Comparison of the Sensitivities of Growth and Digestibility Studies Using Intact, Cecectomized, and Cannulated Roosters1

METABOLISM AND NUTRITION Comparison of the Sensitivities of Growth and Digestibility Studies Using Intact, Cecectomized, and Cannulated Roosters 1 S. D. CRISSEY and O. P. THOMAS Department of Poultry Science, University of Maryland, College Park, Maryland 20742 (Received for publication December 9, 1985)

1987 Poultry Science 66:866-874 INTRODUCTION

Various bioassays have been established in order to test feeds for available amino acids (Payne, 1968). Each method has been shown to have its advantages and disadvantages (McNab, 1979). The growth assay is currently the technique to which all other methods are compared (Varnish and Carpenter, 1975). The fecal analysis method is based on the premise that availability can be determined by measuring digestibility (DeMuelenaere et al., 1967). One technique currently receiving much attention is the amino acid assay by Sibbald (1979b). Recognizing the need for standardization, Engster (1983) and Engster et al. (1985) compared amino acid availability data from six laboratories that used similar procedures to determine amino acids in poultry excreta. Results showed that although there was considerable variability among data, differences between labs were no greater than variation within labs. Because ceca of birds contains high quantities of microorganisms (Barnes, 1972), one method proposed to reduce microbial effects on amino acids in excreta is the use of cecectomized birds. Another method is the ileal analysis assay in which the animal is surgically modified so that

'Scientific Article Number A-4297 Contribution Number 7286 of the Maryland Agriculture Experimental Station (Department of Poultry Science).

digesta can be collected at the terminal ileum (McNab, 1979). This procedure minimizes microbial influence from the ceca and lower intestinal tract.

MATERIALS AND METHODS

Chick Growth Study. The purpose of Experiments 1 and 2 was to determine if the chick growth study could detect differences between the types of soybean meal used and to identify lysine or methionine as the factor made less available when soybean meal is further processed. Commercial soybean meal (49% protein) was blended and divided into portions. One-half was not subject to further processing (SBM); one-fourth was autoclaved for 1 hr 45 min at 121 C, 103.4 x 103 Pa (AUTO-105); and onefourth was processed at the same temperature and pressure for 2 hr 30 min (AUTO-150). Male broiler chicks were fed a low protein, low lysine, and low methionine preliminary diet through Day seven. In Experiment 1, chicks were weighed at seven days of age. The male broiler chicks were distributed so that each pen had eight birds and had approximately the same weight range and mean. Treatments were arranged in a 2 x 2 x 2 factorial. Two types of soybean meal were added to the basal diet (Table 1) at a level of 19.5%: SBM and AUTO-105. Lysine was added 866

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ABSTRACT The sensitivity of several bioassays was determined using soybean meal and two autoclaved soybean meals. Chick growth study results revealed that a four percent difference in weight gain was sufficient to show statistical significance (P<.05) between the diets. Lysine quantities and digestibility values were compared using excreta obtained from intact (excreta collected on mats or in bags), cecectomized, and ileal-cannulated roosters. A 30% difference in lysine quantity in the excreta (collected on mats) was required to detect statistically significant effects of different diets fed to intact birds. Using intact birds (excreta collected in bags), cecectomized, and cannulated birds, differences in lysine quantity of 41, 42, and 67% were required to show statistically significant effects. Results showed that altering birds did not significantly change lysine values obtained nor reduce variability among birds (P>.05). (Key words: lysine, amino acid bioavailability, growth, cecectomy, cannulation, roosters)

LYSINE BIOAVAILABILITY TABLE 1. Composition of experimental diets for Experiment 1 Experiment 1

Basal Corn Defluorinated phosphate Fat blend Limestone Salt 50% Choline chloride Coccidiostat 2 Santoquin Mix 6 3 Vitamin mix 4 Mineral mix 5

(%) 76.12 1.95 1.17 .48 .26 .17 .05 .02 .05 .05

Soybean meal

19.50

'Analyzed lysine content = .76%. Analyzed methionine content = .29%. Diet for Experiment 2 was similar to Experiment 1 except that Lysine was added at .09% and Methionine was added at .09%. 2

Amprol HiE, MSD-AGVET, Rahway, NJ.

