Comparative study of two analytical procedures for the determination of acid insoluble ash for evaluation of nutrient retention in broilers †

Inert markers are routinely used in research to estimate nutrient retention and apparent metabolisable energy nitrogen-corrected (AMEn) content of poultry diets. Acid insoluble ash (AIA) is used as a marker to substitute metal compounds because of environmental concerns. In the current research, two methodologies recommended for determining AIA content in feeds and excretas for the evaluation of total tract apparent retention (TTAR) of nutrients, were compared in 12 broiler diets. The experimental design was completely randomised with 2 AIA analytical techniques and 12 dietary treatments that resulted from a combination of two cereals (corn and rice), two heat processing of the cereal (raw and cooked) and three fiber sources (control with no added fibre, 3% oat hulls and 3% soybean hulls). All diets included 1% celite (diatomaceous earth) as additional source of AIA. The techniques used for AIA determination were coded as VO and GA, respectively. The TTAR of dry matter and organic matter and the AMEn of the feeds differed (p ≤ 0.001) among diets and were lower when using GA than when using VO (p ≤ 0.05). However, no interaction between diet and methodology was observed. Moreover, the TTAR of nutrients as determined by both techniques, were highly correlated (r > 0.98). We concluded that the GA methodology results in lower retention values than the VO methodology but that both methodologies can be used indistinctly to estimate TTAR of nutrients in broiler feeds. Additional key words: analytical methodologies; inert markers; metabolisable energy; nutrient digestibility; poultry.


Introduction
Inert markers are routinely used in nutrient digestibility studies to eliminate the need of total collection of the excreta produced (Sales and Janssens, 2003a).Chromic oxide (Cr 2 O 3 ) has been the main inert marker used in poultry studies for years (Saha and Gilbreath, 1993;Lázaro et al., 2003b).However, Cr 2 O 3 might interfere with other minerals present in the diet (Saha and Gilbreath, 1991a) resulting in low fecal recovery rates of Cr.Saha andGilbreath (1991b) in swine, Hill et al. (1996) in dogs and Titgemeyer (1997) in ruminants, have reported recovery rates of Cr of only 93.3%, 87.0% and 94.0%, respectively.In fact, Lázaro et al. (2003b) showed an incomplete recovery of the Cr in broilers fed rye-based diets, with values varying between 86.4 and 92.3.The voiding of Cr in feces is not uniform (Prigge et al., 1981) with substantial variability in diurnal excretion patterns.In addition, Cr is a heavy contaminant of the environment (Sprinkle et al., 1995) and has carcinogenic properties (Peddie et al., 1982).Thus, the use of Cr 2 O 3 in nutritional research is no longer recommended (Titgemeyer et al., 2001).Problems have been observed also with the use of titanium dioxide (Rymer, 2000;Kavanagh et al., 2001), as a marker.For example, Yin et al. (2000) reported that the recovery of titanium in pigs fed diets high in fibre was very low, ranging between 60.6 and 67.8%, a variability which was probably related to segregation of the marker.Acid insoluble ash (AIA) is a natural alternative to mineral external markers which has shown mean recovery rates close to 100% in several species (Sales and Janssens, 2003a,b).Scott and Boldaji (1997) reported that AIA provided a more accurate estimate of the feeding value of barley in growing chickens than Cr 2 O 3 .Acid insoluble ash occurs naturally in feed ingredients (McCarthy et al., 1974) but the amount of AIA in the final feed is usually low, which makes convenient the inclusion of extra amounts of the marker in the diet to increase the accuracy of the chemical analysis (van Leeuwen et al., 1996;Gracia et al., 2003).Two assays recommended for the determination of AIA in animal digestibility studies are those reported by Vogtmann et al. (1975) and Van Keulen and Young (1977).The Vogtmann et al. (1975) technique (VO) has been successfully used to estimate nutrient digestibility in non-ruminant (Batal and Parsons, 2002;Valencia et al., 2008Valencia et al., , 2009) ) whereas the Van Keulen and Young (1977) technique (VK) has been used more in research conducted with ruminants (Thonney et al., 1979;Sunvold and Cochran, 1991).The VK technique analyses and obtains sequentially, the dry matter (DM), total ash and AIA content of the sample.Consequently, less amount of sample is required.Recently, González-Alvarado et al. (2007) (GA) recommended to introduce some modifications to the VK methodology when used in poultry digestibility studies.The technique of GA has been used consistently in recent studies (De Coca-Sinova et al., 2008;Jiménez-Moreno et al., 2009;González-Alvarado et al., 2010).However, no data have been generated comparing the VO and GA methodologies in broilers.This research was conducted to compare two techniques used in research to estimate AIA content of diets and excretas and nutrient retention in broiler fed diets varying widely in ingredient composition.

