BIOAVAILABILITY OF FE, CU AND MN FROM YEAST ENRICHED IN BIOELEMENTS USED IN LAYING HENS FEEDING

Zbigniew Dobrzański¹, Sebastian Opaliński², Helena Górecka³, Mariusz Korczyński², Roman Kołacz², Tadeusz Trziszka^4
1 Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland
² Department of Environmental Hygiene and Animal Welfare, Wrocław University of Environmental and Life Sciences, Poland
³ Institute of Inorganic Technology and Mineral Fertilizers, Wrocław University of Technology, Poland
⁴ Department of Animal Products Technology and Quality Management, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Standard feed mixture for laying hens was supplemented with dried yeast Saccharomyces cerevisiae enriched in Cu (Y_Cu), Fe (Y_Fe) and Mn (Y_Mn). The availability of investigated organic forms, comparing to inorganic forms (CuSO₄, FeSO₄, and MnO₂), was assessed by analysis of these elements content in diet, droppings and eggs. Following values of apparent absorption were observed: control group 24.04, 29.89 and 20.35 %; Y_Cu group 27.14, 30.0 and 21.9 %; Y_Fe group 27.28, 32.43 and 19.44 %; Y_Mn group 26.01; 24.89 and 31.55 %; Y_Cu+Fe+Mn group: 24.78, 30.84 and 24.65 %; for Cu, Fe and Mn respectively. It was found, that yeast supplementation caused a significant (p<0.05) increase in copper availability (groups receiving Y_Cu and Y_Fe) as well as in manganese availability (groups receiving Y_Mn and Y_Cu+Fe+Mn). The differences in iron availability were not statistically confirmed.

Key words: yeast, copper, iron, manganese, laying hens, bioavailability.

INTRODUCTION

Yeast are used in animal feeding for over 100 years, currently as active yeasts (95 % of dry matter) or alive cultures in a form of feed probiotics or yeast-origin products [2,3,13]. Their importance increased when at the end of 2004 animal origin meals (except fish meals) and, at the beginning of 2006, feed antibiotics were withdrawn from usage.

Baker’s yeast (Sacchoromyces cerevisiae), produced on an industrial scale mainly from molasses, rarely from whey and starch wastes or other organic materials, are known the best [8,9]. To limit or even withdraw mineral premixes from feed, numerous researches, concerning an enrichment of yeast in some macroelements and trace elements, have been conducting [24,30].

According to Noy et al. [22] availability of microelements that play crucial physiological role in poultry is relatively low. Generally the availability for commercial mixtures is on the level of 30 % for zinc, 20 % for cobalt, 15 % for manganese, 12 % for copper, 10 % for iron and only 4% for chromium, whereas macroelements availability in poultry is very different. For example, calcium retention is placed in a range of 36.03–50.60 %, while phosphorus 8.64–23.71 % depending on calcium source and a phase of laying [15]. Moreover, Banks et al. [1] reported that the retention of phosphorus in laying hens was from 13.56 to 23.09 %, depending on copper source; P retention was higher when copper sulfate or chloride was applied in a feed. Different values, especially for organic forms of bioelements, were given by numerous authors dealing with these nutritional-environmental problems [14,17,27,31].

Waste-less production technology of Bioder feed yeast enriched in selenium (Y-Se), zinc (Y-Zn) and chromium (Y-Cr), on a basis of Saccharomyces cerevisiae cultures and molasses, that are well available by poultry, was developed in Poland [5,7]. It is also possible to enrich that yeast in Mn, Fe and Cu [6,9].
The aim of the research was to assess the availability of copper, manganese and iron, from Saccharomyces cerevisiae yeast enriched in these bioelements, in laying hens feeding.

