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Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by cattle

Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by... ABSTRACT Starch is the primary nutrient in ruminant diets used to promote high levels of performance. The site of starch digestion alters the nature of digestive end products (VFA in the rumen vs. glucose in the small intestine) and the efficiency of use. Cereal grain endosperm texture plays a major role in the rate and extent of starch degradation in ruminants. Wheat grain texture is regulated by the starch surface protein complex friabilin that consists primarily of puroindoline (PIN) A and B. Soft kernel texture in wheat is a result of both PIN genes being in the wild type active form and bound to starch. The objective of this study was to investigate the effect of varying PIN content in wheat on the rate of starch digestion in the rumen of beef cattle. In Exp. 1, 6 transgenic soft pin a/b isolines created in a hard wheat background, and 2 hard wheat controls were milled to yield a wide range of mean particle sizes across all lines. Milled samples were incubated in situ for 3 h. Increased expression of both PINA and PINB decreased DM digestibility (DMD) by 29.2% (P < 0.05) and decreased starch digestibility by 30.8% (P < 0.05). Experiment 2 separated the effects of particle size and total PIN content on digestion by milling the hardest and softest lines such that the mean particle size was nearly identical. Increased PIN decreased DMD by 21.7% (P < 0.05) and starch digestibility by 19.9% (P < 0.05) across particle sizes smaller than whole kernel. Experiment 3 addressed the time course of PIN effects in the rumen by observing ground samples of the hardest and softest lines over a 12-h in situ period. Increased PIN decreased DMD by 10.4% (P < 0.05) and starch digestibility by 11.0% (P < 0.05) across all time points. Dry matter and starch digestibility results demonstrated that increased expression of PIN was associated with a decreased rate of ruminal digestion independent of particle size. Puroindolines seem to aid in the protection of starch molecules from microbial digestion in the rumen, potentially increasing the amount of starch entering the small intestine. INTRODUCTION Cereal grains are typically fed to ruminants to increase starch intake. The site of starch absorption along the gastrointestinal tract affects cattle performance and feed efficiency. Slower rates of digestion increase the amount of starch bypassing the rumen. Starch digested in the small intestine can produce up to 42% more energy than fermentation (Owens et al., 1986) because of a more efficient use of digestive end products (glucose vs. VFA). Grain texture plays a major role in the rate and location of starch digestion in ruminants (Philippeau et al., 1999). Small grains (wheat, barley, or oats) are more rapidly fermented than corn and sorghum. Variations in starch granule structure among species of cereal grains may account for distinct rates of digestion patterns. Protein and structural carbohydrates within the cereal kernel may be more important in determining the extent of ruminal starch digestion than the starch type (McAllister et al., 1993). Wheat (Triticum aestivum L.) grain hardness is determined by the degree of adhesion between starch granules and the protein matrix, regulated by the protein complex friabilin. Friabilin, isolated from the surface of starch granules, contains 2 major proteins, puroindolines (PIN) A and B. Puroindolines contain a unique tryptophan-rich domain believed to be involved in their binding to the phospholipids of starch granules (Gautier et al., 1994). Soft wheat results from both pin genes being in the wild type form, whereas hard wheats have an absence or alteration in either pin gene (Morris et al., 2001). Hard wheat transformed with added wild type pina, pinb, or pina and b resulted in isolines with a wide range of grain textures (Hogg et al., 2004). Transgenic PIN experiments have demonstrated that wheat with high levels of one PIN is intermediate in grain texture, and if both PIN are active, then soft wheat texture results (Hogg et al., 2004). The objective of these studies was to investigate the effect of varying PIN content and particle size in wheat on the rate of digestion in the rumen by using PIN isolines that varied only in PIN content. MATERIALS AND METHODS Genetic Material The hard red spring wheat cultivar Hi-Line (Lanning et al., 1992) was used for transformation. Native Hi-Line contains the soft type pina sequence (pina-D/a) and the variant pinb sequence (pinB-D1b), which contains a glycine to serine substitution at the 46th residue of pinb (Giroux et al., 2000). Hi-Line was transformed with vectors containing wild type pina, pinb or both pina and pinb (pin). Pin were under the control of the wheat glutenin promoter (Hogg et al., 2004). Isolines with a wide range of grain textures resulted, and a subset of the lines presented in Hogg et al. (2004) were chosen for this study. Two lines with added pina (HGA1, HGA3) formed the intermediate grain texture HGA group; 2 lines with added pinb (HGB6, HGB12) formed the soft HGB group; 2 lines with added pina and pinb (HGAB12, HGAB18) formed the very soft HGAB group; and 2 hard wheats [native Hi-line and Hi-Line transformed with only bar (line 161 of Hogg et al., 2004)] formed the hard wheat controls (HWC). Seeds used in the study were obtained from a single 4-row plot grown during the 2003 season at the Montana State University-Bozeman Arthur H. Post Field Research Farm under dry-land conditions. Isoline Characterization Isolines used were analyzed for common feed and grain characteristics (Table 1) to demonstrate that lines were nearly identical except for the presence of the pin transgene(s) dictating PIN content and grain hardness. Three independent 100-seed replicate samples per line were analyzed for kernel hardness, kernel weight, and kernel size using the Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL). Dry matter content of each line × treatment was determined using AOAC method 930.15 (2000) for oven drying and replicated twice. Acid detergent fiber for each line was determined using the protocol described by Van Soest et al. (1991) and replicated twice. Crude protein for each line was measured by AACC method 46-30 (2000) using a LECO FP-328 nitrogen analyzer (LECO Corporation, St. Joseph, MI) and replicated 4 times. Starch content for each line was determined using a modified protocol of the Megazyme total starch assay kit (Megazyme International, Brey, Ireland) and was replicated 4 times. Table 1. Puroindoline (PIN) expression, kernel characteristics, and chemical composition (DM basis) of wheat lines varying in PIN expression ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  1 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 2 Level of PINA and PINB relative to the levels in the hard wheat control variety Hi-Line. Data on PINA and PINB levels taken from Hogg et al. (2004). 3 Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL) grain hardness. Hard wheats have values greater than 50 and soft wheats less than 50 units. 4 Compares individual line means. 5 Compares group means. View Large Table 1. Puroindoline (PIN) expression, kernel characteristics, and chemical composition (DM basis) of wheat lines varying in PIN expression ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  1 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 2 Level of PINA and PINB relative to the levels in the hard wheat control variety Hi-Line. Data on PINA and PINB levels taken from Hogg et al. (2004). 3 Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL) grain hardness. Hard wheats have values greater than 50 and soft wheats less than 50 units. 4 Compares individual line means. 5 Compares group means. View Large Line × Milling Treatment Experiment Each isoline was milled using 4 treatments. Treatments were selected to give a wide range of mean particle sizes across all lines to simulate as-fed grain and grain after mastication and rumination. Treatments used were: cracked [Bühler mill (Bühler AG, Uzwil, Switzerland) on setting 11.5] coarse, medium, and fine [Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) settings #6, #3, and #0 with standard grinding wheels, respectively]. Geometric mean particle size analysis was conducted according to the method described by Baker and Herrman (2002). Forty grams of each line was milled per treatment. After milling, samples were placed on a series of 5 International Standards Organization sieves. Sieves used were 2,360, 1,700, 850, 425, and 90 μm in screen opening diameter. The sieve stack was shaken for 5 min using a RoTap shaker (Tyler Co., Mentor, OH). Because of the pore size of the bags used for in situ analysis, particles <90 μm were removed from all samples. Geometric mean particle size (dgw) of each line × treatment was calculated on a weight basis of the geometric mean of the diameter openings in 2 adjacent sieves in a stack using the equation (Pfost and Headley, 1976) (dgw) = log−1 [∑ (Wi log di)/∑ Wi] in which Wi = weight of material in sieve i and di = diameter of the sieve i. The geometric mean particle diameter of each line × treatment is given in Table 2. Table 2. Geometric mean particle size, in μm, of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment and time course experiment)   Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45    Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45  1 Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. Particles < 90 microns were discarded. 2 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety, Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG-designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 3 Compares line means within a milling treatment. 4 Compares group means within a milling treatment. View Large Table 2. Geometric mean particle size, in μm, of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment and time course experiment)   Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45    Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45  1 Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. Particles < 90 microns were discarded. 