Effects of dietary n-6:n-3 PUFA ratios on growth performance
Previous studies have shown that replacing part of the metabolizable energy content of a diet with different fat mixtures does not change the energy content or nutritional value of the diet so does not affect the growth performance and carcass quality of finishing pigs [11, 12]. It has been reported that the absence of a positive effect on growth performance when increasing dietary n-3 PUFA intake may be caused by similar dietary metabolizable energy contents [8]. However, the results of the present study indicated that dietary n-6:n-3 PUFA ratios of 3:1 to 5:1 improved growth performance, which agreed with previous reports showing that a diet with n-6:n-3 PUFA ratios of 2.5 to 5:1 improved the growth performance and health of animals [13, 14]. We also observed that pigs fed the diet with an n-6: n-3 PUFA ratio of 2:1 exhibited a lower ADG and higher FCR than the other groups. This could be explained by the higher content of n-3 PUFA being oxidized preferentially over n-6 PUFA for energy production, therefore decreasing growth performance. The reasons for these inconsistent results may be related to feed ingredients, genetics or the breed of the pigs. Differences in the type of n-3 PUFA, dosage and duration may also have contributed to differences in outcomes.
Effects of dietary n-6:n-3 PUFA ratios on serum lipid and adipokine profiles
Blood TC levels reflect the nutritional status of animals and are positively associated with adiposity [15], while HDL levels are inversely correlated with the risk of coronary artery disease because HDL partially inhibits the uptake and degradation of LDL, and suppresses LDL-induced changes in sterol level [16]. Therefore, it is generally believed that lower concentrations of blood TG, TC and LDL-C and higher concentration of blood HDL-C improve the health of humans and animals [17]. The n-3 PUFA are considered to increase HDL-C and decrease TG by increasing the synthesis of HDL and clearance of TG [18, 19]. As expected, lower serum TG and TC levels and higher HDL-C:LDL-C ratios were observed in pigs fed the diet with lower n-6:n-3 PUFA ratios compared with those in pigs fed a diet with a higher n-6:n-3 PUFA ratio. This agreed with the observations of previous studies, where blended oils with lower n-6:n-3 PUFA ratios decreased the serum concentrations of TG, TC and LDL-C and increased the concentration of HDL-C [20, 21]. It is reported that dietary n-6:n-3 PUFA ratios of 1:1 and 5:1 facilitated the absorption and use of FA and exerted beneficial effects on lipid metabolism in pigs [14]. In terms of the mechanisms underlying the hypolipidemic effects of n-3 PUFA, it can be presumed that n-3 PUFA-mediated reductions in the concentration of the circulating TG included inhibiting hepatic TG synthesis and secretion, reducing the intestinal and hepatic apolipoprotein B species, which are responsible for TG elimination, and increasing LPL activity [8]. The hypolipidemic effect of dietary n-3 PUFA is also associated with the stimulation of FA oxidation in the liver and, to a smaller extent, in skeletal muscle, which may ameliorate dyslipidemia, tissue lipid accumulation and insulin action.
Several secretory adipokines such as LEP, APN and IL-6 are synthesized in adipose tissue. LEP is expressed and secreted in proportion to the number and size of adipocytes and circulates in the body at a concentration highly correlated with the mass of body fat and participates in the regulation of food intake and energy expenditure. Studies have shown that n-3 FA intake is negatively correlated with changes in plasma LEP concentration [22]. Our results showed that the serum concentration of LEP decreased significantly with a decreasing dietary n-6:n-3 PUFA ratio. Similar effects of the n-6:n-3 PUFA ratio on LEP have also been observed in rats [22] and mice [23]. However, other studies on rodents and cultured adipocytes have shown that n-3 PUFA (EPA) increases the production of LEP [24, 25]. Studies assessing the role of n-3 PUFA in weight maintenance have found no significant effect on weight or blood LEP concentrations between n-3 PUFA supplemented subjects [26, 27]. The n-3 PUFA mediated effects on LEP depend on a number of factors, such as diet composition and energy balance, which could thus cause conflicting results. Physical activity is also important for blood LEP concentrations, but independent of fat mass [28].
