Lipid profile and cholesterol of pork with the use of glycerin in feeding

The aim of this work was to evaluate the effects of adding different levels of glycerin in the feeding on the lipid profile and cholesterol of the meat of finishing pigs. The experiment was arranged in a completely randomized design (CRD) with factorial 5x2, being five levels of glycerin in the diet and two genders. Sixty (60) swine of the Topigs genetics (30 barrows and 30 gilts) were used; they presented initial average weight of 79.3+4.0kg and were finished with the weight of 106.2+4.5kg. The levels of glycerin utilized were 0, 50, 100, 150 and 200g/kg in the natural matter. The analyses of lipid and cholesterol profile were conducted in the muscles longissimus dorsi (loin) and semimembranosus (ham) on the left side of the carcasses. Interaction occurred between the levels of crude glycerin in the diet and the sexual category for the profile of fatty acids and cholesterol. On the loin the meat of the barrows presented higher means of C16:0, C16:1, C20:3ω3, C18:1ω9c, Total of saturated (SFA) and monounsaturated (MUFA) fatty acids. On the ham, increased means were found for the fatty acids C18:1ω9c, MUFA, activity of ∆9-desaturase C18 and Elongase C16-18 in barrows. The sows’ meat presented higher proportions of polyunsaturated fatty acids and of the series ω6 for both the cuts. The amount of cholesterol in the sows’ meat presented increase according to the level of glycerin in the meat. The barrows’ meat presented indices of atherogenicity greater than the gilts. The levels of glycerin altered the lipid profile and cholesterol content according to the sexual category, promoting a distinct effect on the loin and ham.


INTRODUCTION
Crude glycerin is one of the by-products of biodiesel production, its use has been growing in animal feeding as a way of reducing the costs of the diets of the several meat-producing species.Crude glycerin contains glycerol contents ranging from 80 to 95 %, this being a precursor for triacylglycerol and phospholipids synthesis in the liver and adipose tissue (Boyle, 2005;Cerrate et al., 2006).
However, the increase in the level of information of the world population as to the importance of a diet with lower levels of saturated fat has led to increased search for meats which present, in their characteristics, better balance between saturated, monounsaturated and polyunsaturated fatty acids and low ratio ω6/ω3.The content in essential fatty acids plays an outstanding role in human nutrition, since they are integrant of cell structures and precursors of the synthesis of several substances essential to the maintenance of the physiological processes (Perini et al., 2010).
In this way, studies should be conducted on crude glycerin, since glycerol is utilized in lipogenesis and even in gluconeogenesis, which could contribute towards the increase in the lipid contents and modification of the lipid profile in pork.

MATERIAL AND METHODS
The animals were reared in the Finishing House of the Swine Culture Sector of the Animal Science Department of the Federal University of Lavras (UFLA) in the municipality of Lavras -MG.Thirty barrows and 30 gilts of the genetics Topigs, with initial average weight of 79.3+4.00kg and finished with weight of 106.2+4.5kg were utilized.This study was approved by the Ethics Committee on Animal Use UFLA with protocol number 011/2010.The experiment was arranged in a completely randomized design (CRD) with factorial 5x2, being five levels of glycerin in the diet (0, 50, 100, 150 and 200g/kg) and two genders (male (barrow) and female (gilt)), with 6 replicates per treatment and each experimental unit consisted of one animal, totaling 60 pigs.The experimental diets were formulated on the basis of corn and soybean meal, supplemented with vitamins, minerals and aminoacids to meet the minimum requirements for barrows of high genetic potential from 70 to 100kg (Table 1) with the utilization of different levels of crude glycerin.
The treatments were carried out in the following way: Treatment 1basal diet without the addition of glycerin; Treatment 2 -50g/kg of glycerin instead of corn in the basal diet in the natural matter; Treatment 3 -100g/kg of glycerin instead of corn in the basal diet in the natural matter; Treatment 4 -150g/kg of glycerin instead of corn in the basal diet in the natural matter and, Treatment 5 -200g/kg of glycerin instead of corn in the basal diet in the natural matter.The animals were given water and feed ad libitum during the experimental period of 30 days.
At the end of the experiment, after fasting and water diets for 12 hours' time, the animals were slaughtered under humanitarian conditions through electric insensibilization (electronarcosis) with later bleeding and evisceration.
After 24 hours' cooling of the carcasses in cold room (+5ºC), the collection of the samples of the muscles Longissimus dorsi (loin) and Semimembranosus (ham) on the left side of the carcasses was done.The samples were carried refrigerated into the Laboratory and then they were frozen for later determination of the lipid profile in the Meat and Fish Technology and Chromatography Laboratory in the Chemistry Department.For determination of the fatty acid profile, samples of the muscle Longissimus dorsi (loin) and Semimembranosus (ham) after thawing at 5ºC for 24 hours in a refrigerator were utilized.For the analysis of cholesterol and fatty acids (FA), lipids were extracted according to the procedures described by Folch et al. (1957) being esterified and separated (Hartman and Lago, 1973).Cholesterol was quantified by colorimetry, with a modification of the method of Bohac et al. (1988)

