The Use of Selenium Enriched Enterococcus faecium as an Alternative Selenium Source for Growing-finishing Pigs

The aim of the study was to evaluate the effect of the feeding of selenium enriched Enterococcus faecium on selenium concentrations in muscle tissue and on meat quality indicators in finishing pigs. Groups of pigs fed Se-enriched E. faecium (n = 12), sodium selenite (n = 12) and Se-enriched yeast (n = 12) were used in the study. The tested feed mixtures were fed for a period of 3 months before the pigs were slaughtered. The selenium concentrations in muscle tissue achieved by the use of Seenriched E. faecium were comparable to those achieved in sodium selenite diets and lower (p ≤ 0.05) compared to Se-enriched yeast. Concentration of Se in serum and glutathione peroxidase (GSH-Px) activities did not differ in animals fed different Se forms. No differences in indices of meat quality were found among the groups. Se-enriched E. faecium bacteria can be recommended as an alternative selenium source for finisher pigs with the similar efficacy as sodium selenite. Pork, meat quality, selenomethionine, selenocysteine Selenium has an important antioxidant role as a part of enzyme glutathione peroxidase (Sunde 1994). Several studies in human medicine provided evidence for the efficacy of selenium as an anticancer agent (Duffield-Lillico et al. 2003). Selenium also enhances immune functions (Beck et al. 2001) and has an important role in the maintenance of fertility (Rayman 2000). It has been documented that pork can contribute significantly to the selenium intake in human population (Kadrabová et al. 1996; Fajt et al. 2009). Selenium deficiency in pigs is manifested as mulberry heart disease, hepatosis dietetica and muscular dystrophy (Lindberg et al. 1972). Selenium requirements of growingfinishing pigs can be achieved by providing selenium either from an inorganic or an organic source. Sodium selenite represents the inorganic form. Selenium from Se-enriched yeast is the most commonly used organic selenium form (Schrauzer 2000). Experiments in growing-finishing pigs revealed higher accumulation of organic Se compared to inorganic form in tissues (Mahan and Parrett 1996; Taylor et al. 2005). Also experiments in sows demonstrated higher Se content in newborn piglets tissues and in colostrum when the Se-enriched yeast was used (Mahan and Peters 2004). Limited data are available so far regarding the use of other organic selenium sources in pigs. For instance Se from Se-enriched alga Chlorella spp. (Svoboda et al. 2009b) and soybean based selenium proteinate (Jang et al. 2006) were used as alternative selenium sources in finishing swine. The aim of the present study was to evaluate the effects of Se from Se-enriched Enterococcus faecium on tissue selenium retention and meat quality indicators in growingfinishing pigs. Materials and Methods Experimental design A total of 36 pigs were used in our experiment. Only female pigs were used. The experiment started at the beginning of the grower phase of production. Before the experiment, all pigs were fed an identical diet ACTA VET. BRNO 2010, 79: 511-517; doi:10.2754/avb201079040511 Address for correspondence: Doc. MVDr. Marin Svoboda, Ph.D. Swine Clinic, Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Palackého 1/3, 612 42 Brno, Czech Republic Phone: +420 541 562 433 E-mail: svobodama@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm supplemented with 0.3 mg·kg-1 Se in the form of sodium selenite. The compositions of experimental mixtures are presented in Table 1 and Table 2. The feed mixtures were composed of a basal diet and a mineral supplement. The pigs received experimental mixtures for the period of 3 months before the slaughter. The feed mixture A1 was administered during the first 30 days. The feed mixture A2 was given to pigs for the rest 60 days. The pigs were divided into 3 equal groups of 12 animals. In group A (n = 12) pigs were fed feed mixtures A1 and A2 supplemented with organic selenium at the dose of 0.3 mg·kg-1 in the form of Se-enriched Enterococcus faecium. The group B (n = 12) had feed mixtures A1 and A2 supplemented with inorganic sodium selenite at the dose of 0.3 mg·kg-1. In group C (n = 12) the pigs received feed mixtures A1 and A2 with the addition of organic selenium in the form of Seenriched yeast (0.3 mg·kg-1). The experiment was approved by the Ethics Committee of the University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic. Sampling The blood samples were taken from all pigs at the beginning of the trial and one day before the slaughter. The pigs were bled from vena cava jugularis. Serum was collected and frozen for further analysis. Tissue samples for selenium determination (heart, musculus biceps femoris, liver and ovaries) were taken after the slaughter and frozen for further analysis. Muscle samples for the evaluation of the meat quality indicators were taken from musculus biceps femoris (300 g). Determination of Se in tissues (heart, liver, muscle, ovaries) The samples for selenium determination were mineralized in a closed system using a microwave (MLS-1200, Milestone, Italy) digestion technique with HNO3 and H2O2. The samples were evaporated and the mineral residue was dissolved in water to which 20% HCl was added. Selenium content was then determined with Solar 939 AA Spectrometer (Unicam, UK) using a hydride AAS technique. Determination of GSH-Px activities in serum The method of Paglia and Valentin (1967) was used for GSH-Px activity measurements in serum. The test kit RANSEL (Randox Laboratories, Ltd., United Kingdom) was employed for this purpose. Meat quality indicators Meat colour was measured 24 h after slaughter. The colour was determined objectively by a Konica Minolta spectrophotometer CM-2600d with 8-mm-diameter measurement area, using the D65 illuminant and the 10 ° standard observer. The instrument was calibrated against a white standard plate. The results were expressed in terms of lightness L*, redness a*, yellowness b*. Samples were placed in a polyethylene bag, and cooked in water bath. Internal temperature of samples was maintained at 70 °C for 60 min. After being cooled to room temperature, samples were weighed. Cooking loss was expressed as percentage weight difference between uncooked and cooked samples. Drip loss percentage was determined as weight loss relative to 24 h. 512 Table 1. Percentage composition of the feed mixture A1 Supplied per kg of diet: 0.30 mg of Se, 0.25 mg of Co, 5.00 mg of Cu, 40.00 mg of Fe, 0.38 mg of I, 15.00 mg of Mn, 45.00 mg of Zn, 5.00 mg of calcium pantothenate, 3500.00 I.U. of vit. A, 625.00 I.U. of vit. D3, 20.00 mg of vit. E, 0.75 mg of vit. K3, 0.75 mg of vit. B1, 2.10 mg of vit. B2, 1.00 mg of vit. B6, 0.013 mg of vit. B12, 7.50 mg of niacinamide, 0.025 mg of biotin, 50.00 mg of cholinchlorid, 0.10mg of folic acid, 25 mg of Natuphos, 0.175 g of L-lysine, 0.076 g of L-threonine, 0.048 g of D-Lmethionine, 0.0045 g of L-tryptophan Ingredient Percentage Zeofeed 1.0 Wheat 24.5 Corn 21.5 Barley 25.0 Soya bean pollard 20.0 Yeast Bitec Q 2.0 Nutrimilk feed 0.8 Soya bean oil 0.9