IL-6, like the tumor necrosis factor -α (TNF-α), is also an important pro-inflammatory cytokine that can stimulate T and B lymphocyte proliferation and initiate the production of several inflammatory mediators, thus leading to an inflammatory response. IL-6 also antagonizes anabolic growth factors and thus suppresses growth [29]. Studies have revealed that most n-3 PUFA are anti-inflammatory and decrease the production of these inflammatory cytokines, whereas n-6 PUFA are pro-inflammatory and increases their production [30, 31]. The present study showed that diets with a lower n-6:n-3 PUFA ratio (2:1) could inhibit the production of IL-6, agreeing with the results of previous studies, which reported that the serum concentrations of IL-6 decreased with decreasing n-6:n-3 PUFA ratio (from 20:1 to 1:1) [13, 32]. In humans and in vitro, n-3 PUFA have also been reported to reduce cytokines, including IL-6 and TNF-α [33], which are both elevated in obesity. The anti-inflammatory effects of n-3 PUFA may help to protect against atherosclerosis and mortality related to cardiovascular diseases [29].
APN is one of the most abundant adipokines and plays a central role in lipid metabolism, exerting protective effects with its insulin-sensitizing, antiatherogenic and anti-inflammatory properties [34]. Studies have also demonstrated that the serum concentration of APN is negatively correlated with body mass index, type 2 diabetes and cardiovascular diseases [35, 36]. In the present study, lower n-6:n-3 PUFA ratios (2:1 and 3:1) promoted the production of APN. Mice tests have found that as the n-6:n-3 PUFA ratio decreases, the serum concentration of APN increases in a dose-dependent manner and also in a PPARγ-dependent manner [37, 38]. Human studies have found significant increases in human blood concentration of APN following the consumption of n-3 PUFA regardless of body weight, leading to speculation that the anti-inflammatory properties of n-3 PUFA may induce an increase in adipocyte APN production [39, 40]. It has also been demonstrated that n-3 PUFA may influence the concentration of APN directly by interacting with transcription factors, or indirectly via unknown mechanisms linked to FA oxidation, synthesis or storage [13, 22].
Effects of dietary n-6:n-3 PUFA ratios on tissue FA composition
FA in tissue play an important role in the flavor, nutritional value and oxidative stability of meat [41]. Therefore, improving FA composition has a vital role in producing high-quality pork meat with a superior nutritional value for human consumption. The FA composition of pork is largely determined by the FA digested by the animal and reflects long-term intake [42]. The relative proportions of these FA are reflected in the FA composition of the deposited fat. Previous studies have confirmed the importance of the n-6:n-3 PUFA ratio and the view that decreasing the ratio leads to increasing the n-3 PUFA content and the protection against obesity and related diseases such as inflammation, hepatic steatosis, coronary heart disease, and cardiovascular diseases [43]. In the present study, as expected, the C18:3n-3 concentration of the LM and SCAT samples was significantly enhanced with decreasing dietary n-6:n-3 PUFA ratios, while the C18:2n-6 and n-6:n-3 PUFA ratios decreased correspondingly. The concentrations of most MUFA were higher in pigs fed a diet with an n-6:n-3 PUFA ratio of 2:1 than in pigs of the other groups. This can be explained by the linseed oil used in the present study being characterized by a high n-3 PUFA content, especially C18:3n-3, which represents about 45–55% of total FA, a moderate MUFA content and low SFA content, whereas soybean oil contains high C18:2n-6 content. Studies have confirmed that reducing dietary n-6 and elevating n-3 PUFA are highly successful at raising the quantities of 18:3n-3 and the n-3 long-chain PUFA in pork, thus supplying valuable n-3 PUFA to the human diet [8]. It has been reported that adding oils rich in n-3 PUFA to pig feed can increase the deposition of these FA in the meat [11, 44]. C18:2n-6 and C18:3n-3 are converted into their derivative n-6 or n-3 long-chain PUFA through Δ6 and Δ5 fatty acyl desaturase (Fad) and elongations [16], respectively. The present study and previous publications suggest that diets with a higher proportion of n-3 PUFA ratio decrease the conversion of C18:2n-6 to n-6 long-chain PUFA, because the enzymes act preferentially on n-3 PUFA. In contrast, diets with a higher n-6 PUFA have adequate Δ6 Fad activity thus ensuring the elongation of C18:2n-6 and their conversion into long-chain PUFA.
