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Background: Enteric methane (CH ) accounts for about 70% of total CH emissions from the ruminant animals. 4 4 Researchers are exploring ways to mitigate enteric CH emissions from ruminants. Recently, nano zinc oxide (nZnO) has shown potential in reducing CH and hydrogen sulfide (H S) production from the liquid manure 4 2 under anaerobic storage conditions. Four different levels of nZnO and two types of feed were mixed with rumen fluid to investigate the efficacy of nZnO in mitigating gaseous production. Methods: All experiments with four replicates were conducted in batches in 250 mL glass bottles paired with RF the ANKOM wireless gas production monitoring system. Gas production was monitored continuously for 72 hat a constant temperatureof39±1°Cinawaterbath. Headspacegas sampleswerecollected using gas-tight syringes from the Tedlar bags connected to the glass bottles and analyzed for greenhouse gases (CH and carbon dioxide-CO )and H S concentrations. CH and CO gas concentrations were analyzed using 4 2 2 4 2 an SRI-8610 Gas Chromatograph and H S concentrations were measured using a Jerome 631X meter. At the same time, substrate (i.e. mixed rumen fluid+ NP treatment+ feed composite) samples were collected from the glass bottles at the beginning and at the end of an experiment for bacterial counts, and volatile fatty acids (VFAs) analysis. Results: Compared to the control treatment the H S and GHGs concentration reduction after 72 h of the tested nZnO levels varied between 4.89 to 53.65%. Additionally, 0.47 to 22.21% microbial population reduction was observed from − 1 the applied nZnO treatments. Application of nZnO at a rate of 1000 μgg have exhibited the highest amount of concentration reductions for all three gases and microbial population. − 1 Conclusion: Results suggest that both 500 and 1000 μgg nZnO application levels have the potential to reduce GHG and H S concentrations. Keywords: Rumen, Feed, Greenhouse gases, Nanoparticle, Concentration Background catalyst . During enteric fermentation, CH and car- The agricultural sector is recognized as one of the great- bon dioxide (CO ) are the two main greenhouse gases est sources of methane (CH ) and other gaseous emis- (GHGs) emitted and contribute to global warming . sions, and it is contributing approximately 250 million Hydrogen sulfide (H S) is another pollutant gas gener- metric ton CO Eq. CH emission per year [1, 2]. Most ated during enteric fermentation, although its amount is 2 4 of the CH emissions from the agricultural sector are not significant compared with CH and CO . Hydrogen 4 4 2 from the livestock industry and manure management. Sulfide might be a potential health hazard to livestock Almost 70% of the agricultural sectors CH emission is and workers depending on the concentration level . from enteric fermentation . Enteric fermentation in- Hence, the reduction of these gas emissions without al- cludes fermentation in the rumen and hindgut paired tering animal productivity is a challenge for a healthy with digestive hydrogen (H ) metabolism by microbial environment and sustainable livestock industries. Fermentation of carbohydrates in the reticulorumen * Correspondence: firstname.lastname@example.org occurs for available hydrogen supply towards volatile Agricultural and Biosystems Engineering Department, North Dakota State fatty acid (VFA) production and eventually leads to CH University, Fargo, ND 58108, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 2 of 8 production [5–9]. Additionally, fermentation and the maximum tolerable level of Zn mineral concentra- neutralization of hydrogen ions (H ), and bicarbonate tion provided by the National Academies of Sciences 3− ions (HCO ) entering the rumen across the ruminal . The specific objective was to characterize the wall during VFA absorption contributes to CO produc- changes in the rumen fluid properties and find the gas- tion in the rumen [10, 11]. Similarly, sulfur-containing eous reduction mechanisms such as by bacterial popula- amino acids and sulfates are the main sources of H S tion reduction. within the