Effects of dietary valine:lysine ratio on the performance, amino acid composition of tissues and mRNA expression of genes involved in branched-chain amino acid metabolism of weaned piglets

Experimental design and diets

This study was conducted to determine the effects of altering the SID valine:lysine ratio on the performance and amino acid metabolism of 144 crossbred (Duroc×Landrace×Large White) pigs weaned at 28±4 days of age and weighing 8.79±0.02 kg body weight. The pigs were randomly allotted to 1 of 4 diets formulated to provide SID valine:lysine ratios of 50%, 60%, 70%, or 80%. The experimental diets were based on corn, extruded corn, soybean meal, and crystalline AA (Table 1). Peanut meal (60 g/kg) was included because of its low valine content which allowed the formulation of a valine deficient diet [12]. The dietary valine concentration of the remaining diets was altered by substituting 1.2, 2.4, or 3.6 g/kg of L-valine for an equivalent amount of corn. The supplemental L-valine (99% purity) was obtained from the CJ CheilJedang Company (Seoul, Korea).

Table 1

Composition and nutrient levels of experimental diets varying in standardized ileal digestible (SID) valine:lysine ratio (% as-fed)

The digestible energy, calcium and phosphorus contents of the diets were calculated using values for the ingredients obtained from NRC [6]. The concentrations of total AA in the diets are the result of an AA analysis conducted in duplicate. The SID AA content of the experimental diets was calculated by multiplying the analyzed AA content of the ingredients by the SID coefficients of the ingredients obtained from NRC [6] and summing the results (Table 1).

Each diet was fed to 6 pens of pigs with 6 pigs per pen (3 gilts and 3 barrows) and the experiment was run as a randomized complete block design. The pigs were housed in 1.8×2.1 m² metabolic cages equipped with fully slatted, plastic floors throughout the experiment. The pigs were weighed after an over-night fast on d 0 and 28 to calculate average daily weight gain (ADG).

The pigs had free access to water via a nipple drinker and were fed 3 times a day (08:00, 14:00, and 18:00 hours) using a single-sided 3-hole feeder. Sufficient feed was added to the feeder to ensure ad libitum access. The amount of feed added was recorded and summed to determine the feed intake for the 28-d experiment. This value was divided by 28 to determine average daily feed intake (ADFI) for the experimental period. Feed efficiency (ADG/ADFI) was then calculated from ADG and ADFI. The temperature in the rooms was decreased weekly from 28°C in the first week to 24°C in the last week. The lights were on from 07:30 until 18:30 hours.

Sample collection

Feed samples were collected and pooled weekly to determine the nutrient levels of the experimental diets. For the determination of plasma biochemical indices, branched-chain keto-acid (BCKA) and AA concentrations, blood was collected from one pig per pen via the jugular vein into 10-mL heparinized vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) on d 28 following an overnight fast. Samples were centrifuged (Biofuge22R; Heraeus, Hanau, Germany) at 3,000×g for 10 min, and the plasma was kept at −80°C until analyzed.

At the end of the 28-day experiment, the 24 pigs used for blood sampling were killed under commercial conditions by electrical stunning followed by exsanguination and then tissue samples were collected. The small intestine was dissected from the mesentery and immediately placed on ice. The duodenum was considered as the part of the intestine from the pylorus to the ligament of Treitz, a segment of the small intestine 10 cm proximal to the ileocecal junction was considered as the ileum, and the remainder of the small intestine was considered as the jejunum [13]. Segments (3 cm in length) of the mid-duodenum, mid-jejunum, and mid-ileum (the entire alimentary canal without any digesta) were fixed in 10% neutral buffered formalin for subsequent histological measurement. In addition, samples (about 10 g) of the liver and longissimus dorsi muscle, dissected from the left side of the carcass of four piglets per treatment, were placed in liquid nitrogen and then stored at −80°C until needed for analysis.

Chemical analysis

All chemical analyses were conducted according to the methods of the AOAC [14]. Analysis of the crude protein content of the ingredients and experimental diets was conducted according to AOAC Method 978.04 (AOAC) [14]. For the analysis of AA except for methionine, cysteine and tryptophan, ingredients and diets were hydrolyzed in 6 N HCl at 110°C for 24 hours (AOAC Method 999.13). Methionine and cysteine were determined after cold performic acid oxidation overnight and hydrolyzed with 7.5 N HCl (AOAC method 994.12). Tryptophan was determined using reverse-phase high-performance liquid chromatography (HPLC, Waters 2690, Milford, MA, USA) after alkaline hydrolysis at 120°C for 16 hours.