3

Monsanto Co., St. Louis, MO.

4 T o supply the following per kilogram of diet: vitamin A 5,500 IU, vitamin D 3 2,200 IU, vitamin E 4.4 IU, vitamin B 12 13.2 jitg, riboflavin 6.6 mg, niacin 33 mg, d calcium pantothenate 11 mg, menadione 1.45 mg, folic acid .22 mg, pyridoxine 1.1 mg. s T o supply the following per kilogram of diet: calcium 150 mg, manganese 165 mg, zinc 88 mg, iron 55 mg, copper 6.6 mg, iodine 1.65 mg.

at levels of 0 and .175%. Methionine was added at levels of 0 and .125%. Each of the eight diets was replicated four times. Glutamic acid was substituted on an equimolar basis for lysine, methionine, or both to keep the diets isonitrogenous. Chick weight gain from 7 to 21 days posthatch was determined. Feed conversion as grams of feed consumed per grams of weight gain also was calculated for this period. Data (in all studies unless stated otherwise) were analyzed by analysis of variance (ANOVA). If an interaction was significant (P<.05), main effects were not interpreted. If main effects were interpreted and found significant (P<.05), Duncan's multiple range test was performed (Sokal and Rohlf, 1981). In addition, a calculation for sensitivity of the assay was performed according to the following formula (Sokal and Rohlf, 1981): n = 2CY! f.o5(df)

+

'.l(df) 2

where: n = number; CV = coefficient of variation; % = percent sensitivity; / = values of the t distribution; and df = degrees of freedom. Experiment 2 was similar to Experiment 1. Procedures were the same except for differences in diet formulation. The two types of soybean meal added to the basal diet (Table 1) were SBM and AUTO-150. Lysine was added at 0 and .085%. Methionine was added at 0 and .038%. Digestibility Assays. The purpose of Experiment 3 was to determine if three different methods of performing the digestibility assay could detect differences in the diets used in the chick studies. The three methods measured lysine values in excreta collected from: 1) intact birds with excreta collected into bags, 2) cecectomized birds with excreta collected into bags, and 3) cannulated birds with digesta collected from the terminal ileum. The means, variability, and sensitivity of each of these methods were compared. General procedures for the total excreta collection method as originally outlined by Sibbald (1980) in his true metabolizable energy studies were adapted for use. A total of 27 adult White Leghorn males was used in Experiment 3: nine intact birds with excreta collection bags, nine cecectomized birds with excreta collection bags, and nine cannulated birds with polypropylene ileal cannulas implanted 10 to 15 cm from the ileal-cecal junction. Treatments were arranged in three 3 x 3 Latin squares where birds represented columns, rows represented replicates in time, and squares represented location of cage. Treatments were repeated three times in each time period. The three treatment groups were force-fed 30 g of SBM, AUTO-105 (used in the growth assay, Experiment 1), or cornstarch. Three hundred Microtaggants-small, blue, plastic chips with a layer of iron (232,800 chips/g, 3M Co., St. Paul, MN)-were added as a marker to each treatment fed the cannulated birds. All roosters were fasted for 24 hr to empty the gastrointestinal tract (Sibbald, 1979a) before feeding the assigned diet. Force-feeding was done through a funnel directly into the crop. Water was given ad libitum throughout the experiment. Excreta were collected for 48 hr. For intact and cecectomized birds, excreta were collected into plastic bags that were secured by a rubber band to excreta collection rings. Rings were attached by a rubber band around the feathers near the cloaca. Bags were changed once during the 48-hr period. Digesta of cannulated birds were collected from cannulas

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Diet component

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CRISSEY AND THOMAS

868

for a total of 5 hr beginning 2 hr after feeding. Additional excreta from cannulated birds were collected on plastic collection mats for 48 hrs during each experiment. A calculation for total lysine from excreta in the cannulas was made using the marker. The calculation was: LYSv=_LYSd_

