Effects of dietary vitamin E on muscle vitamin E and fatty acid content in Aohan fine-wool sheep

Background Increasing the polyunsaturated fatty acid (PUFA) content and decreasing the saturated fatty acid (SFA) content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid (FA) composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep. Methods Forty-two male Aohan fine-wool lambs (5 mo old) with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 (control group), 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the longissimus lumborum (LL) and gluteus medius (GM) muscles. Results Vitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation (P < 0.05). However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA (MUFA) content in the LL and GM (P < 0.05). All six doses of vitamin E significantly increased cis9 trans11-conjugated linoleic acid (c9t11-CLA) content in the LL compared with the control group (P < 0.05). Conclusions Dietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9t11-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.


Introduction
In recent years, an increase in consumer interest in the nutritional aspects of health has resulted in the development of specific health recommendations for food components, especially of dietary fat [1]. The relationship between dietary fat and the incidence of lifestyle diseases, particularly cardiovascular disease and atherosclerosis, is well established. It is recommended that total fat and saturated fatty acids (SFAs) should not exceed 35% and 10% of the total dietary intake, respectively. Additionally, the ratio of polyunsaturated fatty acids (PUFAs) to SFAs (P/S) should be around 0.45, while that of n-6 to n-3 PUFAs should be < 4 [2]. Meat and meat products are important dietary components that provide a major source of macronutrients [3]. Therefore, increasing the PUFA content and decreasing the SFA content in meat may help to improve the nutritional value of this food type to consumers.
Some authors have demonstrated that the fatty acid (FA) profile of meat can be modified by oxidative processes and that the oxidative stability of lipid fractions was associated with their FA composition [4,5]. The susceptibility of unsaturated FAs (UFAs) to oxidation is related to the degree of unsaturation of FAs, because PUFAs are more prone to oxidation than monounsaturated FAs (MUFAs) [6]. It was also demonstrated that lipid oxidation could reduce the content of essential PUFAs (C18:2n-6 and C18:3n-3) and long-chain PUFAs (C20:5n-3 and C22:6n-3); however, oxidation of these PUFAs may be reduced by increasing the availability of natural antioxidants that could delay or inhibit oxidation [7].
Vitamin E is a major antioxidant that protects tissues from oxidative damage. It is deposited in the cell membrane where it protects the membrane PUFAs from oxidation [8]. This protective effect is carried over into the meat and can be enhanced by feeding animals with vitamin E at a level greater than that required for normal growth and reproduction. Demirel et al. reported that high dietary vitamin E supplementation provided additional protection against lipid peroxidation by increasing PUFA content and decreasing monoenoic FA content in muscle, liver, and adipose tissue [9]. Morel et al. also found that dietary vitamin E could reduce lipid oxidation in pork and in processed pork products [10]. It was also reported that vitamin E could stabilize PUFAs and was a major determinant of meat quality, particularly in ruminants [11].
Although the effects of dietary vitamin E on meat quality have been extensively studied in lambs [8,12,13], it is still necessary to determine the optimum tissue concentration for improving meat quality. The efficiency of turning dietary vitamin E into muscle vitamin E is influenced by several factors, including the dietary concentration, muscle type, and the type of vitamin E being used [14]. For these reasons, the muscle vitamin E content is highly variable leading to inconsistencies among prior studies.
Although many researchers have investigated the effects of dietary vitamin E supplementation on meat quality in meat sheep, very few studies have examined the effects of vitamin E supplementation on muscle vitamin E content in wool sheep or evaluated the changes in FA profiles, even though wool sheep are an important source of mutton. Therefore, the objectives of the present study were: 1) to examine the effects of different dietary vitamin E concentrations on muscle vitamin E content; and 2) to determine the relationship between muscle vitamin E content and FA composition in male Aohan fine-wool sheep, a common fine-wool sheep breed in China.