Plasma AA were determined using Ion Exchange Chromatography with a S-433D Amino Acid Analyzer (Sykam, Eresing, Germany) as described by Sedgwick et al [15]. Determination of plasma biochemical indices were carried out using an Automatic Biochemical Analyzer (Hitachi 7020, Tokyo, Japan). Plasma α-keto isocaproate, α-keto isovalerate and α-keto-β-methylvalerate were measured according to the method of Pailla et al [16]. In brief, branched-chain keto acids were derivatized with o-phenylene-diamine to give fluorescent derivates, which were separated chromatographically on a Reverse-Phase Column (Spherisorb ODS-2, 4.6×250 mm column filled with 5 mm particles, Waters, Eschborn, Germany) using a binary gradient (L-7100, Merck-Hitachi, Japan). Detection was performed Fluorometrically (FL Detector L-7480, Merck-Hitachi, Japan). α-keto-valerate was used as an internal standard.

Muscle and liver samples (10 g) were thawed, homogenized within animal and diet, lyophilized in a vacuum-freeze dryer (Tofflon Freezing Drying Systems, Shanghai, China), and ground through a 1-mm screen. Freeze-dried samples (50 mg) of liver and longissimus dorsi muscle were used to measure AA composition using the same methods as described above for plasma. Amino acid analysis was conducted using HPLC (L-8800 AA Analyzer; Hitachi, Japan).

Samples from each intestinal segment were embedded in paraffin and cut into 5-μm serial sections, and five non-successive sections from each tissue sample were selected and stained with hematoxylin–eosin for identification [13]. Six well-oriented villi (determined as the distance between the crypt openings and the end of the villi) and their associated crypt (measured from the crypt-villous junction to the base of the crypt) per section were selected and measured under a light microscope (CK-40, Olympus, Tokyo, Japan) at 40×magnification and analyzed with an Image Analyzer (Lucia Software. Lucia, ZaDrahou, Czechoslovakia). The average of these measurements was calculated to yield a single value for each pig. These procedures were conducted by an observer unaware of the dietary treatments.

Total RNA was isolated from muscle and liver tissue using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality and quantity of RNA were determined by Ultraviolet Spectroscopy using a NanoDrop ND-1000 (Thermo Fisher, Wilmington, DE, USA). The A_260/280 ratios were always between 1.9 and 2.1, and RNA integrity was evaluated using electrophoresis with 1.5% agarose and ethidium bromide staining (1.25 ng/μL; Sigma-Aldrich, St. Louis, MO, USA). Thereafter, about 1 μg of total RNA was incubated with DNase I (Fermentas, Waltham, WI, USA) followed by Reverse Transcription using a First-Strand cDNA Synthesis Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The cDNA was synthesized with Oligo dT and Superscript II Reverse-Transcriptase, and stored at −80°C before further processing. The primer sequences used for the selected genes are listed in Table 9.

Table 9

Sequence of primers used for quantitative real time-polymerase chain reaction and product sizes

The relative expressions of the target genes were determined by real-time polymerase chain reaction (PCR) using an ABI7900HT PCR system (Applied Biosystems, Forrest City, CA, USA). Real-time PCR was performed in duplicate for each cDNA sample, using SYBR Green I as a PCR core reagent in a final volume of 20 μL. The PCR conditions used were incubation for 10 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C with subsequent annealing and extension for 60 s at 56°C to 64°C. β-Actin was used as an internal control to normalize the expression of the target genes. mRNA expression levels of the target genes, expressed as arbitrary units, were acquired from the value of the threshold cycle (C_t) of real-time PCR relative to that of β-actin using the comparative C_t method, according to the following formula: 2^−ΔΔCt(−ΔΔCt = C_{t gene of interest} – C_{t β-actin})_treat – (C_{t gene of interest} – C_{t β-actin})_control.

Statistical analysis

The data were statistically analyzed using the MIXED procedure of SAS (SAS Institute, Cary, NC, USA) with pen as the experimental unit. The model included diet as a fixed effect and sex and replicate as random effects. However, no significant sex effects were observed for any trait measured and therefore sex was removed from the statistical analysis. Polynomial contrasts were conducted to determine the linear and quadratic effects of altering the SID valine:lysine ratios. All values are reported as least square means.

mRNA expression data were analyzed using the relative quantification method that describes the change in target gene expression relative to that of the control group. β-Actin was used as a normalizing internal control to adjust for the amount of RNA added to the reverse transcription reactions. Cycle thresholds of the target genes were subtracted from the control gene index (ΔCt) for normalization and the treatment groups were compared with the control group (ΔΔC_t; [17]). Treatment effects were considered significant if p<0.05 whereas values between 0.05 and 0.10 were considered as indicative of a statistical trend.

Polynomial contrasts were conducted to determine the optimum ratio of SID valine:lysine to maximize specific traits. In order to be selected as a trait to establish the requirement, the trait had to show a significant quadratic effect and be of major economic importance. Based on our experiment, only ADG met these two essential criteria. Therefore, using a quadratic model, the ADG of weaned pigs was plotted against SID valine:lysine ratio. The data were analyzed by a quadratically fitted line [y = a×x²+b×x+c, with the maximum quadratic = −b/(2a)].