MATERIAL AND METHODS

The examination was made in the experimental room (precisely controlled conditions) where laying hens (Lohmann brown) in the first period of egg production were kept in battery system. During the experiment, the standard lighting program was applied; feed and water were available ad libitum. Birds were divided into 5 experimental groups (12 hens in each group) – control group and four groups with supplementation of different yeast (Y_Cu, Y_Fe, Y_Mn and Y_Cu+Fe+Mn) in the diet. Hens were fed with all-mash feed mixture of J-297 type, prepared according to standard recipes (Table 2). Applied concentrations of Cu, Fe and Mn respond, or slightly exceed upper limit of doses recommended in Poultry Feeding Standards [21]. In a basic version of premix, inorganic forms of investigated elements (copper sulfate, iron sulfate and manganese oxide) were used. In premix which were added to the feed for groups Y_Cu, Y_Fe, Y_Mn and Y_Cu+Fe+Mn an organic forms, Saccharomyces cerevisiae yeast enriched in the investigated elements, were supplemented. The contribution of investigated elements originated from organic forms in an overall content of mixture (with premix) in groups Y_Cu, Y_Fe, Y_Mn and Y_Cu+Fe+Mn was as follows: Cu – 47, Fe – 20 and Mn – 57 %. Scheme of the experiment that lasted 8 weeks in total is presented in Table 3.

Saccharomyces cerevisiae yeast were produced on a basis of whey in a laboratory scale according to original waste-less technology described in earlier papers [6,9]. The examined yeast contained almost 40 % of crude protein, a little more than 10 MJ·kg^-1 of metabolizable energy and about 1 % of crude fat. Maximal Fe concentrations were 26.45, Mn – 19.96 and Cu – 18.49 g·kg^-1 d. m. (Table 1). Those concentrations were different from beer yeast [21], Diamond cultures [8] or Yarrowia lipolityca [24].

Table 1. The basic chemical composition of Saccharomyces cerevisiae yeast enriched with Fe, Mn and Cu

Component	YFe	YMn	YCu
Nutrient [%]
Dry matter	96.2%	95.3%	95.2%
Crude ash	6.62%	6.81%	5.09%
Total protein	38.0%	39.75%	39.32%
Crude fat	1.01%	0.93%	0.83%
Crude fibre	trace	trace	trace
Metabolizable energy	10.33 MJ·kg-1	10.48 MJ·kg-1	10.16 MJ·kg-1
Bioelement [g·kg-1]
Fe	1.44-26.45*	0.07	0.026
Mn	0.012	5.33-19.96*	0.011
Cu	0.004	0.004	0.43-18.49*

* maximal values

Feed intake, number and weight of laid eggs were controlled every day. Eggs for the research were collected during the last 5 days of the experiment and yolk was separated from egg white. To express overall retention the percentage share of eggshell, egg white and yolk in overall mass of egg was calculated for particular groups. Every day, droppings were collected from each cage and weighted; after mixing about 10 % of a mass was collected and frozen. From each frozen sample of droppings, 3 samples of material were mineralized and the concentration of investigated elements was determined. Besides, samples of feed mixture from each group were also collected for laboratory analysis.

Table 2. The recipe composition of feed mixture for layer

Feed material	Unit of measure	Content
Maize – meal Soya- extracted meal Wheat + enzyme Wheat – whole grain Sunflower – meal Forage chalk Wheatrye+enzyme Rape meal Plant fats Wheat bran Dicalcium phosphate Neu Sal Liquid** Forage salt Sodium sulfate Methionine Lysine Phytopreparation – Herbiplant CS Enzyme – Rovabio Premix – Finisher***	% % % % % % % % % % % % % % % % % % %	30.0 12.81 12.0 10.0 8.99 8.05 8.00 3.50 2.68 2.00 1.12 0.20 0.18 0.15 0.08 0.03 0.01 0.01 0.20
Nutrients: Crude protein Starch Crude fat Crude fibre Crude ash Salt (NaCl) Sodium – Na Calcium – Ca Total phosphorus Available phosphorus Lysine Methionine Cystine Threonine Tryptophan Isoleucine Arginine Phenylalanine Metabolizable energy	% % % % % % % % % % % % % % % % % % kcal·kg-1	16.5 37.96 4.90 4.04 10.70 0.22 0.15 3.65 0.61 0.30 0.72 0.34 0.30 0.58 0.21 0.69 1.11 0.79 2765

* according to the data from mixture producer ** antibacterial and antifungal preparation *** premix in 1 kg contains: vitamin A – 2000000 jm, Vitamin D3 – 500000 jm, Vitamin E – 4000 jm, Vitamin K3 – 303 mg, Vitamin B1 – 300 mg, Vitamin B2 – 1000 mg, Nicotinic acid – 6000 mg, Panthothenic acid – 2000 mg, Vitamin B6 – 400 mg, Vitamin B12 – 3000 mg, Biotin – 10000 mg, Folic acid – 209 mg, Ca – 31,20 %, Fe – 8000 mg, Mn – 16000 mg, Zn – 10000 mg, I – 204 mg, Co – 100 mg, Se – 50 mg, Cu – 2000 mg, Antioxidant – 3000 mg, Natuphos Enzyme – 16000 mg.

