Practical Dietary Approaches to Increase Longissimus Intramuscular Fat Content and Their Influence on Glycolytic Potential and Pork Quality Characteristics

Introduction

In the pork industry, the trend towards producing lean carcasses is still occurring, concurrently with a demand from certain specific markets for pork products with high intramuscular fat (IMF) in an attempt to ensure palatability. A positive effect of IMF content on the eating quality of pork has been reported by a number of authors (Castell, 1994; Fernandes et al., 1999; Brewer et al., 2001). Fernandez et al. (1999) stated that pork texture and taste are enhanced at IMF levels up to 3.25%. Similarly, Castell et al. (1994), and Brewer et al. (2001) found that highly marbled chops (~3.5% IMF) were juicier, and more tender, and flavorful than leaner chops (~1.0% IMF). Hence, a minimum level of IMF content to improve eating quality, ranging from 2.5 to 3.5%, has been proposed (DeVol et al., 1998; Murray, 2002). However, intensive genetic selection of pigs for high carcass lean content has resulted in a reduction in the IMF content of pork, leading to the perception that it is now tougher, drier, and has reduced flavor (D’Sousa et al., 2004). On the other hand there are reports of little or no association between the IMF content of pork and eating quality characteristics. For example, Ellis et al. (2004) reported that the positive effect of IMF on eating quality has generally only been observed in those studies that used a number of genotypes, especially those that compared a high IMF breed such as the Duroc breed with lean, and low IMF white lines of pigs. Rincker et al. (2007) using pigs of the Duroc breed, reported that the relationship between extractable lipid in the muscle and palatability traits (tenderness, juiciness, and flavor) was not strong (correlation coefficients ~ 0.1). The results of this study (Rincker et al., 2007) also suggested that a large increase in marbling (from 1 to 8%) was associated with a relatively small improvement of only 1 taste panel unit (using a 15-point scale) in palatability measurements. Additionally, consumers appear to select less marbled pork in the retail case, indicating that they prefer to purchase leaner cuts of pork (Rincker et al., 2007). Although there is demand for highly marbled pork by certain export markets (e.g., Japan and Korea) and food services establishments, selection of pork-based only on marbling or IMF content may not ensure a pleasant eating experience.

Using breed substitution, such as the use of Duroc breed in crosses with white lines, has proved to be an effective tool to increase Longissimus intramuscular fat content. Latorre et al. (2003) reported values for the IMF content of the Longissimus muscle of 3.4% and 2.7% for terminal Duroc and white breeds, respectively. Ellis et al. (1996) reported greater marbling scores for Duroc population than lean genotypes that contain Pietrain breeds (3.2 compared to 2.7 on a 5-point scale, for Duroc and lean genotypes, respectively). Thus, even though using the Duroc breed in crossing programs is likely to increase IMF, the magnitude of any increase is not likely to be large. Selection for increased IMF is possible because the heritability of this trait is relatively high (between 0.4 to 0.5; Suzuki et al 2005; Klindt et al., 2006). However, increasing IMF levels using selection is likely to be a slow and expensive process and, at best, a longer term option for the industry.

Different nutritional interventions have been shown to increase IMF content of pork (Castell et al., 1994, Cisneros et al., 1996; Castaneda et al., 2005). The use of low protein-amino acid-deficient diets has been one of the most successful tools to achieve this objective (Castell et al., 1994; Kerr et al., 1995; Cisneros et al., 1996; Stanly and Wahtstron, 1993, Castaneda et al., 2005). Similar results have been achieved when high levels of the ketogenic amino acid leucine has been included in the diet (Cisneros et al., 1996; Hyun et al., 2003 and 2007; Castaneda et al., 2005). These two approaches to increase IMF content operate via different mechanisms: leucine is a branched-chain non-polar amino acid that is metabolized within the muscle and loads AcetilCoa and ketones bodies (acetoacetate), both of which are lipid precursors (Garret and Grisham, 2002; D’Melo, 2003), whereas a dietary lysine deficiency reduces protein synthesis and increases fat deposition. Because these two approaches operate through different mechanisms, they could have additive effects on IMF levels. Another approach that has been evaluated is the utilization of vitamin A deficient diets. However, Castaneda et al. (2006) fed diets that were either adequate (supplemented with 3,900 UI/kg of feed) or deficient (300 UI/kg of feed) in vitamin A to finishing pigs from 20 to 128 kg live weight and found no effect of diet on the IMF content of the Longissimus muscle (3.15 vs. 2.94 % IMF for the diets that were adequate and deficient in vitamin A, respectively).

