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In Vitro Estimation of Dry Matter and Crude Protein Digestibility
In Vitro Estimation of Dry Matter and Crude Protein Digestibility M. CLUNIES and S. LEESON
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N lG 2W 1 (Received for publication January 24, 1983)
1984 Poultry Science 63: 89-96 INTRODUCTION
Traditional in vitro procedures, such as pepsin digestion in acid solutions, have been used to evaluate protein digestibility of feedstuffs for monogastric animals. Johnston and Coon (1978) correlated digestion of protein, using .02% pepsin in .075 N hydrochloric acid solution, to weight gain obtained in 7-day protein quality growth assays. Regression analysis with pepsin digested protein gave a correlation coefficient of .91 for 20 protein ingredients assayed. Two-phase in vitro procedures utilizing rumen fluid followed by pepsin digestion have been used to determine the intake of roughages offered to ruminant animals (Tilley and Terry, 1963). Furuya et al. (1979) proposed a new twostage in vitro method using pepsin and pig intestinal fluid to estimate the digestibility of dry matter (DDM) and crude protein (DCP) of swine diets. The system attempts to simulate gastric and intestinal digestion in swine or other
1
monogastric animals. Samples of diets, ground to pass through a 1 mm screen, were incubated for 4 hr with a commercial pepsin preparation in acid solution followed by 4 hr of incubation with prepared intestinal fluid. Because digestion by monogastrics is quite similar (Hill 1962), Sakamoto et al. (1980) used the two-stage in vitro system to estimate in vivo digestibility of dry matter and crude protein in diets offered to poultry. Regression analysis relating in vivo to in vitro results provided correlation coefficients(r) of .98 and .99 for DDM and DCP, respectively. Furuya et al. (1979) reported that prepared intestinal fluid may be stored at -25 C or lyophilized (Sakamoto et al., 1980) without any significant change in %DDM and %DCP in vitro. Experiments were carried out to examine the method and possible application for routine analysis for quality control of feeds and feed ingredients offered to poultry. MATERIALS AND METHODS
Collection and Preparation of Intestinal Fluid. An 8-month-old Yorkshire gilt weighing approximately 100 kg was fitted with a T-
ICN. K & K Laboratories, Inc., Cleveland, OH.
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ABSTRACT A number of experiments were carried out to determine some of the parameters affecting the estimation of apparent digestibility of dry matter (DDM) and crude protein (DCP) by a two-stage, in vitro method, Experiments investigated the effects of pepsin concentration, time of incubation, pH, the effect of particle size on DDM and DCP, and the relationship between apparent in vivo and in vitro digestibility. Increasing the concentration of pepsin to 1140 units/10 ml .075N Hel in the first phase of incubation increased %DCP (P<'05), while %DDM increased significantly (P<.05) only to 580 pepsin units/10 ml .075N HCI solution. In the first stage of incubation with pepsin there was a significant (P<.05) increase in %DDM and %DCP in the second 1-hr period. In the second stage of incubation, involving porcine intestinal fluid, %DDM increased significantly (P<.05) in the first three 1-hr periods, while no significant (P<.05) increase in %DCP was observed after the second 1-hr period. In the second intestinal stage of incubation, there was no significant (P>.05) difference in %DDM for pH's 6.6 to 6.9. At pH 6.5 there was a significant (P<.05) decrease in %DDM. There was no significant (P>.05) effect of pH on %DCP. By reducing particle size of test material the accuracy of predicting apparent in vivo DDM and DCP was increased for all diets. The in vitro method gave a reliable estimation of apparent DDM and DCP in vivo. The correlations (r) between in vitro and in vivo estimates were high, .99 and .93 for %DDM and %DCP, respectively, when diets were ground to pass through a .4 mm screen. Prediction equations obtained from linear regression analysis were y = .901X + 7.214 and y = 1.331X -26.66 for DM and CP, respectively, where y = in vivo digestibility and X = in vitro digestibility. (Key words: digestibility, dry matter, crude protein in vitro variables)
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CLVNIES AND LEESON
those conditions affecting accuracy of the method. Considerable variation was seen in DDM results and pH of the final incubate with the latter greatly affecting accuracy of results. As the preparation of pepsin in HCl and the sample of intestinal fluid were the same for all treatments, pH variability could only be attributed to neutralization with .20N NaOH solution as recomended by Furuya et al. (1979). Curves constructed from titration of 25 ml of .075N HCl with .20N NaOH showed a rapid rise in pH from approximately 5 to 9 when a few drops of base were added. As a result a slight modification was made to the procedure, in that the NaOH solution used in titration was diluted to a concentration of .1 ON, and a Tris-buffer! [(tris(hydroxymethyl)amino methane) was introduced to stabilize the pH near neutrality in the final stage of incubation. The first stage of incubation was carried out
TABLE 1. Percentage composition of experimental diets l Ingredients Ground corn Ground barley Gr.ound .oats S.oybean meal (48% CP) C.orn glu ten meal (600A. CP) C.orn distillers plus s.olubles Wheat bran Animal-vegetable blend fat Limestone Dicalcium ph.osphate I.odized salt (.01% C.oC0 3 , .15% Ca1 2) Mineral-vitamin mix Mineral-vitamin mix 2 DL-Methi.onine L-Lysine-HCI Chromic .oxide Calculated analysis Metab.olizable energy, kcallkg Crude protein, % Crude fiber, % Ether extract, %
Diet A
DietB
Diet C
Diet D
69.34
57.14
32.00 32.00
11.99 11.99
35.07
13.12
6.50 1.40 1.00 .25
2.50 .90 2.00 .25
3.00 1.44 2.30 .25
.75
.75
.75
52.77
37.13 10.00 .90 1.35 .45
2.70 .06 .20 .30 2976 16.3 7.8 9.7
.28 .30 3259 19.0 2.4 5.5
.30 3029 22.1 2.5 5.3
.30 2762 3 14.5 4.8 2.4
I Provides per kil.ogram diet: 5.0 mg copper, 30.0 mg iron, 55 mg manganesium, .1 mg selenium, and 50 mg zinc, 8,000 IV vitamin A, 1,600 IV vitamin D, 1.0 mg vitamin E, 9.0 mg rib.oflavin, 11.0 mg D-calcium pantohenate, 13 Jolg vitamin B 12 , 26.0 mg niacin, 900 mg ch.oline chl.oride, 1.5 mg vitamin D, 1.5 mg f.olic acid, .20 mg biotin, and 125 mg santoquin.
2Provides per kilogram diet: 19 mg manganese, 220 mg ir.on, 14 mg copper, 58 mg zinc, 6,336 IV vitamin A, 650 IV vitamin D, 18 IV vitamin E, 1.58 mg vitamin K, 1.1 mg thiamine, 2.9 mg riboflavin, 18 mg niacin, 15 mg pantothenic acid, .72 mg folic acid, 1.1 mg pyridoxine, .15 mg biotin, 1.1 mg vitamin B., 22 mg ethoxyquin, 816 mg choline, and .07 mg selenium. 3
Swine ME value.
