Severity of Rolling Reconstituted High Moisture Barley on Ensiling Characteristics and In Vitro Ruminal Fermentation

Barley grain sources with variable kernel sizes makes adequate and consistent processing of kernels challenging. This study evaluated how the severity of processing for reconstituted high moisture (65% on DM basis) barley ( RHB ) affects ensiling characteristics and in vitro ruminal fermentation. Three independent sources of light (<630 g/500 mL) and heavy (>670 g/500 mL) barley were blended to create 4 sources of variable kernel sized barley (646 g/500 mL). Reconstituted barley was rolled through a roller gap width of 1.40 ( RHBF ), 1.86 ( RHBM ), or 2.31 mm ( RHBC ) and ensiled for 1 or 5 mo with dry rolled barley ( DRB ; roller gap width 1.86 mm) used as a control. The 1-mo RHB and the DRB were further evaluated using the artificial rumen technique (RUSITEC) to investigate the effects of severity of processing for RHB on ruminal fermentation, and gas, methane, and microbial protein production. Using a randomized complete block design (n = 4), 16 fermenters from 2 RUSITEC apparatuses were used to assess the 4 sources and 4 processing treatments. The addition of water increased kernel width before rolling and resulted in increased kernel length, width, and thickness for RHB relative to dry rolled barley. Increasing processing severity for RHB linearly increased kernel width. The percentage of fine particles (<1.18 mm) was greater for DRB than RHBF, but did not differ by processing severity for RHB. Dry matter, organic matter, and starch disappearance were not different between DRB and RHBF, but linearly increased with increasing processing severity for RHB. Fermenter pH tended to be less for DRB relative to RHBF. In conclusion, the reduction in fine particles with the addition of water for RHB may prevent a decline in fermenter pH and when processed to achieve the same PI using a smaller roller gap width, yielded similar DM and OM disappearance suggesting a lesser risk for low ruminal pH without compromising digestibility


INTRODUCTION
Barley is an important cereal grain used in western Canada, parts of Europe, and Australia as a starch source in diets for cattle (Nikkhah, 2012).Barley starch granules are rapidly digestible once the endosperm is exposed (Mathison et al., 1997).As such, barley grain must be processed to mechanically damage the hull and pericarp, thus allowing for microbial attachment and degradation of the starch (Wang et al., 2001).However, the size of barley kernels varies considerably among barley varieties and from field to field and year to year (Wang et al., 2001).Variability is exacerbated when light and heavy weight barley sources are blended, resulting in variation in kernel size within a single load.Variability in kernel size makes it difficult to achieve an optimal processing severity with a single roller setting (Ahmad, 2010).While segregating kernels based on size before processing is an option (Ahmad, 2010), applying segregation strategies on a large scale requires substantial added sorting and storage capacity challenging feasibility.
Partial reconstitution, such as that used for tempering, causes swelling of kernels and reduces shattering upon processing (Nixdorff et al., 2020).Tempering of barley grain has been reported to have limited effects on starch digestibility (Kennelly et al., 1988) when tempered grain is processed to the same severity as dry rolled barley; however, tempering may allow for more aggressive processing without the production of fine particles (Yang et al., 1996).Given the rapid rate of barley starch degradation (Yang et al., 1996), however, increasing the severity of processing may increase the risk for low ruminal pH (Ahmad, 2010), thus it is important to determine the optimal ranges for processing severity of tempered barley.
Reconstituting barley before processing may be an alternative method to tempering and dry rolling.With reconstitution, kernels are hydrated to a target DM concentration of 65%, rolled using a roller mill, and then ensiled for storage.However, there are few studies that have evaluated reconstituted high moisture barley (RHB) or high-moisture barley (Kennelly et al., 1988).While reconstituting barley may help improve consistency in processing similarly to tempering (Kennelly et al., 1998), it is possible that rates of starch degradation may also be altered for ensiled barley.Despite this, few studies have reported how processing severity for high moisture barley affects rates of digestion.In one study, Rode et al. (1986) reported in situ starch disappearance was greater for the coarsely rolled high moisture barley than for whole ensiled barley after 42 d of ensiling (Rode et al., 1986), but that study did not compare differing severities of mechanical processing for high moisture barley.Moreover, it is unknown whether RHB processing severity impacts ensiling characteristics and ruminal fermentation, particularly for barley grain with variable kernel sizes.
The objectives of this study were to evaluate how: 1) moisture addition affects kernel characteristics; 2) processing severity and duration of storage for RHB affects starch availability; and 3) severity of rolling RHB relative to dry-rolled barley affects in vitro digestion.It was hypothesized that reconstitution would increase kernel thickness, thus enabling rolling without the production of fines.In addition, it was hypothesized that increasing processing severity for RHB would reduce in vitro ruminal pH, increase short-chain fatty acid (SCFA) and gas production, and increase microbial protein synthesis, such that the most extensively processed RHB would have greater responses than for dry rolled barley.

MATERIALS AND METHODS
The use of cattle in this study was pre-approved by the University of Saskatchewan Animal Research Ethics Board (protocol no.20220028).Cattle were cared for in accordance with the guidelines of the Canadian Council of Animal Care (Canadian Council on Animal Care, 2009).