Selenium has an important antioxidant role as a part of enzyme glutathione peroxidase (Sunde 1994).Several studies in human medicine provided evidence for the efficacy of selenium as an anticancer agent (Duffield-Lillico et al. 2003).Selenium also enhances immune functions (Beck et al. 2001) and has an important role in the maintenance of fertility (Rayman 2000).It has been documented that pork can contribute significantly to the selenium intake in human population (Kadrabová et al. 1996;Fajt et al. 2009).
Selenium deficiency in pigs is manifested as mulberry heart disease, hepatosis dietetica and muscular dystrophy (Lindberg et al. 1972).Selenium requirements of growingfinishing pigs can be achieved by providing selenium either from an inorganic or an organic source.Sodium selenite represents the inorganic form.Selenium from Se-enriched yeast is the most commonly used organic selenium form (Schrauzer 2000).
Experiments in growing-finishing pigs revealed higher accumulation of organic Se compared to inorganic form in tissues (Mahan and Parrett 1996;Taylor et al. 2005).Also experiments in sows demonstrated higher Se content in newborn piglets tissues and in colostrum when the Se-enriched yeast was used (Mahan and Peters 2004).
Limited data are available so far regarding the use of other organic selenium sources in pigs.For instance Se from Se-enriched alga Chlorella spp.(Svoboda et al. 2009b) and soybean based selenium proteinate (Jang et al. 2006) were used as alternative selenium sources in finishing swine.
The aim of the present study was to evaluate the effects of Se from Se-enriched Enterococcus faecium on tissue selenium retention and meat quality indicators in growingfinishing pigs.supplemented with 0.3 mg•kg -1 Se in the form of sodium selenite.The compositions of experimental mixtures are presented in Table 1 and Table 2.The feed mixtures were composed of a basal diet and a mineral supplement.The pigs received experimental mixtures for the period of 3 months before the slaughter.The feed mixture A1 was administered during the first 30 days.The feed mixture A2 was given to pigs for the rest 60 days.
The pigs were divided into 3 equal groups of 12 animals.
In group A (n = 12) pigs were fed feed mixtures A1 and A2 supplemented with organic selenium at the dose of 0.3 mg•kg -1 in the form of Se-enriched Enterococcus faecium.
The group B (n = 12) had feed mixtures A1 and A2 supplemented with inorganic sodium selenite at the dose of 0.3 mg•kg -1 .
In group C (n = 12) the pigs received feed mixtures A1 and A2 with the addition of organic selenium in the form of Seenriched yeast (0.3 mg•kg -1 ).
The experiment was approved by the Ethics Committee of the University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.

