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Comparison of Amino Acid Digestibility Determined Prececally or Based on Total Excretion of Cecectomized Laying Hens

Institut für Agrar- und Ernährungswissenschaften, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle (Saale), Germany

² Corresponding author: te-halle{at}landw.uni-halle.de

	ABSTRACT

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This study investigated the possibility of using cecectomized laying hens for the determination of amino acid (AA) digestibility of protein sources as an alternative to measurements made at the terminal ileum. Toasted soybeans and corn gluten meal were used as test protein sources. A low-protein basal diet was based mainly on corn, wheat gluten, and cornstarch. In 4 other diets toasted soybeans or corn gluten meal were included each at 15% or 30% at the expense of cornstarch, so that the changes in the AA concentrations of the diets resulted from the protein sources only. Titanium dioxide was used as the indigestible marker. In experiment 1, two hundred ten 18-wk-old pullets were used. Digesta from the posterior 2-thirds of the intestine between Meckel’s diverticulum and 2 cm anterior to the ileo-ceca-colonic junction was obtained from killed hens. In experiment 2, fourteen cecectomized hens were used. They were kept individually, and excreta were quantitatively collected. The apparent digestibility of AA was calculated for each diet. Linear regression analysis was used to calculate AA digestibility for the 2 protein sources. Across all diets, apparent AA digestibility was significantly higher in experiment 2 than in experiment 1. However, AA digestibilities of the 2 protein sources were not different between both experiments. Differences in AA digestibility between the protein sources were not significant in experiment 1, but in experiment 2 significant differences between the protein sources were detected for some AA due to a lower standard error of estimate. It was concluded that AA digestibility of protein sources for laying hens can be evaluated based on quantitative excreta collection with cecectomized hens using the regression approach. This needs fewer animals than the determination of digestibility at the terminal ileum. Differences between protein sources are easier to detect because of the lower standard error of estimate.

Key Words: amino acid digestibility • prececal • total excretion • cecectomy • laying hen

	INTRODUCTION

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The digestibility of amino acids (AA) until the end of the ileum [prececal (PC) digestibility] developed as an important tool in poultry nutrition in recent years for the description of the value that a protein source has (Ravindran and Bryden, 1999). Using PC digestibility is suggested because it helps avoiding the influence of postileal microbial activity in protein evaluation. Measurements with both broilers and hens should be based upon digesta collected from the last two-thirds of the small intestine between Meckel’s diverticulum and 2 cm anterior to the ileo-ceca-colonic junction (Kluth et al., 2005; Rezvani et al., 2008). For PC measurements, the digesta of several birds has to be pooled to obtain a sufficient sample size, which is a disadvantage in studies with hens in particular. Repeated measurements with the same animal are impossible. Furthermore, PC measurements depend on the use of an indigestible marker. Often, the variation between replicates is high, which makes it difficult to detect differences between treatments. Digestibility was also studied based on quantitative collection of excreta from hens whose ceca had been surgically removed (Parsons, 1984). Cecectomy has been proposed for reducing the microbial influence on AA digestibility measurements (Parsons, 1984). Furthermore, it is known that the contribution of urine to the AA excretion is negligibly low (Parsons, 1986; Jirjis et al., 1997; Whittow, 2000).

Our objective was to study whether cecectomized laying hens can be used for the determination of AA digestibility of protein sources as an alternative to PC measurements without the need for killing many birds and with the possibility of collecting excreta quantitatively. As model protein sources, toasted soybeans (TS) and corn gluten meal (CG) were chosen. Apparent digestibilities were measured in all experimental diets. Because the relationship between intake and excretion of protein and amino acids is linear (Mitchell and Bert, 1954; Sibbald, 1979; Rodehutscord et al., 2004), a linear regression approach was then used for calculating digestibilities for the 2 protein sources. Prececal digestibility was determined in experiment 1 and total excreta (TE) digestibility with cecectomized hens in experiment 2.

	MATERIALS AND METHODS

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Dietary Treatments

Five diets were used in both experiments. One was a basal diet that contained corn, wheat gluten, and cornstarch as the main ingredients and that met or exceeded the requirements of laying hens recommended by the Gesellschaft für Ernährungsphysiologie (1999). The 4 other diets included TS or CG each at 15 or 30% (Table 1). The 2 protein sources replaced cornstarch in a ratio of 1:1, so that the differences in the AA concentration between the diets resulted from TS and CG alone. Titanium dioxide was included as an indigestible dietary marker. All the dietary ingredients, with the exception of TS, CG, and cornstarch, were mixed in one lot. This mix was subsequently divided in 5 equal parts, and each part was mixed with the respective amounts of TS, CG, and cornstarch. Diets were pelleted without steam through a 3-mm die and later crumbled. Table 2 shows the results of the chemical analyses of the experimental diets and the 2 protein sources.