2 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety, Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG-designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 3 Compares line means within a milling treatment. 4 Compares group means within a milling treatment. View Large In situ DM digestibility (DMD) was determined using the Vanzant et al. (1998) method. Duplicate 5-g samples of each line × treatment combination were placed into preweighed and numbered 10- × 20-cm, 50 μm-pore-size polyester bags (Ankom Technology, Macedon, NY). Thirty experimental bags plus 1 standard and 1 blank were placed in the rumen of each of 2 ruminally cannulated cows fed a grain-based diet at the same time. The 2 ruminally cannulated beef cows, each consuming low quality grass hay ad libitum and 3.6 kg/d of dry-rolled barley, were fed for 14 d before being used for the in situ work. Samples were incubated in the rumen. After incubation, bags were hand washed under cold water to stop microbial digestion. Bags were dried for 48 h at 60° C in a forced air oven. The equation DMD, g/kg = [sample weight in * (mean DM value/100)] − (dried sample weight out − dried blank) * 1,000/sample weight in * (mean DM value/100), was used (Bowman et al., 2001). Starch digestibility was determined using the residues of samples incubated in the rumen. For each line × treatment combination, starting starch content was determined after milling with all particles < 90 μm removed by sifting over a 90-μm screen. Final starch content was determined after the completion of the incubation period. The equation starch digestibility, % = [(initial starch weight − ending starch weight)/initial starch weight] * 100, was used with all samples adjusted for DM recovery after rumen incubation. Similar Particle Size Experiment The hardest (Hi-Line) and the softest (HGAB18) lines were milled such that the geometric mean particle diameter of treatments across lines was nearly identical. This was accomplished by collecting fractions of the milled line from each International Standards Organization sieve in the stack. Sieves used were: 3,350, 2,360, 1,700, 850, 425, and 90 μm in screen opening diameter. Particle size ranges were: 2,360 to 3,349 μm, 1,700 to 2,359 μm, 850 to 1,699 μm, 425 to 849 μm, and 90 to 424 μm. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. Dry matter digestibility and starch digestibility for each line × particle size range combination were determined as described in the line × milling treatment experiment. Time Course Experiment Two hundred grams of Hi-Line and HGAB18 were cracked (Bühler mill on setting 11.5). Hi-Line had a mean particle size of 1,729 μm, whereas HGAB18 had a mean particle size of 1,535 μm. Duplicate samples for each line × time period combination were placed in the rumen of each of 2 ruminally cannulated cows at the same time. Duplicate samples for each line were removed from the rumen of each cow after 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 h. In situ DMD and starch digestibility were determined as described for the line × milling treatment experiment. Starch Granule Visualization Using Scanning Electron Microscope Wheat meal (100 mg) ground through a UDY mill (0.5-mm mesh; Seedburo Equipment Co., Chicago, IL) was placed on top of 1 mL of chloroform in a 2.0-mL tube at 22° C. Samples were allowed to sit for 1 h with occasional stirring of the meal with a small spatula. After 1 h, the supernatant and suspended wheat meal were aspirated off, leaving settled starch on the bottom of the tube. The remaining starch granules were washed in acetone and allowed to dry completely. A thin layer of dried starch was attached to aluminum electron microscope pucks with double-sided tape. The puck was coated with gold. Images were generated with a JEOL Model 6100 Scanning Electron Microscope (JEOL U.S.A. Inc., Peabody, MA) at 1,000× magnification (20 kV). Statistical Analysis Data characterizing the initial grain samples for each genotype were analyzed using 1-way analysis of variance. Duplicate samples for DMD and starch digestibility were averaged before analysis. The model for the line × milling experiment was a factorial treatment structure with factors for group, lines within group, milling treatment and their interactions, and cows as blocks. Data obtained for the similar particle size experiment were analyzed using a 2-factor factorial treatment structure with genotypes and particle size category as factors and cows as blocks. Analyses were accomplished using PROC GLM of SAS (SAS Inst., Inc., Cary, NC). The time course experiment was analyzed using a repeated measures model with time as the repeated factor and a first order autoregressive covariance structure using PROC MIXED of SAS following methods outlined by Littell et al. (1998). The DM and starch disappearance, and rate constant (Kd) and lag time of DM and starch disappearance were calculated as described by Bowman and Firkins (1993) using PROC NLIN of SAS. The basic model used was from Mertens and Loften (1980). Dry matter and starch were partitioned into 3 fractions defined as immediately soluble (Fraction A), disappearing at a measurable rate (Fraction B), and undegradable (Fraction C). The rate constant, lag time, and fractions B and C were determined from the nonlinear model, while Fraction A was calculated as (100 − B − C). RESULTS AND DISCUSSION Scanning Electron Microscope Analysis of Starch Granules Barlow et al. (1973) first reported that starch granules from soft and hard wheat varieties differed in the amount of material adhering to their surface after milling with greater adherence seen in hard textured wheats. Beecher et al. (2002) determined that the amount of material adhered to starch was reduced by complementing the pinb-D1b hardness mutant allele with the wild type pinb-D1a, which also restored soft texture. However, the appearance of super-soft wheat starch granules containing overexpressed wild type pina and pinb had never been examined. Figure 1 (Panels A and B) contains scanning electron microscope photos of starch granules prepared from the hard wheat Hi-Line and the super-soft transgenic HGAB18 (samples described in Table 1). Line HGAB18 is a transgenic version of Hi-Line having high expression levels of PINA and PINB (Hogg et al., 2004). Both samples display some type A (large, oblong) and B (smaller, round) starch granules. However, the amount of material adhering to the surface of type A granules is dramatically different between the samples. Hi-Line granules (Figure 1A) are clumped together along with protein bodies, oblong, and rough in texture with cracks on the surface of the large granules. Line HGAB18 granules (Figure 1B) are single and discrete, smooth on the surface, and have virtually no adhering B granules or protein bodies associated with the type A granules. Figure 1. View largeDownload slide Scanning electron microscope analysis of purified starch granules taken at 1000× magnification. A) Hi-Line, hard wheat control. Granules are oblong and rough in texture with cracks on the surface of the large granules. B) HGAB18, super-soft wheat. Granules are single, discrete, and smooth on the surface. C) Hi-Line after incubating in the rumen for 4 h. Granules are clumped together and show widespread signs of pitting from microbial digestion. D) HGAB18 after incubation in the rumen for 4 h. Granules show less severe and less abundant pitting than Hi-line. Arrows in C) and D) mark pitting of starch granules. Figure 1. View largeDownload slide Scanning electron microscope analysis of purified starch granules taken at 1000× magnification. A) Hi-Line, hard wheat control. Granules are oblong and rough in texture with cracks on the surface of the large granules. B) HGAB18, super-soft wheat. Granules are single, discrete, and smooth on the surface. C) Hi-Line after incubating in the rumen for 4 h. Granules are clumped together and show widespread signs of pitting from microbial digestion. D) HGAB18 after incubation in the rumen for 4 h. Granules show less severe and less abundant pitting than Hi-line. Arrows in C) and D) mark pitting of starch granules. Using scanning electron microscope analysis, the protein matrix of corn was observed to limit access of ruminal bacteria to starch granules (McAllister et al., 1991). Comparing corn and barley ruminal starch digestion, McAllister et al. (1993) concluded that structural components associated with or within the endosperm were responsible for the differences in starch digestion. To determine if physical differences in starch granules during digestion could be seen between hard and soft wheats, Hi-Line and HGAB18 were incubated for 4 h in the rumen and then prepared for scanning electron microscope analysis. Hi-Line granules (Figure 1C) after incubation were large and clumped together with the protein matrix, had deep type A pitting, and showed signs of digestion in all type A granules. Line HGAB18 granules (Figure 1D) after incubation were individual, had shallow type A pitting, and showed signs of digestion in only about 1/4 of type A granules. Starch granules from the soft seeds of HGAB18 appeared more resistant to microbial digestion than the starch granules from the hard textured Hi-Line control variety. The differences seen after digestion may be a result of greater damage to the surface of starch granules in Hi-Line vs. HGAB18. Soft wheats fracture easily, resulting in less starch damage after milling than hard wheats (Symes, 1965). The findings here support current thinking that the starch granule surface is likely the site of functional differences between soft and hard wheats. The lack of interaction of the protein matrix with starch granules in HGAB18 indicates that PIN directly control grain softness by reducing the interaction between starch granules and their surrounding protein matrix. Line × Milling Treatment Experiment To examine the effects of added PIN and grain hardness upon DMD and starch digestibility, we chose a subset of the samples examined by Hogg et al. (2004; Table 1). All transgenic lines were made using explant material from the hard red spring variety Hi-Line. The first group (HWC) consisted of the control variety Hi-Line and a Bar control gene only line termed 161. The HGA group consisted of 2 PINA overexpressing lines, and the HGB group contained 2 PINB overexpressing lines. The final group was HGAB, which consisted of 2 transgenic lines, each with high levels