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Comparison of warm season and cool season forages for dairy grazing systems in continuous culture

Comparison of warm season and cool season forages for dairy grazing systems in continuous culture Comparison of warm season and cool season forages for dairy grazing systems in continuous culture ,2 † ‡ Kathryn E. Ruh,* Bradley J. Heins,* Isaac J. Salfer, Robert D. Gardner, and Marshall D. Stern* *Department of Animal Science, University of Minnesota, St. Paul, MN 55108; Department of Animal Science, The Pennsylvania State University, University Park, PA 16802; Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108 ABSTRACT: The objective of this study was to warm-season annual grasses compared with fresh compare warm-season annual grasses to cool-sea- alfalfa. Total VFA were not affected (P >  0.05) son perennial (CSP) grasses for ruminal nutrient by forage. The NH3-N concentrations were high- digestibility and N metabolism in a dual-flow est (P < 0.05) with alfalfa compared with the continuous culture fermentation system. Dietary other CSP grasses and legumes and warm-season treatments were 1) fresh alfalfa, 2) CSP grasses annual grasses. CP digestibility was not affected and legumes, 3) brown-midrib sorghum-sudan- (P > 0.05) by forage treatment. Flow of NH3-N grass (BMRSS), and 4) teff grass from an organic was greatest (P < 0.05) for alfalfa, reflecting the dairy production system. Eight dual-flow con - greatest NH3-N concentration. Flow of total N tinuous culture fermenters were used during two was greatest (P < 0.05) for alfalfa, intermediate consecutive 10-d periods consisting of 7 d for sta- for teff, and lowest for CSP grasses and legumes bilization followed by 3 d of sampling. Fermenter and BMRSS. Flows of bacterial N, efficiency of samples were collected on days 8, 9, and 10 for bacterial N, non-NH3-N, and dietary N were not analysis of pH, NH3-N, and VFA. Apparent affected (P > 0.05) by forage source. Overall, fer- DM, OM, NDF, and ADF digestibility were on mentation of warm-season grasses was similar to average lesser (P < 0.05) in CSP grasses and leg- the cool-season grasses and legumes which indi- umes and warm-season annual grasses compared cate dairy producers may use warm-season grasses with alfalfa. True DM and OM digestibility were without concerns about negative impact on rumen lesser (P < 0.05) for CSP grasses and legumes and health. Key words: continuous culture fermentation, grazing, sorghum-sudangrass, teff © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2018.2:125–134 doi: 10.1093/tas/txy014 The authors express gratitude to the interns and graduate Food and Agriculture. Financial support was also provided for students at the University of Minnesota West Central Research this project by the Ceres Trust (Chicago, IL). Authors confirm and Outreach Center, Morris, for their assistance in data collec- there is no conflict of interest with this study. tion. This work is supported by Organic Agriculture Research Corresponding author: hein0106@umn.edu and Extension Initiative (grant no. 2012-51300-20015/project Received March 27, 2018. accession no. 0230589) from the USDA National Institute of Accepted April 3, 2018 Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 126 Ruh et al. INTRODUCTION a herbage only diet (Soder et al., 2013a). One study compared fresh alfalfa (Medicago sativa) to alfalfa Profitability of grazing dairy farms relies on hay in continuous culture to determine differences pastures that produce a large quantity of high-qual- in ruminal fermentation when sucrose was added to ity forage for cattle to graze. In the upper Midwest, the alfalfa diet (Ribeiro et al., 2005). There are few cool-season grass and clover species are traditional studies that compare digestibility and ruminal fer- pasture forages for many dairy grazing producers. mentation of pasture diets of different species com- However, cool-season perennial (CSP) grasses and position in continuous culture, although one study legumes experiences a decrease in growth rate during was conducted that used fresh orchardgrass or red periods of high temperatures and low precipitation, clover in combination with different inclusion of as observed in July and August in the upper Midwest corn grain in continuous culture (Loor et al., 2003). (Moore et  al., 2004; Hudson et  al., 2010). Warm- The brown-midrib sorghum-sudangrass season annual grasses, such as sudangrass (Sorghum (BMRSS) and teff grass were chosen as the warm-sea- bicolor × drummondii), sorghum × sudangrass son grasses for the current study, because these grasses (S. bicolor L. Moench × S. bicolor var. sudanense), and are starting to be utilized by farmers in their Midwest Japanese millet (Echinochloa esculenta), have been grazing systems. This study is one of the first to exam - suggested as a potential solution to maintain pasture ine warm-season grasses (BMRSS and teff) as the only production and to overlap a decrease in cool-season forage source grown in a pasture environment to be forage biomass production during the warmest parts utilized in a continuous culture system. Grazing dairy of summer (Najda, 2003). Incorporating warm-sea- producers that utilized warm-season grasses in the son annual grass into a grazing system provides an Midwest U.S. tend to grow the warm-season grasses opportunity to rest CSP when decreased forage qual- in monoculture, and therefore, we chose to mimic cur- ity growth conditions are limiting and to add flexibil - rent producer conditions to be able to provide valua- ity to the grazing system (Moore et al., 2004). ble information back to producers. Fresh alfalfa was There has been interest by grazing producers in chosen as a treatment for the study, because grazing the Upper Midwest and Northeast United States alfalfa is growing in the Upper Midwest of the United to utilize warm-season annual grasses, such as sor- States, and dairy producers that are grazing cattle are ghum (S. bicolor), sudangrass, and their hybrids as adding alfalfa to grass pastures to maintain diversity. feed for dairy cattle. Use of sorghum and sudan- Additionally, by having fresh alfalfa as a treatment, grass and their hybrids are desired for their per- we were able to compare to prior research that has sistent yield in drought conditions in mid to late utilized fresh alfalfa in continuous culture systems summer when CSP grasses may become dormant (Ribeiro et al., 2005). (White et  al., 2002; McCartney et  al., 2009; Tracy The objectives of this study were to compare et  al., 2010). Teff (Eragrostis tef) has also shown nutrient digestibility, ruminal fermentation, and interest from dairy producers during drought stress microbial protein synthesis using different types of periods. Saylor et  al. (2017) reported that teff hay forages that may be used in grazing systems in the may be utilized to replace alfalfa in dairy cow diets Midwest as substrate, and to compare cool-season without any loss of production. grass species to warm-season grasses as the only for- Continuous culture fermentation systems are age source. Specifically, we will compare fermentation in-vitro systems that provide estimates of ruminal of warm-season grasses (BMRSS and teff grass) with fermentation for various dietary sources (Hannah two control diets (CSP and fresh alfalfa) in a dual-flow et  al., 1986). Previous continuous culture studies continuous culture fermentation system. Results from that used pasture-based diets generally compared this study may provide insight into the digestibility and pasture-based diets to diets with feed additives or bacterial protein synthesis of warm-season grasses non–pasture-based diets. Recently, Dillard et  al. used in pasture systems for dairy cattle and how alter- (2017) reported that warm-season grasses (sor- native forages may affect ruminal fermentation. ghum-sudangrass and Japanese millet) in combin- ation with orchardgrass (Dactylis glomerata) may MATERIALS AND METHODS provide an alternative to orchardgrass when sum- mer productivity is lesser. A  study by Soder et  al. (2013a) compared a 100% orchardgrass diet to Experimental Design and Treatments orchardgrass diets including 10% flaxseed, canola, or sunflower seed, and they reported that NH -N This study was conducted at the University of concentrations and flow of NH -N was lowest for Minnesota West Central Research and Outreach Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 127 Center organic dairy, Morris, MN, and the the prior year. The fertility input was from manure University of Minnesota Dairy Cattle Teaching from cattle rotationally grazing the pastures. The CSP and Research Center, St. Paul, MN. All animal samples were harvested every other day in June by procedures involving animal care and management randomly tossing a 0.23 m square into each paddock were approved by the University of Minnesota before grazing and hand clipping to 5 cm above the Institutional Animal Care and Use Committee ground. Three replicates per pasture were collected (#1508-32961A). A  ruminally cannulated lactat- prior to grazing. Because alfalfa was not grazed, a ing crossbred (Swedish Red × Montbéliarde × large quantity of third cutting early-bud alfalfa was Holstein) dairy cow was used as the rumen fluid harvested at one time using hand clippers