Sampling
The blood samples were taken from all pigs at the beginning of the trial and one day before the slaughter.The pigs were bled from vena cava jugularis.Serum was collected and frozen for further analysis.
Tissue samples for selenium determination (heart, musculus biceps femoris, liver and ovaries) were taken after the slaughter and frozen for further analysis.Muscle samples for the evaluation of the meat quality indicators were taken from musculus biceps femoris (300 g).

Determination of Se in tissues (heart, liver, muscle, ovaries)
The samples for selenium determination were mineralized in a closed system using a microwave (MLS-1200, Milestone, Italy) digestion technique with HNO 3 and H 2 O 2 .The samples were evaporated and the mineral residue was dissolved in water to which 20% HCl was added.Selenium content was then determined with Solar 939 AA Spectrometer (Unicam, UK) using a hydride AAS technique.

Determination of GSH-Px activities in serum
The method of Paglia and Valentin (1967) was used for GSH-Px activity measurements in serum.The test kit RANSEL (Randox Laboratories, Ltd., United Kingdom) was employed for this purpose.

Meat quality indicators
Meat colour was measured 24 h after slaughter.The colour was determined objectively by a Konica Minolta spectrophotometer CM-2600d with 8-mm-diameter measurement area, using the D65 illuminant and the 10 ° standard observer.The instrument was calibrated against a white standard plate.The results were expressed in terms of lightness L*, redness a*, yellowness b*.
Samples were placed in a polyethylene bag, and cooked in water bath.Internal temperature of samples was maintained at 70 °C for 60 min.After being cooled to room temperature, samples were weighed.Cooking loss was expressed as percentage weight difference between uncooked and cooked samples.
Drip loss percentage was determined as weight loss relative to 24 h.512 The pH value was measured with a pH-meter 340i WTW (WTW, Germany).The needle probe SenTix Sp was used.
Lipid oxidation-thiobarbituric number (TBARS) was determined spectrophotometricaly at 532 nm after distillation of the sample with vapour and reaction of distillate with 2-thiobarbituric acid in hot water bath (Castellini et al. 2002).

Results
The results of selenium concentrations in serum and of GSH-Px activities in serum are presented in Figs 1 and 2. No differences in serum selenium concentrations were found among the groups at the beginning of the trial.Before the slaughter, the two groups with organic form of selenium (Se-enriched yeast and Se-enriched E. faecium) did not differ  The group of pigs supplemented with inorganic sodium selenite had lower Se concentration in liver, heart and muscle tissues than the pigs fed with Se-enriched yeast.The use of Seenriched E. faecium resulted in Se concentrations in liver, heart and muscle comparable to inorganic sodium selenite group.However they were significantly lower (p ≤ 0.05) than in Se-enriched yeast supplemented group.In ovaries, the Se concentrations in pigs fed with inorganic sodium selenite were comparable to the both organic Se groups.The ovarial Se concentrations of E. faecium supplemented group were comparable to Se-enriched yeast group.The results are presented in Fig. 3.
Major carcass measurements and muscle characteristic are presented in Table 3.We did not find any differences among the tested groups.

Discussion
E. faecium is a nonpathogenic bacteria which can colonize both the human and swine intestine and exert beneficial probiotic effects (Mego et al. 2005).Moreover it positively influences both local and systemic immunity.It has been documented that E. faecium stimulates phagocytosis of peripheral neutrophils and production of antibodies and intestinal IgA secretion (Mikeš et al. 1995).The combination with selenium enhances immunostimulatory properties of the E. faecium preparations (Ferenčík et al. 2000).E. faecium enriched with organic selenium has been used for example for the elimination of pathogenic bacteria of inflammatory bowel disease and colon cancer (Mego et al. 2005) and for the treatment of experimental adjuvant arthritis (Rovenský et al. 2002).

Table 1 .
Percentage composition of the feed mixture A1

Table 3 .
Major carcass and muscle characteristics in pigs