Effect Of Chromium Nicotinate On Oxidative Stability, Chemical Composition And Meat Quality Of Growing-Finishing Pigs

The effect of different organic sources of Cr on growth, feed efficiency and carcass value is known but there is a lack of information between chromium nicotinate (CrNic) and pork quality. Therefore, purpose of this research was to investigate the effects of CrNic on chemical composition, quality and oxidative stability of pork meat. In the study, pigs of Large White breed (40 pcs) were used. The pigs were divided into two groups, namely the control and the experimental of 20 pcs with equal number of barrows and gilts. The pigs were fed the same diet which consisted of three feed mixtures applied at the different growth phases, from 30 - 45 kg OS-03, 45 - 70 kg OS-04 and 70 - 100 kg OS-05. The pigs were allowed ad libitum access to feed and water. The diet of experimental group was supplemented with 0.75 mg.kg -1 CrNic in the form of chromium-inactivated yeast Saccharomyces cerevisiae . The fattening period in pigs lasted from 30 to 100 kg. The chromium supplementation led to a significantly higher content of chromium in longissimus thoracis muscle (LT) of experimental pigs. In addition, the results showed a statistically significant difference ( p ≤0.05) in retention of chromium in the LT, monounsaturated and omega-3 polyunsaturated fatty acids content in experimental group compared with control. Moreover, there was highly significant ( p £0.05) difference in essential fatty acids, as well as in oxidative stability in 7 days, among the groups. The highly significant differences were also observed among sexes, namely in total water, protein and intramuscular fat contents, colour CIE b* in both times, and oxidative stability. However, physical-technological parameters (pH, drip loss, shear force and meat colour) were not affected when pigs were fed the supplement. On the whole, the positive effect of chromium nicotinate in most of investigated parameters may be beneficial not only for pork industry but also for consumers. Normal 0 21 false false false CS JA X-NONE

However, most grains and feedstuffs are deficient in Cr and must be supplemented with a bioavailable source of Cr (Bunting, 1999). Animals cannot utilize glucose when chromium is deficient in feed (Tang et al., 2001).
It is generally accepted that organic sources of Cr like chromium picolinate and chromium nicotinate are utilized more efficiently than inorganic Cr sources Supplemental chromium nanoparticle (CrNano) has shown beneficial effects on carcass characteristics and pork quality in finishing pigs (Wang and Xu, 2004). Research with four organic sources (Cr-tripicolinate, Cr-propionate, Cr-methionine, Cr yeast) in concentration 5000 μg.kg -1 of Cr was reported (Lindemann et al., 2008). The effects of the forms of Cr fed on the meat quality and the carcass measurements were minimal. Selenium-yeast combined with chromium-yeast has positive effect on performance and carcass composition of finishing lambs

Animals and diets
The experiment was carried out in an Experimental Centre near the Department of Animal Husbandry at the Slovak University of Agriculture in Nitra. In the study, 40 pigs of Large White breed were used. The genotype of all pigs on the marker RYR-1 (malignant hyperthermia syndrome) was analysed by the DNA test. All the experimental animals were detected as homozygous dominant (NN).
The pigs were divided into a control group and an experimental group (each of 20 animals) with equal number of barrows (S 1 ) and gilts (S 2 ). Both, control (G 1 = Cont) and experimental group (G 2 = CrNic) of pigs were fed the same diet which consisted of three feed mixtures applied at the different growth phases, from 30 -45 kg OS-03, 45 -70 kg OS-04 and 70 -100 kg OS-05 (Table 1). The diet of experimental group was supplemented with 0.75 mg.kg -1 nicotinate (CrNic) in the form of chromium-inactivated yeast Saccharomyces cerevisiae fermented on the substrate which was from natural resources with a higher content of chromium during the whole fattening period. The pigs were housed in an environmentally controlled finishing barn with two pigs in each pen. They were allowed ad libitum access to feed and water. The fattening period in pigs lasted from 30 to 100 kg. The growth performance of pigs was controlled by weighing with an accuracy of 0.5 kg. The weighing was realised in two-week intervals (30 -90 kg) and one-week intervals (90 -100 kg).

Slaughter and sample collections
The slaughtering and the carcass dissection of pigs were carried out in the slaughterhouse of Experimental Livestock Centre near the Department of Animal Husbandry. The pigs were slaughtered at an average live weight of 102.5 kg and the dissection of carcasses was done according to standard practices STN 466164. Carcasses were chilled at 3 -4 °C overnight. The samples (100 g) for chemical analysis and determination of some meat quality traits were taken from LTon right half-carcass 24 hours post mortem. The place of sampling was above the last thoracic vertebra. After that, the samples were labelled and stored frozen at -19°C ±0.5 °C for 14 days until analysis.