at random donor. The diet fed to the donor cow was formu- locations in the field by cutting the forage to 5  cm lated to meet or exceed requirements of a Holstein above the ground. The BMRSS and teff grass sam- cow producing 25  kg milk/d, with 3.8% fat and ples were harvested on July 14, 2015, before grazing 3.7% protein (NRC, 2001). Diet ingredient com- by cutting sample to 5 cm above the ground. Teff was position was 39.8% alfalfa silage, 21.7% grass hay, not well established in the pastures at the WCROC, so 31.3% corn, and 3.6% each of soybean meal and in order to collect adequate DM for the study, add- vitamins and minerals on a DM basis. itional teff samples were collected from a University A dual-flow continuous culture rumen fermen - of Minnesota research plot in St. Paul, MN. The teff tation system was used to evaluate digestibility and harvested from the two locations was composited microbial fermentation response to four treatments: fresh. Samples were dried in an oven at 60 °C for 48 h 1)  fresh alfalfa (M.  sativa), 2)  CSP grasses and leg- and ground (2-mm screen; Wiley mill, Thompson umes, 3)  BMRSS, and 4)  teff grass. The CSP con- Scientific, Philadelphia, PA). Dried, ground forage sisted of smooth bromegrass (Bromus inermis Leyss), samples were mixed thoroughly in their respective orchardgrass (D.  glomerata), meadow fescue (Festuca treatment and pelleted with a CL-5 California Pellet pratensis), and red (Trifolium pratense) and white clover Mill (California Pellet Mill Co., Crawfordsville, IN) (Trifolium repens). Botanical composition of the CSP to a final dimension of 6 mm diameter × 12 mm long. was 30% smooth bromegrass, 20% orchardgrass, 10% Pelleting facilitated use of an automated feed delivery meadow fescue, 20% red clover, and 20% white clover. system to the fermenters. Pelleted diets were placed in Four experimental forage treatments (fresh alfalfa, shallow trays and allowed to air dry for 96 h before CSP, BMRSS, teff grass) were randomly allocated to storing in plastic containers. Ground forage samples an 8-unit, dual-flow continuous culture fermentation were analyzed with near infrared spectroscopy (NIR), system designed to simulate ruminal fermentation and minerals were analyzed using wet chemistry (Rock and outflow to the small intestine. Four forage dietary River Laboratory, Inc., Watertown, WI). Samples treatments were compared during two experimental were analyzed by AOAC (2005) for CP (method periods. Diet preparation resulted in four treatments 954.01) and ether extract (method 920.39). The Ca, P, arranged in a 2  ×  2 completely randomized block Mg, and K were analyzed using wet chemistry meth- design, because each treatment was assigned to two ods (Schalla et al., 2012). Chemical compositions of fermenters over two periods. The forage collection the four forage treatment diets are in Table 1. was conducted at the West Central Research and Outreach Center (Morris, MN) from May to October Continuous Culture Operation 2015. Perennial grasses and legumes were established in 2012 and rotational grazing began in 2013. Organic An 8-unit, dual-flow, continuous culture fer - dairy cows were used to evaluate the effect of two menter system with a modified pH control and pasture production systems (perennial vs. perennial/ measuring system were used in two consecutive 10-d annual systems) over two grazing seasons with rota- experimental periods, similar to that described by tional grazing. Rotational grazing of lactating cows Hannah et al. (1986) and fermenter operation was was initiated when forages were 20–30  cm tall and similar to Ruiz-Moreno et  al. (2015), Carpenter, strip size was adjusted to leave 7–13  cm of refusals et al. (2017), and Fessenden et al. (2017). Treatments (Ruh et al., 2016). The current study was conducted were randomly assigned and duplicated within with CSP and warm-season grasses during the 2015 experimental period to create a randomized com- grazing year. The BMRSS (Black Hawk 12 Organic, plete block design with four observations per treat- Blue River Hybrids, Ames, IA) and teff grass were ment. Fermenter volumes ranged from 1,055  mL planted on May 28, 2015. The teff grass was planted to 1,103 mL. Ten liters of ruminal fluid and 1.5 kg into a pasture that was BMRSS the prior year and the of ruminal digesta were collected approximately BMRSS was planted into a pasture that was teff grass 4  h after the morning feeding from one ruminally Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 128 Ruh et al. Table  1. Chemical composition (%  DM) of four infused continuously into fermenters and contained forage diets (alfalfa, CSP grasses and legumes, 0.4 g/L of urea to simulate N recycling. The pH was BMRSS, and teff grass) used in continuous culture maintained within a range between 5.0 to 6.7 with fermentation automated influx of 3 M HCl and 5M NaOH as needed and recorded using Daqboard and Dasylab Forage treatment Chemical composition, % software (Daisy Lab, National Instrument Services, of DM* Alfalfa CSP BMRSS Teff Austin, TX). Optimal fiber digestion occurs when OM 88.1 90.5 89.3 85.5 pH is greater, typically above 6.0 (Bach et al., 1999). CP 25.1 18.0 16.9 17.0 Culture pH was recorded every 5 min. Solids dilu- NDF 29.8 50.1 50.0 51.2 tion rate were adjusted daily to 4% per hour, similar ADF 22.0 27.2 25.7 26.3 to Bargo et al. (2003), by regulating artificial saliva Ether extract 1.61 2.54 2.09 2.94 input. Liquid dilution rate was 10% per hour, simi- Lignin 4.5 4.7 5.1 4.2 Ash 11.9 9.5 12.4 14.5 lar to Cerrato-Sánchez et al. (2007). Dilution rates Ca 1.69 0.63 0.71 0.41 were attained by regulation of artificial saliva input Mg 0.51 0.19 0.37 0.30 and filtrate removal. Anaerobic conditions were P 0.38 0.28 0.31 0.42 maintained with constant infusion of N at a rate K 3.84 2.72 3.54 5.78 of 20  mL/min using a digital flow meter (Aalborg TTNDFD 43.9 50.2 56.6 62.4 GFM 17, Orangeburgh, NY). *Chemical composition results from NIR from Rock River Labs, Watertown, WI analyses; CP, NDF, ADF, and Ash represent results Sample Collection and Analysis from Stern lab, St. Paul, MN. Total tract NDF digestibility from NIR. Fermenters were operated for two consecutive a,b Means within a row with different superscripts are different at 10-d periods consisting of a 7-d diet-adaptation P < 0.05. period followed by a 3-d sample collection period. Fermenter pH was recorded automatically every cannulated lactating cow. At the beginning of each 5  min, and mean, minimum, and maximum pH experimental period, ruminal fluid was collected were analyzed for the 3-d sampling time by fer- with a pump (Welch model B2585–50; Welch, Niles, menter for each period. IL) with a hose and a 1-mm stainless-steel filter was On days 8 to 10 of each experimental period, a used. Digesta was collected from the venral, cen- water bath maintained the temperature of the efflu - tral, and dorsal areas of the rumen. Ruminal fluid ent containers at 2 °C to prevent further microbial was collected into a preheated sealed thermos and and enzymatic activity. Solids and liquid effluent transported to the laboratory. Within 20  min of samples were collected on days 8, 9, and 10 and digesta and fluid collection, a 500 mL of liquid and homogenized (PT10/3S homogenizer, Kinematica solid rumen sample were mixed using a PT10/3S GmbH) for 2 min. A subsample of 500 mL of efflu - homogenizer (Kinematica GmbH, Bohemia, ent was taken each day, and the three sample days NY), squeezed through two layers of cheesecloth, were combined within fermenter. This sample was and fermenters were inoculated with 1 liter of kept frozen at −20  °C until analysis for total N, ruminal fluid. NH -N, and VFA. A 500 mL of the combined solids Fermenters were maintained at a constant tem- and liquid effluent sample per fermenter represent - perature of 39 °C and were constantly purged with ing 3 d of collection in each period was lyophilized N gas at a rate of 40  mL/min to maintain anaer- and used for analysis of DM, OM, NDF, ADF, obiosis. Amount of diet (as-fed) was adjusted on ash, and purines. On the final day of sampling at days 0, 4, and 7 for DM content to attain a feeding the end of each period, fermenter contents were rate of 60  g of diet DM/fermenter daily (de Veth squeezed through two layers of cheesecloth, and and Kolver, 2001). Fermenters were fed throughout the liquid was centrifuged at 1,000 × g for 10 min at the day with the automated feeding system, and 4 °C to remove feed particles. The supernatant was the pelleted forage diet was slowly fed into the fer- then centrifuged at 20,000  × g for 20  min at 4  °C menter over eight equally spaced, 90-min periods. to isolate the microbial pellet. The microbial pellet An automated feeding device (Hannah et al., 1986) was suspended in distilled water, frozen at −20 °C controlled by a timer (DT 17, Intermatic, Spring and then lyophilized prior to analysis of DM, ash, Grove, IL) was used to regulate feeding duration total N, and purines. and schedule. Each 90-min feeding period was fol- Pelleted forage samples (dietary forage treat- lowed by 90  min of rest. Artificial saliva was pre - ments), effluent, and microbial pellets were pared according to Weller and Pilgrim (1974) and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 129 analyzed for DM by drying at 105  °C for 24  h. served