Barley sources
Three independent sources of light (<630 g/500 mL) and 4 independent sources of heavy (>670 g/500 mL) barley grain were acquired from barley grain producers in Saskatchewan (Canada).Selection criteria for barley selection was designed to result in barley with differing kernel sizes without consideration for barley variety.The varieties received were Agriculture and Agri-Food Canada (AAC; Lacombe, AB, Canada) Cerveza (grown near Swift Current, SK, Canada), Crop Development Centre (CDC; Saskatoon, SK, Canada) Austenson (grown near Clavet, SK, Canada), CDC Austenson (grown near North Battleford, SK, Canada), CDC Bow, CDC Copeland, CDC Fraser, and AAC Connect (all grown near Swift Current, SK, Canada).All barley sources were grown in 2020.For each source, a subsample was collected to determine bulk density using a Cox Funnel (Johnson et al., 2020;Nixdorff et al., 2020; Table 1).In addition, 20 kernels were randomly selected from each source and kernel dimensions were determined as described by Johnson et al. (2020) and Nixdorff et al. (2020).
Based on bulk density, batches of barley were prepared by combining differing proportions of the heavy and light bulk density barley sources to create sources of variable kernel size (n = 4) barley targeting a final bulk density of density of 646 g/L (Table 1).Blending of light and heavy barley was used to replicate practices that may occur when receiving multiple sources of barley with limited ability to segregate sources.Final kernel dimensions, density, and the percent fines were assessed for each source as described previously.Four experimental units for each treatment was based on a power test for 7-h in vitro starch digestion using Proc Power with a mean difference of 8.5 (equating a difference of 11.3% of the mean) and standard deviation ranging from 2.0 and 2.3 (Johnson et al., 2020) resulting in an n = 4 to achieve 80% power with an α of 0.05.A secondary power test (mean difference of 2, SD = 0.02) was conducted for the starch digestibility measured using the rumen in vitro simulation technique (RUSITEC) using SD values from Sarich et al. (2022).This power test resulted in greater than 80% power providing that the mean difference was greater than 1 and the SD was less than 0.17.

Evaluating processing severity and changes in starch availability for reconstituted high moisture barley ensiled for 1 or 5 mo
Barley sources with variable kernel sizes were used to evaluate whether increasing processing severity for RHB and the duration of ensiling affected starch availability.Before processing, subsamples from each barley source were dried in a forced-air oven at 55°C for 48 h to determine the DM concentration.To prepare RHB, barley was tempered by adding water to achieve a moisture content of 35%.The barley was mixed (5 min) during water addition to achieve consistent hydration and was provided 24 h for tempering at room temperature.The barley was then rolled with roller gaps of 1.40 mm to produce finely processed RHB (RHBF), 1.86 mm to produce medium processed RHB (RHBM), or 2.31 mm to produce coarsely processed RHB (RHBC).Dry rolled barley (DRB) was processed with a roller gap width of 1.86 mm.All processing was conducted using the same roller mill (SvenMill 7, Apollo Mills, Saskatoon, SK, Canada) on the same day.

Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY
The processing index (PI) was measured using a cox funnel with a 500 mL cup (Dimo's Labtronics, MB, Canada) by dividing the weight of a 0.5-L volume of barley after processing by the 0.5-L weight before processing and multiplying it by 100% (Yang et al., 2013).The PI calculations and kernel dimensions for RHB were conducted on a wet basis using the hydrated rolled barley and hydrated whole barley.Samples of the processed barley from each source and PI were collected to determine the DM concentration by drying the sample at 55°C in a forced-air oven until achieving a constant weight.In addition, particle size distribution and percent fines were assessed using a Ro-Tap RX-29 test sieve shaker (Hoskin Scientific, Vancouver, BC, Canada) with samples shaken, in duplicate, for 10 min.The sieves used had aperture openings of 2.36 mm, 1.7 mm, 1.18 mm, 850 μm, 600 μm, 425 μm, 300 μm, 212 μm, 150 μm, 106 μm, and 75 μm and the method followed principles described by Wilcox et al. (1970).
The RHB from each of the 4 barley grain sources was then packed into 1-L pails (Model S-20029, Uline, Milton, ON, Canada) at a constant packing density of 2.15 g/cm 3 (Kung et al., 2004).The pails were then sealed with a screw top lid equipped with a Bunsen valve to allow gas release.The valve was constructed by attaching a rubber tube with a small slit in the side and a stopper at the end of the tube to allow for small releases of gas as pressure accumulated while maintaining an anaerobic environment (Cullison, 1960).For each PI, 2 replicate pails (technical replicates) were prepared to evaluate fermentation characteristics after 1 (n = 4/treatment) and 5 mo (n = 4/treatment).Pails were stored at room temperature for the duration of the ensiling period.As such, this study was conducted as a 3 × 2 factorial design with the main effects of processing severity for RHB and duration of ensiling (1 vs. 5 mo).
After 1 and 5 mo of ensiling, the weight of the RHB was measured to evaluate ensiling shrink by dividing the weight of the RHB after ensiling by the original weight of the RHB added after being corrected for DM.In addition, the RHB from each source was thoroughly mixed and samples were collected to evaluate ensiling characteristics (pH and organic acid concentrations), DM, starch availability, and reactive starch.Ensiling characteristics were analyzed at Cumberland Valley Analytical (Waynesboro, PA, USA) where the extracted sample pH was read using a Mettler DL12 titrator (Mettler-Toledo, Inc., Columbus, OH, USA).Organic acids within the ensiled RHB were measured using a Perkin Elmer Clarus 580/590 Gas Chromatograph with a Stabiliwax-DA column (Shelton, CT, USA).The 7-h in vitro starch digestibility was assessed at Cumberland Valley Analytical Services where 1.0-g samples were incubated in 125-mL Erlenmeyer flasks containing 40 mL of strained ruminal fluid, 2 mL of reducing solution, and 40 mL of buffer (Goering and Van Soest, 1970).Samples were maintained anaerobically at 40°C in a water bath for 7 h.The remaining sample was analyzed for DM and starch concentration (Hall et al., 2015).
Another subsample of the RHB was dried and ground using a Retsch ZM 200 grinder (Haan, Germany) to pass through a 1-mm sieve for determination of starch reactivity as described by Zinn (1990).Enzymatic based starch reactivity assays have been used to evaluate differences in starch availability and solubility in response to cereal grain processing methods.Reactive starch was determined using amyloglucosidase reactive soluble starch (AGR) and 6 h amylase reactive insoluble starch (ARS).These values were utilized to calculate the total insoluble reactive starch (IRS, %) from ARS and AGR as ((ARS-AGR)/6) based on a 6-h incubation.Digestive insoluble starch (ISD) was calculated as (100-AGR) × (IRS/(IRS + 0.05)) where 0.05 is the coefficient representing a 5% passage rate from the rumen (Zinn, 1990).Predicted ruminal starch digestion (PRSD, %) was then calculated as (1.32 × AGR) + (0.93 × ISD) as outlined by (Zinn, 1990).
The remaining grain from the 1-mo ensiling duration was weighed (50 g/bag) into vacuum sealed bags and stored at 4°C for 2 to 16 d until being used in the in vitro study using the RUSITEC.Dry rolled barley was also vacuum sealed and stored at 4°C for consistency.

