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A Comparison of Methods to Determine Amino Acid Availability of Feedstuffs in Cecectomized Ganders¹

College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu Province 225009, P. R. China

² Corresponding author: dkwzy{at}263.net or ssr236{at}163.com

	ABSTRACT

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Experiments were conducted to compare endogenous amino acid losses and the true amino acid availability (TAAA) of 3 feedstuffs by using methods involving a short-term fasting and an N-free diet with cecectomized ganders. Diets were formulated to contain soybean meal, fish meal, and cottonseed meal as the sole source of protein. A precision-fed assay was used in which each feed sample was precise-fed (60 g) to geese and excreta were collected for 48 h. A N-free diet and fasting methods were used to evaluate the endogenous amino acid losses. Endogenous losses of 3 amino acids were significantly different (P < 0.01) with the N-free diet and fasting methods. The TAAA of soybean meal, fish meal, and cottonseed meal determined by N-free diet method ranged from 84.49 to 97.09%, 89.18 to 98.16%, and 77.09 to 98.32%, respectively. The TAAA of these 3 diets determined by the fasting method ranged from 83.50 to 97.77%, 88.08 to 99.60%, and 76.09 to 98.09%, respectively. However, there were only a few small differences (P > 0.05) between methods in each amino acid. In conclusion, there was no difference in determination of the amino acid availability of these feedstuffs using cecectomized ganders between the N-free diet and fasting methods.

Key Words: amino acid availability • endogenous amino acid loss • nitrogen-free diet • cecectomized gander

	INTRODUCTION

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It is generally accepted that the protein requirement of animals is primarily a requirement for amino acids (AA; NRC, 1994; Leeson and Summers, 2001). The amount of AA in the diets and their relative proportions determine the deposition of protein in the animals. However, feed-stuffs used in diets for animals vary not only in their AA composition but also in their digestibility. Depending on the type and source of feedstuff variable amounts of AA disappear from the gastrointestinal tract to be utilized by the animals for maintenance and tissue accretion. As a result, availability of AA between feed components varies considerably (D’Mello, 2003). Thus, determination of AA digestibility of feed ingredients has become crucial. To date, there is a large body of published work on AA digestibility coefficients for different feed ingredients and animals (Parsons, 1991; NRC, 1994; Heartland Lysine, 1996). A number of different methodologies for determining AA digestibility coefficients has been reported in the literature (Ravindran and Bryden, 1999; Lemme et al., 2004). Among these methods, the precision-fed, cecectomized birds assay has been widely used for estimating AA digestibility of feedstuffs, yielding very consistent results (Parsons, 2002). Information gleaned from these studies can be utilized by poultry nutritionists to formulate diets with increased precision to satisfy the AA requirements of birds. However, to the authors’ knowledge, no studies using geese have been reported.

In determining AA availability in poultry, 2 methods that have often been used to measure endogenous AA in excreta: short-term fasting and the N-free diet method. However, with the short-term fasting, doubt exists about whether the metabolic fecal plus endogenous urinary AA contribution to the excreta is the same for fasted controls as for birds fed the test ingredients (McNab and Fisher, 1981; Engster et al., 1985). Also, it may be argued that an N-free diet should be administered to the negative control birds, which introduces the problem of which diet should be used and in what quantity.

Objectives of the present study were to compare endogenous AA losses by using methods involving a short-term fasting and an N-free diet, and to determine the true AA availability (TAAA) in soybean meal, fish meal, and cottonseed meal by using cecectomized ganders, and also to describe a procedure for cecectomy in geese.

	MATERIALS AND METHODS

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Experimental Procedures

All animal handing protocols were approved by The Yangzhou University Animal Care and Use Committee. Eighteen adult Yangzhou ganders that averaged 4,120 g at 24 wk were used in a completely randomized design. All geese were cecectomized according to the procedure outlined by Ragland et al. (1999) for ducks and modified for ganders (Sheng, 2005; Shi et al., 2007) at 24 wk. The birds were individually housed in raised wire cages (75 x 65 x 35 cm), equipped with individual feeders and self-drinking systems, and the data were individually recorded. Housing temperature was maintained at 26°C.