as block with all treatments equally repre- Ash was determined by the weight difference after sented within block. Forages were analyzed as a 24-h combustion at 550 °C (AOAC, 1984; method fixed effect and period (block) was a random effect 967.04). Total N content of diets, effluent, and (Ruiz-Moreno et  al., 2015; Binversie et  al., 2016; bacteria and NH -N of diets and effluent were Carpenter et  al., 2017). All treatment results were determined using the Kjeldahl method (AOAC, reported with least squares means, with signifi - 1984; method 984.13). Ammonia-N concentration cance declared at P  <  0.05. The pH was analyzed in the fermenters was determined on the super- for mean, maximum, and minimum pH for the natant of a centrifuged (5,000  × g for 15  min at 3-d sampling period as repeated measures with an 4 °C) subsample of liquid effluent by steam distilla - autoregressive of order 1 structure of covariance tion (Bremner and Keeney, 1965) with magnesium on the basis of the minimum values of Akaike’s oxide using a 2,300 Kjeltec Analyzer Unit (Foss information criterion. All treatment results were Tecator AB, Höganäs, Sweden). Sequential fiber reported with least squares means, with signifi - analyses (Van Soest, 2015) were used to determine cance declared at P  <  0.05 and trends declared at NDF and ADF concentrations of the diet and P < 0.10. Differences among treatments were tested effluents using an ANKOM A200 fiber analyzer using LSMEANS with the Tukey adjustment for with F57 fiber bags (ANKOM Corp, Fairport, multiple means comparison. Results of the current NY) and lignin content of the diet was measured study are reported as least squared means from four gravimetrically after hydrolysis of acid deter- observations per forage treatment. gent residue using 12 M H SO (Van Soest, 2015). 2 4 Purine concentrations were determined by the RESULTS AND DISCUSSION method of Zinn and Owens (1986). Purine concen- trations of the effluent and bacteria were used to The chemical composition of the forage calculate N metabolism and efficiency of microbial treatments is provided in Table 1. Forage analy- protein synthesis. Effluent samples were prepared ses by NIR were performed by Rock River Labs for VFA analysis using the procedure for rumen Inc., while CP, NDF, and ADF were analyzed fluid preparation described by Erwin et al. (1961). using the same procedures used to analyze fer- Ruminal fluid effluent was centrifuged to remove menter effluent. Dietary protein levels may affect heavy feed particles and clarified, then 2.0 mL was ruminal fermentation patterns and digestibil- mixed with a 25% meta-phosphoric acid solution ity and create confounding results (Bach et  al., (0.5  mL) and centrifuged at 10,000 g for 15  min 1999), which is why many in-vitro studies feed until supernatant was clear. The supernatant was isonitrogenous diets when investigating alter- stored at −20  °C until analyzed. Effluent VFA native treatments. However, the current study concentration was performed via gas chromatog- investigated the differences in ruminal fermen- raphy (Agilent 7890B GC-FID with a G4567A tation between CSP and warm-season annual Autosampler). The Agilent DB-FFAP column was grasses, and it was important to keep the treat- 30 m length, 0.25  mm diameter, and had a film ments at their original protein levels, with the thickness of 0.15 µm. Chromatographic conditions understanding that this may ultimately affect were 1  µL injection with an inlet temperature of fermentation, and may be of interest to reflect a 240 °C, helium as carrier gas at 1 mL/min constant grazing situation (Bach et al., 1999). This differ- o fl w, initial oven temperature of 60 °C with a 2-min ence in N content of the diets was accounted for hold time, ramp at 20 °C/min to 220 °C and a 1-min by expressing results as a percentage of total N hold time at 220 °C. The flame ionization detector intake (Bach et al., 1999). was set at 250 °C. Standard solutions with known concentrations of VFA were analyzed to calibrate Digestibility chromatograph. Apparent and true (corrected for contribu- tion of bacterial flow) digestibility and pH flow Statistical Analysis for forage treatments are provided in Table 2. The CSP, BMRSS, and teff grass had lesser (P < 0.05) Data were analyzed using the MIXED proce- apparent DM and OM digestibility than alfalfa. dure of SAS software 9.4 (SAS Inst. Inc., 2016). The NDF digestibility for CSP and teff was lesser Data from fermenters were analyzed as a rand- (P  <  0.05) than alfalfa, and BMRSS was similar omized complete block (period) design. Period Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 130 Ruh et al. Table  2. Nutrient digestibility and pH of four to the current study. Overall, findings of apparent forage diets (alfalfa, CSP grasses and legumes, digestibility are consistent with previous research BMRSS, and teff grass) during continuous culture comparing alfalfa to grass in vivo, in which alfalfa fermentation disappeared more quickly from the rumen than the perennial ryegrass because of a faster rate of diges- Forage tion and faster particle size reduction of alfalfa Item Alfalfa CSP BMRSS Teff SEM (Waghorn et al., 1989). Results from Ribeiro et al. Apparent digestibility (2005) were different than results from the current -------------------%----------------- study because although there was greater nutrient a b b b DM 69.4 47.1 52.6 49.8 5.2 digestibility in alfalfa, bacterial OM flow was sim - a b b b OM 54.1 32.5 38.1 29.4 4.7 a b ab b ilar across all treatments (Table 4). NDF 75.5 52.6 65.9 56.6 5.3 a b ab ab ADF 75.5 55.4 67.5 59.4 5.3 True digestibility* Volatile Fatty Acids a b b b DM 85.8 64.0 66.2 65.9 5.7 a b b b OM 69.2 47.0 50.4 44.1 4.1 Least square means of VFA for the forage treat- pH ments are in Table  3. Total VFA amount (mM) Mean 6.38 6.18 6.19 6.22 0.10 were similar (P > 0.10) among the forage diets. The Minimum 5.88 5.14 5.41 5.92 0.31 amount of total VFA for CSP was similar to amount Maximum 7.26 6.76 7.23 7.01 0.25 of total VFA found in a previous study for pasture *Corrected for contribution of bacterial flow. intake in continuous culture (Bargo et al., 2003) but a,b Means within a row with different superscripts are different at greater than total VFA with a ryegrass only diet in P < 0.05. continuous culture fed at 60  g DM/d (de Veth and Kolver, 2001). Total VFA for the current study for (P > 0.10) to the other forages. The CSP was lesser the grass species was much greater than reported by (P  <  0.05) for ADF digestibility than alfalfa but Dillard et al. (2017) for mixes of cool- and warm-sea- was similar (P > 0.10) to BMRSS and teff. True son grass species. Quite possibly, differences between DM and OM digestibilities were lesser (P < 0.05) studies were observed, because the current study uti- in grasses compared with alfalfa. Digestibilities lized warm-season grasses as the only forage source, among BMRSS and teff grass were similar (P and Dillard et al. (2017) utilized alternative combi- > 0.10). A  study by de Veth and Kolver (2001) nations of cool-season and warm-season grasses. found a range of apparent DM digestibility There were some differences in individual VFA (44.7% to 56.4%) and OM digestibility (48.1% to concentrations and in molar proportions of VFA. 58.7%) from ryegrass pastures, which was simi- These changes may indicate differences in a shift of lar to results from the current study for DM and the rumen microbial population when alfalfa and OM digestibility. A  study by Soder et  al. (2013b) alfalfa hay are studied (Ribeiro et al., 2005). Similar found much greater apparent digestibility of DM, results of individual VFA production for CSP were OM, and NDF for 100% orchardgrass in a dual- found for a smooth bromegrass and orchardgrass o fl w continuous culture fermenter system, which pasture diets in continuous culture (Bargo et  al., could have been due to a greater CP level or the 2003). Molar proportions of acetate, propionate, greater level of daily diet, which was greater than and butyrate were similar to previous results of a the daily diet level of 60  g DM/d in the current CSP grass and legume diets in continuous culture study. The current study has only 20% orchard- (Bach et al., 1999). grass in the diet, and differences between Soder Bargo et  al. (2003) showed an inverse correl- et  al. (2013b) and the current study may be due ation between pH patterns and VFA production to the composition of orchardgrass in the diet. from smooth bromegrass and orchardgrass pasture A  study evaluating alfalfa in continuous culture diets. Forage treatments were similar (P > 0.10) in found lesser apparent NDF and ADF digestibil- mean pH among the forage treatments in this study ity than results from the current study for alfalfa and differences in individual VFA do not match the (Ribeiro et  al., 2005). Recently, Dillard et  al. pattern of pH. Quite possibly, this insignificant dif - (2017) studied mixes of cool-season (orchard- ferences reported in this study may be due to the fact grass) and warm-season grasses (sorghum × that pH was controlled for in the study. However, sudangrass and Japanese millet) and found simi- another study found no large differences of indi- lar DM digestibilities, greater OM digestibilities, vidual proportions of VFA with change in pH (de and great NDF