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shaped cannula. The cannula, of a rubberized silicone material, was surgically placed in the upper jejunum approximately 25 em distal to the opening of the pancreatic duct. The surgical method was adapted from Furuya and Takahashi (1975). The pig, housed in a gestating stall, was fed 2.5 kg of diet D (Table 1) during the period of collection. Approximately .6 liters of intestinal contents were collected daily 1 to 2 hr after feeding. Intestinal contents collected were tranferred to the laboratory in an ice bath. Intestinal samples were centrifuged at 1700 X g, the supernatant recovered, and supernatant used fresh or stored in polyethylene bottles at -25 C. In Vitro Digestibility Procedures. The method of in vitro digestibility was based on the procedure described by Furuya et al. (1979). Initial studies with this procedure yeilded unpredictable and variable results and, hence, further trials were designed to study
IN VITRO ESTIMA TION OF DIGEST IBILITY
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hemogl obin as substrat e. The first stage of incubat ion was followe d by the second stage of incubat ion with 10 ml of prepare d intestin al fluid for all treatme nts in this experim ent. Experim ent 2. Time of Pepsin Digestion. Four periods were selected for incubat ion: 1, 2, 3, and 4 hr. In this experim ent only the first stage of incubat ion was comple ted. For this first stage the samples were incubat ed with 1140 units of pepsin enzyme in 10 ml of .075N HCI solution . Experim ent 3. Time of Intestinal Digestion. Four periods of incubat ion were investig ated: 1, 2, 3, and 4 hr. In all treatme nts the intestin al stage of incubat ion with 10 ml of prepare d porcine intestin al fluid was precede d-by 4 hr incubatio n at 37 C with 1140 units of pepsin in 10 ml of .75N HCI soln. Experim ent 4. Effect of pH in Intestinal Stage. Followi ng the first stage of incubat ion with 1140 units of pepsin enzyme differin g amount s of NaOH solution were added to the product s of the first phase to effect differen t pH's prior to introdu cing the 10 mls of prepared intestin al fluid. The pH's examin ed were 6.5, 6.6, 6.7, 6.8, and 6.9. The pH of the final incubat e was noted prior to centrifu ging to confirm the average pH measure d. Experim ent 5. Diet Particle Size. Three diets (A, B, and C; Table 1) were chosen to provide an anticipa ted range of DDM and DCP and a includin g supplem ental [(DM feed - DM undiges ted)/ spectru m of ingredie nts %DDM tested in vitro either as were fat. The three diets DM feedl X 100%. mash or regroun d poultry cial commer regular a [(CP feed - CP undiges ted)/ CP %DCP sizes of .84 screen using mill cyclone a through feedl X 100%. ed with incubat were or .40 mm. All three diets C for 4 37 at HCI .075N in pepsin of Where DM feed = grams of dry matter in .50 g 1140 units C with 37 at ion incubat of hr 4 by d followe hr matter sample, DM undiges ted = grams of dry at an average fluid al intestin d prepare of ml 10 in protein crude grams precipit ate, CP feed = .50 g sample, and CP undiges ted = grams of pH 6.9. Experim ent 6. Comparison of In Vivo and In crude protein in precipit ate. DDM and DCP. The in vitro procedu res Vitro dee replicat In Experim ents 1 to 6, five in Experim ent 5 using diets of termina tions were used per treatme nt. The were as outlined For in vivo determi nation sizes. particle same prepara tion of pepsin in HCI and sample three m labeled diets (A, B, chromiu ility digestib of nts of intestin al fluid were used for all treatme Table 1) were each fed ad libitum to in anyone experim ent. Diet C, used in Ex- and C; of 25 male broiler chicken s 7 weeks of perimen ts 1 to 4, was ground to pass through a groups diets were offered to the groups for 5 The age. 1 mm screen. to slaughte r. Immedi ately after prior days Concen Experim ent 1. Effect of Pepsin of ileal digesta were taken tration. Three concent rations of pepsin (Sigma slaughte r, samples to analysis for DM and prior dried, Chemic al Compan y), 290 units, 580 units, or and freeze determi ned by the was oxide c Chromi CPo 1140 units per 10 ml .075N HCI, were tested in (1980). In vivo Fenton and Fenton of method pepsin of unit the first stage of incubat ion. One ned using the determi were %DCP produce s a A 280 of .001/mi n at pH 2.00 at 37 %DDM and e: C, measure d as TCA soluble product s using formula
by mixing .50 g .of a diet pregrou nd to pass through a 1.00 mm screen with 20 mg of powder ed pepsin in 10 ml of .075N HCI solution. The solution was then incubat ed in an agitatin g water bath for 4 hr at 37 C. After Stage 1 incubat ion, 2 ml of .20N Tris-buf fer were added and this mixture subsequ ently neutrali zed with .10N NaOH. Stage 2 incubation of 4 hr at 37 C was carried out after the addition of 10 ml of prepare d porcine intestin al fluid. Followi ng the final stage of incubat ion, content s of the flask were centrifu ged at 1250 x g for 10 min at a tempera ture of 5 C, the superna tant discarde d, and the undiges ted precipit ate washed with distilled water, recentrifu ged, and superna tant again discarde d. The low tempera ture at which the final incubate is centrifu ged is the means by which the action of the added enzyme s is stopped . The undiges ted precipit ate was then transfer red to a dry preweig hed filter paper for dry matter and crude protein determi nations . Dry matter was determi ned by drying- the precipit ate at 60 C in a vacuum oven to constant weight. Crude protein (6.25 X N) was determi ned by nacro-K jeldahl method (AssociatIOn of Official Analyti cal Chemis ts, 1970). In all experim ents in vitro DDM and DCP were determi ned using the followin g formula e:
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%DDM O/oDCP
CLUNIES AND LEESON
=>
[1 - (CrZ03 grams of feed/Crz03 g excreta)] X 100. Hcp gram feed - (CrZ03 g feedl CrZ03 g excreta) X CP gram excreta] CP grams feed} X 100.
Pepsin concentration
%DDM
%DCP
45.44 a ± 2.74 57.44 b ± 2.20 59.64 b ± .68
59.07a ± 2.14 81.35 b ± 1.67 92.56 c ± 1.53
(units/ 10 ml HC!) 270 580 1140
a,b,cWithin columns means (± SEM) followed by different letters are significantly different (P<.05). 'Stage 2 of incubation with 10 ml of prepared porcine intestinal fluid for 4 hr at 37 C.
TABLE 3. Effect of time of first stage (pepsin) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP)
RESULTS
Experiment 1. By increasing pepsin activity from 270 to S80 units there was a significant (P< .OS) increase in %DDM and %DCP in vitro (Table 2). There was no significant (P>.OS) increase in %DDM when the pepsin activity was further increased to 1140 units of enzyme, although there was a significant (P<.OS) increase in %DCP at 1140 units of pepsin enzyme. Experiment 2. A significan t (P<.O S) increase in %DDM was observed in the second 1-hr period, with the increase in the 3rd and 4th hr of incubation being nonsignificant (P> .OS, Table 3). There was a significant (P< .OS) increase in %DCP in the second hour of incubation (Table 3). Experiment 3. A significant (P< .OS) increase in %DDM was observed in each of the first 3 hr of incubation with intestinal fluid; the increase in the last hour was nonsignificnat (P> .OS; Table 4). Crude protein digestibility increased significantly (P< .OS) in the second hour of incubation with increases in subsequent 1-hr periods being nonsignificant (P> .OS). Experiment 4. At a pH of 6.S there was a significant (P<.OS) decrease in the %DDM (Table S). For pH's 6.6 to 6.9 there were no significant (P>.OS) differences in %DDM, although there was a consistent peak in the %DDM at pH 6.6 (Fig 1). The pH had no significant (P>.OS) effect on %DCP Cfable S). Experiment 5. Significant (P< .OS) increases
Time
%DDM
% DCP
(hr) 24.69 a 26.99 b 28.51 b 28.72 b
2 3 4
± ± ± ±
.95 .60 .62 .39
75.F 79.8 b 81.9 b 82.5 b
± ± ± ±
.18 1.96 1.58 1.09
a,bWithin columns means (± SEM) with different letters are significantly different (P<.05).