rumen; H S generation depends on the mi- crobial degradation of amino acids and sulfates [11–13]. Methods Since all of these gaseous emissions pose potential en- Ruminal fluid collection, processing and experimental setup vironmental and safety concerns, scientists are striving Ruminal fluid was collected from two ruminally-fistulated to mitigate the production of these gases. Management mature steers predominately of Angus breeding on a of feeding strategy, application of biotechnology, and the limit-fed grass hay-based diet fed to maintain body weight. introduction of additives are a few of the most common Two hours after morning feeding, approximately one liter approaches that researchers are working on for abating of ruminal fluid was collected from each steer. To ensure enteric gaseous emissions . Similarly, changes in the uniform representation of the liquid and fiber phase, ran- forage species, good forage processing, reduction of for- dom grab samples were collected both from ventral and age maturity, based on your excellent credentials, and dorsal ruminal sacs. Prior to mixing with McDougall’sbuf- increased feeding frequency are a few noteworthy gas fer , ruminal fluid from each steer was combined and mitigation strategies [14–21]. However, all of these ap- strained through four layers of cheesecloth to remove the proaches exhibit a very small amount of gaseous emis- large particulate matter. Five treatments consisting of a sion reduction, and in most of the cases, the mitigation control (no nZnO) and four levels of nZnO (100, 200, strategy focused on the reduction of CH only. So, it is 4 − 1 500, and 1000 μgg of feed), with two different feeds (al- important to develop a new approach that can reduce falfa and maize silage; Table 1) were used. Nutrient com- multiple gaseous emissions without compromising ani- positions of the two base diets are shown in Table 1. mal health and productivity. Levels of nZnO were selected based on the maximum In recent years, nanotechnology has received attention − 1 allowable zinc (Zn) concentration (30 to 500 μgg )in for improving livestock production . In U.S., only 26 − 1 feed recommended by the . The 1000 μgg of nZnO of 160 agri-food nanotechnology research and develop- level was added to investigate the effect of high nZnO ap- ment projects were relevant to livestock facilities . plication level on ruminal gaseous emission. The nZnO Animal health, veterinary medicine, and other animal application levels were weighed on a Sartorius CP2P production facilities are a few of the livestock-related microbalance (Sartorius Corporation, NY, USA) with an sectors on which nanoparticles (NPs) have their promis- accuracy of 1 μg using small aluminum pans (DSC ing footprints [23–25]. For example, silver and zinc NPs Consumables, Inc., AU, USA). The nZnO (US Research have been added to animal feed to control microbial Nanomaterials, Inc., Texas, USA; Particle Size = 35–45 nm proliferation and promote animal growth, respectively. and 99.5% purity) was mixed with two feeds (e.g., alfalfa Similarly, zinc oxide (nZnO) NP is used to enhance RF and maize silage) separately. In each ANKOM gas bot- growth and feed efficiency in piglets and poultry . tle, 1.5 g of ground alfalfa or maize silage (3 to 5 mm size) However, application of nanotechnology in mitigating feed was added. Thereafter, 37.5 mL of the combined gaseous emissions from livestock facilities is still limited. rumen fluid and 150 mL of McDougall’sbuffer were Swain et al.  reported nZnO changes the rumen fer- added to each bottle and a sub-sample of the mixed rumi- mentation kinetics in ruminants and can alter the vola- nal fluid was stored in the freezer for characterization. tile fatty acids, therefore it may affect enteric CH Treatment bottles was purged with CO to create an an- production. Similarly, application levels of NPs may also RF aerobic environment and sealed with the ANKOM pres- alter the microbial population, thus other gaseous emis- sure monitor cap. Thus, in total, twenty (5 treatments × 4 sions. Among the few studies performed with GHGs replications) bottles were used for each