of PINA and PINB. The 4 groups with 2 lines per group and with varying expression of the PIN proteins were milled using 4 milling treatments. The 4 milling treatments resulted in average particle sizes after milling that were smaller (P < 0.05) for the medium and coarse milling treatments (Table 2) for the HGA, HGB, and HGAB groups relative to the HWC group. Subsamples of each milling treatment sample were incubated in the rumen for 3 h. Lines within group source of variation was not significant for DMD (P = 0.378) or starch digestibility (P = 0.167), indicating the 2 lines within a group were similar. Milling treatments did interact with groups for DMD (P = 0.002) and starch digestibility (P = 0.002), but milling treatments did not show interactions with lines within group for either DMD (P = 0.879) or starch digestibility (P = 0.977). Ruminal DMD and starch digestibility milling treatment × group combination means are presented in Table 3. The HWC and added PINA groups had the highest DMD values across all milling treatments and were not different from each other except for the coarse milling treatment (P = 0.002). The HGB and HGAB groups were lower (P = 0.001) in DMD than HWC and HGA groups across all milling treatments. The HGAB group tended to be lower than the HGB group, but that difference reached statistical significance (P = 0.002) only in the medium milling treatment. Results for starch digestibility generally mirrored those for DMD with starch digestibility declining as particle size increased. The HWC group had the greatest (P = 0.001) starch digestibility followed by the HGA, HGB, and HGAB groups. Table 3. In situ DM disappearance (DMD) and starch disappearance of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment) Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  r–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications and starch digestibility values are the mean of 4 replications per particle size × PIN treatment. 2 Particle sizes: Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. 3 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. Lines designated HG contain added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. View Large Table 3. In situ DM disappearance (DMD) and starch disappearance of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment) Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  r–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications and starch digestibility values are the mean of 4 replications per particle size × PIN treatment. 2 Particle sizes: Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. 3 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. Lines designated HG contain added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. View Large The HGAB group showed increased expression of both PINA and PINB, the HGB group had increased expression of PINB, and the HGA group had increased expression of PINA. As a result, the HGAB group had a super-soft texture, the HGB group had a soft texture, the HGA group had an intermediate texture, and the HWC had a hard texture (Table 1). The HGA group had a 7.5× increase in PINA and a 28-unit decrease in hardness when compared with the HWC group. The increase in PINA decreased DMD an average of 5.0% and starch digestibility an average of 5.9% across all treatment levels. The HGB group had a 3.9× increase in PINB and a 54.4 unit decrease in hardness when compared with the HWC group. The increased expression of PINB decreased DMD an average of 22.7% and starch digestibility an average of 22.9% across all treatment levels. The HGAB group had a 6.5× increase in PINA, a 4.4× increase in PINB, and a 58.7 unit decrease in grain hardness when compared with the HWC group. The increased expression of PIN decreased DMD an average of 29.2% and decreased starch digestibility an average of 30.8% across all treatment levels. The line × milling treatment experiment indicated that PIN content affected the rate of wheat starch digestion in the rumen. Increased expression of PINB and both PINA and PINB led to a significant reduction in DMD and starch digestibility across all milling treatments. The largest reduction was achieved by the addition of PINA and PINB. A potential complicating factor in this study is the effect of grain texture on particle size. Soft wheats, having a softer endosperm, fracture easily, requiring less energy to mill than hard wheats (Symes, 1965, 1969). As a result, soft wheats yield smaller particles on the same mill setting, suggesting that the effect of PIN upon digestibility may reflect both particle size variation and PIN expression variation. Similar Particle Size Experiment To separate the effect of particle size and PIN content on DMD and starch digestibility, Hi-Line (hardest) and HGAB18 (softest) were milled such that the mean particle size per treatment was nearly identical, and incubated in the rumen for 3 h. Dry matter digestibility and starch digestibility results are presented in Table 4. A line × particle size treatment interaction was detected (P = 0.001) indicating that lines did not react similarly across particle size treatments. Line HGAB18 had lower DMD than Hi-Line (P = 0.001) among particles ranging in size from 0.09 μm to 2.35 μm. No differences (P = 0.94) in DMD were seen in particles above 2.36 μm in size. Starch digestibility declined with increasing particle size, but HGAB18 also had lower starch digestibility than Hi-Line (P = 0.001) at particles sizes ranging from 0.09 μm to 2.35 μm. No differences (P = 0.85) in starch digestibility were seen in particles above 2.36 μm in size. For wheat, particles above 2.36 μm are generally whole kernels. Seeing no differences between whole kernels of Hi-Line and HGAB18 indicated that PIN proteins had no affect on the seed coat or aleurone layers of the kernel and that differences in DMD and starch digestibility for small particles was a function of PIN interaction with starch. The increased PINA and PINB expression in HGAB18 decreased DMD an average of 21.7% and starch digestibility an average of 19.9% across particle sizes smaller than whole kernel (< 2.36 mm). This experiment indicated that increased PIN expression decreased DMD and starch digestibility, and was largely independent of particle size. Table 4. In situ DM disappearance (DMD) and starch disappearance of 2 wheat lines varying in puroindoline expression and milled to specific particle size ranges Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  t–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications, and starch digestibility values are the mean of 4 replications per line × particle size treatment. 2 Genotypes were the hard red spring variety Hi-Line and HGAB18, which is Hi-Line transformed to have high levels of PINA and PINB expression. 3 Particle size ranges were adjusted to the stated size ranges by sifting after milling using the fine (for 0.09 to 0.424 mm), coarse (for 0.425 to 0.849 and 0.85 to 1.69 mm), and cracked (for 1.7 to 2.35 mm) mill settings. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. View Large Table 4. In situ DM disappearance (DMD) and starch disappearance of 2 wheat lines varying in puroindoline expression and milled to specific particle size ranges Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  t–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications, and starch digestibility values are the mean of 4 replications per line × particle size treatment. 2 Genotypes were the hard red spring variety Hi-Line and HGAB18, which is Hi-Line transformed to have high levels of PINA and PINB expression. 3 Particle size ranges were adjusted to the stated size ranges by sifting after milling using the fine (for 0.09 to 0.424 mm), coarse (for 0.425 to 0.849 and 0.85 to 1.69 mm), and cracked (for 1.7 to 2.35 mm) mill settings. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. View Large Time Course Experiment To investigate the effects of time in the rumen on PIN proteins, Hi-Line and HGAB18 were cracked, and disappearance was observed at various time points over a 12-h period in the rumen. Dry matter and starch digestibility results are presented in Figure 2 and Table 5. Dry matter digestibility increased over time for both lines. Line HGAB18 was consistently lower in DMD than Hi-Line across all time points. However, the difference between lines became less with time after 4 h leading to a line × time interaction (P = 0.001). Similarly, starch digestibility increased over time with HGAB18 being lower than Hi-Line. Again, the difference between HGAB18 and Hi-Line became less with time giving rise to a line × time interaction (P = 0.01). Increased expression of PINA and PINB in HGAB18 decreased DMD by 10.4% and starch digestibility by 11.0% across all time points. Figure 2. View largeDownload slide In situ dry matter (DM) and starch disappearance (0 to 12 h) of Hi-Line (•) and HGAB18 (▪), 2 wheat lines varying in puroindoline expression. A) DM disappearance (%), and B) starch disappearance (%). Wheat line × time interactions were significant for DM (P = 0.002) and starch disappearance (P = 0.006). Within a time period, means without a common superscript letter differ (P < 0.05). Figure 2. View largeDownload slide In situ dry matter (DM) and starch disappearance (0 to 12 h) of Hi-Line (•) and HGAB18 (▪), 2 wheat lines varying in puroindoline expression. A) DM disappearance (%), and B) starch disappearance (%). Wheat line × time interactions were significant for DM (P = 0.002) and starch disappearance (P = 0.006). Within a time period, means without a common superscript letter differ (P < 0.05). Table 5. Characteristics of ruminal in situ disappearance (0 to 12 h) of DM and starch of 2 wheat lines varying in puroindoline expression (time course experiment) Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  1 Hi-Line = untransformed hard red spring control wheat; HGAB18 = Hi-Line transformed to overexpress PINA and PINB and have very soft endosperm texture. 2 Fraction A: immediately soluble fraction. 3 Fraction B: fraction disappearing at a measurable rate. 4 Fraction C: undegraded fraction. 5 Kd: disappearance rate. View Large Table 5. Characteristics of ruminal in situ disappearance (0 to 12 h) of DM and starch of 2 wheat lines varying in puroindoline expression (time course experiment) Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  1 Hi-Line = untransformed hard red spring control wheat; HGAB18 = Hi-Line transformed to overexpress PINA and PINB and have very soft endosperm texture. 