Influence of methodology on the measurement of ileal endogenous calcium losses in broiler chickens

ABSTRACT

An experiment was conducted to determine the ileal endogenous calcium (Ca) losses in four-week old male Ross 308 broiler chickens (mean body weight ± SD, 1.66 ± 0.12 kg) using three assay methodologies. Three experimental diets were used: a Ca and phosphorus (P)-free diet and two other diets by substituting (w/w) 200 g/kg dextrose (w/w) with maize gluten meal or dried egg albumen as Ca-free protein sources. Each of the three experimental diets was randomly allocated and fed for three days to six replicate cages (six birds/cage), and ileal digesta were collected to measure the ileal endogenous Ca losses. The feed intake of birds fed diets containing the two protein sources was higher (P < 0.05) than the Ca and P-free diet. Body weight loss was observed in birds on the Ca and P-free diet and maize gluten meal-based diets. Ileal endogenous Ca losses were influenced (P < 0.05) by the assay methodology. Ileal endogenous Ca losses were determined to be 125, 77 and 46 mg/kg dry matter intake in birds fed the Ca and P-free diet, maize gluten meal diet and dried egg albumen diet, respectively. Ileal endogenous P losses were determined in birds fed Ca and P-free and dried egg albumen based diets. There was no effect (P > 0.05) of methodology on ileal endogenous P losses.

KEYWORDS:

• Broilers
• calcium
• endogenous losses
• methodology
• phosphorus

1. Introduction

Currently there is a move towards the use of digestible phosphorus (P) in feed formulations for poultry and increasing interest in the measurement of P digestibility (Dilger and Adeola 2006; Mutucumarana et al. 2014, 2015; Rodehutscord et al. 2017; Verardi et al. 2019). Maintaining proper ratios between dietary Ca and P is critical for the absorption and utilization of both minerals, and estimation of true Ca digestibility is therefore warranted. For the calculation of true Ca digestibility, it is necessary to determine the endogenous Ca losses. Endogenous Ca originates from saliva, bile, pancreatic juice, gastric juice and damaged intestinal cell lining. Studies have been conducted to determine the endogenous Ca losses in pigs, following the feeding of Ca free diet (Traylor et al. 2001; Gonzalez-Vega et al. 2013a, 2014) or using the regression method (Gonzalez-Vega et al. 2013b). Data on endogenous Ca losses in poultry, however, is scant. The aim of the present work was to determine the endogenous Ca losses in broiler chickens by three different methods, namely feeding a Ca and P-free diet, and diets containing protein sources (maize gluten meal and dried egg albumen) that have negligible amounts of Ca.

2. Materials and methods

The experiment was conducted according to the New Zealand Revised Code of Ethical Conduct for the use of live animals for research, testing and teaching, and approved by the Massey University Animal Ethics Committee (MUAEC 14/01).

2.1. Diets

Three semi-purified diets were developed: a Ca and P-free diet and two other diets by substituting (w/w) 200 g/kg dextrose with maize gluten meal or dried egg albumen (Table 1). Maize gluten meal and dried egg albumen are protein sources that contain negligible amounts of Ca (NRC 2012). All diets contained 3 g/kg of titanium dioxide as an indigestible marker.

Table 1. Ingredient composition and analysis (g/kg as-fed basis) of experimental diets.

	Calcium and phosphorus free	Maize gluten meal	Dried egg albumen
Dextrose	928.7	728.7	728.7
Maize gluten meal^1	-	200.0	-
Dried egg albumen^1	-	-	200.0
Cellulose^2	40.0	40.0	40.0
Soybean oil	20.0	20.0	20.0
Sodium chloride	4.0	4.0	4.0
Sodium bicarbonate	2.0	2.0	2.0
Titanium dioxide	3.0	3.0	3.0
Trace mineral-vitamin premix^3	2.3	2.3	2.3
Calculated analysis
Metabolisable energy (MJ/kg)	15.3	15.3	15.3
Crude protein	-	12.4	16.48
Calcium	-	0.06	0.18
Total phosphorus	-	1.0	0.18
Non-phytate phosphorus	-	0.28	0.18
Analysed values
Calcium	<0.1	<0.1	<0.1
Phosphorus^4	0.1	-	0.3

¹Davis Food Ingredients, Petone, New Zealand.

²Asahi Kasei Chemicals Corporation, Chiyoda-Ku, Tokyo, Japan.

³Supplied per kilogram of diet: vitamin A, 12,000 IU; cholecalciferol, 4,000 IU; thiamine, 3 mg; riboflavin, 9 mg; pyridoxine, 10 mg; folic acid, 3 mg; biotin, 0.25 mg; cyanocobalamin, 0.02 mg; dl-α-tochopherol acetate, 80 mg; niacin, 60 mg; Ca-D pentothenate, 15 mg; menadione, 4 mg; choline chloride, 600 mg; Co, 0.25 mg; I, 1.5 mg; Mo, 0.25 mg; Se, 0.26 mg; Mn, 100 mg; Cu, 10 mg; Zn, 80 mg; Fe, 60 mg; antioxidant, 100 mg.

