Performance , fatty acids digestibility , carcass and muscle composition of pigs fed diets enriched with vitamin E and differing in their MUFA / PUFA ratio *

The effect of dietary vitamin E supplementation (20 vs 200 mg α-tocopheryl acetate/kg) and different MUFA/PUFA ratio on pig performance, digestibility of fatty acids, and carcass and muscle chemical composition was studied. The feed containing higher concentration of C18:2 led to a more (P<0.01) effi cient feed utilization (FCR) in boars and gilts. An effect (P<0.05) of dietary fat on C16:1, C18:0, C18:1 and C18:2 apparent digestibility was found. Apparent digestibility of C16:1 and saturated fatty acids was lower at higher vitamin E supplementation, indicating a possible competition for absorption. The partial substitution of dietary PUFA by MUFA produced a higher proportion of intramuscular fat (P=0.04), and a decrease in total fatty acid apparent digestibility only in the case that a basal level of vitamin E was added to the diet.


INTRODUCTION
Pigs require a certain amount of polyunsaturated fatty acids (PUFA) for optimum health and performance.The Agricultural and Food Research Council (1981) indicated that C18:2 minimum dietary concentration in growing pigs from 30 to 90 kg should be 1.5% of dietary digestible energy (DE), which is approximately equivalent to 7 g/kg diet.The National Research Council (1998) of the United States recommends a minimum concentration of 10 g/kg diet in the same weight range.Whittemore (1993) indicated an ideal concentration of 4% of the gross energy for optimum growth (which is equivalent to 14 g C18:2/kg feed approximately).It might be also that modern pig genotypes with very high growth potential require a higher concentration of essential fatty acids than conventional pigs.
Due to availability and relative low cost in some productive circumstances, it is common that some practical diets for growing pigs provide a level of C18:2 above 15 g/kg.However, increasing the degree of polyunsaturation in animal tissues by dietary manipulation may accelerate oxidative deterioration in pig meat (Monahan et al., 1992) and produce undesirable fl avour (Larick et al., 1992).For this reason, there is growing interest in the utilization of high level of vitamin E in swine nutrition, particularly in combination with dietary unsaturated oils (Asghar et al., 1991;Lopez-Bote, 2000;Rey et al., 2001).Practical recommendations of dietary vitamin E supplementation are approximately 3-6 mg vitamin E/g of PUFA (Putnam and Comben, 1987).In previous researches, it has been reported a negative effect of dietary polyunsaturated fatty acids on α-tocopherol accumulation (Renerre et al., 1999).However, there is little information of the possible interactions between dietary fatty acids and vitamin E concentrations on digestibility in pig nutrition.
The use of vegetable oils high in monounsaturated fatty acids (MUFA) as an alternative to those rich in PUFA (Miller et al., 1990;Myer et al., 1992) has reached an increased interest.This was due to the favourable publicity that monounsaturated fats have received in regard to human health (Grundy, 1986), because of its possibility of decreasing saturated and n-6 fatty acids and its low oxidation rate (Rey et al., 2004).MUFA, not only are coming from the diet but also can be newly synthesised by the animal from saturated fatty acids, as opposite to PUFA which belongs to essential fatty acids.However, dietary PUFA (Warnants et al., 1999) and MUFA (Klingenberg et al., 1995) show different degrees of inhibition of the stearyl-CoA desaturase activity and consequently, affect the synthesis of MUFA in the organism.
The objectives of this research were to evaluate the effect of the utilization of different MUFA/PUFA ratios (maintaining the same concentration of saturated fatty acids) and two levels of vitamin E (20 vs 200 mg/kg) in pig diets, on fatty acid digestibility, pig performance, and carcass and M. longissimus lumborum composition.

