Evaluation of Specific Variables Affecting In Situ Estimates of Ruminal Dry Matter and Protein Digestion James E.

Nocek J ANIM SCI 1985, 60:1347-1358.

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EVALUATION OF SPECIFIC VARIABLES AFFECTING IN SITU ESTIMATES OF RUMINAL DRY MATTER AND PROTEIN DIGESTION 1
James E. Nocek Agway Inc. 2 , Syracuse, NY 13221

Summary

Specific variables associated with the in situ digestion of soybean meal (SBM) were identified and investigated. Soybean meal was incubated in situ for seven periods of time ranging from 1 to 48 h. Bags were introduced into the tureen in two ways: (1) all bags introduced at the same time and (2) bags introduced at different times (all removed at once). The latter procedure resulted in higher rate constants for both dry matter (DM) and nitrogen (N) disappearance. Dry matter disappearance rates of cottonseed meal were not different between four cows in similar stages of lactation. However, differences developed within cows over a 6-wk period. Correction for bacterial N did not significantly alter rate of SBM N disappearance compared with no correction. The amount of SBM DM and N washed from bags before ruminal exposure varied with sample grind and weight. Disappearance was higher for bags with porosities of 80 and 102/am compared with 6, 20, 40 or 59 /am. Grinding SBM at 1, 2, 5 mm or unground did not influence rate constants for DM or N disappearance from polyester bags. As the ratio of sample weight to surface area increased (2.5, 12.6, 25.3 and 37.9 mg/cm2), DM and N disappearance rates decreased. Bag porosities of 6 and 20 ~m gave lowest and 80 and 102 /am highest rate constants of in situ DM and N disappearance. For SBM, a sample weight to surface area ratio of 12.6 mg/cm 2 and use of bags with porosities of 40, 59, 80 and 102/am resulted in acceptable estimates of ruminal protein availability.

(Key Words: Polyester Bag, Ruminal Available Protein, Sample Grind, Sample Weight, Bag Porosity.)
Introduction

Factors influencing in situ measurements have been examined; however, recommendations between laboratories are not consistent. Weakley et al. (1977) indicated that dry matter (DM) and nitrogen (N) degradation was less with coarse (2,000/am) than with a fine particle size (500 gm). Uden et al. (1974) suggested a ratio of sample weight to bag surface area of 3.5 mg/cm 2, which is below the maximum of 10 mg/em 2 recommended by Van Hellen and Ellis (1977). Except for barley, Lindberg and Varvikko (1982) reported that DM disappearance was greatest from bags with 36-/am pores, followed by 20 and 10 #m. The in situ digestion technique cannot characterize the digestion rate of solubles and(or) filterable efflux from bags. The possible confounding effect of this fraction may influence digestion rates of insoluble, potentially available materials. Researchers (Akin and Amos, 1975) have shown that certain ruminal bacteria tenaciously attach to plant particles. The impact of this contamination on N digestion rates has not been investigated. The objective of the present research was to evaluate specific variables associated with in situ DM and N digestion of SBM. These variables included: bag introduction sequence into the rumen, cow and time variation, bacterial N contamination, presoaking, sample weight:bag surface area ratio and bag porosity.

1The author would like to express appreciation to A. Warner, D. G. Braund, R. Ellis, C. Olcott and K. Materials and Methods Nash for technical assistance and J. English for assistance with statistical analyses. Polyester 3 bags measuring 22 x 9 cm with Dept. of Res. and Dev. porosities of 59 -+ 2/am were used in all experi3Tetko, Inc., Elmsford, NY. ments, except where otherwise indicated. Received March 26, 1984. Seams were double-sewn with nylon thread and Accepted January 22, 1985. 1347 JOURNAL OF ANIMAL SCIENCE, Vol. 60, No. 5, 1985

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1348

NOCEK TABLE 1. COMPOSITIONOF CONCENTRATE MIX FED TO LACTATING, RUMINAL-CANNULATEDCOWSa

Ingredient Hominy feed (IFN 4-02-887) Brewers grains (IFN 5-02-141) Dehydrated alfalfa (IFN 1-00-023) Soybean meal (IFN 5-04-612) Soyhulls (IFN 1-O4-560) Molasses (IFN 4-04-560) Ground limestone (IFN 6-02-632) Dynamate c Trace mineral mixture Vitamin ADE mixtured

