All procedures were approved by the Animal and Environment Ethics Committees of the University of São Paulo, College of Agriculture “Luiz de Queiroz” (USP/ESALQ). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Experimental site
Two experiments were carried out at Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, SP, Brazil (22° 42′ S, 47° 38′ W and 546 a.s.l.), during the summer 2017 (January to March). Napier elephant grass (Pennisetum purpureum Schum. cv. Napier) was used as model plant because of its large size, ease of vegetative propagation and the nature of the study. The soil was a high fertility Eutric Kandiudalf with the following chemical characteristics for the 0–20 cm layer: Experiment 1—pH CaCl2 = 5.9; OM = 46.0 g dm−3; P (ion-exchange resin extraction method) = 257.0 mg dm−3; Ca = 148.1 mmolc dm−3, Mg = 80.0 mmolc dm−3; K = 9.1 mmolc dm−3; H + Al = 15.0 mmolc dm−3; sum of bases = 237.1 mmolc dm−3; cation exchange capacity = 252.1 mmolc dm−3; base saturation = 94%; Experiment 2—pH CaCl2 = 5.8; OM = 39.3 g dm−3; P (ion-exchange resin extraction method) = 54.0 mg dm−3; Ca = 62.0 mmolc dm−3, Mg = 22.3 mmolc dm−3; K = 8.6 mmolc dm−3; H + Al = 29.0 mmolc dm−3; sum of bases = 93.1 mmolc dm−3; cation exchange capacity = 122.0 mmolc dm−3; base saturation = 76%. These were considered adequate for the forage species used, with no need for additional fertilisation.
The climate, according to Köppen classification, is Cfa, humid subtropical climate with wet summer40 and an average annual rainfall of 1,328 mm. The average air temperature during the experimental period was 24.2 °C and total precipitation 753.85 mm, from which 424.4 mm corresponded to total precipitation for Experiment 1 (Dec 27, 2016 to Feb 21, 2017) and 502.14.5 mm for Experiment 2 (Dec 09, 2016 to Mar 14, 2017). The greatest precipitation was recorded in January 2017 (336.55 mm).
To avoid soil water deficits, a drip irrigation system was installed in the area used for Experiment 1 and a sprinkler irrigation system was available in the area for Experiment 2. Irrigation in both areas was carried out according to records of precipitation, average air temperature and evapotranspiration. On rainy days, precipitation was recorded and taken into account in calculations for irrigation as a means of ensuring that plants were not submitted to either deficit or excessive soil moisture.
Experiment 1 (leaf morphology and anatomy): establishment and experimental control
Preparation of the experimental area (290 m2) started with the desiccation of previous vegetation (Cynodon dactylon (L.) Pers.) using the broad-spectrum herbicides Glyphosate (N-phosphonomethyl-glycine) and 2.4-D (2.4-Dichlorophenoxyacetic acid) in Sept 09 and Nov 15, 2016, and Paraquat (1.1′-dimethyl-4.4′-bipyridinium dichloride) in Dec 11, 16 days before planting on Dec 27, 2016.
One day before planting of the experimental area (290 m2), planting pits were opened and the desiccated vegetation around them removed. Planting material (stem cuttings with viable lateral buds) was harvested at an 850 m2 pasture of well-established Napier elephant grass41. Stems were fractioned in one-node pieces (one single axillary bud) discarding the basal and the apical portions of the stems to ensure vigorous sprouting from the planting material. Ten buds were placed in each pit at 5 cm depth, covered with soil and gently compressed by hand. The distance between pits in lines was 1.5 m and between planting lines was 2 m, in order to obtain the desired spaced-plant layout. Two weeks after planting, plants were thinned leaving one plant per pit.
Treatments corresponded to phytomer order along the tiller axis and the experimental design was a randomised complete block, with four replications. Plants within blocks were randomised using the statistical package SAS (Statistical Analysis System, v. 9.0).
Weed control during the experiment was carried out manually. Pest (Mocis sp.) and disease (Bipolaris sp.) control was carried out using the water-soluble insecticide Resolva (Lambda-Cyhalothrin) in Jan 07 and 22, 2017 (5 g L−1) and the fungicide Nativo (Trifloxistrobina + Tebuconazol) in Feb 05, 2017 (0.6 L ha−1), respectively.