Experimental design
Thirty male and thirty female Large White × Yorkshire pigs of 70 days age, weighting approximately 25 kg, were randomly located in individual cages and  Cu,22.5;Fe,11.21;Mg,15.3;Zn,88.8;Se, 0.045 and I, 0.573 5 α-tocopheryl acetate supplement = 50% α-tocopheryl acetate+ 50% carrier fed a conventional pig diet until they had approximately 60 kg liveweight.At this moment six experimental diets were randomly assigned to ten pigs (5 male and 5 females).All pigs were fed ad libitum with the appropriate diet, which were formulated to contain three levels of PUFA and MUFA (but maintaining invariable the concentration of saturated) fatty acids.Ingredients, chemical composition and fatty acids of experimental diets are shown in Table 1.Within each dietary fat treatment, one group was fed a basal level of vitamin E (20 mg α-tocopheryl acetate/kg diet; Hoffman La Roche, Switzerland) and the other group received a supplemented level (200 mg α-tocopheryl acetate/kg diet).The feed offered to the pigs was daily weighted and pig weights and feed consumptions were recorded every 3 weeks.
For the digestibility measurement, a parallel experiment using 30 boars (5 per dietary treatment) was carried out.Pigs weighting approximately 65 kg (thus equivalent to the fi rst experimental period) were introduced in metabolic cages for 10 days and offered ad libitum the experimental diets containing 3 g/kg chromic oxide.After a period of accommodation (5 days), faeces were daily collected and storaged at -20°C until analysis.Analysis were carried out within 8 weeks.

Slaughter, sample collection and chemical analysis
Pigs were slaughtered at a local slaughterhouse at an average weight of 97.3 kg.Feeders were emptied 24 h prior to slaughter.
Proximate composition of the dietary ingredients and faeces was carried out according to the AOAC (1990).α-Tocopherol was extracted from feed as previously described by Rey et al. (2001).The analysis was carried out by HPLC (Hewlett Packard 1050, with a UWD, HPIB 10 detector and a RP-18 end-capped column; Waldbronn, Germany).The eluting solvent was methanol: water (97:3) at a fl ow rate of 2 ml/min.Chromic oxide concentrations in the diet and faeces were determined according to the procedure of Fenton and Fenton (1979).
Fatty acids of diets and faeces were extracted and quantifi ed as by the one-step procedure described by Sukhija and Palmquist (1988) in lyophilized samples.Pentadecanoic acid (C15:1) (Sigma, Alcobendas, Madrid) was used as internal standard.Fatty acid methyl esters were identifi ed by gas chromatography using a 6890 Hewlett Packard (Avondale, PA, USA), gas chromatograph equipped with a fl ame ionization detector and a capillary column HP-Innowax (30 m × 0.32 mm × 0.25 µm cross-linked polyethylene glycol) as described by Rey and Lopez-Bote (2001).Concentration of trans fatty acids in fats used in this experiment (hydrogenated palm fat, lard, olive oil and sunfl ower oil) was quantifi ed by gas chromatography using a 6890 Hewlett Packard (Avondale, PA, USA) gas chromatograph equipped with a fl ame ionization detector and a capillary column (Supelco SP2340 60 × 0,25 mm × 0,2 µm fi lm thickness).The carrier gas (helium) fl ow rate was 2,5 ml/min.The injector and detector temperature were 200°C and the oven was maintained at 200ºC.
Chemical and major fatty acid compositions of experimental diets are shown in Tables 1 and 2. All diets were formulated to contain the same concentration of saturated fatty acids (15±0.2g/kg diet) but differed in the concentration of MUFA and PUFA.Therefore, the estimated unsaturated/saturated (U/S) fatty acid ratio was constant in all cases.This fact allowed us to assume that DE was identical in all experimental diets (Powles et al., 1995).Concentration of trans fatty acids in hydrogenated fat was below 0.3% of total fatty acids.The concentration (g/kg) of C18:2 in the diets was 14±0.4,18±0.4 and 21±1.6, which is above the requirements of this nutrient in growing pigs (NRC, 1998).
Intramuscular neutral and polar lipids were extracted by consecutive solvent elution with dichloromethane and dichloromethane/methanol (90/10 v/v) according to the method proposed by Marmer and Maxwell (1981).Chromatographic conditions were similar to those described for feed.

Statistical analysis
The data were analysed using the general lineal model (GLM) procedure of SAS v.8 (SAS Institute., 1999).Source of added fat (3), dietary vitamin E level (2) and sex (2) were the effects studied.In addition, a linear regression procedure was carried out to quantify the effect of dietary fatty acid concentration on feed conversion ratio (FCR) during the fi rst experimental period (21 days) and intramuscular fat content.Data are presented as the mean and standard error of the mean (SEM).