In general, the magnitude of the response in IMF content is likely to be associated with factors such as the degree of protein-amino acid restriction, genetic line, gender, and length of time for which diets are fed. In this context and in order to gain a better understanding of the pattern of intramuscular fat deposition in pigs, the following literature review will focus on evaluating the effect of low protein-amino acid-deficient diets and excess of dietary leucine levels on intramuscular fat content.

Low protein/amino acids deficient diets

Studies that have investigated the impact of feeding lysine/protein-deficient diets on responses in IMF and in growth and carcass characteristics are summarized in Table 1 and will be discussed in this section.

Overview of the experiments using low protein-lysine diets

A total of 10 experiments have been carried out in this area (Table 1). There was considerable variation between these studies in the conditions during the experimental feeding period. For example, the minimum and maximum live body weight at the start of the feeding period was 8.6 and 89.0 kg, respectively, the minimum and maximum live body weights at the end of the feeding period was 92.6 and 135.0 kg, respectively, and the range in live weights over which the experimental diets were fed was from 32 to 88 kg. The degree of lysine restriction below the estimated lysine requirements of the pigs used in the studies varied from 7% to 48%, with reductions in dietary crude protein levels from 0 to 44%. The average IMF content of the control treatment (i.e., those pigs fed at the lysine requirement) was 3.2%, with a minimum of 1.4 and a maximum of 5.5% units. All the diets used in these studies were based on corn and soybean meal.

Effect of lysine deficient diets

Feeding lysine deficient diets (within the range of 7% to 48% below the estimated lysine requirement) increased intramuscular fat levels above that of the control pigs that were fed at the lysine requirement by 13% to 144% (Table 1). For example, Castell et al. (1994) fed low protein-lysine diet (11.9% CP and 0.48% lysine) and a control high protein-lysine diet (17.6% CP and 0.81% of lysine) for 14 weeks (from 25.7 to 95.4 kg live weight) to castrated males and gilts, and increased IMF content from 1.4 percentage units for pigs fed the control treatment to 3.5 percentage units for the pigs fed the lysine deficient diets. However, the low dietary lysine/crude protein level also resulted in reduced daily live weight gain (-22%) and feed efficiency (-21%) compared to the control. These results are in agreement with those reported by Kerr et al. (1995) who fed barrows and gilts from 8 to 96 kg live weight with low protein diets (reduced from 14 to 11% crude protein in the finishing phase), either supplemented with synthetic amino acids to equal the levels in the control diet (14% crude protein and 0.67% total lysine), or unsupplemented (i.e., lysine deficient at 0.45% total lysine). Pigs fed the lysine-deficient, low-protein diet had increased IMF in the Longissimus muscle (increased from 5.5 in the control treatment to 11.2% units) but also had poorer growth performance (11% reduction in daily gain and 13% reduction in feed efficiency). It is important to mention that studies such as those discussed previously were not designed with the objective to study the relationship between IMF and meat quality.

Few studies have investigated the impact of the degree of lysine restriction on responses in IMF. However, it would be expected that, in the absence of any negative effect of dietary lysine restriction on feed intake, the response in IMF would increase with increasing degree of lysine restriction. For example, Castell et al. (1994) and Castaneda et al. (2005) showed that the response in IMF was directly related to the degree of lysine restriction. A summary of the response in IMF (expressed as the percentage increase in IMF of pigs fed the lysine deficient diets relative to those fed at the lysine requirement) plotted against the degree of lysine restriction relative to the requirement is presented in Table 1. The linear regression equation gave almost as good a fit to the data points (R² = 0.61) as the quadratic equation (R² = 0.66) (Figure 1). The slope of the linear regression line presented in Figure 1 indicated that for every one percentage unit of increase in the reduction of dietary lysine level below the estimated requirement was associated with a 2.7 percentage unit increase in IMF content in the Longissimus muscle.

