984) who reported that formaldehyde was quite effective in reducing protein degradation by rumen microorganisms. Protection is afforded by the chemical combination of formaldehyde with the lysine residues of the protein. However, production responses have been mixed, possibly owing to overprotection resulting in reduced protein or lysine digestion in the small intestine. The expeller processed SBM diet had lower (P < 0.05 ) crude protein degradation, bacterial N flow and efficiency of bacterial synthesis and higher (P < 0.05 ) dietary N flow than the control diet. Expeller processing is a method that involves heating to a maximum of 163 °C which results in the Maillard TABLE 5 I n f l u e n c e o f S B M s o u r c e o n n i t r o g e n m e t a b o l i s m in c o n t i n u o u s - c u l t u r e f e r m e n t e r s ~ Item

Ammonia-N ( m g 100 m l ~) Degradation of dietary crude protein (%) E f f l u e n t N flow ( g d a y Total Non-ammonia Bacterial Dietary B a c t e r i a l s y n t h e s i s (g N kg ~OM truly digested)

Diet

SE ~

Control

NaOH

EtOH

CH20

EXPL

PROP

EXTR

LSO:~

21.7 "~

21.3 ~

20.2'

12.9 '~

18.9 'd

18.8 ''d

17.2 "d

14.5 d

0.9

85.5'

79.0 ''d

68.7"'"

32.01

40.9 ~

57.8 de~

65.7 d('

52.3 ~

5.7

~) 2.89 2.28" 1.96"

2.79 2.33 '' 1.95 ~

2.89 2.39 "d 1.56 c'l

2.85 2.53 e 0.68"

2.86 2.39 cd 0.92 de

2.82 2.4@'" 1.27 cd

3.06 2.6ff 1.67 `d

2.83 0.12 2.47 d`' 0.10 1.10 de 0.08

0.32'

0.38'

0.83 `d~"

1.85 ~

1.47 "~

1.13 do~

0.93 ~d~

1.37 `l"j 0.09

38.8 ~

39.6 ~

33.3 ','j

15.8"

20.7 ~l~

27.6 'j

~Each v a l u e is t h e m e a n o f 4 o b s e r v a t i o n s . -'Standard error of the treatment mean. :~Means in t h e s a m e r o w h a v i n g a c o m m o n s u p e r s c r i p t (c, d, e, f) d i f f e r ( P < 0 . 0 5 ) .

35.7 '`d

23.9 d"

2.1

119 reaction between sugar aldehyde groups and free amino groups. If the extent of this reaction can be controlled by regulating the amount of heat applied, ruminal protein degradation can be decreased without adversely affecting intestinal protein digestion. Broderick (1986) reported that expeller processed SBM provided about 65 % more undegraded dietary protein than solvent SBM and improved milk to feed ratio in lactating dairy cows. He also found that smaller amounts of expeller SBM could replace solvent SBM without reducing milk production. This suggests that SBM protected by expeller processing may be available for digestion postruminally. Ammonia N concentration, crude protein degradation, bacterial N flow and efficiency of bacterial synthesis decreased (P < 0.05 ) while non-ammonia and dietary N flows increased (P < 0.05 ) with the lignosulfonate-treated SBM diet compared with the control diet. These results agree with observations in a previous continuous-culture study (Stern, 1984). Lower ruminal ammonia N concentrations, crude protein degradation and efficiency of bacterial synthesis were also observed in vivo with cows fed a diet containing lignosulfonate-treated SBM (Windschitl and Stern, 1988). Lignosulfonate treatment involves the Maillard reaction but utilizes xylose, and possibly other wood sugars, in addition to the sugar aldehydes present in the SBM. The diet containing propionate-treated SBM had lower (P < 0.05) crude protein degradation and efficiency of bacterial synthesis and higher (P < 0.05) dietary N flow than the control diet. However, efficiency of bacterial synthesis was higher (P < 0.05 ) for the propionate-treated SBM than the formaldehydetreated SBM diet. Propionic acid treatment probably reduces protein degradation by altering protein structure and protein solubility. Waltz and Loerch (1986) reported that propionate treatment decreased N solubility, in vitro ammonia N accumulation and in sacco N disappearance without reducing total tract N digestion in lambs. The extruded SBM diet had a higher ( P < 0.05) non-ammonia N flow and a lower ( P < 0.05) crude protein degradation than the control diet, but crude protein degradation was higher (P<0.05) when compared with the formaldehyde diet. Stern et al. (1985) reported that extrusion of whole soybeans reduced crude protein degradation but did not affect non-ammonia N flow from the rumen in vivo. Extrusion, like expeller processing, involves heat but extruded SBM is only heated to 149°C. In this experiment, it did not appear to be as effective as expeller processing. This difference may be owing to the lower temperature or possibly to differences in the length of time that SBM was heated. Sodium hydroxide-treated SBM and ethanol-treated soya flake diets were not different ( P > 0.05) from the control diet in any of the N variables measured. In contrast, Mir et al. (1984) reported that treating SBM with NaOH decreased N disappearance in sacco. In addition, ethanol treatment has been reported to reduce the rate of protein disappearance in sacco and ammonia

