Evaluation of chromium oxide and titanium dioxide as inert markers for calculating apparent digestibility in sheep

ABSTRACT

The objective of the present study was to evaluate two markers: chromium oxide (Cr₂O₃) and titanium dioxide (TiO₂). We evaluate the interaction between Cr₂O₃ and TiO₂, and the techniques used to determine it, using atomic absorption spectrophotometry (AAS) and photometry simple (PS). We used six growing sheep distributed in a replicated Latin square 3 × 3 design, with adjustment for the residual error effect. The TiO₂ and Cr₂O₃–TiO₂ produced values similar to those obtained by total faecal collection (TFC) or the use of Cr₂O₃ alone, determined by AAS and PS. Digestibility of the marker/TFC ratio was similar (p > .05) between markers and technique. The use of TiO₂ alone or in combination with Cr₂O₃ seems to be a suitable alternative to TFC and Cr₂O₃ to calculate apparent digestibility of the total digestive tract determined in sheep by PS and AAS.

KEYWORDS:

• Chromium oxide
• digestibility
• markers
• sheep
• spectrophotometry
• titanium dioxide

1. Introduction

Chromium oxide (Cr₂O₃) has been widely used as an external digestibility marker in digestion trials with ruminants (Amorocho et al. 2009; Delagarde et al. 2010; Al Alami et al. 2014). The recovery of this marker in ruminants’ fed forage has revealed variations in faecal recovery (Titgemeyer et al. 2001); on the other hand, the use Cr₂O₃ is restricted in diets for animals because it possesses carcinogenic properties (Peddie et al. 1982; Sedman et al. 2006). Titanium dioxide (TiO₂) is presented as an alternative, but its study in small ruminants is scarce (Titgemeyer et al. 2001; Glindeman et al. 2009). Additionally, the combined use of these markers in vivo presented differences in digestibility coefficients but had similar recoveries (Kavanagh et al. 2001; Titgemeyer et al. 2001).

Several analyses for these markers differ for colorimetric and spectroscopy techniques, the latter being the most commonly recommended for the least amount of interference present (AAFCO 2004). However, in vivo studies of these two procedures showed no differences (Carciofi et al. 2007). For TiO₂ we do not know whether there are differences between these two methods: colorimetric and spectroscopy.

The aim of this study was to evaluate the use of Cr₂O₃ and TiO₂ as digestibility markers, as well as their interaction Cr₂O₃ + TiO₂, as determined by atomic absorption spectrophotometry (AAS) and colorimetric methods, and to compare digestibility coefficients obtained from total faecal collection (TFC) in sheep.

2. Material and methods

The Institutional Committee for Care and Use of Experimental Animals (CICUAE) approved all procedures of the National Autonomous University of Mexico. Six Pelibuey lambs (mean ± SD) (LW 40.7 ± 5.8 kg) were used to assess recovery of markers: Cr₂O₃ (Prince Minerals, Inc. New York, USA, 1308-31-2; 100% Purity), TiO₂ (Fisher Scientific. Pittsburgh, PA, USA, T315-500; >98% Purity) and the combination Cr₂O₃ + TiO₂. Food intake as dry matter (DM) was restricted to 2.2% of live weight and there were no feed refusals. The diet (10% CP, 11 MJ ME/kg DM; Table 1) was supplemented with 0.4% of the marker (Cr₂O₃, TiO₂ and the combined mixture) and was given at 10:00 and 22:00 h daily. Every day the food was mixed with each marker before being provided to the animals. The animals were housed in metabolic cages, with a harness to collect faeces individually.

Table 1. Ingredients and chemical composition (g/kg DM) in the diet using Cr₂O₃, TiO₂ and their interaction used in growing lambs.

Ingredients (g/kg DM)	Cr2O3	TiO2	Cr2O3 + TiO2
Sorghum grain	720.0	720.0	720.0
Alfalfa hay	61.8	61.8	61.8
Oat hay	123.5	123.5	123.5
Molasses	54.6	54.6	54.6
Urea	10.0	10.0	10.0
Calcium, 38%	11.5	11.5	11.5
Phosphate 18/20	2.0	2.0	2.0
Magnesium oxide	2.1	2.1	2.1
Na Bicarbonate	5.0	5.0	5.0
NaCl	5.0	5.0	5.0
Minerals mix^a	0.5	0.5	0.5
Cr2O3	4.0	ND^b	4.0
TiO2^b	ND	4.0	4.0
Chemical composition
DM	917	921	919
OM	871	870	871
NDF	166	163	164
N	15	15	15

^aMinerals mix: Ca 15.0%, P 8.0%, Mg 0.5%, K 0.035%, S 0.2%, Fe 450 mg/kg, Zn 1900 mg/kg, Mn 1800 mg/kg, Se 15 mg/kg, I 30 mg/kg, Co 25 mg/kg.