and ADF digestibility compared Veth and Kolver, 2001). The molar proportion of Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 131 Table  3. VFA concentration of 4 forage diets Table  4. Nitrogen metabolism of four forage diets (alfalfa, CSP grasses and legumes, BMRSS, and (alfalfa, CSP grasses and legumes, BMRSS, and teff grass) in continuous culture fermentation teff grass) in continuous culture fermentation Forage Forage VFA Alfalfa CSP BMRSS Teff SEM Variable Alfalfa CSP BMRSS Teff SEM a b c c Total VFA, mM 78.6 77.6 75.6 82.3 13.1 N intake, g/d 3.09 2.31 2.18 2.20 0.01 a b b b Individual VFA, mol/100 mol NH3-N, mg/dL 22.5 7.5 7.4 8.9 0.75 a ab b a Acetate 72.1 71.2 67.7 75.0 1.3 CP degradation, % 79.8 77.2 69.1 65.9 4.6 b a ab b Propionate 16.5 20.0 18.4 17.1 0.54 N flows, g/d b b a b a b b ab Butyrate 7.9 7.2 10.1 6.7 0.58 Total N 1.99 1.50 1.51 1.70 0.11 a b b b a b b b Isobutyrate 0.69 0.27 0.25 0.17 0.03 NH -N 0.52 0.17 0.17 0.20 0.02 ab ab a b Isovalerate 0.82 0.30 1.2 0.09 0.31 NAN 1.46 1.33 1.34 1.50 0.12 a b a b Valerate 1.7 0.90 2.1 0.83 0.17 Bacterial N 0.84 0.81 0.67 0.75 0.14 a b a b Caproate 0.29 0.14 0.32 0.10 0.03 Dietary N 0.62 0.53 0.67 0.75 0.12 a b b a A:P ratio 4.4 3.6 3.7 4.4 0.18 N flows, % of total N flow a b b b NH3-N 26.5 11.6 11.4 11.8 1.8 a,b Means within a row with different superscripts are different at a b b b NAN 73.5 88.4 88.6 88.2 1.8 P < 0.05. Bacterial N 41.3 54.1 44.4 43.8 6.1 Dietary N 32.1 34.3 44.2 44.4 6.0 butyrate was greatest for BMRSS compared to the Efficiency of Microbial protein synthesis other forage treatments. g N/kg DM truly digested 15.0 19.9 16.0 18.7 2.7 Isobutyrate was greater in alfalfa than the g N/kg OM truly digested 21.3 30.4 24.0 33.3 4.9 CSP, BMRSS, and teff grass treatments, which is a,b Means within a row with different superscripts are different at similar to the pattern of true and apparent digest- P < 0.05. ibility of DM and OM of the dietary treatments (Table 2). The CP degradation (Table 4) was similar rates of microbial growth and ultimately digestibil- (P > 0.10) among treatments. Molar proportions ity measured in vitro and in vivo (Bach et al., 1999). of acetate, propionate, and butyrate of alfalfa from CP degradation was similar (P > 0.05) among diet- this study were similar to previously reported val- ary treatments in the fermenters. This observation ues of acetate, propionate, and butyrate for fresh is slightly lesser than CP digestibility found in a pre- alfalfa in continuous culture (Ribeiro et al., 2005). vious study using pasture diets in continuous cul- There are no previous studies that evaluated ture (Soder et al. 2013b). warm-season grass as the only forage in continu- The NH -N was greater (P <0.05) in alfal- ous culture. One study investigated warm-season fa-fed fermenters than for CSP, BMRSS, and teff and cool-season grasses fed to cannulated steers fermenters. Similar results of NH -N were found and reported a significant effect for warm-season for cool-season grasses in continuous culture com- vs. cool-season grasses for the molar proportions of pared to CSP in the current study (Bach et  al., propionate, butyrate, and valerate in ruminal fluid 1999). The CSP and BMRSS had similar total N (Bohnert et  al., 2011), which was in agreement to o fl w, while alfalfa had the greatest ( P < 0.05) total the current study. Individual concentrations from N flow and teff was intermediate in total N flow. the current study of acetate and propionate were As a percent of total N flows, NH -N was greater greater and lesser for butyrate for warm-season (P  <  0.05) in alfalfa than the grasses, reflecting grasses (BMRSS) reported by Dillard et al. (2017). the pattern of total NH -N for each treatment. Differences observed between studies may be due to Treatments were similar (P > 0.10) for non–NH - warm-season grass being analyzed as the only for- N, bacterial N, or dietary N flows as a percent of age source compared to warm-season grasses com- total N.  Even though alfalfa had greater NH -N posited with orchardgrass in Dillard et al. (2017). concentrations, treatments were similar (P > 0.10) in efficiency of microbial protein synthesis among Nitrogen Metabolism any of the treatments, on either a DM or OM basis. The N metabolism of fermenters fed the vari- This demonstrates that although there was lesser ous forages is found in Table  4. The N intake was NH -N in all the grass treatments than in alfalfa, highest for alfalfa, intermediate for CSP, and lowest there were still adequate amounts of NH -N for for warm-season grasses based on the dietary CP microbial protein synthesis. The minimum amount of the forage treatments. The N intake can affect of NH -N required for microbial protein synthesis Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 132 Ruh et al. was estimated to be 5  mg/dL for in-vitro systems It is known that grazing cows will experience (Satter and Slyter, 1974), and all treatments in changes in their diet throughout the grazing season this study were well above that value. Efficiency due to weather, the gradually increasing maturity of microbial protein synthesis on an OM basis in of cool season grasses, as well as changes in season the current study was lesser to previous results for in temperate regions of the United States. Cows in alfalfa (Ribeiro et  al., 2005) and pasture in con- a complementary grazing system may have add- tinuous culture (de Veth and Kolver, 2001; Cerrato- itional disruptions to their diet because they often Sánchez et al., 2007; Soder et al., 2013b, 2016). graze one type of grass, then sequentially another Alfalfa, CSP, BMRSS, and teff grass were sim- type of grass in a different pasture. Results from ilar (P > 0.10) for mean, minimum, or maximum the current study found several changes in ruminal pH (Table  2). One continuous culture fermenter fermentation among forage treatments of differ- study researching effects of pH levels determined ent species. Future research could investigate how that the optimal pH for high-quality pasture for- this shift in microbial population, possibly every age is 6.35 (de Veth and Kolver, 2001). We analyzed few days or every few weeks, affects a grazing dairy the amount of time spent below 5.8 as well as the cow. It would be interesting to determine what kind amount of time spent above 6.4. Time spent below of effect a sequential pasture rotation may have pH 5.8 ranged from 1 to 5  min/d. Sauvant et  al. on the rumen microbial population and fermen- (1999) found that it may be possible to experience tation. Further research dealing with warm-sea- a pH below 6.0 for 4 h without affecting microbial son grasses in continuous culture systems should fermentation. pH was analyzed at 5.8 because we include variations of concentrate and total mixed expected lesser values from the CSP examined in ration supplementation. Research on fermentation our study. Our results suggest that fermenters spent characteristics of warm-season grasses should be almost no time below a pH of 5.8. studied further, because there had not been much Because the study was in-vitro system, diets previous research conducted. may be affected differently when actually consumed Dairy farmers that utilize warm-season grasses by cows (Mansfield et  al., 1995 ). Although fresh in pasture rotations for lactating dairy cattle, may grass was used, it underwent heating and pressure benefit from utilizing BMRSS and teff grass in their in the drying and pelleting processes which could rotations. The BMRSS and teff grass had similar alter some components of the grass. These changes digestibility when compared to cool-season pasture should have been uniform across all treatments; grasses and legumes. No differences for total VFA however, it is important to note that dietary values were found for CSP compared to warm-season of pelleted grasses may be different than actual pas- grasses; however, specific individual VFA differed ture forage grasses that cows would be consuming among alfalfa, CSP, and warm-season grasses. in a grazing system. Fresh forages have greater con- Forage quality of the forages utilized in this study centrations of rapidly fermented sugars as well as may have profound effect on the results, and results greater concentrations of more digestible protein, may be different depending on weather conditions which could have been lost during heat processing throughout the year (i.e., drought or heavy rains). of our forages (Van Soest, 1994). In addition, most Overall, fermentation of warm-season grasses was grazing cows do not consume forage-only diets, so similar to the CSP. Results of this study indicate there may be more complex interactions if grazing that warm-season grasses may be successfully cows were to be supplemented with concentrate or grazed by dairy cows in monoculture and may be processed forages while grazing pasture. Previous included in a complementary grazing system with studies have shown alterations in bacterial growth CSP grasses and legumes, without concerns about rates with different combinations of carbohydrates, negative impact on rumen or animal health. which may decrease fiber digestibility ( Russell and Baldwin 1978). Vibart et  al. (2010) demon- LITERATURE CITED strated improvement in N utilization when grazing AOAC. 1984. Official methods of analysis. 14th ed. Arlington cows are supplemented with a total mixed ration. (VA): Association of Official Analytical Chemists. Minimum effective fiber requirements may be dif - AOAC. 2005. Official methods of analysis. 18th ed. 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A rapid procedure for Publishing Services. purine measurement and its use for estimating net ruminal Vibart, R. E., J. C.  Burns, and V.  Fellner. 2010. Effect protein synthesis. Can. J. Anim. Sci. 66:157. doi:10.4141/ of replacing total mixed ration with pasture on cjas86-017 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Comparison of warm season and cool season forages for dairy grazing systems in continuous culture

