Effects of dietary organic selenium and vitamin E supplementation on post mortem oxidative deterioration in muscles of pigs

The effect of feeding high levels of a-tocopherol and organic selenium (Se) to pigs on colour stability and on the susceptibility to oxidative deterioration was investigated. Treatments consisted of supplementation of vitamin E (200 mg/kg diet), organic Se (0.3 mg/kg diet) and both vitamin E and organic Se for the last 60 days to finishing pigs before slaughtering. Longissimus dorsi (LD) and psoas major (PM) muscles were examined after 2 and 7 days of storage on colour stability and on lipid peroxidation (measured as malondialdehyde equivalents). Rate of oxidation by Stimulation with Fe/ascorbate was also estimated in LD samples obtained post mortem. In PM 7 days post mortem we found differences between control and groups of pigs supplemented with vitamin E and Se on refleetance, but significant differences (P<0.05) were found only in the vitamin E + Se group. Positive effects (P<0.05) of vitamin E and Se on colour in psoas major muscle refrigerated for 7 days are supported with significant (P<0.05) lower levels of TBARS values in pigs supplemented with vitamin E and organic Se as well. Supplementation with organic Se does not affect the oxidative stability of muscle tissue (longissimus dorsi) when the rate of iron-induced lipid oxidation was examined. Dietary Se had limited potential for enhancing the quality of pork carcasses (psoas major) and accentuating the effect of vitamin E on the oxidative stability of longissimus dorsi muscle was not found.


Introduction
Oxidative raneidity of muscle Systems begins shortly after death and involves the production of a complex mixture of aldehydes, ketones and alcohols.Non-ham iron, trace elements, and sodium Chloride added to processed meat products initiate pre-ferable the oxidation of highly unsaturated phospholipids.a-tocopherol, when incorporated into animal diets, is a highly effective lipid-soluble chain-breaking antioxidant which is acceptable to the consumer (FAUSTMAN et al., 1989).Vitamin E and selenium (Se) are essential nutritients that are integral components of the antioxidant defence system of cells and tissues.The beneficial effect of vitamin E supplementation on meat quality characteristics is related to lower lipid oxidation (MONAHAN et al., 1992), enhanced colour stability (FAUSTMAN et al., 1989), beneftcal effects on muscle energetic metabolism, electrical conduetivity, drip loss (LAHUCKY et al., 2000) and enhanced the possible storage time ofthe products (BUCKLEY et al., 1995).The formation of metmyoglobin (MetMb) from oxymyoglobin (OxyMb) is positively correlated to lipid oxidation and appears to be dependent on antioxidant Status (YIN et al., 1993).Free radicals produced during lipid oxidation can alter the heme chemistry and initiate pigment oxidation causing loss of desirable colour (MIKKELSEN et al., 1992).Selenium was earlier identified as an integral part of the enzyme glutathione peroxidase (GSH-Px), which destroys lipid peroxides and funetions by protecting of cell membranes against peroxidative damage (HOEKSTRA, 1975).Later, selenium was identified as a component of 5'-iodothyronine deiodinase which converts tetra-to triiodothyronine and funetions in nonshivering thermoregulation (BEHNE et al., 1992).GSH-Px activity is coneidered one ofthe best indices of selenium Status and utilisation (SANKARI, 1985).It has the advantage that it is not affected directly by vitamin E and can be measured quantitatively (for review see MILAD and KOVAC, 1998).Serum glutathione peroxidase activity generally reached a plateau at a dietary level of 0.1 ppm Se when either inorganic or organic Se sources were supplemented to growing or finishing pig diets, but the Se enriched yeast source seemed to be less biologically available (MAHAN and PARRET, 1996).The effects of organic and inorganic Se on pigs have been studied widely (GOEHPJNG et al., 1984;KURKELA and KÄÄNTEE, 1984;MAHAN and PARRET, 1996).No significant differences were found in daily gain, feed/gain ratio or carcass quality of growing pigs fed with 0.1 mg inorganic Se or the same amount of Se bound in yeast (SUOMI and ALAVIUHKOLA, 1992).Selenium in meat ofpigs may also contribute to the Solution of a sufficient supply of this dement into the human organism (KOUTNIK and INGR, 1998).From the literature available it is not yet enough clear if organic bound Se could improve vitamin E effects on properties of meat.The objective of this study was to evaluate the effects of dietary administration of vitamin E alone and in combination with organic bound Se on oxidative stability of muscle tissue ofthe porcine carcass.
Animals and diets Forty Large White x Pietrain were used in this experiment, including 30 gilts and 10 castrates.The RYR-1 genotype of these animals was determined by a DNA based test (Genetic department, RIAP Nitra) described previously (LAHUCKY et al., 1998).To create homogenity of groups on frequency occurence of mutation on RYR-1 gene and on sex of pigs four groups were formed with 5 normal and 5 heterozygotes on MH Syndrome.The pigs were penned in double boxes at institute facilities to minimise the influence of stress.One group was fed a diet (not supplemented with vitamin E) and the other groups received a supplemental level of vitamin E (200 mg kg" 1 ), organic bound selenium (0.3 mg kg" 1 ), and a mix of vitamin E (200 mg kg" 1 ) and organic selenium (0.3 mg kg" 1 ) for 60 days before slaughtered.The vitamin E was provided by Slovakopharma (Hlohovec, Slovakia) and organic selenium (Sel-Plex 50) by Alltech.Differences on MH genotypes and sexes were not evaluated.