Time on feed of lysine deficient diets

The magnitude of the increase in IMF content has been shown to be related with time of feeding of the lysine deficient diets. For example, Cisneros et al. (1996) fed low lysine levels in the diet (0.40%) for two different time intervals (21 or 35 day period) to gilts from a commercial hybrid line and reported an increase in the Semimembranosus IMF content of 0.6 and 1.3% units for the 21 and 35 d period respectively. Most recently Castaneda et al. (2005) reported that IMF content increased with increased time of feeding of a lysine deficient diet (44% below the estimated lysine requirement); feeding this diet for either 21, 42, or 63 days produced increases in Longissimus IMF content ranging from 33 to 78 % of the control treatment (2.74 vs. 3.65 and 4.87% units IMF for 21, 42 and 63 days, respectively).

Dietary approaches to reduce lysine level and their impact on IMF content

Three dietary approaches have been used to achieve the protein and lysine restriction levels. With the first approach, the lower levels of protein and lysine were achieved by gradual removal of soybean meal in the diet; synthetic amino acids (Threonine and Tryptophan) were used to balance other essential amino acid levels (Castell et al., 1994; Kerr et al., 1995; Castaneda et al., 2005). In the literature summarized in Table 1, soybean meal inclusion levels ranged from 29 to 1.4 percentage units which also resulted in lysine reduction levels of 0% to 48% below the estimated requirement (0.81% total lysine levels for 29% of soybean meal and 0.32% total lysine levels for 1.4% of soybean meal inclusion level). Interestingly, the studies that have reported the largest increases in Longissims muscle IMF content (from 19% to 144%) are those that have used soybean meal inclusion levels below 7% resulting in lysine levels of 30% to 44% below the estimated requirement. For example, Castell et al. (1994) fed five diets with lysine content of 0% to 41% below the estimated requirement by reducing soybean meal inclusion levels from 20% to 4% and reported a 144% increase in IMF (from 1.4 to 3.5 percentage units of IMF content, respectively). Castaneda et al. (2005) fed diets with soybean inclusion levels of 16% to 1.5%, resulting in a lysine content 44% below the requirement (from 0.58% to 0.32 % of lysine content for the high and low soybean meal inclusion, respectively), and showed a minimum IMF increase of 19% (from 2.9% to 3.5% units of IMF content). On the other hand it would appear that, with at least 9% soybean meal inclusion and dietary lysine levels 20% below the estimated requirement, the IMF content is affected only when those diets are fed over long periods of time; Castell et al. (1994) fed diets with soybean meal levels content of 20 to 12% which resulted in dietary lysine levels of 20% below the estimated requirement (0.81 vs 0.65% of total lysine for 20% soybean meal and 12% soybean meal diets, respectively) to pigs from 25-99 kg body weight and reported an increase in IMF content of 56% (from 1.4 to 2.3 percentage units). In contrast, Castaneda et al., (2005a) fed diets with 8% of soybean meal and 22% lysine below the requirement (0.67 vs. 0.45% total lysine for the high [16%] and low [8%] soybean meal diets, respectively) over 62 d period, and reported no changes in Longissimus IMF content (2.4% vs. 2.7% units, respectively).