In vitro digestibility using the rumen simulation technique (RUSITEC)
Individual RUSITEC fermenters were randomly assigned to 1 of 4 treatments (n = 4/treatment) to allow for a comparison of DRB, RHBF, RHBM, and RHBC after 1 mo of ensiling.The 4 sources of each variable kernel sized barley were the experimental replicates used for each treatment.Two RUSITEC apparatuses were used for this study, with each apparatus containing 8 fermenters.As such, a total of 16 fermenters were used simultaneously.The experiment included 7 d of fermenter equilibration for microbial adaption and a 7-d sampling period.All fermenters were equipped to facilitate saliva input, effluent outflow, and a 2-L reusable bag for total gas collection (Curity, Covidien Ltd., Mansfield, MA, USA) as previously described by (Sarich et al., 2022).
Ruminal digesta was collected from 4 ruminally cannulated dairy cattle fed a barley silage and barley grainbased diet containing 17.9% CP, 32.0% aNDFom, and 20.8% starch.Approximately 1 L of ruminal digesta was collected using a 250-mL cup from each of the cranial, central, and caudal regions of the rumen.The ruminal fluid was filtered through 4 layers of cheesecloth and placed in pre-heated insulated containers.Ruminal fluid was kept warm during transport and processing in the laboratory, with the procedure taking approximately 1 h.Ruminal fluid samples from the 4 different cows were then combined into a single batch using equal portions to yield a composite ruminal fluid sample (12 L).The composite sample was mixed before use.Each fermenter was filled with a mixture of filtered ruminal fluid (700 mL) and prewarmed artificial saliva (200 mL, pH = 8.2;McDougall, 1948) containing 0.3 g/L of (NH 4 ) 2 SO 4. In addition, one Ankom nylon bag (5 cm × 10 cm; pore size = 50 μm; Ankom Technology Corporation Macedon, NY, USA) containing 10 g (DM basis) of the respective barley grain treatment and one nylon bag with 20 g of ruminal digesta solids (wet basis) were added to each fermenter.The bag with the ruminal digesta solids was removed after 24 h and was replaced with another bag containing the respective treatment.All treatment bags were incubated for 48 h before being removed and replaced.The barley grain treatments incubated represented the original processing imposed as samples were not further dried or ground before being placed in the nylon bags.The barley treatment bags were then prepared and added to each fermenter daily (1000 h) resulting in 48 h of incubation for the substrate in each nylon bag.All fermenters were mixed using vertical oscillation (6 cycles/min) of the bags.During daily feeding, the fermenters were flushed with CO 2 to maintain anaerobic conditions.Subsequently, fermenters were placed in a water-bath under constant circulation to maintain a temperature of 39°C.Artificial saliva (McDougall, 1948) was infused into each fermenter using a peristaltic pump (model 205S, Watson Marlow, Falmouth, Cornwall, UK) at a continuous rate of 29 mL/h to replace 70% of the fermenter volume daily.
Fermenter pH, gas production, and nutrient digestibility.Fermenter pH was measured daily at feeding time (1000 h).The pH meter (AP 110 pH/ORP Meter, Fischer Scientific, Waltham, MA, USA) was calibrated daily using standard buffers at pH 4, 7, and 10.Gas produced was collected into sealed bags (Curity, Covidien Ltd., Mansfield, MA, USA) and the volume produced was measured from d 8 to 11 with the use of a gas meter (model DM3A, Alexander-Wright, London, UK).Before gas volume measurements, a 20-mL sample was collected from each collection bag using a 26-gauge needle and transferred to an evacuated 6.8-mL exetainer vial (Labco Ltd., Wycombe, Bucks, UK) to measure CO 2 and CH 4 concentrations.The methane and carbon dioxide concentrations were determined using a Scion 456-gas chromatograph (Goes, Netherlands, EU) with hydrogen (6 mL/min) used as the carrier gas (Sarich et al., 2022;Fisher Scientific, Hampton, NH, USA).
From d 8 to 11, the nylon bags removed were processed in batches of 5 to 6 bags by placing bags in 1 L of distilled water in a 4-L beaker.Another beaker (1 L) was used as a plunger to gently agitate the water and bags using 10 vertical motions of the top beaker.The distilled water was then emptied, and the entire process was repeated 2 times.After washing, samples were dried in a forced-air oven at 55°C until achieving a constant weight, which was recorded.Residual feed in the nylon bags was com- Short chain fatty acid (SCFA), ammonia-N (NH 3 -N), and microbial protein production.Effluent volumes were recorded at feeding time when effluent flasks were replaced.A clean effluent flask was attached daily to each fermenter with 3 mL of sodium azide solution (20% wt/vol) added from d 7 to 10 to arrest microbial fermentation.From d 8 to 11, 2 10-mL subsamples were collected directly from each effluent flask.One sample was placed in 2 mL of metaphosphoric acid (25% wt/vol) for measurement of SCFA concentrations as described by Khorasani et al. (2000).The second sample was preserved with 1% H 2 SO 4 for determination of NH 3 -N concentration using a colorimetric method as described by Fawcett and Scott (1960).Total production of SCFA and NH 3 -N were calculated using the daily effluent amount and the concentration of the analyte in the effluent.
Starting on d 7, a modified McDougall's buffer was used such that (NH 4 ) 2 SO 4 was replaced with 1.0 g/L of ( 15 NH 4 ) 2 SO 4 (minimum 15 N enrichment 10% ; Cambridge Isotope Laboratories Inc., Andover, MA, USA) and infusion continued until the end of the experiment on d 14 for the measurement of microbial protein.Measurements of microbial protein in the liquid associated and feed particle-bound fractions were used to assess the relative contribution for components contributing to microbial protein production as microbes can pass out of the fermenter in the liquid phase or be attached to feed particles within the nylon bags.From d 12 to 14, nylon bags were removed from the fermenter and gently squeezed by hand with consistent pressure.Bags were then washed in 20 mL of McDougall's buffer (McDougall, 1948) and were processed for 60 s using a Stomacher 400 Laboratory Blender (Seward Medical Ltd., London, England, UK) to dislodge microbes from feed particles.The feed particle associated (FPA) microbial fraction was derived from the liquid effluent as well as the expelled liquid that was captured during the buffer wash.The FPA fraction was combined with the daily liquid outflow present in the effluent flask, and a 35-mL sample was then collected to represent the liquid-associated microbial fraction.Samples for the feed particle bound (FPB) microbial fraction were taken from the washed feed residue.A liquid microbial pellet was produced by centrifuging the 35-mL effluent sample (500 × g, 10 min at 4°C) and retaining the supernatant.The supernatant was then centrifuged (20,000 × g, 30 min at 4°C) and the resulting pellet was washed with distilled water.This last step was repeated 3 times to isolate the liquid-associated microbial pellet.The liquid-associated microbial pellet and FPB fraction were then freeze-dried and ball ground before analysis following the procedure as described by Sarich et al. (2022).The liquid-associated microbial pellet and FPB were subsampled and encapsulated for measurement of N and 15 N abundance using a Flash 2000 Elemental Analyzer (Thermo Fisher Scientific, Voltaweg 22, BC Delft, Netherlands).Total microbial protein production (mg/d) was calculated as a sum of the weight of the solid microbial nitrogen (FPB) and the weight of the liquidassociated microbial nitrogen fraction produced from the effluent and corresponding pellet.