⁴ Dietary phosphorus concentration of maize gluten meal based diet was not analysed and used for ileal endogenous P losses determination, as it was calculated to contain 1.0 g/kg total P (NRC 1994 was used as a reference for P concentration in maize gluten meal).

2.2. Birds and digesta sampling

Day-old male broilers (Ross 308) were obtained from a local hatchery and raised on floor pens (floor space per bird, 520 cm²).. Temperature was maintained at 31°C on day one and gradually reduced to 22 °C by 21 days of age. The birds were fed commercial broiler starter crumbles until day 14. On day 15, birds were moved to colony cages and the diet was gradually changed to mash form as the experimental diets were in mash form. On day 26, birds were individually weighed and allocated to 18 cages (six birds/cage; cage space per bird, 640 cm²) on weight basis so that the average bird weight per cage (average ± SD, 1.66 ± 0.12 kg) was similar. The three experimental diets were then randomly assigned to six replicate cages each. The diets, in mash form, were offered ad libitum and the birds had free access to water. Both the floor pens and cages were located in an environmentally controlled room with 20 h of fluorescent illumination per day. Group body weights and feed intake were recorded on days 26 and 28.

On day 28, all birds were euthanised by intravenous injection (1 ml per 2 kg body weight) of sodium pentobarbitone (Provet NZ Pty. Ltd., Auckland, New Zealand) and the contents of the lower half of the ileum were collected by gently flushing the contents with distilled water into plastic containers (Ravindran et al. 2005). The samples were frozen immediately and subsequently lyophilized (Model 0610, Cuddon Engineering, Blenheim, New Zealand). Lyophilized samples were ground to pass through a 0.5 mm sieve and stored in airtight containers at 4 °C until chemical analysis.

2.3. Chemical analysis

Representative samples of test diets and digesta were analysed for dry matter, Ca, P and titanium dioxide. Dry matter (DM) was determined using the standard AOAC procedure (method 930.15; AOAC 2005).. Titanium was determined by the procedure of Short et al. (1996). Calcium was determined by calorimetric assay (Flexor E, Vital Scientific NV, Spankeren/Dieren, the Netherlands) following digestion with 6M hydrochloric acid to release Ca (method 968.08D; AOAC, 2005). Phosphorus was determined by a colorimetric assay (UV mini1240; Shimadzu Corp., Kyoto, Japan) at 680 nm by standard AOAC procedures (method 968.08D; AOAC, 2005).

2.4. Calculations

Ileal endogenous Ca and P losses were calculated by using the following equation (Anwar et al. 2018). All values were expressed as mg/kg DM and, ileal endogenous Ca and P losses were determined as mg/kg DM intake.$\begin{array}{rl}& \mathrm{I}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{e}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{C}\mathrm{a}\mathrm{o}\mathrm{r}\mathrm{P}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}\mathrm{e}\mathrm{s}=\mathrm{C}\mathrm{a}\mathrm{o}\mathrm{r}\mathrm{P}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\\ & \times (\mathrm{T}\mathrm{i}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}/\mathrm{T}\mathrm{i}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a})\mathrm{s}\end{array}$

2.5. Statistical analysis

Data were analysed as a one-way analysis of variance using the General Linear Models procedure (SAS 2004). Cage served as the experimental unit. Signficant differences between means were separated by the Least Significance Difference test. Significance of treatment effects was declared at P < 0.05.

3. Results

Analysed dietary Ca concentrations are given in Table 1. All experimental diets contained less than 0.1 g Ca/kg diet.

Feed intake and body weight gain of the birds during the experimental period (26-28 days of age) are presented in Table 2. Weight loss was observed in birds fed Ca and P-free diet and the diet supplemented with 200 g/kg of maize gluten meal. This loss was higher (P < 0.05) in birds on the Ca and P-free diet compared to those on the maize gluten meal based diet. Birds fed diets supplemented with 200 g/kg of dried egg albumen gained weight. Feed intake of the birds was lowest (P < 0.05) on the Ca and P-free diet, followed by the maize gluten meal-based diet and was highest (P < 0.05) on the dried egg album based diet.

Table 2. Body weight gain and feed intake (g/bird/day) of the birds fed the three experimental diets (26-28 days of age)¹.

	Body weight gain	Feed intake
Ca and P-free diet	−36^c	47^c
Maize gluten meal diet	−15^b	62^b
Dried egg albumen diet	25^a	79^a
SEM^2	4.13	3.20
Probability, P ≤	0.001	0.001

^{a, b, c} Values with a different superscript within a column differ significantly (P &lt; 0.05).

¹ Each value represents the mean of six replicates (six birds per replicate).

² Pooled standard error of mean.