A second approach to reduce the dietary lysine level consists of the use of a basal diet with low inclusion levels of soybean meal as the lysine deficient treatment. In order to raise the lysine to a level that meets the requirement of the pigs, synthetic lysine was added to the basal low soybean meal diet, additionally, other synthetic amino acids were used to avoid imbalances and deficiencies in the indispensable amino acids. Using this approach, Hyun et al. (2007) fed diets containing 10% soybean meal and two lysine levels of 0.50% and 0.70% (leucine and threonine levels were held constant across diets) and reported no effect on Longissimus muscle IMF content (2.03% vs. 2.38% units for the lysine adequate and deficient diets [0.70% vs. 0.50% of total lysine], respectively). On the other hand, Cisneros et al. (1996) fed a basal diet containing 7% soybean meal and lysine levels of 0.56 and 0.40% (similar synthetic sources of amino acids were used to raise lysine and avoid deficiencies in other essential amino acids) and found that the Longissimus IMF content increased from 3.0 to 4.8 percentage units for the lysine adequate (0.56%) and deficient diets (0.40%), respectively. Interestingly, the experiments that reported a consistent increase in IMF content are those that fed diets with soybean meal inclusions levels of 7% or below, which results in lysine levels of 30% below the estimated requirement and a ~38% reduction in protein levels (from ~16 to ~10 percentage units of total crude protein). This is interesting because in studies where soybean meal has been reduced to ~7% inclusion level, valine (Russell et al., 1983, 1987, Figueroa et al., 2003), histidine, and isoleucine (Figueroa et al., 2003) became limiting. The possible additional restriction in valine, histidine, and isoleucine, may play an additional and important role in the increase of IMF content. Additionally, the low protein level may play an important role in the energy utilization and may, therefore, increase the IMF content. In theory, the increased carcass fatness observed when low protein diets are fed is due to an increase the net energy (Kerr et al., 1995; Knowles et al., 1998; Bellego et al., 2001). Factors associated with higher net energy content in low protein diets include a reduction in deamination of excess of amino acids and excretion of urea (Noblet et al., 1987) and a reduction in the protein turnover and heat production of the animal (van Milgen et al., 2001). As a result, it would be expected that in the absence of any negative effect of dietary protein-lysine restriction on feed intake, the response in IMF would increase with an increasing degree of protein and lysine restriction. However, the main effect of protein and lysine restriction level and the imbalances in other essential amino acids affecting Longissimus IMF content has never been tested. Further research is necessary to understand the independent effects of those nutrients.

A third approach to reduce the lysine level in the diet was used by Castaneda et al. (2005a), who replaced soybean meal with corn gluten meal. A portion of both ingredients (soybean meal and corn gluten meal) was removed to produce a dietary deficiency of lysine from 0 to 44% below the requirement (with 0.58 and 0.32% dietary lysine levels for normal and deficient levels, respectively). In their results, Longissimus IMF content was increased by 181% (from 2.32 to 4.87%) when the control diet (High soybean meal [16% inclusion level], high-lysine [0.58% lysine level] diet) is compared to the low-lysine (0.32% dietary lysine level) corn gluten meal (10% corn gluten meal) based diet. An additional 57% increase (from 3.1 to 4.9 percentage units) in IMF was reported when low-soybean meal (1.5% soybean meal) low-lysine (0.32% of dietary lysine levels) diet was compared to the low-lysine (0.32% dietary lysine level) corn gluten meal (10% corn gluten meal) based diet. This is an interesting finding because it suggests an additional effect of corn gluten meal on Longissimus IMF content, which may be a consequence of a restriction in essential amino acids (tryptophan, arginine and valine) and an excess of leucine, which contributes to the energy supply at the muscle level (Hyun et al., 2007). More detailed concepts of the usage of this ingredient will be discussed in the section of this literature review dealing with the effect of high leucine levels on IMF content.

Effect of lysine deficient diets on muscle deposition

Numerous studies have investigated the effect of lysine deficient diets on protein and lean deposition (Castell et al., 1994; Kerr et al., 1995; Cisneros et al., 1996; Write et al., 2000; Bellego et al., 2001; Andersen et al., 2005; Castaneda et al., 2005; Hyun et al., 2007). Castaneda et al. (2005a) reported that a dietary lysine deficiency 22% below the estimated requirement reduced fat free lean deposition by only 5% (from 400 to 381 g/day fat-free lean gain); however, a greater restriction in dietary lysine (44% below the requirement) reduced lean deposition by 48% (from 342 to 178 g/day fat-free lean gain). Interestingly, Rosenvold et al., (2003) reported that the majority of the glycogen in muscle is associated with muscle proteins (32% as macroglycogen and 68% as proglycogen). Therefore, it is possible that any reduction in muscle protein deposition could result in a reduction in muscle glycogen content, which may be an approach to manipulating muscle glycolytic potential.