Statistical Analysis
Effects of hydrating barley on kernel dimensions before processing were analyzed using the MIXED model of SAS (SAS Institute Inc., Cary, NC, USA) with source (n = 4/treatment) as the experimental unit.The model included the fixed effect of treatment (dry vs. RHB).To evaluate the severity of rolling for RHB and duration of ensiling, the model included the fixed effect of processing treatment with the random effect of source.The 1-mo vs. 5-mo ensiling data were analyzed with the fixed effects of ensiling duration, severity of processing, and the ensiling duration × severity of processing interaction with the random effect of barley source.Polynomial contrasts were constructed to evaluate the linear and quadratic effects of increasing severity of processing RHB.
Data from the RUSITEC experiment were analyzed using the MIXED model of SAS with fermenter as the experimental unit and the fixed effect of treatment.Gas, methane, pH, SCFA, and ammonia were analyzed as repeated measures with sampling day included in the model.A contrast was used to evaluate the difference between DRB and RHBF while polynomial contrasts were used to evaluate the linear and quadratic effects among the RHB processing severity.The comparison between DRB and RHBF was used to test whether processing method at the same processing severity affected in vitro fermentation and digestibility responses.The best fit equal spacing covariance structure was chosen based on the structure with the smallest Akaike's information criterion and Bayesian information criterion values.The equal spacing covariance structures tested included variance components, autoregressive 1, heterogeneous autoregressive, unstructured, compound symmetry, and heterogeneous compound symmetry.
All data and the residuals were tested for normality using visual appraisal of plots and based on the Shapiro-

Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY
Wilke test using the COVTEST statement in SAS.All data were normally distributed and there were no outliers.Significance was declared at P < 0.050 and tendencies are discussed when 0.100 ≥ P ≥ 0.050.

Effects of hydration of barley on kernel dimensions
The DM concentration was 90.7% for the dry barley whereas the RHB had a DM concentration of 63.1% (P < 0.001; Table 2), which was similar to the target DM concentration (65% DM).Bulk density was not different for the whole kernel dry barley (DB) versus whole kernel RHB (P = 0.43).Kernel width after hydrating was greater for the RHB relative to DB (P = 0.018), but kernel length tended to be greater for DB than RHB (P = 0.06).Thickness of the kernels was greater for the RHB than DB (P = 0.033).The proportion of particles retained on the 4-mm sieve and the pan did not differ among DB and RHB, but RHB had a lesser proportion of particles retained on the 1.18-mm sieve (P = 0.004).

Effect of RHB and severity of processing relative to dry-rolled barley
Processing index values (Table 3) were not different between RHBF and DRB (P = 0.73) and the PI linearly decreased from RHBC to RHBF with increased processing severity (P < 0.001).Kernel width after processing was less for DRB compared with RHBF (P < 0.001), and kernel width linearly increased as RHB processing severity increased (P = 0.030).Length and thickness of barley kernels after processing were greater for RHBF relative to DRB (P < 0.001), but severity of processing for RHB did not affect the length or thickness of the kernel.Rela-tive to RHBF, DRB had 36% less particles retained on the 2.36-mm sieve (P < 0.001).Despite a linear reduction in the proportion retained on the 2.36-mm sieve with increased severity of RHB processing (P < 0.001), the vast majority of particles for RHB were retained on the 2.36-mm sieve.The proportion of particles retained on all sieves with aperture openings of 1.70-mm or smaller were greater for DRB than RHBF (P ≤ 0.003), with linear increases in the proportion of particles retained (P ≤ 0.044) on the sieves as the severity of processing increased.That said, a quadratic effect was present for particles retained on the 1.18-mm sieve (P = 0.041) such that as the severity of processing decreased the amount of barley retained on the 1.18-mm sieve decreased at a decreasing rate.The AGR was greater and ARS was less for RHBF relative to DRB (P < 0.001), while severity of processing RHB did not affect either the AGR or ARS response.The IRS and ISD were greater for DRB than RHBF (P < 0.001), resulting in a greater PRSD for DRB than RHBF.Processing severity for RHB did not affect ARS, IRS, ISD, or PRSD.

Nutrient characteristics for RHB processed to differing severities and ensiled for 1 or 5 mo
There were no time × processing severity interactions observed for ensiling characteristics, chemical composition, or starch reactivity (Table 4).The pH of the ensiled RHB was not affected by duration of ensiling or processing severity, with an average pH of 4.4.Total organic acid, lactic, and acetic acid concentrations were greater for RHB ensiled for 5 mo than 1 mo (P < 0.001), with no processing severity effects.The shrink loss increased from 0.9 to 2.8% DM basis as ensiling increased from 1 to 5 mo, but processing severity did not affect shrink loss.While the same sources of RHB were used, the DM concentration was lower for RHB ensiled for 5 mo than for 1 mo but DM concentration was not affected by processing severity.The CP concentration (P = 0.030) and the proportion of soluble CP (8.0 vs. 6.8%;P < 0.001) were greater for RHB ensiled for 5 than 1 mo.Processing severity did not affect CP concentration, but there was a quadratic effect (P = 0.039) on soluble CP where soluble CP decreased from RHBC to RHBM and then increased to RHBF.Ammonia-N concentration was greater for RHB ensiled for 5 than 1 mo, but there was no effect of processing severity.The NDF concentration was greater for the 1-mo than the 5-mo ensiled RHB (P < 0.001), but processing severity did not affect NDF concentration.In contrast, ADF concentration was not affected by ensiling duration or processing severity, with a mean ADF concentration of 7.5%.Starch concentrations were greater for 5-mo than 1-mo ensiled barley (58.8 vs. 57.4;P < 0.001), but there were no effects of processing severity on starch concentration.Calcium and P concentrations were not affected by ensiling duration or processing severity.The 7-h in vitro starch digestibility was nearly 6 percentage units greater for RHB ensiled for 5 mo than 1 mo (P < 0.001) and 7-h in vitro starch digestibility linearly increased with increasing severity of processing (P = 0.005).The AGR was not affected by duration of ensiling although a quadratic response was observed (P = 0.048) such that AGR increased from RHBC to RHBM and then decreased for RHBF.The ARS, IRS, and ISD were greater for barley ensiled for 5 mo than 1 mo while processing severity did not influence these variables.As a result, the PRSD was greater when ensiled for 5 mo than 1 mo, but there were no effects of processing severity.