Percentageb 48.4 14.7 14.0 12.7 6.8 3.0 .23 .10 .05 .02

aThe total diet consisted of 25% corn silage, 25% hay crop silage (alfalfa) and 50% concentrate mix on a dry matter basis. bDry matter basis. CDynamate| registered tradename with International Minerals and Chemicals Corporation for feed grade double sulfate of magnesium and potassium. dprovides 5,200, 2,640 and .5:3 IU of supplemental vitamins A, D and E, respectively, per kg of concentrate mix.

needle holes were sealed with waterproof glue 4. A specific a m o u n t of dry feed was weighed into bags, based on calculated dry matter determined on separate aliquots. Bags were closed and tied with 1~ylon string. The rumen suspension device (RSD) consisted of a 9-gauge wire loop (6.8 x 4.2 cm), sandwiched and brazed between four metal washers (4.4 cm diameter), totaling approximately 150 g in weight. At each time interval (1, 2, 4, 8, 12, 24 and 48 h), quadruplicate sets of bags were tied to one end of the RSD and .5 m of 13.6-kg-test braided fishline was fastened to the other end. Just before ruminal suspension, bags were soaked in a specific a m o u n t of 39 -+ 3 C tap water (1 liter/250 g feed N; Crooker et al., 1978) for 15 rain to remove soluble and(or)59-/~m filterable material. Groups of bags were submersed into the liquid strata of ruminal contents in the mid-ventral region. The fishline was secured to the rumen cannula cover to allow bag movement with contents. An additional bag containing 5 g of cottonseed meal (CSM) ground at 2 mm was used as a control feed to monitor day-today variation of l u m e n function. Cottonseed meal was chosen because it exhibits a linear rate

4Testors; The Testor Corp., Rockford, IL.

of DM disappearance over extended time intervals (100 h), with 15 to 20% residual DM remaining. Test feeds were retested if DM disappearance of CSM varied more than 5% from established standard disappearance curves. Lactating Holstein cows with ruminal cannulae (10 cm id) were used from 2 to 5 mo after the onset of lactation. Cows were fed a total mixed diet (table 1) consisting of 50:50 forage:concentrate mixture. Animals were fed ad libitum at 500, 1000, 1600 and 2100 h. Bags were rinsed immediately after removal with running tap water and a second time (2 min/set of bags) while manipulating the feed sample residue within bags, until the rinse water was clear. Bags were dried to a constant weight at 55 C. Bags at each time period were emptied, composited and residues were ground (1 mm). Nitrogen analysis was by the macro-Kjeldahl method (AOAC, 1975). Percentage residual DM and N data were transformed to natural logarithms (loge) and subjected to linear regression vs time. Residues at the 48-h incubation times were used as the undigestible components (DM and N) for each regression and were subtracted from the residue remaining at each ruminal incubation time (Smith et al., 1971). Residual percentages of DM and N at 48 h were less than 3 and 4%, respectively. The following linear regression equation model was used:

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VARIABLES AFFECTING IN SITU DIGESTION R = - k t + R0, where R = In of percentage residual for DM or Y (Y), k = digestion rate coefficient (slope), t = time (X), R0 = residual DM or N at t = 0 (Y intercept). Each regression equation was calculated with and without the zero-hour time point to correct for lag (Mertens and Loften, 1980). Comparisons for parallelism (Sokal and Rohlf, 1969; Snedecor and Cochran, 1980) revealed no differences (P>.10) in rate constants, so the zero-hour time remained in the regression. Lack-of-fit tests (Sokal and Rohlf, 1969) wereconducted on all regression equations (0 to 24 h) and were nonsignificant for DM and N disappearance, suggesting one linear rate of digestion [Kd -- rate constant (%/h); 1 0 0 x In regression coefficient]. The effects of placing all bags in the rumen at once and removing them at designated time intervals vs introduction of bags in reverse sequence and removing them all at once (i.e., 48-h sample placed in rumen first, 1-h sample last) was investigated. Soybean meal was ground at 2 mm and 5-g (12.6 mg/cm 2) samples were used. Two replications (time series incubations) of each procedure were conducted on the same cow to determine the influence on DM and N disappearance rate constants. The latter procedure for bag introduction was used in all subsequent experiments. Four lactating Holstein cows between 2 and 5 mo postpartum were used to determine the effect of cow and time on CSM DM disappearance rates. All cows were fed the same diet for at least 2 wk before, and continuing throughout, the experiment. Duplicate 5-g samples of CSM were incubated (1, 2, 4, 8, 12, 24 and 48 h) for 6 consecutive wk on the same days of each week in each cow. Soybean meal was used to determine the influence of bacterial N contamination after ruminal exposure and its influence on N disappearance rate. Two replications were conducted within the same week with one cow. Sample