Experimental diets, husbandry and experimental design
The ingredient composition and nutritive value of the 12 experimental diets used have been reported elsewhere (González-Alvarado et al., 2007)  protein concentrate and resulted from the combination of two cereal sources (corn and rice), two heat processing of the cereal (raw vs. cooked) and three fibre sources (control with no additional fibre and 3% of either oat hulls or soy hulls).The fibre sources were included in the diets at expenses (w:w) of sepiolite, a complex magnesium silicate clay added as inert material.Acid washed diatomaceous earth was included as additional inert marker in all the diets.The diets were fed in mash form on ad libitum bases to chicks from 1 to 18 days of age.
All procedures used in this research were in compliance with the Spanish Guidelines for the Care and Use of Animals in Research (BOE, 2005).Details on husbandry and care of chicks have been reported elsewhere (González-Alvarado et al., 2007).Briefly, a total of 576 one day old male chicks (Cobb 500) were randomly placed in groups of 16 in 36 battery cages (3 cages per treatment).The trial was conducted as a completely randomized design with 12 diets arranged factorially with the two analytical methodologies and the 12 dietary treatments previously described.

Laboratory analyses and acid insoluble ash determination
Details on the analytical procedures used to determine the chemical composition and the nutritive value of diets and excreta have been reported elsewhere (González-Alvarado et al., 2007).The AIA of diets and  Shrivastava and Talapatra (1962) and differ in 1) size of sample (12 g for diet and 5 g for excreta vs. 5 g for both diet and excreta), 2) concentration of HCl (4 N vs. 2 N), 3) boiling time (30 min vs. 5 min), 4) ashing after acid treatment vs. before and after acid treatment), 5) duration and temperature applied for ashing of the sample (4 h at 600°C vs. 12 h at 600°C for the first ashing and 6 h at 450°C for the second ashing) and 6) number of analysed variables (AIA exclusively vs. sequential determination of DM, ash and AIA) (Table 2).Briefly, for the VO methodology, ground samples of feed (12 g) and excreta (5 g) were boiled in a 200 mL Erlenmeyer flask with 100 mL of 4 N HCl that was fitted with a reflux condenser to prevent any HCl loss for 30 min.The slurry was then filtered through ash free filter paper (Whatman Nº 541) and the residue washed hot distilled water (85 to 100°C) until free of acid.Then, the residue was transferred to a tared crucible, dried at 103°C for 48 h and ashed at 600°C for 4 h.After ashing, the crucible was cooled in a desiccator to room temperature and weighed.For the GA methodology, ground samples of feed and excreta (5 g) were dried at 103°C for 24 h in a 100 mL Erlenmeyer flask for DM determination.
Then, the dried sample was ashed at 600°C for 12 h for crude ash determination.Afterwards, ashes were hydrolyzed with 40 mL of 2 N HCl, boiled for 5 min and the obtained residues were filtered through ash free filter paper (Whatman Nº 541) and washed until free of acid with hot distilled water (85 to 100°C).The ashes retained in the paper filter were transferred into the same Erlenmeyer flask and dried at 103°C for 48 h.Finally, the ash and paper filter were ashed again at 450°C for 6 h.The flask and its content were cooled in a desiccator to room temperature and weighed to calculate the AIA content.

Total tract apparent retention of dietary components
At 18 days of age of the birds, representative samples of excreta produced during the previous 24 h were collected by replicate, mixed, homogenized, oven-dried (60°C for 72 h) and ground with a hammer mill (Model Z-I, Rechst, Stuttgart, Germany) fitted with a 1-mm screen.Total tract apparent retention (TTAR) of DM, organic matter (OM), nitrogen and ether extract, and the apparent metabolisable energy nitrogen-corrected (AME n ) of the diets were estimated according to VO and GA methodologies using AIA as a marker.The TTAR of nutrients was calculated by the following equation: where Nutrient e and AIA e are the concentrations of dietary components and of AIA in the excreta, respectively, and Nutrient f and AIA f represent the concentrations of the same dietary components and the AIA in the feeds, all of them expressed on g kg -1 of DM.The AME n content of the experimental diets was calculated as indicated by Lázaro et al. (2003a).

Statistical analysis
All data were analysed as a completely randomised design with treatments arranged factorially with two analytical methodologies and 12 diets using the GLM procedure of SAS (SAS Institute, 1990).The main effects of diet and methodology as well as the interaction between diet and methodology on AIA content in excreta and TTAR of nutrients were evaluated.When significant differences were detected, treatment means were separated using the Tukey's test.The experimental unit was the cage for all traits and differences among  treatments were considered significant at p ≤ 0.05.In addition, the REG procedure of SAS (SAS Institute, 1990) was used to determine linear least squares regressions to provide equations between AIA content of the excreta as determined, and between AIA and total ash and gross energy contents of the excreta by the VO and GA methodologies.The correlation coefficients (r) were also calculated.In addition, the CORR procedure of SAS (SAS Institute, 1990) was used to determine the Pearson's correlation coefficient between TTAR of nutrients and AME n of the diets as estimated by the two methodologies.