^bND, no data; the marker is not part of the diet.

Each experimental period lasted 25 d, and consisted of 20 d for diet adaptation and 5 d for data and sample collection. The sample faeces were collected at 09:00 h, weighed and separated at 10% of total, and mixed to obtain a composite sample for each animal per period and frozen (−20°C) for further analysis.

Feed and faeces samples were analysed for DM in a forced air oven (Lindbergh Blue M) at 50°C, 48 h; ash concentration at 660°C, 3 h; organic matter (OM) was determined by difference (AOAC, 1990); neutral detergent fiber (NDF) in feed samples was analysed according to Van Soest et al. (1991) with alpha amylase and uncorrected for ash, and N content by the Kjeldahl method (AOAC 1990); chromium oxide suspension was performed according to Hill and Anderson (1958), and titanium dioxide according to Titgemeyer et al. (2001). Both were analysed through a photometry simple (PS) (Thermo Fisher model Scientyfic Genesys 10 Vis) with a wavelength of 430 nm for Cr₂O₃ and 410 nm for TiO₂; an AAS (Perkin Elmer 3110) was used to determine Cr₂O₃ and TiO₂ with a Perkin Elmer lamp for Cr (part#303-6021 Serial H235571) and Ti (part#303-6075 Serial H167419) for each marker, respectively. The calibration curve was performed with standard solutions for Cr₂O₃ PS with 0.05, 0.01, 0.15 and 0.5 mg/ml (y_mg/ml = 0.481x_(OD) – 0.006; R² = 0.99), and 20, 50 and 100 ppm for AAS (y_ppm = 206.19x_(ABS) – 3.7113; R² = 0.99) (standard for AAS from J.T. Baker 1000 µg/ml CAS 6449-04 for Cr), as for TiO₂ PS with 0.005, 0.01, 0.075, 0.15 and 0.2 mg/ml (y_mg/ml = 0.3226x_(OD) – 0.008; R² = 0.98) and for AAS 10, 20 and 40 ppm (y_ppm =0.175.6x_(ABS) + 1.5; R² = 0.99) (standard for AAS from J.T. Baker 1000 µg/ml CAS 6472-04 for Ti), where ABS is the absorption wavelength value and OD is the optical density value. The combinations of Cr₂O₃ + TiO₂ differ in that the two markers are mixed, but we only suspended and analysed one of the compounds.

The proportion of DM and OM degraded was calculated using the following equation:(1) where intake and excretion are expressed in g/d, and x represents DM and OM content, respectively.

The recovery of the markers was determined using the following equation:(2)

The faecal excretions of chromium and titanium dioxide were estimated by multiplying the total DM excretion by the marker content in the representative faecal sample, according to the following equation:(3) where MFE is the faecal excretion of marker (kg/d), FM is the faecal mass obtained by total collection (g/d) and M_(RS) is the marker content in the representative faecal sample (g/kg).

From the assumptions of Equation (3), the faecal recovery of the markers was calculated as the ratio of faecal excretion to the intake of marker, described as:(4) where FR is the faecal recovery of marker (%), MFE is the faecal excretion of marker (g/d) and D is the daily dose of external marker (g/d).

The faecal excretion was also determined by using the faecal content of markers according to the following equation:(5) where FE_RS is the faecal excretion estimated by using the marker content in the faecal representative sample (kg/d), D is the daily dose of external marker (g/d), and [M]_RS is the marker content in the representative faecal sample (g/kg) (Lippke 2002). The values of total tract apparent digestibility in each treatment were subjected to analysis of variance using a design of two replicated Latin squares with adjustment for residual error, including three treatments in six animals. Treatment sequences within each Latin square were organized to balance the effects of carryover such that each treatment followed every other treatment one time within each square using the GLM procedure SAS (1999), following the model:(6) where Y_jkl is the dependent variable, _µ is the overall mean, P_j is the effect of period j, A_k is the effect of animal k, T_l is the effect of treatment l, and ε_jkl is the residual error.

Furthermore, in order to evaluate the technical values of total tract apparent digestibility and the digestibility of the markers/TFC ratio (DM and OM), a factorial arrangement 4 × 2 with six replicates for each one (Kuehl 2000) was performed using the statistical program Statistical Package for Social Sciences (SPSS, version 15.0, November 2006),(7) where Y_jk is the dependent variable, _µ is the overall mean, M_j is the effect of marker treatment j, T_k is the effect of determination technique k (PS vs. AAS), M × T_jk is the interaction jk, and ε_jk is the residual error. Differences between the means of the least squares were considered significant at p < .05, and differences were considered to indicate a trend towards significance at .05 > p < .10.