1349

s Tetko, Inc., Elmsford, NY.

weights were 5 g/bag with 59-/am porosity bags. Rumen exposure times were those previously described. Diaminopimelic acid (DAPA) analysis (Czerkawski, 1974; el-Shazley and Hungate, 1966) was conducted on composited residues at each fermentation time. Bacteria were harvested twice (1,000 x g for 10 min followed by centrifugation of the supernatant at 16,000 x g for 20 min) to quantify DAPA. Bacterial N content of the sample residue was calculated from DAPA concentration by the ratio of bacterial N:DAPA in isolated rumen bacteria. Recovery of DAPA was 103.2 -+ 6.1% for 80 /ag added and bacterial N:DAPA ratio was 34.2 -+ 1.1. The effect of sample grind, weight and bag porosity on DM and N disappearance were investigated. Samples of soybean meal (SBM) were ground through a 1-, 2- and 5-mm screen in a Wiley Mill, and a fourth size was the unground, commercially processed form. Shown in table 2 are the mean weighted particle sizes of each grind after seiving. Sample weights were to provide 1, 5, 10 and 15 g of DM/bag, with sample DM weight to bag surface area exposures of 2.5, 12.6, 25.3 and 37.9 mg/cm 2. Bags containing SBM were presoaked as described previously. Upon removal from water, bags were lightly blotted and then dried to a constant weight at 55 C. Three replications of each of the 16 treatments were conducted. Additional bags were prepared to determine the influence of bag porosity on DM and N disappearance. Bag porosities were 6, 20, 40, 59, 80 and 102 /~m, according to manufacturers s specifications. Sample weights were 5 g (12.6 mg/cm 2) ground through a 2-mm screen. The presoak procedure was that described previously. Three replications of each of six treatments were conducted. Sample grinds, feed weights to bag surface area and bag porosities were those used to determine water soluble disappearance. Before ruminal suspension, all bags were presoaked. Ruminal suspension times were 1, 2, 4, 8, 12, 24 and 48 h. Three replications of each treatment were conducted. Comparisons for parallelism (Sokal and Rohlf, 1969 and Snedecor and Cochran, 1980) were made between rate constants for the bag introduction procedure and bacterial N contamination experiment. Data for each experiment (i.e., effects of cow-time variation, water soluble disappearance, sample grind, sample weight and bag porosity on DM and N disap-

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1350

NOCEK TABLE 2. PARTICLE SIZE DISTRIBUTION OF SOYBEAN MEAL AFTER GRINDING

Grind, mm a

2.36

2.00

1.68

1.18

Sieve size, mm .841 .590 %d

.420

.297

<.297 b

Mean particle sizec, mm

Unground 5 2 1

2 0 0 0

15 3 0 0

17 7 1 0

28 39 13 1

12 20 41 3

11 16 22 48

5 5 9 20

5 5 6 14

5 5 8 14

1.23 .99 .76 .52

aUnground = material as received; 5, 2, 1 mm = ground through a Wiley Mill. bpercentage of material remaining in the bottom collecting pan. CCalculated from weighted mean, excluding that portion collected in pan. dpercentage of material that remained on the specific size sieve.

pearance rate constants) were subjected to split-plot analyses of variance techniques. When main effects were significant, m e a n differences were tested b y D u n c a n ' s multiple range test.

Estimated

Ruminal

Protein

Availability.

Disappearance rate constants were applied to the prediction model of Van Soest et al. (1982). Other models utilizing in situ digestion rates have been proposed and used to estimate ruminal availability (Orskov and McDonald, 1970; Stern and Satter, 1982; C u m m i n s et al., 1983; and E r d m a n and Vandersall, 1983). R u m i n a l available protein (RAP) was calculated using the following basic e q u a t i o n :

RAP = 1 0 0 -

e BA i [kdi/(kd i + kPi)]), i=3

where e = sum of (1) soluble, (2) insoluble, potentially digestible and (3) undigestible fractions, fractional protein c o n c e n t r a t i o n in feed, A i = a m o u n t of feed dry m a t t e r constit u t i n g each fraction, KPi = rate of passage for each fraction, Kd i = rate of digestion for each fraction. Because all regression analysis for SBM d e m o n s t r a t e d nonsignificant lack-of-fit, it was assumed that SBM has one insoluble, potentially digestible c o m p o n e n t .