The experiments were approved by the animal welfare authorities in accordance with the German Animal Welfare Regulations.

Experiment 1. Two hundred ten 18-wk-old Tetra Brown pullets were housed in individual crates in a temperature-controlled and illuminated room. Each group of 7 neighboring crates was considered one experimental unit. The hens had unrestricted access to the diets from individual troughs and to water from nipple drinkers. In wk 26 of age, feeding the experimental diets was started. Individual feed intake and BW were recorded. In wk 27 of age, experimental diets were offered ad libitum for 7 d. Six experimental units were allocated by random to each of the 5 diets. Feed intake in the 24 h before asphyxiation was separately measured and the number of hens in each experimental unit used for digesta collection reduced to 6, excluding the one with the lowest feed intake. Feed intake was recorded for individual hens but analyzed on an experimental unit basis. Egg production was recorded as well, but not analyzed further due to the short duration of the experiment. On d 7 the birds were asphyxiated by carbon dioxide exposure. The body cavity was immediately opened, the section between Meckel’s diverticulum and 2 cm anterior to the ileo-ceca-colonic junction removed, and its length determined. Only the digesta of the posterior two-thirds of this section were used for digesta collection, as suggested for laying hens by previous results (Rezvani et al., 2008). Digesta was gently flushed out with distilled water, pooled together for the 6 hens of the same experimental unit, frozen immediately at –20°C, and later freeze-dried.

Experiment 2. Fourteen 17-wk-old Lohmann Brown pullets were individually housed in balance crates in a temperature-controlled and illuminated room. Hens were cecectomized as described in detail by Rezvani et al. (2007) when they were between 20 and 30 wk old. The experiment started when the hens were 46 wk old and was conducted in 3 subsequent periods. Diets were allocated between hens in the 3 periods in a way that 7 replicated measures per diet were made in total and each diet considered in each period. Each period consisted of 5 d for adjustment to the diet and 5 d for quantitative excreta collection. Hens were offered 120 g/d of feed, and feed residues were collected daily. Between periods, hens were fed a commercial diet for 4 d. Feed intake and egg production were recorded for each hen. Excreta and feed refusals were collected 3 times per day at about 0800, 1400, and 2000 h, and frozen at –20°C. Excreta from each hen were pooled for the 5 d of the collection period. Feathers were removed before each excreta collection. Thawed excreta were homogenized and a subsample of about 100 g was freeze-dried. Another subsample was used for immediate determination of excreta DM content.

Analyses and Calculations

Diets, freeze-dried digesta, and excreta samples were ground through a 0.5-mm screen. Diets were analyzed for DM, ash, N, crude fat, crude fiber, AA, and TiO₂. Freeze-dried digesta and excreta samples were analyzed for DM, AA, and TiO₂. Concentrations of proximate nutrients in the diets were analyzed according to the official methods in Germany (Naumann and Bassler, 1976). Nitrogen was determined using the Kjeltec Auto 1030 Analyzer (Tecator AB, Höganäs, Sweden). Amino acid analysis was conducted according to standard procedures (Naumann and Bassler, 1976), with laboratory details as described by Rodehutscord et al. (2004). In brief, after an oxidation step, samples were hydrolyzed in 6 M hydrochloric acid. Norleucine was used as the external standard. Amino acids were separated and detected with an AA analyzer (Biochrom 30, Biochrom Ltd., Cambridge, UK) using various buffer solutions and ninhydrin. Because tryptophan is destroyed during acid hydrolysis and histidine and tyrosine are degraded during performic acid oxidation, these AA were not determined this way. Glycine was not measured in excreta samples of experiment 2, because glycine may result from uric acid hydrolysis during sample preparation (Dalgliesh and Neuberger, 1954). Tryptophan analysis followed standard procedures as described in detail by Fatufe et al. (2005). Separation and detection of tryptophan was conducted using HPLC (Agilent Technologies, 1100 series). The concentrations of TiO₂ in diets and digesta were determined spectrophotometrically according to the method described by Brandt and Allam (1987).