Sampling followed the ontogenetic programme of plants, beginning with tiller 1, which corresponded to the anatomical evaluation of the 8th leaf (phytomer 8); tiller 2, which corresponded to the anatomical evaluation of the 9th leaf (phytomer 9) and the morphological evaluation of the 8th leaf; tiller 3, which corresponded to the anatomical evaluation of the 10th leaf (phytomer 10) and the morphological evaluation of the 9th leaf in this sequence until full expansion of the 16th leaf (phytomer 16) for morphological evaluation (tiller 10), totalling 40 tillers (4 tillers for anatomical characterisation of the 8th expanded leaf + 32 tillers for anatomical (9th to 16th leaf) and morphological (8th to 15th expanded leaf) evaluations + 4 tillers for morphological characterisation of the 16th expanded leaf). At each harvest, leaves were carefully removed from the tillers, identified and preserved with ice until processing in the laboratory. Sampled tillers were removed from the experimental area. In order to evaluate the effect of leaf age on the deposition of support tissues, all leaves from 8 tillers (4 for anatomical evaluations (tiller 11) and 4 for morphological evaluations (tiller 12)) were collected at a single harvest when the 16th leaf completed expansion following the same procedure described for each leaf separately. Twenty additional plants were grown (five per block) to ensure that all leaves would be harvested as planned, but they were not necessary.
Leaf anatomy
In the laboratory, the leaf blade was cut at the ligule, its length was measured (distance between the tip of the leaf and the ligule) and fractionated in five segments of similar length designated as: (1) basal—closest portion to the insertion on the tiller; (2) mid-basal—middle portion closest to the basal; (3) middle—middle portion of the blade; (4) mid-apical—middle portion closest to the apical; (5) apical—portion closest to the tip of the leaf. Each of the five segments were fragmented in 1-cm cuts and stored according to methodology described by Johansen42. Sample dehydration was carried out using a progressive alcoholic series with tertiary butyl alcohol43, and fragments infiltrated with paraffin and subsequently with paraplast. In sequence, fragments were sectioned (12-µm width) with a Leica Biosystems manual rotary microtome, followed by a triarch quadruple staining of tissues before permanent blade mounting, following the methodology proposed by Hagquist44. Images were captured using the AxioVision Program (V2.05, Carl Zeiss Vision) attached to a Zeiss Axioskop 2 binocular optical microscope and a Zeiss AxioCam MRc (1.388 × 1.040 pixels) digital camera. Images were captured using 20× objective lens from the first large vascular bundle after the central rib as a means of standardising readings. Estimates of the percentage of each anatomical tissue on the samples were made using the AxioVision software (AxioVs40, release 4.8.2.0, Carl Zeiss Micro Imaging GmbH, Germany). Initially, the whole cross-section area projected on the video was measured (STotal). Next in the measurement sequence were the areas of adaxial (EPIada) and abaxial (EPIaba) epidermis, parenchymatic sheath of vascular bundles (PSV), vascular tissue (VT—including xylem, phloem, mestome sheath and pericyclic fibers27) and sclerenchyma (SCL). The mesophyll area was calculated as the difference between STotal and that of all the other tissues, therefore including airspace. Measurements were made in µm2 and the results expressed as percentage of total area.
Leaf morphology
For the morphology measurements the leaf was cut at the ligule, its length was measured (distance between the tip of the leaf and the ligule) and fractionated in ten segments of similar length. At the mid portion of each segment the fragment width (distance between the opposite borders) was measured in millimetres. In the sequence, the central rib from each segment was removed using a scalpel and its width and length were also measured. The fragment parts (central rib and blade tissue) were passed through a LAI-3100 leaf area integrating device (LI-COR) and put to dry in a forced draught oven at 55 °C until constant weight. The results were used to calculated whole segment mass (mg) and specific leaf area (SLA—cm2.mg−1).
Experiment 2 (nutritive value): establishment and experimental control
Preparation of the experimental area (3,000 m2) started with the desiccation of previous vegetation (Arachis pintoi cv. Belmonte) using the broad-spectrum herbicides Glyphosate (N-phosphonomethyl-glycine) and 2.4-D (2.4-Dichlorophenoxyacetic acid) in Sept 08, Oct 01 and Nov 15, 2016. On Nov 30 the whole area was mowed and a final application of Paraquat (1.1′-dimethyl-4.4′-bipyridinium dichloride) was carried out in Dec 06 (blocks 2 and 3) and Dec 11, 2016 (block 1). The next steps followed the same protocol used in Experiment 1. Six buds were placed in each pit at 5 cm depth, covered with soil and gently compressed by hand. The distance between pits in lines and between lines was 1.0 m (Fig. 8).
General view of the experimental site during the establishment phase showing the layout and distribution of elephant grass plants on the area: (a) Pit opening and (b) planting of the stem cuttings.