feed type. mitigation, nZnO were reported to have an inhibitory action towards CH ,CO and H S from anaerobic stor- 4 2 2 age of manure [27, 28]. Therefore, the objective of this Ruminal pH and redox determination study was to evaluate the efficacy of four different appli- The pH, and redox of the mixed ruminal fluid were − 1 cation rates (100, 200, 500, and 1000 μgg of feed) of determined before and after the ruminal fluid was nZnO in mitigating CO ,CH , and H S emissions from treated with nZnO using a HANNA HI 4522 dual chan- 2 4 2 rumen fluid under anaerobic storage conditions. Other nel benchtop meter (VWR, TX, USA). Both probes were − 1 than the application rate of 1000 μgg , nZnO applica- calibrated following manufacturer standard protocols. tion rates were within the general dietary guideline of The reading of each probe was also confirmed with Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 3 of 8 Table 1 Composition of the feeds (dry matter basis) Feeds % Ash CP NDF ADF Ca P Mg K Zn Cu Alfalfa 13.16 18.33 60.28 42.59 3.99 0.29 0.39 3.26 0.01 0.06 Maize silage 7.06 6.02 53.65 31.42 0.88 0.26 0.20 1.37 0.01 0.08 CP Crude Protein, NDF Neutral Detergent Fiber, ADF Acid Detergent Fiber, Ca Calcium, P Phosphorus, Mg Magnesium, K Potassium, Zn Zinc, Cu Copper respective standard solutions before each measurement analytical instruments and two measurements for indi- to ensure accurate reading of the probes. vidual bottle were taken for each of CH ,CO , and H S 4 2 2 concentration. Nitrogen at 20 psi with a flow rate of − 1 Gas production measurement and monitoring system 250 mL min was supplied to the GC as a carrier gas. All experiments were conducted using 250 mL Additionally, a built-in air compressor and external RF ANKOM gas glass bottles and under the same condi- hydrogen generator were used to supply hydrogen and tions. After proper flushing and sealing of bottles, they air to the GC. Temperatures of 300 and 350 °C were were placed in a water bath (SWBR17 shaking water maintained respectively on the FID and ECD detectors bath, Atkinson NH, USA) that oscillated and heated at before insertion of any sample gas into the GC sample 125 rpm and 39 ± 1 °C, respectively. Once they were loop . Calibration gases were used to check the placed in the water bath, a wireless gas production proper functioning of the instruments and blank samples measurement system (ANKOM Technology Corp., Ma- were used to check any contamination within the instru- cedon, NY, USA) was used for monitoring and measur- ments from previous measurements . ing gas production data. Data obtained from this system were converted from pressure (KPa) units to volume Analysis of microbial populations units (mL) using the ideal gas law as follows: Rumen fluid samples ( ̴ 5 mL) were collected at the be- ginning (just before the experiment) and at the end of the experiment (after 72 h of the experiment) and they n ¼ p Eqn 1 RT were analyzed for the coliforms i.e. potential pathogens (particularly Escherichia coli) that is recommended by Gas producedðÞ mL ¼ n 22:4 1000 Eqn 2 the American Public Health Association (APHA) and Where: n = gas produced in moles (mol), P = pressure the Environmental Protection Agency (EPA). Microbial in kilopascal (kPa), V = head-space volume in the Glass populations (coliforms) density were analyzed by count- Bottle in Liters (L), T = temperature in Kelvin (K), and R ing total coliform bacteria in terms of colony forming − 1 − 1 = gas constant (8.314472 L.kPa.K .mol ). units (CFUs) following the plate count method . All Throughout the experimental period, Each bottle was reagents, labware, and Petri dishes used for microbial connected to a Tedlar bag and once gas pressure inside analysis were handled carefully and the whole experi- a bottle reached a set-limit in the RF pressure sensor mental preparation was conducted in a sterile environ- RF module and recorded by the ANKOM system, the ment. One milliliter of the rumen fluid samples were headspace gas was released in the connected Tedlar bag. collected from each treatment replications, and were di- 2 3 4 5 A typical in vitro study lasts