Material was delivered to the Chemical Laboratory of Multielemental Analysis at Wrocław University of Technology, where Cu, Fe and Mn concentrations were determined in feed mixtures, eggs (eggshell, yolk and egg white) and in droppings. Mass spectrometry method (ICP-MS), with the use of Varian UltraMass-700 device, was applied. Before analysis, samples were digested with rotary microwave oven of MDS type 2000 by CEM (USA) [12].

Obtained results allowed to determine the bioavailability of these elements, expressed as:

• overall retention (difference between intake and excretion in mg/hen/day)

• apparent absorption (ratio of retention to intake in %).

Moreover, the relative biological value (RBV) index was calculated, assuming retention of element in control group as 100%.

Table 3. Scheme of experiment - content of Cu, Fe and Mn in feed mixture for layers

Microelement	Source	Group
		I (Control)	II (YCu)	III (YFe)	IV (YMn)	V (YCu+ Fe+ Mn)
Cu	Feed	11.2	11.2	11.2	11.2	11.2
	Premix**	10	–	10	10	–
	Yeast	–	10	–	–	10
Total		21.2	21.2	21.2	21.2	21.2
*		19.7	24.5	18.9	19.6	25.1
Fe	Feed	160.8	160.8	160.8	160.8	160.8
	Premix**	40	40	–	40	–
	Yeast	–	–	40	–	40
Total		200.8	200.8	200.8	200.8	200.8
*		209.0	215.4	223.2	182.3	198.3
Mn	Feed	60.5	60.5	60.5	60.5	60.5
	Premix**	80	80	80	–	–
	Yeast	–	–	–	80	80
Total		140.5	140.5	140.5	140.5	140.5
*		138.2	156.0	136.9	153.0	127.8

* values determined analytically ** premix contains: CuSO4, FeSO4, MnO2

Results were elaborated statistically; mean values and standard deviations were calculated. Significances of differences between control group and four groups with supplementation of different yeast in the diet were assessed by Duncan’s test (Statgraphics 5.1. software).

RESULTS AND DISSCUSION

Production results
The level of laying was high and similar in all groups, it was from 91.7 to 98.3 % on average (period after peak of laying). No broken or damaged eggs were noted. Feed intake, calculated for one laying hen and also for a production of one egg, did not differ significantly between groups (about 130 g/hen/day). Generally, the influence of yeast supplementation on the average weight of eggs from all experimental groups was not observed. However, the increase of laying and egg weight in groups II and V, groups receiving organic copper (Y_Cu), was determined, but the differences were not statistically confirmed. Obtained production results are typical for that laying line of hens [29], and applied yeast did not have any significant influence on egg laying, feed intake and egg weight.

Intake of microelements
The content of investigated elements in feed mixture (Table 3) according to calculations was as follows: Cu – 21.2, Fe – 206.8, Mn – 140.5 mg·kg^-1. The values do not exceed permissible concentrations established generally for animals feed in EU [11]. Moreover, the content of Cu, Fe and Mn were within the range of concentrations of these bioelements in feed mixtures for laying hens reported by other authors [4,15,20,28]. Determined values were slightly different from values mentioned above, the most in the case of Cu (maximal difference on a level of 18.4%); probably different results were an effect of the sensitivity of applied analytical techniques [12]. Daily intake of copper by laying hens was 2.71–2.76, of iron 26.35–26.84, and of manganese 17.97 – 18.31 mg. However, there was different contribution of organic and inorganic forms of Cu, Fe and Mn in the doses, which was a result of fodder yeast application.

Excretion of microelements
Laying hen’s excreted elements mainly with droppings, in a small degree with laid eggs. An average weight of droppings was from 149.7 to 154.4 g/hen/day. The content of analyzed microelements in droppings was different (Table 4). The rate of excretion of investigated elements by birds in particular groups was determined and varied from 1.93 to 2.04, from 17.02 to 18.64 and from 12.27 to 14.48 mg/hen/day for copper, iron and manganese respectively. Nevertheless, other authors determined quite different content of these elements in droppings of laying hens [26,27,31], taking into consideration the differences of their content in the feed mixture, and moreover chemical forms it is comprehensible.