In experiment 1, endogenous AA losses were determined when ganders were fasted or when they were fed 60 g of N-free diet. Feed was withdrawn from all birds for 24 h to ensure no feed residues remained in the gastrointestinal tract. Eighteen cecectomized ganders were randomly divided into 2 groups. Geese in 1 group were precision-fed (Sibbald, 1987) 60 g of N-free diet (Table 1). Geese in the other group were fasted. Forty-eight hours after crop intubation, excreta were collected into a bottle, dried to a constant weight at 65°C, allowed to reach equilibrium with the atmospheric moisture for a 24 h, weighed, and ground to pass through a 40-mesh sieve. Total collection of excreta was immediately frozen and stored at –20°C for further analysis.

The TAAA values were calculated for each AA using the following equation: TAAA = [AAc– (AAv – AAvf)]/AAc x 100%; where AAc is the total amount of AA consumed by the fed ganders, AAv is the total amount of AA voided in excreta by the fed ganders, and AAvf is the amount of each AA voided by the control (N-free diet or fasted) ganders during 48 h that followed the 24-h fast.

Chemical Analysis

The AA contents were determined using a Waters ion-exchange HPLC system by the method outlined by AOAC methods 994.12 (sulfur and regular; AOAC, 1995), utilizing postcolumn o-phthalaldehyde derivatization and fluorescence detection, following acid hydrolysis. Duplicate samples of diet (5 mg) and digesta (5 mg) were hydrolyzed in 1,000 µL of 6 M HCl, with 0.1% added phenol, for 24 h at 110 ± 2°C in glass tubes sealed under vacuum. Glycine and tryptophan being destroyed under acid hydrolysis were not determined.

Statistical Analysis

All data from the experiments were analyzed using a 1-way ANOVA (SPSS Inc., 1993). When appropriate, differences among treatment means were compared by Duncan’s multiple-range test. Differences in TAAA and endogenous AA in excreta were analyzed according to t-tests.

	RESULTS

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Endogenous AA Losses

Endogenous AA losses values (mg/bird per 48 h) for the N-free diet and fasting methods are presented in Table 2. In general, losses obtained using these 2 methods were not different (P > 0.05), and mean values were 17.56 vs. 16.19 mg/bird per 48 h. Endogenous AA values for histidine, threonine, tyrosine, proline, valine, methionine, cysteine, isoleucine, leucine, and phenylalanine were not different (P > 0.05) between N-free diet and fasting methods. Values for serine, arginine, and lysine in excreta, however, were significantly different (P < 0.01) between 2 groups.

The TAAA of the 3 diets corrected by the N-free diet and fasting method were presented in Table 3. The TAAA of soybean meal, fish meal, and cottonseed meal determined by N-free diet method ranged from 84.49 to 97.09%, 89.18 to 98.16%, and 77.09 to 98.32%, respectively. And TAAA of these 3 diets determined by the fasting method from 83.50 to 97.77%, 88.08 to 99.60%, and 76.09 to 98.09%, respectively.

	DISCUSSION

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The N-free diet and fasting methods have been the most common method for determining the basal endogenous AA excreta, which had been used by many other researchers (Sibbald, 1979a; Boisen and Moughan, 1996; Song et al., 2003). From the results in Table 2, there was no significant difference between the total endogenous AA losses obtained by the N-free diet method and the fasting method, which was similar to the study reported by Parsons (1984). In our study, endogenous AA values for serine, arginine, and lysine in excreta were significantly different (P < 0.01) between the N-free diet and fasting methods, values for other AA were not different (P > 0.05) between the 2 groups. Chung and Baker (1992) reported that compared with the fasting method, the N-free diet method aided body condition, which also generally applies to TAAA determination. Cornstarch and cellulose may stimulate more gastric, pancreatic, bile, and small intestinal secretions. Small intestinal and pancreatic secretions contribute the greatest to total endogenous secretions, and the principal components of these endogenous secretions are mucoproteins and digestive enzymes, which are rich in proline, glutamine, aspartic acid, serine, alanine, threonine, and valine (Chung and Baker, 1992). On the other hand, Crissey and Thomas (1983) reported that there was no difference between the N-free diet and fasting methods.

The difference in endogenous fecal and urinary AA losses by the N-free diet and fasting methods, raises the question of whether energy and fiber content of the diet can affect endogenous losses. The role of these factors inducing endogenous AA losses, such as the protein quantity and quality, dietary fiber content, DMI, AA composition, and the presence of antinutritive factors, has been reviewed in detail elsewhere (Boisen and Moughan, 1996; Nyachoti et al., 1997).