for 24 h, however, in the luted up to five-fold (10, 10 ,10 ,10 and 10 ) to find present study it was continued for 72 h to examine the the optimum dilution for better visibility of the CFUs. effects of nZnO on long term in vitro fermentation. Later on, all treatments were replicated three times with After 72 h of the experimental period, gas samples from the optimum dilution. The 2 mL M-Endo broth ampule the Tedlar bags were drawn using a gas-tight syringe (P/N: 23735–50, HACH LANCH GmbH, Willstatter- (5 mL, Luer-LokTM Tip Syringe, Franklin Lakes, NJ, strasse 11, Dusseldorf, Germany) was used as growth USA) and analyzed for GHGs (CH and CO ), and H S media to culture the bacteria in an incubator. The 4 2 2 concentration. A Jerome Meter (Jerome 631X, Arizona growth media was poured evenly over a gridded sterile Instrument LLC, Arizona, USA) was used to measure membrane filter attached with absorbent pad (47 mm H S concentration and a gas chromatograph (GC, diameter, 0.45 μm pore size, WCN type, Whatman Lim- 8610C, SRI instrument, California, USA) equipped with ited, Maidstone, England, UK) that was placed in a ster- flame ionization detector (FID) and electron capture de- ile petri-dish (Anaerobic, Sterile Petri dishes, 60 mm tector (ECD) detectors were used to measure CH and diameter and 15 mm height, VWR, Radnor, PA, USA). CO concentration. Based on the previous trials, col- Subsequently, 100 μL of the diluted rumen fluid samples lected gas was diluted 100 fold with pure nitrogen to were added to the absorbent pad and smeared evenly keep the concentration in the measurable range of the over the pad using a small sterile glass rod. The petri Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 4 of 8 dishes with the growth media and bacterial culture were combinations ranged between − 296 to − 307 mV (Table then incubated for 24 h at 35 ± 0.5 °C in an incubator 2), which is the preferred range for producing CH and (Lab Companion IB-01E Incubator, San Diego, CA, CO anaerobically . The rumen fluid redox potential USA). After 24 h of incubation, CFUs were counted between two feed types were not significantly different using a manual dark field colony counter with 1.5X mag- (P = 0.748). Additionally, similar to that of pH, no inter- nification (Reichert, Inc. Depew, NY, USA). action among the feed types and nZnO levels was found for the rumen fluid redox potential (P = 0.217), and no Volatile fatty acids (VFAs) analysis significant difference was found among the nZnO levels At the end of the experimental period, Whirl-Pak bags (P = 0.947). (Nasco, Fort Atkinson, WI and Modesto, CA, USA; 532-mL) were used to collect and store the rumen fluid Effect of nZnO application levels on ruminal subsamples at − 20 °C until further analysis. Thereafter, VFA production samples were equally composited using a vortex (Cat: Among the four nZnO levels and the control treatment, 10153–842, VWR® digital vortex mixer, Radnor, PA, the amount of total VFA (TVFA) ranged between USA) and centrifuged (clinical 100 laboratory centrifuge, 136.52 to 194.16 mM for the alfalfa-based rumen fluid, VWR, Rndor, PA, USA) at 2000×g for 20 min. They while it ranged between 161.36 to 192.8 mM for the were filtered through a pore size 0.45 μm to separate out maize silage based rumen fluid. Compared with the the supernatant and analyzed for VFAs using an Agilent other treatments (nZnO levels), after 72 h of the 6890 N gas chromatograph (Agilent Technologies, Inc., experimental period, the control treatments exhibited Wilmington, DE, USA) equipped with an FID and fused the highest TVFA (Table 3). For the acetic acid, no sig- silica column (Supleko brand, NUKUL 15 m × 0.53 mm × nificant difference was found among the feed types (P 0.5 μm, Sigma-Aldrich C., MO, USA), and 7683 series =0.832), and no significant interaction between feed auto-injector following a widely used method . types and nZnO levels (P = 0.172) was found. Moreover, no significant interaction between feed type and nZnO Statistical analysis concentrations (P = 0.688) were found for the propionic The data were analyzed in a 2 × 2 factorial experiment