Growth performance

Table 2 shows the effects of altering the SID valine:lysine ratio on the performance of weaned pigs. The ADG of the pigs was altered quadratically (p<0.05) from d 1 to 28 as the dietary SID valine: lysine ratio increased. Daily SID valine intake was linearly and quadratically altered (p<0.01) while average daily feed intake and feed efficiency of weaned piglets were not influenced by the SID valine:lysine ratio fed (Table 2).

Table 2

Effects of standard ileal digestible (SID) valine:lysine ratio on the performance of weaned piglets (d 0 to 28)

Figure 1 shows the effect of plotting ADG against the dietary SID valine:lysine ratio. The final equation was y = −0.14×(SID valine:lysine)²+19.1×(SID valine:lysine) −214; R² = 0.96]. According to the quadratic model, a dietary SID valine:lysine ratio of 68% maximized growth of weaned pigs (Figure 1).

Figure 1

Quadratic model (---) of average daily gain (ADG) of weaned piglets plotted against standardized ileal digestible (SID) valine to lysine ratio. Using a quadratic model, the SID valine:lysine was 68% [y = −0.14×(SID valine:lysine)²+19.1×(SID valine:lysine)−214; R² = 0.96].

Morphology of small intestine

Table 3 shows the effects of altering the SID valine:lysine ratio on the intestinal morphology of weaned piglets. Pigs fed the valine deficient diet (50% SID valine:lyine) had a lower villous height in the duodenum, jejunum and ileum (p<0.05) than pigs fed the valine supplemented diets. In addition, the villous height to crypt depth ratio in the duodenum of pigs increased (p<0.05) as the SID valine:lysine level increased (Table 3).

Table 3

Effects of standard ileal digestible valine:lysine ratio on the intestinal morphology of weaned piglets (d 28)

Blood metabolites and plasma amino acids

Table 4 shows the effects of altering the SID valine:lysine ratio on plasma biochemical indices. The plasma levels of albumin, globulin, glucose, triglyceride and urea were unaffected by altering the SID valine:lysine ratio (Table 4).

Table 4

Effects of standard ileal digestible (SID) valine:lysine ratio on plasma biochemical indices of weaned piglets (mmol/L)

Table 5 shows the effects of altering the SID valine:lysine ratio on the AA levels in the plasma of weaned pigs. The plasma valine levels of pigs were significantly increased (p<0.01) by an increase in the SID valine:lysine ratio. Plasma aspartate and cysteine were linearly decreased (p<0.01), while asparagine was quadratically decreased (p<0.05) when an increased SID valine:lysine ratio was fed (Table 5).

Table 5

Effects of standard ileal digestible (SID) valine:lysine ratio on amino acid concentration in the plasma of piglets (nmol/mL)

Table 6 shows the effects of altering the SID valine:lysine ratio on branched chain keto AA in the plasma of weaned pigs. The α-keto isovaleric acid concentration in the plasma of pigs was linearly increased (p<0.01) with an increased SID valine:lysine level (Table 6). However, α-keto isocaproate and α-keto-β-methlyvalerate were unaffected by dietary treatment.

Table 6

Effects of standard ileal digestible (SID) valine:lysine ratio on plasma branched-chain keto acids of weaned piglets (μmol/L)

Muscle and liver amino acids

No significant change was observed in the valine concentration of liver tissue due to treatment (Table 7). However, liver cysteine concentrations increased linearly (p<0.01) and liver glutamate showed a trend to decrease linearly (p = 0.06) with increased SID valine:lysine ratio (Table 7).

Table 7

Effects of standard ileal digestible (SID) valine:lysine ratio on the concentration of amino acids in the liver of piglets (nmol/mL)

The effects of SID valine:lysine ratio on the concentration of AA in the muscle of pigs is shown in Table 8. Valine and serine concentrations in pig muscle decreased linearly (p<0.05) and proline showed a trend to decrease (p<0.10) when the SID valine: lysine ratios were increased (Table 8).

Table 8

Effects of standard ileal digestible (SID) valine:lysine ratio on the concentration of amino acids in the muscle of piglets (nmol/mL)

mRNA expression

The sequences of the primers used for real time-PCR and their product sizes are shown in Table 9. The size of the primers ranged from 112 bps for β-actin to 152 bps for the branched chain α-keto acid dehydrogenase subunit E1α.

The effect of SID valine:lysine ratio on the relative mRNA expression of BCAA catabolism genes in the muscle of weaned piglets is shown in Table 10. Increasing the dietary SID valine: lysine supply increased (p<0.05) the mRNA expression of BCAT_m and BCKDH subunit (E1α, E1β, and E2) genes in skeletal muscle (Table 10).

Table 10

Effects of standard ileal digestible (SID) valine:lysine ratio on the relative mRNA expression of branched-chain amino acid catabolism genes in the muscle of weaned piglets