When the concentration of investigated microelements in the content of eggs from experimental groups was compared, only very small differences were found, similarly in case of eggshell. The determined average content in the whole egg was 0.047-0.059 for copper, 1.002-1.242 for iron and 0.026-0.032 mg/hen/day for manganese. Obtained results showed that amount of the investigated elements excreted with laid eggs is very small in comparison with the elements intake with feed and excretion via droppings. Other authors give a bit similar values of these elements in components of egg and also in the whole egg [4,10,20,23].

Elements balance
Determined values of intake and excretion allowed to calculate the retention and apparent absorption of these elements in laying hens and also their relative biological value (RBV) in particular groups (Table 4).

The retention of copper (Cu) was from 0.661 (group I) to 0.753 mg/hen/day (group III). Apparent absorption of copper was within the range of 24.04 – 27.28 %. RBV index was the highest in groups II and III. Thus, the addition of copper in a form of enriched yeast significantly (p<0.05) influenced the increase in retention and apparent absorption in group II (Y_Cu), and also in group III (Y_Fe) comparing to the control one (I). Increase in these indices in group III pointed the phenomenon of synergism between Cu and Fe; however, antagonism phenomenon between these elements might also take place [18].

Um and Paik [31] reported that Cu retention in laying hens varied between 0.4 and 0.98 mg/hen/day depending on the dose of phosphorus and phytase in feed. However, Banks et al. [1], in the case of 3-weeks old broiler chickens, observed apparent retention of copper in the range of 0.5–68.0 %, depending on the level and source of copper in a mixture.

Table 4. The balance of Cu, Fe and Mn in laying hens

Microelement	Group
	I	II	III	IV	V
Intake with feed [mg/hen/day]
Cu	2.75	2.73	2.76	2.71	2.76
Fe	26.72	26.49	26.80	26.35	26.84
Mn	18.22	18.07	18.28	17.97	18.31
Excretion with droppings [mg/hen/day]
Cu	2.036	1.932	1.96	1.95	2.02
Fe	17.73	17.48	17.02	18.64	17.32
Mn	14.48	14.08	14.70	12.27	13.77
Excretion with laid eggs [mg/hen/day]
Cu	0.049	0.059	0.047	0.055	0.056
Fe	1.002	1.064	1.089	1.150	1.242
Mn	0.032	0.032	0.026	0.031	0.029
Retention [mg/hen/day]
Cu	0.661a	0.741b	0.753b	0.705	0.684
Fe	7.988	7.946	8.691	6.560	8.278
Mn	3.708a	3.958	3.554	5.669b	4.511b
Apparent absorption [%]
Cu	24.04a	27.14b	27.28b	26.01	24.78
Fe	29.89	30.00	32.43	24.89	30.84
Mn	20.35a	21.90	19.44	31.55b	24.64b
RBV [%]*
Cu	100.0	112.9	113.5	108.2	103.1
Fe	100.0	100.4	108.5	83.3	103.2
Mn	100.0	107.6	95.5	155.0	121.1

a – b – p<0.05 *relative biological value (RBV)

Jongbloed et al. [17] noted that relative bioavailability (RBV) of copper in broiler chickens was the highest in the case of such forms of that element as CuSO₄·5H₂O, Cu₂O and Cu-lysine chelate, and on similar level for CuSO₄ and copper acetate. However, higher bioavailability of copper from Cu₂(OH)₃Cl comparing to CuCl, and also considerably lower in the case of copper carbonate and copper oxide (II) with relation to CuSO₄·5H₂O was observed. This was confirmed by results obtained by Jackson and Stevenson [14], who compared availability of Cu from oxide form and from CuSO₄·5H₂O during an experiment on laying hens. The RBV index determined for CuO was 40 % lower than the index determined for sulfate form.

The determined retention of Fe varied from 6.56 (group IV) to 8.691 mg/hen/day (group III). Apparent absorption of iron was in a range of 24.89–32.43 %. RBV index was the highest in group III. However, an addition of iron in a form of enriched yeast did not significantly influence the increase in retention and apparent absorption in group III (Y_Fe), although an increasing tendency was observed. It is possible that higher contribution of yeast enriched in iron in a diet of hens would possibly confirm higher availability of that element from organic forms.