In vitro ruminal fermentation characteristics of DRB when compared with RHB processed to differing severities using the RUSITEC
There were no differences for the disappearance of DM, OM, N, and starch (P > 0.15; Table 5) when comparing DRB and RHBF.For DM, OM, and N, disappearance increased linearly with increasing severity of processing while for starch, the response was quadratic (P = 0.027) with a decreasing rate for the increase in starch disappearance with increasing severity of processing.The NDF disappearance was not different between DRB relative to RHBF (P = 0.94), and no effect of processing severity was observed for RHB.Fermenter fluid pH tended to be  3.Effect of altering the processing index for reconstituted high moisture barley (RHB) grain and duration of ensiling (1 vs. 5 mo) relative to dry rolling on kernels dimensions, starch reactivity, and particle size distribution for sources of barley grain with variable barley kernel sizes lower for DRB relative to RHBF (P = 0.100), but there was no effect of processing severity on pH.Total gas production was not different for DRB relative to RHBF (P = 0.14, Table 6) and processing severity of RHB did not affect total gas production.Methane, when reported as a percentage of total gas, in mg/d, and in mmol/d were greater for DRB than RHBF (P ≤ 0.010).Methane production increased linearly with increasing severity of processing.Methane production as a function of OM fermented was greater for DRB relative to RHBF (P = 0.004), and methane production increased linearly with increasing severity of processing for RHB.
Ammonia-N production did not differ between RHBF and DRB (P = 0.62, Table 7), and processing severity of RHB had no effect.Total SCFA production was greater for DRB (P = 0.030) than RHBF and increasing the severity of RHB processing linearly increased SCFA production (P = 0.007).The molar proportions of acetate (P < 0.001) and caproate (P = 0.021) were lower for DRB than RHBF, while the valerate concentration was greater for DRB (P < 0.001).As a result, the acetate: propionate ratio tended (P = 0.062) to be less for DRB than RHBF.
Processing severity tended to linearly (P = 0.051) reduce the molar proportion of acetate with increasing severity of processing.The molar proportion of isobutyrate linearly increased (P = 0.011) and the molar proportion of caproate linearly decreased (P = 0.046) with increasing severity of RHB processing.
The liquid-associated microbial N production was greater for DRB relative to RHBF (P < 0.001; Table 8) and there was a linear increase in liquid-associated microbial N production with increasing severity of processing for RHB (P < 0.001).Production of FPB microbial N was greater for RHBF than for DRB (P = 0.008), but processing severity for RHB had no effect.Consequently, total production of microbial N was greater for DRB (P < 0.001) than RHBF and increasing the severity of processing for RHB linearly increased microbial N production (P < 0.001) When represented as a function of OM fermented, microbial N production was greater for DRB than for RHBF (P < 0.001), and there was a tendency for a linear increase in microbial N production with increasing severity of processing for RHB (P = 0.080).