Estimates of soluble and 59-/~m filterable N digestion ( K d l ) were o b t a i n e d b y multiplying the insoluble N disappearance rate constant b y 3.0 (Nocek et al., 1983). This fraction was assumed to pass with the liquid phase of r u m e n contents. Liquid turnover rate (8.8%/h) was o b t a i n e d from Hartnell and Satter (1979) for cows b e t w e e n 13 and 24 wk of lactation. The value used for t u r n o v e r rate of SBM in the r u m e n was 4.9%/h as d e t e r m i n e d in vivo by Stern et al. (1983). The rate of undigestible passage was assumed to be the same as the insoluble rate. These literature estimates were used because they were d e t e r m i n e d in cows in the stage of lactation that closely approximated those used in the present study. R u m e n available-protein values obtained from these calculations o n t r e a t m e n t mean N disappearance rate constants were evaluated with in vivo d e t e r m i n a t i o n s o b t a i n e d by Santos et al. (1984) for lactating dairy cows.
Results and Discussion

Introducing bags in the r u m e n at specific time intervals and removing them as a group resulted in a faster (P<.05) rate constant of SBM DM and N disappearance t h a n introducing all bags at once and retrieving t h e m at specific s u b s e q u e n t time intervals (figures 1A and B). The relative degree of variation within procedures was slightly lower for bags retrieved all at once. When bags were i n t r o d u c e d all at the same time, a t t a c h m e n t lines became tangled and all bags had to be removed at each incub a t i o n period. More time was required to

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V A R I A B L E S A F F E C T I N G IN S I T U D I G E S T I O N
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Figure 1. Effect of bag introduction sequence on soybean meal dry matter (A) and nitrogen (B) disappearance in the rumen. All bags introduced into the rumen and removed at specific time intervals ( ~ - - ) , sets of bags introduced into the rumen at specific time intervals and retrieved as a group at the end of an incubation sequence (* - - -). Each point represents the mean of triplicate observations. Kd is percentage disappearance per h of DM or N from polyester bags (rate constant). SEB is the standard error of the regression coefficient of slope (R = --kt + R0). Polyester bags (59-t~m porosity, 12.6-rag sample/era 2, 2-mm grind) were soaked in a specific amount of 39 -+ 3 C tap water (1 liter/250 mg N) for 15 rain before ruminal incubation. retrieve bags, and for those bags that were returned to the rumen, the digestion process m a y have been interrupted, perhaps contributing to the slower digestion rate constants and

increased variability. In addition, the final wash for each individual group o f bags was m o r e likely to vary than w h e n all bags were retrieved and washed at once. In subsequent experiments, bags were i n t r o d u c e d at d i f f e r e n t times and all retrieved at the same time. Rates of CSM DM disappearance were n o t significantly d i f f e r e n t a m o n g cows (table 3). Over time, DM disappearance rate constants for 5 of 6 wk were w i t h i n .5%/h of each other. Week 3 t e n d e d to be lowest for all cows. Cows were in similar stages of lactation (2 to 5 too) with no a p p a r e n t seasonal or m a n a g e m e n t effects contributing to this trend. Weakley et al. (1983) showed no significant animal effect for DM disappearance of SBM, with N disappearance o n l y approaching significance at 12 h of ruminal exposure. These researchers also indicated no significant period (time) differences in s o y b e a n meal DM or N disappearance f r o m D a c r o n bags averaged across cows fed diets o f different hay:grain ratios. O t h e r researchers (Varga and H o o v e r , 1983) have used specific standard ingredients to m o n i t o r animal and diurnal variation in r u m e n f e r m e n t a t i o n . Data f r o m the present study suggest that ruminal f e r m e n t a t i o n patterns within animals m a y vary and that the inclusion of a standard ingredient to m o n i t o r variation over t i m e is necessary w h e n using the in situ procedure. No significant differences were d e t e c t e d b e t w e e n N disappearance rate constants with or w i t h o u t correction for bacterial N contamination (figure 2; 10.6 vs 10.3%/h). Bacterial N as a percentage o f t o t a l residual N (figure 3), d e m o n s t r a t e d a quadratic (P<.05) response, peaking at 12 h o f r u m e n exposure. These data suggest t h a t bacteria attach to feed particles at a fast rate up to 4 h. The rate of c o n t a m i n a t i o n and(or) a t t a c h m e n t proceeds at a slower rate until 12 h, t h e n declines. These characteristics m a y be a f u n c t i o n of limiting a t t a c h m e n t sites for continual digestion and(or) substrate availability ( L a t h a m et al., 1978). Blair and C u m m i n s (1983). r e p o r t e d m e a n percentage microbial N c o n t a m i n a t i o n for SBM after 18 h of ruminal e x p o s u r e to be 41.6% of t o t a l N after washing bags with water. F o r d e h y d r a t e d alfalfa, microbial N after 12, 18 and 30 h of r u m e n e x p o s u r e with a s u b s e q u e n t water wash was 4.1, 4.7 and 8.70% of t o t a l N, respectively. F o r SBM, the relative c o m p a r i s o n o f rate constants w o u l d seem permissable w i t h o u t