2.2.
Sample collection and chemical analysis Animals were stunned, slaughtered and exsanguinated in the slaughter house of RIAP Nitra (transportation about 200 m) with a mean live weight of 113 kg.Longissimus muscle samples were collected immediately after exsanguination (0 h), frozen in liquid nitrogen and stored at -70° C until used for estimating of oxidative stability.Following slaughter, the carcasses were chilled at 4° C for 24 h after which longissimus dorsi (LD) and psoas major (PM) muscles were removed from each carcass.A portion ofthe sample was used immediately and the remaining sample was wrapped in aluminium film and stored in a refrigerator at 4° C for 7 days.A portion of LD was also frozen in liquid nitrogen and stored until analysed.
Vitamin E. The concentration of vitamin E in muscle were measured by HPLC (BERLIN et al., 1994).A mixture of 1.5 ml muscle homogenate, 2 ml absolute ethanol and 0.5 ml 10 % ascorbic acid was heated to 70°C for 5 minutes.After adding 1 ml 10 N KOH, the mixture was ineubated at 70°C for 30 minutes.After cooling, 5 ml nhexane was added for extraction.The solvent was removed by evaporation under nitrogen, and the vitamin E was immediately resolved in absolute ethanol and assayed by HPLC.HPLC analysis was performed with the mobile phase methanol with a flow rate of 1 ml/min and a Lichrospher RP 18 column with precolumn (Müder & Wocherle Chromatographietechnik Berlin, 12.5 x 0.4 cm, 5 um).Detection was performed by fluorescence at 292 nm excitation/336 nm emission.Peaks were quantifted upon calibration with authentic samples of vitamin E (Sigiria, Deisenhofen).