Microbial action in the gastrointestinal tract may affect endogenous AA losses using N-free diet method. Cornstarch and cellulose could provide a substrate for microbes in the cecum. Significant differences in all excreted AA except methionine and histidine have been detected between intact roosters and cecectomized roosters (Kessler et al., 1981), suggesting a possible role of the cecum in altering the AA losses values. Salter and Fulford (1974) concluded that the gut microflora of chicks had little influence on the digestion of dietary proteins but might serve an important role in the degradation of endogenous proteins.

Parsons et al. (1983) reported that dietary carbohydrate substantially affects excretion of endogenous AA by poultry and that fasted birds may not provide an accurate estimate of endogenous AA excretion for birds fed high fiber feedstuffs in AA digestibility trials. The difference in endogenous AA losses between the N-free and fasted methods reported in this paper indicates the need for further research in this area. In general, because the cecum interferes with the determination of AA availability (AAA), to determine AAA of protein feedstuffs, it is better to choose the cecectomized birds.

Previous studies have indicated that 24 h is not sufficient for completing intestinal clearance of some feed-stuffs from roosters (Sibbald, 1979b; Chami et al., 1980). Therefore, several researchers have suggested that fasting time should be extended to 30 or 48 h.

Availability of AA

In the present research, AA quantification was carried out in excreta. The TAAA of most AA of soybean meal determined by N-free diet method in this study was higher than the values determined by Ren et al. (1997) in cecectomized cockerels except histidine, threonine, proline, and valine. The TAAA of fish meal determined by N-free diet method in this study was similar to the values determined by Ren et al. (1997) in cecectomized cockerels. The TAAA of most AA of cottonseed meal determined by N-free diet method in this study was higher than the values determined by Ji et al. (2002) in cecectomized broilers except cysteine. Mohamed et al. (1986) reported that there was no difference in the digestibility of AA of soybean meal and cottonseed meal between chicken and Muscovy ducks. However, the result of this experiment on ganders was different; the TAAA of most AA of soybean meal and cottonseed meal determined on ganders was higher than on cockerels. The cause may be the developed cecum of geese, and it suggests that under the same level of dietary protein, the performance of ganders was better than cockerels. Kluth and Rodehutscord (2006) concluded that there are differences in TAAA among poultry species. The underlying mechanisms and reasons still require clarification.

Fasting was the method to estimate endogenous losses and has been used in several studies published as reference values (Sibbald, 1986; Parsons, 1991; NRC, 1994). It has been suggested that this is not an appropriate technique because it creates an abnormal physiological state (Lemme et al., 2004). And the N-free diet method is the most classical method to determine the endogenous AA losses at present. When compared with the fasting method, other nutriments are offered in the diet except N-containing compounds. And all AA found in the excreta are assumed to be of endogenous origin. The studies of de Lange et al. (1989a, b) and Chung and Baker (1992) suggest that the N-free diet method may be a valid approach for estimating TAAA for protein sources. However, as indicated by Butts et al. (1993) and Donkoh et al. (1995), an N-free diet, as well as fasting method, may lack the stimulatory effect on endogenous gut protein secretions. This may lead to an underestimation of endogenous N. In the present study, there was no significant difference between these 2 methods. So, further attention should be given to the practicality of conducting repeated assays and the choice of better methods for evaluation of feed ingredients.

In summary, when taking cecectomized ganders as the experimental animals, there was no difference in determining the endogenous AA losses and the AAA of these feedstuffs between the N-free diet and fasting methods. However, due to more normal physiological conditions, the N-free diet method should be used in determination the endogenous AA losses and the AAA of these feedstuffs in geese.

	ACKNOWLEDGMENTS

 
This work was financially supported by Key Scientific and Technological Project of Jiangsu Province in the Tenth Five-Year, PR China (Grant No.: BE2001369).

	FOOTNOTES

 
¹ Supported by the Key Scientific and Technological Project of Jiangsu Province in the Tenth Five-Year, P. R. China (Grant No. BE2001369).

Received for publication February 13, 2007. Accepted for publication September 11, 2007.

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