Chemical analysis
The chemical composition of pork in LT was determined from samples of muscle homogenate (50 g) using the FT IR method (Nicolet 6700). The analysis of infrared spectra of muscle homogenate was done by the method of molecular spectroscopy.
The principle of the method was the absorption of infrared radiation by the transition the sample, in which were changes in rotation vibrational energy states of molecules in response to changes in dipole moment of the molecule. The analytical output was an infrared spectrum which was a graphical display of functional dependence of the energy, usually expressed in percentage of the transmittance (T) or in units of absorbance (A) on the wavelength of the incident radiation. The transmittance (throughput) was defined as the ratio of the intensity of the radiation that passed through the sample (I) and the intensity of the radiation emitted by the source (Io). The absorbance was defined as the common logarithm of 1/T. The energy dependence on the wavelength was logarithmic; the wave number was used, which was defined as the reciprocal of the wavelength, and thus the energy dependence of the wave number will be a linear function. Individual groups of fatty acids (g.100g -1 FAME, Fatty Acid Methyl Ester) were determined from the muscle homogenate of LT in the Laboratory of gas chromatography at Faculty of Natural Sciences (Comenius University, Bratislava, Slovakia).

Preparation of fatty acid methyl esters
Small amount (4 -5 g) of muscle tissue was sampled and homogenized by grinding. From the obtained homogeneous mixture, 1 g sample was collected. After that, 4 mL of mixture for the extraction was used, chloroform: methanol (2:1) and the sample was shaken for 1h. After extraction, 2 mL of saline solution (0.9% NaCl) was added and shakedagain for 10 minutes. After few minutes, it was taken approximately 2 mL of lower layer which was subsequently centrifuged. From the adjusted sample, it was collected 1 mL for the transesterification.
Discovery Ag-Ion SPE preseparation columns were developed for the separation of methyl esters according to the degree of saturation of fatty acids using a method of Kramer et al., (2008).

Meat quality measurements
The physical characteristics of meat quality were measured in the laboratory conditions of the Experimental Centre near the Department of Animal Husbandry, SUA in Nitra. The meat colour was determined on the cut of the LTabove the last thoracic vertebra 24 h post mortem using a spectrophotometer CM-2600d. Commission Internationale de IʼEclaire (CIE) L*, a*, and b* values were determined using the CIE Lab space with a D65 illuminate.
For determination of drip loss, the methodology described by Honikel (1998) was used. In time 24 h to 48 h post mortem, a sample (approximately 50 g) was taken from the LT, placed in vacuum plastic bagsand hung in the refrigerator at 4 -6 °C.
After 7 day-storage at temperature 4 ±1 °C, the Warner-Bratzler shear force was analysed. The samples were heated to temperature of 71 ±1 °C for 30 minutes and then cut for chips in 1x1 cm across fibers. Shear force was determined using the device Chatillon.

TBA method
Procedure for the sample preparation and determination of MDA was done according to the method of Marcinčák et al., (2009). A ground sample (1.5 g) was weighed in a 50 ml centrifuge tube and 1 mL EDTA (complex-forming agent) was added immediately. After gentle agitation, 5 mL 0.8% BHT was added, and the tube was gently shaken again. Just before homogenization, 8 mL 5% TCA was added to the tube and homogenization was carried out for 30 s at maximum speed. After homogenization, the sample stood for 10 minutes and then it was centrifuged for 5 min (3500 rpm, 4 °C). After centrifugation, the top hexane layer was discarded and the bottom layer was filtered through Whatman filter paper No. 4 into a 10 mL volumetric flask and diluted to volume with 5% TCA. After that, a 1 mL of TBA was added to the tube of 4 mL sample. The samples and MDA standards were incubated in a water bath for 90 min at 70 °C. After cooling in an ice bath, samples were incubated at room temperature for 30 min and extinction of samples was measured by UVspectrophotometer at a wavelength of 532 nm.

Preparation of calibration curve
From the stored MDA solution, 1 mL was pipetted to 25 mL volumetric flask and added 0.1 mol -1 HCl. The resulting MDA working solution with a concentration of 0.1748 g.mL -1 was used to preparation of the calibration curve.

Statistical analysis
The parameters of meat quality were statistically evaluated by statistical methods described by Grofík and Fľak (1990) and by statistical package Statistix, Version 8 and 9 (Anonymous, 2001). At first, the basic statistical characteristics, means (ӯ) and standard deviations (SD) of analysed traits were computed. The differences of analysed traits between studied groups (G i ), sex (S j ), their interactions (GS) and pens (P k ) were evaluated by twofactor analysis of variance (AOV) with repeated measurements/animals on pens factor. The colour of meat was evaluated by three-factor AOV, with these same factors and factor time (24 hours and 7 days). The linear regression method was used for describing the dependence of oxidative stability of LT muscle after Cr-supplementation on time of storage.