TABLE 4. Effect of time of the second stage (intestinal) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP) Time
%DDM
% DCP
(hr) 0' 1 2 3 4
28.72 a 37.9S b 52.02 c 57.98 d 60.50 d
± ± ± ± ±
.39 3.01 3.02 1.72 1.49
82.5 a 81. 7 a 84.5 b 84.9 b 85.9 b
± ± ± ± ±
1.09 .98 1.92 1.72 1.01
a,b,c,dWithin columns means (± SEM) followed by different letters are significantly different (P<.05). 'Time 0 values taken from Table 3 for time 4 hr.
in %DDM were observed by reducing particle size for all three diets (Table 6). Significant (P<.OS) increases in the %DCP of diets A and B
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Where Crz 0 3 gram feed = parts per million Cr2 0 3 Ig feed, Crz 0 3 gram excreta = ppm Crz 0 3 Igram excreta, CP gram feed gram CP/gram feed and CP gram excreta = gram CP Igram excreta. Statistical Analysis. Analyses of variance were carried out to determine any treatment effects in Experiments 1 to S (Snedecor and Cochran, 1967). Data from Experiment 6 were analyzed by regression analysis according to the method of Cochran and Cox (19S7). Regression analysis was used to regress in vitro digestibility on in vivo apparent digestibility. Planned means comparisons were made by Student's paired t test (Snedecor and Cochran, 1967).
TABLE 2. Effect of pepsin concentration on digestibility of dry matter (DDM) and digestibility of crude protein (CDP) in vitro'
IN VITRO ESTIMATION OF DIGESTIBILITY
65 ,--:.
60
:11 >
55
~
t
50
i5
6.6
6.5
68
6.7
69
pH
FIG. 1. Effect of pH in second (intestinal) stage of incubation on percent digestibility of dry matter.
TABLE 5. Effect of pH in the second stage (intestinal) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP) pH
%DDM
6.5 6.6 6.7 6.8 6.9
49.0P 60.19 b 59.67 b 57.52 b 57.95 b
% DCP ± ± ± ± ±
3.42 1.45 1.49 2.36 1.17
87.39 87.56 87.50 87.55 87.58
(Table 6). No significant (P> .05) difference in %DDM was observed between diets Band C when diets were ground to pass through a .84-mm screen. All diets differed significantly (P<.05) in %DCP when tested unground as regular mash or ground through a .84-mm screen. When a .40-mm screen was used diets B and C were higher in DCP than diet A, although Band C were themselves not significantly (P> .05) different. Experiment 6. Increasing fineness of grind increased the accuracy of estimation of in vivo digestibility by the in vitro technique (Table 7). Actual in vivo and in vitro DDM and DCP values for the .40 mm diet are shown in Table 8. As anticipated from ingredient composition (Table 1) diet A to C differed significantly (P< .05) in %DDM. Irrespective of diet composition there was close agreement between in vitro and in vivo estimates of DDM and DCP (Table 8). Use of the regression equations, shown in Table 7, for .40 mm treatment to predict in vivo DDM and DCP substantially improves the accuracy of determination (Table 8).
± .76 ± .91 ± .36 ± .59 ± 1.02
DISCUSSION
a,bWithin columns means (± SEM) followed by different letters are significantly different (P<.05).
were observed when particle size of these diets were reduced. There was no significant (P> .05) increase in %DCP of diet C when particle size was reduced from .84 to .4 mm. All diets differed significantly (P<.05) in %DDM when tested unground or as .40 mm particle size
Furuya et al. (1969) reported that an increase in pepsin concentration from 1 to 2 mg in 10 ml of HCI increased digestibility in the first stage of incubation for both DM and CP in diets of lower digestibility (approximately 56% DDM). No effect was observed in diets of high digestibility (90% DDM). Work with a diet of intermediate digestibility indicated a significant (P<.05) increase in DDM and DCP when pepsin was increased from 1 to 2 mg using the two stages of incubation. There may be some effect of the pepsin digestion on the results of the second stage of incubation.
TABLE 6. Effect of particle size on in vitro digestibility of dry matter (DDM) and digestibility of crude protein (DCP) % DCP
%DDM
Particle size
Diet A
Diet B
DietC
Diet A
Diet B
Diet C
Unground .84mm .40mm
43.07a,A 47.14 b ,A 52.90c ,A
48.99 a ,B 64.91 b ,B 68.95 c ,B
57.7S a ,C 66.89 b ,B 70.94c ,C
66.67 a ,A 76.30 b ,A 81.99 c ,A
70.12 a,B 85.01 b ,B 88.16 c ,B
81.67 a,C 87.4S b ,C 88.39 b ,B
a,b,cWithin columns means followed by different letters are significantly different (P<.05). A,B,CWithin rows means followed by different letters are significantly different (P<.OS).
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Q
93
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CLUNIE S AND LEESON TABLE 7. Regression analysis relating in vitro digestibi lity to in vivo digestibi lity of dry matter (DM) and crude protein (CP)
Particle size
Regressio n equation
r-value
RSD
Ungroun d
DM CP
y = .5933x + 36.25 Y = .4800x + 53.13
.37 .48
<.84mm
6.28 3.50
DM CP
y = .8070x + 16.97 Y = .8564x + 17.02
.97 .89
1.46 1.57
<.40mm
DM CP
y = .9010x + 7.21 Y = 1.3310x -26.66
.98 .88
1.06 1.65
TABLE 8. Compari son of digestibi lity (DDM) and digestibi lity of crude protein (DCP) in vivo, in vitro, or by predictio n equation based on in vitro techniqu e using diets ground through a.40 mm screen. %DDM
% DCP
Diet
In vitro
SEM
In vivo
In vitro' predicte d
A B C
52.90 a 68.95 b 70.92 c
.53 .68 .16
54.37 68.89 71.76
54.88 69.34 71.13
In vitro
SEM
In vivo
In vitro 2 predicted
81.99 a 88.16 b 88.39 b
.48 .53 .07
81.66 90.93 91.54
82.47 90.69 90.99
a,b,CWithin columns means followed by different letters are significa ntly different (P<.05). 'Predicte d values generate d with the equation : y = .9010x + 7.21, where x = in vitro %DDM. 2 Predicted values generate d with the equation : y = 1.3310x -26.66, where x = in vitro % DCP.