Animals and management
Aohan fine-wool sheep, which were initially bred in the Inner Mongolia Autonomous Region of China, are well known for their high-grade fleece weight (16.2 kg), staple length (7.5-9.8 cm), fiber diameter (21.6-25 μm), and exceptional meat quality. We purchased 42 male Aohan lambs (5 mo old) with similar initial body weights from the Aohan fine-wool sheep breeding farm (Chifeng, Inner Mongolia Autonomous Region, China). The sheep were initially fed a diet with a forage/concentrate ratio of 6/4. The formulation developed according to the National Research Council (NRC) feeding standard is shown in Table 1 [15]. The animals were housed individually, had free access to fresh, clean water, and were fed ad libitum.
Three sheep of each group were randomly selected for slaughter at the end of the feeding period and samples of the longissimus lumborum (LL) and gluteus medius (GM) muscles were collected and stored at −20°C to measure vitamin E and FA content.
All of the animal procedures were conducted with approval from the China Agricultural University Animal Care and Use Committee.

Chemical analysis
Muscle vitamin E content was measured using a commercially available vitamin E assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The principal of this kit of that, in the presence of phenanthroline, Fe 3+ is reduced to Fe 2+ by vitamin E. Phenanthroline then forms a complex with Fe 2+ forming a colored adduct. Vitamin E content can be calculated using the following equation: where OD U = absorbance of the assay tube, OD S = absorbance of the standard tube, OD B = absorbance of the blank tube, C S = standard concentration, and N = dilution ratio. Muscle FA content was analyzed by gas chromatography according to the method described previously [17]. Briefly, the LL and GM were freeze-dried and 300 mg of each sample was placed into tissue culture tubes fitted with a rubber stopper and screw cap. Next, 4 mL of methanol: chloroacetyl solution (v/v 10:1) and 5 mL of normal hexane solution (1 mg/mL) were added to each tube. The normal hexane solution was prepared as follows: 1 g of nonadecanoic acid (Sigma, St. Louis, MO, USA) was transferred to a 1,000 mL volumetric flask and the flask was filled to a specified volume with normal hexane. The flask was covered tightly with the rubber stopper and screw cap to prevent gas leakage and was incubated in a water bath for 2 h at 80°C. After cooling to room temperature, 5 mL of 7% potassium carbonate was added, the flask was thoroughly mixed and left to stand. After the formation of distinct layers in the solution, the upper layer was used for FA analysis. FA methyl esters were analyzed by gas chromatography (HP6890) using a capillary column (HP-INNOWAX [19091N-213, 60.0 m × 320 μm × 0.5 μm], Agilent, Santa Clara, California, America). Gas chromatography was performed using the following conditions: the temperature was held at 220°C for 10 min and then increased to 250°C at a rate of 10°C/min; the temperature of the FID detector was set at 280°C; the split flow ratio was 20:1; the injection volume was 1 μL; the injection temperature was 250°C; and nitrogen (3.0 mL/ min) was used as the flow gas.

Statistical analysis
The effects of dietary vitamin E content on muscle vitamin E and FA contents were analyzed by one-way analysis of variance followed by Duncan's new multiple range test. We used SAS 9.1 software (SAS Institute, Cary, NC, USA) for all analyses. Results are expressed as the mean ± standard error (SE). FA content was compared between the two muscles using paired t tests. Differences at P < 0.05 were considered statistically significant.