MTd/MTf

LYSc - (LYSvt - LYSvc) X 100 LYSc where: LYSc = milligrams of lysine consumed. LYSvt = milligrams of lysine voided in test group excreta, and LYSvc = milligrams of lysine voided in control group excreta. The purpose of Experiment 4 was to determine if four different methods of performing the digestibility assay could detect differences between SBM and a severely processed meal (AUTO-150). The procedures followed were similar to those in Experiment 3 with the modification of one excreta collection method. The same nine intact birds were used for collecting excreta in bags or on mats. Collection on mats was performed similarly to the other methods except that excreta were collected by scraping into collection cups after scales and feathers were removed. The F-max test for determining

RESULTS AND DISCUSSION

In Experiment 1 (Table 2), the weight gain of chicks fed AUTO-105 averaged 328 g whereas birds fed SBM gained at a greater rate and averaged 342 g by the end of the study. Mean weight gain of chicks fed SBM or AUTO105 without added lysine was 298 g. However, mean weight gain of birds fed diets supplemented with .175% lysine was 373 g. Thus, there was a growth response to the addition of lysine (P<.05). Feed conversion also was measured in Experiment 1. Data (Table 2) show that feed conversion was markedly improved when lysine was added to the diet of AUTO-105 groups. A type of soybean meal (SBM, AUTO-105) by lysine interaction was found for feed conversion (P< .05). No difference was found between methionine groups when either weight gain or feed conversion was measured (P>.05). Results were similar in Experiment 2 (Table 3) to those of Experiment 1. Groups fed SBM had a mean weight gain of 390 g. Mean weight gain in groups fed AUTO-150 was 360 g (P< .05). Birds responded significantly in weight gain with the addition of lysine (P<.05); the weight gain was more pronounced when lysine was added to the AUTO-150 diet than to the SMB diet. Thus, lysine was affected by the autoclaving. As in Experiment 1, there was no significant difference in weight gain or feed conversion found with the addition of methionine (P>.05). Effects of the diets on feed conversion paralleled those of weight gain. The AUTO-150 treatment was inferior to the SBM treatment (P< .05). A response to lysine addition also was seen in feed conversion values (P<.05). Groups fed treatments without added lysine showed lower weight gains and higher feed conversion means in Experiment 1 vs. Experiment 2. This was because the basal diet used in Experiment 1 was more deficient in lysine (.76 vs. .85% in Experiment 2). Chicks performed better at the lower levels of lysine in Experiment 2. However, at maximum supplementation levels, chicks in both experiments performed similarly. Growth studies showed that chick performance decreased 4% when SBM was autoclaved

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where: LYSv = calculated total milligrams of lysine voided, LYSd = mg of lysine voided in the digesta sample collected from the cannula, MTd = number of Microtaggants in the digesta, and MTf = number of Microtaggants in the feed. In all collection studies, samples were freeze dried, weighed, ground, and stored in a dessicator until hydrolyzed. Approximately 175 mg of the sample, in duplicate, was hydrolyzed under nitrogen in 4 ml of 6 NHC1 for 24 hr in individually sealed vacuum tubes at 110 C (Moore and Stein, 1963). Samples were analyzed for amino acids on a Durrum amino acid analyzer (Moore, 1972; Dionex Corp., 1979). Percentage of lysine found in the sample was multiplied by dry sample weight to obtain mg per sample. Two other measurements that were determined are: 1) the quantity of lysine in the excreta after correction was made for endogenous controls, and 2) percent digestibility, calculated with the following formula (Likuski and Dorrell, 1978):

homogeneity of variance was carried out to compare variability among methods (Sokal and Rohlf, 1981); statistical methods for data analysis cited in Experiment 1 were also used.

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LYSINE BIOAVAILABILITY

Digestibility Studies: 1 hr 45 min Intact Roosters. In Experiment 3 (Table 4) the mean value for lysine in the excreta of birds fed AUTO-105 was 77 mg; the mean value for lysine in the excreta of birds fed SBM was 63 mg (P>.05). Cornstarch-fed roosters had significantly lower quantities of lysine in excreta than other groups. The average was 30 mg (P<.05). Bird and replicate variations were taken into account in the model for the ANOVA so that any differences found by the method were due to diet alone. Corrected lysine values were 33 mg for SBM

TABLE 2. Weight gain and feed conversion of chicks fed diets containing either soybean meal (SBM) or soybean meal autoclaved (AUTO) for 105 min with or without added lysine and methionine: Experiment I