2 Fraction A: immediately soluble fraction. 3 Fraction B: fraction disappearing at a measurable rate. 4 Fraction C: undegraded fraction. 5 Kd: disappearance rate. View Large Line HGAB18 had a 27.9% lower (P = 0.001) immediately soluble fraction of DM than did Hi-Line, and a 24.5% slower (P = 0.003) DM disappearance rate (Table 5). The immediately soluble fraction of starch tended (P = 0.118) to be lower for HGAB18 than for Hi-Line, and HGAB18 had a 30.2% slower (P = 0.003) starch disappearance rate. In addition, HGAB18 had a lower (P = 0.050) undegraded starch fraction compared with Hi-Line, indicating that slowing starch digestibility via increasing PIN expression will not necessarily reduce extent of starch digestion. The rate of passage of feedstuffs out of the rumen is affected by specific gravity and particle size. Grains, although rapidly fermented, are small and dense, and thus pass rapidly compared with roughages (Firkins et al., 2001). The time course experiment demonstrated that a difference in starch digestibility could still be seen after 6 h in the rumen, indicating that starch supply to the small intestine may be increased with increased PIN levels. Differences in starch digestibility after incubation suggest that starch granules of HGAB18 possess a type of protection that Hi-Line does not. Being isogenic lines, expression of PIN proteins is the only difference between Hi-Line and HGAB18. The time course experiment led to the conclusion that PIN proteins aid in the protection of starch molecules from ruminal fermentation. Conclusions Comparisons between cereal species have shown that wheat starch is fermented rapidly in the rumen when compared with barley, maize, and sorghum (Herrera-Saldana et al., 1990; Owens et al., 1997). Research also has shown that variations in starch digestion exist between cultivars of the same species (Philippeau et al., 1999; Bowman et al., 2001). However, previous research has relied on the use of grains within the same market class or named varieties for comparison. Genetic factors controlling starch digestion cannot be determined by such comparisons due to the diversity of genetic backgrounds. Little work has been done with near-isogenic or transformed isolines. The use of such genetic material allows the nutritional implication of specific traits to be evaluated against a consistent genetic background. At least 2 studies in wheat have been done with near-isogenic lines. Short et al. (2000) studied the effects of grain hardness on AA digestion in poultry. They indicated that hard wheat endosperm was associated with decreased AA digestibility. Chickens, being a nonruminant animal, cannot be easily compared with ruminants and generally are considered to be particularly sensitive to changes in quality of the diet. Garnsworthy and Wiseman (2000) used near isogenic lines to evaluate the ruminal digestibility of wheat starch; no differences were seen between hard and soft wheats. However, actual grain hardness and PIN content were not stated. Typical soft wheats have at most twice the PIN content of typical hard wheats (Giroux and Morris, 1997; 1998). One may therefore expect that differences due to PIN content are too small to be detected among native wheat varieties. Overall, our present experiment demonstrated that PIN proteins affected DM and starch digestibility of wheat in the rumen. Decreasing wheat grain hardness by increasing PIN expression slowed DMD and starch digestibility in the rumen and was largely independent of particle size. Data indicated that PIN proteins aid in the protection of starch molecules from fermentation type digestion in the rumen. In barley, low in situ DMD values are correlated with increased feed efficiency, increased ADG, and increased NE content (Bowman et al., 2001). Slower or lower ruminally digestible starch shifts more starch digestion from the rumen to the small intestine. Starch digestion in the small intestine theoretically could provide up to 42% more energy than starch fermented in the rumen (Owens et al., 1986) because of reduction in energy loss via methane production and more efficient use of glucose as an energy source compared with VFA. In addition, lower DMD could reduce excessive fermentation acid production and reduce the incidence of bloat, acidosis, and laminitis (Hunt, 1996). IMPLICATIONS Although wheat is not a predominant cattle feed in the United States, it is an ideal model system to study the effect of grain hardness and puroindoline content on digestion in the rumen. In wheat, the puroindoline genes control the majority of grain hardness variation. Isolines that differed only in puroindoline content illustrated that the presence of additional puroindoline proteins slowed the digestion of starch. Using current transformation methods, it should be possible to reduce the rate of starch digestion in other cereals such as barley via puroindoline overexpression. Barley is an attractive cereal in which to conduct further research because no true soft barleys seem to exist, nor are there any barley varieties that have soft wheat levels of friabilin bound to starch. Perhaps addition of puroindoline proteins to highly fermentable feeds such as oats and barley would decrease the rate of digestion and increase feed efficiency and beef cattle performance. LITERATURE CITED AACC 2000. Approved Methods of the American Association of Cereal Chemists.  10th ed. Am. Assoc. Cereal Chem., St. Paul, MN. AOAC 2000. Official Methods of Analysis. 17th ed. Assoc. Offic. Anal. Chem., Gaithersburg, MD. Baker, S., and T. Herrman 2002. Evaluating Particle Size.  Kansas State Univ. Ag. Exp. Sta. Bull. No. MF-2051, Manhattan, KS. Barlow, K. K., M. S. Buttrose, D. H. Simmonds, and M. Vesk 1973. The nature of the starch-protein interface in wheat endosperm. Cereal Chem.  50: 443– 454. Beecher, B., A. Bettge, E. Smidansky, and M. J. Giroux 2002. Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype. Theor. Appl. Genet.  105: 870– 877. Google Scholar CrossRef Search ADS PubMed  Bowman, J. G. P., T. K. Blake, L. M. M. Surber, D. K. Habernicht, and H. Bockelman 2001. Feed-quality variation in the barley core collection of the USDA National Small Grains Collection. Crop Sci.  41: 863– 870. Google Scholar CrossRef Search ADS   Bowman, J. G. P., and J. L. Firkins 1993. Effects of forage species and particle size on bacterial cellulolytic activity and colonization in situ. J. Anim. Sci.  71: 1623– 1633. Google Scholar CrossRef Search ADS PubMed  Firkins, J. L., M. L. Eastridge, N. R. St-Pierre, and S. M. Noftsger 2001. Effects of grain variability and processing on starch utilization by lactating dairy cattle. J. Dairy Sci.  79(E. Sup-pl.): E218– E238. Garnsworthy, P. C., and J. Wiseman 2000. Rumen digestibility of starch and nitrogen in near-isogenic lines of wheat. Anim. Feed Sci. Technol.  85: 33– 40. Google Scholar CrossRef Search ADS   Gautier, M. F., M. E. Aleman, A. Guirao, D. Marion, and P. Joudrier 1994. Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression. Plant Mol. Biol.  25: 43– 57. Google Scholar CrossRef Search ADS PubMed  Giroux, M. J., and C. F. Morris 1997. A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin. Theor. Appl. Genet.  95: 857– 864. Google Scholar CrossRef Search ADS   Giroux, M. J., and C. F. Morris 1998. Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b. Proc. Natl. Acad. Sci. USA  95: 6262– 6266. Google Scholar CrossRef Search ADS   Giroux, M. J., L. Talbert, D. K. Habernicht, S. Lanning, A. Hemphill, and J. M. Martin 2000. Association of puroindoline sequence type and grain hardness in hard red spring wheat. Crop Sci.  40: 370– 374. Google Scholar CrossRef Search ADS   Herrera-Saldana, R. E., J. T. Huber, and M. H. Poore 1990. Dry matter, crude protein, and starch degradability of five cereal grains. J. Dairy Sci.  73: 2386– 2393. Google Scholar CrossRef Search ADS   Hogg, A. C., T. Sripo, B. Beecher, J. M. Martin, and M. J. Giroux 2004. Wheat puroindolines interact to form friabilin and control wheat grain hardness. Theor. Appl. Genet.  108: 1089– 1097. Google Scholar CrossRef Search ADS PubMed  Hunt, C. W. 1996. Factors affecting the feeding quality of barley for ruminants. Anim. Feed Sci. Technol.  62: 37– 48. Google Scholar CrossRef Search ADS   Lanning, S. P., L. E. Talbert, F. H. McNeal, W. L. Alexander, C. F. McGuire, H. Bowman, G. Carlson, G. Jackson, J. Eckhoff, G. Kushnak, V. Stewart, and G. Stallknecht 1992. Registration of ‘Hi-Line’ wheat. Crop Sci.  32: 283– 284. Google Scholar CrossRef Search ADS   Littell, R. C., P. R. Henry, and C. B. Ammerman 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci.  76: 1216– 1231. Google Scholar CrossRef Search ADS PubMed  McAllister, T. A., R. C. Phillippe, L. M. Rode, and K. J. Cheng 1993. Effect of the protein matrix on the digestion of cereal grains by ruminal microorganisms. J. Anim. Sci.  71: 205– 212. Google Scholar CrossRef Search ADS PubMed  McAllister, T. A., L. M. Rode, K. J. Cheng, and C. W. Forsberg 1991. Selection of a sterilization method of the study of cereal grain digestion. J. Anim. Sci.  69: 3039– 3043. Google Scholar CrossRef Search ADS PubMed  Mertens, D. R., and J. R. Loften 1980. The effect of starch on forage fiber digestion kinetics in vitro. J. Dairy Sci.  63: 1437– 1446. Google Scholar CrossRef Search ADS PubMed  Morris, C. F., M. Lillemo, G. M. Simeone, M. J. Giroux, S. L. Babb, and K. K. Kidwell 2001. Prevalence of puroindoline grain hardness genotypes among historically significant North American spring and winter wheats. Crop Sci.  41: 218– 228. Google Scholar CrossRef Search ADS   Owens, F. N., D. S. Secrist, W. J. Hill, and D. R. Gill 1997. The effect of grain source and grain processing on performance of feedlot cattle: A review. J. Anim. Sci.  75: 868– 879. Google Scholar CrossRef Search ADS PubMed  Owens, F. N., R. A. Zinn, and Y. K. Kim 1986. Limits to starch digestion in the ruminant small intestine. J. Anim. Sci.  63: 1634– 1648. Google Scholar CrossRef Search ADS PubMed  Pfost, H., and V. Headley 1976. Methods of determining and expressing particle size. Page 517 in Feed Manufacturing Technology II.  H. Pfost ed. Anim. Feed Manuf. Assoc., Arlington, VA. Philippeau, C., F. Le Deschault de Monredon, and B. Michalet-Doreau 1999. Relationship between ruminal starch degradation and the physical characteristics of corn grain. J. Anim. Sci.  77: 238– 243. Google Scholar CrossRef Search ADS PubMed  Short, F. J., J. Wiseman, and K. N. Boorman 2000. The effect of the 1B/1R translocation and endosperm texture on amino acid digestibility in near-isogenic lines of wheat for broilers. J. Agric. Sci.  134: 69– 76. Google Scholar CrossRef Search ADS   Symes, K. J. 1965. The inheritance of grain hardness in wheat as measured by particle size index. Aust. J. Agric. Res.  16: 113– 123. Google Scholar CrossRef Search ADS   Symes, K. J. 1969. Influence of a gene causing hardness on the milling and baking quality of two wheats. Aust. J. Agric. Res.  20: 971– 979. Google Scholar CrossRef Search ADS   Van Soest, P. J., J. B. Robertson, and B. A. Lewis 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci.  74: 3583– 3597. Google Scholar CrossRef Search ADS PubMed  Vanzant, E. S., R. C. Cochran, and E. C. Titgemeyer 1998. Standardization of in situ techniques for ruminal feedstuff evaluation. J. Anim. Sci.  76: 2717– 2729. Google Scholar CrossRef Search ADS PubMed  Copyright 2006 Journal of Animal Science http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science Oxford University Press

Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by cattle

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Copyright 2006 Journal of Animal Science
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0021-8812
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1525-3163
DOI
10.2527/2006.843641x
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Abstract

ABSTRACT Starch is the primary nutrient in ruminant diets used to promote high levels of performance. The site of starch digestion alters the nature of digestive end products (VFA in the rumen vs. glucose in the small intestine) and the efficiency of use. Cereal grain endosperm texture plays a major role in the rate and extent of starch degradation in ruminants. Wheat grain texture is regulated by the starch surface protein complex friabilin that consists primarily of puroindoline (PIN) A and B. Soft kernel texture in wheat is a result of both PIN genes being in the wild type active form and bound to starch. The objective of this study was to investigate the effect of varying PIN content in wheat on the rate of starch digestion in the rumen of beef cattle. In Exp. 1, 6 transgenic soft pin a/b isolines created in a hard wheat background, and 2 hard wheat controls were milled to yield a wide range of mean particle sizes across all lines. Milled samples were incubated in situ for 3 h. Increased expression of both PINA and PINB decreased DM digestibility (DMD) by 29.2% (P < 0.05) and decreased starch digestibility by 30.8% (P < 0.05). Experiment 2 separated the effects of particle size and total PIN content on digestion by milling the hardest and softest lines such that the mean particle size was nearly identical. Increased PIN decreased DMD by 21.7% (P < 0.05) and starch digestibility by 19.9% (P < 0.05) across particle sizes smaller than whole kernel. Experiment 3 addressed the time course of PIN effects in the rumen by observing ground samples of the hardest and softest lines over a 12-h in situ period. Increased PIN decreased DMD by 10.4% (P < 0.05) and starch digestibility by 11.0% (P < 0.05) across all time points. Dry matter and starch digestibility results demonstrated that increased expression of PIN was associated with a decreased rate of ruminal digestion independent of particle size. Puroindolines seem to aid in the protection of starch molecules from microbial digestion in the rumen, potentially increasing the amount of starch entering the small intestine. INTRODUCTION Cereal grains are typically fed to ruminants to increase starch intake. The site of starch absorption along the gastrointestinal tract affects cattle performance and feed efficiency. Slower rates of digestion increase the amount of starch bypassing the rumen. Starch digested in the small intestine can produce up to 42% more energy than fermentation (Owens et al., 1986) because of a more efficient use of digestive end products (glucose vs. VFA). Grain texture plays a major role in the rate and location of starch digestion in ruminants (Philippeau et al., 1999). Small grains (wheat, barley, or oats) are more rapidly fermented than corn and sorghum. Variations in starch granule structure among species of cereal grains may account for distinct rates of digestion patterns. Protein and structural carbohydrates within the cereal kernel may be more important in determining the extent of ruminal starch digestion than the starch type (McAllister et al., 1993). Wheat (Triticum aestivum L.) grain hardness is determined by the degree of adhesion between starch granules and the protein matrix, regulated by the protein complex friabilin. Friabilin, isolated from the surface of starch granules, contains 2 major proteins, puroindolines (PIN) A and B. Puroindolines contain a unique tryptophan-rich domain believed to be involved in their binding to the phospholipids of starch granules (Gautier et al., 1994). Soft wheat results from both pin genes being in the wild type form, whereas hard wheats have an absence or alteration in either pin gene (Morris et al., 2001). Hard wheat transformed with added wild type pina, pinb, or pina and b resulted in isolines with a wide range of grain textures (Hogg et al., 2004). Transgenic PIN experiments have demonstrated that wheat with high levels of one PIN is intermediate in grain texture, and if both PIN are active, then soft wheat texture results (Hogg et al., 2004). The objective of these studies was to investigate the effect of varying PIN content and particle size in wheat on the rate of digestion in the rumen by using PIN isolines that varied only in PIN content. MATERIALS AND METHODS Genetic Material The hard red spring wheat cultivar Hi-Line (Lanning et al., 1992) was used for transformation. Native Hi-Line contains the soft type pina sequence (pina-D/a) and the variant pinb sequence (pinB-D1b), which contains a glycine to serine substitution at the 46th residue of pinb (Giroux et al., 2000). Hi-Line was transformed with vectors containing wild type pina, pinb or both pina and pinb (pin). Pin were under the control of the wheat glutenin promoter (Hogg et al., 2004). Isolines with a wide range of grain textures resulted, and a subset of the lines presented in Hogg et al. (2004) were chosen for this study. Two lines with added pina (HGA1, HGA3) formed the intermediate grain texture HGA group; 2 lines with added pinb (HGB6, HGB12) formed the soft HGB group; 2 lines with added pina and pinb (HGAB12, HGAB18) formed the very soft HGAB group; and 2 hard wheats [native Hi-line and Hi-Line transformed with only bar (line 161 of Hogg et al., 2004)] formed the hard wheat controls (HWC). Seeds used in the study were obtained from a single 4-row plot grown during the 2003 season at the Montana State University-Bozeman Arthur H. Post Field Research Farm under dry-land conditions. Isoline Characterization Isolines used were analyzed for common feed and grain characteristics (Table 1) to demonstrate that lines were nearly identical except for the presence of the pin transgene(s) dictating PIN content and grain hardness. Three independent 100-seed replicate samples per line were analyzed for kernel hardness, kernel weight, and kernel size using the Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL). Dry matter content of each line × treatment was determined using AOAC method 930.15 (2000) for oven drying and replicated twice. Acid detergent fiber for each line was determined using the protocol described by Van Soest et al. (1991) and replicated twice. Crude protein for each line was measured by AACC method 46-30 (2000) using a LECO FP-328 nitrogen analyzer (LECO Corporation, St. Joseph, MI) and replicated 4 times. Starch content for each line was determined using a modified protocol of the Megazyme total starch assay kit (Megazyme International, Brey, Ireland) and was replicated 4 times. Table 1. Puroindoline (PIN) expression, kernel characteristics, and chemical composition (DM basis) of wheat lines varying in PIN expression ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  1 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 2 Level of PINA and PINB relative to the levels in the hard wheat control variety Hi-Line. Data on PINA and PINB levels taken from Hogg et al. (2004). 3 Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL) grain hardness. Hard wheats have values greater than 50 and soft wheats less than 50 units. 4 Compares individual line means. 5 Compares group means. View Large Table 1. Puroindoline (PIN) expression, kernel characteristics, and chemical composition (DM basis) of wheat lines varying in PIN expression ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  ID1  PINA level2  PINB level2  SKCS grain hardness3  Kernel diameter,3 mm  Kernel wt,3 mg  ADF, %  Protein, %  Starch, %  161  1.00  1.00  75.97  2.19  26.55  4.16  18.34  61.00  Hi-Line  1.00  1.00  78.86  2.05  25.10  4.79  18.14  60.62      HWC avg  1.00  1.00  77.41  2.12  25.82  4.48  18.24  60.80  HGA1  7.00  1.00  55.36  2.25  27.93  4.27  17.11  58.52  HGA3  8.00  1.00  41.48  2.00  24.85  4.78  18.52  60.31      HGA avg  7.50  1.00  48.42  2.12  26.39  4.52  17.82  59.42  HGB6  1.00  3.75  26.88  1.90  23.99  4.84  18.50  60.24  HGB12  1.00  4.00  19.10  2.08  24.88  4.73  18.00  61.96      HGB avg  1.00  3.88  22.99  1.99  24.44  4.78  18.25  61.10  HGAB12  5.75  3.75  21.43  1.98  25.52  4.94  19.66  59.27  HGAB18  7.25  5.00  15.93  1.94  24.71  4.61  19.85  61.05      HGAB avg  6.50  4.38  18.68  1.96  25.12  4.78  19.75  60.16  LSD4 (0.05)  0.41  0.41  3.27  0.10  2.69  0.13  0.02  0.36  LSD5 (0.05)  0.29  0.29  2.31  0.07  1.91  0.09  0.01  0.26  1 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 2 Level of PINA and PINB relative to the levels in the hard wheat control variety Hi-Line. Data on PINA and PINB levels taken from Hogg et al. (2004). 3 Single Kernel Characterization System 4100 (Perten Instruments, Springfield, IL) grain hardness. Hard wheats have values greater than 50 and soft wheats less than 50 units. 4 Compares individual line means. 