Arq
For the loin, there was distinct behavior of the glycerin levels according to the gender, revealing that effect in the barrows for the fatty acid oleic (C18:0) and activity of enzyme Δ9-desaturase C16 with 50g/kg and 100g/kg glicerin levels, respectively.In the gilts, poorer amounts of oleic acid (C18:1ω9) were found, which showed a decrease along with the activity of the enzyme Δ9-desaturase C18 and Elongase C16-C18 according to the levels of glycerin of 150g/kg in the diet, which brought about similar behavior and the reduction of the total of monounsaturated fatty acids (MUFA) compared to barrows and glicerin levels.
Interaction was verified between the glycerin levels and gender, which revealed the C15:0, PUFA, Σω6, C18:2ω6, C20:4ω6, Ratio PUFA/SFA for the 150g/kg glicerin level means for gilts (Table 2), which showed that dietary levels of glycerin interfered in the fatty acids composition of the meat of gilts in relation to the series w6 fatty acids to increase the amounts of polyunsaturated fatty acids, while in barrows this effect did not occur.In general barrows present increased lipogenesis activity than the gilts.That effect is due to the absence of anabolic stimuli, since androgens exercise a direct effect upon the musculature and estrogens promote a greater release of growth hormone and insulin-like growth factor (IGF-I), increasing the deposition of aminoacids and glucose into the muscle (Clapper et al., 2000).The results found in this study may be related to the increased rate of intramuscular and subcutaneous deposition of lipids that is supported by emasculation, promoting a greater deposition of saturated fatty acids, whereas in the gilts, higher concentrations of polyunsaturated fatty acids are found in total lipids or triacylglycerols (Alonso et al., 2009;Mas et al., 2010).
There was an influence of the glycerin levels on the values of fatty acids identified in the ham, regardless of gender (Table 3).For the ham, greater means of the fatty acids oleic (C18:1ω9c), MUFA and ∆9-desaturase C18 activity were found in barrows.These results may be related to a greater activity of the enzymes Elongase C16-C18 (Table 3) and ∆9desaturases in the barrows meat, since this acts in the removal of hydrogen molecules of the carbonated chains of saturated fatty acids, increasing the formation of monounsaturated fatty acids cis-9, such as palmitoleic and oleic (Hayashi et al., 2007;Metz et al., 2009).
In the metabolism of polyunsaturated fatty acids ω-6 and ω-3 PUFA, the desaturation and elongation reactions are mediated through enzymes desaturase and elongase (Δ4, Δ5, Δ6), resulting in the synthesis of highly unsaturated fatty acids, which afterwards are esterified into phospholipids and will go to constitute the cell membranes, contributing to its fluidity, in addition to being precursors of prostaglandins, thromboxanes and leukotrienes (Nakamura and Nara, 2004).As the affinity of enzyme Δ6 desaturase is greater for fatty acids ω-3, it will need smaller amounts of these in relation to ω-6 for the production of the same amount of the product (Palmquist, 2009).Thus, in this work, in both cuts of swine gilts' meat, we found a greater amount of the fatty acids linoleic (C18:2ω6c) and arachidonic (C20:4ω6) (Table 2.3).These results suggest that in the gilts there is possibly a poorer activity of Δ6 desaturase and consequent greater activity of Δ5 desaturase, where as in the Barrows that behavior would be inverse, since smaller amounts of linoleic acid and greater of 11,14,17-Eicosatrienóico (C20:3ω3) were found in these animals.
The sows' meat presented higher proportions of polyunsaturated fatty acids in relation to the saturated ones (PUFA/SFA) for both the cuts (Table 2.3) according to the glycerin level of 150g/kg in the diets in the loin for the total of polyunsaturated fatty acids, the series ω6, arachidonic acid with an average superior proportion of 1.79 as high as this component in relation to the amount found in the barrows.This behavior can be related to increased activity of the enzymes desaturase (Δ6) and elongase in gilts, since the increase of the glycerol levels would promote greater synthesis of triglycerides, these being utilized for the production of polyunsaturated fatty acids in this category, while for the barrows there would be greater formation of SFA (Nuernberg et al. (2005).
In the ham there was an interaction between the levels of glycerin and gender for the amount of cholesterol in the meat (Table 4).For the gilts, there was an effect and the amount of cholesterol increased according to the level of glycerin in the diet, while in the barrows high means were found in the 150g/kg.This increase in the amount of cholesterol in the meat of the gilts according to the dietary glycerin level may be related to the increased need of synthesis of progesterone and/or estradiol, since from cholesterol sex hormones are synthesized.The process of biosynthesis of cholesterol and triacylglycerols is similar and depends on a source of glicerol-3-phosphate (glicerol-3-P), which is obtained through the action of the enzyme glycerol kinase upon glycerol, with further production of pyruvate, where this is utilized to produce acetyl-coA, which is the precursor for the cholesterol synthesis (Boyle, 2005).As in the barrows, there is no production of steroid hormones, possibly the different amounts of glycerol fed in the diet determine that range since acetyl-coA can be utilized for synthesis of other compounds as lipids, ketone bodies and energy production via the Krebs cycle.

