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The impact of maturity stages on yield, quality, and nutritive value of ensiled Johnsongrass [Sorghum halepense (L.) Pers]

The impact of maturity stages on yield, quality, and nutritive value of ensiled Johnsongrass... Translational Animal Science, 2022, 6, 1–9 https://doi.org/10.1093/tas/txac118 Advance access publication 26 August 2022 Forage Based Livestock Systems The impact of maturity stages on yield, quality, and nutritive value of ensiled Johnsongrass [Sorghum halepense (L.) Pers] †,1 ‡, †,2 †, Camila S. da Silva, Jennifer J. Tucker, Fabio J. Maia, Jeferson M. Lourenço, †,3 † †, ║,4 Morgan L. Bass, Darren S. Seidel, T odd R. Callaway, Dennis W. Hancock, and †,5, R. Lawton, Stewart Jr Animal and Dairy Science Department, University of Georgia, Athens, GA 30602, USA Animal and Dairy Science Department, University of Georgia, Tifton, GA 31793, USA Crop and Soils Sciences Department, University of Georgia, Athens, GA 30602, USA Present address: Federal Rural University of Pernambuco, Recife 52171-900, Brazil Present address: Federal Rural University of Paraná, Dois Vizinhos 85660-000, Brazil Present address: University of Glasgow, Glasgow G61 1QH, UK Present address: U.S. Dairy Forage Research Center, Madison WI 53706, USA Corresponding author: lawtons@uga.edu ABSTRACT Johnsongrass [Sorghum halepense (L.) Pers.] is a non-native, invasive species that causes substantial losses in row crops and hay fields, which could be minimized by using Johnsongrass as a conserved forage. Two experiments were conducted to evaluate the yield and quality of Johnsongrass ensiled at four maturities: harvested every 3 weeks (3WK), boot stage (BOOT), flower stage (FLOWER), and dough (DOUGH) stages. In experiment 1, yield, botanical composition, nutritive value, and fermentation characteristics of Johnsongrass were measured. In ex- periment 2, Johnsongrass silage was incubated for 48 h for assessment of gas production, pH, in vitro dry matter digestibility (IVDMD), and volatile fatty acids. The experimental area consisted of 16 plots (2.74 m × 4.57 m) divided into four blocks, and treatment was randomly assigned to plot within block. Each year, silage was prepared for each plot from the two cutting closest to July 1. After 10 weeks, the silos were opened, and silage samples were frozen for further analysis. Data from both experiments were tested for the effects of maturity stage and harvest timing (first and second harvest). The results from experiment 1 showed an increase ( P < 0.0001) in dry matter yield from 3WK stage to DOUGH. Johnsongrass, as a proportion of the total botanical composition, declined at the end of the growing season for 3WK but increased in FLOWER (P = 0.0010). In the first harvest, 3WK and BOOT stage silages had the greatest concentrations of crude protein and total digestible nutrients and lowest of fiber (neutral detergent fiber and acid detergent fiber; P < 0.0001). In the second harvest, differences in nutrient content were significant only for 3WK silages, which showed the best nutritive value ( P < 0.0001). In experiment 2, IVDMD of silage followed the same trends described for nutritive value from experiment 1. Overall, these results demonstrate that Johnsongrass can be successfully ensiled, but to opti- mize forage nutritive value and quantity, Johnsongrass should be ensiled before it reaches the flower stage. Key words: ensiled forage, invasive species, Johnsongrass, plant maturity INTRODUCTION requires longer drying time or additional steps to crush or cut the stem for hay production. Johnsongrass is similar in growth Johnsongrass [Sorghum halepense (L.) Pers.] is a perennial characteristics and belongs to the same genus as forage sorghum warm-season grass that was originally introduced to the United [Sorghum bicolor (L.) Moench]; an annual forage option that States as a forage in the 1830s in South Carolina. If managed is commonly conserved as silage. There is potential to utilize properly in a grazing system, Johnsongrass can serve as a quality Johnsongrass as silage, however, research is limited (Rude and grazeable forage, however, it is intolerant of heavy grazing due Rankins, 1993). Therefore, the objective of this study was to eval- to the depletion of rhizome reserves and will not persist under uate the effect of maturity stage at harvest on herbage accumula- these conditions (Howard, 2004). Nevertheless, since its intro- tion, ensiling characteristics, and nutritive value of Johnsongrass. duction, Johnsongrass has become a problematic weed in hay We hypothesized that Johnsongrass can be successfully ensiled fields and row crops in the Southeast. Johnsongrass has been as a stored forage, and nutritive value and fermentation quality identified as a major weed in 53 countries around the world will be determined by plant maturity at harvest for ensiling. (Hartzler et al., 1991). In the United States, the occurrence of Johnsongrass has been reported in all states except Minnesota, Maine, and Alaska (USDA Plants Database, 2017). MATERIALS AND METHODS Although Johnsongrass is palatable to cattle and has a sim- ilar nutritive value to bermudagrass (Dillard et al., 2012), the This study was divided into two experiments: Experiment thick culm (0.5–2.0 cm in diameter; Warwick and Black, 1983) 1 was carried out from May to September of 2015 (YR1), Received May 17, 2022 Accepted August 24, 2022. Published by Oxford University Press for the American Society of Animal Science 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US. 2 da Silva et al. AprilMay June July AugustSeptember 2015 2017 2018 70 yr AVG AprilMay June JulyAugust September 2015 2017 2018 70 yr AVG Figure 1. Monthly mean temperature(°C) and monthly total precipitation (cm) for 2015, 2017, and 2018, and 70-yr average for the experimental period at the J. Phil Campbell, Sr. Research and Education Center, Watkinsville, GA. 2017 (YR2), and 2018 (YR3) to evaluate the effect of matu- Prior to project initiation, baseline soil samples were taken rity stage on Johnsongrass herbage accumulation, nutritive at the research site and sent to the University of Georgia value, and parameters of fermentation of Johnsongrass silage. Agricultural and Environmental Services Lab (Athens, GA). The experiment was not carried out in 2016 due to a severe Each year, prior to designating plots, the experimental area drought. Experiment 2 was conducted in May 2018 as an (200 m ) was mowed to a 7.5 cm stubble height, and the res- in vitro study where parameters of fermentation and in vitro idue was removed to simulate a hay harvest on May 8, 2015, dry matter digestibility (IVDMD) of Johnsongrass silage were May 23, 2017, and May 18, 2018. Since no recommended assessed. levels specific for Johnsongrass were found, each year, the ex - perimental area was fertilized with 22.7 kg of N (in the form of urea) at plant emergence based on N recommendations for Experiment 1 forage sorghum (Vendramini et al., 2010). Phosphorus and Location, treatments, and forage management. K were applied following recommendations given by the soil Plots were established in a preexisting stand of Johnsongrass test report (23.0 kg/ha of P O , 90.8 kg/ha of K O). 2 5 2 located at the J. Phil Campbell, Sr. Research and Education The total experimental area was divided into four blocks Center in Watkinsville, GA (33°52ʹ20.02″N, 83°25ʹ28.50″W; separated by a 1.5 m boundary and subdivided into four 340 m elevation). Weather information of this location (av- 4.57  ×  2.74 m plots within each block, totaling 16 experi- erages of monthly temperature, precipitation, and 70-yr av- mental units. Plots were randomly assigned to one of four erage) for the months of field evaluations were recorded by treatments, which consisted of different maturity stages of weather instruments operated by the University of Georgia Johnsongrass: harvested every 3 weeks (3WK), boot stage Automated Environmental Monitoring Network (UGA- (BOOT), flower stage (FLOWER), and dough (DOUGH) AEMN, 2018) and are provided in Figure 1. The soil type of stages. For the 3WK treatment, the first harvest occurred the research site is classified as Cecil sandy loam and Pacolet when plants reached approximately 60  cm in height and sandy-clay loam (USDA Web Soil Survey, 2022). every 3 weeks thereafter. For BOOT, FLOWER, and DOUGH Precepitation, mm Temperature, °C Impact of maturity stages on Johnsongrass 3 stage treatments, plots were observed approximately twice a Silo preparation. As expected by design, target maturity week and harvest timing was set based on the morphological did not align such that all treatments could be harvested and characteristics of plants at each stage. Plants were considered ensiled on the same day (Table 1). Therefore, the two harvests as being at the boot stage when an enlargement of the top- closest to the end of July 31 were ensiled in each treatment. portion of the stem was observed due to the development and Due to an infestation of sugarcane aphids (Melanaphis preemergence of the seed head (inflorescence) enclosed in the sacchari), FLOWER and DOUGH plots were not harvested stem. At the flower stage, the seed head had emerged, and the on the second date in 2018. Thirty-two mini-silos were pre- peduncle was fully elongated. The dough stage represented pared and consisted of 90 cm lengths of 10-cm diameter poly- plants harvested at the soft dough when seeds were filled and vinyl chloride pipes sealed with air-tight rubber caps at either could be squeezed between fingers but had little or no liquid end. The target DM for packing was 55%. After plots were present. harvested, the forage was evenly spread on tarps to allow drying in the