DISCUSSION
This study confirmed that hydrating kernels caused them to swell before processing with greater kernel width and thickness for the RHB than the original dry whole barley.Thicker and wider kernels are expected to increase the likelihood that the pericarp of each kernel is damaged during processing, which is a necessary step for adequate starch digestibility (Ahmad, 2010).Addition of moisture before processing also reduced kernel shattering as indicated by greater kernel width, and length, and a reduction in the proportion of particles retained on the bottom pan of the PSPS.While we are unaware of other studies evaluating processing characteristics for RHB, others have reported that hydrated kernels, as occurs with tempering, reduces shattering of kernels at processing (Mathison et al., 1997;Nikkhah, 2012;Nixdorff et al., 2020).That said, increasing the severity of processing for RHB linearly increased the proportion of fines, thus supporting the results of Beauchemin et al. (2001) indicating that increasing the extent of processing increased kernel breakage and therefore the proportion of fines.As barley starch is rapidly digested (Yang et al., 2013), minimizing the proportion of fine particles while ensuring the pericarp of kernels is damaged may reduce risk for low ruminal pH while maintaining adequate starch digestibility.In partial support, we observed that RHBF had lesser proportions of fine particles despite an equal PI, and consequently a lower PRSD than DRB.Moreover, when assessed using the RUSITEC, RHBF tended to have greater fermenter fluid pH but starch disappearance was not different when compared with DRB.
While starch availability results may slightly differ depending on in vitro or enzyme-based metrics (Zinn, 1990;Hall et al., 2015), the 7-h in vitro starch digestibility was greater after 5-mo of ensiling and increased with increasing processing severity for RHB.These results support the concept that greater severities of processing should increase starch digestibility (Mathison et al., 1997;Johnson et al., 2020) and that increasing the duration of ensiling may increase starch availability.There are limited data available on high moisture ensiled barley  grain starch reactivity or digestibility, but for high moisture corn researchers have reported a 13.2% increase for in vitro starch digestion relative to dry ground corn (Ferraretto et al., 2014).Along this line, Ferraretto et al. (2014) and Kung et al. (2014) reported that maximum starch digestibility occurs after 6 to 12 mo of storage for high moisture corn.The increase in starch digestibility for ensiled high moisture corn is thought to be related to a reduction in zein protein cross linkages (Hoffman et al., 2011).However, starch-protein matrices do not limit digestibility of barley starch (Engstrom et al., 1992), at least to the same extent as for corn.That said, initial digestion of barley requires microbial colonization and degradation of protein before starch digestion (Yang, 2017).As such, it may be possible that increasing the duration of storage for ensiled barley further alters the protein structure increase starch access.Nevertheless, our results for increased starch reactivity with a longer ensiling period supports previous research by Rode et al. (1986), who observed that ensiling high moisture barley for at least 42 d increased starch digestibility relative to rolled high moisture barley that was not ensiled.Disappearance of all chemical components measured using the RUSITEC (DM, OM, CP, starch), were not different for RHBF relative to DRB.Despite a lack of differences for nutrient disappearance, DRB had greater total SCFA, ammonia, and microbial protein production relative to RHBF and these results are consistent outcomes indicating more extensive microbial fermentation (Yang et al., 2000).The lesser fermentation intensity for RHBF may be attributed to DRB having a greater proportion of fine particles which can potentially increase microbial access for attachment, thus allowing a greater rate and extent of nutrient degradation (Beauchemin 2 Greatest SEM for the interaction is present. 3 Linear and quadratic contrasts for processing severity for RHB. 4 Liquid-associated microbial fraction (from liquid effluent). 5FPB, feed particle-bound microbial fraction (from residue of forage bags).et al., 2001).The previous suggestion is supported by greater fluid phase microbial protein production for DRB relative to RHBF.The increase in total SCFA production with increasing severity of processing with RHB reflect greater nutrient disappearance.The lack of difference for propionate production with increasing processing severity for RHB was unexpected as propionate production is typically increased with greater starch digestion (Yang et al., 2000).However, Beauchemin et al. (2001) only detected a tendency for propionate to change with differing intensities of barley grain processing.The lack of a propionate production response is supported with the increase in total gas and methane production for DRB relative to RHBF and with increasing processing severity for RHB.These data suggest that there was an overall increase in fermentation intensity when comparing DRB and RHBF and with increasing severity of barley processing rather than a shift in the pathways of fermentation.
High-starch diets that contain rapidly fermentable carbohydrates typically reduce CH 4 production due to the drop in ruminal pH inhibiting the growth of protozoa (Hook et al., 2011), along with increased disposal of reducing equivalents in fermentation pathways that produce propionate (Janssen, 2010;Hatew et al., 2015).However, a greater proportion of small particles for barley processed to a greater extent has previously been reported to increase total gas production while also increasing the rate of fermentation (Yang et al., 2014;Naghadeh et al., 2020;Saleem et al., 2020) and in this study with DRB versus RHBF.Despite an increase in fine particles and a lower pH for DRB than RHBF, there were no differences in propionate production and no difference in digestibility.Methane production (mg/d) was greater for DRB than RHMF as the fine particles are more rapidly degradable, thus increasing substrate utilization (Getachew et al., 2000) and microbial protein production (Janssen, 2010).In the present study, we observed that DRB had greater fluid associated and lesser feed particle bound microbial protein production than RHBF further supporting that fine particles associated with dry rolling may have altered fermentation responses and that these particles passed out of the bags and the fermenter in the fluid phase.Consequently, DRB had greater total microbial protein production, and this was still greater than RHBF when expressed as microbial protein production per unit of OM fermented.Thus, the increased CH 4 intensity produced per g of OM fermented for DRB over RHBF is likely related to the increased rate, but not the extent of fermentation.
Shrink, calculated as the change in DM weight before and after ensiling (Robinson et al., 2016), can represent a significant loss of nutrients and concerns over storage of high-moisture barley have been documented (Rode et al., 1996).For ensiling, microorganisms utilize nutrients resulting in microbial growth and organic acid production that reduce pH and preserve the feed (Kung et al., 2001).The ensiling process typically takes approximately 21 d to reach stable conditions, but data form the present study indicates that shrink increased from 1 to 5 mo suggesting that ensiling may not have been complete within 1 mo.Further support for an extended ensiling duration is provided by the increase in total SCFA, lactic acid, acetic acid, and starch digestibility from 1 mo of ensiling to 5 mo of ensiling (Kung et al., 2001).Likewise, the total DM and NDF concentrations were decreased from 1 to 5 mo if ensiling.These data suggest that RHB digestibility may change with advancing duration of storage, as occurs for high moisture corn, and that shrink increases from 1 to 5 mo of ensiling.