3. Results and discussion

Diets (g/kg DM) averaged 919 g/kg DM; OM, 871 g/kg DM; NDF, 165 g/kg DM, and N, 15 g/kg DM, consisting of the same amount of ingredients for each marker (Table 1).

The recovery percentage (Table 2) was similar between markers (p > .05), and the techniques used do not affect the TiO₂ determination (this may be due to the inclusion of the Cr₂O₃ level used in the present study). Therefore, it can be assumed that chromic oxide and titanium dioxide achieve the necessary requirement for an ideal marker, because faecal recovery of both markers seems to be unaffected by different feeding conditions. From this, assuming that there is total faecal recovery and there are no diet effects on recovery, both external markers can be presumed to be similar to each other. Thus, titanium dioxide can be assuredly used as a substitute for chromic oxide, which is more desirable because it has not been reported as a carcinogenic precursor.

Table 2. Markers recovery percentage in faeces, using Cr₂O₃, TiO₂ and Cr₂O₃+TiO₂, estimated by PS and AAS.

Markers^a	CrPS^b	CrAAS^c	TiPS^d	TiAAS^e
Cr2O3	140.8	138.3	ND^h	ND
TiO2	ND	ND	148.7	121.1
Cr2O3 + TiO2	148.2	162.9	143.5	128.4
SEM^f	8.0	6.5	8.3	10.8
p value^g	.388

^aMarkers, 0.4% Cr₂O₃, 0.4% TiO₂, 0.4% + 0.4% TiO₂ Cr₂O₃.

^bCr-PS: Cr₂O₃ determined by PS.

^cCr-AAS: Cr₂O₃ determined by AAS.

^dTi-PS: TiO₂ determined by PS.

^eTi-AAS: TiO₂ determined by atomic absorption spectrophotometry.

^fSEM, standard error of the mean.

^gp value: value of general significance.

^hND, no data; the marker is not part of the mixture.

Although the recovery of Cr₂O₃ in the presence of TiO₂ is the highest numerically (Table 2), no statistical difference was found (p = .388) from those markers analysed by PS and AAS. It remains unclear as to how the presence of Ti could improve the assessment of Cr recovery. The values differ from Titgmeyer et al. (2001), who recovered 112% of Cr₂O₃+ TiO_2, analysed by AAS using the technique proposed by Williams et al. (1962). In another study, Kavanagh et al. (2001) showed 96% recovery in pig diets adding Cr₂O₃; however, Jagger et al. (1992) obtained recoveries of 74% and 80% for diets supplemented with 1 g and 5 g Cr₂O₃/kg respectively, not in combination with TiO₂, using the colorimetric method of Fenton and Fenton (1979). Using the same technique, Carciofi et al. (2007) recovered 106 ± 0.044 and 101 ± 0.045% of Cr₂O₃ with AAS and PS, respectively. The recovery percentages differ from those of Jagger et al. (1992), who reported recoveries of uncombined Cr₂O₃ with TiO₂ of 98.3% and 96.9%, respectively, for additions of 1 g and 5 g/kg of marker; moreover, Kavanagh et al. (2001) found 92.3% recoveries with 1 g/kg when combined with Cr₂O₃; Titgmeyer et al. (2001) found recoveries of 95% adding 5 g TiO₂/kg DM. Hafez et al. (1988) obtained 99% recovery in concentrate-based diets and corn silage forage diets.

Different methodologies for determining TiO₂ vary according to the use of substance for the wet samples. Leone (1973) recommended 10 ml of concentrated H₂SO₄, but Jagger et al. (1992) and Short et al. (1996) modified this technique, using twice as much H₂SO₄. Titgmeyer et al. (2001) modified this technique using 7.4 M H₂SO₄ and 10 ml of 30% H₂O₂. However, Myers et al. (2004) changed the use of the ashing procedure prior to the suspension, by the wet suspension of the sample with concentrated H₂SO₄. The wavelength used was different in each technique, varying from 400 nm by Short et al. (1996) and Kavanagh et al. (2001), to 408 nm by Jagger et al. (1992) and Leone (1973) and 410 nm by Titgmeyer et al. (2001) and Myers et al. (2004). The wavelength used in each technique may affect the sensitivity obtained for TiO₂. Myers et al. (2004) founded better recoveries when using 409 vs. 410 nm; in the present study TiO₂ was determined at 410 nm.