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13 5 2

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V A R I A B L E S A F F E C T I N G IN SITU DIGESTION
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procedure. Presoaking did not result in uniform DM disappearance for sample weight and grind. There were interactions (P<.05) between grind and weight for both DM and N disappearance. With the exception of 10-~and 15-g samples at 1-mm grind, DM disappearance tended to d e c r e a s e w i t h increasing particle size (figure 4A). Clumping of feed was often observed in l-ram grind bags, which became more n o t i c e a b l e as sample weight increased. Other r e s e a r c h e r s (Figroid et al., 1972; Stern et al., 1983) have observed clumping with different feedstuffs. Ehle et al. (1982) demonstrated no effect of

RUMINAL EXPOSURE TIME Ihl

Figure 2. Effect of bacterial N c o n t a m i n a t i o n on t h e rate of s o y b e a n meal N disappearance. Nitrogen disappearance w i t h o u t (e - - -) and w i t h (0 ) correction for bacterial N. Kd is percentage disappearance of N or N corrected for bacterial N from polyester bags. SEB is the standard error of the regression coefficient or slope (R = - k t + R0). Polyester bags (59-/am porosity, 12.6-mg sample/era ~ , 2-ram grind) were soaked in a specific amount of 39 -+ 3 C tap water (1 liter/250 mg feed N) for 15 min before ruminal incubation.

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correction for bacterial N, because they were not different. However, extrapolation to time zero, for quantification of N associated with a particular rate, may be questionable. More
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Figure 3. Bacterial nitrogen contamination of soybean meal at specific times after tureen exposure in polyester bag.

Figure 4. Effect of sample grind and sample weight on soybean meal dry matter (A) and nitrogen (B) disappearance from polyester bags (59-~m porosity) soaked for 15 min in 39 C water (1 liter/250 mg feed N). Each point is the mean of triplicate replications.

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1354
32Dry Matter
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Figure 5. Effect of bag porosity on soybean meM dry" matter and nitrogen disappearance from polyester bags (12.6-mg sample/cm 2 , 2-ram grind) soaked for 15 min in 39 C water (1 liter/250 mg feed N). Each point is the mean of triplicate replication.

SBM particle size on DM disappearance from bags, with no systematic effect on N disappearance. Presoaking produced nonuniform N disappearance values (figure 4B). Bags containing 1 and 5 g of SBM demonstrated a tendency for decreasing N disappearance as particle size increased (except 5 mm). Bags containing 10 and 15 g tended to decrease, then increase in N disappearance as sample grind increased. The 5-g sample showed a consistent trend for both DM and N disappearance (figure 4A, B). All sample weights (except 15 g) decreased in N disappearance from 1- to 2-mm grind. There were no differences (P>.10) between pore sizes up to 59 /am for DM and N disappearance (figure 5), however, disappearance tended to increase with increased bag pore size. Bag porosities of 80 and 102/am demonstrated more (P<.05) DM disappearance than smaller pore sizes and were not different from each other. Nitrogen washout for 6 and 20/am was lower (P<.05) than that for 80 or 102/am, with 4-0 /am not being different from all others. Lindberg and Knutsson (1981) showed increased loss of SBM DM as bag porosity increased from