sun, and the weight of each tarp was monitored Data collection. Plots were evaluated for plant maturity, every 30–45 min. Drying time varied from plot to plot, from botanical composition, and herbage accumulation. Plant ma- 1 to 4 hrs, depending on forage mass and weather conditions. turity was assessed in boot, flower, and dough stage plots at The target density for each silo was 0.24 kg/L, which resulted each harvest by collecting Johnsongrass tillers prior to har- in 2.16 kg of forage being packed at 55% DM. vest. Two 91 cm-grazing sticks (used in practical estimation Approximately 100 g of forage was used for determination of forage availability for grazing) were placed in parallel of DM prior to ensiling through the microwave technique on the ground (approximately 12  cm apart), and all tillers (Gay et al., 2020). In this method, the forage material was growing within that segment were collected. Next, the growth placed in a microwave-safe container along with a mug filled stage was determined based on the calculation of the mean with water (for trapping excess moisture), and its weight re- stage count (MSC) proposed by Moore et al. (1991). Briefly, corded initially and after consecutively drying for 2 min. The this method attributes a numerical index (0 to 5) to primary final weight is recorded when the weight difference becomes (from germination to seed ripening) and secondary events less than 1 g. In the present study, the objective of performing (e.g., emergence of the first leaf, the onset of stem elongation) this procedure was to determine, by weight difference, how that occur similarly throughout growth of most grass species. much the fresh forage from each plot had to dry to achieve Once the indices have been obtained, the MSC can be calcu- appropriate moisture for ensiling. lated. The target MSC was 3.0 for boot stage, 3.5 for flower, Once the forage had reached adequate moisture, a small and 4.3 for dough stage plots. The average calculated MSC layer of forage was placed at the bottom of the silo and cov- for boot, flower, and dough stage plots were 2.9, 3.2, and 4.0, ered with a section of plastic. Then, the forage remaining respectively. was packed by putting in small amounts at a time and Botanical composition (vegetative cover estimates and compacting it using a steel-stick. Once the silo was packed manual separations) was evaluated using a 0.1-m quadrat within 5  cm of the top of the silo, another layer of plastic randomly tossed to three locations within plot. Botanical was placed at the top, followed by an extra layer of forage, composition was first assessed subjectively by recording veg - and the silo was closed. The extra layers on each end were etative cover (visual estimation of percentage of ground cov- used to ensure that fermentation and quality of packed ered by actively growing forage to the nearest 1%) of the forage would be minimally impacted by air that may enter species components of Johnsongrass, other grasses, legumes, the silo. and weeds. Then all forage material within quadart was The silos were allowed to ferment outside under a cov- harvested via clipping at 7.5 cm with scissors. Different plant ered structure for 10 weeks and monitored for 4 days post- species identified in the quadrat were manually separated and packing, then weekly, to check for pressure buildup and similar components (Johnsongrass, other grasses, legumes, potential leaks. After 10 weeks, the silos were opened, and and weeds) from the three quadrats were combined to rep- their contents frozen at −20 °C. Half of the Johnsongrass resent an individual sample per component per plot. Samples silage samples were transferred to paper bags and freeze- were placed in a paper bag and were weighed fresh and after dried in a VirTis Freeze mobile lyophilizer (SP Industries, drying at 60 °C for 48 hrs for estimation of the botanical Warminster, PA) at −50 °C for 24 to 48 hrs depending on composition. Botanical composition was calculated as the the thickness and maturity of the sample. The freeze-dried amount of Johnsongrass or other species in the plots in rela- samples were ground at 2  mm using a Model 4 Wiley Mill tion to the total sample mass collected (i.e., % Johnsongrass (Thomas Scientific, Swedesboro, NJ), then ground through = (Johnsongrass (g)/Total sample mass (g)*100). Plots were harvested using a push lawn mower with grass catcher attachment (Troy-Bilt, Valley City, OH) set to leave Table 1. Harvest dates from Experiment 1 a stubble of 7.5 cm. When forage was cut at flower stage and dough stage, a trimmer (Husqvarna 122HD60) was used be- Year fore the push mower to aid in the harvesting of stalks and Maturity stage 2015 2017 2018 maximize the amount of forage collected. All material col- lected from each plot was placed on an individual tarp (1.2 3 weeks 05/29–07/31 06/13–09/07 06/08–07/30 m × 1.8 m), which had been previously tared, and weighed Boot 06/18, 08/10 06/26, 09/05 06/22, 08/13 on a digital hanging scale (CS25 Pentair, USA). Grab samples Flower 07/01, 09/02 07/12, 09/18 07/04 were taken for each plot, weighed initially and dried at 60 Dough 07/10, 09/18 07/24, 09/29 07/10 °C for 48 hrs to determine dry matter (DM) percentage ((dry wt/wet wt) × 100)) and subsequent determination of DM Flower and dough-stage plots had only one cut in 2018 due to infestation yield. by sugarcane aphids. 4 da Silva et al. a CT 193 Cyclotec Sample Mill (FOSS, Eden Prairie, MN) VFA analysis was performed according to methodology fitted with 1-mm screen for in vitro analysis. The remaining described by Henry et al. (2015). Rumen fluid samples half of the frozen samples were shipped to Cumberland Valley were defrosted at room temperature, transferred to 2.0  mL Analytical Services (Hagerstown, MD) in quart-size reseal- microcentrifuge tubes, and centrifuged at 10,000 g for 15 min. able freezer bags for analysis of fermentation characteristics. 1.0 mL of the supernatant was collected from the tubes and mixed with 200 microliters of 25% metaphosphoric acid (5:1 ratio). These subsamples were kept at −20 °C overnight. Experiment 2 On the next day, subsamples were defrosted and centrifuged Ensiled samples collected in YR2 of Experiment 1 were one more time at 10,000 g. 500 μL of the supernatant were subjected to in vitro fermentation to calculate IVDMD and added to 1 mL of ethyl acetate (2:1 ratio) and shaken vigor- to measure pH, gas production, and volatile fatty acid (VFA) ously. Samples were allowed to settle for about 3 min or until production. the supernatant portion could be collected. The supernatant was transferred to 1.8 screw cap vials and analyzed in a gas Animal care and use. All procedures were approved by chromatographer (GC-2010 Plus; Shimadzu Corporation, the University of Georgia’s Institutional Animal Care and Use Japan) through a flame ionization detector and a capillary Committee (AUP # A2021 11-008-Y1-A0) column (Zebron ZB-FFAP GC Cap. Column 30 m × 0.32 mm Substrate preparation and inoculation process. The × 0.25 μm; Phenomenex Inc., Torrance, CA). The retention in vitro assay was performed in May of 2018. Samples were time for major VFAs (acetic acid, propionic acid, butyric acid) simultaneously analyzed for IVDMD, gas production, pH, were 2.874, 3.305, and 3.826 min. and VFA concentration. On the day of the incubation, ap- proximately 2L of ruminal fluid was collected from three Statistical analysis. Herbage accumulation, botanical cannulated crossbred Angus steers at approximately 0800 h. composition, nutrient content, and fermentation characteris- The donor animals were grazing a pasture of tall fescue tics evaluated in Experiment 1 were analyzed using PROC [Schedonorus arundinaceus (Schreb.) Dumort., nom. cons.] MIXED procedure of SAS (SAS Institute Inc., Cary, NC). and white clover (Trifolium repens) for the previous 30 days. Plots were arranged in a randomized complete block de- The ruminal fluid was collected by grabbing the ruminal sign with four plots in each treatment, replicated four times. contents through the canula and straining it through a nylon Treatment was considered the fixed effect and year and period strainer into a 1-L plastic bottle. The bottles were placed in a were considered random effects. Data collected in Experiment cooler with warm water for transportation to the lab. 2 were analyzed by PROC MIXED procedure of SAS treat- Upon arrival to the lab, the bottles were inverted slowly ment as fixed effects and period and tube as random effects. to mix stratified layers of the fluid and the pH was meas - Least square means from both experiments were generated ured with a portable pH meter (pH = 6.35). Ruminal fluid by LSMEANS and compared with Tukey’s HSD test of mul- was mixed with a minimal medium described by Wells et al. tiple comparisons. Differences were considered significant at (1997) at a 3:1 ratio (3 parts of medium, 1 part of ruminal α < 0.05. fluid). The medium was prepared, adjusted to a final pH of 6.5, and saturated with CO 24 hrs prior incubation. Ten mil- RESULTS AND DISCUSSION liliter of ruminal fluid-media mix were delivered to 20-mL Experiment 1 crimp-top glass tubes containing 0.1 g of Johnsongrass silage There was a significant effect on Johnsongrass herbage ac - samples at the different stages. Blank samples (containing cumulation by maturity stage in that material harvested at only ruminal fluid-media mix) were also incubated. Five the dough stage (2,930 kg/ha) was greater than at boot stage tubes per sample were used as replicates. The tubes were con- (2,031  kg/ha), with flower stage showing intermediate DM stantly bubbled with CO before receiving the mix and im- accumulation (2570  kg/ha); all of these values were greater mediately