Genotype effects on IMF content

The genotype of the pigs used is likely to have an effect on the extent of the response in IMF to lysine deficiency. However, none of the experiments carried out in this area have investigated this concept. Genetic variation in factors such as muscle fiber type (Chang et al., 2003), the incidence of the Rendement Napole Gene mutation (Hamilton et al., 2000), feed intake, and lean growth potential (Hamilton et al., 2003) could potentially affect the response in IMF levels.

Impact of leucine on intramuscular fat deposition

Studies that have investigated the impact of feeding high dietary leucine levels on the growth response and IMF content in pigs are summarized in Table 2. These reports will be discussed in this section.

Overview of the experiments using high dietary leucine levels to increase intramuscular fat levels.

A total of 5 experiments have been carried out in this area (Table 2). Within these studies, the minimum and maximum live body weight at the start of the feeding period was 66 and 89 kg respectively, the minimum and maximum live body weights at the end of the feeding period was 113 and 134 kg, respectively, and the range in live weights over which the experimental diets were fed was from 37 to 64 kg. The average IMF content of the control treatment (i.e., those pigs fed at the lysine and leucine requirement), was 2.9%, ranging from 2.0% to 3.8%. A wide range of total dietary leucine levels was used (between 1.10 and 3.35% units), and two approaches were employed to achieve these dietary leucine levels. In the first approach, soybean meal was kept constant and synthetic leucine (from 0 to 2% of added leucine) and other amino acids (lysine, methionine, threonine and tryptophan) were added to the diet (Cisneros et al., 1996; Hyun et al., 2003 and 2007). In this approach, the experimental diets had digestible leucine levels from 1.2 to 3.5% for the control and diets with added synthetic leucine, respectively.

A second approach was used by Castaneda et al. (2005ab), where soybean meal was replaced with corn gluten meal. In this approach, the experimental diets reached digestible leucine levels from 1.2 to 1.8% for soybean meal and corn gluten meal based diets, respectively. Also a number of synthetic amino acids (lysine, threonine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan) were used to maintain equal levels of all essential amino acids across diets. The differences between both approaches will be discussed later in this paper.

Impact of synthetic leucine on intramuscular fat deposition

Feeding high dietary leucine levels (from 1.1 to 3.35% of total leucine for the control [no added leucine] and experimental diet [with added synthetic leucine], respectively) to finishing pigs increased IMF fat levels above that of the control pigs that were fed at the lysine requirement by 26 to 120% (Table 2). Hyun et al. (2003), fed diets with two leucine levels (1.35 and 3.35% of dietary leucine) over a 39-day period. In their results, pigs fed 3.35% leucine had higher marbling score (3.9 vs. 3.2, respectively, on a continuous scale of 1-10) and more IMF content (3.4 vs. 2.4% units, respectively) in the Longisimus muscle than pigs fed 1.35% dietary leucine; however, pigs fed the high leucine diets grew 11% slower than the pigs fed the control diet (ADG = 0.93 and 0.83 for control and high leucine diets, respectively). In a following experiment, Hyun et al. (2007) fed three levels of dietary leucine (1.2, 1.9 and 2.6% of leucine) over a 46 day period, and reported that Longissimus muscle IMF content was increased for the two higher leucine levels (2.4%, 4.4%, and 3.7% for dietary leucine levels of 1.2, 1.9 and 2.6%, respectively). The maximum response in IMF content was observed at 1.9% of dietary leucine level, which suggests that there is an upper limit of dietary leucine to increase IMF content. In contrast to the previous study, these authors did not report any negative effect of high dietary leucine levels on growth performance. The results of Hyun et al. (2003 and 2007) contrast with Cisneros et al. (1996) who fed high levels of leucine (1.0 vs. 3.0% of dietary leucine) for a 21 or 35 day feeding period, and found non-significant effects of dietary leucine level on IMF content (Longissimus IMF content of 3.5 and 4.4% units for low and high leucine levels, respectively, and Semimembranosus IMF content of 3.8 to 4.5%, units for low and high leucine levels, respectively), similarly these authors did not report any negative effect of high dietary leucine levels on growth performance.