10 to 36/am, after soaking bags in water for 6 h. They observed disappearance values considerably less than the present study (36 /am, 2.6%), using a 1-g sample weight ground to pass a 1-mm screen with agitation. A major limitation associated with the bag technique is the inability to characterize ruminal availability of soluble and(or) filterable material that may be degraded to a greater or lesser extent than the insoluble, digestible material (Mahadevan et al., 1980; Nocek et al., 1983). These preruminal disappearance data indicate that even before ruminal exposure, there are several physical characteristics of both feeds and bags that must be considered. Test for homogeneity of regression profiles indicated sample grind had no effect (P>.10) on DM or N disappearance rates from polyester bags (table 4). These data agree with Ehle et al. (1982), who demonstrated no significant effect of SBM particle size (1, 180, 600, 300 and 150 /am) on DM disappearance using 1-g samples (10.7 mg/cm 2). Weakley et al. (1977) indicated that SBM DM and N degradation was less with a coarse particle size (2,000/am) than with a fine size (500 /am). Presoaking could minimize differences between grinds because large particles may become hydrated and swollen, allowing more accessibility for microbial attachment and digestion. Rate constants for DM and N were higher (P<.01) for 1-g compared with all other sample weights. There were no mean differences in rate constants for DM and N among 5-, 10- and 15-g samples; however, those for 5-g samples tended to be consistently higher than for 10- or 15-g samples. The importance of sample weight to bag surface area ratio has been emphasized by several researchers (Uden et al., 1974; Van Hellen and Ellis, 1977). More recently, Varga and Hoover (1983) compared DM disappearance from various concentrates and forage with two bag sizes (9 17 and 13 x 21 cm) and two sample weights (25 and 59 g). Dry matter disappearance was depressed by 3 and 8 percentage units for sample weight to bag surface area ratios of 9.2 vs 20.3 and 18.3 vs 32.7 mg/cm 2 . Generally, as bag pore size increased, rate constants for SBM DM and N disappearance also increased; however, the increases were mainly associated with two porosity changes, 20 to 4 0 / a m and 59 to 80/am (table 5). Rate constants from bags with pororisites of 6 and

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VARIABLES AFFECTING IN SITU DIGESTION

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1356

NOCEK TABLE 5. THE EFFECT OF BAG POROSITY ON SOYBEAN MEAL DRY MATTER AND NITROGEN DISAPPEARANCE RATES FROM POLYESTER BAGS a DM N r2d .95 .93 .95 .94
.97

Porosity, tam 6 20 40 59 80 102

Kd b 6.1 ef 5.0 f 10.8 g 11.0g 12.9 h 12.6 h

SEB c .3 .3 .6 .5 .6 .5

Kd 5.2 f 4.2 f 10.2g 10.4g 12.8 h 12.2 h

SEB .4 .3 .7 .9 .7 .5

r2 .91 .90 .93 .92 .95 .97

.98

apolyester bags (12.6-mg sample/era 2 , 2-mm grind) were soaked in a specific amount of 39 + 3 C tap water (1 liter/250 mg feed N) for 15 min before ruminal incubation. bKd is percentage disappearance per h of DM or N from polyester bags (rate constant). CSEB = Standard error of regression coefficient or slope (R = --kt + R 0 ). dr2 = Coefficient of determination. eEach value is the mean of triplicate incubation replications in time for the same cow. f'g'hMean Kd in the same column that do not have a common superscript differ (P<.05).

20 # m w e r e slower ( P < . 0 1 ) t h a n t h o s e o f o t h e r sizes, a n d n o t d i f f e r e n t b e t w e e n e a c h o t h e r . R a t e c o n s t a n t s o b t a i n e d w i t h bag p o r o s i t i e s o f 4 0 a n d 5 9 / ~ m were s l o w e r ( P < . 0 5 ) t h a n 8 0 a n d 102 gtm. O t h e r r e s e a r c h e r s ( U d e n et al., 1 9 7 4 ; L i n d b e r g a n d V a r v i k k o , 1 9 8 2 ) have d e m o n s t r a t e d similar findings. R a t e o f d i g e s t i o n is a n i m p o r t a n t ' c r i t e r i o n for determining optimum combinations of p h y s i c a l f a c t o r s t o use f o r in situ experimentation, but both depressed and stimulated rates m a y b e e q u a l l y e r r o n e o u s . A m o s t logical