acid. However, rumen fluid with alfalfa had significantly using PROC GLM (SAS Inst. Inc., Cary, NC), which cal- lower propionic acid concentration than rumen fluid culated the statistics for general linear models. Both of with maize silage (P < 0.001). Propionic acid was also the feed types and five levels of nZnO were used as fixed found to be affected by nZnO levels (Table 3), although, effects models. Means were declared statistically signifi- no definite trend was found. Propionic acid to acetic cant at P ≤ 0.05 using Duncan multiple range test. acid (P/A) ratio was higher for maize silage based fer- mentation than the alfalfa-based fermentation. Alfalfa- Results based rumen fermentation’s P/A ratio varied from 0.29 Effect of nZnO application levels on ruminal pH and to 0.32, whereas this ratio varied from 0.38 to 0.55 for redox the maize silage-based fermentation. The pH of the rumen fluid incubated 72 h with alfalfa ranged between 7.20 to 7.25, whereas the pH of the Effect of nZnO application levels on ruminal gaseous maize silage based rumen fluid ranged between 6.92 to emission and CH ,CO , and H S concentrations 4 2 2 6.96 (Table 2). Alfalfa based rumen fluid showed signifi- Table 4 represents the amount of total gas produced, and RF cantly higher pH than of maize silage (P < 0.0001).No gas concentrations in the ANKOM bottles over 72 h of interaction was found between feed types and nZnO incubation with four different nZnO application levels levels (P = 0.401). Additionally, none of the zinc levels and two feed types. Produced total gas from the maize sil- − 1 (100 to 1000 μgg ) were found to indicate a significant age fermentation was two times higher than that of alfalfa difference in pH values (P = 0.644). Redox potential fermentation (P < .0001). However, no significant differ- among the treated rumen fluid and two different feed ence in terms of total gas production among different Table 2 Effect of nZnO levels on ruminal pH and redox (after 72 h of incubation) −1 − 1 − 1 − 1 a Effects Alfalfa Maize silage Control 100 μgg 200 μgg 500 μgg 1000 μgg SEM P value Feed nZnO nZnO Feed pH 7.22x 6.94y 7.09a 7.07a 7.08a 7.07a 7.08a 0.001 <.0001 0.644 0.401 Redox -300x -301x -301a -301a -300a -302a -299a 9.004 0.748 0.947 0.217 Data’s are presented as least square means per treatment ± SEM Means followed by the same letters (x/y/a/b/c/d) in each row are not significantly different at P ≤ 0.05 Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 5 of 8 Table 3 Effect of nZnO levels on the rumen fluid VFA (n = 4 observations/treatment) −1 − 1 − 1 − 1 a Effects Alfalfa Maize silage Control 100 μgg 200 μgg 500 μgg 1000 μgg SEM P value Feed nZnO nZnO Feed Acetic Acid (mM) 108x 107x 122a 97.1b 98b 108ab 114a 14.15 0.832 0.005 0.172 Propionic Acid (mM) 33.05x 49.53y 50.88a 34.01c 36.94bc 41.11bc 43.50ab 7.68 <.0001 0.002 0.688 P/A ratio 0.306x 0.444y 0.423a 0.389a 0.369a 0.379a 0.380a 0.058 0.189 0.004 0.007 Total VFA (mM) 161x 173x 193a 149c 152bc 166bc 174ab 21.51 0.086 0.002 0.743 Data are presented as least square means per treatment ± SEM Means followed by the letters (x/y/a/b/c/d) in each VFA type are not significantly different at P ≤ 0.05 VFA Volatile Fatty Acid, P/A Propionic acid to Acetic acid ratio applied nZnO levels was found (P = 0.875).Moreover, significant interaction between feed level and zinc was there was no significant interaction between feed types found for CH (P = 0.479) and CO (P = 0.948). 4 2 and zinc levels were evidential (P = 0.542). Measured total gas volume from the maize silage Effect of nZnO application levels on ruminal microbial based rumen fluid was significantly higher than that of population alfalfa based rumen fluid (P < .0001), although maize sil- Plate counts were done in terms of CFUs from pre- and age based rumen fluid produced lower CH ,CO , and post-treated rumen fluid samples to determine the 4 2 H S gas concentrations than that of alfalfa based rumen effects of applied nZnO on coliforms (Table 5). The fluid. However, all of the nZnO levels irrespective of the average initial CFUs were 88.4 counts with alfalfa feed feed types showed a similar