General procedure

3.2.2.1 Experiment 1

The same three high-producing Holstein-Friesian dairy cows were used in three consecutive trials each of four days (spring 2009, then summer and autumn 2010). Mean live weight of the cows were 584 + _{5 kg; 552} + _{9 kg and 579 ± 6 kg for spring summer and autumn, respectively.}

Milk production 8week after calving was 34.7 + _{3.8 litres milk/day and 4week before drying off}

was 18.1 + _{3.3 litres milk/day. Every day the cows were removed from their paddock at 0430h}

for morning milking and returned to their paddock at 0700h. For afternoon milking the cows were removed from the paddock at 1430h and returned at 1700h, where they received a new fresh pasture allocation.

Pasture mass was measured daily for each break and pasture samples were taken by walking diagonal lines, and taking a 10 cm2 _{hand-plucked sample to 2.5cm height (1450 kgDM/ha)}

within the leaf horizon, every 6th _{step. Samples were immediately frozen at -20°C for later}

chemical analysis. A summary of the general procedure for the experiment 1 is shown in Figure 3.1.

3.2.2.1.1Rumen fill and fluid fractional disappearance rate

To quantify diurnal variations of the rumen pools, three rumen evacuations were carried out for each animal at 16h intervals to preclude any effects on rumen function, in each of the three seasons. The first rumen evacuation was performed at 0900h (day 3); the second and third rumen evacuations were carried out at 0100h and 1700h, respectively (day 4). The evacuation times were chosen to be representative of the assumed minimal (0900 and 1700h) and maximal (0100h) periods of rumen fill. Cows were weighed prior to, and immediately after rumen evacuation. Rumen evacuations were executed as described by Taweel et al (2005b) with some minor modifications as explained below.

Rumen contents were emptied manually, using portable yards at the site of grazing, into an insulated container inside a bucket of sufficient warm water to maintain the temperature of the removed rumen contents at 38ºC ± 1.5. All the digesta was weighed, fully mixed and sampled for later analyses. Two of the fistulated cows were rumen-evacuated simultaneously and the time required for each evacuation was recorded and kept under 30min. Before and after each rumen evacuation the animals had full access to pasture and water to mimic as close as possible the typical grazing management of the herd. Standardisation of the grazing allocations was ensured using individual hot wire confinement for each cow with pre and post grazing pasture mass assessment.

To estimate rumen fluid fractional disappearance rate, the external marker PEG 4000 was added to the rumen contents during the first evacuation on day 3 0900h (80g of PEG 4000 in 500ml of water). A second dose of PEG was added directly to the rumen through the rumen cannula 24h after the first application (day 4 at 0900h).

3.2.2.1.2Rumen environment, faecal and urine assessment

To determine the diurnal pattern of rumen metabolites and rumen pH, on day 2 rumen content samples (solid and fluid) were taken from the ventral sac every two hours for a period of 24h. The pH of the rumen fluid samples was immediately measured using a pH probe (Ionode IJ-44, Brisbane, Australia) and temperature sensor (PT-100. Bell Technology, Auckland, NZ). Additionally to the two hourly sampling of rumen content, representative samples of faeces and urine were simultaneously sampled for later analyses.

Figure 3.1. General schedule in all seasons for experiment one of sampling, rumen evacuations (R.E) and, infusions of the external markerpolyethylene glycol (PEG). Rumen fluid fractional disappearance rate (%/h) was calculated between 0900h (day 3) and 1700h; and 0900h (day 3) and 0100h. Ventral sac, faeces and urine sampling was conducted (24h sampling).

3.2.2.2 Experiment 2

Three high-producing Holstein-Friesian dairy cows (556 + _{74 kg BW) in mid lactation were}

used. The cows produced 21.7 ± 5.2 kg/day of milk a day at the start, and 22 ± 3.1 kg/day at the end of the experiment during spring over the five days of the experiment. Pasture allocation and milking management were described in section 3.2.1.

3.2.2.2.1Rumen fill and rumen fluid fractional disappearance rate

Four rumen evacuations, performed as in experiment one, were carried out for each cow every 8h, from 0100h to 0900h and from 1700h to 0100h (Figure 3.2). Within the two consecutive rumen evacuations the cows were fasted with water available all the time. Prior to and between periods of rumen evacuations cows were grazed as described in section 3.2.1. In brief, for the

51 first period of rumen evacuations (0100 – 0900h) the cows were removed from the paddock at 0100h and returned to their paddock just after the second rumen evacuations (0900h). In the second period of rumen evacuations, the cows left their paddock at 1430h for afternoon milking and they returned to their paddock after the second rumen evacuation (0100h).

To estimate rumen fluid fractional disappearance rate within each rumen evacuation period PEG was added to the mixed rumen content in day 2 (0100h) and day 4 (1700h) as described for experiment one (section 3.2.2.1.1). Rumen samples for PEG assessment were taken after adding the external marker at 0100 and 1700h and 8h after the addition of the marker at 0900 and 0100h, respectively.

Figure 3.2. General schedule of adaption, rumen evacuation (R.E.), mixing of polyethylene glycol with rumen contents and access to feed during experiment two. Rumen fluid fractional disappearance rate (%/h) was calculated between 0100h and 0900h (day 3); and 1700h and 0100h.