Results from Experim ent 2 show a significant (P< .05) increase in %DDM and %DCP in the second I-hr period, althoug h Furuya et al. (1979) reported only a slight increase in %DDM in the first 4 hr of incubat ion. Approx imately 29% DDM can be attribut ed to the pepsin stage of incubat ion (Table 3). This is compar able with data from Furuya et al. (1979) who reporte d 30% DDM could be attribut ed to the pepsin stage in diets with digestibilities ranging from 56 to 68% DDM. It is also evident that the majorit y of the digestio n of CP is due to the action of pepsin in the first stage of incubat ion. Results from Experim ent 3 indicate that the 4-hr incubat ion period propose d by Furuya et at. (1979) appears correct, since after 3 hr of incubat ion, there is no signific ant increase in either %DDM and %DCP. Thus, digestio n appears comple te after 3-hr incubat ion with intestin al fluid in contras t to the 4 hr reported by Furuya et al. (1979). An importa nt factor determi ning in vitro DDM results is pH of the incubat e. The pan-
creatic enzyme alpha-am ylase contain ed in the intestin al fluid is probabl y primaril y respons ible for disappe arance of DM in the second stage of incubat ion. Long (1961) reported an optimal pH of 6.9 for the enzyme alpha amylase. Results from Experim ent 4 showed no decreas e in DDM until pH fell to 6.5 in the final stage of incubat ion. Thus, it would seem that when pH remains above 6.6 no effect on %DDM would be observed, and any differen ces that occur appear nonsign ificant (P> .05). A pH in the range 6.6 to 6.9 in the final stage is thus recommen ded. When selecting a pH one must also conside r that the product s of digestio n cause a decrease in pH. Because diets are likely to differ in their bufferin g capacity , the amount of base required to obtain the desired pH may also differ from diet to diet. Also, decreasing the particle size increases the amount of base required in the neutrali zation process. Particle size is a very importa nt factor affectin g both in vivo and in vitro digestib ility. The particle size determi nes the degree of access that enzyme s have to digestib le nutrien ts
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Nutrient
IN VITRO ESTIMATION OF DIGESTIBILITY
able to differentiate DDM between two diets of similar digestibility ground to pass through a .40-mm screen. A greater degree of accuracy may be obtained by using the regression equation obtained from diets of known digestibility to predict in vivo values. If regression equations are to be used then it is necessary to assay a number of samples o(known digestibility along with the test ingredients for the calculations to be valid. Also, diets tested should fall within the range of those used to standardize the equation. Although the in vitro results agree quite well with results obtained from in vivo ileal samples, there are questions as to how well the system can predict the overall digestibility of DM and CP in fowl. Data reported by Sakamoto et al. (1980) indicate that the in vitro results accurately predict the overall digestibility of DM and CP in the colostomized fowl. This would seem to indicate that the majority of digestion takes place in the precolonic and prececal areas of the digestive tract. Riesenfeld et al. (1980) reported that about 97% of starch was digested at the terminal end of the ileum. Data from Payne et al. (1971) shows no significant difference in the digestion of protein for cecectomized versus noncecectomized adult chickens. Thus, any digestion or fermentation taking place in the cecal and colonic areas would appear to be of less importance in the chicken. In monogastric animals, digestion of the major dietary components starts in the stomach and is essentially completed in the small intestine (Keyes and Debarthe, 1974). Although there are some differences between in vivo and in vitro results, the method does seem to simulate gastric and small intestinal digestion of monogastric animals (Furuya et al., 1979). There appears to be limited application to predicting overall digestibility in swine diets due to a larger proportion of fermentation in their large intestine. In poultry, the amount of fermentation in the hindgut is very limited with the diets offered today; thus, the in vitro method may be able to give a fair approximation of the digestibility of such diets offered to chickens. Due to the high correlation between DDM and metabolizable energy (Nelson et al., 1975), the in vitro technique may also have application as a rapid means of assaying available energy in poultry diets.
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by increasing surface area relative to mass when particle size is reduced. In the bird, the gizzard, through its abrasive actions, functions to grind the feed and effect an increase in surface area. The finer the diets were ground, the greater the accuracy of the in vitro technique in estimating in vivo apparent digestibility. Of interest is that while the r value for DDM analysis increased when the .84-mm screen was replaced with the .40 mm screen, the r value for DCP analysis was decreased slightly (Table 7). This indicates that although accuracy of the method to simulate the digestive process may be increased, the ability to rank and differentiate proteins of similar digestibilities appears to decrease with fineness of grind. The in vitro method gives a good estimation of in vivo digestibility of DM and CP for complete diets offered to poultry when diets are ground to pass through a .40 mm screen (Table 8). The low residual standard deviation (RSD) value also indicates the method to be precise. The regression coefficient obtained from DM data analysis shows that in vitro results are highly correlated to in vivo digestibility (r = .99). The r value for the regression analysis of crude protein digestibility was also very high (r = .93). These r values are similar to those obtained by Sakamoto et al. (1980) of .98 and .99 for DM and CP, respectively. The lower accuracy and correlation coefficient obtained with CP may be due to differences in the pepsin preparations as well as an effect of diet on the enzymes contained in the intestinal fluid. Partridge et al. (1982) reported that diet composltlon may affect the amount of pancreatic enzymes, trypsin and chymotrypsin, secreted. In vitro results predict in vivo digestibility of DM and CP for diets of high digestibility as well as low digestibility, although in vitro results obtained with highly digestible diets more accurately simulates in vivo DDM than is the case with diets of low digestibility. This may be because fermentation plays a more important role in diets of lower digestibility or because the level of fat affects in vitro digestibility. Although not quite as obvious, the same trends are seen when comparing the means of in vitro and in vivo data presented by Sakamoto et al. (1980) and Furuya et al. (1979) for diets of high versus low digestibility. For DCP the in vitro estimate was more accurate for diets of low versus high digestibility. The method is also
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CLUNIES AND LEESON ACKNOWLEDGMENTS
REFERENCES Association of Official Analytical Chemists, 1970. Official Methods of Analysis. 11 th ed. Washington, DC. Cochran, W. G., and G. M. Cox, 1967. Experimental Designs. 7th ed. John Wiley & Sons, Inc., New York, NY. Fenton, T. W., and M. Fenton, 1980. Research notes. An improved procedure for determination of chromic oxide in feed and faeces. Can. J. Anim. Sci. 59:631-634. Furuya, S., K. Sakamoto, and S. Takahashi, 1979. A new in vitro method for the estimation of digestibility using the intestinal fluid of the pig. Br. J. Nutr. 41:511-520. Furuya, S., and S. Takahashi, 1975. Absorption of L-histidine and glucose from the jujenum segment of the pig and its diurnal fluctuation. Br. J. Nutr.34:267-277. Hill, F. W., 1962. Nutrition of pigs and pOUltry. J. T. Morgan and D. Lewis, ed. Butterworths, London. Johnston, J., and C. N. Coon, 1978. A comparison of six protein quality assays using commercially available protein meals. Poultry Sci. 58: 919-
927. Keys, J. E. Jr., and J. V. DeBarthe, 1974. Site and extent of carbohydrate, energy and protein digestion and the rate of passage of grain diets in swine. J. Anim. Sci. 39:57-62. Long, G., 1961. Biochemists Handbook. D. Van Nostrand Co. Inc., New York, NY. Nelson, T. S., E. L. Stephenson, A. Burgos, J. Floyd, and J. O. York, 1975. Effect of tannin content and dry matter digestion on energy utilization and average amino acid availability of hybrid sorghum grains. Poultry Sci. 54:1620-1623. Partridge, I. G., A. G. Glow, I. E. Sam brook, and T. Corring, 1982. The influence of diet on the exocrine pancreatic secretion of growing pigs. Br. J. Nutr. 48: 137-145. Payne, W. L., R. R. Kifer, D. G. Snyder, and G. F. Combs, 1971. Studies of protein digestion in the chicken. 1. Investigation of apparent amino acid digestibility of fish meal protein using cecectomized, adult male chickens. Poultry Sci. 50: 143-150. Riesenfeld, G., D. Sklan, A. Bar, U. Eisner, and S. Hurwitz, 1980. Glucose absorption and starch digestion in the intestine of the chick. J. Nutr. 110:117-121. Sakamoto, K., T. Asano, S. Furuya, and S. Takahashi, 1980. Estimation of in vivo digestibility with the laying hen by an in vitro method using the intestinal fluid of the pig. Br. J. Nutr. 43: 389391. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 7th ed. Iowa State Univ. Press, Ames IA .. Tilley. J.M.A., and R. A. Terry, 1963. A two-stage technique for in vitro digestion of forage crops. J. Br. Grassl. Soc. 18: 104-111.
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This work was supported by the Ontario Ministry of Agriculture and Food and the Natural Sciences and Engineering Research Council of Canada. The authors would like to express appreciation to B. O. Friendship of the Ontario Veterinary College for performing surgery.