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Comparison of warm season and cool season forages for dairy grazing systems in continuous culture ,2 † ‡ Kathryn E. Ruh,* Bradley J. Heins,* Isaac J. Salfer, Robert D. Gardner, and Marshall D. Stern* *Department of Animal Science, University of Minnesota, St. Paul, MN 55108; Department of Animal Science, The Pennsylvania State University, University Park, PA 16802; Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108 ABSTRACT: The objective of this study was to warm-season annual grasses compared with fresh compare warm-season annual grasses to cool-sea- alfalfa. Total VFA were not affected (P >  0.05) son perennial (CSP) grasses for ruminal nutrient by forage. The NH3-N concentrations were high- digestibility and N metabolism in a dual-flow est (P < 0.05) with alfalfa compared with the continuous culture fermentation system. Dietary other CSP grasses and legumes and warm-season treatments were 1) fresh alfalfa, 2) CSP grasses annual grasses. CP digestibility was not affected and legumes, 3) brown-midrib sorghum-sudan- (P > 0.05) by forage treatment. Flow of NH3-N grass (BMRSS), and 4) teff grass from an organic was greatest (P < 0.05) for alfalfa, reflecting the dairy production system. Eight dual-flow con - greatest NH3-N concentration. Flow of total N tinuous culture fermenters were used during two was greatest (P < 0.05) for alfalfa, intermediate consecutive 10-d periods consisting of 7 d for sta- for teff, and lowest for CSP grasses and legumes bilization followed by 3 d of sampling. Fermenter and BMRSS. Flows of bacterial N, efficiency of samples were collected on days 8, 9, and 10 for bacterial N, non-NH3-N, and dietary N were not analysis of pH, NH3-N, and VFA. Apparent affected (P > 0.05) by forage source. Overall, fer- DM, OM, NDF, and ADF digestibility were on mentation of warm-season grasses was similar to average lesser (P < 0.05) in CSP grasses and leg- the cool-season grasses and legumes which indi- umes and warm-season annual grasses compared cate dairy producers may use warm-season grasses with alfalfa. True DM and OM digestibility were without concerns about negative impact on rumen lesser (P < 0.05) for CSP grasses and legumes and health. Key words: continuous culture fermentation, grazing, sorghum-sudangrass, teff © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2018.2:125–134 doi: 10.1093/tas/txy014 The authors express gratitude to the interns and graduate Food and Agriculture. Financial support was also provided for students at the University of Minnesota West Central Research this project by the Ceres Trust (Chicago, IL). Authors confirm and Outreach Center, Morris, for their assistance in data collec- there is no conflict of interest with this study. tion. This work is supported by Organic Agriculture Research Corresponding author: hein0106@umn.edu and Extension Initiative (grant no. 2012-51300-20015/project Received March 27, 2018. accession no. 0230589) from the USDA National Institute of Accepted April 3, 2018 Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 126 Ruh et al. INTRODUCTION a herbage only diet (Soder et al., 2013a). One study compared fresh alfalfa (Medicago sativa) to alfalfa Profitability of grazing dairy farms relies on hay in continuous culture to determine differences pastures that produce a large quantity of high-qual- in ruminal fermentation when sucrose was added to ity forage for cattle to graze. In the upper Midwest, the alfalfa diet (Ribeiro et al., 2005). There are few cool-season grass and clover species are traditional studies that compare digestibility and ruminal fer- pasture forages for many dairy grazing producers. mentation of pasture diets of different species com- However, cool-season perennial (CSP) grasses and position in continuous culture, although one study legumes experiences a decrease in growth rate during was conducted that used fresh orchardgrass or red periods of high temperatures and low precipitation, clover in combination with different inclusion of as observed in July and August in the upper Midwest corn grain in continuous culture (Loor et al., 2003). (Moore et  al., 2004; Hudson et  al., 2010). Warm- The brown-midrib sorghum-sudangrass season annual grasses, such as sudangrass (Sorghum (BMRSS) and teff grass were chosen as the warm-sea- bicolor × drummondii), sorghum × sudangrass son grasses for the current study, because these grasses (S. bicolor L. Moench × S. bicolor var. sudanense), and are starting to be utilized by farmers in their Midwest Japanese millet (Echinochloa esculenta), have been grazing systems. This study is one of the first to exam - suggested as a potential solution to maintain pasture ine warm-season grasses (BMRSS and teff) as the only production and to overlap a decrease in cool-season forage source grown in a pasture environment to be forage biomass production during the warmest parts utilized in a continuous culture system. Grazing dairy of summer (Najda, 2003). Incorporating warm-sea- producers that utilized warm-season grasses in the son annual grass into a grazing system provides an Midwest U.S. tend to grow the warm-season grasses opportunity to rest CSP when decreased forage qual- in monoculture, and therefore, we chose to mimic cur- ity growth conditions are limiting and to add flexibil - rent producer conditions to be able to provide valua- ity to the grazing system (Moore et al., 2004). ble information back to producers. Fresh alfalfa was There has been interest by grazing producers in chosen as a treatment for the study, because grazing the Upper Midwest and Northeast United States alfalfa is growing in the Upper Midwest of the United to utilize warm-season annual grasses, such as sor- States, and dairy producers that are grazing cattle are ghum (S. bicolor), sudangrass, and their hybrids as adding alfalfa to grass pastures to maintain diversity. feed for dairy cattle. Use of sorghum and sudan- Additionally, by having fresh alfalfa as a treatment, grass and their hybrids are desired for their per- we were able to compare to prior research that has sistent yield in drought conditions in mid to late utilized fresh alfalfa in continuous culture systems summer when CSP grasses may become dormant (Ribeiro et al., 2005). (White et  al., 2002; McCartney et  al., 2009; Tracy The objectives of this study were to compare et  al., 2010). Teff (Eragrostis tef) has also shown nutrient digestibility, ruminal fermentation, and interest from dairy producers during drought stress microbial protein synthesis using different types of periods. Saylor et  al. (2017) reported that teff hay forages that may be used in grazing systems in the may be utilized to replace alfalfa in dairy cow diets Midwest as substrate, and to compare cool-season without any loss of production. grass species to warm-season grasses as the only for- Continuous culture fermentation systems are age source. Specifically, we will compare fermentation in-vitro systems that provide estimates of ruminal of warm-season grasses (BMRSS and teff grass) with fermentation for various dietary sources (Hannah two control diets (CSP and fresh alfalfa) in a dual-flow et  al., 1986). Previous continuous culture studies continuous culture fermentation system. Results from that used pasture-based diets generally compared this study may provide insight into the digestibility and pasture-based diets to diets with feed additives or bacterial protein synthesis of warm-season grasses non–pasture-based diets. Recently, Dillard et  al. used in pasture systems for dairy cattle and how alter- (2017) reported that warm-season grasses (sor- native forages may affect ruminal fermentation. ghum-sudangrass and Japanese millet) in combin- ation with orchardgrass (Dactylis glomerata) may MATERIALS AND METHODS provide an alternative to orchardgrass when sum- mer productivity is lesser. A  study by Soder et  al. (2013a) compared a 100% orchardgrass diet to Experimental Design and Treatments orchardgrass diets including 10% flaxseed, canola, or sunflower seed, and they reported that NH -N This study was conducted at the University of concentrations and flow of NH -N was lowest for Minnesota West Central Research and Outreach Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 127 Center organic dairy, Morris, MN, and the the prior year. The fertility input was from manure University of Minnesota Dairy Cattle Teaching from cattle rotationally grazing the pastures. The CSP and Research Center, St. Paul, MN. All animal samples were harvested every other day in June by procedures involving animal care and management randomly tossing a 0.23 m square