Población diana

4.2.1

Herbage sampling

Details of sward establishment and management are given in sections 3.2.1 and 3.2.3. Briefly, replicated (n = 3) monoculture swards of five forage species were sown in a randomised split-plot design (plot size 9 x 6 m) in October 2014 at DairyNZ’s Scott Farm property, near Hamilton, New Zealand (37°47′S, 75°19′E; 40 m.a.s.l) on a Horotiu silt loam. They were: perennial ryegrass, white clover, lucerne, chicory, and plantain.

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The herbage material used in this study was obtained from harvests of plots receiving 200 kg N/ha/y, corresponding with the last month of each season: February (summer), May (autumn), August (winter) and November (spring) during the 2015 year. Collection occurred when each forage reached target harvest criteria (described in Section 3.2.3) and because the forages differed in growth rate, they were not necessarily on the same date. Harvest was conducted between 0900 and 1100 h to minimise variation in chemical composition and turgor pressure in the material. Once sampling was complete, samples were stored in a refrigerator (4°C) until processing was complete.

4.2.2

Herbage physical characterisation

Herbage DM content was determined by weighing and oven-drying three subsamples of 200 ± 5 g wet weight at 95°C for 48 h.

Botanical composition of the herbage was determined by hand sorting sub-samples (200 g) into green leaf, dead leaf, stem, petiole, and unsown species. Sorted samples were dried in a forced- draught oven at 95°C for 48 h to determine botanical composition on a dry weight basis.

For morphological measurement, from each sample, 100 pieces were randomly sampled and the length of the stem, and the length and width leaf blade were measured using a standard ruler or digital callipers where the leaf or stem was less than 20 mm in size (Mitutoyo digimatic CD-8"CS, Mitutoyo Corp, Japan).

Leaf and stem strength was described by measuring the force (Newtons) required to punch a 2 mm diameter hole in the material using a digital force gauge (DS2 Digital force gauge, Imada Inc., Japan). Detailed methodology is given in Chapter 3 (section 3.3.1). In brief, for each sample 50 punch force measurements were taken for each component (leaf, stem and petiole) present in the sample. The physical strength of the herbage was estimated by multiplying the average force required to punch each component by the percentage of that component in the herbage, as determined by the botanical composition measurement.

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The relative toughness of the herbage was measured by determining the amount of energy required to mechanically macerate the forage material using a Kreft Compact mincer (Kreft, Germany) (methodology detailed in Chapter 3, section 3.3.2). Macerated material was collected and frozen, for evaluation of the extent of comminution (particle size distribution) and nutrient release by maceration (section 4.2.4).

4.2.3

Herbage chemical characterisation

A 200 g sub-sample was frozen at -20°C, freeze dried and ground to pass through a 1 mm sieve (Christy & Norris Mill, United Kingdom) for chemical analysis. Structural carbohydrates, neutral detergent fibre (NDF) was measured using the methods of Van Soest et al., (1991), and acid detergent fibre (ADF) by method 973.18 (AOAC Int. 2000), using Whatman 934-AH glass micro- fibre filters with a 1.5 µm particle retention. Fibre recovered from the ADF measurement was washed in sulphuric acid, then dried and ashed for 2 h to determine lignin content as per Goering and Van Soest (1970). Water soluble carbohydrate content (WSC) was measured colorimetrically with sucrose as a standard, by the method of DuBois et al. (1956). Total N concentration was determined by combustion (method 990.03; AOAC Int. 2000) in a LECO FP-528 Nitrogen analyser (Leco, St Joseph, MI, USA). Nitrogen in the NDF residue (neutral detergent insoluble N, NDIN) was measured in the residue from the NDF procedure also using the LECO FP-528 analyser. Concentration of total non-protein N components was assessed by the urease method (941.04; AOAC Int. 2000) and nitrate by the potentiometric method (986.31; AOAC Int. 2000). Total ash concentration was determined by heating 1.5 g of sample to 600°C in porcelain crucibles for 4 h (method 942.05; AOAC Int. 2000).

4.2.4

Herbage comminution and cell content release

The effect of maceration on the physical breakdown (comminution) of the herbage was determined by evaluating the particle size distribution (PSD) of the macerated herbage using a wet sieving apparatus (Turner and Newell Ltd) with four counter-rotating sieves. Sieve sizes (as the length of the size of the square hole) were 4, 2, 1, 0.5 and 0.075 mm. Duplicate subsamples of 25 g (fresh weight) were placed on the top sieve, then 1500 ml of water recirculated through all sieves at a flow

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rate of 4 litres/minute for 5 minutes. Material retained on each sieve was transferred on to weighed filter papers then oven-dried at 60 ºC for 48 h before weighing to determine PSD on a DM basis. The soluble fraction was defined as the DM passing the 0.075 mm sieve, and was calculated as the difference between the total DM sieved and the sum of the DM retained on the five sieves.

For determination of nutrient release from maceration, 100 g sub-samples of macerated herbage were placed in weighed dacron bags (mean pore size 35 µm; Ankom Technology, USA), the bags sealed and then rinsed carefully by hand until no further colour appeared in the rinsing solution. Rinsed samples were freeze-dried, weighed, ground to pass a 1 mm sieve (Christy & Norris Mill, United Kingdom), then analysed for NDF, N and WSC using the methodology described in section 4.2.3. The percentage of N and WSC released was defined as the difference between the quantity of N or WSC in the unmacerated forage material and the quantity in the macerated material divided by the quantity in the unmacerated material. The weight of the sample pre- and post-rinsing was used to correct for loss of DM.

4.2.5

Statistical analyses

Data for each variable were analysed in a repeated measures analysis using a restricted maximum likelihood (REML) approach with mixed models including forage and season as fixed effects, and replicate and plot within replicate as random effects. The data for each season were then analysed using REML including forage as a fixed effect. When a significant forage effect was observed, multiple comparisons were made using Fishers protected least significant difference test.

Relationships between variables were investigated using regression analysis to fit linear equations, first using the mean values for each forage to test for associations between variables using between forage variation, then using individual replicates to test the association within forage using environmental variation including seasonal variation. The regression analysis provided slope, SE of the slope, P-value and the R2 _{coefficient. A multiple regression analysis was conducted using an}

all sub-sets regression to find the best fitting model to predict CP loss and mincing energy. The predictive models were determined using a maximum of five variables. One forage was selected at

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random and omitted from the analyses; data from the other four forages were then included to test predictions for the excluded forage. GenStat version 16.2 (VSN International, Hemel Hempstead, UK) was used for all statistical analyses.