Food intake and muscle vitamin E concentrations
The DMI and nutrient intake are shown in Table 2; there were no differences among the seven groups in these factors (P > 0.05). The muscle vitamin E levels are presented in Table 3. Muscle vitamin E concentrations were significantly increased by dietary vitamin E supplementation. Interestingly, the increase was not dosedependent, because vitamin E levels were highest in the E200 group in the LL and GM. In the LL, vitamin E concentrations in the E200, E1,000, and E2,400 groups were significantly higher than that in the control group (E0). Vitamin E content was also significantly higher in the E200 group than in the E20 and E100 groups (P < 0.05). In the GM, the vitamin E concentration was significantly higher in the E200 group than in the other groups (P < 0.05) except for the E1,000 and E2,400 groups. Tables 4 and 5 show the muscle SFA composition in the LL and GM, respectively. In the LL, the total SFA content was significantly lower in the vitamin E-supplemented groups than in the control group (P < 0.05). In terms of individual SFAs, we found significant differences in C12:0, C14:0, C16:0, C18:0, and C20:0 FAs (P < 0.05) ( Table 4). Of note, the total SFA content was lowest in the E200 group, mainly because of the lower levels of C12:0, C14:0, C16:0, C18:0, and C20:0 FAs. Similarly, the ratio of C14:0 +C16:0 was also significantly lower in the vitamin Esupplemented groups, except in the E2,000 group, than in the control group (P < 0.05).

Muscle SFA composition
In the GM, the total SFA content was significantly lower in the vitamin E-supplemented groups, except for the E20 group, than in the control group (P < 0.05). The total SFA content was lowest in the E200 group. Regarding individual SFAs, the C10:0 content was significantly lower in the vitamin E-supplemented groups, except in the E1,000 and E2,400 groups, than in the control group (P < 0.05). The C16:0 content was also significantly lower in each vitamin E-supplemented group than in the control group (P < 0.05) ( Table 5). The relative C10:0 and C16:0 contents were lower in the E200 than in the other groups. As in the LL, the ratio of C14:0+C16:0 FAs in the GM were significantly lower in the vitamin E-supplemented groups than in the control group (P < 0.05), and were lowest in the E200 group.

Muscle UFA composition
The UFA profile of the LL is shown in Table 6. The total MUFA content was significantly higher in each vitamin Esupplemented group than in the control group (P < 0.05), which was mainly due to the significantly higher levels of C14:1, C16:1, C18:1n9, C18:1n7, and C20:1 in the vitamin E-supplemented groups (P < 0.05) ( Table 6). The index (C18:0+C18:1):C16:0 was significantly increased by vitamin E supplementation (P < 0.05), except in the E2,000 group, and was highest in E200 group. Unexpectedly, vitamin E supplementation did not affect the total PUFA content, or the n-6 and n-3 PUFA content (P > 0.05). For individual PUFAs, only cis9 trans11-conjugated linoleic acid (c9t11-CLA) content was significantly increased by vitamin E supplementation, except in the E20 group, compared with the control group (P < 0.05). c9t11-CLA content was highest in the E200 group. Vitamin E supplementation significantly increased the MUFA:SFA ratio (P < 0.05) but not the PUFA:SFA ratio (P > 0.05).
The UFA profile of GM is presented in Table 7. Vitamin E supplementation significantly increased the total MUFA content, mainly because of changes in C18:1n9 (P < 0.05). The (C18:0+C18:1):C16:0 ratio was significantly higher in the E200 group than in the control group (P < 0.05). However, there were no significant differences in total PUFA, individual PUFA, or in the MUFA:SFA and PUFA:SFA ratios in the GM (P > 0.05).
Finally, we compared the effects of dietary vitamin E supplementation on FA deposition on FA concentration between the GM and LL. However, we found no significant differences in any of the FA parameters between the two muscles (P > 0.05).