c o n s i d e r a t i o n w o u l d b e t o select criteria t h a t w o u l d p r e d i c t in vivo r u m i n a l d i g e s t i o n m o s t a c c u r a t e l y . H o w e v e r , very f e w in vivo e s t i m a t e s of R A P f o r SBM in l a c t a t i n g cows have b e e n r e p o r t e d . S a n t o s et al. ( 1 9 8 4 ) i n d i c a t e s in vivo R A P f o r SBM was 7 0 . 5 % f o r l a c t a t i n g cows. O t h e r in vivo studies f o r c a t t l e s h o w ranges f r o m 63 t o 79% ( P o t t e r e t al., 1 9 7 1 ; K r o p p e t al., 1 9 7 7 ; M e r c h e n et at., 1 9 7 9 ; L o e r c h e t al., 1 9 8 3 ; W e a k l e y a n d O w e n s , 1983). C a l c u l a t e d e s t i m a t e s o f R A P for SBM s u b j e c t e d t o d i f f e r e n t in situ variables are s h o w n in t a b l e 6.

TABLE 6. ESTIMATION OF IN SITU SOYBEAN MEAL RUMINAL AVAILABLE PROTEIN a AS INFLUENCED BY SAMPLE GRIND, FEED TO BAG SURFACE AREA RATIO AND BAG POROSITY Feed weight to bag surface area ratio mg/cm 2 2.5 12.6 25.3 37.9 Estimated RAP, % 79 70 65 65 tam 6 20 40 59 80 102

Grind
mm

Porosity Estimated RAP, % 53 48 69 72 73 73

n

Estimated RAP, % 72 70 71 71

2 5 Unground

aEstimated by the method of Van 8oest et al. (1982), rumen available protein (RAP) = 100 - ( i_e3_BA i [kdi/(kd i + kPi)] ), where e -- sum of (1) soluble, (2) insoluble, potentially digestible and (3) undigestible fractions. Santos et al. (1984) in vivo estimate of ruminal protein availability for soybean meal in lactating cows = 70.5%.

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VARIABLES AFFECTING IN SITU DIGESTION Estimated RAP for different SBM grinds was similar, and within 1 to 2 percentage units of the in vivo estimate by Santos et al. (1984). A feed to bag surface area ratio of 2.5 mg/cm 2 overestimated and ratios of 25.3 and 37.9 mg/cm 2 underestimated in vivo RAP. A ratio of 12.6 mg/cm 2 resulted in a RAP that most closely approximated in vivo. It is realized that there may be quality differences between the SBM used b y Santos et al. (1984) and the present trial. Ultimately, size of bags and sample weight will be dictated by the number and type of analyses one desires to conduct on the residue, and density of the feed ingredient. Bag porosities of 6 and 20 /am underestimated, whereas porosity of 40, 59, 80 and 102 /am compared favorably with in vivo RAP. Weakley et al. (1983) concluded that bag materials of small pore (5 /am) size limit influx of digesting agents, regardless of restriction on efflux of digested residues. Van Hellen and Ellis (1974) tested porosities of 1.2, 5 and 10 /am and reported no significant influx of neutral detergent fiber (NDF) into bags after 48 h of rumen exposure. However, there was a significant influx of NDF into bags with porosities of 100/am. These researchers recommended a pore size of no larger than 10 /am. It may be that different pore sizes are necessary, depending on the specific ingredient and nutrient being investigated. Under the conditions and criteria used in this study, the following conclusions can be made for in situ digestion variables and techniques involving SBM or like ingredients. 1) Bag Introduction: Ruminal introduction of bags in reverse order (i.e., 100 h first, 1 h last) decreased variation for in situ digestion profiles. 2) Cow and Time Variation: Use of a standard ingredient (CSM) detected time-related variation for in situ digestion profiles. 3) Bacterial N Contamination: Did not affect N digestion rate constant. 4) Presoaking: Disappearance of soluble DM and N are affected by particle size, sample weight to bag surface area and bag porosity. 5) Particle Size: Grinds of 1, 2, 5 mm, or unground, had no influence on DM or N digestion rate constants. 6) Feed Weight to Bag Surface Area Ratio: 12.6 mg/cm 2 compared most favorably

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with literature in vivo estimates for ruminal N digestion. 7) Pore Size: 40 to 102 /am were similar and higher in estimated ruminal protein availability than smaller pore sizes, and compared more favorably with in vivo literature estimates.
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