The reduction in ADG reported by Hyun et al. (2003) contrast with other studies that have fed high leucine levels, for example, Taylor et al. (1984) and Edmonds and Baker (1987ab) did not find any deleterious effect on growth performance in young pigs from feeding diets containing 4% of supplemental leucine. The results of these experiments suggest that IMF and marbling levels of pork can be increased by feeding high levels of leucine to finishing pigs. However, more research is required to determine the impact of high dietary leucine levels on growth performance.

Impact of natural leucine on intramuscular fat deposition

Experimental evidence (Castaneda et al., 2005ab) suggests an effect of corn gluten meal on IMF content. As previously stated in the review of the effect of lysine deficient diets on IMF content, Castaneda et al. (2005a) showed that after a 63 d feeding period, corn gluten meal based diets increased Longissimus IMF by 181% (from 2.32 to 4.87 percentage units) compared with the control diet (High soybean meal [16% inclusion level], high-lysine [0.58% lysine level] diet). When a diet with low soybean meal (1.5% soybean meal) and low-lysine (0.32% of dietary lysine levels) was compared with a diet based on corn gluten meal (10% corn gluten meal) with the same low lysine level (0.32%), the Longissimus IMF content was increased by 57% (from 3.10 vs. 4.87 percentage units (Castaneda et al., 2005a), which suggest an additive effect of corn gluten meal on Longissimus IMF content (Figure 2). This additive effect was corroborated later by the same authors; Castaneda et al. (2005b) fed two protein sources (soybean meal and corn gluten meal with a dietary leucine content of 1.1 to 1.8%, respectively) and found that corn gluten meal based diets gave a 50% increase in Longissimus IMF content (3.9%) when compared with the Longissimus IMF content (2.6%) of a soybean meal-based diet with the same total lysine level. These additional 1.7% of IMF content observed with corn gluten meal based diets, could be a consequence of a restriction in the levels of essential amino acids and/or an excess of leucine. Peter et al. (2000) and van Milgen et al. (2001) reported that corn gluten meal based diets have severe deficiencies in lysine and other amino acids (tryptophan, arginine, threonine, valine, isoleucine and histidine) inherent to the imbalanced protein present in corn gluten meal. Besides those imbalances, this ingredient provide high levels of the ketogenic amino acid leucine which contributes to the energy supply at the muscle level by producing AcetilCoA and ketone bodies (acetoacetate), both of which are lipid precursors (D’Melo, 2003;) (Hyun et al., 2007). On the other hand, high levels of leucine can have an antagonistic effect with isoleucine and valine which may produce an increase in fat depots. Working with chicks D’Mello and Lewis (1970) sowed that excess dietary leucine depressed plasma valine concentrations and then this reduction can be exacerbated with the addition of isoleucine. From studies principally with rats, Harper et al (1984) attributed the leucine-induced changes in plasma levels of isoleucine and valine to increased oxidation of these two amino acids, having discounted any effects emanating from competition for intestinal or renal transport. Limited studies with chickens support this view, Calvert et al. (1982) demonstrated that excess leucine failed to influence excretion of ¹⁴C-labeled isoleucine or valine, but markedly increased the oxidation of these amino acids as indicated by enhanced in vivo output of ¹⁴CO₂. The catabolism of these branched-chain amino acids is initiated by an aminotransferase reaction that loads branched-chain keto acids, and then, by oxidative decarboxilation, yields Acetyl-CoA which is a lipid precursor that may enhance fat deposition (D’Mello, 2003). However, none of this has been tested in pigs. Further research is required to determine the effect of the imbalanced amino acids and leucine dietary level of corn gluten meal based diets on IMF content.