2.3.
Meat quality Colour changes after refrigerated storage were measured on the freshly cut surface of the sample by means of spectrophotometer (Specol, Germany) at 520, 580 and 640 nm as external reflectance.Values at 580 and 640 nm were used for calculation of R (reflectance at 580 -reflectance at 640 nm) which should be related to metmyoglobin production (RENERE et al., 1987).Lipid oxidation was assessed at 24 h and 7 days by the 2-thiobarbituric acid method of SALIH et al. (1987).For evaluating the peroxidative stability of longissimus homogenates the determination of thiobarbituric acid reactive substances (TBARS) was used.TBARS were expressed in terms of malondialdehyde, a breakdown product formed during peroxidation.To stimulate lipid peroxidation 3 ml of muscle homogenate were ineubated in 0.1 mM ascorbate and 5 uM FeSO^ From this 0.5 ml were immediately removed and pipetted into 0.25 ml of 20 % trichloracetic acid (TCA) in 100 mM KCl.The remaining homogenate was placed in a water bath at 37° C and after 30, 60, and 120 min 0.5 ml each of this ineubated homogenate were transferred into the TCA medium (see above).These samples were centrifuged at 10000 g for 10 min and 0.5 ml ofthe supernatants were mixed with 0.5 ml thiobarbituric acid (0.67 %) and boiled for 15 min in a water bath.The absorbance at 535 nm was determined immediately after cooling.Standard MDA Solution was prepared by hydrolysis of 1,1,3,3-tetraethoxypropane and the results were expressed as nM MDA/ mg" 1 homogenate protein.Protein content of homogenates was estimated by a modified biuret method.Statistical analyses were calculated as mean values and Standard deviation and differences were evaluated by t-test.