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N lG 2W 1 (Received for publication January 24, 1983)
1984 Poultry Science 63: 89-96 INTRODUCTION
Traditional in vitro procedures, such as pepsin digestion in acid solutions, have been used to evaluate protein digestibility of feedstuffs for monogastric animals. Johnston and Coon (1978) correlated digestion of protein, using .02% pepsin in .075 N hydrochloric acid solution, to weight gain obtained in 7-day protein quality growth assays. Regression analysis with pepsin digested protein gave a correlation coefficient of .91 for 20 protein ingredients assayed. Two-phase in vitro procedures utilizing rumen fluid followed by pepsin digestion have been used to determine the intake of roughages offered to ruminant animals (Tilley and Terry, 1963). Furuya et al. (1979) proposed a new twostage in vitro method using pepsin and pig intestinal fluid to estimate the digestibility of dry matter (DDM) and crude protein (DCP) of swine diets. The system attempts to simulate gastric and intestinal digestion in swine or other
1
monogastric animals. Samples of diets, ground to pass through a 1 mm screen, were incubated for 4 hr with a commercial pepsin preparation in acid solution followed by 4 hr of incubation with prepared intestinal fluid. Because digestion by monogastrics is quite similar (Hill 1962), Sakamoto et al. (1980) used the two-stage in vitro system to estimate in vivo digestibility of dry matter and crude protein in diets offered to poultry. Regression analysis relating in vivo to in vitro results provided correlation coefficients(r) of .98 and .99 for DDM and DCP, respectively. Furuya et al. (1979) reported that prepared intestinal fluid may be stored at -25 C or lyophilized (Sakamoto et al., 1980) without any significant change in %DDM and %DCP in vitro. Experiments were carried out to examine the method and possible application for routine analysis for quality control of feeds and feed ingredients offered to poultry. MATERIALS AND METHODS
Collection and Preparation of Intestinal Fluid. An 8-month-old Yorkshire gilt weighing approximately 100 kg was fitted with a T-
ICN. K & K Laboratories, Inc., Cleveland, OH.
89
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ABSTRACT A number of experiments were carried out to determine some of the parameters affecting the estimation of apparent digestibility of dry matter (DDM) and crude protein (DCP) by a two-stage, in vitro method, Experiments investigated the effects of pepsin concentration, time of incubation, pH, the effect of particle size on DDM and DCP, and the relationship between apparent in vivo and in vitro digestibility. Increasing the concentration of pepsin to 1140 units/10 ml .075N Hel in the first phase of incubation increased %DCP (P<'05), while %DDM increased significantly (P<.05) only to 580 pepsin units/10 ml .075N HCI solution. In the first stage of incubation with pepsin there was a significant (P<.05) increase in %DDM and %DCP in the second 1-hr period. In the second stage of incubation, involving porcine intestinal fluid, %DDM increased significantly (P<.05) in the first three 1-hr periods, while no significant (P<.05) increase in %DCP was observed after the second 1-hr period. In the second intestinal stage of incubation, there was no significant (P>.05) difference in %DDM for pH's 6.6 to 6.9. At pH 6.5 there was a significant (P<.05) decrease in %DDM. There was no significant (P>.05) effect of pH on %DCP. By reducing particle size of test material the accuracy of predicting apparent in vivo DDM and DCP was increased for all diets. The in vitro method gave a reliable estimation of apparent DDM and DCP in vivo. The correlations (r) between in vitro and in vivo estimates were high, .99 and .93 for %DDM and %DCP, respectively, when diets were ground to pass through a .4 mm screen. Prediction equations obtained from linear regression analysis were y = .901X + 7.214 and y = 1.331X -26.66 for DM and CP, respectively, where y = in vivo digestibility and X = in vitro digestibility. (Key words: digestibility, dry matter, crude protein in vitro variables)
90
CLVNIES AND LEESON
those conditions affecting accuracy of the method. Considerable variation was seen in DDM results and pH of the final incubate with the latter greatly affecting accuracy of results. As the preparation of pepsin in HCl and the sample of intestinal fluid were the same for all treatments, pH variability could only be attributed to neutralization with .20N NaOH solution as recomended by Furuya et al. (1979). Curves constructed from titration of 25 ml of .075N HCl with .20N NaOH showed a rapid rise in pH from approximately 5 to 9 when a few drops of base were added. As a result a slight modification was made to the procedure, in that the NaOH solution used in titration was diluted to a concentration of .1 ON, and a Tris-buffer! [(tris(hydroxymethyl)amino methane) was introduced to stabilize the pH near neutrality in the final stage of incubation. The first stage of incubation was carried out
TABLE 1. Percentage composition of experimental diets l Ingredients Ground corn Ground barley Gr.ound .oats S.oybean meal (48% CP) C.orn glu ten meal (600A. CP) C.orn distillers plus s.olubles Wheat bran Animal-vegetable blend fat Limestone Dicalcium ph.osphate I.odized salt (.01% C.oC0 3 , .15% Ca1 2) Mineral-vitamin mix Mineral-vitamin mix 2 DL-Methi.onine L-Lysine-HCI Chromic .oxide Calculated analysis Metab.olizable energy, kcallkg Crude protein, % Crude fiber, % Ether extract, %
Diet A
DietB
Diet C
Diet D
69.34
57.14
32.00 32.00
11.99 11.99
35.07
13.12
6.50 1.40 1.00 .25
2.50 .90 2.00 .25
3.00 1.44 2.30 .25
.75
.75
.75
52.77
37.13 10.00 .90 1.35 .45
2.70 .06 .20 .30 2976 16.3 7.8 9.7
.28 .30 3259 19.0 2.4 5.5
.30 3029 22.1 2.5 5.3
.30 2762 3 14.5 4.8 2.4
I Provides per kil.ogram diet: 5.0 mg copper, 30.0 mg iron, 55 mg manganesium, .1 mg selenium, and 50 mg zinc, 8,000 IV vitamin A, 1,600 IV vitamin D, 1.0 mg vitamin E, 9.0 mg rib.oflavin, 11.0 mg D-calcium pantohenate, 13 Jolg vitamin B 12 , 26.0 mg niacin, 900 mg ch.oline chl.oride, 1.5 mg vitamin D, 1.5 mg f.olic acid, .20 mg biotin, and 125 mg santoquin.
2Provides per kilogram diet: 19 mg manganese, 220 mg ir.on, 14 mg copper, 58 mg zinc, 6,336 IV vitamin A, 650 IV vitamin D, 18 IV vitamin E, 1.58 mg vitamin K, 1.1 mg thiamine, 2.9 mg riboflavin, 18 mg niacin, 15 mg pantothenic acid, .72 mg folic acid, 1.1 mg pyridoxine, .15 mg biotin, 1.1 mg vitamin B., 22 mg ethoxyquin, 816 mg choline, and .07 mg selenium. 3
Swine ME value.