sealed with a rubber stopper and crimp thereafter. than the herbage accumulation of Johnsongrass harvested at After sealing, the tubes were placed in an incubator at 39 °C 3WK (747 kg/ha; P < 0.0001; Figure 2). for 48 hrs and were gently shaken every 6 hrs throughout the In the current study, Johnsongrass produced less DM incubation period. compared to sorghum species used for grain or forage produc- When the 48-hour incubation was completed, the tubes tion. DM yield of Sorghum sp. Has been reported as 12 to 20 were removed from the incubator and immediately placed t/ha (Miron et al., 2005; Amer et al., 2012; Bean et al., 2013), on ice to stop fermentation. After 20 min, gas volume was a fourfold increase compared to the highest DM accumulation measured from each tube using a 16-gauge needle coupled of Johnsongrass in the current trial. In addition, the highest to a 60-mL disposable syringe. Next, the seal was broken herbage accumulation of Johnsongrass is substantially lower and pH was recorded. Samples for VFA analysis were than the annual accumulation expected from bermudagrass obtained by filtering the content of the glass tubes into a (Cynodon dactylon (L.) Pers) hay under different irrigation previously weighed 10 × 50 Dacron bag, positioned on top levels or no irrigation. Zhou et al. (2014) reported that the of a V-shaped bottom plastic storage tube. Plastic storage 5-year average of expected yield of bermudagrass hay baled tubes were immediately capped and stored at −20 °C. The at 15% moisture was 6.84 t/ha without irrigation, 8.09 t/ glass tubes were rinsed with deionized water to remove −1 ha at medium irrigation level (1.52 cm/wk ), and 8.43 t/ha particles stuck to the tube walls. The residue present inside −1 under high irrigation (3.05 cm/wk ). the bags after filtration was rinsed with tap water until the Botanical composition of Johnsongrass plots is water from wash was clear. The bags containing the residue summarized in Figure 3. The percentage of Johnsongrass did post-incubation were placed in a dryer at 60 °C for 48 hrs not differ (P > 0.05) between the first and second harvest for and weighed at the end of this period for determination of boot and dough stages. However, Johnsongrass decreased DM degradation. Impact of maturity stages on Johnsongrass 5 ab 3 weeks Boot FlowerDough Maturity Stages Figure 2. Herbage accumulation (kg/ha) of Johnsongrass harvested at four maturity stages (3 weeks, boot, flower, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.0001). ab abcd abc abcd abcd bcde Inial Final 3 weeksBootFlowerDough Maturity Stages Figure 3. Initial and final percentage of Johnsongrass in plots harvested at four maturity stages of Johnsongrass (3 weeks, boot, flower, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.05). (P = 0.0460) from the first to the last harvest in 3-week the second cutting, the 3-week stage had the highest crude plots and increased (P = 0.0010) between the two harvests protein (CP) concentration (P < 0.0001). The first harvest in flower-stage plots. This shift in composition of species in also resulted in higher content of total digestible nutrients 3-week stage plots indicates that the growth of Johnsongrass (TDN) for 3-week and boot stage compared to flower and was suppressed by short harvest intervals preventing rhi- dough. As with the CP values, the second harvest resulted zome growth and seed production (Warwick and Black, in highest TDN for forage at the 3-week stage (P = 0.0015). 1983). In fact, the employment of continuous grazing or fre- Conversely, the flower and dough stage had the highest neu- quent mowing has been often suggested as one of the few ef- tral detergent fiber (NDF) and acid detergent fiber (ADF) ficient methods for controlling infestations of Johnsongrass values in comparison to the 3-week and boot stage within (Ceseski et al., 2017; Rocateli and Manuchehri, 2017). the first harvest, and the lowest for 3-week in the second ( P Except for the 3-week treatment, there was an average of < 0.001). over 60% Johnsongrass in all of the treated plots at the end The negative impact of advanced plant maturity on nutri- of the experimental period. tive value observed in this study is consistent with the changes Plant maturity also had a great impact on nutritive value previously reported in grain sorghum, forage sorghum, and of Johnsongrass pre-ensiling, resulting in a significant harvest sudangrass [Sorghum bicolor (L.) Moench ssp. drummondii x maturity stage interaction (P < 0.05) for all parameters of (Nees ex Steud.) de Wet & Harlan; Snyman and Joubert, nutritive value evaluated; however, results will be discussed 1996; Abdelhadi and Tricarico, 2009; Beck et al., 2013], within harvest. Lower crude protein was detected in plots that which indicate that as plant growth progresses, protein con- reached the flower and dough stages, and greater values were centration decreases while fiber content increases due to the found for 3-week and boot stage within the first harvest. On deposition of lignified tissues as part of the structural support Johnsongrass (%) Herbage Accumulation (kg/ha) 6 da Silva et al. of the plant (Crowe et al., 2017). Mature Johnsongrass plants al. (2013) for silage made from forage sorghum. However, can reach 2.5 m tall and have stems of up to 2.0  cm in di- Johnsongrass silage in this experiment resulted in higher ameter (Warnick and Black, 1983), which reflects in the high CP values compared to that of sorghum silages treated or NDF and ADF values found in the present study (around untreated with inoculants, which were all below 7.6%. 70% and 44%, respectively). Overall, our results indicate that ensiling Johnsongrass We also detected a significant decline in nutritive value after the boot stage compromises the amount of nutrients for the boot-stage treatment (less protein, more fiber) from available. the first to the second cutting. This observation may be re - Harvest timing and maturity stage (P < 0.0001) affected all lated to the delayed second harvest of this treatment in 2017 parameters of fermentation (Table 3; P < 0.05). On the first (Table 1), which happened during dryer weather conditions. cutting, higher VFA levels were detected in boot-stage silages Viciedo et al (2019) reported changes in concentrations of compared to the other treatments (P < 0.01). Despite this carbon, nitrogen, and C:N ratio for forage plants subjected result, silages from boot-stage plots were similar to 3-week to different water availabilities and ambient temperatures. silages in lactic acid and acetic acid concentrations. On the Harvesting time and maturity stages had similar effects on second harvest, 3-week silages showed the highest VFA con- the nutritive value of ensiled Johnsongrass (Table 2). The DM centration (P < 0.001) but did not differ from later stages in content was lowest for 3-week and boot treatments within the lactic and acetic acid levels. Butyric acid levels were undetect- first harvest ( P < 0.0001). In addition, CP content of 3-week able in all samples. Silage pH did not differ across treatments silages was greater than boot, flower, or dough-stage silages in (P > 0.05) on the first cutting; on the second, pH was lower the first harvest which is different than the results obtained pre- in 3-week and flower compared to boot and dough (P < 0.05; ensiling. Moreover, flower and dough-stage silages had compa - Table 3). rable CP levels (P > 0.05) but different NDF and ADF content. According to Wanapat et al. (2013), cellulose and hemi- These were highest for silages prepared from Johnsongrass at cellulose are broken down to simple sugars (nonstructural the dough stage and lowest from 3-week plants. carbohydrates) during the ensiling process and those simple On the second harvest, DM content of silages from boot sugars are further utilized by certain groups of bacteria, and dough stage plots did not differ, nor did 3-week and such as Lactobacillus spp. (Dogi et al., 2013; Rahman et al., flower stage silages ( P > 0.05). As for the second harvest nu- 2017), which generate organic acids as their final products tritive value, 3-week silages values were better (higher CP and (mainly lactic, acetic, butyric acid). Therefore, our results lower NDF and ADF content) compared to all other growth suggest that Johnsongrass plants ensiled at 3-week or at stages (P < 005). Protein and fiber levels reported for silage the boot stage provide more substrates for fermentation, from all other stages (boot, flower, and dough) from the which favor pH drop and preservation of the silage (Zhang second cutting were similar (P > 0.05). et al., 2009). The concentrations of NDF and ADF in Johnsongrass The boot stage presented one of the lowest pH values silages are comparable to values reported by Thomas et but also the highest pH (Table 3). The significant rise in pH Table 2. Nutritive value of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS e e abc abc cd ab bcd a DM 38.2 33.5 48.2 45.8 42.8 51.5 44.6 52.0 <0.0001 <0.0001 <0.0001 a b c c b c c c CP 14.7 12.6 9.9 8.6 12.5 8.3 9.9 8.8 <0.0001 <0.0001 <0.0001 d c b a cd b b ab NDF 56.0 60.3 65.3 69.6 57.0 65.8 65.0 67.4 <0.001 <0.0001 <0.001 d c b a d ab b a ADF 36.0 38.8 42.2 44.8 36.3 42.5 42.0 43.9 0.10 <0.0001 <0.002 ADF, acid detergent fiber; CP, crude protein; H, harvest; MS, maturity stage; NDF, neutral detergent fiber. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). Table 3. Fermentation characteristics of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, o fl wer, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H x MS bcd a cd bc ab d cd cd tVFA 6.46 6.92 4.62 4.92 5.56 4.21 5.17 3.70 <0.0001 <0.0001 <0.001 ab a bc abc abc c abc c Lac 5.10 5.40 3.80 4.10 4.33 3.34 4.07 3.14 <0.001 <0.01 0.0153 ab a bc bc abc c abc c Ace 1.37 1.52 0.84 0.78 1.20 0.64 1.23 0.59 0.0446 0.0018 0.0055 c c abc c c ab abc a pH 4.40 4.32 4.51 4.42 4.40 4.70 4.50 4.78 <0.0001 0.0163 <0.001 tVFAs, total volatile fatty acids (VFAs); Lac, lactic acid, % total VFAs; Ace = acetic acid, % total VFAs. H, harvest; MS, maturity stage. a,b,c,d,e Means followed by different lowercase letters within a row are statistically significant (P < 0.05). Impact of maturity stages on Johnsongrass 7 observed from the first to the second harvest in this partic - was near this desirable value. However, pH values above 4.0 ular treatment may be associated with growth inhibition of can be observed for dry silages (>40% DM), as silages were lactic acid-producing bacteria by low moisture content of that made in the present study. Lactic acid should range from 4% silage in the second cutting (Table 4). In addition, the nega- to 7% of DM, which was observed for most Johnsongrass tive impact of low moisture content on those microorganisms silages in this evaluation. For acetic acid, acceptable levels may explain the low production of total acids detected for are below 3%, as it was seen for Johnsongrass silages at all boot and dough treatments. growth stages. Lastly, non-detectable levels of butyric acid in- As explored by Troller and Stinson (1981) and later dicate that minimum undesirable fermentation occurred in demonstrated by Zheng et al. (2011), the growth of the silos. Lactobacillus plantarum can be completely suppressed by low moisture levels in their environment. Zheng et al. Experiment 2 (2011) tested the effects of moisture content on microbial Total gas volume produced over 48 hrs of incubation is activity and the quality of silage produced from tomato presented in Figure 4. There was an interaction (P = 0.0185) pomace and sugar beet pulp and found that ensiling tomato between harvesting time and maturity stages of Johnsongrass; pomace and sugar beet pulp at 10% and 30% moisture results will also be discussed within harvest. Gas volume was (90% and 70% of DM, respectively) did not support mi- greater for Johnsongrass ensiled at the boot stage compared crobial activity and acidification of the silage. In the present to dough stage on the first harvest ( P = 0.0028). On the second study all moisture contents were above 30%, but it can be harvest, gas production did not differ among treatments (P > suggested that the higher DM of the boot and dough-stage 0.05). The difference in gas production between the boot and silages on the second harvest impaired the fermentation to dough stage in the first harvest ( Figure 4) corroborate with some extent, whereas the lower DM on the first harvest fa - the findings of Ribeiro et al. (2014) who investigated, among vored fermentation. other traits, in vitro gas production of Andropogon gayanus The analysis of fermentation characteristics indicates that grass harvested at different maturities and preserved as hay Johnsongrass can be utilized for ensiling. According to refer- or silage. They reported that both hay and silage exhibited a ence values of fermentation quality of silage provided by Ward decline in gas production as maturity of the plant advanced. and Ondarza (2008), pH values around 4.0 are indicators of According to these authors, a decrease in gas production well-fermented silages. Apart from boot and dough-stage occurs in response to a reduction in cell wall degradability in silages from the second harvest, pH of Johnsongrass silages mature plants. Table 4. Nutritive value of fresh Johnsongrass in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS a ab cd d ab cd c d CP 14.6 13.5 8.5 7.2 13.9 7.8 9.1 7.2 <0.0001 <0.0001 <0.0001 ab abc e de a cde bcd de TDN 59.4 58.6 55.4 55.7 60.2 56.4 57.7 55.9 0.4326 <0.0001 0.0015 ef de ab a f abcd abcd abc NDF 60.7 63.3 69.2 70.1 58.7 69.1 66.9 69.1 0.8460 <0.0001 0.0006 ef e abc a f abcd abcd ab ADF 35.6 37.7 43.4 44.4 33.9 43.2 41.3 44.1 0.5034 <0.0001 0.0002 ADF, acid detergent fiber; CP, crude protein; H, harvest; MS, maturity stage; NDF, neutral detergent fiber; TDN, total digestible nutrients. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). 16.0 ab 14.0 abc abc abc abc bc 12.0 10.0 8.0 First 6.0 Second 4.0 2.0 0.0 3 weeksBoot Flower Dough Maturity Stages Figure 4. Volume of gas (mL) produced in vitro by Johnsongrass ensiled in two harvest timings (first, second) and four maturity stages (3 weeks, boot o fl wer, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.05). Gas volume (mL) 8 da Silva et al. Table 5. Digestibility and production of volatile fatty acids (VFAs) in vitro of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS abc a cd d ab d d d IVDMD 65.7 68.8 59.0 53.0 68.5 56.9 56.8 52.8 0.0313 <0.0001 0.0013 VFAs (mM) bc a a bcd ab bcde def f Ace 17.45 17.82 17.74 17.30 17.57 17.29 17.03 16.87 <0.0001 0.0007 0.0074 a b bcd cde bc cde cde e Prop 8.40 8.00 7.67 7.55 7.69 7.47 7.45 7.23 <0.0001 <0.0001 0.1945 a a a a b b b b IsoBut 0.54 0.51 0.50 0.54 0.49 0.50 0.49 0.50 0.0008 0.1386 0.1390 a bc bc bc bc c b bc But 5.22 4.93 4.84 4.83 4.77 4.71 4.99 4.76 0.0196 0.1065 0.0136 a ab c c bc c c c Val 0.60 0.58 0.55 0.55 0.56 0.55 0.55 0.54 0.0004 0.0018 0.0711 a a a a b b b b Cap 0.30 0.21 0.33 0.30 0.22 0.21 0.19 0.14 0.0033 0.4834 0.1956 d c abc abc abc ab abc a A:P Ratio 2.08 2.22 2.31 2.29 2.29 2.31 2.29 2.34 0.0009 0.0008 0.0056 Ace, acetate; Prop, propionate; IsoBut, isoburyrate; But, butyrate; Val, valeric acid; Cap, caproic acid; tVFAs, total volatile fatty acids (VFAs). H, harvest; MS, maturity stage. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). The IVDMD was affected by both harvest and treatments Butyrate production was affected by harvest and matu- (P = 0.0013; Table 5). On the first harvest, the boot stage rity stages (P = 0.0136). The first harvest resulted in higher showed the highest digestibility along with the 3-week stage, proportion of butyrate in the 3-week stage and there was no which was both different from the digestibility of dough-stage difference between boot, flower, and dough. On the second silages. On the second harvest, 3-week had greater digesti- harvest, flower-stage silages yielded more butyrate than boot bility than any other treatment (P = 0.0013). stage, but they both did not differ from 3-week and dough Our results suggest that NDF and ADF concentrations stage. were the key factors affecting digestibility of Johnsongrass. Overall, the changes in individual VFAs corroborate with Bean et al. (2013) reported a negative correlation of r ≤ −0.72 the greater concentration of protein and digestibility of DM between NDF, ADF, lignin, and true digestibility of sorghum at the earliest stages of growth, indicating that younger plants classes cultivated for grain and forage yield. Additionally, the of Johnsongrass will optimize fermentation. pronounced difference in IVDMD between boot and dough stages followed the general trends observed for nutritive value CONCLUSION of fresh and ensiled Johnsongrass in the first harvest ( Tables 2 and 3), suggesting that ensiling Johnsongrass early in its It can be concluded that Johnsongrass can be ensiled and development will provide more substrates for degradation by potentially used for cattle feeding. Harvesting and ensiling rumen bacteria (which reflect in increased gas production), Johnsongrass before it reaches the flower stage will provide and consequently enhance nutrient utilization by the animal. the best balance between yield, nutritive value, and quality of In regard to specific VFAs, acetate production was higher fermentation. In addition, 3-week and boot-stage silages will for Johnsongrass at boot and flower stage in comparison be more digestible and generate more energy in the form of to 3-week and dough stage within the first harvest, On the VFAs for the animal. Therefore, producers can benefit from second harvest, the highest acetate value was observed in the ensiling Johnsongrass in areas of high infestation. 3-week stage. The boot stage showed higher acetate produc- tion than dough (P = 0.0074), but did not differ from the flower stage. Acetate production generally increases to a Acknowledgment certain level as maturity of forages progresses (Rinne et al., Funding for this project was provided by the Georgia 1997; Vanhatalo et al., 2009; Sarmadi et al., 2016), as shown Agricultural Commodity Commission for Beef, Atlanta, GA. in our results. Nonetheless, it is worth mentioning that the differences observed in acetate production among treatments and across harvests would likely not promote different bio- Conflict of interest statement logical effects. The authors declare no conflict of interest. Propionate production was highest in silages from Johnsongrass at 3-week on the first harvest, but no statis- tical difference was seen on the second cutting when 3-week, LITERATURE CITED boot, and flower stages were compared (P < 0.0001). When Abdelhadi, L. O., and J. M. Tricarico. 2009. 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The impact of maturity stages on yield, quality, and nutritive value of ensiled Johnsongrass [Sorghum halepense (L.) Pers]