Um and Paik [31] reported, that Fe retention in laying hens varied from 12.96 to 19.33 mg/hen/day depending on doses of phosphorus and microbiological phytase. Moreover, Jongbloed et al. [17] determined the highest relative bioavailabilities (RBV) of that element in poultry for such forms of Fe like iron chlorides (II), Fe-ZnSO₄·H₂O or iron ammonium citrate (III). Not much lower RBVs were obtained for FeSO₄·7H₂O and FeSO₄·H₂O or FeSO₄. Bioavailability of iron from iron carbonate (II), iron sodium pyrophosphate (III) or iron orthophosphate (III) in poultry was very low, and in the case of iron (III) in a form of chloride, pyrophosphate of sulfate was moderate.

Manganese (Mn) retention varied from 3.708 (group I) to 5.669 mg/hen/day (group IV). Apparent absorption of manganese was within the range of 19.44–31.55 %. RBV value was the highest in groups IV and V. Thus, the addition of manganese in a form of enriched yeast significantly (p<0.05) influenced the increase in retention and apparent absorption in group IV (Y_Mn) and also in group V, what indicated a good availability of manganese from organic forms. It was confirmed also in research by Ji et al. [16], who observed significantly higher absorption of manganese in intestines of broilers that were fed with amino acids Mn complex (mainly with methionine) comparing to hydrated MnSO₄. The highest percentage of absorption was observed in ileal loops, while in duodenal and jejunal loops it was similar.

It may be concluded from data published by Jongbloed et al. [17] that manganese bioavailability in poultry is various. The highest RBV index was characteristic for MnSO₄·H₂O, MnCl₂·H₂O, protein form of Mn, and also manganese – methionine chelate, while Mn availability from the latter form was the highest, about 10 % higher than in the case of sulfate. The bioavaliability rate of Mn from compounds like MnO, MnCO₃ and MnO·Mn₂O₃ was on a lower level, and it was the lowest in the case of MnO₂. Results reported by Jongbloed et al. [17] confirm results of our research due to the fact that manganese oxide was applied to the feed during experiment.

Obtained results were difficult to compare, as there were no similar works in available literature. Only Korniewicz et al. [19] estimated availability of elements from the same yeast in fatteners. Values of apparent absorption for copper and manganese were similar to the present study, while considerably higher in the case of iron.

CONCLUSIONS

In comparison to inorganic forms (copper sulfate and manganese oxide) manganese and copper were significantly better available from used in poultry feeding Saccharomyces cerevisiae dried yeast enriched in bioelements. Moreover, it was found that iron availability from Y_Fe and from inorganic form (iron sulfate) was on the same level. Determined values of apparent absorption for investigated elements were in a range of 19.44–32.43%. The highest differences of RBV (relative biological value) index were noted between control group and groups receiving Y_Mn and Y_Cu.

ACKNOWLEDGEMENTS

The study was financed by the Ministry of Science and Higher Education (scientific project no. 3 T09B 09328).

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Accepted for print: 4.04.2008

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Zbigniew Dobrzański
Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland
J. Chełmońskiego 38 C
51-630 Wrocław
Poland
Phone: +48 71 320 5865
email: zbigniew.dobrzanski@up.wroc.pl

Sebastian Opaliński
Department of Environmental Hygiene and Animal Welfare,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 38 C, 51-630 Wrocław, Poland
Phone: (+48 71) 32 05 865

Helena Górecka
Institute of Inorganic Technology and Mineral Fertilizers,
Wrocław University of Technology, Poland
Smoluchowskiego 25, 50-372 Wrocław, Poland
Phone: (+48 71) 32-02-486
email: helena.gorecka@pwr.wroc.pl

Mariusz Korczyński
Department of Environmental Hygiene and Animal Welfare,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 38 C, 51-630 Wroclaw, Poland
Phone (+48 71) 32 05 865,

Roman Kołacz
Department of Environmental Hygiene and Animal Welfare,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 38 C, 51-630 Wrocław, Poland
Phone: (+48 71) 32 05 865

Tadeusz Trziszka
Department of Animal Products Technology and Quality Management,
Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 25, 51-630 Wrocław, Poland
Phone: (+48 71) 32 05 121
email: trziszka@ozi.ar.wroc.pl

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