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shaped cannula. The cannula, of a rubberized silicone material, was surgically placed in the upper jejunum approximately 25 em distal to the opening of the pancreatic duct. The surgical method was adapted from Furuya and Takahashi (1975). The pig, housed in a gestating stall, was fed 2.5 kg of diet D (Table 1) during the period of collection. Approximately .6 liters of intestinal contents were collected daily 1 to 2 hr after feeding. Intestinal contents collected were tranferred to the laboratory in an ice bath. Intestinal samples were centrifuged at 1700 X g, the supernatant recovered, and supernatant used fresh or stored in polyethylene bottles at -25 C. In Vitro Digestibility Procedures. The method of in vitro digestibility was based on the procedure described by Furuya et al. (1979). Initial studies with this procedure yeilded unpredictable and variable results and, hence, further trials were designed to study
IN VITRO ESTIMA TION OF DIGEST IBILITY
91
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hemogl obin as substrat e. The first stage of incubat ion was followe d by the second stage of incubat ion with 10 ml of prepare d intestin al fluid for all treatme nts in this experim ent. Experim ent 2. Time of Pepsin Digestion. Four periods were selected for incubat ion: 1, 2, 3, and 4 hr. In this experim ent only the first stage of incubat ion was comple ted. For this first stage the samples were incubat ed with 1140 units of pepsin enzyme in 10 ml of .075N HCI solution . Experim ent 3. Time of Intestinal Digestion. Four periods of incubat ion were investig ated: 1, 2, 3, and 4 hr. In all treatme nts the intestin al stage of incubat ion with 10 ml of prepare d porcine intestin al fluid was precede d-by 4 hr incubatio n at 37 C with 1140 units of pepsin in 10 ml of .75N HCI soln. Experim ent 4. Effect of pH in Intestinal Stage. Followi ng the first stage of incubat ion with 1140 units of pepsin enzyme differin g amount s of NaOH solution were added to the product s of the first phase to effect differen t pH's prior to introdu cing the 10 mls of prepared intestin al fluid. The pH's examin ed were 6.5, 6.6, 6.7, 6.8, and 6.9. The pH of the final incubat e was noted prior to centrifu ging to confirm the average pH measure d. Experim ent 5. Diet Particle Size. Three diets (A, B, and C; Table 1) were chosen to provide an anticipa ted range of DDM and DCP and a includin g supplem ental [(DM feed - DM undiges ted)/ spectru m of ingredie nts %DDM tested in vitro either as were fat. The three diets DM feedl X 100%. mash or regroun d poultry cial commer regular a [(CP feed - CP undiges ted)/ CP %DCP sizes of .84 screen using mill cyclone a through feedl X 100%. ed with incubat were or .40 mm. All three diets C for 4 37 at HCI .075N in pepsin of Where DM feed = grams of dry matter in .50 g 1140 units C with 37 at ion incubat of hr 4 by d followe hr matter sample, DM undiges ted = grams of dry at an average fluid al intestin d prepare of ml 10 in protein crude grams precipit ate, CP feed = .50 g sample, and CP undiges ted = grams of pH 6.9. Experim ent 6. Comparison of In Vivo and In crude protein in precipit ate. DDM and DCP. The in vitro procedu res Vitro dee replicat In Experim ents 1 to 6, five in Experim ent 5 using diets of termina tions were used per treatme nt. The were as outlined For in vivo determi nation sizes. particle same prepara tion of pepsin in HCI and sample three m labeled diets (A, B, chromiu ility digestib of nts of intestin al fluid were used for all treatme Table 1) were each fed ad libitum to in anyone experim ent. Diet C, used in Ex- and C; of 25 male broiler chicken s 7 weeks of perimen ts 1 to 4, was ground to pass through a groups diets were offered to the groups for 5 The age. 1 mm screen. to slaughte r. Immedi ately after prior days Concen Experim ent 1. Effect of Pepsin of ileal digesta were taken tration. Three concent rations of pepsin (Sigma slaughte r, samples to analysis for DM and prior dried, Chemic al Compan y), 290 units, 580 units, or and freeze determi ned by the was oxide c Chromi CPo 1140 units per 10 ml .075N HCI, were tested in (1980). In vivo Fenton and Fenton of method pepsin of unit the first stage of incubat ion. One ned using the determi were %DCP produce s a A 280 of .001/mi n at pH 2.00 at 37 %DDM and e: C, measure d as TCA soluble product s using formula
by mixing .50 g .of a diet pregrou nd to pass through a 1.00 mm screen with 20 mg of powder ed pepsin in 10 ml of .075N HCI solution. The solution was then incubat ed in an agitatin g water bath for 4 hr at 37 C. After Stage 1 incubat ion, 2 ml of .20N Tris-buf fer were added and this mixture subsequ ently neutrali zed with .10N NaOH. Stage 2 incubation of 4 hr at 37 C was carried out after the addition of 10 ml of prepare d porcine intestin al fluid. Followi ng the final stage of incubat ion, content s of the flask were centrifu ged at 1250 x g for 10 min at a tempera ture of 5 C, the superna tant discarde d, and the undiges ted precipit ate washed with distilled water, recentrifu ged, and superna tant again discarde d. The low tempera ture at which the final incubate is centrifu ged is the means by which the action of the added enzyme s is stopped . The undiges ted precipit ate was then transfer red to a dry preweig hed filter paper for dry matter and crude protein determi nations . Dry matter was determi ned by drying- the precipit ate at 60 C in a vacuum oven to constant weight. Crude protein (6.25 X N) was determi ned by nacro-K jeldahl method (AssociatIOn of Official Analyti cal Chemis ts, 1970). In all experim ents in vitro DDM and DCP were determi ned using the followin g formula e:
92
%DDM O/oDCP
CLUNIES AND LEESON
=>
[1 - (CrZ03 grams of feed/Crz03 g excreta)] X 100. Hcp gram feed - (CrZ03 g feedl CrZ03 g excreta) X CP gram excreta] CP grams feed} X 100.
Pepsin concentration
%DDM
%DCP
45.44 a ± 2.74 57.44 b ± 2.20 59.64 b ± .68
59.07a ± 2.14 81.35 b ± 1.67 92.56 c ± 1.53
(units/ 10 ml HC!) 270 580 1140
a,b,cWithin columns means (± SEM) followed by different letters are significantly different (P<.05). 'Stage 2 of incubation with 10 ml of prepared porcine intestinal fluid for 4 hr at 37 C.
TABLE 3. Effect of time of first stage (pepsin) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP)
RESULTS
Experiment 1. By increasing pepsin activity from 270 to S80 units there was a significant (P< .OS) increase in %DDM and %DCP in vitro (Table 2). There was no significant (P>.OS) increase in %DDM when the pepsin activity was further increased to 1140 units of enzyme, although there was a significant (P<.OS) increase in %DCP at 1140 units of pepsin enzyme. Experiment 2. A significan t (P<.O S) increase in %DDM was observed in the second 1-hr period, with the increase in the 3rd and 4th hr of incubation being nonsignificant (P> .OS, Table 3). There was a significant (P< .OS) increase in %DCP in the second hour of incubation (Table 3). Experiment 3. A significant (P< .OS) increase in %DDM was observed in each of the first 3 hr of incubation with intestinal fluid; the increase in the last hour was nonsignificnat (P> .OS; Table 4). Crude protein digestibility increased significantly (P< .OS) in the second hour of incubation with increases in subsequent 1-hr periods being nonsignificant (P> .OS). Experiment 4. At a pH of 6.S there was a significant (P<.OS) decrease in the %DDM (Table S). For pH's 6.6 to 6.9 there were no significant (P>.OS) differences in %DDM, although there was a consistent peak in the %DDM at pH 6.6 (Fig 1). The pH had no significant (P>.OS) effect on %DCP Cfable S). Experiment 5. Significant (P< .OS) increases
Time
%DDM
% DCP
(hr) 24.69 a 26.99 b 28.51 b 28.72 b
2 3 4
± ± ± ±
.95 .60 .62 .39
75.F 79.8 b 81.9 b 82.5 b
± ± ± ±
.18 1.96 1.58 1.09
a,bWithin columns means (± SEM) with different letters are significantly different (P<.05).