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Translational Animal Science, 2022, 6, 1–9 https://doi.org/10.1093/tas/txac118 Advance access publication 26 August 2022 Forage Based Livestock Systems The impact of maturity stages on yield, quality, and nutritive value of ensiled Johnsongrass [Sorghum halepense (L.) Pers] †,1 ‡, †,2 †, Camila S. da Silva, Jennifer J. Tucker, Fabio J. Maia, Jeferson M. Lourenço, †,3 † †, ║,4 Morgan L. Bass, Darren S. Seidel, T odd R. Callaway, Dennis W. Hancock, and †,5, R. Lawton, Stewart Jr Animal and Dairy Science Department, University of Georgia, Athens, GA 30602, USA Animal and Dairy Science Department, University of Georgia, Tifton, GA 31793, USA Crop and Soils Sciences Department, University of Georgia, Athens, GA 30602, USA Present address: Federal Rural University of Pernambuco, Recife 52171-900, Brazil Present address: Federal Rural University of Paraná, Dois Vizinhos 85660-000, Brazil Present address: University of Glasgow, Glasgow G61 1QH, UK Present address: U.S. Dairy Forage Research Center, Madison WI 53706, USA Corresponding author: lawtons@uga.edu ABSTRACT Johnsongrass [Sorghum halepense (L.) Pers.] is a non-native, invasive species that causes substantial losses in row crops and hay fields, which could be minimized by using Johnsongrass as a conserved forage. Two experiments were conducted to evaluate the yield and quality of Johnsongrass ensiled at four maturities: harvested every 3 weeks (3WK), boot stage (BOOT), flower stage (FLOWER), and dough (DOUGH) stages. In experiment 1, yield, botanical composition, nutritive value, and fermentation characteristics of Johnsongrass were measured. In ex- periment 2, Johnsongrass silage was incubated for 48 h for assessment of gas production, pH, in vitro dry matter digestibility (IVDMD), and volatile fatty acids. The experimental area consisted of 16 plots (2.74 m × 4.57 m) divided into four blocks, and treatment was randomly assigned to plot within block. Each year, silage was prepared for each plot from the two cutting closest to July 1. After 10 weeks, the silos were opened, and silage samples were frozen for further analysis. Data from both experiments were tested for the effects of maturity stage and harvest timing (first and second harvest). The results from experiment 1 showed an increase ( P < 0.0001) in dry matter yield from 3WK stage to DOUGH. Johnsongrass, as a proportion of the total botanical composition, declined at the end of the growing season for 3WK but increased in FLOWER (P = 0.0010). In the first harvest, 3WK and BOOT stage silages had the greatest concentrations of crude protein and total digestible nutrients and lowest of fiber (neutral detergent fiber and acid detergent fiber; P < 0.0001). In the second harvest, differences in nutrient content were significant only for 3WK silages, which showed the best nutritive value ( P < 0.0001). In experiment 2, IVDMD of silage followed the same trends described for nutritive value from experiment 1. Overall, these results demonstrate that Johnsongrass can be successfully ensiled, but to opti- mize forage nutritive value and quantity, Johnsongrass should be ensiled before it reaches the flower stage. Key words: ensiled forage, invasive species, Johnsongrass, plant maturity INTRODUCTION requires longer drying time or additional steps to crush or cut the stem for hay production. Johnsongrass is similar in growth Johnsongrass [Sorghum halepense (L.) Pers.] is a perennial characteristics and belongs to the same genus as forage sorghum warm-season grass that was originally introduced to the United [Sorghum bicolor (L.) Moench]; an annual forage option that States as a forage in the 1830s in South Carolina. If managed is commonly conserved as silage. There is potential to utilize properly in a grazing system, Johnsongrass can serve as a quality Johnsongrass as silage, however, research is limited (Rude and grazeable forage, however, it is intolerant of heavy grazing due Rankins, 1993). Therefore, the objective of this study was to eval- to the depletion of rhizome reserves and will not persist under uate the effect of maturity stage at harvest on herbage accumula- these conditions (Howard, 2004). Nevertheless, since its intro- tion, ensiling characteristics, and nutritive value of Johnsongrass. duction, Johnsongrass has become a problematic weed in hay We hypothesized that Johnsongrass can be successfully ensiled fields and row crops in the Southeast. Johnsongrass has been as a stored forage, and nutritive value and fermentation quality identified as a major weed in 53 countries around the world will be determined by plant maturity at harvest for ensiling. (Hartzler et al., 1991). In the United States, the occurrence of Johnsongrass has been reported in all states except Minnesota, Maine, and Alaska (USDA Plants Database, 2017). MATERIALS AND METHODS Although Johnsongrass is palatable to cattle and has a sim- ilar nutritive value to bermudagrass (Dillard et al., 2012), the This study was divided into two experiments: Experiment thick culm (0.5–2.0 cm in diameter; Warwick and Black, 1983) 1 was carried out from May to September of 2015 (YR1), Received May 17, 2022 Accepted August 24, 2022. Published by Oxford University Press for the American Society of Animal Science 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US. 2 da Silva et al. AprilMay June July AugustSeptember 2015 2017 2018 70 yr AVG AprilMay June JulyAugust September 2015 2017 2018 70 yr AVG Figure 1. Monthly mean temperature(°C) and monthly total precipitation (cm) for 2015, 2017, and 2018, and 70-yr average for the experimental period at the J. Phil Campbell, Sr. Research and Education Center, Watkinsville, GA. 2017 (YR2), and 2018 (YR3) to evaluate the effect of matu- Prior to project initiation, baseline soil samples were taken rity stage on Johnsongrass herbage accumulation, nutritive at the research site and sent to the University of Georgia value, and parameters of fermentation of Johnsongrass silage. Agricultural and Environmental Services Lab (Athens, GA). The experiment was not carried out in 2016 due to a severe Each year, prior to designating plots, the experimental area drought. Experiment 2 was conducted in May 2018 as an (200 m ) was mowed to a 7.5 cm stubble height, and the res- in vitro study where parameters of fermentation and in vitro idue was removed to simulate a hay harvest on May 8, 2015, dry matter digestibility (IVDMD) of Johnsongrass silage were May 23, 2017, and May 18, 2018. Since no recommended assessed. levels specific for Johnsongrass were found, each year, the ex - perimental area was fertilized with 22.7 kg of N (in the form of urea) at plant emergence based on N recommendations for Experiment 1 forage sorghum (Vendramini et al., 2010). Phosphorus and Location, treatments, and forage management. K were applied following recommendations given by the soil Plots were established in a preexisting stand of Johnsongrass test report (23.0 kg/ha of P O , 90.8 kg/ha of K O). 2 5 2 located at the J. Phil Campbell, Sr. Research and Education The total experimental area was divided into four blocks Center in Watkinsville, GA (33°52ʹ20.02″N, 83°25ʹ28.50″W; separated by a 1.5 m boundary and subdivided into four 340 m elevation). Weather information of this location (av- 4.57  ×  2.74 m plots within each block, totaling 16 experi- erages of monthly temperature, precipitation, and 70-yr av- mental units. Plots were randomly assigned to one of four erage) for the months of field evaluations were recorded by treatments, which consisted of different maturity stages of weather instruments operated by the University of Georgia Johnsongrass: harvested every 3 weeks (3WK), boot stage Automated Environmental Monitoring Network (UGA- (BOOT), flower stage (FLOWER), and dough (DOUGH) AEMN, 2018) and are provided in Figure 1. The soil type of stages. For the 3WK treatment, the first harvest occurred the research site is classified as Cecil sandy loam and Pacolet when plants reached approximately 60  cm in height and sandy-clay loam (USDA Web Soil Survey, 2022). every 3 weeks thereafter. For BOOT, FLOWER, and DOUGH Precepitation, mm Temperature, °C Impact of maturity stages on Johnsongrass 3 stage treatments, plots were observed approximately twice a Silo preparation. As expected by design, target maturity week and harvest timing was set based on the morphological did not align such that all treatments could be harvested and characteristics of plants at each stage. Plants were considered ensiled on the same day (Table 1). Therefore, the two harvests as being at the boot stage when an enlargement of the top- closest to the end of July 31 were ensiled in each treatment. portion of the stem was observed due to the development and Due to an infestation of sugarcane aphids (Melanaphis preemergence of the seed head (inflorescence) enclosed in the sacchari), FLOWER and DOUGH plots were not harvested stem. At the flower stage, the seed head had emerged, and the on the second date in 2018. Thirty-two mini-silos were pre- peduncle was fully elongated. The dough stage represented pared and consisted of 90 cm lengths of 10-cm diameter poly- plants harvested at the soft dough when seeds were filled and vinyl chloride pipes sealed with air-tight rubber caps at either could be squeezed between fingers but had little or no liquid end. The target DM for packing was 55%. After plots were present. harvested, the forage was evenly spread on tarps to allow drying in the sun, and the weight of each tarp was monitored Data collection. Plots were evaluated for plant maturity, every 30–45 min. Drying time varied from plot to plot, from botanical composition, and herbage accumulation. Plant ma- 1 to 4 hrs, depending on forage mass and weather conditions. turity was assessed in boot, flower, and dough stage plots at The target density for each silo was 0.24 kg/L, which resulted each harvest by collecting Johnsongrass tillers prior to har- in 2.16 kg of forage being packed at 55% DM. vest. Two 91 cm-grazing sticks (used in practical estimation Approximately 100 g of forage was used for determination of forage availability for grazing) were placed in parallel of DM prior to ensiling through the microwave technique on the