into each paddock were approved by the University of Minnesota before grazing and hand clipping to 5 cm above the Institutional Animal Care and Use Committee ground. Three replicates per pasture were collected (#1508-32961A). A  ruminally cannulated lactat- prior to grazing. Because alfalfa was not grazed, a ing crossbred (Swedish Red × Montbéliarde × large quantity of third cutting early-bud alfalfa was Holstein) dairy cow was used as the rumen fluid harvested at one time using hand clippers at random donor. The diet fed to the donor cow was formu- locations in the field by cutting the forage to 5  cm lated to meet or exceed requirements of a Holstein above the ground. The BMRSS and teff grass sam- cow producing 25  kg milk/d, with 3.8% fat and ples were harvested on July 14, 2015, before grazing 3.7% protein (NRC, 2001). Diet ingredient com- by cutting sample to 5 cm above the ground. Teff was position was 39.8% alfalfa silage, 21.7% grass hay, not well established in the pastures at the WCROC, so 31.3% corn, and 3.6% each of soybean meal and in order to collect adequate DM for the study, add- vitamins and minerals on a DM basis. itional teff samples were collected from a University A dual-flow continuous culture rumen fermen - of Minnesota research plot in St. Paul, MN. The teff tation system was used to evaluate digestibility and harvested from the two locations was composited microbial fermentation response to four treatments: fresh. Samples were dried in an oven at 60 °C for 48 h 1)  fresh alfalfa (M.  sativa), 2)  CSP grasses and leg- and ground (2-mm screen; Wiley mill, Thompson umes, 3)  BMRSS, and 4)  teff grass. The CSP con- Scientific, Philadelphia, PA). Dried, ground forage sisted of smooth bromegrass (Bromus inermis Leyss), samples were mixed thoroughly in their respective orchardgrass (D.  glomerata), meadow fescue (Festuca treatment and pelleted with a CL-5 California Pellet pratensis), and red (Trifolium pratense) and white clover Mill (California Pellet Mill Co., Crawfordsville, IN) (Trifolium repens). Botanical composition of the CSP to a final dimension of 6 mm diameter × 12 mm long. was 30% smooth bromegrass, 20% orchardgrass, 10% Pelleting facilitated use of an automated feed delivery meadow fescue, 20% red clover, and 20% white clover. system to the fermenters. Pelleted diets were placed in Four experimental forage treatments (fresh alfalfa, shallow trays and allowed to air dry for 96 h before CSP, BMRSS, teff grass) were randomly allocated to storing in plastic containers. Ground forage samples an 8-unit, dual-flow continuous culture fermentation were analyzed with near infrared spectroscopy (NIR), system designed to simulate ruminal fermentation and minerals were analyzed using wet chemistry (Rock and outflow to the small intestine. Four forage dietary River Laboratory, Inc., Watertown, WI). Samples treatments were compared during two experimental were analyzed by AOAC (2005) for CP (method periods. Diet preparation resulted in four treatments 954.01) and ether extract (method 920.39). The Ca, P, arranged in a 2  ×  2 completely randomized block Mg, and K were analyzed using wet chemistry meth- design, because each treatment was assigned to two ods (Schalla et al., 2012). Chemical compositions of fermenters over two periods. The forage collection the four forage treatment diets are in Table 1. was conducted at the West Central Research and Outreach Center (Morris, MN) from May to October Continuous Culture Operation 2015. Perennial grasses and legumes were established in 2012 and rotational grazing began in 2013. Organic An 8-unit, dual-flow, continuous culture fer - dairy cows were used to evaluate the effect of two menter system with a modified pH control and pasture production systems (perennial vs. perennial/ measuring system were used in two consecutive 10-d annual systems) over two grazing seasons with rota- experimental periods, similar to that described by tional grazing. Rotational grazing of lactating cows Hannah et al. (1986) and fermenter operation was was initiated when forages were 20–30  cm tall and similar to Ruiz-Moreno et  al. (2015), Carpenter, strip size was adjusted to leave 7–13  cm of refusals et al. (2017), and Fessenden et al. (2017). Treatments (Ruh et al., 2016). The current study was conducted were randomly assigned and duplicated within with CSP and warm-season grasses during the 2015 experimental period to create a randomized com- grazing year. The BMRSS (Black Hawk 12 Organic, plete block design with four observations per treat- Blue River Hybrids, Ames, IA) and teff grass were ment. Fermenter volumes ranged from 1,055  mL planted on May 28, 2015. The teff grass was planted to 1,103 mL. Ten liters of ruminal fluid and 1.5 kg into a pasture that was BMRSS the prior year and the of ruminal digesta were collected approximately BMRSS was planted into a pasture that was teff grass 4  h after the morning feeding from one ruminally Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 128 Ruh et al. Table  1. Chemical composition (%  DM) of four infused continuously into fermenters and contained forage diets (alfalfa, CSP grasses and legumes, 0.4 g/L of urea to simulate N recycling. The pH was BMRSS, and teff grass) used in continuous culture maintained within a range between 5.0 to 6.7 with fermentation automated influx of 3 M HCl and 5M NaOH as needed and recorded using Daqboard and Dasylab Forage treatment Chemical composition, % software (Daisy Lab, National Instrument Services, of DM* Alfalfa CSP BMRSS Teff Austin, TX). Optimal fiber digestion occurs when OM 88.1 90.5 89.3 85.5 pH is greater, typically above 6.0 (Bach et al., 1999). CP 25.1 18.0 16.9 17.0 Culture pH was recorded every 5 min. Solids dilu- NDF 29.8 50.1 50.0 51.2 tion rate were adjusted daily to 4% per hour, similar ADF 22.0 27.2 25.7 26.3 to Bargo et al. (2003), by regulating artificial saliva Ether extract 1.61 2.54 2.09 2.94 input. Liquid dilution rate was 10% per hour, simi- Lignin 4.5 4.7 5.1 4.2 Ash 11.9 9.5 12.4 14.5 lar to Cerrato-Sánchez et al. (2007). Dilution rates Ca 1.69 0.63 0.71 0.41 were attained by regulation of artificial saliva input Mg 0.51 0.19 0.37 0.30 and filtrate removal. Anaerobic conditions were P 0.38 0.28 0.31 0.42 maintained with constant infusion of N at a rate K 3.84 2.72 3.54 5.78 of 20  mL/min using a digital flow meter (Aalborg TTNDFD 43.9 50.2 56.6 62.4 GFM 17, Orangeburgh, NY). *Chemical composition results from NIR from Rock River Labs, Watertown, WI analyses; CP, NDF, ADF, and Ash represent results Sample Collection and Analysis from Stern lab, St. Paul, MN. Total tract NDF digestibility from NIR. Fermenters were operated for two consecutive a,b Means within a row with different superscripts are different at 10-d periods consisting of a 7-d diet-adaptation P < 0.05. period followed by a 3-d sample collection period. Fermenter pH was recorded automatically every cannulated lactating cow. At the beginning of each 5  min, and mean, minimum, and maximum pH experimental period, ruminal fluid was collected were analyzed for the 3-d sampling time by fer- with a pump (Welch model B2585–50; Welch, Niles, menter for each period. IL) with a hose and a 1-mm stainless-steel filter was On days 8 to 10 of each experimental period, a used. Digesta was collected from the venral, cen- water bath maintained the temperature of the efflu - tral, and dorsal areas of the rumen. Ruminal fluid ent containers at 2 °C to prevent further microbial was collected into a preheated sealed thermos and and enzymatic activity. Solids and liquid effluent transported to the laboratory. Within 20  min of samples were collected on days 8, 9, and 10 and digesta and fluid collection, a 500 mL of liquid and homogenized (PT10/3S homogenizer, Kinematica solid rumen sample were mixed using a PT10/3S GmbH) for 2 min. A subsample of 500 mL of efflu - homogenizer (Kinematica GmbH, Bohemia, ent was taken each day, and the three sample days NY), squeezed through two layers of cheesecloth, were combined within fermenter. This sample was and fermenters were inoculated with 1 liter of kept frozen at −20  °C until analysis for total N, ruminal fluid. NH -N, and VFA. A 500 mL of the combined solids Fermenters were maintained at a constant tem- and liquid effluent sample per fermenter represent - perature of 39 °C and were constantly purged with ing 3 d of collection in each period was lyophilized N gas at a rate of 40  mL/min to maintain anaer- and used for analysis of DM, OM, NDF, ADF, obiosis. Amount of diet (as-fed) was adjusted on ash, and purines. On the final day of sampling at days 0, 4, and 7 for DM content to attain a feeding the end of each period, fermenter contents were rate of 60  g of diet DM/fermenter daily (de Veth squeezed through two layers of cheesecloth, and and Kolver, 2001). Fermenters were fed throughout the liquid was centrifuged at 1,000 × g for 10 min at the day with the automated feeding system, and 4 °C to remove feed particles. The supernatant was the pelleted forage diet was slowly fed into the fer- then centrifuged at 20,000  × g for 20  min at 4  °C menter over eight equally spaced, 90-min periods. to isolate the microbial pellet. The microbial pellet An automated feeding device (Hannah et al., 1986) was suspended in distilled water, frozen at −20 °C controlled by a timer (DT 