Chemical parameters
Supplementation of the diet with 0.75 mg.kg -1 chromium nicotinate resulted in a significantly higher content of chromium in the LT of experimental pigs than that of control pigs (0.199 vs. 0.153 mg) as shown in Table 2. The percentage of total water content in LT muscle was the same in both control pigs and pigs fed chromium. The percentage of total protein content was lower in the control group compared with the experimental, but the effect was According to study of Štefanka et al., (2013), addition of selenium with chromium nicotinate has reduced cholesterol content in the pork muscles. In our experiment, the differences in the fatty acids content in intramuscular fat of LT (g.100 g -1 FAME) are presented in Table 2. There are highly significant differences caused by sex in total water, protein and intramuscular fat content. The content of monounsaturated fatty acids was significantly lower in the control (51.63 g) compared with the experimental group (53.61 g). Also, the content of the essential fatty acids in the control group was significantly lower than that of experimental group (6.65 g vs. 7.80 g), see Tables 2 and 3. On the other hand, the chromium supplementation resulted in the significant increase (p ≤0.05) of omega 3-polyunsaturated fatty acids in experimental pigs compared with the control ones (0.47 vs. 0.42 g). Lien et al., (2001) suggest that the carcass of the pigs that received the chromium picolinate supplemented diet (400 μg/kg) contained less oleic acid (C18:1) and total unsaturated fatty acids (p ≤0.05). The total saturated fatty acid content in chromium fed group was higher than that in control.

Physical and technological quality of pork
The results for the physical and technological quality of pork are presented in Table 2 and Table 3  115 Note: ӯmean; SDstandard deviation; G 1control group; G 2experimental group; G 1 S 1 , G 1 S 2 , G 2 S 1 , G 2 S 2subgroups. y y y y In our study, an effect of chromium supplementation on some meat colour parameters 7 days post mortem was determined. The values of meat colour were not significantly different between experimental and control group. The differences between sexes in CIE b* in 24 hours and 7 days were significant. The means of meat colour CIE L*, a* and b* in LT 24 hours and 7 days by groups, sex and time are presented in Table 6. It was found out that the differences between analysed groups, sexes and time were not significant for CIE L*. The results showed that there was a highly significant difference between Time and interaction Group x Time in parameter CIE a*. Highly significant difference in CIE b* was caused by differences of Sex and Time. Also, there was a significant interaction in Group x Sex.

Oxidative stability
The effect of dietary chromium supplementation on the antioxidative stability of LT muscle is presented in Tables 2, 3 and 5. It was showed a highly significant difference between groups in 7 th day (0.161 mg.kg -1 in G 2 group vs. 0.314 mg.kg -1 in G 1 group). The means and standard deviations of the oxidative stability of LT for total observations in the 1 st , 3 rd , 5 th and 7 th day, for groups and sex and also for subgroups G i S j are presented in Table 5.
The linear regression parameter estimates, the corresponded analyses of variance and significance of differences between elevations and slopes of oxidative stability of LT muscle are presented in Table 4.
The dependence of oxidative stability on time of storage (x = t = 1, 3, 5, and 7 days) was highly significant. The deviations from linearity, i. e. nonlinearity (NonLin) were not significant. The dependence of oxidative stability had a pure linearity form. The comparison between groups and sexes showed significant or highly significant differences between elevations andslopes (Table 4).
There was a similar situation by comparison of sexes (S j ) in analysed groups (G i ). Figure 1 and 2 illustrate the linear functions. The coefficients of determination R 2 were highly significant. The slope in the G i = Cont was two times higher thanslope in the G 2 = CrNic group (b 1 = 0.0478 vs. 0.0229 MDA). The reverse situation was observed by comparison of sexes.
Recent research indicates that there are two mechanisms for chromium to affect the pork quality. It is an effect on carbohydrate metabolism or effect on stress. The investigation has shown that Cr-propionate and Cr-picolinate increases insulin sensitivity (Amoikon et al., 1995;Matthews et al., 2001). We can assume that Note: nnumber; ӯmean; SDstandard deviation; G 1control group, G 2experimental group; S 1 -barrows,S 2gilts; G 1 S 1 , G 1 S 2 , G 2 S 1 , G 2 S 2subgroups.

Figure 2
Linear regressions of oxidative stability of longissimus thoracis muscle after Cr-supplementation for subgroups G i S j , (G 1 = Contr, G 2 = CrNic, S 1 = barrows, S 2 = gilts). These findings would be indicated that the Cr may affect glycolytic potential in muscles and subsequently impact the pork quality. The glycolytic potential of muscle tissue also plays an important role in the preslaughter stress. Some research has mentioned that Cr may partially mitigate the effect of short-term stress (National Research Council, 1997).

CONCLUSION
According to the results obtained in vivo experiment, it can be concluded that the supplementation of organic chromium as chromium nicotinate (0.75 mg.kg -1 ) in the pig diet resulted in a higher retention of chromium in LT. The dietary addition of organic chromium to growing and finishing diets for pigs caused a higher content of monounsaturated fatty acids and essential fatty acids in intramuscular fat of LT. However, the feeding with the supplementation increased the content of polyunsaturated and omega 3 fatty acids in the experimental group of pigs. On the other hand, the chromium nicotinate have no effect on the chemical composition of meat and meat quality traits, except with some colour parameters after 7-days storage. The Cr-addition had significantly positive impact on the oxidative stability of pork during its storage. It could be demonstrated that Cr consistently affects pork quality, so that may be beneficial from pork industry and consumer point of view. However, more research is needed to investigate the consistency in which the chromium nicotinate may improve the pork quality.