Arq
Considering the nutrient allowances of the World Health Organization (World Health Organization, 2003) stating that the PUFA/SFA ratio should be higher than 0.4; it is found in this work that in both the cuts, the gilts presented better results (Table 4) and that only in the loin of barrows no values above that index were found.For human diets, a consumption proportion of 4:1 in relation to the fatty acids ω6/ω3 is desirable.Nevertheless, in the meat of the animals in this study the results showed average values of ω6/ω3 ratio of 34.69 in the loin and of 39.89 in the ham, which stands for 9 to 10 times the recommended, which constitutes a risk factor for the development of certain types of cancer and heart diseases if considering only its consumption (World Health Organization, 2003).
According to Wood et al. (2003), this is one of the undesirable aspects of pork, brought about by the high amount of linoleic acid present in the ingredients of the feeds which are given in the raising system, which has led many workers to seek alternatives such as the supplementation in swine feeding with soil sources rich in w3 (Nuernberg et al., 2005;Tikk et al., 2007), conjugated with linoleic acid (Martin et al., 2008) and monounsaturated fatty acids (Mas et al., 2010, Realini et al., 2010) .
The determination of the indices of atherogenicity and thrombogenicity in the loin showed the poorest indices for the meat of barrows; while in the ham there was a difference for the index of atherogenicity with best results for the gilts (Table 4).These indices are related to the amounts of saturated, polyunsaturated fatty acids and those of the series ω6, being a health indicator associated with the risk of cardiovascular disease according to the lipid composition of the food (Turan et al., 2007).In that way, the lower the indices of atherogenicity and thrombogenicity of a given products are, the better its consumption will be for one's health.
The indices of atherogenicity and thrombogenicity found in this work are similar to the results reported by Tonial et al. (2010) for salmon (0.56 and 0.23) and lower than the ones cited by Arruda et al. (2012) for lambs (0.64 and 1.37), demonstrating in that way characteristics intermediary to the meat of the two species.
In general, regardless of the gender and cut, the meat of the pigs fed crude glycerin offered characteristics favorable to its consumption, since gilts presented higher values of polyunsatured fatty acids and barrows presented higher values of monounsaturated fatty acids, mainly oleic acid which aids in the reduction of serum cholesterol, possesses anti-thrombogenic effect and inhibits the platelet aggregation.

CONCLUSION
The use of glycerin in swine feeding promoted modification in relation to the lipid profile, promoting modifications in the deposition of fatty acids, with higher amounts of glycerin in swine feeding promoting modifications regarding the lipid profile, promoting modifications in the fatty acid deposition with greater amounts of saturated and monounsaturated acids in the barrows meat and polyunsaturated acids and cholesterol in the sows.

Table 1 .
Centesimal composition and values calculated in the experimental diets

Table 2 .
Composition of the fatty acids of the loin of pigs depending on the glycerine level of feeding and gender

Table 4 .
Mean of the index and cholesterol according to the level of glycerin and gender in the muscle Longissimus dorsi (loin) and Semimembranosus (ham)