ground (approximately 12  cm apart), and all tillers (Gay et al., 2020). In this method, the forage material was growing within that segment were collected. Next, the growth placed in a microwave-safe container along with a mug filled stage was determined based on the calculation of the mean with water (for trapping excess moisture), and its weight re- stage count (MSC) proposed by Moore et al. (1991). Briefly, corded initially and after consecutively drying for 2 min. The this method attributes a numerical index (0 to 5) to primary final weight is recorded when the weight difference becomes (from germination to seed ripening) and secondary events less than 1 g. In the present study, the objective of performing (e.g., emergence of the first leaf, the onset of stem elongation) this procedure was to determine, by weight difference, how that occur similarly throughout growth of most grass species. much the fresh forage from each plot had to dry to achieve Once the indices have been obtained, the MSC can be calcu- appropriate moisture for ensiling. lated. The target MSC was 3.0 for boot stage, 3.5 for flower, Once the forage had reached adequate moisture, a small and 4.3 for dough stage plots. The average calculated MSC layer of forage was placed at the bottom of the silo and cov- for boot, flower, and dough stage plots were 2.9, 3.2, and 4.0, ered with a section of plastic. Then, the forage remaining respectively. was packed by putting in small amounts at a time and Botanical composition (vegetative cover estimates and compacting it using a steel-stick. Once the silo was packed manual separations) was evaluated using a 0.1-m quadrat within 5  cm of the top of the silo, another layer of plastic randomly tossed to three locations within plot. Botanical was placed at the top, followed by an extra layer of forage, composition was first assessed subjectively by recording veg - and the silo was closed. The extra layers on each end were etative cover (visual estimation of percentage of ground cov- used to ensure that fermentation and quality of packed ered by actively growing forage to the nearest 1%) of the forage would be minimally impacted by air that may enter species components of Johnsongrass, other grasses, legumes, the silo. and weeds. Then all forage material within quadart was The silos were allowed to ferment outside under a cov- harvested via clipping at 7.5 cm with scissors. Different plant ered structure for 10 weeks and monitored for 4 days post- species identified in the quadrat were manually separated and packing, then weekly, to check for pressure buildup and similar components (Johnsongrass, other grasses, legumes, potential leaks. After 10 weeks, the silos were opened, and and weeds) from the three quadrats were combined to rep- their contents frozen at −20 °C. Half of the Johnsongrass resent an individual sample per component per plot. Samples silage samples were transferred to paper bags and freeze- were placed in a paper bag and were weighed fresh and after dried in a VirTis Freeze mobile lyophilizer (SP Industries, drying at 60 °C for 48 hrs for estimation of the botanical Warminster, PA) at −50 °C for 24 to 48 hrs depending on composition. Botanical composition was calculated as the the thickness and maturity of the sample. The freeze-dried amount of Johnsongrass or other species in the plots in rela- samples were ground at 2  mm using a Model 4 Wiley Mill tion to the total sample mass collected (i.e., % Johnsongrass (Thomas Scientific, Swedesboro, NJ), then ground through = (Johnsongrass (g)/Total sample mass (g)*100). Plots were harvested using a push lawn mower with grass catcher attachment (Troy-Bilt, Valley City, OH) set to leave Table 1. Harvest dates from Experiment 1 a stubble of 7.5 cm. When forage was cut at flower stage and dough stage, a trimmer (Husqvarna 122HD60) was used be- Year fore the push mower to aid in the harvesting of stalks and Maturity stage 2015 2017 2018 maximize the amount of forage collected. All material col- lected from each plot was placed on an individual tarp (1.2 3 weeks 05/29–07/31 06/13–09/07 06/08–07/30 m × 1.8 m), which had been previously tared, and weighed Boot 06/18, 08/10 06/26, 09/05 06/22, 08/13 on a digital hanging scale (CS25 Pentair, USA). Grab samples Flower 07/01, 09/02 07/12, 09/18 07/04 were taken for each plot, weighed initially and dried at 60 Dough 07/10, 09/18 07/24, 09/29 07/10 °C for 48 hrs to determine dry matter (DM) percentage ((dry wt/wet wt) × 100)) and subsequent determination of DM Flower and dough-stage plots had only one cut in 2018 due to infestation yield. by sugarcane aphids. 4 da Silva et al. a CT 193 Cyclotec Sample Mill (FOSS, Eden Prairie, MN) VFA analysis was performed according to methodology fitted with 1-mm screen for in vitro analysis. The remaining described by Henry et al. (2015). Rumen fluid samples half of the frozen samples were shipped to Cumberland Valley were defrosted at room temperature, transferred to 2.0  mL Analytical Services (Hagerstown, MD) in quart-size reseal- microcentrifuge tubes, and centrifuged at 10,000 g for 15 min. able freezer bags for analysis of fermentation characteristics. 1.0 mL of the supernatant was collected from the tubes and mixed with 200 microliters of 25% metaphosphoric acid (5:1 ratio). These subsamples were kept at −20 °C overnight. Experiment 2 On the next day, subsamples were defrosted and centrifuged Ensiled samples collected in YR2 of Experiment 1 were one more time at 10,000 g. 500 μL of the supernatant were subjected to in vitro fermentation to calculate IVDMD and added to 1 mL of ethyl acetate (2:1 ratio) and shaken vigor- to measure pH, gas production, and volatile fatty acid (VFA) ously. Samples were allowed to settle for about 3 min or until production. the supernatant portion could be collected. The supernatant was transferred to 1.8 screw cap vials and analyzed in a gas Animal care and use. All procedures were approved by chromatographer (GC-2010 Plus; Shimadzu Corporation, the University of Georgia’s Institutional Animal Care and Use Japan) through a flame ionization detector and a capillary Committee (AUP # A2021 11-008-Y1-A0) column (Zebron ZB-FFAP GC Cap. Column 30 m × 0.32 mm Substrate preparation and inoculation process. The × 0.25 μm; Phenomenex Inc., Torrance, CA). The retention in vitro assay was performed in May of 2018. Samples were time for major VFAs (acetic acid, propionic acid, butyric acid) simultaneously analyzed for IVDMD, gas production, pH, were 2.874, 3.305, and 3.826 min. and VFA concentration. On the day of the incubation, ap- proximately 2L of ruminal fluid was collected from three Statistical analysis. Herbage accumulation, botanical cannulated crossbred Angus steers at approximately 0800 h. composition, nutrient content, and fermentation characteris- The donor animals were grazing a pasture of tall fescue tics evaluated in Experiment 1 were analyzed using PROC [Schedonorus arundinaceus (Schreb.) Dumort., nom. cons.] MIXED procedure of SAS (SAS Institute Inc., Cary, NC). and white clover (Trifolium repens) for the previous 30 days. Plots were arranged in a randomized complete block de- The ruminal fluid was collected by grabbing the ruminal sign with four plots in each treatment, replicated four times. contents through the canula and straining it through a nylon Treatment was considered the fixed effect and year and period strainer into a 1-L plastic bottle. The bottles were placed in a were considered random effects. Data collected in Experiment cooler with warm water for transportation to the lab. 2 were analyzed by PROC MIXED procedure of SAS treat- Upon arrival to the lab, the bottles were inverted slowly ment as fixed effects and period and tube as random effects. to mix stratified layers of the fluid and the pH was meas - Least square means from both experiments were generated ured with a portable pH meter (pH = 6.35). Ruminal fluid by LSMEANS and compared with Tukey’s HSD test of mul- was mixed with a minimal medium described by Wells et al. tiple comparisons. Differences were considered significant at (1997) at a 3:1 ratio (3 parts of medium, 1 part of ruminal α < 0.05. fluid). The medium was prepared, adjusted to a final pH of 6.5, and saturated with CO 24 hrs prior incubation. Ten mil- RESULTS AND DISCUSSION liliter of ruminal fluid-media mix were delivered to 20-mL Experiment 1 crimp-top glass tubes containing 0.1 g of Johnsongrass silage There was a significant effect on Johnsongrass herbage ac - samples at the different stages. Blank samples (containing cumulation by maturity stage in that material harvested at only ruminal fluid-media mix) were also incubated. Five the dough stage (2,930 kg/ha) was greater than at boot stage tubes per sample were used as replicates. The tubes were con- (2,031  kg/ha), with flower stage showing intermediate DM stantly bubbled with CO before receiving the mix and im- accumulation (2570  kg/ha); all of these values were greater mediately sealed with a rubber stopper and crimp thereafter. than the herbage accumulation of Johnsongrass harvested at After sealing, the tubes were placed in an incubator at 39 °C 3WK (747 kg/ha; P < 0.0001; Figure 2). for 48 hrs and were gently shaken every 6 hrs throughout the In the current study, Johnsongrass produced less DM incubation period. compared to sorghum species used for grain or forage produc- When the 48-hour incubation was completed, the tubes tion. DM yield of Sorghum sp. Has been reported as 12 to 20 were removed from the incubator and immediately placed t/ha (Miron et al., 2005; Amer et al., 2012; Bean et al., 2013), on ice to stop fermentation. After 20 min, gas volume was a fourfold increase compared to the highest DM accumulation