Dietary treatment

0% Added lysine

n

.175% Added lysine (n=16)

Weight gain' SMB

0% Added methionine .125% Added methionine X

369 384 376

342 d

8

310 307 309

372 366 369

328"

8

287 288 287

298 c

373d

AUTO 105 min 0% Added methionine .125% Added methionine X X

16

Feed conversion 2 ' 3 SBM

0% Added methionine .125% Added methionine X

1.64 1.60 1.62

1.66

8

1.71 1.68 1.70

1.60 1.60 1.60

1.70

8

1.84 1.75 1.80

16

1.74

1.61

AUTO 105 min 0% Added methionine .125% Added methionine X X

Values with different superscripts within columns or within rows are significantly different (P<.05). 'Mean weight gain in grams ± SEM based on a pooled estimate of means squared error, SEM = ± 2.4 g (n=32); ± 3.4g(n=16);±4.9g(n=8). 2 Mean feed conversion (g feed eaten/g weight gain) ± SEM based on a pooled estimate of means squared error, SEM = + .013 (n=32); ± .018 (n=18); ± .026 (n=8). 3

An interaction was significant for type of soybean meal X lysine (P<.05).

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and 47 mg for AUTO-105, and did not differ significantly (P>.05). Since lysine was made less available to the roosters by autoclaving, it follows that more lysine was found in the excreta of birds fed AUTO-105. Digestibilities were calculated to be 97 and 95% for SBM and AUTO105, respectively. Cecectomized Roosters. No significant difference was found between the mean quantity of lysine in excreta of birds fed SBM and excreta of those fed AUTO-105 (P>.05) (Table 4). The lowest mean quantity of lysine was excreted from the cornstarch-fed group (P<.05). Cannulated Roosters. Results obtained using cannulated roosters (Table 4) showed no significant differences between SBM and AUTO-105fed groups (P>.05) in amount of lysine in excreta. The mean for the cornstarch-fed group was lower (P<.05). Digestibilities were 96% for both. Values of lysine in excreta of cannu-

for 1 hr 45 min and 8% when SBM was autoclaved for 2 hr 30 min. The actual sensitivity of the growth assay was calculated to be 4%. Thus, the assay was capable of detecting a 4% or greater difference in weight gain (P<.05).

870

CRISSEY AND THOMAS

hypothesized that collection of digesta from the terminal ileum should increase assay sensitivity as microbial contributions from the ceca and large intestine are largely avoided. In Experiment 3, however, the type of bird used (intact, cecectomized, or cannulated) did not alter the results significantly and none of these methods significantly increased the accuracy or precision for detecting differences beween SBM and AUTO-105-fed birds (P>.05) in lysine contents of excreta. Digestibility Studies: 2 hr 30 min Intact Roosters: Collection on Mats. In Experiment 4, (Table 5) the mean quantity of excreted lysine was 124 mg in roosters fed AUTO150. This value was significantly different from the quantities of lysine in the excreta obtained from the SBM-fed group (87 mg) and the cornstarch-fed group (40 mg) (P<.05). The dif-

TABLE 3. Weight gain and feed conversion of chicks fed diets containing either soybean meal (SBM) or soybean meal autoclaved (AUTO) for 150 min with or without added lysine or methionine: Experiment 2 0% Added lysine

Dietary treatment

.085% Added lysine (n=16)

Weight gain' SBM 0% Added methionine _.038% Added methionine X

387 397 392

389 387 388

390 a

AUTO 150 min 0% Added methionine .038% Added methionine X

340 358 349

373 367 370

360u

371 c

379 u

16

X 2

Feed conversion SBM 0% Added methionine _.038% Added methionine X

1.61 1.61 1.61

1.59 1.56 1.57

1.59w

AUTO 150 min 0% Added methionine _.038% Added methionine X

1.70 1.68 1.69

1.64 1.67 1.65

1.67"

i.esy

1.62z

a—d,w—z

Values with different superscripts within columns or rows are significantly different (P<.05).

'Mean weight gain in grams ± SEM based on a pooled estimate of means squared error, SEM = ± 2.5 g (n=32) ; 3.5 g (n=16); 5.0 g(n=8). 2 Mean feed conversion (g feed eaten/g weight gain) ± SEM based on a pooled estimate of means squared error, SEM = .014 (n=32); ± .020 (n=16); ± .028 (n=8).