DM and OM intake were similar among treatments (Table 3), due to the feed intake being restricted to 2.2% of live weight; DM digestibility (g/d) was higher (p < .05) for the combination TiO₂ + CrO₃ than for either CrO₃ or TiO₂ alone. The OM digestibility showed a rising trend between markers (p = .087), being higher for the combination TiO₂ + CrO₃. The DM and OM digestibility (g/g) and the digestibility marker/TFC ratio were similar between markers (p > .05). DM digestibility (g/d) comparing PS vs. AAS showed a rising trend (p = .062), being higher (1.8%) for AAS than PS, and similar (p > .05) when data are expressed in OM; the DM and OM digestibility and the digestibility marker/TFC ratio was similar between techniques (PS and AAS; p > .05). Across treatments, DM digestion averaged 74.35%; this agrees closely with values reported previously (74.1%, Almaraz et al. 2010 and 75.25%, Cabral Filho et al. 2013) with lambs fed a sorghum-based diet, even though the chemical composition of the diets was different.

Table 3. Intake (g DM/d), digestibility (g/g) and digestibility marker/TFC¹ ratio, obtained by Cr₂O₃, TiO₂ or combined determined by PS and AAS.

Item	Markers (M)^2				Technique (T)		SEM^5	p value
	Cr2O3	TiO2	Cr2O3 + TiO2^3	TiO2 + Cr2O3^4	PS	AAS		M	T	M × T
Intake, g/d
DM	909	909	909	909
OM	863	860	862	862
Digestibility, g/d
DM	746^a	751^a	752^ab	774^b	749	763	3.8	.041	.062	.625
OM	727	731	729	748	729	739	3.3	.087	.115	.569
Digestibility, g/g
DM	0.83	0.81	0.84	0.83	0.83	0.84	0.004	.070	.258	.264
OM	0.85	0.83	0.86	0.85	0.85	0.85	0.004	.101	.290	.243
Digestibility, marker/TFC ratio
DM	1.00	0.99	1.02	1.02	1.01	1.01	0.007	.367	.927	.271
OM	1.00	0.99	1.02	1.02	1.00	1.01	0.006	.360	.913	.204

Note: Means with the different letter in the same row are significantly (p < 0.05) different.

¹TFC: total faecal collection.

²Markers, 0.4% de Cr₂O₃, 0.4% TiO₂, 0.4% Cr₂O₃ + 0.4% TiO₂.

³Estimated digestion based on chromium.

⁴Estimated digestion based on titanium.

⁵SEM, standard error of mean.

This results are similar to Carciofi et al. (2007), who obtained no differences in digestibility using Cr₂O₃ compared with TFC determined by PS or AAS, while Titgmeyer et al. (2001) overestimate the digestibility using Cr₂O₃ and underestimate with TiO₂ when compared with TFC. Kavangh et al. (2001) obtained similar values for TFC and Cr₂O₃, while the values obtained with TiO₂ were lower compared with TFC and Cr₂O₃.

The observed difference between DM digestibility (g/d) by ASS vs. PS may be due to variations in the estimation of digestibility caused by a lack of certainty in the analysis of the marker (TiO₂ + CrO₃), as the use of the wet sample proposed by Hill and Anderson (1958) includes HClO_4, which could saturate the solutions with potassium perchlorate and cause errors in the readings when it is mixed with TiO₂. Kavanagh et al. (2001) and Titgmeyer et al. (2001) found TFC values similar to those using Cr₂O₃ mixed with TiO₂, determined by the technique proposed by Williams et al. (1962).

The results of the present study regarding titanium dioxide agree with Ferreira et al. (2009), Batista Sampaio et al. (2011), and Glindeman et al. (2009), who reported similar results for the marker/TFC ratio obtained with titanium dioxide. Additionally, Marcondes et al. (2008) and Ferreira et al. (2009) observed that titanium dioxide could be accurately used for estimating the individual intake of concentrate in group feeding when it was associated with other markers.

4. Conclusions

These experiments show that the use of TiO₂ or TiO₂ with Cr₂O₃ is a reliable marker for calculating TFC and for determining the apparent digestibility of the total digestive tract in growing lambs, as determined by AAS and colorimetric methods.

Acknowledgments

We thank Ms. Penelope Krumm for the critical review of this paper.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Luis Corona http://orcid.org/0000-0002-6640-7626

Manuel Gonzalez-Ronquillo http://orcid.org/0000-0003-3616-4157

Additional information

Funding

Guzman, MSc, was granted for a CONACyT fellowship during his studies in the University National Autonomous of Mexico. Dr. Gonzalez Ronquillo was granted for a CONACyT fellowship ‘Estacias sabaticas en el Extranjero, 2014’. This project was supported by UNAM, DGAPA - PAPIIT [IN206006-3].