reduction trend for both based rumen fluid, and it was 85.2 counts with the maize CH and CO concentrations. Regardless of the feed and silage feed based rumen fluid. Initial CFUs were similar 4 2 nZnO levels, CO concentrations were around five times regardless of feed types (P = 0.231) or nZnO inclusion higher than that of CH concentrations. In contrast, al- (P = 998). In contrast, final CFU numbers exhibited a dif- though the evidential significant interaction between ferent trend than the initial number of CFUs (Table 5). At feed types and zinc levels (P < .0001) was found for H S the end of the 72 h experimental period, CFU numbers in- concentration (Table 4), but the reduction trends were creased by̴ 98% for all of the treatments including control, similar to that of CH and CO .H S concentration from and they ended up with an average of 4630, and 5155 4 2 2 the maize silage was ̴ 60% less than that of alfalfa. Re- counts for alfalfa and maize silage feeds, respectively. Irre- gardless of the feed used, compared to control treatment spective of the nZnO application levels, final CFU counts higher nZnO application levels reduced higher amount were higher with maize silage compared with the alfalfa. of CH ,CO , and H S concentrations. Compare to the Although, lower application levels of nZnO exhibited very 4 2 2 control, pooled average of the gas concentrations small CFU reduction efficiency compared with the higher showed that applied levels of nZnO reduced CH ,CO , levels. The greatest reduction in microbial population was 4 2 and H S concentration by 9.14 to 46.85%, 4.89 to observed at the highest nZnO level. 42.79%, and 9.33 to 53.65%, respectively. Among the − 1 treatments, the 1000 μgg of nZnO application level Discussions produced the highest reduction in CH CO ,and Lower pH of the rumen fluid incubated with maize 4, 2 H Sconcentration (P < .0001). Additionally, both 500 silage based treatments might affect/inhibit acidogenic − 1 and 1000 μgg nZnO levels reduced significant (P bacteria those are responsible for anaerobic digestion. In < .0001) amount CO and H S concentrations com- contrast, higher pH in alfalfa feed based treatments 2 2 pared to other treatments (Table 4). However, no might increase the rate of fermentation, and contribute Table 4 Effect of nZnO levels on cumulative gas volume and gas concentrations (n = 4 observations/treatment) − 1 − 1 − 1 − 1 a Effects Alfalfa Maize silage Control 100 μgg 200 μgg 500 μgg 1000 μgg SEM P value Feed nZnO NZnO Feed Total Gas (mL) 41.09x 72.77y 58.17a 55.88a 55.88a 57.99a 58.17a 5.71 <.0001 0.875 0.542 CH (%) 10.69x 9.47y 12.68a 11.52ab 10.18bc 9.26c 6.74d 1.44 0.0114 <.0001 0.479 CO (%) 48.78x 45.55y 56.21a 53.46ab 50.06b 43.93c 32.16d 4.51 0.0314 <.0001 0.948 H S (ppm) 3420x 1314y 3150a 2856a 2431b 1936c 1460d 298 <.0001 <.0001 <.0001 Data are presented as least square means per treatment ± SEM Means followed by the letters (x/y/a/b/c/d) in each row are not significantly different at P ≤ 0.05 Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 6 of 8 Table 5 Effect of nZnO levels on ruminal microbial populations (n = 4 observations/treatment) − 1 − 1 − 1 − 1 a Effects Alfalfa Maize silage Control 100 μgg 200 μgg 500 μgg 1000 μgg SEM P value Feed nZnO nZnO Feed Initial_CFU 88.40x 85.20x 87.63a 86.50a 86.38a 86.88a 86.63a 8.27 0.231 0.998 0.923 Final_CFU 4630x 5155y 5350a 5325a 4900a 4725ab 4162b 590 0.009 0.002 0.887 Data are presented as least square means per treatment ± SEM Means followed by the letters (x/y/a/b/c/d) in each row are not significantly different at P ≤ 0.05 CFU Colony Forming Unit to the growth of spoilage microbes [36–38]. Moreover, significantly, even 1000 μg g-1 of nZnO was not enough the higher pH in the post-treated alfalfa-based rumen to reduce a significant amount of cumulative gas pro- fluid would likely produce a higher amount of soluble duction. Therefore, nZnO at this application rate does protein, carbohydrate, and volatile fatty acids . not appear to decrease the digestibility of feed by the Hence, higher concentrations of all three gases (CH , animal, and therefore, should not decrease productivity