17, Intermatic, Spring and then lyophilized prior to analysis of DM, ash, Grove, IL) was used to regulate feeding duration total N, and purines. and schedule. Each 90-min feeding period was fol- Pelleted forage samples (dietary forage treat- lowed by 90  min of rest. Artificial saliva was pre - ments), effluent, and microbial pellets were pared according to Weller and Pilgrim (1974) and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 129 analyzed for DM by drying at 105  °C for 24  h. served as block with all treatments equally repre- Ash was determined by the weight difference after sented within block. Forages were analyzed as a 24-h combustion at 550 °C (AOAC, 1984; method fixed effect and period (block) was a random effect 967.04). Total N content of diets, effluent, and (Ruiz-Moreno et  al., 2015; Binversie et  al., 2016; bacteria and NH -N of diets and effluent were Carpenter et  al., 2017). All treatment results were determined using the Kjeldahl method (AOAC, reported with least squares means, with signifi - 1984; method 984.13). Ammonia-N concentration cance declared at P  <  0.05. The pH was analyzed in the fermenters was determined on the super- for mean, maximum, and minimum pH for the natant of a centrifuged (5,000  × g for 15  min at 3-d sampling period as repeated measures with an 4 °C) subsample of liquid effluent by steam distilla - autoregressive of order 1 structure of covariance tion (Bremner and Keeney, 1965) with magnesium on the basis of the minimum values of Akaike’s oxide using a 2,300 Kjeltec Analyzer Unit (Foss information criterion. All treatment results were Tecator AB, Höganäs, Sweden). Sequential fiber reported with least squares means, with signifi - analyses (Van Soest, 2015) were used to determine cance declared at P  <  0.05 and trends declared at NDF and ADF concentrations of the diet and P < 0.10. Differences among treatments were tested effluents using an ANKOM A200 fiber analyzer using LSMEANS with the Tukey adjustment for with F57 fiber bags (ANKOM Corp, Fairport, multiple means comparison. Results of the current NY) and lignin content of the diet was measured study are reported as least squared means from four gravimetrically after hydrolysis of acid deter- observations per forage treatment. gent residue using 12 M H SO (Van Soest, 2015). 2 4 Purine concentrations were determined by the RESULTS AND DISCUSSION method of Zinn and Owens (1986). Purine concen- trations of the effluent and bacteria were used to The chemical composition of the forage calculate N metabolism and efficiency of microbial treatments is provided in Table 1. Forage analy- protein synthesis. Effluent samples were prepared ses by NIR were performed by Rock River Labs for VFA analysis using the procedure for rumen Inc., while CP, NDF, and ADF were analyzed fluid preparation described by Erwin et al. (1961). using the same procedures used to analyze fer- Ruminal fluid effluent was centrifuged to remove menter effluent. Dietary protein levels may affect heavy feed particles and clarified, then 2.0 mL was ruminal fermentation patterns and digestibil- mixed with a 25% meta-phosphoric acid solution ity and create confounding results (Bach et  al., (0.5  mL) and centrifuged at 10,000 g for 15  min 1999), which is why many in-vitro studies feed until supernatant was clear. The supernatant was isonitrogenous diets when investigating alter- stored at −20  °C until analyzed. Effluent VFA native treatments. However, the current study concentration was performed via gas chromatog- investigated the differences in ruminal fermen- raphy (Agilent 7890B GC-FID with a G4567A tation between CSP and warm-season annual Autosampler). The Agilent DB-FFAP column was grasses, and it was important to keep the treat- 30 m length, 0.25  mm diameter, and had a film ments at their original protein levels, with the thickness of 0.15 µm. Chromatographic conditions understanding that this may ultimately affect were 1  µL injection with an inlet temperature of fermentation, and may be of interest to reflect a 240 °C, helium as carrier gas at 1 mL/min constant grazing situation (Bach et al., 1999). This differ- o fl w, initial oven temperature of 60 °C with a 2-min ence in N content of the diets was accounted for hold time, ramp at 20 °C/min to 220 °C and a 1-min by expressing results as a percentage of total N hold time at 220 °C. The flame ionization detector intake (Bach et al., 1999). was set at 250 °C. Standard solutions with known concentrations of VFA were analyzed to calibrate Digestibility chromatograph. Apparent and true (corrected for contribu- tion of bacterial flow) digestibility and pH flow Statistical Analysis for forage treatments are provided in Table 2. The CSP, BMRSS, and teff grass had lesser (P < 0.05) Data were analyzed using the MIXED proce- apparent DM and OM digestibility than alfalfa. dure of SAS software 9.4 (SAS Inst. Inc., 2016). The NDF digestibility for CSP and teff was lesser Data from fermenters were analyzed as a rand- (P  <  0.05) than alfalfa, and BMRSS was similar omized complete block (period) design. Period Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 130 Ruh et al. Table  2. Nutrient digestibility and pH of four to the current study. Overall, findings of apparent forage diets (alfalfa, CSP grasses and legumes, digestibility are consistent with previous research BMRSS, and teff grass) during continuous culture comparing alfalfa to grass in vivo, in which alfalfa fermentation disappeared more quickly from the rumen than the perennial ryegrass because of a faster rate of diges- Forage tion and faster particle size reduction of alfalfa Item Alfalfa CSP BMRSS Teff SEM (Waghorn et al., 1989). Results from Ribeiro et al. Apparent digestibility (2005) were different than results from the current -------------------%----------------- study because although there was greater nutrient a b b b DM 69.4 47.1 52.6 49.8 5.2 digestibility in alfalfa, bacterial OM flow was sim - a b b b OM 54.1 32.5 38.1 29.4 4.7 a b ab b ilar across all treatments (Table 4). NDF 75.5 52.6 65.9 56.6 5.3 a b ab ab ADF 75.5 55.4 67.5 59.4 5.3 True digestibility* Volatile Fatty Acids a b b b DM 85.8 64.0 66.2 65.9 5.7 a b b b OM 69.2 47.0 50.4 44.1 4.1 Least square means of VFA for the forage treat- pH ments are in Table  3. Total VFA amount (mM) Mean 6.38 6.18 6.19 6.22 0.10 were similar (P > 0.10) among the forage diets. The Minimum 5.88 5.14 5.41 5.92 0.31 amount of total VFA for CSP was similar to amount Maximum 7.26 6.76 7.23 7.01 0.25 of total VFA found in a previous study for pasture *Corrected for contribution of bacterial flow. intake in continuous culture (Bargo et al., 2003) but a,b Means within a row with different superscripts are different at greater than total VFA with a ryegrass only diet in P < 0.05. continuous culture fed at 60  g DM/d (de Veth and Kolver, 2001). Total VFA for the current study for (P > 0.10) to the other forages. The CSP was lesser the grass species was much greater than reported by (P  <  0.05) for ADF digestibility than alfalfa but Dillard et al. (2017) for mixes of cool- and warm-sea- was similar (P > 0.10) to BMRSS and teff. True son grass species. Quite possibly, differences between DM and OM digestibilities were lesser (P < 0.05) studies were observed, because the current study uti- in grasses compared with alfalfa. Digestibilities lized warm-season grasses as the only forage source, among BMRSS and teff grass were similar (P and Dillard et al. (2017) utilized alternative combi- > 0.10). A  study by de Veth and Kolver (2001) nations of cool-season and warm-season grasses. found a range of apparent DM digestibility There were some differences in individual VFA (44.7% to 56.4%) and OM digestibility (48.1% to concentrations and in molar proportions of VFA. 58.7%) from ryegrass pastures, which was simi- These changes may indicate differences in a shift of lar to results from the current study for DM and the rumen microbial population when alfalfa and OM digestibility. A  study by Soder et  al. (2013b) alfalfa hay are studied (Ribeiro et al., 2005). Similar found much greater apparent digestibility of DM, results of individual VFA production for CSP were OM, and NDF for 100% orchardgrass in a dual- found for a smooth bromegrass and orchardgrass o fl w continuous culture fermenter system, which pasture diets in continuous culture (Bargo et  al., could have been due to a greater CP level or the 2003). Molar proportions of acetate, propionate, greater level of daily diet, which was greater than and butyrate were similar to previous results of a the daily diet level of 60  g DM/d in the current CSP grass and legume diets in continuous culture study. The current study has only 20% orchard- (Bach et al., 1999). grass in the diet, and differences between Soder Bargo et  al. (2003) showed an inverse correl- et  al. (2013b) and the current study may be due ation between pH patterns and VFA production to the composition of orchardgrass in the diet. from smooth bromegrass and orchardgrass pasture A  study evaluating alfalfa in continuous culture diets. Forage treatments were similar (P > 0.10) in found lesser apparent NDF and ADF digestibil- mean pH among the forage treatments in this study ity than results from the current study for alfalfa and differences in individual VFA do not match the (Ribeiro et  al., 