measured from each tube using a 16-gauge needle coupled of Johnsongrass in the current trial. In addition, the highest to a 60-mL disposable syringe. Next, the seal was broken herbage accumulation of Johnsongrass is substantially lower and pH was recorded. Samples for VFA analysis were than the annual accumulation expected from bermudagrass obtained by filtering the content of the glass tubes into a (Cynodon dactylon (L.) Pers) hay under different irrigation previously weighed 10 × 50 Dacron bag, positioned on top levels or no irrigation. Zhou et al. (2014) reported that the of a V-shaped bottom plastic storage tube. Plastic storage 5-year average of expected yield of bermudagrass hay baled tubes were immediately capped and stored at −20 °C. The at 15% moisture was 6.84 t/ha without irrigation, 8.09 t/ glass tubes were rinsed with deionized water to remove −1 ha at medium irrigation level (1.52 cm/wk ), and 8.43 t/ha particles stuck to the tube walls. The residue present inside −1 under high irrigation (3.05 cm/wk ). the bags after filtration was rinsed with tap water until the Botanical composition of Johnsongrass plots is water from wash was clear. The bags containing the residue summarized in Figure 3. The percentage of Johnsongrass did post-incubation were placed in a dryer at 60 °C for 48 hrs not differ (P > 0.05) between the first and second harvest for and weighed at the end of this period for determination of boot and dough stages. However, Johnsongrass decreased DM degradation. Impact of maturity stages on Johnsongrass 5 ab 3 weeks Boot FlowerDough Maturity Stages Figure 2. Herbage accumulation (kg/ha) of Johnsongrass harvested at four maturity stages (3 weeks, boot, flower, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.0001). ab abcd abc abcd abcd bcde Inial Final 3 weeksBootFlowerDough Maturity Stages Figure 3. Initial and final percentage of Johnsongrass in plots harvested at four maturity stages of Johnsongrass (3 weeks, boot, flower, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.05). (P = 0.0460) from the first to the last harvest in 3-week the second cutting, the 3-week stage had the highest crude plots and increased (P = 0.0010) between the two harvests protein (CP) concentration (P < 0.0001). The first harvest in flower-stage plots. This shift in composition of species in also resulted in higher content of total digestible nutrients 3-week stage plots indicates that the growth of Johnsongrass (TDN) for 3-week and boot stage compared to flower and was suppressed by short harvest intervals preventing rhi- dough. As with the CP values, the second harvest resulted zome growth and seed production (Warwick and Black, in highest TDN for forage at the 3-week stage (P = 0.0015). 1983). In fact, the employment of continuous grazing or fre- Conversely, the flower and dough stage had the highest neu- quent mowing has been often suggested as one of the few ef- tral detergent fiber (NDF) and acid detergent fiber (ADF) ficient methods for controlling infestations of Johnsongrass values in comparison to the 3-week and boot stage within (Ceseski et al., 2017; Rocateli and Manuchehri, 2017). the first harvest, and the lowest for 3-week in the second ( P Except for the 3-week treatment, there was an average of < 0.001). over 60% Johnsongrass in all of the treated plots at the end The negative impact of advanced plant maturity on nutri- of the experimental period. tive value observed in this study is consistent with the changes Plant maturity also had a great impact on nutritive value previously reported in grain sorghum, forage sorghum, and of Johnsongrass pre-ensiling, resulting in a significant harvest sudangrass [Sorghum bicolor (L.) Moench ssp. drummondii x maturity stage interaction (P < 0.05) for all parameters of (Nees ex Steud.) de Wet & Harlan; Snyman and Joubert, nutritive value evaluated; however, results will be discussed 1996; Abdelhadi and Tricarico, 2009; Beck et al., 2013], within harvest. Lower crude protein was detected in plots that which indicate that as plant growth progresses, protein con- reached the flower and dough stages, and greater values were centration decreases while fiber content increases due to the found for 3-week and boot stage within the first harvest. On deposition of lignified tissues as part of the structural support Johnsongrass (%) Herbage Accumulation (kg/ha) 6 da Silva et al. of the plant (Crowe et al., 2017). Mature Johnsongrass plants al. (2013) for silage made from forage sorghum. However, can reach 2.5 m tall and have stems of up to 2.0  cm in di- Johnsongrass silage in this experiment resulted in higher ameter (Warnick and Black, 1983), which reflects in the high CP values compared to that of sorghum silages treated or NDF and ADF values found in the present study (around untreated with inoculants, which were all below 7.6%. 70% and 44%, respectively). Overall, our results indicate that ensiling Johnsongrass We also detected a significant decline in nutritive value after the boot stage compromises the amount of nutrients for the boot-stage treatment (less protein, more fiber) from available. the first to the second cutting. This observation may be re - Harvest timing and maturity stage (P < 0.0001) affected all lated to the delayed second harvest of this treatment in 2017 parameters of fermentation (Table 3; P < 0.05). On the first (Table 1), which happened during dryer weather conditions. cutting, higher VFA levels were detected in boot-stage silages Viciedo et al (2019) reported changes in concentrations of compared to the other treatments (P < 0.01). Despite this carbon, nitrogen, and C:N ratio for forage plants subjected result, silages from boot-stage plots were similar to 3-week to different water availabilities and ambient temperatures. silages in lactic acid and acetic acid concentrations. On the Harvesting time and maturity stages had similar effects on second harvest, 3-week silages showed the highest VFA con- the nutritive value of ensiled Johnsongrass (Table 2). The DM centration (P < 0.001) but did not differ from later stages in content was lowest for 3-week and boot treatments within the lactic and acetic acid levels. Butyric acid levels were undetect- first harvest ( P < 0.0001). In addition, CP content of 3-week able in all samples. Silage pH did not differ across treatments silages was greater than boot, flower, or dough-stage silages in (P > 0.05) on the first cutting; on the second, pH was lower the first harvest which is different than the results obtained pre- in 3-week and flower compared to boot and dough (P < 0.05; ensiling. Moreover, flower and dough-stage silages had compa - Table 3). rable CP levels (P > 0.05) but different NDF and ADF content. According to Wanapat et al. (2013), cellulose and hemi- These were highest for silages prepared from Johnsongrass at cellulose are broken down to simple sugars (nonstructural the dough stage and lowest from 3-week plants. carbohydrates) during the ensiling process and those simple On the second harvest, DM content of silages from boot sugars are further utilized by certain groups of bacteria, and dough stage plots did not differ, nor did 3-week and such as Lactobacillus spp. (Dogi et al., 2013; Rahman et al., flower stage silages ( P > 0.05). As for the second harvest nu- 2017), which generate organic acids as their final products tritive value, 3-week silages values were better (higher CP and (mainly lactic, acetic, butyric acid). Therefore, our results lower NDF and ADF content) compared to all other growth suggest that Johnsongrass plants ensiled at 3-week or at stages (P < 005). Protein and fiber levels reported for silage the boot stage provide more substrates for fermentation, from all other stages (boot, flower, and dough) from the which favor pH drop and preservation of the silage (Zhang second cutting were similar (P > 0.05). et al., 2009). The concentrations of NDF and ADF in Johnsongrass The boot stage presented one of the lowest pH values silages are comparable to values reported by Thomas et but also the highest pH (Table 3). The significant rise in pH Table 2. Nutritive value of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS e e abc abc cd ab bcd a DM 38.2 33.5 48.2 45.8 42.8 51.5 44.6 52.0 <0.0001 <0.0001 <0.0001 a b c c b c c c CP 14.7 12.6 9.9 8.6 12.5 8.3 9.9 8.8 <0.0001 <0.0001 <0.0001 d c b a cd b b ab NDF 56.0 60.3 65.3 69.6 57.0 65.8 65.0 67.4 <0.001 <0.0001 <0.001 d c b a d ab b a ADF 36.0 38.8 42.2 44.8 36.3 42.5 42.0 43.9 0.10 <0.0001 <0.002 ADF, acid detergent fiber; CP, crude protein; H, harvest; MS, maturity stage; NDF, neutral detergent fiber. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). Table 3. Fermentation characteristics of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, o fl wer, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H x MS bcd a cd bc ab d cd cd tVFA 6.46 6.92 4.62 4.92 5.56 4.21 5.17 3.70 <0.0001 <0.0001 <0.001 ab a bc abc abc c abc c Lac 5.10 5.40 3.80 4.10 4.33 3.34 4.07 3.14 <0.001 <0.01 0.0153 ab a bc bc abc c abc c Ace 1.37 1.52 0.84 0.78 1.20 0.64 1.23 0.59 0.0446 0.0018 0.0055 c c abc c c ab abc a pH 4.40 4.32 4.51 4.42 4.40 4.70 4.50 4.78 <0.0001 0.0163 <0.001 tVFAs, total volatile fatty acids (VFAs); Lac, lactic acid, % total VFAs; Ace = acetic acid, % total VFAs. H, harvest; MS, maturity stage. a,b,c,d,e Means followed by different lowercase letters within a row are statistically significant (P < 0.05). Impact of maturity stages on Johnsongrass 7 observed from the first to the second harvest in this partic - was near this desirable value. However, pH values above 4.0 ular treatment may be associated with growth inhibition of can be observed for dry silages (>40% DM), as silages were lactic acid-producing bacteria by low moisture content of that made in the present study. Lactic acid should range from 4% silage in the second cutting (Table 4). In addition, the nega- to 7% of DM, which was observed for most Johnsongrass tive impact of low moisture content on those microorganisms silages in this evaluation. For acetic acid, acceptable levels may explain the low production of total acids detected for are below 3%, as it was seen for Johnsongrass silages at all boot and dough treatments. growth stages. Lastly, non-detectable levels of butyric acid in- As explored by Troller and Stinson (1981) and later dicate that minimum undesirable fermentation occurred in demonstrated by Zheng et al. (2011), the growth of the silos. Lactobacillus plantarum can be completely suppressed by low moisture levels in their environment. Zheng et al. Experiment 2 (2011) tested the effects of moisture content on microbial Total gas volume produced over 48 hrs of incubation is activity and the quality of silage produced from tomato presented in Figure 4. There was an interaction (P = 0.0185) pomace and sugar beet pulp and found that ensiling tomato between harvesting time and maturity stages of Johnsongrass; pomace and sugar beet pulp at 10% and 30% moisture results will also be discussed within harvest. Gas volume was (90% and 70% of DM, respectively) did not support mi- greater for Johnsongrass ensiled at the boot stage compared crobial activity and acidification of the silage. In the present to dough stage on the first harvest ( P = 0.0028). On the second study all moisture contents were above 30%, but it can be harvest, gas production did not differ among treatments (P > suggested that the higher DM of the boot and dough-stage 0.05). The difference in gas production between the boot and silages on the second harvest impaired the fermentation to dough stage in the first harvest ( Figure 4) corroborate with some extent, whereas the lower DM on the first harvest fa - the findings of Ribeiro et al. (2014) who investigated, among vored fermentation. other traits, in vitro gas production of Andropogon gayanus The analysis of fermentation characteristics indicates that grass harvested at different maturities and preserved as hay Johnsongrass can be utilized for ensiling. According to refer- or silage. They reported that both hay and silage exhibited a ence values of fermentation quality of silage provided by Ward decline in gas production as maturity of the plant advanced. and Ondarza (2008), pH values around 4.0 are indicators of According to these authors, a decrease in gas production well-fermented silages. Apart from boot and dough-stage occurs in response to a reduction in cell wall degradability in silages from the second harvest, pH of Johnsongrass silages mature plants. Table 4. Nutritive value of fresh Johnsongrass in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS a ab cd d ab cd c d CP 14.6 13.5 8.5 7.2 13.9 7.8 9.1 7.2 <0.0001 <0.0001 <0.0001 ab abc e de a cde bcd de TDN 59.4 58.6 55.4 55.7 60.2 56.4 57.7 55.9 0.4326 <0.0001 0.0015 ef de ab a f abcd abcd abc NDF 60.7 63.3 69.2 70.1 58.7 69.1 66.9 69.1 0.8460 <0.0001 0.0006 ef e abc a f abcd abcd ab ADF 35.6 37.7 43.4 44.4 33.9 43.2 41.3 44.1 0.5034 <0.0001 0.0002 ADF, acid detergent fiber; CP, crude protein; H, harvest; MS, maturity stage; NDF, neutral detergent fiber; TDN, total digestible nutrients. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). 16.0 ab 14.0 abc abc abc abc bc 12.0 10.0 8.0 First 6.0 Second 4.0 2.0 0.0 3 weeksBoot Flower Dough Maturity Stages Figure 4. Volume of gas (mL) produced in vitro by Johnsongrass ensiled in two harvest timings (first, second) and four maturity stages (3 weeks, boot o fl wer, and dough). Bars identified by different lowercase letters are statistically significant (P < 0.05). Gas volume (mL) 8 da Silva et al. Table 5. Digestibility and production of volatile fatty acids (VFAs) in vitro of Johnsongrass silage prepared in two harvest timings (first, second) and at four maturity stages (3 weeks, boot, flower, and dough) First harvest Second harvest P-value Item 3 Weeks Boot Flower Dough 3 Weeks Boot Flower Dough H MS H × MS abc a cd d ab d d d IVDMD 65.7 68.8 59.0 53.0 68.5 56.9 56.8 52.8 0.0313 <0.0001 0.0013 VFAs (mM) bc a a bcd ab bcde def f Ace 17.45 17.82 17.74 17.30 17.57 17.29 17.03 16.87 <0.0001 0.0007 0.0074 a b bcd cde bc cde cde e Prop 8.40 8.00 7.67 7.55 7.69 7.47 7.45 7.23 <0.0001 <0.0001 0.1945 a a a a b b b b IsoBut 0.54 0.51 0.50 0.54 0.49 0.50 0.49 0.50 0.0008 0.1386 0.1390 a bc bc bc bc c b bc But 5.22 4.93 4.84 4.83 4.77 4.71 4.99 4.76 0.0196 0.1065 0.0136 a ab c c bc c c c Val 0.60 0.58 0.55 0.55 0.56 0.55 0.55 0.54 0.0004 0.0018 0.0711 a a a a b b b b Cap 0.30 0.21 0.33 0.30 0.22 0.21 0.19 0.14 0.0033 0.4834 0.1956 d c abc abc abc ab abc a A:P Ratio 2.08 2.22 2.31 2.29 2.29 2.31 2.29 2.34 0.0009 0.0008 0.0056 Ace, acetate; Prop, propionate; IsoBut, isoburyrate; But, butyrate; Val, valeric acid; Cap, caproic acid; tVFAs, total volatile fatty acids (VFAs). H, harvest; MS, maturity stage. a,b,c,d,e,f Means followed by different lowercase letters within a row are statistically significant (P < 0.05). The IVDMD was affected by both harvest and treatments Butyrate production was affected by harvest and matu- (P = 0.0013; Table 5). On the first harvest, the boot stage rity stages (P = 0.0136). The first harvest resulted in higher showed the highest digestibility along with the 3-week stage, proportion of butyrate in the 3-week stage and there was no which was both different from the digestibility of dough-stage difference between boot, flower, and dough. On the second silages. On the second harvest, 3-week had greater digesti- harvest, flower-stage silages yielded more butyrate than boot bility than any other treatment (P = 0.0013). stage, but they both did not differ from 3-week and dough Our results suggest that NDF and ADF concentrations stage. were the key factors affecting digestibility of Johnsongrass. Overall, the changes in individual VFAs corroborate with Bean et al. (2013) reported a negative correlation of r ≤ −0.72 the greater concentration of protein and digestibility of DM between NDF, ADF, lignin, and true digestibility of sorghum at the earliest stages of growth, indicating that younger plants classes cultivated for grain and forage yield. Additionally, the of Johnsongrass will optimize fermentation. pronounced difference in IVDMD between boot and dough stages followed the general trends observed for nutritive value CONCLUSION of fresh and ensiled Johnsongrass in the first harvest ( Tables 2 and 3), suggesting that ensiling Johnsongrass early in its It can be concluded that Johnsongrass can be ensiled and development will provide more substrates for degradation by potentially used for cattle feeding. Harvesting and ensiling rumen bacteria (which reflect in increased gas production), Johnsongrass before it reaches the flower stage will provide and consequently enhance nutrient utilization by the animal. the best balance between yield, nutritive value, and quality of In regard to specific VFAs, acetate production was higher fermentation. In addition, 3-week and boot-stage silages will for Johnsongrass at boot and flower stage in comparison be more digestible and generate more energy in the form of to 3-week and dough stage within the first harvest, On the VFAs for the animal. Therefore, producers can benefit from second harvest, the highest acetate value was observed in the ensiling Johnsongrass in areas of high infestation. 3-week stage. The boot stage showed higher acetate produc- tion than dough (P = 0.0074), but did not differ from the flower stage. Acetate production generally increases to a Acknowledgment certain level as maturity of forages progresses (Rinne et al., Funding for this project was provided by the Georgia 1997; Vanhatalo et al., 2009; Sarmadi et al., 2016), as shown Agricultural Commodity Commission for Beef, Atlanta, GA. in our results. Nonetheless, it is worth mentioning that the differences observed in acetate production among treatments and across harvests would likely not promote different bio- Conflict of interest statement logical effects. The authors declare no conflict of interest. Propionate production was highest in silages from Johnsongrass at 3-week on the first harvest, but no statis- tical difference was seen on the second cutting when 3-week, LITERATURE CITED boot, and flower stages were compared (P < 0.0001). When Abdelhadi, L. O., and J. M. Tricarico. 2009. Effects of stage of ma- looking at butyrate on the first harvest, the most apparent turity and microbial inoculation at harvest on nutritive quality difference occurred for silages from the 3-week treat- and degradability of grain sorghum whole-plant and head chop ment, which showed the highest molar proportion of all silages. Anim. Feed Sci. Technol. 152:175–185. doi:10.1016/j. comparisons. As a consequence, the lowest A:P ratio was anifeedsci.2009.04.014. observed with Johnsongrass from the first harvest ensiled at Amer, S., F. Hassanat, R. Berthiaume, P. Seguin, and A. F. Mustafa. 2012. 3-week (P = 0.0056), reflecting greater concentrations of rap - Effects of water soluble carbohydrate content on ensiling char- idly fermented carbohydrates and protein in this treatment. acteristics, chemical composition and in vitro gas production of Impact of maturity stages on Johnsongrass 9 forage millet and forage sorghum silages. Anim. Feed Sci. Technol. 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Journal

Translational Animal ScienceOxford University Press

Published: Aug 26, 2022

Keywords: ensiled forage; invasive species; Johnsongrass; plant maturity

References