CO , and H S) were likely from the alfalfa based treat- or growth. However, further studies are needed to 2 2 ments compared with its counterpart. The resulted understand the process and to verify if the productivity consistent redox potential among the treatments was is sustained when nZnO is included in the diet. preferred for anaerobic fermentation [40–43]. Additionally, It is noteworthy that CH concentrations with alfalfa redox potential among the treated rumen fluid and two dif- were higher than those of maize silage (Table 4), ferent feed combinations were in the preferred range for although higher cumulative gas production was observed producing CH ,CO ,and H S anaerobically . in maize silage-based fermentation (Table 4). This was 4 2 2 Volatile fatty acids are considered as one of the most likely due to appropriate P/A ratio and subsequent bal- important parameters for ensuring anaerobic fermenta- anced fermentation with alfalfa-based rumen fluid that tion. Higher TVFA amount in maize silage based rumen might prompt higher CO and H S concentration as 2 2 fluid compared with the alfalfa forage types might be an well . Generally, a group of archaea belonging to the indication of the higher amount of digestible carbohy- phylum Euryarcheota, and collectively known as metha- drate in the maize silage . Subsequently, a higher nogens are responsible for CH production within the amount of cumulative gas production from the maige animal rumen and hindgut . Reduction of the CH silage-based fermentation was likely. The resulted pooled concentration from rumen fluid at the highest applica- average of P/A ratio in the present study was 0.304 and tion level of nZnO was likely due to the impact of exces- 0.459 for the alfalfa and maize silage, respectively. The sive nZnO application rate (which was almost two-fold P/A ratio from the maize silage was 26% higher than the of the allowable limit as recommended by NAS as feed) previously reported value, while the P/A ratio for the al- specifically on methanogens . As mentioned previ- − 1 falfa was identical to the reported value (Ghimire, 2015). ously, the highest application rate (1000 μgg )of Higher P/A ratio might be an indication of imbalanced nZnO did not affect total gas production, but likely re- anaerobic fermentation with the maige silage-based duced the enteric CH concentration due to inhibitory rumen fluid fermentation . Application of nZnO was action on the CH -producing methanogenic microbial hypothesized to affect either hydrolysis, acetogenesis, community. Additionally, adsorption of the produced fermentation, methanogenesis or a combination of these methane on the NPs surface might also contribute to processes in the fermentation process. In some cases, the reduction in CH4 when nZnO was added to the the bactericidal action of the applied higher nZnO level rumen fluid. This situation warrants further study for in- might kill the higher amount of methanogens, and hence vestigating the effect of higher levels of zinc as a feed a higher amount of unconverted TVFA was likely. Fur- additive on animal growth and productivity. thermore, increased energy utilization followed by rumi- CO concentration was five times higher than the nal microbial protein synthesis by the microbes in the CH , which might be an indication of biocidal action of early stages of fermentation might have increased the nZnO on methanogen archaea. During anaerobic diges- TVFA with the applied higher nZnO level as indicated tion process, methanogenic archaea utilize CO , and H 2 2 by others . to produce CH . Nano zinc oxide might leave only a Higher gas production from the maize silage fermenta- small amount of methanogenic archaea active, and thus tion might be due to probable higher carbohydrate con- higher amount of unconverted CO was likely. Further- tent and subsequent higher fermentability of maize more, CO emission from rumen is directly related to silage compared to alfalfa. None of the applied nZnO ap- the degradation of the organic constituents present in plication levels were able to reduce total gas volume the feed, hence the decreasing trend in the