Muscle vitamin E concentrations
Previous studies have demonstrated increases in tissue α-tocopherol concentrations following dietary vitamin E supplementation in lambs [18,19]. Liu et al. fed steers with diets supplemented with 250, 500 or 2,000 mg all-racα-tocopherol acetate/calf/d and reported that the mean muscle α-tocopherol concentrations were 1.39, 2.27 and 4.95 μg/g, respectively, in the LL, semimembranosus, and GM [20]. Lauzurica et al. reported that α-tocopherol concentrations in meat increased with increasing dietary vitamin E content [21]. Kasapidou et al. also reported that the muscle α-tocopherol content increased with increasing dietary vitamin E content, and that muscle vitamin E concentrations were positively correlated with dietary vitamin E levels [8,13]. In our study, muscle vitamin E content increased significantly with increasing dietary vitamin E content, although the relationship was not dose-dependent because the muscle vitamin E content in the LL and GM was greatest in the E200. Muscle vitamin E content was lower in the E1,000, E2,000, and E2,400 groups than in E200, probably because the uptake and deposition of vitamin E were saturated at E200. It has been reported that, when animal diets are supplemented with 'supranutritional' vitamin E levels, the effects of dietary vitamin E on its tissue content diminishes [13]. Slow hydrolysis was also reported as a cause of low plasma tocopherol levels in animals fed supranutritional levels of α-tocopherol acetate [22].
The duration of dietary supplementation is another factor that influences α-tocopherol deposition. In general, greater dietary vitamin E content and/or longer supplementation time are associated with higher α-tocopherol concentrations in meat [23]. The muscle vitamin E content in our study was higher than that in other studies, possibly because our study was conducted over 12 mo or because we used a different animal model. Álvarez et al. reported lower semimembranosus vitamin E concentrations in younger lambs fed a diet supplemented with vitamin E for only 37 d [12]. However, in the present study, muscle vitamin E levels were highly variable among sheep in the same group (Table 3). Wide variability was also   [8].
The small number of animals used in these studies might also contribute to the high variability in muscle vitamin E content [26]. Nevertheless, greater understanding of the mechanisms controlling the uptake and metabolism of vitamin E is necessary to explain the differences observed among prior studies.