5 Compares group means. View Large Line × Milling Treatment Experiment Each isoline was milled using 4 treatments. Treatments were selected to give a wide range of mean particle sizes across all lines to simulate as-fed grain and grain after mastication and rumination. Treatments used were: cracked [Bühler mill (Bühler AG, Uzwil, Switzerland) on setting 11.5] coarse, medium, and fine [Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) settings #6, #3, and #0 with standard grinding wheels, respectively]. Geometric mean particle size analysis was conducted according to the method described by Baker and Herrman (2002). Forty grams of each line was milled per treatment. After milling, samples were placed on a series of 5 International Standards Organization sieves. Sieves used were 2,360, 1,700, 850, 425, and 90 μm in screen opening diameter. The sieve stack was shaken for 5 min using a RoTap shaker (Tyler Co., Mentor, OH). Because of the pore size of the bags used for in situ analysis, particles <90 μm were removed from all samples. Geometric mean particle size (dgw) of each line × treatment was calculated on a weight basis of the geometric mean of the diameter openings in 2 adjacent sieves in a stack using the equation (Pfost and Headley, 1976) (dgw) = log−1 [∑ (Wi log di)/∑ Wi] in which Wi = weight of material in sieve i and di = diameter of the sieve i. The geometric mean particle diameter of each line × treatment is given in Table 2. Table 2. Geometric mean particle size, in μm, of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment and time course experiment)   Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45    Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45  1 Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. Particles < 90 microns were discarded. 2 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety, Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG-designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 3 Compares line means within a milling treatment. 4 Compares group means within a milling treatment. View Large Table 2. Geometric mean particle size, in μm, of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment and time course experiment)   Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45    Milling treatment1  ID2  Fine  Medium  Coarse  Cracked  161  161  426  829  1,717  Hi-Line  169  442  839  1,729      HWC avg  165  434  834  1,721  HGA1  144  366  772  1,688  HGA3  141  358  756  1,674      HGA avg  143  362  764  1,681  HGB6  127  318  613  1,586  HGB12  120  315  610  1,581      HGB avg  124  312  607  1,583  HGAB12  113  304  596  1,567  HGAB18  101  276  574  1,535      HGAB avg  107  290  584  1,551  LSD3 (0.05)  64  64  64  64  LSD4 (0.05)  45  45  45  45  1 Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. Particles < 90 microns were discarded. 2 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety, Hi-Line. HWC = hard wheat controls. All lines contain the Bar marker gene, with 161 containing only Bar, and all other HG-designated lines containing added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. 3 Compares line means within a milling treatment. 4 Compares group means within a milling treatment. View Large In situ DM digestibility (DMD) was determined using the Vanzant et al. (1998) method. Duplicate 5-g samples of each line × treatment combination were placed into preweighed and numbered 10- × 20-cm, 50 μm-pore-size polyester bags (Ankom Technology, Macedon, NY). Thirty experimental bags plus 1 standard and 1 blank were placed in the rumen of each of 2 ruminally cannulated cows fed a grain-based diet at the same time. The 2 ruminally cannulated beef cows, each consuming low quality grass hay ad libitum and 3.6 kg/d of dry-rolled barley, were fed for 14 d before being used for the in situ work. Samples were incubated in the rumen. After incubation, bags were hand washed under cold water to stop microbial digestion. Bags were dried for 48 h at 60° C in a forced air oven. The equation DMD, g/kg = [sample weight in * (mean DM value/100)] − (dried sample weight out − dried blank) * 1,000/sample weight in * (mean DM value/100), was used (Bowman et al., 2001). Starch digestibility was determined using the residues of samples incubated in the rumen. For each line × treatment combination, starting starch content was determined after milling with all particles < 90 μm removed by sifting over a 90-μm screen. Final starch content was determined after the completion of the incubation period. The equation starch digestibility, % = [(initial starch weight − ending starch weight)/initial starch weight] * 100, was used with all samples adjusted for DM recovery after rumen incubation. Similar Particle Size Experiment The hardest (Hi-Line) and the softest (HGAB18) lines were milled such that the geometric mean particle diameter of treatments across lines was nearly identical. This was accomplished by collecting fractions of the milled line from each International Standards Organization sieve in the stack. Sieves used were: 3,350, 2,360, 1,700, 850, 425, and 90 μm in screen opening diameter. Particle size ranges were: 2,360 to 3,349 μm, 1,700 to 2,359 μm, 850 to 1,699 μm, 425 to 849 μm, and 90 to 424 μm. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. Dry matter digestibility and starch digestibility for each line × particle size range combination were determined as described in the line × milling treatment experiment. Time Course Experiment Two hundred grams of Hi-Line and HGAB18 were cracked (Bühler mill on setting 11.5). Hi-Line had a mean particle size of 1,729 μm, whereas HGAB18 had a mean particle size of 1,535 μm. Duplicate samples for each line × time period combination were placed in the rumen of each of 2 ruminally cannulated cows at the same time. Duplicate samples for each line were removed from the rumen of each cow after 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 h. In situ DMD and starch digestibility were determined as described for the line × milling treatment experiment. Starch Granule Visualization Using Scanning Electron Microscope Wheat meal (100 mg) ground through a UDY mill (0.5-mm mesh; Seedburo Equipment Co., Chicago, IL) was placed on top of 1 mL of chloroform in a 2.0-mL tube at 22° C. Samples were allowed to sit for 1 h with occasional stirring of the meal with a small spatula. After 1 h, the supernatant and suspended wheat meal were aspirated off, leaving settled starch on the bottom of the tube. The remaining starch granules were washed in acetone and allowed to dry completely. A thin layer of dried starch was attached to aluminum electron microscope pucks with double-sided tape. The puck was coated with gold. Images were generated with a JEOL Model 6100 Scanning Electron Microscope (JEOL U.S.A. Inc., Peabody, MA) at 1,000× magnification (20 kV). Statistical Analysis Data characterizing the initial grain samples for each genotype were analyzed using 1-way analysis of variance. Duplicate samples for DMD and starch digestibility were averaged before analysis. The model for the line × milling experiment was a factorial treatment structure with factors for group, lines within group, milling treatment and their interactions, and cows as blocks. Data obtained for the similar particle size experiment were analyzed using a 2-factor factorial treatment structure with genotypes and particle size category as factors and cows as blocks. Analyses were accomplished using PROC GLM of SAS (SAS Inst., Inc., Cary, NC). The time course experiment was analyzed using a repeated measures model with time as the repeated factor and a first order autoregressive covariance structure using PROC MIXED of SAS following methods outlined by Littell et al. (1998). The DM and starch disappearance, and rate constant (Kd) and lag time of DM and starch disappearance were calculated as described by Bowman and Firkins (1993) using PROC NLIN of SAS. The basic model used was from Mertens and Loften (1980). Dry matter and starch were partitioned into 3 fractions defined as immediately soluble (Fraction A), disappearing at a measurable rate (Fraction B), and undegradable (Fraction C). The rate constant, lag time, and fractions B and C were determined from the nonlinear model, while Fraction A was calculated as (100 − B − C). RESULTS AND DISCUSSION Scanning Electron Microscope Analysis of Starch Granules Barlow et al. (1973) first reported that starch granules from soft and hard wheat varieties differed in the amount of material adhering to their surface after milling with greater adherence seen in hard textured wheats. Beecher et al. (2002) determined that the amount of material adhered to starch was reduced by complementing the pinb-D1b hardness mutant allele with the wild type pinb-D1a, which also restored soft texture. However, the appearance of super-soft wheat starch granules containing overexpressed wild type pina and pinb had never been examined. Figure 1 (Panels A and B) contains scanning electron microscope photos of starch granules prepared from the hard wheat Hi-Line and the super-soft transgenic HGAB18 (samples described in Table 1). Line HGAB18 is a transgenic version of Hi-Line having high expression levels of PINA and PINB (Hogg et al., 2004). Both samples display some type A (large, oblong) and B (smaller, round) starch granules. However, the amount of material adhering to the surface of type A granules is dramatically different between the samples. Hi-Line granules (Figure 1A) are clumped together along with protein bodies, oblong, and rough in texture with cracks on the surface of the large granules. Line HGAB18 granules (Figure 1B) are single and discrete, smooth on the surface, and have virtually no adhering B granules or protein bodies associated with the type A granules. Figure 1. View largeDownload slide Scanning electron microscope analysis of purified starch granules taken at 1000× magnification. A) Hi-Line, hard wheat control. Granules are oblong and rough in texture with cracks on the surface of the large granules. B) HGAB18, super-soft wheat. Granules are single, discrete, and smooth on the surface. C) Hi-Line after incubating in the rumen for 4 h. Granules are clumped together and show widespread signs of pitting from microbial digestion. D) HGAB18 after incubation in the rumen for 4 h. Granules show less severe and less