2005). Recently, Dillard et  al. pattern of pH. Quite possibly, this insignificant dif - (2017) studied mixes of cool-season (orchard- ferences reported in this study may be due to the fact grass) and warm-season grasses (sorghum × that pH was controlled for in the study. However, sudangrass and Japanese millet) and found simi- another study found no large differences of indi- lar DM digestibilities, greater OM digestibilities, vidual proportions of VFA with change in pH (de and great NDF and ADF digestibility compared Veth and Kolver, 2001). The molar proportion of Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 Warm season annuals and continuous culture 131 Table  3. VFA concentration of 4 forage diets Table  4. Nitrogen metabolism of four forage diets (alfalfa, CSP grasses and legumes, BMRSS, and (alfalfa, CSP grasses and legumes, BMRSS, and teff grass) in continuous culture fermentation teff grass) in continuous culture fermentation Forage Forage VFA Alfalfa CSP BMRSS Teff SEM Variable Alfalfa CSP BMRSS Teff SEM a b c c Total VFA, mM 78.6 77.6 75.6 82.3 13.1 N intake, g/d 3.09 2.31 2.18 2.20 0.01 a b b b Individual VFA, mol/100 mol NH3-N, mg/dL 22.5 7.5 7.4 8.9 0.75 a ab b a Acetate 72.1 71.2 67.7 75.0 1.3 CP degradation, % 79.8 77.2 69.1 65.9 4.6 b a ab b Propionate 16.5 20.0 18.4 17.1 0.54 N flows, g/d b b a b a b b ab Butyrate 7.9 7.2 10.1 6.7 0.58 Total N 1.99 1.50 1.51 1.70 0.11 a b b b a b b b Isobutyrate 0.69 0.27 0.25 0.17 0.03 NH -N 0.52 0.17 0.17 0.20 0.02 ab ab a b Isovalerate 0.82 0.30 1.2 0.09 0.31 NAN 1.46 1.33 1.34 1.50 0.12 a b a b Valerate 1.7 0.90 2.1 0.83 0.17 Bacterial N 0.84 0.81 0.67 0.75 0.14 a b a b Caproate 0.29 0.14 0.32 0.10 0.03 Dietary N 0.62 0.53 0.67 0.75 0.12 a b b a A:P ratio 4.4 3.6 3.7 4.4 0.18 N flows, % of total N flow a b b b NH3-N 26.5 11.6 11.4 11.8 1.8 a,b Means within a row with different superscripts are different at a b b b NAN 73.5 88.4 88.6 88.2 1.8 P < 0.05. Bacterial N 41.3 54.1 44.4 43.8 6.1 Dietary N 32.1 34.3 44.2 44.4 6.0 butyrate was greatest for BMRSS compared to the Efficiency of Microbial protein synthesis other forage treatments. g N/kg DM truly digested 15.0 19.9 16.0 18.7 2.7 Isobutyrate was greater in alfalfa than the g N/kg OM truly digested 21.3 30.4 24.0 33.3 4.9 CSP, BMRSS, and teff grass treatments, which is a,b Means within a row with different superscripts are different at similar to the pattern of true and apparent digest- P < 0.05. ibility of DM and OM of the dietary treatments (Table 2). The CP degradation (Table 4) was similar rates of microbial growth and ultimately digestibil- (P > 0.10) among treatments. Molar proportions ity measured in vitro and in vivo (Bach et al., 1999). of acetate, propionate, and butyrate of alfalfa from CP degradation was similar (P > 0.05) among diet- this study were similar to previously reported val- ary treatments in the fermenters. This observation ues of acetate, propionate, and butyrate for fresh is slightly lesser than CP digestibility found in a pre- alfalfa in continuous culture (Ribeiro et al., 2005). vious study using pasture diets in continuous cul- There are no previous studies that evaluated ture (Soder et al. 2013b). warm-season grass as the only forage in continu- The NH -N was greater (P <0.05) in alfal- ous culture. One study investigated warm-season fa-fed fermenters than for CSP, BMRSS, and teff and cool-season grasses fed to cannulated steers fermenters. Similar results of NH -N were found and reported a significant effect for warm-season for cool-season grasses in continuous culture com- vs. cool-season grasses for the molar proportions of pared to CSP in the current study (Bach et  al., propionate, butyrate, and valerate in ruminal fluid 1999). The CSP and BMRSS had similar total N (Bohnert et  al., 2011), which was in agreement to o fl w, while alfalfa had the greatest ( P < 0.05) total the current study. Individual concentrations from N flow and teff was intermediate in total N flow. the current study of acetate and propionate were As a percent of total N flows, NH -N was greater greater and lesser for butyrate for warm-season (P  <  0.05) in alfalfa than the grasses, reflecting grasses (BMRSS) reported by Dillard et al. (2017). the pattern of total NH -N for each treatment. Differences observed between studies may be due to Treatments were similar (P > 0.10) for non–NH - warm-season grass being analyzed as the only for- N, bacterial N, or dietary N flows as a percent of age source compared to warm-season grasses com- total N.  Even though alfalfa had greater NH -N posited with orchardgrass in Dillard et al. (2017). concentrations, treatments were similar (P > 0.10) in efficiency of microbial protein synthesis among Nitrogen Metabolism any of the treatments, on either a DM or OM basis. The N metabolism of fermenters fed the vari- This demonstrates that although there was lesser ous forages is found in Table  4. The N intake was NH -N in all the grass treatments than in alfalfa, highest for alfalfa, intermediate for CSP, and lowest there were still adequate amounts of NH -N for for warm-season grasses based on the dietary CP microbial protein synthesis. The minimum amount of the forage treatments. The N intake can affect of NH -N required for microbial protein synthesis Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/2/125/4969817 by Ed 'DeepDyve' Gillespie user on 19 June 2018 132 Ruh et al. was estimated to be 5  mg/dL for in-vitro systems It is known that grazing cows will experience (Satter and Slyter, 1974), and all treatments in changes in their diet throughout the grazing season this study were well above that value. Efficiency due to weather, the gradually increasing maturity of microbial protein synthesis on an OM basis in of cool season grasses, as well as changes in season the current study was lesser to previous results for in temperate regions of the United States. Cows in alfalfa (Ribeiro et  al., 2005) and pasture in con- a complementary grazing system may have add- tinuous culture (de Veth and Kolver, 2001; Cerrato- itional disruptions to their diet because they often Sánchez et al., 2007; Soder et al., 2013b, 2016). graze one type of grass, then sequentially another Alfalfa, CSP, BMRSS, and teff grass were sim- type of grass in a different pasture. Results from ilar (P > 0.10) for mean, minimum, or maximum the current study found several changes in ruminal pH (Table  2). One continuous culture fermenter fermentation among forage treatments of differ- study researching effects of pH levels determined ent species. Future research could investigate how that the optimal pH for high-quality pasture for- this shift in microbial population, possibly every age is 6.35 (de Veth and Kolver, 2001). We analyzed few days or every few weeks, affects a grazing dairy the amount of time spent below 5.8 as well as the cow. It would be interesting to determine what kind amount of time spent above 6.4. Time spent below of effect a sequential pasture rotation may have pH 5.8 ranged from 1 to 5  min/d. Sauvant et  al. on the rumen microbial population and fermen- (1999) found that it may be possible to experience tation. Further research dealing with warm-sea- a pH below 6.0 for 4 h without affecting microbial son grasses in continuous culture systems should fermentation. pH was analyzed at 5.8 because we include variations of concentrate and total mixed expected lesser values from the CSP examined in ration supplementation. Research on fermentation our study. Our results suggest that fermenters spent characteristics of warm-season grasses should be almost no time below a pH of 5.8. studied further, because there had not been much Because the study was in-vitro system, diets previous research conducted. may be affected differently when actually consumed Dairy farmers that utilize warm-season grasses by cows (Mansfield et  al., 1995 ). Although fresh in pasture rotations for lactating dairy cattle, may grass was used, it underwent heating and pressure benefit from utilizing BMRSS and teff grass in their in the drying and pelleting processes which could rotations. The BMRSS and teff grass had similar alter some components of the grass. These changes digestibility when compared to cool-season pasture should have been uniform across all treatments; grasses and legumes. No differences for total VFA however, it is important to note that dietary values were found for CSP compared to warm-season of pelleted grasses may be different than actual pas- grasses; however, specific individual VFA differed ture forage grasses that cows would be consuming among alfalfa, CSP, and warm-season grasses. in a grazing system. Fresh forages have greater con- Forage quality of the forages utilized in this study centrations of rapidly fermented sugars as well as may have profound effect on the results, and results greater concentrations of more digestible protein, may be different depending on weather conditions which could have been lost during heat processing throughout the year (i.e., drought or heavy rains). of our forages (Van Soest, 1994). In addition, most Overall, fermentation of warm-season grasses was grazing cows do not consume forage-only diets, so similar to the CSP. 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Published: Apr 13, 2018

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