Muscle FA profile
SFAs are implicated in the etiology of several diseases, and there is a strong positive correlation between SFA  intake and the rate of coronary artery heart disease [27]. Grundy and Denke also reported that high dietary levels of long-chain SFAs increase plasma cholesterol level whereas high levels of MUFAs and PUFAs do not [28]. However, not all SFAs have equivalent effects. It was suggested that C14:0 and C16:0 exerted differential, dose-dependent effects on cholesterol and lipoprotein metabolism, even in animals fed low-cholesterol diets, while C18:0 did not appear to have such an effect [29]. It was also reported that high concentrations of palmitic acid (C16:0) were toxic to mitochondria [30] and that the concentration of stearic acid (C18:0) was positively correlated with the odor and flavor intensity of mutton [31]. In this study, the C16:0, C14:0+C16:0, and total SFA levels in the LL and GM, and C12:0, C14:0, and C18:0 levels in the LL were significantly lower in the vitamin E-supplemented groups than in the control group (P < 0.05), which is consistent with the results of other studies [7,32]. In our previous study, 24 Boer male goats were fed diets supplemented with 0, 80, 320 and 880 IU/goat/d vitamin E for 5 mo, which tended to decrease the C18:0 and total SFA levels, especially in the 320 IU/goat/d group [33]. However, our results did not agree with those of another study in which vitamin E supplementation (45 mg/lamb/d) significantly increased intermuscular fat stearic acid (C18:0) content but did not affect other FAs [34]. Salvatori et al. also studied the effects of vitamin E supplementation on FA composition of lamb meat and found no differences between the dietary supplementation groups [35]. The reasons for these discrepancies remain unclear, and warrant further studies. In our study, muscle total MUFA and C18:1n9 contents were significantly higher in the vitamin E-supplemented groups than in the control group (P < 0.05). The higher tissue MUFA content might be partially related to the activation of their biosynthesis in the Δ 9 d-catalysed reaction. Δ 9 d also catalyzes the tissue synthesis of c9t11-CLA from trans11-C18:1 (t11-C18:1) [36]. In this study, Δ 9 d activity was estimated by calculating the product/substrate ratios of Δ 9 d-catalysed reactions (i.e. MUFA/SFA and c9t11-CLA/t11-C18:1) [37]. The MUFA/SFA ratio in the LL was higher in the vitamin E-supplemented groups, indicating changes in Δ 9 d activity; however, we found no differences in the muscle c9t11-CLA/t11-C18:1 among the experimental groups. The vitamin E-supplemented diet significantly affected the MUFA/SFA index but not the c9t11-CLA/t11-C18:1 index, which is somewhat surprising because both indices should reflect Δ 9 d activity. A possible explanation for this inconsistency is that CLA formation and MUFA biosynthesis might be catalyzed by different Δ 9 d isoforms that are regulated by different mechanisms [36]. Other animal species were reported to express more than one Δ 9 d isoform [38], but the number of Δ 9 d isoforms in ruminant tissues has not been clarified. To date, only one isoform has been identified in cattle and sheep [39]. However, in a recent study using bovine tissues, two Δ 9 d-immunoreactive bands at close molecular weights were found, which might indicate the presence of more than one Δ 9 desaturase proteins [36]. Total MUFA content in the GM was also higher in the vitamin E-supplemented groups because of the higher MUFA/SFA ratio, although the differences among the experimental groups were not significant (P = 0.0556). As in the LL, dietary vitamin E supplementation did not affect the c9t11-CLA/t11-C18:1 index in the GM. Bonanome and Grundy reported that only C16:0 increases blood cholesterol levels, whereas C18:0 does not, and C18:1 decreases blood cholesterol levels [40]. Because these molecules represent the majority of FAs, the ratio of (C18:0+C18:1):C16:0 could help to evaluate the possible clinical effects of different lipids. As shown in Tables 6 and 7, the (C18:0+C18:1):C16:0 ratio in the LL was significantly increased by vitamin E supplementation (P < 0.05), and was significantly higher in the E200 group than in the control group in the GM (P < 0.05).
We found no significant differences in muscle PUFA content between the vitamin E-supplemented groups and the control group (P > 0.05). These findings are consistent with those reported by Kasapidou et al. who found that vitamin E levels did not affect phospholipid content in the muscle or FA composition, and that low vitamin E levels were sufficient to protect lipids from oxidation in sheep [8,13]. However, in other studies, PUFA content in meat was affected by vitamin E. For example, it was reported that lambs with muscle vitamin E concentrations of 0.27 mg/g had lower PUFA content in muscle than lambs with muscle vitamin E concentrations of 0.52 mg/g [9]. Álvarez et al. also reported that increasing the concentration of α-tocopherol in meat via dietary supplementation protected against PUFA oxidation [7]. The differences in findings reported to date suggest to us that further studies are needed to better understand how vitamin E affects the PUFA composition in meat. Regarding individual PUFAs, only c9t11-CLA content in the LL was significantly higher in the vitamin E-supplemented groups than in the control group (P < 0.05). This is consistent with the findings reported by Gabryszuk et al. who found that dietary supplementation with Se, Zn, and vitamin E improved the lipid profile and increased the concentration of CLA in the meat of Polish Merino ram-lambs [41]. Chen et al. also reported that diets supplemented with soybean oil and vitamin E increased the c9t11-CLA content in lamb meat [42].
It was notable that the dietary vitamin E content of 200 IU/sheep/d seemed to be the best dose in this study because it achieved the highest muscle vitamin E content, the lowest SFA content, and the highest MUFA and c9t11-CLA contents. In our previous study, we found that semen quality, as well as the antioxidant capacity of the testicular cell membrane and mitochondria of Aohan fine-wool sheep showed the greatest improvements when the diet was supplemented with 200 IU/sheep/d vitamin E [16]. These results suggest that this level of vitamin E supplementation has particularly beneficial health effects and provides the basis for future studies aimed at investigating the benefits of dietary supplementation with this antioxidant in sheep.

Conclusions
Overall, the present study showed that dietary vitamin E supplementation increases muscle vitamin E content in sheep. Dietary vitamin E also reduced SFA content and increased MUFA and c9t11-CLA contents in the muscle of Aohan fine-wool sheep. These effects of vitamin E were greatest in sheep fed a diet supplemented with 200 IU/sheep/d vitamin E.