TABLE 4. Effect of time of the second stage (intestinal) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP) Time
%DDM
% DCP
(hr) 0' 1 2 3 4
28.72 a 37.9S b 52.02 c 57.98 d 60.50 d
± ± ± ± ±
.39 3.01 3.02 1.72 1.49
82.5 a 81. 7 a 84.5 b 84.9 b 85.9 b
± ± ± ± ±
1.09 .98 1.92 1.72 1.01
a,b,c,dWithin columns means (± SEM) followed by different letters are significantly different (P<.05). 'Time 0 values taken from Table 3 for time 4 hr.
in %DDM were observed by reducing particle size for all three diets (Table 6). Significant (P<.OS) increases in the %DCP of diets A and B
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Where Crz 0 3 gram feed = parts per million Cr2 0 3 Ig feed, Crz 0 3 gram excreta = ppm Crz 0 3 Igram excreta, CP gram feed gram CP/gram feed and CP gram excreta = gram CP Igram excreta. Statistical Analysis. Analyses of variance were carried out to determine any treatment effects in Experiments 1 to S (Snedecor and Cochran, 1967). Data from Experiment 6 were analyzed by regression analysis according to the method of Cochran and Cox (19S7). Regression analysis was used to regress in vitro digestibility on in vivo apparent digestibility. Planned means comparisons were made by Student's paired t test (Snedecor and Cochran, 1967).
TABLE 2. Effect of pepsin concentration on digestibility of dry matter (DDM) and digestibility of crude protein (CDP) in vitro'
IN VITRO ESTIMATION OF DIGESTIBILITY
65 ,--:.
60
:11 >
55
~
t
50
i5
6.6
6.5
68
6.7
69
pH
FIG. 1. Effect of pH in second (intestinal) stage of incubation on percent digestibility of dry matter.
TABLE 5. Effect of pH in the second stage (intestinal) of incubation on digestibility of dry matter (DDM) and digestibility of crude protein (DCP) pH
%DDM
6.5 6.6 6.7 6.8 6.9
49.0P 60.19 b 59.67 b 57.52 b 57.95 b
% DCP ± ± ± ± ±
3.42 1.45 1.49 2.36 1.17
87.39 87.56 87.50 87.55 87.58
(Table 6). No significant (P> .05) difference in %DDM was observed between diets Band C when diets were ground to pass through a .84-mm screen. All diets differed significantly (P<.05) in %DCP when tested unground as regular mash or ground through a .84-mm screen. When a .40-mm screen was used diets B and C were higher in DCP than diet A, although Band C were themselves not significantly (P> .05) different. Experiment 6. Increasing fineness of grind increased the accuracy of estimation of in vivo digestibility by the in vitro technique (Table 7). Actual in vivo and in vitro DDM and DCP values for the .40 mm diet are shown in Table 8. As anticipated from ingredient composition (Table 1) diet A to C differed significantly (P< .05) in %DDM. Irrespective of diet composition there was close agreement between in vitro and in vivo estimates of DDM and DCP (Table 8). Use of the regression equations, shown in Table 7, for .40 mm treatment to predict in vivo DDM and DCP substantially improves the accuracy of determination (Table 8).
± .76 ± .91 ± .36 ± .59 ± 1.02
DISCUSSION
a,bWithin columns means (± SEM) followed by different letters are significantly different (P<.05).
were observed when particle size of these diets were reduced. There was no significant (P> .05) increase in %DCP of diet C when particle size was reduced from .84 to .4 mm. All diets differed significantly (P<.05) in %DDM when tested unground or as .40 mm particle size
Furuya et al. (1969) reported that an increase in pepsin concentration from 1 to 2 mg in 10 ml of HCI increased digestibility in the first stage of incubation for both DM and CP in diets of lower digestibility (approximately 56% DDM). No effect was observed in diets of high digestibility (90% DDM). Work with a diet of intermediate digestibility indicated a significant (P<.05) increase in DDM and DCP when pepsin was increased from 1 to 2 mg using the two stages of incubation. There may be some effect of the pepsin digestion on the results of the second stage of incubation.
TABLE 6. Effect of particle size on in vitro digestibility of dry matter (DDM) and digestibility of crude protein (DCP) % DCP
%DDM
Particle size
Diet A
Diet B
DietC
Diet A
Diet B
Diet C
Unground .84mm .40mm
43.07a,A 47.14 b ,A 52.90c ,A
48.99 a ,B 64.91 b ,B 68.95 c ,B
57.7S a ,C 66.89 b ,B 70.94c ,C
66.67 a ,A 76.30 b ,A 81.99 c ,A
70.12 a,B 85.01 b ,B 88.16 c ,B
81.67 a,C 87.4S b ,C 88.39 b ,B
a,b,cWithin columns means followed by different letters are significantly different (P<.05). A,B,CWithin rows means followed by different letters are significantly different (P<.OS).
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Q
93
94
CLUNIE S AND LEESON TABLE 7. Regression analysis relating in vitro digestibi lity to in vivo digestibi lity of dry matter (DM) and crude protein (CP)
Particle size
Regressio n equation
r-value
RSD
Ungroun d
DM CP
y = .5933x + 36.25 Y = .4800x + 53.13
.37 .48
<.84mm
6.28 3.50
DM CP
y = .8070x + 16.97 Y = .8564x + 17.02
.97 .89
1.46 1.57
<.40mm
DM CP
y = .9010x + 7.21 Y = 1.3310x -26.66
.98 .88
1.06 1.65
TABLE 8. Compari son of digestibi lity (DDM) and digestibi lity of crude protein (DCP) in vivo, in vitro, or by predictio n equation based on in vitro techniqu e using diets ground through a.40 mm screen. %DDM
% DCP
Diet
In vitro
SEM
In vivo
In vitro' predicte d
A B C
52.90 a 68.95 b 70.92 c
.53 .68 .16
54.37 68.89 71.76
54.88 69.34 71.13
In vitro
SEM
In vivo
In vitro 2 predicted
81.99 a 88.16 b 88.39 b
.48 .53 .07
81.66 90.93 91.54
82.47 90.69 90.99
a,b,CWithin columns means followed by different letters are significa ntly different (P<.05). 'Predicte d values generate d with the equation : y = .9010x + 7.21, where x = in vitro %DDM. 2 Predicted values generate d with the equation : y = 1.3310x -26.66, where x = in vitro % DCP.