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lated birds were not significantly different from mean quantities of lysine in excreta of either intact or cecectomized birds (P<.05). This also was true for corrected lysine quantities and digestibility values. Since AUTO-105 feed was significantly inferior to SBM as measured by its effects on chick growth (Experiment 1), digestibility values obtained by the above methods may be expected to reflect these differences. In the chick growth study, a 4% difference in weight between chicks fed SBM and AUTO105 was found (Experiment 1). Comparison of digestibility measures for birds fed SBM and AUTO-105 yielded a 2% difference using intact birds with excreta collection bags, a 1% difference using cecectomized birds, but no difference using cannulated birds. It is hypothesized that the use of cecectomized birds should decrease microbial influence on lysine quantities in the excreta and increase assay sensitivity. Further, it was

LYSINE BIOAVAILABILITY

871

TABLE 4. Lysine in the excreta of intact roosters fed soybean meal (SEM), soybean meal autoclaved (AUTO) for 105 min, or cornstarch: Experiment 3

Lysine

Corrected 2

Digestibility1

Intact"

SBM AUTO-105 min Cornstarch

63a 77a 30 b

33 a 47a

97 95

Cecectomized 5

SBM AUTO-105 min Cornstarch

64 a 67a 22b

42a 45a

96 95

Cannulated 6

SBM AUTO-105 min Cornstarch

62a 66 a 24b

38 a 42a

96 96

a ' b Values with different superscripts within columns are significantly different (P<.05). 1 Means (milligrams on a dry weight basis) based on least squares with SEM based on an estimate of means squared error. 2

Values from controls fed cornstarch were subtracted from test diet values.

3

Percent digestibility = 100X [lysine fed — (lysine from test group excreta — lysine from control group)] /lysine fed. "Intact birds with collection into bags, SEM = 5.4 mg. 5 6

Cecectomized birds with collection into bags, SEM = 4.9 mg.

Cannulated birds with digesta collected into bags, SEM = 10.5 mg.

TABLE 5. Lysine in the excreta of roosters fed soybean meal (SBM), soybean meal autoclaved (AUTO) for 150 min, or cornstarch: Experiment 4 Dietary treatment

Lysine

Corrected 2

Digestibility'

Intact"

SBM AUTO-150 min Cornstarch

87 a 124 b 40 c

47a 84b

95 92

Intact s

SBM AUTO-150min Cornstarch

67d 79d 23e

44d 56 d

96 94

Cecectomized6

SBM AUTO-150min Cornstarch

81d 96d 25e

56d 71d

94 92

Cannulated 7

SBM" AUTO-150min Cornstarch

73d 78d 23e

50d 55d

95 94

Group

a—f Values with different superscripts within columns are significantly different (P<.05). Means (milligrams on a dry weight basis) based on least squares with SEM based on a pooled estimate of means squared error. 1 Values from controls fed cornstarch were subtracted from test diet values. 1

'Percent digestibility = 100 X [lysine fed — (lysine from test group excreta — lysine from control group)] / lysine fed. "Intact birds with collection on mats, SEM = 8.8 mg. 5

Intact birds with collection into bags, SEM = 7.5 mg.

6

Cecectomized birds with collection into bags, SEM = 9.3 mg.

'Cannulated birds with digesta collected into bags, SEM = 14.5 mg.

