Animal care
The study followed all procedures approved by the University of Florida Institutional Animal Care and Use Committee (Protocol #201810218) and all methods were carried out in accordance with relevant guidelines and regulations. This manuscript is reported in accordance with ARRIVE guidelines.
The experiment was conducted at North Florida Research and Education Center (NFREC), from University of Florida, located in Marianna, FL (3052N, 8511 W, 35m a.s.l.) in a pasture of Pensacola bahiagrass (Paspalum notatum Flgg). The soil at the experimental site is classified as an Orangeburg loamy sand (fine-loamy-kaolinitic, thermic Typic Kandiudults), with an average pH of 6.5. Average Mehlich-I extractable P, K, Mg, and Ca concentrations were 13, 45, 31, and 245mgkg1, respectively. Soil organic matter was 6.3gkg1, and the estimated cation-exchange capacity was 2.8meq 100g1. The study was carried out for two experimental periods of 32days each, separated by a 15-day interval (Period 1: from 06/08/2018 to 07/10/2018; Period 2: from 07/25/2018 to 08/27/2018). The average, maximum and minimum temperatures and rainfall for the experimental periods are represented in Fig.5.
Marianna (FL) station of the Florida Automated Weather Network (FAWN) data of (a) weekly rainfall and temperature data from two experimental periods and (b) accumulated monthly rainfall (mm) and average temperature (C). Period 1: from 06/08/2018 to 07/10/2018; Period 2: from 07/25/2018 to 08/27/2018.
Fifteen BrahmanAngus crossbred steers [Period 1: 32426kg initial body weight (BW); Period 2: 33630kg BW] were randomly distributed into three experimental diets: 0, 50, or 100% (as fed) inclusion of AU Grazer sericea lespedeza hay [SL; Lespedeza cuneata (Dum. Cours.) G. Don] into Tifton 85 bermudagrass hay (BG; Cynodon spp.) diets (Table 3) and used as donors of excreta (urine and feces). Steers were fed for 21days for two feeding periods in a concomitant study17 and excreta used in the current study was collected in the last two experimental days of each. All methods were carried out in accordance with relevant guidelines and regulations.
Emissions of GHG were evaluated using the static chamber (non-steady state) technique14. Chambers were circular with 30cm radius and made of a base and a lid, both built out of PVC47. The lids were wrapped with reflective tape to provide insulation and a rubber septum was added for gas sampling48. The base was fitted with a 10-cm length copper venting tube to ensure adequate air pressure inside the chamber during measurements49,50. Lids and bases were kept closed for gas sampling by fitting a bicycle tire inner tube that tightly sealed the parts together.
Bases of chambers were installed in the non-grazed pasture of bahiagrass twoweeks prior to excreta application to avoid any effect of soil disturbance on GHG emissions51. Bases were installed at 8-cm depth, with 5cm extending above ground level. Depth for installation was determined based on Clough et al.48. Chamber tops were 22cm height, which when summed with 5cm of base totaled 27cm, in agreement with the indication of40cm of chamber height per hour of deployment48. New bases were installed in a near location of the same pasture for the second experimental period, also twoweeks prior to starting new gas sampling.
Treatments applied to the chambers consisted of either feces or urine within one of the three levels of inclusion of SL hay fed to the beef steers and were distributed as a complete randomized block design. Urine and feces were collected directly from each animal by spontaneous or stimulated urinations and defecations and applied at a rate of 2 L of urine and 2kg of feces, as typical amounts excreted by cattle for the area of the chamber5,52. To obtain quantities required of excreta, sampling occurred twice a day (700 and 1500h) and samples were kept refrigerated at 4C until next morning (day 0 of gas sampling). Samples of each excreta type were composited across all five animals within each SL diet resulting in three final subsamples (urine and feces from each of three SL diets). Excreta samples were kept at room temperature 2h prior to application to the chambers and their chemical composition is described on Table 1.
Application of excreta to the chambers was made one time in each experimental period on the soil surface inside the area determined by the base of the chamber (0.28 m253). Grass inside the chamber area was cut at ground level before each sampling day, when appropriate. Gas sampling occurred between 0900 and 1100h, when temperature is considered more representative of the daily average47 on days 0, 1, 3, 5, 7, 14, 18, 25, and 32 after excreta application for both experimental periods. One subsample was taken per deployment time per chamber, separated by 15-min intervals (T0, T15, and T30). At T0, a sample was collected from the area directly above the soil surface54. Immediately thereafter, chambers were tightly closed by fitting the lid to the base with the bicycle inner tube, followed by the next sample deployment times. All samples were collected with the use of a 60-mL syringe and immediately flushed into a pre-vacuumed 30-mL glass vial. The vial was equipped with a butyl rubber stopper and sealed with an aluminum septum. Samples were analyzed immediately after finishing each experimental period.