Results and discussion
The AIA content of the excreta differed (p ≤ 0.001) among diets and was lower (p ≤ 0.001) when determined by the GA technique than when determined by the VO technique (Table 3).The differences observed in the AIA content of the experimental diets reflected differences in HCl-insoluble ash content of the ingredients used.The main reason for the higher AIA content of the control diets without fibre supplementation was the inclusion of 3% sepiolite in these diets.negative correlation between AIA and gross energy, and positive between AIA and total ash observed with both methodologies (-0.972 and 0.974 for VO and -0.972 and 0.970 for GA), is conf irmed by the Pearson's coefficients values obtained (Table 4).These results suggest that the concentration of HCl used might affect the AIA determination with lower values when lower HCl concentration was used.However, Furuichi and Takahashi (1981) did not find any significant difference in AIA concentration in feeds and faeces of rabbits when the boiling process was performed using 2, 4 or 6 N HCl.Similarly, Bergero et al. (2009) did not find any difference in AIA values in feeds and faeces of horses when using 2 N or 4 N HCl.However, Van Keulen and Young (1977) reported in sheep that the use of 4 N HCl provided higher (p ≤ 0.05) estimates of nutrients digestibility than the use of 2 N HCl.This information suggests that in addition to the concentration of the HCl used, other factors, such as the characteristics of the excreta of the different animal species, could affect the effects of HCl on AIA recovery.When the GA technique is used, samples of diets and excreta are ashed for longer time than when the VO technique is used.

Analytic methodologies of acid insoluble ash 765
Table 3. Acid insoluble ash content (AIA, % dry matter) in feeds and excreta as determined by VO and GA methodologies1 in broilers at 18 days of age Therefore, the conditions applied when using the GA technique might be better adapted to all types of excreta than the VO technique, ensuring the complete ashing of the organic material contained in the original samples.For AIA content of the excreta the regression equation between the two techniques was: y = 0.972x (± 0.0066) -0.112 (R 2 = 0.998; residual standard deviation = 0.1219; p < 0.0001) where y and x were the percentage of AIA in the excreta on DM basis using the GA and VO technique, respectively (Fig. 1).The correlation coefficient for AIA content of the excreta between VO and GA techniques was high (r = 0.999) suggesting that both techniques can be used indistinctly for the determination of AIA and the estimation of nutrient retention in broiler studies.Nutrients digestibility differed among diets and in general was higher with diets containing 3% oat hulls than with containing 3% soy hulls (Table 5).Similarly, nutrient retention values were higher when the VO methodology was used.However, no interactions between methodology and diet were detected for any of the variables studied.The TTAR of nutrients as determined by both techniques was highly correlated (r > 0.98; data not shown).The TTAR of DM, OM and the AME n of the diets were lower (p ≤ 0.05) with the GA than with the VO technique.The CV values of CTTAR of DM and OM and of AME n of the diets were low (0.92%, 1.01% and 0.71%, respectively) for both methodologies.However, for ether extract (1.20%) and especially for total nitrogen retention (4.43%), the CV values were less acceptable.Probably, these high CV values precluded the detection of significant differences between methodologies for such fractions (ether extract and nitrogen).
The sequential analysis of DM, total ash and AIA content, as recommended by González-Alvarado et al. (2007), minimizes sample losses that inevitably occur when analysis is not performed using the same flask.Therefore, GA might be the technique of choice for estimating AIA when the size of the sample is very small.Thus, the GA methodology might have advantages over the VO methodology in ileal digestibility studies carried out in chicks, in which AIA and nutrient contents have to be determined in the digesta from different intestinal segments of individual birds and in which the amount of collected sample is generally small.Under these circumstances, the GA technique allows to analyse more variables per sample or to perform more analyses per each variable of interest.Therefore, the GA technique might improve the assessment of the feeding value of ingredients or diets, over the VO technique.
We conclude that nutrient retention values are higher when the VO methodology is used in comparison to the GA methodology.However, both techniques are highly correlated and can be used indistinctly for efficient quantification of AIA in comparative studies on nutrient retention in broilers.Moreover, the GA me- 764A. de Coca-Sinova et al. / Span J Agric Res (2011) 9(3), 761-768 766A. de Coca-Sinova et al. / Span J Agric Res (2011) 9(3), 761-768 time and effort and less sample size to perform the same number of analytical measurements per sample.Therefore, this technique may be more convenient in nutrient ileal digestibility studies than the VO technique.
and are shown in Table1.The diets were based on cereals and soy Se concluye que la metodología GA proporciona valores de retención de nutrientes inferiores a los obtenidos con VO, aunque ambas pueden ser utilizadas indistintamente para estimar la digestibilidad fecal de nutrientes en avicultura.

Table 1 .
Analytic methodologies of acid insoluble ash763 Ingredient and chemical composition of the experimental diets (% as fed-basis, unless otherwise indicated)

Table 2 .
Analytical conditions applied for the determination of acid insoluble ash (AIA) in feed and excreta

Table 4 .
Regression equations between acid insoluble ash (AIA, % dry matter) and ash and gross energy (GE) contents of the excreta according to the methodology 1 used See Table2. 2 Standard error of estimated slope. 3 Residual standard deviation.

Table 5 .
Total tract apparent retention (%) of nutrients and apparent metabolisable energy nitrogen-corrected (AME n , kcal kg -1 ) of the diets as estimated by VO and GA methodologies 1 in broilers at 18 days of age