120

release in vitro (van der Aar et al., 1982) and improve N retention in lambs (Lynch et al., 1987). It is unclear why NaOH and ethanol treatments had no significant effect on protein degradation in this experiment. Amino acids Table 6 shows the influence of treatment method on effluent flow of amino acids. Total amino acids and nonessential amino acid flows were highest (P < 0.05) for the formaldehyde-treated SBM. Expeller processed, lignosulfonate and propionate-treated SBM had higher ( P < 0.05) total amino acid and nonessential amino acid flows than the control diet. Essential amino acid flows were higher ( P < 0.05) for formaldehyde-treated and expeller-processed SBM than the control diet. Formaldehyde treatment increased (P < 0.05 ) the effluent flow of all essential amino acids compared with the control diet except for lysine, valine, methionine and isoleucine. Expeller processing increased ( P < 0.05) the flow of arginine, leucine and isoleucine flows compared with the control diet. LignoTABLE 6

Influence of SBM source on effluent flow (mg d a y - l ) of amino acids I Item

Diet Control

Total amino acids Intake Effluent flow Essential amino acids Intake

Effluent flow Nonessential amino acids Intake

Effluent flow Effluent flow of individual amino acids Lysine Histidine Arginine Threonine Valine Methionine Isoleucine Leucine Phenylalanine

SE 2 NaOH

11406 11468 9905 e3 9859 e

EtOH

CH20

EXPL

PROP

EXTR

LSO3

11264 10101 de

11581 12246 ¢

11330 11141 d

11672 10872 d

12059 10479 de

11273 10708 d

557

5092 4450 d

5137 44736

4995 4593 d

5102 5406 ¢

5092 5123 ¢

5141 4885 Cd

4871 4786 cd

4963 4873 cd

283

6314 5355 e

6331 5386 e

6269 5508 e

6479 6840 c

6238 60186

6531 5987 de

7188 5693 de

6310 58356

302

646 c 163 d 411 e 557 d 5416

548 d 175 d 438 e 564 d 536 d

536 d 159 d 426 e 557 d 541 d

632 ¢ 228 c 639" 654 ¢ 502 d

627 c 196 cd 508 d 607 cd 594 Cd

627 c 162 d 485 d~ 622 cd 608 ¢

62V 173 d 449 e 574 d 556 d

282 5746 872 ~ 504 d

280 559 d 874 ~ 499 d

299 605 d 949 d~ 521 d

296 649 ~d 1152 c 654 ¢

321 688 ~ 1003 Cd 579 cd

277 604 d 951 d 549 d

306 63V d 935 de 541 d

1Each value is the mean of 4 observations. 2Standard error of the treatment mean. ~Means in the same row not having a common superscript (c, d, e) d i f f e r ( P < 0.05 ).

643 ~ 201Cd

32 13

539 d 580 d

25 38

566 d

29

257 579 d

28 35

958 d 550 d

61 54

121 sulfonate t r e a t m e n t increased ( P < 0.05 ) arginine and leucine flows. Propionate treatment increased ( P < 0 . 0 5 ) leucine flow while ethanol-treated soya flakes and N a O H treatment reduced ( P < 0 . 0 5 ) the flow of lysine from the fermenters. CONCLUSION In this experiment, 5 of the 7 treatments including formaldehyde, expeller processing, lignosulfonate, propionate and extrusion reduced protein degradation by ruminal bacteria and with the exception of extrusion, increased total amino acid flow. The extent to which they reduced protein degradation differed, with formaldehyde appearing to be the most effective followed by expeller and lignosulfonate treatment. Propionate and extrusion appeared to be the least effective treatments for reducing bacterial degradation. Different protection methods had varying effects on individual amino acids. The most obvious difference was between formaldehyde and expeller treatments. T h e y both increased arginine and leucine flows but formaldehyde also increased histidine, threonine and phenylalanine, whereas expeller processing increased valine and isoleucine. Because the amino acid composition of the protein escaping degradation could be as important as the total amount of amino acids in deciding which protection method to use, differences in individual amino acid flow and availability in the small intestine between methods should be investigated further. ACKNOWLEDGEMENT Published as Paper No. 16 093 of the Scientific Journal Series of the Minnesota Agric. Exp. Stn. on research conducted under Project No. 16-048 supported by the College of Agriculture.

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