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Dietary treatment

Group

872

CRISSEY AND THOMAS

intact birds using collection bags, but lower values than those obtained using intact birds with excreta collection on mats (P<.05). This is consistent with results found by Isshiki et al. (1974) and Picard et al. (1983). In addition, Payne et al. (1971) found no significant differences between excreta amino acid values from intact vs. cecectomized birds given fish meal. Kessler et al. (1981) found increased amino acids in the excreta of fasted cecectomized birds. Lysine in the excreta after a 24-hr collection period averaged 24 mg for intact birds and 28 mg for cecectomized birds. Likewise, Parsons (1981) found lysine in the excreta of fasted chickens averaged 17 and 24 mg for intact and cecectomized birds, respectively. When he fed these birds a source of fiber, no difference in lysine quantity was found between bird types. The use of cecectomized birds in excreta collection studies for determining lysine digestibility did not improve the precision of the method. Cannulated Roosters. When SBM and AUTO-150 were tested on ileal-cannulated birds (Table 5), no significant difference was found in mean lysine quantities from the ileal digesta of birds fed the two diets (P>.05). The mean lysine quantity in the digesta from roosters fed cornstarch was 23 mg. This was significantly different from the quantity in digesta of roosters given either SBM or AUTO-150 (P<.05). It was calculated that there must be a 67% difference in lysine values from the digesta before significance (P<.05) would be shown. Overall mean lysine values from cannulated birds were similar to those obtained using the total excreta collection bag and the cecectomy methods (P< .05), but were significantly lower than those determined using total excreta collection mats (P<.05). Corrected lysine and digestibility values for Experiment 4 also showed trend similarities. It is expected that the ileal cannulation method should be able to detect differences between the lysine digestibility of SBM and AUTO. Results of Experiment 4 showed a difference in digestibility between SBM and AUTO-150-fed groups of 1 %. The growth study (Experiment 2) showed an 8% higher weight gain for the SBM vs. the AUTO-150 diet. These results are congruent to those obtained by Soares and Kifer (1971) who examined digesta from the ileum of sacrificed chickens and found differences in growth rates produced by the feeding of good, fair, and poor quality herring and anchovy meal. In their experiments, the range of

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ference in lysine in the excreta of the SBM and AUTO-150-fed birds was 30%. When the correction factor (40 mg) was subtracted from the test diet values, means were 47 and 84 mg of lysine in the excreta of birds fed SBM and AUTO-150, respectively (P<.05). These results are reflected in digestibility values of 95% for SBM and 92% for AUTO-150-fed birds. Intact Roosters: Collection into Bags. Though excreta collection on mats detected differences among treatments, scale and feather contamination problems were still possible (Sibbald, 1983). Feathers and scales were analyzed and found to contain .96% lysine. To eliminate feather and scale contamination in the excreta sample, excreta collection bags were used. Results when using excreta collection bags (Table 5) showed the mean quantity of lysine excreted by roosters fed SBM was not significantly different (P>.05) from that of the group fed AUTO150. Total amounts of excreta collected on mats and in collection bags were significantly different. The highest lysine means were obtained when excreta was collected on mats (P<.05). Comparison of results revealed no treatment by method interaction (P>.05). Thus, trends were similar. The method using collection on mats gave lower values of lysine digestibilities than when collection bags were used. Use of collection bags may be superior to collection on mats because the former avoids scale and feather contamination. For data collected by the bag method, no significant differences in lysine content of excreta of SBM and AUTO-fed birds were found. When lysine availability was determined by measuring digestibility using the excreta collection method with bags, the difference was 3% between values obtained in groups fed SBM vs. those fed AUTO-150. A 41% difference in excreta lysine is needed before the method can detect a significant difference between effects of SBM and AUTO diets. Cecectomized Roosters. Average values for excreted lysine using cecectomized birds (Table 5) were 81 mg for birds fed SBM, 96 mg for birds fed AUTO-150 (P>.05), and 25 mg (P< .05) for birds fed cornstarch. Corrected lysine values from the cecectomized birds were 56 and 71 mg for excreta from SBM and AUTO-150fed birds, respectively (P>.05). Calculated digestibilities were 94% for SBM and 92% for AUTO-150 data. The cecectomy method yielded values similar to those obtained with