Gas sample analyses were conducted using a gas chromatograph (Trace 1310 Gas Chromatograph, Thermo Scientific, Waltham, MA). For N2O, an electron capture detector (350C) and a capillary column (J&W GC packed column in stainless steel tubing, length 6.56 ft (2M), 1/8 in. OD, 2mm ID, Hayesep D packing, mesh size 80/100, pre-conditioned, Agilent Technologies) were used. Methane was analyzed using a flame ionization detector (250C) and a capillary column (J&W PoraBOND Q GC Column, Agilent Technologies). For CO2, a thermal conductivity detector (200C) and capillary column [J&W GC packed column in stainless steel tubing, length 7 ft (2.13M), 1/8 in. OD, 2mm ID, Haysep N packing, mesh size 60/80, pre-conditioned, Agilent Technologies] were used. Temperature of the injector and columns were 80 and 200C, respectively.
The hourly gases fluxes (mg of N2O or CH4 or CO2 per m2h1) were calculated according to Cardoso et al.55:
$${text{F}}_{{{text{GHG}}}} = left( {updelta {text{C }}/ updelta {text{t}}} right) , times , left( {{text{V}}/{text{A}}} right) , times , left( {{text{M}}/{text{Vm}}} right),$$
where C/t is the change in gas concentration in the chamber during the deployment time; V and A are the chamber volume and soil area covered by the chamber, respectively; M is the molecular weight of the gas; and Vm is the molecular volume of gas. The Vm parameter was corrected to the standard conditions of temperature and pressure as Vm=0.02241(273.15+Tc/273.15)p0/p1, where 0.02241 is the molar volume (m3), Tc is the chamber headspace temperature at sampling time (C), p0 is the air pressure at sea level, and p1 is the local pressure calculated using the barometric equation. The minimal detectable flux was 0.012ppbmin1 for N2O, 0.004ppmmin1 for CH4, and 1.40ppmmin1 for CO2.
Daily N2O, CO2, and CH4 emissions were calculated by multiplying the fluxes by 24h and cumulative emissions were estimated by integrating the fluxes over each day (area under the curve) and averaged per period. The fraction of N applied in the excreta lost as N2O, named as emission factor (EF), was calculated according to the equation:
$${text{EF}}_{{{text{N}}_{{2}} {text{O}}}} left( % right) , = , left[ {left( {{text{N}}_{{2}} {text{O}} - {text{N}}_{{{text{emitted}}}} } right) , - , left( {{text{N}}_{{2}} {text{O}} - {text{N}}_{{{text{blank}}}} } right)} right]/{text{N}}_{{{text{applied}}}} times { 1}00,$$
where ({text{EF}}_{{{text{N}}_{{2}} {text{O}}}}) is the emission factor of N2O; N2O-Nemitted is the cumulative N2O-N emissions from the chamber with excreta (mgm2); N2O-Nblank is the cumulative N2O-N emissions from the blanks (chamber with no excreta deposited; mgm2); and Napplied is the urine or feces N application rate (mgm2).
A subsample (12ml) of the collected gas was transferred to evacuated exetainers (Labco, UK). Exetainers were pierced with a double needle and flushed with an ultrapure stream of He (12mLmin1) for 6min using an autosampler (Gilson GX-271, Gilson Inc, WI). During flushing, samples were transferred to a preconcentration unit (Trace Gas, Hanau, Germany) equipped with glass traps (OD 10mm, length 20cm) filled with Ascarite, Sofnocat and Mg(ClO4)2to scavenge CO2, CO, and water, respectively. Remaining N2O was cryo-focused on a capillary column submerged in liquid nitrogen for 12min and transferred to an isotope ratio mass spectrometer (IsoPrime 100, IsoPrime, Manchester, UK) for 15N analysis, using He (2mlmin1)as a carrier. The isotope ratio for 15N/14N was calculated as:
$$updelta^{{{15}}} {text{N }} = , left( {^{{{15}}} {text{N}}/^{{{14}}} {text{N}}_{{{text{sample}}}} -^{{{15}}} {text{N}}/^{{{14}}} {text{N}}_{{{text{reference}}}} } right)/left( {^{{{15}}} {text{N}}/^{{{14}}} {text{N}}_{{{text{reference}}}} times { 1}000} right),$$
where 15N is the N isotope ratio of the sample relative to atmospheric nitrogen, 15N/14Nsample is the N isotope ratio of the sample, and 15N/14Nreference is the N isotope ratio of atmospheric N (standard). The stable isotopic composition of nitrogen was reported using the conventional delta per mill notation. 15N values are expressed relative to the international standard (AIR-N2).
The experiment was analyzed as a completely randomized block design, with feces and urine data computed separately due to differences in the magnitude of responses53. There were three replicates (chamber) of each treatment, and day was considered the repeated measurement for all variables. Glimmix procedure of SAS (SAS Inst., Inc., Cary, NC, version 9.4) was used, in which the chamber was considered the experimental unit. Graphs were drawn using Microsoft Excel (version 16.61). Normality of distribution and homogeneity of variances were evaluated using the Univariate procedure of SAS. Covariance structures were based upon the smallest Akaike Information Criterion value. The model included the fixed effect of level of SL inclusion and day after excreta application and their interaction, and the random effects of block, period, and their interactions. Means were compared using the PDIFF adjusted by Tukeys test at 5% significance.