Results and Discussion
The content of vitamin E (a-tocopherol) of the longissimus dorsi muscles was determined and the results are presented in Table 1.The levels of vitamin E in muscles were found to be higher in groups of pigs supplemented with vitamin E and highly correlated with the dietary intake (PO.01).The amount of vitamin E analysed in longissimus dorsi in the present study when pigs were fed 200 mg vitamin E/kg feed was more than twice higher if compared to control and with Se supplemented pigs.This is comparable to previously reported results (BUCKLEY et al., 1995).Lower levels of vitamin E in LD in supplemented and control pigs, found in our experiments, if compared to results which were introduced by another authors (MONAHAN et al 1992;BUCKLEY et al., 1995;HONIKEL et al, 1998;LAURIDSEN et al, 1999) could account for no supplemented vitamin E in basal diet and/or longer time stored and manipulation of samples (6 months stored and once thawed and frozen).The level of vitamin E in longissimus dorsi muscle of control and supplemented pigs with our experimental conditions were twice lower as introduced by HONIKEL et al. (1998).Numerous studies have found dietary vitamin E to improve the colour of chill-stored pork chops (MONAHAN et al, 1992;BUCKLEY et al, 1995).Results vary considerably and findings of non-significant effects of dietary vitamin E on colour stability have also been reported (JENNIES et al, 1997).In the present study by administration of vitamin E and Se colour stability (7 days storage) was unaffected in LD muscle using surface reflectance measurements and difference values (630nm -580 nm).Using psoas major muscle we found differences between control and group of pigs supplemented with vitamin E and Se on reflectance at 630 nm but significant differences (PO.05) were in group vitamin E + Se.It is supposed that higher differences on reflectance values between 630 nm and 580 nm are connected with a higher level of methmyoglobin (RENERE, 1987).We found significant (PO.05) lower differences between reflectance at 630 and 580 nm in psoas major if comparing control and with vitamin E and Se administered pigs.Our results are in agreement with findings of no significant changes in colour stability in porcine longissimus dorsi (LANARI et al, 1995;JENSEN et al, 1998).When administered vitamin E colour of psoas major could be stabilised as was shown mainly in beef (CHAN et al, 1996).Dietary supplementation with vitamin E has been shown to reduce lipid oxidation and accumulation of metmyoglobin in fresh beef (FAUSTMAN et al, 1989).In agreement with our results on longissimus dorsi muscle the changes in colour stability as a consequence of feeding high levels of vitamin E in pork is not as evident as in beef.ASGHAR et al. (1991) showed that 'a' values (an indicator of surface redness) of pork chops from pigs fed vitamin E (200 mg/kg feed) were significantly higher than those of chops from pigs fed the basal level (10 mg/kg feed) after 6 days of refrigerated storage.MONAHAN et al. (1992) reported a higher 'a' value in chops from pigs fed the higher level of vitamin E after 2, 4, 6 and 8 days of refrigerated storage.An effect on surface-redness ('a' value) of biceps femoris slices from cured hams from pigs supplemented with vitamin E (200 mg/kg" 1 diet) was recently reported (ISABEL et al, 1999).Results of these authors are more in agreement with our results when a lower reflectance at 630 nm and significant (PO.05) difference between reflectance at 580 and 630 nm were received from psoas major muscle after refrigerated storage for 7 days of pigs supplemented with 200 mg vitamine E/kg" 1 feed for 60 days before slaughtering.Different muscles (psoas major with overall lower reflectance values when compare to longissimus dorsi muscle in our experiment, Table 2) could exhibit different rates of oxymyoglobin oxidation (i.e.discoloration) when displayed under different condition and vitamin E treatment increased oxymyoglobin stability in ränge psoas major, gluteus medius and longissimus dorsi beef muscles stored at 4° C (CHAN et al, 1996).Colour stability and lipid oxidative processes in fresh meat are known to be closely related and low muscle vitamin E levels resulted in low 'a' values and concurrent high rates of lipid oxidation (JENSEN et al, 1998).Our findings of positive vitamin E effects on colour differences in psoas major muscle (refrigerated storage for 7 days) are supported by significant (PO.05) lower TBARS values (Table 2) in pigs supplemented with vitamin E and organic Se as well.We did not receive significant differences (P>0.05) on TBARS values of longissimus dorsi muscle (refrigerated storage for 7 days) of pigs supplemented with vitamin E and organic Se (Table 2) but the rate of iron-induced lipid oxidation of LD was strongly influenced by dietary vitamin E. However, as shown in the Figure, the oxidative changes on LD were not influenced by dietary addition of organic Se.The effect of vitamin E was statistically significant (PO.05) in muscle (longissimus dorsi) homogenate prepared from samples post mortem.It was shown previously that the oxidative changes were large in LD ofpigs fed diets not supplemented with dl-a-tocopheryl acetate compared with LD ofpigs supplemented with dl-a-tocopheryl acetate at incubation for 120 and 160 min (LAURIDSEN et al, 1999).Supplementation of organic Se does not affect the oxidative stability of muscle tissue (longissimus dorsi) when the rate of ironinduced lipid oxidation of LD was examined and the time course of Stimulation with Fe 2 * does not differ from control pigs (Fig. ).GRADY et al. (1998) were not able to find differences on muscle Se-GSH-Px activity between control and the dietary Se supplemented pigs.Elevating dietary Se (0.3 mg/kg" 1 diet), while adhering to regulations relating to dietary Se does not affect the oxidative stability of beef.
In the literature there are data about more efficiency on clinical signs of cows when supplemented with both vitamin E and Se but their action was not additive and it is supposed glutathione peroxidase activity (related to Se administration) may spare the requirement for vitamin E in the membranes (SMITH et al, 1997).From our results follows that dietary Se has limited potential for enhancing the quality of pork carcass (psoas major) and accentuating the effect of vitamin E on oxidative stability of longissimus dorsi muscle was not found.

Conclusion
The results showed that administration of organic bound Se (0,3 mg/kg feed for 60 days) could in some extend positively influence colour and oxidative stability but as expected supplementation with vitamin E (200 mg/kg" 1 feed for 60 days) to finishing pigs is more effective in improving the antioxidative defence system in carcasses of pigs.We concluded that high dietary level of vitamin E to pigs increases the color stability and stability offat in carcasses ofpigs.

Table 1
Groups of supplementation with vitamin E and organic selenium in diet and concentration of vitamin E (cctocopherol) in longissimus dorsi muscle (Fütterungsgruppen und Konzentrationen von Vitamin E im M.I. d.) Means with different superscripts differ (P < 0.001)

Table 2 Colour
measured as reflectances and amount of thiobarbituric acid (TBA) related Compounds in muscles longissimus dorsi (LD) and psoas major (PM) of pigs during chill storage (Reflexionswerte und