Effects of dietary n-6:n-3 PUFA ratios on gene expression of tissue
Lipogenesis and lipolysis are major factors affecting fat accumulation in tissues. Dietary supplementation with fat or FA can modify the FA composition of tissues by altering the expression of fat metabolism genes [13]. FA or their derivatives may interact with nuclear receptor proteins that bind to certain regulatory regions of DNA and thereby alter the transcription of the target genes [22]. Both n-3 and n-6 PUFA can bind and/or regulate transcriptional factors that control genes involved in pre-adipocyte differentiation. PPARγ, a member of the ligand-activated PPAR subfamily of nuclear hormone receptors, is highly expressed during the late stage of white adipocyte differentiations. The PPARγ signaling pathway is mainly associated with lipid metabolism, dipocyte differentiation, thermogenesis and gluconeogenesis via the modulation of a number of target genes. When PUFA are added to the diet, the main effect of PPARγ on glucose metabolism is to increase the activity of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) involved in TG synthesis rather than the induction of de novo lipogenesis [45]. Furthermore, PPARγ senses incoming non-esterified long-chain PUFA and regulates the genes for long-chain PUFA oxidation then induces the pathways to store long-chain PUFA as TG during fasting and endurance exercise in skeletal muscle. Thus, the decrease in PPARγ expression of the LM and SCAT samples in the present study may be consistent with the trend towards lower concentrations of serum TG, TC with decreasing dietary n-6:n-3 PUFA ratios. This indicates that n-3 PUFA can suppress the transcription of lipogenic genes and lipogenesis by functioning as natural ligands for PPARγ [46]. However, the present study showed that an n-6:n-3 PUFA ratio of 5:1 rather than 8:1 led to the highest expression level of PPARγ, suggesting that an optimal n-6:n-3 PUFA ratio can promote adipogenesis and a healthy expansion of adipose tissue during positive energy balance, and a metabolically healthy phenotype. Studies on clonal adipocytes (3T3-L1) have also demonstrated the up-regulation of PPARγ expression, adipogenesis and lipid droplet formation after supplementation with n-3 PUFA [47].
aP2 and LPL are two important target genes in the PPARγ signaling pathway and play important role in the transport of FA [48, 49]. aP2 is secreted from both macrophages and adipocytes, functions as an adipokine, and participates in FA uptake and the transport process of intramuscular adipocytes [50]. LPL is the enzyme mainly responsible for the hydrolysis of TG from triacylglycerol-rich lipoprotein particles from cycling and releasing free FA for muscle uptake [51], so is responsible for fat mobilization and deposition of muscle. In the present study, a lower n-6:n-3 PUFA ratio down-regulated the expression level of LPL in both the LM and SCAT samples, as well as aP2 in the SCAT sample, indicating that a higher proportion of n-3 PUFA could stimulate FA β-oxidation resulting in a lower synthesis of TG and non-esterified fatty acid (NEFA) levels [18]. However, the n-6:n-3 PUFA ratio only affected the expression level of aP2 in the SCAT and not the LM samples. This may have been because aP2 is an adipocyte-specific gene which is known to be a later marker of adipogenesis, whereas LPL is widely expressed in most cell types, not just functioning in adipocytes [52]. Similar studies have reported that the gene expression levels of LPL and aP2 were negatively correlated with concentrations of n-3 PUFA. We can thus speculate that a lower n-6:n-3 PUFA ratio could reduce the amount of FA being delivered to the muscle by decreasing the transcription of genes involved in the muscle [53]. However, these results were not consistent with the previous study where LPL and aP2 gene expression levels increased with the content of n-3 PUFA in the muscle of pigs [54]. In addition, another study has reported that the LPL expression level increased with the concentrations of C16:1 and C18:1, but decreased with those of C16:0 and C18:0 [49]. It therefore seems that the LPL expression level was associated with not only the concentrations of PUFA but also with those of MUFA and SFA. These results again indicate that the composition of dietary FA is a factor influencing the genes regulating the endogenous synthesis of FA.