abundant pitting than Hi-line. Arrows in C) and D) mark pitting of starch granules. Figure 1. View largeDownload slide Scanning electron microscope analysis of purified starch granules taken at 1000× magnification. A) Hi-Line, hard wheat control. Granules are oblong and rough in texture with cracks on the surface of the large granules. B) HGAB18, super-soft wheat. Granules are single, discrete, and smooth on the surface. C) Hi-Line after incubating in the rumen for 4 h. Granules are clumped together and show widespread signs of pitting from microbial digestion. D) HGAB18 after incubation in the rumen for 4 h. Granules show less severe and less abundant pitting than Hi-line. Arrows in C) and D) mark pitting of starch granules. Using scanning electron microscope analysis, the protein matrix of corn was observed to limit access of ruminal bacteria to starch granules (McAllister et al., 1991). Comparing corn and barley ruminal starch digestion, McAllister et al. (1993) concluded that structural components associated with or within the endosperm were responsible for the differences in starch digestion. To determine if physical differences in starch granules during digestion could be seen between hard and soft wheats, Hi-Line and HGAB18 were incubated for 4 h in the rumen and then prepared for scanning electron microscope analysis. Hi-Line granules (Figure 1C) after incubation were large and clumped together with the protein matrix, had deep type A pitting, and showed signs of digestion in all type A granules. Line HGAB18 granules (Figure 1D) after incubation were individual, had shallow type A pitting, and showed signs of digestion in only about 1/4 of type A granules. Starch granules from the soft seeds of HGAB18 appeared more resistant to microbial digestion than the starch granules from the hard textured Hi-Line control variety. The differences seen after digestion may be a result of greater damage to the surface of starch granules in Hi-Line vs. HGAB18. Soft wheats fracture easily, resulting in less starch damage after milling than hard wheats (Symes, 1965). The findings here support current thinking that the starch granule surface is likely the site of functional differences between soft and hard wheats. The lack of interaction of the protein matrix with starch granules in HGAB18 indicates that PIN directly control grain softness by reducing the interaction between starch granules and their surrounding protein matrix. Line × Milling Treatment Experiment To examine the effects of added PIN and grain hardness upon DMD and starch digestibility, we chose a subset of the samples examined by Hogg et al. (2004; Table 1). All transgenic lines were made using explant material from the hard red spring variety Hi-Line. The first group (HWC) consisted of the control variety Hi-Line and a Bar control gene only line termed 161. The HGA group consisted of 2 PINA overexpressing lines, and the HGB group contained 2 PINB overexpressing lines. The final group was HGAB, which consisted of 2 transgenic lines, each with high levels of PINA and PINB. The 4 groups with 2 lines per group and with varying expression of the PIN proteins were milled using 4 milling treatments. The 4 milling treatments resulted in average particle sizes after milling that were smaller (P < 0.05) for the medium and coarse milling treatments (Table 2) for the HGA, HGB, and HGAB groups relative to the HWC group. Subsamples of each milling treatment sample were incubated in the rumen for 3 h. Lines within group source of variation was not significant for DMD (P = 0.378) or starch digestibility (P = 0.167), indicating the 2 lines within a group were similar. Milling treatments did interact with groups for DMD (P = 0.002) and starch digestibility (P = 0.002), but milling treatments did not show interactions with lines within group for either DMD (P = 0.879) or starch digestibility (P = 0.977). Ruminal DMD and starch digestibility milling treatment × group combination means are presented in Table 3. The HWC and added PINA groups had the highest DMD values across all milling treatments and were not different from each other except for the coarse milling treatment (P = 0.002). The HGB and HGAB groups were lower (P = 0.001) in DMD than HWC and HGA groups across all milling treatments. The HGAB group tended to be lower than the HGB group, but that difference reached statistical significance (P = 0.002) only in the medium milling treatment. Results for starch digestibility generally mirrored those for DMD with starch digestibility declining as particle size increased. The HWC group had the greatest (P = 0.001) starch digestibility followed by the HGA, HGB, and HGAB groups. Table 3. In situ DM disappearance (DMD) and starch disappearance of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment) Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  r–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications and starch digestibility values are the mean of 4 replications per particle size × PIN treatment. 2 Particle sizes: Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. 3 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. Lines designated HG contain added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. View Large Table 3. In situ DM disappearance (DMD) and starch disappearance of 4 wheat groups varying in puroindoline expression and milled to 4 particle sizes (line × milling treatment experiment) Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  Item    DMD, %  Starch disappearance, %  No. of observations1    128  64  Fine2  HWC3  70.42z  96.17y    HGA  67.06y  94.48y    HGB  59.75x  82.03x    HGAB  58.15wx  74.87w  Medium  HWC  67.73yz  94.70y    HGA  66.29y  92.83y    HGB  57.67wx  77.67wx    HGAB  49.43v  63.75v  Coarse  HWC  58.90x  84.81x    HGA  54.94w  76.69w    HGB  40.45u  58.35u    HGAB  39.46u  55.02tu  Cracked  HWC  33.78t  51.16t    HGA  31.59t  45.76s    HGB  23.78s  37.02r    HGAB  20.42r  34.10r  SE    1.369  2.012  Particle size P-value    0.001  0.001  PIN expression P-value    0.001  0.001  Line (PIN expression) P-value    0.378  0.167  Particle size × PIN P-value    0.002  0.002  Particle size × line (PIN) P-value    0.879  0.977  r–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications and starch digestibility values are the mean of 4 replications per particle size × PIN treatment. 2 Particle sizes: Fine = Perten Laboratory Mill 3303 on setting #0, Medium = Perten Laboratory Mill 3303 on setting #3, Coarse = Perten Laboratory Mill 3303 on setting #6, and Cracked = Bühler mill on setting 11.5. 3 Lines grown in 2003 under nonirrigated conditions in Bozeman, MT. All lines are isolines created using the hard red spring variety Hi-Line. HWC = hard wheat controls. Lines designated HG contain added PINA (HGA), PINB (HGB), or both PINA and PINB (HGAB). Two independently created lines make up each PIN expression group. View Large The HGAB group showed increased expression of both PINA and PINB, the HGB group had increased expression of PINB, and the HGA group had increased expression of PINA. As a result, the HGAB group had a super-soft texture, the HGB group had a soft texture, the HGA group had an intermediate texture, and the HWC had a hard texture (Table 1). The HGA group had a 7.5× increase in PINA and a 28-unit decrease in hardness when compared with the HWC group. The increase in PINA decreased DMD an average of 5.0% and starch digestibility an average of 5.9% across all treatment levels. The HGB group had a 3.9× increase in PINB and a 54.4 unit decrease in hardness when compared with the HWC group. The increased expression of PINB decreased DMD an average of 22.7% and starch digestibility an average of 22.9% across all treatment levels. The HGAB group had a 6.5× increase in PINA, a 4.4× increase in PINB, and a 58.7 unit decrease in grain hardness when compared with the HWC group. The increased expression of PIN decreased DMD an average of 29.2% and decreased starch digestibility an average of 30.8% across all treatment levels. The line × milling treatment experiment indicated that PIN content affected the rate of wheat starch digestion in the rumen. Increased expression of PINB and both PINA and PINB led to a significant reduction in DMD and starch digestibility across all milling treatments. The largest reduction was achieved by the addition of PINA and PINB. A potential complicating factor in this study is the effect of grain texture on particle size. Soft wheats, having a softer endosperm, fracture easily, requiring less energy to mill than hard wheats (Symes, 1965, 1969). As a result, soft wheats yield smaller particles on the same mill setting, suggesting that the effect of PIN upon digestibility may reflect both particle size variation and PIN expression variation. Similar Particle Size Experiment To separate the effect of particle size and PIN content on DMD and starch digestibility, Hi-Line (hardest) and HGAB18 (softest) were milled such that the mean particle size per treatment was nearly identical, and incubated in the rumen for 3 h. Dry matter digestibility and starch digestibility results are presented in Table 4. A line × particle size treatment interaction was detected (P = 0.001) indicating that lines did not react similarly across particle size treatments. Line HGAB18 had lower DMD than Hi-Line (P = 0.001) among particles ranging in size from 0.09 μm to 2.35 μm. No differences (P = 0.94) in DMD were seen in particles above 2.36 μm in size. Starch digestibility declined with increasing particle size, but HGAB18 also had lower starch digestibility than Hi-Line (P = 0.001) at particles sizes ranging from 0.09 μm to 2.35 μm. No differences (P = 0.85) in starch digestibility were seen in particles above 2.36 μm in size. For wheat, particles above 2.36 μm are generally whole kernels. Seeing no differences between whole kernels of Hi-Line and HGAB18 indicated that PIN proteins had no affect on the seed coat or aleurone layers of the kernel and that differences in DMD and starch digestibility for small particles was a function of PIN interaction with starch. The increased PINA and PINB expression in HGAB18 decreased DMD an average of 21.7% and starch digestibility an average of 19.9% across particle sizes smaller than whole kernel (< 2.36 mm). This experiment indicated that increased PIN expression decreased DMD and starch digestibility, and was largely independent of particle size. Table 4. In situ DM disappearance (DMD) and starch disappearance of 2 wheat lines varying in puroindoline expression and milled to specific particle size ranges Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  t–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications, and starch digestibility values are the mean of 4 replications per line × particle size treatment. 