Ileal endogenous Ca losses in 28-day old broilers determined by the three assay methodologies are presented in Table 3. Ileal endogenous Ca losses differed (P < 0.05) among the three methods. Ileal endogenous Ca losses determined for the Ca and P-free diet, maize gluten meal diet and dried egg albumen diet were 125, 77 and 46 mg/kg DMI, respectively. Ileal endogenous Ca losses were significantly higher (P < 0.001) in birds fed the Ca and P-free diet as compared to the maize gluten diet and dried egg albumen based diet. Corresponding losses were higher (P < 0.001) in the maize gluten-based diet as compared to the dried egg albumen-based diet.

Table 3. Ileal endogenous calcium and phosphorus losses (mg/kg dry matter intake) in broilers¹

	Ileal endogenous calcium losses	Ileal endogenous phosphorus losses
Ca and P-free diet	125^a	133
Maize gluten meal diet	77^b	-
Dried egg albumen diet	46^c	110
SEM^2	10.26	12.31
Probability, P ≤	0.001	0.21

^{a, b, c} Values with a different superscript within a column differ significantly (P < 0.05).

¹ Each value represents the mean of six replicates (six birds per replicate).

² Pooled standard error of mean.

Ileal endogenous P losses were also determined in birds fed the Ca and P-free and dried egg albumen-based diets. The Ca and P-free and dried egg albumen based diets contained negligible amount of P, enabling the determination of endogenous P flow. The maize gluten meal based diet was calculated to contain 1.0 g/kg total P, so was not used for estimation of endogenous P losses. Ileal endogenous P losses were 133 and 110 mg/kg DMI on Ca and P-free and dried egg albumen based diets, respectively. There was no effect (P > 0.05) of methodology on ileal endogenous P losses in broiler chickens.

4. Discussion

Body weight loss and lower feed intake in birds fed the Ca and P-free diet was as expected as the diet did not contain any protein. Birds fed the maize gluten meal-based diet also lost body weight possibly because the diet was still deficient in protein compared to the recommended level of 190–230 g/kg (Ross 2007). However, the weight loss on the maize gluten meal-based diet was less than on Ca and P-free diet, which may be attributed to the higher feed intake and the presence of protein in the maize gluten diet. The higher dietary protein content (160 g/kg) and feed intake stimulated the weight gain observed in birds on the dried egg albumen-based diet compared to those in birds fed maize gluten meal based and Ca and P-free diets.

Saliva (Tryon and Bibby 1966), gastric secretions (Moore and Tyler 1955), bile, pancreatic secretions and cellular debris (Bronner 1997) are all sources of endogenous Ca. In the present study, ileal endogenous Ca loss estimates differed depending on the methodology used. The determined values were 125, 77 and 46 mg/kg DMI on the Ca and P-free, maize gluten and dried egg albumen diets, respectively. No comparative data are available for the ileal endogenous Ca losses in poultry. Markedly higher endogenous Ca losses, of 349 (Traylor et al. 2001) and 420 mg/kg DMI (Gonzalez-Vega et al. 2013a) have been reported in pig studies, wherein casein and maize gluten and potato protein were used as Ca-free protein sources, respectively. Digestive enzyme secretions are known to vary with changes in the dietary nutrient composition and feed intake. Increases in dietary protein (Zhao et al. 2007; Ravindran et al. 2009) and feed intake (Sklan 2001) have been reported to increase the digestive enzyme secretions that can contribute to higher endogenous Ca. The current findings were contrary to expectations and difficult to explain because the feed intake of the birds and dietary protein concentration of dried egg albumen and maize gluten meal diets were higher while respective endogenous Ca losses were lower than the birds on the Ca and P-free diet. No explanation can be provided from the present data, but one possibility may be that the reabsorption of endogenous Ca may have been higher in diets containing the protein sources.

Ileal endogenous P losses were measured only for the Ca and P-free and dried egg albumen-based diets, as the maize gluten meal-based diet was calculated to contain 1.0 g/kg total P that could have caused an overestimation of endogenous P values. Variable results have been reported previously regarding ileal endogenous P losses in broiler chickens. Ileal endogenous P losses have been reported to be 272 and 446 mg/kg DMI in broiler chickens fed P-free synthetic amino acid-based diets in two different studies (Rutherfurd et al. 2002; 2004). In the current study, much lower ileal endogenous P losses were determined in birds fed the Ca and P-free diet (133 mg/kg DMI) and egg albumen-based diet (110 mg/kg DMI). These differences may be attributed to dissimilarities in diet composition between the studies.

5. Conclusions

The present data showed that estimates of ileal endogenous Ca losses in broiler chickens differed depending on the assay methodology used. Ileal endogenous Ca losses in broiler chickens were highest on the Ca and P-free diet, intermediate on the maize gluten meal based diet and lowest on the dried egg albumen based diet. No differences were observed for ileal endogenous P losses in birds fed the Ca and P-free diet or dried egg albumen-based diet.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.