Digestibility of AA in the Diets. Apparent digestibilities of AA for each diet were calculated in both experiments according to the following equation:

where TiO_{2 Diet} and TiO₂ Digesta or Excreta = concentrations of TiO₂ in the diet and digesta or excreta samples (g/kg); and AA _Diet and AA _{Digesta or Excreta} = concentrations of AA in the diet and digesta or excreta samples (g/kg). Calculations were made for each experimental unit in experiment 1 and each individual hen in experiment 2; thus, the number of replicates per treatment was 6 in experiment 1 and 7 in experiment 2. Marker-based digestibility was used in experiment 2 although the amount of excreta was quantified to have the comparison of results from both experiments remain unaffected by the way of calculation of digestibility.

Digestibility of AA in the Protein Sources. The daily intake of each AA (g/d) was calculated as feed intake multiplied by the analyzed AA concentration in the diet. The quantity of digested AA (g/d) was calculated as AA intake multiplied by its digestibility determined for the respective diet. The digestibility of AA from the 2 protein sources was obtained as the slope of linear regressions of the type y = a + bx, calculated between quantitative AA intake (x) and digested amount of AA (y) as described by Rodehutscord et al. (2004). The calculated slope (b) multiplied by 100 is taken as the digestibility in percentage. By this regression the effects of the 2 protein sources were separated from the protein of the basal diet. These calculations were made using data for the basal diet and both diets including the respective protein source; hence, the number of data sets used in regression analysis for each protein source was 18 (experiment 1) and 21 (experiment 2). Regressions were calculated using GraphPad Prism 4.02 (GraphPad Software, Inc., San Diego, CA). Differences between TS and CG in AA digestibility were tested for significance by t-test, which is implemented in this software. The chosen level of significance for all comparisons was P < 0.05.

Other Statistical Analyses. The data for feed intake, BW, ileum lengths, and AA digestibility of the diets were analyzed by ANOVA using the software package SAS (V 9.1, SAS Institute Inc., Cary, NC) and taking into account the fixed effect of dietary treatment. Differences between means were assessed by Student’s t-test with Tukey adjustment. The chosen level of significance for all comparisons was P 0.05. A significant effect of period or an interaction between period and diet were not detected in experiment 2.

	RESULTS

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In both experiments hens responded to the experimental diets by a reduction in feed intake in comparison to the preexperimental period (Table 3). Feed intake was highest when the diets that contained TS were fed, but the differences to the other treatments were significant only in experiment 1. Hens weighed 1.9 to 2.0 kg before the experiments started. The sampled section of the ileum in experiment 1 was shorter when the basal diet was fed in comparison with the diets containing the protein sources.

Experiment 1. Prececal digestibility of most AA was improved with increasing inclusion of the protein sources in the diets (Table 4). Differences between means were significant, however, only for Ala, Arg, Asp, Leu, Ser, and Thr. The average digestibility of all AA for the diets containing TS was 86%, at a minimum for Thr (76%) and a maximum for Glu (94%). The average digestibility of all AA for the diets containing CG was also 86%. It was at a minimum for Trp (76%) and at a maximum for Glu (93%).

The digestibilities for the 2 protein sources, determined by linear regression analysis, are summarized in Table 7, and the regression for Met is shown as an example in Figure 1. In experiment 1, the linear regression model explained 97 to 99% of the variance observed for TS and 87 to 99% for CG (Table 7). The differences in the digestibilities calculated between the 2 protein sources were not greater than 3% with the exception of Lys, where the difference was 6%. None of the differences was statistically significant. Prececal digestibility ranged from 85% (Cys) to 95% (Arg, Glu) in TS and from 83% (Trp) to 95% (Pro) in CG. In experiment 2, r² was very high (0.99) for all AA from both protein sources (Table 7). Significant differences in TE digestibility between the 2 protein sources were detected for Ala, Leu, Lys, Ser, and Thr. Total excreta digestibility ranged from 81% (Cys) to 96% (Arg, Glu) for TS and from 80% (Cys) to 97% (Leu) for CG.

View larger version (14K): [in this window] [in a new window]   Figure 1. Relationship between intake and digested amounts of Met from toasted soybeans (TS) and corn gluten meal (CG), determined in experiment 1 (upper panel) and experiment 2 (lower panel). PC = prececal; TE = total excreta.

	DISCUSSION

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The digestibilities of all AA studied for the data pooled across all diets were significantly higher in experiment 2 than in experiment 1. This may have been caused by postileal degradation of AA by gastrointestinal tract enzymes or microorganism activity (Kadim et al., 2002) although the ceca had been removed. Furthermore, the hens in experiment 2 were older than those in experiment 1, and a previous study indicated that age could affect AA digestibility in hens (Rezvani et al., 2007). Such an age effect may be related to changes in AA absorption or endogenous secretion, as Ravindran and Hendriks (2004) reported.