2 Genotypes were the hard red spring variety Hi-Line and HGAB18, which is Hi-Line transformed to have high levels of PINA and PINB expression. 3 Particle size ranges were adjusted to the stated size ranges by sifting after milling using the fine (for 0.09 to 0.424 mm), coarse (for 0.425 to 0.849 and 0.85 to 1.69 mm), and cracked (for 1.7 to 2.35 mm) mill settings. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. View Large Table 4. In situ DM disappearance (DMD) and starch disappearance of 2 wheat lines varying in puroindoline expression and milled to specific particle size ranges Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  Item    DMD, %  Starch disappearance, %  No. of observations1    40  20  Hi-Line2  0.09 to 0.424 mm3  68.59z  94.69z    0.425 to 0.849 mm  65.81z  94.42z    0.85 to 1.69 mm  59.44y  85.31y    1.7 to 2.35 mm  33.42v  54.66v    > 2.36 mm  2.78t  24.27t  HGAB18  0.09 to 0.424 mm  62.28y  88.90y    0.425 to 0.849 mm  52.23x  73.04x    0.85 to 1.69 mm  42.49w  62.48w    1.7 to 2.35 mm  23.93u  41.54u    > 2.36 mm  2.74t  24.47t  Line P -value    0.001  0.001  Particle size P-value    0.001  0.001  Line × particle size P-value    0.001  0.001  SE    1.159  1.552  t–z Within a column, means without a common superscript letter differ (P < 0.05). 1 DMD values are the mean of 8 replications, and starch digestibility values are the mean of 4 replications per line × particle size treatment. 2 Genotypes were the hard red spring variety Hi-Line and HGAB18, which is Hi-Line transformed to have high levels of PINA and PINB expression. 3 Particle size ranges were adjusted to the stated size ranges by sifting after milling using the fine (for 0.09 to 0.424 mm), coarse (for 0.425 to 0.849 and 0.85 to 1.69 mm), and cracked (for 1.7 to 2.35 mm) mill settings. The mills used were: fine = Perten Laboratory Mill 3303 (Perten Instruments, Springfield, IL) on setting #0, coarse = Perten Laboratory Mill 3303 on setting #6, and cracked = Bühler mill on setting 11.5. View Large Time Course Experiment To investigate the effects of time in the rumen on PIN proteins, Hi-Line and HGAB18 were cracked, and disappearance was observed at various time points over a 12-h period in the rumen. Dry matter and starch digestibility results are presented in Figure 2 and Table 5. Dry matter digestibility increased over time for both lines. Line HGAB18 was consistently lower in DMD than Hi-Line across all time points. However, the difference between lines became less with time after 4 h leading to a line × time interaction (P = 0.001). Similarly, starch digestibility increased over time with HGAB18 being lower than Hi-Line. Again, the difference between HGAB18 and Hi-Line became less with time giving rise to a line × time interaction (P = 0.01). Increased expression of PINA and PINB in HGAB18 decreased DMD by 10.4% and starch digestibility by 11.0% across all time points. Figure 2. View largeDownload slide In situ dry matter (DM) and starch disappearance (0 to 12 h) of Hi-Line (•) and HGAB18 (▪), 2 wheat lines varying in puroindoline expression. A) DM disappearance (%), and B) starch disappearance (%). Wheat line × time interactions were significant for DM (P = 0.002) and starch disappearance (P = 0.006). Within a time period, means without a common superscript letter differ (P < 0.05). Figure 2. View largeDownload slide In situ dry matter (DM) and starch disappearance (0 to 12 h) of Hi-Line (•) and HGAB18 (▪), 2 wheat lines varying in puroindoline expression. A) DM disappearance (%), and B) starch disappearance (%). Wheat line × time interactions were significant for DM (P = 0.002) and starch disappearance (P = 0.006). Within a time period, means without a common superscript letter differ (P < 0.05). Table 5. Characteristics of ruminal in situ disappearance (0 to 12 h) of DM and starch of 2 wheat lines varying in puroindoline expression (time course experiment) Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  1 Hi-Line = untransformed hard red spring control wheat; HGAB18 = Hi-Line transformed to overexpress PINA and PINB and have very soft endosperm texture. 2 Fraction A: immediately soluble fraction. 3 Fraction B: fraction disappearing at a measurable rate. 4 Fraction C: undegraded fraction. 5 Kd: disappearance rate. View Large Table 5. Characteristics of ruminal in situ disappearance (0 to 12 h) of DM and starch of 2 wheat lines varying in puroindoline expression (time course experiment) Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  Item  Hi-Line1  HGAB18  SEM  P-value  DM disappearance      Fraction A,2 g/kg  11.1  8.0  0.41  0.001      Fraction B,3 g/kg  68.9  75.3  1.35  0.014      Fraction C,4 g/kg  20.0  16.6  1.67  0.204  Kd5 h−1  0.269  0.203  0.0099  0.003      Lag time, h  0  0  —  —  Starch disappearance      Fraction A, g/kg  11.3  8.1  1.24  0.118      Fraction B, g/kg  84.1  87.9  1.64  0.146      Fraction C, g/kg  4.6  2.2  0.69  0.050      Kd, h− 1  0.368  0.257  0.0165  0.003      Lag time, h  0  0  —  —  1 Hi-Line = untransformed hard red spring control wheat; HGAB18 = Hi-Line transformed to overexpress PINA and PINB and have very soft endosperm texture. 2 Fraction A: immediately soluble fraction. 3 Fraction B: fraction disappearing at a measurable rate. 4 Fraction C: undegraded fraction. 5 Kd: disappearance rate. View Large Line HGAB18 had a 27.9% lower (P = 0.001) immediately soluble fraction of DM than did Hi-Line, and a 24.5% slower (P = 0.003) DM disappearance rate (Table 5). The immediately soluble fraction of starch tended (P = 0.118) to be lower for HGAB18 than for Hi-Line, and HGAB18 had a 30.2% slower (P = 0.003) starch disappearance rate. In addition, HGAB18 had a lower (P = 0.050) undegraded starch fraction compared with Hi-Line, indicating that slowing starch digestibility via increasing PIN expression will not necessarily reduce extent of starch digestion. The rate of passage of feedstuffs out of the rumen is affected by specific gravity and particle size. Grains, although rapidly fermented, are small and dense, and thus pass rapidly compared with roughages (Firkins et al., 2001). The time course experiment demonstrated that a difference in starch digestibility could still be seen after 6 h in the rumen, indicating that starch supply to the small intestine may be increased with increased PIN levels. Differences in starch digestibility after incubation suggest that starch granules of HGAB18 possess a type of protection that Hi-Line does not. Being isogenic lines, expression of PIN proteins is the only difference between Hi-Line and HGAB18. The time course experiment led to the conclusion that PIN proteins aid in the protection of starch molecules from ruminal fermentation. Conclusions Comparisons between cereal species have shown that wheat starch is fermented rapidly in the rumen when compared with barley, maize, and sorghum (Herrera-Saldana et al., 1990; Owens et al., 1997). Research also has shown that variations in starch digestion exist between cultivars of the same species (Philippeau et al., 1999; Bowman et al., 2001). However, previous research has relied on the use of grains within the same market class or named varieties for comparison. Genetic factors controlling starch digestion cannot be determined by such comparisons due to the diversity of genetic backgrounds. Little work has been done with near-isogenic or transformed isolines. The use of such genetic material allows the nutritional implication of specific traits to be evaluated against a consistent genetic background. At least 2 studies in wheat have been done with near-isogenic lines. Short et al. (2000) studied the effects of grain hardness on AA digestion in poultry. They indicated that hard wheat endosperm was associated with decreased AA digestibility. Chickens, being a nonruminant animal, cannot be easily compared with ruminants and generally are considered to be particularly sensitive to changes in quality of the diet. Garnsworthy and Wiseman (2000) used near isogenic lines to evaluate the ruminal digestibility of wheat starch; no differences were seen between hard and soft wheats. However, actual grain hardness and PIN content were not stated. Typical soft wheats have at most twice the PIN content of typical hard wheats (Giroux and Morris, 1997; 1998). One may therefore expect that differences due to PIN content are too small to be detected among native wheat varieties. Overall, our present experiment demonstrated that PIN proteins affected DM and starch digestibility of wheat in the rumen. Decreasing wheat grain hardness by increasing PIN expression slowed DMD and starch digestibility in the rumen and was largely independent of particle size. Data indicated that PIN proteins aid in the protection of starch molecules from fermentation type digestion in the rumen. In barley, low in situ DMD values are correlated with increased feed efficiency, increased ADG, and increased NE content (Bowman et al., 2001). Slower or lower ruminally digestible starch shifts more starch digestion from the rumen to the small intestine. Starch digestion in the small intestine theoretically could provide up to 42% more energy than starch fermented in the rumen (Owens et al., 1986) because of reduction in energy loss via methane production and more efficient use of glucose as an energy source compared with VFA. In addition, lower DMD could reduce excessive fermentation acid production and reduce the incidence of bloat, acidosis, and laminitis (Hunt, 1996). IMPLICATIONS Although wheat is not a predominant cattle feed in the United States, it is an ideal model system to study the effect of grain hardness and puroindoline content on digestion in the rumen. In wheat, the puroindoline genes control the majority of grain hardness variation. 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Journal of Animal Science – Oxford University Press

Published: Mar 1, 2006

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