LYSINE BIOAVAILABILITY

currently available techniques, is not as sensitive as the growth study. It is possible that forcefeeding 50 g of feed instead of 30 g might reduce some of the variability in digestibility data. It is evident that more research is needed before the amino acid digestibility assay can produce accurate and reliable results. REFERENCES Barnes, E. M., 1972. The avian intestinal flora with particular reference to the possible ecological significance of the cecal anaerobic bacteria. Am. J. Clin. Nutr. 25:1475-1479. DeMuelenaere, H.J.H., H-L. Chen, and A. E. Harper, 1967. Assessment of factors influencing estimation of lysine availability in cereal products. Agric. Food Chem. 15:310-317. Dionex Corp., 1979. Amino acid analyzer manual. Sunnyvale, CA. Engster, H.M., 1983. Report on a collaborative study to evaluate the precision-fed rooster assay for determining true amino acid availability of feed ingredients. Pages 1-23 in: Proc. Anim. Nutr. Res. Council. Engster, H. M., N. A. Cave, H. Likuski, J. M. McNab, C. A. Parsons, and F. E. Pfaff, 1985. A collaborative study to evaluate a precision-fed rooster assay for true amino acid availability in feed ingredients. Poultry Sci. 64:487^*98. Isshiki, Y., H. Yokota, Y. Nakahiro, and I. Tasaki, 1974. Digestion and absorption in the cecum of chickens. Jpn. J. Zootech. Sci. 45:488^193. Kessler, J. W., T. H. Nguyen, and O. P. Thomas, 1981. The amino acid excretion values in intact and cecectomized negative control roosters used for determining metabolic plus endogeneous urinary losses. Poultry Sci. 60:1576-1577. Likuski, H.J.A., and H. G. Dorrell, 1978. A bioassay for rapid determinations of amino acid availability values. Poultry Sci. 57:1658-1660. McNab, J. M., 1979. The concept of amino acid availability in farm animals. Pages 1-9 in: Recent Advances in Animal Nutrition. Butterworths Publ. Co., Boston, MA. Moore, S., 1972. The precision and sensitivity of amino acid analysis, in: Chemistry and Biology of Peptides. Ann Arbor Sci. Publ., Ann Arbor, MI. Moore, S., and W. H. Stein, 1963. Acid hydrolysis. Page 819 in: Methods in Enzymology VI. Academic Press, New York, NY. Parsons, C. M., 1981. Evaluation of a procedure for determination of amino acid digestibility and metabolizable energy of feedstuffs for poultry. Ph.D. Diss. Virginia Polytechnic Inst, and State Univ., Blacksburg, VA. Payne, W. L., 1968. An investigation of intestinal amino acids as a method to determine protein quality. Ph.D. Diss. Univ. of Maryland, College Park, MD. 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. Picard, M., S. Bertrand, M. Duron, and R. Maillard, 1983. Comparative digestibility of amino acids using five

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values found in the ileum was narrow and did not reflect the larger differences obtained by chick growth assay. Summers and Robblee (1984, 1985) determined the availability of soybean meal from excreta of intact chickens compared with digesta sampled at the terminal ileum of sacrificed birds. Soybean meal had an availability of 89% in the excreta of intact birds compared with 82.5% in samples collected from the ileum. In Experiment 4 variance did not differ among excreta of intact, cecectomized, or cannulated birds. Engster et al. (1985) also found problematic variabilities using intact birds. Sibbald (1982) suggests that the addition of a marker in digestibility studies increases variability. A marker (Microtaggants) was used with the cannulation studies and may explain why the variance of cannulated birds was not significantly less than the variance of intact or cecectomized birds. When Microtaggants were used in Experiment 4, percentage recovery of the marker from the digesta averaged 30% for birds fed SBM, 19% for birds fed AUTO-150, and 40% for birds fed cornstarch. These values were not significantly different (P<.05) from each other. The variability of marker recovery is reflected in values obtained in digestibility determinations using cannulated birds, as the marker is used in the calculation for lysine quantities. The effect can be seen in the standard errors of the mean obtained from studies using cannulated birds in Experiments 3 and 4 (Tables 4 and 5). The standard errors were 10.5 mg and 14.5 mg lysine, respectively. This may explain why differences in lysine quantities in the excreta need to be at least 67% before significance occurs. The expected decrease in variance from this method may be negated by the use of the marker. In all the digestibility studies, data variability was high regardless of the type of bird used: intact, cecectomized, or cannulated. Significant differences were not found among bird types and the assays were not sensitive enough to detect differences between soybean meal or two autoclaved soybean meals. The digestibility assays also were not as sensitive as the chick growth study. An exception was found when intact birds were used and their excreta were collected on mats. However, with this bird type there was the added problem of feather and scale contamination of the excreta sample. This detailed examination of amino acid digestibility assays shows that problems still exist in the method. The digestibility assay, with its

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