By contrast, AA digestibilities of the 2 protein sources were not significantly different between both experiments (Table 7). When digestibilities are determined with linear regressions, the estimates of the slopes are unaffected by the AA contained in the basal diet and basal endogenous AA losses, which are part of the estimated intercept (Rodehutscord et al., 2004). If differences in basal endogenous gut losses had existed between the experiments, they would have not been relevant to the digestibility values calculated for the protein sources. Also, possible basal urinary AA losses in experiment 2 were without any effect on the calculated digestibilities because they were part of the estimated intercepts. Jirjis et al. (1997) reported that increasing the protein content of diets fed to turkeys from 228 to 330 g per kg did not influence the urinary excretion of AA; hence, we assume that urinary excretion was without relevance to the calculated slopes. Rezvani et al. (2008) have already used the regression approach to study AA digestibilities with laying hens at the end of the ileum. To our knowledge, the data from experiment 2 of the present study and the strong linear relationship between AA intake and digested amounts (Figure 1) show for the first time that the linear regression approach can be used in studies with cecectomized hens and based on total excreta collection. From the absence of any significant difference between experiment 1 and 2, we conclude that cecectomized laying hens can be used to study the AA digestibility of individual protein sources, and that the results are comparable with those from PC measurements.

Using cecectomized hens has some advantages. By using cecectomized hens, only few birds are needed. The sample that can be obtained from one animal is much bigger than when taken from the terminal ileum. Repeated measurements can be made with each hen for several protein sources. Thus, killing a great number of birds in the process of sample collection can be avoided. Further, existing differences between different protein sources are easier to detect than in PC studies because the SE of measurement is lower although fewer birds are used. In experiment 1, r² of all regression lines was high. The amount of individual AA digested prececally depended linearly on its respective intake for all AA studied. These findings agree with earlier reports (Short et al., 1999; Ishibashi and Yonemochi, 2003; Rodehutscord et al., 2004). No significant differences in PC digestibility between the 2 protein sources were detected although the differences between the 2 protein sources were up to 6% (Lys). In experiment 2, r² of the calculated linear regressions was also high, and the relation between intake and digested amounts of AA strongly linear. In contrast to experiment 1, differences in digestibility between the 2 protein sources were significant for several AA. Lysine digestibility was higher in toasted soybeans than in corn gluten meal, but digestibility of Ala, Leu, and Ser was lower in toasted soybeans than in corn gluten meal. In this context, it is important to note that the SE of the predicted slopes was different between the 2 experiments. It ranged from 1.9 to 7.9% in experiment 1, but only 0.4 to 2.4% in experiment 2 (Figure 2). This made it easier in experiment 2 to detect existing differences between the protein sources than in experiment 1. The lower SE in experiment 2 was probably possible because the sample collected from the cecectomized hens over time was more representative of the digestive process than the spot sample that was taken from the terminal ileum of killed hens. Results from an earlier study showed that the length of the ileum used for sample collection affects the measured digestibility (Rezvani et al., 2008). This suggests that the process of AA digestion is not completed in all hens in the same section of the small intestine, which may also contribute to the high SE of AA digestibility measurements on the PC level.

View larger version (22K): [in this window] [in a new window]   Figure 2. Standard error of amino acids digestibility of toasted soybeans (TS) and corn gluten meal (CG) measurement in experiment 1 and experiment 2.

It can be concluded that cecectomized laying hens and their excreta can be used to study AA digestibility for protein sources. The suitability of the regression approach as a standard approach for considering basal endogenous AA losses can be confirmed for cecectomized hens. Although PC digestibility appears the method of choice in young and growing birds, the use of cecectomized animals and total excreta collection has advantages in comparison with the determination of PC digestibility in hens. Fewer animals are needed, repeated measurements with each hen are possible, and existing differences between protein sources are easier to detect because of lower variation.

	ACKNOWLEDGMENTS

 
Mohammad Rezvani is grateful to the Iranian General Office of Scholarships and Overseas Students for the postgraduate grant supporting his studies at the University of Halle-Wittenberg.

	FOOTNOTES

 
¹ Actual address: Animal Science Department, School of Agriculture, Shiraz University, Bajgah, Isfahan Freeway, Shiraz, Iran.

Received for publication April 4, 2008. Accepted for publication July 8, 2008.

	REFERENCES

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