A total of 1,320 plants were cultivated. These were divided in three homogeneous blocks with 440 plants each. The number of plants per block was dimensioned to provide the necessary amount of dried and ground samples for the nutritive value analysis. Twenty days after planting, plants were thinned leaving one plant per pit.
Treatments corresponded to phytomer order along the tiller axis and the experimental design was a randomised complete block, with three replications. Plants within blocks were randomised using the statistical package SAS (Statistical Analysis System, v. 9.0). Weed, pest and disease control was the same as for Experiment 1.
Sampling was carried out when the 16th leaf (phytomer 16) complete its expansion and exposed the ligule. Main tillers had all their leaves identified with permanent marker before harvest. The leaves were stored in ice and taken to the laboratory. Processing involved removal of the sheaths and blades stored in freezer for future segmentation. Leaves 5, 6 and 7 were discarded because they were in advanced stage of senescence and decay, leaving leaves 8 to 16 for the analysis.
Nutritive value
After all field work was finished, leaf blades had their length measured (distance between the tip of the leaf and the ligule) and were fractionated in five segments of similar length, as described for leaf anatomy measurements in Experiment 1 (i.e. chemical analyses included all tissues of the leaf blade). These were pooled into a composite sample per leaf hierarchical order, totalling 135 samples (9 leaves × 5 segments × 3 blocks). These were put to dry into forced draught oven at 55 °C until constant weight.
After drying, because the apical portion of the leaves was too delicate, the dried material was ground in a micro mill with a 1 mm sieve (Wiley Mill, Thomas Scientific, Philadelphia, PA, USA) as a means of reducing dry matter losses. Ground samples were subjected to the following chemical analysis: in vtiro dry matter digestibility (IVDMD), using the artificial rumen fermentation device DAISYII from ANKOM Technology Corporation45; total nitrogen (Ntotal), determined by the Dumas combustion method using the Leco FP 528 System (Leco Instruments Inc., St. Joseph, MI, USA); and ashes (ASH) determined according to Silva & Queiroz46. Crude protein (CP) was calculated as Ntotal × 6.25. For the in vitro trial, rumen liquid was collected from one rumen-cannulated Nellore steer fed only with Tifton-85 (Cynodon dactylon spp.) haylage.
Statistical analysis
Leaf blade morphology and anatomy data were initially analysed using descriptive statistical analysis (means and standard error of the mean). Leaf blade total mass and central rib total mass were obtained by adding values for all ten segments from each leaf and were subjected to a regression analysis as a means of identifying the correlation between these two variables. Regression was performed using the procedure PROC REG of SAS (Statistical Analysis System, v. 9.0).
Nutritive value data were subjected to ANOVA using the procedure PROC GLM of SAS (Statistical Analysis System, v. 9.0), and means compared by Tukey test (P < 0.05). The statistical model considered phytomer and block as fixed effects:
$$Y_{ij} = mu + B_{i} + F_{j} + varepsilon_{ij}$$
where Yij : average response of the jth phytomer in the ith block; µ : mean; Bi : block i; Fj : phytomer j; εij : random error ~ NID (0;σ2ε). All assumptions for these analyses were verified as homogeneity of variances, error normality, outliers, and need for transformation by Box–Cox47.
A Principal Components Analysis (PCA) was carried out with the objective of integrating the leaf morphology, leaf anatomy and nutritive value results. The data set was comprised of the following response-variables: Morphology—blade length, segment length, blade width, central rib width, total width (blade + central rib), blade mass, central rib mass, total mass (blade + central rib) and specific leaf area; Anatomy (percentage of the cross sectional areas for each tissue)—mesophyll, vascular bundles (parenchymatic sheath of vascular bundles + vascular tissue), total epidermis and sclerenchyma; and Nutritive Value—crude protein, in vitro dry matter digestibility and ash (data from Experiment 2 when all leaves were harvested at full expansion of leaf 16). Because leaf blades were fractioned in ten segments for morphology measurements and five segments for anatomy and nutritive value measurements, segments from the morphology measurements were combined two-by-two to balance segment number from all measurements (1 + 2, 3 + 4, 5 + 6, 7 + 8 and 9 + 10 to form 1, 2, 3, 4 and 5, respectively). As a result, all response variables had blade segments associated with them. PCA was performed using the procedure PROC PRINCOMP and the biplots were generated using the procedure PROC PRINQUAL of SAS (Statistical Analysis System, v. 9.0).
Cluster analysis was performed using the same PCA data set with the unweighted pair-group method with arithmetic mean (UPGMA) using the procedure PROC CLUSTER of SAS (Statistical Analysis System, v. 9.0) as a means of identifying phytomers groups according to the similarity of their elements.
Source: Ecology - nature.com