CO 2 Sarker et al. Journal of Animal Science and Technology (2018) 60:27 Page 7 of 8 concentration was likely to indicate lower degradation lower enteric GHG emission from grass fed beef. How- rate of the organic matter in the rumen. Application ever, additional microbial studies are necessary to deter- of NPs might have an adverse impact on the micro- mine the mode of action. Additionally, further work is bial community and as a consequence lower degrad- needed to assess the effect of nZnO inclusion on animal ation of organic compounds might occur. However, performance when cattle are fed ingredients commonly additional microbial studies are needed to understand used in beef feedlot diets. the in-depth process. Acknowledgments The Higher amount of H S concentration from the al- Thanks to Debra Baer, Technical Communication Specialist, Agricultural and falfa based feed compared with the maize silage was Biosystems Engineering Department, NDSU, USA, for reviewing the manuscript. likely to be an indication of higher activity of the micro- Funding organisms. Since, in absence of oxygen (O ) sulfate- This study was conducted using the discretionary funding of the corresponding reducing bacteria utilize sulfate to oxidize organic com- author. No specific funding was involved. pounds present in the feed and ends up with the H S Availability of data and materials production as a byproduct, hence the reduction trend of The data generated or analyzed during the current study are available upon H S concentration might be due to the reduced activity a reasonable request to the corresponding author. of the sulfate-reducing bacteria . However, the Authors’ contributions concentration reduction mechanism needs to be ex- Shafiqur Rahman was the PI for the project and designed the experiment. plored to investigate the adverse effect of the nZnO on Niloy Chandra Sarker did the experiment, drafted and wrote this manuscript, the microbial community. did statistical analysis and statistical work. Faithe Keomanivong, Md. Borhan, Shafiqur Rahman, and Kendall Swanson helped to set up the experiment Initial CFUs were measured right after the application and data collection. All the authors read and approved the final manuscript. of the nZnO in the system, therefore, there was little or no effects of nZnO levels on CFUs. In this circumstance, Ethics approval and consent to participate irrespective of the nZnO application levels, the number The authors have an IACUC approval specifically for doing the in vitro digestion studies at North Dakota State University (NDSU) and the protocol of microbial populations was most likely to represent approval number is A15038. the similar number of the populations present in the rumen fluid. In contrast, addition of fresh feed was most Consent for publication All authors agreed to submit the manuscript to this journal. likely to contribute towards the increasing amount of final CFUs. Compared with the control (final), lower Competing interests CFU numbers in the nZnO treated samples were most The authors declare that they have no competing interest. likely due to the biocidal effect of nZnO. An insignifi- cant amount of CFUs reduction from the treatments Publisher’sNote with lower application levels of nZnO might be an indi- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. cation of the lower amount of available biocides. In contrast, a higher reduction in CFUs was observed with Author details higher application levels of nZnO and the reduction was Agricultural and Biosystems Engineering Department, North Dakota State − 1 University, Fargo, ND 58108, USA. Animal Sciences Department, North significant only with 1000 μgg inclusion level. Fur- Dakota State University, Fargo, ND 58108, USA. thermore, the presence of higher CFUs in the maize silage based treatments were likely to validate the higher Received: 21 September 2018 Accepted: 29 October 2018 gas production from those treatments, and vice versa. 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Journal of Animal Science and Technology – Springer Journals
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