RESULTS
The effect of dietary treatment on growth, performance and carcass composition is shown in Tables 3 and 4. No effect of dietary fat was observed for weight gain or feed intake, although a signifi cant effect of dietary fat was observed on FCR during the fi rst three weeks of the experiment (P=0.034).The feed containing higher concentration of C18:2 led to a more effi cient feed utilization.To quantify this effect, the following equation was calculated FCR (0-21 days)=2.74(±0.208)-0.03(±0.034) × dietary C18:2 (g/kg feed) (P<0.05)(R 2 = 0.21) (n=10).Moreover, an interaction effect of dietary fat and vitamin E was also observed (P<0.01), in which the effect of dietary C18:2 concentration on feed effi ciency was evident in pigs fed diets containing the basal level of vitamin E, but not in those receiving the supplemented diet (Figure 1).Boars showed a higher weight than gilts from the beginning of the experiment.Also boars showed a higher carcass length (P<0.001) and ham perimeter (P=0.011)(Table 4).A signifi cant effect of dietary fat was also observed for ham perimeter (P=0.044).Since no signifi cant effect of sex was found in chemical composition of muscle Longissimus lumborum, this was removed from the model.
No effect of dietary treatment was observed for dry matter digestibility (Table 5), but differences were found for C16:1, C18:0, C18:1 and C18:2 fatty acid apparent digestibility.Moreover, a signifi cant effect of vitamin E supplementation was observed on C16:1 and saturated fatty acid digestibility.In both cases, apparent digestibility was lower at higher vitamin E supplementation.Apparent digestibility of total saturated fatty acids was lower in pigs receiving a diet containing supplemented level of vitamin E (P=0.042).
No signifi cant effect of vitamin E or dietary treatment was observed on Longissimus lumborum muscle composition (Table 6), except for intramuscular fat content that was higher in pigs receiving a diet containing higher concentration of C18:1 (instead of C18:2).Moreover, a linear effect of dietary MUFA concentration was found, which could be fi tted to the following equation: % in fat=0.35(± 0.199)+0.17(± 0.076) Dietary MUFA (g/kg) (P<0.05)(R 2 = 0.24) (n=10).total saturated (C14:0, C16:0, C18:0, C20:0) 3 total unsaturated (C16:1, C18:1, C18:2, C18:3) 4 total fatty acids (C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0) DISCUSSION Sex differences in weight gain and carcass composition are in agreement with previous researches (Lopez-Bote et al., 1997).The signifi cant effect of dietary fat on FCR in the initial phase of the experiment is in agreement with results from Morgan et al. (1992), who reported a more effi cient feed utilization in pigs fed a diet containing higher concentration of C18:2.A possible explanation for this effect could be due to the higher digestibility or/and different metabolic utilization of PUFA than MUFA to obtain energy, particularly at young ages (Soares and Lopez-Bote, 2001).According to Sanz et al. (2000a,b), saturated fats are used less for yielding energy in broiler chickens than unsaturated fats.On the other hand, Eder et al. (2001) found no effect of altering the oleic to linoleic acid ratio on weight gain and feed utilization in growing pigs.
Considering the whole experimental period, no effect of dietary supplementation with vitamin E was observed in the growth or feed intake of pigs, which is in agreement with Monahan et al. (1992).However, during the initial phase of the experiment (3 weeks), daily gains of pigs fed the vitamin E supplemented diets were lower than in those receiving a basal diet (P=0.042).These results disagree with those of Asghar et al. (1991), who reported improved average daily gain and feed effi ciency during the early phases of growth for pigs supplemented with vitamin E and diets with a high content of PUFA (C18:2) (soyabean oil).According to the results of Table 3, when using pigs of high productivity in a healthy environment, vitamin E supplementation may impair productivity, probably because it interfere absorption of other lipo soluble compounds.On the other hand, the interaction between dietary fat and vitamin E on feed effi ciency during the fi rst experimental period (Figure 1) suggest that high dietary vitamin E concentration may interfere with the absorption or metabolic utilization to obtain energy of PUFA, thus counteracting the effect of C18:2 concentration on pig performance.In order to explain the effect of dietary α-tocopheryl acetate supplementation on fatty acids absorption, a parallel experiment was carried out.Fatty acid digestibility was measured (Table 5).As expected, we found marked differences in the apparent digestibility of individual fatty acids.C18:2 showed higher values than C18:1 and this, higher digestibility than C18:0 and other saturated fatty acids.This is in agreement with literature (Soares and Lopez-Bote, 2001) and may be a possible reason for the higher effi ciency in feed utilization of pigs fed diets containing higher concentration of C18:2.The negative effect of vitamin E supplementation on fatty acid digestibility mainly affects on saturated fatty acids.It might be that tocopherol molecules compete with dietary fatty acids (mostly saturated) for micelle incorporation.We also observed an interaction effect of dietary fat source and vitamin E supplementation on total fatty acid digestibility.An increase in C18:2 concentration produced an increase in total fatty acid apparent digestibility only in the case that a basal level of vitamin E was incorporated in the diet (Figure 2).This result reinforce the hypothesis that dietary vitamin E supplementation may interfere with fatty acid absorption when both vitamin E and C18:2 are included at high dietary concentrations.Meluzzi et al. (2000) reported a reduction in α-tocopherol concentration in egg yolk as the dietary n-3 polyunsaturated fatty acids rises, indicating a possible competition for absorption and/or distribution.Also, King et al. (1995) reported a muscle α-tocopherol lowering effect of dietetic β-carotene.According to these results, vitamin E supplementation in pigs could interfere with fatty acid digestion, particularly at high C18:2 dietary concentration, thus reducing the feed effi ciency during the initial periods of growth.No effect of dietary treatment was observed on carcass characteristics (Table 4), except for ham perimeter (P=0.044), which is an indirect measurement of lean content in the carcass.Therefore, this results indicate a tendency to higher muscle mass in carcasses from pigs receiving diets with higher concentration of C18:2.
The effect of dietary fat composition on intramuscular lipids (Table 6) is a matter of interest because lean meat with a high content of intramuscular fat has better sensory properties and higher palatability than meat with low fat content (Bejerholm and Barton-Gade, 1986).However, as far as the authors are aware, there is little information of the dietary fat type effect on intramuscular fat content.Miller et al. (1990) and Madsen et al. (1992) reported a signifi cant effect of dietary fatty acids on intramuscular fat content, but the relationship between concentration of individual fatty acids and intramuscular fat concentration seems complicated.On the other hand, Eder et al. (2001) found no effect of altering the dietary oleic to linoleic fatty acid ratio on intramuscular lipids, although they utilized a different experimental design than the one used in this experiment, and varied the dietary concentration of short chain saturated fatty acids, which could have a possible effect also.Enhanced fat catabolism and reduced fat synthesis have been reported to occur in rats (Shimomura et al., 1990) and broiler chickens (Sanz et al., 2000a) fed PUFA enriched diets when compared to rats fed diets enriched with MUFA or saturated fatty acids.Moreover, Sanz et al. (2000b), recently evidenced in broilers that blood circulating PUFA are preferentially taken up by muscle tissue as an immediate energy source rather than being stored in intramuscular lipids.
In conclusion, a higher MUFA/PUFA ratio in pig diets may produce a decrease on fatty acid digestibility, a higher feed conversion ratio (FCR) during the initial period of fattening, and a higher concentration of intramuscular fat at slaughter.Vitamin E supplementation may affect mainly the apparent digestibility of saturated fatty acids, and total fatty acids (P=0.062) when diets have higher C18:2 proportion.