CONCLUSION
Applying moisture before processing swelled barley kernels and reduced the proportion of fines resulting from rolling variable kernel size barley grain relative to dry rolled barley grain.Starch availability increases for RHB from 1 to 5 mo of storage and increases with increasing severity of processing.When processed to the same processing index, the in vitro starch digestion measured using the RUSITEC was not different for RHBF than DRB.This study provides new information for how processing severity and duration of storage affect nutrient digestibility for RHB but given the in vitro approach implemented, further research is necessary to evaluate the effects of feeding RHB and the subsequent effects on ruminal fermentation and performance responses for lactating cows.

NOTES
The use of cattle in this study was pre-approved by the University of Saskatchewan Animal Research Ethics Board (protocol no.20220028).Cattle were cared for in accordance with the guidelines of the Canadian Council of Animal Care (Canadian Council on Animal Care, 2009).Funding for this research was provided by the Saskatchewan Barley Development Commission (Saskatoon, SK, Canada), SaskMilk (Regina, SK, Canada), and the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada) through the Alliance program.Data associated with this manuscript is held by the corresponding author at the Department of Animal and Poultry Science at the University of Saskatchewan (Saskatoon, SK).Requests for access can be made through the corresponding author.
posited from d 8 to 11. Original feed samples and residue samples were analyzed for dry matter (AOAC, 2006; method 930.15), ash (AOAC, 2006; method 942.05), and crude protein (AOAC, 2006; method 990.03) using combustion (FP-528, LECO corporation, St. Joseph, MI, USA).Neutral detergent fiber (AOAC 1977; method 973.18) was analyzed using the ANKOM 200 Fiber Analyzer (Ankom Technology Corporation, Macedon, NY, USA) with the use of sodium sulfite (S430-500 so-Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY dium sulfite anhydrous, Fisher Scientific, Hampton, NH, USA) and heat-stable α-amylase (ANKOM Technology Corporation., Macedon, NY, USA).Starch was measured with the Megazyme kit (Megazyme, Lansing USA) according to AOAC (2014) method 996.11 and 76-13.01.Digestibility of each nutrient was then calculated using the weight of the original diet nutrient minus the weight of the residual feed nutrient divided by the weight of the nutrient in the original diet, with all values corrected for DM.
Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEYTable Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY

Table 1. Bulk density, kernel dimensions, and DM concentration for the original barley grain sources and the combination of individual sources to create barley samples with variable kernel sizes
Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY

Table 2 .
Effects of hydrating barley grain to 65% DM (RHB) relative to unaltered barley on kernel dimensions for sources of barley grain with variable kernel sizes

Table 4 .
Effect of altering the processing index and ensiling duration (1 vs. 5 mo) for reconstituted high moisture barley (RHB) grain on ensiling characteristics for sources of barley grain with variable barley kernel sizes

Table 5 .
Effect of altering the processing index for reconstituted high moisture barley (RHB) grain ensiled for 1 mo relative to dry rolling on nutrient disappearance and pH in the RUSITEC for sources of barley grain with variable barley kernel sizes

Table 6 .
Effect of altering the processing index for reconstituted high moisture barley (RHB) grain ensiled for 1 mo relative to dry rolling on gas production, methane, and carbon dioxide in the RUSITEC for sources of barley grain with variable barley kernel sizes DRB = dry rolled barley using a 1.86-mm roller gap width, RHBF = reconstituted high moisture barley rolled using a 1.40-mm roller gap width, RHBM = reconstituted high moisture barley rolled using a 1.86-mm roller gap width, and RHBC = reconstituted high moisture barley rolled with a 2.31-mm roller gap width.
1 2 Greatest SEM for the interaction is present.3Linearand quadratic contrasts done on RHB data only.

Table 7 .
Lynch et al.: ROLLING RECONSTITUTED HIGH MOISTURE BARLEY Effect of altering the processing index for reconstituted high moisture barley (RHB) grain ensiled for 1 mo relative to dry rolling on ammonia and SCFA production and concentrations in the RUSITEC for sources of barley grain with variable barley kernel sizes 3Linear and quadratic contrasts done on RHB data only.

Table 8 .
Effect of altering the processing index for reconstituted high moisture barley (RHB) grain ensiled for 1 mo relative to dry rolling on microbial nitrogen flow in the RUSITEC for sources of barley grain with variable barley kernel sizes RHBM = reconstituted high moisture barley rolled using a 1.86-mm roller gap width, and RHBC = reconstituted high moisture barley rolled with a 2.31-mm roller gap width.