Results from Experim ent 2 show a significant (P< .05) increase in %DDM and %DCP in the second I-hr period, althoug h Furuya et al. (1979) reported only a slight increase in %DDM in the first 4 hr of incubat ion. Approx imately 29% DDM can be attribut ed to the pepsin stage of incubat ion (Table 3). This is compar able with data from Furuya et al. (1979) who reporte d 30% DDM could be attribut ed to the pepsin stage in diets with digestibilities ranging from 56 to 68% DDM. It is also evident that the majorit y of the digestio n of CP is due to the action of pepsin in the first stage of incubat ion. Results from Experim ent 3 indicate that the 4-hr incubat ion period propose d by Furuya et at. (1979) appears correct, since after 3 hr of incubat ion, there is no signific ant increase in either %DDM and %DCP. Thus, digestio n appears comple te after 3-hr incubat ion with intestin al fluid in contras t to the 4 hr reported by Furuya et al. (1979). An importa nt factor determi ning in vitro DDM results is pH of the incubat e. The pan-
creatic enzyme alpha-am ylase contain ed in the intestin al fluid is probabl y primaril y respons ible for disappe arance of DM in the second stage of incubat ion. Long (1961) reported an optimal pH of 6.9 for the enzyme alpha amylase. Results from Experim ent 4 showed no decreas e in DDM until pH fell to 6.5 in the final stage of incubat ion. Thus, it would seem that when pH remains above 6.6 no effect on %DDM would be observed, and any differen ces that occur appear nonsign ificant (P> .05). A pH in the range 6.6 to 6.9 in the final stage is thus recommen ded. When selecting a pH one must also conside r that the product s of digestio n cause a decrease in pH. Because diets are likely to differ in their bufferin g capacity , the amount of base required to obtain the desired pH may also differ from diet to diet. Also, decreasing the particle size increases the amount of base required in the neutrali zation process. Particle size is a very importa nt factor affectin g both in vivo and in vitro digestib ility. The particle size determi nes the degree of access that enzyme s have to digestib le nutrien ts
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Nutrient
IN VITRO ESTIMATION OF DIGESTIBILITY
able to differentiate DDM between two diets of similar digestibility ground to pass through a .40-mm screen. A greater degree of accuracy may be obtained by using the regression equation obtained from diets of known digestibility to predict in vivo values. If regression equations are to be used then it is necessary to assay a number of samples o(known digestibility along with the test ingredients for the calculations to be valid. Also, diets tested should fall within the range of those used to standardize the equation. Although the in vitro results agree quite well with results obtained from in vivo ileal samples, there are questions as to how well the system can predict the overall digestibility of DM and CP in fowl. Data reported by Sakamoto et al. (1980) indicate that the in vitro results accurately predict the overall digestibility of DM and CP in the colostomized fowl. This would seem to indicate that the majority of digestion takes place in the precolonic and prececal areas of the digestive tract. Riesenfeld et al. (1980) reported that about 97% of starch was digested at the terminal end of the ileum. Data from Payne et al. (1971) shows no significant difference in the digestion of protein for cecectomized versus noncecectomized adult chickens. Thus, any digestion or fermentation taking place in the cecal and colonic areas would appear to be of less importance in the chicken. In monogastric animals, digestion of the major dietary components starts in the stomach and is essentially completed in the small intestine (Keyes and Debarthe, 1974). Although there are some differences between in vivo and in vitro results, the method does seem to simulate gastric and small intestinal digestion of monogastric animals (Furuya et al., 1979). There appears to be limited application to predicting overall digestibility in swine diets due to a larger proportion of fermentation in their large intestine. In poultry, the amount of fermentation in the hindgut is very limited with the diets offered today; thus, the in vitro method may be able to give a fair approximation of the digestibility of such diets offered to chickens. Due to the high correlation between DDM and metabolizable energy (Nelson et al., 1975), the in vitro technique may also have application as a rapid means of assaying available energy in poultry diets.
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by increasing surface area relative to mass when particle size is reduced. In the bird, the gizzard, through its abrasive actions, functions to grind the feed and effect an increase in surface area. The finer the diets were ground, the greater the accuracy of the in vitro technique in estimating in vivo apparent digestibility. Of interest is that while the r value for DDM analysis increased when the .84-mm screen was replaced with the .40 mm screen, the r value for DCP analysis was decreased slightly (Table 7). This indicates that although accuracy of the method to simulate the digestive process may be increased, the ability to rank and differentiate proteins of similar digestibilities appears to decrease with fineness of grind. The in vitro method gives a good estimation of in vivo digestibility of DM and CP for complete diets offered to poultry when diets are ground to pass through a .40 mm screen (Table 8). The low residual standard deviation (RSD) value also indicates the method to be precise. The regression coefficient obtained from DM data analysis shows that in vitro results are highly correlated to in vivo digestibility (r = .99). The r value for the regression analysis of crude protein digestibility was also very high (r = .93). These r values are similar to those obtained by Sakamoto et al. (1980) of .98 and .99 for DM and CP, respectively. The lower accuracy and correlation coefficient obtained with CP may be due to differences in the pepsin preparations as well as an effect of diet on the enzymes contained in the intestinal fluid. Partridge et al. (1982) reported that diet composltlon may affect the amount of pancreatic enzymes, trypsin and chymotrypsin, secreted. In vitro results predict in vivo digestibility of DM and CP for diets of high digestibility as well as low digestibility, although in vitro results obtained with highly digestible diets more accurately simulates in vivo DDM than is the case with diets of low digestibility. This may be because fermentation plays a more important role in diets of lower digestibility or because the level of fat affects in vitro digestibility. Although not quite as obvious, the same trends are seen when comparing the means of in vitro and in vivo data presented by Sakamoto et al. (1980) and Furuya et al. (1979) for diets of high versus low digestibility. For DCP the in vitro estimate was more accurate for diets of low versus high digestibility. The method is also
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CLUNIES AND LEESON ACKNOWLEDGMENTS
REFERENCES Association of Official Analytical Chemists, 1970. Official Methods of Analysis. 11 th ed. Washington, DC. Cochran, W. G., and G. M. Cox, 1967. Experimental Designs. 7th ed. John Wiley & Sons, Inc., New York, NY. Fenton, T. W., and M. Fenton, 1980. Research notes. An improved procedure for determination of chromic oxide in feed and faeces. Can. J. Anim. Sci. 59:631-634. Furuya, S., K. Sakamoto, and S. Takahashi, 1979. A new in vitro method for the estimation of digestibility using the intestinal fluid of the pig. Br. J. Nutr. 41:511-520. Furuya, S., and S. Takahashi, 1975. Absorption of L-histidine and glucose from the jujenum segment of the pig and its diurnal fluctuation. Br. J. Nutr.34:267-277. Hill, F. W., 1962. Nutrition of pigs and pOUltry. J. T. Morgan and D. Lewis, ed. Butterworths, London. Johnston, J., and C. N. Coon, 1978. A comparison of six protein quality assays using commercially available protein meals. Poultry Sci. 58: 919-
927. Keys, J. E. Jr., and J. V. DeBarthe, 1974. Site and extent of carbohydrate, energy and protein digestion and the rate of passage of grain diets in swine. J. Anim. Sci. 39:57-62. Long, G., 1961. Biochemists Handbook. D. Van Nostrand Co. Inc., New York, NY. Nelson, T. S., E. L. Stephenson, A. Burgos, J. Floyd, and J. O. York, 1975. Effect of tannin content and dry matter digestion on energy utilization and average amino acid availability of hybrid sorghum grains. Poultry Sci. 54:1620-1623. Partridge, I. G., A. G. Glow, I. E. Sam brook, and T. Corring, 1982. The influence of diet on the exocrine pancreatic secretion of growing pigs. Br. J. Nutr. 48: 137-145. Payne, W. L., R. R. Kifer, D. G. Snyder, and G. F. Combs, 1971. Studies of protein digestion in the chicken. 1. Investigation of apparent amino acid digestibility of fish meal protein using cecectomized, adult male chickens. Poultry Sci. 50: 143-150. Riesenfeld, G., D. Sklan, A. Bar, U. Eisner, and S. Hurwitz, 1980. Glucose absorption and starch digestion in the intestine of the chick. J. Nutr. 110:117-121. Sakamoto, K., T. Asano, S. Furuya, and S. Takahashi, 1980. Estimation of in vivo digestibility with the laying hen by an in vitro method using the intestinal fluid of the pig. Br. J. Nutr. 43: 389391. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 7th ed. Iowa State Univ. Press, Ames IA .. Tilley. J.M.A., and R. A. Terry, 1963. A two-stage technique for in vitro digestion of forage crops. J. Br. Grassl. Soc. 18: 104-111.
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This work was supported by the Ontario Ministry of Agriculture and Food and the Natural Sciences and Engineering Research Council of Canada. The authors would like to express appreciation to B. O. Friendship of the Ontario Veterinary College for performing surgery.