Figure 1 .
Figure 1.Effect of dietary C18:2 concentration on feed conversion ratio (FCR) in pigs fed a basal ( ) or supplemented ( ) level of vitamin E during the fi rst three weeks of the experiment

Figure 2 .
Figure 2. Effect of dietary C18:2 concentration on total fatty acid apparent digestibility in pigs fed a basal ( ) or supplemented ( ) level of vitamin E

TABLE 1
Ingredients and chemical composition of experimental diets 1 (with a high concentration of monounsaturated fatty acids (19 g/kg dry matter, mono), a high concentration of polyunsaturated fatty acids (21.3-23.2g/kg,poly) or an intermediate concentration(14.7-16.2g monounsaturated and 19.0-19.6

TABLE 2
Fatty acid composition of experimental diets (with a high concentration of monounsaturated fatty acids (19 g/kg dry matter, mono), a high concentration of polyunsaturated fatty acids (21.3-23.2g/kg, poly) or an intermediate concentration (14.7-16.2g monounsaturated and 19.0-19.6 g polyunsaturated fatty acids/kg, medium), g per kg DM cant effect of vit.E, fat × vit.E, fat × sex, vit.E × sex and fat × vit.E × sex was observed in any case 2 not signifi

TABLE 6
Longissimus lumborum composition (g/100 g weight) from pigs fed on diets with a high concentration of monounsaturated fatty acids (19 g/kg dry matter, mono), a high concentration of polyunsaturated fatty acids (21.3-23.2g/kg, poly) or an intermediate concentration (14.7-16.2g monounsaturated and 19.0-19.6 g polyunsaturated fatty acids/kg, medium), in each case with a basal (20 mg/kg diet) or supplemented level (200 mg/kg diet) of vitamin E