Interaction of dietary lysine and leucine level on IMF content

Only one study has reported an interaction between low dietary lysine levels and high dietary leucine levels for muscle IMF content. Hyun et al. (2007) fed two lysine levels (0.70% and 0.50% of total lysine in the diet) in combination with three levels of leucine (low 1.2%, moderate 1.9% and high 2.7% units) over a 46 day period. They reported an interaction between lysine and leucine level for Longissimus muscle IMF content (Figure 3). There was no effect of dietary leucine level on IMF content in pigs fed 0.70% lysine (2.03, 1.97 and 2.07% IMF content for low, moderate, and high leucine levels, respectively); however, for pigs fed 0.50% lysine, the Longissimus muscle IMF content was increased for the moderate (1.9% leucine) and high (2.7% leucine) leucine levels (4.43% and 3.65 percentage units, respectively) compared with the 1.2% dietary leucine treatment (2.38% of IMF content). In general, this study suggests an interaction of high leucine and low lysine levels. Both approaches to increasing IMF operate by increasing the availability of energy for fat deposition within the muscle. Feeding lysine levels below the estimated requirement increases the total pool of energy within the body; therefore, this approach is likely to result in an increase in fat deposition in all fat depots including intramuscular fat. On the other hand, it has been proposed that excess dietary leucine is catabolized within the muscle and contributes to the energy supply locally (Hyun et al., 2007). Because these two approaches operate through different mechanisms, an additive effect on increasing in IMF content might be expected. Additionally, it is possible that an excess of dietary leucine may induce a delay in glycogen replenishment during or after muscle glycogen depletion because it contributes energy in the form of lipid precursors (Garret and Grisham, 2002) at the muscle level. In general, these studies suggest that the response of intramuscular fat to dietary leucine level is dependent on the lysine level fed, more research is necessary to fully understand this interaction.

Unanswered questions

What are the dietary factors that can affect IMF content?

Low lysine diets. In general, the greatest the lysine restriction the greater the increase in IMF content. Different dietary approaches have been used to achieve the protein and lysine restriction levels, in those cases were the lysine restriction level (0.40% of lysine level or below) results in soybean meal inclusion levels of 7% or below, total crude protein and other essential amino acids (lysine, valine, histidine, and isoleucine) (Russell et al., 1993; Figueroa et al., 2003) may be limiting. Further research is necessary to understand the combined and independent effects of those nutrients in order to improve the formulation strategies to achieve the response in IMF content.
Time on feed. From the research reported in this review, we can conclude that the degree of lysine restriction interact with time on feed (Castaneda et al., 2005a). In theory as the degree of lysine restriction and the time on feed increase, the magnitude of the response in IMF will also increase. From this literature review, it is difficult to clearly establish this relationship, more research is necessary to fully understand the relationship of low protein diets and the period of time in which those diets are feed and their impact on IMF.
Lysine-leucine interaction. There has been limited research to test for interactions between high dietary leucine levels and low dietary lysine levels. Both nutrients operate by increasing the availability of energy for fat deposition within the muscle, therefore the degree of restriction of one nutrient may an important factor for the expression of the other on IMF content. More research is needed to understand the combined effects of these two approaches.
High dietary leucine levels and glycogen replenishment. An excess of dietary leucine may induce a delay in glycogen replenishment during or after muscle depletion because an excess of this amino acid can contribute to the energy supply at the muscle level. However, this hypothesis has never been evaluated.

Hypothesis:

We hypothesize that the deposition of intramuscular fat in different genotypes of finishing pig would be a function of the protein-lysine intake and the growth potential of those genotypes. The highest level of intramuscular fat deposition will be reached when the fattest genotype feed the low lysine diets.
We hypothesize that different approaches to reduce dietary lysine (which loads different levels of protein, lysine and other essential amino acids) will have different impact on IMF content. The highest level of IMF content will be reached with pigs fed the lowest soybean meal level due to a low protein and lysine intake and imbalances in other amino acids.
We hypothesize that in the absence of any negative effect of dietary protein-lysine restriction on feed intake (i.e. energy intake), the response in IMF would increase with increasing degree of protein-lysine restriction.
We hypothesize that as the degree of lysine restriction and the time on feed increase, the magnitude of the response in IMF will also increase.

We hypothesize that the deposition of intramuscular fat in the finishing pig would be a function of both lysine and leucine intake. The highest level of intramuscular fat deposition will be reached when the optimum inclusion levels for lysine and leucine are achieved.
We also hypothesize that an excess of leucine may induce a delay in glycogen replenishment during or after muscle depletion.