HSL is an intracellular enzyme that catalyzes the hydrolysis of TG in adipose tissue and regulates the release of NEFA from lipid stores [55]. It has been reported that blood TC level increased markedly in HSL-deficient animals [16]. In the present study, compared with higher dietary n-6:n-3 PUFA ratios, a lower dietary n-6:n-3 PUFA ratio increased the expression level of HSL in the LM sample, but decreased it in the SCAT sample, suggesting that the fat in the LM required a lower n-6: n-3 PUFA ratio for lipid hydrolysis. In contrast, the fat in the SCAT, with its higher lipid content and higher capacity for lipogenesis, required a higher n-6:n-3 PUFA ratio for hydrolysis, which was possibly related to metabolic specificities of the different tissues [56]. It has also been reported that a lower dietary n-6:n-3 PUFA ratio was more effective for increasing the expression of lipoclastic genes HSL in both LM and SCAT samples [14]. In contrast, regulation of HSL may be affected by the type of n-3 PUFA, as well as by the fat depot. For example, the expression level of HSL in retroperitoneal fat was reported to decrease on supplementation with DHA or a mixture of EPA and DHA but not with EPA supplementation alone [57].
In the present study, the effects of a higher n-6:n-3 PUFA ratio on genes involved in lipid metabolism were similar to those of a lower n-6:n-3 PUFA ratio. In a similar study in rats, it has been demonstrated that PPARγ expression was down-regulated by either high n-6 PUFA (soybean oil) or high n-3 PUFA (linseed oil) [58]. Studies have shown that an elevated intake of n-3 PUFA decreased tissue levels of n-6 PUFA, and vice versa. However, it is worth noting that diets adequate in n-3 PUFA but extremely deficient in n-6 PUFA significantly decreased the whole-body utilization of not only n-3 PUFA but also n-6 PUFA [59]. This suggests that n-3 PUFA can reduce the requirement for n-6 PUFA or act synergistically with n-6 PUFA. Therefore, a balanced n-6:n-3 PUFA ratio may be more important than the intake levels of n-3 PUFA for lipid synthesis and catabolism, lipid homeostasis, and thus preventing obesity and inflammation related diseases [14]. In the present study, the optimal n-6:n-3 PUFA ratios are 3:1 and 5:1, which are also close to those commonly recommended [20]. These ratios could provide sufficient PUFA to ensure adequate energy and nutrients for lipid homeostatic metabolism and high growth performance. In contrast, the gene transcriptional regulation of FA synthesis and catabolism could be more long-term and correlated with specific tissues as well as with the enrichment of tissues with n-3 and n-6 PUFA. Dietary FA may affect lipid metabolism through many potential mechanisms, such as regulating transcriptional genes, gene interactions with different signaling pathways and the activities of proteins and enzymes. [60]. Therefore, further research is need to determine the optimal n-6:n-3 PUFA ratio for growth, immunity, lipid metabolism and gene expressions and to elucidate the underlying metabolic pathways and mechanisms.